Development of a nanoparticle-based immunotherapy targeting PD-L1 and PLK1 for lung cancer treatment

Abstract

Immune checkpoint inhibitors (ICIs) targeting PD-L1 and PD-1 have improved survival in a subset of patients with advanced non-small cell lung cancer (NSCLC). However, only a minority of NSCLC patients respond to ICIs, highlighting the need for superior immunotherapy. Herein, we report on a nanoparticle-based immunotherapy termed ARAC (Antigen Release Agent and Checkpoint Inhibitor) designed to enhance the efficacy of PD-L1 inhibitor. ARAC is a nanoparticle co-delivering PLK1 inhibitor (volasertib) and PD-L1 antibody. PLK1 is a key mitotic kinase that is overexpressed in various cancers including NSCLC and drives cancer growth. Inhibition of PLK1 selectively kills cancer cells and upregulates PD-L1 expression in surviving cancer cells thereby providing opportunity for ARAC targeted delivery in a feedforward manner. ARAC reduces effective doses of volasertib and PD-L1 antibody by 5-fold in a metastatic lung tumor model (LLC-JSP) and the effect is mainly mediated by CD8+ T cells. ARAC also shows efficacy in another lung tumor model (KLN-205), which does not respond to CTLA-4 and PD-1 inhibitor combination. This study highlights a rational combination strategy to augment existing therapies by utilizing our nanoparticle platform that can load multiple cargo types at once.

Fig. 1. PLK1 knockdown by siRNA induces PD-L1 expression.

a PLK1 and PD-L1 mRNA expression in A549 (human NSCLC) at 48 h post treatment with PLK1 siRNA (siPLK1) or scrambled siRNA (siSCR) normalized to HPRT housekeeping gene. Dharmafect 1 transfection agent was used. Data presented as mean ± SD from 3 independent samples; ****P < 0.0001 (Unpaired t-test; two-tailed). PD-L1 surface expression of (b) A549 and (c) LLC-JSP at 72 h post treatments assessed by flow cytometry. Data presented as histogram of individual events (cells); ****P < 0.0001 for A549 siPLK1 vs. untreat; ****P < 0.0001 for LLC-JSP siPLK1 vs. untreat (Unpaired t-test; two-tailed). Source data are provided as a Source Data file.

Fig. 2. Volasertib-induced PD-L1 upregulation is dependent on MAPK.

a PD-L1 surface expression of NSCLC cells (H460) treated with vehicle control (0.1% DMSO in PBS), SCH772984 (ERK1/2 small molecule inhibitor, 1 μM), volasertib (100 nM), or SCH772984 (SCH; 1 μM) + volasertib (vol; 100 nM) – (left) representative histograms, (right) MFI quantification. Data presented as mean MFI (median fluorescent Intensity) from biological duplicates, 10,000 events collected per sample. b PD-L1 surface expression of H460 cells treated with vehicle control (0.1% DMSO in PBS), SC75741 (NF-kB inhibitor, 1 μM), volasertib (100 nM), or SC75741 (SC75; 1 μM) + volasertib (vol; 100 nM) – (left) representative histograms, (right) MFI quantification. Data presented as mean MFI from biological duplicates, 10,000 events collected per sample. Source data are provided as a Source Data file.

Fig. 3. PLK1 inhibition potentiates PD-L1 blockade in syngeneic lung tumors.

a C57BL/6 mice were injected with 200,000 LLC-JSP cells on the right flank. On day 0 (8 post tumor inoculation), mice were grouped (n = 7–8) and received i.p. treatments of control vehicles (PBS and HCl/saline), PLK1 inhibitor volasertib (20 mg/kg), PD-L1 antibody (10 mg/kg), or combination of PLK1 inhibitor and PD-L1 antibody at the same dose. Treatments were administered every 5 days for 3 doses. b Tumor growth of mice. Data presented as mean ± SEM; ****P < 0.0001 (mixed model Two-Way repeated measures ANOVA with Tukey’s correction for multiple comparisons). c Kaplan–Meier Survival curve. ***P = 0.0007 vs. saline (Log-rank Mantel–Cox test). Source data are provided as a Source Data file.

Fig. 4. PD-L1 antibody conjugated NP for delivering PLK1 inhibitor volasertib (p-iPLK1-NP; ARAC).

a Synthesis scheme. Nanoparticle images are re-printed with permission from Wiley; © 2022 Wiley-VCH GmbH. b Hydrodynamic size by dynamic light scattering (DLS). c Cell viability by CTG assay of LLC-JSP cells treated with volasertib (iPLK1) or iPLK1-NP; all having equivalent volasertib dose as specified. Data presented as mean ± SD from 4 independent samples; ****P < 0.0001 (Unpaired t-test; two-tailed). d Cell viability by CTG assay of LLC-JSP cells treated with iPLK1-NP or ARAC (p-iPLK1-NP) at equivalent NP and volasertib dose. Data presented as mean ± SD from 5 independent samples; ns – not significant (Unpaired t-test; two-tailed). (e, f) PD-L1 surface expression of the cells treated with PBS, PD-L1 antibody (50 µg/ml), iPLK1-NP (with 210 ng/ml volasertib), or ARAC (with 210 ng/ml volasertib and 1.68 µg/ml antibody) for 2 h and 2 days. Data presented as mean MFI from biological duplicates, 10,000 events collected per sample. Source data are provided as a Source Data file.

Fig. 5. ARAC elicits anti-tumor immune effects.

a 100,000 LLC-JSP cells were injected in right flank and 40,000 cells were injected in left flank of C57BL/6 mice. On day 12 post tumor inoculation, mice (n = 7 per treatment group) received intratumoral treatments of saline, p-NP, iPLK1-NP, or ARAC to the right (local) tumor. Each dose consists of 0.5 mg NP (containing 2.5 µg volasertib and/or 20 µg PD-L1 antibody) in 50 µl per dose for 3 doses total. b Local (treated) tumor growth. Data presented as mean ± SEM; *P = 0.0104, **P = 0.0017, ****P < 0.0001 (Two-Way repeated measures ANOVA with Tukey’s correction for multiple comparisons). c Distant (untreated) tumor growth of individual mice (distant tumors developed in 6/7 saline, 3/7 p-NP, 3/7 iPLK1-NP, and 2/7 ARAC at shown time-points). d Kaplan–Meier Survival curve (mice were euthanized when a combined tumor size reached 2000 mm³). **P = 0.0036 for ARAC vs. saline (Log-rank Mantel–Cox test). e–g Mice (n = 7) were inoculated with 250,000 and 100,000 LLC-JSP cells for bilateral tumors and treated as shown in (a) with ARAC or saline. One day post 3rd injection, tumors were harvested and processed into single cell suspensions for flow cytometry analysis. e PD-L1 expression (median fluorescent intensity; MFI) in CD45+ and CD45− cells. f Proliferative effector T cells (%Ki67 of CD44+ CD8+ cells) in tumor-draining lymph nodes. g CD45+ (% of live cells), CD8+ (% of CD45+ CD3+ cells), CD4+ (% of CD45+ CD3+ cells), and CD8+/Treg (Regulatory T cells; CD4+ FoxP3+ (% of CD45+ CD3+ cells)) in tumors. Data presented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001 (One-Way ANOVA with Tukey’s correction for multiple comparisons). Source data are provided as a Source Data file.

Fig. 6. ARAC improves survival of mice bearing metastatic lung tumors.

a C57BL/6 mice were injected with 200,000 LLC-JSP cells intravenously. After 3 days, mice were treated with saline, free drugs (i.p., at 1x dose: 2.5 µg volasertib and 20 µg PD-L1 and 5x dose: 12.5 µg volasertib and 100 µg PD-L1 antibody), ARAC (i.v., containing 2.5 µg volasertib and 20 µg PD-L1), or ARAC + anti-CD8 (200 μg i.p. twice weekly). Treatments were administered every 3 days for a total of 4 doses. b, c Kaplan–Meier Survival curve. *P = 0.0120, **P = 0.0033, ***P = 0.0009, ns – not significant (Log-rank Mantel–Cox test). d Mouse weight change post first treatment; data presented as mean ± SEM. Source data are provided as a Source Data file.

Fig. 7. ARAC reduces tumor growth and prolongs survival of mice bearing KLN-205 murine lung tumors.

a DBA/2 mice were injected with 500,000 KLN-205 cells in 100 μL PBS on the right flank. 13 days post inoculation, mice were grouped (n = 7) and treated with saline, ICIs (PD-1 and CTLA-4 antibodies – i.p. 200 μg/dose and 100 μg/dose respectively – for 6 doses), or ARAC (i.v. 50 mg/kg for 4 doses). a KLN-205 tumor growth; blue arrows specify ARAC dosing days, red arrows specify ICI dosing days. Data presented as mean + SEM; **P = 0.0011 for ARAC vs. saline, **P = 0.0041 for ARAC vs. ICIs (mixed model Two-Way repeated measures ANOVA with Tukey’s correction for multiple comparisons). b Kaplan–Meier Survival curve. *P = 0.0101 for ARAC vs. saline, *P = 0.0231 for ARAC vs. ICIs (Log-rank Mantel–Cox test). Source data are provided as a Source Data file.

Fig. 8. Proposed mechanism of action of ARAC nanoconstruct.

(Left cell) ARAC binds to PD-L1 on cancer cell surface and is internalized via receptor-mediated endocytosis. Endosomal escape of ARAC is mediated by PEI polymer and volasertib is released to inhibit PLK1 activity, leading to G2/M cell cycle arrest and apoptotic cell death. However, G2/M arrest induced by volasertib upregulates PD-L1 levels in surviving cancer cells, thereby rendering them unresponsive to immune-mediated effects (due to PD-L1-mediated immunosuppression). (Right cell) We capitalize on this property by utilizing elevated PD-L1 levels in surviving cancer cells as the homing target for subsequent ARAC delivery, leading to cancer targeting in a feedforward manner (i.e., higher targeting with increased doses of the treatment). Enhanced delivery of ARAC results in loss of PD-L1, which allows for cytotoxic CD8+ T cells to effectively kill cancer and generate an anti-tumor immune response.