Antibody-drug conjugate therapy entails targeted killing of cancer cells with cytotoxic compounds covalently linked to tumor-specific antibodies and shows promise in the treatment of several human cancers. Current antibody-drug conjugate designs that incorporate a disulfide linker between the antibody and cytotoxic drug are inspired by indirect evidence suggesting that the redox potential within the endosomal system is reducing. It is presumed that antigen-dependent endocytosis leads to disulfide linker reduction and intracellular release of free drug, but direct demonstration of such a mechanism is lacking. To determine whether the disulfide N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP) linker would be reduced during endocytic recycling of the anti-HER2 antibody trastuzumab (Herceptin, Genentech), we synthesized a trastuzumab-SPP-Rhodamine red conjugate and developed a linker cleavage assay by using the self-quenching property of this fluorophore. In breast carcinoma SKBr3 cells, no SPP linker cleavage was observed, as detected by fluorescence dequenching upon internalization. By contrast, the conjugate did display fluorescence dequenching when diverted to the lysosomal pathway by geldanamycin, an effect partly due to proteolytic degradation rather than disulfide reduction. To understand why linker reduction was inefficient, we measured redox potentials of endocytic compartments by expressing a redox-sensitive variant of GFP fused to various endocytic proteins. Unexpectedly, we found that recycling endosomes, late endosomes, and lysosomes are not reducing, but oxidizing and comparable with conditions in the endoplasmic reticulum. These results suggest that intracellular reduction is unlikely to account for the potency of disulfide-linked antibodydrug conjugates.disulfide linker ͉ redox potential ͉ endocytosis ͉ HER2 ͉ Herceptin O ne approach to the treatment of cancer is to specifically target cytotoxic drugs to tumor cells by linking them via a cleavable linker to antibodies that recognize a tumor-restricted antigen. Such linkers include hydrazone linkers, designed to hydrolyze upon internalization into acidic endosomes and lysosomes (1-3); peptide linkers optimized for cleavage by certain lysosomal proteases (3-5); and disulfide linkers, thought to be cleaved by the reducing environment within the endocytic pathway (6-10). In the latter category, the monoclonal antibody C242 against CanAg (a glycotope on the mucin1 (MUC1) colorectal tumor antigen) has shown efficacy against colorectal xenograft models in vivo when disulfide-linked to the ribosomal inhibitor ricin A chain via a 4-succinimdyloxycarbonyl-methyl-␣-[2-pyridyldithio]-toluene (SMPT) linker (11) and to the maytansinoid-derived microtubule active drug DM1 via an N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP) linker (12). The latter conjugate is now humanized and in clinical trials as cantuzumab mertansine (9, 13). An ideal linker should be cleaved only upon internalization of the antibody-drug conjugate into the tumor cell, thereby specifical...

Antibody-drug conjugates (ADC), potent cytotoxic drugs covalently linked to antibodies via chemical linkers, provide a means to increase the effectiveness of chemotherapy by targeting the drug to neoplastic cells while reducing side effects. Here, we systematically examine the potential targets and linker-drug combinations that could provide an optimal ADC for the treatment for non-Hodgkin's lymphoma. We identified seven antigens (CD19, CD20, CD21, CD22, CD72, CD79b, and CD180) for potential treatment of non-Hodgkin's lymphoma with ADCs. ADCs with cleavable linkers mediated in vivo efficacy via all these targets; ADCs with uncleavable linkers were only effective when targeted to CD22 and CD79b. In target-independent safety studies in rats, the uncleavable linker ADCs showed reduced toxicity, presumably due to the reduced release of free drug or other toxic metabolites into the circulation. Thus, our data suggest that ADCs with cleavable linkers work on a broad range of targets, and for specific targets, ADCs with uncleavable linkers provide a promising opportunity to improve the therapeutic window for

Delivery of siRNA is a key hurdle to realizing the therapeutic promise of RNAi. By targeting internalizing cell surface antigens, antibody–siRNA complexes provide a possible solution. However, initial reports of antibody–siRNA complexes relied on non-specific charged interactions and have not been broadly applicable. To assess and improve this delivery method, we built on an industrial platform of therapeutic antibodies called THIOMABs, engineered to enable precise covalent coupling of siRNAs. We report that such coupling generates monomeric antibody–siRNA conjugates (ARCs) that retain antibody and siRNA activities. To broadly assess this technology, we generated a battery of THIOMABs against seven targets that use multiple internalization routes, enabling systematic manipulation of multiple parameters that impact delivery. We identify ARCs that induce targeted silencing in vitro and extend tests to target prostate carcinoma cells following systemic administration in mouse models. However, optimal silencing was restricted to specific conditions and only observed using a subset of ARCs. Trafficking studies point to ARC entrapment in endocytic compartments as a limiting factor, independent of the route of antigen internalization. Our broad characterization of multiple parameters using therapeutic-grade conjugate technology provides a thorough assessment of this delivery technology, highlighting both examples of success as well as remaining challenges.

