Joseph Z. Hamilton

Development of novel quaternary ammonium linkers for antibody-drug conjugates

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.

Optimization of a PEGylated Glucuronide-Monomethylauristatin E Linker for Antibody–Drug Conjugates

The emergence of antibody–drug conjugates (ADC), such as brentuximab vedotin and ado-trastuzumab emtansine, has led to increased efforts to identify new payloads and develop improved drug-linker technologies. Most antibody payloads impart significant hydrophobicity to the ADC, resulting in accelerated plasma clearance and suboptimal in vivo activity, particularly for conjugates with high drug-to-antibody ratios (DAR). We recently reported on the incorporation of a discrete PEG24 polymer as a side chain in a β-glucuronidase-cleavable monomethylauristatin E (MMAE) linker to provide homogeneous DAR 8 conjugates with decreased plasma clearance and increased antitumor activity in xenograft models relative to a non-PEGylated control. In this work, we optimized the drug-linker by minimizing the size of the PEG side chain and incorporating a self-stabilizing maleimide to prevent payload de-conjugation in vivo. Multiple PEG-glucuronide-MMAE linkers were prepared with PEG size up to 24 ethylene oxide units, and homogeneous DAR 8 ADCs were evaluated. A clear relationship was observed between PEG length and conjugate pharmacology when tested in vivo. Longer PEG chains resulted in slower clearance, with a threshold length of PEG8 beyond which clearance was not impacted. Conjugates bearing PEG of sufficient length to minimize plasma clearance provided a wider therapeutic window relative to faster clearing conjugates bearing shorter PEGs. A lead PEGylated glucuronide-MMAE linker was identified incorporating a self-stabilizing maleimide and a PEG12 side chain emerged from these efforts, enabling highly potent, homogeneous DAR 8 conjugates and is under consideration for future ADC programs. Mol Cancer Ther; 16(1); 116–23. ©2016 AACR.

Abstract 56: Antibody-drug conjugates containing glucuronide-tubulysin payloads display activity in MDR+ and heterogeneous tumor models

While antibody-drug conjugates (ADCs) find increasing application in cancer treatment regimens, de novo or treatment-emergent resistance mechanisms could impair clinical benefit. Two resistance mechanisms that emerge under continuous ADC exposure in vitro include upregulation of transporters that confer multidrug resistance (MDR+) and loss of cognate antigen expression. New technologies that circumvent these resistance mechanisms may serve to extend the utility of next generation ADCs. Recently, we developed the quaternary ammonium linker system to expand the scope of conjugatable payloads to include tertiary amine-containing compounds and applied the linker to tubulysins, a highly potent class of microtubule disrupting agents that maintain activity in MDR+ cell lines. Quaternary ammonium-linked glucuronide-tubulysin drug-linkers were synthesized and evaluated as ADCs. The resulting conjugates were potent and immunologically specific across a panel of cancer cell lines, including those displaying the MDR phenotype. The ADCs also demonstrate potent bystander activity in a co-culture model containing a mixture of antigen-positive and -negative cell lines. Incorporation of a PEG12 side chain in the linker enabled loading at 8-drugs/Ab for increased in vivo potency while maintaining suitable ADC pharmacokinetic properties. In vivo, the glucuronide-tubulysin conjugates displayed activity in MDR+ xenograft models and bystander activity in an admixed Ag+/Ag- heterogeneous tumor model. Thus, the glucuronide-tubulysin drug-linkers represent a promising new payload for ADCs, combining conjugate potency in the presence of the MDR phenotype with robust activity in models of tumor antigen heterogeneity. Citation Format: Patrick J. Burke, Joseph Z. Hamilton, Joshua H. Hunter, Julia H. Cochran, Thomas A. Pires, Christopher I. Leiske, Kim K. Emmerton, Peter D. Senter, Robert P. Lyon, Scott C. Jeffrey. Antibody-drug conjugates containing glucuronide-tubulysin payloads display activity in MDR+ and heterogeneous tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 56. doi:10.1158/1538-7445.AM2017-56

Abstract 648: Optimization of a PEGylated glucuronide-auristatin linker for antibody-drug conjugates

