Cell Antibody-dependent Cellular Cytotoxicity Research Articles

Abstract 83: NK cell ADCC assays: Leveraging flow cytometry and reporter cell lines for enhanced biological relevance and throughput

Abstract Amid the growing arsenal of cancer treatments, antibody-dependent cellular cytotoxicity (ADCC) assays have emerged as a significant contributor to the quest for more effective therapies. The ADCC-based antibody therapy often exploits Natural Killer (NK) cells as effectors. This immunotherapeutic approach harnesses the innate cytotoxic potential of NK cells, offering a promising solution for the precision targeting of malignant cells. In this study, we employed two methods that can be used to measure ADCC potency and specificity during preclinical drug discovery. The first was a flow cytometry-based approach that focused on enhancing biological relevance by utilizing primary human peripheral blood mononuclear cells (PBMCs) as a source for NK effector cells. To this end, target cells were fluorescently labeled with CFSE and cultured with freshly isolated PBMCs at 3 effector to target (E:T) ratios, in the presence of ADCC antibody. After 16h incubation, a viability dye was added, and percent cytotoxicity was measured in CFSE+ targets cells by flow cytometry. The second method offered a high-throughput plate reader-based approach (ADCC Reporter Bioassay; Promega), that utilized an ADCC reporter effector cell line harboring a luciferase cassette driven by an NFAT promoter, which is activated when effector and targets cells are bridged by a therapeutic antibody. Cytotoxicity was read out by measuring light output on a Cytation 3 plate imaging reader (Agilent Technologies) after a 6h cell culture incubation using the recommended 6:1 E:T ratio. Preclinical application for both assays was demonstrated using an anti-HER2 therapeutic antibody (trastuzumab) against BT-474, which is a HER2 positive cell line. To demonstrate specificity, an anti-VEGF antibody (bevacizumab) and Hs 578T cells (HER2 low) were used as controls. Our results revealed trastuzumab induced a strong dose-dependent increase in cytotoxicity in the BT-474 cells but not the Hs 578T cell line (range: 40-70%) while bevacizumab failed to induce any cytotoxicity above background levels at any concentration. Furthermore, the flow cytometry and plate reader methodologies produced similar trends. In summary, both applications can be used to produce reliable ADCC drug screening assessments. Additional considerations include investigational needs surrounding high throughput capability versus biological relevance. Unlike traditional models using immortalized cell lines, primary human PBMCs more faithfully recapitulate the physiological interactions between effector cells and target cells. On the other hand, the more streamlined reporter cell line approach not only accelerates assay throughput but also ensures consistent and reliable results in an attractive option for large-scale screening and drug development efforts. Citation Format: David William Draper, Yewei Xing, Scott Wise. NK cell ADCC assays: Leveraging flow cytometry and reporter cell lines for enhanced biological relevance and throughput [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 83.

Efbalropendekin Alfa enhances human natural killer cell cytotoxicity against tumor cell lines in vitro.

IL-15 has shown preclinical activity by enhancing the functional maturation of natural killer (NK) cells. Clinical evaluation of the potential anticancer activity of most cytokines, including IL-15, has been limited by low tolerability and rapid in vivo clearance. Efbalropendekin Alfa (XmAb24306) is a soluble IL15/IL15-receptor alpha heterodimer complex fused to a half-life extended Fc domain (IL15/IL15Rα-Fc), engineered with mutations to reduce IL-15 affinity for CD122. Reduced affinity drives lower potency, leading to prolonged pharmacodynamic response in cynomolgus monkeys. We show that in vitro, human NK cells treated with XmAb24306 demonstrate enhanced cytotoxicity against various tumor cell lines. XmAb24306-treated NK cells also exhibit enhanced killing of 3D colorectal cancer spheroids. Daratumumab (dara), a monoclonal antibody (mAb) that targets CD38 results in antibody-dependent cellular cytotoxicity (ADCC) of both multiple myeloma (MM) cells and NK cells. Addition of XmAb24306 increases dara-mediated NK cell ADCC against various MM cell lines in vitro. Because NK cells express CD38, XmAb24306 increases dara-mediated NK cell fratricide, but overall does not negatively impact the ADCC activity against a MM cell line likely due to increased NK cell activity of the surviving cells. These data show that XmAb24306 increases direct and ADCC-mediated human NK cell cytotoxicity in vitro.

Generation and optimization of off-the-shelf immunotherapeutics targeting TCR-Vβ2+ T cell malignancy

Current treatments for T cell malignancies encounter issues of disease relapse and off-target toxicity. Using T cell receptor (TCR)Vβ2 as a model, here we demonstrate the rapid generation of an off-the-shelf allogeneic chimeric antigen receptor (CAR)-T platform targeting the clone-specific TCR Vβ chain for malignant T cell killing while limiting normal cell destruction. Healthy donor T cells undergo CRISPR-induced TRAC, B2M and CIITA knockout to eliminate T cell-dependent graft-versus-host and host-versus-graft reactivity. Second generation 4-1BB/CD3zeta CAR containing high affinity humanized anti-Vβ scFv is expressed efficiently on donor T cells via both lentivirus and adeno-associated virus transduction with limited detectable pre-existing immunoreactivity. Our optimized CAR-T cells demonstrate specific and persistent killing of Vβ2+ Jurkat cells and Vβ2+ patient derived malignant T cells, in vitro and in vivo, without affecting normal T cells. In parallel, we generate humanized anti-Vβ2 antibody with enhanced antibody-dependent cellular cytotoxicity (ADCC) by Fc-engineering for NK cell ADCC therapy.

