Site-specific conjugation of small molecules and enzymes to monoclonal antibodies has broad utility in the formation of conjugates for therapeutic, diagnostic, or structural applications. Precise control over the location of conjugation would yield highly homogenous materials that could have improved biological properties. We describe for the first time chemical reduction and oxidation methods that lead to preferential cleavage of particular monoclonal antibody interchain disulfides using the anti-CD30 IgG1 monoclonal antibody cAC10. Alkylation of the resulting cAC10 cysteine thiols with the potent antimitotic agent monomethyl auristatin E (MMAE) enabled the assignment of drug conjugation location by purification with hydrophobic interaction chromatography followed by analysis using reversed-phase HPLC and capillary electrophoresis. These analytical methods demonstrated that treating cAC10 with reducing agents such as DTT caused preferential reduction of heavy-light chain disulfides, while reoxidation of fully reduced cAC10 interchain disulfides caused preferential reformation of heavy-light chain disulfides. Following MMAE conjugation, the resulting conjugates had isomeric homogeneity as high as 60−90%, allowing for control of the distribution of molecular species. The resulting conjugates are highly active both in vitro and in vivo, and are well tolerated at efficacious doses.Monoclonal antibodies (mAbs) have been used extensively as carriers of fluorophores, radionuclides, cytotoxic agents, and enzymes, yielding conjugates that find utility in therapeutic (1-3) and imaging applications (4,5), ELISA-based assays (6), as well as for the investigation of protein structure and dynamics (7). The methods employed for making mAbbased conjugates can be classified in two general categories: those that involve the random modification of mAb amino acid residues, and those that are highly regioselective. Examples of random modification procedures include the acylation of lysine ε-amino groups (8), alkylation of tyrosines (9), and amidation of carboxylates (10). The biological and functional properties of these conjugates are often acceptable, however random modification of mAbs may impair antigen binding and leads to conjugate heterogeneity.In the past several years, a number of selective methods have been described to introduce molecules of interest onto mAbs. The ability to control the location and stoichiometry of conjugation can significantly improve the properties of mAb conjugates in some applications. The greatest selectivities are obtained using recombinant technologies for the production of fusion proteins (11)(12)(13)(14). Selective modification has also been reported for such chemically based methods as reductive amination of oxidized mAb carbohydrates (15), photoaffinity labeling of unconventional mAb binding sites (16), and reduction-alkylation of antibody interchain disulfides (17,18 We have previously described the preparation of mAb-drug conjugates for use as antitumor agents (17,19). The potent a...
Modification of the potent fibrinogen receptor (alpha(IIb)beta(3)) antagonist 1 generated compounds with high affinity for the vitronectin receptor alpha(v)beta(3). Sequential modification of the basic N-terminus of 1 led to the identification of the 5,6,7, 8-tetrahydro[1,8]naphthyridine moiety (THN) as a lipophilic, moderately basic N-terminus that provides molecules with excellent potency and selectivity for the integrin receptor alpha(v)beta(3). The THN-containing analogue 5 is a potent inhibitor of bone resorption in vitro and in vivo. In addition, the identification of a novel, nonpeptide radioligand with high affinity to alpha(v)beta(3) is also reported.
The design, synthesis, and pharmacological evaluation of L-734,217, a potent, low-molecular weight, orally active fibrinogen receptor antagonist, is reported. A strategy for producing low-molecular weight inhibitors from the peptide c-[(Ac)CRGDC] A, previously reported from these laboratories, is outlined. This strategy combines a retrodesign analysis of the conformationally defined cyclic peptide A with stereochemical information present in the arginine-glycine-aspartic acid (RGD) tripeptide sequence, culminating with the discovery of L-734,217. L-734,217 inhibited the aggregation of human, dog, and chimpanzee platelets at concentrations below 100 nM and was found to be > 15000-fold less effective at inhibiting the attachment of human umbilical vein endothelial cells to fibrinogen, fibronectin, and vitronectin than it was at inhibiting the aggregation of platelets. L-734,217 showed significant ex vivo antiplatelet activity following oral administration in dogs and chimpanzees at doses of 1.0 and 2.0 mg/kg, respectively, and has been selected as a clinical candidate for development as an antithrombotic agent.
Features which recur in the active site of enzymes, unrelated by evolution, are particularly worthy of chemical modelling. It is essential, however, that these small organic molecules maintain the spatial relationships found in the enzymic system. Models of the serine proteases' (e.g., I23, 22b, 32c) orient the anti lone pair of the carboxylate toward the imidazole, in contrast to the serine proteases,3 malate and lactate dehydrogenase,4 thermolysin,5 and
The nickel(0)-catalyzed oligomerization of 1,3-dienes holds a prominent place in the history and practice of organometallic chemistry as one of the first and most extensively studied transition-metal-catalyzed, C-C bond-forming reactions.1 In the case of buta-1,3-diene, this process selectively affords four-, six-, eight-, or twelve-membered rings, depending on catalyst ligands and reaction conditions.13 Notwithstanding its enormous synthetic potential, this chemistry has not been applied to complex molecule synthesis, due in part to the low regio-and/or stereoselectivity observed in the reactions of unsymmetrical dienes,2 the inefficiency of crossed oligomerizations,1 and the general observation that substituted dienes are far less reactive than butadiene.1 We now report a solution to these problems in the form of the first intramolecular nickel(0)-catalyzed cycloaddition and the initial study of stereoinduction in this reaction class. This investigation provides the basis for an efficient and direct [4 + 4] cycloaddition3 approach to cyclooctane-containing polycycles which are generally accessible only through multistep sequences based on fragmentation and expansion reactions.4 Illustrative of the efficacy of this method for complex polycycle synthesis, tetraene l,5 quantitatively prepared from diethyl malonate and bromopentadiene, when treated in toluene at 60 °C with 11 mol % Ni(COD)2 and 33 mol % Ph3P, gave cyclooctadienes 2a and 2b6 (19:1, respectively) in 70% yield. The products of six-membered ring formation (3) and of /3-hydride elimination (4) were also obtained in 2.6% and 12% yields, respectively. Variations in the nickel-phosphine ratio and in the ligand structure resulted in lower yields of cyclooctadiene products and preferential formation of 3 and 4. For example, reactions conducted with Ni(COD)2 and tri-o-tolyl phosphite (1:3, retí) For reviews and lead references, see: (a) Jolly, P.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.