Generation and evaluation of antibody agents for molecular imaging of CD44v6-expressing cancers

Haylock, Anna-Karin; Nilvebrant, Johan; Mortensen, Anja C.; Velikyan, Irina; Nestor, Marika; Falk, Ronny

doi:10.18632/oncotarget.17996

Cited by 9 publications

(9 citation statements)

References 40 publications

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“…The higher CD44v6-density on HCT116 cells was in line with results from LigandTracer analyses, where a clear signal was detected from HCT116 cells, whereas the signal from UM-SCC-74B cells was below the detection limit of the LigandTracer instrument. This supports previous studies assessing the CD44v6-antigen expression of these cell lines, verifying that CD44v6-antigen density on HCT116 and UM-SCC-74B cells can be considered moderate and low, respectively [27][28][29].…”

Section: Discussionsupporting

confidence: 91%

Enhancing the therapeutic effects of in vitro targeted radionuclide therapy of 3D multicellular tumor spheroids using the novel stapled MDM2/X-p53 antagonist PM2

et al. 2020

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Background: Precision therapeutics continuously make advances in cancer therapy, and a field of growing interest is the combination of targeted radionuclide therapy (TRNT) with potential radiosensitizing agents. This study evaluated whether the effects of in vitro TRNT, using the 177 Lu-labeled anti-CD44v6 antibody AbN44v6, were potentiated by the novel stapled MDM2/X-p53 antagonist PM2. Materials and methods: Two wt p53 cell lines, HCT116 (colorectal carcinoma) and UM-SCC-74B (head and neck squamous cell carcinoma), expressing different levels of the target antigen, CD44v6, were used. Antigen-specific binding of 177 Lu-AbN44v6 was initially verified in a 2D cell assay, after which the potential effects of unlabeled AbN44v6 on downstream phosphorylation of Erk1/2 were evaluated by western blotting. Further, the therapeutic effects of unlabeled AbN44v6, 177 Lu-AbN44v6, PM2, or a combination (labeled/unlabeled AbN44v6 +/− PM2) were assessed in 3D multicellular tumor spheroid assays. Results: Radiolabeled antibody bound specifically to CD44v6 on both cell lines. Unlabeled AbN44v6 binding did not induce downstream phosphorylation of Erk1/2 at any of the concentrations tested, and repeated treatments with the unlabeled antibody did not result in any spheroid growth inhibition. 177 Lu-AbN44v6 impaired spheroid growth in a dose-dependent and antigen-dependent manner. A single modality treatment with 20 μM of PM2 significantly impaired spheroid growth in both spheroid models. Furthermore, the combination of TRNT and PM2based therapy proved significantly more potent than either monotherapy. In HCT116 spheroids, this resulted in a two-and threefold spheroid growth rate decrease for the combination of PM2 and 100 kBq 177 Lu-AbN44v6 compared to monotherapies 14-day post treatment. In UM-SCC-74B spheroids, the combination therapy resulted in a reduction in spheroid size compared to the initial spheroid size 10-day post treatment. Conclusion: TRNT using 177 Lu-AbN44v6 proved efficient in stalling spheroid growth in a dose-dependent and antigen-dependent manner, and PM2 treatment demonstrated a growth inhibitory effect as a monotherapy. Moreover, by combining TRNT with PM2-based therapy, therapeutic effects of TRNT were potentiated in a 3D multicellular tumor spheroid model. This proof-of-concept study exemplifies the strength and possibility of combining TRNT targeting CD44v6 with PM2-based therapy.

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Section: Discussionsupporting

confidence: 91%

Enhancing the therapeutic effects of in vitro targeted radionuclide therapy of 3D multicellular tumor spheroids using the novel stapled MDM2/X-p53 antagonist PM2

et al. 2020

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“…What is more, the single chain antibody showed a very good tumor-to-tissue ratio since it was shown to accumulate within the neoplastic pancreas and the metastatic liver and no relevant fluorescent signal was detected in the heart. The tumor-to-organ ratios we obtained are compatible with what reported with other scFvs used for in vivo imaging applications [ 31 – 33 ] and better compared to whole antibody molecules [ 34 ].…”

Section: Discussionsupporting

confidence: 86%

Generation and characterization of novel recombinant anti-hERG1 scFv antibodies for cancer molecular imaging

