Quantitative Conjugated Payload Measurement Using Enzymatic

Jan 31, 2017 - As antibody-drug conjugate (ADC) design is evolving with novel payload, linker, and conjugation chemistry, the need for sensitive and p...
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Quantitative Conjugated Payload Measurement Using Enzymatic Release of Antibody−Drug Conjugate with Cleavable Linker Brian Rago,† L. Nathan Tumey,† Cong Wei,‡,# Frank Barletta,*,‡ Tracey Clark,§ Steven Hansel,‡ and Xiaogang Han*,‡,¶ †

Medicine Design, ‡Biomedicine Design, and §Pfizer Essential Health, Pfizer Inc., Eastern Point Road, Groton, Connecticut 06340, United States ABSTRACT: As antibody-drug conjugate (ADC) design is evolving with novel payload, linker, and conjugation chemistry, the need for sensitive and precise quantitative measurement of conjugated payload to support pharmacokinetics (PK) is in high demand. Compared to ADCs containing noncleavable linkers, a strategy specific to linkers which are liable to pH, chemical reduction, or enzymatic cleavage has gained popularity in recent years. One bioanalytical approach to take advantage of this type of linker design is the development of a PK assay measuring released conjugated payload. For the ADC utilizing a dipeptide ValCit linker studied in this report, the release of payload PF-06380101 was achieved with high efficiency using a purified cathepsin B enzyme. The subsequent liquid chromatography mass spectrometry (LC/MS) quantitation leads to the PK profile of the conjugated payload. For this particular linker using a maleimide-based conjugation chemistry, one potential route of payload loss would result in an albumin adduct of the linker-payload. While this adduct’s formation has been previously reported, here, for the first time, we have shown that payload from a source other than ADC contributes only up to 4% of total conjugated payload while it accounts for approximately 35% of payload lost from the ADC at 48 h after dosing to rats.

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INTRODUCTION The study of ADC absorption, distribution, metabolism, and excretion (ADME)1 is more complex relative to other therapeutic modalities due to the unique challenges associated with simultaneous analysis of both the large molecule (monoclonal antibody) and small molecule (payload and linker) components. These challenges include the identification of the most pertinent analytes, the measurement of the clearance and exposure of the most pharmacologically relevant species, and the associated technical feasibility to achieve this. One particular aspect, highlighted by Gorovits et al.,2 is the unique bioanalytical challenges of working with ADCs that are dosed as heterogeneous mixtures, often requiring characterization by drug−antibody ratios (DAR) and/or the quantitative measurement of small molecule moieties while conjugated to the antibody. At different stages of novel linker-payload discovery, it is often critical to qualitatively measure the most prominent released small molecule species using LC/MS, thus providing insight into exposure−response relationships and structure− activity relationships (SAR). The cytotoxic payload or linker− payload components are themselves amenable to well established quantitative bioanalytical methods employing an LC/MS/MS tandem quadrupole platform. It is generally believed that antibody-conjugated payload levels provide a valuable measure of the potentially active circulating entity © XXXX American Chemical Society

which presumably drives both ADC efficacy and toxicity. Intact protein analysis of the ADC can be used to quantitate conjugated payload and to characterize any biotransformation of the payload in its conjugated form.3,4 The approach described herein can also be used to determine conjugated payload PK for conjugates that utilize chemical5 or enzymatic6 labile linkers and potentially can be applied both in late stage discovery as well as in regulated studies.7,8 The payload, PF-06380101,9 is an auristatin derivative which is conjugated to trastuzumab via an mcValCitPABC linker (Scheme 1A,B), resulting in an ADC with an average drug load to antibody ratio of 3.8. The quantitation of the conjugated payload takes advantage of the protease cleavable ValCit linker. In short, the total payload level was quantitated by LC/MS/MS following incubation of the plasma sample with cathepsin B, which resulted in ∼90% cleavage efficiency. Subsequently the conjugated payload can be obtained by subtracting the unconjugated payload concentration measured in the absence of enzyme treatment. LC/MS/MS measurement of the enzymatically released analyte, PF-06380101, is far more sensitive than the alternative DAR measurement by intact protein MS analysis. The conjugated payload levels determined Received: December 4, 2016 Revised: January 6, 2017

