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In Vitro and In Vivo Evaluation of Cysteine Rebridged TrastuzumabMMAE Antibody Drug Conjugates With Defined DARs Antony Godwin, Penny Bryant, Martin Pabst, George Badescu, Matthew Bird, William McDowell, Estera Jamieson, Julia Swierkosz, Kosma Jurlewicz, Rita Tommasi, Korinna Henseleit, XiaoBo Sheng, Nicolas Camper, Anais Manin, Kasia Kozakowska, Karolina Peciak, Emmanuelle Laurine, Ruslan Grygorash, Andrew Kyle, David Morris, Vimal Parekh, Amrita Abhilash, Ji-won Choi, Jeff Edwards, Mark Frigerio, and Matthew Paul Baker Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00116 • Publication Date (Web): 20 Apr 2015 Downloaded from http://pubs.acs.org on April 26, 2015

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Molecular Pharmaceutics

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In Vitro and In Vivo Evaluation of Cysteine Rebridged TrastuzumabMMAE Antibody Drug Conjugates With Defined DARs

Penny Bryant1, Martin Pabst1, George Badescu1, Matthew Bird1, William McDowell1, Estera Jamieson1, Julia Swierkosz1, Kosma Jurlewicz1, Rita Tommasi1, Korinna Henseleit1, XiaoBo Sheng1, Nicolas Camper1, Anais Manin2, Kasia Kozakowska1, Karolina Peciak1, Emmanuelle Laurine1, Ruslan Grygorash1, Andrew Kyle1, David Morris1, Vimal Parekh1, Amrita Abhilash1, Ji-won Choi1, Jeff Edwards1, Mark Frigerio1, Matthew P. Baker1,2, Antony Godwin1* 1

PolyTherics Ltd. (an Abzena company), Babraham Research Campus, CB22 3AT, Cambridge, United Kingdom

2

Antitope Ltd. (an Abzena company), Babraham Research Campus, CB22 3AT, Cambridge, United Kingdom *Correspondence to: [email protected]

The conjugation of monomethyl auristatin E (MMAE) to trastuzumab using a reduction bisalkylation approach that is capable of rebridging reduced (native) antibody interchain disulfide bonds has been previously shown to produce a homogeneous and stable conjugate with a drug to antibody ratio (DAR) of 4 as the major product. Here, we further investigate the potency of the DAR 4 conjugates prepared by bis-alkylation by comparing to lower drug loaded variants and to maleimide linker based conjugates possessing typical mixed DAR profiles. Serum stability, HER2 receptor binding, internalization, in vitro cytotoxicity and in vivo efficacy were all evaluated. Greater stability compared with maleimide conjugation was observed with no significant decrease in receptor/FcRn binding. A clear dose-response was obtained based on drug loading (DAR) with the DAR 4 conjugate showing the highest potency in vitro and a much higher efficacy in vivo compared with the lower DAR conjugates. Finally, the DAR 4 conjugate demonstrated superior efficacy compared to Trastuzumab-DM1 (T-DM1, Kadcyla®), ABSTRACT.

as evaluated in a low HER2 expressing JIMT-1 xenograft model. Key words: Antibody drug conjugates (ADC), drug loading, disulfide rebridging, bis-sulfone, bis-alkylation, trastuzumab, auristatin, MMAE, JIMT-1 xenograft model.

INTRODUCTION. The specificity of monoclonal antibodies (mAbs) to surface markers on target cells has led to their use as effective new treatments for cancer.1, 2 Trastuzumab (TRA) is considered as a standard of care both in early and metastatic human epidermal growth factor receptor 2 (HER2) over-expressing breast cancer. Around 20% of breast cancer cases overexpress HER2, however, many patients ultimately show resistance to TRA and therefore disease progression during treatment.3, 4 The properties of TRA and other mAbs have been further exploited by their covalent conjugation to highly potent cytotoxic drugs to produce antibody drug conjugates (ADCs). ADCs enhance the antibody’s overall potency and also may address issues due to developed resistance to the mAb.5, 6 ADCs are designed to selectively deliver highly potent cytotoxic drugs to tumors and minimize systemic toxicity caused by exposure of the drugs to healthy tissue.6 Both currently approved 1


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ADCs use linkers that are reactive towards either the amino groups in the side chains of lysine residues, as in the case of adotrastuzumab emtansine (T-DM1, ® 7 Kadcyla ), or to accessible thiol side chains from cysteines created from reduction of interchain disulfide bonds, as is the case with brentuximab vedotin (Adcetris®).8 These approaches necessarily result in heterogeneous mixtures comprising multiple positional isomers, significant amounts of unconjugated mAb and conjugates with sub-optimal drug to antibody ratio (DAR) of greater than 4.9 Highly drug loaded antibodies show an increased hydrophobicity and tend therefore to clear very quickly from circulation as well as appear to be less well tolerated.10, 11 Overall, the higher loaded species provide no further improvement in efficacy over the lower DAR variants.12 Conjugates with a higher MMAE drug load were also reported in a recent study to be less stable and more prone to aggregation than the corresponding conjugates with lower drug loads.13 Ideally therefore, the drug to antibody linking strategy should avoid excessive or unspecific drug loading as well as prevent the generation of heterogeneous mixtures and be efficient in terms of antibody and reagent requirements.14, 15 The ideal linker chemistry is also extremely stable in circulation and allows release of the drug only upon reaching the target cancer cell. Auristatins and maytansinoids, that are currently extensively used to produce ADCs, are extremely toxic and are not suitable as standalone chemotherapeutic drugs.6, 16, 17 Release of these payloads from an ADC prior to reaching the tumor therefore can result in decreased tolerability.12 In addition, the unconjugated antibody binds competitively to the tumor cells, reducing efficacy by blocking binding

sites for the intact ADCs. The classical maleimide chemistry widely used for conjugating at reduced interchain disulfide bonds is only capable of mono-alkylation, leaving the disulfide bond unbridged and potentially introducing structural instability to the antibody. Maleimide based ADCs have also been shown to be capable of releasing the payload in serum, which may reduce overall efficacy by lowering the DAR as well as introduce the potential for increased off-site toxicity. In vivo the released maleimide linker payload can reconjugate to the reactive free cysteine (Cys34) of serum albumin14, 18 and potentially also to other thiol containing species. Recently maleimide conjugates with improved stability were introduced by Lyon et al. through self-hydrolyzing maleimide derivatives and by Tumey et al. through developing a mild method for selective hydrolysis of the succinimidethioether.15, 19 Those ADCs with a “ringopened” linker were found to possess improved stability and pharmacological properties although the typically high DAR heterogeneity obtained with maleimide chemistry remains unaddressed.15, 19 The use of engineered mAbs has gained interest as an approach to overcome the issues of both heterogeneity and, depending on the chemistry employed, instability. THIOMABS, for example, are mAbs that have been engineered to contain a defined number of unpaired cysteine residues, typically one on each heavy or light chain, specifically for conjugation of a cytotoxic payload with a final DAR close to 2.20, 21 While this strategy overcomes issues of drug loading heterogeneity, it may not be an appropriate strategy where the optimal drug loading is greater than 2. Drug conjugation can also be achieved by enzymatic conjugation approaches, such as with 2



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microbial transglutaminase (MTGase) which forms an isopeptidic bond between the amino acids glutamine and lysine. MTGase selectively recognises Gln295 from the IgG heavy chain as a substrate and therefore can produce a homogeneous conjugate with a DAR of 2, 22, 23 and also higher DARs with additional engineering of Gln residues. An alternative approach uses protein re-engineering to incorporate nonnatural amino acids into the antibody as sites for conjugation.10, 11, 22, 24 The nonnatural amino acids have functionalities that can be targeted using orthogonal conjugation chemistries that avoid native amino acids. This approach provides a promising alternative to production of homogeneous and potentially stable ADCs. Re-engineering antibodies requires additional development time and the sites of conjugation may need to be optimised by experimentation in vivo for optimal performance. Some of the conjugation chemistries employed may be less efficient than thiol based conjugation with some reported methods requiring large excesses of reagent and long conjugation times for sufficient reaction.24 Our interests are in addressing the issues of ADC heterogeneity and instability without having to reengineer the antibody for site-specific conjugation. We have previously reported the use of a bis-sulfone based linker that can be used to produce stable and homogeneous ADCs by specifically targeting the accessible interchain disulfide bonds of the native antibody after disulfide reduction. The linker consisted of a bis-alkylating bissulfone group, a cathepsin cleavable valine citrulline p-aminobenzyl ether linker, and MMAE.9 This approach was used to successfully attach the cytotoxic drug to the four interchain disulfides of TRA generating an ADC (TRA-bisAlk-vc-

