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. 2020 Aug;29(8):1774-1783.
doi: 10.1002/pro.3894. Epub 2020 Jun 16.

Design of a surrogate Anticalin protein directed against CD98hc for preclinical studies in mice

Friedrich-Christian Deuschle  1 , André Schiefner  1 , Corinna Brandt  1 , Arne Skerra  1
Affiliations

Design of a surrogate Anticalin protein directed against CD98hc for preclinical studies in mice

Friedrich-Christian Deuschle et al. Protein Sci. 2020 Aug.

Abstract

The human CD98 heavy chain (CD98hc) offers a promising biomedical target both for tumor therapy and for drug delivery to the brain. We have previously developed a cognate Anticalin protein with picomolar affinity and demonstrated its effectiveness in a xenograft animal model. Due to the lack of cross-reactivity with the murine ortholog, we now report the development and X-ray structural analysis of an Anticalin with high affinity toward CD98hc from mouse. This binding protein recognizes the same protruding epitope loop-despite distinct structure-in the membrane receptor ectodomain as the Anticalin selected against human CD98hc. Thus, this surrogate Anticalin should be useful for the preclinical assessment of CD98hc targeting in vivo and support the translational development for medical application in humans.

Keywords: CD98hc; cancer theranostics; lipocalin; mouse model; protein engineering.

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Conflict of interest statement

A. Skerra is founder and shareholder of Pieris Pharmaceuticals, Inc.

Figures

FIGURE 1
FIGURE 1
Biophysical characterization of the αmCD98hcED Anticalin C1B12. (a) Amino acid sequence alignment of the selected lipocalin variant C1B12 compared to wtLcn2 and to the αhCD98hc Anticalin P3D11. The central randomized gene cassette flanked by two BstXI sites is underlined; β‐strands and structurally hypervariable loops are labeled with letters A–H and numbers #1–#4, respectively. Randomized positions in the Lcn2 sequence are highlighted bold. (b) Analytical SEC profile of the mCD98hc‐specific Anticalin C1B12 in comparison with recombinant wtLcn2, revealing monodispersity and a monomeric oligomerization status. From calibration runs with protein size standards a molecular weight of 20.7 kDa was deduced for C1B12 (inset). (c) Real‐time SPR analysis of the Anticalin C1B12 versus mCD98hcED (produced in E. coli), demonstrating a picomolar dissociation constant. As expected, no binding activity of wtLcn2 toward this molecular target was detectable
FIGURE 2
FIGURE 2
Structural comparison of the Anticalin complexes with the murine and human CD98hc target proteins. (a) Contact analysis of the two Anticalins C1B12 and P3D11. Residues that interact with mCD98hc and hCD98hc, respectively, are highlighted in the amino acid sequence with the same color (yellow or pink, respectively) as used for each Anticalin in the crystal structures shown below. Stars denote the randomized positions in the wtLcn2 sequence. (b, d) Crystal structure of C1B12 (yellow) in complex with mCD98hc (light blue) (Table 1) in comparison with P3D11 (pink) in complex with hCD98hc (light green) (PDB ID: 6S8V). The two Anticalins are shown in the same orientation and the residues in both CD98hc epitopes are highlighted red (Table S3). (c, e) View of the buried molecular surfaces for C1B12·mCD98hc and P3D11·hCD98hc, respectively, after separation of the complex partners. The contacting residues are colored like the individual molecules in (b) and (d), whereas noncontacting residues are shown in light gray. Hydrogen bond as well as salt bridge donor/acceptors are highlighted blue and red, respectively. (f) Superposition of the murine and human CD98hcED target proteins reveals differences in the mode of recognition by their cognate Anticalins, C1B12 and P3D11, as evident from their relative rotation by almost 180° and the mutual shift
FIGURE 3
FIGURE 3
Effect of N‐glycosylation on the interaction between mCD98hc and C1B12. (a) mCD98hcED with modeled glycan structures and the bound Anticalin C1B12 in the crystallized complex, 23 illustrating potential interference of the oligosaccharides attached to Asn385 and, in particular, Asn399. (b, c) Close‐up views of contacts between the Asn385 (b) and Asn399 (c) side chains in the unglycosylated mCD98hcED and the bound Anticalin C1B12
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