A Phase I Study of Intravenous NCI IL-15 to Enhance Adoptively Transferred NK Cells Uncovers Defects in CD16 Triggered IFNγ Production and Competition Between Donor NK and Recipient T Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 566-566 ◽  
Author(s):  
Jeffrey S. Miller ◽  
Michael R. Verneris ◽  
Julie Curtsinger ◽  
Todd E. DeFor ◽  
David McKenna ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Target Cell ◽  
Adoptive Transfer ◽  
Cell Expansion ◽  
Nk Cell ◽  
Donor Chimerism ◽  
The Mean ◽  
Stimulation Of

Abstract Adoptive transfer of IL-2 driven NK cells can induce remissions in patients with refractory AML but this may be limited by IL-2 induced expansion of regulatory T cells (Tregs). Because IL-15 does not stimulate Tregs, we performed a phase I dose escalation trial of recombinant human IL-15 (NCI Frederick) to enhance adoptive NK cell transfer. Patients with refractory AML received a lymphodepleting regimen of cyclophosphamide and fludarabine, followed by infusion of haploidentical NK cells activated overnight with IL-15 and intravenous IL-15 for 12 daily doses in cohorts of 0.25, 0.5, 1 and 0.75 mcg/kg. We treated 24 patients and deterimined the maximum tolerated dose (MTD) to be 0.75 mcg/kg. IL-15 serum levels peaked 1 hour after dosing with a significant drop at 4 hours and no evidence of drug accumulation between doses 1 and 6 (Figure, A). Fevers were common, possibly attributable to the rise in multiple inflammatory cytokines 4 hours after IL-15 dosing (Figure, B). Higher fevers occurred after 6 doses, corresponding to higher cytokine levels. No patients experienced hypotension or vascular leak. Dose limiting toxicity was observed in 2 subjects at 1.0 mcg/kg who did not recover neutrophils 42 days after NK cell infusion, one of whom died of infection 60 days after adoptive NK cell transfer at which time donor NK cells persisted in blood and pleural fluid. Clearance of refractory leukemia was seen in 38% of patients 14 days after adoptive transfer. Three patients with CRp went on to best donor allogeneic transplant with overall survival of those receiving IL-15 (n=24) of 32% at 12 months. We observed two patterns of donor cell expansion and autologous count recovery. In the 5 of 24 (21%) patients with detectable NK cell donor chimerism, the mean ALC measured at day 7 after NK infusion was 160/µL with an mean of 56% donor chimerism. By day 14 the mean ALC increased to 1200/µL with 87% donor chimerism, where virtually all donor lymphocytes were NK cells. The remaining 19 patients who did not expand NK cells had a mean ALC of 68/µL with 27% donor chimerism at day 7, which increased to 900/µL and only 2.5% donor chimerism by day 14. In the non-NK expanders, nearly all of the day 14 lymphocytes were recipient CD8+ T cells, suggesting host T cell mediated rejection of donor NK cells. We extensively evaluated the function of the successfully in vivo expanded donor NK cells collected from the recipient day 14 post-NK infusion and IL-15 administration, and directly compared it to that of NK cells at steady state from the same donor. Although we expected degranulation to be increased in the IL-15 expanded NK cells, CD107a expression was similar in steady state donor and in vivo IL-15 expanded NK cells (Figure, C). In contrast, IFNγ production triggered by CD16 signalling with Rituxan against antibody-dependent cellular cytotoxicity sensitive Raji targets was significantly decreased in the in vivo IL-15 expanded NK cells (Figure, D). This did not reflect an inherent defect in IFNγ production as IL-12/IL-18 stimulation of day 14 in vivo IL-15 expanded donor NK cells induced significantly more IFNγ compared to the study state donor cells (Figure, E). These characteristics (decreased target cell-induced INFg and increased IL-12/IL-18 induced IFNγ) suggest that donor NK cells expanded in vivo with IL-15 are functionally similar to the CD56bright population. This effect is not unique to IL-15 as a similar functional pattern was observed in patients with in vivo IL-2 expanded NK cells. This suggests that robust, cytokine driven in vivo NK proliferation may limit the target cell-induced cytokine production of the expanded population. In summary, NCI IL-15 led to robust donor NK cell expansion in some patients, but competitive stimulation of recipient CD8+ T cells was common. Even when in vivo expansion of donor NK cell was successful, the cells exhibited defects in target cell and CD16-induced IFNγ production. Long-lived CMV-driven adaptive NK cells with memory like properties are associated with enhanced anti-leukemia activity and CD16 signalling. Novel methods to expand adaptive NK cells may be of value for the next generation of NK cell therapy. Additionally, the NCI CITN trial of subcutaneous IL-15 administration suggests that compared to IV dosing, it may favor donor NK vs. host T cell expansion based on different affinities of trans-presented IL-15 to these cell populations, and these strategies should be tested to optimize NK cell adoptive transfer. Figure 1. Figure 1. Disclosures Miller: Coronado: Speakers Bureau; BioSciences: Speakers Bureau; Celegene: Speakers Bureau.

