Jocelyn R. Setter

Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates

Many antibody-drug conjugates (ADCs) are unstable in vivo because they are formed from maleimide-containing components conjugated to reactive thiols. These thiosuccinimide linkages undergo two competing reactions in plasma: elimination of the maleimide through a retro-Michael reaction, which results in loss of drug-linker from the ADC, and hydrolysis of the thiosuccinimide ring, which results in a derivative that is resistant to the elimination reaction. In an effort to create linker technologies with improved stability characteristics, we used diaminopropionic acid (DPR) to prepare a drug-linker incorporating a basic amino group adjacent to the maleimide, positioned to provide intramolecular catalysis of thiosuccinimide ring hydrolysis. This basic group induces the thiosuccinimide to undergo rapid hydrolysis at neutral pH and room temperature. Once hydrolyzed, the drug-linker is no longer subject to maleimide elimination reactions, preventing nonspecific deconjugation. In vivo studies demonstrate that the increased stability characteristics can lead to improved ADC antitumor activity and reduced neutropenia.

Reducing hydrophobicity of homogeneous antibody-drug conjugates improves pharmacokinetics and therapeutic index

The in vitro potency of antibody-drug conjugates (ADCs) increases with the drug-to-antibody ratio (DAR); however, ADC plasma clearance also increases with DAR, reducing exposure and in vivo efficacy. Here we show that accelerated clearance arises from ADC hydrophobicity, which can be modulated through drug-linker design. We exemplify this using hydrophilic auristatin drug linkers and PEGylated ADCs that yield uniform, high-DAR ADCs with superior in vivo performance.

Conjugation of Anticancer Drugs Through Endogenous Monoclonal Antibody Cysteine Residues

Many methods have been described for the conjugation of drugs to monoclonal antibodies. The presence of a discrete number of readily reducible disulfides in the common IgG subtypes presents a convenient opportunity for conjugation to cysteine residues with thiol-reactive drug-linkers. Such conjugates can be prepared by a straightforward two-step reaction scheme involving the reduction of the antibody disulfides to the desired number of average thiols per antibody, followed by addition of the drug-linker, ideally with a maleimido functionality for rapid, selective reaction. In a discovery setting, this basic method can be scaled down to produce microgram quantities of conjugate for early screening, and in a manufacturing setting can be scaled up to produce grams or kilograms of conjugate for clinical trials and commercialization. The resulting conjugates are readily characterized using common HPLC methods.

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.

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.

Pironetin Binds Covalently to αCys316 and Perturbs a Major Loop and Helix of α-Tubulin to Inhibit Microtubule Formation

Microtubule-targeting agents are among the most powerful drugs used in chemotherapy to treat cancer patients. Pironetin is a natural product that displays promising anticancer properties by binding to and potently inhibiting tubulin assembly into microtubules; however, its molecular mechanism of action remained obscure. Here, we solved the crystal structure of the tubulin-pironetin complex and found that the compound covalently binds to Cys316 of α-tubulin. The structure further revealed that pironetin perturbs the T7 loop and helix H8 of α-tubulin. Since both these elements are essential for establishing longitudinal tubulin contacts in microtubules, this result explains how pironetin inhibits the formation of microtubules. Together, our data define the molecular details of the pironetin binding site on α-tubulin and thus offer a promising basis for the rational design of pironetin variants with improved activity profiles. They further extend our knowledge on strategies evolved by natural products to target and perturb the microtubule cytoskeleton.

Abstract 3694: Relationship between in vivo antitumor activity of ADC and payload release in preclinical models

Antibody-drug conjugates (ADC) deliver cytotoxic payloads to target cells via receptor-mediated internalization. We have demonstrated that SGN-35 (cAC10-vcMMAE) binds to CD30 on target cells and releases intracellular monomethyl auristatin E (MMAE) in vitro, contributing to potent killing of CD30+ cells. Using a set of auristatin-based ADCs, we evaluated whether the intratumoral concentration of the released payload is a predictive indicator of antitumor activity in preclinical models. We first treated CD30+, anaplastic large cell lymphoma (ALCL) Karpas 299 tumors with SGN-35 or a non-binding control ADC (IgG-vcMMAE). The plasma ADC and MMAE PK measurements were similar in SGN-35 and IgG-vcMMAE treated tumor-bearing animals. In contrast, SGN-35 generated 5-fold greater intratumoral MMAE exposure than IgG-vcMMAE, which correlated with tumor regression observed in the SGN-35- treated group. We then examined the relationship between released MMAE and in vitro cytotoxicity by targeting different receptors expressed on the same cells. The lymphoma cell line L82 expresses CD30, CD70, and CD71 receptors. Although auristatin-based ADCs targeting each of these targets has a different IC50, the intracellular MMAE was similar when 50% growth inhibition was achieved (approximately 200 nM). In vivo, CD30, CD70, or CD71-directed ADCs demonstrated similar antitumor activity for a given dose level (0.5, 1, or 3 mg/kg). Concordantly, equal intratumoral MMAE concentrations were observed for a given dose level, regardless of the antigen target. The cell permeable MMAE has been shown to mediate bystander killing in vitro. To evaluate whether intratumoral MMAE mediates bystander killing in vivo, an admixed tumor model consisting CD30+ and CD30- Karpas 299 cells was treated with SGN-35. As predicted, both CD30+ and CD30- tumor cells were killed, presumably due to intratumoral diffusion of MMAE across cell membranes. In contrast, a conjugate with a highly polar form of auristatin (MMAF) was unable to kill the CD30-negative cell population of the admixed tumors. These preclinical results provide evidence that the concentration of released MMAE within the tumor correlates with the ADC antitumor activity in vitro and in vivo. Moreover, the in vivo bystander activity of MMAE may improve utility of this chemotype in tumors with heterogeneous antigen expression. Citation Format: Fu Li, Xinqun Zhang, Kim Emmerton, Mechthild Jonas, Jocelyn Setter, Bill Arthur, Nicole Okeley, Robert Lyon, Dennis Benjamin, Che-Leung Law. Relationship between in vivo antitumor activity of ADC and payload release in preclinical models. [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 3694. doi:10.1158/1538-7445.AM2014-3694