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DOI: 10.1021/acs.bioconjchem.6b00695 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Scheme 1. (A) Payload PF-06380101; (B) Payload Release via Enzymatic Cleavage of Maleimidocaproyl-L-valine-L-citrulline-paminobenzyl (mcValCit-PABC) Linker; (C) Retro-Michael Reaction Followed by Maleimide Transfer; (D) Proposed Clearance Pathways of an ADC with Cys-mcValCit Conjugation Chemistry in Systemic Circulation

by these two methods are in very good agreement considering that two different sample preparations and MS platforms are involved as well as the incorporation of LBA data used to calculate DAR derived conjugated payload for the latter approach.10 In order to further investigate the clearance pathway for this ADC, we used an immunocapture procedure to remove ADC from the plasma sample and then treated the remaining plasma with cathepsin B in order to quantitate any PF-06380101 payload originating from sources other than circulating ADC. It is well established that retro-Michael reaction of the maleimide could be a significant cause of payload loss and results in the formation of adducts with albumin.4,11,12 The goal of the present study is to quantitate the exposure contribution from these non-ADC components that formed in circulation.

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Figure 1. Release kinetics of PF-06380101 from ADC spiked into rat plasma and incubated with cathepsin B. The appearance of the payload level is measured as a function of in vitro incubation time with cathepsin B.

RESULTS Kinetics of Payload Release by Cathepsin B. The ADC was prepared by conjugating partially reduced trastuzumab to mcValCit-PABC-PF-06380101. Cathepsin B efficiently cleaves the ValCit linker6 resulting in the rapid appearance of the payload, as shown in Figure 1. The release kinetic was plotted against two standard curves, one using the payload itself and

the other using spiked ADC samples with DAR of 3.80 which underwent the same enzymatic cleavage. Based on theoretical total conjugated payload of a sample at 50 μg/mL with a DAR of 3.8, the recovery of the payload is calculated at 90%. When B

DOI: 10.1021/acs.bioconjchem.6b00695 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry using ADC as the standard curve for release payload quantitation, a precise measurement of DAR is required in addition to having an ADC stock of known concentration that is free of any unconjugated payload. The knowledge of the kinetic profile provided the basis for finalizing the sample incubation protocol. Detailed studies of the cleavage kinetics of other ADCs with the same linker payload reveals that the cleavage efficiency is conjugation site-dependent and therefore ADC dependent (data not shown). The released entity, PF06380101, was found to be completely stable in the presence of cathepsin B during the time course of the incubation (data not shown). For the remainder of the experiments described herein, the ADC was used to prepare the standard curve. Total Payload and Unconjugated Payload Measurements. The ADC was dosed intravenously at 3 mg/kg in Sprague−Dawley rats. The direct measurement of PF06380101 in plasma after cathepsin B treatment reflects the total payload amount in plasma. It encompasses the unconjugated payload, antibody-conjugated payload, and other possible adducts formed by any deconjugated linker− payload due to maleimide exchange. Unconjugated payload can be isolated from plasma by organic solvent precipitation and quantitatively measured by LC/MS/MS. As shown in Figure 2, the unconjugated payload level is much lower than the total plasma payload, with AUC of 8 ng mL/h vs 39800 ng mL/h, respectively (Figure 2A, Table 1). This indicates that conjugated is the most dominant payload form in plasma,

Table 1. PK Parameters of Conjugated and Unconjugated Payload

LBA ADCb Total CatB Release DAR × mAbb ADC Bound ADC Depleted Unconjugated

T1/2 (h)

Cmax (ng/ mL)

1.10 1.30

60 53

1080 1100

1.30 1.80 NCc N/A

60 54 63 N/A

1130 708 9.7 N/A

AUC (ng h/mL)

% total CatB AUCa

clearance (mL/min/kg)

44100 39800

N/A N/A

39400 27200 1390 8.00

99.2 68.4 3.5 0.03

a

Based on total AUC as calculated from cathepsin b cleavage. bData converted to ng/mL. cNot calculated, ADC depleted is a measurement of adduct formation, not given as dosed drug.

