CD98hc is a target for brain delivery of biotherapeutics

Abstract

Brain exposure of systemically administered biotherapeutics is highly restricted by the blood-brain barrier (BBB). Here, we report the engineering and characterization of a BBB transport vehicle targeting the CD98 heavy chain (CD98hc or SLC3A2) of heterodimeric amino acid transporters (TV^CD98hc). The pharmacokinetic and biodistribution properties of a CD98hc antibody transport vehicle (ATV^CD98hc) are assessed in humanized CD98hc knock-in mice and cynomolgus monkeys. Compared to most existing BBB platforms targeting the transferrin receptor, peripherally administered ATV^CD98hc demonstrates differentiated brain delivery with markedly slower and more prolonged kinetic properties. Specific biodistribution profiles within the brain parenchyma can be modulated by introducing Fc mutations on ATV^CD98hc that impact FcγR engagement, changing the valency of CD98hc binding, and by altering the extent of target engagement with Fabs. Our study establishes TV^CD98hc as a modular brain delivery platform with favorable kinetic, biodistribution, and safety properties distinct from previously reported BBB platforms.

Fig. 1. Transport vehicles engineered at an Fc β-sheet surface bind with a range of affinities to CD98hc.

a Cartoon of the ATV^CD98hc bound to the CD98hc-LAT1 complex with the relative position of the membrane highlighted (dotted line). b Outline of the steps used to identify and engineer TVs. Positions in the naïve and maturation libraries are modeled onto the human IgG1 Fc domain (PDB ID: 1HZH) with positions randomized in the original library (orange), randomized expanded positions (green), unaltered original positions (tan), and unaltered peripheral positions (gray) highlighted. An example of one of ten patch libraries is provided. c Representative TV sequences from each stage are shown with corresponding affinities to human and cynomolgus CD98hc of the ATV^CD98hc:DNP clone, as measured by surface-plasmon resonance. d A compilation of affinities of ATV^CD98hc:DNP variants to human and cynomolgus CD98hc across rounds of maturation. e Immunocytochemistry detection of huIgG 1 h after incubation of ATV^CD98hc:DNP (62.5 nM), anti-CD98hc (62.5 nM) and anti-DNP (500 nM) to human (HeLa), cynomolgus CD98hc-expressing cells (CHO: cynomolgus CD98hc) and parental cells (CHO). Representative images are shown from at least 15 images per well, n = 2 independent experiments. Scale bars = 50 µm. Source data are provided as a Source Data file.

Fig. 2. Crystal structure of the TV6.6 and human CD98hc co-complex provides molecular details of the interaction.

a Model of Fc with TV6.6 (red and blue) in complex with two copies of CD98hc (tan); PDB ID: 8G0M b Zoom in of the TV6.6 surface with engineered residues (blue sticks) and wild-type positions (gray sticks) that contact CD98hc. Surface model of CD98hc indicating residues in contact with the TV (orange) and surface exposed positions that differ between human and cynomolgus (pink). c, d Model of monovalent (c) and bivalent (d) TV binding to the CD98hc-LAT1 complex (PDB ID: 6JMQ) on the cell surface.

Fig. 3. Monovalent and bivalent ATV^CD98hc.6.8:DNP have prolonged brain exposure after a single dose in CD98^mu/hu KI mice.

a Cartoons of monovalent and bivalent ATV^CD98hc6.8:DNP, with (EF−) and without (EF+) mutations to mitigate FcγR binding. b–c Brain and plasma concentrations of ATV^CD98hc6.8:DNP variants (170 nM K_D) after a single intravenous (IV) 50 mg/kg dose in CD98hc^mu/hu KI mice. Plasma clearance values were 19.6–21.9 mL/d/kg and 43–49 mL/d/kg for monoATV^CD98hc.6.8:DNP and biATV^CD98hc.6.8:DNP, respectively, compared to 7.1 and 8.3 mL/d/kg for mono- or biATV^CD98hc.6.8:DNP in WT mice (Supplementary Table 1). All ATV^CD98hc variants had higher plasma clearance and brain exposure compared to the anti-DNP control, and biATV^CD98hc6.8:DNP variants had higher plasma clearance and brain exposure compared monoATV^CD98hc6.8:DNP variants. FcγR binding did not impact plasma clearance and was associated with a small increase in brain exposure. d–e Concentrations of huIgG in brain parenchymal (d), vascular fractions (e), and cell-associated and non-cell associated fractions (f) obtained by capillary depletion at 7 days post dose. There were increased concentrations of all ATV^CD98hc TV variants in the parenchymal and vascular fractions. Consistent with whole-brain exposure, biATV^CD98hc6.8:DNP EF+ had the highest concentration in the parenchyma and biATV^CD98hc6.8:DNP variants were found at higher concentrations in the vascular fraction. biATV^CD98hc6.8:DNP variants were also more cell associated than monoATV^CD98hc6.8:DNP variants. b–e Two-way ANOVA. f One-way ANOVA. b–f Graphs display mean ± SEM, n = 4–5, see Source Data for exact n/group. *p < 0.05, ****p < 0.0001. See Supplementary Table 6 for exact p values. g Immunohistochemical localization of ATV^CD98hc6.8:DNP variants in brain cortical sections as assessed with anti-huIgG across timepoints. Scale bars = 15 µm. Representative image shown from n = 5/group, n = 2 stained sections per animal. Source data are provided as a Source Data file.

