Homogeneous Antibody-drug Conjugates Research Articles

Abstract 2960: Potent in vivo activity of site-specific indolino-benzodiazepine antibody-drug conjugates (ADCs) generated via engineered cysteine conjugation

Abstract ADCs are widely studied for cancer therapy, with numerous agents in preclinical and clinical development embodying a wide array of targets, linker chemistries, and cytotoxic effector classes. A fourth element of ADC design that has received much attention recently is the site of conjugation of the cytotoxic molecule to the antibody. Historically, lysine- or interchain cysteine-directed conjugation has been used, but site-specific chemistries have become increasingly popular. Our previous evaluation of site-specific and lysine-linked ADCs utilizing a tubulin-acting maytansinoid effector molecule found the lysine-linked version was more active in vivo (Yoder et al., AACR 2015 #645). Here we present a comparison of engineered cysteine site-specific and lysine-linked ADCs utilizing the previously described indolino-benzodiazepine (henceforth referred to as IGN) effector IGN-P1 (Miller et al., AACR 2015 #652) which is designed to undergo proteolytic cleavage upon cell uptake to release a potently cytotoxic DNA alkylator. We show that HC-S442C mutants of human IgG1 can be conjugated via maleimide chemistry to IGN-P1 to give stable, potent, and homogeneous ADCs with drug to antibody ratio (DAR) of 2. The in vitro potency of engineered-cysteine IGN-P1 ADCs is largely dependent on the DAR of the ADC, although some difference is observed between HC-S442C and other cysteine mutants used for conjugation. Pharmacokinetic study of C442 maleimide conjugates suggests that the chemical linkage between effector and antibody is stable upon administration in mice. Further, and in contrast to our previous observations utilizing maytansinoid ADCs, the site-specific and Lys-linked IGN-P1 ADCs showed comparable efficacy in vivo on a molar drug basis. This effect was observed across two different antibodies targeting two different cell surface antigens. These results suggest that, in certain cases, site-specific conjugation chemistry can offer comparable activity to heterogeneous conjugation at well-tolerated doses. Citation Format: Nicholas C. Yoder, Chen Bai, Alan Wilhelm, Erin K. Maloney, Olga Ab, Emily E. Reid, Manami Shizuka, Daniel Tavares, Rassol Laleau, Xiuxia Sun, Megan E. Bogalhas, Lintao Wang, Jan Pinkas, Michael L. Miller, Ravi Chari, Thomas A. Keating. Potent in vivo activity of site-specific indolino-benzodiazepine antibody-drug conjugates (ADCs) generated via engineered cysteine conjugation. [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 2960.

Abstract 2967: In vitro and in vivo activity of site-specific antibody-drug conjugates (ADCs) with 2 and 4 maytansinoid molecules per antibody prepared through conjugation to SeriMabs (N-terminal serine engineered Abs)

