To fulfill the increased demand for clinical and commercial grade lentiviral vectors (LV), considerable efforts arebeing 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 regulatoryrequirements for clinical and commercial supply in a cost-effective manner. However, unlike AAV vectors, whereit has been possible to establish standardized, cost-effective downstream processes (DSP) built around an highlyefficient immunoaffinity purification step that specifically enriches for the AAV vector, DSP for LV has remaineda major bottleneck owing to the intrinsic fragility of the virus that makes it highly susceptible to degradation and/orinactivation 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 purificationmethods for LV. These would require currently not available materials which allow for very mild conditions todesorb the fragile vector particles from the immunoaffinity matrix. The objective of this proposal is to assess thefeasibility to selectively isolate and enrich for functional lentiviral vectors from production supernatants via theuse of Switchable Affinity Mild-Elution (SAFE) immune-affinity ligands that can be formatted into matrices for thepurification of lentiviral vectors under mild non-destructive conditions. This will be a disruptive achievement forLV DSP as it will allow an easily scalable and cost-effective method to obtain high yields of infective LV particlesfor therapeutic applications. Specifically, in Aim 1 we will develop switchable forms of an antibody scFv withallosterically regulatable affinity to a common lentivirus glycoprotein. The allosteric regulation does not requirethe large changes in pH or salt concentration typically needed for immunoaffinity chromatography. We willproduce 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 affinityswitch built into the antibody fragment to evaluate the feasibility of selective enrichment of the solubleglycoprotein as well as respectively pseudotyped lentiviral particles from a typical LV production supernatant. Atthe conclusion of this work, we will have demonstrated that the incorporation of a ligand controlled affinity switchinto the binding domain of an antibody scFv that recognizes a lentivirus glycoprotein allows for high yieldingimmunoaffinity purification of the soluble glycoprotein and the respective pseudotyped lentiviral vectors undermild conditions with minimal inactivation of the lentivirus. Future work will be directed at generating affinitymatrices for scalable mild elution immunoaffinity purification of LV and improving the stability of the best variantsusing established in-vitro evolution approaches, with the aim to improve on-site regeneration capability of theaffinity matrix. The work will also be a first in class case study to demonstrate the usefulness of our approach formany other fragile biotherapeutic reagents where currently immunoaffinity purification is not yet feasible.
Public Health Relevance Statement: Project narrative:
Downstream processing has remained a major bottleneck for the high-yield production of Lentiviral vectors 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 purification matrix. In this project,
Mabswitch will develop LentiSAFE Ligands which are Switchable Affinity Mild-Elution immuno-affinity ligands
based on a commercially available anti-lentivirus antibody, with the aim to assess the feasibility to purify
functional lentiviral vectors from production supernatants under mild non-destructive conditions. Importantly
owing to the universal character of the approach it will serve as a template for purification of many other fragile
biotherapeutic reagents where immunoaffinity purification is currently not feasible.
Project Terms:
