Stable and Potent Selenomab-Drug Conjugates Li, Xiuling; Nelson, Christopher G.; Nair, Rajesh R. ...
Cell chemical biology,
04/2017, Letnik:
24, Številka:
4
Journal Article
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Selenomabs are engineered monoclonal antibodies with one or more translationally incorporated selenocysteine residues. The unique reactivity of the selenol group of selenocysteine permits ...site-specific conjugation of drugs. Compared with other natural and unnatural amino acid and carbohydrate residues that have been used for the generation of site-specific antibody-drug conjugates, selenocysteine is particularly reactive, permitting fast, single-step, and efficient reactions under near physiological conditions. Using a tailored conjugation chemistry, we generated highly stable selenomab-drug conjugates and demonstrated their potency and selectivity in vitro and in vivo. These site-specific antibody-drug conjugates built on a selenocysteine interface revealed broad therapeutic utility in liquid and solid malignancy models.
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•Selenomab-drug conjugates are antibody-drug conjugates based on selenocysteine•Iodoacetamide-based selenomab-drug conjugates have excellent stability•Selenomab-drug conjugates are potent and specific cancer therapeutics•The location of selenocysteine in selenomab-drug conjugates impacts their potency
Li et al. harness the high reactivity of the 21st natural amino acid selenocysteine to rapidly and efficiently assemble antibody-drug conjugates that reveal excellent stability, potency, and selectivity in diverse in vitro and in vivo models of human cancers.
Mammalian cells acquire cholesterol, a critical membrane constituent, through multiple mechanisms. We synthesized mimics of cholesterol, fluorescent N-alkyl-3β-cholesterylamine-glutamic acids, that ...are rapidly incorporated into cellular plasma membranes compared with analogous cholesteryl amides, ethers, esters, carbamates, and a sitosterol analogue. This process was inhibited by ezetimibe, indicating a receptor-mediated uptake pathway.
Reduced Adipose Tissue Oxygenation in Human Obesity
Evidence for Rarefaction, Macrophage Chemotaxis, and Inflammation Without an Angiogenic Response
Magdalena Pasarica ,
Olga R. Sereda ,
Leanne M. ...Redman ,
Diana C. Albarado ,
David T. Hymel ,
Laura E. Roan ,
Jennifer C. Rood ,
David H. Burk and
Steven R. Smith
From the Pennington Biomedical Research Center, Baton Rouge, Louisiana
Corresponding author: Steven R. Smith, smithsr{at}pbrc.edu
Abstract
OBJECTIVE— Based on rodent studies, we examined the hypothesis that increased adipose tissue (AT) mass in obesity without an adequate
support of vascularization might lead to hypoxia, macrophage infiltration, and inflammation.
RESEARCH DESIGN AND METHODS— Oxygen partial pressure (AT pO 2 ) and AT temperature in abdominal AT (9 lean and 12 overweight/obese men and women) was measured by direct insertion of a
polarographic Clark electrode. Body composition was measured by dual-energy X-ray absorptiometry, and insulin sensitivity
was measured by hyperinsulinemic-euglycemic clamp. Abdominal subcutaneous tissue was used for staining, quantitative RT-PCR,
and chemokine secretion assay.
RESULTS— AT pO 2 was lower in overweight/obese subjects than lean subjects (47 ± 10.6 vs. 55 ± 9.1 mmHg); however, this level of pO 2 did not activate the classic hypoxia targets (pyruvate dehydrogenase kinase and vascular endothelial growth factor VEGF).
AT pO 2 was negatively correlated with percent body fat ( R = −0.50, P < 0.05). Compared with lean subjects, overweight/obese subjects had 44% lower capillary density and 58% lower VEGF, suggesting
AT rarefaction (capillary drop out). This might be due to lower peroxisome proliferator–activated receptor γ1 and higher collagen
VI mRNA expression, which correlated with AT pO 2 ( P < 0.05). Of clinical importance, AT pO 2 negatively correlated with CD68 mRNA and macrophage inflammatory protein 1α secretion ( R = −0.58, R = −0.79, P < 0.05), suggesting that lower AT pO 2 could drive AT inflammation in obesity.
CONCLUSIONS— Adipose tissue rarefaction might lie upstream of both low AT pO 2 and inflammation in obesity. These results suggest novel approaches to treat the dysfunctional AT found in obesity.
Footnotes
Published ahead of print at http://diabetes.diabetesjournals.org on 15 December 2008.
Clinical trials reg. no. NCT00704197, clinicaltrials.gov.
Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work
is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore
be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Accepted December 9, 2008.
Received August 11, 2008.
DIABETES
Although macromolecules on cell surfaces are predominantly targeted and drugged with antibodies, they harbor pockets that are only accessible to small molecules and constitutes a rich subset of ...binding sites with immense potential diagnostic and therapeutic utility. Compared to antibodies, however, small molecules are disadvantaged by a less confined biodistribution, shorter circulatory half‐life, and inability to communicate with the immune system. Presented herein is a method that endows small molecules with the ability to recruit and activate chimeric antigen receptor T cells (CAR‐Ts). It is based on a CAR‐T platform that uses a chemically programmed antibody fragment (cp‐Fab) as on/off switch. In proof‐of‐concept studies, this cp‐Fab/CAR‐T system targeting folate binding proteins on the cell surface mediated potent and specific eradication of folate‐receptor‐expressing cancer cells in vitro and in vivo.
