High-avidity reversible engager proteins efficiently redirect the entry of intravenously delivered lentiviral vectors, simplifying the direct in vivo delivery of CAR-T cell therapy

We have developed engager proteins that reversibly block VSV-G, directing VSV-G-pseudotyped lentiviral vectors (LVV) into resting CD3-positive T cells. Following selective binding of the engager-modified LVV particles to CD3, they are internalized into endosomes, where the engager is detached by the low pH and the absence of calcium ions, releasing unmodified LVV. Free LVV particles then fuse with the endosomal membrane and efficiently deliver their genetic cargo to the cytoplasm. These engager proteins are stable and easily produced in high yields, making them suitable for practical use. Reversible bi-specific engagers that bind to the homotrimeric VSV-G protein and redirect it to a targeted receptor were initially developed by genetically fusing a cell-targeting polypeptide to the VSV-G binding domains CR2 and CR3 of the LDL receptor (Israel Patent application No. 296547, 2022. Inventors: M. Rubinstein, G. Schreiber, D. Gataulin, Weizmann Institute). When pre-incubated with VSV and VSV-G-LVV, these engagers mediated selective vector entry into mammalian cells via the targeted receptor while preventing non-specific entry through LDLR. The monomeric engagers blocked binding of VSV-G to LDLR with an ED 50 of 3 nM. Trimeric engager versions exhibited an ED 50 of 20 pM. To increase the binding avidity of the engager to VSV-G, we engineered and produced trimeric engagers consisting of CD3-specific scFv fused through a trimerizing peptide to VSV-G binding domains of LDLR. When co-incubated with LVV, the engagers bound stably to LVV, preventing transduction through LDLR or its other family members, while mediating efficient transduction via the targeted CD3 receptor. CD3-directed engager-modified LVVs efficiently transduced and activated primary human T cells in PBMC cultures, exhibiting stable CD3 targeting even after prolonged storage at room temperature and freeze-thaw cycling. CD3-engager-modified LVV particles encoding an anti-BCMA CAR were administered intravenously to (human) PBMC-engrafted NSG-DKO mice bearing established BCMA-positive human myeloma xenografts. Rapid and complete tumor regressions were observed in all animals treated with the engager-modified LVVs. In summary, we have generated trimerizing bifunctional engagers that redirect the attachment and entry of VSV-G-pseudotyped lentiviral vectors into resting human T cells. Engager-modified LVVs encoding a CAR transgene were therapeutically effective when administered intravenously to tumor-bearing animals. Clinical translation is planned.