MMAE), with DAR 4 as the major product (≥ 78%) and a narrow DAR profile. The mAb to payload linkage produced using this reagent was demonstrated to be stable in the presence of free thiols and in sera of different species. To further evaluate the DAR 4 conjugate produced by the reduction bis-alkylation approach, we modified the conjugation conditions to prepare sufficient amounts of lower loaded species with defined DARs of 1, 2 and 3 for comparison. Serum stability, HER2 receptor binding, internalization, in vitro cytotoxicity and in vivo efficacy in a BT474 xenograft model were evaluated for each single DAR conjugate. Comparisons were also made with maleimide based MMAE conjugates possessing either a typical maleimide mixed DAR profile or a purified DAR 4 profile. Finally, a JIMT-1 mouse xenograft study was performed showing good efficacy of the MMAE DAR 4 conjugate. MATERIALS AND METHODS Materials. Maleimide-val-cit-PABMMAE was purchased from Concortis Biosystems. PEG precursors were purchased from IRIS Biotech Gmbh. All other reagents and solvents were purchased from Fisher Scientific, Acros Organics or Sigma-Aldrich and used as received. All cell lines were purchased from the American Type Culture Collection (ATCC), except for A549 which was purchased from the Health Protection Agency Culture Collection (HPACC) and the JIMT-1 cells which were sourced from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen). Cells were maintained in McCoy’s 5a (SK-BR3), DMEM:F12 (BT-474, JIMT-1), Dulbecco’s modified Eagle medium (DMEM) supplemented with 200 mM glutamine (A549), or Minimum Essential 3


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Medium (MEM) supplemented with 10 μg/mL insulin (MCF-7). All media were complemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. All media were obtained from Life Technologies. Cell lines were grown at 37 °C and 5% CO2 in a CO2 air-jacket incubator (Binder). TRA (Herceptin) and T-DM1 (Kadcyla®) were supplied from a pharmacy for research purposes. The extracellular domain of the HER2 receptor (ECD/HER2) was purchased from Stratech Scientific Ltd., UK. Nunc Maxisorp microtiter plates were purchased from Thermo Scientific. Anti 6xHis antibody conjugated to HRP was purchased from Clontech Laboratories Inc. Reagent Synthesis. Synthesis of the bisalkylating reagent was carried out using the methods described previously.9 The reagent comprised either a 6 unit or 24 unit PEG, followed by a cathepsin B cleavable val-cit PAB linker (-vc-) to MMAE (Figure 1). Conjugation of MMAE to TRA using the bis-alkylating reagent. Typically, to a solution of TRA (5.2 mg/mL, 20 mg TRA) in reaction buffer (20 mM sodium phosphate, pH 7.5, 150 mM NaCl, 20 mM EDTA), TCEP was added (6 equivalents with respect to mAb) and the resulting mixture was incubated at 40 °C for 1 h. The reduced TRA was cooled to room temperature and diluted to 4.4 mg/mL with reaction buffer. MMAE reagent was dissolved in MeCN (3.2 mM). The reagent (6 equivalents with respect to mAb, 5% MeCN v/v) was added to the reduced TRA solution and the resulting conjugation reaction was carefully mixed and incubated at 22 °C for 22 h. After this, 50 mM Nacetyl-L-cysteine was added (3.0 mM) and the resulting mixture was incubated for a further 1 h at 22 °C. Finally, the conjugation mixture was subjected to purification by

preparative HIC to generate a single purified DAR 4 species. Lower DAR variants were produced by HIC and SEC purification from a larger scale conjugation (250 mg TRA), where the reaction was stopped with an average DAR of 2.3. The reaction mixture was buffer exchanged by gel filtration to remove any unreacted reagent and then treated with 10 mM dehydroascorbic acid (DHA) for 1 h at room temperature. Conjugation using maleimide reagent. Maleimide-val-cit-PAB-MMAE was purchased from Concortis Biosystems and TRA-mc-vc-MMAE conjugates were finally prepared as described previously in Badescu et al., 2014. 9 Preparative HIC. TRA-bisAlk-vc-MMAE and TRA-mc-vc-MMAE conjugates were purified by HIC using ToyoPearl Phenyl650S columns attached to an ÄKTA Prime system. Conjugates were eluted by either a step or a linear gradient from 100% buffer A (50 mM sodium phosphate pH 7.0, 2 M NaCl) to 100% buffer B (50 mM sodium phosphate pH 7.0, 20% isopropanol v/v). Fractions were analysed by analytical HIC and pooled to obtain pure DAR species with greater than 95% purity. Characterization of individual DAR variants. The extent of drug loading was based on measuring the ratio of absorbance at 248 nm relative to 280 nm (Figure 2b), which corresponded to maxima for the drug and the antibody components 12 respectively. The purities of the individual DAR variants were determined by analytical HIC (Figure 2a) and further analysed by SEC for the presence of aggregates as described previously, in Badescu et al., 2014.9 Finally, isolation of the DAR 4 conjugate was also confirmed by