and the unconjugated payload component is cleared relatively fast and its presence is negligible for the sake of discussion below. Payload Level in ADC-Depleted Samples. It has been reported that deconjugation of mcValCit linker from the ADC can result in the formation of adducts, particularly with thiolcontaining molecules4,11−13 such as cysteine, glutathione, or plasma albumin. To what extent there is payload contribution from sources other than ADC has not been quantitatively assessed in an in vivo setting. These adducts would be subject to cathepsin B cleavage during sample preparation. Here, ADC was first removed from plasma using beads with anti-human Fc antibody and the remaining plasma samples were subjected to cathepsin B treatment and subsequent payload quantitation. The data shows that there is a measurable amount of proteinbound payload after removal of ADC. The Tmax of payload in the ADC-depleted samples is 24 h (Figure 2A) indicating that it is being generated after dosing. Comparison of the payload level in the ADC-depleted samples at the 5 min time point (i.e., adduct species) to the total payload level (Figure 2A) indicates more than 99% removal of the ADC. The concentrations of payload detected after ADC removal was less than 1% of the total payload cleaved in the 5 min sample. The AUC ratio of payload from ADC depleted compared to that of total payload conjugated to the ADC is small and approximately 4% (Table 1). An estimate of the amount of payload lost due to maleimide exchange deconjugation is obtained from the total antibody concentration (molar) multiplied by the change of DAR at the relevant time points: ΔDAR × [mAb] × 742 Da. This denominator does not include those cleared from circulation by catabolism. Accordingly, the payload from adducts measured in ADCdepleted plasma accounted for as much as 35% of payload loss due to deconjugation (Figure 2B) at 48 h. The Tmax for the payload level from the ADC depleted sample is in the range of 48 to 72 h, suggesting that the payload adducts form more slowly than the appearance of systemic unconjugated payload. Evidence has shown that a retro-Michael process leads to deconjugation and ultimately to the formation of albuminadducts.4,11,12 In order to attenuate this process, we have described conditions for the hydrolysis of the maleimide ring resulting in ring-opened conjugates wherein the retro-Michael reaction is prevented.13 With this in mind, a ring-opened trastuzumab-vc0101 conjugate was prepared by the previously described method13and evaluated in a PK study dosed in rats at 10 mg/kg. As anticipated, the resulting payload level in the ADC-depleted plasma samples are considerably lower in the

Figure 2. (A) Overlay of total, unconjugated, and ADC depleted payload profiles (n = 3). (B) Relative non-ADC contribution to total plasma payload and due to retro-Michael deconjugation. The ratio of payload in ADC depleted against total (■) was calculated by the curves (▲/⧫) in (A). C

DOI: 10.1021/acs.bioconjchem.6b00695 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry ring-opened maleimide group as compared to the ring-closed maleimide group (Figure 3). The AUC ratio of ADC-depleted

Figure 4. Comparison of conjugated payload PK measured by intact mass (DARxmAb), LBA, and enzyme release methodologies for in vivo samples at 3 mg/kg. For comparison all measurements are shown in ng/mL conjugated payload. Figure 3. Hydrolysis of maleimide ring prevents the retro-Michael deconjugation thus reducing significantly the formation of albumin adduct. Reduced payload levels in the ADC depleted samples for ringopened arm (×) as compared to nonhydrolyzed ADC (▲).

support at different stages of ADC discovery and development when different bioanalytical platforms might be best suited.

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DISCUSSION Assay Development and Performance. The measurement of conjugated payload for ADCs with cleavable linkers has been reported for Mylotarg,5 DM1 with N-succinimidyl 4-(2pyridyldithio) butanoate (SPDB) linker15 and vcMMAE ADCs.7,8 Analysis of Mylotarg and the DM1 conjugate relied upon chemical reduction of the disulfide bond in order to release the payload for quantitation. For the enzymatically liable linker, ValCit-PABC, a LC/MS/MS quantitative payload assay was developed based on ex vivo cleavage of the dipeptide linker. The method described here is based on that of Sanderson et al.6 Optimization was carried out to ensure the integrity of the released payload during the incubation period while maximizing the cleavage efficiency. When using either payload or cathepsin treated ADC for standard curve quantitation, the resulting payload levels of total antibody-conjugated payload from cathepsin cleavage are in excellent agreement, which suggests good release efficiency (Figure 1). Using an ADC reference material as a standard curve requires material that has a known DAR and is free of residual (unconjugated) payload. The lower limit of quantitation of PF-06380101 was 10 pg/mL. More recently, the same group that originally reported a cathepsin B cleavage assay in 2005 described a release treatment using papain.8 The authors showed that this enzyme was robust in cleaving the ValCit-PABC linker, thereby releasing MMAE. Comparison of Three Bioanalytical Methods for PK. Our lab has recently described a conjugated payload assay combining LBA measured total antibody concentration and LC/MS DAR.10 In the present study, the same set of PK samples (3 mg/kg dose) were analyzed by three assays: by

