mini review

Tuning the Diels−Alder Reaction for Bioconjugation to Maleimide Drug-Linkers Andre H. St. Amant,† Daniel Lemen,‡ Stelios Florinas,‡ Shenlan Mao,§ Christine Fazenbaker,§

Haihong Zhong,§ Herren Wu,‡ Changshou Gao,‡ R. James Christie,*,‡ and Javier Read de Alaniz*,†

†Department of Chemistry and Biochemistry, University of California, Santa Barbara, California 93106-9510, United States ‡Antibody Discovery and Protein Engineering, MedImmune, Gaithersburg, Maryland 20878, United States §Oncology Research, MedImmune, Gaithersburg, Maryland 20878, United States

*S Supporting Information

ABSTRACT: The thiol−maleimide linkage is widely used for antibody−drug conjugate (ADC) production; however, con- jugation of maleimide−drugs could be improved by simplified procedures and reliable conjugate stability. Here, we report the evaluation of electron-rich and cyclic dienes that can be appended to antibodies and reacted with maleimide-containing drugs through the Diels−Alder (DA) reaction. Drug con- jugation is fast and quantitative due to reaction acceleration in water, and the linkage is more stable in serum than in the corresponding thiol−maleimide adduct with the same drug. ADCs produced using the DA reaction (DAADCs) are effective in vitro and in vivo, demonstrating the utility of this reaction in producing effective biotherapeutics. Given the large number of commercially available maleimide compounds, this conjugation approach could be readily applied to the production of a wide range of antibody (or protein) conjugates.

■ INTRODUCTION Bioconjugation reactions have a unique and essential role in life science research and drug development, due to their ability to couple biomolecules with drugs, reporter molecules, polymers, surfaces, and other biologically active compounds.1,2

In particular, bioconjugation technologies are used for production of ADCs that provide a delivery platform for selectively targeting anticancer drugs to tumor cells.3,4 The impact of this approach has been validated with four ADCs that have been approved by the US Food and Drug Administration (FDA), as well as more than 65 that are under clinical evaluation.5 Among the classes of available bioconjugation methods, the Michael addition reaction between thiols and maleimides (Figure 1A) is one of the most widely used; two FDA-approved ADCs (brentuximab vedotin and trastuzumab emtansine) utilize this chemistry for attaching drugs to antibodies. Of the various reactive groups that are available for coupling

drugs to antibodies, maleimide holds unique potential. The use of maleimides for bioconjugation is routine in the laboratory and in certain clinical drugs; as a result, many compounds bearing a maleimide group are available. For example, the maleimide−drug maleimidocaproyl-valine-citrulline-p-amino- benzyloxy-carbonyl-monomethyl-auristatin-E (vedotin or vcMMAE) is synthesized on an industrial scale to produce brentuximab vedotin and several other vedotin ADCs in clinical trials. Additionally, maleimide compounds are routinely

used for labeling biomolecules with fluorophores,6 metal chelators,7,8 and polymers.9 Despite the widespread use of maleimide in bioconjugation chemistries, a significant challenge is to identify conjugation strategies that leverage the properties of maleimide without relying on the thiol- maleimide Michael reaction, which suffers from several limitations. For instance, conjugation to maleimide requires a

Received: May 9, 2018 Revised: June 4, 2018 Published: June 22, 2018

Figure 1. (A) Brentuximab vedotin: reduced hinge-region thiols reacted with maleimide−drug (Michael addition). (B) Conjugation approach with randomly distributed electron-rich dienes which takes advantage of available maleimide−drugs. The DA conjugation is fast and results in adducts stable in serum.

Article

pubs.acs.org/bcCite This: Bioconjugate Chem. 2018, 29, 2406−2414

© 2018 American Chemical Society 2406 DOI: 10.1021/acs.bioconjchem.8b00320 Bioconjugate Chem. 2018, 29, 2406−2414

