Abstracts

Calibrant Biosystems, Inc.
Academic Partner: University of Southern California

1 R41 CA122715-01 (STTR)

Because of the long history of the use of formalin as the standard fixative for tissue processing in histopathology, there are a large number of archival formalin-fixed and paraffin-embedded (FFPE) tissue banks worldwide. These FFPE tissue collections, with the attached clinical and outcome information, present invaluable resources for conducting retrospective protein biomarker investigations. However, the high degree of covalently cross-linked proteins in FFPE tissues hinders efficient extraction of proteins from tissue sections and prevents subsequent bioanalytical efforts from opening the door to a veritable treasure trove of information sequestered in archival tissue banks. Thus, this project aims to address methodological optimization for achieving effective protein extraction from FFPE tissues together with technological development for performing comprehensive and comparative studies of protein expression profiles within FFPE tissue specimens. By combining Calibrant’s ability to enable proteomic profiling from minute protein samples with the technology and expertise offered by Professor Clive R. Taylor (University of Southern California School of Medicine) in antigen retrieval (AR)-immunohistochemistry (IHC) and tumor pathology, the proposed research represents a synergistic effort toward the evaluation and validation of a novel biomarker discovery paradigm on the basis of years of archived FFPE tissue collections. The specific aims for R41 Phase and the respective scientific milestones are: Specific Aim 1. Evaluation and optimization of protein recovery from FFPE tissues using AR approaches coupled with Gemini proteome platform (Months 1-12). Scientific Milestone: Selection of an optimal protein recovery protocol to be employed in proteome investigations using a “test battery” AR approach and IHC staining for a panel of 10-20 proteins. Specific Aim 2. Validation and quantification of antigens retrieved from FFPE tissue specimens (Months 13-24). Scientific Milestone: Demonstration of a sample consumption of 5 microgram total protein or less for an average of five peptides in each protein identification and the identification of at least 3,000 high confidence proteins with greater than 80% reproducibility in identified proteins among triplicates of identical FFPE tissue using combined AR and Gemini technologies. Direct comparison of IHC results and proteomic display will be attempted for a panel of 10-20 proteins.

Calibrant Biosystems, Inc.
Academic Partner: Yale University

1 R41 CA122745-01 (STTR)

The generation of biologically relevant proteomics data requires samples consisting of homogenous cell populations, in which no unwanted cells of different types and/or development stages obscure the results. The problem is compounded for the analysis of tissue biopsies, since many different cell types are typically present, and small numbers of abnormal cells may lie within or adjacent to unaffected areas. While methods such as laser capture microdissection (LCM) enable the isolation of homogeneous subpopulations of cells, proteomic analysis of LCM-procured specimens is severely constrained by the very low amounts of sample generated. To avoid the limitations of established proteome techniques for analyzing protein extracts obtained from microdissection-procured tissue specimens, an effective discovery-based proteome platform has recently been developed at Calibrant. This proteome platform, called Gemini, combines a unique multidimensional separation system with customized back-end bioinformatics tools, and allows ultrasensitive analysis of minute protein amounts extracted from cells captured by tissue microdissection. This project further aims to employ a novel, laser-free microdissection technique pioneered by our collaborator, Dr. Zhengping Zhuang at the National Institute of Neurological Disorders and Stroke (NINDS), capable of providing enriched, high quality, and reproducible tissue samples. By combining Calibrant’s ability to perform proteomic profiling from minute samples with the technology and expertise offered by Dr. Zhuang (NINDS) in tissue microdissection and tumor pathology, the proposed research represents a synergistic effort toward the evaluation and validation of a novel biomarker discovery paradigm for enabling the proteome analysis of cancer cells and their micro-environment in support of cancer research, diagnosis, and treatment. Application of the resulting biomarker discovery platform for studying the molecular mechanisms associated with breast carcinoma at the global level will be realized through a collaboration with Professor Fattaneh A. Tavassoli (Yale University School of Medicine), who will apply more than 30 years of research experience in breast cancer pathology and biology and provide access to a collection of fresh frozen human breast cancer biopsies for biomarker discovery.

Cell Preservation Services, Inc.

1 R43 CA118537-01A1 (SBIR)

The intent of this project is to develop improved methods for the cryopreservation of human cancer cell lines and tumor biopsies. While there has been a current emphasis on the importance of improving biobanking of tissues (GEN, 2/1/05), there has been little research dedicated to improving the archival storage of cancer samples. Cell Preservation Services Inc. (CPSI) develops hypothermic storage (HypoThermosol, “HTS”) and cryopreservation (CryoStor) solutions marketed by CPSI’s partner, BioLife Solutions. These solutions are used as the shipping/storage solutions in Regenerative Medicine applications such as cellular cardiomyoplasty. The fully defined, serum-free CryoStor platform is designed for cell and tissue storage in liquid nitrogen, requires reduced DMSO levels, and improves cryopreservation efficacy up to 50% compared to traditional protocols. Yet, even the CryoStor series can only protect the viability of cancer cells to a maximum of 70%. Thus, this “cryopreservation cap” must be attacked so as to achieve optimal preservation. CPSI is able to develop improved HTS and CryoStor solutions given its ability to investigate and modulate the cell death cascades that can be initiated due to extended storage. The purpose of this Phase I Project is to develop a new platform of solutions called CryoStor-CANCER - a series of solutions expressly designed for the improved cryopreservation of human cancer cells and tumor biopsies. CPSI will (1) use cDNA microarrays to determine if the analysis of stress pathways activated by cancer cells undergoing cryopreservation can lead to improved preservation solutions for cancer cells and tumor biopsies; (2) determine if the same analysis of stress pathways might lead to “rescue solutions” that can reverse the adverse effects of sub-optimal cryopreservation and; (3) determine if sub-optimal preservation results in a long term change to the expression of any of the 400+ genes that are most often studied in cancer biology. Phase 2 studies will test the prototype CryoStor solutions on a wider variety of cancer cell and tumor types, and determine if cryopreservation and the proposed “rescue solution” preserve or rescue the oncological fingerprint of cancer cell lines and tumor tissues. This work will be important to agencies that are currently developing improved methods for biobanking of cryopreserved cancer specimens. As a result, archival storage of cancer tissues will be improved and poorly preserved specimens can be rescued.

Ambion, Inc.