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mass spectrometry using a Waters Q-TOF mass spectrometer (S.I. Figure 1). Stability in the presence of free thiol containing HSA. Stability in the presence of free thiols was analysed using a previously described method.26 Briefly, TRA-bisAlk-vc-MMAE DAR 4 and TRAmc-vc-MMAE DAR 4 conjugates were diluted with 50% IgG depleted serum (SCIPAC # SF142-2) at a final concentration of 1 mg/mL in sterile tubes. Sodium azide was added (final concentration 1 mM) then the mixtures were split into 10 equal aliquots. Aliquots were removed from the incubator after 0, 24, 48, 96 and 168 h and transferred to a 80 °C freezer. After the final time point, mixtures were analysed by analytical HIC using a ProPac® 2.1 mm × 100 mm HIC10 column (Fisher Scientific) connected to a Dionex Ultimate 3000RS HPLC system. The method consisted of a linear gradient from 100% buffer A (50 mM sodium phosphate pH 7.0 containing 1.5 M ammonium sulfate) to 100% buffer B (50 mM sodium phosphate pH 7.0, 20% isopropanol) over 60 min. The flow rate was 0.2 mL/min and the temperature was maintained at 30 °C throughout the analysis. Detection was carried out by following UV absorption at 280 nm. An aliquot of 40 µg of ADC was used per analysis. HER2 ELISA. The affinity of the binding between the extracellular domain of the HER2 receptor (ECD/HER2) and TRA and TRA-bisAlk-vc-MMAE conjugates with a DAR of 1, 2, 3 and 4 was carried out using previously described methods.9 FcRn binding assay. Recombinant human FCGRT His tag protein (Biorbyt Limited, orb 84388) (50 μg) was re-suspended in sterile PBS pH 7.4 to a final concentration of 0.2 mg/mL. An aliquot (25 μg) was then biotinylated using EZ-Link® Micro NHS-

PEG4-biotinylation Kit (Pierce Biotechnology #21955, Rockford, USA) using 100 equivalents of biotin reagent. HER2 extracellular domain receptor (HER2 ECD; Sino Biological Inc., Stratech Scientific Ltd, UK) was used to coat 96well microtiter plates (Maxisorp, Thermo Scientific nunc) at a concentration of 2 µg/mL diluted in coating buffer (0.05 M carbonate/bicarbonate buffer, pH 9.6) overnight at 4 °C. Following incubation, the plates were washed with washing buffer (PBS, 0.05% Tween® 20) and blocked with blocking buffer (1.5% (w/v) BSA in PBS, 0.05% Tween 20) for 1 h at room temperature. The plates were then incubated with TRA (13.3 - 5.1 × 10-3 nM) and TRA-bisAlk-vc-MMAE ADCs (20.0 1.0 × 10-3 nM) serially diluted in diluent (0.1% (w/v) BSA in PBS, 0.05% Tween 20) for 3 h at room temperature. Following the incubation, the plates were washed with PBS - 0.05% Tween® 20 pH 6.0 and incubated for 2 h at room temperature with biotinylated FCGRT His tag protein (0.2 μg/mL in PBS containing 0.1% (w/v) BSA, 0.05% Tween 20, pH 6). The plates were then washed with washing buffer and incubated with streptavidin conjugated to horse radish peroxidase (HRP) (GE Healthcare; RPN1231VS) in diluent buffer, pH 6.0. Following incubation for 1 h at room temperature, the plates were washed again and the binding of HRP conjugated streptavidin to the biotinylated FcRn bound to TRA and ADCs compounds was detected with tetramethyl benzidine substrate (Sigma Aldrich; T0440/100). The reaction was stopped with stop reagent for TMB substrate (Sigma Aldrich; S 5689) and the absorbance was read at 630 nm using a Spectramax M3 microplate reader. The data was analysed with Graphpad Prism 5 software using ‘One site-Specific binding with hill slope’ fit.


