Current cell and gene therapy medicines for oncology have reshaped how cancer is treated. Specifically, chimeric antigen receptor (CAR)-T cells have demonstrated that cell therapy can achieve durable remissions in hematologic malignancies. However, CAR-T cell therapies have limited efficacy in solid tumors and are often associated with severe toxicity, highlighting the need for novel cell therapies that are safer and more efficacious. With their intrinsic killing capacity of tumor cells and few, if any, treatment related toxicities, natural killer (NK) cell therapies represent an attractive alternative therapy option to CAR-T cells. In addition, NK cells can be generated from allogeneic donors and given to patients off-the-shelf without causing graft versus host disease. Of the various sources of donor types to generate NK cells from, induced pluripotent stem cells (iPSCs) have the unique advantage of being a renewable source. A clone with any desired edits to enhance the effector function of NK cells can be derived, fully characterized, and expanded indefinitely, to generate large quantities of a naturally allogeneic medicine, therefore streamlining the manufacturing process and increasing scalability. Here, a bicistronic cargo encoding CD16 and a membrane-bound IL-15 (mbIL-15) was knocked into iPSCs at the GAPDH locus using an engineered and highly active AsCas12a. The promoter at the GAPDH locus drives robust constitutive expression of inserted cargos and avoids the promoter silencing that often occurs during differentiation with other strategies. CD16 and mbIL-15 were selected as Knock-Ins (KI) to specifically enhance NK cell therapy in two areas, namely NK cell deactivation caused by CD16 downregulation, and the reliance of co-administration of cytokines such as IL-15 or IL-2 for persistence. CD16 (FcRyIII) can bind the Fc portion of IgG antibodies triggering the lysis of targeted cells. This mechanism of cytotoxicity is known as antibody dependent cellular cytotoxicity (ADCC), and is an innate immune response largely mediated by NK cells through CD16. ADCC is severely impaired when surface CD16 is cleaved by a metalloprotease known as ADAM17. By having CD16 expressed from the GAPDH locus, there is consistent CD16 protein expression to replace what is shed. This hypothesis was demonstrated by performing flow cytometry before and after a cytotoxicity assay. WT cells showed a marked reduction in the surface level expression of CD16 compared to CD16 KI cells after tumor cell exposure. Using a lactate dehydrogenase (LDH) release assay as a measure of cytotoxicity, only the iNK cells expressing the CD16 construct showed statistically significant increases in cytotoxicity when trastuzumab was added. Furthermore, to better model a solid tumor, a 3D tumor spheroid killing assay was utilized where CD16 KI cells showed an increase in ADCC capacity. The benefit of increased effector function via CD16 KI cannot be fully realized without iNK cells persisting. IL-2 or IL-15 is needed for NK maintenance but the administration of either cytokine is associated with acute clinical toxicities. mbIL-15 allows NK cells to survive for a prolonged period without the support of homeostatic cytokines. An in vitro persistence assay was performed that demonstrated IL-15 KI cells showed an increase in persistence compared to WT cells. Specifically, during the three-week in vitro assay, WT cells became undetectable by Day 14 while IL-15 KI NK cells remained stable over time. In summary, to overcome two shortfalls of NK cell therapies, a bicistronic construct encoding CD16 and a mbIL-15 was knocked into the GAPDH locus of iPSCs. The strong GAPDH promoter drove constitutive expression of CD16 that mitigated CD16 shedding, enhanced ADCC of iNK cells, which can be used in combination with any ADCC enabling IgG1 and IgG3 antibodies, such as trastuzumab and rituximab, for tumor-specific targeting. In addition, mbIL-15 KI allowed iNK cells to persist without exogenous cytokine administration and thus can circumvent exogeneous cytokine-induced clinical toxicities. CD16 and mbIL-15 double KI iNKs, with enhanced ADCC and increased cytokine-independent persistence, can potentially be developed into a safe and efficacious therapy for the treatment of a variety of liquid and solid tumors with high unmet medical needs. Disclosures Allen: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Pattali: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Izzo: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Getgano: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Wasko: Editas Medicine: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Blaha: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Zuris: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Zhang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Shearman: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Chang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company.
