Yijiu Tong

Natural killer (NK) cells are innate lymphocytes recognized for their important role against tumor cells. NK cells expressing chimeric antigen receptors (CARs) have enhanced effector function against various type of cancer and are attractive contenders for the next generation of cancer immunotherapies. However, a number of factors have hindered the application of NK cells for cellular therapy, including their poor in vitro growth kinetics and relatively low starting percentages within the mononuclear cell fraction of peripheral blood or cord blood (CB). To overcome these limitations, we genetically-engineered human leukocyte antigen (HLA)-A − and HLA-B − K562 cells to enforce the expression of CD48, 4-1BBL, and membrane-bound IL-21 (mbIL21), creating a universal antigen presenting cell (uAPC) capable of stimulating their cognate receptors on NK cells. We have shown that uAPC can drive the expansion of both non-transduced (NT) and CAR-transduced CB derived NK cells by >900-fold in 2 weeks of co-culture with excellent purity (>99.9%) and without indications of senescence/exhaustion. We confirmed that uAPC-expanded research- and clinical-grade NT and CAR-transduced NK cells have higher metabolic fitness and display enhanced effector function against tumor targets compared to the corresponding cell fractions cultured without uAPCs. This novel approach allowed the expansion of highly pure GMP-grade CAR NK cells at optimal cell numbers to be used for adoptive CAR NK cell-based cancer immunotherapy.

Abstract Natural killer (NK) cells are a major component of the innate immune system, possessing the ability to lyse their targets without the need for prior sensitization or specificity for antigen. Besides their classical role in providing potent antitumor and antiviral immunity, NK cells can reduce the risk of graft-versus-host disease (GVHD) by targeting host antigen-presenting cells, as well as activated alloreactive donor T cells, indicating that NK-mediated graft-versus-leukemia (GVL) responses may occur in the absence of GVHD. Although most groups have relied on autologous or adult peripheral blood donor-derived NK cells, we have studied umbilical CB as a potential source of NK cells because of their availability as an "off-the-shelf" frozen product and their potent preclinical activity against leukemia cells. To overcome the obstacle of limited numbers of NK cells in a single CB unit, we have established GMP-compliant conditions for the ex vivo expansion of clinically relevant doses of such cells. By using GMP grade K562-based artificial antigen-presenting cells (aAPCs), which express membrane-bound IL-21 (clone 9.mbIL21), to numerically expand highly functional and mature CB-derived NK cells. To further enhance the GVL effect independent of KIR-ligand mismatch, we have genetically modified human CB-derived NK cells with a retroviral vector, CAR19-CD28-zeta-2A-IL15 (CAR19/IL15), which incorporates the genes for CAR-CD19, IL-15 to enhance proliferation and survival, and the inducible caspase-9 molecule. CB-NK cells could be stably transduced with CAR19/IL15, proliferated efficiently in vitro and maintained superior effector function against CD19-expressing leukemia cell lines and primary CLL cells. Moreover, the effector functions of both NK-CAR and nontransduced NK cells against K562 were comparable, indicating that the genetic modification of CB-NK cells does not alter their intrinsic cytotoxicity against NK-sensitive targets. Because of concerns over autonomous, uncontrolled NK cell growth due to autocrine production of IL15, we also incorporated into our construct a suicide gene based on the inducible caspase-9 (IC9) gene. The addition of as little as 10 nM of the small molecule dimerizer CID AP20187 to cultures of iC9/CAR19/IL15+ NK cells induced apoptosis/necrosis of >60% of transgenic cells within 4 hours as assessed by annexin-V-7AAD staining. The infusion of CAR.CD19.IL15-transduced CB-NK cells into a NOD-SCID-gamma null model of lymphoblastic lymphoma (Raji model) resulted in impressive anti-tumor responses (Fig. 1). Moreover, adoptively infused CAR-transduced CB NK persisted for up to 70 days post-infusion (Fig. 2), supporting our hypothesis that IL-15 enhances the proliferation and survival of the engineered CB-NK cells. Based on these promising data, we now propose to manufacture a GMP-grade CAR19-CD28-zeta-2A-IL15 vector for a phase 1 dose escalation trial in patients with high risk B-cell leukemia. Disclosures Wierda: Celgene Corp.: Consultancy; Glaxo-Smith-Kline Inc.: Research Funding. Rezvani:Pharmacyclics: Research Funding.

International audience

Abstract NK cells are potent cellular immunotherapeutic agents against a wide array of human malignancies. Ex vivo expansion of NK cells to achieve clinically relevant numbers must overcome poor in vitro growth kinetics, low starting percentages within the mononuclear cell fraction (especially from autologous donors with active disease), and limited in vivo life span. We targeted three universally critical NK signaling pathways, namely IL-21, 4-1BB, and SLAM family member 4 (SLAMF4), to increase NK cell proliferation and enhance survival. We genetically-engineered HLA-A-ve and -B-ve K562 cells to enforce expression of membrane-bound IL-21 (mbIL21), 4-1BB-L, and CD48, forming a universal antigen presenting cell (UAPC) to generate highly potent clinical-grade umbilical cord blood (CB-NK) or peripheral blood NK cells (PB-NK). While the mbIL21 and 4-1BB signaling nexuses have been utilized previously, we highlight here SLAMF-mediated immunological sculpting of NK cells for clinical applications. SLAMF triggering of co-receptors modulate NK cell activation, in particular through high-avidity interactions between SLAMF4 (2B4/CD244) and its heterophilic, robust affinity, and physiological ligand CD48, a glycosyl-phosphatidyl-inositol (GPI)-anchored cell surface protein. Upon ligand binding and receptor phosphorylation, SLAMF4 recruits PTPN11/SHP-2 and SH2D1A/SAP for downstream signaling, including significant increases in NK cell-mediated cytotoxicity, granule exocytosis, and production of IFN-γ and IL-2. Other than a subset of low surface density expressers from aging subjects consistently associated with inefficient and impaired activating signal transduction, the majority of NK cells express SLAMF4. The functional prominence of SLAMF4, presently recognized as an activating co-receptor, is also evidenced by loss-of-function mutations associated with X-linked lymphoproliferative (XLP) disease. We achieved log-scale expansion of NK cells with UAPC (>1000 fold in 2 weeks), with excellent purity (>99% CD56+ve/CD3−ve and < 1% CD3+ve cells), without indications of senescence/exhaustion, even after 4 weeks of culture. Surface molecular phenotypes of UAPC-expanded CB-NK cells exhibited a phenotype similar to CB-NK cells expanded without SLAMF4. Synergistic signals from IL-21/STAT3, 4-1BB/4-1BBL, and SLAMF4 drive tonic NK propagation, supporting their clinical application. Our novel and clinically accessible platform technology for generation of high purity CB-NKs, a promising source of fresh and cryopreserved allogeneic NK cells, is well-suited for adoptive cancer immunotherapy. Disclosures Champlin: Sanofi: Research Funding; Otsuka: Research Funding. Shpall:Affirmed GmbH: Research Funding. Rezvani:Affirmed GmbH: Research Funding.