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. 2020 May;4(5):531-543.
doi: 10.1038/s41551-020-0549-2. Epub 2020 Apr 13.

Collagen-binding IL-12 enhances tumour inflammation and drives the complete remission of established immunologically cold mouse tumours

Aslan Mansurov #  1 , Jun Ishihara #  2 , Peyman Hosseinchi  1 , Lambert Potin  1 , Tiffany M Marchell  1   3 , Ako Ishihara  1 , John-Michael Williford  1 , Aaron T Alpar  1 , Michal M Raczy  1 , Laura T Gray  1 , Melody A Swartz  1   3   4 , Jeffrey A Hubbell  5   6
Affiliations

Collagen-binding IL-12 enhances tumour inflammation and drives the complete remission of established immunologically cold mouse tumours

Aslan Mansurov et al. Nat Biomed Eng. 2020 May.

Abstract

Checkpoint-inhibitor (CPI) immunotherapy has achieved remarkable clinical success, yet its efficacy in 'immunologically cold' tumours has been modest. Interleukin-12 (IL-12) is a powerful cytokine that activates the innate and adaptive arms of the immune system; however, the administration of IL-12 has been associated with immune-related adverse events. Here we show that, after intravenous administration of a collagen-binding domain fused to IL-12 (CBD-IL-12) in mice bearing aggressive mouse tumours, CBD-IL-12 accumulates in the tumour stroma due to exposed collagen in the disordered tumour vasculature. In comparison with the administration of unmodified IL-12, CBD-IL-12 induced sustained intratumoural levels of interferon-γ, substantially reduced its systemic levels as well as organ damage and provided superior anticancer efficacy, eliciting complete regression of CPI-unresponsive breast tumours. Furthermore, CBD-IL-12 potently synergized with CPI to eradicate large established melanomas, induced antigen-specific immunological memory and controlled tumour growth in a genetically engineered mouse model of melanoma. CBD-IL-12 may potentiate CPI immunotherapy for immunologically cold tumours.

