Fc-engineered large molecules targeting blood-brain barrier transferrin receptor and CD98hc have distinct central nervous system and peripheral biodistribution

Abstract

Blood brain barrier-crossing molecules targeting transferrin receptor (TfR) and CD98 heavy chain (CD98hc) are widely reported to promote enhanced brain delivery of therapeutics. Here, we provide a comprehensive and unbiased biodistribution characterization of TfR and CD98hc antibody transport vehicles (ATV^TfR and ATV^CD98hc) compared to control IgG. Mouse whole-body tissue clearing reveals distinct organ localization for each molecule. In the brain, ATV^TfR and ATV^CD98hc achieve enhanced exposure and parenchymal distribution even when brain exposures are matched between ATV and control IgG in bulk tissue. Using a combination of cell sorting and single-cell RNAseq, we reveal that control IgG is nearly absent from parenchymal cells and is distributed primarily to brain perivascular and leptomeningeal cells. In contrast, ATV^TfR and ATV^CD98hc exhibit broad and unique parenchymal cell-type distribution. Finally, we profile in detail brain region-specific biodistribution of ATV^TfR in cynomolgus monkey brain and spinal cord. Taken together, this in-depth multiscale characterization will guide platform selection for therapeutic targets of interest.

Fig. 1. Whole body mouse fluorescence imaging reveals unique peripheral biodistribution patterns of ATV^TfR, ATV^CD98hc, and control IgG.

a Schematic of the molecules used with non-targeting control Fabs. The orange patch in ATV^TfR Fc region binds to TfR and blue patch in ATV^CD98hc binds to CD98hc. b Schematic of the experimental paradigm. TfR^mu/hu KI, CD98hc^mu/hu KI, or WT mice were dosed with 30 mg/kg AF647-conjugated control IgG and ATV^TfR for 1 day and ATV^CD98hc for 5 days. c Ventral and dorsal view 3D immunofluorescence images AF647-conjugated control IgG, ATV^TfR, or ATV^CD98hc in the whole mouse. Representative immunostaining from n = 3/group. Quantification of mean fluorescence intensity of AF647-ATV^TfR (d) or AF647-ATV^CD98hc (e) normalized to AF647-conjugated control IgG. n = 3/group, mean ± sem, two-tailed t-test with Benjamini–Hochberg false discovery rate, vertebrae adjusted p value 0.045, brain cerebrum adjusted p value 0.045, brain hippocampus adjusted p value 0.045, mean fluorescence intensity of ATV compared to control IgG. 3D reconstructed images of select peripheral organs with high ATV^TfR (f) or ATV^CD98hc (g) uptake. VNO vomeronasal organ, intra. l.g. intraorbital lacrimal gland, extra. l.g. extraorbital lacrimal gland. Representative images from n = 3/group.

Fig. 2. Enhanced brain uptake and biodistribution of ATV^TfR and ATV^CD98hc in the mouse brain and spinal cord by the whole-body fluorescence imaging.

a Representative images from AI-segmented 3D-reconstructed brains obtained from the whole-body tissue cleared mice dosed with AF647-conjugated control IgG, ATV^TfR, or ATV^CD98hc. arc. hypoth nuc arcuate hypothalamic nucleus, ventromed. hypoth nuc. ventromedial hypothalamic nucleus, median em. median eminence. Representative images from n = 3/group. b Mean fluorescence intensity (a.u.) of control IgG, ATV^TfR, and ATV^CD98hc as a function of brain depth from the cortical surface (0 μm) moving into deeper cortical tissues (1350 μm). n = 3/group, mean ± sem, two-tailed t-test with Benjamini–Hochberg false discovery rate control per depth bin, *p < 0.05, **p < 0.01, compared to control IgG, exact values provided in Source Data. Mean fluorescence intensity (a.u.) of AF647-conjugated control IgG (c), ATV^TfR (d), or ATV^CD98hc (e) across different mouse brain regions from the whole-body tissue cleared mice. n = 3/group, mean ± sem, two-tailed t-test with Benjamini–Hochberg false discovery rate control, all regions nonsignificant p value > 0.05 (mean fluorescence intensity of each brain region compared to whole brain average per treatment group). Top ten regions with highest fold change in fluorescence intensity compared to brain average fluorescence intensity for AF647-conjugated IgG (f), ATV^TfR (g), or ATV^CD98hc (h). Dotted line denotes the average brain intensity. n = 3/group, mean ± sem, two-tailed t-test with Benjamini–Hochberg false discovery rate control, subgeniculate nucleus adjusted p value 0.043, stria terminalis adjusted p value 0.024, arcuate hypothalamic nucleus adjusted p value 0.012 (mean fluorescence intensity of each brain region compared to whole brain average per treatment group). Optical slice longitudinal (i) or transverse (j) views of fluorescence images from mouse spinal cord from whole-body tissue cleared mice. Representative images from n = 3/group.