Recombinant Human IL-15 Promotes in Vivo Expansion of Adoptively Transferred NK Cells in a First-in-Human Phase I Dose Escalation Study in Patients with AML

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 894-894 ◽  
Author(s):  
Sarah Cooley ◽  
Michael R. Verneris ◽  
Julie Curtsinger ◽  
David McKenna ◽  
Daniel J. Weisdorf ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Board Of Directors ◽  
Adoptive Transfer ◽  
Cell Expansion ◽  
Nk Cell ◽  
Advisory Committees ◽  
Dose Cohort ◽  
Refractory Aml

Abstract Abstract 894 Adoptive transfer of haploidentical NK cells can induce remissions in patients with refractory AML. However, many do not expand NK cells and fail to respond. While IL-2 can promote NK cell proliferation, it also promotes the expansion of regulatory T cells, which impede NK cell expansion. Because IL-15 has different effects on regulatory T cells than IL-2, we initiated a phase I dose escalation trial of recombinant human IL-15 to enhance adoptive transfer of NK cells. Patients with refractory AML receive a lymphodepleting preparative regimen of fludarabine 25 mg/m2 × 5 days and cyclophosphamide 60 mg/kg × 2 days. Haploidentical NK cells (CD3- and CD19-depleted and overnight activated with IL-15 10 ng/ml) were infused Day 0, followed by 12 daily doses of intravenous IL-15 (Biopharmaceutical Development Program, NCI Frederick) in planned dosing cohorts of 0.25, 0.5, 1, 2 and 3 mcg/kg. To date 9 patients have been treated (Table). The first 6 patients (0.25 and 0.5 mcg/ml cohorts) received all 12 planned doses of IL-15, with no dose-limiting toxicities (DLTs). Apart from transient fevers, the IL-15 was well tolerated. While donor-derived NK cells were detected at Day 7 in all patients, none achieved the primary endpoint of >100 donor-derived NK cells/μl circulating in blood at Day 14. At the completion of IL-15 dosing, all patients in cohorts 1–2 had a lymphocytosis comprised of host T cells, which were mainly CD8+ (mean 81±6%). Five of 6 recovered neutrophils by Day 28 (range 15–26 days), and one with residual leukemia remained neutropenic leading us to conclude that IL-15 does not impede neutrophil recovery. Three of the 6 cleared their leukemic blasts, fully recovered and proceeded to allogeneic hematopoietic cell transplant (HCT). In contrast to the first 2 cohorts, the first patient at the 1 mcg/kg dose experienced a DLT (grade 4 dyspnea due to diffuse alveolar hemorrhage requiring high dose steroids) and received only 8 does of IL-15 making him not evaluable for in vivo expansion. Due to the DLT, the 1 mcg/kg dose cohort will expand to 6 evaluable patients, 2 of whom have completed IL-15 dosing. Both are evaluable, each having received the required minimum of 9 doses, but additional planned doses 10–12 were held in both patients due to high fevers and transient hypoxia possibly related to infection that did not constitute DLT. Both cleared refractory leukemia at Day 14 and successfully expanded donor NK cells (2094 and 448 cells/ml) at the end of IL-15 dosing. The in vivo expanded NK cells exhibited potent function, with 81.5% and 82.3% cytotoxicity against K562 targets at a 20:1 E:T ratio. Thus 1 mcg/ml dosing of IL-15 is significantly more likely to induce successful donor NK cell expansion at day 14 than the 0.25 or 0.5 mcg/ml doses (p = 0.04). Since endogenous IL-15 may heterodimerize with its receptor, IL-15Ra, to provide more stability and potent signalling to NK cells, batched serum samples are in process to measure free and IL-15Ra complexed IL-15. In summary, this platform of adoptive transfer of haploidentical NK cells with IL-15 has, in this early experience shown to be an effective treatment for refractory AML allowing patients to achieve remission and subsequent allogenenic HCT. The 1 mcg/kg dose is associated with more toxicity and limited ability to deliver all 12 doses, but toxicity was transient. 9 daily doses were sufficient to promote robust in vivo expansion of highly functional donor-derived NK cells. Further dose modifications (perhaps with continuous infusion or using fewer doses given subcutaneously) may be required to enhance safety. Based on this preliminary experience, IL-15 should emerge as the optimal cytokine to promote expansion and activation of adoptively transferred NK cells without Treg stimulation, which should be effective therapy for AML. In vivo expansion of donor-derived NK cells is dependent on IL-15 dosing. Subject IL-15 Dose Cohort (mcg/kg) # IL-15 Doses DLT Day 7 Day 11–14 WBC (cells/ml blood) %NK % Donor DNA Max ALC (cells/ml blood) %NK % Donor DNA %T cells %CD8+ T cells 1 0.25 12 No 100 37 QNS 1800 0.3 0% 95 86 2 0.25 12 No 100 38 25% 1200 16 0% 82 72 3 0.25 12 No 100 12 17% 1500 5.5 0% - - 4 0.5 12 No 100 44 43% 3200 0.3 2% 96 91 5 0.5 12 No <100 54 41% 100 0.2 0% 98 61 6 0.5 12 No <100 67 36% 1400 12 2% 57 93 7* 1.0 8 Yes <100 35 37% 600 0.1 0% 96 85 8 1.0 9 No 200 80 97% 2300 98 93% 1 - 9 1.0 9 No <100 92 86% 700 94 97% 1 - * Not evaluable for in vivo NK expansion due to DLT requiring IL-15 discontinuation and steroids Disclosures: Miller: Celgene: Membership on an entity's Board of Directors or advisory committees; Coronado Bioscience: Membership on an entity's Board of Directors or advisory committees.

Optimal NK Cell Expansion Depends on Accessory Cells, Synergy between Physiologic Concentrations of IL-2 and IL-15, and Umbilical Cord Blood (UCB) NK Cell Precursors Expand Better Than Adult NK Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3642-3642 ◽  
Author(s):  
Purvi Gada ◽  
Michelle Gleason ◽  
Valarie McCullar ◽  
Philip B. McGlave ◽  
Jeffrey S. Miller
Keyword(s):  
T Cells ◽  
B Cells ◽  
Nk Cells ◽  
Cell Expansion ◽  
Nk Cell ◽  
High Dose ◽  
Unexpected Result ◽  
Accessory Cells

Abstract Allogeneic NK cells may play a therapeutic role in treating patients with AML. We have previously shown that high dose cyclophosphamide (120 mg/kg × 1 day) and fludarabine (125 mg/m2 × 5 days) can clear lymphoid space and induce a surge of endogenous IL-15 to expand haploidentical NK cells obtained from CD3-depleted lymphapheresis products from adult donors. In this initial study, 5 of 19 patients achieved remissions and in vivo NK cell expansion. Limitations of this therapy includeinability of NK cells to expand in most patients,development of PTLD (in one patient) andinadequate disease control.We hypothesized that contaminating T cells could compete for NK cell expansion, that B-cells may contribute to PTLD, and that a 2-step NK cell purification method using CD3 depletion followed by CD56 selection (CliniMacs) may overcome these problems. We tested this in 9 patients with advanced AML. The purified NK cells, activated with 1000 U/ml IL-2 (16–20 hours), were infused 48 hours after the last fludarabine dose. Patients then received subcutaneous IL-2 (10 MU) every other day × 6 doses to expand NK cells in vivo. None of the 9 pts treated on this protocol achieved remission or exhibited evidence of in vivo expansion. Several studies were designed to investigate this unexpected result. First, we found that the more extensive processing resulted in approximately 1/3 the NK cell recovery compared to CD3 depletion alone (38±% viable NK cells vs. 91±2% respectively). In addition, we questioned whether the contaminating B cells and monocytes that were removed in the 2-step depletion strategy had served a critical role in NK cell activation or expansion. Cytotoxicity assays performed against K562 targets showed that the killing was about 3-fold higher with the purified (CD3-CD56+) product compared the CD3-depleted product alone (P=0.001 at E:T of 6.6:1). Proliferation, measured by a 6-day thymidine assay, was higher in proportion to the higher NK cell content. The only difference between the two NK products was their expansion after 14 days of culture, where the CD3-depleted product, with contaminating B-cells and monocytes, gave rise to greater NK cell expansion (14 ±3-fold) compared to the 2-step purified product (4.5±0.9, n=6, P=0.005). If this finding holds true in vivo, the co-infusion of accessory cells may be required for NK cell expansion. We next developed in vitro assays using very low concentrations (0.5 ng/ml) of IL-2 and IL-15 to understand their role in expansion. IL-2 or IL-15 alone induced low proliferation and the combination was synergistic. Lastly, UCB, a rich source of NK cell precursors, was compared to adult NK cells. In a short term proliferation assay, CD56+ NK cells stimulated with IL-2 + IL-15 expanded better from adult donors (61274±12999, n=6) than from UCB (20827± 6959, n=5, P=0.026) but there was no difference after 14 days in expansion culture suggesting that the only difference is in kinetics. However, UCB depleted of T-cells (enriching for NK cell precursors) exhibited higher fold expansion over 14 days under different culture conditions conducive to NK cell progenitors. In conclusion, NK cell expansion in vitro depends on cell source, IL-2 and IL-15 (increased in vivo after lymphoid depleting chemotherapy) as well as accessory cells. The role of these factors to enhance in vivo expansion is under clinical investigation to further exploit the NK cell alloreactivity against AML targets.

Successful Haploidentical Hematopoietic Cell Engraftment Using a Non-Myeloablative Preparative Regimen Including Natural Killer (NK) Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 827-827 ◽  
Author(s):  
Sarah Cooley ◽  
Purvi Gada ◽  
David McKenna ◽  
Valarie McCullar ◽  
Susan Fautsch ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Cell Expansion ◽  
Nk Cell ◽  
Cell Transfer ◽  
Haploidentical Transplantation ◽  
Preparative Regimen ◽  
The Mean ◽  
Refractory Aml

Abstract We have previously shown that adoptive transfer of haploidentical natural killer (NK) cells can induce remissions in 27% of patients with refractory or relapsed acute myeloid leukemia (AML) [Miller et al., Blood 2005, 105 (8)]. Aiming to optimize NK cell expansion, which we hypothesize is required for therapeutic efficacy, we tested additional CD56-positive selection (N=10) versus the CD3-depletion method used for our earlier NK cell infusions (N=10). Donor-derived NK cells were not measurable immediately after infusion. Successful in vivo NK cell expansion, defined as >100 donor-derived NK cells/ml at 14 days (by VNTR chimerism and flow cytometry) was not improved with CD56-selection (11% vs. 11%; mean 131±3 NK cells/ml), and was associated with poorer outcomes (10% vs. 27% AML remissions). Because the remissions induced by adoptive NK cell transfer were not durable, we added a CD34+ stem cell infusion to create a nonmyeloablative haploidentical transplantation protocol for older and less fit patients. We also added radiation to the NK cell-based preparative regimen to further improve NK cell expansion. The lymphodepleting chemoradiation plus NK cell preparative regimen included fludarabine 25 mg/m2 × 5 (day -18 through day -14), cyclophosphamide 60 mg/kg × 2 (days -16 and -15), and 200 cGy of total body irradiation (twice a day on day -13). The NK cell product, prepared by cliniMACS (Miltenyi) CD3-depletion of a single leukapheresis collection from a haploidentical related donor, was incubated overnight in 1000 U/ml IL-2 and then infused on day -12 followed by 6 doses subcutaneous IL-2 (10 million units) given every other day to promote in vivo NK cell expansion. The mean NK cell dose was 1.85 × 107 cells/kg and the mean CD3+ cell dose was 4.8 × 104 cells/kg (maximum permitted 3 × 105 CD3+ cells/kg). A CD34-selected filgrastim-mobilized peripheral blood graft from the same donor (target dose >3 × 106 CD34 cells/kg) was given with Thymoglobulin 3 mg/kg days 0, +1 and +2 as the only additional immunosuppression. In the 13 patients treated to date a significantly higher rate of NK cell expansion (75% [9/12 evaluable]; mean 607±184 NK cells/ml) was achieved compared to the adoptive NK cell transfer regimen, which did not include radiation. Plasma IL-15, which is critical for NK expansion, was highest on day -12 (the NK infusion day) after the preparative regimen (64 ± 8 pg/ml [day -12] vs. 6 ± 1 pg/ml [baseline pre-chemo]; p <.0001). This adoptive NK cell plus allograft protocol led to 66% of relapsed or refractory AML patients (8/12 evaluable) clearing leukemia by day -1, with only one late relapse (day +93). Patients who did not clear leukemia (N=4) did not engraft, and it was not evaluable in 3 patients with early (pre-day +13) treatment related mortality (TRM). All others (N=6), engrafted quickly (defined by an absolute neutrophil count >500/ml and 100% donor chimerism: median 17 days [range 11–31]). None developed graft vs. host disease (GVHD), but infections were common (3 fatal EBV/PTLD; 1 Fusarium). To prevent EBV reactivation NK products are now CD19 depleted and patients receive prophylactic Rituxan to prevent PTLD. The other deaths were due to persistent disease (N=4) or relapse (N=1). One patient is alive in remission beyond day +100. No clear associations between killer immunoglobulinlike receptor (KIR) ligand mismatch between donor and recipient were detected. In this series of patients with refractory AML, addition of haploidentical NK cells to a nonmyeloablative haploidentical transplantation yields NK cell expansion in a majority of patients, achievement of complete remission, and quick engraftment without GVHD. This is a promising platform upon which to add other strategies aimed at improving disease free survival in patients with refractory AML.

