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. 2016 Jul 15;22(14):3440-50.
doi: 10.1158/1078-0432.CCR-15-2710. Epub 2016 Feb 4.

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

Daniel A Vallera  1 , Martin Felices  2 , Ron McElmurry  3 , Valarie McCullar  2 , Xianzheng Zhou  4 , Joerg Uwe Schmohl  5 , Bin Zhang  2 , Alexander J Lenvik  2 , Angela Panoskaltsis-Mortari  3 , Michael R Verneris  3 , Jakub Tolar  3 , Sarah Cooley  2 , Daniel J Weisdorf  2 , Bruce R Blazar  3 , Jeffrey S Miller  2
Affiliations

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

Daniel A Vallera et al. Clin Cancer Res. .

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 IL15 crosslinker, producing a 161533 trispecific killer engager (TriKE) to induce expansion, priming, and survival, which we hypothesize will enhance clinical efficacy.

Experimental design: Reagents were tested in proliferation and functional assays and in an in vivo xenograft model of AML.

Results: When compared with 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 antitumor 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. Clin Cancer Res; 22(14); 3440-50. ©2016 AACRSee related commentary by Talmadge, p. 3419.

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Conflict of interest statement

Conflict-of-interest disclosure: Drs. Vallera and Miller are members of the Oxis Biotech Scientific Advisory Board and hold equity in the company. This relationship has been reviewed and managed by the University of Minnesota in accordance with its conflict of interest policies.

