Anticalins directed against vascular endothelial growth factor receptor 3 (VEGFR-3) with picomolar affinities show potential for medical therapy and in vivo imaging.

Members of the vascular endothelial growth factor receptor (VEGFR) family play a central role in angiogenesis as well as lymphangiogenesis and are crucial for tumor growth and metastasis. In particular, VEGFR-3 expression is induced in endothelial cells during tumor angiogenesis. We report the design of anticalins that specifically recognize the ligand-binding domains 1 and 2 of VEGFR-3. To this end, a library of the lipocalin 2 scaffold with 20 randomized positions distributed across its binding site was subjected to phage display selection and enzyme linked immunosorbent assay (ELISA) screening using the VEGF-C binding fragment (D1-2) or the entire extracellular region (D1-7) of VEGFR-3 as target proteins. Promising anticalin candidates were produced in Escherichia coli and biochemically characterized. Three variants with different receptor binding modes were identified, and two of them were optimized with regard to target affinity as well as folding efficiency. The resulting anticalins show dissociation constants down to the single-digit picomolar range. Specific recognition of VEGFR-3 on cells was demonstrated by immunofluorescence microscopy. Competitive binding versus VEGF-C was demonstrated for two of the anticalins with Ki values in the low nanomolar range. Based on these data, VEGFR-3 specific anticalins provide promising reagents for the diagnosis and/or therapeutic intervention of tumor-associated vessel growth.

Anticalins directed against the fibronectin extra domain B as diagnostic tracers for glioblastomas.

The standard of care for diagnosis and therapy monitoring of gliomas is magnetic resonance imaging (MRI), which however, provides only an indirect and incomplete representation of the tumor mass, offers limited information for patient stratification according to WHO-grades and may insufficiently indicate tumor relapse after antiangiogenic therapy. Anticalins are alternative binding proteins obtained via combinatorial protein design from the human lipocalin scaffold that offer novel diagnostic reagents for histology and imaging applications. Here, the Anticalins N7A, N7E and N9B, which possess exquisite specificity and affinity for oncofetal fibronectin carrying the extra domain B (ED-B), a well-known proangiogenic extracellular matrix protein, were applied for immunohistochemical studies. When investigating ED-B expression in biopsies from 41 patients with confirmed gliomas of WHO grades I to IV, or in non-neoplastic brain samples, we found that Anticalins specifically detect ED-B in primary glioblastoma multiforme (GBM; WHO IV) but not in tumors of lower histopathological grade or in tumor-free brain. In primary GBM samples, ED-B specific Anticalins locate to fibronectin-rich perivascular areas that are associated with angiogenesis. Anticalins specifically detect ED-B both in fixed tumor specimen and on vital cells, as evidenced by cytofluorometry. Beyond that, we labeled an Anticalin with the γ-emitter (123) I and demonstrated specific binding to GBM-tissue samples using in vitro autoradiography. Overall, our data indicate that ED-B specific Anticalins are useful tools for the diagnosis of primary GBM and related angiogenic sites, presenting them as promising tracers for molecular tumor imaging.

Automated synthesis of [18F]Ga-rhPSMA-7/ -7.3: results, quality control and experience from more than 200 routine productions.

