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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

A Antigen ELISA using anti-MUC1 scFv-Fc fusion proteins and anti-MUC1 IgGs.50 µg MUC1 peptide antigen (32mer cys) were immobilised in each well. Antibodies were incubated in different concentrations. A goat-anti-human-IgG (Fc spec.) HRP conjugate (1∶10,000) was used for detection of bound anti-MUC1 antibodies. B Flow cytometry analysis of anti-MUC1 IgG and scFv-Fc antibodies titrated on T47D. Antibodies were detected with goat anti-human IgG Alexa488 conjungate (1∶200) (Invitrogen, Darmstadt, Germany). 10000 cells were analysed per run. MFI was plotted against the antibody concentration.
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pone-0015921-g010: A Antigen ELISA using anti-MUC1 scFv-Fc fusion proteins and anti-MUC1 IgGs.50 µg MUC1 peptide antigen (32mer cys) were immobilised in each well. Antibodies were incubated in different concentrations. A goat-anti-human-IgG (Fc spec.) HRP conjugate (1∶10,000) was used for detection of bound anti-MUC1 antibodies. B Flow cytometry analysis of anti-MUC1 IgG and scFv-Fc antibodies titrated on T47D. Antibodies were detected with goat anti-human IgG Alexa488 conjungate (1∶200) (Invitrogen, Darmstadt, Germany). 10000 cells were analysed per run. MFI was plotted against the antibody concentration.

Mentions: These antibodies and the hHMFG1 IgG control were compared by titration ELISA using the 32 amino acid MUC1 peptide (figure 10A). The hHMFG1 showed weaker binding to MUC1 when compared to the three HT186 antibodies. The scFv-Fc variants bound slightly better than the IgG variants of HT186-D11 and HT186-B7. In case of HT186-G2, the recloning into the IgG format led to an affinity decrease of about 10fold. The binding to MUC1 positive cells was analysed by FACS (figure 10B). Here, all IgG varants bound weaker than their scFv-Fc analog with HT186-D11 showing the best binding to T47D cells, and huHMFG1 (only analysed as IgG) showing the lowest binding to MUC1.


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)

A Antigen ELISA using anti-MUC1 scFv-Fc fusion proteins and anti-MUC1 IgGs.50 µg MUC1 peptide antigen (32mer cys) were immobilised in each well. Antibodies were incubated in different concentrations. A goat-anti-human-IgG (Fc spec.) HRP conjugate (1∶10,000) was used for detection of bound anti-MUC1 antibodies. B Flow cytometry analysis of anti-MUC1 IgG and scFv-Fc antibodies titrated on T47D. Antibodies were detected with goat anti-human IgG Alexa488 conjungate (1∶200) (Invitrogen, Darmstadt, Germany). 10000 cells were analysed per run. MFI was plotted against the antibody concentration.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3021526&req=5

pone-0015921-g010: A Antigen ELISA using anti-MUC1 scFv-Fc fusion proteins and anti-MUC1 IgGs.50 µg MUC1 peptide antigen (32mer cys) were immobilised in each well. Antibodies were incubated in different concentrations. A goat-anti-human-IgG (Fc spec.) HRP conjugate (1∶10,000) was used for detection of bound anti-MUC1 antibodies. B Flow cytometry analysis of anti-MUC1 IgG and scFv-Fc antibodies titrated on T47D. Antibodies were detected with goat anti-human IgG Alexa488 conjungate (1∶200) (Invitrogen, Darmstadt, Germany). 10000 cells were analysed per run. MFI was plotted against the antibody concentration.
Mentions: These antibodies and the hHMFG1 IgG control were compared by titration ELISA using the 32 amino acid MUC1 peptide (figure 10A). The hHMFG1 showed weaker binding to MUC1 when compared to the three HT186 antibodies. The scFv-Fc variants bound slightly better than the IgG variants of HT186-D11 and HT186-B7. In case of HT186-G2, the recloning into the IgG format led to an affinity decrease of about 10fold. The binding to MUC1 positive cells was analysed by FACS (figure 10B). Here, all IgG varants bound weaker than their scFv-Fc analog with HT186-D11 showing the best binding to T47D cells, and huHMFG1 (only analysed as IgG) showing the lowest binding to MUC1.

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