MABS 2020, VOL. 12, NO. 1, e1702262 (11 pages) https://doi.org/10.1080/19420862.2019.1702262 REPORT Homogeneous antibody-drug conjugates: DAR 2 anti-HER2 obtained by conjugation on isolated light chain followed by mAb assembly Mercè Farràs a, Joan Miret b, Marc Camps a, Ramón Román b, Óscar Martínez a, Xavier Pujol a, Stéphane Erbc, Anthony Ehkirch c, Sarah Cianferani c, Antoni Casablancas b, and Jordi Joan Cairó b a Research&Development and New Business Development, Farmhispania SA, Montmeló, Spain; bDepartment of Chemical, Biological and Environmental Engineering, Autonomous University of Barcelona, Barcelona, Spain; cLaboratoire de Spéctrometrie de Masse BiorOganique, Université de Strasbourg, Strasbourg, France ABSTRACT ARTICLE HISTORY Despite advances in medical care, cancer remains a major threat to human health. Antibody-drug Received 23 September 2019 conjugates (ADCs) are a promising targeted therapy to overcome adverse side effects to normal tissues. Revised 11 November 2019 In this field, the current challenge is obtaining homogeneous preparations of conjugates, where Accepted 4 December 2019 a defined number of drugs are conjugated to specific antibody sites. Site-directed cysteine-based KEYWORDS conjugation is commonly used to obtain homogeneous ADC, but it is a time-consuming and expensive ADC; mAb; trastuzumab; approach due to the need for extensive antibody engineering to identify the optimal conjugation sites assembly; disulfide bonds; and reduction – oxidation protocols are specific for each antibody. There is thus a need for ADC Cys-based conjugation; platforms that offer homogeneity and direct applicability to the already approved antibody therapeutics. homogeneous conjugation; Here we describe a novel approach to derive homogeneous ADCs with drug-to-antibody ratio of 2 from affinity chromatography; any human immunoglobulin 1 (IgG1), using trastuzumab as a model. The method is based on the cancer production of heavy chains (HC) and light chains (LC) in two recombinant HEK293 independent cultures, so the original amino acid sequence is not altered. Isolated LC was effectively conjugated to a single drug-linker (vcMMAE) construct and mixed to isolated HC dimers, in order to obtain a correctly folded ADC. The relevance of the work was validated in terms of ADC homogeneity (HIC-HPLC, MS), purity (SEC- HPLC), isolated antigen recognition (ELISA) and biological activity (HER2-positive breast cancer cells cytotoxicity assays). Introduction Different approaches have been developed to obtain homo- geneous ADCs by site-specific conjugation, including cysteine Antibody-drug conjugates (ADCs) are a class of targeted residue(s) addition, non-natural amino acids, enzyme- therapy used in the treatment of various cancers. As of late mediated conjugation (including transglutaminase, sortase A, 2019, the US Food and Drug Administration (FDA) has glycosyltransferase or endoglycosidase) and linker-based approved four ADCs, three ADCs are undergoing regulatory conjugation.5-12 review, and over 100 ADCs are in the clinical pipeline.1 Their Cysteine-engineered residues have been introduced into medical potential is based on their structure, since it combines different antibodies by site-directed mutation to provide the ability of a monoclonal antibody (mAb) to specifically free thiol groups for conjugation using conventional thiol- target a tumor-associated antigen2 with the cell-killing ability specific maleimide linkers,13 a technology developed by of potent cytotoxic drugs via a chemical linker. Seattle Genetics and used to produce the heterogeneous These unique targeted drugs, however, pose both technological ADC brentuximab vedotin (Adcetris), which was approved and developmental challenges. One key characteristic of an ADC is by FDA in 2011. The process generates almost homoge- the drug:antibody ratio (DAR), which is the number of cytotoxic neous ADCs containing approximately two drug molecules drug molecules per molecule of antibody. The DAR defines the two per antibody, but additional antibody reduction/oxidation major types of ADCs: heterogeneous (conjugated in random posi- steps are required for selective conjugation of the added tions that vary between mAbs) and homogeneous (conjugation at cysteines.14-16 specific sites that are defined in each mAb molecule). The four Non-recombinant methods for reducing ADC heterogene- commercially available ADCs are heterogeneous due to limitations ity that enable the production of homogeneous ADCs based in the technology available when they were originally developed. on existing antibodies have also been developed. For example, The lack of efficient methods for producing completely homoge- disubstituted maleimide linkers have been used for synthesiz- neous ADCs has compelled pharmaceutical companies to develop ing homogeneous ADCs via interchain cysteine cross-linking site-specific conjugation methods, which improves ADC through disulfide bonds rebridging.17 Godwin and coworkers homogeneity,3 batch-to-batch consistency, and is more desirable presented an alternative thiol conjugation approach where from a regulatory perspective.4 bis-sulfone-functionalized reagents are used to form a stable CONTACT Mercè Farràs mfcosta@fhrt.com Farmhispania SA, Costa. Passeig Riu Besòs, 9-10, Montmeló (Barcelona) 08160, SPAIN © 2019 Farmhispania S.A. Published with license by Taylor & Francis Group, LLC. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. e1702262-2 M. FARRÀS ET AL. three-carbon bridge with disulfides.18 In addition, the struc- Results ture of the antibody remains intact and the ligand-to-antibody Selection of the components of the study ratio can be tightly controlled, with a 78% conversion to DAR 4 ADCs.19 Badescu and coworkers used a bis-sulfone linker To evaluate the technology described, trastuzumab designed to react with two antibody cysteines to conjugate an (Herceptin) was selected as a model. This therapeutic anti- auristatin payload (monomethyl auristatin E (MMAE)) to body has been successfully used in the treatment of HER2- trastuzumab and its antigen-binding fragments (Fabs).20 The positive breast cancer, and it is the antibody component of approaches described above are time consuming and specific FDA-approved ADC trastuzumab emtansine (Kadcyla). for each construct, as they often require extensive antibody Anticancer drug MMAE and valine-citruline linker were engineering to identify the optimal conjugation sites9,21 where selected for their commercial availability and their proven unique side chains can be introduced for conjugation, or they activity in another FDA-approved ADC, brentuximab vedo- require complex linker and payload modifications that have tin. The method described could be applied to alternative not been clinically validated.8 As a result, they are not suitable linkers conjugated to reactive thiols with improved stability for converting existing antibodies directly into ADCs.5,7 characteristics.24 In vivo, heavy chains (HCs) and light chains (LCs) are co- translationally translocated into the endoplasmic reticulum and folding begins even before the polypeptide chains are Structure of independently produced HC and LC completely translated. Most IgGs assemble first as HC dimers Anti-HER2 LC and HC were produced in separate HEK293 to which LCs are added covalently via a disulfide bond cultures and purified by affinity chromatography. Using between the LC constant domain and the first HC constant sodium dodecyl sulfate-polyacrylamide electrophoresis gel domain (CH1) domain.22 Most FDA-approved mAbs are (SDS-PAGE) under denaturing conditions (Figure 2a), HC is human(ized) immunoglobulin (Ig) G1. detected as a single band of approximately 100 KDa, indicat- Here, we describe a novel platform to generate totally ing that the transfected HEK293 cell line expresses the protein homogeneous DAR 2 ADCs, without antibody engineer- forming a covalently bonded dimer. The single band together ing, that could potentially be applied to any human IgG1 with the single peak under native conditions by SEC-HPLC and IgG3 antibodies by using conventional maleimide lin- (Figure 2b) confirm that no trimers or other structures are ker. Trastuzumab, an IgG1 mAb that targets human epi- formed Further characterization of HC by HPLC-MS (Figure dermal growth factor receptor-2 (HER2), was used as 5) shows two peaks that correspond with 2xHC (100 KDa). a model. The method described is based on the produc- This could be explained because the unpaired cysteines of the tion of HCs and LCs in two independent recombinant heavy chain dimers (dcHC) are capped by small cysteine- HEK293 cultures, obtained by transfection,23 and the sub- containing molecules from the culture broth, mainly glu- sequent conjugation of isolated LC in the only solvent- tathione, therefore avoiding unwanted reactions of the HC. accessible cysteine residue available, obtaining Unexpectedly, LC appears in 2 bands in the SDS-PAGE gel, a homogeneous solution of LC-MMAE. MAb assembly representing 55% the 42 KDa band and 45% the 21 KDa band. was subsequently achieved by combining LC-MMAE with The same sample under reducing conditions shows a single an isolated HC to generate a non-engineered Cys-based lane at 21 KDa (data not shown) and the SEC-HPLC analysis homogeneous ADC (Figure 1). shows that there is only one peak of 42 KDa under native Figure 1. Strategy to obtain DAR 2 homogeneous ADCs. anti-HER2 chains were independently produced in recombinant HEK293 cultures. Then, each chain was purified by affinity chromatography and light chain (LC) was conjugated to vcMMAE. Finally, the complete mAb was assembled. MABS e1702262-3 Figure 2. HC and LC conformation of proteins expressed by recombinant HEK293. (a) SDS-PAGE gel (denaturing conditions). (b) SEC-HPLC (native conditions) for HC. (c) SEC-HPLC for LC. conditions (Figure 2c). Therefore, the anti-HER2 LC stable than the independent sub-units; SEC-HPLC profile expressed forms dimers, but whereas 55% of them are cova- confirmed the presence of 90.1% of monomer (Table 1). The lently bonded, 45% of LC forms dimers without covalent resulting product recognized isolated HER2 antigen in an unions. A small amount of aggregates (3%) are observed in ELISA assay in the same proportion as in-house anti-HER2 the produced LC (Figure 2c). antibody (Table 2) produced by HEK293 by using a tricistronic vector, as previously described.23 In the reduction-oxidation approach, a lower yield was mAB assembly from independently produced HC and LC obtained, since residual LC was detected in the final product. Since both residual HC and LC are present, a more complex Reduction-oxidation assembly approach purification sequence must be used, as compared with the Prior to conjugation, the mAb assembly was evaluated follow- spontaneous approach, where only residual HC was detected. ing a sequential reduction-oxidation protocol of a LC and The final mixture of monomer, aggregates and dcHC dcHC mixture (see Materials and Methods