Haploidentical Natural Killer (NK) Cells Expanding In Vivo After Adoptive Transfer Exhibit Hyperfunction That Partially Overcomes Self Tolerance and Leads to Clearance of Refractory Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 355-355 ◽  
Author(s):  
Sarah Cooley ◽  
Bree Foley ◽  
Michael R Verneris ◽  
David McKenna ◽  
Xianghua Luo ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Adoptive Transfer ◽  
Cell Expansion ◽  
Nk Cell ◽  
Color Flow ◽  
Self Tolerance ◽  
Hla Ligands ◽  
Better Than

Abstract Abstract 355FN2 We have previously shown that adoptive transfer of haploidentical natural killer (NK) cells can induce remissions in patients with refractory or relapsed acute myeloid leukemia (AML). We hypothesize that in vivo expansion of functional NK cells is required for therapeutic efficacy. To achieve the adequate host immune suppression required for expansion we added total body irradiation (TBI) to our lymphodepleting chemotherapy regimen, giving patients fludarabine (Flu) 25 mg/m2 × 5 days, cyclophosphamide (Cy) 60 mg/kg × 2 days, and 400 cGy of TBI. The NK cell product, a CD3- and CD19-depleted lymphapheresis from a haploidentical related donor, was incubated overnight in 1000 U/ml IL-2 and infused followed by 6 doses of alternate day subcutaneous IL-2 (10 million units) to promote in vivo expansion. Because of the increased myelosuppression following the TBI, a CD34-selected filgrastim-mobilized peripheral blood graft from the same donor (target dose >3 × 106 CD34 cells/kg) was given for hematopoietic rescue. Successful in vivo NK cell expansion was prospectively defined as >100 donor-derived NK cells/ml at 14 days after adoptive transfer (by analysis of STR chimerism, % NK and the clinical absolute lymphocyte count). In the 38 evaluable patients, robust in vivo expansion was induced in 50% (absolute donor NK count of 666 ± 134 cells/μL blood), a rate considerably higher than the 10% we observed in a cohort receiving Cy/Flu alone without TBI. Successful NK cell expansion correlated with leukemia clearance (<1% marrow blasts 14 days after NK cell infusion) and remission (leukemia free with donor neutrophil engraftment at day +30; 42 days after NK infusion). Of the 19 patients who achieved NK cell expansion, 89% cleared their leukemia compared to 42% of the non-expanders (p=0.002); and 84% achieved remission vs. 10% of non-expanders (p <.0001). The robust in vivo expansion of adoptively transferred NK cells gave us the unique opportunity to study their function. We studied blood collected from patients 14 days after NK cell infusion and compared it to paired donor samples obtained at steady state from the apheresis products prior to IL-2 stimulation. Using multi-color flow cytometry, we measured CD107a expression (a surrogate marker for NK cell cytotoxicity) on NK cells which we could subset by expression of single inhibitory killer cell immunoglobulin-like receptors (KIR) (CD158a, CD158b and CD158e) or NKG2A. We defined NK subsets as self-KIR+ or non-self KIR+ based on the cognate HLA ligands (C2, C1, Bw4) present in the donor or recipient. The bulk population of in vivo expanded donor NK cells exhibited hyperfunction with 62.4±4.4% degranulation in response to class I negative K562 targets compared to 36.6±3.0% in the donor product samples (N=15; p=0.0043). As expected, the most potent NK cells in the unstimulated donor product were the self-KIR+ subset, which expressed 39.5±3.0% CD107a after incubation with K562 (N=23) compared to either the non-self KIR+subset (13.1±4.0%, N=6; p=0.0001), or the uneducated KIR−/NKG2A− subset (12.4±5.8%, N=10; p<0.0001). Remarkably, all 3 subsets exhibited even greater degranulation activity after 14 days of in vivo expansion where they were exposed to homeostatic factors and the IL-2 administered to the patient. While all subsets expressed more CD107a, the rules of education were maintained. The subset expressing self-KIR that recognized donor HLA ligands degranulated significantly better than the non-self KIR+ subset (53.5±14.1% vs. 34.3±13.6%, p<0.01). Interestingly, the in vivo expanded NK cells with KIR recognizing cognate ligands unique to the recipient also functioned better (53.1±14.3% [recipient self KIR+] vs. 32.4±12.0% [non-self KIR+], N=25 and N=6; p<0.0045), showing that the education status of adult NK cells is dynamic, not fixed. Importantly, the KIR−/NKG2A− subset functioned better after in vivo expansion (39.5±115.3%, N=12), demonstrating that adoptively transferred NK cells can acquire function by two separate mechanisms: 1) acquisition of function through NK cell education; and 2) acquisition of function by homeostatic expansion and cytokine activation. These data suggest that while hyperfunctioning NK cells that expand in vivo after adoptive transfer partially overcome self tolerance, which may augment their anti-leukemic effects, they still follow the rules of NK cell education where self KIR+ cells kill better than non-self KIR+ cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Cytokine-Induced Memory-like NK Cells Have a Distinct Single Cell Transcriptional Profile and Persist for Months in Adult and Pediatric Leukemia Patients after Adoptive Transfer

