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Rise and fall of an anti-MUC1 specific antibody.

Thie H, Toleikis L, Li J, von Wasielewski R, Bastert G, Schirrmann T, Esteves IT, Behrens CK, Fournes B, Fournier N, de Romeuf C, Hust M, Dübel S - PLoS ONE (2011)

Bottom Line: Half-life in serum was improved from below 1 day to more than 4 weeks and was correlated with the dimerisation tendency of the individual scFvs.However, the experiments showed no significant decrease in tumour growth or increase in the survival rates.Despite these "best in class" binding parameters, the therapeutic success of this antibody was prevented by the target biology.

View Article: PubMed Central - PubMed

Affiliation: Technische Universität Braunschweig, Institut für Biochemie und Biotechnologie, Braunschweig, Germany.

ABSTRACT

Background: So far, human antibodies with good affinity and specificity for MUC1, a transmembrane protein overexpressed on breast cancers and ovarian carcinomas, and thus a promising target for therapy, were very difficult to generate.

Results: A human scFv antibody was isolated from an immune library derived from breast cancer patients immunised with MUC1. The anti-MUC1 scFv reacted with tumour cells in more than 80% of 228 tissue sections of mamma carcinoma samples, while showing very low reactivity with a large panel of non-tumour tissues. By mutagenesis and phage display, affinity of scFvs was increased up to 500fold to 5,7×10(-10) M. Half-life in serum was improved from below 1 day to more than 4 weeks and was correlated with the dimerisation tendency of the individual scFvs. The scFv bound to T47D and MCF-7 mammalian cancer cell lines were recloned into the scFv-Fc and IgG format resulting in decrease of affinity of one binder. The IgG variants with the highest affinity were tested in mouse xenograft models using MCF-7 and OVCAR tumour cells. However, the experiments showed no significant decrease in tumour growth or increase in the survival rates. To study the reasons for the failure of the xenograft experiments, ADCC was analysed in vitro using MCF-7 and OVCAR3 target cells, revealing a low ADCC, possibly due to internalisation, as detected for MCF-7 cells.

Conclusions: Antibody phage display starting with immune libraries and followed by affinity maturation is a powerful strategy to generate high affinity human antibodies to difficult targets, in this case shown by the creation of a highly specific antibody with subnanomolar affinity to a very small epitope consisting of four amino acids. Despite these "best in class" binding parameters, the therapeutic success of this antibody was prevented by the target biology.

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Related in: MedlinePlus

Size exclusion chromatography analysis to analyse the dimerisation tendency of the anti-MUC1 scFvs.80 µg purified scFv fragments were separated on a Superdex200 10/300 column using PBS as running buffer with a flow rate of 0.5 mL min−1. The UV-absorption (A280) was drawn against the retention volume.
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pone-0015921-g009: Size exclusion chromatography analysis to analyse the dimerisation tendency of the anti-MUC1 scFvs.80 µg purified scFv fragments were separated on a Superdex200 10/300 column using PBS as running buffer with a flow rate of 0.5 mL min−1. The UV-absorption (A280) was drawn against the retention volume.

Mentions: To evaluate the tendency of the scFv fragments to form dimers, immobilized metal ion affinity chromatography (IMAC) purified scFvs were analysed by SEC (Figure 9). The original IIB6 and HT220-M-G8 showed a strong tendency to form dimers with approximately 50% of the scFvs being dimerised. The other scFvs showed a strongly reduced dimerisation tendency.


Rise and fall of an anti-MUC1 specific antibody.

Thie H, Toleikis L, Li J, von Wasielewski R, Bastert G, Schirrmann T, Esteves IT, Behrens CK, Fournes B, Fournier N, de Romeuf C, Hust M, Dübel S - PLoS ONE (2011)

Size exclusion chromatography analysis to analyse the dimerisation tendency of the anti-MUC1 scFvs.80 µg purified scFv fragments were separated on a Superdex200 10/300 column using PBS as running buffer with a flow rate of 0.5 mL min−1. The UV-absorption (A280) was drawn against the retention volume.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3021526&req=5

pone-0015921-g009: Size exclusion chromatography analysis to analyse the dimerisation tendency of the anti-MUC1 scFvs.80 µg purified scFv fragments were separated on a Superdex200 10/300 column using PBS as running buffer with a flow rate of 0.5 mL min−1. The UV-absorption (A280) was drawn against the retention volume.
Mentions: To evaluate the tendency of the scFv fragments to form dimers, immobilized metal ion affinity chromatography (IMAC) purified scFvs were analysed by SEC (Figure 9). The original IIB6 and HT220-M-G8 showed a strong tendency to form dimers with approximately 50% of the scFvs being dimerised. The other scFvs showed a strongly reduced dimerisation tendency.

Bottom Line: Half-life in serum was improved from below 1 day to more than 4 weeks and was correlated with the dimerisation tendency of the individual scFvs.However, the experiments showed no significant decrease in tumour growth or increase in the survival rates.Despite these "best in class" binding parameters, the therapeutic success of this antibody was prevented by the target biology.

View Article: PubMed Central - PubMed

Affiliation: Technische Universität Braunschweig, Institut für Biochemie und Biotechnologie, Braunschweig, Germany.

ABSTRACT

Background: So far, human antibodies with good affinity and specificity for MUC1, a transmembrane protein overexpressed on breast cancers and ovarian carcinomas, and thus a promising target for therapy, were very difficult to generate.

Results: A human scFv antibody was isolated from an immune library derived from breast cancer patients immunised with MUC1. The anti-MUC1 scFv reacted with tumour cells in more than 80% of 228 tissue sections of mamma carcinoma samples, while showing very low reactivity with a large panel of non-tumour tissues. By mutagenesis and phage display, affinity of scFvs was increased up to 500fold to 5,7×10(-10) M. Half-life in serum was improved from below 1 day to more than 4 weeks and was correlated with the dimerisation tendency of the individual scFvs. The scFv bound to T47D and MCF-7 mammalian cancer cell lines were recloned into the scFv-Fc and IgG format resulting in decrease of affinity of one binder. The IgG variants with the highest affinity were tested in mouse xenograft models using MCF-7 and OVCAR tumour cells. However, the experiments showed no significant decrease in tumour growth or increase in the survival rates. To study the reasons for the failure of the xenograft experiments, ADCC was analysed in vitro using MCF-7 and OVCAR3 target cells, revealing a low ADCC, possibly due to internalisation, as detected for MCF-7 cells.

Conclusions: Antibody phage display starting with immune libraries and followed by affinity maturation is a powerful strategy to generate high affinity human antibodies to difficult targets, in this case shown by the creation of a highly specific antibody with subnanomolar affinity to a very small epitope consisting of four amino acids. Despite these "best in class" binding parameters, the therapeutic success of this antibody was prevented by the target biology.

Show MeSH
Related in: MedlinePlus