Development and Testing of a Glycoengineered Anti-ROR1 Antibody with Enhanced Capacity for Directing Antibody-Dependent Cellular Cytotoxicity (ADCC) of Chronic Lymphocytic Leukemia Cells

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic surface antigen expressed on the neoplastic cells of patients with chronic lymphocytic leukemia (CLL) or other types of cancer, but not on virtually all normal postpartum tissues (Fukuda T., et al., PNAS, 105:3047, 2008). Zilovertamab (also known as UC-961 or cirmtuzumab) is a humanized anti-ROR1 monoclonal antibody (mAb) currently under clinical investigation; this mAb is capable of blocking ROR1-signaling, which can contribute to cancer-cell migration, proliferation, survival, and self-renewal. We generated a glycoengineered form of zilovertamab (GE- zilovertamab) and examined whether it could mediate enhanced antibody-dependent cellular cytotoxicity (ADCC) against neoplastic cells that express ROR1. Initial studies used Jurkat-Lucia™ NFAT-CD16 cells (Invivogen, San Diego, CA, USA) which express high-affinity CD16A (FcγRIIIA, V158 allotype) as effector cells (EC), and the CLL-cell line MEC1, or MEC1 cells transduced to express ROR1 (MEC1-ROR1), as target cells (TC), which each also express CD20. Co-culture of Jurkat-Lucia cells for 6 hours with the anti-CD20 mAb rituximab and MEC1 or MEC1-ROR1 cells induced Jurkat-Lucia cells to express a luciferase reporter gene under the control of an ISG54 minimal promoter fused to six NFAT response elements; this endowed the Jurkat-Lucia cells with high luminescence activity that was not observed in co-cultures of EC and TC without added mAb. Neither zilovertamab nor GE-zilovertamab endowed Jurkat-Lucia cells with higher than background luminescence activity when co-cultured with MEC1 cells. However, GE-zilovertamab could induce high levels of luminescence activity in Jurkat-Lucia cells co-cultured with MEC1-ROR1 cells that was comparable to that of rituximab, and significantly greater than that of zilovertamab. We generated an orthogonal system with which to examine the relative ADCC activity of each of these mAb, using EC generated from the NK cell line, NK92, which we transduced with a lentivirus encoding a newly-generated high-affinity variant of CD16A (CD16v), which has been modified to block its cleavage by ADAM17. NK92 or NK92-CD16v were used as EC to effect cytolysis of Cr 51-labeled MEC1, MEC1-ROR1, or primary CLL cells in a chromium-release assay. Addition of rituximab to co-cultures of EC with Cr 51-labeled MEC1, MEC1-ROR1, or primary CLL caused mAb-dose-dependent ADCC of each of these TC by NK92-CD16v that was significantly greater than that by parental NK92 cells. Again, neither zilovertamab nor GE-zilovertamab could direct ADCC by NK92-CD16v or NK92 ADCC of MEC1 cells, which lack ROR1. However, GE-zilovertamab directed dose-dependent ADCC of MEC1-ROR1 or primary ROR1 + CLL cells by either NK92-CD16v or NK92 at levels comparable to that directed by rituximab for either EC population; such levels were significantly greater than that directed by zilovertamab. ROR1 + CLL cells harboring del(17p) or mutations in TP53 (del(17p)/m TP53) and/or that were resistant to targeted therapies (e.g., inhibitors of BTK or BCL2), were as susceptible to GE-zilovertamab-directed ADCC as were CLL cells without del(17p)/m TP53 from patients who had not had prior therapy. These data demonstrate that GE-zilovertamab can direct high-level ADCC lysis of ROR1-expressing neoplastic cells with greater activity than zilovertamab, encouraging development of clinical studies to evaluate GE-zilovertamab for therapy of patients with CLL or other ROR1-positive cancers.

Impact of HIV-1 Vpu-mediated downregulation of CD48 on NK-cell-mediated antibody-dependent cellular cytotoxicity.

HIV-1 evades antibody-dependent cellular cytotoxicity (ADCC) responses not only by controlling Env conformation and quantity at the cell surface but also by altering NK cell activation via the downmodulation of several ligands of activating and co-activating NK cell receptors. The signaling lymphocyte activation molecule (SLAM) family of receptors, which includes NTB-A and 2B4, act as co-activating receptors to sustain NK cell activation and cytotoxic responses. These receptors cooperate with CD16 (FcγRIII) and other activating receptors to trigger NK cell effector functions. In that context, Vpu-mediated downregulation of NTB-A on HIV-1-infected CD4 T cells was shown to prevent NK cell degranulation via an homophilic interaction, thus contributing to ADCC evasion. However, less is known on the capacity of HIV-1 to evade 2B4-mediated NK cell activation and ADCC. Here, we show that HIV-1 downregulates the ligand of 2B4, CD48, from the surface of infected cells in a Vpu-dependent manner. This activity is conserved among Vpu proteins from the HIV-1/SIVcpz lineage and depends on conserved residues located in its transmembrane domain and dual phosphoserine motif. We show that NTB-A and 2B4 stimulate CD16-mediated NK cell degranulation and contribute to ADCC responses directed to HIV-1-infected cells to the same extent. Our results suggest that HIV-1 has evolved to downmodulate the ligands of both SLAM receptors to evade ADCC. IMPORTANCE Antibody-dependent cellular cytotoxicity (ADCC) can contribute to the elimination of HIV-1-infected cells and HIV-1 reservoirs. An in-depth understanding of the mechanisms used by HIV-1 to evade ADCC might help develop novel approaches to reduce the viral reservoirs. Members of the signaling lymphocyte activation molecule (SLAM) family of receptors, such as NTB-A and 2B4, play a key role in stimulating NK cell effector functions, including ADCC. Here, we show that Vpu downmodulates CD48, the ligand of 2B4, and this contributes to protect HIV-1-infected cells from ADCC. Our results highlight the importance of the virus to prevent the triggering of the SLAM receptors to evade ADCC.

The human cytomegalovirus-specific and UL40-mediated imprint in the natural killer cell repertoire is associated with antibody-mediated rejection in lung transplant recipients.

CD16+ natural killer (NK-) cells play, together with donor-specific antibodies (DSA) and via antibody-dependent cellular cytotoxicity (ADCC), an important role in the pathogenesis of antibody-mediated rejection (ABMR) in lung-transplant recipients (LTRs). Cytotoxic CD16+NKG2C+ NK cells proliferate in response to human Cytomegalovirus (HCMV) infections via the presentation of HCMV-encoded and highly polymorphic UL40 peptides. In our study, we aimed to clarify whether infections with HCMV-strains carrying different UL40 peptide variants are associated with the shift of the NK cell repertoire and the development of ABMR in LTRs. We included 30 DSA+ABMR+, 30 DSA+ABMR- and 90 DSA-ABMR- LTRs. In all patients, 1 episode of high-level HCMV-replication occurred. In all DSA+ABMR+ LTRs, HCMV-replication occurred prior to ABMR diagnosis. The association of HCMV UL40 variants with the expansion of CD16+ NK cell subsets and ABMR was assessed in NK cell proliferation and ADCC assays. Our study revealed that the VMAPRTLIL and VMTPRTLVL UL40 variants were significantly overrepresented in DSA+ABMR+ LTRs. Both peptides were associated with a pronounced proliferation of cytotoxic and proinflammatory CD16+NKG2C+ NK cells. The stimulation with both peptides led to a shift of the NK cell repertoire towards CD16+NKG2C+ NK cells, which was associated with strong ADCC responses after stimulation with endothelial cells and plasma from DSA+ABMR+ LTRs. Distinct UL40 peptide variants of the infecting HCMV-strain are associated with the development of ABMR after lung transplantation, due to a shift towards a highly cytotoxic CD16+NKG2C+ NK cell population. These peptides are thus potential prognostic markers for ABMR.

Humanized T Cell Receptor-like Antibody Hu8F4 Reduces Leukemia By Fc-Mediated Mechanism

8F4, a T cell receptor (TCR) - like antibody which targets the leukemia-associated antigen PR1 (VLQELNVTV) when presented in the context of MHC class I (HLA-A2:01), is a mouse IgG2a monoclonal antibody (mAb) which mediates both complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) in vitro and eliminates AML in PDX (patient derived xenograft) mouse models. Recognizing the potential therapeutic value of 8F4, we produced a humanized IgG1 (Hu8F4) as a first-in-class mAb for preclinical development and clinical use. Characterization of Hu8F4 by ELISA and surface plasmon resonance showed high affinity (KD=6.5 nM) and specificity of Hu8F4 binding to PR1/HLA-A2 monomer. Staining of peptide-pulsed T2 cells confirmed Hu8F4 specificity to PR1, but not to control peptide MART1. To study anti-leukemia activity of Hu8F4 we used HLA-A2-transfected leukemia cell lines U937 (U937-A2) and MV411 (MV411-A2), as well as THP1 (endogenous HLA-A2+) as targets. In contrast to mouse 8F4, Hu8F4 did not induce CDC in vitro. Rather, Hu8F4 mediated both potent antibody-dependent cellular phagocytosis (ADCP) and ADCC of U937-A2 and THP1 cells in NFAT reporter assays, using FcgRI- (EC50 = 0.002 µg/ml), FcgRII- (EC50 = 1.49 µg/ml), and FcgRIII- (EC50 = 0.04 µg/ml) transfected Jurkat cells, as well as pooled human PBMC (EC50 = 0.95 µg/ml) and donor-derived macrophages (EC50 = 1.1 µg/ml), as effectors. Based on the ADCP activity of Hu8F4, we sought to examine the potential synergy of anti-CD47 antibody, a macrophage checkpoint inhibitor, with Hu8F4. Treatment of U937-A2 and THP1 target cells with anti-CD47 F(ab')2 neutralizing antibody made cells susceptible to Hu8F4-mediated phagocytosis by NSG mouse bone marrow derived macrophages (BMDM) in vitro. To confirm the critical role of CD47 in Hu8F4-mediated ADCP we created CD47 KO U937-A2 and THP1 AML lines. Within 96 hours of treatment with Hu8F4 both CD47 KO cell lines were completely phagocytosed by NSG BMDM, thus confirming the role of phagocytosis as a mechanism. To test Hu8F4 activity in vivo, we established xenografts using luciferase-transfected U937-A2 and MV411-A2 cell lines in NSG mice. Hu8F4 was then administered starting on either day 3 (U937-A2) or day 7 (MV411-A2). Treatment with Hu8F4 resulted in elimination of leukemia as measured by FACS and BLI and increased median survival (from 33 days to >85 days for U937-A2, p=0.0075; and from 125 days to >225 days for MV411-A2, p=0.0033). To further test Hu8F4 efficacy, we established PDX in NSG mice using cells from HLA-A2+ refractory/relapsed AML patients. Hu8F4 treatment reduced leukemia by 90.3% to 99.9% when assessed by FACS in four out of five PDX and resulted in increased median survival of the mice (from 41 days to 49 days, p=0.0002) in one PDX tested. To study the mechanism of action of Hu8F4, we produced a 3 aa mutant (Hu8F4 PG LALA), which completely abrogated binding to Fcg receptors. Hu8F4 PG LALA, as well as Hu8F4 F(ab')2, showed no in vivo activity against U937-A2 or MV411-A2 xenografts, confirming that the anti-leukemia activity of Hu8F4 is dependent on its ability to bind Fc receptors. Because FcgR may be expressed on AML we tested fratricide as a possible in vivo mechanism of Hu8F4. We utilized the CRISPR /Cas9 system to edit out both FcgRI and II in the U937-A2 cell line that is naturally deficient in FcgRIII (DKO U937-A2). We then used a retroviral system to stably express FcgRI or FcgRII in U937-A2 to create FcgRI-U937-A2 and FcgRII-U937-A2, respectively, and tested their susceptibility to Hu8F4 in the xenograft model. Compared to FcR-expressing wild-type U937-A2, Hu8F4 showed similar anti-leukemia activity in all three xenografts. There was no significant difference noted in the anti-leukemia potency of Hu8F4 between DKO U937-A2 or single FcR-expressing xenografts compared with FcgRI/FcgRII expressing wild-type U937-A2 xenografts, suggesting that fratricide is an unlikely mechanism in this model. Here we demonstrate high specificity, affinity, and remarkable anti-AML activity of Hu8F4, a first-in-class humanized TCR mimic monoclonal antibody that is currently being tested in a phase I clinical trial for HLA-A2+ refractory/relapsed AML and MDS. This study provides evidence for the importance of FcgR-mediated mechanisms (ADCC and ADCP) in the activity of Hu8F4. In addition, our results support future combination trials with treatments that promote phagocytosis.