to total is approximately 0.8% for the former and ∼4% for the latter (Table 2) suggesting that lower levels of maleimide exchange, thus adduct formation, occurred in the ring-opened cohort. It should be noted that preparation process resulted in about 80% ring hydrolysis (data not shown); thus there is still is a small amount of linker−payload deconjugation and adduct formation observed. Antibody-Conjugated Payload. The PK of antibodyconjugated payload can be assessed by three different methods: LBA measurement of ADC,14 combination of total antibody measurement (by LBA) with an intact protein based DAR analysis,10and the presently described cathepsin B cleavage approach (Figure 4). Conjugated payload level was calculated form LBA ADC by multiplying ADC concentration in molar with DAR = 3.8 by the molecular weight of PF-06380101. The reason we use the same DAR throughout the time curve is that the ADC standard curve for LBA measurement was prepared with DAR = 3.8. Immuno-extracted ADC was also subjected to cathepsin B release; the PF-06380101 level is listed in Table 1. The DAR × total mAb approach can be applied to both cleavable or noncleavable ADCs, and is particularly valuable in the early discovery stage when anti-payload reagents are not yet available to support PK studies. When the ADC is conjugated with a cleavable linker, the enzymatic protocol described above can be employed to measure antibody-conjugated payload. The agreement among the three PK profiles suggests that there is cross-platform consistency and ensured continuity of PK

Table 2. PK Parameters of Conjugated and Unconjugated Payload for Ring-Open and Conventional ADC Total CatB Release ADC Depleted Unconjugated

a

ADC

AUC (ng h/mL)

% total CatB AUC

clearance (mL/min/kg)

T1/2 (h)

Cmax (ng/mL)

Conventional ADC Ring-open ADC Conventional ADC Ring-open ADC Conventional ADC Ring-open ADC

188000 169000 6460 1360 30.0 56.0

N/A N/A 3.5 0.8 N/A N/A

0.900 1.00 NCa NCa N/A N/A

58 64 60 56 N/A N/A

4100 3350 51.0 12.0 N/A N/A

Not calculated; ADC depleted is a measurement of adduct formation, not given as dosed drug. D

DOI: 10.1021/acs.bioconjchem.6b00695 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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less payload loss and further minimize exposure from non-ADC species.17 In a recent report of an in vitro rat plasma study, close to 90% of payload loss ended up being due to formation of albumin adducts.12 The difference noted between the present in vivo and prior in vitro data is that during an in vitro incubation the catabolism of ADC is very limited, while during an in vivo study the clearance of the ADC as a whole will contribute to overall payload loss. The clearance of ADC and payload involves a variety of routes (Scheme 1). In addition to maleimide transfer and catabolism of ADC, linker stability may also contribute to the overall stability profile.18 For example, in the case of the mcValCitPABC linker shown herein, the ValCit site is a substrate of cathepsin B and similar enzymes which may result in the release of PF-06380101, which will be cleared by metabolic enzymes9 and renal mechanisms. At this point no plasma-mediated cleavage of the ValCit linker was observed by intact mass LC/MS analysis. Recent literature and the work herein shows that the majority of payload lost from the ADC is actually due to the formation of adducts with thiol-containing species in circulation, with the albumin adduct being the most dominant form.11 Albumin typically has a long half-life, and has been used in drug development as a half-life extender for small molecules and peptides.19 Therefore, it is important to have analytical methods that can quantify the albumin-bound payload. In Figure 2B, the plot also details the non-ADC bound payload as compared to the theoretical loss of payload as ADC undergoes retro-Michael deconjugation over the in vivo time course. This represents approximately the loss of payload excluding ADC catabolism pathway (Scheme 1D). Using this method, we found that at a maximum, the payload adduct found in circulation can account for ∼35% of the total payload lost from the ADC, although the payload adduct is a very small percentage (4%) of the systemic exposure when compared to the total conjugated payload including the ADC. As has been previously discussed, stabilization of the ADC via site-specific conjugation reduces this loss of payload presumably by slowing the retro-Michael reaction. Similarly, we evaluated the stability of an ADC with a hydrolyzed (ring opened) maleimide and showed that the amount of non-ADC payload in circulation was considerably lower (12 vs 51 ng/mL at Cmax; Figure 3, Table 2), resulting in a 5-fold lower AUC in payload from ADC depleted samples as compared to that of unmodified (ring-closed) ADC. Assuming the same DAR profile at both 3 and 10 mg/kg doses, the AUC contribution from payload-adducts for this ring-opened ADC would account for close to 10% of the deconjugation payload loss as compared to 34.8% for that of the ring-closed (parent) form. This result directly shows that ring-opening results in lower levels of albumin-payload adducts in vivo. Peter Senter’s lab has reported using the maleimide self-hydrolyzing chemistry to improve ADC stability.20 In summary, due to the complex nature of ADCs and the desire to explore more potent linker payload design, the challenges surrounding the bioanalysis of ADCs requires the analyst to rely on a variety of methods for characterizing the PK of this modality. We provide here a sensitive and precise assay for the quantitative measure of conjugated payload to support the PK profiling of an ADC. In addition, this assay was employed to further investigate the fate of the payload during an in vivo PK assessment as an ADC undergoes deconjugation.

