RESUMO
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.
Assuntos
Alcinos/química , Aminoácidos/química , Azidas/química , Imunoconjugados/química , Radioisótopos de Índio/química , Radioisótopos/química , Zircônio/química , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Reação de Cicloadição , Humanos , Imunoconjugados/uso terapêutico , Marcação por Isótopo , CamundongosRESUMO
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.
RESUMO
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.
Assuntos
Antioxidantes/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glutamato-Cisteína Ligase/genética , Hidroquinonas/farmacologia , Região 5'-Flanqueadora/genética , Animais , Sequência de Bases , Cádmio/farmacologia , DNA/análise , Genoma , Glutationa/metabolismo , Peróxido de Hidrogênio/farmacologia , Camundongos , Dados de Sequência Molecular , Estresse Oxidativo , Regiões Promotoras Genéticas/efeitos dos fármacos , Subunidades Proteicas , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Homologia de Sequência do Ácido Nucleico , TATA Box , Sítio de Iniciação de Transcrição , Transcrição Gênica/efeitos dos fármacos , Células Tumorais Cultivadas , Vitamina K 3/farmacologiaRESUMO
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.
Assuntos
Anticorpos/sangue , Anticorpos/imunologia , Sítios de Ligação de Anticorpos/imunologia , Imunoconjugados/química , Imunoconjugados/imunologia , Imunoglobulina G/química , Engenharia de Proteínas , Aminobenzoatos/química , Aminobenzoatos/imunologia , Animais , Anticorpos/química , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular , Cisteína/química , Humanos , Imunoconjugados/administração & dosagem , Imunoglobulina G/imunologia , Macaca fascicularis , Maleimidas/química , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/imunologia , Maitansina/química , Maitansina/imunologia , Camundongos , Camundongos Nus , Modelos Moleculares , Oligopeptídeos/química , Oligopeptídeos/imunologia , Conformação Proteica , Ratos , Relação Estrutura-Atividade , Compostos de Sulfidrila/química , TrastuzumabRESUMO
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.