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free thiol that is produced through a partial reduction of native disulfide bonds or introduction of cysteine residues by protein engineering.10,11 Although maleimide conjugation to antibody thiols is an efficient reaction, preparation of antibody for conjugation can be a cumbersome process. For example, reducing agents such as tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT) used to activate cysteines also inactivate maleimide and so must be removed before conjugation.12 Site-specific maleimide−thiol conjugation to engineered cysteines (nonhinge) allows for controlled drug:antibody ratios (DAR), but the conjugation process requires an additional oxidation step to reform native disulfides in the antibody hinge region.11 Furthermore, thiosuccinimide adducts often undergo a site-dependent retro-Michael reaction, leading to deconjugation in vivo with a half-life of approximately 4−5 days in rats13 which decreases the efficacy of an ADC.14 To overcome challenges, several innovative strategies have been developed to increase the stability of the thiol-maleimide linkage; for example, engineering antibodies with cysteine residues in stable conjugation positions14 or designing a thiosuccinimide that undergoes a stabilizing succinimide hydrolysis.13,15−18 Additionally, new reagents for thiol specific conjugation have also been developed; however, these new linkages for in vivo applications remain relatively unexplored and do not harness the proven applications of maleimide compounds.19−21 One appealing solution would be to develop new maleimide-linkage strategies for antibody− drug conjugation that are stable regardless of location, while simultaneously using a simplified conjugation protocol. The DA reaction offers unrealized potential in this context.22

Maleimide is an excellent dienophile due to its electron- deficient and sterically unhindered π-system. As a result, maleimide is one of the most commonly used dienophiles in DA reactions; it has been used to chemically tag proteins,23

oligonucleotides,24 oligosaccharides,25 and nanoparticles.26

Because of synthetic ease, these reactions typically rely on furan and electron-rich 2,4-hexadiene derivatives that can be readily incorporated and used for a given application. Although these results clearly demonstrate the potential of the DA- maleimide bioconjugation, the sluggish reaction rates con- stitute a serious drawback for production of ADCs. For example, reactions between furans and maleimide typically have a second-order rate constant on the order of 10−5 M−1

s−1.27 These rate constants can be offset by high concentrations of the maleimide, long reaction times, or elevated temper- atures.28 Although this is feasible for materials chemistry or organic synthesis, such reaction conditions for ADC production are prohibitive due to antibody sensitivity, stability, and cost. High concentrations cannot be used to overcome slow conjugation, as antibodies can undergo aggregation, and many maleimide-drugs have low water solubility. Furthermore, sophisticated drug-linker molecules used for conjugation to antibodies are expensive and minimizing equivalents is desirable.3,4,29 Thus, fast kinetics are needed to overcome limitations. Improved kinetics for the DA reaction with maleimide have

been achieved by modifying the diene component or the reaction solvent. For example, a study of the DA reaction of maleic anhydride (which has similar reactivity to maleimide) has shown that the electron-rich and cyclic cyclopentadiene has about a 1,300-fold rate enhancement compared to unsubstituted butadiene.30 In conjugation studies, Barner- Kowollik,31 our group,32 and others33−37 have also reported

examples of highly efficient and rapid DA or hetero-DA cycloadditions with substituted cyclopentadienes. The DA reaction rate also depends on the solvent, as rates are accelerated in water due to hydrophobic aggregation and hydrogen-bonding.38,39 For instance, Grandas used an aqueous DA reaction to produce peptide−oligonucleotide conjugates with good efficiency.40 We envisioned developing an ADC system utilizing electron-rich dienes that, although historically considered a liability due to their high reactivity, could be incorporated onto an antibody and then reacted with a commercially available maleimide−drug to form stable ADCs (Figure 1B). Such a system would allow properties of the DA reaction (reaction rate, selectivity, stability, and synthetic ease) to be exploited in the context of antibodies and clinically validated drug-linkers. Here we report the preparation of normal electron-demand

DAADCs. This conjugation approach takes advantage of widely available maleimide−drugs with established manufac- turing processes. In addition, design of the conjugation strategy is centered on the drug-linkers typically used for ADC production. Antibodies were modified with electron-rich and cyclic dienes and then subsequently reacted with the therapeutically relevant maleimide drug-linker vcMMAE. Reaction rates were found to be 2.6−77 M−1 s−1, which is suitable for ADC production. The DA cycloadducts are more stable in serum than thiol−maleimide linkages formed with the same maleimide−drug. The resulting DAADCs produced using trastuzumab exhibited potencies similar to analogous thiol−maleimide conjugates in vitro and in vivo, demonstrating that this conjugation approach maintains drug potency.