Abstract 4333: Self-stabilizing ADCs: antibody-drug conjugates prepared with maleimido drug-linkers that catalyze their own thiosuccinimide ring hydrolysis.

Many antibody-drug conjugates (ADCs) currently in clinical trials employ maleimide-containing drug-linkers which are conjugated to antibody cysteine residues to form thiosuccinimide linkages. It is now known that these thiosuccinimide linkages can undergo two competing reactions while in plasma: elimination of the maleimide resulting in undesirable loss of drug from the ADC, and hydrolysis of the thiosuccinimide ring resulting in a succinic acid derivative which cannot undergo elimination. Thus, thiosuccinimide ring hydrolysis is a reaction which stabilizes the chemical linkage of the drug to the antibody. We have engineered a new class of drug-linkers which incorporate a basic amino group adjacent to the maleimide, providing intramolecular base catalysis of the ring hydrolysis. This basic group induces the thiosuccinimide to undergo rapid self-catalyzed hydrolysis at neutral pH and room temperature, with complete hydrolysis achieved in less than 2 hours. Once hydrolyzed, the drug-linker is no longer subject to maleimide elimination reactions, preventing loss of drug from the antibody by this mechanism and forming a highly stable ADC. To compare these new drug-linkers to traditional maleimido drug-linkers, a number of in vitro and in vivo studies have been conducted. Results of these studies will be presented to demonstrate a robust improvement in drug-linker stability compared to traditional drug-linker formats. Citation Format: Robert P. Lyon, Jocelyn R. Setter, Tim D. Bovee, Svetlana O. Doronina, Martha E. Anderson, Chris L. Leiske, Peter D. Senter. Self-stabilizing ADCs: antibody-drug conjugates prepared with maleimido drug-linkers that catalyze their own thiosuccinimide ring hydrolysis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4333. doi:10.1158/1538-7445.AM2013-4333

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 4470: Elucidating the role of drug-linker hydrophobicity in the disposition of antibody-drug conjugates

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The in vitro potency of antibody-drug conjugates (ADCs) is generally proportional to the level of drug loading, with higher drug per antibody levels producing more potent ADCs. However, this intuitive relationship often fails to translate in vivo, presumably because plasma clearance of the ADC also increases with drug loading, resulting in substantially reduced exposure to highly loaded ADCs (Hamblett, KJ et al, Clin. Cancer Res. 2004, 10: 7063-7070). We postulated that this accelerated clearance effect is not purely a function of the number of drugs per antibody, but also of the intrinsic hydrophobicity of each drug-linker. Collectively, drug-linker hydrophobicity and number of drugs per antibody determine the overall hydrophobicity of the ADC. To test this hypothesis, we designed and prepared a series of drug-linkers within the auristatin class that varied in hydrophobicity, and used them to produce a corresponding series of homogeneous 8-loaded ADCs. The pharmacokinetic profiles of these ADCs were determined and found to correlate well with their relative retention times by hydrophobic interaction chromatography. We further postulated that hydrophobicity-driven clearance is likely to be mediated by cells of the mononuclear phagocytic system (MPS). To identify the specific cell populations responsible for accelerated ADC clearance, we performed an immunohistochemistry-based biodistribution study which has provided clear evidence for the cellular sites of hydrophobic ADC clearance which are not operative on native antibodies or hydrophilic ADCs. Using the information gained from these studies, we have successfully prepared a hydrophilic auristatin drug-linker which enables the preparation of ADCs uniformly loaded with 8 drugs per antibody at defined cysteine sites. These ADCs evade clearance by the MPS, maintain native antibody pharmacokinetics, and translate their high in vitro potency into the in vivo setting. Citation Format: Svetlana O. Doronina, Jocelyn R. Setter, Tim D. Bovee, Martha E. Anderson, Mechtild Jonas, Steven Daniho, Heather Kostner, Peter D. Senter, Robert P. Lyon. Elucidating the role of drug-linker hydrophobicity in the disposition of antibody-drug conjugates. [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 4470. doi:10.1158/1538-7445.AM2014-4470