Plasma stability of quaternary ammonium linker in rodent plasma.

Pharmacokinetic profile of clearance for three ADCs.

Experimental details for the chemical synthesis of the drug-linkers.

The recent clinical success of antibody-drug conjugates (ADCs) has spawned an increased effort to identify new technologies, and the development of new drug-linker chemistry is vital to expand the scope of conjugatable payloads. The tertiary amine functional group is a common structural motif present in many bioactive compounds, including antimitotics of the auristatin and tubulysin classes. Traditionally, conjugation of tertiary amines required drug derivatization or modification to remove an N-alkyl group, thus creating a readily conjugatable secondary amine. However, identifying appropriate modifications that do not compromise the activity of the drug is frequently time consuming and often unsuccessful. To eliminate the need for such structural modifications, we sought a method for stable conjugation and facile release through the tertiary amine functional group by creating linkers with a quaternary amine point of attachment. To validate the linker strategy, quaternary amine-based cleavable linkers bearing auristatin E were synthesized and evaluated as ADCs. The conjugates were stable in rodent plasma, and were potent and immunologically specific both in vitro and in vivo in a Hodgkin lymphoma xenograft model. A second application of this technology has been demonstrated with tubulysins, another class of potent antimitotics containing a tertiary amine at the N-terminus. A cleavable quaternary amine linker containing a tubulysin analog was synthesized and ADCs were prepared and evaluated. The tubulysin conjugates were potent and immunologically specific across a panel of cancer cell lines, including multiple MDR-positive lines. Furthermore, the tubulysin conjugate displayed ‘bystander activity’ in an in vitro co-culture assay. The quaternary amine linkers represent an advance in linker technology and will enable the evaluation of drug classes previously inaccessible as ADCs. Citation Format: Patrick J. Burke, Joseph Z. Hamilton, Thomas A. Pires, Jocelyn R. Setter, Joshua H. Hunter, Julia H. Cochran, Brian E. Toki, Peter D. Senter, Robert P. Lyon, Scott C. Jeffrey. Development of quaternary amine linkers for ADCs: Application to auristatin E and tubulysin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2056.

Abstract A quaternary ammonium-based drug-linker has been developed to expand the scope of antibody–drug conjugate (ADC) payloads to include tertiary amines, a functional group commonly present in biologically active compounds. The linker strategy was exemplified with a β-glucuronidase–cleavable auristatin E construct. The drug-linker was found to efficiently release free auristatin E (AE) in the presence of β-glucuronidase and provide ADCs that were highly stable in plasma. Anti-CD30 conjugates comprised of the glucuronide-AE linker were potent and immunologically specific in vitro and in vivo, displaying pharmacologic properties comparable with a carbamate-linked glucuronide-monomethylauristatin E control. The quaternary ammonium linker was then applied to a tubulysin antimitotic drug that contained an N-terminal tertiary amine that was important for activity. A glucuronide-tubulysin quaternary ammonium linker was synthesized and evaluated as an ADC payload, in which the resulting conjugates were found to be potent and immunologically specific in vitro, and displayed a high level of activity in a Hodgkin lymphoma xenograft. Furthermore, the results were superior to those obtained with a related tubulysin derivative containing a secondary amine N-terminus for conjugation using previously known linker technology. The quaternary ammonium linker represents a significant advance in linker technology, enabling stable conjugation of payloads with tertiary amine residues. Mol Cancer Ther; 15(5); 938–45. ©2016 AACR.

The role that carbohydrates play in antibody function and pharmacokinetics has made them important targets for modification. The terminal fucose of the N-linked glycan structure, which has been shown to be involved in modulation of antibody-directed cellular cytotoxicity, is a particularly interesting location for potential modification through incorporation of alternative sugar structures. A library of fucose analogues was evaluated for their ability to incorporate into antibody carbohydrates in place of the native fucose. A number of efficiently incorporated molecules were identified, demonstrating the ability of fucosyltransferase VIII to utilize a variety of non-natural sugars as substrates. Among these structures was a thiolated analogue, 6-thiofucose, which was incorporated into the antibody carbohydrate with good efficiency. This unnatural thio-sugar could then be used for conjugation using maleimide chemistry to produce antibody-drug conjugates with pronounced cytotoxic activities and improved homogeneity compared to drug attachment through hinge disulfides.

The key role played by fucose in glycoprotein and cellular function has prompted significant research toward identifying recombinant and biochemical strategies for blocking its incorporation into proteins and membrane structures. Technologies surrounding engineered cell lines have evolved for the inhibition of in vitro fucosylation, but they are not applicable for in vivo use and drug development. To address this, we screened a panel of fucose analogues and identified 2-fluorofucose and 5-alkynylfucose derivatives that depleted cells of GDP-fucose, the substrate used by fucosyltransferases to incorporate fucose into protein and cellular glycans. The inhibitors were used in vitro to generate fucose-deficient antibodies with enhanced antibody-dependent cellular cytotoxicity activities. When given orally to mice, 2-fluorofucose inhibited fucosylation of endogenously produced antibodies, tumor xenograft membranes, and neutrophil adhesion glycans. We show that oral 2-fluorofucose treatment afforded complete protection from tumor engraftment in a syngeneic tumor vaccine model, inhibited neutrophil extravasation, and delayed the outgrowth of tumor xenografts in immune-deficient mice. The results point to several potential therapeutic applications for molecules that selectively block the endogenous generation of fucosylated glycan structures.

