Antibody Structural Integrity of Site-Specific Antibody-Drug Conjugates

May 4, 2015 - A side-by-side comparison of the HC Ser239 wild type (wt) monoclonal antibody (mAb) and the engineered Cys239 mAb indicates that site di...
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Antibody Structural Integrity of Site-Specific Antibody-Drug Conjugates Investigated by Hydrogen/Deuterium Exchange Mass Spectrometry Lucy Yan Pan,* Oscar Salas-Solano, and John F. Valliere-Douglass Department of Analytical Sciences, Seattle Genetics, 21823 30th Drive SE, Bothell, Washington 98021, United States ABSTRACT: We present the results of a hydrogen/ deuterium exchange mass spectrometric (HDX-MS) investigation of an antibody−drug conjugate (ADC) comprised of drug-linkers conjugated to cysteine residues that have been engineered into heavy chain (HC) fragment crystallizable (Fc) domain at position 239. A side-by-side comparison of the HC Ser239 wild type (wt) monoclonal antibody (mAb) and the engineered Cys239 mAb indicates that site directed mutagenesis of Ser239 to cysteine has no impact on the HDX kinetics of the mAb. According to the crystal structure of a homologous immunoglobulin G1 (IgG1) antibody (PDB: 1HZH), the backbone amide of Ser239 is hydrogen-bonded to Val264 backbone amide in the wt-mAb studied here. Replacing Ser239 with a Cys residue does not alter the exchange kinetics of the backbone amide of Val264 suggesting that either Ser or Cys at position 239 has similar amide-hydrogen bonding with Val264. However, a small segment in CH2 domain of the ADC (264VDVS) was found to have a slightly increased HDX rate compared to the wt- and C239-mAb constructs. The slightly increased HDX rate of the segment 264VDVS in ADCs indicates that the further modification of Cys239 with drug-linkers only attenuates the local backbone amide hydrogen-bonding network between Cys239 and Val264. All other regions which are proximal to the site of drug conjugation are unaffected. The results demonstrate that the site-specific drug conjugation at the engineered Cys residue at the position 239 of HC does not impact the structural integrity of antibodies. The results also highlight the utility of applying HDX-MS to ADCs to gain a molecular level insight into the impact of site-specific conjugation technologies on the higher-order structure (HOS) of mAbs. The methodology can be applied generally to site-specific ADC modalities to understand the individual contributions of site-mutagenesis and drug-linker conjugation on the HOS of therapeutic candidate ADCs.

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lysine side chains.4,5 Conjugation of cysteine or lysine residues in mAbs typically results in ADCs which have a heterogeneous composition of drug-loaded forms.5 More recently, site-specific conjugation modalities have emerged that yield ADCs with a homogeneous drug-load and distribution.6 In some cases, sitespecific conjugation has been shown to improve ADC stability and to minimize undesirable attributes such as aggregation.7,8 Development of site-specifically conjugated ADCs typically involves some prior manipulation of the cell-line or the primary sequence of the mAb. Site-directed mutagenesis has been used to introduce unpaired cysteine residues into the amino acid backbone of mAbs to provide a scaffold for the attachment of drug-linkers at predefined locations in the primary sequence.9,10 Some site-specific conjugation strategies utilize consensus amino acid sequences to formylglycine-generating enzyme to introduce aldehydes into the primary sequence of mAbs during cell culture which are subsequently conjugated with druglinkers.11 A more direct enzymatic approach involves using microbial transglutaminase to conjugate amine-containing drugs to specific glutamine residues in mAbs.12,13 Others

umor specific monoclonal antibody (mAb) intended for therapeutic use generally has exquisite specificity for tumor antigens but modest antitumor activity. Conversely, small-molecule cytotoxic agents are highly potent but untargeted and thus can cause collateral damage to noncancerous tissues. The liabilities of stand-alone mAb and smallmolecule-based cancer treatments can be overcome by attaching cytotoxic agents to the amino acid backbone of tumor specific mAbs to constitute an antibody−drug conjugate (ADC). The recent approvals of ADCETRIS (brentuximab vedotin) for the treatment of relapsed Hodgkin lymphoma and systemic anaplastic large cell lymphoma and KADCYLA (adotrastuzumab emtansine) for the treatment of HER2 positive metastatic breast cancer highlight the effectiveness of this approach. Additionally, improved therapeutic indices for ADCs compared to untargeted therapies have contributed to the development of highly potent drug-linkers that incorporate cytotoxic agents as ADC payloads that would otherwise have limited benefit as stand-alone treatments due to systemic toxicity.1−3 Several strategies have been developed to attach drug-linkers to mAbs. Drug-linkers have been attached to mAbs by reductive alkylation of the thiols on endogenous interchain cysteine residues or by conjugation of the epsilon-amino groups on © 2015 American Chemical Society

Received: February 25, 2015 Accepted: May 4, 2015 Published: May 4, 2015 5669

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varying lengths of time under native conditions and subsequently measuring the extent of hydrogen exchange at protein backbone amides by MS. HDX of backbone amides is mediated by solvent exposure and hydrogen bonding networks that involve the amides, and the rate of exchange can vary by as much as 108 depending on the local amide environment.33−35 The use of proteolytic enzymes to cleave the exchanged samples into small peptides while the deuterium label is present allows for the resolution of differential exchange to specific local structures in the protein.36,37 The peptide-level resolution can be further refined to specific amino acids using available overlapping peptides.38−40 HDX-MS has been applied to several different protein therapeutics to study conformational changes brought about by chemical and posttranslational modifications,38,41,42 aggregation,43,44 and the choice of formulation excipients.45 More recently, HDX-MS has been used to gain an understanding of the impact that conjugation of drug-linkers to interchain Cys residues has on the conformation of mAbs.46 In our current work, we expand on our previous structural investigation of immuno-conjugates46 by applying HDX-MS to site-specific ADCs in which pyrrolobenzodiazepine dimer (PBD) or maleimidocaproyl-monomethyl Auristatin F (mcMMAF) drug-linkers have been specifically conjugated to the engineered Cys residues at position 239 in Fc domain.7,10 We believe that insights gained from this approach can be applied proactively to inform one on the development of promising therapeutic ADC modalities.

have shown that unnatural amino acids can be site specifically introduced into the primary sequence of mAbs during protein synthesis by utilizing amber codon suppression methodologies. Drug-linkers are then attached to the unnatural amino acids in a subsequent conjugation step.14,15 N-Glycans found in the fragment crystallizable (Fc) domain of mAbs can be used as a conjugation scaffold by converting the terminal monosaccharides to conjugatable aldehydes through chemical treatment.16 Similarly, thiolate forms of monosaccharides can be incorporated into N-glycan structures and subsequently conjugated to drug-linkers via maleimide chemistry.17 The diversity of conjugation strategies used to attach drugs to the mAbs, and the multitude of drug-linker payloads in preclinical and clinical development, suggests that there is no single best approach to developing ADCs. From the perspective of molecule testing and characterization, the choice of drug-linker type and conjugation modality greatly impacts the analytical approach that is taken to gain a thorough understanding of the relevant ADC quality attributes. Analytical approaches to ADC characterization have been reviewed previously.18 Methods that can provide an accurate assessment of ADC drug-load are very important because drugload is directly linked to cytotoxicity, the mechanism of action for ADCs.19−22 In general, analytical assessments of ADC quality are most informative when the parent unconjugated mAb can be analyzed side by side with the resulting ADC, using the same analytical methods. The comparative assessment of the parent mAb and the ADC can shed light on how conjugation modalities, drug-linker type, and conjugation processes may or may not impact mAb quality attributes such as aggregate, charge variant distribution, and conformation/ higher-order structure (HOS). While minor differences in the quality attributes of the therapeutic resulting from drug conjugation may not have any impact on safety and efficacy, it is still important to understand the source or cause of these differences. Among the attributes listed above, HOS assessment of mAbs and ADCs continues to be challenging. X-ray crystallography and nuclear magnetic resonance (NMR) are high-resolution techniques which have traditionally been used to provide atomic-level structural information about protein conformation. However, these techniques are not easily applied to large protein therapeutics such as mAbs and ADCs due to their relatively large molar mass (∼150 kDa). Traditional biophysical techniques such as circular dichroism (CD), Fourier transform infrared (FT-IR), and fluorescence spectroscopy can be run on a routine basis to generate averaged HOS information across all proteins in a sample,23−25 but these techniques provide little insight into local structural differences that may be existing between protein samples. Differential scanning calorimetry (DSC) is capable of providing domain specific heat capacities for mAbs and ADCs,23,26,27 but the technique cannot be used to pinpoint alterations in the local structural features within a given subunit that might manifest as a change in mAb or ADC heat capacity. Due in part to the apparent shortcomings of traditional biophysical techniques, hydrogen/deuterium exchange mass spectrometry (HDX-MS) is being increasingly applied in the pharmaceutical industry to study the conformation of protein therapeutics.28−32 HDX-MS bridges the gap between low and high resolution approaches aimed at studying protein conformation and is ideal for comparing local protein structures across samples. A typical HDX-MS experiment involves incubating a protein in D2O for

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MATERIALS AND METHODS Materials. Dithiothreitol (DTT), tris (2-carboxyethyl) phosphine (TCEP) hydrochloride, sodium cyanoborohydride (NaCNBH3), pepsin from porcine stomach mucosa, urea, formic acid, trifluoroacetic acid (TFA), sodium phosphate, and sodium chloride were purchased from Sigma (St. Louis, MO). Deuterium oxide (D2O) was obtained from Cambridge Isotope Laboratories (Andover, MA). NAP-5 columns were obtained from GE Healthcare (Pittsburgh, PA). All chemicals were used as received. The wild type (wt)-mAb and C239-mAb are humanized immunoglobulin G1 (IgG1) kappa monoclonal antibodies, expressed in Chinese hamster ovary (CHO) cells. The C239mAb has the same amino acid sequence as the wt-mAb except that the Ser239 residue in the mAb heavy chain (HC) was replaced with a cysteine residue to facilitate site-specific drug conjugation. The ADC is composed of C239-mAb conjugated with PBD or mcMMAF drug-linkers produced at Seattle Genetics (Bothell, WA) by established procedures.7,10 Reduced Liquid Chromatography/Mass Spectrometry (LC/MS) Analysis. mAbs and ADCs were reduced with 10 mM DTT. Before reduction, PBD-ADCs were chemically treated according to previous reports.7,47 The resulting light chain (LC) and heavy chain (HC) subdomains of the ADC or mAb were bound to a 2.1 × 150 mm PLRP-S reversed-phase column (Agilent, Santa Clara, CA) and eluted with an acetonitrile/TFA gradient. The column eluent was delivered to an Agilent 6510 QTOF MS (Agilent) operating in positive electrospray ionization (ESI) mode. The mass spectra were deconvoluted with a maximum entropy deconvolution algorithm within the MassHunter workstation software version B.03.01. Hydrogen/Deuterium Exchange Mass Spectrometry. Prior to isotope labeling, all protein samples were buffer5670