■ RESULTS AND DISCUSSION Design and Synthesis of Electron-Rich and Cyclic

Diene Compounds. The DA reaction rate and the stability of both diene and DA adducts are crucial for efficient production of DAADCs. The diene must be highly reactive in conditions suitable for antibodies (i.e., aqueous solution, ≤37 °C) and yet avoid dimerization or polymerization during synthesis, storage, and conjugation with maleimide. Identification of such a diene is challenging because reagents that incorporate this functional group into antibodies are not readily available. To develop a general and practical synthetic approach to evaluating DA reactions with highly reactive, electron-rich and cyclic dienes on antibodies, we designed a modular approach that could be systematically investigated. We synthesized heterobifunctional linkers incorporating a range of diene functionality for reaction with maleimide and an N-hydroxysuccinimide (NHS) ester to react with antibody lysine amines. We focused on furan and cyclopentadiene-containing linkers, for which the sterics and electronics could be easily tuned (Figure 2). We began with NHS-ester 1, using a strategy similar to that

described for the conjugation of furan-containing nanoparticles with maleimide-containing antibodies.26,41,42 Furan NHS- esters have been widely utilized in bioconjugation approaches, and therefore this strategy provides an easy way to evaluate and compare the reactivity to known systems. The reactivity of 1 with maleimide was low under typical antibody conjugation conditions (1.3% conjugation, 20 h, 2.7 mg/mL antibody, pH 5.5, room temperature), which motivated us to explore other more reactive dienes. We designed 2a to take advantage of cyclopentadiene’s fast kinetics in the DA reaction.30 In addition, the endo DA adduct does not undergo a retro-DA until reaching approximately 275 °C.43,44 This feature

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increases the thermal stability of the DA conjugate and distinguishes it from the DA adduct between 1 and maleimide, which yields a mixture of endo and exo adducts, with the retro- DA occurring at approximately 50 °C for the endo adduct.45,46

The synthesis of 2a was obtained in an interconverting mixture of 1- and 2-cyclopentadiene isomers, as monosubstituted cyclopentadienes undergo a facile [1,5]-hydride shift.47 Like many monosubstituted cyclopentadienes with a low molecular weight, 2a was found to dimerize at room temperature (approximately 55% dimer after 5 days; data not shown) and required storage at −20 °C, where it was stable for several months. To provide access to dienes with enhanced stability while maintaining similar reactivity, spiroheptadiene 348 and pentamethylcyclopentadiene 449,50 were synthesized; these electron-rich dienes have not been reported to dimerize at room temperature,51 thus simplifying the synthesis and handling of the material. In addition to cyclopentadienes 2− 4, we designed the more reactive furan derivative, 3- methoxyfuran 5.52 Compared to 3H furan, the DA reaction of 3-alkoxyfuran with maleimide is reported to have a lower kinetic barrier due to reduced aromaticity.53 Altogether, the synthesis of a series of diene-NHS bifunctional linkers was straightforward and provided a means for incorporating various diene functionalities onto antibodies for evaluation of the DA reaction with maleimide−drugs. Production of Diene-Functionalized Antibodies. With

a series of dienes bearing NHS-esters in hand, we next sought to functionalize antibodies through the well-known reaction between lysine amines and NHS-activated esters. A typical IgG1 molecule bears approximately 40 surface-exposed reactive lysine residues,54 thus, lysine modification in solution or on solid support55 is random, and the distribution of product obtained is controlled by the antibody:NHS-ester ratio used in the reaction. Conjugation of 1, 2a, 3, 4, and 5 to antibody lysine amines was confirmed and quantified by mass spectrometry, yielding antibody-dienes mAb-1 to mAb-5 with average linker-to-antibody ratios (LARs) between 2 and 4 (see the Supporting Information). A distribution of products was obtained ranging from one to eight modified amines per antibody. In all cases examined, there was no observed antibody

aggregation or dimerization for the diene after antibody conjugation (see the Supporting Information). Of note, this includes mAb-2a, the conjugate formed with the dimerization-

prone cyclopentadiene 2a (Figure 3B). Antibody species with high LAR values are the most susceptible to deleterious

properties such as aggregation and/or dimerization of diene functional groups, which were not observed. Ester cleavage of 2a was observed following incubation at pH 7.4 and 37 °C for several days (data not shown); this is likely to have occurred via reaction with water or a neighboring lysine ε-amine. We designed cyclopentadiene 2b, which lacks an internal ester, for production of ADCs for use in vivo and in vitro (vide infra).