section). SEC- obtained in the spontaneous assembly approach was purified HPLC results shown in Table 1 demonstrate that a 73% of mAb can successfully be reassembled. Table 1. Molecules detected under native conditions (SEC-HPLC). Spontaneous assembly approach Molecule Aggregates Monomer mAb dcHC dcLC Isolated HC and LC were mixed in 50 mM citrate pH 6 buffer Reduction-oxidation approach 2.1 73.0 14.5 10.4 and similar results were obtained without a reduction–oxida- (% Area) tion procedure. In this approach, the mAb original structure Spontaneous assembly approach 3.2 90.1 6.7 ND (% Area) was assembled because it may be thermodynamically more e1702262-4 M. FARRÀS ET AL. Table 2. Isolated antigen HER2 recognition in the ELISA test. In vivo folded In vivo folded In vivo folded trastuzumab In vitro reassembled trastuzumab Homogeneous T-MMAE Molecule LC HC (control) (spontaneous approach) DAR 2 Isolated antigen HER2 ND ND 51 ± 5 UA/µg 54 ± 6 UA/µg 59 ± 4 UA/µg recognition Table notes. ND: not detected. The error of the method is around 11%. by Capto L affinity chromatography (specific for LC), since no antibody produced by HEK293 by using a tricistronic vector, residual LC was detected by SEC-HPLC so only the assembled as previously described23 and shown in Table 2. mAb would be captured. DAR analysis: MS MAb and ADC were analyzed with direct hyphenation of HIC ADC assembly from independently produced HC and to native MS through a HICxSEC-native MS setup.25 Results MMAE-conjugated LC obtained confirm that only one main peak is present in HIC LC conjugation and online detected with native MS as DAR 2 for the ADC Purified LC was completely reduced with TCEP and conju- without the presence of D0 and under/over conjugate, high- gated with valine-citrulline monomethyl auristatin lighting a homogeneous ADC with an average DAR of 2.0 E (vcMMAE) at the only solvent-accessible thiol group of (Figure 4). Also, dcHC were detected by confirming the excess cysteine in the LC. Since only one thiol is available, each LC of this sub-unit and the necessity of purification by protein is conjugated to a single vcMMAE molecule and L affinity chromatography. a homogeneous solution of LC-MMAE is obtained, as shown by HIC-HPLC (Figure 3), which shows the differences Identification of cysteine linked payload position in hydrophobicity between LC and conjugated LC due to the In order to confirm the homogeneous and specific conjuga- presence of the hydrophobic cytotoxic drug. A single peak was tion of the ADC, structural information based on the identi- detected in the conjugated LC, showing a conjugation effi- fication of drug-loaded peptides was performed after classical ciency close to 100%. tryptic digestion followed by a nanoLC-MS/MS analysis as previously described. To perform this manual identification, ADC assembly the first step was to explore the extracted ion chromatogram The selected assembly strategy for ADC generation was the (XIC) of one of the two characteristic in-source fragmentation spontaneous approach due to its simplicity and efficacy. The ions (m/z 718.51 or 762.51) of the MC-vc-PAB-MMAE- obtained monomer recognized isolated HER2 antigen in an conjugate.15 Due to the high resolution MS1, conjugated ELISA assay in the same proportion as in-house anti-HER2 peptides were identified based on accurate mass Figure 3. HIC-HPLC chromatogram. LC (dotted line) appears in two peaks, corresponding to the (un)covalently bonded LC dimers. LC-MMAE (continuous line) appears as a single peak at 8.1 minutes. MABS e1702262-5 Figure 4. Comparison of HICxSEC-native MS chromatograms of ADC and naked mAb. a) DAR 2 HIC profile. b) DAR 2 deconvoluted spectra. c) Naked mAb HIC profile. d) Naked mAb deconvoluted spectra. Figure 5. MS and MS/MS spectra of the two peptides containing the MMAE payload linked to the Cys214 localized on the LC part of the ADC. (a) [SFNRGEC] + 1 payload, (b) [GEC] + 1 payload. measurement. Then, confirmation of the drug-loaded pep- MC-vc-PAB-MMAE payload was highlighted by the detection tides was achieved through the detection of seven additional of the conjugated peptide with the loss of the m/z 762.51 signature ions of MC-vc-PAB-MMAE-conjugate (m/z 686.49, species. Through this manual interpretation, the identification 506.36, 411.26, 321.22, 170.11 and 134.09)26 in all MS/MS of two drug-loaded peptides ([SFNRGEC] + 1 payload and spectra. Moreover, the presence of the peptide linked to the [GEC] + 1 payload) containing the same cysteine at position e1702262-6 M. FARRÀS ET AL. Figure 6. Biological activity of the in vitro assembled mAb and homogeneous DAR 2 ADC compared to in vivo folded mAb and heterogeneous DAR 4 ADC, respectively. Cell inhibition assay. Orange: DAR 2 homogeneous T-MMAE assembled by the strategy described. Black: assembled non-conjugated mAb by the strategy described. Green: DAR 4 heterogeneous T-MMAE (reference). Blue: in vivo folded trastuzumab. The concentration indicated is referred to the complete ADC (mainly mAb) and not to toxin:payload. 