Svetlana O. Doronina

Development of potent monoclonal antibody auristatin conjugates for cancer therapy

We describe the in vitro and in vivo properties of monoclonal antibody (mAb)-drug conjugates consisting of the potent synthetic dolastatin 10 analogs auristatin E (AE) and monomethylauristatin E (MMAE), linked to the chimeric mAbs cBR96 (specific to Lewis Y on carcinomas) and cAC10 (specific to CD30 on hematological malignancies). The linkers used for conjugate formation included an acid-labile hydrazone and protease-sensitive dipeptides, leading to uniformly substituted conjugates that efficiently released active drug in the lysosomes of antigen-positive (Ag+) tumor cells. The peptide-linked mAb-valine-citrulline-MMAE and mAb-phenylalanine-lysine-MMAE conjugates were much more stable in buffers and plasma than the conjugates of mAb and the hydrazone of 5-benzoylvaleric acid-AE ester (AEVB). As a result, the mAb-Val-Cit-MMAE conjugates exhibited greater in vitro specificity and lower in vivo toxicity than corresponding hydrazone conjugates. In vivo studies demonstrated that the peptide-linked conjugates induced regressions and cures of established tumor xenografts with therapeutic indices as high as 60-fold. These conjugates illustrate the importance of linker technology, drug potency and conjugation methodology in developing safe and efficacious mAb-drug conjugates for cancer therapy.We describe the in vitro and in vivo properties of monoclonal antibody (mAb)-drug conjugates consisting of the potent synthetic dolastatin 10 analogs auristatin E (AE) and monomethylauristatin E (MMAE), linked to the chimeric mAbs cBR96 (specific to Lewis Y on carcinomas) and cAC10 (specific to CD30 on hematological malignancies). The linkers used for conjugate formation included an acid-labile hydrazone and protease-sensitive dipeptides, leading to uniformly substituted conjugates that efficiently released active drug in the lysosomes of antigen-positive (Ag+) tumor cells. The peptide-linked mAb-valine-citrulline-MMAE and mAb-phenylalanine-lysine-MMAE conjugates were much more stable in buffers and plasma than the conjugates of mAb and the hydrazone of 5-benzoylvaleric acid-AE ester (AEVB). As a result, the mAb-Val-Cit-MMAE conjugates exhibited greater in vitro specificity and lower in vivo toxicity than corresponding hydrazone conjugates. In vivo studies demonstrated that the peptide-linked conjugates induced regressions and cures of established tumor xenografts with therapeutic indices as high as 60-fold. These conjugates illustrate the importance of linker technology, drug potency and conjugation methodology in developing safe and efficacious mAb-drug conjugates for cancer therapy.

Lysosomal Trafficking and Cysteine Protease Metabolism Confer Target-specific Cytotoxicity by Peptide-linked Anti-CD30-Auristatin Conjugates *

The chimeric anti-CD30 monoclonal antibody cAC10, linked to the antimitotic agents monomethyl auristatin E (MMAE) or F (MMAF), produces potent and highly CD30-selective anti-tumor activity in vitro and in vivo. These drugs are appended via a valine-citrulline (vc) dipeptide linkage designed for high stability in serum and conditional cleavage and putative release of fully active drugs by lysosomal cathepsins. To characterize the biochemical processes leading to effective drug delivery, we examined the intracellular trafficking, internalization, and metabolism of the parent antibody and two antibody-drug conjugates, cAC10vc-MMAE and cAC10vc-MMAF, following CD30 surface antigen interaction with target cells. Both cAC10 and its conjugates bound to target cells and internalized in a similar manner. Subcellular fractionation and immunofluorescence studies demonstrated that the antibody and antibody-drug conjugates entering target cells migrated to the lysosomes. Trafficking of both species was blocked by inhibitors of clathrin-mediated endocytosis, suggesting that drug conjugation does not alter the fate of antibody-antigen complexes. Incubation of cAC10vc-MMAE or cAC10vc-MMAF with purified cathepsin B or with enriched lysosomal fractions prepared by subcellular fractionation resulted in the release of active, free drug. Cysteine protease inhibitors, but not aspartic or serine protease inhibitors, blocked antibody-drug conjugate metabolism and the ensuing cytotoxicity of target cells and yielded enhanced intracellular levels of the intact conjugates. These findings suggest that in addition to trafficking to the lysosomes, cathepsin B and perhaps other lysosomal cysteine proteases are requisite for drug release and provide a mechanistic basis for developing antibody-drug conjugates cleavable by intracellular proteases for the targeted delivery of anti-cancer therapeutics.

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.

Novel peptide linkers for highly potent antibody-auristatin conjugate.