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Figure 1. (A) LC/UV profiles of the partially reduced wt-mAb (top), C239-mAb (middle), and PBD-ADC (bottom). (B) Deconvoluted masses of the heavy chains from the wt-mAb (top), C239-mAb (middle), and PBD-ADC (bottom). The structure of the heavy chains is shown as a cartoon to the right of the MS. (C) Chemical structures of drug-linkers PBD and mcMMAF.

exchanged into the labeling buffer (50 mM sodium phosphate, 100 mM sodium chloride, pH 7.0) using NAP-5 columns. Deuterium labeling was initiated at room temperature by mixing 100 μL of protein samples (10 mg/mL) with 900 μL of D2O-labeling buffer (50 mM sodium phosphate and 100 mM sodium chloride, pH 7.0 in D2O). After incubation for various labeling times ranging from 30 s to 22 h, 50 μL aliquots were removed and mixed with 50 μL of quenching buffer (210 mM TCEP, 7.4 M urea, pH 2.5), followed by flash freezing in liquid nitrogen. Prior to LCMS analysis, the quenched samples were thawed and digested by addition of 50 μL of pepsin stock solution (3 mg/mL, pH 4.5) at a final pH of 2.5 and incubated for 6 min on ice. 50 μL of the resulting digests were loaded onto a Waters (Milford, MA) BEH C8 column (2.1 × 30 mm, 1.7 μm) held at 0 °C. To minimize back exchange, the column, accessories, injector, and solvent delivery lines were submerged in an ice bath. Solvent A was water with 0.1% formic acid and 0.02% TFA, and solvent B was acetonitrile with 0.1% formic acid and 0.02% TFA. Chromatographic separations were carried out at a flow rate of 140 μL/min. Most of the peptides were eluted from the column within 10 min. Mass spectrometry measurements were performed on a Thermo Scientific Q-Exactive high resolution MS with an ESI interface (San Jose, CA). Peptide mass spectra were acquired in positive ion mode with a resolution of 70 000 (at m/z 400). Peptides were identified from data-dependent MSMS scans on the basis of the SEQUEST score and from MS scans by correlation of experimental mass with theoretical-exact mass and isotopic distribution. No adjustments were made for deuterium back exchange. All results are reported as relative deuteration level. The relative deuteration percent of the individual peptides was determined as

deuteration% =

m − m0 × 100 N − 2 − Np

(1)

where m and m0 are the centroid mass value of the labeled and unlabeled peptide(s), respectively. N is the number of residues in the peptide, and Np is the number of proline residues which are not located in the first two positions of the peptide. It is assumed that the first two residues of any peptide will not retain any deuterium after HPLC separation due to their rapid back exchange.48 HDExaminer analysis software (Sierra Analytics, Modesto, CA) was employed to calculate the centroid mass of each peptide as a function of labeling time. The mass difference ΔD of individual peptides between the ADC, C239-mAb, and the wt-mAb was determined as ΔD = mADC − mC239‐mAb or

ΔD = mC239‐mAb − mmAb

(2)

where mADC, mC239‑mAb, and mmAb are the centroid mass of the same peptide from ADC, C239-mAb, and wt-mAb, respectively. Calculation of Intrinsic HDX Profiles. In the case of zero protection (in the absence of HOS), every individual amide hydrogen in a peptide will undergo HDX with its intrinsic exchange rate constant k ch .48 EXCEL Spreadsheets to determine the kch value of each residue are available on Prof. Englander’s Web site (http://hx2.med.upenn.edu/download. html). As previously reported,49 for a peptide consisting of N nonproline residues, the intrinsic HDX profile can be calculated as deuterationlevel (t ) =

1 (N − 2)

N

∑ (1 − exp(−kch(i) × t )) i=3

(3) 5671

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Figure 2. HDX comparison of the wt-mAb and corresponding variant C239-mAb. (A) Mirror plot of the HDX kinetics of wt-mAb (top) versus C239-mAb (bottom). The x-axis is the position of individual peptides. Heavy and light chain peptides are designated as H or L, respectively, followed by the numerical position of the peptide in the linear amino acid sequence. The y-axis is the deuteration percent of the individual peptides calculated from eq 1. The black, blue, light blue, green, orange, magenta, and red lines correspond to data acquired at 0.5, 1, 5, 20, 60, and 240 min and 22 h of deuterium labeling, respectively, for both samples. Each data point is an average of three experiments. Error bars are not shown. (B) Mass difference (ΔD) plot of the individual peptides from the C239-mAb and wt-mAb at each labeling time point. The ΔD was calculated from the average data shown in (A) using eq 2. The dotted lines at the y-axis values of ±0.5 Da represent the threshold for identifying significant differences between the C239-mAb and wt-mAb peptides.

Contributions of the first two residues of any peptide are omitted because they undergo quick back exchange during analysis.