INTRODUCTION: The radiohybrid (rh) prostate-specific membrane antigen (PSMA)-targeted ligand [18F]Ga-rhPSMA-7 has previously been clinically assessed and demonstrated promising results for PET-imaging of prostate cancer. The ligand is present as a mixture of four stereoisomers ([18F]Ga-rhPSMA-7.1, - 7.2, - 7.3 and - 7.4) and after a preclinical isomer selection process, [18F]Ga-rhPSMA-7.3 has entered formal clinical trials. Here we report on the establishment of a fully automated production process for large-scale production of [18F]Ga-rhPSMA-7/ -7.3 under GMP conditions (EudraLex). METHODS: [18F]Fluoride in highly enriched [18O]H2O was retained on a strong anion exchange cartridge, rinsed with anhydrous acetonitrile and subsequently eluted with a solution of [K+ ⊂ 2.2.2]OH- in anhydrous acetonitrile into a reactor containing Ga-rhPSMA ligand and oxalic acid in DMSO. 18F-for-19F isotopic exchange at the Silicon-Fluoride Acceptor (SiFA) was performed at room temperature, followed by dilution with buffer and cartridge-based purification. Optimum process parameters were determined on the laboratory scale and thereafter implemented into an automated synthesis. Data for radiochemical yield (RCY), purity and quality control were analyzed for 243 clinical productions (160 for [18F]Ga-rhPSMA-7; 83 for [18F]Ga-rhPSMA-7.3). RESULTS: The automated production of [18F]Ga-rhPSMA-7 and the single isomer [18F]Ga-rhPSMA-7.3 is completed in approx. 16 min with an average RCY of 49.2 ± 8.6% and an excellent reliability of 98.8%. Based on the different starting activities (range: 31-130 GBq, 89 ± 14 GBq) an average molar activity of 291 ± 62 GBq/µmol (range: 50-450 GBq/µmol) was reached for labeling of 150 nmol (231 µg) precursor. Radiochemical purity, as measured by radio-high performance liquid chromatography and radio-thin layer chromatography, was 99.9 ± 0.2% and 97.8 ± 1.0%, respectively. CONCLUSION: This investigation demonstrates that 18F-for-19F isotopic exchange is well suited for the fast, efficient and reliable automated routine production of 18F-labeled PSMA-targeted ligands. Due to its simplicity, speed and robustness the development of further SiFA-based radiopharmaceuticals is highly promising and can be of far-reaching importance for future theranostic concepts.

Synthesis and Preclinical Evaluation of a 68Ga-Labeled Adnectin, 68Ga-BMS-986192, as a PET Agent for Imaging PD-L1 Expression.

Blocking the interaction of the immune checkpoint molecule programmed cell death protein-1 and its ligand, PD-L1, using specific antibodies has been a major breakthrough for immune oncology. Whole-body PD-L1 expression PET imaging may potentially allow for a better prediction of response to programmed cell death protein-1-targeted therapies. Imaging of PD-L1 expression is feasible by PET with the adnectin protein 18F-BMS-986192. However, radiofluorination of proteins such as BMS-986192 remains complex and labeling yields are low. The goal of this study was therefore the development and preclinical evaluation of a 68Ga-labeled adnectin protein (68Ga-BMS-986192) to facilitate clinical trials. Methods:68Ga labeling of DOTA-conjugated adnectin (BXA-206362) was performed in NaOAc-buffer at pH 5.5 (50°C, 15 min). In vitro stability in human serum at 37°C was analyzed using radio-thin layer chromatography and radio-high-performance liquid chromatography. PD-L1 binding assays were performed using the transduced PD-L1-expressing lymphoma cell line U-698-M and wild-type U-698-M cells as a negative control. Immunohistochemical staining studies, biodistribution studies, and small-animal PET studies of 68Ga-BMS-986192 were performed using PD-L1-positive and PD-L1-negative U-698-M-bearing NSG mice. Results:68Ga-BMS-986192 was obtained with quantitative radiochemical yields of more than 97% and with high radiochemical purity. In vitro stability in human serum was at least 95% after 4 h of incubation. High and specific binding of 68Ga-BMS-986192 to human PD-L1-expressing cancer cells was confirmed, which closely correlates with the respective PD-L1 expression level determined by flow cytometry and immunohistochemistry staining. In vivo, 68Ga-BMS-986192 uptake was high at 1 h after injection in PD-L1-positive tumors (9.0 ± 2.1 percentage injected dose [%ID]/g) and kidneys (56.9 ± 9.2 %ID/g), with negligible uptake in other tissues. PD-L1-negative tumors demonstrated only background uptake of radioactivity (0.6 ± 0.1 %ID/g). Coinjection of an excess of unlabeled adnectin reduced tumor uptake of PD-L1 by more than 80%. Conclusion:68Ga-BMS-986192 enables easy radiosynthesis and shows excellent in vitro and in vivo PD-L1-targeting characteristics. The high tumor uptake combined with low background accumulation at early imaging time points demonstrates the feasibility of 68Ga-BMS-986192 for imaging of PD-L1 expression in tumors and is encouraging for further clinical applications of PD-L1 ligands.