Abstract Site-specific attachment of cell-killing agents to antibodies directed against tumor-associated antigens has continued to be an active area of innovation in the field of ADCs. Most reports focus on homogeneous ADCs that have a DAR (cytotoxic molecules per antibody ratio) of 2. Here we describe the preparation, biochemical characterization, and biological evaluation of ADCs made through conjugation of maytansinoids (DM1, DM4) to aldehydes derived from chemically oxidized N-terminal serines (SeriMab) engineered onto the antibody heavy chain (2 DAR) or both light and heavy chain simultaneously (4 DAR). ADCs prepared with a non-cleavable linker or a cleavable disulfide linker were homogeneous 2 or 4 DAR by MS analysis, and were produced in high yield with a monomer content of >98%. Despite conjugation at the N-termini of both the light and heavy chain variable regions, FACS analysis showed the 4 DAR SeriMab conjugates maintained binding to the target antigen. The ADCs showed antigen-specific potency in vitro on a panel of target-expressing cancer cell lines. In the disulfide cleavable linker series, the 2 DAR SeriMab conjugate was 2-5 fold less active than lysine-conjugated Ab-SPDB-DM4 (3.4 DAR), while the 4 DAR SeriMab conjugate was comparably active on an antibody basis. The SeriMab conjugates also displayed strong bystander killing. Surprisingly, in the non-cleavable linker series, the 2 DAR SeriMab conjugate was up to 17-fold more active (depending on cell line) than lysine-conjugated Ab-SMCC-DM1 (3.5 DAR), and the 4 DAR SeriMab conjugate was up to 100-fold more potent than the SMCC-DM1 conjugate on an antibody concentration basis. In a P-gp-positive multi-drug resistant cell line, the non-cleavable 4 DAR SeriMab-maytansinoid conjugate was highly active while the 2 DAR SeriMab ADCs and lysine-conjugated maytansinoid ADCs were >100-fold less potent. The unique oxime bond formed with the non-cleavable SeriMab-maytansinoid conjugate was found to be stable in circulation in mice for >3 days as assayed by affinity capture LC-MS. Polar carboxylic acid containing metabolites were identified which may lead to high cellular retention of maytansinoid species in cancer cells, yielding higher in vitro potency than lysine-linked ADCs in some cell lines. The in vivo anti-tumor activity of disulfide cleavable 2 and 4 DAR SeriMab-DM4 ADCs was evaluated in a clinically relevant cancer xenograft model. The 4 DAR conjugate was active at 60 μg/kg (maytansinoid payload dose) and was more active than the 2 DAR conjugate at this same payload dose. Using the SeriMab conjugation platform we show that in vitro and in vivo activity of site-specific ADCs can be dependent on amount of cytotoxic agent attached per antibody. Citation Format: Luke Harris, Leanne Lanieri, Jose Ponte, Erin Maloney, Laura Bartle, Olga Ab, Juliet Costoplus, Lingyun Rui, Jan Pinkas, Ravi Chari, Thomas Keating, Daniel Tavares, Nathan Fishkin. In vitro and in vivo activity of site-specific antibody-drug conjugates (ADCs) with 2 and 4 maytansinoid molecules per antibody prepared through conjugation to SeriMabs (N-terminal serine engineered Abs). [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 2967.

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

Abstract 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.

ChemInform Abstract: Construction of Homogeneous Antibody—Drug Conjugates Using Site‐Selective Protein Chemistry

AbstractReview: 98 refs.

ChemInform Abstract: Processes for Constructing Homogeneous Antibody Drug Conjugates

AbstractReview: 91 refs.

Processes for Constructing Homogeneous Antibody Drug Conjugates

Antibody drug conjugates (ADCs) are synthesized by conjugating a cytotoxic drug or “payload” to a monoclonal antibody. The payloads are conjugated using amino or sulfhydryl specific linkers that react with lysines or cysteines on the antibody surface. A typical antibody contains over 60 lysines and up to 12 cysteines as potential conjugation sites. The desired DAR (drugs/antibody ratio) depends on a number of different factors and ranges from two to eight drugs/antibody. The discrepancy between the number of potential conjugation sites and the desired DAR, combined with use of conventional conjugation methods that are not site-specific, results in heterogeneous ADCs that vary in both DAR and conjugation sites. Heterogeneous ADCs contain significant fractions with suboptimal DARs that are known to possess undesired pharmacological properties. As a result, new methods for synthesizing homogeneous ADCs have been developed in order to increase their potential as therapeutic agents. This article will review re...

Antibody–drug conjugates for targeted anticancer drug delivery

Antibody–drug conjugates (ADCs) are tumor-targeted therapeutic agents that combine the specificity of monoclonal antibodies (mAbs) with the potent anti-tumor effects of cytotoxic drugs. Over the past few years, ADCs have become a powerful tool in the field of cancer chemotherapy. Recently, two ADC products, brentuximab vedotin (Adcetris®) and trastuzumab emtansine (Kadcyla®), have received FDA approval and there are more than 40 ADC candidates in clinical trials for the treatment of various cancers. Despite the success of some products, considerable interests for the next generation of ADCs have focused on the development of homogeneous conjugates because most of the current ADCs are highly heterogeneous with different drug-to-antibody ratios and drug conjugation sites. Recent studies have demonstrated that the site-specific conjugation of drugs to mAbs could produce homogeneous ADC with better pharmacokinetic properties and improved therapeutic index. A number of approaches, including the use of engineered cysteines, the insertion of unnatural amino acids, and enzymatic ligation, have addressed the challenging issues for the synthesis of homogeneous ADC. This review discusses the limitations of current ADC technologies and describes recent site-specific conjugation methods that can be used to prepare homogeneous ADCs for targeted anticancer drug delivery.