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3825-3825
Author(s):  
Jennifer A. Foltz ◽  
Melissa M. Berrien-Elliott ◽  
David A. Russler-Germain ◽  
Carly C. Neal ◽  
Jennifer Tran ◽  
...  
Keyword(s):  
Clinical Trial ◽  
T Cells ◽  
Transcription Factors ◽  
Nk Cells ◽  
Single Cell ◽  
Adoptive Transfer ◽  
Research Funding ◽  
Nk Cell ◽  
Mass Cytometry

Abstract Natural killer (NK) cells are innate lymphoid cells that mediate anti-tumor responses and exhibit innate memory following stimulation with IL-12, IL-15, and IL-18, thereby differentiating into cytokine-induced memory-like (ML) NK cells. ML NK cells have well-described enhanced anti-tumor properties; however, the molecular mechanisms underlying their enhanced functionality are not well-understood. Initial reports of allogeneic donor ML NK cellular therapy for relapsed/refractory (rel/ref) acute myeloid leukemia (AML) demonstrated safety and a 47% CR/CRi rate (PMID32826231). In this setting, allogeneic ML NK cells are rejected after 3 weeks by recipient T cells, which precludes long-term evaluation of their biology. To address this limitation, we conducted a clinical trial for rel/ref AML patients that added adoptive transfer of same-donor ML NK cells on day +7 of a reduced-intensity conditioning (RIC) MHC-haploidentical HCT, followed by 4 doses of IL-15 (N-803) over 2 weeks (NCT02782546). Since the ML NK cells are from the HCT donor, they are not rejected, but remain MHC-haploidentical to the patient leukemia. Using samples from these patients, we profiled the single cell transcriptomes of NK cells using multidimensional CITE-seq, combining scRNAseq with a custom NK panel of antibodies. To identify donor ML NK cells in an unbiased fashion, we developed a CITE-seq ML NK classifier from in vitro differentiated paired conventional NK (cNK) and ML NK cells. This classifier was applied via transfer learning to CITE-seq analyzed samples from the donor (cNK cells) and patients at days +28 and +60. This approach identified 28-40% of NK cells as ML at Day +28 post-HCT. Only 1-6% of donor peripheral blood NK cells and 4-7% of NK cells in comparator leukemia patients at day +28 after conventional haplo-HCT alone were identified as ML NK cells (Fig 1A). These ML NK cells had a cell surface receptor profile analogous to a previously reported mass cytometry phenotype. Within the CITE-seq data, ML NK cells expressed a transcriptional profile consistent with enhanced functionality (GZMK, GZMA, GNLY), secreted proteins (LTB, CKLF), a distinct adhesome, and evidence of prior activation (MHC Class II and interferon-inducible genes). ML NK cells had a unique NK receptor repertoire including increased KIR2DL4, KLRC1(NKG2A), CD300A, NCAM1(CD56) , and CD2 with decreased expression of the inhibitory receptor KLRB1(CD161). Furthermore, ML NK cells upregulated HOPX, a transcription factor implicated in memory T cells and murine CMV adaptive NK cells. Additionally, ML NK cells downregulated transcription factors related to terminal maturation (ZEB2) and exhaustion (NR4A2). We next sought to identify changes during ML differentiation in patients post-HCT from day +28 to +60 post-HCT. Trajectory analysis identified a ML NK cell state distinct from cNK cells that was present at least 60 days post-HCT (Fig 1B). The ML transcriptional phenotype continued to modulate during late differentiation, including downregulation of GZMK and NCAM1, and upregulation of maturation related transcription factors, while maintaining high expression of HOPX. ML NK cells retained their enhanced functionality during in vivo differentiation, as patient ML NK cells had significantly increased IFNγ production compared to cNK cells after restimulation with leukemia targets or cytokines using mass cytometry (Fig. 2). Subsequently, we confirmed the ML CITE-seq profile in an independent clinical trial treating pediatric AML relapsed after allogenic HCT with same-donor ML NK cells (NCT03068819). In this setting, ML NK cells expressed a similar transcriptional signature and persisted for at least 2 months in the absence of exogenous cytokine support. Thus, ML NK cells possess a distinct transcriptional and surface proteomic profile and undergo in vivo differentiation while persisting within patients for at least 2 months. These findings reveal novel and unique aspects of the ML NK cell molecular program, as well as their prolonged functional persistence in vivo in patients, assisting in future clinical trial design. Figure 1 Figure 1. Disclosures Foltz: Kiadis: Patents & Royalties: TGFbeta expanded NK cells; EMD Millipore: Other: canine antibody licensing fees. Berrien-Elliott: Wugen: Consultancy, Patents & Royalties: 017001-PRO1, Research Funding. Bednarski: Horizon Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Fehniger: Wugen: Consultancy, Current equity holder in publicly-traded company, Patents & Royalties: related to memory like NK cells, Research Funding; ImmunityBio: Research Funding; Kiadis: Other; Affimed: Research Funding; Compass Therapeutics: Research Funding; HCW Biologics: Research Funding; OrcaBio: Other; Indapta: Other.

scholarly journals 722 INBRX-121 is an NKp46-targeted detuned IL-2 with antitumor activity as a monotherapy or in combination with multiple cancer immunotherapy modalities