Fig. 4. Biodistribution of ATV^CD98hc6.8:DNP variants in the brain is dependent on valency and FcγR binding in CD98hc^mu/hu KI mice.

a Immunohistochemical cell-specific localization of ATV^CD98hc6.8:DNP variants (170 nM K_D) in brain cortical sections with antibodies against huIgG (purple) and AQP4 (a, green, scale bars = 15 µm) or Iba1 (b, green, scale bars = 20 µm). Overlays are shown with colocalization pseudocolored in white. Representative image shown from n = 5/group, n = 2 stained sections per animal.

Fig. 5. Pharmacokinetics and distribution of ATV^CD98hc are distinct from ATV^TfR.

a, b PK of ATV^CD98hc.6.8:DNP (170 nM K_D) and ATV^TfR.35.23.4:DNP (620 nM K_D) in plasma (a) and brain (b) following a single 50 mg/kg IV dose. Plasma clearances for both ATVs were increased relative to anti-DNP (5.3 mL/d/kg), and plasma clearance for ATV^TfR.35.23.4:DNP (39.0 mL/d/kg) was increased compared to ATV^CD98hc.6.8:DNP (17.7 mL/d/kg). Both ATV^CD98hc.6.8:DNP and ATV^TfR.35.23.4:DNP had increased brain exposure compared to control and had different exposure profiles compared to each other. c, d PK of mono- and biATV^CD98hc6.39:BACE1 (94 nM K_D) in plasma (c) and brain (d) following a single 50 mg/kg IV dose. Both mono- and biATV^CD98hc6.39:BACE1 had higher plasma clearance compared to control, and biATV^CD98hc6.39:BACE1 (35.9 mL/d/kg) had higher plasma clearance compared to monovalent (19.9 mL/d/kg). Compare to the plasma clearance measured for mono- and biATV^CD98hc.6.8:DNP (19.6 and 43.9 mL/d/kg, respectively Fig. 3b). Both mono- and biATV^CD98hc6.39:BACE1 had increased brain exposure compared to control, and were not significantly different from each other. a–d Graphs display mean ± SEM, n = 5/group. Two-way ANOVA. Data points not shown were <LLOD. *p < 0.05, ****p < 0.0001. See Supplementary Table 6 for exact p values. e Immunohistochemical localization of huIgG for mono- and biATV^CD98hc.6.8:BACE1 (purple) in cortical brain sections, LAMP2 (green), and NeuN (yellow) 7 days after a single 50 mg/kg IV dose. Scale bars = 7 µm. Overlays are shown with colocalization of huIgG and Lamp2 within NeuN positive neurons is pseudocolored in white. Representative images are shown from n = 5 animals/treatment group, n = 2 IHC sections per animal. f, g Simulated brain uptake, clearance, and total net flux for mono- and biATV^CD98hc:DNP (f) and ATV^CD98hc: BACE1(g). Simulations are performed for a 50 mg/kg IV dose of ATV^CD98hc, CD98hc K_D = 170 nM. Source data are provided as a Source Data file.

Fig. 6. Multidose of ATV^CD98hc6.8:DNP variants in CD98hc^mu/hu KI mice accumulate in brain without impacting circulating cells.

a, c Plasma PK of mono- and biATV^CD98hc6.8:DNP (170 nM K_D) with (EF−) and without (EF+) mutations to mitigate FcγR binding after 5 weekly IV doses (50 mg/kg). Plasma concentrations were assessed at 30 min after each dose (C_max) and 24 h before the next dose (C_trough). Two-way ANOVA. b, d Concentrations of ATV^CD98hc:DNP variants in brain 24 h after the final dose. One-way ANOVA ****p < 0.0001. See Supplementary Table 6 for exact p values. e–j Circulating cell counts for monocytes (e, h), lymphocytes (f, i) and reticulocytes (g, j) as measured by complete blood count 24 h after the final dose. Cell numbers compared with a one-way ANOVA. a–j All graphs display n = 4–5/group (see Source Data for exact n/group), mean ± SEM. Source data are provided as a Source Data file.

Fig. 7. ATV^CD98hc uptake is enhanced in brain and localize to astrocytes and microglia in cynomolgus monkeys.

a, b Plasma and brain exposure of mono- and biATV^CD98hc6.29:DNP (205 nM K_D to cyno CD98hc) EF+ and monoATV^CD98hc6.29:DNP EF- in cynomolgus monkeys 4 days after a single 30 mg/kg IV dose. Graphs display mean ± SEM (for n = 2 or 3 as noted in figure) c, d Localization of ATV^CD98hc6.29:DNP variations in cortical brain sections assessed by IHC with antibodies against for huIgG (purple) and AQP4 (c, green) or Iba1 (d, green). Scale bars = 30 µm. Overlays are shown with colocalization pseudocolored in white. Representative image shown from n = 3 animals/treatment group (except for biATV^CD98hc.6.29:DNP, where one animal was excluded), n = 3 stained sections per animal. Source data are provided as a Source Data file.