Article pubs.acs.org/bc
Cathepsin B Cleavage of vcMMAE-Based Antibody−Drug Conjugate Is Not Drug Location or Monoclonal Antibody Carrier Specific Benson Gikanga,† Nia S. Adeniji,‡ Thomas W. Patapoff,§ Hung-Wei Chih,‡ and Li Yi*,‡ †
Pharmaceutical Processing and Technology Development, ‡Late Stage Pharmaceutical Development, §Early Stage Pharmaceutical Development, Genentech Inc., 1 DNA Way, South San Francisco, California 94080, United States W Web-Enhanced Feature * S Supporting Information *
ABSTRACT: Antibody−drug conjugates (ADCs) require thorough characterization and understanding of product quality attributes. The framework of many ADCs comprises one molecule of antibody that is usually conjugated with multiple drug molecules at various locations. It is unknown whether the drug release rate from the ADC is dependent on drug location, and/or local environment, dictated by the sequence and structure of the antibody carrier. This study addresses these issues with valine-citrulline-monomethylauristatin E (vc-MMAE)-based ADC molecules conjugated at reduced disulfide bonds, by evaluating the cathepsin B catalyzed drug release rate of ADC molecules with different drug distributions or antibody carriers. MMAE drug release rates at different locations on ADC I were compared to evaluate the impact of drug location. No difference in rates was observed for drug released from the VH, VL, or CH2 domains of ADC I. Furthermore, four vc-MMAE ADC molecules were chosen as substrates for cathepsin B for evaluation of Michaelis−Menten parameters. There was no significant difference in KM or kcat values, suggesting that different sequences of the antibody carrier do not result in different drug release rates. Comparison between ADCs and small molecules containing vc-MMAE moieties as substrates for cathepsin B suggests that the presence of IgG1 antibody carrier, regardless of its bulkiness, does not impact drug release rate. Finally, a molecular dynamics simulation on ADC II revealed that the val-cit moiety at each of the eight possible conjugation sites was, on average, solvent accessible over 50% of its maximum solvent accessible surface area (SASA) during a 500 ns trajectory. Combined, these results suggest that the cathepsin cleavage sites for conjugated drugs are exposed enough for the enzyme to access and that the drug release rate is rather independent of drug location or monoclonal antibody carrier. Therefore, the distribution of drug conjugation at different sites is not a critical parameter to control in manufacturing of the vc-MMAE-based ADC conjugated at reduced disulfide bonds.
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INTRODUCTION Antibody−drug conjugates (ADCs) allow delivery of highly potent drugs to targeted cells via specific antibody−antigen binding. The framework of ADCs comprises an antibody carrier and one or multiple drug-linker moieties that conjugate the drug to the antibody carrier. As an emerging approach to treat cancer diseases, the ADC technology has been optimized on many key parameters, such as linker chemistry and payload potency, in the past decades.1−3 While a handful of ADC molecules have been approved for market, more are currently under evaluation in clinical development, among which the valine-citrulline-monomethylauristatin E (vc-MMAE)-based ADC is one of the leading types.1,4 This category of ADC is designed to deliver microtubule destabilizing monomethylauristatin E (MMAE) to cancer cells. The cytotoxic agent can be conjugated to antibody carrier at either reduced disulfide bonds or engineered cycsteines. Within this category, a CD30targeting molecule, brentuximab vedotin (Adcetris), was approved in 2011 for use in the United States, and at least eight of the ADCs currently in phase I and II clinical trials are using the same linker drug.4 For the vc-MMAE based ADC conjugated at reduced disulfide bonds, whether the drug release rate is conjugation site specific or monoclonal antibody (mAb) carrier specific remains unknown. This information could © 2016 American Chemical Society
provide guidance for the rational design of ADC constructs, and/or apply appropriate controls for ADC manufacturing. In the vc-MMAE based ADCs, MMAE drugs are conjugated onto mAbs through a protease-cleavable dipeptide valinecitrulline (vc) linker. This linker evolved from natural substrates of lysosomal proteases (such as phenylalaninearginine dipeptide with lysosomal cysteine proteases) and was modified to reduce nonspecific plasma cleavage while maintaining a fast cleavage rate.5 Compared with hydrolyzable linkers, such as hydrazones and disulfides, the vc linker has a significantly longer half-life in plasma.6,7 This is mostly because the proteases are primarily located in the lysosome such that the drug is only released after the ADC is internalized within the cell. The enzyme most widely used to study vc linker cleavage is cathepsin B (EC 3.4.22.1), although other lysosomal enzymes may have similar activity.8 In order for cathepsin B to cleave the vc linker, a spacer is required between the drug and the linker to allow accessibility of vc linker into the enzyme active site.9 The p-aminobenzyloxycarbonyl (PABC) is chosen as the spacer because it undergoes self-immolation upon Received: January 28, 2016 Revised: February 24, 2016 Published: February 25, 2016 1040