214 located on the LC part of the ADC was achieved without required to achieve a functional mAb conformation despite ambiguity (Figure 5). the fact that the expressed LCs form dimers. We hypothesize that, as it is stated to occur in vivo,22 it is thermodynamically Biological activity – In vitro assay more favorable for LC to be assembled with HC than to HER2-positive breast cancer cells SKBR3 were used to assess remain as dimers. The reaction is, therefore, biologically dis- the cytotoxicity of the homogeneous DAR 2 trastuzumab placed to generate a monomer antibody. conjugated to MMAE (T-MMAE) we obtained. Its biological Most ADCs in current clinical development were obtained by activity was compared to an in-house heterogeneous DAR conjugation to endogenous lysine or cysteine residues of the anti- 4 T-MMAE (control) using an in vitro MTS cell-based assay. body, carefully controlling the average degree of modification to Our results (Figure 6) show that homogeneous DAR yield an average DAR in the range of 3.5–4.0.6 This ratio was 2 T-MMAE has an antiproliferative effect on the target cells, selected in order to minimize the amount of non-conjugated anti- and it exhibits a significantly lower cytotoxic activity than body and to avoid very high DAR species in the mixture, which heterogeneous DAR 4 T-MMAE (IC50 DAR 2: 51.5 pM; may be problematic in manufacturing and formulation because of DAR 4: 25.5 pM). These differences correspond to the differ- higher hydrophobicity and lower solubility.6 ences in the drug load. Furthermore, in vitro assembled mAb Furthermore, it has been stated that despite the fact that in vitro behave similarly to trastuzumab folded in vivo (Figure 6). potency of ADCs increases with the DAR, ADC plasma clearance and aggregation also increase in species with a high DAR, reducing exposure and in vivo efficacy.27,28 An optimal DAR value and Discussion a homogeneous ADC are therefore crucial to maximize the balance Several methods to generate homogeneous ADC have of efficacy, tolerability and cytotoxic activity.29 Engineered cysteine- reported,5,7 but they require either genetic engineering of based methods to produce homogeneous ADC targeted a DAR the sequence of the original mAb or complex linker technol- value of 2 or 4. ThioMabs enabled the generation of 90% DAR 2 ogy, which have not been clinically evaluated. Here, we ADCs30 and dibromomaleimide linkers targeted DAR 4.30 Some describe a novel method to obtain a homogeneous ADC approaches were developed to reduce clearance of highly hydro- based on cysteine conjugation without genetic engineering phobic ADCs, but they require complex drug-linked design.27,28 of the mAb sequence and by using a conventional linker- Our work provides a novel strategy to obtain cysteine- drug structure. The use of genetic engineering remains at based completely homogeneous ADC (no other DAR values the expression level since HCs and LCs are expressed and were detected) that involves selective conjugation of the iso- folded in two different cell lines, and the complete antibody lated mAb light chain. In this method, the antibody sequence with the original amino acid sequence is generated after they is preserved, but HC and LC are expressed in independent cell are isolated. The in vitro assembled mAb behaves like its cultures. In vivo folded and isolated chains are assembled to correspondent in vivo assembled mAb in terms of isolated obtain a functional ADC. DAR 2 trastuzumab conjugated to antigen binding capacity and biological activity (Table 2). The MMAE was used as a model, but the applicability of the proposed method shows that no HC or LC reduction is method might include any cysteine-based conjugation to any MABS e1702262-7 human IgG1 or IgG3. This method offers a generic strategy to expression vector derived from the commercial pIRESpuro3 vector obtain homogeneous ADCs with high efficiency and, to the (Clontech, CA, USA) between NheI-AgeI for LC DNA sequence best of the authors’ knowledge, such an approach has not and BamHI-EcoRI for HC DNA sequence. HEK293SF-F6 cells been previously described. were then transfected with the construct and selected by puromycin The two cell lines used in this work were not industrial cell antibiotic resistance. The new anti-HER2 antibody producer cell lines, so further work will include their improvement as well lines were referred to as HEK293_T10. as optimization of the equimolar proportion of the chains in HEK293SF-3F6 producing anti-HER2 HC or anti-HER2 order to enhance process yield, which was outside the scope of LC were obtained by cloning the above-mentioned synthetic our study. Other future steps include HC conjugation to DNA sequences in the expression vector pIRESpuro3 obtain DAR 2 homogeneous ADCs with another drug-linker (Clontech, CA, USA) between BamHI and NotI for HC and construct in the CH1 region cysteines and preserving the in the vector pIRESneo3 (Clontech) between NheI and AgeI hinge disulfide bonds followed by either LC or LC-MMAE for LC. HEK293 cells were then transfected with the different assembly (Figure 7), which would allow the generation of constructs and selected as described previously.30 The new homogeneous ADCs taking advantage of 2 different payloads. producer cell lines were referred to as HEK293_THC and These new ADC should be assessed in vivo and compared in HEK293_TLC. terms of bioavailability and pharmacokinetics Cells were subcultured at 0.3 × 106 cell/mL three times per week to keep them in exponential growing phase. Cell maintenance was performed in 125 mL polycarbonate shake flasks (Corning Inc., NY, Materials and methods USA), with a working volume of 12 mL, and maintained at 37°C in Cell lines and maintenance an incubator with a 5% CO2 humidity saturated atmosphere (Steri- cult 2000 Incubator, Forma Scientific). Flasks were continuously Trastuzumab protein sequence for LC and HC were obtained agitated at 110 rpm on an orbital shaking platform (Stuart SSL110). from the Drugbank