The clinical success of antibody-drug conjugates (ADCs) brentuximab vedotin and ado-trastuzumab emtansine has led to an increased effort to identify new antibody payloads and develop improved linker technologies. Most antibody payloads impart significant hydrophobicity to the ADC resulting in accelerated plasma clearance and diminished in vivo activity, particularly for conjugates with high drug to antibody ratios (DAR). Consequently, drug-linkers have been designed to minimize or mask hydrophobicity, enabling a higher DAR and improved pharmacokinetic properties, translating to enhanced in vivo potency. We recently reported on the incorporation of a discrete PEG polymer as a side chain in the β-glucuronide-monomethylauristatin E (MMAE) linker to provide homogeneous DAR 8 conjugates with decreased plasma clearance and increased antitumor activity in xenograft models relative to a non-PEGylated control. The PEG-glucuronide-MMAE lead has now been optimized by minimizing the size of the PEG side chain and incorporating a self-stabilizing maleimide to prevent payload de-conjugation in vivo. Multiple PEG-glucuronide-MMAE linker constructs were prepared with PEG size varying from 0 to 24 ethylene oxide units, and uniform DAR 8 ADCs were evaluated. PEG size had small but variable effects in vitro, with diminished activity observed in some cell lines. In contrast, a clear relationship was observed between PEG length and plasma pharmacokinetics. Longer PEG chains resulted in slower clearance, with a ‘threshold’ length beyond which clearance was not impacted. Conjugates bearing PEG of sufficient length to minimize ADC plasma clearance provided a wider therapeutic window relative to conjugates bearing shorter PEGs. Thus, sufficient masking of drug hydrophobicity is a viable strategy to provide uniform DAR 8 ADCs that preserve ADC pharmacokinetics and increase potency through higher drug loading. Citation Format: Patrick J. Burke, Joseph Z. Hamilton, Scott C. Jeffrey, Joshua H. Hunter, Svetlana O. Doronina, Nicole M. Okeley, Martha E. Anderson, Peter D. Senter, Robert P. Lyon. Optimization of a PEGylated glucuronide-auristatin linker for antibody-drug conjugates. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 648. doi:10.1158/1538-7445.AM2015-648

Abstract 4465: Differential MMAE delivery from ADCs utilizing the valine-citrulline-PAB and β-glucuronide cleavable linker systems

The previously described β-glucuronide linker system (Jeffrey S.C. et al, Bioconj. Chem. 17, 831-840, 2006) was designed to release the toxic payload from an antibody-drug conjugate via the activity of β-glucuronidase, a known lysosomal enzyme. This linker system was shown to be suitable for the generation of potent ADCs utilizing the microtubule disrupting agent monomethylauristatin E (MMAE) as the delivered payload, and is thus an alternative to the protease-cleavable linker valine-citrulline-p-amino benzyl alcohol (vc-PAB). We have recently observed that ADCs utilizing the β-glucuronide linker system to deliver an MMAE payload exhibit potent cytotoxic activity in some cell lines that are resistant to conjugates utilizing the vc-PAB linker to deliver the same drug. To better understand this phenomenon, we have used mass spectrometry to compare the concentration and distribution of MMAE released from both glucuronide and vc-PAB linked ADCs in cell culture. Our data demonstrate that cellular accumulation of released MMAE is considerably greater when delivered by a glucuronide ADC relative to a vc-PAB ADC. We have also employed live-cell fluorescence microscopy to monitor the process of enzymatic cleavage of glucuronide and vc-PAB conjugates. The results of these studies suggest that the two linker systems deliver drug via distinct cellular mechanisms, possibly due to unique intracellular locations of drug release. We propose a model for how these unique linker systems can lead to differences in cellular accumulation of free drug and ultimately increased cytotoxicity. Citation Format: Nicole M. Okeley, Heather A. VanEpps, Xinqun Zhang, Jocelyn R. Setter, Patrick J. Burke, Joseph Z. Hamilton, Robert P. Lyon. Differential MMAE delivery from ADCs utilizing the valine-citrulline-PAB and β-glucuronide cleavable linker systems. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4465. doi:10.1158/1538-7445.AM2014-4465

Abstract 1786: Development and pharmacological properties of PEGylated glucuronide-auristatin linkers

