Vivovec™ Surface-Engineered Lentiviral Particles Mediate In Vivo CAR T Generation with Potent and Highly Durable Activity in Non-Human Primates and Tumor-Bearing Humanized Mice
Authors: Parker Maura , Ulrich-Lewis Justin , Tang Weiliang , Nicolai Chris , Michels Kathryn , Hernandez Susana , Parrilla Don , Cooper Sara , Perez Anai , McDonnell Mollie , Hamilton Greyson , Wu Chang-Chih , Shin Seungjin , Scharenberg Andrew M. , Larson Ryan P. , Beitz Laurie , Ryu Byoung Y. 
Published: December 2023

Introduction: Ex-vivo generated autologous CAR T cell therapy has transformed the treatment of hematologic malignancies, driving deep and durable responses in patients refractory to conventional therapies. However, multiple challenges including complex manufacturing, high cost, and toxic pre-conditioning regimens limits access to these therapies. To overcome these challenges, a novel lentiviral vector platform, VivoVec™, is being developed. The VivoVec™ platform comprises lentiviral particles surface-engineered with a multi-domain fusion (MDF) protein assembled from T cell activating and costimulatory ligands along with a CAR transgene payload, designed to provide an off-the-shelf solution for generation of CAR T cells in vivo. Here we have evaluated the performance of VivoVec™ particles in multiple preclinical models. Following a single VivoVec™ particle injection, CAR T cells are efficiently generated in vivo and expand in response to cognate antigen, eradicate target antigen expressing cells, and form CAR T cell memory populations, in the absence of lymphodepleting chemotherapy or exogenous supportive cytokines.
Methods:
Mice (NSG MHCI/II knockout): Mice were engrafted with CD19+ Nalm6 tumors and resting human PBMCs, followed by intraperitoneal administration of varying doses of VivoVec™ carrying an aCD19 CAR payload. Absolute CAR T-cell numbers in peripheral blood were quantified by flow cytometry, and Nalm6 tumor burden was assessed by bioluminescence imaging.
Non-Human Primates ( Macaca nemestrina): VivoVec™ surface engineered with NHP-specific surrogate MDF and carrying a human/NHP cross-reactive aCD20 CAR payload. VivoVec™ was administered via intranodal injection to 3 animals at doses of 0.77e8/kg TU in 1 animal and 2.50 e8/kg TU in 2 animals. aCD20 CAR T expansion and B-cell depletion were continuously evaluated by flow cytometry approximately weekly over ~4 months. Body weight, body temperature, neurological exams, serum chemistry panels, and complete blood counts were assessed weekly for the duration of the study.
Results:
VivoVec™ cultured with resting PBMCs selectively bind T cells and some NK cells in an MDF surface engineering-dependent manner, resulting in T cell activation, transduction, and CAR expression. The resultant CAR T cells demonstrate polyfunctional activity through antigen-specific tumor cell killing of Nalm6 tumor cells, cytokine secretion, and proliferation in response to serial antigen exposure. VivoVec™ administration led to dose-dependent generation of CAR T cells in Nalm6-tumor bearing humanized NSG MHCI/II knockout mice. In vivo generated CAR T cells expanded and mediated potent and durable anti-Nalm6 tumor activity.
Non-Human Primates: In the two animals receiving 2.50e8 TU/kg of particles, aCD20 CAR T cells were detected in peripheral blood by flow cytometry with an early peak expansion by day 10 (representing 25.9% or 61.3% of total T cells, respectively). In the first animal, B-cell aplasia was observed from day 7 to day 42, when a small recovering B-cell population was detectable. This was closely followed by a secondary CAR T-cell expansion peaking at day 51: the CAR T cell population represented 43.2% of T cells present in the blood and resulted in recurrent B-cell aplasia sustained through day 70. Persistent B cell aplasia in the second animal receiving the 2.50e8 TU/kg dose began at day 7 and continues to be monitored.
VivoVec™ was well-tolerated with no evidence of toxicity associated within, or shortly after the period of administration. Mild CRS was observed in association with both early and late CAR T cell expansions.
Conclusions: VivoVec™ with MDF surface engineering enables potent and specific generation of functional CAR T cells in vivo in multiple preclinical models, including immune competent NHPs. Importantly, the generation and expansion of CAR T cells occurs in the absence of lymphodepleting chemotherapy, or supportive exogenous cytokines, thus the MDF engineering could impart properties consistent with a natural T cell effector response followed by formation of functional memory T cells. VivoVec™ represents a novel therapeutic platform in oncology that has the potential to overcome many of the challenges associated with the current ex vivo CAR T cell approaches. Additional data from ongoing NHP studies will be provided at the time of presentation.