doi: 10.1038/s41467-020-20220-1.
An IL-2-grafted antibody immunotherapy with potent efficacy against metastatic cancer
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- PMID:33353953
- PMCID: PMC7755894
- DOI: 10.1038/s41467-020-20220-1
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An IL-2-grafted antibody immunotherapy with potent efficacy against metastatic cancer
Dilara Sahin et al. Nat Commun..
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Abstract
Modified interleukin-2 (IL-2) formulations are being tested in cancer patients. However, IL-2 immunotherapy damages IL-2 receptor (IL-2R)-positive endothelial cells and stimulates IL-2Rα (CD25)-expressing lymphocytes that curtail anti-tumor responses. A first generation of IL-2Rβ (CD122)-biased IL-2s addressed some of these drawbacks. Here, we present a second-generation CD122-biased IL-2, developed by splitting and permanently grafting unmutated human IL-2 (hIL-2) to its antigen-binding groove on the anti-hIL-2 monoclonal antibody NARA1, thereby generating NARA1leukin. In comparison to hIL-2/NARA1 complexes, NARA1leukin shows a longer in vivo half-life, completely avoids association with CD25, and more potently stimulates CD8+ T and natural killer cells. These effects result in strong anti-tumor responses in various pre-clinical cancer models, whereby NARA1leukin consistently surpasses the efficacy of hIL-2/NARA1 complexes in controlling metastatic disease. Collectively, NARA1leukin is a CD122-biased single-molecule construct based on unmutated hIL-2 with potent efficacy against advanced malignancies.
Conflict of interest statement
O.B. is a founder and shareholder of Anaveon AG. The remaining authors declare no competing interests.
Figures
a Graphical representation of strategy followed for generation of NARA1leukin. hIL-2: helix A, green; helix B, yellow; helix C, dark orange; helix D, pink; and NARA1: heavy chain, blue; light chain, cyan.b Schematic representation of strategy followed for generation of NARA1leukin showing the engineered protein (color code as ina).
a,b Sandwich ELISA coating anti-hIL-2 mAb 5344 (a) or hCD25 (b) and detecting with anti-hIL-2 mAb MAB202 shows binding of titrated hIL-2, hIL-2/NARA1 complexes, and NARA1leukin.c,d Phosphorylated STAT5 (pSTAT5) levels of human (c) and mouse (d) immune cell subsets responding to titrated hIL-2, hIL-2/NARA1 complexes, and NARA1leukin. 1:1 and 2:1 indicate molar ratios of cytokine to antibody of hIL-2/NARA1 complexes. Dotted lines indicate mean fluorescent intensity (MFI) of pSTAT5 of each cell subset following incubation in media for 15 min. Half maximal effective concentration (EC50) of each stimulant is presented in the table below. Differences between curves were analyzed using one-way ANOVA followed by Tukey’s multiple comparison test (c,d). (c) *p = 0.0112, **p = 0.0082, ****p < 0.0001, ***p = 0.0002. (d) ****p < 0.0001, ***p = 0.0001 (hIL-2), ***p = 0.0006 (hIL-2/NARA1). Data are presented as mean ± SD of two (a,b), four (c), or three (d) independent experiments. Source data are provided as a Source Data file.
a Mice received a single injection of hIL-2 (1 µg), hIL-2/NARA1 complexes (1 µg/5 µg), or NARA1leukin (0.5 µg or 1 µg hIL-2 equivalent). Blood samples were collected at the indicated time points and the presence of hIL-2 in serum was assessed using a sandwich ELISA coating anti-hIL-2 mAb 5344 and detecting with anti-hIL-2 mAb MAB202. Data are represented as mean ± SD of two independent experiments,n = 2–6 mice/group.b Experimental scheme. Mice received a single injection (arrowhead) of hIL-2 (1.5 µg), hIL-2/NARA1 complexes (1.5 µg/15 µg), or NARA1leukin (0.5 µg hIL-2 equivalent) and were assessed at indicated time points after the injection (h: hours, d: days).c–e Spleens were analyzed by flow cytometry to determine pSTAT5, Ki67, and total cell counts of CD8+ T cells (c), CD3- NK1.1+ NK cells (d), and CD4+ CD25+ T cells (e). Differences between groups at the same time point were analyzed using two-way ANOVA followed by Tukey’s multiple comparison test (c–e). The comparisons of hIL-2/NARA1 and NARA1leukin to hIL-2 are indicated by asterisks (*), the differences between hIL-2/NARA1 and NARA1leukin are indicated by hashtags (#) on top of the columns; non-significantp-values are not indicated. (c) ****p < 0.0001,###p = 0.0002, **p = 0.0082 (d1) **p = 0.0085 (d2)#p = 0.0168. (d) ****p < 0.0001 ***p = 0.0006,##p = 0.0032 (2 h), *p = 0.0412 (pSTAT5),##p = 0.013 (d1),####p < 0.0001, *p = 0.0252 (Ki67) (e) ****p < 0.0001,####p < 0.0001, **p = 0.0016, *p = 0.0227 (d2),#p = 0.0302, *p = 0.0337 (d4). Data are presented as mean ± SD of three independent experiments,n = 5 mice/group. Source data are provided as a Source Data file.