1 R43 CA116218-01 (SBIR)

Cervical cancer, caused by human papillomaviruses (HPVs), is a major public health problem, worldwide. About 230,000 women die of cervical cancer every year, the majority in developing countries. Although early detection via routine cytological screenings (Pap smears) and HPV testing have lowered both the incidence and mortality of cervical cancer, significant problems and barriers remain, including the low predictive value of current testing. As many as 3 million Pap smears are classified as inconclusive in the US every year leading to costly and invasive follow-up procedures and emotional stress in patients. MicroRNAs (miRNAs) are small, regulatory RNAs encoded by the plant, animal, and fungal genomes that act to inhibit expression of specific target genes. Recent studies have shown that miRNAs play key roles in many cellular processes, including development and differentiation. The role of miRNAs in the development of diseases and cancers has just begun to emerge. Furthermore, recent data show that many viruses encode miRNAs that regulate both viral and cellular gene expression to establish and maintain productive infection. Work at Ambion and in Golub’s lab has shown that miRNAs are differentially expressed in specific types of cancers, providing the first evidence that miRNAs can be used to classify human tumors and develop diagnostic assays. Our hypothesis is that miRNAs are involved in the host-cell response to HPV infection and in HPV- induced cellular transformation, and that HPVs, themselves, encode miRNAs that are involved in these processes. We propose to investigate host cell and viral miRNAs involved in HPV infection for the purpose of better understanding the natural history of HPV infections and the early events that lead to the onset of cervical cancer. In Phase I, we will use a miRNA profiling system to analyze the host cell miRNA response to HPV infection and transformation in HPV-positive cell models and cervical biopsies. We will also explore the identification and validation of HPV-encoded miRNAs and determine their relevance to HPV infection and transformation. Phase II will encompass wider evaluation of the identified cellular and viral miRNAs in clinical samples as potential biomarkers and a diagnostic assay will be developed based on these miRNAs.

Ambion, Inc.

1 R44 CA118785-01 (SBIR)

During Phase I of our proposed research, we will develop and validate procedures for recovering, labeling, and analyzing miRNAs from fixed tissue samples. The procedures will be based on the miRNA microarray and fixed tissue RNA isolation systems that we developed in other SBIR-funded programs. The development of our miRNA isolation and labeling procedures will be accomplished using a model system wherein mouse organs will be split and half is flash-frozen and the other half formalin-fixed using a procedure that is commonly employed in hospitals. The frozen and fixed samples will be processed to recover the miRNAs. The miRNAs from the fixed and frozen tissues will be independently labeled and analyzed using miRNA microarrays. The isolation, labeling, and hybridization procedures will be varied until the fixed samples yield the same miRNA expression profiles as the equivalent frozen samples. The fixed sample procedures will) then be used to analyze formalin fixed, human tissue samples to analyze miRNA profiles from multiple organs. The fixed tissue miRNA profiles will be compared to the profiles generated from frozen samples to verify that the fixed tissue miRNA profiling process can be used for stored, human fixed tissue samples. During Phase II of our research project, we will use the miRNA isolation, labeling and microarray analysis procedures developed during Phase I to analyze archived, fixed human cancer tissues to identify miRNAs with expression profiles that are significantly different from equivalent, normal tissues. The most interesting miRNAs or miRNA signatures might provide opportunities for diagnostic/prognostic assay development or even an intervention point for therapeutic agents.

Bionanomatrix

1 R43 CA120611-01 (SBIR)

We are developing a nanochip device for manipulating long genomic DNA for high-resolution (kilobase), whole-genome analysis of cancer biomarkers such as gene amplifications, deletions, and translocations. These chromosome structural aberrations are strongly implicated in the process of malignant transformation and are important diagnostic, prognostic, and therapeutic indicators for many types of cancer. Although PCR offers the ultimate (single-base) resolution for detecting and analyzing these anomalies, it is impractical for scanning the entire genome in a comprehensive, linear fashion. Techniques that rely on probing chromosomes, such as metaphase FISH, while providing a pan-genomic view, cannot resolve structures below the Mb range. By probing uncompressed interphase DNA, resolution can be improved, but spatial organization of the genome is lost, so multiplexed and quantitative information is difficult to obtain. By stretching out (linearizing) interphase DNA, using techniques such as “molecular combing” or “optical mapping,” it is possible to probe specific loci in a spatially significant way, and with resolutions in the kb range. However, techniques for mechanically linearizing DNA are inherently variable, leading to inconsistent stretching of molecules, which often cross over and retract upon themselves. This makes it difficult to standardize such techniques as high-throughput methods for the biomedical community. We are developing an innovative alternative to mechanical stretching of DNA. We have found that individual DNA molecules, because of the self-avoiding nature of the DNA polymer, will elongate and straighten in a consistent manner when streamed into confining nanometer-scale channels (nanochannels). We have used a novel nanoimprint lithography technique to reliably manufacture nanochannel structures in silicon chips and have demonstrated that DNA in these nanochannels can be visualized and their dimensions measured. We now ask the question, can we quantitatively interrogate this linearized DNA with locus-specific probes for the detection of chromosome structural aberrations associated with cancer? Our product, the nanochannel array chip, will comprise part of an integrated platform for the routine and standardized quantitative analysis of DNA structure that will enable archiving and cross-laboratory comparison of data.

Department of Clinical Pathology, Cleveland Clinic Foundation
Industrial Partner: Cleveland Biolabs, Inc.

1 R41 CA110584-01A2 (STTR)

The development of cancer patient pre-treatment screenings which utilize genetic markers to stratify the patient to a particular therapy is one of the main new directions in improving quality of anti-cancer therapies. Gliomas, the most common primary brain tumors, represent a specific type of cancer with well-characterized prognostic genetic markers that predict patient survival and response to chemotherapy. These alterations include loss of chromosomes 1 p, 19q and 10q, gain of chromosome 7 and 19q, amplification of oncogenes, and deletions/mutations of tumor suppressor genes PTEN, TP53, and CDKN2A. Routine genotyping of glioma patients for loss of 1 p/19q and amplification of the epidermal growth factor receptor (EGFR) gene has been available at the Cleveland Clinic since 2001 and has already affected the treatment of oligodendroglioma patients. Still, only few molecular markers are routinely tested in a number of leading clinics in the United States. Current genotyping assays are based on loss of heterozygosity (LOH), fluorescent in situ hybridization (FISH), and direct sequencing techniques which are time consuming and focused on only a handful of molecular markers. We propose the development of a high-throughput genotyping assay which will provide simultaneous analysis of multiple genetic alterations, including mutations of selected genes, allelic imbalances, and copy number alterations. The assay will be focused on previously identified molecular prognostic markers including both well-validated, as well as recently discovered markers with potential prognostic value. This combination will accelerate validation of novel markers through fast accumulation of data on genetic alteration in tumor specimens. The assay technology is based on custom oligonucleotide microarrays manufactured by NimbleGen Systems, Inc., using recently developed maskless lithography which allows easy adjustment and replacement of the oligonucleotide probes as necessary. The sample preparation procedures will be adjusted for use of tumor DNA derived from formalin-fixed paraffin-embedded tissue from the pathology archive. Analysis of archival tissues will enable us to perform retrospective studies using clinically and genetically characterized tumor specimens. The significant increase in the number of analyzed genetic markers will result in more accurate prognosis at lower cost, will lead to the discovery and validation of additional prognostic markers, and will improve treatment stratification of glioma patients.

SynBiogen, Inc.

1 R43 CA120937-01 (SBIR)

The ability to monitor and measure protein kinase activity in tumorigenesis and cancer can be indicative of and critical to the transformation process and therefore represents an attractive diagnostic strategy, with a multibillion dollar market opportunity. In this proposal, we aim to develop a novel, high-affinity fluorescent nanosensor and homogeneous assay system for monitoring the phosphorylation of a bona fide peptide substrate by c-Abl, an important kinase involved in the etiology of chronic myelogenous leukemia (CML), using FRET. The technology deployed during this phase of the proposal will directly translate into the development of a sensitive platform for the diagnosis of CML. In specific Aim 1, we will synthesize the high-affinity nanosensor. In specific Aim 2, we will develop a sensitive one-step assay using this reagent. Specific Aim 3 will elaborate on this and test the effectiveness of the nanosensor in measuring c-Abl activity in CML-positive cell lysates. Given the fact that ~400 disorders such as cancer have been associated with protein kinases, the development of a family of sensitive nanosensors such as the one proposed in this grant application will facilitate the diagnosis of these diseases sensitively and selectively and therefore becomes of paramount importance and will find immense utility in all the various facets of our health care, from drug discovery to patient health and point-of-care diagnostics.

S20/20 Gene Systems, Inc.
Academic Partner: University of Kentucky

1 R41 CA118625-01A1 (STTR)

Tumor markers, measured in peripheral blood, could assist in diagnosis and management of non-small cell lung cancer (NSCLC) and potentially improve historically dismal outcomes. Circulating antibodies, generated to a wide range of tumor-associated proteins, can be translated into a valuable blood test for lung cancer. Preliminary data supports this hypothesis. We have successfully used phage-display, biopan enrichment techniques and high throughput fluorescent array screening to identify multiple known and unknown tumor-associated proteins specifically recognized by circulating tumor-associated antibodies NSCLC patients but not in normals. A panel of phage-expressed proteins arrayed on a glass slide microarray used to measure tumor-associated antibodies in serum from a cohort of cancer patients and risk-matched controls affords predictive accuracy that exceeds that of currently available circulating NSCLC-associated protein markers. Although fluorescent microarray system is an ideal tool for identifying proteins recognized by tumor-associated antibodies, it is not a commercial-ready platform. The intent of this application is to incorporate these markers into a layered protein array (LPA), a 96-well ELISA type platform that has been developed for clinical diagnostics. The high-throughput format of the LPA allows measurement of multiple antibody markers simultaneously will be central to the application is a perfect complement to biomarker identification. The LPA will be initially constructed and tested using a panel of proteins that have already been identified. Our initial application will be early detection of lung cancer, although multiple applications in lung cancer management are rational. Data shows feasibility and proof of concept that supports the rationale for further development and testing of this approach. Subsequent .Phase II application will evaluate an assay developed in this Phase I project for application to screening of NSCLC. Thus the primary goal of this application is to develop a novel blood test for NSCLC that can be rapidly translated into clinical practice. Success in this project will herald similar development in other malignant diseases. Relevance to Public Health. A blood test for lung cancer could improve the capability and cost- effectiveness of early detection as a viable strategy for reducing mortality from this disease. Relevance to Public Health. A blood test for lung cancer could improve the capability and cost-effectiveness of early detection as a viable strategy for reducing mortality from this disease.

Cellonix Corporation

1 R43 CA120619-01 (SBIR)

The main objective of this proposal is to develop a microfluidic platform for cancer drug toxicity screening in cultured human cells. While it is believed that improved information on a patient’s individual cancer signature can aid diagnosis and treatment, the technology available to validate this claim is currently limiting. The long term goal of this work is to commercialize a microfluidic screening platform to provide a compact, low cost, automated screening system that can be used in the clinical setting. The specific aims of this proposal are to automate a previously developed microfluidic cell culture array and to demonstrate the feasibility and reproducibility of cancer drug toxicity screening in the microfluidic format. The design and fabrication of the addressable 8x3 unit microfluidic array will leverage expertise developed within the company related to soft lithography technology. Automation of fluidic delivery through the array will be accomplished through implementation of novel microfluidic valves controlled with an industrial pneumatic interface. Initial demonstration of cancer cell cytotoxicity will be collected on HeLa cells over 7 days exposure to anticancer drugs such as etoposide. Cell viability as well as apoptosis kinetics (quantified by fluorescence assay) will be collected in the array and experimental robustness determined. Response and statistical uniformity will be compared to the same assay performed in a 96-well plate. The commercialization of the microfluidic platform can improve public health by providing a reliable, cost effective instrument that can be used for personalized cancer diagnosis in the clinical setting. This technology overcomes current limitations by reducing the cost of automated cell analysis through the scalability of microfabrication, and by enabling multiplexed assays on a small amount of patient tissue through reduced sample volume. A similar platform can also be adapted for molecular screening in cancer cell biology and for improved high throughput drug discovery.

Arbor Vita Corporation

2 R44 CA121155-01 (SBIR)

Arbor Vita Corporation (AVC) has developed a novel cervical cancer diagnostic based on an understanding of the biology of human papillomavirus (HPV). HPV infection is one of the most common sexually transmitted diseases with an estimated 5.5 million new infections per year in the United States alone. High-risk (oncogenic) HPV types are correlated with virtually all cervical cancers. Pap smear and liquid-based cytology screening has greatly reduced the incidence of cervical cancer, but the Pap test has both high false-negative and false-positive rates and requires an extensive infrastructure of trained cytologists to interpret the results. A cheaper test with greater reliability and predictive value would be of great clinical benefit. The virally encoded E6 and E7 proteins of high-risk HPV types have been shown to be essential for cell transformation and cancer progression and E6 proteins from high-risk HPV types, but not low-risk HPV types, are known to bind cellular PDZ domains. AVC has extended those studies and demonstrated a perfect correlation between high-risk HPV and E6-PDZ binding. Based on these findings, we have developed a novel cervical cancer diagnostic assay of HPV E6 using PDZ protein capture. In our SBIR Phase I, AVC developed a novel PDZ capture sandwich ELISA test for HPV E6 that detects over 75% of high-risk HPV types and demonstrated its utility with human cancer samples. We were able to begin quantifying E6 from cells and improved sensitivity to allow E6 detection in a much smaller sample than typically collected in a Pap test. In Phase II, we propose to complete development of our prototype PDZ-based cervical cancer test in preparation for clinical trials. Specifically, we propose to (1) Expand our antibody detection to include 95% of known high-risk HPV types, (2) Optimize clinical sample handling for E6 protein detection, (3) Optimize the PDZ/antibody sandwich ELISA for clinical laboratory implementation, and (4) Extend the studies correlating high-risk E6 protein levels and clinical cytology staging.