website (ref. DB00072).31 The optimized signal peptides for trastuzumab were added to LC and HC.32 The Trastuzumab DNA sequence for both LC and HC were HC and LC production obtained from the mentioned protein sequence, after being optimized to mammalian codon usage using the Codon Culture media used in shake flasks and 5 L wave bag cultures Optimization On-line (COOL) method,33 and were synthe- for HEK293_THC and HEK293-TLC cell lines was sized by Genscript (NJ, USA). SFM4Transfx-293 (HyClone, UT, USA) supplemented with HEK293SF-3F6 producing anti-HER2 antibody were obtained 4 mM GlutaMAX (Gibco, Invitrogen, CA, USA), 10% (v/v) by cloning the above synthetic DNA sequence in a tricistronic Cell Boost 5 (60 g/L solution) (HyClone), 2% (v/v) Kolliphor Figure 7. Future proposed strategies based on the described method to obtain homogeneous ADCs. The same conjugation could occur in either LC or HC. After assembly, the ADC could be loaded with 2 different site-directed payloads. e1702262-8 M. FARRÀS ET AL. Figure 8. Reduction-oxidation assembly approach. In vivo produced and purified LC and HC are reduced to free disulfide bonds and deoxidized to obtain the assembled mAb. P188 (100 g/L solution) (Sigma, MO, USA) and 0.5% (v/v) ADC assembly from independently produced HC and Antifoam C (10 g/L solution) (Sigma). Selection pressure was MMAE-conjugated LC maintained during the production step by adding 0.2% (v/v) LC conjugation puromycin (1 mg/mL) (Merck, NJ, USA) for HEK293_THC MAb assembly approach 2 was easiest, and it was applied to and 20 mL/L of neomycin (G-418 solution, Roche, Basel, MMAE-conjugated LC. Since DTT reduction power is not Switzerland) for HEK293_TLC. optimal at pH 6, tris(2-carboxyethyl)phosphine (TCEP) in Bioreactor cell cultures were performed using Flexsafe RM a 100:1 molar proportion was used to reduce purified LC. 10 L bags (DFB010L, Sartorius, Göttingen, Germany) in Under this condition, LC did not form covalently bonded a WAVE 20/50 EHT (GE Healthcare, IL, USA) with dimers (analyzed by SDS-PAGE) and was conjugated to a working volume of 5 L. Temperature was set at 37ºC. vcMMAE construct (MedchemExpress, NJ, USA). Agitation and angle were set at 22 rpm and 8º, respectively. Purification steps were performed when maximum cell ADC assembly density was reached, including solids separation by depth Working at a concentration range of 1–5 mg/mL of HC in the filtration (Clarisolve 40MS, Merck) and a bioburden reduc- final mixture leads to a successful reassembly of the mAb. We tion filter (Millipore Express SHF, Merck), broth concen- applied a relation of 5.54 g dcHC/g scLC. Thereafter, affinity tration by tangential flow filtration (Hydrosart 30 KDa chromatography (GE) is used in the ADC purification so membrane, Sartorius), protein A affinity chromatography unassembled LC-MMAE will not bind to protein A ligand. for HC purification (MAb Select Sure, GE) and protein This procedure is described in Figure 9. L affinity chromatography for LC purification (Capto L, GE), as described by the supplier and buffer exchange by desalting columns (PD Minitrap G-25, GE). The final buffer Heterogeneous T-MMAE DAR 4 (control for MTS test) was 50 mM sodium citrate pH 6 for both HC and LC. Heterogeneous T-MMAE DAR 4 was obtained by trastuzumab reduction with 4:1 molar proportion of TCEP with respect to mAB assembly from independently produced HC and LC trastuzumab followed by the addition of diethylenetriaminepen- taacetic acid (Sigma) at a final concentration of 1 mM. Next, the Reduction-oxidation assembly approach conjugation step occurred by the reaction of free thiol groups of The reduction-oxidation mAb assembly approach involves reduced trastuzumab to vcMMAE (MedchemExpress), used in mixing HC and LC. Later, reduction of the disulfide bonds a 10:1 molar proportion, as described before.15 of the mixed chains was achieved by the addition of 16:1 molar proportion of dithiothreitol (DTT) (Sigma) with respect to dcHC, and the mixture was incubated at Analytical methods 37ºC for 1 h. Next, a buffer exchange column allowed SDS-PAGE gels glutathione/cysteine and DTT removal while the product Protein mixture composition was analyzed by SDS-PAGE gels remained in 50 mM citrate buffer. Finally, 2:1 molar pro- (MiniProtean TGX Stain-free gels 4-20%, Biorad, CA, USA). Band portion of dehydroascorbic acid (Sigma) with respect to densitometry to determine the relative amount of each band in the DTT was added to promote disulfide bonds reoxidation sample was performed using the software GelDoc EZ (Biorad). for 3 hours at room temperature. This procedure is detailed in Figure 8. SEC-HPLC SEC experiments were performed in Waters Alliance 2695 sys- Spontaneous assembly approach tem using Zenix-C SEC-300 (4,6 x 300 mm, Sepax, DE, USA). A simpler approach involved the incubation of a mixture of The mobile phase was PBS 1x (Sigma) at a flow rate of LC and HC in 50 mM citrate pH 6 buffer for 3 hours at room 0.35 ml/min. The UV absorbance was measured at temperature. a wavelength of 214 nm. MABS e1702262-9 Figure 9. Spontaneous assembly approach to generate homogeneous ADC. LC are reduced to free interchain cysteines and allow the site-directed payload:linker conjugation. Then, dcHC is mixed to obtain the assembled ADC. HIC-HPLC HIC-SEC native MS HIC experiments were performed using a Waters Alliance 2695 The direct hyphenation of HIC to native MS consists of system using Proteomix HIC Butyl-NP5 (4,6 x 50 mm, Sepax). The a combination of H-Class and