David W. Meyer

SGN-CD33A: a novel CD33-targeting antibody–drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML

Outcomes in acute myeloid leukemia (AML) remain unsatisfactory, and novel treatments are urgently needed. One strategy explores antibodies and their drug conjugates, particularly those targeting CD33. Emerging data with gemtuzumab ozogamicin (GO) demonstrate target validity and activity in some patients with AML, but efficacy is limited by heterogeneous drug conjugation, linker instability, and a high incidence of multidrug resistance. We describe here the development of SGN-CD33A, a humanized anti-CD33 antibody with engineered cysteines conjugated to a highly potent, synthetic DNA cross-linking pyrrolobenzodiazepine dimer via a protease-cleavable linker. The use of engineered cysteine residues at the sites of drug linker attachment results in a drug loading of approximately 2 pyrrolobenzodiazepine dimers per antibody. In preclinical testing, SGN-CD33A is more potent than GO against a panel of AML cell lines and primary AML cells in vitro and in xenotransplantation studies in mice. Unlike GO, antileukemic activity is observed with SGN-CD33A in AML models with the multidrug-resistant phenotype. Mechanistic studies indicate that the cytotoxic effects of SGN-CD33A involve DNA damage with ensuing cell cycle arrest and apoptotic cell death. Together, these data suggest that SGN-CD33A has CD33-directed antitumor activity and support clinical testing of this novel therapeutic in patients with AML.

A Potent Anti-CD70 Antibody-Drug Conjugate Combining a Dimeric Pyrrolobenzodiazepine Drug with Site-Specific Conjugation Technology

A highly cytotoxic DNA cross-linking pyrrolobenzodiazepine (PBD) dimer with a valine-alanine dipeptide linker was conjugated to the anti-CD70 h1F6 mAb either through endogenous interchain cysteines or, site-specifically, through engineered cysteines at position 239 of the heavy chains. The h1F6239C-PBD conjugation strategy proved to be superior to interchain cysteine conjugation, affording an antibody-drug conjugate (ADC) with high uniformity in drug-loading and low levels of aggregation. In vitro cytotoxicity experiments demonstrated that the h1F6239C-PBD was potent and immunologically specific on CD70-positive renal cell carcinoma (RCC) and non-Hodgkin lymphoma (NHL) cell lines. The conjugate was resistant to drug loss in plasma and in circulation, and had a pharmacokinetic profile closely matching that of the parental h1F6239C antibody capped with N-ethylmaleimide (NEM). Evaluation in CD70-positive RCC and NHL mouse xenograft models showed pronounced antitumor activities at single or weekly doses as low as 0.1 mg/kg of ADC. The ADC was tolerated at 2.5 mg/kg. These results demonstrate that PBDs can be effectively used for antibody-targeted therapy.

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.

Design, Synthesis, and Biological Evaluation of Antibody-Drug Conjugates Comprised of Potent Camptothecin Analogues

Antibody-drug conjugates (ADCs) were prepared with potent camptothecin analogues attached to monoclonal antibodies (mAbs) via dipeptide or glucuronide-based linkers. Aniline-containing camptothecin analogues were employed to provide a site of linker attachment via carbamate bonds that would be stable in circulation. The camptothecin analogues, 7-butyl-10-amino-camptothecin and 7-butyl-9-amino-10,11-methylenedioxy-camptothecin, are generally 10-1000 times more potent than camptothecin. Dipeptide and glucuronide drug linkers were employed containing self-immolative spacers that release drug following lysosomal degradation upon ADC internalization into antigen-positive cell lines. The camptothecin drug linkers were conjugated to three antibodies: chimeric BR96, chimeric AC10, and humanized 1F6, which bind to the Lewis-Y antigen on carcinomas, CD30 on hematologic malignancies, and CD70 present on hematologic malignancies and renal cell carcinoma, respectively. ADCs bearing the potent camptothecin analogue, 7-butyl-9-amino-10,11-methylenedioxy-camptothecin, were highly potent and immunologically specific on a panel of cancer cell lines in vitro, and efficacious at well-tolerated doses in a renal cell carcinoma xenograft model.

Novel immunoconjugates comprised of streptonigrin and 17-amino-geldanamycin attached via a dipeptide-p-aminobenzyl-amine linker system.

