Antitumor immunity induced by antibody-based natural killer cell engager therapeutics armed with not-alpha IL-2 variant

Abstract

Harnessing innate immunity is emerging as a promising therapeutic approach in cancer. We report here the design of tetraspecific molecules engaging natural killer (NK) cell-activating receptors NKp46 and CD16a, the β-chain of the interleukin-2 receptor (IL-2R), and a tumor-associated antigen (TAA). In vitro, these tetraspecific antibody-based natural killer cell engager therapeutics (ANKETs) induce a preferential activation and proliferation of NK cells, and the binding to the targeted TAA triggers NK cell cytotoxicity and cytokine and chemokine production. In vivo, tetraspecific ANKETs induce NK cell proliferation and their accumulation at the tumor bed, as well as the control of local and disseminated tumors. Treatment of non-human primates with CD20-directed tetraspecific ANKET leads to CD20⁺ circulating B cell depletion, with minimal systemic cytokine release and no sign of toxicity. Tetraspecific ANKETs, thus, constitute a technological platform for harnessing NK cells as next-generation cancer immunotherapies.

Keywords: Cancer immunotherapy; Natural Killer cells; cytokine; multispecific antibodies.

Graphical abstract

Figure 1

In vitro characterization of ANKET-IL-2v efficacy (A) pSTAT5 monitoring on NK cells (CD3⁻CD56⁺), Tregs (CD3⁺CD4⁺CD25^hiFoxP3⁺), CD4⁺ T cells (CD3⁺CD4⁺CD25^lowFOXP3⁻), and CD8⁺ T cells (CD3⁺CD8⁺) gated on peripheral blood mononuclear cells (PBMCs) activated by the IL-2v/aNKp46/Fc/aCD20 molecule or the IL-2v/IC/Fc-null/aCD20 molecule. The picture is representative of at least 3 independent experiments. (B) pSTAT5 monitoring on NK cells (CD3⁻CD56⁺) gated on PBMCs activated with IL-2v/aNKp46/Fc/aCD20, IL-2v/aNKp46/Fc-null/aCD20, IL-2v/IC/Fc/aCD20, and IL-2v/IC/Fc-null/aCD20 molecules. The dose response of a representative donor is presented in the left panel, and a boxplot, with whiskers showing minimal and maximal value, depicting the EC₅₀ obtained for three to seven donors is shown in the right panel. (C) Purified NK cell proliferation induced by IL-2v/aNKp46/Fc/aCD20, IL-2v/IC/Fc-null/IC, and aNKp46/Fc/aCD20 molecules in the absence of the target tumor cells. Left, the percentage of NK cells displaying proliferation induced by a dose response to the indicated molecules. Right, CellTrace Violet (CTV) dilution profile induced by the indicated molecules at a concentration of 3.7 nM. The data shown are representative of results obtained for five donors. (D) IFN-γ and MIP-1β production by purified NK cells in the presence of Raji tumor cells induced by IL-2v/aNKp46/Fc/aCD20, IL-2v/aNKp46/Fc-null/aCD20, IL-2v/IC/Fc/aCD20, and IL-2v/aNKp46/Fc/IC molecules. The data shown are the mean ± SD for three donors. (E) NK cell cytotoxicity toward Raji tumor cells induced by IL-2v/aNKp46/Fc/aCD20, IL-2v/aNKp46/Fc-null/aCD20, IL-2v/IC/Fc/aCD20, and IL-2v/aNKp46/Fc/IC molecules. Left, NK cell-mediated specific lysis from a representative donor analyzed with experimental duplicate ± SD. Right, boxplot, with whiskers showing minimal and maximal value, depicting the EC₅₀ of specific lysis obtained for five donors. The statistical analysis is described in the STAR methods, ∗p < 0.05; ∗∗p < 0.01.

Figure 2

Transcriptomic landscape of NK cells activated by ANKET-IL-2v (A and B) Purified human NK cells were activated for 4 h in the presence of the murine huCD20-B16F10 target cells, with 0.1 μg/mL IL-2v/aNKp46/Fc/aCD20, IL-2v/IC/Fc-null/IC, Obinutuzumab, or aNKp46/Fc/aCD20. RNA sequencing was performed, and reads were aligned with the human genome, n = 4 to 6 donors. (A) Venn diagram representing the number of genes significantly regulated by each of the indicated stimulation conditions relative to unstimulated conditions (false discovery rate [FDR] = 0.05). (B) Bar plot showing the number of genes regulated (FDR = 0.05) for each stimulation treatment relative to unstimulated conditions.