Cy Vera Corporation

1 R43 CA118536-011 (SBIR)

CyVera Corporation proposes to develop and validate the feasibility of a rapid, robust, and inexpensive method for performing multiplexed protein expression measurements. These measurements are needed for the early detection, diagnosis, and management of patients with cancer. This cancer diagnostic platform will be based on the combination of (i) CyVera’s newly developed holograpically encoded, multiplexed microparticle assays, (ii) self-assembly, and (iii) microfluidics. The format we propose will allow rapid and highly sensitive detection of protein expression patterns in small sample volumes, and will ultimately lead to a high-throughput instrument platform for cancer diagnostics. In Phase I of this project, prototype microfluidic devices will be constructed with antibody functionalized particles. Batches of individually encoded glass particles will be antibody functionalized, pooled, and self-assembled into microfluidic devices. Once assembled, the identity of each type of particle will be read via its holographic code. Five detection analytes in Phase I will be chosen from a set of putative cancer biomarkers. These commercially available markers will include von Willebrand factor (vWF), C-reactive protein (CRP), albumin, free Prostate Specific Antigen (fPSA), and complexed PSA (cPSA), all of which have been reported as prostate cancer biomarkers in the literature. The limit of detection and repeatability of each analyte will be assessed via spike-in experiments in serum samples. The goals of Phase I will be (1) to demonstrate <10 pg/mL sensitivity of each multiplexed analyte in a complex sample in under 1 hour, (2) low sample volume requirements of <10 microliters, and (3) ease of fabrication and replication of the microfluidic devices. Success in Phase I will pave the way for the development of an affordable tool for molecular cancer diagnostics and follow-up patient therapy monitoring.

Biochemistry and Cancer Biology, Medical University of Ohio, Toledo
Industrial Partner: Innotech Biopharma, Inc.

1 R41 CA118540-01 (STTR)

Ovarian cancer is the most common cause of death among cancers of the female genital tract owing to the lack of methods for early detection of the cancer. Despite the identification of a number of serum markers for ovarian cancer, the clinical application of such proteins is confounded by their low tumor expression levels, false-positive tests, and limitations of the assay methods. The glycosyl phosphatidylinositol (GPI)-anchored folate receptor (FR) type A is consistently expressed by non-mucinous adenocarcinomas of the ovary and uterus and in 20- 30 percent of breast carcinomas. In contrast to the receptor expressed in these tumors, the expression of FR-a in normal tissues is restricted to the luminal surface of certain epithelial cells that is not in contact with the bloodstream. The soluble form of FR-a is a serum marker for the receptor-rich tumors because a large proportion of the membrane anchored form is shed into the circulation both by proteolysis and by the action of serum phospholipase. Our recent studies strongly support a new methodological concept for the utilization of FR-a that addresses the current limitations of serum markers. Based on mechanistic studies in vitro and tumor xenograft model studies in vivo , we have established that brief treatment with innocuous doses of certain well-tolerated transcription modulators profoundly increases the levels of both membrane associated and serum FR-a produced by FR-a-positive malignant cells but not by the receptor-negative tissues. This finding offers a more effective means for early detection of FR-a -positive tumors. In addition, the ability of FRa to simultaneously bind both specific antibodies and folate conjugates will allow the development of superior assay methods (increased signal to noise ratio) incorporating two levels of selectivity for the protein in the serum. This proposal represents a major step in the development of an innovative technology for the application FR-a as a serum marker by manipulating its expression and also improving its assay. Upon detection by a serum assay, the tumor may be localized using an available FDA-approved FR-a-targeted imaging agent. In Aim 1, we will further develop antibody and folate conjugate probes to develop simple and high-throughput fluorescent assays for soluble FR-a. In Aim 2, we will utilize malignant cell lines in murine tumor xenograft models to establish optimal treatment regimens for induction of serum FR-a. This initial phase of developing the technology will provide a basis for future clinical studies.

GlycoFi, Inc.

1 R43 CA118539-01 (SBIR)

With antibody based drugs emerging as a powerful new source for cancer treatment, the ability to create specifically targeted, highly efficacious antibodies quickly and cost-effectively is vital to improving patient outcomes. GlycoFi’s novel yeast-based protein production platform is an emerging technology primed for development and delivery of clinical glycoprotein-based therapeutics. This project seeks to apply GlycoFi’s technology toward the production of a known cancer therapeutic with the long term objective of bringing new antibody based cancer drugs to the clinic. The influence of the structure of Asn297linked oligosaccharides on IgGs is well known with elements such as pharmacokinetic stability, antibody mediated cell cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) all affected by glycosylation. Through highly targeted metabolic engineering of the Pichia pastoris glycosylation pathway, GlycoFi has developed unique strains of yeast capable of secreting monoclonal antibodies with homogeneous, human glycostructures. Phase I explores the feasibility of producing three different human glycoforms of anti-CD20 mAb in GlycoFi’s “humanized” yeast. In Aim 1, the gene encoding anti-CD20 mAb will be cloned and expressed in strains of P. pastoris to produce three distinct glycoforms. The functionality of these glycoforms will be tested in vitro and compared to the commercially produced anti-CD20 mAb rituximab. Aim 2 looks at antibody binding to CD20-presenting Raji cells. Both ADCC and CDC have been implicated as mechanisms by which rituxan exerts its antitumor effect. As activation of ADCC requires antibody binding to Fcy receptors, Aim 3 analyzes the potential in vivo efficacy of these antibodies through in vitro receptor binding assays. Activation of the complement pathway requires antibody binding to C1 q, thus Aim 4 focuses on comparing C1 q binding abilities of the different GlycoFi-produced antibodies with rituximab. Phase II will focus on proving the clinical potential of GlycoFi’s anti-CD20 mAbs through ADCC and CDC assays and in vivo pharmacokinetic and efficacy studies. Relevance to public health: GlycoFi’s technology allows unprecedented control over glycosylation, providing the ability to improve the biological function of therapeutic proteins including monoclonal antibodies used for cancer treatment. In addition, P. pastoris is a robust protein expression host, secreting large volumes of therapeutic drugs that will translate into increased speed to the clinic and lower patient costs.

Microcosm, Inc.