HOS of the mAb, side-by-side HDX comparisons of the C239mAb and the wt-mAb were performed. Deuterium uptake was assessed over 7 time points from 0.5 min to 22 h by pepsin digestion as described in the Materials and Methods section. With the exception of the IgG1 hinge and CH2 domain Nlinked glycopeptide, 94% of the amino acid sequence in both samples could be monitored on the basis of the recovery of overlapping peptic peptides. The deuterium uptake kinetics of approximately 120 peptic peptides was assessed by monitoring the mass increase of each peptide as a function of labeling time. A subset of 60 representative peptides distributed contiguously along HC and LC were selected for the purpose of monitoring the exchange kinetics of the antibodies. The deuteration kinetics of the 60 peptides from the wt-mAb and C239-mAb are illustrated in Figure 2A as Mirror plots.29 The individual peptides show kinetic differences in deuterium uptake which are reflective of different local HOS in the antibody. In order to identify peptides that have differential exchange kinetics in the wt-mAb and C239-mAb, a plot of mass difference (ΔD) for each peptide is shown in Figure 2B and the ΔD of individual peptides was calculated according to eq 2. Similar to previous reports,46 a significance threshold of ±0.5 Da was adopted for the purpose of assessing similarity. For each time point, if the mass difference (absolute values) of a peptide from the two samples is more than 0.5 Da, then the difference is attributed to different deuterium incorporation levels, not measurement errors. The ΔD of all peptides is within ±0.2 Da at any given time point. The ΔD values are much lower than the significance threshold indicating that the C239-mAb and wt-mAb have very similar HDX behavior over these peptides which cover 94% of the primary sequence.

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RESULTS AND DISCUSSION HPLC/MS Analysis. The ADC was conjugated sitespecifically with PBD (or mcMMAF) drug-linkers at HC position 239 in the C239-mAb. The chemical structures of both drug-linkers are shown in Figure 1C. The conjugation strategy results in a conjugated ADC with a molar ratio of drugs to antibody (MRD) of ∼2. To confirm the presence of the druglinker on the HC, the wt-mAb, C239-mAb, and the ADC were partially reduced with DTT and the resulting LCs and HCs were analyzed by reversed phase HPLC/MS. Figure 1 shows the LC/UV profiles (A) of these 3 samples and the measured masses of the HCs (B). As anticipated, the LCs of the 3 samples have the same retention time and the same mass (data not shown). The HCs of wt-mAb and C239-mAb have similar retention time since they only differ by a single amino acid, but the PBD-ADC HCs eluted much later due to the incorporation of hydrophobic PBD drug-linkers. As expected, the mass of C239-mAb heavy chains is 16 Da higher than the wt-mAb and is consistent with the replacement of Ser239 with cysteine. The mass of PBD-ADC heavy chains is 1093 Da higher than the C239-mAb, which is consistent with the incorporation of the PBD drug-linker into HCs in the ADC. Peptide mapping confirms that the drug-linker is linked to the engineered cysteine residue at position 239 in HC (data not shown). HDX-MS Analysis of the C239-mAb and mAb. To understand the impact of the site-directed mutagenesis on the 5672

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assumption of zero protection (complete absence of HOS) and shown in Figure 3 as dotted lines. The calculation was based on published reference values48 (Materials and Methods). The theoretical HDX kinetics of the sequence LGGPCV do not differ substantially from those of the LGGPSV, and there is only a slight increase in the exchange rate after the replacement of Ser with Cys. The experimental HDX characteristics (Figure 3, solid lines) trend in the same manner as the theoretical data with LGGPSV and LGGPCV displaying similar HDX kinetics. On the basis of these data, it is very likely that the two peptides from the wt-mAb and C239-mAb have similar conformational features. HDX-MS Analysis of the ADC and C239-mAb. The HDX comparison of the PBD-ADC and C239-mAb was performed in the same manner as the comparison of C239mAb and wt-mAb, and the mass difference (ΔD) plot of the HDX kinetics of individual peptides from the PBD-ADC and C239-mAb is shown in Figure 4. It is evident from the ΔD plot that there is a high degree of similarity between the PBD-ADC and C239-mAb. For each time point, the ΔD of 59 peptides is within ±0.3 Da. One peptide, H262-277, had positive ΔD values just above the significance threshold for some time points indicating that the PBD-ADC had increased deuterium uptake relative to the C239-mAb in that region (Figure 5A). It should be noted, however, that the first two residues of any peptide will not retain deuterium due to their rapid back exchange, so the actual difference in the PBD-ADC and the C239-mAb exchange kinetics can be further narrowed down to residues 264−277. We also detected the overlapping peptide H266-277, and this peptide had the same HDX kinetics in the C239-mAb and PBD-ADC (Figure 5B). The results from the overlapping peptide indicated that residues 268−277 in the primary sequence of the C239-mAb and the PBD-ADC display the same exchange behavior. Taken together, the similar HDX exchange kinetics for peptide H266-277 and the differential kinetics displayed by the longer peptide H262-277 indicate that region of dissimilarity between the C239-mAb and the PBDADC can be localized to the regions on the two peptides which do not overlap and do not include the first two N-terminal amino acids. On the basis of this rationale, we conclude that the small segment (264VDVS) located in the CH2 domain is more structurally dynamic and/or more solvent exposed in the PBDADC. The subtle differences observed for the PBD-ADC relative to the C239-mAb prompted further inquiry into whether the differences were specific to the PBD-type drug-linker used here.

