Acyl Fluorides: Fast, Efficient, and Versatile Lysine-Based Protein

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Acyl Fluorides: Fast, Efficient, and Versatile Lysine-Based Protein Conjugation via Plug-and-Play Strategy Igor Dovgan,† Sylvain Ursuegui,† Stéphane Erb,‡ Chloé Michel,§ Sergii Kolodych,§ Sarah Cianférani,‡,∥ and Alain Wagner*,† †

Laboratory of Functional ChemoSystems (UMR 7199), LabEx Medalis, University of Strasbourg, 67087 Strasbourg, France BioOrganic Mass Spectrometry Laboratory (LSMBO), IPHC, University of Strasbourg, 25 rue Becquerel, 67087 Strasbourg, France § Syndivia SAS, 650 Boulevard Gonthier d’Andernach, 67400 Illkirch, France ∥ IPHC, CNRS, UMR7178, University of Strasbourg, 67087 Strasbourg, France ‡

S Supporting Information *

ABSTRACT: We report a plug-and-play strategy for the preparation of functionally enhanced antibodies with a defined average degree of conjugation (DoC). The first stage (plug) allows the controllable and efficient installation of azide groups on lysine residues of a native antibody using 4-azidobenzoyl fluoride. The second step (play) allows for versatile antibody functionalization with a single payload or combination of payloads, such as a toxin, a fluorophore, or an oligonucleotide, via copper-free strainpromoted azide−alkyne cycloaddition (SPAAC). It is notable that in comparison to a classical N-hydroxysuccinimide ester (NHS) strategy, benzoyl fluorides show faster and more efficient acylation of lysine residues in a PBS buffer. This translates into better control of the DoC and enables the efficient and fast functionalization of delicate biomolecules at low temperature.

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INTRODUCTION The development of new chemistry and conjugation techniques is of great interest in the construction of functionally enhanced proteins, such as antibody−drug conjugates (ADCs), which have been used successfully as cancer therapeutics.1,2 One of the oldest and most versatile methods for protein modification is through lysine conjugation,3,4 due to solvent accessibility and the high abundance of these residues on a protein surface. In general, this is achieved through the formation of an amide bond between a lysine residue and activated esters, such as Nhydroxysuccinimide (NHS),5 pentafluorophenol,6 or 4-sulfotetrafluorophenyl esters.7 However, the reaction is usually performed directly between two complex molecules, i.e., a protein and the activated ester of the payload molecule. This classical approach makes the outcome of the conjugation uncertain, due to the unpredictable stability and reactivity profile of activated esters bearing different linkers and payload molecules. Consequently, an optimization of conjugation conditions is needed in each case to obtain a desirable degree of conjugation (DoC), which is a laborious and timeconsuming procedure. A possible strategy to overcome these limitations is to perform a two-step coupling, by first reacting the lysine residues of the protein with a bifunctional linker, bearing an activated ester and a maleimide group, followed by the reaction of the modified protein with the thiol-containing molecule.4,8 However, selectivity issues can arise during the first © XXXX American Chemical Society

step of the bioconjugation, with lysine residues reacting with maleimide groups; this side reaction is a significant drawback of this strategy, as exemplified by the case of trastuzumab emtansine, ADC.9 In attempt to improve selectivity of bioconjugation on ADC, protein engineering and enzymatic approaches have been actively developed.2,10,11 Although these processes have been successfully applied for the preparation of homogeneous ADC, most of them are not applicable to native antibodies and require costly protein engineering techniques. In searching for more efficient procedure for lysine conjugation, we envisioned that a plug-and-play strategy, as introduced by Chudasama et al. for ADCs construction via disulfide bond rebridging,12−16 could be of great interest. This could allow for tight control of the DoC in the plug stage and further versatile protein functionalization in the play stage using biorthogonal chemistry. Here, we report that compared to a classical Nhydroxysuccinimide ester (NHS) strategy, 4-azidobenzoyl fluoride shows faster and more efficient acylation of lysine residues of a native antibody in PBS buffer, which translates into better control of the DoC at the plug stage (Figure 1). We Received: March 14, 2017 Revised: April 13, 2017

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DOI: 10.1021/acs.bioconjchem.7b00141 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

Bioconjugate Chemistry

Figure 1. Plug-and-play strategy for the preparation of the antibody conjugates with defined DoC using 4-azidobenzoyl fluoride (ABF).

further illustrated the versatility of the plug-and-play strategy by preparing antibodies conjugated with toxin, oligonucleotide, and fluorophore derivatives. We also showed that the disconnection of the conjugation step and the functionalization step enabled a straightforward dual modification of the antibody with straightforward control of the relative amount of the two payloads.

