Core-shell nanoscale coordination polymers combine chemotherapy and photodynamic therapy to potentiate checkpoint blockade cancer immunotherapy

Abstract

Advanced colorectal cancer is one of the deadliest cancers, with a 5-year survival rate of only 12% for patients with the metastatic disease. Checkpoint inhibitors, such as the antibodies inhibiting the PD-1/PD-L1 axis, are among the most promising immunotherapies for patients with advanced colon cancer, but their durable response rate remains low. We herein report the use of immunogenic nanoparticles to augment the antitumour efficacy of PD-L1 antibody-mediated cancer immunotherapy. Nanoscale coordination polymer (NCP) core-shell nanoparticles carry oxaliplatin in the core and the photosensitizer pyropheophorbide-lipid conjugate (pyrolipid) in the shell (NCP@pyrolipid) for effective chemotherapy and photodynamic therapy (PDT). Synergy between oxaliplatin and pyrolipid-induced PDT kills tumour cells and provokes an immune response, resulting in calreticulin exposure on the cell surface, antitumour vaccination and an abscopal effect. When combined with anti-PD-L1 therapy, NCP@pyrolipid mediates regression of both light-irradiated primary tumours and non-irradiated distant tumours by inducing a strong tumour-specific immune response.

Figure 1. Chemotherapy and PDT of NCP@pyrolipid potentiate PD-L1 blockade to induce systemic antitumour immunity.

Chemotherapy and PDT of NCP@pyrolipid induce ICD and an inflammatory environment at the primary tumour site, leading to the release of tumour-associated antigens (TAAs). TAAs are processed and presented by infiltrated antigen-presenting cells, to elicit the proliferation of tumour-specific effector T cells in lymphoid organs, such as tumour-draining lymph nodes. Combined with PD-L1 checkpoint blockade, the NCP@pyrolipid chemotherapy/PDT significantly promoted the generation of tumour-specific effector T cells and enhanced their infiltration in both primary and distant tumours, resulting in not only tumour eradication in the primary sites but also a systemic antitumour immune response to reject distant tumours.

Figure 2. Preparation and characterization of NCP@pyrolipid.

(a) Schematic presentation showing the structure of NCP@pyrolipid and its three combined therapeutic modalities. (b) Transmission electron microscopy image showing the spherical and monodispersed morphology of NCP@pyrolipid. Scale bar, 200 nm. (c) Number-average diameters of DOPA-NCP in THF and NCP@pyrolipid in PBS by DLS measurements. Data are expressed as means±s.d. (n=3).

Figure 3. NCP@pyrolipid induces ICD and acute inflammation.

(a) CRT exposure on the cell surface of CT26 cells was assessed after the treatments of PBS, free oxaliplatin, NCP, porphysome and NCP@pyrolipid with or without light irradiation (90 J cm⁻²) by flow cytometry analysis. The fluorescence intensity was gated on PI-negative cells. ‘+' and ‘−' in the figure legends refer to treatments with and without irradiation, respectively. (b–d) Pro-inflammatory cytokine levels in the serum of mice treated with PDT of NCP@pyrolipid. Syngeneic CT26 tumour-bearing mice were i.v. injected with PBS, NCP or NCP@pyrolipid at an oxaliplatin dose of 2 mg kg⁻¹, followed by light irradiation at a dose of 180 J cm⁻² (670 nm, 100 mW cm⁻²). The blood was collected daily from Day 7, when the mice received their first i.p. injections of nanoparticles, to Day 10, 2 days after the first light irradiation treatment. The serum was separated and the concentrations of IFN-γ (b), IL-6 (c) and TNF-α (d) were determined by enzyme-linked immunosorbent assay. Data are expressed as means±s.d. (n=3).

Figure 4. Pharmacokinetics and biodistribution of NCP@pyrolipid.