The exchange kinetics of the wt- and C239-mAb peptides containing the amino acid substitution is important for understanding the impact of the substitution on the HOS of the mAb. The deuteration kinetics of the peptic peptide 235 LGGPSV from the wt-mAb and 235LGGPCV from C239mAb are shown in Figure 3 (solid lines). A direct comparison

Figure 3. Comparison of the experimental HDX kinetics of two peptides 235LGGPSV in the wt-mAb (solid line, black circles) and 235 LGGPCV in the C239-mAb (solid line, red triangles). The black and red dotted lines represent the theoretical HDX kinetics of the peptides LGGPSV and LGGPCV, respectively, calculated in the hypothetical case of zero protection. For the theoretical data points, it is assumed that HDX at every amide proceeds with its intrinsic rate constant determined on the basis of published reference values.48,49

of the deuteration behavior of these two peptides is not straightforward because they have one different amino acid due to the substitution, and previous studies have shown that primary sequence has an impact on amide HDX. The intrinsic HDX kinetics of unstructured peptides (assuming a complete absence of HOS) can vary by as much as 102 depending on the amino acid sequence differences.48 Experimental HDX kinetics of a protein segment or peptide depends on two factors: the conformation and dynamics of the protein segment and the amino acid sequence. In the case of the wt-mAb and the C239mAb, we do not know the actual impact that the amino acid substitution by itself has on the experimental HDX kinetics. Thus, we assumed that any observable differences are a summation of the local conformational differences and the amino acid (primary sequence) differences. For reference purposes, the theoretical or intrinsic HDX profiles of the two sequences LGGPSV and LGGPC are calculated under the

Figure 4. Mass difference (ΔD) plot of the individual peptides from the PBD-ADC and C239-mAb at each labeling time point. The x-axis is the position of individual peptides. The ΔD was calculated from eq 2. The dotted lines at the y-axis values ±0.5 Da represent the threshold for identifying significant differences between the C239-mAb and PBD-ADC peptides. 5673

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Figure 5. HDX comparison of two overlapping peptides from the C239-mAb and the corresponding PBD-ADC (A and B) or mcMMAF-ADC (C and D). A and C represent HDX kinetics of the peptide 262VVVDVSHEDPEVKFNW; B and D represent HDX kinetics of a smaller overlapping peptide 266VSHEDPEVKFNW.

Figure 6. Crystal structure of the CH2 domain of a model humanized IgG1 (PDB: 1HZH). (A) Residues 235−240 (LGGPSV) and 264−267 (VDVS) in the CH2 domain are highlighted in magenta color. The intrachain disulfide is depicted as spheres. (B) Zoomed-in view of backbone amide H-bonding of the CH2 domain. The amide hydrogen is depicted in blue and carbonyl oxygen is depicted in red. Green dotted lines represent stable H-bonds identified by Swiss PDB Viewer with default values.50

The PBD drug molecule contains reactive imine functional groups which potentially may interact with the mAb. To evaluate this question, the C239-mAb was also conjugated with another drug-linker, mcMMAF, where the drug molecule is mainly a polypeptide, negatively charged, and less hydrophobic than the PBD molecule. The resulting site-specific conjugate mcMMAF-ADC was then compared to the C239-mAb in a similar manner as the previously described PBD-ADC/C239mAb comparison. As was the case for the ADC conjugated with the PBD drug-linker, the HDX behavior of the mcMMAF-ADC is very similar to C239-mAb throughout 90% of the amino acid

sequence (data not shown) and only one segment, H262-277, displayed increased HDX kinetics relative to the C239-mAb (Figure 5C). Similar to the PBD-ADC, an overlapped peptide, H266-277, was found to have the same deuterium uptake in the mcMMAF-ADC and C239-mAb (Figure 5D). Subtracting peptide H266-277 from H262-277, it is apparent that the same small segment (264VDVS), which was pinpointed to have increased deuterium uptake in the PBD-ADC, also has increased deuterium uptake in the mcMMAF-ADC. These results indicate that the segment (264VDVS) in the CH2 domain becomes more structurally dynamic and/or solvent exposed 5674

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followed by drug conjugation at the altered residue(s). Sitespecifically conjugated ADCs have emerged as an attractive therapeutic modality due in part to their reduced heterogeneity and the potential to optimize the number and the site of druglinker incorporation on the mAb.7,8 An important consideration for site-specific conjugation strategies is ensuring that the amino acid sites selected for substitution and subsequent drugconjugation are not leading to significant changes in protein structure and function. It has been shown previously that chemical modifications on the primary structure of mAb can impact HOS and stability.38,41,42,52 These prior results led us to question whether an ADC composed of cytotoxic agents linked to engineered amino acids in the mAb backbone might also manifest significant HOS changes as a consequence and whether there is potential for robust analytical methods to facilitate the optimization of the conjugation site and druglinkers incorporated. This work, which is the first application of HDX-MS for understanding the impact of site-specific conjugation on the HOS of mAb, was undertaken to shed light on these questions. The HDX kinetics of the wt-mAb and the corresponding engineered cysteine variant C239-mAb were assessed over 7 time-points spanning 0.5 min to 22 h. The highly similar HDX behavior of the wt-mAb and C239-mAb demonstrates that the replacement of Ser239 with Cys does not change the local and long-range backbone amide H-bonding networks. Subsequent HDX comparison between the ADC and C239-mAb indicated that the incorporation of drug-linkers (either PBD or mcMMAF) at Cys239 did not induce significant conformational changes. Taken together, the HDX data indicates that the site-specific drug conjugation strategies studied here are largely structurally benign. However, we did observe minor and highly localized HDX changes in the ADCs confined to a small segment (264VDVS). The backbone amide of Ver264 is supposed to be H-bonded with Cys239 which is the site of drug conjugation. The increased HDX rate of the segment 264 VDVS in both PBD and mcMMAF-ADCs indicates that drug conjugation on the side chain of Cys239 can impact the backbone amide H-bonding network of the conjugation site Cys239. It is reasonable to expect that HOS changes could be minor or substantial depending on the site of conjugation, the chemical properties of the drug-linkers, and other factors. The current work demonstrates the utility of HDX-MS for pinpointing the structural impact of site-specific conjugation technologies on parent mAbs that would be missed by traditional biophysical methods. The methodology has great potential for use as an analytical tool for screening and optimizing the conjugation sites and drug-linkers for sitespecific ADC modalities.