Fig. 3. ATV^TfR and ATV^CD98hc exhibit enhanced brain uptake and biodistribution compared to control IgG.

a Schematic of the experimental design. WT, TfR^mu/hu KI, and CD98hc^mu/hu KI mice were dosed with 50 mg/kg of AF647-conjugated control IgG and ATV^TfR for 1 day and ATV^CD98hc for 5 days. b Brain concentration of AF647-conjugated control IgG, ATV^TfR, or ATV^CD98hc as measured by huIgG ELISA in bulk brain lysates after a single 45 mg/kg IV dose. n = 4/ group, mean ± SEM, one-way ANOVA ****p < 0.0001 compared to control IgG, 95% confidence interval of ATV^TfR vs. control IgG −34.76 to −23.5, 95% confidence interval of ATV^CD98hc vs. control IgG −24.72 to −13.47. c Representative immunofluorescence in sagittal brain sections of AF647-conjugated control IgG, ATV^TfR, and ATV^CD98hc in mouse. Representative immunostainings from n = 2/group. d Brain lysate concentration measured by ELISA 1 day after a single 100 mg/kg IV dose of control IgG or 15 mg/kg of ATV^TfR (n = 4 mice/group), unpaired two-tailed t-test, nonsignificant (n.s.) p value > 0.05. e Immunodetection of huIgG in whole sagittal brain sections by widefield imaging. Experiment conducted once with n = 4/group. f Magnified examples of brain regions from (e) including the superior colliculus, neocortex, and thalamus. Examples of putative perivascular huIgG signal indicated by arrowheads; notably darker alpha-smooth muscle actin (αSMA) + arteriole indicated by arrows (see Supplementary Fig. 4e–g). Insets show immunodetected huIgG (magenta) and vascular marker caveolin-1 (green), scalebar 50 μm. g Ventral volume view of immunodetected huIgG in tissue-cleared whole mouse hemibrain (n = 6 mice/group). Arrowheads indicate huIgG associated with the large surface arteries, asterisk indicates the median eminence, arrow indicates cranial nerves (region of trigeminal, facial, and vestibulocochlear nerves). h Higher magnification light sheet imaging of immunodetected huIgG and vascular marker lectin around the middle cerebral artery (MCA) and anterior cerebral artery (ACA) branching from the circle of Willis. Circumferential banding pattern around putative smooth muscle cells indicated by arrowheads; arrow indicates putative perivascular profile of a penetrating vessel. For micrographs, display settings were optimized independently for each region of interest and were identical for both treatment groups.

Fig. 4. ATV^TfR and ATV^CD98hc localize to BBB vascular and CNS parenchymal cells while control IgG localizes to blood-CSF and perivascular barrier cells.

a Schematic of the experimental design: single cells obtained from dissociated brains of WT, TfR ^mu/hu KI, and CD98hc^mu/hu KI mice dosed with 45 mg/kg AF647-conjugated antibodies were sorted based on AF647 signal and then loaded onto the 10x Genomics platform for scRNA sequencing. b Representative flow cytometry plots showing the percentage of AF647-positive cells and their percent distribution across the negative, low, mid, and high AF647 intensity bins. c UMAP of cell clusters captured from the dissociated brains of mice from all three treatment groups and across all four bins. d Bar graphs represent mean percent of total huIgG distribution across indicated cell types captured per treatment group across fluorescence intensity bins. Points represent individual mice. Numbers displayed represent the total number of cells captured for the respective cell type and fluorescent intensity bin. Control IgG (n = 2/group); ATV^TfR and ATV^CD98hc (n = 4/group).

Fig. 5. ATV^TfR and ATV^CD98hc exhibit distinctly different localization to BEC subtypes compared to control IgG across the arteriovenous axis.

a–c Bar graph represent mean percent of total huIgG distribution across arterial, capillary, and venous endothelial cells captured per treatment group across fluorescence intensity bins. Points represent individual mice. Numbers displayed represent the total number of BEC subtypes captured. Control IgG (n = 2/group); ATV^TfR and ATV^CD98hc (n = 4/group).

Fig. 6. Enhanced exposure and biodistribution of ATV^TfR in cynomolgus monkey brain and spinal cord.

a Schematic of the in vivo experimental design. Immunofluorescence images of 3D reconstructed hemibrains (1x objective) (b, c), coronal slice view of hemibrain (200 μm thick, 1x objective) (d, e), and cortical slice view of temporal lobe (ventral cortex) (200 μm thick, 4x objective) (f, g) from cynomolgus monkeys dosed with 23 mg/kg AF647-conjugated control IgG or ATV^TfR (IV) for 2 days. Arrow heads in (f) highlight localization of control IgG to large penetrating vessels, while arrow heads in (g) highlight localization of ATV^TfR within large vessels as well as capillaries. Experiment conducted once with from n = 1/group. Mean fluorescence intensity of ATV^TfR and control IgG in vessels (h) and non-vessels (i) of cynomolgus monkey brains across regions of different depths from surface of the brain. j Mean fluorescence intensity of ATV^TfR and control IgG within vessels of different radii in the cynomolgus monkey brains. k Mean fluorescence intensity of ATV^TfR and control IgG across different brain regions in cynomolgus monkeys. 3D Immunofluorescence images of spinal cords from cynomolgus monkeys dosed with AF647-conjugated control IgG (l) or ATV^TfR (m). Immunostaining image from n = 1/group.