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.

The efficacy of protein-based medicines can be compromised by their rapid clearance from the blood circulatory system. Achieving optimal pharmacokinetics is a key requirement for the successful development of safe protein-based medicines. Protein PEGylation is a clinically proven strategy to increase the circulation half-life of protein-based medicines. One limitation of PEGylation is that there are few strategies that achieve site-specific conjugation of PEG to the protein. Here, we describe the covalent conjugation of PEG site-specifically to a polyhistidine tag (His-tag) on a protein. His-tag site-specific PEGylation was achieved with a domain antibody (dAb) that had a 6-histidine His-tag on the C-terminus (dAb-His(6)) and interferon α-2a (IFN) that had an 8-histidine His-tag on the N-terminus (His(8)-IFN). The site of PEGylation at the His-tag for both dAb-His(6)-PEG and PEG-His(8)-IFN was confirmed by digestion, chromatographic, and mass-spectral studies. A methionine was also inserted directly after the N-terminal His-tag in IFN to give His(8)Met-IFN. Cyanogen bromide digestion studies of PEG-His(8)Met-IFN were also consistent with PEGylation at the His-tag. By using increased stoichiometries of the PEGylation reagent, it was possible to conjugate two separate PEG molecules to the His-tag of both the dAb and IFN proteins. Stability studies followed by in vitro evaluation confirmed that these PEGylated proteins retained their biological activity. In vivo PK studies showed that all of the His-tag PEGylated samples displayed extended circulation half-lives. Together, our results indicate that site-specific, covalent PEG conjugation at a His-tag can be achieved and biological activity maintained with therapeutically relevant proteins.

Lipoyl synthase (LipA) is required for the final step in the biosynthesis of lipoyl groups, the insertion of sulfur atoms at C6 and C8 of octanoyl groups (Scheme 1).[1] The octanoyl groups are found attached through an amide linkage to a lysine residue in a sequence motif that is conserved within a small family of protein domains [2] that include the H-protein of the glycine cleavage system [3] and the E2 subunit of oxoacid dehydrogenases. [4] After attachment of the octanoyl groups, [5][6][7] the substrates undergo sulfur insertion by LipA, [1] a member of the "radical S-adenosyl l-methionine" family.[8] Biochemical studies have shown that it contains two essential [4Fe-4S] clusters [9] and that one of these clusters is used to generate 5'-deoxyadenosyl radicals (AdoC) through the reductive cleavage of S-adenosyl l-methionine (AdoMet). The lipoyl forming reaction requires two hydrogen atoms to be removed from the octanoyl group, one from C6 and the other from C8, so that the overall reaction utilizes two equivalents of AdoMet to form each lipoyl group. [10] LipA has been shown to donate both of the inserted sulfur atoms [11] that were proposed to originate from a [4Fe-4S] cluster.[12] Recently, we reported that a short octanoyl peptide, which corresponds in sequence to the lipoylation site of the E2 subunit, could function as a substrate for LipA.[13] Peptide substrates have now been used to investigate the order of sulfur-insertion steps and hence clarify the structure of a key intermediate on the reaction pathway.The formation of lipoyl groups was monitored in a reaction by using Sulfolobus solfataricus P2 LipA with an octanoyl substrate tripeptide (0.5 mole equiv; 3, ), AdoMet, and dithionite as a reductant. LipA is not catalytic during assays in vitro, producing substoichiometric quantities of lipoyl products with either octanoyl-protein [10] or octanoylpeptide substrates.[13] To investigate the formation of any intermediate species, the reaction was stopped by acidification before reaching completion (after 20 min), the precipitated protein was pelleted by centrifugation, and the supernatant analyzed by LCMS. Four peptide species were eluted over the time range from 22 to 25 min (Table 1). The peptide at 24.4 min corresponds to the unreacted octanoyl substrate 3, whereas new species at 22.4 and 23.4 min correspond to the expected protonated product masses of lipoyl 5 and dihydrolipoyl products 4, respectively. The final species at 23.2 min coelutes with the dihydrolipoyl product and corresponds to a monothiolated species (either 6 or 7). Analysis of later time points (up to 2 h) showed that the amount of this monothiolated species decreased with time and that there was a Scheme 1. Insertion of sulfur atoms into octanoyl substrates to form lipoyl groups.

Many clinically used protein therapeutics are modified to increase their efficacy. Example modifications include the conjugation of cytotoxic drugs to monoclonal antibodies or poly(ethylene glycol) (PEG) to proteins and peptides. Monothiol-specific conjugation can be efficient and is often accomplished using maleimide-based reagents. However, maleimide derived conjugates are known to be susceptible to exchange reactions with endogenous proteins. To address this limitation in stability, we have developed PEG-mono-sulfone 3, which is a latently reactive, monothiol selective conjugation reagent. Comparative reactions with PEG-maleimide and other common thiol-selective PEGylation reagents including vinyl sulfone, acrylate, and halo-acetamides show that PEG-mono-sulfone 3 undergoes more efficient conjugation under mild reaction conditions. Due to the latent reactivity of PEG-mono-sulfone 3, its reactivity can be tailored and, once conjugated, the electron-withdrawing ketone is easily reduced under mild conditions to prevent undesirable deconjugation and exchange reactions from occurring. We describe a comparative stability study demonstrating a PEG-maleimide conjugate to be more labile to deconjugation than the corresponding conjugate obtained using PEG-mono-sulfone 3.