Diels−Alder Reaction. To examine whether mAb-dienes mab-1 to mAb-5 were amenable to facile DA cycloaddition, we reacted them with an excess of the therapeutically relevant maleimide-containing drug vcMMAE. For the initial studies, 15 equiv of vcMMAE was added to the diene-functionalized antibody (note that the diene:maleimide ratio was from 4.5:1 to 6.5:1 due to variable LAR values, see below). Reaction conversion was monitored by mass spectrometry, which can be used to quantify drug conjugated to antibodies56,57 (see the Supporting Information). Dimethyl sulfoxide (DMSO) was added to all reaction mixtures to solubilize vcMMAE and ensure homogeneous reaction conditions. The DA reaction of mAb-1 with vcMMAE (6 equiv relative to diene) produced mAb-1-MMAE in a very low yield (<2%, Table 1). With extended reaction times and elevated temperature (20 h, 37 °C), the yield was not improved, and nonselective conjugation (about 1%) was observed (Figure S52). As expected for the production of DAADCs, 3H furan does not react at sufficient rates for practical use. In contrast to mAb-1, the antibody- dienes mAb-2a, mAb-4, and mAb-5 underwent complete conversion after 4 h at both room temperature and 37 °C when reacted with 4−6 equiv of vcMMAE per diene (Figure 4), indicating promising reaction kinetics for these more reactive dienes. Although spiroheptadiene mAb-3’s conversion was lower under these conditions (73% at room temperature or 88% at 37 °C), nearly quantitative conversion could be obtained with longer reaction times (48 h, Figure 5). No nonselective conjugation (i.e., conjugation to a heavy or light chain that does not contain diene) was observed for mAb-2a

Figure 2. Overview of diene-NHS esters and dienophile compounds used to investigate stability and reactivity. Compounds 2a and 2b are obtained as an interconverting mixture of 1- and 2-substituted cyclopentadienes.

Figure 3. Mass spectrometry analysis of (A) mAb and (B) mAb-2a conjugates. Antibodies were deglycosylated with EndoS and analyzed intact. Peak masses and linker to antibody ratio (LAR) are indicated (average LAR for this sample is 3.7). Only one isomer of cyclopentadiene is shown.

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to mAb-5, indicating high specificity for the diene. Importantly, other than slow ester hydrolysis observed while DAADCs of 2a, 3, and 4 were incubated, the DAADCs were found to be stable over the course of the reaction, during purification, and upon storage in buffer (phosphate-buffered saline (PBS), pH 7.4). The DA reaction was further measured at a stoichiometric ratio of 1:1 diene:maleimide to determine second-order rate constants (see below). Kinetics. To discern differences in diene reactivity and

understand the effects of solvent on the DA reaction, we measured the conjugation rate in aqueous solution (with diene-modified mAbs) and in organic solvent (with the NHS- esters). The rate of aqueous conjugation reactions is a critical feature for the development of ADCs. If a reaction is too slow, large quantities of drug-linker or long reaction times must be

used to drive the reaction to completion. However, this can be cost prohibitive, can lead to aggregation, and may not be possible for drug-linkers with low solubility in aqueous buffer. We began by monitoring the rates in organic solvent to evaluate the reactivity trend. Second-order rate constants in organic conditions for the DA reaction between dienes 1, 2b, 3, 4, and 5 and N-ethylmaleimide were determined in deuterated chloroform (Table 2, see the Supporting Information for full details). Reactions were monitored using 1H nuclear magnetic

resonance (NMR), which provides a rapid way to screen the dienes. Furan 1’s second-order rate at room temperature was the slowest in the series with 4.9 × 10−5 M−1 s−1, with a calculated half-life of 7,873 days (30 μM at room temperature, conditions in the range commonly used for ADC conjugation). The reactivity of 3-methoxyfuran 5 was markedly greater than that of 3H furan, with a half-life of 31 days. The half-lives of cyclopentadiene derivatives 3 and 4 are 44 and 32 days, respectively. The least sterically hindered cyclopentadiene in our series, 2b, reacted the fastest, having a calculated half-life of 4 days. Although the reaction rates in organic solvent are unacceptable for ADC production, the reactivity trend is important, and the DA reaction would be accelerated in aqueous buffer.38,39

Diene−maleimide DA reaction rates were further evaluated in aqueous conditions using mAb-dienes and vcMMAE. Cycloaddition reactions showed reaction rates ranging from 2.6 to 77 M−1 s−1 (Figure 5, Table 2). The second-order rate constant for mAb-2a at 22 °C was the fastest at k2 = 77 M

−1

s−1, whereas cycloadditions rates for mAb-3, mAb-4, and mAb-5 were slightly slower with rate constants from 2.6 to 18 M−1 s−1. Enhanced reactivity for mAb-2a is probably due to reduced steric interactions compared to dienes 3 and 4. Although 4 is more sterically hindered than 3, the rate of cycloaddition benefits from five electron-donating methyl groups and greater hydrophobic aggregation than 3. Analyses of the cycloaddition reaction also revealed that dienophiles based on furan could be tuned to increase their rate of cycloaddition. Furan 5 bearing the electron donating methyl ether group in the 3 position dramatically increased the rate compared to unsubstituted furan 1.53 In fact, the rate constant