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Figures

Fig. 1 |
Fig. 1 |. CBD-IL-12 binds to collagen with high affinity while retaining bioactivity.
a, Schematic illustrating the fusion sites of the von Willebrand factor A3 CBD to murine p35 and p40 subunits. CBD was fused to each of the subunits via a (GGGS)2 linker. b, Dose-response relationship of phosphorylated STAT4 with IL-12 and CBD-IL-12 in preactivated primary mouse CD8+ T cells (n = 3, mean ± SD). c,d, Affinity (KD values are shown) of CBD-IL-12 against collagen I (c) and collagen III (d) as measured by SPR. CBD-IL-12 was flowed over the chips at indicated concentrations. Curves represent the specific responses (in resonance units, RU) to CBD-IL-12. Experimental curves were fitted with 1:1 Langmuir model. Dissociation constants (KD) and rate constants (kon and koff) determined from the fitted curves are shown. e,f, Binding of unmodified IL-12 (e) or CBD-IL-12 (f) to human melanoma cryosections was imaged by fluorescence microscopy. Scale bars, 100 μm. Experiments were performed twice (b,c,d,e,f), with similar results. Representative data are shown.
Fig. 2 |
Fig. 2 |. CBD-IL-12 induces regression of B16F10 melanoma and EMT6 mammary carcinoma.
a,b, 5 × 105 B16F10 melanoma cells were inoculated intradermally on back skin and mice were treated with either PBS (p.t., n = 9), 25 μg IL-12 (p.t., n = 10), 25 μg IL-12 (i.v., n = 7), equimolar CBD-IL-12 (p.t., n = 10), or equimolar CBD-IL-12 (i.v., n = 15) once on day 7. Individual tumour curves (a) and survival curves (b) are shown. c,d, 5 × 105 EMT6 mammary carcinoma cells were inoculated into the left mammary fat pad and mice were treated i.v. with either PBS (n = 7), 25 μg IL-12 (n = 15) or equimolar CBD-IL-12 (n = 15) once on day 7. Individual tumour curves (c) and survival curves (d) are shown. Data are compiled from two independent experiments. Statistical analyses were done using log-rank (Mantel-Cox) test.
Fig. 3 |
Fig. 3 |. CBD-IL-12 induces intratumoral inflammation by rapidly localizing into the tumour.
a, Mice bearing EMT6 tumours were injected i.v. with 25 μg of DyLight 650-labeled IL-12 (n = 3) or CBD-IL-12 (n = 4). Fluorescence intensity in each tumour was measured using IVIS 1 hr post injection and normalized to the weight of the tumour. b, Naïve C57BL/6 mice were administered 25 μg IL-12 (n = 3) or equimolar CBD-IL-12 (n = 3) via i.v. injection. Blood was collected at the indicated time points, plasma was separated and analysed for IL-12p70 concentration via ELISA. c, Mice bearing established EMT6 tumours were injected i.v. with either PBS (left, n = 3) or 25 μg CBD-IL-12 (IL-12 molar eq., right, n = 3) and 3 days after injection, tumours were, fixed and stained with H&E and anti-mouse CD8 (brown). Scale bar = 400 μm. d, B16F10 melanoma-bearing mice were treated with either 25 μg IL-12 or equimolar CBD-IL-12 i.v. once on day 7 and tumours were harvested 2, 3 and 4 days after treatment. Tumours were homogenized for protein extraction and IFNγ levels were quantified using ELISA and normalized by total tumour protein content. For day 2, n = 9. For day 3, n = 10. For day 4, n = 5. f-i, Luminex assay was performed on day 4 tumour lysates and analysed for indicated cytokines/chemokines (n = 5). Data are mean ± SEM. Experiments in a,b,c were performed twice, with similar results. Representative data are shown. In d, data were compiled from two independent experiments. Luminex assay was performed once on independent biological samples. Statistical analyses were done using unpaired, two-tailed t-test with Welch correction for a,f,g,h and two-tailed Mann-Whitney test for d,e,i due to nonparametric data.
Fig. 4 |
Fig. 4 |. CBD-IL-12 minimizes irAEs in tumour-bearing and non-tumour-bearing mice.
a,b, 7 days before cytokine treatment, 5 × 105 B16F10 melanoma cells were inoculated and mice were treated i.v. with either 10, 25, 50 μg IL-12 or with either 10, 25 or 50 μg CBD-IL-12 (IL-12 molar eq.) once on day 0. a, Kinetics of IFNγ levels (left) and day 2 comparison (right) in the serum are shown. b, Kinetics of serum ALT activity (left) and day 3 comparison (right) are shown. Data are represented as fold of PBS-treated. For 10 μg groups, n = 4. For 25 μg groups, n = 11. For 50 μg groups, n = 5. c,d, Naïve C57BL/6 mice received either 25 μg IL-12 or 25 μg CBD-IL-12 (IL-12 molar eq.) once on day 0 and bled on days 2 and 3 for IFNγ and ALT activity measurements, respectively. n = 5 per group. Data are mean ± SEM. Data are compiled from three independent experiments. Statistical analyses between two groups were done using unpaired, two-tailed t-test with Welch correction.
Fig. 5 |
Fig. 5 |. CBD-IL-12 decreases metastatic tumour burden by triggering activation of innate and adaptive compartments of the immune system in the pulmonary metastatic model of B16F10 melanoma.
a, 5 × 105 B16F10 cells were injected i.v. on day 0. Mice were treated with either 25 μg IL-12 (n = 3) or with equimolar CBD-IL-12 (n = 4) i.v. once on day 8 and sacrificed on day 17. Metastatic burden was quantified using ImageJ software and normalized by total area of the lung. b-i, 2.5 × 105 B16F10 cells were injected i.v. on day 0. Mice were treated with either PBS (n = 6), 25 μg IL-12 (n = 12) or with equimolar CBD-IL-12 (n = 12) i.v. once on day 9 and lungs were collected on day 18. 2 × 106 live cells/well were plated for flow cytometric analysis. b, Percentages of CD3+CD8+ T cells within live cells. c, Frequency of CD3+CD4+CD25+Foxp3+ Tregs within lung-infiltrating immune cells (% of CD45+). d, Frequency of CD3+CD8+CD44+CD62L− effector CD8+ T cells within total CD8+ T cells. e, Ratio of effector CD8+ T cells to Tregs. f-h, Percentages of DCs (CD11c+MHCII+F4/80−) (f), CD103+ DCs (g), and CD11b+ DCs (h) within live cells. i, Frequency of MHCII+CD80+ macrophages within total macrophages (defined as CD11b+F4/80+). Lines represent mean ± SEM. Antitumor efficacy experiment (a) was performed twice, with similar results. Flow analysis was performed once on independent biological samples. Statistical analyses were done using unpaired, two-tailed t-test with Welch’s correction (a), ordinary one-way ANOVA with Tukey’s test for parametric data (b,c,d,f,g,h,i) and Kruskal-Wallis test followed by Dunn’s multiple comparison for nonparametric data (e).
Fig. 6 |
Fig. 6 |. CBD-IL-12 synergizes with CPI and elicits tumour antigen-specific response.
a,b, 5 × 105 B16F10 cells were inoculated intradermally on day 0. PBS (n = 5), α-PD-1 + α-CTLA-4 (CPI, 100 μg each; n = 5), 25 μg CBD-IL-12 (IL-12 molar eq., n = 11) or CBD-IL-12 + CPI (n = 12) were administered on days 9 and 14. CBD-IL-12 was administered i.v. and CPI was administered i.p. Tumour growth curves (a) and survival curves (b) are shown. c-f, Tyr:Cre-ER+/LSL-BrafV600E/PTENfl/fl mice received 50 μg of 4-OH-tamoxifen on their back. Mice were treated with CPI (n = 6) or CBD-IL-12 + CPI (n = 7) on days 25, 30, 39 and 44 post tamoxifen application. d-f, On day 50, mice were bled for the analysis of circulating T cells. Box plots (median, min to max) for total CD8+ T cells (% of CD3+) (d), effector CD8+ T cells (% of CD8+ T cells) (e), and PD-1+ cells (% of effector CD8+ T cells) (f) are shown. g-i, Tyr:Cre-ER+/LSL-BrafV600E/PTENfl/fl/βCatSTA mice received 50 μg of 4-OH-tamoxifen on their back. Mice were treated with CPI (n = 4) or CBD-IL-12 + CPI (n = 4) on days 25, 30, 35, 40, 45 post tamoxifen application. h,i, On day 50, tumours from CPI (n = 4) and CBD-IL-12 + CPI (n = 3) were excised, fixed and stained with DAPI (blue) and anti-mouse CD8 (purple). For each tumour sample, CD8+ T cells were counted within two different fields and average was calculated (mean ± SEM). Representative images (i) are shown. Scale bars, 200 μm. Tumour curves are represented as mean ± SEM. Experiments in a,b, were performed twice, with similar results. BrafV600E/PTENfl/fl and BrafV600E/PTENfl/fl/βCatSTA experiments were performed once on distinct biological samples. Statistical analysis for a,c,d,e,g,h was done using unpaired, two-tailed t-test with Welch correction. For f, two-tailed Mann-Whitney test was applied due to nonparametric data. Statistical analysis for survival curve was done using log-rank test.
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