First In-Human Medical Imaging with a PASylated 89Zr-Labeled Anti-HER2 Fab-Fragment in a Patient with Metastatic Breast Cancer.

PURPOSE: PASylation® offers the ability to systematically tune and optimize the pharmacokinetics of protein tracers for molecular imaging. Here we report the first clinical translation of a PASylated Fab fragment (89Zr∙Df-HER2-Fab-PAS200) for the molecular imaging of tumor-related HER2 expression. METHODS: A patient with HER2-positive metastatic breast cancer received 37 MBq of 89Zr∙Df-HER2-Fab-PAS200 at a total mass dose of 70 µg. PET/CT was carried out 6, 24, and 45 h after injection, followed by image analysis of biodistribution, normal organ uptake, and lesion targeting. RESULTS: Images show a biodistribution typical for protein tracers, characterized by a prominent blood pool 6 h p.i., which decreased over time. Lesions were detectable as early as 24 h p.i. 89Zr∙Df-HER2-Fab-PAS200 was tolerated well. CONCLUSION: This study demonstrates that a PASylated Fab tracer shows appropriate blood clearance to allow sensitive visualization of small tumor lesions in a clinical setting.

Can the Injected Dose Be Reduced in 68Ga-PSMA-11 PET/CT While Maintaining High Image Quality for Lesion Detection?

Our purpose was to define a clinically useful lower limit of injected dose for 68Ga-prostate-specific membrane antigen (PSMA)-11 PET/CT imaging of prostate cancer. Methods:68Ga-PSMA-11 PET/CT was performed on 11 patients. PET was acquired in list mode and reconstructed using a 3-min full acquisition, a 2-min acquisition, and a 1-min acquisition to generate images obtained with three thirds (standard dose), two thirds (low dose), and one third (very low dose) of the injected dose, respectively. Overall image quality (5-point scale) was assessed, and the detectability of PSMA-positive lesions was determined by 3 readers and compared with the reference standard. Results: Image quality declined with decreasing dose (mean score of 4.1 ± 0.4 for the standard dose, 3.4 ± 0.7 for the low dose, and 1.9 ± 0.4 for the very low dose; all P < 0.05). Readers 1, 2, and 3 correctly identified the lesions (n = 21) at a rate of 100%, 100%, and 95% with the standard dose; 95%, 81%, and 90% with the low dose; and 71%, 76%, and 59% with the very low dose, respectively. Conclusion:68Ga-PSMA-11 dose reduction is not feasible without a negative impact on image quality and lesion detectability.

FluoroCalins: engineered lipocalins with novel binding functions fused to a fluorescent protein for applications in biomolecular imaging and detection.

FluoroCalins represent novel bifunctional protein reagents derived from engineered lipocalins fused to a fluorescent reporter protein, here the enhanced green fluorescent protein (eGFP). We demonstrate the construction, facile bacterial production and broad applicability of FluoroCalins using two Anticalin® molecules directed against the tumor vasculature-associated extra domain B of fibronectin (ED-B) and the vascular endothelial growth factor receptor 3, a marker of tumor and lymphangiogenesis. FluoroCalins were prepared with two different spacers: (i) a short Ser3Ala linker and (ii) a long hydrophilic and conformationally unstructured PASylation® polypeptide comprising 200 Pro, Ala and Ser residues. These FluoroCalins were applied for direct target quantification in enzyme-linked immunosorbent assay as well as target detection by flow cytometry and fluorescence microscopy of live and fixed cells, respectively, demonstrating high specificity and signal-to-noise ratio. Hence, FluoroCalins offer a promising alternative to antibody-based reagents for state of the art fluorescent in vitro detection and biomolecular imaging.

Molecular Imaging of Fibroblast Activity After Myocardial Infarction Using a 68Ga-Labeled Fibroblast Activation Protein Inhibitor, FAPI-04.