Table 1. Diels−Alder Conjugation of mAb-Dienes with an Excess of vcMMAEa,b

conversion (%)c

mAb-diene LAR reaction time (h) 22 °C 37 °C

mAb-1d 2.5 20 1.3e

mAb-2a 2.3 4 100 100 mAb-3 3.3 4 73 88 mAb-4 3.04 4 100 100 mAb-5 2.95 4 100 99

aAll conjugation reactions were performed in PBS supplemented with 100 mM sodium phosphate monobasic, 20% DMSO, pH 5.5. The antibody used was R347. bAll reactions were performed with 15 mol equiv of vcMMAE relative to mAb. mAb concentration was 2.7 mg/ mL (18 μM) for all reactions. Diene concentration for each reaction can be obtained by multiplying LAR by 18 μM. cConversion was calculated from the disappearance of mAb-diene peaks following deglycosylated, reduced mass spectrometry analysis. Both heavy and light chains were analyzed. dAntibody used was 5T4. eAn additional 1.1% nonspecific conjugation was observed (see Figure S52).

Figure 4. Mass spectrometry analysis of mAb-2a before (A) and after (B) reaction with vcMMAE (15 equiv relative to mAb, 22 °C, 4 h). Samples were deglycosylated and reduced prior to analysis; spectra are zoomed to show the heavy chain (HC) species only. Only one isomer of cyclopentadiene is shown.

Figure 5. Kinetic analysis of the reaction of mAb-diene (1 equiv of diene) with vcMMAE (1 equiv). All reactions were performed at 22 °C; unreacted diene concentration was determined by mass spectrometry. Data are plotted as the average value ± standard deviation, n = 3, for mAb-2a and mAb-3 samples. Data are plotted as the average value ± absolute error, n = 2, for mAb-4 and mAb-5 samples. Kinetic experiments were repeated with independent samples.

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for mAb-1 was not determined because the rate was prohibitively slow. Note that reaction rates reported here cannot discern the potential effect of antibody position on DA reaction rate due to the random nature of lysine modification. It is currently unknown if antibody position affects DA reaction rate; thus, the reaction rates reported here should be considered average values. Overall, the diene-maleimide cycloaddition rates for mAb-2a

to mAb-5 enable nearly quantitative conversion of diene groups in a time frame of hours, with one equivalent of maleimide:diene, at antibody concentrations that could be used for routine conjugations. Although slower than the thiol- maleimide Michael addition (k2 ∼ 500−700 M

−1 s−1),15,58 this DA strategy avoids the challenges of selectively reducing and handling thiols. The normal electron-demand DA reaction is slower than the inverse electron-demand DA reaction (k2 up to 2.8 × 106 M−1 s−1 with trans-cyclooctene and tetrazine),59−61

but it uses a commercially available maleimide−drug, and the cyclopentadiene-NHS ester 2b can be readily synthesized in three steps. Overall, the DA reaction rate is comparable to that of other commonly used ligation chemistries such as the strain- promoted azide−alkyne cycloaddition (k2 ∼ 10

−2 to 1 M−1

s−1) or the copper-catalyzed version (k2 ∼ 10 to 200 M −1

s−1).62 This DAADC strategy may have been overlooked for ADC production because reported organic reaction rates were too slow at concentrations typically encountered for bioconjugation to antibodies, as well as the propensity of electron-rich and cyclic dienes to undergo self-dimerization. However, our findings demonstrate that maleimide−diene aqueous reaction rates are accelerated approximately 300− 1500-fold and the dienes are stable, allowing efficient conjugation at diene concentrations in the micromolar range. The rate acceleration is consistent with previous literature reports demonstrating acceleration of the DA reaction in aqueous phase up to 10,000-fold.63,64

