Combining CAR Engineering and CIS Checkpoint Deletion in NK Cells for the Treatment of B Cell Hematologic Malignancies
Authors: Daher May , Basar Rafet , Gokdemir Elif , Baran Natalia , Uprety Nadima , Mendt Mayela Carolina , Kerbauy Lucila , Shanley Mayra , Cortes Ana Karen Nunez , Li Li , Banerjee Pinaki P , Mohanty Vakul , Lu Junjun , Ozcan Gonca , Ensley Emily , Nandivada Vandana , Kaplan Mecit , Bdaiwi Mustafa H , Liu Enli , Ang Sonny , Chen Ken , Konopleva Marina Y , Champlin Richard E. , Shpall Elizabeth J. , Rezvani Katayoun 
Published: November 2019

Immune checkpoint-based therapies, which target the regulatory pathways of immunocompetent cells to enhance anti-tumor responses, have been at the heart of many recent clinical advances and have led to long-term remissions and possible cures. Most of this success was achieved with T-cells, but there are compelling reasons to predict that checkpoint ablation could modify NK cells in ways that would facilitate their antitumor activity.
The suppressor of cytokine signaling (SOCS) family of proteins plays an important role in NK cell biology by attenuating cytokine signaling and effector function against cancer. One of its members, cytokine inducible SH2 containing protein (CIS), encoded by the CISH gene, is as an important checkpoint molecule in NK cells and is upregulated in response to IL-15. We hypothesized that CIS may act as a potent checkpoint in our iC9/CAR19/IL15 NK cells given the fact that they continuously produce IL-15, and that targeting this pathway would enhance their potency against B cell malignancies.
In a series of in vitro studies, we showed that CISH is induced in iC9/CAR19/IL15 NK cells in a time dependent manner. To examine the functional consequences of CISH deletion in our CAR-NK cells, we developed a protocol for combined Cas9 ribonucleoprotein (Cas9 RNP)-mediated gene editing to silence CISH and retroviral transduction with the iC9/CAR19/IL15 construct. On day 7 we nucleofected the CAR transduced NK cells with Cas9 alone (Cas9 control) or Cas9 pre-loaded with crRNA:tracrRNA duplex targeting CISH exon 4. Gene editing efficiency was >90% as quantified by PCR and western blot. CISH knockout induced a phenotype characterized by the increased expression of markers of activation and cytotoxicity. These included granzyme-b, perforin, TRAIL and CD3z; transcription factors such as eomesodermin and T-bet; adaptor molecules such as DAP12; and activating coreceptors/proliferation markers such as DNAM, CD25 and Ki67. CISH knockout resulted in significantly enhanced function of iC9/CAR19/IL15 NK cells against Raji lymphoma evident by increased cytokine production (TNFa p=0.007, IFNg p=0.033) and degranulation (CD107a p=0.003) compared to Cas9 control cells. Moreover, CISH KO iC9/CAR.19-IL15 NK cells killed Raji lymphoma more efficiently than Cas9 control cells and formed a stronger immunologic synapse (p=0.037). RNA sequencing with gene set enrichment analysis (GSEA) confirmed enrichment of JAK/STAT signaling, TNFα and IFN-γ inflammatory response, mTORC1, and MYC hallmark pathways in CISH KO iC9/CAR19/IL15 NK cells compared to Cas9 control counterparts, providing a molecular mechanism for their enhanced effector function. Moreover, in an in-vivo NSG mouse model of Raji lymphoma, the antitumor activity of a single dose of CISH KO iC9/CAR19/IL15 transduced CB NK cells was significantly better than that of Cas9 control cells leading to a significant survival advantage (p=0.003) without evidence of increased toxicity.
Thus, we demonstrate for the first time, that silencing a critical checkpoint in CAR-NK cells improves their potency, permitting greater cytotoxic effector function than seen with unmodified CAR-NK cells. Our data support the merging of CAR-engineering and immune checkpoint gene editing to enhance the therapeutic potential of NK cells. We are in the process of scaling up this approach in our GMP facility for translation to the clinic for the treatment of relapsed/refractory B cell hematologic malignancies.

Disclosures
Konopleva: Calithera: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria; Eli Lilly: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Amgen: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Ascentage: Research Funding; Kisoji: Consultancy, Honoraria; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Ablynx: Research Funding; Astra Zeneca: Research Funding; Agios: Research Funding. Champlin:Sanofi-Genzyme: Research Funding; Actinium: Consultancy; Johnson and Johnson: Consultancy.