CD4-Targeted Fusosomes Are Capable of Transducing Resting T Helper Cells to Generate Highly Potent CAR-T Cells

Introduction: Chimeric antigen receptor T cell therapy (CAR T) is a successful treatment for B cell malignancies; however, the time, complexity and cost of manufacturing autologous CAR T products limits the availability of these therapies to patients. Furthermore, ex vivo manipulation of T cells is likely to have a negative impact on quality. In vivo gene delivery of CAR T transgenes by systemic infusion of standard lentiviral vectors may increase therapeutic accessibility but is limited by off-target transduction and the requirement for T cell activation. Here, we demonstrate that a paramyxovirus-based integrating vector (fusosome) engineered with a CD4 re-targeted envelop (CD4 fusogen) can efficiently and specifically transduce resting and activated CD4+ T cells to generate functional CD4+ CD19-specific CAR T cells capable of eliminating CD19+ lymphoma cells.

Methods: Anti-CD4 single chain variable fragments () and single variable domain (VHHs) were screened for CD4 binding, specificity, and NHP cross-reactivity and inserted into receptor binding paramyxovirus fusogen. CD4-targeted fusosomes expressing GFP were screened for high on-target titer against the CD4+ SupT1 cell line and low off-target transduction on non-CD4 expressing cells. Subsequently, a CD19-specific CAR encoding 4-1BB and the CD3z endo-domains (CD19 CAR) was generated to examine CD4+ CAR T transduction efficiency and functionality. PBMCs were thawed and activated with anti-CD3/anti-CD28 beads and exposed to GFP, CD4-targeted fusosomes and specificity of targeting CD4+ T cells was measured by flow cytometry. Subsequently, CD19 CAR fusosomes targeting CD4 were used to test transduction efficiency against activated (CD3/CD28 or IL-7 treated) or resting T cells, and to measure T cell function against CD19+ and CD19 knockout (CRISPR/Cas9-edited) Nalm-6 lymphoma cells (e.g., tumor co-culture and rechallenge assays and cytokine production) in vitro. Vector copy number (VCN) was determined by a multiplex ddPCR assay and reported as copies per diploid genome (c/dg).

Results: To target CD4+ T cells, we generated fusogens encoding scFvs and VHHs specific to the CD4 T cell co-receptor (n = 399). Using fusosomes carrying the GFP transgene, NHP cross-reactive CD4-targeted fusogens that efficiently transduced CD4+ SupT1 cells were selected (n = 12 with crude SupT1 titers >1E6). Activated PBMCs transduced with a CD4-targeted fusosomes exhibited specific CD4 T cell transduction whereas VSV-G pseudotyped vectors showed broad transduction including CD4+ and CD8+ T cells. CD4-targeted CD19 CAR fusosomes could efficiently transduce both activated (34% ± 1.5% CD4+CAR+; 0.54 ± 0.18 c/dg) and resting T cells, albeit at a lower expression and integration rate (20% ± 0.5% CD4+CAR+; 0.28 ± 0.14 c/dg). Resting CD4-transduced CAR T cells demonstrated specific cytotoxicity and cytokine production (GM-CSF, IFN-g, TNF-a, IL-2, IL-6, and IL-10) against CD19+ Nalm-6 but did not recognize CD19 knockout tumor cells. In long-term co-culture assays with repetitive stimulation with fresh tumor cells, resting CD4+ CD19 CAR T cells continued to show potent tumor cell killing. Future experiments will evaluate the efficacy of CD4 fusosomes against CD19+ tumors in vivo.

Summary: CD4-specific fusosomes can efficiently deliver an integrating CAR payload to resting and activated CD4+ T cells. Modified CD4+ CAR T cells demonstrate potent anti-tumor activity against CD19+ tumor cells. These data suggest that targeting the CD4 co-receptor through in vivo delivery using a novel pseudotyped integrating vector can produce functional CAR T cells to target cancer.