Construction of homogeneous antibody-drug conjugates using site-selective protein chemistry.

Systemic chemotherapy, the current standard of care for the treatment of cancer, is rarely curative and is often accompanied by debilitating side effects. Targeted drug delivery stands as an alternative to chemotherapy, with the potential to improve upon its low efficacy and systemic toxicity. Among targeted therapeutic options, antibody-drug conjugates (ADCs) have emerged as the most promising. These conjugates represent a new class of biopharmaceuticals that selectively deliver potent cytotoxic drugs to cancer cells, sparing healthy tissue throughout the body. Despite this promise, early heterogenous ADCs suffered from stability, pharmacokinetic, and efficacy issues that hindered clinical development. Recent advances in antibody engineering, linkers for drug-release, and chemical site-selective antibody conjugation have led to the creation of homogenous ADCs that have proven to be more efficacious than their heterogeneous predecessors both in vitro and in vivo. In this minireview, we focus on and discuss recent advances in chemical site-selective modification strategies for the conjugation of drugs to antibodies and the resulting potential for the development of a new generation of homogenous ADCs.

Selective Coupling of Click Anchors to Proteins via Trypsiligase.

The combination of pure chemical methods with enzymatic approaches offers a kit system with maximum flexibility for site-specifically tagging proteins with a broad variety of artificial structures. Trypsiligase, a recently introduced designer enzyme for both N- and C-terminal site-specific labeling of peptides and proteins, has been used to introduce click anchors into the human protein cyclophilin 18 and the antibody Fab fragments anti-TNFα and anti-Her2. The subsequent click reactions with tetrazine or norbornene moieties lead to quantitative conversions to the corresponding dihydropyridazine products, thereby forming a stable covalent linkage between the label and the protein of interest. With this technology, cyclophilin 18 has been efficiently modified with the fluorescent dansyl moiety and the pharmaceutically relevant polymer PEG exclusively at its N-terminus. With the same methodology, the Fab fragments of anti-TNFα and anti-Her2 were derivatized exclusively at their C-terminal ends with PEG and the fluorescent dye carboxyfluorescein in the case of anti-TNFα or with the cytotoxic payload DM1 in the case of anti-Her2, to form a homogeneous antibody-drug conjugate (ADC).

Efficient Preparation of Site-Specific Antibody-Drug Conjugates Using Phosphopantetheinyl Transferases.

Post-translational modification catalyzed by phosphopantetheinyl transferases (PPTases) has previously been used to site-specifically label proteins with structurally diverse molecules. PPTase catalysis results in covalent modification of a serine residue in acyl/peptidyl carrier proteins and their surrogate substrates which are typically fused to the N- or C-terminus. To test the utility of PPTases for preparing antibody-drug conjugates (ADCs), we inserted 11 and 12-mer PPTase substrate sequences at 110 constant region loop positions of trastuzumab. Using Sfp-PPTase, 63 sites could be efficiently labeled with an auristatin toxin, resulting in 95 homogeneous ADCs. ADCs labeled in the CH1 domain displayed in general excellent pharmacokinetic profiles and negligible drug loss. A subset of CH2 domain conjugates underwent rapid clearance in mouse pharmacokinetic studies. Rapid clearance correlated with lower thermal stability of the particular antibodies. Independent of conjugation site, almost all ADCs exhibited subnanomolar in vitro cytotoxicity against HER2-positive cell lines. One selected ADC was shown to induce tumor regression in a xenograft model at a single dose of 3 mg/kg, demonstrating that PPTase-mediated conjugation is suitable for the production of highly efficacious and homogeneous ADCs.

Hydrolytically Stable Site-Specific Conjugation at the N-Terminus of an Engineered Antibody.

Antibody-drug conjugates (ADCs) have emerged as an important class of therapeutics for cancer treatment that combine the target specificity of antibodies with the killing activity of anticancer chemotherapeutics. Early conjugation technologies relied upon random conjugation to either lysine or cysteine residues, resulting in heterogeneous ADCs. Recent technology advancements have resulted in the preparation of homogeneous ADCs through the site-specific conjugation at engineered cysteines, glycosylated amino acids, and bioorthogonal unnatural amino acids. Here we describe for the first time the conjugation of an anti-mitotic drug to an antibody following the mild and selective oxidation of a serine residue engineered at the N-terminus of the light chain. Using an alkoxyamine-derivatized monomethyl auristatine E payload, we have prepared a hydrolytically stable ADC that retains binding to its antigen and displays potent in vitro cytotoxicity and in vivo tumor growth inhibition.

