SBIR-STTR Award

Autologous hematopoietic stem cell transplantation (HSCT) has been an important treatment option for many patients with hematologic malignancies and solid tumors such as acute myeloid leukemia, myelodysplastic syndromes, chronic myeloid leukemia, systemic mastocytosis and breast cancer. During the treatment, anti CD34 antibodies are used to enrich the CD34+ hematopoietic stem cells from mobilized peripheral blood, which are re-infused after a high-dose radio/chemotherapy. Unfortunately, due to the large sample volumes required to obtain sufficient number of stem cells (100-300 mL), the antibody reagents are prohibitively expensive, typically in excess of $10,000 per therapy. To address this problem, we propose to develop an alternate, nucleic acid-based affinity reagents (i.e. aptamers) against the CD34 antigen, which will be stable, exhibit high affinities and specificities, significantly less immunogenic, and considerably less expensive (~10X less) compared to protein-based reagents. Toward this end, we propose three specific aims: first, in order to circumvent the traditional time-consuming and labor-intensive SELEX process, we will adopt and optimize our high throughput microfluidic aptamer discovery platform (Pro-SELEX) to isolate high affinity aptamers (Kd < 1 nM) for the CD34 antigen. The selection will be carried out using purified extracellular domain of the CD34 protein from mammalian cell expression system (Chinese Hamster Ovary cells) such that the structure and post-translational modifications are conserved. Secondly, we note that the overwhelming reason for treatment failure is highly correlated to the degree of contamination of circulating tumor cells during the purification of CD34+ cells. Thus, in order to significantly raise the specificity of the aptamers against tumor cells, we will develop a novel, cell-surface counter selection process using live leukemia and other tumor cells. Finally, we will evaluate and compare the performance of the anti-CD34 aptamers with commercially available anti-CD34 antibodies. We will measure the binding efficiency and specificity to CD34+ cells with flow cytometry, and perform magnetic cell selection of spiked CD34+ cells in whole blood. If successful, the project will have significant ramifications because 1) it will significantly lower the cost of HSCT and, 2) it will advance the aptamer selection technology to rapidly generate high affinity aptamers against a wide range of cell surface markers, which have far-reaching application in clinical diagnosis and therapies.

Public Health Relevance:
Autologous hematopoietic stem cell transplantation (HSCT) has served as an important treatment option for many patients with hematologic malignancies and solid tumors including acute myeloid leukemia, myelodysplastic syndromes, chronic myeloid leukemia, systemic mastocytosis and breast cancer. Unfortunately, the antibody-based reagents used in the therapy to isolate CD34+ hematopoietic stem cells remain prohibitively expensive (> $10,000 per procedure). To address this issue, we aim to develop a novel system capable of rapidly generating aptamers, and utilize it to isolate high affinity aptamers that bind to the CD34 surface antigen. The success of this project has important ramifications as it will significantly reduce the cost for many cell-based transplantation therapies, and also establish a universal method to generate high-affinity aptamers for a wide range of diagnostic and therapeutic applications.

Thesaurus Terms:
"1h-Thieno(3,4-D)Imidazole-4-Pentanoic Acid, Hexahydro-2-Oxo-, (3as-(3aalpha,4beta,6aalpha))-; Aml - Acute Myeloid Leukemia; Address; Adopted; Affinity; Antibodies; Assay; Autologous; Band Shift Mobility Assay; Bandshift Mobility Assay; Binding; Binding (Molecular Function); Bioassay; Biologic Assays; Biological Assay; Biotin; Blood (Leukemia); Blood Precursor Cell; Blood Sample; Blood Specimen; Cd34; Cd34 Antigens; Cd34 Gene; Cd34 Molecule; Cho Cells; Cancer Of Breast; Cell Isolation; Cell Segregation; Cell Separation; Cell Separation Technology; Cell Surface Antigens; Cell Surface; Cells; Chinese Hamster Ovary Cell; Chronic Myeloid Leukemia; Cytofluorometry, Flow; Dna; Deoxyribonucleic Acid; Diagnostic; Disease; Disorder; Dose; Dysmyelopoietic Syndromes; Electrophoretic Mobility Shift Assay; Ensure; Exhibits; External Domain; Extracellular Domain; Flow Cytofluorometries; Flow Cytometry; Flow Microfluorimetry; Fluorescence; Generations; Goals; Hpca1; Hsc Transplantation; Hematologic Cancer; Hematologic Malignancies; Hematologic Neoplasms; Hematological Malignancies; Hematological Neoplasms; Hematological Tumor; Hematopoietic Cancer; Hematopoietic Progenitor Cell Antigen Cd34; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; K562 Cells; Leukemia, Granulocytic, Chronic; Leukemia, Myelocytic, Acute; Leukemias, General; Life; Magnetism; Malignant Hematologic Neoplasm; Malignant Mastocytosis; Malignant Tumor Of The Breast; Malignant Neoplasm Of Breast; Mammalian Cell; Mastocytosis, Systemic; Measures; Methods; Microfluidic; Microfluidics; Microfluorometry, Flow; Moab, Clinical Treatment; Mobility Shift Assay; Molecular Interaction; Monoclonal Antibodies; Mother Cells; Myeloblastic Leukemia, Acute; Myelocytic Leukemia, Chronic; Myelodysplastic Syndromes; Myelogenous Leukemia, Acute; Myelogenous Leukemia, Chronic; Myeloid Leukemia, Chronic; Nucleic Acids; Patients; Performance; Post-Translational Modifications; Post-Translational Protein Processing; Posttranslational Modifications; Procedures; Process; Progenitor Cells; Progenitor Cells, Hematopoietic; Protein Modification; Protein Modification, Post-Translational; Protein Processing, Post-Translational; Protein Processing, Posttranslational; Protein/Amino Acid Biochemistry, Post-Translational Modification; Proteins; Radio; Reagent; Recovery; Recurrence; Recurrent; Sampling; Smoldering Leukemia; Solid Neoplasm; Solid Tumor; Specificity; Stem Cells; Strepavidin; Streptavidin; Structure; Surface; Surface Antigens; Surface Markers, Immunologic; Surface Markers, Immunological; System; System, Loinc Axis 4; Systemic Mast-Cell Disease; Systemic Mastocytosis; Systemic Tissue Mast Cell Disease; Technology; Therapeutic; Time; Transplantation; Treatment Failure; Tumor Cell; Vitamin H; Whblood; Whole Blood; Work; Acute Granulocytic Leukemia; Acute Myeloid Leukemia; Acute Nonlymphocytic Leukemia; Aptamer; Base; Cell Sorting; Chemotherapy; Clinical Diagnosis; Coenzyme R; Cost; Cross Reactivity; Disease/Disorder; Flow Cytophotometry; Gel Shift Assay; Gene Product; Gp105-120; Immunogenic; Improved; Innovate; Innovation; Innovative; Leukemia; Magnetic; Magnetic Beads; Malignant Breast Neoplasm; Myelodysplasia; Neoplastic Cell; Novel; Peripheral Blood; Public Health Relevance; Success; Transplant"

Aptamers are nucleic acid based affinity reagents that can be selected in vitro from large combinatorial libraries. As an affinity reagent they have inherent advantages as they are chemically synthesized, thermostable, immunologically neutral, significantly cheaper than antibody reagents and can be evolved for both specificity and affinity. Conversely, the process to generate useful aptamer reagents is lengthy, requiring significant experience and time to generate a useful reagent;frequently months for 8-15 rounds of library selection. Therefore, despite the promise of aptamer technology and the notable success of aptamer therapeutics in the short time the technology has been available, the threshold for entry into this field is inhibited by this labor-intensive process and mixed success. Phase I feasibility studies showed that DNA aptamers with low nanomolar dissociation constants (Kd <3 nM) can be generated in just 3 rounds of selection, since extended to 5 proteins. This work has clearly shown the feasibility of our technology. From this foundation, this application will help fund commercialization of this aptamer selection platform, the reagents and protocol. We expect that successful commercialization will have a major impact on the advancement of diagnostics, therapeutics, and the emerging proteomics applications where reagent demands far outstrip supply. Provide key words (8 maximum) to identify the research or technology. aptamer, Pro-SELEX, affinity, selection, Provide a brief summary of the potential commercial applications of the research. The Pro-SELEX platform and sequencing protocol 60-80% both the workload and chance of failure for selection of aptamer affinity reagents. This generates a significantly more appealing approach to aptamer selection likely to expand the application of this technology into proteomics and diagnistic reagents.

Public Health Relevance:
Aptamers are molecular recognition molecules analogous to monoclonal antibodies except that they are made in test tubes using nucleic acid libraries. They have demonstrated enormous diversity and potential since they were first used 20 years ago, but they are very time consuming and troublesome to develop. This project describes a platform that standardizes and automates generation of aptamer reagents. It has to potential to significantly accelerate the development of aptamer tools for use in drug delivery, therapeutics as well as basic research and diagnostic sciences.