Abstract 4216: Tipifarnib inhibits the secretion of tumor-derived small extracellular vesicles and enhances the immunotherapeutic efficacy of dinutuximab in neuroblastoma

Abstract Anti-GD2 monoclonal antibody (Dinutuximab) immunotherapy has significantly improved the overall survival rate for high-risk neuroblastoma patients. However, 40% of patients fail to respond or develop resistance to the treatment, and the molecular mechanisms by which this occurs remain poorly understood. Here we utilize the syngeneic 9464D-GD2 mouse model to investigate the role of neuroblastoma tumor-derived small extracellular vesicles (sEVs) in developing resistance to the anti-GD2 monoclonal antibody Dinutuximab. Neuroblastoma-derived sEVs significantly attenuated the efficacy of Dinutuximab treatment in vivo. Mechanistically, both RNA-sequencing and flow cytometry analysis of whole tumors demonstrated that neuroblastoma-derived sEVs modulate immune cells tumor infiltration upon Dinutuximab treatment to create an immunosuppressive tumor microenvironment, which contains more tumor-associated macrophages (TAMs) and fewer tumor-infiltrating NK cells, in addition to suppression of splenic NK cell maturation in vivo. Moreover, neuroblastoma-derived sEVs suppressed Dinutuximab-mediated NK cell antibody-dependent cellular cytotoxicity in vitro. Importantly, Tipifarnib, a farnesyltransferase inhibitor that inhibits sEV secretion, drastically enhanced the efficacy of Dinutuximab in vivo and reversed the immunosuppressive effects of neuroblastoma-derived sEVs. Notably, Tipifarnib modulated immature myeloid cells in the bone marrow to disfavor the formation of CD11b+Ly6ChighLy6Glow subpopulations, which are the precursors for TAMs. Taken together, these preclinical findings uncover a novel mechanism by which neuroblastoma-derived sEVs modulate the immune system to promote resistance to Dinutuximab and suggest that Tipifarnib-mediated inhibition of sEV secretion may serve as a viable treatment strategy to improve immunotherapy in high-risk patients. Citation Format: Xiaoming Liu, Carson A. Wills, Longgui Chen, Jiawen Zhang, Yuanjun Zhao, Mi Zhou, Jeffrey M. Sundstrom, Todd D. Schell, Vladimir S. Spiegelman, Megan M. Young, Hong-Gang Wang. Tipifarnib inhibits the secretion of tumor-derived small extracellular vesicles and enhances the immunotherapeutic efficacy of dinutuximab in neuroblastoma [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 4216.

Therapeutic efficacy of humanized monoclonal antibodies targeting dengue virus nonstructural protein 1 in the mouse model.

Dengue virus (DENV) which infects about 390 million people per year in tropical and subtropical areas manifests various disease symptoms, ranging from fever to life-threatening hemorrhage and even shock. To date, there is still no effective treatment for DENV disease, but only supportive care. DENV nonstructural protein 1 (NS1) has been shown to play a key role in disease pathogenesis. Recent studies have shown that anti-DENV NS1 antibody can provide disease protection by blocking the DENV-induced disruption of endothelial integrity. We previously demonstrated that anti-NS1 monoclonal antibody (mAb) protected mice from all four serotypes of DENV challenge. Here, we generated humanized anti-NS1 mAbs and transferred them to mice after DENV infection. The results showed that DENV-induced prolonged bleeding time and skin hemorrhage were reduced, even several days after DENV challenge. Mechanistic studies showed the ability of humanized anti-NS1 mAbs to inhibit NS1-induced vascular hyperpermeability and to elicit Fcγ-dependent complement-mediated cytolysis as well as antibody-dependent cellular cytotoxicity of cells infected with four serotypes of DENV. These results highlight humanized anti-NS1 mAb as a potential therapeutic agent in DENV infection.

Sodium stibogluconate and CD47-SIRPα blockade overcome resistance of anti-CD20–opsonized B cells to neutrophil killing

AbstractAnti-CD20 antibodies such as rituximab are broadly used to treat B-cell malignancies. These antibodies can induce various effector functions, including immune cell-mediated antibody-dependent cellular cytotoxicity (ADCC). Neutrophils can induce ADCC toward solid cancer cells by trogoptosis, a cytotoxic mechanism known to be dependent on trogocytosis. However, neutrophils seem to be incapable of killing rituximab-opsonized B-cell lymphoma cells. Nevertheless, neutrophils do trogocytose rituximab-opsonized B-cell lymphoma cells, but this only reduces CD20 surface expression and is thought to render tumor cells therapeutically resistant to further rituximab-dependent destruction. Here, we demonstrate that resistance of B-cell lymphoma cells toward neutrophil killing can be overcome by a combination of CD47-SIRPα checkpoint blockade and sodium stibogluconate (SSG), an anti-leishmaniasis drug and documented inhibitor of the tyrosine phosphatase SHP-1. SSG enhanced neutrophil-mediated ADCC of solid tumor cells but enabled trogoptotic killing of B-cell lymphoma cells by turning trogocytosis from a mechanism that contributes to resistance into a cytotoxic anti-cancer mechanism. Tumor cell killing in the presence of SSG required both antibody opsonization of the target cells and disruption of CD47-SIRPα interactions. These results provide a more detailed understanding of the role of neutrophil trogocytosis in antibody-mediated destruction of B cells and clues on how to further optimize antibody therapy of B-cell malignancies.