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A751-A751
Author(s):  
Florian Sulzmaier ◽  
Heather Kinkead ◽  
Anya Polovina ◽  
Nadja Kern ◽  
Angelica Sanabria ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Cell ◽  
Nk Cells ◽  
Cancer Immunotherapy ◽  
Cell Expansion ◽  
Nk Cell ◽  
Human Lymphocytes ◽  
Multiple Cancer

BackgroundNatural Killer (NK) cells play a pivotal role in cancer immunosurveillance due to their potent cytolytic activity and NK cell-centric therapies have emerged as safer alternatives to targeting T cells.1 2 Interleukin 2 (IL-2) drives NK cell expansion and activity, but its therapeutic utility is limited by rapid clearance, expansion of immunosuppressive regulatory T cells, and by severe dose-limiting toxicities.3 INBRX-121 overcomes these liabilities through specific targeting of an affinity-detuned IL-2 variant to cells expressing NKp46.MethodsAn IL-2 variant was engineered to eliminate binding to CD25 and to have attenuated affinity for CD122. This detuned cytokine was fused to a high-affinity single-domain antibody targeting NKp46 to generate INBRX-121. The ability of INBRX-121 to target IL-2-like signaling specifically to NKp46-expressing cells was evaluated in vitro using human lymphocytes by measuring STAT5 signaling and cytotoxic activity in tumor cell co-cultures. Characterization of the pharmacokinetic/pharmacodynamic relationship of INBRX-121 was completed in non-human primates across escalating dose levels, while anti-tumor activity as a monotherapy and in combination with Rituximab or PD-1 checkpoint blockade was tested in Raji xenografts and syngeneic CT-26 mouse models, respectively.ResultsINBRX-121 induces a STAT5 signal equal to that of wild-type IL-2 in human lymphocytes but shows an NK cell-centric activity profile. Cells targeted by INBRX-121 have increased proliferative capacity and improved cytotoxicity in antibody-dependent and -independent tumor cell killing assays. INBRX-121 shows prolonged pharmacokinetic exposure in vivo and is well-tolerated in mice and cynomolgus monkeys. The NKp46-specific IL-2 stimulus in these models results in a robust, dose-dependent NK cell expansion. As predicted by its in vitro activity, INBRX-121 also enhances the cytotoxic capacity of NK cells in vivo measured via elevated intracellular levels of Granzyme B. In a Raji xenograft model, INBRX-121 slows tumor growth as a single agent and synergizes with Rituximab to induce complete tumor regression. Similarly, co-treatment with INBRX-121 improves the incomplete suppression of CT-26 tumor growth by a PD-1 blocking antibody to yield complete responses that show immunological memory upon re-challenge.ConclusionsINBRX-121 offers a unique approach to overcoming the limitations of current IL-2 therapeutics. NKp46-targeting of a detuned IL-2 variant helps to avoid IL-2-mediated toxicity while enhancing the antitumor activities of NK cells. Through its novel therapeutic concept INBRX-121 provides a promising treatment option for multiple cancer indications both as a monotherapy and in combination with a variety of frontline agents.ReferencesShimasaki N, Jain A, Campana D. NK cells for cancer immunotherapy. Nat Rev Drug Discov 2020;19:200–218.Liu S, Galat V, Galat Y, Lee Y, Wainwright D, Wu J. NK cell-based cancer immunotherapy: from basic biology to clinical development. J Hematol Oncol 2021;14:7.Overwijk W, Tagliaferri M, Zalevsky J. Engineering IL-2 to give new life to T Cell immunotherapy. Annu Rev Med 2021;72:281–311.Ethics ApprovalAll animal studies were conducted in accordance with AAALAC regulations and were approved by the IACUC for Explora BioLabs (#SP17-010-013) and BTS Research (20-015 Enrollment 05).

scholarly journals Natural Killer Cells as Allogeneic Effectors in Adoptive Cancer Immunotherapy

Cancers ◽  
2019 ◽  
Vol 11 (6) ◽  
pp. 769 ◽  
Author(s):  
Kyle B. Lupo ◽  
Sandro Matosevic
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Cancer Immunotherapy ◽  
Natural Killer ◽  
Adoptive Transfer ◽  
Nk Cell ◽  
Human Leukocyte ◽  
Hematologic Malignancies ◽  
Published Evidence

Natural killer (NK) cells are attractive within adoptive transfer settings in cancer immunotherapy due to their potential for allogeneic use; their alloreactivity is enhanced under conditions of killer immunoglobulin-like receptor (KIR) mismatch with human leukocyte antigen (HLA) ligands on cancer cells. In addition to this, NK cells are platforms for genetic modification, and proliferate in vivo for a shorter time relative to T cells, limiting off-target activation. Current clinical studies have demonstrated the safety and efficacy of allogeneic NK cell adoptive transfer therapies as a means for treatment of hematologic malignancies and, to a lesser extent, solid tumors. However, challenges associated with sourcing allogeneic NK cells have given rise to controversy over the contribution of NK cells to graft-versus-host disease (GvHD). Specifically, blood-derived NK cell infusions contain contaminating T cells, whose activation with NK-stimulating cytokines has been known to lead to heightened release of proinflammatory cytokines and trigger the onset of GvHD in vivo. NK cells sourced from cell lines and stem cells lack contaminating T cells, but can also lack many phenotypic characteristics of mature NK cells. Here, we discuss the available published evidence for the varying roles of NK cells in GvHD and, more broadly, their use in allogeneic adoptive transfer settings to treat various cancers.