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Cell viability assays (Cell Titer Glo®). The in vitro activity of TRA-bisAlk-vcMMAE DAR 1-4 using SK-BR-3 cells and TRA-bisAlk-vc-MMAE DAR 4, Kadcyla and Trastuzumab in JIMT-1 cells was assessed by Cell Titer Glo® luminescence assay (Promega) as described previously.9 In-vivo efficacy studies. Efficacy against BT-474 human breast carcinoma. Xenografts were initiated in female severe combined immunodeficient mice with BT-474 human breast carcinomas maintained by serial subcutaneous transplantation (Fox Chase SCID®, C.B-17/Icr-Prkdcscid, Charles River Laboratories). Each test mouse received a 1 mm3 BT-474 tumor fragment implanted subcutaneously in the right flank. At a mean tumor volume of 100 mm3, mice were randomized into treatment groups (n = 10), and dosing was initiated. All agents were administered intravenously (i.v.) into the tail vein in a dosing volume of 200 µL per 20 g of body weight (based on 10 mL/kg), scaled to the body weight of the individual animal. The treatment schedule was once per day on days 1, 8 and 15. Tumors were measured in two dimensions using calipers twice per week (for tumor size calculations, see formula below). Efficacy against JIMT-1 human breast carcinoma. Female NMRI nude mice (Janvier, Le Genest St Isle, France) were inoculated subcutaneously with 5 x 106 JIMT-1 tumor cells into their right flanks. At a mean tumor volume of 150 mm3 animals were randomized into groups (n = 10) according their tumor sizes and treatment was initiated (Heidelberg Pharma GmbH, 68526 Ladenburg, Germany). For a 30 g mouse a test substance volume of 300 µL was applied (based on 10 mL/kg) by intravenous injection to the tail vein. The total conjugate amount administered was either 5 mg conjugate per kg mouse body

weight or 10 mg conjugate per kg mouse body weight. A 6 unit PEG was used in the reagent to prepare the conjugate as in vitro the ADC had equivalent potency to the 24 unit PEG conjugate. The complete study was performed with a single dose injection. Tumor volumes were measured twice per week by caliper measurements (for tumor size calculations, see formula below). 𝑇𝑢𝑚𝑜𝑟 𝑣𝑜𝑙𝑢𝑚𝑒 (𝑚𝑚3 ) =

𝑤2 × 𝑙 2

Where w = width and l = length of the tumor, in mm. RESULTS Analytical Characterisation of individual DAR species. Each of the DAR species were produced in greater than 95% purity as determined by HIC, SEC and MS. Figure 2b shows the hydrophobic interaction chromatography analysis for the purified DAR species. The drug to antibody ratio was further confirmed by monitoring the adsorption at 248 and 280 nm respectively. Only small amounts of DAR 2 and 3 were present in the DAR 3 and DAR 4 samples respectively. Size exclusion chromatography (SEC) revealed approximately 0.5% and 2.0% aggregation in the DAR 1 and DAR 3 samples respectively. No aggregation was observed for the DAR 2 and for the DAR 4 variants. DAR 4 identity was further confirmed using mass spectrometry. See supplemental information for spectra and chromatograms. Stability of purified TRA-bisAlk-vcMMAE DAR 4 ADC. The stability of DAR 4 TRA-bisAlk-vc-MMAE ADC was first compared with a purified, homogenous DAR 4 TRA-mc-vc-MMAE in the presence of the free thiol containing protein, human serum albumin (HSA). Samples were incubated at 37 ºC in HSA solution and changes in DAR distribution over time 6



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were monitored by HIC. Similar to results seen previously with a mixed DAR9, minimal degradation was observed for the bisAlk DAR 4 conjugate after 120 h as the average was measured at 3.9. Conversely, the HIC profile of the maleimide conjugate changed with time with the average DAR falling to below 3.5 after 120 h, indicating loss of MMAE (Figure 3). In addition, we tested the serum stability of both TRAbisAlk-vc-MMAE DAR 4 and TRA-mcvc-MMAE DAR 4, by incubating in IgG depleted human serum. Changes in the DAR profile were then analysed by HIC. IgG depleted human serum was used in place of whole serum in order to avoid chromatographic interference of endogenous IgG type antibodies during the analysis. A HIC analysis method was chosen where the majority of the serum derived proteins eluted before the unconjugated antibody. More hydrophobic species, i.e., the TRA-bisAlk-vc-MMAE and TRA-mc-vc-MMAE eluted later, between 30 and 60 min respectively. There was no significant change in the peak intensity for DAR 4 TRA-bisAlk-vcMMAE after 120 h and also no new peaks that could be identified as degradation products were observed. At time zero, the maleimide DAR 4 conjugate was also observed as a single peak. However, unlike for the TRA-bisAlk-vc-MMAE conjugate, the peak corresponding to the intact TRAmc-vc-MMAE was reduced by approximately 35% in area after 120 h. In addition, a series of new peaks appeared in the chromatogram, several of which had corresponding elution times as the purified lower DAR and free mAb standards (Figure 4). In vitro binding of DAR 1 to 4 variants to HER2. The binding of purified TRAbisAlk-vc-MMAE individual DAR variants (DARs 1 to 4) to the extracellular domain of ECD/HER2 was compared in a direct

