Development and evaluation of β-galactosidase-sensitive antibody-drug conjugates

Accepted Manuscript Development and evaluation of β-galactosidase-sensitive antibody-drug conjugates Sergii Kolodych, Chloé Michel, Sébastien Delacroix, Oleksandr Koniev, Anthony Ehkirch, Jitka Eberova, Sarah Cianférani, Brigitte Renoux, Wojciech Krezel, Pauline Poinot, Christian D. Muller, Sébastien Papot, Alain Wagner PII:

S0223-5234(17)30611-6

DOI:

10.1016/j.ejmech.2017.08.008

Reference:

EJMECH 9652

To appear in:

European Journal of Medicinal Chemistry

Received Date: 28 June 2017 Revised Date:

25 July 2017

Accepted Date: 2 August 2017

Please cite this article as: S. Kolodych, Chloé. Michel, Sé. Delacroix, O. Koniev, A. Ehkirch, J. Eberova, S. Cianférani, B. Renoux, W. Krezel, P. Poinot, C.D. Muller, