Rise and Fall of an Anti-MUC1 Specific Antibody

Abstract

BackgroundSo 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.ResultsA 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.ConclusionsAntibody 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.

MUC1 and metastatic cancer

“…MUC1 is typically found to be expressed in greater than 60% of captured CTCs from metastatic breast, lung, pancreatic and colon cancer patients, among others. [33][34][35] The detection of MUC1 (which is also defined as serum antigens CA 15-3, KL-6 and BM7 36,37 ) in patient sera is currently used clinically as a marker of response to therapy and as a prognostic indicator for survival. 38 In fact, the serum antigen CA 15-3 is currently one of the most widely used serum antigens in breast cancer, with high CA 15-3 levels correlating with higher grade tumors, lymph node involvement, and presence of distant metastases.…”

Biomedicals from a soil bug

“…6,7 Newly introduced inducible scFv expression plasmid constructs expand the established production host range. Phage display selected human scFvs 7,8 directed against a human cancer marker protein and potential therapeutic target, Mucin1, 9,10 an "acutephase" inflammation indicator in the human blood, C-reactive protein 11 and a murine anti-hen egg-white lysozyme model antibody, D1.3 7,[12][13][14][15] were used to assess the functionality of those new constructs. A feature of the plasmids is the chassis that is based on the wellstudied RK2 broad host range plasmid which therefore should allow replication, maintenance and expression of scFvs in the majority of Gram-negative bacteria.…”

Recombinant antibody fragments allow repeated measurements of C-reactive protein with a quartz crystal microbalance immunosensor

“…6). In the first experiment, two other scFvs were used as controls: the scFv IIB6 specific for human MUC1 was described to have a low stability of less the 24 h at 37°C; 13 the second control scFv was derived from the hen egg white lysozyme specific antibody D1.3 and was chosen for its reportedly good stability. 14 The scFv LA13-IIE3 showed extraordinary good stability after prolonged incubation at 37°C and also after treatment with GuHCl.…”