binding ELISA. Each of the purified DAR 1, 2, 3 and 4 ADCs retained antigen binding compared to the parent mAb and there were no significant differences between the different DAR conjugates (Figure 2a). The effect of conjugation of MMAE on FcRn binding was also investigated. The FcRn binding assay made use of a heterodimer consisting of FcRn ECD and the β2microglobulin domain. Binding to this heterodimer was measured in a direct binding ELISA. FcRn binding was not found to be affected by conjugation using TRA-bisAlk-vc-MMAE and increasing DAR did not have a noticeable effect (Figure 2b). In vitro cytotoxicity of DAR 1 to 4 variants. The in vitro cytotoxicity of the conjugates against HER2-positive human breast cancer cell line SK-BR-3 was tested using a Cell Titre Glo® assay. The concentration range of each DAR ADC species tested in the assay was matched by the amount of MMAE. When results were plotted as a function of drug concentration (Figure 5a) similar potencies were observed for all DAR species. The data were also plotted as a function of ADC concentration (Figure 5b). In this case, a trend of increasing potency with increased drug loading was observed. The lowest IC50 of 0.04 nM was determined for the DAR 4 conjugate, while the DAR 1 variant was least active with an IC50 of 0.12 nM (Figure 4c). The cytotoxicity of TRA-bisAlk-vcMMAE DAR 4 conjugate was further compared to T-DM1 (Kadcyla®) using the HER2 low expression cell line JIMT-1. Cell proliferation was measured by means of Cell Titer Glo® viability assay after 96 h in the presence of conjugates and unconjugated TRA. The TRA-bisAlk-vcMMAE conjugate inhibited proliferation of JIMT-1 cells in a dose dependent manner 7


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with an IC50 value of 0.1 nM. In comparison, Kadcyla® showed an IC50 value of 4.7 nM and unconjugated TRA showed no effect on cell viability (S.I. Figure 2). In vivo efficacy of DAR 1 to 4 variants in a BT-474 xenograft model. The effect of changes in drug loading on the ability of the TRA-bisAlk-vc-MMAE conjugates to inhibit tumor growth in vivo was evaluated in a BT-474 breast cancer xenograft model. Purified rebridged conjugates with a DAR of 1, 2, 3 and 4 were administered to female CB.17 SCID mice bearing subcutaneous BT-474 tumor xenografts. Each DAR conjugate was injected at a dose based on the total mAb content. Dosing was carried out weekly with up to three doses. The DAR 1 and DAR 2 conjugates produced measurable tumor growth delays (TGDs) of 14.4 d and 16.9 d respectively (Figure 6a), but no significant survival difference from controls (P > 0.05). Treatment with the DAR 3 and DAR 4 conjugates each produced the maximum TGD (26.1 d, 75%), and a significant survival difference versus controls (P < 0.001). Differences in regression responses were observed for the higher DAR regimens. Treatment with DAR 3 conjugate produced three partial regressions, whereas DAR 4 conjugate was the most active ADC and elicited 10/10 tumor free survivors. All ADCs were well tolerated. No treatment related or nontreatment related deaths were recorded in the study. Declining body weights (BW) were observed in all groups, including controls, over the course of the study (Figure 6b). There was no significant difference in changes in body weight between different DAR species. The declining BWs observed in the study were likely SCID mouse strain-related rather than treatment-related based on the weight loss in the control group.

