Rise and Fall of an Anti-MUC1 Specific Antibody

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.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.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

Neonates differ adults in their responsiveness to TLR-ligands

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Neonatal goats display a stronger TH1-type cytokine response to TLR ligands than adults.

International audienceIn mammals, Toll-like receptors play a critical role in initiating innate immune responses and modulating adaptive immunity, by recognizing conserved microbial molecular patterns. This study was undertaken to identify specific features of the responses to synthetic toll-like receptor (TLR) agonists in goats, for the definition of tailored immunostimutation strategies. We show here, in contrast to what has been shown in mice, that mesenteric lymph nodes (MLNs) cells and splenocytes from neonatal goats produce much higher levels of TH1-type cytokines than adults in response to various TLR agonists. IL-12 was identified as a critical cytokine for IFN gamma production by CD8(+) neonatal cells. The higher level of IL-12 production by neonatal MLN and spleen cells than by adult cells was not correlated with a higher level of TLR expression or lower levels of production of the regulatory cytokine IL-10. In neonates, two cell popuLations-class II+ CD8(+) and class II+ CD8(-) cells-produce IL-12 in response to R848 and Poly I:C, respectively. Thus, goat kids have characteristics that could be exploited to favor development of the TH1-type responses critical for the control of intracellular pathogens. (C) 2008 Elsevier Ltd. All rights reserved

Flow cytometry analysis of anti-MUC1 IgG and scFv-Fc antibodies on different tumour cell lines HEK293T (A), SKOV3 (B) and MCF-7 (C).

<p>Different anti-MUC1 antibody concentrations were incubated on two different MUC1 positive cell lines and one MUC1 negative cell line. Detection was performed as given for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015921#pone-0015921-g008" target="_blank">figure 8B</a>.</p

Analysis of the IHC staining with non tumour tissues.

<p>Eight slides of each tissue type were analysed.</p><p>*suspected reaction of the detection system with endogenous biotin.</p

Internalisation of anti-MUC1 HT186-D11 and huHMFG1 IgGs.

<p>Internalisation was analysed by incubation of MCF-7 cells with anti-MUC1 antibodies at 37°C or on ice (on ice or 4°C, should be consistent throughout the paper) for 1h. Remaining cell surface-associated mAbs were detected by staining with PE-conjugated mouse anti-human IgG mab.</p