A Bicistronic Vector Expressing CD16 and a Membrane Bound IL-15 Construct in iPSC Derived NK Cells Increased Cytotoxicity and Persistence

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. DisclosuresAllen: 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.

Efficient Targeting of CD38 in Mature T-Cell Neoplasms with Daratumumab and Allogeneic NK Cells

Introduction: Mature T-cell neoplasms (MTCN) are a heterogeneous and aggressive group of lymphoid neoplasms with very limited options for precision immunotherapy. Targeted immunotherapy with antibodies directed against surface markers on tumor cells has emerged as an effective treatment for B-cell neoplasms, but the development of immunotherapy strategies for MTCN has been much slower. CD38 is expressed at low levels in a subset of normal resting T-cells. Data on the frequency and level of CD38 expression is MTCN are lacking. Our goal was to study the expression and stability of CD38 on a spectrum of neoplastic T-cell populations and assess the potential anti-tumor effect of anti-CD38 monoclonal antibodies in combination with allogeneic natural killer (NK) cells in MTCN.Methods and results: We searched the Thomas Jefferson University Hospital (TJUH) pathology records for all cases of MTCN for which immune-phenotypical characterization of the neoplastic T-cell population by multi-color flow cytometry (FC) was available. CD38 expression was evaluated in cases where an abnormal T-cell population (defined as loss of one on more pan T-cell markers and/or a skewed CD4 to CD8 ratio). A total of 103 unique patients with MTCN were identified. Of these, 51 had at least one biopsy whereby tumor cells had an abnormal immunophenotype which could then be assessed for CD38 expression. CD38 was expressed to some extent in all but 2 cases with expression levels on peripheral T-cell lymphoma-not other specified (PTCL-NOS)(% Mean±SEM = 80.84±10.26, N=11), angioimmunoblastic T-cell lymphoma (AITL) (% Mean±SEM = 80.56±7.34, N=6), nodal PTCL with T follicular-helper (T FH) phenotype (% Mean±SEM = 55.00±12.72, N=7), anaplastic large cell lymphoma (ALCL) (% Mean±SEM = 77.38±10.75, N=3), large granular lymphocytic leukemia (LGLL) (% Mean±SEM = 80.27±7.49, N=4), T-cell prolymphocytic leukemia (T-PLL) (% Mean±SEM = 88.26±4.20, N=7), cutaneous T-cell lymphoma (CTCL) (% Mean±SEM = 49.52±14.77, N=7), adult T‐cell leukemia/lymphoma (ATLL) (% Mean±SEM = 76.68±10.45, N=3), hepatosplenic T-cell lymphoma/monomorphic epitheliotropic intestinal T-cell lymphoma (HSTCL/MEITL) (% Mean±SEM = 75.00±11.37, N=3). The medians and ranges of the MFI of CD38 on CD38+ tumor cells were the following: PTCL-NOS 29.46 (3.1-115.62), AITL 13.64 (2.67-29.41), other PTCL-T FH 5.87 (0-48.67), ALCL 11.13 (3.6-11.28), LGL (13.52 (9.72-18.25), T-PLL 6.49 (3.09-18), CTCL 8.36 (0-116.75), ATLL 27.17 (3.54-60.5), HSTCL/MEITL 15.96 (6.96-167.85). We also measured surface expression of CD38 on the patient-derived MTCN cell lines HuT-78, HuT-102, Jurkat, H9, HH, and MOTN1, all of which expressed CD38, to determine which of these cell lines could be used for in vitro experiments. We next evaluated if the CD38 molecule is an effective target for antibody-mediated therapy in MTCN, by testing the ability of daratumumab (dara) to enhance antibody-dependent cellular cytotoxicity (ADCC) elicited by NK cells. For this, we purified normal NK-cells from TJUH Blood Bank leukoreduction filters and cultured with recombinant IL-15 for 48 hours prior to all experiment. T-cell lines and primary MTCN cells were treated with increasing concentrations (0.1 µg/mL - 2 µg/mL) of dara or isotype control. NK-cells were added at Effector:Target ratio of 5:1 and incubated for 4 hours at 37⁰ Celsius. Cytotoxicity was measured by LDH release assay. Dara induced significant cell lysis starting at doses as low as 0.1μg/mL in both T-cell lines and primary MTCN cells, reaching maximum cytotoxicity at 0.5-2μg/mL (mean±SEM cytotoxicity in isotype vs dara treated cells= 50.0±5.05% vs 97.5±2.5%, N=4, p-value=0.0002). The degree of ADCC induction also correlated with interferon-gamma (IFN-g) release by NK cells in vitro for both T-cell lines and primary MTCN cells.Conclusions: The majority of MTCN analyzed (N=49, 96%) showed any degree of CD38 expression by FC with a wide variation of intensity, including within the same subtype. Allogeneic NK cells efficiently elicited dara-mediated ADCC of tumor cells from all MTCN subtypes and produced abundant IFN-g. These data highlight the potential of targeting CD38 in MTCN with anti-CD38 antibodies and allogeneic NK cells. The strong CD38 expression observed in most tumor cells from ultra-rare and very aggressive subtypes of MTCL opens the door to much needed new treatment strategies. DisclosuresBrammer: Celgene: Research Funding; Kymera Therapeutics: Consultancy; Seattle Genetics: Speakers Bureau. Chakravarti: Kiadis Pharma: Patents & Royalties. Porcu: Viracta: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Innate Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BeiGene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Research Funding; Daiichi: Honoraria, Research Funding; Kiowa: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Spectrum: Consultancy; DrenBio: Consultancy.