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RESULTS AND DISCUSSION Efficacy Assessment of Antibody Modification with 4Azidobenzoyl Fluoride. In order to evaluate the efficacy of the lysine acylation of the antibody with 4-azidobenzoyl fluoride (ABF) we used trastuzumab as our model platform. Trastuzumab is an anti-HER2 monoclonal IgG1 antibody used in the clinic for breast cancer therapy and a component of FDA-approved ADC (Kadcyla). Trastuzumab (T) was reacted with various amounts of ABF in a PBS 1× buffer (pH 7.3) at 25 °C for 18 h (Figure 2). The resulting trastuzumab−azide conjugates (T-N3) were purified by gel filtration chromatography and analyzed by mass spectrometry (MS)17−19 in nondenaturing conditions to determine the average degree of conjugation for each sample, i.e., average number of conjugated add-on molecules per antibody. Plotting the measured DoC vs the number of ABF equivalents added per mAb resulted in a linear correlation, from which an efficacy of 72% could be deduced. To our surprise, a corresponding NHS ester of 4-azidobenzoic acid (ABNHS) under the same conditions proved to be much less efficient (49% efficacy; see SI for details, Figure S3). Moreover, benzoyl chloride derivative was found to be inactive, even when 10 equiv was employed; only unmodified antibodies were observed by mass spectrometry after reaction. It has been known for decades that the acylation reactivity of acyl fluorides is unlike that reported for other acyl halides, and instead can be compared to that of activated esters in aminolysis reactions.20 Most applications of acyl fluoride electrophiles have been reported in organic solvents,21 where they served as peptide coupling reagents,22−24 and peptidomimetics,25,26 or in the construction of heterocycles,27 and the synthesis of biologically active molecules.28,29 Recently, Sintes et al. and Kielland et al. have reported water-stable mesoionic acyl fluorides for the fluorescent derivatization of aminecontaining biomolecules under biological conditions.30,31 Consequently, it was decided to measure the stability of ABF in a PBS 1× buffer (pH 7.3) used for the conjugation. In contrast to our expectations, based on the report by Sintes et al., ABF proved to be quite unstable in PBS buffer with a pseudo-first-order hydrolysis constant k1 = 2.46 × 10−3 s−1 (t1/2

Figure 2. (A) Efficacy evaluation of antibody (T) modification with 4azidobenzyl fluoride ABF. (B) Example of a deconvoluted mass spectrum of T-N3 conjugate with DoC = 4.20. (C) Plot of DoC vs amount of ABF (72% efficacy).

= 4.7 min). We then hypothesized that the high efficacy of ABF for lysine acylation could be explained by a much faster reaction rate with amines. Indeed, the second-order rate constant of the acylation of benzylamine with ABF in PBS 1× (pH 7.3) was found to be k2 = 87.9 M−1 s−1 (t1/2 = 1.9 min; see SI for details, Table S1). Notably, we did not observe any degradation of ABF over months, when it was kept in a solid state at −20 °C. For comparison, NHS ester ABNHS showed a pseudo-firstorder hydrolysis constant k1 = 7.70 × 10−5 s−1 (t1/2 = 150 min) under the same conditions and a second-order rate constant for benzylamine acylation of k2 = 2.72 M−1 s−1 (t1/2 = 62 min; see SI for details, Table S1). In the case of the ABNHS hydrolysis, an unusual peak of N-(4-azidobenzyloxy)-succinamic acid was detected by LC-MS (see SI for details, Figure S1). As previously reported by Romieu et al. the latter did not react with excess amounts of benzylamine and was slowly converted into its benzoic acid derivative.32 Kinetic Study of the Modification of an Antibody with 4-Azidobenzoyl Fluoride. It was then decided to evaluate whether the high reactivity of 4-azidobenzoyl fluoride toward amines would translate into more efficient antibody conjugation by measuring the DoC at different reaction times. We therefore carried out a plug-and-play conjugation in parallel with ABF and ABNHS (Figure 3A). In the first step, trastuzumab (1 mg/mL at 4 or 25 °C) was treated separately with 4 equiv of each reagents. The aliquots were then withdrawn at regular time intervals (1, 7.5, 15, 30, 60, and 120 min). At each point in time the aliquot was quickly purified by gel filtration chromatography to stop the reaction. The resulting conjugates T-N3 were subjected to SPAAC B


