Current cytometry technologies query cell phenotype and protein expression but do not reliably measure key, clinically-relevant proteins that are challenging to detect in single cells due to their low basal levels and/or diminished levels following drug treatment (e.g. phosphoproteins, immune regulatory proteins). As a result, many drug targets are not evaluated and the absence of detection cannot be reliably interpreted as a negative result. To overcome major bottlenecks in sensitive protein detection, OHSU and Becton Dickinson (BD) will commercially translate an imaging technology that will enable investigators to perform drug screening with unprecedented molecular-level sensitivity in primary patient samples. We have built a platform, the DigiCount, that implements a new strategy of counting discrete immunolabeled proteins in the presence of diffuse background noise (e.g. cell autofluorescence) in single cells (Jacob et al, Nat Sci Reports 2016; OHSU: 1 patent filed, 2 pending). Proprietary digitized molecular-localization algorithms discriminate discrete protein complexes tagged with ultrabright antibody-fluorophore probes, offering up to 1-2 log times increased signal to noise over conventional flow cytometry/fluorescence microscopy. We will scale up the DigiCount throughput for efficient clinical screening. We will also generate additional probe panels that include markers of clinically-relevant/low abundance proteins (stem cell, immune regulatory, phosphoproteins) to further increase target screening information. Probe panels leverage a new class of conductive polymer dyes with efficient light harvesting amplification chemistry and ultrabright emission (Nobel Prize, 2000). The probes are selected from BD's compound library, and specifically optimized for digitized molecular detection. In pilot screening of acute myeloid leukemia 1 patients enrolled in the Beat AML study, an ongoing 900 patient study to identify improved AML targeted therapies (OHSU Knight Cancer Institute), we have identified synergistic small molecule/immune checkpoint inhibitor combinations that produce AML cell kill and induce immune cell proliferation more effectively than single molecule agents. We will apply the DigiCount to screen hundreds of combinations per patient to determine if there are main classes of small-molecule/immune checkpoint inhibitor combinations that are more effective than single agents. We will also profile the effects of promising combinations to characterize their effect on immune and myeloid cell targets. The research team consists of the innovator of single cell imaging technologies (PI: Vu, OHSU), a lead scientist with expertise in tandem polymer fluorophores and novel/HCA assay development from the commercial sector (PI: Martin, Becton Dickinson), and clinician- scientists leading efforts to identify AML targeted therapeutics for clinical deployment (co-PIs: Tyner, Lind, OHSU). Letters of support from key opinion leaders attest to the innovation and clinical significance of the DigiCount technology and support of relationships at BD and OHSU for commercial translation.
To improve targeted therapy approaches for hematological malignancies, we will translate a drug screening platform for commercial application which will enable investigators to perform direct drug screening in primary patient cells with unprecedented molecular-level sensitivity. We will scale up the platform for clinical application-based screening. The platform will be applied to screen hundreds of small molecule/immune checkpoint agents in order to determine the most effective therapeutics for treating individuals diagnosed with acute myeloid leukemia.