Heart failure remains a major source of late morbidity and mortality after myocardial infarction (MI). The temporospatial presence of activated fibroblasts in the injured myocardium predicts the quality of cardiac remodeling after MI. Therefore, monitoring of activated fibroblasts is of great interest for studying cardiac remodeling after MI. Fibroblast activation protein (FAP) expression is upregulated in activated fibroblasts. This study investigated the feasibility of imaging activated fibroblasts with a new 68Ga-labeled FAP inhibitor (68Ga-FAPI-04) for PET imaging of fibroblast activation in a preclinical model of MI. Methods: MI and sham-operated rats were scanned with 68Ga-FAPI-04 PET/CT (1, 3, 6, 14, 23, and 30 d after MI) and with 18F-FDG (3 d after MI). Dynamic 68Ga-FAPI-04 PET and blocking studies were performed on MI rats 7 d after coronary ligation. After in vivo scans, the animals were euthanized and their hearts harvested for ex vivo analyses. Cryosections were prepared for autoradiography, hematoxylin and eosin (H&E), and immunofluorescence staining. Results:68Ga-FAPI-04 uptake in the injured myocardium peaked on day 6 after coronary ligation. The tracer accumulated intensely in the MI territory, as identified by decreased 18F-FDG uptake and confirmed by PET/MR and H&E staining. Autoradiography and H&E staining of cross-sections revealed that 68Ga-FAPI-04 accumulated mainly at the border zone of the infarcted myocardium. In contrast, there was only minimal uptake in the infarct of the blocked rats, comparable to the uptake in the remote noninfarcted myocardium (PET image-derived ratio of infarct uptake to remote uptake: 6 ± 2). Immunofluorescence staining confirmed the presence of FAP-positive myofibroblasts in the injured myocardium. Morphometric analysis of the whole-heart sections demonstrated 3- and 8-fold higher FAP-positive fibroblast density in the border zone than in the infarct center and remote area, respectively. Conclusion:68Ga-FAPI-04 represents a promising radiotracer for in vivo imaging of post-MI fibroblast activation. Noninvasive imaging of activated fibroblasts may have significant diagnostic and prognostic value, which could aid clinical management of patients after MI.

Anticalins Reveal High Plasticity in the Mode of Complex Formation with a Common Tumor Antigen.

We describe the comparative X-ray structural analysis of three Anticalin proteins directed against the extra-domain B (ED-B) of oncofetal fibronectin (Fn), a validated marker of tumor neoangiogenesis. The Anticalins were engineered from the human lipocalin 2 (Lcn2) scaffold via targeted randomization of the structurally variable loop region and selection by phage display, resulting in 15-19 exchanged residues. While the four reshaped loops exhibit diverse conformations (with shifts in Cα positions up to 20.4 Å), the ß-barrel core of the lipocalin remains strongly conserved, thus confirming the extraordinary robustness of this scaffold. All three Anticalins bind the cc' hairpin loop of ED-B, the most exposed motif in the context of its neighboring Fn domains, but reveal entirely different binding modes, with orientations differing by up to 180°. Hence, each Anticalin recognizes its molecular target in an individual manner, in line with the distinct epitope specificities previously seen in binding experiments.

ANTICALIgN: visualizing, editing and analyzing combined nucleotide and amino acid sequence alignments for combinatorial protein engineering.