In vivo efficacy of TRA-bisAlk-vcMMAE DAR 4 in a JIMT-1 xenograft model. We further evaluated the in vivo efficacy of TRA-bisAlk-vc-MMAE DAR 4 in a HER2 low expressing JIMT-1 human breast cancer xenograft mouse model. This model was used as it is difficult to treat with an ADC and would potentially allow any efficacy differentiation between the different conjugates in the study to be seen.25 Two different single dose regimens at 5 mg/kg and 10 mg/kg were performed. At 5 mg/kg, similar efficacy was seen to TDM1 (Figure 6 and S.I. Figure 3). However, at the higher 10 mg/kg dose, there was an improved tumor response, which was not observed for Kadcyla®. DISCUSSION. We have previously shown that bis-sulfone based bis-alkylation reagents can be used for conjugation at reduced disulfide bonds as a stable alternative to maleimide chemistry. These reagents conjugate at native antibody interchain disulfides and yield ADCs with a very narrow DAR distribution containing DAR 4 as the major product. The intrachain disulfides of the mAb do not conjugate as these disulfides are not as readily reduced and are not accessible to the bis-sulfone linker. 26, 27 Consequently, no cross-linked mAb is observed in the ADC reaction mixtures. To further characterise the TRA-bisAlk-vcMMAE ADC and investigate the effect of drug loading, conjugates with a uniform DAR of 1, 2, 3 and 4 were isolated. First, the effect of drug loading on binding to the HER2 receptor was investigated. The results of a direct binding ELISA showed that conjugation of 1, 2, 3 or 4 MMAE molecules had no impact on HER2 binding as binding was similar to native TRA. No significant impact on FcRn binding due to drug loading was observed compared to TRA. These results indicate that the sites of 8



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conjugation, i.e., at the interchain disulfides, are very tolerant of derivatization by bis-alkylation and the antibody is not functionally perturbed or sterically inhibited by conjugation. The DAR 4 conjugate formed using the bisalkylating reagent was found to be stable in the presence of albumin concentrations close to physiological conditions. A comparative study with a maleimidederived conjugate showed the maleimide conjugate was less stable, with a significant decrease in average DAR over time. Although high drug loads correlate with increased potency in vitro, it is known that excessive drug loading can result in reduced efficacy in vivo. A study by Shen et al. has shown that more stable ADCs attain better efficacy and improved survival rates within in vivo cancer models18 Antibody-doxorubicin conjugates have also been shown to display a clear correlation between drug loading and cell cytotoxicity,28, 29 however, the more highly loaded conjugates cleared faster from circulation.12 Here, we investigated the influence of the drug loading specifically for highly stable conjugates produced by a bis-alkylating strategy. Thereby, we found an in vitro potency directly correlating with the drug loading when results of an in vitro cell viability assay were plotted against ADC concentration. However, when results were normalised based on MMAE concentration no significant difference was observed between different DARs. This indicates that binding, internalization and drug release are not affected by increased drug conjugation using the bis-alkylating reagent. All DAR species were found to be well tolerated in vivo. In a BT-474 mouse xenograft study the TRA-bisAlk-vcMMAE conjugates demonstrated an increase in efficacy with increasing DAR. The lower DAR species (1 and 2) were found to have minimal activity whereas the

higher DAR species (3 and 4) demonstrated the best efficacy (Figure 6). The DAR 4 conjugate resulted in 100% tumor-free survival. The site of conjugation has been shown to be a factor that can influence the PK and therefore efficacy of an ADC.18 However, in this study, drug location and drug loading would not appear to influence the PK to such an extent to affect the order of efficacy of DAR 1 to DAR 4 (Figure 6). This order also correlated strongly with the observed in vitro potencies. In a single dose JIMT-1 xenograft study, we evaluted the efficacy of the DAR 4 conjugate in a low HER2 expressing cell line observing a strong tumour response at 10 mg/kg (Figure 7). T-DM1 was used a comparator ADC in the study, which showed a weak tumour response at the same 10 mg/kg dose. While there are clear limitations in comparing the maytansine based ADC, T-DM1, with an MMAE based conjugate using a different linker system, the results indicate the potential of the bis-akylation approach to produce efficacious ADCs.

ADCs have begun to have a tremendous impact on the treatment of cancer. A current limitation in ADC development is that much effort and time is needed to fully optimise the combination of antibody, linker and drug. New linker strategies are required that ensure more homogeneous and stable ADCs can be produced with more predictable in vivo behaviour without the need for extensive re-optimization, especially if one component of the ADC is changed. As the major product of the reduction bisalkylation approach is a DAR 4 ADC conjugated at the interchain disulfides, different payloads and release mechanisms can be evaluated while keeping the site of conjugation and DAR profile consistent. 9


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This allows efficient screening of new ADCs without the need for simultaneous re-engineering of the antibody.

ACKNOWLEDGEMENT. We thank our PolyTherics and Antitope colleagues for their help and advice during this study.

In summary, our results show that bisalkylation conjugation at reduced interchain disulfides is a promising approach to produce stable and more homogeneous ADCs without the need to reengineer the antibody for conjugation.

SUPPORTING INFORMATION AVAILABLE. This information is available free of charge via the Internet at http://pubs.acs.org/

CORRESPONDENCE TO: [email protected]. CONFLICT OF INTEREST. The authors declare no competing ﬁnancial interest.