CT26 tumour-bearing BALB/c mice received i.v. injections of NCP@pyrolipid at an oxaliplatin dose of 3 mg kg⁻¹ (or pyrolipid dose of 2.1 mg kg⁻¹). Pt and pyrolipid concentrations were measured by ICP-MS and ultraviolet–visible spectroscopy, respectively. (a) Biodistribution of Pt in mice. A one-compartment model was used to fit the blood concentration of Pt (b) and pyrolipid (c) over time. Data are expressed as means±s.d. (n=3).

Figure 5. In vivo antitumour activity of NCP@pyrolipid.

PBS, NCP or NCP@pyrolipid was i.v. injected into a syngeneic CT26 mouse model and an HT29 xenograft mouse model at an oxaliplatin dose of 2 mg kg⁻¹, followed by irradiation (670 nm, 100 mW cm⁻²) for 30 min, 24 h after each injection. Tumour growth inhibition curves in CT26 (a) and HT29 (c) models. Weights of excised tumours at the endpoint of the experiment for CT26 (b) and HT29 (d) models. Black and red arrows in a and c represent the time of drug administration and irradiation, respectively. ‘+' and ‘−' in the figure legends refer to treatments with and without irradiation, respectively. Data are expressed as means±s.d. (n=6).

Figure 6. The abscopal effect of NCP@pyrolipid in combination with anti-PD-L1.

Bilateral tumour models of MC38 and CT26 were developed by subcutaneously injecting cancer cells into both the right and left flank regions of each animal. The right tumours were designated the primary tumours for light irradiation and the left tumours were designated the distant tumours and did not receive light irradiation. For the MC38 model, PBS, porphysome, oxaliplatin plus porphysome or NCP@pyrolipid was i.p. injected into mice, followed by light irradiation at a dose of 180 J cm⁻² (670 nm, 100 mW cm⁻²) and i.p. injection of anti-PD-L1 at a dose of 50 μg per mouse. The treatment was carried out every 3 days for a total of three treatments. For the CT26 model, PBS or NCP@pyrolipid was i.p. injected into the mice, followed by light irradiation at a dose of 180 J cm⁻² (670 nm, 100 mW cm⁻²) and i.p. injection of anti-PD-L1 at a dose of 75 μg per mouse. The treatment was carried out every other day for a total of two treatments. Tumour growth inhibition curves in MC38 (a,b) and CT26 (c,d) models. The arrows represent the times of drug administration (black) and irradiation (red). ‘+' and ‘−' in the figure legends refer to with and without irradiation, respectively. Data are expressed as means±s.d. (n=6).

Figure 7. Tumour-specific immune responses and the abscopal effect.

Bilateral tumour models of MC38 were established and treated as described in Figs 6a,b. On Day 19 (12 days after the first treatment), the splenocytes were harvested and stimulated with 10 μg ml⁻¹ KSPWFTTL peptide for 48 h. ELISPOT assay was performed to detect IFN-γ producing T cells (n=4 or 5) (a). The primary (right) and distant (left) tumours were collected for flow cytometry analysis (n=5). The cells were stained with CD45⁺PI^- (b), CD45⁺CD3e⁺CD4⁺PI⁻ (c) and CD45⁺CD3e⁺CD8⁺PI⁻ (d), and gated from total tumour cells. Data are expressed as means±s.d. (n=5). *P<0.05 from control, **P<0.01 from control and ***P<0.001 from control by t-test.

Figure 8. CD8⁺ T cells immunofluorescence assay.

Bilateral tumour models of MC38 were established and treated with PBS with irradiation or NCP@pyrolipid with irradiation plus anti-PD-L1. On Day 19 (12 days after the first treatment), primary (right) and distant (left) tumours were collected, sectioned and subjected to immunofluorescence staining. (a) Representative CLSM images of tumours after immunofluorescence staining. White arrows indicate CD8⁺ T cells. Tumour cell nuclei in treated primary tumours appear to be smaller, probably due to the effects of PDT treatment. Scale bar, 50 μm. (b) The densities of CD8⁺ T cells in the whole tumours. Data are expressed as means±s.d. (n=3). ***P<0.001 from control by t-test.