Mechanisms involved in IL-15 superagonist enhancement of anti-PD-L1 therapy

Abstract

Background: Immunotherapy targeting PD-1/PD-L1 fails to induce clinical responses in most patients with solid cancers. N-803, formerly ALT-803, is an IL-15 superagonist mutant and dimeric IL-15RαSushi-Fc fusion protein complex that enhances CD8+ T and NK cell expansion and function and exhibits anti-tumor efficacy in preclinical models. Previous in vitro studies have shown that IL-15 increases PD-L1 expression, a negative regulator of CD8+ T and NK cell function. Most reported preclinical studies administered N-803 intraperitoneally not subcutaneously, the current clinical route of administration. N-803 is now being evaluated clinically in combination with PD-1/PD-L1 inhibitors. However, the mechanism of action has not been fully elucidated. Here, we examined the anti-tumor efficacy and immunomodulatory effects of combining N-803 with an anti-PD-L1 antibody in preclinical models of solid carcinomas refractory to anti-PD-L1 or N-803.

Methods: Subcutaneous N-803 and an anti-PD-L1 monoclonal antibody were administered as monotherapy or in combination to 4T1 triple negative breast and MC38-CEA colon tumor-bearing mice. Anti-tumor efficacy was evaluated, and a comprehensive analysis of the immune-mediated effects of each therapy was performed on the primary tumor, lung as a site of metastasis, and spleen.

Results: We demonstrate that N-803 treatment increased PD-L1 expression on immune cells in vivo, supporting the combination of N-803 and anti-PD-L1. N-803 plus anti-PD-L1 was well-tolerated, reduced 4T1 lung metastasis and MC38-CEA tumor burden, and increased survival as compared to N-803 and anti-PD-L1 monotherapies. Efficacy of the combination therapy was dependent on both CD8+ T and NK cells and was associated with increased numbers of these activated immune cells in the lung and spleen. Most alterations to NK and CD8+ T cell phenotype and number were driven by N-803. However, the addition of anti-PD-L1 to N-803 significantly enhanced CD8+ T cell effector function versus N-803 and anti-PD-L1 monotherapies, as indicated by increased Granzyme B and IFNγ production, at the site of metastasis and in the periphery. Increased CD8+ T cell effector function correlated with higher serum IFNγ levels, without related toxicities, and enhanced anti-tumor efficacy of the N-803 plus anti-PD-L1 combination versus either monotherapy.

Conclusions: We provide novel insight into the mechanism of action of N-803 plus anti-PD-L1 combination and offer preclinical proof of concept supporting clinical use of N-803 in combination with checkpoint inhibitors, including for patients non- and/or minimally responsive to either monotherapy.

Keywords: ALT-803; N-803; PD-L1; Tumor microenvironment.

Conflict of interest statement

Ethics approval and consent to participate

All in vivo experiments were approved by the NCI/NIH Animal Care and Use Committee (ACUC).

Consent for publication

Not applicable.

Competing interests

Authors from the National Cancer Institute do not have any competing interests to disclose. S.A. is an employee and shareholder of Altor Bioscience (a NantWorks company).

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1

N-803 treatment increases PD-L1 expression on total CD45+ cells and MDSC populations in the primary tumor, lung, and spleen. 5 × 104 4T1 tumor cells were orthotopically implanted into female Balb/c mice. When tumor volumes reached ~50mm3, mice were treated at days 9 and 13 with 1 μg N-803 (s.c.). Twenty-four hours after the last treatment, PD-L1 expression (geometric mean fluorescence intensity (gMFI)) in the primary tumor, lung parenchyma and vasculature, and spleen was determined by flow cytometry on total CD45+ or CD45− cells (a), G-MDSC/Granulocytes (b), or M-MDSC/Monocytes (c). Graphs show mean ± SD. Data combined from 2 independent experiments, n = 5 mice/group per experiment

Fig. 2

Combination of N-803 and αPD-L1 decreases 4T1 lung metastasis and improves survival. a-c Mice were implanted with 4T1 tumors as in Fig. 1 and treated on days 8 and 14 with 1 μg N-803 (s.c.) and/or 200 μg αPD-L1 (i.p.) on days 8, 11, and 14. Graphs of tumor volumes of individual animals at day 25 post-tumor implant in (a) and number of lung metastases in individual mice 26 days after tumor implant (b) show mean ± SD. c Table denotes the distribution of number of lung metastases and % reduction in mean versus PBS. Data are representative of 2 independent experiments, n = 20–23 mice. d-g Mice were implanted with 4T1 tumors as in Fig. 1 and treated at days 9 and 13 with N-803 and/or αPD-L1 on days 9, 11, and 13. The primary tumor was surgically resected at day 15. Graphs of tumor volumes of individual animals at day 14 post-tumor implant (d) and number of lung metastases in individual mice at day 28 (e) show mean ± SD. f Table denotes the distribution of number of lung metastases per mouse and % reduction in mean versus PBS. Data are representative of 2 independent experiments, n = 15–19 mice. g Survival curves (inset: mOS) show % survival. Data are representative of 2 independent experiments, n = 13–19 mice

