Previous investigations on antibody-drug conjugate (ADC) stability have focused on drug release by linker-deconjugation due to the relatively stable payloads such as maytansines. Recent development of ADCs has been focused on exploring technologies to produce homogeneous ADCs and new classes of payloads to expand the mechanisms of action of the delivered drugs. Certain new ADC payloads could undergo metabolism in circulation while attached to antibodies and thus affect ADC stability, pharmacokinetics, and efficacy and toxicity profiles. Herein, we investigate payload stability specifically and seek general guidelines to address payload metabolism and therefore increase the overall ADC stability. Investigation was performed on various payloads with different functionalities (e.g., PNU-159682 analog, tubulysin, cryptophycin, and taxoid) using different conjugation sites (HC-A118C, LC-K149C, and HC-A140C) on THIOMAB antibodies. We were able to reduce metabolism and inactivation of a broad range of payloads of THIOMAB antibody-drug conjugates by employing optimal conjugation sites (LC-K149C and HC-A140C). Additionally, further payload stability was achieved by optimizing the linkers. Coupling relatively stable sites with optimized linkers provided optimal stability and reduction of payloads metabolism in circulation in vivo.

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A novel strategy to attach indole-containing payloads to antibodies through a carbamate moiety and a self-immolating, disulfide-based linker is described. This new strategy was employed to connect a selective estrogen receptor down-regulator (SERD) to various antibodies in a site-selective manner. The resulting conjugates displayed potent, antigen-dependent down-regulation of estrogen receptor levels in MCF7-neo/HER2 and MCF7-hB7H4 cells. They also exhibited similar antigen-dependent modulation of the estrogen receptor in tumors when administered intravenously to mice bearing MCF7-neo/HER2 tumor xenografts. The indole-carbamate moiety present in the new linker was stable in whole blood from various species and also exhibited good in vivo stability properties in mice.

A number of cytotoxic pyrrolobenzodiazepine (PBD) monomers containing various disulfide-based prodrugs were evaluated for their ability to undergo activation (disulfide cleavage) in vitro in the presence of either glutathione (GSH) or cysteine (Cys). A good correlation was observed between in vitro GSH stability and in vitro cytotoxicity toward tumor cell lines. The prodrug-containing compounds were typically more potent against cells with relatively high intracellular GSH levels (e.g., KPL-4 cells). Several antibody-drug conjugates (ADCs) were subsequently constructed from PBD dimers that incorporated selected disulfide-based prodrugs. Such HER2 conjugates exhibited potent antiproliferation activity against KPL-4 cells in vitro in an antigen-dependent manner. However, the disulfide prodrugs contained in the majority of such entities were surprisingly unstable toward whole blood from various species. One HER2-targeting conjugate that contained a thiophenol-derived disulfide prodrug was an exception to this stability trend. It exhibited potent activity in a KPL-4 in vivo efficacy model that was approximately three-fold weaker than that displayed by the corresponding parent ADC. The same prodrug-containing conjugate demonstrated a three-fold improvement in mouse tolerability properties in vivo relative to the parent ADC, which did not contain the prodrug.

Azithromycin has been widely used for the treatment of . However, the drug resistance of for azithromycin is currently increasing. The aim of the present study was to analyze the association between gene subtypes of and drug resistance for azithromycin. The gene subtypes of were assayed by a polymerase chain reaction technique. Drug resistance of was analyzed using an antimicrobial susceptibility test. The results demonstrated that gene type presented higher drug resistance compared with and gene types of . Gene type was identified as eight gene subtypes (14a/f, 14e/f, 12e/f, 12d/f, 6d/f, 11d/f, 14j/f and 8d/f) among 324 cases. It was identified that 23S rRNA A2058G mutation was observed in gene subtypes 14a/f, 14e/f and 12e/f. A2059G mutation occurred in the gene subtypes 8d/f, 12d/f, 6d/f, 11d/f and 14j/f. The proportions of azithromycin-resistant genotypes harboring either the A2058G or the A2059G mutation among the strains were 34.2 and 65.8%, respectively. The antimicrobial susceptibility test demonstrated that A2059G mutations exhibited a higher drug resistance for azithromycin compared with A2058G mutations. In conclusion, these results indicate that azithromycin resistance in is associated with gene subtype, which may contribute to the treatment of .