A Site-Specific MiniAp4-Trastuzumab Conjugate Prevents Brain Metastasis

Abstract

Monoclonal antibodies (mAbs) are changing cancer treatments. However, the presence of the blood-brain barrier (BBB) and the blood-tumor barrier (BTB) limits the use of mAbs to treat brain cancer or brain metastasis. Molecules that hijack endogenous transport mechanisms on the brain endothelium (brain shuttles) have been shown to increase the transport of large molecules and nanoparticles across the BBB. Among these shuttles, protease-resistant peptides such as MiniAp-4 are particularly efficient. Here, we report the synthesis, characterization, and evaluation of site-specific mAb-brainshuttle antibody conjugates (ASC) based on the anti-HER2 mAb trastuzumab (Tz) and four molecules of MiniAp-4. The ASCs preserve the binding and cell cycle arrest capacity of Tz. MiniAp-4 ASC displays enhanced transport across an in vitro BBB cellular model with respect to Tz and Tz conjugated to Angiopep-2, the brain shuttle that has advanced the most in clinical trials. More importantly, evaluation of Tz-MiniAp4 in a murine brain metastasis model demonstrated that the protease-resistant peptide showed preferential transport across the BBB/BTB, displaying a marked therapeutic effect and protecting against metastasis development. The technology described herein could be applied to any antibody of interest to treat central nervous system-related diseases. MiniAp-4 enhances the brain transport of the monoclonal antibody trastuzumab, preventing brain metastasis.

Keywords: brain metastasis; brain shuttle peptide; trastuzumab.

Figure 1

(a) ASC synthetic scheme. (b) Mass characterization of Tz-Ang2 (top) and Tz-MiniAp4 (bottom) by LCT-Premier. Deconvoluted spectra are shown. Mcal for Tz-Ang2 = 159,244; Mfound: 79,537, 159,396; Mcal for Tz-MiniAp4= 153,684; Mfound: 76,918, 153,678.

Figure 2

Binding of Tz, Tz-Ang2, or Tz-MiniAp4 to HER2-overexpressing cells: (a) BT-474; (b) SK-BR-3. Error bars represent the standard deviation (n = 3). Cell cycle arrest analysis of Tz-, Tz-Ang2-, or Tz-MiniAp4-treated cells: (c) SK-BR-3, (d) BT-474, and (e) MDA-MB-231. Error bars represent the standard deviation (n = 3). **p < 0.005; ***p < 0.0005.

Figure 3

(a) Permeability of Tz, Tz-Ang2, and Tz-MiniAp4 (100 nM) in the human in vitro BBB cellular model. (b) Brain concentration of Tz, Tz-Ang2, and Tz-MiniAp4 after i.v. bolus injection. Results are expressed in terms of nmol/g of tissue. Error bars represent the standard deviation (n = 3). (c) Brain-to-plasma ratio. Results are expressed in terms of brain/serum ratio for mAbs. In all graphs, error bars represent the standard deviation (n = 3). P value was calculated using one-way ANOVA test. ****p < 0.001; *<0.05.

Figure 4

BBB-shuttle peptides allow for higher brain accumulation of Tz in metastatic mouse brains. (a) Schema of experimental design. Upon intracardial injection of the brain metastatic H2030-BrM cancer cell line expressing GFP and luciferase, the indicated compounds, labeled with NIR antibodies, were administered systemically and mice were killed 4 h later. (b) Representative ex vivo images of bioluminescence (cancer cells) and fluorescence (antibodies) in the brains of inoculated mice. (c) Three anterior-posterior levels of the cerebral cortex were analyzed by histology to measure fluorescence derived from antibody accumulation in areas affected by metastases. (d) Representative low and high magnifications of metastatic cells (green, GFP) and the various therapeutic antibodies (red, NIR antibodies). Blue, bis-benzamide. Scale bars: 100 μm, low magnification; 50 μm, high magnification.

Figure 5

(a) Schema of the experimental design. (b) Representative images of brains obtained from mice at the end point of the experiment showing their bioluminescence signal. (c) Quantification of ex vivo bioluminescent images (BLI) of brains at the end point of the experiment (vehicle n = 7; Tz n = 16; Tz-MiniAp4 n = 16 mice per experimental condition). (d) Representative images of thorax obtained from mice at the end point of the experiment showing their bioluminescence signal. (e) Quantification of ex vivo BLI of thoracic regions at the end point of the experiment. (f) Representative sections of brains from vehicle-, Tz-, and Tz-Miniap4-treated mice. Scale bar, 1000 μm. (g) Quantification of the number of established metastases found in vehicle-, Tz-, and Tz-Miniap4-treated brains (vehicle n = 4; Tz n = 5; Tz-MiniAp4 n = 4 mice per experimental condition). (h) Representative images showing established metastases with different sizes (big, >50,000 μm²; medium, 10,000–50,000 μm²; and small, <10,000 μm²) at the end point of the experiment. Scale bars, 200 μm. (i) Quantification of the number of established metastases according to the size (big, medium, and small) in each experimental condition (vehicle, Tz, Tz-MiniAp4). In vehicle slices (n = 4), 3.4% were big, 27.6% were medium, and 69% were small. In Tz slices (n = 5), 2% were big, 28% were medium, and 70% were small. In Tz-MiniAp4-slices (n = 4), 0% were big, 11.5% were medium, and 88.5% were small. (j) Quantification of the accumulative area of established metastases found in vehicle-, Tz-, and Tz-Miniap4-treated brains (vehicle n = 4; Tz n = 5; Tz-MiniAp4 n = 4 mice per experimental condition). P value was calculated using two-tailed t test (vehicle vs Tz p = 0.0213; vehicle vs Tz-MiniAp4 p = 0.02; Tz vs Tz-MiniAp4 p = 0.0315).