2 R44 CA118466-01 (SBIR)

The fight against cancer, heart disease, HIV infection, and all diseases is slow. Lives are being lost. The public spends over $1 trillion a year on health care. The government spends that much again. The tool used most in the fight against cancer employs fluorescent dyes to indicate the presence of cancer, to discover new ways to test for cancer, and to discover medicinal cures. Millions of tests are performed each year using thousands of tiny spots on a microscope slide, called a microarray. These spots fluoresce when a sought-after biological indication is present. The problem is that this ubiquitous method of research and diagnosis can detect less than half of the biological information needed to end cancer because the fluorescent light signal is very weak. The proposed project will increase the sensitivity of fluorescence assays 1,000-fold. This innovation will revolutionize the battle against cancer and all diseases. The mission of NCI is to discover and develop new technology for the fight against cancer. This project will continue the development of metal-enhanced fluorescence (MEF) begun under a previous Phase I SBIR grant from NCI. MEF uses thin-film technology to deposit layers of metal particles and dielectrics on microarray substrates. MEF has been repeatedly proven to increase the fluorescence assay sensitivity 40- to 100-fold. This project will transfer MEF technology to a manufacturing environment, optimize the manufacturing protocols, and pilot test the first products. Cancer DNA assays will be performed at the NCI Microarray Center to evaluate and validate the performance of the new product. In addition, this project will integrate two commercially available assay technologies with the MEF microarray. The active surface of a microarray substrate must be able to bind biological material efficiently. GenTel BioSurfaces, Inc., provides surface chemistry which is proven to increase binding by a factor of 7-10 compared to all other available substrates. Their surface chemistry combines with MEF to produce an assay sensitivity increase in the range of 100 to 1,000. Martek sells super bright fluors that are proven to increase the fluorescence signal 20- to 300-fold compared to conventional fluors. Microcosm has proven that MEF increases the light output of Martek fluors another 40-fold. The integration of Martek labeled assay reagents, Gentel’s surface chemistry, and Microcosm MEF substrates promises a multiplicative increase in assay sensitivity exceeding 1,000-fold. Martek and Gentel products are on the market. Based on over 3 years of development at Microcosm, this integrated MEF substrate is ready for commercialization. With this project, a universally needed and revolutionary new microarray assay product line can enter the market in less than 2 years.

OneCell Systems, Inc.

1 R43 CA120297-01 (SBIR)

Development of improved procedures for sample preparation and storage of biological specimens are critical for diagnosis of human diseases, particularly cancer, for which positive cells are rare. Recent improvements in sample preparation and storage of cervical specimens led to significant increase in sensitivity and accuracy of cervical cancer screening. Although important, there have been few improvements in sample preparation, handling and storage of other biological specimens (e.g. fine needle aspirates or other biopsy materials). Problems associated with specimen handling include cell loss and cell clumping. Sample loss resulting from conventional biological specimen processing conditions such as fixation and permeabilization, used for intracellular immunophenotyping, is a well documented phenomenon. If the sample is limited, or cells of interest are rare, cell loss is a significant problem hampering research, diagnosis, patient monitoring, and clinical studies. This SBIR aims to develop a simple procedure to reduce cell loss associated with sample processing and intracellular immunophenotyping, as well as cell clumping.

Armed Forces Institute of Pathology

1 R43 CA115041-01A2 (SBIR)

The primary goal of Bio-Quick is to provide a vehicle to transform the innovative biotechnology available in research labs from Armed Forces Institute of Pathology into marketable and profitable medical instruments that can greatly benefit the health care, food market safety control, and the advancement in medical community. Formalin-fixation and paraffin-embedding (FFPE) is a time consuming but standard tissue preservation and processing method used in over 90 percent cases in hospitals and clinical settings for routine histology diagnosis. Our proposed project is to design and develop an ultrasound-facilitated processor (DTP) for rapid tissue fixation and processing for histology diagnosis and any further molecular study if necessary. The implementation of the technique will allow a significant reduction in processing time from at least 24 hours by conventional FFPE to less than 1 hour. We also need funding to support collaboration with outside and independent researchers to provide objective evaluation of the technique. Our specific aims of this SBIR phase I project are: 1) Development of a commercialized intensity adjustable bench top fixer/processor for rapid formalin fixation and paraffin embedding, 2) Evaluate and validate the DTP method in comparison to conventional FFPE method based on preservation of morphological details and molecular analyses. During the past 7 years, we have compared the DTP method with conventional FFPE method on over 100 human tissue specimens of 14 tissue types. Our preliminary data have demonstrated that compared to conventional FFPE, US-facilitated FFPE not only significantly reduces the total fixation/processing time from over 24 hours to within 1 hour, but also preserves similar or better tissue morphology, much improved protein antigen properties and mRNA integrity. As a result of improved preservation of macromolecules, antigen retrieval treatment prior to IHC staining may be reduced, much reduced (20X or more) antibody concentration and shortened IHC reaction time are used. Long term stability of tissue morphology and mRNA integrity in USFFPE tissues is slightly better than that in conventional FFPE tissues.

Vivonetics, Inc.
Academic Partner: Department of Biomedical Engineering, Georgia Tech

2 R42 CA103103-02 (STTR)

We propose to develop a novel dual FRET molecular beacons technology for living cell detection and analysis of cancer. Molecular beacons are dual-labeled oligonucleotide probes with a stem-loop hairpin structure. Hybridization of molecular beacons with target mRNAs corresponding to cancer markers results in fluorescence of the cell. Thus, cancer cells (bright) can be distinguished from normal cells (dark). However, the conventional design of molecular beacons may induce a significant amount of false positives in cancer cell detection due to probe degradation by nucleases and non-specific interactions. To overcome this difficulty, we have developed the dual FRET molecular beacons approach in which a pair of molecular beacons with respectively donor and acceptor fluorophores hybridizes to adjacent regions of the same target mRNA and results in a FRET signal upon proper excitation, which is readily differentiable from non-FRET false-positive signals due to probe degradation and non-specific probe opening. In our Phase I STTR studies, we have demonstrated that, using dual FRET molecular beacons in living cell mRNA detection, false positive signals can be significantly reduced. We have also developed new molecular beacon delivery methods with high efficiency and fast kinetics for live-cell studies, and examined the sensitivity and specificity of detecting Kras and survivin mRNAs in living cells. In Phase II STTR studies, we will demonstrate the quantitative capability of molecular beacons in detecting and analyzing cancer genes in living cells. We will use dual FRET molecular beacons to detect the up-regulation of specific genes and compare the mRNA levels detected using molecular beacons and RT-PCR. To further increase the detection sensitivity and specificity, we will use molecular beacons to target multiple sites on the same mRNA molecule and target multiple tumor markers in the same pancreatic cancer cells. We will demonstrate the capability of molecular beacons to detect mutant mRNAs in fixed or live cells and the sensitivity of detecting a small number of cancer cells in a sample. The goals are to develop the dual FRET molecular beacons technology for early cancer detection and diagnosis, and to commercialize this technology for a wide range of biomedical applications including cancer analysis, drug discovery, and in vivo detection of gene expression in basic biological studies.