Paralysis of the cytotoxic granule machinery is a new cancer immune evasion mechanism mediated by chitinase 3-like-1

BackgroundNatural killer (NK) cells require a functional lytic granule machinery to mediate effective antitumor responses. Evading the lytic cargo deployed at the immune synapse (IS) could be a critical step...

606 SAR444245 (THOR-707), an engineered non-alpha IL-2, enhances NK mediated antibody-dependent cellular cytotoxicity

BackgroundSAR444245 is a non-alpha IL-2 Synthorin TM molecule designed with a site-specific non-natural amino acid serving as a bioconjugation site for a single PEG. The non-natural amino acid is positioned to enable the PEG bioconjugation to obscure block binding to the IL-2 alpha receptor, while retaining near-native affinity with the intermediate affinity βγ IL-2 receptor. The non-alpha features of SAR444245 minimize activation of immune suppressive regulatory CD4+ T cells, while retaining activity on CD8+ T cells and NK cells expressing the IL-2 βγ receptors. NK cells exert anti-tumor activity through antibody dependent cellular cytotoxicity (ADCC) of IgG antibodies as well as antibody independent mechanisms.MethodsHere, we utilized a panel of human primary PBMC based immunoassays and transcriptomic analysis to evaluate whether SAR444245 may improve ADCC function of IgG1 anti-tumor target antibodies.ResultsWe characterized the ability of SAR444245 to enhance the cytolytic function of NK cells towards the prototypic NK target cell K562 as well as to modulate NK cell ADCC in combination with EGFR or CD20-targeting antibodies. In vitro assays demonstrated that SAR444245 can activate NK cells, promote NK cell proliferation and improve cytotoxicity of NK cells against K562 cells and across a panel of human EGFR and CD20 positive cell lines. In PBMC based ADCC assays with 1ug/ml of antibody, SAR444245 improved ADCC function maximally by 9-fold for an anti-EGFR antibody and at 5-fold for an anti-CD20 antibody. SAR444245 exhibited dose-dependent enhancement of NK cell ADCC function. Notably, this activity was observed in cell lines expressing varying levels of EGFR and CD20. SAR444245 treatment was associated with dose dependent increases in NK cell degranulation and IFN-γ production. Transcriptomic profiling revealed that SAR444245 had broad effects on NK cell biology leading to changes in inhibitory and activating receptors.ConclusionsIn summary, these results indicate that SAR444245 can enhance the cytolytic activity of NK cells and enhance the ADCC effect of tumor-directed antibodies by activating NK cells.

Abstract 1306: Primary NK cells and luciferase expressing reporter cell lines for use in developing ADCC assays for immuno-oncology drug screening

Abstract Immuno-oncology is one of the fastest growing fields in oncology, and cancer immunotherapy in recent years represents a milestone in the treatment of cancer. Natural killer (NK) cells are potent innate effectors capable of targeting and killing virally infected and malignant cells. The use of monoclonal antibodies (mAbs) targeting tumor antigens triggers antibody-dependent cellular cytotoxicity (ADCC) by NK cells, which has shown prominent clinical efficacy in clearing tumors in cancer patients. Therefore, the development of tumor cell targeting mAbs and biologics dictate a clear need for human cell-based models to evaluate antibody efficacy. In this study, human PBMCs and purified NK cells from healthy donors are used to develop an ADCC assay. The NK cells purified from PBMCs are >90% CD45+ CD56+, express high levels of CD16, and NK activation markers such NKG2A, KIR2DL1, NKp44 upon activation. The activated NK cells exhibit both strong direct and antibody-dependent cytotoxicity. Genotyping for FcγRIIIa receptor F and V variant is also performed for cells isolated from different donors, which is the genetic polymorphism that affects ADCC response. In addition, we employ a portfolio of luciferase reporter tumor cell lines to evaluate NK cell direct killing and ADCC targeted killing. These include K562, Wil2S, BT474, A431, A375 parental, and CRISPR edited A375 KRAS mutant cell lines engineered to express luc2 reporter. The commonly targeted cell surface markers, such as CD20, Her2, and EGFR, are highly expressed within this panel of target tumor cell lines. We validate the ADCC assay system through testing the efficacy for some of the well-known mAb biologics such as anti-CD20, anti-HER2, and anti-EGFR. After co-culture of activated primary NK cells with luciferase reporter target cell lines and the addition of dilutions of ADCC and isotype control antibodies, we are able to show target cell specific and dose-dependent killing effects. The luciferase reporter provides great convenience in measuring target cell killing without having to use radioactive 51Cr release assay or pre-label the cells with calcein. This data demonstrates the utility of primary NK cells along with a portfolio of well characterized luciferase reporter tumor cell line to develop “custom” designed ADCC assays for studying NK cell immune response and screening for biological drugs for cancer immunotherapy. Citation Format: Haiyun Liu, Brian Della Fera, John Foulke, Luping Chen, Fang Tian. Primary NK cells and luciferase expressing reporter cell lines for use in developing ADCC assays for immuno-oncology drug screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1306.

Balancing the CD38 Expression on Effector and Target Cells in Daratumumab-Mediated NK Cell ADCC against Multiple Myeloma.

