Yanan Yue

Clinical application of autologous chimeric antigen receptor (CAR)-T cells is complicated by limited targeting of cancer types, as well as the time-consuming and costly manufacturing process. We develop CD70-targeted, induced pluripotent stem cell-derived CAR-natural killer (NK) (70CAR-iNK) cells as an approach for universal immune cell therapy. Besides the CD70-targeted CAR molecule, 70CAR-iNK cells are modified with CD70 gene knockout, a high-affinity non-cleavable CD16 (hnCD16), and an interleukin (IL)-15 receptor α/IL-15 fusion protein (IL15RF). Multi-gene-edited 70CAR-iNK cells exhibit robust cytotoxicity against a wide range of tumors. In vivo xenograft models further demonstrate their potency in effectively targeting lymphoma and renal cancers. Furthermore, we find that recipient alloreactive T cells express high levels of CD70 and can be eliminated by 70CAR-iNK cells, leading to improved survival and persistence of iNK cells. With the capability of tumor targeting and the potential to eliminate alloreactive T cells, 70CAR-iNK cells are potent candidates for next-generation universal immune cell therapy.

Immune cell therapy has become a cornerstone in cancer therapy. However, the needs for new therapies targeting acute myeloid leukemia (AML) and solid tumors remains high. Natural Killer (NK) cells derived from genetically engineered human induced Pluripotent Stem Cells (iPSCs) hold great potential to become the next-generation allogeneic cell therapy products. NK cells exert anti-tumor activity through the net balance of stimulatory and repressive signals from various activating and inhibitory receptors. Some of the activating ligands for NK cell activation are highly expressed on the AML blasts as well as the solid tumors. Thus, the off-the-shelf iPSC-derived NK cell product is a promising clinical drug candidate for the treatment of AML and solid tumors. In this study, we demonstrated that human iPSCs-derived NK (iNK) cells mediate potent cytotoxicity to several AML cell lines and solid tumor cell lines that highly express NKG2D ligands. Further, we developed CD33-CAR iNK (QN-023a) with 4 anti-tumor functional modalities, including a CAR optimized for NK cell biology that targets AML antigen CD33, a high-affinity, non-cleavable CD16 (hnCD16) to enhance antibody-dependent cell-mediated cytotoxicity (ADCC), an IL-15 molecule to increase the persistence of allogeneic cells in transplant patients, a CD38 knockout to effectively avoid fratricide of CAR-NK cells and acquire synergetic function with daratumumab. Here, we first engineered a small library of human iPSC clones with desired genetic modifications. We then differentiated the engineered human iPSC clones into NK cells and conducted both in vitro and in vivo screening by using a variety of AML cell lines. We identified QN-023a as the lead, who demonstrated superior anti-tumor activity among the candidates both in vitro and in NOG mouse model. QN-023a show potent anti-tumor activity against a variety of AML cell lines and primary AML blasts from patients, regardless of the expression level of NKG2D ligands on these tumor cells. QN-023a cells can be mass-produced in a cGMP process, have phenotype comparable to healthy donor NK cells, and are functionally potent against multiple AML tumor models. When administered in combination with daratumumab, QN-023a demonstrated superior ADCC compared to unmodified iPSC-derived NK cells against primary AML blasts from patients. Together, we have engineered iPSC-derived QN-023a NK cells as promising clinical drug candidates for treatment of AML.

iPSC-derived natural killer cells (iNKs) have emerged as a promising cellular therapy, especially for the refractory or relapsed acute myeloid leukemia (R/R AML) patients, but limited research focused on the chemotaxis of iNKs. Here we demonstrate that C-X-C chemokine receptor type 4 (CXCR4) is significantly reduced in iNKs, resulting in impaired bone marrow (BM) infiltration, which cannot be rescued by constitutively expressed CXCR4 in iPSC due to CXCR4-induced differentiation failure. To address this, we developed a strategy to allow specific expression of CXCR4 during the iNK maturation stage without compromising the final iNK yield and function. The engineered iNKs exhibited enhanced BM infiltration, resulting in improved therapeutic effects in AML murine models. This, brought attention to iNK chemotaxis, provided a meaningful strategy by incorporating well-designed gene editing with stem cells for cell product development, and obtained improved effective NK cells for AML therapy.

Abstract Engineered human induced pluripotent stem cells (hiPSC)-derived allogeneic Natural Killer (NK) cells are emerging as promising safe and effective off-the-shelf cell therapy drugs. Repeated dosing of NK cells can partly compensate the relatively shorter persistence of NK cells compared with chimeric antigen receptor T cells (CAR-T); However, with each repeat, the persistence and efficacy are increasingly challenged by activation of host innate and adaptive immunity. Thus, reducing immunogenicity of allogeneic NK cells holds promise for better efficacy. In this study, we report a combination of “hypoimmunity” gene edits including multiple transgene knock-ins and both class I and class II MHC knockouts (2KO). We show that hiPSC engineered by the hypoimmunity combo can be efficiently differentiated into NK cells, comparable to the non-engineered ones. This is contrary to the notion from mouse studies that NK cells devoid of class I molecules are hypo-responsive, these engineered NK cells can be expanded by aAPC (artifitial Antigen Presenting Cells, a K562 cell line with surface expression of mbIL-21 and CD137L) and are capable of killing tumor cells, Additionally, we show that the engineered NK cells can evade T cell immunity, as examined by in vitro T cell proliferation and cytotoxicity assays. A humanized mouse model with transplanted CD34-positive human hematopoietic stem cells confirmed that the engineered NK cells can persist longer than WT and 2KO NK cells. Hypoimmune-QN-019 NK cells are generated by incorporating hypoimmunity gene combo edits into our hiPSC-derived NK cell clinical candidate QN-019 which has 3 transgenes including membrane-bound IL-15, high affinity non-cleavable CD16 and CAR targeting CD19. Comparing to QN-019, hypoimmune-QN-019 displayed better efficacy in humanized CDX mouse model in clearing CD19-positive cancer cells. Taken together, we have engineered hiPSC-derived allogeneic NK cells with hypoimmunity features, which hold great promise in becoming an off-the-shelf drug with improved in vivo persistence and efficacy for repeated dosing. Citation Format: Jiabiao Hu, Xiangjun He, Jing Xu, Yixuan Zhou, Yanan Yue, Yangbin Gao, George Church, Luhan Yang. Functional natural killer cells derived from engineered hiPSC with hypoimmunity gene combo demonstrate hypoimmunity features in evading host attacks [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6070.