Antibody-drug conjugates (ADCs) were prepared with potent camptothecin analogues attached to monoclonal antibodies (mAbs) via dipeptide or glucuronide-based linkers. Aniline-containing camptothecin analogues were employed to provide a site of linker attachment via carbamate bonds that would be stable in circulation. The camptothecin analogues, 7-butyl-10-amino-camptothecin and 7-butyl-9-amino-10,11-methylenedioxy-camptothecin, are generally 10-1000 times more potent than camptothecin. Dipeptide and glucuronide drug linkers were employed containing self-immolative spacers that release drug following lysosomal degradation upon ADC internalization into antigen-positive cell lines. The camptothecin drug linkers were conjugated to three antibodies: chimeric BR96, chimeric AC10, and humanized 1F6, which bind to the Lewis-Y antigen on carcinomas, CD30 on hematologic malignancies, and CD70 present on hematologic malignancies and renal cell carcinoma, respectively. ADCs bearing the potent camptothecin analogue, 7-butyl-9-amino-10,11-methylenedioxy-camptothecin, were highly potent and immunologically specific on a panel of cancer cell lines in vitro, and efficacious at well-tolerated doses in a renal cell carcinoma xenograft model.

Cytotoxic agents streptonigrin and 17-amino-geldanamycin were linked to monoclonal antibodies (mAbs), forming antibody-drug conjugates (ADCs) for antigen-mediated targeting to cancer cells. The drugs were conjugated with a linker construct that is labile to lysosomal proteases and incorporates a valine-alanine-p-aminobenzyl (PAB)-amino linkage for direct attachment to the electron-deficient amine functional groups present in both drugs. The resulting ADCs release drug following internalization into antigen-positive cancer cells. The drug linkers were conjugated to mAbs cAC10 (anti-CD30) and h1F6 (anti-CD70) via alkylation of reduced interchain disulfides to give ADCs loaded with 4 drugs/mAb. The streptonigrin ADCs were potent and immunologically specific on a panel of cancer cell lines in vitro and in a Hodgkin lymphoma xenograft model. We conclude that streptonigrin ADCs are candidates for further research, and that the novel linker system used to make them is well-suited for the conjugation of cytotoxic agents containing electron-deficient amine functional groups.

623 A great deal of interest has surrounded the use of monoclonal antibodies (mAbs) for the selective delivery of cytotoxic agents to tumor cells. Although the approach is conceptually appealing, several limitations have been identified, including the physiological barriers to mAb extravasation and intratumoral penetration, conjugate immunogenicity, non-specific conjugate uptake, low drug potency, and inefficient release of active drug. In previous work (Doronina, et al. Nature Biotech. 7, 778-784, 2003; Francisco, et al. Blood, 102, 1458-1465, 2003), we addressed some of these issues in developing cAC10-valine-citrulline-MMAE, a cathepsin B cleavable conjugate that delivers the highly potent antimitotic agent monomethyl auristatin E (MMAE) inside CD30 positive tumor cells recognized by the chimeric mAb, cAC10. One of the advantages in using MMAE as the drug component, is that being totally synthetic, it is amenable to extensive modification. Here, we describe novel analogues of MMAE that are designed for enhanced efficacy and lower toxicity. MMAF is an acidic form of MMAE that has reduced cytotoxic activity compared to MMAE, most likely due to impaired intracellular uptake. MMAF is significantly less potent than MMAE on a diverse panel of tumor cell lines, and has a >6-fold increased MTD in mice. However, mAb-valine-citrulline-MMAF conjugates are >10 times more potent than corresponding MMAE conjugates, presumably due to intracellular drug entrapment. Further evidence that this mechanism of enhanced potency stems from the finding that mAb-valine-citrulline-MMAF conjugates are as much as 400-fold more potent on antigen positive cells than the free MMAF drug. A phenylenediamine adduct of the auristatin acid, AFP, was also found to be highly potent when linked to mAbs through the cathepsin cleavable valine-citrulline linker. Upon intracellular uptake the mAb-valine-citrulline-AFP conjugates are subject to hydrolysis of the phenylenediamine unit, leading to the release of active AF. These new conjugates, comprised of an active drug that has impaired cellular permeability, are highly active and potent. Treatment of mice with low and well-tolerated doses of these conjugates leads to cures and regressions of established carcinoma and hematologic tumor xenografts. The results illustrate how mAbs can be used to enhance the potency of relatively impermeable drugs through active intracellular delivery.