NKTR-255, a novel polymer-conjugated rhIL-15 with potent antitumor efficacy

Abstract

Background: NKTR-255 is a novel polyethylene glycol-conjugate of recombinant human interleukin-15 (rhIL-15), which was designed to retain all known receptor binding interactions of the IL-15 molecule. We explored the biologic and pharmacologic differences between endogenous IL-15 receptor α (IL-15Rα)-dependent (NKTR-255 and rhIL-15) and IL-15Rα-independent (precomplexed rhIL-15/IL-15Rα) cytokines.

Methods: In vitro pharmacological properties of rhIL-15, NKTR-255 and precomplex cytokines (rhIL-15/IL-15Rα and rhIL-15 N72D/IL-15Rα Fc) were investigated in receptor binding, signaling and cell function. In vivo pharmacokinetic (PK) and pharmacodynamic profile of the cytokines were evaluated in normal mice. Finally, immunomodulatory effect and antitumor activity were assessed in a Daudi lymphoma model.

Results: NKTR-255 and rhIL-15 exhibited similar in vitro properties in receptor affinity, signaling and leukocyte degranulation, which collectively differed from precomplexed cytokines. Notably, NKTR-255 and rhIL-15 stimulated greater granzyme B secretion in human peripheral blood mononuclear cells versus precomplexed cytokines. In vivo, NKTR-255 exhibited a PK profile with reduced clearance and a longer half-life relative to rhIL-15 and demonstrated prolonged IL-15R engagement in lymphocytes compared with only transient engagement observed for rhIL-15 and precomplexed rhIL-15 N72D/IL-15Rα Fc. As a consequent, NKTR-255 provided a durable and sustained proliferation and activation of natural killer (NK) and CD8+ T cells. Importantly, NKTR-255 is more effective than the precomplexed cytokine at inducing functionally competent, cytotoxic NK cells in the tumor microenvironment and the properties of NKTR-255 translated into superior antitumor activity in a B-cell lymphoma model versus the precomplexed cytokine.

Conclusions: Our results show that the novel immunotherapeutic, NKTR-255, retains the full spectrum of IL-15 biology, but with improved PK properties, over rhIL-15. These findings support the ongoing phase 1 first-in-human trial (NCT04136756) of NKTR-255 in participants with relapsed or refractory hematologic malignancies, potentially advancing rhIL-15-based immunotherapies for the treatment of cancer.

Keywords: hematologic neoplasms; immunotherapy; natural killer T-cells.

Conflict of interest statement

Competing interests: All authors are current or former employees of Nektar Therapeutics and/or have Nektar Therapeutics stock ownership interests to disclose. PKu is currently employed by Gilead Sciences. MA is currently employed by HIBC Biopharma. PKi is currently employed by Immunocore. LM is currently employed by Invivoscribe.

© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

Figures

Figure 1

NKTR-255 is an IL-15Rα-dependent polymer-conjugated cytokine with an improved pharmacokinetic profile versus rhIL-15. (A) Representative flow cytometry histogram plots of surface IL-15 binding on IL-15Rα-overexpressing 293-F cells after incubation with rhIL-15, NKTR-255 or rhIL-15 N72D/IL-15Rα Fc (10 nM) for 30 min from two independent experiments. Solid line: cytokine treatment; dotted line: vehicle control. (B) pSTAT5 expression in CD8+ T cells derived from human peripheral blood mononuclear cells (n=3 donors indicated by a different color dot) in co-culture with cytokine pre-incubated IL-15Rα-overexpressing 293-F cells. ***p≤0.001, ****p≤0.0001 (Dunnett’s multiple comparisons test vs vehicle). (C) Cytokine-induced pSTAT5 expression in CD8+ T cells derived from C57BL/6 wild-type or IL-15Rα KO mice. Results are shown from two independent experiments. (D) Log-linear plasma concentration versus time profiles for rhIL-15 and NKTR-255 in mice (n=3). IL, interleukin; IL-15Rα, IL-15 receptor α; KO, knock out; pSTAT5, STAT5 phosphorylation; rhIL-15, recombinant human IL-15; STAT5, signal transducer and activator of transcription.