Evaluation of Trastuzumab DAADCS. Two DA dienes, the cyclopentadiene and furan with fastest reaction rates (2b and 5 respectively), were further evaluated in functional DAADCs using trastuzumab (T). DAADCs were prepared by functionalizing the antibody with NHS-linkers 2b or 5, followed by reaction with vcMMAE (as described in Table 1) with DARs of approximately 3−4 as determined by mass spectrometry and rRP-HPLC (Table 3 and the Supporting Information). Reaction of vcMMAE with T-diene resulted in

nearly quantitative conversion of the diene groups. Monomer content in the DAADCs remained high, indicating that diene functionalization, DA reaction, and purification did not cause aggregation (Supporting Information). As a control, ADCs were also prepared by Michael addition to reduced native hinge thiols (T-Cys-MMAE), with DARs targeted to be the same as the DAADCs (DAR = 3−4). In vitro, the ADCs T-2b-MMAE and T-5-MMAE exhibited

similar potency to T-Cys-MMAE in gastric carcinoma N87 and adenocarcinoma SKBr3 cell lines (Table 3). ADCs produced with nonbinding control antibody (mAb, R347) had very low inhibition, indicating low nonspecific activity. Overall, in vitro cytotoxicity values were in the expected

range for a vedotin ADC, and the DA conjugation approach did not result in lower potency.

Serum Stability. The stability of the ADCs prepared via DA linkage or thiosuccinimide linkage was evaluated ex vivo in both rat and mouse serum (Figure 6, 7 days at 37 °C). Total human antibody was recovered from serum by immunocapture and analyzed by mass spectrometry to determine the amount of drug remaining on ADCs.65 We did not expect the DAADCs to deconjugate in serum through the retro-DA reaction because the adduct of cyclopentadiene and maleimide does not undergo a retro-DA reaction until temperatures exceed 275 °C,43,44 and the 3-alkoxyfuran−maleimide adducts are reported to be irreversible.53 Indeed, both DAADCs mAb- 2b-MMAE and mAb-5-MMAE were stable in rat serum (Figure 6A), having 90% and 94% drug retention, respectively. In mouse serum there was significant loss of drug through

Table 2. Kinetic Data for the Diels−Alder Reaction in Water and Deuterated Chloroform

mAb-diene reaction with vcMMAE in watera,b NHS-diene reaction with N- ethylmaleimide in CDCl3

c

mAb- diene LAR

[diene] (μM)

k2(H2O) (M−1 s−1)d,e

half-life (min)

k2(CDCl3) (1000 × M−1 s−1)f

half-life (days)h

aqueous rate acceleration k2(H2O)/ k2(CDCl3)

mAb-1 ND ND 0.0491 ± 0.0005 7873 ND mAb-2a 3.4 ± 0.3 31 ± 2 77 ± 7 7.0 ± 0.4 97 ± 7g 4 794 mAb-3 2.9 ± 0.4 27 ± 3 2.6 ± 0.2 237 ± 50 8.7 ± 0.6 44 299 mAb-4 2.9 ± 0.2 27 ± 2 18 ± 8 34 ± 20 12.1 ± 0.1 32 1488 mAb-5 3.0 ± 0.1 26 ± 1 4 ± 2 160 ± 108 12.6 ± 0.7 31 317 aAll conjugation reactions were performed in PBS supplemented with 100 mM sodium phosphate monobasic, 20% DMSO, pH 5.5, at 22 °C. bAll reactions were performed with 1 equiv of vcMMAE relative to diene. mAb concentration was 1.3 mg/mL for all reactions. cReactions were performed in CDCl3 with 2b, 3, 4, or 5 (0.01 M) and N-ethylmaleimide (0.01 M) at room temperature. 1 was performed at 0.05 M.

dk2(H2O) was calculated from the concentration of unreacted mAb-diene peaks (deglycosylated and reduced mass spectrometry analysis). Both heavy and light chains were analyzed. eErrors reported for k2 values were derived from the best fit line of the inverse concentration plot, n = 3 for mAb-2a and mAb-3, n = 2 for mAb-4 and mAb-5. fk2(CDCl3) was calculated from the integration of diagnostic peaks in

1H NMR spectra, n = 3. For the endo:exo or syn:anti ratios and characterization of the conjugates see the Supporting Information. g2b was used. hHalf-life calculated from the k2(CDCl3) at 30 μM.

Table 3. Summary of Trastuzumab ADCs

DAR EC50 (ng/mL)

mAb-linker-druga MS rRP- HPLC

monomer (%) N87 SKBr3

T-2b-MMAE 3.9 3.5 98.7 1.98 1.98 mAb-2b-MMAE 3.2 3.4 98.4 8230 1811 T-5-MMAE 3.5 4.5 97.7 3.02 1.57 mAb-5-MMAE 3.2 3.5 98.1 9990 ∼2000 T-Cys-MMAEb 3.2 3.5 97.4 3.61 2.78 mAb-Cys-MMAEb 2.9 3.1 98.5 8177 >2000 aADCs were prepared using trastuzumab (T) or a control antibody (mAb). bPrepared using the thiol-maleimide reaction of native hinge cysteines.