Abstract 645: Stability and efficacy comparison of site-specific and lysine-linked maytansinoid antibody-drug conjugates

Abstract As with any therapeutic molecule, antibody-drug conjugates (ADCs) exhibit structure-activity relationships, and medicinal chemistry efforts in this field strive to optimize structure to give the maximum therapeutic index. Recent interest in ADCs as cancer therapy has led to a number of different combinations of linker, payload, and conjugation chemistry.In particular, site-specific methods of payload conjugation have been suggested to generally improve therapeutic properties as compared with more established approaches directed toward lysines or endogenous cysteines.We have investigated the preparation, stability, and activity of anti-folate receptor alpha (FRα) ADCs carrying the microtubule inhibitor, DM1, and conjugated to engineered cysteine mutants utilizing different sites, and compared these ADCs with lysine-directed heterogeneous conjugates. In both embodiments, the DM1 is linked with a protease-cleavable linker. We show that highly homogeneous DM1 ADCs can be produced using engineered cysteine chemistry, enabling assessment of the effects of site-specific conjugation in cells and in animal models. We find that in vitro potency of both lysine-linked and engineered cysteine-linked ADCs against FRα-positive KB cells scales with the total DM1 delivered to cells. Buffer stability experiments in the presence of excess thiol suggest that most engineered cysteine conjugates are comparable in stability to the lysine-linked ADC. A notable exception shows about twice as much fractional DM1 loss upon 3 days of incubation as the other conjugates. Comparison of in vivo activity of two site-specific DM1 ADCs in a KB xenograft model shows measurable activity differences between different conjugation sites. However, a lysine-linked conjugate using almost identical linker chemistry shows approximately 2-fold superior activity to either site-specific construct on a molar DM1 basis. We conclude that, while site-specific conjugation of ADCs may provide a benefit in certain contexts, in other contexts, it may lead to decreased activity, such as in the anti-FRα/KB model examined here. We also observe that different conjugation sites may offer significant differences in activity. It is therefore advisable to evaluate each unique combination of payload, linker, drug:antibody ratio, conjugation site(s), and antibody to the maximum extent possible. Citation Format: Nicholas C. Yoder, Chen Bai, Daniel Tavares, Wayne C. Widdison, Olga Ab, Kathleen R. Whiteman, Alan Wilhelm, Erin K. Maloney, Hans K. Erickson, Thomas A. Keating. Stability and efficacy comparison of site-specific and lysine-linked maytansinoid 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 645. doi:10.1158/1538-7445.AM2015-645

Site-Specific Dual Antibody Conjugation via Engineered Cysteine and Selenocysteine Residues.

Site-specific conjugation technologies enable the production of homogeneous antibody-drug conjugates (ADCs) with improved therapeutic indices compared to conventional ADCs. However, current site-specific conjugation methods can only attach one type of drug to a single antibody. Given the emergence of acquired resistance to current ADCs, arming single antibodies with different drugs may provide an attractive option in the development of next-generation ADCs. Here, we describe a site-specific dual conjugation strategy as a platform for dual warhead ADCs.

Glycans of Antibodies as a Specific Site for Drug Conjugation Using Glycosyltransferases.

The therapeutic cargo molecules conjugated to a specific site on a monoclonal antibody (mAb), called antibody-drug conjugates (ADCs), are becoming powerful tools in cancer treatment. Generally, the cargo molecules conjugate at the cysteine or lysine residue of the mAb, which generally results in a highly heterogeneous ADC. Therapeutic cargo molecules need to be conjugated in a site-specific manner to the mAb so that the bioefficacy of these molecules is not compromised. The mAb (IgG1) are N-glycosylated at the conserved residue Asn(297), which is present in each heavy chain of the IgG1, near the CH2 domain of the Fc fragment. The mutant or wild-type glycosyltransferases transfer sugars with a chemical handle to the glycan molecule of IgG1, making the site-specific linking of cargo molecules possible via the chemical handle, and thus making the process an invaluable technique for the production of homogeneous ADCs.

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.

