Humanization of a mouse anti-human complement C6 monoclonal antibody as a potential therapeutic for certain complement-mediated diseases.

The assembly of tissue-damaging membrane attack complexes (MACs; C5b-9) is a major mechanism by which excessive complement activation causes diseases. We previously developed a mouse anti-human C6 monoclonal antibody (mAb) 1C9 that selectively inhibits the assembly of MACs in human and non-human primates. In this project, we found that 1C9 also cross-reacted with rat and guinea pig C6, and determined its binding domains on C6 using different truncated C6 proteins. We then humanized the anti-C6 mAb by molecular modeling and complementarity-determining region grafting. After screening a library of 276 humanized variants with different combinations of humanized light and heavy chains in biophysical assays, we identified clone 3713 with the best developability profile, and an increased affinity against C6 when compared with the parental 1C9 mAb. This humanized 3713 mAb inhibited human, monkey, and rat complement-mediated hemolysis in vitro, and more importantly, it significantly reduced complement-mediated hemolysis in vivo in rats. These results demonstrated the successful humanization of the anti-C6 mAb and suggested that the humanized 3713 mAb could be further developed as a new therapeutic that selectively targets MAC for certain complement-mediated pathological conditions.

Co-crystallisation and humanisation of an anti-HER2 single-domain antibody as a theranostic tool.

Human epidermal growth factor receptor-2 (HER2) is a well-recognised biomarker associated with 25% of breast cancers. In most cases, early detection and/or treatment correlates with an increased chance of survival. This study, has identified and characterised a highly specific anti-HER2 single-domain antibody (sdAb), NM-02, as a potential theranostic tool. Complete structural description by X-ray crystallography has revealed a non-overlapping epitope with current anti-HER2 antibodies. To reduce the immunogenicity risk, NM-02 underwent a humanisation process and retained wild type-like binding properties. To further de-risk the progression towards chemistry, manufacturing and control (CMC) we performed full developability profiling revealing favourable thermal and physical biochemical 'drug-like' properties. Finally, the application of the lead humanised NM-02 candidate (variant K) for HER2-specific imaging purposes was demonstrated using breast cancer HER2+/BT474 xenograft mice.

Discovery of a novel pseudo ß-hairpin structure of N-truncated amyloid-ß for use as a vaccine against Alzheimer's disease.

One of the hallmarks of Alzheimer's disease (AD) are deposits of amyloid-beta (Aß) protein in amyloid plaques in the brain. The Aß peptide exists in several forms, including full-length Aß1-42 and Aß1-40 - and the N-truncated species, pyroglutamate Aß3-42 and Aß4-42, which appear to play a major role in neurodegeneration. We previously identified a murine antibody (TAP01), which binds specifically to soluble, non-plaque N-truncated Aß species. By solving crystal structures for TAP01 family antibodies bound to pyroglutamate Aß3-14, we identified a novel pseudo ß-hairpin structure in the N-terminal region of Aß and show that this underpins its unique binding properties. We engineered a stabilised cyclic form of Aß1-14 (N-Truncated Amyloid Peptide AntibodieS; the 'TAPAS' vaccine) and showed that this adopts the same 3-dimensional conformation as the native sequence when bound to TAP01. Active immunisation of two mouse models of AD with the TAPAS vaccine led to a striking reduction in amyloid-plaque formation, a rescue of brain glucose metabolism, a stabilisation in neuron loss, and a rescue of memory deficiencies. Treating both models with the humanised version of the TAP01 antibody had similar positive effects. Here we report the discovery of a unique conformational epitope in the N-terminal region of Aß, which offers new routes for active and passive immunisation against AD.

Alzheimer therapy with an antibody against N-terminal Abeta 4-X and pyroglutamate Abeta 3-X.

Full-length Aß1-42 and Aß1-40, N-truncated pyroglutamate Aß3-42 and Aß4-42 are major variants in the Alzheimer brain. Aß4-42 has not been considered as a therapeutic target yet. We demonstrate that the antibody NT4X and its Fab fragment reacting with both the free N-terminus of Aß4-x and pyroglutamate Aß3-X mitigated neuron loss in Tg4-42 mice expressing Aß4-42 and completely rescued spatial reference memory deficits after passive immunization. NT4X and its Fab fragment also rescued working memory deficits in wild type mice induced by intraventricular injection of Aß4-42. NT4X reduced pyroglutamate Aß3-x, Aßx-40 and Thioflavin-S positive plaque load after passive immunization of 5XFAD mice. Aß1-x and Aßx-42 plaque deposits were unchanged. Importantly, for the first time, we demonstrate that passive immunization using the antibody NT4X is therapeutically beneficial in Alzheimer mouse models showing that N-truncated Aß starting with position four in addition to pyroglutamate Aß3-x is a relevant target to fight Alzheimer's disease.

N-Hydroxyimides and hydroxypyrimidinones as inhibitors of the DNA repair complex ERCC1-XPF.

A high throughput screen allowed the identification of N-hydroxyimide inhibitors of ERCC1-XPF endonuclease activity with micromolar potency, but they showed undesirable selectivity profiles against FEN-1. A scaffold hop to a hydroxypyrimidinone template gave compounds with similar potency but allowed selectivity to be switched in favour of ERCC1-XPF over FEN-1. Further exploration of the structure-activity relationships around this chemotype gave sub-micromolar inhibitors with >10-fold selectivity for ERCC1-XPF over FEN-1.