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.

CD22 represents a promising target for antibody-drug conjugate therapy in the context of B cell malignancies since it rapidly internalizes, importing specifically bound antibodies with it. To determine the pharmacokinetic parameters of anti-CD22-MCC-DM1 and MC-MMAF conjugates, various approaches to quantifying total and conjugated antibody were investigated. Although the total antibody assay formats gave similar results for both conjugates, the mouse pharmacokinetic profile for the anti-CD22-MCC-DM1 and MC-MMAF appeared significantly different depending on the conjugated antibody assay format. Since these differences significantly impacted the PK parameters determination, we investigated the effect of the drug/antibody ratio on the total and conjugated antibody quantification using multiple assay formats. Our investigations revealed the limitations of some assay formats to quantify anti-CD22-MCC-DM1 and MC-MMAF with different drug load and in the context of a heterogeneous ADC population highlight the need to carefully plan the assay strategy for the total and conjugated antibody quantification in order to accurately determine the ADC PK parameters.

Based on available data, patients with melanomas between 0.75 and 1.00 mm are appropriate candidates to be considered for sentinel node biopsy after discussing the likelihood of finding evidence of nodal progression, the risks of sentinel node biopsy (including the risk of a false-negative result), and the lack of proven survival benefit from any form of surgical nodal staging.

The preparation of KH as a one:one homogenate with paraffin, termed KH(P), is reported. KH(P), a solid at room temperature, is stable without special handling. On suspension in THF with a phosphonium salt, KH(P) rapidly generates the ylide. Wittig condensation with aromatic, aliphatic and α, β-unsaturated aldehydes proceeds with high Z-selectivity. KH(P) should be a generally useful base for organic synthesis.Sodium hydride (NaH), potassium t-butoxide and n-BuLi have long been the workhorse bases for organic synthesis. Potassium hydride (KH), although it is a powerful base and much faster kinetically than NaH, has not been so widely used. 1-3 Largely, this is because it comes commercially as a slurry in mineral oil, and it is operationally difficult to dispense precisely. We report the preparation of KH in a more convenient form, as a 50% by weight homogenate in paraffin. 4 We have termed this new reagent "KH(P)".There was the real concern that the heat from the reaction of surface KH with ambient moisture would be sufficient to initiate melting, leading to rapid decomposition. In fact, we have found that the KH(P) is stable to normal handling. It is easily cut and weighed in the air. Indeed, we have stored a sample in air for four months, and have observed no loss of titer. The titer was determined by addition of n-butanol and volumetric measurement of the evolved hydrogen.We were pleased to observe (Table 1) that the KH(P) so prepared readily generated ylides from phosphonium salts. 5 The alkenes formed from the non-stabilized phosphoranes were predominantly Z, while the alkene (Entry 5) from the stabilized phosphorane was E. The yields in Table 1 refer to reactions employing 1.8 equivalents of KH(P) and 2.0 equivalents of phosphonium salt.We expect that KH(P), easily handled and measured and stable to storage, will have wide utility in organic synthesis. 6 * Taberdf@udel.edu . Supporting Information available:General experimental procedures, and 1 H and 13 C spectra and other data for all new compounds. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public AccessAuthor Manuscript J Org Chem. Author manuscript; available in PMC 2011 December 30. Experimental Section Potassium hydride in paraffinIn a nitrogen atmosphere (grade 4.8) drybox, KH (35 % w/w dispersion in mineral oil) was washed with cyclohexane and filtered. Paraffin wax (paraffin wax for canning, mp = 48 -50°C , 2.00 g) was warmed to melting in a small cylindrical clear glass vial via a heating mantle. To the melted wax was added KH (2.00 g, 50 mmol) and the slurry was rapidly stirred to homogeneity with a Teflon® coated magnetic stir bar while still warm. Magnetic stirring was continued until the mixture began to solidify, at which time the vial was rolled on its side on a flat surface until the mixture was completely solid. The homogenous 50% w/ w dispersion of KH in paraffin was then capped and removed from the drybox for storage in the laboratory without further protection. [Caution: While we hav...

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