Site-Specific 89Zr- and 111In-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 para-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. The incorporation of pAMF enables the conjugation of payloads functionalized for strain-promoted azide-alkyne cycloaddition. Here, we introduce two dibenzylcyclooctyne-functionalized bifunctional chelators that enable the incorporation of radioisotopes for positron emission tomography with 89Zr (t1/2 = 78.4 h, ß+ = 395 keV (22%), γ = 897 keV) or single photon emission computed tomography with 111In (t1/2 = 67.3 h, γ = 171 keV (91%), 245 keV (94%)) under physiologically compatible conditions. We show that the corresponding radiolabeled conjugates with site-specifically functionalized antibodies targeting HER2 are amenable to targeted molecular imaging of HER2+ expressing tumor xenografts in mice and exhibit a favorable biodistribution profile in comparison with conventional, glycosylated antibody conjugates generated by stochastic bioconjugation.

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. CD74 mRNA was detectable in CD138+ enriched plasma cells from 100% (892/892) of patients with newly diagnosed MM. Immunohistochemistry confirmed CD74 expression in 35/36 BM biopsies from patients with newly diagnosed and relapsed/refractory MM. Cytotoxicity assays demonstrated nanomolar STRO-001 potency in 4/6 MM cell lines. In ARP-1 and MM.1S tumor-bearing mice, repeat STRO-001 dosing provided significant antitumor activity with eradication of malignant hCD138+ BM plasma cells and prolonged survival. In a luciferase-expressing MM.1S xenograft model, dose-dependent STRO-001 efficacy was confirmed using bioluminescent imaging and BM tumor burden quantification. Consistent with the intended pharmacodynamic effect, STRO-001 induced dose-responsive, reversible B-cell and monocyte depletion in cynomolgus monkeys, up to a maximum tolerated 10 mg/kg, with no evidence of off-target toxicity. Collectively, these data suggest that STRO-001 is a promising therapeutic agent for the treatment of MM.

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. In contrast, a partially accessible site with a positively charged environment promoted hydrolysis of the succinimide ring in the linker, thereby preventing this exchange reaction. The site with partial solvent-accessibility and neutral charge displayed both properties. In a mouse mammary tumor model, the stability and therapeutic activity of the antibody conjugate were affected positively by succinimide ring hydrolysis and negatively by maleimide exchange with thiol-reactive constituents in plasma. Thus, the chemical and structural dynamics of the conjugation site can influence antibody conjugate performance by modulating the stability of the antibody-linker interface.

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 22.3 kb; all exons, intron-exon junctions, and 4.7 kb of 5'-flanking region were sequenced. By RNase protection analysis, we identified two major and several minor transcription start-site clusters over a 300-bp region. The Gclm 5'-flanking region is GC-rich and lacks a canonical TATA box. Transient and stable transfection studies, using luciferase reporter constructs containing incremental Gclm 5'-flanking deletions (4.7-0.5 kb), showed high basal activity but only modest ( approximately 2-fold) inducibility by tBHQ. The only candidate motif for oxidative stress regulation (in the 4.7-kb region we sequenced) is a putative inverted electrophile response element (EPRE) 9 bp upstream from the 5'-most transcription start-site. Site-directed mutagenesis of this -9 EPRE demonstrated minimal (30-40%) decreases in tBHQ induction and no effect on basal activity-suggesting that this EPRE might be necessary but not sufficient. The nuclear erythroid factor-2 (NEF2)-related factor-2 (NRF2) is known to transactivate via EPRE motifs. In the presence of co-transfected NRF cDNA expression vector, however, no increase in Gclm promoter activity was observed. Thus, the endogenous Gclm gene shows robust transcriptional activation by tBHQ in the intact Hepa-1 cell, but reporter constructs containing up to 4.7 kb of promoter (having only the one EPRE at -9) demonstrate a disappointing response, indicating that the major tBHQ-responsive regulatory element of the mouse Gclm gene must exist either further 5'- or 3'-ward of the 4.7-kb region studied.

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. Molecular dissection of MTF1 has been hindered by its high constitutive trans-activity following transient transfection and the failure of these systems to examine genes packaged in native chromatin. In developing a system to avoid these problems, we employed a high-efficiency retroviral transduction system to reintroduce MTF1 into mouse Mtf1(-/-) knockout cells (dko7). Electrophoretic mobility shift assays demonstrated that MTF1 retrovirally transduced dko7 cells (MTF1dko7) possess levels of inducible MTF1-MRE binding activity similar to that seen in mouse hepatoma Hepa-1 cells, and MTF1 binding could be modulated over a 20-fold range by varying the concentration of Zn(2+) present in the culture medium. The dko7 cells exhibited no change in Mt1 gene expression upon Zn(2+) or cadmium (Cd(2+)) treatment; in contrast, in MTF1dko7 cells, Zn(2+) or Cd(2+) induced MT1 mRNA accumulation in a dose-dependent manner. Interestingly, MTF1dko7 cells showed resistance to Zn(2+) toxicity, but negligible resistance to Cd(2+). Concomitantly, MT1 protein levels in MTF1dko7 cells were inducible to the same degree as that in Hepa-1 cells when treated with Zn(2+), but not with Cd(2+). Together, our studies suggest that MTF1-mediated regulation of gene expression is sufficient to protect cells against Zn(2+) toxicity and may be necessary but not sufficient to protect cells against Cd(2+) toxicity.