after conjugating either PBD or mcMMAF drug-linkers on the side chain of Cys239. The observed conformational changes may be a general consequence of the site-specific drug conjugation at Cys239 but not exclusive to the PBD-ADC. Structural Interpretation. The crystal structure of CH2 domain of a homologous IgG1 antibody (PBD: 1HZH)50 was examined in order to understand the structural implications of the HDX kinetics observed in the wt-mAb, C239-mAb, and ADCs. The 1HZH Fc domain primary sequence is identical to that of the wt-mAb examined here. The residues of interest, 235 LGGPSV and 264VDVS, are highlighted in magenta color in Figure 6A. The crystal structure indicates that these regions are proximally adjacent and that they are both involved in the secondary structure of beta strands. Backbone amide hydrogenbonding (H-bonding) of the CH2 domain was detected using the Swiss PDB Viewer with default values,51 i.e., a donor− acceptor distance between 2.195 and 3.3 Å, respectively, and a minimum angle of 90°. A zoomed-in view of the CH2 domain amide H-bonding network is shown in Figure 6B with green dotted lines representing identified stable H-bonds and green numbers representing the donor−acceptor distance. It is apparent that Ser239 and Val264 in the wt-mAb form backbone amide H-bonds which stabilize the local beta-sheet secondary structure. If there was significant conformational disruption in the CH2 domain caused by replacing Ser239 with Cys239, then we would expect to see significantly different HDX behavior in the regions containing, or proximally adjacent to, Cys239 and Val264. After carefully comparing all potentially interacting primary sequence regions, we found that all relevant regions (H241-252, H253-262, H262-277, H264-277, etc.) had identical HDX behavior in the wt-mAb and C239-mAb. These results indicate that Cys239 forms stable amide Hbonds with Val264 in a similar manner as the Ser239 in the wtmAb and that replacement of Ser239 with Cys239 does not change the local or long-range backbone amide H-bonding network. The incorporation of PBD or mcMMAF drug-linkers to Cys239 resulted in a slight increase in the deuterium uptake of a small region 264VDVS relative to the corresponding unconjugated C239-mAb, but all other regions were unchanged. This result indicates that alkylation of Cys239 with drug-linkers weakened the backbone amide H-bonding and/or solvent protection between the CH2 domain residues Cys239 and Val264. The HDX behavior of proximally adjacent sequences in the C239-mAb and ADCs are equivalent which indicates that conjugation of the C239-mAb with drug-linkers does not significantly alter the local amide H-bonding network. Also, the ADCs conjugated with either PBD or mcMMAF drug-linkers share very similar conformational features despite the considerable difference in chemistry and hydrophobicity for the two drug molecules. The equivalent HDX kinetics displayed by the PBD-ADC and the mcMMAF-ADC suggests that the two different drug-linkers have similar impacts on the HOS of the Fc domain, but it should be noted that both PBD and mcMMAF drug-linkers utilize the same maleimide-based conjugation chemistry. It will be interesting to explore the impact of other ADC linkers which involve different conjugation chemistry.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: 425-527-2456. Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Alley, S. C.; Zhang, X.; Okeley, N. M.; Anderson, M.; Law, C. L.; Senter, P. D.; Benjamin, D. R. J. Pharmacol. Exp. Ther. 2009, 330, 932−938. (2) Ikeda, H.; Hideshima, T.; Fulciniti, M.; Lutz, R. J.; Yasui, H.; Okawa, Y.; Kiziltepe, T.; Vallet, S.; Pozzi, S.; Santo, L.; Perrone, G.; Tai, Y. T.; Cirstea, D.; Raje, N. S.; Uherek, C.; Dalken, B.; Aigner, S.;

CONCLUSIONS Many site-specific ADCs are developed by modifying the primary sequence of mAbs via site-directed mutagenesis or through incorporation of unnatural amino acids or tags 5675

