Natural Killer and Gamma Delta T Cells Derived from Engineered Induced Pluripotent Stem Cells Have Potent Preclinical Activity to Treat B Cell-Mediated Autoimmune Diseases

B-lymphocyte antigen CD19 specific chimeric antigen receptor (CAR)-expressing T-cell therapy has been established as an effective treatment for CD19+ malignancies. These therapies are being evaluated as treatments for subsets of autoimmune diseases thought to be mediated by B cells producing auto-reactive antibodies. Emerging data, largely using autologous CAR-T cells have demonstrated durable drug-free remissions in patients enrolled in early phase studies. Long-term safety, specifically due to prolonged B cell aplasia often seen when used in oncology, is unknown. CAR-engineered induced pluripotent stem cell (iPSC)-derived effector cells are a potential treatment of these autoimmune diseases as an off-the-shelf allogeneic cell therapy. Gamma delta (γδ) T cells and natural killer (NK) cells exhibit the potency and cytolytic features of conventional alpha beta (αβ) CAR-T cells, with a lower expansion capacity and persistence upon antigen engagement, mitigating the risk of prolonged B cell aplasia. Through Allo-Evasion™ engineering, iPSC-derived γδ T and NK (iT and iNK, respectively) cells can be repeat-dosed, allowing for tighter control of drug exposure and subsequent B cell depletion. Here we describe preclinical development of iPSC-derived CAR γδ T and CAR NK cell platforms for use in autoimmune diseases. iPSCs derived by re-programming peripheral γδ T cells (TiPSC) and peripheral blood mononuclear cells (PiPSC) were engineered using MAD7, a Class 2 Type V Crispr-Cas nuclease, to express a CD19-targeting (FMC63) CAR, membrane-bound IL-15/IL15Ra, and Allo-Evasion™ edits including expression of Human Leukocyte Antigen alpha-chain E (HLA-E) and G (HLA-G), accompanied by a knock-out of B2M and CIITA resulting in abrogated expression of Major Histocompatibility Complex Class I and II on the cell surface. Engineered TiPSC and PiPSC were then differentiated into hematopoietic progenitor cells (HPC), and further differentiated into γδ T cells and NK cells respectively. Leukopaks were obtained from normal donors and autoimmune disease patients, and peripheral blood mononuclear cells (PBMCs) isolated. From these, B cells were isolated for in vitro assays. Normal T cells were transduced for CD19 CAR expression, and these primary CD19 CAR-T were used as a positive control in experiments. iT and iNK killing efficiency of B cells and acute lymphoblastic leukemia NALM-6 cells were tested in an in vitro single round killing assay. Effector cells and target cells were plated at multiple effector-to-target (E:T) cell ratios, and incubated for 24 hours, at which point cells were stained and analyzed by flow cytometry for target cell cytolysis. A PBMC-humanized NOD SCID Gamma (NSG) mouse model was utilized to test the B cell killing capacity of iT and iNK. Mice were intravenously engrafted with PBMCs, and 14 days later received an intravenous injection of iT, iNK, or primary CAR-T. Mice were humanely euthanized, and blood and tissues collected for Fluorescence-Activated Cell Sorting (FACS) and immunohistochemical (IHC) detection of B cells. IPSC-derived γδ T and NK cells were generated using differentiation processes yielding >90% pure CD19 CAR+ γδ T and NK cells. At the completion of differentiation, cells were confirmed by FACS to be positive for IL-15/IL15Ra, HLA-E and HLA-G transgenes, and negative for B2M and CIITA. In vitro, both iT and iNK had near equivalent and significant potency in killing of B cells and NALM-6 target cells. Cytolysis by iNK was observed to be slightly higher than that by iT, especially at greater E:T ratios. Mouse blood and tissues were evaluated by FACS and IHC for presence of human B cells following CD19 CAR+ effector cell treatment. B cell depletion was observed in blood and tissues of iT and iNK treated mice, at levels equivalent to or greater than that observed in primary CD19 CAR-T treated mice. In blood and bone marrow, near complete B cell depletion was observed with iT and iNK treatment. Splenic B cell depletion was incomplete but nearly equivalent across all treatments including CAR-T. We have demonstrated iPSC-derived CD19 CAR+ γδ T and NK cells are potentially potent cell therapies for the treatment of autoimmune disease. The ability to repeat dose as-needed as a result of Allo-Evasion™ technology, and the ability to manufacture large, clonal batches of cells, has the potential to enable a true off-the-shelf allogenic cell therapy for autoimmune disease.