Fig. 3

CD8+ T cells and NK cells contribute to the anti-tumor efficacy of N-803 + αPD-L1 combination. Mice were implanted with 4T1 tumors as in Fig. 1 and treated on days 13 and 17 with N-803 and αPD-L1 on days 13, 15, and 17. CD8-expressing cells and NK cells were depleted on days 10, 11, 12, 16, and 19 using 100 μg anti-CD8 and/or 25 μl anti-asialo-GM1 (i.p.). Graphs show tumor volumes of individual mice (a) or number of lung metastases in individual mice at day 23 post-tumor implant (b) as mean ± SD. c Table denotes the distribution of the number of lung metastases per mouse and % reduction in mean versus PBS. Data combined from 2 independent experiments, n = 23–25 mice/group total

Fig. 4

N-803 monotherapy and N-803 + αPD-L1 combination promote an activated NK cell phenotype and increase function. Mice were implanted with 4T1 tumors as in Fig. 1 and treated on days 9 and 13 with N-803 and/or αPD-L1 on days 9, 11, and 13. a-d NK cells were examined by flow cytometry in the primary tumor, lung vasculature, and spleen 24 h after the last treatment. Graphs show NK cell number (a) and frequencies of NKG2D+ (b), Ki67+ (c), and Granzyme B+ (d) NK cells. e Purified splenic NK cells were co-cultured with 111In-labeled Yac-1 target cells at a 100:1 effector-to-target (E:T) ratio for 18 h. 111In release was measured to determine cytotoxic function. All graphs show mean ± SD. Data combined from 2 to 3 independent experiments, n = 4–5 mice/group per experiment

Fig. 5

Combination of N-803 + αPD-L1 induces an activated CD8+ T cell phenotype. Mice were implanted with 4T1 tumors as in Fig. 1 and treated on days 9 and 13 with N-803 and/or αPD-L1 on days 9, 11, and 13. CD8+ T cells were examined by flow cytometry in the primary tumor, lung parenchyma and vasculature, and spleen 24 h after the last treatment. Graphs of CD8+ T cell number (a), frequency of CD44hi (b), CD44hiCD62Lhi TCM (c), and Ki67+ CD44hi (d) CD8+ T cells show mean ± SD. Data combined from 2 to 3 independent experiments, n = 5 mice/group per experiment

Fig. 6

Combination of N-803 + αPD-L1 increases Granzyme B and effector cytokine production by CD8+ T cells. Mice were implanted with 4T1 tumors as in Fig. 1 and treated on days 12 and 16 with N-803 and/or αPD-L1 on days 12, 14, and 16. CD8+ T cell effector molecule and cytokine production were examined by flow cytometry in the primary tumor, lung parenchyma and vasculature, and spleen 24 h after last treatment. a Graphs show frequency of Granzyme B+ CD44hi CD8+ T cells. b-d Immune cells were stimulated with 1 μg/ml αCD3 + 1 μg/ml αCD28 for 4 h. Graphs show frequency of total IFNγ+ CD44hi CD8+ T cells (b) and frequency of IFNγ-single producing (SP) (c) or IFNγ/TNFα-double producing (DP) (d) CD44hi CD8+ T cells. All graphs show mean ± SD. Data combined from 2 independent experiments, n = 5 mice/group per experiment

Fig. 7

N-803 + αPD-L1 combination promotes the generation of an immunostimulatory milieu in the serum. Mice were implanted with 4T1 tumors as in Fig. 1 and treated on days 9 and 13 with N-803 and/or αPD-L1 on days 9, 11, and 13. Serum was obtained at days 14 and 21 post-tumor implant and analyzed for level of IFNγ (a), TNFα (b), IL-10 (c), and IL-6 (d). Level of serum cytokines from individual animals at day 14 (left panels) and curves showing kinetics of serum cytokine levels (right panels) show mean ± SD. Data are combined from 2 experiments, n = 4–5 mice/group per experiment