Engineering Anti-AML Antibodies for Improved NK Cell ADCC
Authors: Senyukov Vladimir , Kelton William , Mehta Nishant , Georgiou George , Lee Dean 
Published: October 2019

Abstract
Abstract 3629
Acute myeloid leukemia (AML) is an aggressive malignancy for which current therapy fails to provide durable remission in approximately half of cases. Natural killer (NK) cells, as a key component of innate immunity, have recently shown clinical potential for adoptive immunotherapy against AML, particular when the donor and recipient are KIR mismatched. In addition to patients who do not have a suitable related donor, approximately 30% of patients bear all three families of KIR ligands and therefor cannot benefit from KIR mismatch. Thus, finding a related donor with predicted KIR mismatch is a major obstacle for adoptive NK cell immunotherapy. The majority of peripheral blood NK cells express CD16a (FcγRIIIa), which is the most potent receptor among the activating receptors that NK cells posses. NK cells express CD16a in association with disulflde-linked homo- or hetero-dimers of FcRγ or CD3ζ. Clustering of CD16a initiated by binding to the Fc-portion of IgG1 or IgG3 that opsonize target cells induces signals strong enough to overcome KIR inhibition. Thus, combining NK cell adoptive immunotherapy with Abs against tumor antigens could help overcome the limitations of KIR mismatching. Indeed, many promising anticancer Abs have failed in clinical trials because of insufficient efficacy, which, at least in part, may result from low affinity CD16a binding. Indeed, it was shown that the affinity between Fc and FcγRs correlates with cytotoxicity in cell-based assays and that the Abs with optimized FcγR affinity induced strong cytotoxicity against targeted tumor cells.
CD33 is expressed on the blast cells of most cases of AML and represents a suitable antigen for antibody-based therapies. Lintuzumab, an unconjugated, humanized anti-CD33 mAb (HuM195), failed to improve patient outcomes in two randomized trials when combined with conventional chemotherapy. Gemtuzumab ozogamicin, an anti-CD33 mAb conjugated to the calicheamicin, in combination with chemotherapy, improved survival in a subset of AML patients, but has been withdrawn from US market by safety concerns. We optimized the FcγR affinity of HuM195 mAb (mNuM195) by cloning into pMaz-IgH Herceptin recipient vector containing S239D, A330L, I332E mutations that, as previously shown, leads to significant improvement of IgG1 binding to CD16a. To generate control wild type variant (wHuM195) we cloned the variable domains of HuM195 into pMaz-IgH Herceptin. Plasmids were transfected into HEK293F, and Abs were purified from cell culture supernatant with protein A resin, eluted with glycine HCL, and then the samples were buffer exchanged into PBS pH 7.4 for long-term storage. This S239D-A330L-I332E triple mutation in Fc portion of IgG1 did not affect antigen-biding affinity for CD33 target protein but showed more than 14-fold higher binding to CD16a than the wild type variant. The mHuM195 Abs increased cytotoxic activity of expanded human NK cells in Calcein AM-release assay when used in concentration as low as 0.01 μg/ml to pretreat murine thymoma EL-4 cells gene-modified to express human CD33 (ADCC, Mean±SD: 38.7±2.25% vs 11.7±3.49% for optimized vs wild type HuM195, and 5±3.15% without Abs, E:T ratio 2:1). We obtained the similar results when using K562 as targets, which naturally express CD33. K562 cells pretreated with mHuM195 Abs induced degranulation in 34±5.25% of NK cells where wHuM195 did so only in 17±4.6% of NK cells. Thus, optimization of HuM195 Ab to improve CD16a affinity results in dramatic increases NK cell cytotoxic activity.

Disclosures:
No relevant conflicts of interest to declare.