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Bioconjugate Chemistry Scheme 1. (A) Locational Isomers of ADC-vc-MMAE; (B) All Possible Conjugation Sites
interaction with the linker-drug moiety and ultimately affect the cleaving reaction or accessibility of the vc linker to lysosomal enzymes. In addition, the presence of mAb carriers, instead of having a fully exposed vc-MMAE molecule, may simply create bulkiness that can reduce the accessibility of the vc linker to lysosomal enzymes. To address these questions, the Michaelis−Menten parameters of cathepsin B can be compared when using various substrates: vc-MMAE-containing ADC with various mAb carriers, and vc-MMAE conjugating to N-acetyl cysteine. These substrates for Cathepsin B were not compared in controlled experiments before, though there was Cathepsin B activity reported on a variety of short peptides, with a typical kcat ranging from 1 to 364 s−1 and KM from 108 to 1190 μM, which vary by enzyme source and test conditions (Table S1).13−26 These comparisons would help identify whether mAb carrier is a factor impacting drug release rate and, therefore, inform the design of ADC constructs.
cleavage of the vc linker, and an intact MMAE drug is released from the ADC. Production of a vcMMAE-based ADC with drugs conjugating to cysteine (Cys) reduced from interchain disulfides always results in a heterogenic product containing antibody with various amounts of drug (i.e., different drug−antibody ratio; DAR), and drugs at various locations (i.e., different isomers).10 Conjugation occurs between presynthesized vcMMAE linker− drug complexes and Cys residues on the antibody that are reduced from interchain disulfide bonds.6 Depending on the average availability of free thiol, the conjugation product can result in a different DAR. Specifically, the prevailing species for an ADC with an average DAR of 3.5 are species with DARs of 2, 4, and 6, which can be separated by hydrophobic interaction chromatography (HIC).11 Within each DAR group, there are isomers with drugs on different locations, and they are usually not resolved on HIC (Scheme 1). It is known that ADC molecules with different DAR values have distinctive therapeutic windows, which are calculated from maximumtolerated dose and efficacy.11 Based on this fact, the average DAR value and the distribution of the DAR species are important parameters of an ADC drug. However, whether the distribution of isomers within the same DAR species is a critical parameter that affects pharmacokinetics or efficacy is not yet thoroughly understood. If the drug release rates are different for different positional isomers, the overall drug release rate would be affected by distribution of positional isomers. Several in vivo works indicated that cytotoxicity and mouse pharmacokinetics are indifferent for isomers,7,12 but there are no published studies that provide direct evidence of enzymatic-cleavage rates for drugs conjugated at different sites on the antibody. One goal of this study is to address whether cathepsin B preferentially cleaves the linker when it is conjugated at one drug location over another in a vcMMAE-based ADC by monitoring the MMAE-release rate using peptide mapping. In addition to conjugate site locations on an ADC molecule, it is also not understood whether different mAb carriers would have an impact on the drug release rate. MAbs with different complementarity determining region (CDR) sequences may render different surface properties, which may lead to
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RESULTS AND DISCUSSION For the vc-MMAE-based ADC conjugated at reduced disulfide bonds, an earlier study showed that ADC molecules with different average drug loads (i.e., DARs) exhibited different half-maximal inhibitory concentrations (IC50s) and maximumtolerated doses.11 As such, DAR is a critical parameter to control in ADC manufacturing. For ADCs with the same DAR values, there may be a distribution of multiple positional isomers, especially for DAR species of 2, 4, and 6 (Scheme 1). Methods are available to characterize such a distribution.10 However, in order to determine whether the distribution of a positional isomer should be included into the critical parameters for control or characterization in ADC production, its impact on the pharmacological properties must be better understood. In vivo studies have been carried out to evaluate the cytotoxic activity of pure positional isomers. Sun et al.27 used specific chemical reduction and reoxidation procedures to generate different isomers at high purity and demonstrated no difference in cytotoxic activity of the isomers with the same DAR value. McDonagh et al.12 also reached the same conclusion using homogeneous isomers prepared by mutating selected Cys residues to serine. These results provided support 1041
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Bioconjugate Chemistry that the in vivo cleavage of drug is similar at various drug locations. However, direct evidence to demonstrate drug release rates from different locations are similar has not yet been provided. Furthermore, the impact of the protein carrier structure on drug release is also unknown. This study addresses these issues by quantifying drug release from different locations on ADC molecules, and comparing the Michaelis−Menten parameters of cathepsin B using various ADC-molecule (ADC I−IV) and small-molecule substrates. Cathepsin B Mediated MMAE Release from ADC. An in vitro enzymatic reaction model was constructed to mimic the in vivo cleavage of an ADC molecule. This model reaction is representative but can deviate from in vivo conditions in terms of enzyme and substrate concentrations, the actual lysosomal enzyme ensemble (since cathepsin B is not the only one with such activity), and the availability of cofactors, such as metal ions. Cathepsin B isolated from human liver was used in this study. As demonstrated by Moin et al.,26 the human liver cathepsin B closely mimics the cathepsin B expressed in tumors, in terms of model substrate cleavage activity. The results from this study may apply to other lysosomal proteases such as cathepsin L and cathepsin S, due to their similarity in catalytic activity to cathepsin B,28,29 but additional experiments are needed to definitively demonstrate this. These lysosomal proteases are also overexpressed in some types of cancer tissue, and the extracellular microenvironment of some cancer tissue is also slightly acidic.30 The optimal pH of cathepsin B, which was determined in this study to be pH 5.5 (Figure S1), is close to the pH in the lysosome31 and that of the extracellular region of cancer tissue.32 The enzymatic reaction temperature was set at 37 °C to mimic physiological conditions. The reaction mixture contained 25 mM sodium acetate, and 1 mM EDTA, at pH 5.5, and the Cys protease inhibitor, E-64,33 was used to quench the reaction. The released MMAE-related molecules were quantified using a reversed-phase high-performance liquid chromatography (RPHPLC) assay (Figure 1). A co-mix of MMAE, NAC-vc-MMAE, and vc-MMAE was injected as a standard to identify the corresponding species in the reaction mixture. As shown in Figure 1, under the given reaction conditions, only the MMAE drug moiety was enzymatically released from the ADC, which is consistent with the proposed mechanism that (1) cathepsin B cleaved the ADC specifically at the C-terminus of the dipeptide vc linker and that (2) self-immolation of the PABC spacer linker released MMAE, the active form of drug.34 A typical time course of free MMAE released from ADC I by cathepsin B is shown in Figure 2. In this representative time course, 70% of the MMAE drug was released from ADC I within 80 min. Within the first 20 min, the drug release rate approximated a constant value, which suggested a steady state of the enzyme kinetics. Based on this data, a reaction time of 5 min was chosen to obtain initial velocities for the Michaelis− Menten measurements. The ADC molecules used in this study were shown to be stable for months at refrigerated conditions without degradation or release of the drug (data not shown). In this study, control experiments without cathepsin B also demonstrated the stability of ADC molecules, including the linker, throughout the enzymatic reaction and sample preparation. Impact of Drug Conjugate Location on Drug Release. The rate of drug released by cathepsin B at different locations on ADC I was measured by a peptide mapping assay following an enzymatic reaction that was quenched at predetermined
Figure 1. Quantification of free drug using the RP-HPLC assay. The chromatograms are shown for (A) the co-mixture of standard compounds MMAE, NAC-vc-MMAE, and vc-MMAE, (B) the cathepsin B digested ADC I, (C) the intact ADC I control without enzyme digestion, and (D) a blank injection.
Figure 2. Typical time course of MMAE cleaved from ADC I by cathepsin B. An enzyme to substrate ratio of 1:1000 was used in this example. The average of triplicate data is shown ± standard deviation.
