A Fab of trastuzumab to treat HER2 overexpressing breast cancer brain metastases

Abstract

Despite major therapeutic advances for two decades, including the most recently approved anti-HER2 drugs, brain metastatic localizations remain the major cause of death for women with metastatic HER2 breast cancer. The main reason is the limited drug passage of the blood-brain barrier after intravenous injection and the significant efflux of drugs, including monoclocal antibodies, after administration into the cerebrospinal fluid. We hypothesized that this efflux was linked to the presence of a FcRn receptor in the blood-brain barrier. To overcome this efflux, we engineered two Fab fragments of trastuzumab, an anti-HER2 monoclonal antibody, and did a thorough preclinical development for therapeutic translational purpose. We demonstrated the safety and equal efficacy of the Fabs with trastuzumab in vitro, and in vivo using a patient-derived xenograft model of HER2 overexpressing breast cancer. For the pharmacokinetic studies of intra-cerebrospinal fluid administration, we implemented original rat models with catheter implanted into the cisterna magna. After intraventricular administration in rats, we demonstrated that the brain-to-blood efflux of Fab was up to 10 times lower than for trastuzumab, associated with a two-fold higher brain penetration compared to trastuzumab. This Fab, capable of significantly reducing brain-to-blood efflux and enhancing brain penetration after intra-cerebrospinal fluid injection, could thus be a new and original effective drug in the treatment of HER2 breast cancer brain metastases, which will be demonstrated by a phase I clinical trial dedicated to women in resort situations.

Keywords: Brain metastases; Fab; HER2 breast cancer; Pharmacokinetic; Trastuzumab.

Fig. 1

In vitro and vivo anti-tumor and toxic effects of anti-HER2 Fab compared to trastuzumab. (A) SDS-PAGE with the anti-HER2 Fab#1 under reducing conditions with the light and heavy chains are identified at ~ 25 kDa (left panel), or under non-reducing conditions with the total Fab fragment identified at ~ 40-45 kDa (right panel). (B) Affinity test using fluorescence on HER2-overexpressing BT-474 breast cancer cells (left panel) and triple negative MDA-231 breast cancer cells as control (right panel). Cell nuclei are stained in blue (DAPI). Anti-HER2 Fab#2 (top panel) and trastuzumab (bottom panel) are coupled with Alexa Fluor 488 fluorophore (green). (C) In vivo antitumor effect of trastuzumab, anti-HER2 Fab#1 and anti-HER2 Fab#2 after intravenous administration (N = 20 for each antibody). Tumor growth is expressed in percentage of the tumor volume at day 0 (D0) corresponding to the first day of treatment (black arrow), *P < 0.001. (D-E-F) Histological studies of tumors analyzed at Day 21 in untreated mice as control group, mice treated with trastuzumab or with anti-HER2 Fab#2. Proliferation index is assessed with the percentage of Ki67-expressing cancer cells using immunostaining (D), microvessel density with the number of CD31-expressing vessels at high power field (hpf) using immunostaining (E), and necrosis (in yellow) with the percentage of delineated necrotic area on tissue sections (F). ns: not significant, * P < 0.001

Fig. 2

Pharmacokinetic studies after intra-CSF administration of anti-HER2 antibodies in rats. (A-B) Pharmacokinetic study with a total dose of 36.5 µg administered for each antibody (N = 4 rats for trastuzumab, N = 6 rats for Fab#2), in the CSF (A) and in the serum (B). (C-D) Pharmacokinetic study with a total dose of 1400 µg administered for each antibody (N = 4 rats for trastuzumab, N = 3 rats for Fab#2), in the CSF (C) and in the serum (D). (E) CSF-to-blood efflux for trastuzumab and anti-HER2 Fab#2 obtained on pooled data (N = 8 rats for trastuzumab and 9 rats for Fab#2). (F) Schematic of the population pharmacokinetic model used to describe concentrations of each antibody after administration in the cisterna magna. V1: volume of distribution of the CSF compartment, V2: volume of distribution of the serum compartment, k₁₀: diffusion constant from CSF to brain; k₁₂: diffusion constant from CSF to blood; k₂₀: elimination from serum constant. (G) Brain concentration assessment of anti-HER2 Fab#2 and trastuzumab from 0 to 4 h after intra-CSF administration. Kp_{u, ubrain0−4 h} = AUC_u,_{brain0−4 h} /AUC_u,_csf0−4 h. (H) Schematic dissection of rat brains. CE1 represents deeper brain area, including basal ganglia. CE2 represents posterior area including cerebellum and brainstem. CE3 represents cortical area