a Treatment scheme. Mice were injected with 106 B16-F10 melanoma cells intradermally and treated with PBS, hIL-2/NARA1 complexes (0.5 µg/2.5 µg or 1.5 µg/15 µg, three times a week), or NARA1leukin (0.5 µg hIL-2 equivalent, two times a week) from day 4 to 14. Spleens, tumor-draining lymph nodes (TDLNs), and tumor-infiltrating lymphocytes (TILs) were analyzed at day 15 by flow cytometry for the indicated immune cell subsets.b Tumor growth curves of intradermal B16-F10 melanoma until day 15. ****p < 0.0001, ***p = 0.0001.c Change in tumor volumes in individual mice between days 4 and 15.d Cells counts of CD44+ CD8+ T, CD3− CD122+ NK1.1+ NK, and CD4+ CD25+ Foxp3+ Treg cells per mm3 of tumor are indicated. CD8: ***p = 0.001 (NARA1leukin), ***p = 0.0005 (hIL-2/NARA1), *p = 0.0166; NK: **p = 0.0069, ***p = 0.0003; Treg: ****p < 0.0001.e Ratios of CD44+ CD8+ T to CD4+ CD25+ Foxp3+ Treg cells for indicated organs are shown. TILs: *p = 0.0333, **p = 0.0043; TDLNs: ***p = 0.0002, *p = 0.0238 (PBS), *p = 0.0166 (hIL-2/NARA1); Spleen: ***p = 0.0004 (PBS), *p = 0.0389, ***p = 0.0001 (hIL-2/NARA1).f–i Intratumoral CD8+ T cells were analyzed by flow cytometry for CD44 versus TOX (f,g) and PD-1 versus TIM-3 (h,i). Shown are representative flow cytometry plots (f,h) and percentages (g,i) of CD8+ T cells.g **p = 0.0049, *p = 0.0171.i ****p < 0.0001, **p = 0.0011 (PBS), **p = 0.003 (●), **p = 0.0089 (▲). Data are presented as mean ± SD of four (b–e,h andi) or three (f,g) independent experiments,)n = 15 (PBS and hIL-2/NARA1 0.5 µg),n = 12 (hIL-2/NARA1 1.5 µg),n = 16 (NARA1leukin) (b),n = 13 (PBS),n = 14 (hIL-2/NARA1 0.5 µg),n = 12 (hIL-2/NARA1 1.5 µg),n = 16 (NARA1leukin) (d,e),n = 7 (PBS),n = 8 (hIL-2/NARA1 0.5 µg/1.5 µg and NARA1leukin) (g),n = 10 (PBS and hIL-2/NARA1 0.5 µg),n = 12 (hIL-2/NARA1 1.5 µg and NARA1leukin) (i) mice/group. Differences between groups at the same time point were analyzed using mixed-effects analysis with Greenhouse–Geisser correction followed by Tukey’s multiple comparison test (b). Differences in immune cell subsets between groups were analyzed using Kruskal–Wallis test followed by Dunn’s multiple comparison test (d,e, andg) or by two-way ANOVA followed by Tukey’s multiple comparison test (i). ns, not significant. Source data are provided as a Source Data file.