Duranti

Carraresi²,

Sette

et al. 2018

Oncotarget

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Modern molecular imaging techniques have greatly improved tumor detection and post-treatment follow-up of cancer patients. In this context, antibody-based imaging is rapidly becoming the gold standard, since it combines the unique specificity of antibodies with the sensitivity of the different imaging technologies. The aim of this study was to generate and characterize antibodies in single chain Fragment variable (scFv) format directed to an emerging cancer biomarker, the human ether-à-go-go-related gene-1 (hERG1) potassium channel, and to obtain a proof of concept for their potential use for in vivo molecular imaging.The anti-hERG1scFv was generated from a full length monoclonal antibody and then mutagenized, substituting a Phenylalanine residue in the third framework of the VH domain with a Cysteine residue. The resulting scFv-hERG1-Cys showed much higher stability and protein yield, increased affinity and more advantageous binding kinetics, compared to the “native” anti-hERG1scFv. The scFv-hERG1-Cys was hence chosen and characterized: it showed a good binding to the native hERG1 antigen expressed on cells, was stable in serum and displayed a fast pharmacokinetic profile once injected intravenously in nude mice. The calculated half-life was 3.1 hours and no general toxicity or cardiac toxic effects were detected. Finally, the in vivo distribution of an Alexa Fluor 750 conjugated scFv-hERG1-Cys was evaluated both in healthy and tumor-bearing nude mice, showing a good tumor-to-organ ratio, ideal for visualizing hERG1-expressing tumor masses in vivo.In conclusion, the scFv-hERG1-Cys possesses features which make it a suitable tool for application in cancer molecular imaging.

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“…Recombinant Fab-fragment AbD15179 was generated using a peptide that overlaps with the epitopes of U36 and BIWA 1 [ 7 ]. CD44v6-specific scFvs recognize an epitope in the same region (Haylock et al [ 8 ]). It is noticeable that murine CD44v6 has a low homology with the human sequence in the region where common antibody epitopes overlap, which is indicated in the alignment of a 14-residue region from the two species.…”

Section: Figurementioning

confidence: 99%

“…More recently we have generated a panel of scFvs that target an epitope that overlaps with Fab AbD15179 (Haylock et al [ 8 ], Figure 2 ). CD44v6-specific scFvs were selected by phage display with negative selection on a CD44v6-negative isoform of CD44, which represented more ubiquitously expressed CD44 isoforms.…”

Section: Introductionmentioning

confidence: 99%

“…The smaller size of scFvs versus, for example, F(ab′) 2 combined with high affinity monovalent binding yielded advantageous tumor to organ ratios already at 24 h p.i. (Haylock et al [ 8 ]), which is half the time required to reach similar ratios for the F(ab′) 2 fragment [ 92 ]. For imaging, the contrast between tumor and surrounding tissue is more important than the total tumor uptake and a high affinity is generally advantageous for radioimmunodiagnostic applications [ 25 ].…”

Section: Introductionmentioning

confidence: 99%

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CD44v6-Targeted Imaging of Head and Neck Squamous Cell Carcinoma: Antibody-Based Approaches

Spiegelberg

Nilvebrant

2017

Contrast Media & Molecular Imaging

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Head and neck squamous cell carcinoma (HNSCC) is a common and severe cancer with low survival rate in advanced stages. Noninvasive imaging of prognostic and therapeutic biomarkers could provide valuable information for planning and monitoring of the different therapy options. Thus, there is a major interest in development of new tracers towards cancer-specific molecular targets to improve diagnostic imaging and treatment. CD44v6, an oncogenic variant of the cell surface molecule CD44, is a promising molecular target since it exhibits a unique expression pattern in HNSCC and is associated with drug- and radio-resistance. In this review we summarize results from preclinical and clinical investigations of radiolabeled anti-CD44v6 antibody-based tracers: full-length antibodies, Fab, F(ab′)2 fragments, and scFvs with particular focus on the engineering of various antibody formats and choice of radiolabel for the use as molecular imaging agents in HNSCC. We conclude that the current evidence points to CD44v6 imaging being a promising approach for providing more specific and sensitive diagnostic tools, leading to customized treatment decisions and functional diagnosis. Improved imaging tools hold promise to enable more effective treatment for head and neck cancer patients.

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Generation and evaluation of antibody agents for molecular imaging of CD44v6-expressing cancers

Cited by 9 publications

References 40 publications

Enhancing the therapeutic effects of in vitro targeted radionuclide therapy of 3D multicellular tumor spheroids using the novel stapled MDM2/X-p53 antagonist PM2

Enhancing the therapeutic effects of in vitro targeted radionuclide therapy of 3D multicellular tumor spheroids using the novel stapled MDM2/X-p53 antagonist PM2

Generation and characterization of novel recombinant anti-hERG1 scFv antibodies for cancer molecular imaging

CD44v6-Targeted Imaging of Head and Neck Squamous Cell Carcinoma: Antibody-Based Approaches

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