Cytotoxic agents streptonigrin and 17-amino-geldanamycin were linked to monoclonal antibodies (mAbs), forming antibody-drug conjugates (ADCs) for antigen-mediated targeting to cancer cells. The drugs were conjugated with a linker construct that is labile to lysosomal proteases and incorporates a valine-alanine-p-aminobenzyl (PAB)-amino linkage for direct attachment to the electron-deficient amine functional groups present in both drugs. The resulting ADCs release drug following internalization into antigen-positive cancer cells. The drug linkers were conjugated to mAbs cAC10 (anti-CD30) and h1F6 (anti-CD70) via alkylation of reduced interchain disulfides to give ADCs loaded with 4 drugs/mAb. The streptonigrin ADCs were potent and immunologically specific on a panel of cancer cell lines in vitro and in a Hodgkin lymphoma xenograft model. We conclude that streptonigrin ADCs are candidates for further research, and that the novel linker system used to make them is well-suited for the conjugation of cytotoxic agents containing electron-deficient amine functional groups.

Engineered cysteine antibodies: an improved antibody-drug conjugate platform with a novel mechanism of drug-linker stability

Antibody-drug conjugates (ADCs) are fulfilling the promise of targeted therapy with meaningful clinical success. An intense research effort is directed towards improving pharmacokinetic profiles, toxicity and chemical stability of ADCs. The majority of ADCs use amide and thioether chemistry to link potent cytotoxic agents to antibodies via endogenous lysine and cysteine residues. While maleimide-cysteine conjugation is used for many clinical stage ADC programs, maleimides have been shown to exhibit some degree of post-conjugation instability. Previous research with site-directed mutagenic incorporation of cysteine residues for conjugation revealed that the stability of the drug-antibody linkage depends on the site of conjugation. Here we report on a collection of engineered cysteine antibodies (S239C, E269C, K326C and A327C) that can be site-specifically conjugated to potent cytotoxic agents to produce homogenous 2-loaded ADCs. These ADCs confirm that site of conjugation impacts maleimide stability and present a novel mechanism of thioether stabilization, effectively unlinking stability from either local chemical environment or calculated solvent accessibility and expanding the current paradigm for ADC drug-linker stability. These ADCs show potent in vitro and in vivo activity while delivering half of the molar equivalent dose of drug per antibody when compared to an average 4-loaded ADC. In addition, our lead engineered site shields highly hydrophobic drugs, enabling conjugation, formulation and clinical use of otherwise intractable chemotypes.

Abstract 4634: Engineered cysteine drug conjugates show potency and improved safety

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Antibody-drug conjugates (ADCs) are emerging as an important therapeutic option for new oncology drugs. In this abstract we present data on engineered cysteine antibodies (EC-mAbs) with an additional unpaired cysteine residue introduced at position 239 on each heavy chain to facilitate more uniform drug loading while interfering with Fc gamma receptor function. With the EC-mAb, we have identified a site that blocks Fc gamma receptor III (CD16) binding and has a number of additional desirable biochemical and biophysical properties. We demonstrate that EC-mAb-drug conjugates, with two drugs per antibody, retain antitumor activity in mouse xenograft experiments and are well tolerated in non-human primates. These data suggest that engineered cysteine antibodies are a potential alternative to conjugation of native cysteines for future ADCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4634. doi:1538-7445.AM2012-4634

Abstract 1786: Development and pharmacological properties of PEGylated glucuronide-auristatin linkers

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Antibody-drug conjugates (ADCs) are an emerging therapeutic modality for the treatment of cancer, exemplified by the recent clinical success of brentuximab vedotin and ado-trastuzumab emtansine. The majority of ADCs currently in development include functionalities that are hydrophobic, resulting in increased plasma clearance. Consequently, there has been significant interest in generating new linkers that compensate for this potential liability. In an attempt to mitigate the pharmacokinetic impact of monomethylauristatin E (MMAE) when conjugated to an antibody, we prepared drug-linkers incorporating a polyethylene glycol (PEG) polymer. Initial work demonstrated that incorporation of a discrete PEG24 polymer into the cleavable β-glucuronide-MMAE linker system could either increase or decrease the plasma clearance of the resulting ADCs, depending upon the configuration of the PEG. When inserted into the drug-linker as a stretcher unit between the maleimide and the cleavage site, PEG24 elicited increased ADC plasma clearance. Conversely, incorporation of the PEG24 as a side chain from a modified lysine residue adjacent to the maleimide resulted in ADCs with slower clearance. These differences in ADC pharmacokinetics translated into corresponding differences in antitumor activity in two xenograft models. Thus, incorporation of PEG24 into ADC linkers impacts both pharmacokinetics and activity in a structure-dependent manner. Citation Format: Patrick J. Burke, Joseph Z. Hamilton, Joshua H. Hunter, Scott C. Jeffrey, Svetlana O. Doronina, Nicole M. Okeley, David W. Meyer, Peter D. Senter, Robert P. Lyon. Development and pharmacological properties of PEGylated glucuronide-auristatin linkers. [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 1786. doi:10.1158/1538-7445.AM2014-1786