Simple SummaryWe tracked the cytotoxic potential of NK cells towards multiple myeloma cells in daratumumab-mediated antibody-dependent cellular cytotoxicity assays. These cytotoxicity levels could be directly correlated to the expression of the target antigen (CD38) and to the percentage of fratricide between effector cells. Increasing the expression of CD38 on target cells or neutralizing CD38 on effector cells changed the equilibrium between target and effector cell lysis and promoted multiple myeloma cell death. This study highlights the importance of a balanced CD38 expression on target and effector cells and attempts to alter this balance will affect the susceptibility of MM cells towards daratumumab-mediated cellular toxicity. Multiple myeloma (MM) is an incurable cancer characterized by the proliferation and accumulation of monoclonal plasma cells in the bone marrow. The monoclonal anti-CD38 daratumumab has taken a central place in the different treatment regimens for newly diagnosed and relapsed, refractory myeloma. In this study, we correlated the NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) and potential fratricide induced by daratumumab with CD38-expression levels on both effector and target cells. We show that CD38 expression can be modulated by adding all-trans retinoic acid (ATRA) or interferon-α to MM cells to further fine-tune these effects. In addition, we observed that ADCC becomes inefficient when fratricide occurs and both ADCC and fratricide depend on the balance between CD38 expression on effector and target cells. However, the addition of adjuvants (retinoic acid or interferon-α) to myeloma cells or the inhibition of fratricide using a CD38-blocking nanobody on NK-cells can reverse this balance towards ADCC and thus promote lysis of target cells by ADCC. ATRA and interferon-α increased the CD38 expression at the surface of MM cells about three-fold and two-fold, respectively. This increase was of interest for MM cells with low CD38 expression, that became susceptible to daratumumab-mediated ADCC after preincubation. A CD38-blocking nanobody prevented the binding of daratumumab to these NK-cells and blunted the fratricidal effect on effector NK cells. In conclusion, our study highlights the importance of a balanced CD38 expression on target and effector cells and attempts to alter this balance will affect the susceptibility of MM cells towards daratumumab-mediated ADCC.

T cells, particularly activated CD4+ cells, maintain anti-CD20-mediated NK cell viability and antibody dependent cellular cytotoxicity

Anti-CD20 monoclonal antibody (mAb) therapy is a mainstay of therapy for B cell malignancies, however many patients fail to respond or eventually develop resistance. The current understanding of mechanisms responsible for this resistance is limited. When peripheral blood mononuclear cells of healthy donors were cultured with Raji cells for 7 days, rituximab (RTX) induced NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC), enhanced NK cell viability and increased or maintained NK expression of CD56, CD16, CD57 and KIR. T cells, mainly CD4+, mediated these changes in a contact-dependent manner, with local T cell production of IL2 playing a central role. Similar findings were found when autologous B cells were used as target cells demonstrating the need for T cell help was not due to allogenic reaction. Results with other anti-CD20 and anti-EGFR antibodies were consistent. Small numbers of T cells activated by anti-CD3/CD28 beads or bispecific antibody enhanced RTX-mediated NK cell ADCC, viability and phenotypical changes. Pathway analysis of bulk NK cell mRNA sequencing after activation by RTX with and without T cells was consistent with T cells maintaining the viability of the activated NK cells. These findings suggest T cell help, mediated in large part by local production of IL2, contributes to NK cell ADCC and viability, and that activating T cells in the tumor microenvironment, such as through the use of anti-CD3 based bispecific antibodies, could enhance the efficacy of anti-CD20 and other mAb therapies where NK-mediated ADCC is a primary mechanism of action.

Low number of KIR ligands in lymphoma patients favors a good rituximab-dependent NK cell response

ABSTRACT The antibody-dependent cellular cytotoxicity (ADCC) effector function of natural killer (NK) cells is one of the known mechanisms of action for rituximab-based anti-cancer immunotherapy. Inhibition of the ADCC function of NK cells through interactions between inhibitory killer cell immunoglobulin-like receptors (KIRs) and HLA class I ligands is associated with resistance of cancers to rituximab. In this study, we deeply investigated the impact of KIR, HLA class I, and CD16 genotypes on rituximab-dependent NK cell responses in both an in vitro cellular model from healthy blood donors and ex vivo rituximab-treated non-Hodgkin lymphoma (NHL) patients. We highlight that an HLA environment with limited KIR ligands is beneficial to promoting a higher frequency of KIR+ NK cells including both educated and uneducated NK cells, two NK cell compartments that demonstrate higher rituximab-dependent degranulation than KIR− NK cells. In contrast, a substantial KIR ligand environment favors a higher frequency of poorly effective KIR− NK cells and numerous functional KIR/HLA inhibitions of educated KIR+ NK cells. These phenomena explain why NHL patients with limited KIR ligands respond better to rituximab. In this HLA environment, CD16 polymorphism appears to have a collateral effect. Furthermore, we show the synergic effect of KIR2DS1, which strongly potentiates NK cell ADCC from C2− blood donors against C2+ target cells. Taken together, these results pave the way for stronger prediction of rituximab responses for NHL patients. HLA class I typing and peripheral blood KIR+ NK cell frequency could be simple and useful markers for predicting rituximab response.

Abstract 5658: A homogenous PBMC ADCC bioassay enables bridging studies with ADCC reporter bioassays in immunotherapy monoclonal antibody development

