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.

In vitro dose response curves

Supplementary Table 1: Surface antigen density impacts in vitro potency of anti-LIV-1 ADC . MCF-7 ATCC cells from three sources with varying levels of LIV-1 expression were tested with SGN-LIV1A for cytotoxicity. Lower antigen density resulted in decreased potency.

Additional PK measurements and antibody sequences

While antibody-drug conjugates (ADCs) are advancing through clinical testing and receiving new marketing approvals, improvements to the technology continue to be developed in both academic and industrial laboratories. Among the key ADC attributes that can be improved upon with new technology are their biodistribution and pharmacokinetic properties. During the course of ADC development, it has become apparent that conjugation of drugs to the surface of a monoclonal antibody can alter its physicochemical characteristics in a manner that results in increased nonspecific interactions and more rapid elimination from plasma. Researchers in the field have typically relied upon in vivo studies in preclinical models to understand how a particular ADC chemistry will impact these biological characteristics. In previous work, we described how animal studies have revealed a relationship between ADC hydrophobicity, pharmacokinetics, and nonspecific hepatic clearance, particularly by sinusoidal endothelium and Kupffer cells. Here, we describe a fluorescence-based assay using cultured Kupffer cells to recapitulate the nonspecific interactions that lead to ADC clearance in an in vitro setting with the aim of developing a tool for predicting the pharmacokinetics of novel ADC designs. Output from this assay has demonstrated an excellent correlation with plasma clearance for a series of closely related ADCs bearing discrete PEG chains of varying length and has proven useful in interrogating the mechanism of the interactions between ADCs and Kupffer cells.

Abstract To provide a better understanding of the pharmacokinetics–pharmacodynamics relationships of antibody-based drugs, we analyzed several chimeric and humanized monoclonal antibodies or antibody–drug conjugates (ADC) for PK and efficacy among four strains of mice. Notably, antibodies and ADCs displayed a dose-dependent drug disposition profile in the plasma of NSG mice. The increased clearance rate in NSG mice resulted in the reduction of antitumor activity of ADCs. Furthermore, we identified that the abnormal clearance was mediated by Fc–FcγR interaction by comparing antibodies that lack FcγR binding capacity. We also found a high percentage of FcγR-expressing macrophages in the bone marrow, spleen, and liver of NSG mice, which may be responsible for the abnormal distribution of antibodies. Overall, these findings suggest that preclinical evaluation of efficacy and pharmacokinetics of antibodies and ADCs need to consider mouse strain-induced variations.

Native size-exclusion chromatography-mass spectrometry (nSEC-MS) is an analytical methodology that is appropriate for accurately quantitating the drug-to-antibody ratio (DAR) on a wide variety of interchain cysteine-linked antibody-drug conjugates (ADCs), irrespective of chemotype. In the current preclinical environment, novel ADCs conjugated with unique drug-linkers need to progress toward the clinic as quickly as possible. Platform analytical approaches can reduce time-to-clinic because key process development and optimization activities can be decoupled from the development of bespoke, molecule-specific analytical methods. In this work, we assessed the potential of nSEC-MS as a platformable, quantitative DAR method. The nSEC-MS method was evaluated according to performance characteristics and parameters described in the ICH guideline Validation of Analytical Procedures: Text and Methodology Q2(R1). In order to comprehensively assess the accuracy and bias of nSEC-MS DAR quantitation, ADCs were generated using three different drug-linker chemotypes with DARs ranging from 2 to 8. These molecules were tested by hydrophobic interaction chromatography (HIC) and nSEC-MS, and DARs obtained from both methods were compared to assess the degree to which nSEC-MS quantitation aligned with the HIC release assay. Our results indicated that there is no bias introduced by nSEC-MS quantitation of DAR and that SEC-MS data can be bridged to HIC data without the need for a correction factor or offset. nSEC-MS was also found to be suitable for unbiased DAR quantitation in the other ADC chemotypes that were evaluated. Based on the totality of our work, we conclude that, used as intended, nSEC-MS is well suited for quantitating DAR on a variety of interchain cysteine-linked ADCs in an accurate, unbiased manner.

Abstract 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 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 Cmax 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

AbstractAlthough 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 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

Abstract 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.

AbstractA 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.

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 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.

Abstract SGN-LIV1A is an antibody-drug conjugate (ADC) currently being evaluated in a phase 1 clinical trial for metastatic breast cancer. SGN-LIV1A consists of the microtubule disrupting agent, monomethyl auristatin E (MMAE), conjugated to the anti-LIV-1 humanized monoclonal antibody hLIV22. LIV-1, as a downstream target of STAT3, promotes the epithelial to mesenchymal transition that is important in the malignant progression to metastasis. We have previously shown that as a single agent, SGN-LIV1A displays target specific internalization and cytotoxic activity against a breast cancer cell line in vitro and also demonstrates antitumor activity in in vivo preclinical xenograft models with significant delay of tumor growth. We report here additive and synergistic effects when combining SGN-LIV1A with current chemotherapeutic modalities used in the treatment of metastatic breast cancer. Specifically, we show synergy between SGN-LIV1A in combination with either doxorubicin or Abraxane in MCF-7 breast cancer tumor model. In addition, we show additive effects when carboplatin or protein kinase inhibitors are dosed in combination with SGN-LIV1A in this tumor model. These findings support further evaluation and development of SGN-LIV1A in combination with standard of care chemotherapeutic agents for the treatment of breast cancer. Citation Format: Fu Li, Martha Anderson, Joshua Hunter, Jaime Miyamoto, Michelle Ulrich, Ana Kostic, Che-Leung Law, Django Sussman. Preclinical combinations of the antibody-drug conjugate SGN-LIV1A with chemotherapies show increased activity. [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 2966.