Julia H. Cochran

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.

The Methylene Alkoxy Carbamate Self‐Immolative Unit: Utilization for the Targeted Delivery of Alcohol‐Containing Payloads with Antibody–Drug Conjugates

A strategy for the conjugation of alcohol-containing payloads to antibodies has been developed and involves the methylene alkoxy carbamate (MAC) self-immolative unit. A series of MAC β-glucuronide model constructs were prepared to evaluate stability and enzymatic release, and the results demonstrated high stability at physiological pH in a substitution-dependent manner. All the MAC model compounds efficiently released alcohol drug surrogates under the action of β-glucuronidase. To assess the MAC technology for ADCs, the potent microtubule-disrupting agent auristatin E (AE) was incorporated through the norephedrine alcohol. Conjugation of the MAC β-glucuronide AE drug linker to the anti-CD30 antibody cAC10, and an IgG control antibody, gave potent and immunologically specific activities in vitro and in vivo. These studies validate the MAC self-immolative unit for alcohol-containing payloads within ADCs, a class that has not been widely exploited.

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 60: Reducing toxicity of antibody-drug conjugates through modulation of pharmacokinetics

Antibody-drug conjugates (ADCs) continue to emerge as effective therapeutics in a variety of oncology indications. Research on ADCs has revealed that the physicochemical properties of the drug-linker component can exert a significant impact on the disposition of the ADCs, particularly at higher levels of drug loading. We have recently reported (Nature Biotechnology 33, 733-735 (2015); Molecular Cancer Therapeutics, manuscript accepted) that these properties can be modulated through the judicious incorporation of small, discrete PEG chains of varying lengths into a monomethyl auristatin E (MMAE) drug-linker. Homogeneous DAR 8 ADCs prepared with these drug-linkers using native cysteine conjugation display a continuum of pharmacokinetic behaviors that mirror the length of the incorporated PEG chain. We selected four of these drug-linkers that span the range of observed pharmacokinetics and used them as model compounds to evaluate the impact of ADC clearance on the concentration profile of released MMAE in normal tissues and consequent toxicology in the Sprague-Dawley rat. Faster clearing ADCs (prepared with drug-linkers containing very short or no PEG modifier) produced higher tissue MMAE C max values at early post-dose time points relative to slower clearing ADCs that incorporate longer PEG chains. This finding indicates that MMAE concentrations in tissues are proportional to the rate at which the ADC is catabolized, a process which converts the conjugated payload into free drug. Faster clearing ADCs also exhibited diminished tolerability, with greater histologic depletion of bone marrow and more dramatic decreases and/or delayed recovery in select peripheral hematology parameters. These results provide a strategy for reducing the non-antigen-mediated toxicity of ADCs through modulation of pharmacokinetics. Citation Format: Jessica Simmons, Francisco Zapata, Haley Neff-Laford, Joshua Hunter, Julia Cochran, Patrick Burke, Robert P. Lyon. Reducing toxicity of antibody-drug conjugates through modulation of pharmacokinetics [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 60. doi:10.1158/1538-7445.AM2017-60

Lactone Stabilization is Not a Necessary Feature for Antibody Conjugates of Camptothecins

Camptothecins exist in a pH-dependent equilibrium between the active, closed lactone and the inactive open-carboxylate forms. Several previous reports underscore the need for lactone stabilization in generating improved camptothecins, and indeed, such designs have been incorporated into antibody-drug conjugates containing this drug. Here, we demonstrate that lactone stabilization is not necessary for camptothecin-based ADC efficacy. We synthesized and evaluated camptothecin SN-38 drug linkers that differed with respect to lactone stability and released SN-38 or the hydrolyzed open-lactone form upon cleavage from the antibody carrier. An α-hydroxy lactone-linked SN-38 drug linker preserved the closed-lactone ring structure, while the phenol-linked version allowed conversion between the closed-lactone and open-carboxylate structures. The in vitro cytotoxicity, pharmacokinetic properties, and in vivo efficacy in the L540cy Hodgkins lymphoma model of the corresponding ADCs were found to be indistinguishable, leading us to conclude that camptothecin-based antibody-drug conjugates possess pronounced activity regardless of the lactone state of the bound drug. This is most likely a result of ADC processing within acidic intracellular vesicles, delivering camptothecin in its active closed-lactone form.

Abstract 4075: Effect of PEG chain length on antibody-drug conjugate tumor and tissue distribution in tumor bearing xenograft mice

Antibody-drug conjugates (ADCs) are a promising new therapeutic modality having demonstrated clinical efficacy in the treatment of cancer. While ADCs have better exposures than their constitutive chemotypes, it is important that they maintain the favorable pharmacokinetics of their antibody backbones. Previous work incorporating PEG into the drug-linker as a sidechain helped to mask the hydrophobicity of the chemotypes, resulting in improved ADC exposures in rats compared to ADCs with non-PEGylated drug-linkers. The current studies were aimed at understanding the effect of PEG chain length on ADC efficacy, tumor and tissue distribution in tumor bearing mice. Efficacy and biodistribution of radiolabeled ADCs with 2, 4, 8, 12 and 24 PEG units in the drug-linker as a side chain were investigated in SCID mice bearing a subcutaneous xenograft of L540cy tumor cells. A non-PEGylated ADC was used as control. Antibody based radioactivity in plasma, tumor, liver, lung, kidney, spleen and brain were measured over 14 days. MMAE, the free drug released from the ADC, was measured by mass spectrometry. Reduction in tumor weight was determined as a measure of efficacy. Compartmental analysis of the plasma exposure data was conducted to understand the change in rate constants with change in PEG chain length. ADCs with side chain PEGylated drug-linkers had greater plasma and tumor exposures than the non-PEGylated control ADC. Increasing PEG chain length in the linker led to increased plasma and tumor exposures, and lower plasma clearances. Tumor distribution was binary with ADCs with 2 and 4 PEG units in the linker showing similar tumor exposures while the ADCs with 8, 12 and 24 PEG units providing similar but significantly higher tumor exposures. ADCs with 8, 12 and 24 PEG units in the linker had significantly higher tumor to plasma exposure ratios than ADCs with 2 and 4-PEG units in the drug-linker. The reduction in tumor weights was also binary. ADCs with 2 and 4 PEG units provided a 35-45% decrease in tumor weights while ADCs with 8, 12 and 24 PEG units in the linker provided 75-85% reduction in tumor weights. The non-PEGylated control ADC decreased the tumor weights by 11%. In general, PEGylated ADCs increased the extent of tissue distribution, but the tissue to plasma ratios remained the same indicating that the increased tissue exposures was a function of increased plasma exposures. However, PEGylated ADC tumor exposure increased beyond that expected based solely on the increase in plasma exposure. Incorporation of PEG as a side chain into the drug-linker improved the tumor disposition of ADCs with little alteration in tissue disposition. ADCs with 8, 12 and 24 PEG units in the drug- linker showed preferential uptake into tumor, but not the tissues as evidenced by tumor (or tissue) to plasma exposure ratios. Over all, the ADCs with 8, 12 and 24 PEG units in the linker provided the optimal combination of tumor distribution, efficacy and tissue distribution. Citation Format: Nagendra V. Chemuturi, Martha Anderson, Julia Cochran, Thomas Pires, Josh Hunter, David Meyer, Jason Neale, Alice Chin, Paul Pittman, Cindy Balasubramanian, Francisco Zapata, Jessica Simmons, Robert Lyon, Steve Alley. Effect of PEG chain length on antibody-drug conjugate tumor and tissue distribution in tumor bearing xenograft mice [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 4075. doi:10.1158/1538-7445.AM2017-4075

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.