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cleavage of the Val-Cit linker; however, the DA linkage remained intact (Supporting Information). Enzymatic cleavage of the Val-Cit dipeptide is a known property of mouse plasma and is especially prevalent for ADCs prepared by conjugation to solvent-exposed positions.66 By combining the intact DAADCs and the Val-Cit cleaved species that retained the DA-adduct, the stability of the DA linkage in mouse serum was confirmed (Figure 6B). The thiol-conjugated drug in mAb-Cys-MMAE incurred

significant deconjugation in both rat and mouse serum (38% and 41% respectively), with the remaining drug connected through a hydrolyzed thiosuccinimide linkage. The amount of drug loss observed here for thiol-linked ADCs is consistent with previous reports and highlights the potential liability of

stability for ADCs produced through thiol−maleimide coupling.14,19,67 Drug loss for thiosuccinimide-linked ADCs occurs via the retro-Michael reaction, in which regenerated maleimide can subsequently react with endogenous thiols in serum such as serum albumin.16,19 The amount of drug loss for thiosuccinimide-linked ADCs is position dependent, with solvent-exposed positions typically undergoing higher decon- jugation; in contrast, positions that are buried or that promote thiosuccinimide hydrolysis can be highly stable.68 Thiosucci- nimide linkage stability is also influenced by linker properties, such that hydrophilic and/or cationic linkers capable of promoting thiosuccinimide hydrolysis yield stable thiol-linked constructs.13,17 Interestingly, mAb-Cys-MMAE did not under- go significant Val-Cit cleavage in mouse serum, possibly due to the sheltered nature of the thiols in the hinge region. Effective stabilization of the drug−mAb linkage in ADCs

prepared with maleimide drug-linkers using the DA con- jugation strategy represents an alternative approach to current methods. To date, most strategies for stabilizing maleimide− mAb conjugates involve identifying stable antibody conjuga- tion positions or altering chemistry in the drug-linker reaction partner to promote thiosuccinimide hydrolysis. In the DA conjugation strategy, the maleimide conjugate stability was improved by using an antibody-diene as the reaction partner, which to date has not been demonstrated.

In Vivo Study. Functional ADCs T-2b-MMAE, T-5- MMAE, and T-Cys-MMAE and control ADC mAb-2b- MMAE were evaluated for antitumor activity in vivo using mice bearing N87 xenograft tumor models (Figure 7). The

dose of 3 mg/kg was chosen based on a recent literature report for a trastuzumab-MMAE ADC that showed a durable tumor response for a DAR 3.8 Cys-ADC dosed at 5 mg/kg and partial tumor growth inhibition and complete regrowth after 35 days when dosed at 1 mg/kg in a NCI-N87 model.69 Thus, at 3 mg/ kg dose, a clear tumor response is expected, and a drastic loss in ADC potency should also be evident. As seen in Figure 7, all functional ADCs evaluated in this work resulted in complete tumor regression for 30 days after a single injection of ADC. T- Cys-MMAE and T-5-MMAE ADCs showed continued strong tumor growth inhibition for up to 40 days, whereas tumors treated with T-2b-MMAE ADC grew slightly from days 30 to 40; however, tumor volume did not exceed the tumor volume at the beginning of treatment, and the differences at day 40

Figure 6. Serum stability of ADCs. Samples were added to normal rat (A) or mouse (B) serum and maintained at 37 °C for 7 d prior to analysis. Samples were recovered from serum by immunocapture, reduced, deglycosylated, and analyzed by mass spectrometry. Quantification of intact ADC (including stable thiosuccinimide- hydrolyzed species) or intact DA adduct only (C, ADC + the Val-Cit cleaved species) was determined from relative peak heights of mass spectra. Intact linker in mouse serum was calculated as the sum of intact ADC and the valine-citrulline cleaved species. Each analysis is reported as the average value ± standard deviation, n = 3.

Figure 7. NCI-N87 tumor growth inhibition by ADCs. Treated mice received a single dose (IV) of T-Cys-MMAE (3 mg/kg), T-2b- MMAE (3 mg/kg), or T-5-MMAE (3 mg/kg) 5 days after tumor inoculation. Tumor volumes are plotted as the average volume ± SD, n = 5.

