Kim K. Emmerton

Intracellular Released Payload Influences Potency and Bystander-Killing Effects of Antibody-Drug Conjugates in Preclinical Models

Antibody-drug conjugates (ADC) comprise targeting antibodies armed with potent small-molecule payloads. ADCs demonstrate specific cell killing in clinic, but the basis of their antitumor activity is not fully understood. In this study, we investigated the degree to which payload release predicts ADC activity in vitro and in vivo ADCs were generated to target different receptors on the anaplastic large cell lymphoma line L-82, but delivered the same cytotoxic payload (monomethyl auristatin E, MMAE), and we found that the intracellular concentration of released MMAE correlated with in vitro ADC-mediated cytotoxicity independent of target expression or drug:antibody ratios. Intratumoral MMAE concentrations consistently correlated with the extent of tumor growth inhibition in tumor xenograft models. In addition, we developed a robust admixed tumor model consisting of CD30(+) and CD30(-) cancer cells to study how heterogeneity of target antigen expression, a phenomenon often observed in cancer specimens, affects the treatment response. CD30-targeting ADC delivering membrane permeable MMAE or pyrrolobenzodiazepine dimers demonstrated potent bystander killing of neighboring CD30(-) cells. In contrast, a less membrane permeable payload, MMAF, failed to mediate bystander killing in vivo, suggesting local diffusion and distribution of released payloads represents a potential mechanism of ADC-mediated bystander killing. Collectively, our findings establish that the biophysical properties and amount of released payloads are chief factors determining the overall ADC potency and bystander killing. Cancer Res; 76(9); 2710-9. ©2016 AACR.

Orthogonal Cysteine Protection Enables Homogeneous Multi‐Drug Antibody–Drug Conjugates

Abstract A strategy for the preparation of homogeneous antibody–drug conjugates (ADCs) containing multiple payloads has been developed. This approach utilizes sequential unmasking of cysteine residues with orthogonal protection to enable site‐specific conjugation of each drug. In addition, because the approach utilizes conjugation to native antibody cysteine residues, it is widely applicable and enables high drug loading for improved ADC potency. To highlight the benefits of ADC dual drug delivery, this strategy was applied to the preparation of ADCs containing two classes of auristatin drug‐linkers that have differing physiochemical properties and exert complementary anti‐cancer activities. Dual‐auristatin ADCs imparted activity in cell line and xenograft models that are refractory to ADCs comprised of the individual auristatin components. This work presents a facile method for construction of potent dual‐drug ADCs and demonstrates how delivery of multiple cytotoxic warheads can lead to improved ADC activities. Lastly, we anticipate that the conditions utilized herein for orthogonal cysteine unmasking are not restricted to ADCs and can be broadly utilized for site‐specific protein modification.

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 982: Development of homogeneous dual-drug ADCs: Application to the co-delivery of auristatin payloads with complementary antitumor activities

A common theme in treating cancer is the use of combination chemotherapy, where multiple drugs with different mechanisms of action are combined to elicit synergistic activity or overcome differential drug sensitivities. Antibody-drug conjugates (ADCs) have emerged as a powerful approach for treating cancer, combining the tumor targeting specificity of monoclonal antibodies with the potent cell-killing activity of cytotoxic drugs. Like other therapies, these agents are increasingly being tested in combination with unconjugated, clinically approved anticancer agents. In addition, emerging data demonstrates that insensitivity to a particular ADC can be overcome through delivery of a different payload using the same antibody. For these reasons, the development of ADCs that can deliver two complementary payloads to a tumor would likely be a significant advancement in ADC technology. To enable dual-drug conjugation, we utilized a multiplexing drug carrier that contains cysteine residues with orthogonal protecting groups and identified novel conditions for utilization of these protecting groups on a folded protein. Sequential cysteine unmasking enables discrimination between conjugation sites to allow for site-specific drug conjugation. This strategy provides homogeneous ADCs bearing 16 total drugs per antibody, split evenly between the two drug linkers. Importantly, this strategy is flexible, as it does not require engineered antibodies or custom enzymes for drug-linker conjugation. To demonstrate the potential benefits of ADC dual drug delivery, this strategy was applied to the construction of ADCs bearing two classes of auristatin drug linkers that have different physiochemical properties and complementary anti-cancer activities. Dual-auristatin ADCs were tested in cell line and xenograft models that have differential sensitivities to the individual auristatin components, including those with heterogeneous antigen expression or high levels of drug efflux transporters. The data from these studies demonstrate that the dual-auristatin ADCs were active on cells and tumors that are refractory to treatment with either of the individual component drugs. This work highlights the potential for delivering two synergistic or complementary payloads on a single ADC and presents a flexible method for constructing dual-drug ADCs with site-specific and homogeneous drug loading. Citation Format: Matthew R. Levengood, Xinqun Zhang, Kim K. Emmerton, Joshua H. Hunter, Peter D. Senter. Development of homogeneous dual-drug ADCs: Application to the co-delivery of auristatin payloads with complementary antitumor activities [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 982. doi:10.1158/1538-7445.AM2017-982

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.

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 5507: Characterization of ADC bystander killing in admixed tumor model

We have previously described an admixed tumor model, consisting of the CD30+ ALCL cell line Karpas 299 and CD30- Karpas-35R (SGN-35 resistant) cells, to study the phenomenon of ‘bystander killing’ of antigen negative tumor cells by antibody-drug conjugates (ADCs) in tumors with a heterogeneous target expression profile. Here we study the contribution of payload membrane permeability and target expression in bystander killing. We hypothesize that membrane permeability of payloads may impact bystander killing in addition to their intrinsic potency. Monomethyl auristatin E (MMAE)-based ADCs utilizing the cleavable linkers valine-citrulline (vc) or glucuronide (gluc) release the membrane permeable MMAE. As a result, antiCD30-vcMMAE and antiCD30-glucMMAE showed similar bystander killing in vivo. We then compared the more membrane permeable payloads, MMAE and pyrrolobenzodiazepine dimer (PBD), to those that are less permeable, monomethyl auristatin F (MMAF) and auristatin T (AT), as antiCD30 conjugates in this model. We found that antiCD30-AT and antiCD30-vcMMAF killed only CD30+ tumor cells in admixed tumors, while antiCD30-MMAE, antiCD30-GlucMMAE and antiCD30-PBD killed both CD30+ and CD30- cells. These results suggest that membrane permeability is one of the key factors determining in vivo bystander killing. To elucidate the role of antigen positive cells in bystander killing in this model, we varied the implant ratio of CD30+ cells and evaluated ADC activity in vivo. Flow cytometric analysis and CD30 immunohistochemistry confirmed a corresponding change of CD30+ cell percentage in the resulting admixed tumors (∼40%, 70%, or 80%). Treatment with ADCs in these tumors demonstrated that antiCD30-vcMMAE induced remissions in tumors with 70 or 80% CD30+ cells, whereas antiCD30-PBD produced remissions in all three admixed tumor ratios. Collectively, these data confirm that membrane permeable payloads can kill antigen negative cells in vivo. We hypothesize that the enhanced bystander killing by PBD-based ADCs may result from the intrinsic potency of the small molecule and/or membrane permeability. This information will be useful to consider when designing future ADCs. Citation Format: Fu Li, Kim Kissler Emmerton, Mechthild Jonas, Xinqun Zhang, Che-Leung Law. Characterization of ADC bystander killing in admixed tumor model. [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 5507. doi:10.1158/1538-7445.AM2015-5507