BackgroundNatural killer (NK) cells have emerged as an alternative cell type for clinical utility given the low propensity for graft-versus-host disease, thereby making NK cells a potential off-the-shelf cell therapy. One critical pathway NK cells use to target tumor cells is through expression of Fc gamma receptor III alpha (CD16). Antibodies that bind tumor antigens are recognized by CD16 on NK cells, promoting NK-mediated tumor cell killing. High-affinity CD16 variants in the human population correlate with better clinical outcome and anti-tumor response. One mechanism tumors use to evade NK cell recognition is through down-regulation of CD16 expression on the NK cell surface. After being activated, CD16 is cleaved by A Disintigrin and Metalloprotease-17 (ADAM-17). By using a highly-active engineered AsCas12a to knock-in high-affinity CD16 (hCD16KI) at the GAPDH locus, hCD16 is constitutively expressed, continuously replacing hCD16, thereby allowing for repeated ADCC mediated killing.Methods iPSCs were edited at the GAPDH locus with an engineered AsCas12a along with the CD16 donor construct. The bulk edited population was then plated at clonal density and single clones were selected and screened. iPSC clones were then differentiated into NK cells. A 3D tumor spheroid killing assay was used to demonstrate NK cell cytotoxicity against an ovarian cancer cell line (SKOV-3). In addition, a serial killing assay was used to better model NK cell serial killing.ResultsBi-allelic CD16KI iPSC clones were successfully generated. These iPSCs exhibited normal morphology and were able to differentiate into iNK cells. hCD16KI iNK cells showed normal differentiation and surface marker expression, such as CD45/CD56, compared to unedited iNK cells. CD16KI iNK cells demonstrated significantly increased cytotoxicity in the presence of antibody against tumor cells when compared with unedited iNK cells, as measured by reduction in tumor spheroid size in a 3D tumor spheroid killing assay. Importantly, enhanced surface expression of hCD16 on iNK cells after tumor exposure was detected, demonstrating the replenishment of cleaved hCD16. Notably, hCD16KI iNK cells demonstrated prolonged and enhanced tumor cell killing after being subjected to repeated tumor cell exposure in a serial killing assay.ConclusionsThis work demonstrates a powerful new method to drive high-level constitutive hCD16 expression on the surface of iNK cells through transgene knock-in at the GAPDH locus using an engineered AsCas12a. The high level constitutive hCD16 expression enhances ADCC of iNK cells and enables enhanced serial tumor killing and is expected to exert enhanced anti-tumor activity in the clinic.
Current cell and gene therapy medicines for oncology have reshaped how cancer is treated. Chimeric antigen receptor (CAR)-T cells have demonstrated that cell therapy can achieve durable remissions in hematologic malignancies. CAR-T cell therapies, however, have limited efficacy in solid tumors and are associated with severe toxicity, highlighting the need for safer and more efficacious novel cell therapies. With their intrinsic tumor killing capacity, few treatment-related toxicities, and the ability to be given to patients off-the shelf, natural killer (NK) cells are an attractive alternative therapy option to CAR-T cells. While most NK cell therapies are produced from healthy donor cells, deriving NK cells from induced pluripotent stem cells (iPSCs) has the unique advantage that a clone with any desired edits can be generated. We aim to leverage our iPSC platform in combination with our proprietary gene editing technologies to create highly differentiated off-the-shelf treatments for solid tumors. Using our proprietary engineered AsCas12a, we generated double knocked-in (DKI) iPSC clones in which a bicistronic cargo encoding CD16 and a membrane-bound IL-15 (mbIL-15) was knocked into the GAPDH locus to increase the effector function and persistence of iNKs. Constitutive surface expression of CD16 and mbIL-15 by the DKI iNKs was demonstrated. DKI iNKs showed significantly increased natural and antibody dependent cellular cytotoxicity when compared to wild type (WT) iNKs in a SKOV3 tumor spheroid assay in vitro. Furthermore, in the absence of exogeneous cytokines, DKI iNKs persistence in vitro was dramatically improved over WT iNKs. The anti-tumor efficacy of the DKI iNKs in vivo was evaluated using a SKOV3 ovarian cancer model. Tumor bearing mice were treated with WT or DKI iNKs intraperitoneally in combination with trastuzumab or treated with trastuzumab alone. No exogenous cytokines were administered. DKI iNKs combined with a single dose or multiple doses of trastuzumab exerted greater tumor control compared to WT iNKs with trastuzumab, or trastuzumab alone. A single dose of DKI iNKs combined with three doses of trastuzumab induced tumor clearance in 6 out of 8 mice and significantly prolonged survival. Importantly, DKI iNKs were detected in the peritoneum of the treated animals for greater than 3 months, demonstrating that the mbIL-15 maintained iNK survival for a prolonged period of time in the absence of exogeneous cytokine support. In summary, knocking-in CD16 and mbIL-15 to the GAPDH locus of iPSCs dramatically increased the persistence of the DKI iNKs which exhibited robust anti-tumor activities in a solid tumor mouse model. These data demonstrate that our platform enables the development of off-the-shelf iNK cell medicines that may be highly effective for treating solid tumors. Citation Format: Alexander G. Allen, Samia Q. Khan, Kaitlyn M. Izzo, Mrunali Jagdale, Alexandra Gerew, Nadire R. Cochran, Jared Getgano, Stephen Sherman, Laura Blaha, Mark Shearman, Kate Zhang, Kai-Hsin Chang. AsCas12a gene-edited iPSC-derived NK cells constitutively expressing CD16 and membrane-bound IL-15 demonstrate prolonged persistence and robust anti-tumor activities in a solid tumor mouse model [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 562.
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