a Treatment scheme. Mice were injected intravascularly with 3 × 105 B16-F10 melanoma cells and treated with PBS, hIL-2/NARA1 complexes (0.5 µg/2.5 µg or 1.5 µg/15 µg, three times weekly), or NARA1leukin (0.5 µg hIL-2 equivalent, twice weekly) from day 4 to 15.b–d Representative images of the lungs (b), quantification of lung nodules (c), and percentage distributions according to severity of metastasis categorized by nodule counts (d) are shown for day 18. ****p < 0.0001, **p = 0.0456 (PBS), **p = 0.0024 (hIL-2/NARA1).e Experimental scheme. Mice were injected with 106 B16-F10 melanoma cells intradermally and treated as described in (a). On day 12 the intradermal tumor was surgically removed, and mice were observed for 35–50 days for lymph node metastasis.f Metastasis-free survival curves after surgery. Numbers indicate surviving mice out of total mice at endpoint. ***p = 0.0005, **p = 0.0041.g Experimental scheme. Mice were injected both intravascularly and intradermally with 3 × 105 and 106 B16-F10 melanoma cells, respectively. Mice were treated with PBS, hIL-2/NARA1 complexes (1.5 µg /15 µg, three times weekly), or NARA1leukin (0.5 µg hIL-2 equivalent, twice weekly) from day 4 to 14 with or without peptide vaccination given on day 4.h Tumor growth curves of intradermal B16-F10 melanoma. ****p < 0.0001, **p = 0.0087, *p = 0.0457.i,j Quantification of lung nodules (i) and percentage distribution according to severity of metastasis categorized by nodule counts (j) are shown for day 15. ****p < 0.0001, ***p = 0.0006. Data are presented as mean ± SD of three independent experiments,n = 10 (PBS, hIL-2/NARA1 0.5 µg and NARA1leukin),n = 12 (hIL-2/NARA1 1.5 µg) (b–d),n = 16 (PBS and hIL-2/NARA1 0.5 µg),n = 15 (hIL-2/NARA1 1.5 µg),n = 14 (NARA1leukin) (f),n = 9 (PBS),n = 10 (hIL-2/NARA1, NARA1leukin, and Vaccine),n = 11 (Vaccine + hIL-2/NARA1 and Vaccine + NARA1leukin) (h–j) mice/group. Differences in nodule counts between groups were analyzed using Kruskal–Wallis test followed by Dunn’s multiple comparison test (c,i). Differences in survival curves were analyzed by pairwise Mantel-Cox test (f). Differences between groups at the same time point were analyzed using two-way ANOVA followed by Tukey’s multiple comparison test (h). ns, not significant. Source data are provided as a Source Data file.
(a) Experimental scheme. Mice were intradermally injected with 106 LLC cells and treated with PBS, hIL-2/NARA1 complexes (1.5 µg/15 µg, three times weekly), or NARA1leukin (0.5 µg hIL-2 equivalent, twice weekly) from day 4 to 13. On day 14 the intradermal tumor was surgically removed, and mice were observed for 28 days. (b,c) Change in tumor volume in individual mice between day 4 and 14 (b) and tumor growth curves until day 14 (c) are shown. **p = 0.0018, *p = 0.0392. (d,e) Quantification of spontaneous lung nodules on day 28 (d), percentage distribution according to severity of metastasis categorized by nodule counts (e) are shown. ****p < 0.0001, *p = 0.0325. (f) Percentages of metastasis-free and metastasis-bearing mice at endpoint. (g) Experimental scheme. Mice were injected with 4T1 cells within mammary fat pad and treated as described in (a) until day 18. (h,i) Change in tumor volume in individual mice between day 7 and 28 (h) and tumor growth curves until day 28 (i) are shown. *p = 0.0338. (j,k) Quantification of spontaneous lung nodules on day 28 (j), percentage distribution according to severity of metastasis categorized by nodule counts (k) are shown. ****p < 0.0001, **p = 0.0079. (l) Percentage of metastasis-free and metastasis-bearing mice at endpoint. Data are presented as mean ± SD of three independent experiments (two for hIL-2/NARA1 group of 4T1),n = 12 (PBS),n = 11 (hIL-2/NARA1),n = 13 (NARA1leukin) (b–f),n = 13 (PBS),n = 7 (hIL-2/NARA1),n = 15 (NARA1leukin) (h–l) mice/group. Differences between groups at the same time point were analyzed using mixed effects analysis with Greenhouse-Geisser correction followed by Tukey’s multiple comparison test (c, i). Differences in nodule counts between groups were analyzed using Kruskal-Wallis test followed by Dunn’s multiple comparison test (d,j). ns, not significant. Source data are provided as a Source Data file.
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