LBA, by total payload with cathepsin B cleavage, and by intact protein analysis derived DAR × mAb. The PK profiles are recorded in Table 1 and are overlaid in Figure 4. The excellent agreement of these measurement reflects the compatibility and robustness of these approaches. Compared to the intact protein MS based drug loading measurements,3 an LC/MS/MS approach to profile released payload to support PK offers the considerable advantages of sensitivity, reliability, and a path toward assay validation. The lower limit of quantitation that can be reached is around the low ng/mL ADC level, while a DAR based assay measurement will encounter challenges when ADC concentrations are found below single digit μg/mL levels. The advantage of sensitivity can be especially valuable as many recently developed DNA-damaging payloads are exceptionally potent and are being dosed at sub-mg per kg levels. Note that the first two methodologies (LBA and DAR × mAb) do not measure payload that has been transferred to any nonmonoclonal antibody (mAb) components, such as albumin, by virtue of the specificity of the LBA reagents and MS monitoring settings. The LC/MS/MS platform can also deliver a superior specificity of the analyte, which is particularly valuable for the quantitation of payload metabolites which may also be required during the development process. The beadcaptured ADC was recovered and subjected to the same cathepsin B treatment, and the resulting mAb-bound payload was measured with LC/MS/MS as well. The difference between the conjugated payload levels derived from DAR × mAb and from extracted ADC with cathepsin B treatment is noticeable. Although there is higher than 99% removal of ADC, the recovery of ADC for the latter cleavage is approximately ∼70%; it is possible error in the ADC bound measurement that may have contributed to the difference observed. Payload from Albumin Adduct. When assessing in vitro ADC stability, a major discrepancy was observed when using different bioanalytical assays, as reported in an IND report of a mcValCit-MMAE ADC.16 Only trace amounts of unconjugated payload were observed in human, monkey, and rat plasma, suggesting that the ADC was largely intact. However, at the same time point, 11−30% loss of payload was measured by LBA and 35−45% loss of payload was measured by LC/MS/ MS following enzymatic release of the ADC-bound MMAE. Other studies have noted similar discrepancies.12 As various reports have noted, and this study confirms, the major loss of payload as detected by LBA and by LC/MS DAR analysis is due to maleimide deconjugation and not due to ValCit cleavage, which does not result in the formation of unconjugated payload, but rather in the formation of protein adduct species which were not detected by the LBA. In order to quantify the payload contribution from sources other than ADC, we removed the ADC using an immunocapture protocol and the resulting ADC-depleted sample was treated with cathepsin B (Figure 2A, Table 1). It was found that at maximum, only 4% of the total conjugated payload is related to those non-ADC components (adducts) (Figure 2B, Table 1). This quantitative assessment demonstrated that the levels of non-ADC payload measured in this study were found to be relatively low and are not likely to significantly impact the overall exposure. It should be noted that the site of conjugation and the structure of the payload itself may have a considerable impact on the susceptibility toward the retro-Michael related deconjugation. Site specific conjugation can improve the stability of the maleimide linkage and it would be reasonable to assume that this would lead to E

DOI: 10.1021/acs.bioconjchem.6b00695 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry For the first time, the amount of non-ADC bound payload in this in vivo setting was quantified and the impact on payload exposure assessed with clearance route described in more detail. The loss of payload due to Retro-Michael induced ADC deconjugation can be minimized by maleimide ring hydrolization.

mAb and ADC PK by LBA and LC/MS. PK analysis for total mAb as well as ADC using a Gyrolab (Warren, NJ) LBA platform was previously described.10,14 Briefly, the dosed lot of ADC was used to prepare calibration standards and quality controls in a minimum required dilution (MRD) of 1/10 for the 0.3 mg/kg dose group or 1/40 for the 1 and 3 mg/kg dose groups. Biotinylated sheep anti-human IgG (H + L) was captured onto streptavidin coated beads located in the affinity capture column of the Gyrolab CD microstructure, and the total mAb was detected with Alexa Fluor 647 labeled goat antihuman IgG (H + L). For ADC measurement, anti-PF06380101 monoclonal antibody with fluor label was used. The ADC DAR time profile analyzed by intact mass LC/MS analysis was also described previously.10,14 Calculation of conjugated payload using the DAR × mAb approach was determined by DAR (ratio) multiplied by mAb (μg/mL) and then converted to ng/mL − DAR × mAb*742 Da(MW of Payload)/150 kDa (MW of ADC). Cathepsin B Incubation. In vivo samples and ADC spiked samples at 50 μg/mL in plasma were diluted 40 times into 50 mM sodium acetate buffer, pH 4.8, containing 5 mM TCEP and approximately10 units/mL Cathepsin B. For in vitro testing the samples were collected over a time course, and each 25 μL aliquot was quenched with 100 μL ice-cold acetonitrile containing deuterated PF-06380101 as internal standard; the quench solution also includes a cathepsin B specific inhibitor (CA-074). Method optimization was performed regarding incubation time, amount of enzyme used, pH of buffer, and temperature of incubation (data not shown). For in vivo sample analysis the cathepsin B incubation was terminated at 4 h. A standard curve was prepared using the payload reference material and also with the ADC reference material, while the latter was treated the same with cathepsin B incubation. Unconjugated Payload Analysis. In vivo samples were prepared for unconjugated payload analysis using protein precipitation. Briefly, 25 μL of sample or standard was diluted into 25 μL of control Sprague−Dawley plasma, then precipitated with 200 mL of acetonitrile containing 0.2 ng/ mL of deuterated Internal Standard. 150 μL of supernatant was removed, transferred to fresh plate, dried under a stream of nitrogen, and reconstituted in 90 μL of 90/10 water/ acetonitrile, briefly mixed, and injected on LC/MS/MS. LC/MS/MS Analysis. Separation was performed on a Waters Acquity UPLC System (Milford, MA, USA). The autosampler and column were kept at 4 and 60 °C, respectively, with a MacMod ACE C18 column (3.0 × 30 mm, 3 μm) and a gradient of 5 mM ammonium acetate (mobile phase A) and acetonitrile (mobile phase B) at a flow rate of 0.35 mL/min. An initial mobile phase composition of 35% B was held for 0.6 min, then ramped to 95% B over 2.5 min, held at 90% B for 0.5 min, and then returned to initial 35% B for re-equilibration. Total analysis time for each sample was 4.5 min. Data was collected on an AB Sciex API5500 (QTRAP) mass spectrometer (Foster City, CA, USA) using positive Turbo IonSpray electrospray ionization (ESI) and multiple reaction monitoring (MRM) mode. The ionization source temperature was 500 °C with ion source gas 1 and 2 setting of 40 (nitrogen) and curtain gas set to 10 (nitrogen). The transitions of 743.6 → 188.0 and 751.6 → 188.0 were used for the analyte and deuterated internal standard, respectively. Data acquisition and processing were carried out with Analyst software v 1.5.2 (Applied Biosystems/MDS Sciex, Canada).