Figure 3

A specific transcriptomic effector program associated with enhanced NK cell responses is induced by ANKET-IL-2v stimulation (A–D and F) Purified human NK cells were activated for 4 h in the presence of the murine huCD20-B16F10 target cells, with 0.1 μg/mL IL-2v/aNKp46/Fc/aCD20, IL-2v/IC/Fc-null/IC, Obinutuzumab, or aNKp46/Fc/aCD20 or a combination of 0.1 μg/mL IL-2v/IC/Fc-null/IC + 0.1 μg/mL aNKp46/Fc/aCD20 molecules. RNA sequencing was performed, and reads were aligned with the human genome, N = 4 to 6 donors. (A) Volcano plot representing the results of the analysis of differential expression between NK cells activated with IL-2v/aNKp46/Fc/aCD20 and those treated with a mixture of IL-2v/IC/Fc-null/IC + aNKp46/Fc/aCD20 molecules. In green: differentially expressed genes (FDR = 0.05); in gray: genes not differentially expressed. (B) Gene sets displaying significant enrichment in NK cells stimulated with IL-2v/aNKp46/Fc/aCD20 relative to cells stimulated with a combination of the trispecific aNKp46/Fc/aCD20 and the IL-2v polypeptide, based on GSEA. (C) Venn diagram representing the number of genes upregulated in NK cells following activation with the indicated molecules, relative to unstimulated conditions (FDR = 0.05). (D) Heatmap representing the expression of a selection of differentially regulated genes. (E) CD69 monitored by flow cytometry on purified human NK cells activated for 24 h in the presence of the murine huCD20-B16F10 target cells with 0.1 μg/mL IL-2v/aNKp46/Fc/aCD20, IL-2v/IC/Fc-null/IC, Obinutuzumab, or aNKp46/Fc/aCD20 or a combination of equal doses of IL-2v/IC/Fc-null/IC + aNKp46/Fc/aCD20. Picture is a boxplot obtained with NK cells from four donors. Whiskers show minimal and maximal value. Symbols are conserved for each donor across the conditions of stimulation. (F) Gene set score for CD56^dimCD16a⁺CD57⁺ NK1 cells and CIML NK cells regulated by each of the indicated condition of stimulation.

Figure 4

In vivo characterization of the antitumor efficacy of ANKET-IL-2v (A–C and E) Raji B cell lymphoma cells were subcutaneously engrafted in CB17 SCID mice. (A) Efficacy of 70 μg IL-2v/aNKp46/Fc/aCD20 or 1,500 μg Obinutuzumab injected i.v. once, 9 days after tumor engraftment. (B) Efficacy of 25 μg IL-2v/aNKp46/Fc/aCD20 or 25 μg IL-2v/aNKp46/Fc/IC injected i.v. once, 9 days after tumor engraftment. (C) Efficacy of two weekly i.v. injections of 25 μg IL-2v/aNKp46/Fc/aCD20, IL-2v/IC/Fc null/aCD20, or IL-2v/IC/Fc/aCD20 molecules. (D) Immunostaining for human NKp46 and Gzmb on sections of Raji tumors grown subcutaneously in RAG1ko huNKp46Tg mice, 3 days after treatment with 25 μg IL-2v/aNKp46/Fc/aCD20 or vehicle. The images shown are representative of n = 5 tumors. (E) Efficacy of a single i.v. injection of 25 μg IL-2v/aNKp46/Fc/aCD20 in the context of NK cell depletion with anti-Asialo-GM1. Dotted vertical lines represent the day of treatment. Each curve represents data from a single mouse. The data shown are representative of at least two independent experiments. The statistical analysis is described in the STAR methods, ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Figure 5

In vivo activity of ANKET-IL-2v in immunocompetent mice (A) NK cell number and percentage of CD69⁺ NK cells in spleen of huCD20-B16F10 tumor bearing C57BL6 mice treated with 25 μg tetraspecific ANKET (IL2v/aNKp46/Fc/aCD20) i.v. injection for 4 days. Picture is a boxplot obtained with NK cells from five mice. Whiskers show minimal and maximal value. (B) Efficacy of a single i.v. injection of 70 μg IL-2v/aNKp46/Fc/aCD20, 70 μg aNKp46/Fc/aCD20, or 600 μg obinutuzumab in the huCD20-B16F10 disseminated melanoma model in C57BL6 wild-type (WT) mice. (C) Efficacy of i.v. injections performed at days 1, 9, and 16 of 25 μg IL-2v/aNKp46/Fc/aCD20 or 25 μg IL-2v/IC/Fc-null/aCD20 in the model of huCD20-B16F10 melanoma cells engrafted subcutaneously in C57BL6 μM mice. The data shown are representative of at least two independent experiments. The statistical analysis is described in the STAR methods, ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Figure 6

Efficacy and safety of ANKET-IL-2v in non-human primates (A) Counts of circulating B cells, expressed as a percentage of the baseline counts in NHPs receiving a single injection of vehicle (n = 6, black) or IL-2v/aNKp46/Fc/aCD20 at a dose of 0.05 (n = 4, orange) or 0.5 mg/kg (n = 4, green). (B) Cytokine concentrations in the plasma of NHPs receiving a single injection of vehicle (n = 6, black) or IL-2v/aNKp46/Fc/aCD20 at a dose of 0.05 (n = 4, orange) or 0.5 mg/kg (n = 4, green).