Site-Specific Zr- and In-Radiolabeling and In Vivo Evaluation of Glycan-free Antibodies by Azide-Alkyne Cycloaddition with a Non-natural Amino Acid.

Antibody-drug conjugates (ADCs) are a class of targeted therapeutics consisting of a monoclonal antibody coupled to a cytotoxic payload. Various bioconjugation methods for producing site-specific ADCs have been reported recently, in efforts to improve immunoreactivity and pharmacokinetics and minimize batch variance-potential issues associated with first-generation ADCs prepared via stochastic peptide coupling of lysines or reduced cysteines. Recently, cell-free protein synthesis of antibodies incorporating -azidomethyl phenylalanine (pAMF) at specific locations within the protein sequence has emerged as a means to generate antibody-drug conjugates with strictly defined drug-antibody-ratio, leading to ADCs with markedly improved stability, activity, and specificity.

Targeting CD74 in multiple myeloma with the novel, site-specific antibody-drug conjugate STRO-001.

STRO-001 is a site-specific, predominantly single-species, fully human, aglycosylated anti-CD74 antibody-drug conjugate incorporating a non-cleavable linker-maytansinoid warhead with a drug-antibody ratio of 2 which was produced by a novel cell-free antibody synthesis platform. We examined the potential pharmacodynamics and anti-tumor effects of STRO-001 in multiple myeloma (MM). CD74 expression was assessed in MM cell lines and primary bone marrow (BM) MM biopsies.

Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates.

The reactive thiol in cysteine is used for coupling maleimide linkers in the generation of antibody conjugates. To assess the impact of the conjugation site, we engineered cysteines into a therapeutic HER2/neu antibody at three sites differing in solvent accessibility and local charge. The highly solvent-accessible site rapidly lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione.

Glutamate-cysteine ligase modifier subunit: mouse Gclm gene structure and regulation by agents that cause oxidative stress.

Glutamate-cysteine ligase is a heterodimer comprising a modifier (GCLM) and a catalytic (GCLC) subunit. In mouse Hepa-1c1c7 hepatoma cell cultures, we found that tert-butylhydroquinone (tBHQ; 50 microM) induces the GCLM and GCLC mRNAs approximately 10- and approximately 2-fold, respectively, and that these increases primarily reflect de novo transcription. We determined that the mouse Gclm gene has seven exons, spanning 22.

Retrovirally expressed metal response element-binding transcription factor-1 normalizes metallothionein-1 gene expression and protects cells against zinc, but not cadmium, toxicity.

Metal response element (MRE) transcription factor-1 (MTF1), a member of the Cys2-His2 class of zinc-finger transcription factors, is best known for its robust transcriptional regulation of mammalian metallothionein (MT) genes. MTF1 is also believed to play a generalized role in regulating genes involved in protection against heavy metals and oxidative stress. MTF1 binding to MRE motifs is regulated by changes in intracellular zinc (Zn(2+)) concentration.