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. 2024 Jan 24;25(3):1406.
doi: 10.3390/ijms25031406.

GT-00AxIL15, a Novel Tumor-Targeted IL-15-Based Immunocytokine for the Treatment of TA-MUC1-Positive Solid Tumors: Preclinical In Vitro and In Vivo Pharmacodynamics and Biodistribution Studies

Johanna Gellert  1 , Anika Jäkel  1 , Antje Danielczyk  1 , Christoph Goletz  1 , Timo Lischke  1 , Anke Flechner  1 , Laura Dix  1 , Alexandra Günzl  2 , Patrik Kehler  1
Affiliations

GT-00AxIL15, a Novel Tumor-Targeted IL-15-Based Immunocytokine for the Treatment of TA-MUC1-Positive Solid Tumors: Preclinical In Vitro and In Vivo Pharmacodynamics and Biodistribution Studies

Johanna Gellert et al. Int J Mol Sci. .

Abstract

GT-00AxIL15 is a novel interleukin-15-based immunocytokine targeting a tumor-specific, glycosylated epitope of MUC1 (TA-MUC1). We characterized mode of action, pharmacokinetic (PK) and pharmacodynamic (PD) properties and investigated the relevance of TA-MUC1 binding for the concept of delivering IL-15 to solid tumors. In vitro pharmacology was analyzed in binding and cell-based assays. The in vivo PK profile and IL-15-mediated PD effects of GT-00AxIL15 were investigated in tumor-free mice. Tumor accumulation, immune infiltration and anti-tumor activity were assessed in TA-MUC1+ syngeneic and xenogeneic murine tumor models. GT-00AxIL15 was shown to specifically bind TA-MUC1 on tumor cells via its mAb moiety, to IL-15 receptors on immune cells via its IL-15 fusion modules and to FcγRs via its functional Fc-part. In vitro, NK, NKT and CD8+ T cells were activated and proliferated, leading to anti-tumor cytotoxicity and synergism with antibody-dependent cellular cytotoxicity (ADCC)-mediating mAbs. In vivo, GT-00AxIL15 exhibited favorable PK characteristics with a serum half-life of 13 days and specifically accumulated in TA-MUC1+ tumors. In the tumor microenvironment, GT-00AxIL15 induced robust immune activation and expansion and mediated anti-metastatic and anti-tumor effects in syngeneic and xenograft tumor models. These results support the rationale to improve PK and anti-tumor efficacy of IL-15 by increasing local concentrations at the tumor site via conjugation to a TA-MUC1 binding mAb. The tumor-selective expression pattern of TA-MUC1, powerful immune activation and anti-tumor cytotoxicity, long serum half-life and tumor targeting properties, render GT-00AxIL15 a promising candidate for treatment of solid tumors with high medical need, e.g., ovarian, lung and breast cancer.

Keywords: TA-MUC1; anti-tumor cytotoxicity; cancer; immunocytokine; immunotherapy; interleukin-15; monoclonal antibody.

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Conflict of interest statement

All authors except for A.G. are current or former employees of Glycotope. A.D. is a shareholder of Glycotope. A.G. was paid as consultant by Glycotope.