Figure 2

The rhIL-15 and NKTR-255 have different signaling and leukocyte degranulation properties in vitro compared with precomplexed rhIL-15/IL-15Rα cytokines. (A) Dose-response curves for pSTAT5, pAKT and pERK in NK, CD8+ T and CD4+ T cells following stimulation with rhIL-15, NKTR-255, rhIL-15/IL-15Rα or rhIL-15 N72D/IL-15Rα in human whole blood (n=6). (B) pSTAT5 EC50 ratios for CD8+ T/NK cells and CD4+ T/NK cells and (C) EC50 ratios for pAKT/pSTAT5 or pERK/pSTAT5 in NK cells (*p<0.05, **p<0.01; Dunnett’s multiple comparisons test vs rhIL-15). Human peripheral blood mononuclear cells (n=3) were stimulated in vitro with rhIL-15, NKTR-255, rhIL-15/IL-15Rα and rhIL-15 N72D/L-15Rα Fc overnight to assess (D) CD107a surface expression by flow cytometry (n=3) and (E) secreted granzyme B by Meso Scale Discovery (n=3; *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001; Dunnett’s multiple comparisons test vs vehicle). EC50, half maximal effective concentration; IL, interleukin; IL-15Rα, IL-15 receptor α; NK, natural killer; pAKT, phosphorylated AKT; pERK, phosphorylated ERK; pSTAT5, STAT5 phosphorylation; rhIL-15, recombinant human IL-15; STAT5, signal transducer and activator of transcription.

Figure 3

Improved pharmacokinetic profile of NKTR-255 relative to rhIL-15 results in sustained IL-15R engagement and proliferation of NK and CD8+ T cells. (A) Time-course of pSTAT5 induction in peripheral blood NK, CD8+ T and CD4+ T cells in mice after a single dose of rhIL-15 (0.3 mg/kg intraperitoneally) or NKTR-255 (0.3 mg/kg intravenously; n=3 per time point in each group). (B) Time-course of fold change (relative to vehicle) in absolute number of NK and CD8+ T cells after a single dose or three consecutive daily doses of rhIL-15 (0.3 mg/kg intraperitoneally) or a single dose of NKTR-255 (0.03 or 0.3 mg/kg intravenously) in mice. IL, interleukin; NK, natural killer; pSTAT5, STAT5 phosphorylation; rhIL-15, recombinant human IL-15; STAT5, signal transducer and activator of transcription.

Figure 4

NKTR-255 induces similar proliferation responses after single and repeated doses. Time-course of Ki67 positivity in NK and CD8+ T cells after a single or a third repeat intravenous dose of NKTR-255 (treatment repeated once every 7 days for three doses (Q7D×3) in mice). NK, natural killer; Q7D, once every 7 days.

Figure 5

NKTR-255 has more sustained IL-15R engagement and prolonged NK cell increase compared with precomplexed rhIL-15 N72D/IL-15Rα Fc NK and CD8+ T cell time-course of (A) pSTAT5 and pAKT induction, (B) cell number increase (fold change relative to vehicle) and Ki67 positivity, and (C) intracellular granzyme B and cell surface IL-2Rβ (CD122) expression after a single dose of NKTR-255 (0.1 mg/kg intravenously) or rhIL-15 N72D/IL-15Rα Fc (0.1 mg/kg intravenously) in mice. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way analysis of variance, Sidak’s post-test treatment versus matching vehicle (A) or NKTR-255 versus rhIL-15 N72D/IL-15Rα Fc (Ki67 positivity) (C), and unpaired t-test (B). IL, interleukin; IL-15Rα, IL-15 receptor α; NK, natural killer; pAKT, phosphorylated AKT; pSTAT5, STAT5 phosphorylation; rhIL-15; recombinant human IL-15; STAT5, signal transducer and activator of transcription.

Figure 6

NKTR-255 has superior antitumor activity and NK cell cytotoxic function activation compared with precomplexed rhIL-15 N72D/IL-15Rα Fc and rhIL-15 in a Daudi lymphoma model. (A) Survival plot of Daudi Burkitt lymphoma xenograft model. *p+ [HLA-DR+] Daudi lymphoma cell count) on days 17 and 21 after tumor inoculation. (C) NK cell expansion, (D) granzyme B induction and (E) CD107a induction in the femoral bone marrow of tumor-bearing mice at 3, 6 and 10 days after treatment with vehicle, NKTR-255 (0.3 mg/kg, intravenously), rhIL-15 N72D/IL-15Rα Fc (0.3 mg/kg, intravenously) or rhIL-15 (0.3 mg/kg, intravenously). **p<0.01, ***p<0.001, ****p<0.0001, one-way analysis of variance, Sidak’s post-test; NKTR-255 versus IL-15 N72D/IL-15Rα Fc,. IL, interleukin; IL-15Rα, IL-15 receptor α; NK, natural killer; rhIL-15, recombinant human IL-15.