IL15 Trispecific Killer Engagers (TriKE) Make Natural Killer Cells Specific to CD33+ Targets While Also Inducing Persistence,In VivoExpansion, and Enhanced Function

Abstract

Purpose The effectiveness of NK cell infusions to induce leukemic remission is limited by lack of both antigen specificity and in vivo expansion. To address the first issue we previously generated a bispecific killer engager (BiKE) containing single chain scFV against CD16 and CD33 to create an immunologic synapse between NK cells and CD33+ myeloid targets. We have now incorporated a novel modified human IL-15 crosslinker, producing a 161533 trispecific killer engager (TriKE) to induce expansion, priming, and survival, which we hypothesize will enhance clinical efficacy. Methods Reagents were tested in proliferation and functional assays and in an in vivo xenograft model of AML. Results When compared to the 1633 BiKE, the 161533 TriKE induced superior NK cell cytotoxicity, degranulation, and cytokine production against CD33+ HL-60 targets and increased NK survival and proliferation. Specificity was shown by the ability of a 1615EpCAM TriKE to kill CD33-EpCAM+ targets. Using NK cells from patients after allogeneic stem cell transplantation when NK cell function is defective, the 161533 TriKE restored potent NK function against primary AML targets and induced specific NK cell proliferation. These results were confirmed in an immunodeficient mouse HL-60-Luc tumor model where the 161533 TriKE exhibited superior anti-tumor activity and induced in vivo persistence and survival of human NK cells for at least 3 weeks. Conclusions Off-the-shelf 161533 TriKE imparts antigen specificity and promotes in vivo persistence, activation, and survival of NK cells. These qualities are ideal for NK cell therapy of myeloid malignancies or targeting antigens of solid tumors.

STAT5 Loss Awakens the Dark Force in Natural Killer Cells

“…Thus, the data presented in this study support current NK-based therapeutic strategies, including injections of IL2 or IL15 into patients with cancer or targeted therapies, including cytokines such as trispecifi c killer engagers ( 10 ). The cytokines IL12, IL18, IL21, and IFN β decreased STAT5 activity and induced VEGF expression in NK cells; however, these cytokines are also known to be potent activators of NK cell cytotoxicity or cytokine production.…”

Trispecific killer engager CD16xIL15xCD33 potently induces NK cell activation and cytotoxicity against neoplastic mast cells

“…PBMCs were collected, and NK cells were enriched as described previously. 4 HL-60, K562, 5 HMC-1.1, HMC-1.2, and ROSA KIT D816V cell lines were maintained in culture as described. 6,7 Functional assays Fluorochrome-conjugated antibodies were from BD Bioscience (anti-CD3), Biolegend (anti-CD117, CD34, CD45, CD2, CD25, CD30, CD56, CD16, CD57, CD158a, CD158b, NKB1, NKG2A, CD107a, tumor necrosis factor-a [TNF-a], interferon-g [IFN-g]), and the CellTrace Violet Cell Proliferation Dye kit and the Live/Dead Fixable Aqua Dead Cell Stain Kit were from Invitrogen.…”

“…Flow cytometry and analysis were as described. 4 PBMCs were treated at 30 nmol/L of 161533 TriKE or anti-CD16 scFv as indicated. rhIL-15 was diluted to provide equivalent biological activity to the IL-15 linker of 161533 TriKE.…”

Bispecific antibodies in cancer immunotherapy

“…Recently, to take advantage of the stimulation and proliferation capacity of IL-15, 80 a trispecific killer engager (TriKE) construct was proposed using IL-15 to link the 2 antibody arms for the purpose of boosting anti-tumor activity. 88 …”