Geneprism, Inc.

1 R43 CA112612-01A1 (SBIR)

Technical innovation has transformed our ability to analyze genetic information in a comprehensive fashion. DNA biochips and related technologies now permit the simultaneous measurement of the structures and activities of essentially all human genes. This comprehensive capability has given us our first glimpse of the complexity of the underlying molecular events that define metabolism and disease pathogenesis. To complement this information and translate its findings to the diagnosis and treatment of human disease, these observations must be translated and extended by direct measurement of the proteins that are encoded by this genetic information. Within the pharmaceutical, biotechnology, and research communities, there is currently a large unmet need for multiplexed protein detection and quantification technologies. The current techniques for multiplexed protein profiling rely heavily on application of sandwich-ELISA format in miniaturized scale. However, these techniques suffer limited multiplexing capabilities and variable performance including specificity, sensitivity, and accuracy. The innovation described in this proposal is designed to alleviate these limitations by eliminating the need for the use of sandwich-ELISA in microarray assays and introducing signal amplification mechanism to improve the assay performance. This innovation, termed the protein footprint scanning technology, is founded on a novel immunochemical detection method which combines the specificity of antibodies with regiospecific amino acid cross-linking chemistry to produce analyte-specific quantification. The successful validation and implementation of this technology will catalyze the development of highly sensitive protein microarray assays using only one antibody per target analyte. This will in turn enable microarrays with higher content multiplexity, diversity, and novelty. The expanded content will be especially important for cancer research since the multi-factorial nature of oncogenesis will likely require parallel examination of large numbers of proteins in cellular and extracellular proteome compartments. As a proof-of-concept, the feasibility of applying this innovative technology for protein detection and quantification will be validated using multiplexed protein microarray assays consisting of 10 validated cancer biomarkers.

Expression Pathology, Inc.

1 R43 CA116217-01 (SBIR)

The objective of this Phase I proposal is to evaluate the usefulness of the emerging technology of mass spectrometry for proteomic analysis of formalin-fixed cancer tissue for initial application to the discovery/analysis of cancer biomarkers. The model system chosen for this proposal is Head and Neck Squamous Cell Carcinoma (HNSCC). Because HNSCC is directly related to tobacco use, this proposal will be an excellent model to demonstrate cancer biomarkers that can be linked to tobacco-induced cancer. This Phase I evaluation will be accomplished by integrating the technologies of tissue microdissection, Liquid Tissue(tm) methodology, and higher order mass spectrometry to demonstrate application for high content proteomic analysis of the cancer microenvironment of formalin-fixed archival cancer tissue. Laser capture microdissection (LCM) will be employed to separately obtain normal and tumorigenic epithelium directly from formalin-fixed archival HNSCC tissue blocks. In addition, normal- and tumor-associated stromal components supporting these epithelial regions will also be obtained by LCM and analyzed in parallel. A searchable database will be developed based on mass spectrometric analysis of normal, tumor, and associated stroma from five separate HNSCC cases and bioinformatics conclusions drawn to demonstrate the ability to discover biomarkers of cancer directly from formalin-fixed archival HNSCC cancer tissue. In addition, it is hypothesized that analysis of stromal components will lead to discovery of stroma-associated biomarkers that could potentially develop a role for surrounding stroma in the diagnosis and treatment of cancer. If this demonstration Phase I is successful, a Phase II proposal will be submitted to apply this platform to discovery of proteins critical to HNSCC, resulting in commercial applications to the discovery/analysis of biomarkers of not only HNSCC but all cancers. Additional commercial applications that might result from a Phase II are; a) formalin-fixed tissue-based mass spectrometry contract service, b) development of an internal cancer biomarkers discovery core for license to the biopharmaceutical industry, and c) a proteomic contract service for analysis of animal models of toxicology with strategic biopharmaceutical partners. The following Specific Aims will be addressed in this Phase I proposal: (1) Assess the ability of mass spectrometry to reproducibly profile the proteome by analysis of Liquid Tissue(tm) protein preparations obtained from different and distinct histological regions of formalin-fixed, paraffin-embedded HNSCC tissue. (2) Assess the ability to use mass spectrometry data to develop a database reflecting proteomic similarities and differences between normal and tumorigenic HNSCC epithelium. (3) Assess the ability to use mass spectrometry data to develop a database reflecting proteomic similarities and differences between stroma surrounding normal epithelium and stroma surrounding tumorigenic epithelium of HNSCC tissue.

Rubicon Genomics, Inc.

1 R43 CA114128-01 (SBIR)

A proprietary OmniPlex(tm) technology will be optimized to prepare amplified DNA and RNA samples from formalin-fixed, paraffin-embedded (FFPE) tissues. These OmniPlex products will have predictable performance in downstream applications and allow meaningful genome-wide genetic and gene expression analyses of formalin-fixed tumor specimens for cancer research and diagnostics. These analyses are currently challenging, because no technology can effectively amplify FFPE DNA and RNA samples of variable quality and produce amplified samples with predictable, standardized quality. In the basic OmniPlex process, high quality DNA and RNA samples from fresh tissues and cultured cells are amplified with robust and reproducible efficiencies, but FFPE samples are frequently damaged and/or degraded and amplify with unpredictable and highly variable efficiencies. In Phase I the basic OmniPlex process will be modified in order to produce more robust and reproducible FFPE sample amplification efficiencies, which are required for producing amplified products with predictable, standardized quality. Specific Aim 1 is to identify a real-time quantitative PCR assay that will normalize input FFPE DNA sample amounts better than mass measurements. Specific Aim 2 is to improve the conversion of FFPE samples to amplifiable OmniPlex libraries by modifying reagent and incubation conditions. Finally, Specific Aim 3 is to use in-house quantitative PCR assays to develop quality parameters that predict amplified FFPE sample performance in genetic and gene expression studies. Successful Phase I project results will lead to Phase II studies that test the robustness of the improved process on a large set of normal and tumor samples, uniquely allow DNA and RNA amplification from the same samples, and demonstrate the utility of amplified FFPE samples for cancer research on major commercial genetic and gene expression platforms. The potential commercial applications of this research will be OmniPlex FFPE amplification kits and amplification service projects.

Ambion, Inc.

