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The Thomsen-Friedenreich disaccharide as antigen for in vivo tumor targeting with multivalent scFvs

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Abstract

The Thomsen-Friedenreich disaccharide (TFα) is a promising antigen for tumor immunotargeting, since it is almost exclusively expressed on carcinoma tissues. So far, an obstacle preventing the exploitation of TF for immunotargeting has been the lack of suitable (non-IgM) antibodies with high affinity and specificity. Recently we reported on a novel strategy for generating antibodies toward small uncharged carbohydrates and the generation of recombinant antibodies toward TF. Among them, two multivalent scFv antibodies showed sub-micromolar functional affinities and appeared well suited for immunotargeting. In the present study, the trimeric scFv(1aa) and the tetrameric scFv(0aa) have been further developed for radioimmunotargeting. The scFvs were radiolabeled with 111In using DTPA as chelator without losing binding activity or molecular stoichiometry. Binding affinities as high as 1 × 10−7 M toward TF displayed on living cells were determined. Antibody biodistribution and tumor targeting efficacy were studied in TF-positive human breast cancer (ZR-75-1) bearing mice. TF was successfully targeted in vivo with tumor uptakes of ∼11 and 8% ID/g after 24 h for the trimeric and tetrameric scFv, respectively. These results validate TF as a potent antigen for tumor targeting. The biodistribution of the scFvs was comparable to that reported for IgGs. In contrast to the IgGs, the serum clearance of the scFvs was very fast, which could be an advantage in a therapeutic setting. Furthermore, kidney uptake, which is often critical for small recombinant antibodies labeled with radio-metals, was low with the tetramer (11% ID/g). We conclude that the multimeric anti-TF scFvs are promising candidates to be further developed toward therapeutic application.

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Abbreviations

aa:

Amino acid

DTPA:

Diethylenetriamine pentaacetic acid

PAA:

Polyacrylamide

RIA:

Radioimmunoassay

scFv:

Single chain fragment variable

TF:

Thomsen-Friedenreich antigen

TLC:

Thin layer chromatography

VH and VL :

Variable domain of heavy chain and light chain, respectively

% ID/g:

Per cent injected dose per gram organ

N :

Number of individual experiments

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Acknowledgments

The work was supported by the Danish Research Training Council (Forskeruddannelsesrådet), NEMOD Biotherapeutics GmbH & Co.KG, and Glycotope GmbH.

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Authors and Affiliations

  1. Glycotope GmbH, Robert-Rössle-Str. 10, 13125, Berlin-Buch, Germany

    Peter Ravn, Renate Stahn, Antje Danielczyk, Uwe Karsten & Steffen Goletz

  2. NEMOD Biotherapeutics GmbH & Co.KG, Berlin, Germany

    Peter Ravn & Dorian Faulstich

  3. Max Delbrück Center for Molecular Medicine, Berlin, Germany

    Uwe Karsten

  4. Royal Free & University College Medical School, London, UK

    Peter Ravn

Authors
  1. Peter Ravn
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  2. Renate Stahn
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  3. Antje Danielczyk
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  6. Steffen Goletz
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Correspondence to Steffen Goletz.

Electronic supplementary material

Fig7

Supplementary Fig. 1 DTPA conjugation conditions and quality analysis after conjugation. a–c Binding activity of the scFv(1aa) after incubation in three different buffers used during the DTPA conjugation at different temperatures (N = 2). d Size exclusion chromatography of the chelated versus the nonchelated scFv(1aa). e Binding activity of the chelated versus the nonchelated scFv(1aa) to asialoglycophorin

Fig8

Supplementary Fig. 2 Quality analysis of 111In labeling. a Thin layer chromatography of labeled protein. Lanes: 1, free 111In; 2, 111In-labeled unbound DTPA (111In in excess); 3, 111In-labeled scFv(1aa). b Radiography of SDS-PAGE. 111In-labeled scFv(1aa) was loaded on SDS-PAGE and visualised via radiography. c Size exclusion chromatography. 111In-labeled scFv(1aa) and an 111In-labeled control mouse IgG were examined in size exclusion chromatography

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Ravn, P., Stahn, R., Danielczyk, A. et al. The Thomsen-Friedenreich disaccharide as antigen for in vivo tumor targeting with multivalent scFvs. Cancer Immunol Immunother 56, 1345–1357 (2007). https://doi.org/10.1007/s00262-007-0292-5

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