The goal of this Phase I SBIR project is to develop affinity reagents useful for the sub-fractionation of specific exosomal populations. Because exosomal makeup and cargo reflect the type, developmental stage, and the disease state of the cells they are shed from, they are attractive targets for non-invasive diagnostics. Exosomes are also being investigated for therapeutic applications such as vaccines and drug delivery. Because exosomes from bodily fluids generally contain exosomes from many different cells, tools are needed to isolate specific populations. Current exosome purification techniques like differential centrifugation and filtration techniques are effective for isolating exosomes from cell culture and biological fluids, but do not discriminate between exosomes from different sources. Affinity purification of exosomes based on unique surface markers is capable of separating subpopulations, but is expensive, cumbersome, and hard to scale. Exosomes isolated by affinity purification using traditional antibodies are usually difficult to elute, a property making sub-fractionation of the primary elution infeasible. Affinity reagents that would allow the sequential isolation of exosomes based on different surface markers would enable unprecedented sorting of exosomal populations. nanoCLAMPs are a newly described class of single domain affinity reagents with high affinity, high specificity and the rare property of polyol-responsiveness. nanoCLAMPs have the ability to release functionally and structurally intact targets upon exposure to buffer containing polyol and salt. Panels of nanoCLAMPs to new protein targets are easily generated from our validated synthetic phage display library in a few weeks, a process that eliminates the need for animal immunization and provides multiple binders per target. We hypothesize that nanoCLAMP affinity resins to exosomal surface proteins will allow the sequential sub-fractionation of exosomes based on surface proteins specific to those populations. We propose to develop a panel of nanoCLAMP affinity resins to the extracellular domains of several common exosomal surface proteins; demonstrate their utility in binding to and purifying exosomes; compare their performance to current techniques; characterize the eluted populations of exosomes for surface proteins, yield, and morphology; and finally utilize the nanoCLAMPs to purify and sub-fractionate exosomes from bodily fluids. The successful completion of this project will result in a platform for the facile production of novel tools for the isolation of exosomes based on the ever increasing identification of unique surface markers related to a wide field of cellular origins and conditions.
The development of novel tools for the specific isolation and sub-fractionation of exosomal populations will enable investigators to precisely isolate and analyze exosomes from complex mixtures in bodily fluids. These tools will support the development of innovative assays and diagnostic tests providing information about the specific cell type, developmental stage, disease state, and drug-response of the cells under investigation.