IL-2 Stimulated Treg Inhibit in Vitro Expansion of Haploidentical Natural Killer (NK) Cells, Which Is Partially Overcome with An IL-2-Diphtheria Toxin Fusion Protein In Vivo,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3611-3611
Author(s):  
Sarah Cooley ◽  
Veronika Bachanova ◽  
Melissa Geller ◽  
Michael R Verneris ◽  
Bin Zhang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Nk Cells ◽  
Natural Killer ◽  
Adoptive Transfer ◽  
Cell Expansion ◽  
Nk Cell ◽  
Treg Depletion ◽  
Cfse Dilution

Abstract Abstract 3611 Adoptive transfer of haploidentical natural killer (NK) cells can induce remissions in patients with refractory myeloid leukemia (AML). However, NK cells do not expand and persist in all patients despite lymphodepleting chemotherapy. In trials of adoptive NK cell therapy in solid tumors or lymphoma, host regulatory T cells (Treg) often expand in response to IL-2 given to stimulate donor NK cell expansion. Although murine studies report that Tregs inhibit NK cells, the influence of human Treg on NK cell proliferation and function is not well characterized. We studied the effect of allogeneic Tregs that were derived from human umbilical cord blood (UCB) as described by our group. Resting CFSE labelled NK cells or Teff were purified from healthy donors, and mixed with UCB Treg at various ratios. Unstimulated NK cells did not proliferate and thus IL-2 or IL-15 were added to the media at concentrations of 0.1, 0.25 and 0.5 ng/ml. In the absence of Treg, both cytokines induced equal NK cell proliferation at 5 days as measured by CFSE dilution in a concentration dependent manner. CFSE dilution was inhibited by Treg at a 1:1 ratio, especially at low cytokine concentrations. There were marked differences between the two cytokine conditions. Following IL-15 induced stimulation, the reduction in NK cell proliferation by Treg ranged from 1–35% (at different concentrations tested), whereas the inhibition of IL-2 stimulated NK cell proliferation ranged from 65–85%. Treg inhibition of NK cell proliferation could be measured at ratios as low as 1:8 in the presence of IL-2, but not IL-15. This inhibitory effect was partially explained by competition from CD25+ Tregs for IL-2. We measured Treg utilization of IL-2 by incubating NK cells with or without Treg in 0.5 ng/ml IL-2 for 4 days. The level of IL-2 with NK cells alone was 40 pg/ml vs. 17 pg/ml with Treg (compared to 330 pg/ml in IL-2-supplemented media without cells). Based on this data, we have incorporated host Treg depletion to enhance NK expansion after adoptive transfer to treat patients with refractory AML. As murine data from Blazar's group shows that CTL therapy is enhanced by Treg depletion, we added one dose of denileukin diftitox (ONTAK®, Eisai Inc) at 12 mg/kg to our lymphodepleting preparative regimen of fludarabine 25 mg/m2 × 5 days, cyclophosphamide 60 mg/kg × 2 days for 12 AML patients. Haploidentical NK cells (CD3- and CD19-depleted PBMCs and overnight activated with IL-2 1000 U/ml) were infused on Day 0, followed by 6 doses subcutaneous IL-2 (9 million units) given every other day to promote in vivo NK cell expansion. Eleven of 12 patients were evaluable, having received at least 4 of 6 planned doses of IL-2. Blood and marrow were collected 7 and 14 days after infusion to assess NK cell and Treg expansion, as well as leukemia clearance. Of the 10 patients with interpretable day 7 chimerism data, 9 had detectable donor DNA (median 68% donor DNA). At day 14, 4 of the 12 patients (33%) had successfully expanded NK cells in vivo, with absolute donor derived NK cell counts of 480, 530, 1470 and 12390 cells/μL blood, improving on our previous 10% rate of in vivo NK cell expansion which was observed with the same regimen, without Treg depletion. In the 4 patients who expanded NK cells in vivo, there were no detectable Treg (defined as a CD25+CD4+FoxP3+ lymphocyte population) at either day 7 or day 14. In contrast, the presence of a bona fide Treg population at either day 7 [range 9.5–53%] or day 14 [27–71%] correlated with a lack of in vivo NK cell expansion at day 14. Clinically, 8 of the 11 evaluable subjects cleared leukemia (72%), 7 of whom recovered neutrophils (63% CRp) and 6 of whom went on to best donor transplant (45%). In summary, we demonstrate in vitro and in vivo suppression of NK cell proliferation by IL-2 stimulated Treg. This effect is not seen in vitro with IL-15. We have shown that the absence of host Treg correlates with in vivo NK cells expansion. Although an increased rate of donor NK expansion was observed with a single dose of denileukin diftitox, it did not completely overcome the IL-2 induced host Treg expansion. Future trials testing additional doses of denileukin difitox or other methods of Treg depletion, as well as the use of IL-15 are planned. Disclosures: No relevant conflicts of interest to declare.

Trispecific Killer Engagers (TriKEs) that contain IL-15 to make NK cells antigen specific and to sustain their persistence and expansion

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 232-232 ◽  
Author(s):  
Jeffrey S. Miller ◽  
Martin Felice ◽  
Ron McElmurry ◽  
Valerie McCullar ◽  
Xianzheng Zhou ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Nk Cells ◽  
Target Cell ◽  
Adoptive Transfer ◽  
Inclusion Bodies ◽  
Cytokine Production ◽  
Nk Cell ◽  
Antigen Specificity ◽  
Single Chain