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Article

Analytical Chemistry

(23) Guo, J.; Kumar, S.; Prashad, A.; Starkey, J.; Singh, S. K. Pharm. Res. 2014, 31, 1710−1723. (24) Hawe, A.; Kasper, J. C.; Friess, W.; Jiskoot, W. Eur. J. Pharm. Sci. 2009, 38, 79−87. (25) Nagarkar, R. P.; Murphy, B. M.; Yu, X.; Manning, M. C.; AlAzzam, W. A. Curr. Pharm. Biotechnol. 2013, 14, 199−208. (26) Ionescu, R. M.; Vlasak, J.; Price, C.; Kirchmeier, M. J. Pharm. Sci. 2008, 97, 1414−1426. (27) Wakankar, A. A.; Feeney, M. B.; Rivera, J.; Chen, Y.; Kim, M.; Sharma, V. K.; Wang, Y. J. Bioconjugate Chem. 2010, 21, 1588−1595. (28) Houde, D.; Arndt, J.; Domeier, W.; Berkowitz, S.; Engen, J. R. Anal. Chem. 2009, 81, 2644−2651. (29) Houde, D.; Berkowitz, S. A.; Engen, J. R. J. Pharm. Sci. 2011, 100, 2071−2086. (30) Houde, D.; Engen, J. R. Methods Mol. Biol. 2013, 988, 269−289. (31) Huang, R. Y.; Chen, G. Anal. Bioanal. Chem. 2014, 406, 6541− 6558. (32) Marciano, D. P.; Dharmarajan, V.; Griffin, P. R. Curr. Opin. Struct. Biol. 2014, 28C, 105−111. (33) Smith, D. L.; Deng, Y.; Zhang, Z. J. Mass Spectrom. 1997, 32, 135−146. (34) Dharmasiri, K.; Smith, D. L. Anal. Chem. 1996, 68, 2340−2344. (35) Englander, S. W.; Sosnick, T. R.; Englander, J. J.; Mayne, L. Curr. Opin. Struct. Biol. 1996, 6, 18−23. (36) Konermann, L.; Pan, J.; Liu, Y. H. Chem. Soc. Rev. 2011, 40, 1224−1234. (37) Wang, L.; Pan, H.; Smith, D. L. Mol. Cell Proteomics 2002, 1, 132−138. (38) Houde, D.; Peng, Y.; Berkowitz, S. A.; Engen, J. R. Mol. Cell. Proteomics 2010, 9, 1716−1728. (39) Mayne, L.; Kan, Z. Y.; Chetty, P. S.; Ricciuti, A.; Walters, B. T.; Englander, S. W. J. Am. Soc. Mass Spectrom. 2011, 22, 1898−1905. (40) Zhang, Z.; Zhang, A.; Xiao, G. Anal. Chem. 2012, 84, 4942− 4949. (41) Burkitt, W.; Domann, P.; O’Connor, G. Protein Sci. 2010, 19, 826−835. (42) Bobst, C. E.; Abzalimov, R. R.; Houde, D.; Kloczewiak, M.; Mhatre, R.; Berkowitz, S. A.; Kaltashov, I. A. Anal. Chem. 2008, 80, 7473−7481. (43) Iacob, R. E.; Bou-Assaf, G. M.; Makowski, L.; Engen, J. R.; Berkowitz, S. A.; Houde, D. J. Pharm. Sci. 2013, 102, 4315−4329. (44) Zhang, A.; Singh, S. K.; Shirts, M. R.; Kumar, S.; Fernandez, E. J. Pharm. Res. 2011, 29, 236−250. (45) Manikwar, P.; Majumdar, R.; Hickey, J. M.; Thakkar, S. V.; Samra, H. S.; Sathish, H. A.; Bishop, S. M.; Middaugh, C. R.; Weis, D. D.; Volkin, D. B. J. Pharm. Sci. 2013, 102, 2136−2151. (46) Pan, L. Y.; Salas-Solano, O.; Valliere-Douglass, J. F. Anal. Chem. 2014, 86, 2657−2664. (47) Cheung, A.; Struble, E.; He, J.; Yang, C.; Wang, E.; Thurston, D. E.; Liu, P. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2005, 822, 10−20. (48) Bai, Y.; Milne, J. S.; Mayne, L.; Englander, S. W. Proteins 1993, 17, 75−86. (49) Pan, Y.; Piyadasa, H.; O’Neil, J. D.; Konermann, L. J. Mol. Biol. 2012, 416, 400−413. (50) Saphire, E. O.; Parren, P. W.; Pantophlet, R.; Zwick, M. B.; Morris, G. M.; Rudd, P. M.; Dwek, R. A.; Stanfield, R. L.; Burton, D. R.; Wilson, I. A. Science 2001, 293, 1155−1159. (51) Guex, N.; Peitsch, M. C. Electrophoresis 1997, 18, 2714−2723. (52) Majumdar, R.; Esfandiary, R.; Bishop, S. M.; Samra, H. S.; Middaugh, C. R.; Volkin, D. B.; Weis, D. D. MAbs 2015, 7, 84−95.