time points (Figure 3). After treatment with cathepsin B, mAb I and ADC I were subjected to trypsin digestion followed by separation of peptides by RP-HPLC. Trypsin likely does not cleave the vc linker, since valine is not passively charged, and citrulline was shown to be not a substrate of trypsin.35 Most of the tryptic peptides without the drug attached were resolved within retention time 0−152 min, while peptides with drug still attached were eluted at 152−205 min. This is most likely due to the hydrophobicity contributed by the MMAE drug. The peaks were assigned by mass spectroscopy (data not shown). Peptide mapping chromatograms are shown in Figure 3. Compared to unconjugated mAb I, the ADC I showed decreased peak area for tryptic peptides with empty conjugation sites (peaks 1 and 2, for example, in Figure 3A), and increased peak area of tryptic peptides conjugated with drugs (peaks 3−5 in Figure 3B), indicating occupancy of conjugation sites with drug. There are also minor peaks in Figure 3B which could be the ring-opening species of the conjugated tryptic peptides, since the succinimide ring tends to get hydrolyzed to a linear 1042
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Figure 3. Peptide mapping chromatograms of cathepsin B-treated ADC I and mAb I. (A) Full view. Fractions eluted prior to 150 min are peptides without drug. Peak 1 corresponds to a light chain peptide (highlighted in the antibody illustration). Peak 2 is a heavy chain peptide. (B) Expanded view of retention time 150−208 min. Peptides with drug attached are resolved in this region. Peak 3 corresponds to a heavy chain peptide with one drug attached. Peak 4 is a light chain peptide with one drug attached. Peak 5 is the hinge region of the heavy chain peptide with two drugs attached. The locations of peptides on the antibody that correspond to peaks 1−5 are shown in the antibody illustrations.
format under the alkaline pH conditions that were used during digestion. Since the ring-opening of succinimide is pH dependent and favors the alkaline pH, these species were minimized by lowering the tryptic digestion pH from 8.3 to 7.5. A typical sample preparation resulted in 50% exposed). All these results point to 1046
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between NAC-vc-MMAE and ADC I, the ADC I was also assayed in a reaction mixture containing 5% or 10% DMA. Circular Dichroism. ADC I was prepared at 1.25 mg/mL as three solutions: (1) in enzymatic reaction buffer (20 mM sodium acetate, 1 mM EDTA, pH 5.0); (2) in enzymatic reaction buffer with 10% DMA; and (3) in enzymatic reaction buffer with 6 M guanidine HCl. The guanidine HCl-denatured sample was incubated at room temperature for 2 h prior to taking the measurement. Samples were loaded into quartz cuvettes with a path length of 10 mm, and measured on the Jasco J-815CD spectrometer (Jasco Analytical Instruments) at 340−250 nm with a bandwidth of 1.0 nm. The spectra were averaged from three measurements and with buffer blank signal subtracted. Molecular Dynamic Simulation of ADC. In silico molecular dynamics simulations were performed using the Amber Molecular Dynamics Package v12.37 A protein data bank (pdb) file of the unconjugated precursor of ADC II was made by replacing the Fv of a trastuzumab template generated previously38 with an Fv containing the correct CDRs. This Fv was generated by homology modeling using the “modeller” program.39 It was then “reduced” by unlinking the interchain Cys bonds relevant to each DAR isoform. The “reduced” molecule was then simulated at pH 5.0 in water (TIP3P) at 300 K for at least 300 ns in order to allow the sulfur residues to move far enough apart from one another to facilitate the drug linker addition. A Cys residue containing the vc-MMAE drug linker was created using the Amber work package, and subsequently added to this “reduced” and relaxed ADC II protein backbone using Swiss-PDBViewer version 4.1.0. ADC II species with DAR 4 (four drugs in the hinge), DAR 6 (four drugs in the hinge, two between the heavy and light chains), and DAR 8 (all interchain sites conjugated) were created and modeled in silico at pH 5 in water for 500 ns. The SASA values for each component of vc-MMAE were calculated using the AREAIMOL program,40−42 which calculates the SASA values by rolling a sphere of radius 1.4 Å over the specified surface area. This calculation was performed for every 5000 steps (0.01 ns) in the 213 ns trajectory. Since the absolute SASA values scale with the size of the specified surface, a fractional SASA (with respect to the maximum SASA) was deemed necessary for comparison purposes. The average maximum SASA was determined by modeling a vc-MMAE residue capped with Cand N-terminal glycine residues under the aforementioned simulation conditions. Error bars on all average fractional SASA values represent ±1 standard deviation from the average value in a trajectory.
