Molecular Design of 68Ga- and 89Zr-Labeled Anticalin Radioligands for PET-Imaging of PSMA-Positive Tumors.

Anticalin proteins directed against the prostate-specific membrane antigen (PSMA), optionally having tailored plasma half-life using PASylation technology, show promise as radioligands for PET-imaging of xenograft tumors in mice. To investigate their suitability, the short-circulating unmodified Anticalin was labeled with 68Ga (τ1/2 = 68 min), using the NODAGA chelator, whereas the half-life extended PASylated Anticalin was labeled with 89Zr (τ1/2 = 78 h), using either the linear chelator deferoxamine (Dfo) or a cyclic derivative, fusarinine C (FsC). Different PSMA targeting Anticalin versions (optionally carrying the PASylation sequence) were produced carrying a single exposed N- or C-terminal Cys residue and site-specifically conjugated with the different radiochelators via maleimide chemistry. These protein conjugates were labeled with radioisotopes having distinct physical half-lives and, subsequently, applied for PET-imaging of subcutaneous LNCaP xenograft tumors in CB17 SCID mice. Uptake of the protein tracers into tumor versus healthy tissues was assessed by segmentation of PET data as well as biodistribution analyses. PET-imaging with both the 68Ga-labeled plain Anticalin and the 89Zr-labeled PASylated Anticalin allowed clear delineation of the xenograft tumor. The radioligand A3A5.1-PAS(200)-FsC·89Zr, having an extended plasma half-life, led to a higher tumor uptake 24 h p.i. compared to the 68Ga·NODAGA-Anticalin imaged 60 min p.i. (2.5% ID/g vs 1.2% ID/g). Pronounced demetallation was observed for the 89Zr·Dfo-labeled PASylated Anticalin, which was ∼50% lower in the case of the cyclic radiochelator FsC (p < 0.0001). Adjusting the plasma half-life of Anticalin radioligands using PASylation technology is a viable approach to increase radioisotope accumulation within the tumor. Furthermore, 89Zr-ImmunoPET-imaging using the FsC radiochelator is superior to that using Dfo. Our strategy for the half-life adjustment of a tumor-targeting Anticalin to match the physical half-life of the applied radioisotope illustrates the potential of small binding proteins as an alternative to antibodies for PET-imaging.

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

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.

Development of a high affinity Anticalin® directed against human CD98hc for theranostic applications.

Enhanced amino acid supply and dysregulated integrin signaling constitute two hallmarks of cancer and are pivotal for metastatic transformation of cells. In line with its function at the crossroads of both processes, overexpression of CD98hc is clinically observed in various cancer malignancies, thus rendering it a promising tumor target. Methods: We describe the development of Anticalin proteins based on the lipocalin 2 (Lcn2) scaffold against the human CD98hc ectodomain (hCD98hcED) using directed evolution and protein design. X-ray structural analysis was performed to identify the epitope recognized by the lead Anticalin candidate. The Anticalin - with a tuned plasma half-life using PASylation® technology - was labeled with 89Zr and investigated by positron emission tomography (PET) of CD98-positive tumor xenograft mice. Results: The Anticalin P3D11 binds CD98hc with picomolar affinity and recognizes a protruding loop structure surrounded by several glycosylation sites within the solvent exposed membrane-distal part of the hCD98hcED. In vitro studies revealed specific binding activity of the Anticalin towards various CD98hc-expressing human tumor cell lines, suggesting broader applicability in cancer research. PET/CT imaging of mice bearing human prostate carcinoma xenografts using the optimized and 89Zr-labeled Anticalin demonstrated strong and specific tracer accumulation (8.6 ± 1.1 %ID/g) as well as a favorable tumor-to-blood ratio of 11.8. Conclusion: Our findings provide a first proof of concept to exploit CD98hc for non-invasive biomedical imaging. The novel Anticalin-based αhCD98hc radiopharmaceutical constitutes a promising tool for preclinical and, potentially, clinical applications in oncology.