Abstract Natural killer (NK) cells are capable of immune surveillance mediated by a balance of activating and inhibitory receptors. We have shown that adoptive transfer of NK cells can induce complete remissions in patients with refractory AML when combined with lymphodepleting chemotherapy and IL-2 to stimulate survival and in vivo expansion of the NK cells. Using this approach, 30-50% of patients with refractory attain clinical remissions. We hypothesize that clinical benefit is limited by the lack of antigen specificity and because IL-2 induces regulatory T cells (Treg) that inhibit NK cell proliferation. We have developed trispecific killer engagers (TriKE) to overcome these limitations. We have previously shown that bispecific killer engagers (BiKEs) are capable of creating immunologic synapses between NK cells and CD33 antigens on AML and MDS targets leading to NK cell signaling through the highly potent CD16 (FcγRIII) receptor. We observed that although CD16 engagement leads to enhanced killing and cytokine production by NK cells, there is no effect on proliferation (designated 1633 in Figure, Panel B). Because IL-15 is the homeostatic factor for NK cell proliferation, survival, activation, and development and because unlike IL-2, it does not stimulate CD4+ CD25+ Treg, we developed a TriKE that includes a modified human IL-15 crosslinker sandwiched between single chain Fv against CD16 and CD33 (designated 161533). The final target gene was spliced into pET21d vector expression plasmid and transformed into the Escherichia coli and compared to a 1633 BiKE with the identical Fv regions as above. TriKEs were recovered from inclusion bodies, refolded, and purified. The addition of the IL-15 crosslinker to the molecule reduced its isoelectric point by two pH units creating more favorable conditions for purification and enhancing yield. Despite identical amounts of starting inclusion bodies, the final yield of 161533 TriKE was twice the yield of 1633 BiKE indicating more favorable purification dynamics. To establish the selectivity of the anti-CD33 binding moiety, BiKEs and TriKEs were analyzed using flow cytometry and both bound to CD16 and CD33 selectively without interference from the IL-15 crosslinker. To determine the capacity of BiKE and TriKE reagents to mediate NK cell killing, activity was compared in a 4-hour chromium release assay (representative of 3 separate experiments) against CD33+ HL60 targets (Figure Panel A) or CD33- HT29 (not shown). The highest specific activity is with the 161533 TriKE containing a modified IL-15 linker as control samples of anti-CD16 and anti-CD33 alone did not augment cytotoxicity while 1633 BiKE mediating killing was intermediate. CD33- HT29 was not killed. We next tested BiKE and TriKE in a proliferation assay using CSFE labeled primary NK cells. NCI produced recombinant human IL-15 was used as a positive control. The TriKE exhibited potent proliferation of primary NK cells compared to BiKE (Figure Panel B). As target cell induced NK cell production of cytokines is an important aspect of the anti-leukemia response, we compared the BiKE and TriKE for their ability to mediate cytokine production with primary NK cells and HL60 targets. TriKE induced greater target cell specific TNFα, INFγ, IL-6, MIP1α, GM-CSF and IL-8 compared to BiKE. We then tested the activity of the TriKE in a xenogeneic model of human NK cell adoptive transfer where NSG mice are given 0.75 million HL60-Luc cells followed by 250 cGy XRT ± 1 million NK cells 3 days later from a CD3 and CD19 depleted clinical product and daily IP injection of 50 mcg/kg TriKE. Significant anti-tumor activity without toxicity was seen in the NK and TriKE treated mice (P<0.002). In addition, the 161533 TriKE had potent in vivo IL-15 activity to support the in vivo persistence and expansion of NK cells while BiKE did not (data not shown). In summary, we have shown that a novel TriKE construct can mediate CD16 directed cytotoxicity against CD33 targets and this is specifically maintained when adding a modified IL-15 linker. The IL-15 activity is fully preserved in vitro and in vivo and unexpectedly enhances the purification product. This will allow us to redirect NK cells to malignant targets while providing cytokine stimulation with an easily exportable off-the-shelf non-gene therapy strategy using a drug that also maintains NK cell persistence, survival and in vivo expansion. Drug production is in progress for clinical testing in 2016. Figure 1. Figure 1. Disclosures Miller: Coronado: Speakers Bureau; BioSciences: Speakers Bureau; Celegene: Speakers Bureau.

scholarly journals Anti-tumor effects of RTX-240: an engineered red blood cell expressing 4-1BB ligand and interleukin-15

2021 ◽  
Author(s):  
Shannon L. McArdel ◽  
Anne-Sophie Dugast ◽  
Maegan E. Hoover ◽  
Arjun Bollampalli ◽  
Enping Hong ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Blood Cell ◽  
Nk Cells ◽  
Red Blood Cell ◽  
Safety Profile ◽  
Nk Cell ◽  
Cell Activity ◽  
In Vivo Studies

AbstractRecombinant agonists that activate co-stimulatory and cytokine receptors have shown limited clinical anticancer utility, potentially due to narrow therapeutic windows, the need for coordinated activation of co-stimulatory and cytokine pathways and the failure of agonistic antibodies to recapitulate signaling by endogenous ligands. RTX-240 is a genetically engineered red blood cell expressing 4-1BBL and IL-15/IL-15Rα fusion (IL-15TP). RTX-240 is designed to potently and simultaneously stimulate the 4-1BB and IL-15 pathways, thereby activating and expanding T cells and NK cells, while potentially offering an improved safety profile through restricted biodistribution. We assessed the ability of RTX-240 to expand and activate T cells and NK cells and evaluated the in vivo efficacy, pharmacodynamics and tolerability using murine models. Treatment of PBMCs with RTX-240 induced T cell and NK cell activation and proliferation. In vivo studies using mRBC-240, a mouse surrogate for RTX-240, revealed biodistribution predominantly to the red pulp of the spleen, leading to CD8 + T cell and NK cell expansion. mRBC-240 was efficacious in a B16-F10 melanoma model and led to increased NK cell infiltration into the lungs. mRBC-240 significantly inhibited CT26 tumor growth, in association with an increase in tumor-infiltrating proliferating and cytotoxic CD8 + T cells. mRBC-240 was tolerated and showed no evidence of hepatic injury at the highest feasible dose, compared with a 4-1BB agonistic antibody. RTX-240 promotes T cell and NK cell activity in preclinical models and shows efficacy and an improved safety profile. Based on these data, RTX-240 is now being evaluated in a clinical trial.

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