I-Class liquid chromatography mobile phase was a gradient of 25 mM sodium phosphate pH 7 systems hyphenated to a Synapt G2 HDMS Q-TOF mass (Sigma) with decreasing ammonium sulfate (2170, Merck) concen- spectrometer. The HIC column was a MAbPac HIC-10 tration (1.8 M to 0M) and increasing concentration of isopropanol (100 mm x 4.6 mm, 5 µm, 1000 Å) (Thermo Scientific, (Scharlab, Barcelona, Spain) to improve peaks resolution, as recom- Cheshire, UK). The SEC column used in the second dimen- mended by the column manufacturer, at a flow rate of 0.8 ml/min. sion was an AdvanceBio SEC (50 mm x 4.6 mm, 2.7 µm, The UV absorbance was measured at a wavelength of 214 nm. 300 Å) (Agilent Technologies, Wilmington, DE, USA). For the HIC first dimension, the mobile phase A was composed of 2.5 M of ammonium acetate and 0.1 M phos- ELISA assay phate buffer (Na2HPO4), pH 7.0 (adjusted with phosphoric Assembly was evaluated by comparing in vitro isolated anti-HER2 acid), while the mobile phase B was composed of 0.1 M recognition. Namely, 50 µL of 50 ng/µL anti-HER2 antibody in phosphate buffer (Na2HPO4 and NaH2PO4) with pH 7.0 phosphate-buffered saline (PBS, P5493, Sigma Aldrich) solution (adjusted with sodium hydroxide solution). The following (SinoBiological, Beijing, China) was placed in the wells of 96-well gradient was employed in HIC: 0 to 90% B in 36 min, 90 to Maxisorp plates for its adsorption at 4ºC overnight. After removal of 100% B in 21 min, followed by an isocratic step at 100% B for the supernatants, 100 µL of 2% skim milk were added and allowed to 8 min at a flow rate of 100 µL/min. For the second chromato- stand at room temperature for 1 h. The plates were washed three graphic dimension in SEC, the separation was carried out in times with PBS-Tween20 (Sigma) and 50 µL of samples at different isocratic mode with an aqueous mobile phase composed of dilutions in PBS were placed in each well. Samples were analyzed in 100 mM ammonium acetate at a flow-rate of 700 µL/min. duplicates. After standing at room temperature for 1 h, the plates were The Synapt G2 HDMS was operated in sensitive mode and washed three times with PBS-Tween20. After the addition of 50 µL of positive polarity with a capillary voltage of 3.0 kV. The sample a solution of 0.1 µg/mL of polyclonal anti-IgG1 conjugated to horse- cone and pressure in the interface region were set to 180 V radish peroxidase (A00166, Genscript), the plates were allowed to and 6 mbar, respectively. Source and desolvation temperature stand at room temperature for 1 h. After three washings with PBS- were set to 100 and 450°C, respectively. Desolvation and cone Tween20, 50 µL of TMB were added. The staining reaction was gas flows were set at 750 and 60 L/hr, respectively. carried out at room temperature for 10–15 minutes and stopped by Acquisitions were performed in the m/z range of adding 50 µL of 20% sulfuric acid addition. The absorbance at 1000–10000 with a 1.5 s scan time. External calibration was 450–630 nm was measured with a Labtech LT-4000 microplate reader performed using singly charged ions produced by a 2 g/L (Labtech, MI, USA). solution of cesium iodide in 2-propanol/water (50/50 v/v). e1702262-10 M. FARRÀS ET AL. Sample preparation for peptide mapping analysis was measured with a Labtech LT-4000 microplate reader. The Fifteen micrograms of conjugated antibody were solubilized in concentration indicated is referred to the complete ADC 150 mM NH4HCO3, 0.1% RapiGestTM (Waters, Milford, USA) at (mainly mAb) and not to toxin:payload. pH 7.4. Disulfide reduction was performed by incubating the ADC solution with 5 mM DTT for 30 min at 60°C. Alkylation was performed with 15 mM IAA for 30 min in the dark. Digestion Abbreviations was performed by adding trypsin (Promega, Madison, USA) to a 1:50 enzyme:substrate ratio. Sample was incubated overnight at ADC antibody-drug conjugate dhAA dehydroascorbic acid 37°C. The reaction was quenched by adding 1% of TFA. IgG immunoglobulin RapiGestTM was eliminated by centrifugation at 10,000 g for 5 min. mAb monoclonal antibody LC light chain Peptide mapping analysis HC heavy chain NanoLC-MS/MS analysis was performed using a nanoAcquity dcHC heavy chains dimers SDS-PAGE dodecyl sodium sulfate polyacrylamide electrophoresis gel Ultra-Performance-LC (Waters, Milford, USA) coupled to the vcMMAE valine-citrulline monomethyl auristatin E Q-Exactive HF-X mass spectrometer (Thermo Scientific, TCEP tris(2-carboxyethyl)phosphine Bremen, Germany) with a nanoSpray source. The sample SEC-HPLC size exclusion chromatography - high performance liquid was trapped on a nanoACQUITY UPLC precolumn (C18, chromatography 180 µm x 20 mm, 5 µm particle size), and the peptides were HIC-HPLC hydrophobic interaction column – high performance liquid chromatography separated on a nanoACQUITY UPLC column (C18, DAR drug antibody ratio 75 µm x 250 mm with 1.7 µm particle size, Waters, Milford, MS mass spectrometry USA) maintained at 60°C. Mobile phase A was 0.1% (v/v) T-MMAE trastuzumab conjugated to monomethyl auristatin E formic acid in water and mobile phase B was 0.1% (v/v) formic acid in acetonitrile. A gradient (1–8% B for 2 min, 8–35% B for 58 min, 35–90% B for 1 min, 90% B for 5 min, Acknowledgments 90–1% B for 1 min and maintained 1% B for 20 min) was This work was supported by Farmhispania SA with funding from CDTI used at a flow rate of 450 nL/min. The Q-Exactive HF-X (Spanish Government). source temperature was set to 250°C and spray voltage to This work was supported by the