Figures

Figure 1
Figure 1
GT-00AxIL15 binds to its targets TA-MUC1 and IL-15R on human tumor cell lines and immune cells. (A) Structure of GT-00AxIL15: Fc- (dark grey) and Fab- (light grey) part of the parental mAb GT-00A, human IL-15 (dark red), N54Q mutation to remove N-glycosylation site in the Fab domain, Fc N-glycosylation site N297, K447A mutation at transition sequence to IL-15 modules to remove potential protease site; red line depicts 6xPAPAP linker between IL-15 and the heavy chain. (B) Binding to cellular TA-MUC1 on human MCF-7 and ZR-75-1 tumor cell lines. (C) Binding to cellular IL-15R on murine CTLL-2 cell line. (D) Binding of AF647-labeled GT-00AxIL15 to human NK, NKT, CD4+ and CD8+ T cells of a representative PBMC donor. Mean ± SD (n = 2) of representative experiments.
Figure 2
Figure 2
GT-00AxIL15 induces proliferation of human NK cell line and primary PBMCs and activates human immune effector cells. (A) IL-15 bioactivity assay: Proliferation of KHYG-1 cells after 48 h treatment with test antibodies as determined by viability assay; mean ± SD (n = 3) from a representative experiment. (B) Proliferation and (C) activation of primary human immune cell subsets determined by flow cytometry. Mean ± SD (n = 2) from representative experiments.
Figure 3
Figure 3
GT-00AxIL15 shows enhanced anti-tumor cytotoxicity compared to untargeted IL-15 variants and parental anti-TA-MUC1 mAb. Europium release assays using TA-MUC1+ cell lines as target cells in the presence of primary human PBMCs as effector cells to determine anti-tumor cytotoxicity (mean ± SD; n = 3 from representative experiments) with test items titrated at fixed E:T ratio (80:1) in ZR-75-1 cells (A) or titration of E:T ratio at fixed test item concentration (20 nM) in ZR-75-1 (left), MCF-7 (mid) and T-47D (right) tumor cells (B).
Figure 4
Figure 4
GT-00AxIL15 has a prolonged half-life and induces activation and expansion of NK, NKT and CD8+ effector T cells in tumor-free mice. (A) Mouse PK profiles after single i.v. administration of 2 mg/kg GT-00AxIL15 or equivalent dose of rhIL-15 (0.29 mg/kg). (B,C) PD effects in spleen after single i.v. injection of 1 mg/kg GT-00AxIL15: Relative proportions of (B) Ki-67+ cells in NK, NKT, CD8+, Foxp3-CD4+ and Foxp3+CD4+ T cells after d3 and (C) ratio of CD8+ T effector cells to CD4+Foxp3+ regulatory T cells. (D,E) Dose-dependency of PD effects on peripheral blood NK (D) and CD8+ T cells (E) as determined by relative expansion (left), Ki-67 (middle) and GzmB (right) expression after single doses of up to 2.5 mg/kg GT-00AxIL15. Mean ± SEM (n = 3 mice).
Figure 5
Figure 5
GT00AxIL15 shows improved tumor accumulation compared to an untargeted immunocytokine. (A) Biodistribution in respective organs and tissues (in %ID/g) at final timepoint (72 h) after i.v. injection of 89Zr-GT-00AxIL15 and 89Zr-MOPCxIL15 into hMUC1-B16.F10 tumor-bearing mice. (B) Tumor accumulation and (C) Tumor-to-Blood ratio at 72 h; One-Way ANOVA, * p < 0.05, ** p < 0.01, Mean ± SEM from n = 5 mice.
Figure 6
Figure 6
GT-00AxIL15 shows in vivo efficacy in syngeneic hMUC1-B16.F10 tumor models. (A) Metastasis model (i.v.): mice were treated with vehicle, 0.1 mg/kg or 0.5 mg/kg GT-00AxIL15 i.v. on d1 and 8. On d15, organs were collected and analyzed; percentage of organs containing metastases relative to vehicle group (left) and luciferase activity in lung (mean ± SEM, n = 12) (right). (B) Solid tumor model (mfp): TV until day 20 (mean ± SEM, n = 12), and corresponding spider plots after treatment with vehicle, 0.5 mg/kg GT-00AxIL15, 200 µg/mouse anti-PD-L1 antibody, or combination thereof on d1, 8 and 15 (dotted vertical lines); 2-way ANOVA, Bonferroni post-test, *** p < 0.001. (C,D) Proliferation and expansion of tumor-infiltrating NK and CD8+ T cells in solid tumor model (mfp): Mice with established tumors (~130 mm3) were treated once with vehicle, 0.1 mg/kg or 0.5 mg/kg GT-00AxIL15 i.v.; Percentage of Ki-67+ cells and absolute counts of NK cells (C) and CD8+ T cells (D) in tumors after d3; Mean ± SEM from n = 7–8 mice, 1-way ANOVA * p < 0.05; *** p < 0.001.
Figure 7
Figure 7
GT-00AxIL15 shows in vivo efficacy in humanized DU-145 prostate cancer model (challenge setting). DU-145 tumor cells and PBMC from 5 donors were mixed and implanted in NCG mice. Tumor volume (mean ± SEM, n = 3–4) after i.v. treatment with GT-00AxIL15 or vehicle on d1, 8 and 15 (dotted vertical lines); 2-way ANOVA, Bonferroni post-test, * p < 0.05, ** p < 0.01, *** p < 0.001.
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