Abstract 351: Uptake of antibody-drug conjugates by cultured Kupffer cells can predict pharmacokinetics

Interest in antibody-drug conjugates (ADCs) has increased rapidly in the oncology field over the past several years. Recent advances in the ADC field have been achieved in part by an improved understanding of how conjugation with drug-linkers impacts the biophysical, pharmacokinetic (PK), and biodistribution properties of a monoclonal antibody. Most of these advances have been driven by in vivo preclinical observations, an inevitably low throughput endeavor. A higher throughput in vitro method that would allow some level of predictive power for ADC disposition in vivo would be highly advantageous, requiring less test article and allowing for much faster turnaround. Thus far no such methods for ADCs have been reported, in contrast to the situation for the prediction of the metabolic clearance of small molecule therapeutics, for which cultured hepatocytes are widely used as an in vitro tool. It has been generally assumed that clearance of ADCs is driven by cells of the monocyte phagocytic system, and we recently published immunohistochemistry (IHC) data implicating hepatic sinusoidal endothelium and the resident liver macrophages, Kupffer cells (KCs), in the accelerated clearance of highly-loaded ADCs. We sought to use this observation to develop a convenient assay that might correlate ADC uptake in vitro with in vivo PK. Initial fluorescent microscopy experiments revealed that cultured rat KCs stain intensely when incubated with fluorescently labeled ADCs, but much less so with unconjugated antibody, similar to the observed IHC results. In an effort to further improve the throughput of this method to allow the rapid comparison of many ADCs, we moved to a FACS-based platform to quantify the amount of KC-associated fluorescent conjugate. To quantify the effect of different drug-linkers, we prepared homogeneous ADCs with 8 drugs per antibody and observed that the KC staining intensity varied widely depending upon the characteristics of the drug-linker that was conjugated. KCs incubated with ADCs loaded with vc-PAB-MMAE had a mean fluorescent intensity (MFI) >20 fold higher than unconjugated Antibody. Additionally, ADCs loaded with MMAE that contain a polyethylene glycol (PEG) masking moiety in the linker had a much lower MFI than those without PEG. This FACS method allowed us to compare fluorescent uptake by KCs with ADC clearance determined in vivo. We have observed that ADC uptake by cultured KCs in vitro correlates well with ADC PK, allowing for more rapid assessment of the PK impact of new drug classes, drug-linker designs, and conjugation methodologies. Citation Format: David Meyer, Sara Shum, Mechthild Jonas, Martha Anderson, Joshua Hunter, Nagendra Chemuturi, Nicole Okeley, Robert Lyon. Uptake of antibody-drug conjugates by cultured Kupffer cells can predict pharmacokinetics. [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 351.

Abstract 4321: Development of pyrrolobenzodiazepine-based antibody-drug conjugates for cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Pyrrolobenzodiazepine (PBD) dimers are highly cytotoxic minor groove binding DNA crosslinking agents derived from the anthramycin class of natural products. In our efforts to develop highly effective and well-tolerated treatment options for cancer patients, we have investigated several antibody-drug conjugates (ADCs) based on a PBD dimer. The fully synthetic PBD-linker, comprised of a protease-cleavable val-ala sequence and a reactive maleimide group, was conjugated via engineered cysteine residues to humanized monoclonal antibodies specific for their binding to the well-characterized cancer antigen targets, CD33 and CD70. The resulting ADCs were well-defined, monomeric and showed pronounced in vitro and in vivo activity against both hematologic and solid tumor cancer models. These included models of acute myelogenous leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma and renal cell carcinoma. The ADCs were immunologically specific, and showed potent activity in MDR-positive models, including models resistant to other ADCs. These results underscore the importance of the drug component in developing highly efficacious ADCs for cancer therapy. Citation Format: Scott C. Jeffrey, Patrick J. Burke, May K. Sutherland, David W. Meyer, Robert P. Lyon, Julie A. McEarchern, Che-Leung Law, Peter D. Senter. Development of pyrrolobenzodiazepine-based antibody-drug conjugates for cancer. [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 4321. doi:10.1158/1538-7445.AM2013-4321