Article


Figure 3. (A) Kinetic study of antibody modification with ABF or ABNHS in PBS 1× at 4 or 25 °C. Over time, the aliquots of the reaction mixture were withdrawn, purified, and subjected to SPAAC with TAMRA-BCN. (B) Visual comparison of the reactivity of ABF and ABNHS by color change of T-TAMRA conjugates over time. (C) SDS PAGEs analysis of the resulting conjugates: gel fluorescence and Coomassie Blue staining. (D) Example of a deconvoluted mass spectrum of T-TAMRA (DoC = 2.3) prepared using ABF (3 equiv).

reaction with TAMRA-BCN (1.5 equiv per azide group) overnight at 25 °C, to give trastuzumab−TAMRA conjugates T-TAMRA after purification by gel filtration chromatography. Visually, the more intense pink color of the ABF-based TTAMRA conjugates indicated the higher efficacy and reactivity of ABF compared to ABNHS both at 4 and 25 °C (Figure 3B). For a quantitative comparison of the conjugation efficacy, the antibody−dye conjugates (0.1 mg/mL) were analyzed using SDS PAGE (Figure 3C). Coomassie Blue staining showed the same intensity of lines corresponding to the antibody in all sets of the experiment. As expected, the negative control (lanes T and T+TAMRABCN) did not show any fluorescence, while the fluorescence signal of the ABF-based T-TAMRA indicated an almost complete conversion of acyl fluoride after 1 min at both 4 and 25 °C. In contrast, the gel fluorescence of the ABNHS-based TTAMRA revealed that conjugation did not take place at 4 °C, while only a moderate efficacy could be obtained after 30 min at 25 °C. It thus appeared that despite a fast hydrolysis rate, the higher reactivity of acyl fluoride toward amine nucleophiles resulted in more efficient conjugation. In order to quantify this efficacy more precisely, we performed a native MS analysis of the T-TAMRA conjugates. The native MS analysis first confirmed the complete SPAAC modification of trastuzumab−azide conjugates T-N3 with TAMRA-BCN, the DoC of the ABF conjugates was found to be 2.9 (72% efficacy) at both 15 min and 2 h at 25 °C, indicating completion of the reaction after 15 min. In accordance with SDS PAGE analysis, the DoC measured using native MS for ABNHS conjugates was only 0.3 (7% efficacy) and 1.8 (45% efficacy) after 15 min and 2 h at 25 °C, respectively, thus confirming a much slower reaction (Table S2). ADC Affinity. In order to test whether our chemistry would affect antigen recognition, we measured the affinity of antibodydye conjugate T-TAMRA (DoC = 2.9) using flow cytometry on two breast adenocarcinoma cell lines: HER2+ SKBR-3 (Figure 4) and HER2− MDA-MB-231 cell lines (see SI, Figure S4). Trastuzumab, the native antibody, was used as a reference

Figure 4. Estimate of antibody affinities of T-TAMRA (pink), T-DM1 (blue), trastuzumab (black), and the isotype control rituximab (gray). The plot on the left displays the superimposition of the fluorescence profile of each compound. The bar-plot on the right displays the median fluorescence intensity (MFI) of these profiles. These plots are representative of two independent experiments.

in the expected median fluorescence intensity (MFI) and trastuzumab emtansine (T-DM1), a commercialized ADC, was used as a benchmark. As clearly shown by the MFIs, no significant change in antibody binding affinity was observed for T-TAMRA compared to our reference (Figure 4). Versatility of the Plug-and-Play Strategy. We then turned our attention to the “play” stage of the process and found that the copper-free azide−alkyne click reaction with bicyclononynes (BCN) was both efficient and appropriate for biological conditions.33,34 To illustrate the versatility of the plug-and-play strategy, it was decided to utilize different types of payloads bearing BCN groups at this step. Antibody−Oligonucleotide Conjugates. First, an antibody−oligonucleotide conjugate C1 was prepared (Figure 5A). In order to do this, trastuzumab (1 mg/mL) was conjugated with 3 equiv of ABF followed by functionalization in the presence of 4.5 equiv of oligonucleotide-BCN ON1 at 25 C


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Figure 5. (A) Construction of antibody−oligonucleotide conjugates C1 using SPACC of T-N3 with BCN-Oligonucleotide (ON1) and hybridization of the resulting conjugate C1 with complementary oligonucleotide (ON2). (B) Denaturing SDS PAGEs analysis showed successful antibody− oligonucleotide conjugation and hybridization (M: molecular weight marker; T: trastuzumab; T+ON1: mixture of trastuzumab with ON1; C1: antibody−oligonucleotide conjugate; C2: hybridized antibody−oligonucleotide conjugate).