Figures

Figure 1
Figure 1. 161533 TriKE elicits superior purification properties over 1633 BiKE
A) Schematic of gene placement of the BiKE (left) and TriKE (right) moieties in the pET expression vector. B) Absorbance tracings for 1633 BiKE (left) and 161533 TriKE (right), eluted from the ion exchange column as the first phase in drug purification using a 3-step elution protocol. For both agents a similar quantity of inclusion bodies was refolded and purified. The first peak eluted from the column represents the product. C) SDS-PAGE gel and Coomasie Blue staining of the BiKE and TriKE agents after a second step purification over a size exclusion column.
Figure 2
Figure 2. 161533 TriKE elicits superior NK cell function against CD33+ targets
A) Freshly isolated PBMCs were cultured with chromium loaded HL-60 cells for 4 hours at E:T ratios of 20:1, 6.6:1, and 2:1. The listed reagents were added at the beginning of co-culture at a 20 nM concentration. Data are displayed as percent NK cell cytolytic activity. Statistical significance only shown for 161533 TriKE and 1633 BiKE comparison (n=3). B) To evaluate the specificity of 161533 TriKE, a chromium release assay was performed against CD33−EpCAM+ HT29 targets. EpCAM1533 TriKE was used as a positive control (n = 2). Data are displayed as percent NK cell cytolytic activity. C) NK cells were enriched from normal donor PBMCs utilizing magnetic beads and placed in culture with HL-60 targets at an E:T ratio of 2:1 alone, in the presence of 1633 BiKE or in the presence of 161533 TriKE for 24 hours. Supernatants collected and frozen at the end of the incubation were later assessed for levels of of secreted IFNγ, TNFα, GM-CSF, and MIP1a using a Luminex-based multiplex assay (n=5). Points and bars represent mean ± SEM.
Figure 3
Figure 3. The 161533 TriKE mediates NK cell survival and proliferation
Post-HSCT recipient PBMCs were loaded with CellTrace proliferation dye and co-cultured with HL-60 Targets at a 5:1 (E:T) ratio for 7 days in the presence of 50 nM 1633 BiKE or 161533 TriKE. At the end of the incubation CD56+CD3− NK cells were assessed for viability through Live/Dead Near IR staining. A) An individual histogram and B) pooled analyses of viability in the NK cell population treated with the 1633 BiKE (gray) or 161533 TriKE (black) are shown. Individual dots represent separate post-HSCT samples treated with noted reagents (n=8). C) After 7 days, significantly fewer live cells remained in the 1633 BiKE condition precluding proliferation analysis. Proliferation in the 161533 TriKE condition was assessed by CellTrace dilution on live CD56+CD3− NK cells (black) and CD56−CD3+ T cells (gray). D) Pooled analyses depict the percent of dividing cells (top) and expansion index (bottom). The expansion index is calculated based on fold expansion within the population for a given amount of CellTrace dilution. Individual dots represent separate post-HSCT samples for noted populations (n=8).
Figure 4
Figure 4. 161533 TriKE potently restores function in defective post-HSCT recipient NK cells
The NK cell function induced by 161533 TriKE was compared to1633 BiKE using post-HSCT recipient PBMCs that were thawed and rested overnight with no cytokines. The next night they were incubated with no drug, 1633 BiKE (50 nM), or 161533 TriKE (50 nM). The next morning they were washed and reincubated with the specified agent immediately prior to addition of targets. A) Cytotoxicity against chromium loaded HL-60 targets was measured after 4 hours and the percent cytolytic activity was calculated. Dots denote mean ± SEM (n=9). B) Target cell-induced function is shown in representative histograms and C) pooled data of CD56+CD3− NK cell expression of CD107a (left panels), IFNγ (center panels), and TNFα (right panels) after 4 hour incubation with HL-60 targets. Dots denote individual patient samples treated with noted reagents (n=10).
Figure 5
Figure 5. 161533 TriKE induces enhanced NK cell function against primary AML blasts
Post-transplant patient PBMCs were thawed and rested overnight. The next night they were incubated with 1633 BiKE (50 nM), or 161533 TriKE (50 nM). The next morning they were washed and reincubated with the specified agent immediately prior to addition of targets. Primary AML blasts from apheresis products of two separate patients were thawed and rested overnight. Treated post-transplant patient PBMCs (n = 6) were incubated with the two different primary AML blasts (n = 12 total) for four hours and NK cell function was assessed by flow cytometry. A) Representative histograms denoting CD107a (left), IFNγ (center), and TNFα (right) expression on post-transplant patient NK cells treated with 1633 BiKE (gray) or 161533 TriKE (black) after 4 hour incubation with primary AML blasts. C) Pooled data for CD107a (left), IFNγ (center), and TNFα (right) expression on post-transplant patient NK cells treated with 1633 BiKE and 161533 TriKE and incubated with primary AML blasts. Each box represents a separate post-transplant patient sample incubated against two separate patient AML blast targets, denoted by filled and open boxes (n=12 total).
Figure 6
Figure 6. 161533 TriKE limits HL-60 tumor growth in vivo better than 1633 BiKE through enhanced maintenance of NK cells
An in vivo tumor model was established by conditioning NSG mice (275 cGy) and then injecting HL-60-luc cells intravenously (7.5 × 105 cells/mouse). Three days later 1×106 normal human donor NK cells (calculated from a magnetically depleted CD3/CD19 product) activated overnight with IL-15 were infused. 1633 BiKE and 161533 TriKE (50 ug) was administered MTWThF through the next two weeks of the study (10 doses total), and a control group only received HL-60-luc cells. A) Individual mouse photoluminscence after 2 minute exposures on day 14 (left) and day 21 (right) after NK infusion are shown (n=5 per treatment group,). B) Quantification of luminescence in mice from the three treatment groups at day 14 (top) and day 21 (bottom). Each dot represents a different mouse and bars denote mean ± SEM (n=5 per treatment group, representative of two separate experiments). C) Blood was collected on day 20 from the mice in each of the experiential treatment groups. Circulating CD45+CD56+CD3− human NK cells were quantified by flow cytometry. Events were collected over 60 seconds and the number of human NK cell events was calculated. Representative dot plots are shown denoting the number of NK (CD56+CD3−) cell events within the CD45+ gate. D) Aggregate data demonstrating the number of human NK cell events in each treatment group at day 20. Individual dots represent different mice and bars denote mean ± SEM (n=3 for HL-60-luc group [two mice died], n=5 for the 1633 BiKE and 161533 TriKE groups).
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