Abstract Measurement of antibody-dependent cellular cytotoxicity (ADCC) is critical for characterizing antibody Fc effector functions during monoclonal antibody development. Classic ADCC assays measure the short-term cytotoxicity of target cells, typically pre-loaded with radioactive or fluorescent dyes, after exposure to antibody bound primary PBMCs or NK cells. These assays are widely used in antibody discovery and characterization during early drug development. However, these assays are relatively low throughput, present a biosafety risk due to radioactivity, and are prone to donor variability and weak assay signal. Therefore, these assays are not well-suited for implementation in quality-controlled drug development environments. Previously, we developed a mechanism of action (MOA)-based ADCC reporter bioassay using genetically engineered Jurkat effector cells. When co-cultured with antigen-expressing target cells and a relevant antigen-specific antibody, engagement of FcγRIIIa expressed on the effector cells leads to NFAT-mediated expression of luciferase and a bioluminescent signal. The effector cells are optimized for use in thaw-and-use format and can accurately measure antibody potency for a wide range of therapeutic antibodies targeted to hematopoietic or solid tumors. The bioassay can distinguish effector functions for different antibody IgG isotypes, is stability indicating, and prequalified according to ICH guidelines (precision, accuracy, linearity, and robustness). The ADCC reporter bioassay can be developed as a QC lot release assay for antibody therapeutics that function via an ADCC MOA. However, prior to implementation, a bridging study with a primary cell-based cytotoxicity assay must be performed. To enable this analysis, we developed a PBMC-based ADCC assay that overcomes limitations of traditional primary cell ADCC assays. First, we established a workflow to prequalify donor PBMCs for ADCC activity, significantly reducing the level of variability introduced with random donor PBMCs. We then developed a platform to genetically engineer target cells with a cytosolic protein (HaloTag) fused to HiBiT, an 11 amino acid protein tag. When cultured with a relevant antibody and ADCC-qualified PBMCs, the target cells are killed and HaloTag-HiBiT is released into the extracellular medium containing the protein LgBiT. Binding of HiBit and LgBiT reconstitutes a luciferase enzyme and produces a bioluminescent signal. The use of HaloTag as a binding partner offers the additional opportunity to image the target cells using fluorescent HaloTag ligands. This improved PBMC ADCC assay is simple, homogenous, highly sensitive, and yields a robust assay window. Importantly, we have demonstrated that this assay accurately measures the potency of a range of antibody therapeutics including rituximab, cetuximab, and trastuzumab, and correlates with the surrogate ADCC reporter bioassay. Citation Format: Pete Stecha, Denise Garvin, Julia Gilden, Jun Wang, Jamison Grailer, Jim Hartnett, Vanessa Ott, Frank Fan, Mei Cong, Zhijie Jey Cheng. A homogenous PBMC ADCC bioassay enables bridging studies with ADCC reporter bioassays in immunotherapy monoclonal antibody development [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5658.

Abstract 1630: FceR1g negative NK-cells (g-NK) enhance antibody-dependent cellular cytotoxicity and in vivo efficacy of therapeutic monoclonal antibodies against hematologic malignanices

Abstract Monoclonal antibodies (mAbs) are a central component of hematologic malignancy therapy; however, not all patients initially respond to these agents and resistance is a major clinical issue. A major mechanism of action for these mAbs in vivo is NK cell antibody-dependent cellular cytotoxicity (ADCC). Recent studies led to the discovery of a novel subset of human NK cells that lack expression of FceR1g (“g-NK cells”) and have a multi-fold increase in ADCC activity after CD16 crosslinking. Here, we validate the potency of g-NKs as an off-the shelf cellular immunotherapy to enhance the efficacy of therapeutic mAbs. g-NK cells were enriched and expanded using a proprietary method from CMV-positive blood donors selected for high levels of circulating g-NKs. For ADCC cytotoxicity assays, expanded NK cells were sorted by flow cytometry into populations of “conventional” NK cells (cNK), adaptive (NKG2C+) NK cells, and g-NK cells. After sorting, NK-cells were co-cultured 4 hr at 37° C with Raji or MM.1S target cells (1e4 cells) at varying NK-cell:target cell ratios. For in vivo persistence studies a single dose of 1e7 g-NK or cNK cells was injected into NOD scid gamma (NSG) mice. For in vivo efficacy studies with rituximab (RIT), NSG mice were inoculated with 5e5 luciferase-labeled Raji lymphoma cells and dosed weekly with 15e6 g-NK cells +/-200 ug RIT or vehicle. For in vivo efficacy studies with daratumumab (DARA) and elotuzumab (ELO), NSG mice were inoculated with 1e6 luciferase-labeled MM.1S cells and dosed every 6d with expanded g-NKs (20e6), resting cNKs (3e5, equivalent to physiological levels of NK cells/kg in humans, and +/- 10 ug DARA or 10 ug ELO. In all studies, mice received IL-15 intraperitoneally every 3 days. Our expansion method was able to expand g-NKs from selected CMV-seropositive donors ~800-fold (n=8). By flow cytometry we found an increase in the percentage of g-NK from 28% of NK cells at input to 82% post-expansion. In vitro ADCC assays demonstrated that g-NKs enhanced cytotoxicity of RIT to Raji cells (70% at 1:1 E:T) when compared to both NKG2C+ NKs (29%) and cNKs (27%) (p<0.001). Notably, we saw similar ADCC of g-NK cells whether used fresh or thawed after viable freezing (70% vs. 63%, p=0.71). In addition, g-NK cells enhanced cytotoxicity of DARA and ELO to MM.1S cells (58% and 54% at 1:1 E:T) when compared to cNK (14% and 12% at 1:1 E:T) (p<0.001). We confirmed in NSG mice that g-NK persistence was markedly improved (>90%) over cNKs in blood at multiple time points (p<0.001), and spleen at Day 22 (p<0.001). Efficacy studies using Raji cells showed the combination of g-NK and RIT led to a >90% reduction in tumor burden versus RIT alone (p<0.001). Evaluation of expanded g-NKs in a disseminated orthotopic xenograft model of multiple myeloma found a dramatic reduction (>70%) in tumor burden with DARA+g-NK vs. DARA alone or DARA+cNK (p<0.001). Similar results were found with ELO. Here we demonstrate that adoptive transfer of expanded g-NK cells markedly enhances anti-tumor ADCC of therapeutic mAbs in several preclinical models. Importantly, because adoptive transfer of NK-cells in humans does not result in severe graft-versus-host disease, we propose that this long-lived, highly potent NK-cell therapy could be administered in an “off-the-shelf” manner to supercharge mAb efficacy. Citation Format: Austin Basil Bigley, Nadia H. Agha, Shanae Spade, Gaetano Dipierro, Ronald Martell, Byron C. Hann, Nina Shah, Arun P. Wiita. FceR1g negative NK-cells (g-NK) enhance antibody-dependent cellular cytotoxicity and in vivo efficacy of therapeutic monoclonal antibodies against hematologic malignanices [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1630.