1 R44 CA097482-02A1 (SBIR)

This Phase II proposal aims to simplify, expedite, and stabilize the recovery of high quality RNA from tissue samples. In Phase I, we demonstrated the feasibility of a hands-off, closed tube tissue disruption method termed “MELT” (Multi-Enzymatic Liquefaction of Tissue). MELT enlists potent catabolic enzymes to liquefy tissue within minutes without invasive mechanical force. High yields of intact RNA are obtained after MELT. Importantly, MELT enzymes destroy cellular RNases and stabilize RNA in tissue lysates for up to 5 days at ambient temperatures. Additionally, MELT is compatible with freshly harvested, flash-frozen, or RNAlater® treated tissues, both mouse and human, and including tumor specimens. Taken together, these advances promise faster, simpler, safer, and more robust methods for stabilizing and quantifying gene expression in tissues through innovation in RNA stability, closed-tube tissue disruption, and rapid single-tube sample preparation. In Phase II we will integrate continuing MELT enhancements with new ways to facilitate RNA processing. First, we will accelerate MELT tissue digestions and maximize the quality of the resulting RNA. Second, we will link MELT improvements with novel magnetic beads that can enable the purification of DNA-free RNA in as little as 20 minutes. This method will secure the recovery of large amounts of RNA for analysis by any expression profiling method, including microarrays. Last, we will enable an ultra rapid RNA sample preparation strategy specifically suited for qRT-PCR (“Tissue-to-RT-PCR”) that skips RNA isolation altogether. Using novel approaches for tissue disruption, RNase control, DNA removal, and the management of RT-PCR inhibition, we will enable Tissue-to-RT-PCR in less than 10 minutes, with all of the steps but the RT-PCR reaction itself occurring in the same tube. Success in these objectives will result in easy-to-use products that offer improved RNA yields, greater sample throughput, more ready automation, and reduced variability, contamination, and biohazard risk compared to current methods. The beneficiaries of MELT technology will include life science researchers and clinical diagnostic labs, where emerging RNA biomarkers can be combined with simpler sample preparation methods to hasten the adoption of molecular diagnostics procedures.

Ambergen, Inc.

1 R43 CA114126-01 (SBIR)

Significant advances have been made in the development of a new generation of molecularly targeted cancer drugs, many of which are only now emerging from the drug development pipeline. Examples of small molecule drugs are imatinib (Gleevec), used to treat CML, and gefinitib (Iressa), used to treat lung cancer. Other drugs, such as cetuximab (Erbitux) for colorectal cancer, are monoclonal antibodies. All of these drugs selectively modulate the activity of specific target proteins such as BCR-ABL tyrosine kinase (imatinib) and EGFR (gefitinib and cetuximab) that are critical for the proliferation and survival of cancer cells. However, clinical studies are revealing that patients often develop drug resistance due to overproduction or mutant forms of the target protein. For this reason, it is likely that future treatment with single or multiple targeted cancer drugs will require testing for preexisting or acquired resistance at the level of the target protein. The principal objective of this proposal is to develop and evaluate a sensitive and low-cost technology known as PC-SNAG for monitoring resistance, to one or more drugs, in patients at the level of individual target proteins. A key feature of PC-SNAG is the ability to rapidly isolate native/active proteins from crude biological samples using photocleavable antibodies (PC-antibodies). During Phase I, proprietary high-efficiency photocleavable linkers will be used to produce PC-antibodies immobilized on beads or other solid surfaces. The PC-antibodies are used to isolate and concentrate the target proteins from a heterogeneous biological sample. The target protein is then rapidly and gently photo released into solution in a native and highly pure form for functional analyses that will be evaluated to detect resistant forms of the drug target and to determine optimal drug therapy on a per patient basis. In Phase I, the technology will be tested using model kinase-directed small molecule and antibody drugs followed by a focus on detection of drug-resistant forms of the BCR-ABL tyrosine kinase. One goal is detection of <5% of the resistant form of the target, which is difficult to achieve using conventional DNA sequencing. In Phase II, clinical evaluation of the technology will be performed in collaboration with Dr. Daniel Write, Chief of Hematology/Oncology at the Boston University Medical Center.

Genaco Biomedical Products, Inc.

1 R43 CA112735-01 (SBIR)

The aim of this SBIR application is to develop a bead-based array system for the specific detection and classification of microRNAs (miRNAs). The discovery of miRNAs represents a paradigm shift that suggests the existence of many unknown cellular function and regulation mechanisms. Already, miRNAs have been found implicated in different cancers and leukemia. However, the existing methods for studying the expression of miRNAs are labor intensive, time consuming, and also lack specificity and sensitivity. A more efficient and accurate research and development tool is much in demand. We have developed a bead-based array method that integrates the xMAP technology platform with the locked nucleic acid (LNA) technology. The method, called xMAP-MP for xMAP-based miRNA profiling, uses beads coupled to a capture oligonucleotide (oligo) and a biotin labeled detecting oligo to quantitatively detect and classify miRNA. With this method, multiple miRNAs can be studied together in one reaction. Preliminary studies have shown that the assay is highly specific and sensitive: miRNAs can be detected using only 100 ng of total RNA. This is more than 100 times more sensitive than the traditional Northern blot method. The xMAP-MP is also very efficient: samples do not need to be labeled; hybridization takes only 30 minutes; and detection and data acquisition takes only a minute. The entire procedure can be finished within an hour. Furthermore, the multiplex capability of the xMAP technology platform allows the study of up to 100 miRNAs in one assay. The proposed study has four specific aims: (1) Select 20 cancer related miRNA targets and design a multiplexed detection system for these miRNAs; (2) Design internal and external controls for the assay system; (3) Develop and optimize a standard assay protocol, and (4) Use the prototype assay to study cancer samples.

One Cell Systems, Inc.

1 R43 CA113926-01 (SBIR)

Human papillomavirus (HPV) infection is the cause of virtually all genital warts (condyloma), and cervical and anal cancers. The Company has developed a novel assay for detecting integrated HPV DNA in cervical cells, which has the potential to more accurately predict disease progression and prognosis. Using this novel assay format on cervical cell samples from HIV-positive women, this SBIR will investigate HPV types involved and whether HPV DNA has integrated into the host genome. A rapid cell-based in situ hybridization assay for screening cervical cell samples from HIV-infected women is urgently needed because conventional HPV diagnostic methods such as simply determining whether an infection involves high-risk or low-risk families are not as useful in this patient population. In immunocompetent women, most infections, regardless of viral type, resolve spontaneously over several months. HPV prevalence in asymptomatic immunocompetent women ranges from 10% to 20% and, in most cases, involves infection only by a single viral type. In contrast, typically >70% of HIV-positive women have HPV infection which results in cervical and anal cell abnormalities. Viral persistence is greatly increased, as is the frequency of multiple type infection. Higher relapse rates after condyloma treatment are seen, as well as differences in HPV strain prevalence.