order to fully separate the protein content, the methanol precipitated sample was frozen at −20 °C for 30 min prior to centrifugation at 15000 rpm for 30 min at 5 °C. The resulting supernatant was diluted 4 times with purified water and then subjected to RP-HPLC assay on an Xterra MS c18 3.5 μm, 2.1 × 100 mm column (Waters). The method was run at a column temperature of 50 °C. Buffer A was 0.1% trifluoroacetic acid (TFA) in water, and buffer B was 0.1% TFA in acetonitrile. Samples were eluted with a gradient from 25% to 65% buffer B in 20 min at the flow rate of 0.3 mL/min. A standard curve of known concentrations of MMAE was generated for quantitation of the free drug. A co-mixed sample of MMAE, vc-MMAE, and NAC-MMAE was used to provide markers for possible released drug formats. Peptide Mapping. The cathepsin B digested sample was adjusted to pH 7.2 using 500 M Tris-HCl at pH 8.0 and purified through the Protein A HP SpinTrap (GE Healthcare) columns to remove cathepsin B, E-64, and free MMAE. The sample was loaded to the Protein A column in 200 mM sodium phosphate at pH 7 and eluted with 1 M glycine buffer at pH 2.7. The resulting eluents were adjusted to pH 7.5 with 1 M Tris-HCl at pH 9.0 and mixed with denaturing buffer (6 M guanidine chloride, 360 mM Tris-HCl, and 2 mM EDTA at pH 8.6) to a final protein concentration of 1 mg/mL. The mixture was incubated at 37 °C for 30 min followed by the addition of iodoacetamide and incubation at ambient temperature in the dark for another 20 min. The denatured and reduced sample was then buffer exchanged to trypsin digestion buffer (0.025 M Tris-HCl, 0.001 M CaCl2 at pH 7.5) using PD-10 columns (GE Healthcare). Trypsin enzyme was added to the sample (trypsin to protein ∼695:1). The digestion was carried out overnight at 37 °C before being quenched by 10% TFA in water. The digested protein was analyzed with a ZORBAX 300SB-C8 (3.5 μm, 4.6 × 150 mm) column (Agilent Technologies). Column temperature was set to 42 °C during analysis. Buffer A was 0.1% TFA in water while buffer B was 0.1% TFA in acetonitrile. Samples were eluted with a gradient run from 1% to 40% buffer B for 140 min followed by 40% to 60% buffer B for another 15 min. The UV signal at 214 and 248 nm were monitored. The identity of the peaks was determined by linear ion trap mass spectrometry (LTQ MS, Thermo Scientific) equipped with XCalibur software (LC-MS data not included). Decay curves of drug-loaded tryptic peptide peaks were plotted. Data were fit to the first-order decay equation y = Ae−kt using Scientist software (MicroMath). Michaelis−Menten Kinetics of Cathepsin B on ADC and Small-Molecule Substrates. ADC I−IV at 50−2200 μM were incubated for 5 min at 37 °C with 0.2 μM cathepsin B in 25 mM sodium acetate and 1 mM EDTA at pH 5.5. Reactions were quenched by adding E-64 to a final concentration of 0.2 μM. The samples were analyzed by RPHPLC for free MMAE. The initial velocity of MMAE release was plotted against substrate concentration. Data was nonlinearly fitted to the Michaelis−Menten equation using the JMP Software to derive kcat and KM values. In addition to ADCs, NAC-vc-MMAE (a vc-MMAE fragment capped with an acetyl group at the N-terminus) was also tested as a substrate of cathepsin B. A stock solution of NAC-vc-MMAE was prepared at 20 mM in 100% dimethylamine (DMA), and diluted to 0.05−2 mM in enzymatic reaction mixtures. Due to limited solubility of NAC-vc-MMAE in aqueous solution, the reaction mixture was compensated to a final concentration of 5% or 10% of DMA. When comparing the Michaelis−Menten plots
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00055. Table S1: Literature reported Michaelis−Menten parameters of cathepsin B on various peptide-based substrates. Figure S1: pH dependence of cathepsin B activity using ADC I-vc-MMAE as substrate. Figure S2: In silico frame of a DAR 8 ADC during a 213 ns trajectory. Figure S3: vc factional SASA time course for select ADC DAR 8 conjugation sites. Figure S4: Average vc fractional SASA values ADC DAR 8 conjugation sites. (PDF) 1047
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The 360° rotation of the DAR8 ADC structure.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors would like to thank Nancy Chen (Protein Analytical Chemistry, Genentech Inc., South San Francisco, CA) for developing the peptide mapping method used in this work. The authors are also grateful to Seattle Genetics (Bothell, WA) for their collaboration. The authors acknowledge the editorial assistance of Eileen Y. Ivasauskas of Accuwrit Inc.
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DOI: 10.1021/acs.bioconjchem.6b00055 Bioconjugate Chem. 2016, 27, 1040−1049
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DOI: 10.1021/acs.bioconjchem.6b00055 Bioconjugate Chem. 2016, 27, 1040−1049