Auristatins are highly potent antimitotic agents that have received considerable attention because of their activities when targeted to tumor cells in the form of antibody-drug conjugates (ADCs). Our lead agent, SGN-35, consists of the cAC10 antibody linked to the N-terminal amino acid of monomethylauristatin E (MMAE) via a valine-citrulline p-aminobenzylcarbamate (val-cit-PABC) linker that is cleaved by intracellular proteases such as cathepsin B. More recently, we developed an auristatin F (AF) derivative monomethylauristatin F (MMAF), which unlike MMAE contains the amino acid phenylalanine at the C-terminal position. Because of the negatively charged C-terminal residue, the potency of AF and MMAF is impaired. However, their ability to kill target cells is greatly enhanced through facilitated cellular uptake by internalizing mAbs. Here, we explore the effects of linker technology on AF-based ADC potency, activity, and tolerability by generating a diverse set of dipeptide linkers between the C-terminal residue and the mAb carrier. The resulting ADCs differed widely in activity, with some having significantly improved therapeutic indices compared to the original mAb-Val-Cit-PABC-MMAF conjugate. The therapeutic index was increased yet further by generating dipeptide-based ADCs utilizing new auristatins with methionine or tryptophan as the C-terminal drug residue. These results demonstrate that manipulation of the C-terminal peptide sequence used to attach auristatins to the mAb carrier can lead to highly potent and specific conjugates with greatly improved therapeutic windows.

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.

Lymphocyte Activation Antigen CD70 Expressed by Renal Cell Carcinoma Is a Potential Therapeutic Target for Anti-CD70 Antibody-Drug Conjugates

Metastatic renal cell carcinoma (RCC) is an aggressive disease refractory to most existing therapeutic modalities. Identifying new markers for disease progression and drug targets for RCC will benefit this unmet medical need. We report a subset of clear cell and papillary cell RCC aberrantly expressing the lymphocyte activation marker CD70, a member of the tumor necrosis factor superfamily. Importantly, CD70 expression was found to be maintained at the metastatic sites of RCC. Anti-CD70 antibody-drug conjugates (ADC) consisting of auristatin phenylalanine phenylenediamine (AFP) or monomethyl auristatin phenylalanine (MMAF), two novel derivatives of the anti-tubulin agent auristatin, mediated potent antigen-dependent cytotoxicity in CD70-expressing RCC cells. Cytotoxic activity of these anti-CD70 ADCs was associated with their internalization and subcellular trafficking through the endosomal-lysosomal pathway, disruption of cellular microtubule network, and G2-M phase cell cycle arrest. The efficiency of drug delivery using anti-CD70 as vehicle was illustrated by the much enhanced cytotoxicity of antibody-conjugated MMAF compared with free MMAF. Hence, ADCs targeted to CD70 can selectively recognize RCC, internalize, and reach the appropriate subcellular compartment(s) for drug release and tumor cell killing. In vitro cytotoxicity of these ADCs was confirmed in xenograft models using RCC cell lines. Our findings provide evidence that CD70 is an attractive target for antibody-based therapeutics against metastatic RCC and suggest that anti-CD70 ADCs can provide a new treatment approach for advanced RCC patients who currently have no chemotherapeutic options.

Efficient elimination of B-lineage lymphomas by anti-CD20-auristatin conjugates.

The anti-CD20 antibody rituximab is useful in the treatment of certain B-cell malignancies, most notably non-Hodgkin’s lymphoma. Its efficacy has been increased when used in combination with chemotherapy, yet anti-CD20 monoclonal antibodies (mAbs) directly conjugated with drugs such as doxorubicin (Dox) have failed to deliver drug or to demonstrate antitumor activity. We have produced anti-CD20 antibody-drug conjugates that possess potent antitumor activity by using the anti-mitotic agent, monomethyl auristatin E (MMAE), linked via the lysosomally cleavable dipeptide, valine-citrulline (vc). Two anti-CD20 conjugates, rituximab-vcMMAE and 1F5-vcMMAE, were selectively cytotoxic against CD20+ B-lymphoma cell lines, with IC50 values ranging from 50 ng/mL to 1 μg/mL. Unlike rituximab, which showed diffuse surface localization, rituximab-vcMMAE capped and was internalized within 4 hours after binding to CD20+ B cells. Internalization of rituximab-vcMMAE was followed by rapid G2-M phase arrest and onset of apoptosis. Anti-CD20 antibody-drug conjugates prepared with Dox were internalized and localized as with rituximab-vcMMAE, yet these were not effective for drug delivery (IC50 > 50 μg/mL). Consistent with in vitro activity, rituximab-vcMMAE showed antitumor efficacy in xenograft models of CD20-positive lymphoma at doses where rituximab or rituximab-Dox conjugates were ineffective. These data indicate that anti-CD20–based antibody-drug conjugates are effective antitumor agents when prepared with a stable, enzyme-cleavable peptide linkage to highly potent cytotoxic agents such as MMAE.

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 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 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