CNRS, the University of Strasbourg, the 1.8 kV. Full scan MS spectra (300–1800 m/z) were acquired “Agence Nationale de la Recherche” (ANR) and the French Proteomic Infrastructure (ProFI; ANR-10-INBS-08-03). The authors thank GIS IBiSA in positive mode at a nominal resolution of 60,000 with and Région Alsace for financial support in purchasing a Synapt G2 HDMS a maximum injection time of 50 ms and an AGC target instrument. A.E. acknowledges the “Association Nationale de la Recherche et value of 3 × 106 charges, with lock-mass option being enabled de la Technologie” (ANRT) and Syndivia for funding his PhD fellowship. (polysiloxane ion from ambient air at 445.12 m/z). The 10 most intense multiply charged peptides per full scan were isolated using a 2 m/z window and fragmented using higher Disclosure of potential conflicts of interest energy collisional dissociation (normalized collision energy of ADC are a promising therapeutic tool because they combine the selec- 27). MS/MS spectra were acquired with at a nominal resolu- tivity of the antibody (vehicle) with the cell killing ability of cytotoxic tion of 15,000 with a maximum injection time of 50 ms and drugs, minimizing chemo-associated side effects. The method described provides a solution to generate homogeneous compositions of ADCs an AGC target value of 1 x 105, and dynamic exclusion was set without antibody sequence engineering. Results also demonstrate that to 60 sec. The system was fully controlled by XCalibur soft- ADC and mAbs can be folded in vitro from their subunit parts (LC and ware v3.0.63, 2013 (Thermo Scientific) and NanoAcquity HC) which can be prior assembly conjugated to a cytotoxic drug. The UPLC console v1.51.3347 (Waters). work described herein could pave the way to easily obtaining homoge- neous ADCs that could be tested in clinical trials for existing described antibodies or approved to target a disease. MTS assay SK-BR-3 cells at the logarithmic growth phase were washed with sterile PBS 1x (HyClone), treated with trypsin (Sigma), Funding re-suspended in 10% fetal bovine serum (Sigma) DMEM medium (Sigma) with cell density at 2 × 105 cells/ml and This work was supported by the Centre for Industrial Technological Development (ES). seeded in a 96-well cell culture plate (Corning) by applying 50 µL of cell suspension per well. The plates were incubated at 37ºC in a humidified 5% CO2 incubator for 24 hours. Next, ORCID samples to be analyzed were diluted to the concentration Mercè Farràs http://orcid.org/0000-0003-3099-9313 range to be tested and 50 µL were applied to the cell culture Joan Miret http://orcid.org/0000-0001-7918-8963 wells under sterile conditions by using decreasing sample Marc Camps http://orcid.org/0000-0003-2729-2587 concentrations. The last row of each plate was used as control, Ramón Román http://orcid.org/0000-0003-2311-2737 which consisted of cells in medium without sample to be Óscar Martínez http://orcid.org/0000-0001-5323-276X tested. The plates were incubated at 37ºC in a humidified 5% Xavier Pujol http://orcid.org/0000-0003-0699-1178 Anthony Ehkirch http://orcid.org/0000-0002-5616-5280 CO2 incubator for 72 hours. After incubation, each well was Sarah Cianferani http://orcid.org/0000-0003-4013-4129 treated with 20 µL MTS solution (Promega, WI, USA) fol- Antoni Casablancas http://orcid.org/0000-0002-0773-9364 lowed by incubation for 3 hours. The absorbance at 405 nm Jordi Joan Cairó http://orcid.org/0000-0001-7661-0858 MABS e1702262-11 References 19. Badescu G, Bryant P, Bird M, Henseleit K, Swierkosz J, Parekh V, Tommasi R, Pawlisz E, Jurlewicz K, Farys M, et al. Bridging 1. Persistence Market Research. Antibody drug conjugates market: disulfides for stable and defined antibody drug conjugates. global industry analysis and forecast 2017–2025. [Accessed 04 Apr Bioconjug Chem. 2014 18;25(6):1124–36. doi:10.1021/bc500148x. 2018]. https://www.persistencemarketresearch.com/market- 20. Badescu G, Bryant P, Swierkosz J, Khayrzad F, Pawlisz E, Farys M, research/antibody-drug-conjugates-market.asp Cong Y, Muroni M, Rumpf N, Brocchini S, et al. A new reagent 2. Maruani A, Smith MEB, Miranda E, Chester KA, Chudasama V, for stable thiol-specific conjugation. Bioconjug Chem. 2014;25 Caddick S, et al. A plug-and-play approach to antibody-based thera- (3):460–469. doi:10.1021/bc400245v. peutics via a chemoselective dual click strategy. Nat Commun. 2015 21. Shinmi D, Taguchi E, Iwano J, Yamaguchi T, Masuda K, Mar 31;6:6645. doi:10.1038/ncomms7645. PMID: 25824906. Enokizono J, Shiraishi Y. One-step conjugation method for site- 3. Panowski S, Bhakta S, Raab H, Polakis P, Junutula JR. Site-specific specific antibody–drug conjugates through reactive cysteine- antibody drug conjugates for cancer therapy. MAbs. 2014 Jan-Feb engineered antibodies. Bioconjug Chem. 2016;27(5):1324–1331. ;6(1):34–45. doi:10.4161/mabs.27022. PMID: 24423619. doi:10.1021/acs.bioconjchem.6b00133. 4. Storz U. Antibody-drug conjugates: intellectual property 22. Feige MJ, Hendershot LM, Buchner J. How antibodies fold. Trends considerations. mAbs. 2015 7;6:989–1009. doi:10.1080/ Biochem Sci. 2010;35(4):189–98. doi:10.1016/j.tibs.2009.11.005. 19420862.2015.1082019. 