To improve both the homogeneity and the stability of ADCs, we have developed site-specific drug-conjugating reagents that covalently rebridge reduced disulfide bonds. The new reagents comprise a drug, a linker, and a bis-reactive conjugating moiety that is capable of undergoing reaction with both sulfur atoms derived from a reduced disulfide bond in antibodies and antibody fragments. A disulfide rebridging reagent comprising monomethyl auristatin E (MMAE) was prepared and conjugated to trastuzumab (TRA). A 78% conversion of antibody to ADC with a drug to antibody ratio (DAR) of 4 was achieved with no unconjugated antibody remaining. The MMAE rebridging reagent was also conjugated to the interchain disulfide of a Fab derived from proteolytic digestion of TRA, to give a homogeneous single drug conjugated product. The resulting conjugates retained antigen-binding, were stable in serum, and demonstrated potent and antigen-selective cell killing in in vitro and in vivo cancer models. Disulfide rebridging conjugation is a general approach to prepare stable ADCs, which does not require the antibody to be recombinantly re-engineered for site-specific conjugation.

The conjugation of monomethyl auristatin E (MMAE) to trastuzumab using a reduction bis-alkylation approach that is capable of rebridging reduced (native) antibody interchain disulfide bonds has been previously shown to produce a homogeneous and stable conjugate with a drug-to-antibody ratio (DAR) of 4 as the major product. Here, we further investigate the potency of the DAR 4 conjugates prepared by bis-alkylation by comparing to lower drug loaded variants to maleimide linker based conjugates possessing typical mixed DAR profiles. Serum stability, HER2 receptor binding, internalization, in vitro potency, and in vivo efficacy were all evaluated. Greater stability compared with maleimide conjugation was observed with no significant decrease in receptor/FcRn binding. A clear dose-response was obtained based on drug loading (DAR) with the DAR 4 conjugate showing the highest potency in vitro and a much higher efficacy in vivo compared with the lower DAR conjugates. Finally, the DAR 4 conjugate demonstrated superior efficacy compared to trastuzumab-DM1 (T-DM1, Kadcyla), as evaluated in a low HER2 expressing JIMT-1 xenograft model.

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Runoff and pond water samples from a container nursery that recycles water for irrigation were tested for movement of herbicides from the site of application. Residues of oryzalin [4‐(dipropylamino)‐3,5‐dinitrobenzene‐sulfonamide], pendimethalin [N‐(1‐ethylpropyl)‐3,4‐dimethyl‐2,6‐dinitrobenzamine]), and oxyfluorfen [2‐chloro‐1‐(3‐ethoxy‐4‐nitrophenoxy)‐4‐(trifluoromethyl)benzene], active ingredients in the granular formulations of Rout [2‐chloro‐1‐(3‐ethoxy‐4‐nitrophenoxy)‐4‐(trifluoromethyl)benzene+3,5‐dinitro‐N4,N4‐dipropylsulfanilamide] and OH‐2 (Ornamental Herbicide‐2), were evaluated. Herbicides were applied at labeled rates and followed by irrigation. Water samples were collected, herbicides were extracted by solid phase extraction and analyzed by reverse phase high pressure liquid chromatography with ultraviolet (UV) detection. Maximum herbicide residues were detected within the first 15 min of water runoff; oryzalin residues were the greatest of the three herbicides evaluated (4 mg L−1 water at 15 min) and showed rapid decreases thereafter. Herbicide residues detected in pond samples decreased over time until the detection limit was reached (2 wk following application). A microplot study was conducted to evaluate the effects of plastic, woven fabric or gravel bedcovers on herbicide movement. Plastic and fabric allowed greatest movement of oryzalin and pendimethalin, while gravel significantly retained and retarded movement of all three herbicides. Results indicate that bedcover composition plays a significant role in the movement of herbicide from the site of application. Release of active ingredient from granular formulations was evaluated; dinitroanilines (oryzalin and pendimethalin) release faster than oxyfluorfen. Oryzalin in Rout was the most rapidly released, is the most water soluble, and 71% of total active ingredient was accounted for after 3 wk.