°C for 20 h to give conjugate C1. The conjugate C1 was further hybridized with complementary oligonucleotide ON2 to yield trastuzumab-ON1-ON2 C2. To verify the efficacy of the play stage, the conjugates were analyzed using SDS PAGE (Figure 5B). On the Coomassie Blue stained gel, the conjugate C1 had a number of lines corresponding to the expected species of ON1-antibody loads ranging from 0 to 4. Interestingly, calculations based on the integration of the fluorescence intensity of these lines after Cy5 excitation indicated a DoC value of 2.4, in close correlation with the DoC of the plug stage (for comparison, see SI). This again indicates the high efficacy of the click reaction (the nonfluorescent line corresponding to load 0 accounts for the slightly higher DoC). The conjugates C2 had the same number of lines as C1; however, these were more widely spread, because of the higher mass of the payload. The successful hybridization could be estimated by the fluorescence of these lines under Atto 488 excitation. Antibody−Drug Conjugate. To expand the scope of the functionalization step, the antibody−drug conjugate trastuzumab-MMAE was prepared. For this purpose, the antibody conjugate T-N3 (DoC = 2.16, using 3 equiv of ABF) was reacted with the BCN derivative of monomethyl auristatin E (MMAE-BCN) connected through a cleavable valine-citrulline linker (Figure 6). After purification, the resulting conjugates TMMAE were subjected to native MS analysis, which confirmed the complete SPAAC modification with preservation of the DoC (DoC = 2.10, see SI for details). Dual Antibody Functionalization. Recently, dual modification of biomolecules has gained a great deal of attention from the scientific community.35 Finally, it was decided to demonstrate the controllable dual modification of the antibody by employing various ratios of two payloads in the play step. Our assumption was that two payloads bearing the same strain of alkyne would react with similar kinetic rates; thus, their relative amounts in the reaction media would determine their final ratio on the resulting bioconjugate. The trastuzumab−

Figure 6. Preparation of ADC, trastuzumab-MMAE.

azide conjugate T-N3 (DoC = 2.2) was therefore reacted with a solution containing a mixture of TAMRA-BCN and Cy5-BCN at different ratios (Figure 7A). The excess of the reagents was removed by gel filtration chromatography after 20 h, and the absorptions at both 558 and 650 nm were measured for each antibody conjugate prepared at different fluorophore ratios (Figure 7B, red lines). A standard curve was obtained by measuring the absorption of the mixture of two fluorophores in corresponding ratio (Figure 7B, black lines). The standard lines agreed well with the lines corresponding to the absorption of antibody conjugates, showing that the bioconjugates displayed the same ratio of fluorophores as in the mixture used for the conjugation. This simple procedure opens up interesting D


Article


Figure 7. (A) Dual modification of antibody−azide conjugate T-N3 (DoC = 2.2) with a mixture of TAMRA-BCN and Cy5-BCN. (B) Normalized absorption of fluorophore mixture (black lines) and normalized absorption of T-TAMRA/Cy5 conjugates (red lines) at 558 and 660 nm.

prospects for the routine preparation of multilabeled antibodies.

Notes

CONCLUSIONS In summary, we introduce 4-azidobenzoyl fluoride (ABF) as an efficient and fast reagent for the lysine modification of native antibodies. The efficient and rapid acylation of the antibody with ABF in biological conditions makes acyl fluoride chemistry interesting for bioconjugate chemistry and biological applications. The decoupling of the conjugation and functionalization steps allows for antibody modification with azide groups in a controllable and reliable manner at the plug stage, using highly reactive acyl fluoride. These prefunctionalized antibodies can then be stored and further derivatized on demand, in a versatile way. For this purpose, the copper-free SPACC reaction with BCN derivatives appears to be highly practical and facile because of the good chemical stability/reactivity/bioorthogonality balance of both the azide and BCN functions. This approach was illustrated using a variety of representative payloads such as fluorophores, toxin, and oligonucleotide, with good reproducibility of the DoC. Moreover, this direct strategy for dual conjugation opens up interesting prospects for the routine preparation of multilabeled antibodies.

ACKNOWLEDGMENTS International Center for Frontier Research in Chemistry (icFRC), Region Alsace and the French Proteomic Infrastructure (ProFI; ANR-10-INBS-08-03) are acknowledged for financial support.

The authors declare no competing financial interest.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.7b00141. Materials and instrumentation, experimental procedures, analytical data (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Alain Wagner: 0000-0003-3125-601X E


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Acyl Fluorides: Fast, Efficient, and Versatile Lysine-Based Protein

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