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23. Dennler, P.; Chiotellis, A.; Fischer, E.; Bregeon, D.; Belmant, C.; Gauthier, L.; Lhospice, F.; Romagne, F.; Schibli, R., Transglutaminase-based chemo-enzymatic conjugation approach yields homogeneous antibody-drug conjugates. Bioconjugate chemistry 2014, 25, 569-78. 24. Axup, J. Y.; Bajjuri, K. M.; Ritland, M.; Hutchins, B. M.; Kim, C. H.; Kazane, S. A.; Halder, R.; Forsyth, J. S.; Santidrian, A. F.; Stafin, K.; Lu, Y.; Tran, H.; Seller, A. J.; Biroc, S. L.; Szydlik, A.; Pinkstaff, J. K.; Tian, F.; Sinha, S. C.; Felding-Habermann, B.; Smider, V. V.; Schultz, P. G., Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proceedings of the National Academy of Sciences of the United States of America 2012, 109, 16101-6. 25. Koninki, K.; Barok, M.; Tanner, M.; Staff, S.; Pitkanen, J.; Hemmila, P.; Ilvesaro, J.; Isola, J., Multiple molecular mechanisms underlying trastuzumab and lapatinib resistance in JIMT-1 breast cancer cells. Cancer letters 2010, 294, 211-9. 26. Liu, H.; Chumsae, C.; Gaza-Bulseco, G.; Hurkmans, K.; Radziejewski, C. H., Ranking the susceptibility of disulfide bonds in human IgG1 antibodies by reduction, differential alkylation, and LC-MS analysis. Analytical chemistry 2010, 82, 5219-26. 27. Liu, H.; May, K., Disulfide bond structures of IgG molecules: structural variations, chemical modifications and possible impacts to stability and biological function. mAbs 2012, 4, 17-23. 28. King, H. D.; Yurgaitis, D.; Willner, D.; Firestone, R. A.; Yang, M. B.; Lasch, S. J.; Hellstrom, K. E.; Trail, P. A., Monoclonal antibody conjugates of doxorubicin prepared with branched linkers: A novel method for increasing the potency of doxorubicin immunoconjugates. Bioconjugate chemistry 1999, 10, 279-88. 29. Firestone, R. A.; Willner, D.; Hofstead, S. J.; King, H. D.; Kaneko, T.; Braslawsky, G. R.; Greenfield, R. S.; Trail, P. A.; Lasch, S. J.; Henderson, A. J.; Casazza, A. M.; Hellström, I.; Hellström, K. E., Synthesis and antitumor activity of the immunoconjugate BR96-Dox. Journal of Controlled Release 1996, 39, 251-259.



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GRAPHICAL ABSTRACT


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FIGURES

Figure 1. Structure of the bisAlk-vc-MMAE reagent.

Figure 2. Characteristaion of individual DAR 1 to 4 variants by (A) Hydrophobic interaction chromatography (HIC) (B) UV absorption, where absorption at 248 nm indicates a stepwise increase in drug loading (C) FcRn binding and (D) HER2 receptor binding.



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Figure 3. Stability of purified DAR 4 bisAlk (blue) and maleimide (red) conjugates in the presence of the free thiol containing human serum albumin, HSA.

Figure 4. Stability of DAR 4 conjugates incubated in IgG depleted serum. HIC chromatograms for (a) TRAbisAlk-vc-MMAE. (b) TRA-mc-vc-MMAE. Peaks marked with an asterisk correspond to degraded DAR products.


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Figure 5. In vitro potency of TRA-bisAlk-vc-MMAE ADCs with different drug loading (DAR 1 to 4) on SK-BR3 cells. Results were plotted based on (a) MMAE concentrations and (b) based on ADC concentration. IC50 values were 0.22 nM and 0.12 nM, 0.07 nM, 0.05 nM and 0.04 nM for MMAE, DAR 1, DAR 2, DAR 3 and the DAR 4 conjugate respectively.

Figure 6. Administration (20 mg/kg based on TRA, dosing on days 1, 8 and 15) of DAR 1 to 4 TRA-bisAlk-vcMMAE (PEG24 spacer) to SCID mice bearing BT-474 xenografts. (a) Changes in tumor volume and (b) Changes in body weight.



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Figure 7. Changes in average tumor volume of TRA-bisAlk-vc-MMAE (PEG6 spacer) in a JIMT-1 xenograft model. Single doses were administered at 5 and 10 mg/kg respectively.


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