ANTIC ALIGN: is an interactive software developed to simultaneously visualize, analyze and modify alignments of DNA and/or protein sequences that arise during combinatorial protein engineering, design and selection. ANTIC ALIGN: combines powerful functions known from currently available sequence analysis tools with unique features for protein engineering, in particular the possibility to display and manipulate nucleotide sequences and their translated amino acid sequences at the same time. ANTIC ALIGN: offers both template-based multiple sequence alignment (MSA), using the unmutated protein as reference, and conventional global alignment, to compare sequences that share an evolutionary relationship. The application of similarity-based clustering algorithms facilitates the identification of duplicates or of conserved sequence features among a set of selected clones. Imported nucleotide sequences from DNA sequence analysis are automatically translated into the corresponding amino acid sequences and displayed, offering numerous options for selecting reading frames, highlighting of sequence features and graphical layout of the MSA. The MSA complexity can be reduced by hiding the conserved nucleotide and/or amino acid residues, thus putting emphasis on the relevant mutated positions. ANTIC ALIGN: is also able to handle suppressed stop codons or even to incorporate non-natural amino acids into a coding sequence. We demonstrate crucial functions of ANTIC ALIGN: in an example of Anticalins selected from a lipocalin random library against the fibronectin extradomain B (ED-B), an established marker of tumor vasculature. Apart from engineered protein scaffolds, ANTIC ALIGN: provides a powerful tool in the area of antibody engineering and for directed enzyme evolution.

Selection and characterization of Anticalins targeting human prostate-specific membrane antigen (PSMA).

Although prostate carcinoma (PCa) is by far the most commonly diagnosed neoplasia in men, corresponding diagnostic and therapeutic modalities have limited efficacy at present. Anticalins comprise a novel class of binding proteins based on a non-immunoglobulin scaffold that can be engineered to specifically address molecular targets of interest. Here we report the selection and characterization of Anticalins that recognize human prostate-specific membrane antigen (PSMA), a membrane-tethered metallopeptidase constituting a disease-related target for imaging and therapy of PCa as well as solid malignancies in general. We used a randomized lipocalin library based on the human lipocalin 2 (Lcn2) scaffold together with phage display and ELISA screening to select PSMA-specific variants. Five Anticalin candidates from the original panning were expressed in Escherichia coli as soluble monomeric proteins, revealing affinities toward PSMA down to the low nanomolar range. Binding characteristics of the most promising candidate were further improved via affinity maturation by applying error-prone PCR followed by selection via phage display as well as bacterial surface display under more stringent conditions. In BIAcore measurements, the dissociation constant of the best Anticalin was determined as ∼500 pM, with a substantially improved dissociation rate compared with the first-generation candidate. Finally, immunofluorescence microscopy revealed specific staining of PSMA-positive tumor cell lines while flow cytometric analysis confirmed the ability of the selected Anticalins to detect PSMA on live cells. Taken together, Anticalins resulting from this study offer a viable alternative to antibody-based PSMA binders for biomedical applications, including in vivo imaging of PCa or neovasculature of solid tumors.

Inefficient boosting of antitumor CD8(+) T cells by dendritic-cell vaccines is rescued by restricting T-cell cytotoxic functions.

Dendritic cells (DCs) are powerful activators of primary and secondary immune responses and have promising activity as anticancer vaccines. However, various populations of immune cells, including natural killer cells, regulatory T cells and especially cytotoxic T lymphocytes (CTLs), can inhibit DC function through cytotoxic clearance. Spontaneous tumor-specific CTL responses are frequently observed in patients before immunotherapy, and it is unclear how such pre-existing responses may affect DC vaccines. We used an adoptive transfer model to show that DC vaccination fail to induce the expansion of pre-existing CTLs or increase their production of interferon γ (IFNγ). The expansion and effector differentiation of naïve host CD8(+) T cells was also suppressed in the presence of CTLs of the same specificity. Suppression was caused by the cytotoxic functions of the adoptively transferred CTLs, as perforin-deficient CTLs could respond to DC vaccination by expanding and increasing IFNγ production. Proliferation and effector differentiation of host CD8(+) T cells as well as resistance to tumor challenge were also significantly increased. Expression of perforin by antitumor CTLs was critical in regulating the survival of vaccine DCs, while FAS/FASL and TRAIL/DR5 had a significant, but comparatively smaller, effect. We conclude that perforin-expressing CTLs can suppress the activity of DC-based vaccines and prevent the expansion of naïve and memory CD8(+) T cells as well as antitumor immune responses. We suggest that, paradoxically, temporarily blocking the cytotoxic functions of CTLs at the time of DC vaccination should result in improved vaccine efficiency and enhanced antitumor immunity.