Osterroth, F.; Munshi, N.; Richardson, P.; Anderson, K. C. Clin. Cancer Res. 2009, 15, 4028−4037. (3) Lutz, R. J.; Whiteman, K. R. MAbs 2009, 1, 548−551. (4) Doronina, S. O.; Toki, B. E.; Torgov, M. Y.; Mendelsohn, B. A.; Cerveny, C. G.; Chace, D. F.; DeBlanc, R. L.; Gearing, R. P.; Bovee, T. D.; Siegall, C. B.; Francisco, J. A.; Wahl, A. F.; Meyer, D. L.; Senter, P. D. Nat. Biotechnol. 2003, 21, 778−784. (5) Lewis Phillips, G. D.; Li, G.; Dugger, D. L.; Crocker, L. M.; Parsons, K. L.; Mai, E.; Blattler, W. A.; Lambert, J. M.; Chari, R. V.; Lutz, R. J.; Wong, W. L.; Jacobson, F. S.; Koeppen, H.; Schwall, R. H.; Kenkare-Mitra, S. R.; Spencer, S. D.; Sliwkowski, M. X. Cancer Res. 2008, 68, 9280−9290. (6) Panowski, S.; Bhakta, S.; Raab, H.; Polakis, P.; Junutula, J. R. MAbs 2014, 61, 34−45. (7) Jeffrey, S. C.; Burke, P. J.; Lyon, R. P.; Meyer, D. W.; Sussman, D.; Anderson, M.; Hunter, J. H.; Leiske, C. I.; Miyamoto, J. B.; Nicholas, N. D.; Okeley, N. M.; Sanderson, R. J.; Stone, I. J.; Zeng, W.; Gregson, S. J.; Masterson, L.; Tiberghien, A. C.; Howard, P. W.; Thurston, D. E.; Law, C. L.; Senter, P. D. Bioconjugate Chem. 2013, 24, 1256−1263. (8) Shen, B. Q.; Xu, K.; Liu, L.; Raab, H.; Bhakta, S.; Kenrick, M.; Parsons-Reponte, K. L.; Tien, J.; Yu, S. F.; Mai, E.; Li, D.; Tibbitts, J.; Baudys, J.; Saad, O. M.; Scales, S. J.; McDonald, P. J.; Hass, P. E.; Eigenbrot, C.; Nguyen, T.; Solis, W. A.; Fuji, R. N.; Flagella, K. M.; Patel, D.; Spencer, S. D.; Khawli, L. A.; Ebens, A.; Wong, W. L.; Vandlen, R.; Kaur, S.; Sliwkowski, M. X.; Scheller, R. H.; Polakis, P.; Junutula, J. R. Nat. Biotechnol. 2012, 30, 184−189. (9) Junutula, J. R.; Raab, H.; Clark, S.; Bhakta, S.; Leipold, D. D.; Weir, S.; Chen, Y.; Simpson, M.; Tsai, S. P.; Dennis, M. S.; Lu, Y.; Meng, Y. G.; Ng, C.; Yang, J.; Lee, C. C.; Duenas, E.; Gorrell, J.; Katta, V.; Kim, A.; McDorman, K.; Flagella, K.; Venook, R.; Ross, S.; Spencer, S. D.; Lee Wong, W.; Lowman, H. B.; Vandlen, R.; Sliwkowski, M. X.; Scheller, R. H.; Polakis, P.; Mallet, W. Nat. Biotechnol. 2008, 26, 925−932. (10) Kung Sutherland, M. S.; Walter, R. B.; Jeffrey, S. C.; Burke, P. J.; Yu, C.; Kostner, H.; Stone, I.; Ryan, M. C.; Sussman, D.; Lyon, R. P.; Zeng, W.; Harrington, K. H.; Klussman, K.; Westendorf, L.; Meyer, D.; Bernstein, I. D.; Senter, P. D.; Benjamin, D. R.; Drachman, J. G.; McEarchern, J. A. Blood 2013, 122, 1455−1463. (11) Drake, P. M.; Albers, A. E.; Baker, J.; Banas, S.; Barfield, R. M.; Bhat, A. S.; de Hart, G. W.; Garofalo, A. W.; Holder, P.; Jones, L. C.; Kudirka, R.; McFarland, J.; Zmolek, W.; Rabuka, D. Bioconjugate Chem. 2014, 25, 1331−1341. (12) Rachel, N. M.; Pelletier, J. N. Biomolecules 2013, 3, 870−888. (13) Strop, P. Bioconjugate Chem. 2014, 25, 855−862. (14) Axup, J. Y.; Bajjuri, K. M.; Ritland, M.; Hutchins, B. M.; Kim, C. H.; Kazane, S. A.; Halder, R.; Forsyth, J. S.; Santidrian, A. F.; Stafin, K.; Lu, Y.; Tran, H.; Seller, A. J.; Biroc, S. L.; Szydlik, A.; Pinkstaff, J. K.; Tian, F.; Sinha, S. C.; Felding-Habermann, B.; Smider, V. V.; Schultz, P. G. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 16101−16106. (15) Liu, W.; Brock, A.; Chen, S.; Chen, S.; Schultz, P. G. Nat. Methods 2007, 4, 239−244. (16) Zhou, Q.; Stefano, J. E.; Manning, C.; Kyazike, J.; Chen, B.; Gianolio, D. A.; Park, A.; Busch, M.; Bird, J.; Zheng, X.; SimondsMannes, H.; Kim, J.; Gregory, R. C.; Miller, R. J.; Brondyk, W. H.; Dhal, P. K.; Pan, C. Q. Bioconjugate Chem. 2014, 25, 510−520. (17) Okeley, N. M.; Toki, B. E.; Zhang, X.; Jeffrey, S. C.; Burke, P. J.; Alley, S. C.; Senter, P. D. Bioconjugate Chem. 2013, 24, 1650−1655. (18) Wakankar, A.; Chen, Y.; Gokarn, Y.; Jacobson, F. S. MAbs 2011, 3, 161−172. (19) Chen, J.; Yin, S.; Wu, Y.; Ouyang, J. Anal. Chem. 2013, 85, 1699−1704. (20) Valliere-Douglass, J. F.; McFee, W. A.; Salas-Solano, O. Anal. Chem. 2012, 84, 2843−2849. (21) Wang, L.; Amphlett, G.; Blattler, W. A.; Lambert, J. M.; Zhang, W. Protein Sci. 2005, 14, 2436−2446. (22) Xu, K.; Liu, L.; Saad, O. M.; Baudys, J.; Williams, L.; Leipold, D.; Shen, B.; Raab, H.; Junutula, J. R.; Kim, A.; Kaur, S. Anal. Biochem. 2011, 412, 56−66. 5676

DOI: 10.1021/acs.analchem.5b00764 Anal. Chem. 2015, 87, 5669−5676