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EXPERIMENTAL PROCEDURES Materials and Reagents. Acetonitrile (HPLC grade), nanopure water, sodium acetate, ammonium acetate, tris(2carboxyethyl)phosphine (TCEP), and formic acid were purchased from Sigma (St. Louis, MO). Cathepsin B human liver enzyme was purchased from Millipore (Taunton, MA) Goat anti-human FC was obtained from Jackson ImmunoResearch (West Grove, PA). Streptavidin T1 beads were purchased from Life Technologies (Grand Island, NY). Dulbecco Phosphate Buffered Solution (PBS) was obtained from Sigma. Superblock was purchased from Scytek (West Logan, UT). Biotinylated sheep anti-human IgG (H+L) was obtained from The Binding Site (San Diego, CA). Alexa Fluor 647 labeled goat anti-human IgG (H+L) was obtained from Bethyl Laboratories (Montgomery, TX) and labeled in-house. Alexa Fluor 647 anti-PF-06380101 reagent was generated inhouse. Sprague−Dawley K2EDTA rat plasma was obtained from Bioreclamation (Hicksville, NY). Superblock was purchased from Scytek (West Logan, UT). Biotinylated sheep anti-human IgG was obtained from The Binding Site (San Diego, CA). Alexa 647 labeled goat anti-human IgG was obtained from Bethyl Laboratories (Montgomery, TX). Payload, Linker−Payload, and ADC Preparation. PF06380101 and its deuterated internal standard were synthesized by Pfizer Global Research and Development and their structures are shown in Scheme 1. The antibody is commercially available Trastuzumab (Genentech Inc.). Maleimide conjugation protocol was used as previously described.13 Briefly, antibody is prepared in PBS containing 5 mM 2,2′,2″,2‴-(ethane-1,2-diyldinitrilo) tetraacetic acid (EDTA) at pH 7, and was treated with 2−2.5 equiv of (TCEP, 5 mM in distilled water) and allowed to stand at 37 °C for 1−2 h. Dimethylacetamide (DMA) was added to achieve 10% (v/v) total organic, and 8−10 equiv of the vc0101 as a 10 mM stock solution in DMA was added. The resulting ADC was purified by size exclusion chromatography (SEC) and buffer exchanged for storage at 4 °C. Preparation of a ring-opened version of this ADC has been previously described.13 In Life PK Study. The in-life portion of the rat pharmacokinetic study was conducted at Pfizer, Inc., in compliance with National Institutes of Health guidelines for the care and use of laboratory animals and approved by the Institutional Animal Care and Use Committee (IACUC). The study was conducted in male Sprague−Dawley rats from Charles River Laboratories (Wilmington, MA) that were 8−10 weeks old or 300−350 g. Rats randomized in 3 groups of 6 individuals were administered the ADC in phosphate-buffered saline (PBS) at 3 and 10 mg/kg as a single intravenous dose via the tail vein. After dosing, rats (6 rats per dose group, 9 time points per rat) were anesthetized with isoflurane inhalation and blood (200 μL) was collected from the jugular vein at 5 min, 2, 6, 24, 48, 72, 168, 336, and 504 h post-dose. Blood was transferred to lithium heparin tubes and immediately placed on ice until centrifugation for collection of the plasma samples. Plasma samples were then stored at −70 °C until analysis. F

DOI: 10.1021/acs.bioconjchem.6b00695 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

Bioconjugate Chemistry

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ADC Depletion. ADCs from in vivo plasma samples were depleted with an immunocapture protocol. Briefly, 20 μL of plasma sample was diluted with PBS, and then incubated with 5 μL of goat anti-human FC capture agent for 1 h at room temperature. Streptavidin T1 magnetic beads were added to the samples and then mixed on vortex-shaker at room temperature for 1 h. ADCs were retained on beads and supernatant was removed. 2.5 μL of the ADC depleted sample was added to the cathepsin B reaction buffer with 40-fold of dilution. The beadbound ADC was eluted with 50 μL of 2% formic acid in water, and payload was released by cathepsin B with similar conditions as described in a previous section.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: frank.barletta@pfizer.com. Tel: 1 845-602-3629. *E-mail: [email protected]. Tel: 1 617-444-5203. ORCID

Frank Barletta: 0000-0001-6018-2131 Present Addresses #

Drug Metabolism & Pharmacokinetics, Vertex Pharmaceuticals Inc., 50 Northern Ave, Boston, Massachusetts 02210, United States. ¶ PKDM, Amgen, Inc., 360 Binney Street, AMA 1, Cambridge, Massachusetts 02142, United States. Notes

The authors declare the following competing financial interest(s): All authors are/or were full time employees of Pfizer Inc. when research was performed and own company stock options and/or units.

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ACKNOWLEDGMENTS The authors are grateful to members of the Oncology In Vivo Pharmacology Group and vivarium staff in Pearl River, NY, and Groton, CT, for the animal studies.

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REFERENCES

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DOI: 10.1021/acs.bioconjchem.6b00695 Bioconjugate Chem. XXXX, XXX, XXX−XXX