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. 2019 Feb 19;116(8):3100-3105.
doi: 10.1073/pnas.1815087116. Epub 2019 Feb 4.

Second-generation IL-2 receptor-targeted diphtheria fusion toxin exhibits antitumor activity and synergy with anti-PD-1 in melanoma

Laurene S Cheung  1 , Juan Fu  2   3 , Pankaj Kumar  1 , Amit Kumar  1 , Michael E Urbanowski  1 , Elizabeth A Ihms  1 , Sadiya Parveen  1 , C Korin Bullen  1 , Garrett J Patrick  1 , Robert Harrison  4 , John R Murphy  5 , Drew M Pardoll  6   3 , William R Bishai  5
Affiliations

Second-generation IL-2 receptor-targeted diphtheria fusion toxin exhibits antitumor activity and synergy with anti-PD-1 in melanoma

Laurene S Cheung et al. Proc Natl Acad Sci U S A. .

Abstract

Denileukin diftitox (DAB-IL-2, Ontak) is a diphtheria-toxin-based fusion protein that depletes CD25-positive cells including regulatory T cells and has been approved for the treatment of persistent or recurrent cutaneous T cell lymphoma. However, the clinical use of denileukin diftitox was limited by vascular leak toxicity and production issues related to drug aggregation and purity. We found that a single amino acid substitution (V6A) in a motif associated with vascular leak induction yields a fully active, second-generation biologic, s-DAB-IL-2(V6A), which elicits 50-fold less human umbilical vein endothelial cell monolayer permeation and is 3. 7-fold less lethal to mice by LD50 analysis than s-DAB-IL-2. Additionally, to overcome aggregation problems, we developed a production method for the fusion toxin using Corynebacterium diphtheriae that secretes fully folded, biologically active, monomeric s-DAB-IL-2 into the culture medium. Using the poorly immunogenic mouse B16F10 melanoma model, we initiated treatment 7 days after tumor challenge and observed that, while both s-DAB-IL-2(V6A) and s-DAB-IL-2 are inhibitors of tumor growth, the capacity to treat with higher doses of s-DAB-IL-2(V6A) could provide a superior activity window. In a sequential dual-therapy study in tumors that have progressed for 10 days, both s-DAB-IL-2(V6A) and s-DAB-IL-2 given before checkpoint inhibition with anti-programmed cell death-1 (anti-PD-1) antibodies inhibited tumor growth, while either drug given as monotherapy had less effect VSports手机版. s-DAB-IL-2(V6A), a fully monomeric protein with reduced vascular leak, is a second-generation diphtheria-toxin-based fusion protein with promise as a cancer immunotherapeutic both alone and in conjunction with PD-1 blockade. .

Keywords: Tregs; cancer immunotherapy; fusion toxin. V体育安卓版.

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Conflict of interest statement

Conflict of interest statement: J. R. M. , D. M. P. , and W V体育ios版. R. B. hold positions in Sonoval, LLC, which holds rights to develop V6A.

Figures

Fig. 1.
Fig. 1.
Production of s-DAB or V6A from C. diphtheriae generates monomeric and highly purified fusion toxin. (A) Schematic of fusion toxin production from E. coli versus C. diphtheriae. (B) Supernatant harvested from fermenter-grown C7s(−)tox- secreting V6A was analyzed by Coomassie-stained SDS/PAGE gel before (lane 2) and after purification (lane 4). Lanes 1 and 3 are molecular weight markers. Arrow indicates expected size of V6A at 56 kDa. (C) Elution profile of S-100 Superdex gel permeation chromatographic analysis of s-DAB, V6A, and proteins of known molecular weight.
Fig. 2.
Fig. 2.
V6A exhibits comparable cytotoxic activity and induces less vascular permeability than s-DAB in vitro. (A) Activity of V6A and s-DAB against the CD25+ cell line MT-2 (7) in an MTS cell-killing assay showed an IC50 of 0.33 and 0.12 pM for V6A and s-DAB, respectively. (B) FITC-labeled dextran permeation assay in HUVEC cells. Cells were seeded in the upper well of a Transwell chamber and treated with V6A, s-DAB, or LPS for 19 h. The amount of FITC-dextran that permeates to the lower well was measured by fluorescence. Values represent the percentage of fluorescence of LPS-positive control-treated cells. Three independent experiments were performed in duplicate. Statistical analysis of the results was carried out using ANOVA. Error bars show SEM. ***P < 0.001 and *P < 0.05.
Fig. 3.
Fig. 3.
V6A treatment induces less toxicity than s-DAB in mice. Mice were treated daily with PBS, s-DAB, or V6A at indicated doses for 20 d (n = 5). (A) Weights of treated mice. Error bars are mean ± SEM. (B) Survival of treated mice. Statistical analysis by log-rank test. **P < 0.01.
Fig. 4.
Fig. 4.
Antitumor activity of V6A is equivalent to s-DAB and enhances the efficacy of anti–PD-1 in the B16F10 melanoma model. (A and B) C57BL/6 mice were treated with 5 μg of s-DAB or V6A on day 7 and day 10 post B16F10 inoculation. (A) Percentage of CD25+FoxP3+ Tregs of CD4+ T cells in tumor-draining lymph nodes (LN) and spleens at day 24 post B16F10 inoculation. Statistical significance was measured by one-way ANOVA (n = 6–8). (B) Tumor size was measured by electronic caliper (n = 8). Statistical significance for tumor growth experiments was assessed by repeated measures two-way ANOVA on day 18 (PBS vs. s-DAB, P < 0.001; PBS vs. V6A, P < 0.01). (C) C57BL/6 mice were treated with 10 μg V6A on day 10 and day 13 post B16F10 inoculation. Anti–PD-1 or isotype control was begun on day 11 and given twice per week for the duration of the experiment (n = 8) (isotype vs. V6A + anti–PD-1, P < 0.05). (D) C57BL/6 mice were treated with 5 μg s-DAB on days 10 and 13 post B16F10 inoculation. Anti–PD-1 or isotype control was begun on day 11 post B16F10 inoculation and given two times per week for the duration of the experiment. (isotype vs. s-DAB; P < 0.01; isotype vs. anti–PD-1, anti–PD-1 vs. s-DAB + anti–PD-1; P < 0.001; s-DAB vs. s-DAB + anti–PD-1; P < 0.05). Tumor size was measured by electronic caliper. Arrows indicate day treatments were given; green: V6A; blue: anti–PD-1; orange: s-DAB. Data are shown as mean ± SEM. Statistical significance for tumor growth experiments was assessed by repeated measures two-way ANOVA with Bonferroni posttest for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 5.
Fig. 5.
Effects of fusion toxin treatment on CD8+ lymphocytes in tumors and spleens. Mice were treated with 5 μg s-DAB or V6A on days 7 and 10 post B16F10 inoculation (n = 6–8). Tumors and spleens were harvested on day 24 and analyzed by flow cytometry. Statistical significance determined by one-way ANOVA. *P < 0.05, **P < 0.01.
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