NK Cell Line Killing of Leukemia Cells Is Enhanced By Reverse Antibody Dependent Cell Mediated Cytotoxicity (R-ADCC) Via NKp30 and NKp44 and Target Fcγ Receptor II (CD32)
Authors: Williams Brent A. , Wang Xinghua , Routy Bertrand , Cheng Richard , Maghera Sonam , Keating Armand 
Published: October 2019

Abstract
Introduction: We are studying NK cell immunotherapy to treat acute myeloid leukemia (AML) and have focused on NK-92 and KHYG-1, CD16(-) human malignant NK cell lines. Phase I NK-92 trials show minimal toxicity; KHYG-1 has not been tested in humans. Here, we investigated modulation of cytotoxicity of NK cell lines against primary AML blasts and cell lines with monoclonal antibodies (mAb) directed against natural cytotoxicity receptors.
Methods: NK cytotoxicity was assessed with a standard 4 hour Cr51 release assay at an effector to target (E:T) ratio of 10:1. NK lines were incubated with and without isotype control and mAbs against NKp30, NKp44 at various doses (0.001-10 µg/ml) for 1 hour and washed with medium prior to cytotoxicity assays. The student’s t-test was used to compare cytotoxicity data. Target cells were incubated with 100 µCi Cr51 and cell supernatants assayed on a gamma counter. NK targets (leukemic and esophageal cancer) were evaluated for Fcγ receptor expression by flow cytometry. To test the cytotoxic effect on in vivo proliferation, OCI/AML5 cells were co-incubated with irradiated KHYG-1 (iKHYG-1) +/-1 µg/ml NKp30 pretreatment for 4 hours at a 10:1 E:T ratio and injected ip into NOD/SCID gamma null (NSG) mice with survival as an endpoint analyzed with the log rank test.
Results: NK-92 and KHYG-1 were both highly cytotoxic against K562 with moderate killing of OCI/AML3 and KG1 and KG1a. OCI/AML5 was highly sensitive to killing by NK-92, but resistant to KHYG-1. Pretreatment of NK-92 with mAbs against NKp30, NKp44 (10 µg/ml) yielded small increases in cytotoxicity against leukemic cell lines with NKp30 pretreatment only. Pretreatment of KHYG-1 with 10 µg/ml of anti-NKp30 or anti-NKp44 mediated fold increases in cytotoxicity above isotype control against 4 leukemia cell line targets and 4 primary AML samples (Table1). Anti-NKp30 and anti-NKp44 pretreatment of NK-92 and KHYG-1 did not enhance killing of a panel of esophageal cancer cell lines. Immunophenotyping cancer cell lines showed high expression of Fcγ receptor II (CD32), but very low expression of Fcγ receptor I (CD64) or III (CD16) on leukemia lines (K562, OCI/AML3, OCI/AML5, KG1 and KG1a), and no expression of Fcγ receptors on esophageal lines (OE-33, FLO-1, KYAE-1, SKGT-4). Regression analysis of the relationship between cytotoxic enhancement and CD32 expression of targets revealed a strong correlation for NKp30 (p<0.01; R2=0.71) and NKp44 (p<0.01; R2=0.64) pretreated KHYG-1. NSG mice injected with 2x106 OCI/AML5 cells developed progressive malignant ascites at 9 weeks requiring sacrifice, unaffected by iKHYG-1 (p=0.92). However, NKp30 pretreated iKHYG-1 improved survival versus no therapy (p<0.05) or iKHYG-1 (p<0.05) cohorts.
Conclusion: We show a novel means to enhance cytotoxicity of NK cell lines many fold against primary AML cells by pretreatment with mAbs against NKp30 and NKp44. The mechanism of enhanced KHYG-1 cytotoxicity is from bound NKp30 or NKp44 becoming crosslinked when the Fc portion binds the Fcγ receptor II (CD32) on targets. This is the first demonstration of reverse antibody-dependent cell-mediated cytotoxicity (R-ADCC) with a NK cell line leading to enhanced killing of AML primary blasts in vitro and the first demonstration of R-ADCC in an in vivo model.

Table 2: Effect of NKp30 or NKp44 pretreatment on KHYG-1 cytotoxicity against primary AML samples Fold lysis and p values Primary AML samples 5890 080078 0909 080179 Fold change lysis NKp30 1.7 15.7 2.7 4.9 Fold change lysis NKp44 0.9 16.3 2.8 6.2 p value NKp30 <0.05 =0.0001 <0.0001 <0.001 p value NKp44 0.9 <0.001 <0.001 <0.001

Disclosures
No relevant conflicts of interest to declare.