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were not statistically significant (p > 0.05). Considering the ex vivo mouse serum stability results, both DAADCs and Cys- linked ADCs are expected to lose approximately the same amount of drug 7 days after injection, albeit through different mechanisms (Val-Cit linker cleavage and retro-Michael addition respectively). Thus, current results suggest that DA conjugation does not lead to deleterious loss of drug function in vivo compared to cysteine-based ADCs. Avoiding Val-Cit linker cleavage by incorporating the diene at nonexposed positions or by use of other maleimide−drugs could yield ADCs with higher potency than cysteine-based surrogates, which is a focus of our future studies. Altogether, randomly lysine-conjugated DAADCs have similar potency to hinge Cys- linked ADCs in vitro and in vivo, resulting in ADCs with potent antitumor activity.

■ CONCLUSION In summary, we have developed the first systematic investigation of a normal-demand DA reaction to produce ADCs. This method involves easily synthesized diene compounds, facile antibody incorporations, and was studied both in vitro and in vivo. Of the five electron-rich dienes that were incorporated onto mAbs, four underwent conjugation with maleimide with rates viable for ADC production. To our knowledge, this is the first systematic investigation of normal- demand DA reaction rates on the surface of antibodies. In general, cycloaddition rates for these reactions were found to be comparable with those of ligation reactions such as the strain-promoted or copper-catalyzed azide−alkyne cyclo- addition. It was also demonstrated that enhancement of the DA reaction rate in water is a key factor for bringing this reaction into the realm of practical application. An approximately 300−1500-fold rate acceleration was deter- mined for the aqueous maleimide−diene reaction, which supports the potential for application of this ligation approach to a wide range of bioconjugation strategies. DAADCs produced with cyclopentadiene 2b and 3-methoxyfuran 5 are more stable to deconjugation than is the classic thiol− maleimide linkage in serum, which is desirable for production of clinical compounds that require predictable properties. Functional DAADCs of trastuzumab have activities in vitro and in vivo that are comparable to those of thiol−maleimide ADCs, demonstrating that attaching drugs to antibodies through a DA linkage does not lead to drastic loss of drug function. A key design feature of the DA conjugation strategy as

compared with other methodologies is its capitalization on the large number of commercially available maleimide compounds, such as maleimide−drugs, fluorophores, and chelators. This avoids the need to develop new entities to facilitate bioconjugation reactions and represents a key step toward a general DA bioconjugation method with maleimide. Moreover, in comparison with the thiol-maleimide Michael addition reaction, which is the most widely used reaction with maleimides, the DA reaction avoids the technical challenges associated with generating free thiols. Overall, we have shown that the normal-demand DA reaction strategy can serve as a useful bioconjugation platform when fast kinetics and high conjugate stability are required.

■ ASSOCIATED CONTENT *S Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconj- chem.8b00320.

Synthesis of dienes, biochemistry procedures, character-

ization data, and supporting figures (PDF)

■ AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]. *E-mail: [email protected]. ORCID Andre H. St. Amant: 0000-0002-4842-1918 Javier Read de Alaniz: 0000-0003-2770-9477 Author Contributions The manuscript was written through contributions of A.H.S., J.R., and R.J.C. All authors contributed experimental data, analysis, and/or scientific guidance. Notes The authors declare the following competing financial interest(s): D.L., S.F., S.M., C.F., H.Z., H.W., C.G., and R.J.C. are current or past employees of MedImmune, a division of AstraZeneca.

■ ACKNOWLEDGMENTS A.H.S. thanks the Natural Sciences and Engineering Research Council of Canada (NSERC) for a postgraduate scholarship (PGS-D). NMR instrumentation was supported by an NIH Shared Instrumentation Grant (1S10OD012077-01A1). We thank Vineela Aleti for expressing and purifying trastuzumab. We thank Nazzareno Dimasi and Ryan Fleming for their support with ADC analytical characterization. Editorial support was provided by Jacquelyn Beals, Ph.D. This study was financially supported by MedImmune, the global biologics R&D arm of AstraZeneca.

■ ABBREVIATIONS ADC, antibody-drug conjugate; DA, Diels−Alder; DAADC, Diels−Alder antibody−drug conjugate; DAR, drug:antibody ratio; DMSO, dimethyl sulfoxide; LAR, linker-to-antibody ratio; mAb, monoclonal antibody; NHS, N-hydroxysuccini- mide; NMR, nuclear magnetic resonance; PBS, phosphate- buffered saline; vcMMAE, maleimidocaproyl-valine-citrulline- p-aminobenzyloxy-carbonyl-monomethyl-auristatin-E (vedo- tin)

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