Reduction-rebridging strategy for the preparation of ADPN-based antibody-drug conjugates.

The reduction-rebridging strategy is a powerful method for the preparation of stable and homogeneous antibody-drug conjugates (ADCs). In this communication, we describe the development of the arylene-dipropiolonitrile (ADPN) functional group for the rebridging of reduced disulphide bonds and its application in the preparation of potent and selective ADCs.

Development and evaluation of ß-galactosidase-sensitive antibody-drug conjugates.

The selective destruction of tumour cells while sparing healthy tissues is one of the main challenges in cancer therapy. Antibody-drug conjugates (ADCs) are arguably the most rapidly expanding class of targeted cancer therapies. Efficient drug conjugation and release technologies are essential for the development of these new therapeutic agents. In response to the ever-increasing demand for efficient drug release systems, we have developed a new class of ß-galactosidase-cleavable linkers for ADCs. Within this framework, novel payloads comprising a galactoside linker, the monomethyl auristatin E (MMAE) and cysteine-reactive groups were synthesized, conjugated with trastuzumab and evaluated both in vitro and in vivo. The ADCs with galactoside linkers demonstrated superior therapeutic efficacy in mice compared to the marketed trastuzumab emtansine used for the treatment of breast cancer.

MAPN: First-in-Class Reagent for Kinetically Resolved Thiol-to-Thiol Conjugation.

Thiols are among the most frequently used functional groups in the field of bioconjugation. While there exists a variety of heterobifunctional reagents that allow for coupling thiols to other functions (e.g., amines, carboxylic acids), there is no specific reagent for creating heteroconjugates using two different thiols. In response to the ever-increasing demand for bioconjugation tools, we have developed p-(maleimide)-phenylpropionitrile (MAPN)-an efficient reagent for kinetically resolved thiol-to-thiol coupling. In a comparative study with its closest commercially available analogue, p-phenylenedimaleimide, MAPN has shown substantial advantages for the preparation of thiol-thiol heteroconjugates. Namely, an antibody-drug conjugate (ADC) with mertansine (DM1), conjugated to the cysteine residues of Trastuzumab, was prepared for the first time.

Histone methyltransferase DOT1L drives recovery of gene expression after a genotoxic attack.

UV-induced DNA damage causes repression of RNA synthesis. Following the removal of DNA lesions, transcription recovery operates through a process that is not understood yet. Here we show that knocking-out of the histone methyltransferase DOT1L in mouse embryonic fibroblasts (MEF(DOT1L)) leads to a UV hypersensitivity coupled to a deficient recovery of transcription initiation after UV irradiation. However, DOT1L is not implicated in the removal of the UV-induced DNA damage by the nucleotide excision repair pathway. Using FRAP and ChIP experiments we established that DOT1L promotes the formation of the pre-initiation complex on the promoters of UV-repressed genes and the appearance of transcriptionally active chromatin marks. Treatment with Trichostatin A, relaxing chromatin, recovers both transcription initiation and UV-survival. Our data suggest that DOT1L secures an open chromatin structure in order to reactivate RNA Pol II transcription initiation after a genotoxic attack.

Glucocerebrosidase gene has an alternative upstream promoter, which has features and expression characteristic of housekeeping genes.

Database searches have shown that a part of glucocerebrosidase (GBA) transcripts may originate at an alternative upstream promoter (P2) located 2.6 kb upstream of the known (P1) GBA promoter. The putative alternative transcripts contained one or two extra exons (exon -2 or exons -2, -1, respectively), but the first ATG codon and predicted amino-acid sequence are the same as in the transcript from P1. Luciferase assays confirmed promoter activity of both sites in HepG2 cells: the P1 construct exhibited the highest activity of luciferase (17.82±1.10 relative luciferase units), while the P2 construct reached 3.01±0.43 relative luciferase units. Serial 5' deletions of P2 led to changes in reporter activity, the most prominent decreases were observed in deletion constructs carrying bases -353 to -658, and -353 to -920 (numbered as in NM_001005750.1), respectively. This suggests that the P2 core promoter is contained within the region of -920bp to -1311bp. Three P2 transcription initiation sites were found by 5' RACE at positions 347, 380, and 413bp upstream of the +1 ATG. The expression stability of transcripts from P2, P1 was studied in 20 human tissues and was higher than that of GAPDH and ACTB, which are commonly used as reference housekeeping genes. The P2 contains an unmethylated CpG island, multiple Sp-1 consensus binding sites and, unlike P1, does not contain a TATA box, features all common to the majority of housekeeping gene promoters. We have examined DNA samples from a phenotypically diverse group of twenty Ashkenazi Jewish Gaucher patients homozygous for the common mild mutation N370S. Both P1 and P2, as well as exons -2 and -1, did not contain any sequence variations, with the exception of the known polymorphism rs10908459 found on one allele. The phenotypical differences in the patients were thus not explained by nucleotide variations in both promoters.

Disruption of OTC promoter-enhancer interaction in a patient with symptoms of ornithine carbamoyltransferase deficiency.

In a female patient with signs of ornithine carbamoyltransferase deficiency (OTCD), the only variation found was a heterozygous single nucleotide substitution c.-366A>G. Determination of transcription start sites of human OTC 95, 119 and 169 bp upstream of the initiation codon located the variation upstream of the 5'-untranslated region. We predicted the human promoter and enhancer elements from homology with rat and mouse, performed function analysis of both regulatory regions and assessed the impact of the promoter variation in functional studies using dual luciferase reporter assay. Our data indicate that: (i) Full transcriptional activity of human OTC promoter depends on an upstream enhancer, as do the rodent promoters. (ii) The promoter variation c.-366A>G does not affect the function of the promoter alone but it disrupts the interaction of the promoter with the enhancer. (iii) The promoter-enhancer interaction contributes to tissue specific expression of OTC in the liver. We conclude that mutations in the regulatory regions of OTC can lead to OTCD and should be included in genetic testing.

Urinary and fecal immunoglobulin A, cortisol and 11-17 dioxoandrostanes, and serum cortisol in metabolic cage housed female cynomolgus monkeys (Macaca fascicularis).

BACKGROUND AND METHODS: Quantitative enzyme-immunoassays of urinary and fecal immunoglobulin A (IgA), cortisol and 11-17-dioxoandrostanes (11,17-DOA), and serum cortisol in eight metabolic-cage-housed female cynomolgus monkeys were performed. The monkeys were divided into two groups, B and NB. Group B animals were blood sampled every 6 hours, whereas Group NB animals were not handled/blood sampled. RESULTS: No differences were recorded between the amounts of feces and urine excreted by the two groups. Group B animals excreted more urinary cortisol than did Group NB animals indicating that restraint-blood sampling resulted in a stress response. Excreted amounts of IgA and 11,17-DOA (urine and feces) did not differ between the groups. CONCLUSIONS: Urinary cortisol was a reliable marker of the stress associated with repeated blood sampling. Declining amounts of excreted urinary cortisol indicated that cynomolgus monkeys acclimated quickly to repeated blood sampling in metabolism cages. Within and between animal variation in amounts of feces voided demonstrated the importance of expressing fecal markers as 'amounts excreted per time unit per kg body weight' rather than just measuring the concentrations in fecal samples.