Catechols and 3-hydroxypyridones as inhibitors of the DNA repair complex ERCC1-XPF.

Catechol-based inhibitors of ERCC1-XPF endonuclease activity were identified from a high-throughput screen. Exploration of the structure-activity relationships within this series yielded compound 13, which displayed an ERCC1-XPF IC50 of 0.6 µM, high selectivity against FEN-1 and DNase I and activity in nucleotide excision repair, cisplatin enhancement and γH2AX assays in A375 melanoma cells. Screening of fragments as potential alternatives to the catechol group revealed that 3-hydroxypyridones are able to inhibit ERCC1-XPF with high ligand efficiency, and elaboration of the hit gave compounds 36 and 37 which showed promising ERCC1-XPF IC50 values of <10 µM.

Inhibition of the ERCC1-XPF structure-specific endonuclease to overcome cancer chemoresistance.

ERCC1-XPF is a structure-specific endonuclease that is required for the repair of DNA lesions, generated by the widely used platinum-containing cancer chemotherapeutics such as cisplatin, through the Nucleotide Excision Repair and Interstrand Crosslink Repair pathways. Based on mouse xenograft experiments, where ERCC1-deficient melanomas were cured by cisplatin therapy, we proposed that inhibition of ERCC1-XPF could enhance the effectiveness of platinum-based chemotherapy. Here we report the identification and properties of inhibitors against two key targets on ERCC1-XPF. By targeting the ERCC1-XPF interaction domain we proposed that inhibition would disrupt the ERCC1-XPF heterodimer resulting in destabilisation of both proteins. Using in silico screening, we identified an inhibitor that bound to ERCC1-XPF in a biophysical assay, reduced the level of ERCC1-XPF complexes in ovarian cancer cells, inhibited Nucleotide Excision Repair and sensitised melanoma cells to cisplatin. We also utilised high throughput and in silico screening to identify the first reported inhibitors of the other key target, the XPF endonuclease domain. We demonstrate that two of these compounds display specificity in vitro for ERCC1-XPF over two other endonucleases, bind to ERCC1-XPF, inhibit Nucleotide Excision Repair in two independent assays and specifically sensitise Nucleotide Excision Repair-proficient, but not Nucleotide Excision Repair-deficient human and mouse cells to cisplatin.

Novel heterozygous OTX2 mutations and whole gene deletions in anophthalmia, microphthalmia and coloboma.

Severe ocular malformations, including anophthalmia-microphthalmia (AM), are responsible for around 25% of severe visual impairment in childhood. Recurrent interstitial deletions of 14q22-23 are associated with AM and a wide range of extra-ocular phenotypes including brain anomalies. The homeobox gene OTX2 is located at 14q22.3 and has recently been identified as mutated in AM patients. Eight human OTX2 mutations have been reported in subjects with severe eye malformations, including AM, and variable developmental delay. We screened a novel AM cohort for mutations and deletions in OTX2, and identified four new mutations in six individuals and two cases of whole gene deletions. Our data suggest that OTX2 mutations and deletions account for 2-3% of AM cases.

Mutations in BMP4 cause eye, brain, and digit developmental anomalies: overlap between the BMP4 and hedgehog signaling pathways.

Developmental ocular malformations, including anophthalmia-microphthalmia (AM), are heterogeneous disorders with frequent sporadic or non-Mendelian inheritance. Recurrent interstitial deletions of 14q22-q23 have been associated with AM, sometimes with poly/syndactyly and hypopituitarism. We identify two further cases of AM (one with associated pituitary anomalies) with a 14q22-q23 deletion. Using a positional candidate gene approach, we analyzed the BMP4 (Bone Morphogenetic Protein-4) gene and identified a frameshift mutation (c.226del2, p.S76fs104X) that segregated with AM, retinal dystrophy, myopia, brain anomalies, and polydactyly in a family and a nonconservative missense mutation (c.278A-->G, p.E93G) in a highly conserved base in another family. MR imaging and tractography in the c.226del2 proband revealed a primary brain developmental disorder affecting thalamostriatal and callosal pathways, also present in the affected grandmother. Using in situ hybridization in human embryos, we demonstrate expression of BMP4 in optic vesicle, developing retina and lens, pituitary region, and digits strongly supporting BMP4 as a causative gene for AM, pituitary, and poly/syndactyly. Because BMP4 interacts with HH signaling genes in animals, we evaluated gene expression in human embryos and demonstrate cotemporal and cospatial expression of BMP4 and HH signaling genes. We also identified four cases, some of whom had retinal dystrophy, with "low-penetrant" mutations in both BMP4 and HH signaling genes: SHH (Sonic Hedgehog) or PTCH1 (Patched). We propose that BMP4 is a major gene for AM and/or retinal dystrophy and brain anomalies and may be a candidate gene for myopia and poly/syndactyly. Our finding of low-penetrant variants in BMP4 and HH signaling partners is suggestive of an interaction between the two pathways in humans.