A Triple-Transgenic Immunotolerant Mouse Model

ABSTRACTAvoiding unwanted immunogenicity is of key importance in the development of therapeutic drug proteins. Animal models are of less predictive value because most of the drug proteins are recognized as foreign proteins. However, different methods have been developed to obtain immunotolerant animal models. So far, the immunotolerant animal models have been developed to assess one protein at a time and are not suitable for the assessment of combination products. Our aim was to develop an animal model for evaluating the impact of manufacturing and formulation changes on immunogenicity, suitable for both single protein and combination products. We constructed two lines of transgenic mice expressing the three human coagulation factors, II, VII, and X, by inserting a single vector containing the three coagulation factors encoding sequences separated by insulator sequences derived from the chicken beta-globin locus into the mouse genome. Immunization of transgenic mice from the two lines and their wild-type littermates showed that transgenic mice from both lines were immunotolerant to the expressed human coagulation factors. We conclude that transgenic mice immunotolerant to multiple proteins can be obtained, and that these mice are potentially useful as animal models in the assessment of immunogenicity in response to manufacturing changes. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:1116-1124, 201

Transcriptional regulation of the human carboxyl ester lipase gene in THP-1 monocytes: an E-box required for activation binds upstream stimulatory factors 1 and 2.

The bile salt-stimulated carboxyl ester lipase (CEL) is important for the digestion and absorption of dietary lipids, and is expressed at high levels by the exocrine pancreas and the lactating mammary gland. However, the presence of CEL in human plasma suggests that the role of CEL in lipid metabolism may stretch beyond its function in the intestinal lumen, and possibly include interactions with cholesterol and oxidized lipoproteins to modulate the progression of atherosclerosis. We have used the CEL-expressing human monocytic cell line THP-1 to investigate the transcriptional regulation of the human CEL in monocytes. Analyses of the promoter region revealed that an E-box located at -47/-52 is necessary for CEL expression. Point mutations in the E-box almost completely abolish the transcriptional activity. Electrophoretic mobility-shift assay analyses reveal that the E-box binds the upstream stimulatory factors 1 and 2, and the binding of an upstream stimulatory factor-containing complex in THP-1 cells also requires the presence of a putative nuclear receptor-binding site at -60/-66. Furthermore, we demonstrate that the E-box is also necessary for CEL expression in the pancreas and the mammary gland, although there are tissue-specific requirements for additional activating elements

HLA-DR7 and HLA-DQ2 : Transgenic mouse strains tested as a model system for ximelagatran hepatotoxicity

The oral thrombin inhibitor ximelagatran was withdrawn in the late clinical trial phase because it adversely affected the liver. In approximately 8% of treated patients, drug-induced liver injury (DILI) was expressed as transient alanine transaminase (ALT) elevations. No evidence of DILI had been revealed in the pre-clinical in vivo studies. A whole genome scan study performed on the clinical study material identified a strong genetic association between the major histocompatibility complex alleles for human leucocyte antigens (HLA) (HLA-DR7 and HLA-DQ2) and elevated ALT levels in treated patients. An immunemediated pathogenesis was suggested. Here, we evaluated whether HLA transgenic mice models could be used to investigate whether the expression of relevant HLA molecules was enough to reproduce the DILI effects in humans. In silico modelling performed in this study revealed association of both ximelagatran (pro-drug) and melagatran (active drug) to the antigen-presenting groove of the homology modelled HLA-DR7 molecule suggesting "altered repertoire" as a key initiating event driving development of DILI in humans. Transgenic mouse strains (tgms) expressing HLA of serotype HLA-DR7 (HLA-DRB1*0701, -DRA*0102), and HLA-DQ2 (HLA-DQB1*0202, -DQA1*0201) were created. These two lines were crossed with a human (h) CD4 transgenic line, generating the two tgms DR7xhCD4 and DQ2xhCD4. To investigate whether the DILI effects observed in humans could be reproduced in tgms, the mice were treated for 28 days with ximelagatran. Results revealed no signs of DILI when biomarkers for liver toxicity were measured and histopathology was evaluated. In the ximelagatran case, presence of relevant HLA-expression in a preclinical model did not fulfil the prerequisite for reproducing DILI observed in patients. Nonetheless, for the first time an HLA-transgenic mouse model has been investigated for use in HLA-associated DILI induced by a low molecular weight compound. This study shows that mimicking of genetic susceptibility, expressed as DILI-associated HLA-types in mice, is not sufficient for reproducing the complex pathogenesis leading to DILI in man.Funding Agencies|AstraZeneca; AstraZeneca R&amp;D as part of safety problem-solving activities initiated for ximelagatran (Exanta(R))</p

Identified antigen-binding groove binding-site.

<p>Antigen-binding groove binding-site identification of homology modelled HLA-DR7 using SiteMap (A–front view, B–top view). Blue–hydrogen bond donor region, red–hydrogen bond acceptor region, yellow–hydrophobic region, white–binding site grid.</p

Study design of ximelagatran exposure study.

<p>Study design of ximelagatran exposure study.</p

Surface markers.

<p>Surface markers.</p

Verification of normal and comparable profiles between wt and tg mice.

<p>Representation of lymphoid cell subsets from PBMC (A) and spleen (B) to compare wt and tg mice using mouse specific tracer antibodies. *^/** significant difference, * p≤0.05 ** p≤0.01.</p

Representative surface-marker expression on cells from wt and tg mice.

<p>A and B illustrate the expression surface markers hCD4 and HLA DR/DQ on PBMCs, respectively. C displays the T-cell population and shows that hCD4 in the tgms is almost exclusively expressed on T-cells also expressing mCD4.</p

ALT levels in animals treated with ximelagatran for 28 days.

<p>ALT levels in mice compared before and after 28 days of ximelagatran treatment. A fold change of one equals no change between start of treatment and end of treatment; fold change of two equals two times higher ALT levels.</p