Fab‐ulous! A novel small‐molecule‐controlled chimeric antigen receptor T cell (CAR‐T) therapy was developed based on a chemically programmed antibody fragment (Fab) as an on/off switch. As a proof‐of‐concept, a folate‐programmed Fab switch mediated potent and specific eradication of folate‐receptor‐expressing cancer cells by engaging CAR‐T cells in both in vitro and in vivo models of ovarian cancer.
Over the past several years, the Peterson group has developed synthetic mimics of cholesterol that rapidly incorporate into the plasma membrane of mammalian cells. These N-alkyl-3β-cholesterylamines ...cycle between the plasma membrane and early/recycling endosomes similar to natural cell surface receptors. Because of this unique activity, cholesterylamines have been linked to protein-binding and other motifs to generate artificial cell surface receptors. More recently, they have been conjugated to membrane disruptive peptides to generate synthetic agents that selectively permeabilize early endosomes and deliver cell-impermeable small molecules to the cytosol. Although this pioneering work has significant potential as a system to deliver cell-impermeable small molecule drugs and/or therapeutic agents, additional research is needed to understand the mechanism of action and develop therapeutic applications. To further evaluate this system, we first investigated the structure-activity relationships of cholesterylamines using fluorescent analogues designed to probe the mechanism of cellular uptake. This study produced probes with robust activity that suggests an unprecedented mechanism of cholesterol uptake on mammalian cell surfaces. Second, we investigated novel endosome disruptive peptides that provided insights into their mechanism of action. Additionally, we applied this knowledge to obtain proof-of-concept with antibody conjugates and endosome disruptive peptides as a new strategy to selectively deliver small molecules to tumor cells that overexpress specific cell surface receptors. Another project involves the development of a fluorescence-based method to detect protein-protein interactions in complex biological systems. This method utilizes a novel fluorinated fluorophore that undergoes proximity-driven exchange between lysine residues at the interface of a protein complex. Transfer of this fluorophore from donor to acceptor lysine residues produces a fluorescent protein partner that can be detected by gel electrophoresis or proteomics methods. Since lysine is prevalent at the interface of numerous protein complexes, this method may be useful to identify novel protein-protein interactions and/or factors that affect these interactions.
A 26-year-old male presented with three weeks of jaundice after the self-initiation of the injectable anabolic steroid, Mastabol Dromastanolone Di-Propionate (17 ...beta-Hydroxy-2alpha-methyl-5alpha-androstan-3one propionate). He reported dark urine, light stools, and pruritus. He denied abdominal pain, intravenous drug use, intranasal cocaine, blood transfusions, newly placed tattoos, or sexually transmitted diseases. He used alcohol sparingly. Physical exam revealed jaundice with deep scleral icterus. The liver was palpable 2 cm below the right costal margin with no ascites. The peak bilirubin was 23.6 mg/dL, alkaline phosphatase was 441 units/L, and aspartate aminotransferase/alanine aminotransferase were 70 units/L and 117 units/L respectively. A working diagnosis of acute intrahepatic cholestasis was made. Liver biopsy revealed a centrilobular insult with neutrophilic infiltrates and Ito cell hyperplasia consistent with acute drug induced cholestasis. The patient’ s clinical symptoms resolved and his liver enzymes, bilirubin, and alkaline phosphatase normalized. Anabolic steroids with 17 alpha carbon substitutions have been associated with a bland variety of cholestatic injury with little hepatocellular injury. Cholestasis, under these circumstances, may be secondary to the binding of drugs to canalicular membrane transporters, accumulation of toxic bile acids from canalicular pump failure, or genetic defects in canalicular transport proteins. Mastabol is an injectable, 17 beta hydroxyl compound with no alpha alkyl groups at the 17 carbon position. As such, it has been reported to have little potential toxic effects on the liver. This is the first known reported case of Mastabolinduced cholestatic liver injury. It highlights the need for physicians to consider such widely available substances when faced with hepatic injury of unclear etiology.
Critical protein-protein interactions are ubiquitous in biology. To provide a new method to detect these interactions, we designed and synthesized fluorinated bromopyronins as molecular probes. These ...electrophilic compounds rapidly react with amines via a S(N)Ar mechanism to form modestly electrophilic aminopyronin fluorophores. To investigate whether proteins modified with aminopyronins might selectively transfer these fluorophores between proximal lysine residues at protein-protein interfaces, immunoglobulin-G (IgG) was conjugated to fluorinated pyronins and added to unlabeled Protein A (SpA) from S. aureus. Analysis by gel electrophoresis and mass spectrometry revealed transfer of this fluorophore from IgG to specific lysines of its binding partner SpA but not to bovine serum albumin (BSA) as a nonbinding control. Examination of an X-ray structure of IgG bound to SpA revealed that the fluorophore was selectively transferred between amino groups of lysines that reside within ~10 Å at the protein-protein interface. To evaluate whether this approach could be used to identify interactions with endogenous cellular proteins, pyronin-modified Rnase A was added to crude extracts of human HeLa cells. Analysis of interacting proteins by gel electrophoresis revealed the endogenous ribonuclease inhibitor as the primary cellular target. Given that proximal lysine residues frequently reside at protein-protein interfaces, this method may facilitate identification of diverse protein-protein interactions present in complex biological matrices.
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