Immunotope, Inc.

1 R43 CA114194-01 (SBIR)

Early diagnosis and immunotherapeutic stimulation of a patient’s own immune system to detect and destroy tumors is the best hope for the treatment, prevention, and eventual cure for ovarian cancer. The overall goal of this proposal is to conduct proof-of-concept studies to identify tumor-associated antigens (TAA) that will lead to the development of (1) early stage diagnostics and (2) immunotherapeutics that induce strong cellular (T cell) and humoral (B cell) responses against ovarian tumors. The novelty of this program is in the identification of tumor-specific antigens that are both reactive to auto antibodies in the serum of ovarian cancer patients at progressive stages of disease and are also processed through the immune system Major Histocompatibility Complex (MHC) class I pathway and recognized by cytotoxic T lymphocytes. Discover and preliminary evaluation of the antigens will begin in Project Phase I. the first Aim of this Phase I proposal is to conduct a comprehensive proteomic analysis to identify tumor-associated antigens reactive to serum immunoglobulin from primary ovarian cancer patients at different stages of disease, with the goal of identifying antigens common to the majority of patients sampled. This Aim will be achieved by immunoprecipitation of TAA from primary tumor lysates using patient serum as the source of autoantibodies and control serum to identify antigens found only in patient serum. The TAA will be fully characterized by mass spectroscopy and a TAA proteomics database will be generated. The second Aim is to identify the epitopes in these TAA that are recognized by the autoantibodies in cancer patient serum. Since native conformation and post-translational processing can have profound effects on antibody recognition, we will further characterize glycosylation patterns on these antigens and determine whether they play a role in antibody recognition. This emphasis on native proteins has important implications for our future work on the design of diagnostics and immunotherapeutics, as it takes into account the specificity of the interactions between native antigens and autoantibodies.

Columbus Nanoworks, Inc.

1 R43 CA116048-01 (SBIR)

The aim of this proposal is to develop high-resolution magnetic nanoparticles for use in Quadruple Magnetic Sorting (QMS). QMS is a flow-through immunomagnetic separation system that can provide sensitive enrichment of circulating cancer cells in blood as well as other biological fluids. Optimal cellular separation by QMS requires immunomagnetic particles having high magnetic susceptibility, narrow particle size distribution and high density attachment sites for antibodies. Current commercial immunomagnetic beads are either too large, lack size uniformity, or have too low magnetic susceptibility. We will make the necessary improvements in the magnetic immunobeads using recent advances in nano-particle technology and evaluate their performance by enriching ovarian cancer cells from a complex population of cultured cells. In Specific Aim 1 we will synthesize and physically characterize monodisperse paramagnetic nanoparticles using sol-gel methods to encapsulate Fe nano-crystals. These nanoparticles will have uniform size distribution and shape, and possess a high weight percentage of iron (Milestone 1). In Specific Aim 2 we will use siloxane treatments and heterobifunctional coupling agents to create high-density attachment sites for antibodies and streptavidin (Milestone 2). The resulting particles will be analyzed for their particle field interaction parameter values which is a major determinant of the effectiveness of magnetic nanoparticles in cellular separation (Milestone 3). Finally in Specific Aim 3 we will evaluate our magnetic nanoparticles for immunomagnetic detection and separation of ovarian tumor cells by QMS. The human ovarian tetracarcinoma PA-1 tumor cells will be diluted into whole blood and subjected to QMS using antibodies against TAG-72, a surface-expressed protein present on ovarian cancer cells. Demonstrating the detection of tumor cells in whole blood cells will be taken as the proof of principal for this project.

Cell Signaling Technology, Inc.

2 R44 CA101106-02 (SBIR)

Among post-translational modifications, protein phosphorylation is particularly relevant to cancer biology and therapy. However, despite advances in proteomics, it is still difficult to pinpoint phosphorylation sites in proteins. The long-term goal of this project is to develop and commercialize a multiplexed method for isolating, identifying, and quantifying phosphorylation sites using phosphorylation-specific antibodies. This method would contribute to the development of a new generation of drugs tailored to inhibit specific protein kinases with roles in cancer by identifying new phosphorylation sites that could become targets for cancer diagnosis and treatment. During our IMAT-funded Phase I, we established the feasibility of using an immunoaffinity method to isolate phosphopeptides from complex mixtures, which were then identified by tandem mass spectrometry. In this Phase II application, we will optimize the immunoaffinity method so it can be used to analyze low-level samples, using cell numbers that are comparable to what would be available from patient samples. Once we have optimized the method and established the repertory of antibodies that can be used productively in the method, we will demonstrate the method’s utility by applying it to a variety of cancer cell lines, to show the method can probe the major signal transduction networks involved in cancer and can identify oncogenic lesions. We will bring a quantitative dimension to the method by merging it with mass spectrometry methods such as SILAC and Aqua, which are needed for later biomarker discovery and biomarker assay efforts.

Bioren, Inc.

1 R43 CA116053-01 (SBIR)

Prostate cancer is one of the most commonly diagnosed cancer in men, accounting for over 30,000 deaths annually in the U.S. The current lack of predictive tests and treatment methodologies illustrates a need for improved detection, prognostics, and therapies. The goal of this proposal is to generate improved antibodies against Prostate Stem Cell Antigen (PSCA) to facilitate enhanced diagnostics and immunotherapeutics. PSCA is a prostate-specific membrane protein with increased expression correlating with prostate cancer progression and metastasis. Anti-PSCA antibodies are of diagnostic utility and, in preclinical animal models, able to prevent prostate cancer metastases and inhibit tumor growth. These findings make PSCA a great candidate for antibody-based therapeutics. For potential human applications, anti-PSCA monoclonal antibodies have been humanized through complementarity determining region (CDR) grafting but however, exhibits more than a 50% loss in affinity for PSCA. Bioren's patented proprietary Look Through Mutagenesis(tm) (LTM) and Walk Through Mutagenesis(tm) (WTM) technology is based on synthetic oligonucleotide chemistry to create defined antibody mutations. Using Bioren's LTM/WTM strategies, an efficient, high-throughput mutagenesis system can systematically explore the chemical modalities involved in the PSCA antigen binding pocket. The advantages of our approach allow a hypothesis-driven rational replacement of codons necessary to determine and optimize amino acid functionality in all the VH and VL CDRs of the antibody. By focusing on generating targeted small diversity libraries for screening, our process is both accelerated and economically efficient, unlike other mutagenesis strategies. In combining mapped LTM/WTM beneficial mutations through an iterative permutation process, we have been able to achieve 1000x fold (low pM KD) affinity improvements. The technology developed under this proposal can be applied in the affinity maturation of many other immunotherapy antibody candidates.