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Antibody-drug conjugates (ADCs) are an emerging therapeutic modality for the treatment of cancer, exemplified by the recent clinical success of brentuximab vedotin and ado-trastuzumab emtansine. The majority of ADCs currently in development include functionalities that are hydrophobic, resulting in increased plasma clearance. Consequently, there has been significant interest in generating new linkers that compensate for this potential liability. In an attempt to mitigate the pharmacokinetic impact of monomethylauristatin E (MMAE) when conjugated to an antibody, we prepared drug-linkers incorporating a polyethylene glycol (PEG) polymer. Initial work demonstrated that incorporation of a discrete PEG24 polymer into the cleavable β-glucuronide-MMAE linker system could either increase or decrease the plasma clearance of the resulting ADCs, depending upon the configuration of the PEG. When inserted into the drug-linker as a stretcher unit between the maleimide and the cleavage site, PEG24 elicited increased ADC plasma clearance. Conversely, incorporation of the PEG24 as a side chain from a modified lysine residue adjacent to the maleimide resulted in ADCs with slower clearance. These differences in ADC pharmacokinetics translated into corresponding differences in antitumor activity in two xenograft models. Thus, incorporation of PEG24 into ADC linkers impacts both pharmacokinetics and activity in a structure-dependent manner. Citation Format: Patrick J. Burke, Joseph Z. Hamilton, Joshua H. Hunter, Scott C. Jeffrey, Svetlana O. Doronina, Nicole M. Okeley, David W. Meyer, Peter D. Senter, Robert P. Lyon. Development and pharmacological properties of PEGylated glucuronide-auristatin linkers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1786. doi:10.1158/1538-7445.AM2014-1786

Glucuronide-Linked Antibody–Tubulysin Conjugates Display Activity in MDR+ and Heterogeneous Tumor Models

Although antibody–drug conjugates (ADCs) find increasing applications in cancer treatment, de novo or treatment-emergent resistance mechanisms may impair clinical benefit. Two resistance mechanisms that emerge under prolonged exposure include upregulation of transporter proteins that confer multidrug resistance (MDR+) and loss of cognate antigen expression. New technologies that circumvent these resistance mechanisms may serve to extend the utility of next-generation ADCs. Recently, we developed the quaternary ammonium linker system to expand the scope of conjugatable payloads to include tertiary amines and applied the linker to tubulysins, a highly potent class of tubulin binders that maintain activity in MDR+ cell lines. In this work, tubulysin M, which contains an unstable acetate susceptible to enzymatic hydrolysis, and two stabilized tubulysin analogues were prepared as quaternary ammonium-linked glucuronide-linkers and assessed as ADC payloads in preclinical models. The conjugates were potent across a panel of cancer cell lines and active in tumor xenografts, including those displaying the MDR+ phenotype. The ADCs also demonstrated potent bystander activity in a coculture model comprised of a mixture of antigen-positive and -negative cell lines, and in an antigen-heterogeneous tumor model. Thus, the glucuronide–tubulysin drug-linkers represent a promising ADC payload class, combining conjugate potency in the presence of the MDR+ phenotype and robust activity in models of tumor heterogeneity in a structure-dependent manner. Mol Cancer Ther; 17(8); 1752–60. ©2018 AACR.

Abstract 2056: Development of quaternary amine linkers for ADCs: Application to auristatin E and tubulysin

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 2956: Optimal PEGylation of an auristatin linker provides ADCs with improved pharmacological properties

As antibody-drug conjugates (ADCs) continue to emerge as an important therapeutic modality for the treatment of cancer, there is an increased effort to elucidate critical design parameters and devise improved linker technologies. The impact of drug-to-antibody ratio (DAR) on conjugate plasma pharmacokinetics (PK) is known to be an important attribute, and accelerated clearance induced by high levels of drug loading has served as a barrier to translating increased in vitro potency to in vivo xenografts. We have recently demonstrated that the incorporation of a discrete PEG24 unit into an auristatin drug-linker can greatly diminish the impact of drug loading on ADC PK. In an effort to optimize the antibody-mediated delivery of monomethylauristatin E (MMAE) as a homogeneous DAR 8 conjugate, we prepared a series of MMAE linkers using PEG units of varying lengths to identify constructs that preserve antibody PK properties and provide enhanced in vivo activity. The extent of PEGylation and linker chemistry was found to impact conjugate PK properties, biodistribution, antitumor activity, and tolerability. From that effort, a cleavable MMAE linker incorporating the glucuronide-based trigger, a self-stabilizing maleimide, and 12 PEG units emerged as the optimal design. ADCs prepared with this linker have now undergone further preclinical characterization in activity and toxicology models in which they have demonstrated an increase in therapeutic index relative to other MMAE-based ADCs. Citation Format: Patrick J. Burke, Joseph Z. Hamilton, Scott C. Jeffrey, Joshua H. Hunter, Julia H. Cochran, Nagendra Chemuturi, Martha E. Anderson, Peter D. Senter, Robert P. Lyon. Optimal PEGylation of an auristatin linker provides ADCs with improved pharmacological properties. [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 2956.