Site-Specific Conjugation of Monomethyl Auristatin E to Anti-CD30 Antibodies Improves Their Pharmacokinetics and Therapeutic Index in Rodent Models.

Antibody-drug conjugates (ADCs) have demonstrated clinical benefits that have led to the recent FDA approval of KADCYLA and ADCETRIS. Most ADCs that are currently in clinical use or development, including ADCETRIS, are produced by chemical conjugation of a toxin via either lysine or cysteine residues, inevitably leading to heterogeneous products with variable drug-to-antibody ratios (DARs). Here, we describe the in vitro and in vivo characterization of four novel ADCs that are based on the anti-CD30 antibody cAC10, which has the same polypeptide backbone as ADCETRIS, and compare the results with the latter. Bacterial transglutaminase (BTG) was exploited to site-specifically conjugate derivatives of monomethyl auristatin E (all comprising a cleavable linker) to the glutamine at positions 295 and 297 of cAC10, thereby yielding homogeneous ADCs with a DAR of 4. In vitro cell toxicity experiments using two different CD30-positive cell lines (Karpas 299 and Raji-CD30(+)) revealed comparable EC50 values for ADCETRIS (1.8 ± 0.4 and 3.6 ± 0.6 ng/mL, respectively) and the four cAC10-based ADCs (2.0 ± 0.4 to 4.9 ± 1.0 ng/mL). Quantitative time-dependent in vivo biodistribution studies (3-96 h p.i.) in normal and xenografted (Karpas 299 cells) SCID mice were performed with a selected (125)I-radioiodinated cAC10 ADC and compared with that of (125)I-ADCETRIS. The chemo-enzymatically conjugated, radioiodinated ADC showed higher tumor uptake (17.84 ± 2.2% ID/g 24 h p.i.) than (125)I-ADCETRIS (10.5 ± 1.8% ID/g 24 h p.i.). Moreover, (125)I-ADCETRIS exhibited higher nontargeted liver and spleen uptake. In line with these results, the maximum tolerated dose of the BTG-coupled ADC (>60 mg/kg) was significantly higher than that of ADCETRIS (18 mg/kg) in rats. These results suggest that homogeneous ADCs display improved pharmacokinetics and better therapeutic indexes compared to those of chemically modified ADCs with variable DARs.

Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry.

Antibody drug conjugates (ADCs) have emerged as an important pharmaceutical class of drugs designed to harness the specificity of antibodies with the potency of small molecule therapeutics. The three main components of ADCs are the antibody, the linker, and the payload; the majority of early work focused intensely on improving the functionality of these pieces. Recently, considerable attention has been focused on developing methods to control the site and number of linker/drug conjugated to the antibody, with the aim of producing more homogenous ADCs. In this article, we review popular conjugation methods and highlight recent approaches including "click" conjugation and enzymatic ligation. We discuss current linker technology, contrasting the characteristics of cleavable and non-cleavable linkers, and summarize the essential properties of ADC payload, centering on chemotherapeutics. In addition, we report on the progress in characterizing to determine physicochemical properties and on advances in purifying to obtain homogenous products. Establishing a set of selection and analytical criteria will facilitate the translation of novel ADCs and ensure the production of effective biosimilars.

Methods to Make Homogenous Antibody Drug Conjugates.

Antibody drug conjugates (ADCs) have progressed from hypothesis to approved therapeutics in less than 30 years, and the technologies available to modify both the antibodies and the cytotoxic drugs are expanding rapidly. For reasons well reviewed previously, the field is trending strongly toward homogeneous, defined antibody conjugation. In this review we present the antibody and small molecule chemistries that are currently used and being explored to develop specific, homogenous ADCs.

A chemoenzymatic approach toward the preparation of site-specific antibody–drug conjugates

An efficient chemical synthesis of UDP-N-azidoacetylgalactosamine (UDP-GalNAz) is presented, while the value of this molecule was demonstrated through its attachment to an antibody Fc domain. Thus, the antibody was first degalactosylated, which was followed by loading of the UDP-GalNAz with a recombinant galactosyltransferase. This engineered Azide-Fc-N-glycan antibody was subsequently ‘clicked’ by a strain-promoted alkyne-azide cycloaddition reaction for site-specific attachment of a fluorescent probe. The principles detailed will allow for the facile preparation of chemically defined homogeneous antibody–drug conjugates (ADCs).

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

Abstract 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