LentiSAFE Ligands: Mild-Elution Immunoaffinity Ligands for Enrichment and Purification of Lentiviral Vectors

Project Summary: To fulfill the increased demand for clinical and commercial grade lentiviral vectors (LV), considerable efforts are being made to transition in-efficient and often crude laboratory scale LV production processes to more robust, high yielding and scalable production processes that will satisfy the more restrictive quality and regulatory requirements for clinical and commercial supply in a cost-effective manner. However, unlike AAV vectors, where it has been possible to establish standardized, cost-effective downstream processes (DSP) built around an highly efficient immunoaffinity purification step that specifically enriches for the AAV vector, DSP for LV has remained a major bottleneck owing to the intrinsic fragility of the virus that makes it highly susceptible to degradation and/or inactivation by the often harsh elution conditions that are required to desorb the lentivirus from the affinity matrix. Consequently, there is a significant unmet need for high-yielding and scalable immunoaffinity purification methods for LV. These would require currently not available materials which allow for very mild conditions to desorb the fragile vector particles from the immunoaffinity matrix. The objective of this proposal is to assess the feasibility to selectively isolate and enrich for functional lentiviral vectors from production supernatants via the use of Switchable Affinity Mild-Elution (SAFE) immune-affinity ligands that can be formatted into matrices for the purification of lentiviral vectors under mild non-destructive conditions. This will be a disruptive achievement for LV DSP as it will allow an easily scalable and cost-effective method to obtain high yields of infective LV particles for therapeutic applications. Specifically, in Aim 1 we will develop switchable forms of an antibody scFv with allosterically regulatable affinity to a common lentivirus glycoprotein. The allosteric regulation does not require the large changes in pH or salt concentration typically needed for immunoaffinity chromatography. We will produce sufficient quantities for testing in Aim 2 for initial affinity, affinity changes, specificity, stability and yield. In Aim 3 we will undertake ligand induced release studies under mild conditions utilizing the allosteric affinity switch built into the antibody fragment to evaluate the feasibility of selective enrichment of the soluble glycoprotein as well as respectively pseudotyped lentiviral particles from a typical LV production supernatant. At the conclusion of this work, we will have demonstrated that the incorporation of a ligand controlled affinity switch into the binding domain of an antibody scFv that recognizes a lentivirus glycoprotein allows for high yielding immunoaffinity purification of the soluble glycoprotein and the respective pseudotyped lentiviral vectors under mild conditions with minimal inactivation of the lentivirus. Future work will be directed at generating affinity matrices for scalable mild elution immunoaffinity purification of LV and improving the stability of the best variants using established in-vitro evolution approaches, with the aim to improve on-site regeneration capability of the affinity matrix. The work will also be a first in class case study to demonstrate the usefulness of our approach for many other fragile biotherapeutic reagents where currently immunoaffinity purification is not yet feasible.