23. Miret RRJ, Roura A, Moreno C, Arboix G, Farràs M, Cancelliere D, Di 5. Jackson DY. Processes for constructing homogeneous antibody Gesù C, Casablancas A, Lecina M, Cairó JJ, “PO276 comparison of drug conjugates. Org Process Res Dev. 2016:205852–866. bicistronic and tricistonic expression strategies for trastuzumab and doi:10.1021/acs.oprd.6b00067. trastuzumab-interferon-a2b production in CHO and HEK293 cells; 6. Grawunder U, Barth S. Next generation antibody drug conjugates 2017; 25th ESACT meeting in Lausanne. (ADCs) and immunotoxins. Chapter 5 Enzyme-based strategies to 24. Lyon R, Setter J, Bovee T, Doronina SO, Hunter JH, generate site-specifically conjugated antibody drug conjugates. Anderson ME, Balasubramanian CL, Duniho SM, Leiske CI, Springer; 2017. Li F, et al. Self-hydrolyzing maleimides improve the stability and 7. Kline T, Steiner AR, Penta K, Sato AK, Hallam TJ, Yin G. pharmacological properties of antibody-dug-conjugates. Nat Methods to make homogenous antibody drug conjugates. Pharm Biotechnol. 2014;32:1059–1062. doi:10.1038/nbt.2968. Res. 2015;32(11):3480–93. doi:10.1007/s11095-014-1596-8. 25. Ehkirch A, D’Atri V, Rouviere F, Hernandez-Alba O, Goyon A, 8. Jain N, Smith SW, Ghone S, Tomczuk B. Current ADC linker Colas O, Sarrut M, Beck A, Guillarme D, Heinisch S, et al. An online chemistry. Pharm Res. 2015;32(11):3526–40. doi:10.1007/s11095-015- four-dimensional HIC×SEC-IM×MS methodology for proof-of- 1657-7. concept characterization of antibody drug conjugates. Anal Chem. 9. Sochaj AM, Świderska KW, Otlewski J. Current methods for the 2018;90(3):1578–1586. doi:10.1021/acs.analchem.7b02110. synthesis of homogeneous antibody–drug conjugates. Biotechnol 26. Said N, Gahoual R, Kuhn L, Beck A, Yannis-Nicolas F. Structural Adv. 2015;33(6):775–784. doi:10.1016/j.biotechadv.2015.05.001. characterization of antibody drug conjugate by a combination of 10. Agarwal P, Bertozzi CR. Site-specific antibody-drug conjugates: intact, middle-up and bottom-up techniques using sheathless the nexus of bioorthogonal chemistry, protein engineering, and capillary electrophoresis - Tandem mass spectrometry as drug development. Bioconjug Chem. 2015;26:176–192. nanoESI infusion platform and separation method. Anal Chim doi:10.1021/bc5004982. Acta. 2016;918:50–9. doi:10.1016/j.aca.2016.03.006. 11. Behrens CR, Liu B. Methods for site-specific drug conjugation to 27. Lyon RP, Bovee TD, Doronina SO, Burke PJ, Hunter JH, Neff- antibodies. mAbs. 2014;6:46–53. doi:10.4161/mabs.26632. LaFord HD, Jonas M, Anderson ME, Setter JR, Senter PD, et al. 12. Dan N, Setua S, Kashyap V, Khan S, Jaggi M, Yallapu M and Reducing hydrophobicity of homogeneous antibody-drug conju- Chauhan S. Antibody-drug conjugates for cancer therapy: chem- gates improves pharmacokinetics and therapeutic index. Nat istry to clinical implications. Pharmaceuticals (Basel). 2018;11 Biotechnol. 2015;33:733–735. doi:10.1038/nbt.3212. (2):32. doi:10.3390/ph11020032. 28. Burke PJ, Hamilton JZ, Jeffrey SC, Hunter JH, Doronina SO, 13. Bhakta S, Raab H, Junutula JR. Engineering THIOMABs for Okeley NM, Miyamoto JB, Anderson ME, Stone IJ, Ulrich ML, site-specific conjugation of thiol-reactive linkers. Methods Mol et al. Optimization of a PEGylated glucuronide- Biol. 2013;1045:189–203. monomethylauristatin E linker for antibody–drug conjugates. 14. Junutula J, Hans Erickson Rachana V, Ohri S, Bhakta H, Raab R, Mol Cancer Ther. 2017;16:116–123. doi:10.1158/1535-7163. Vandlen JJ, Erickson H, A high-throughput conjugation strategy for 29. Tsuchikama K, An Z. Antibody-drug conjugates: recent advances the selection of THIOMABTM antibodies with desired properties for in conjugation and linker chemistries. Protein Cell. 2018;9:33–46. antibody-drug conjugation; 2015; AACR 106th Annu. Meet. in doi:10.1007/s13238-016-0323-0. Philadelphia 30. Román R, Miret J, Scalia F, Casablancas A, Lecina M, Cairó JJ. 15. Junutula JR, Raab H, Clark S, Bhakta S, Leipold DD, Weir S, Chen Y, Enhancing heterologous protein expression and secretion in Simpson M, Tsai SP, Dennis MS, et al. Site-specific conjugation of HEK293 cells by means of combination of CMV promoter and a cytotoxic drug to an antibody improves the therapeutic index. Nat IFNα2 signal peptide. J Biotechnol. 2016;239:57–60. doi:10.1016/j. Biotechnol. 2008;26:925–932. doi:10.1038/nbt.1480. jbiotec.2016.10.005. 16. Kim EG, Kim KM. Strategies and advancement in antibody-drug 31. Wishart DS, Feunang YD, Guo AC, Lo EJ, Marcu A, Grant JR, conjugate optimization for targeted cancer therapeutics. Sajed T, Johnson D, Li C, Sayeeda Z, et al. DrugBank 5.0: Biomolecules Ther. 2015;23(6):493–509. doi:10.4062/ a major update to the DrugBank database for 2018. Nucleic biomolther.2015.116. Acids Res. 2018;46(D1):D1074–D1082. doi:10.1093/nar/ 17. Behrens CR, Ha EH, Chinn LL, Bowers S, Probst G, Fitch-Bruhns gkx1037. M, Monteon J, Valdiosera A, Bermudez A, Liao-Chan S, et al. 32. Haryadi R, Ho S, Kok YJ, Pu HX, Zheng L, Pereira NA, Li B, Bi X, Antibody–drug conjugates (ADCs) derived from interchain Goh L-T, Yang Y, et al. Optimization of heavy chain and light cysteine cross-linking demonstrate improved homogeneity and chain signal peptides for high level expression of therapeutic other pharmacological properties over conventional heteroge- antibodies in CHO cells. PLoS One. 2015 Feb 23;10(2):e0116878. neous ADCs. Mol Pharm. 2015;12(11):3986–3998. doi:10.1021/ doi:10.1371/journal.pone.0116878. acs.molpharmaceut.5b00432. 33. Chin JX, Chung BKS, Lee DY. Codon Optimization OnLine 18. Dennler P, Fischer E, Schibli R. Antibody conjugates: from het- (COOL): a web-based multi-objective optimization platform for erogeneous populations to defined reagents. Antibodies. synthetic gene design. Bioinformatics. 2014;30(15):2210–2212. 2015;4:197–224. doi:10.3390/antib4030197. doi:10.1093/bioinformatics/btu192.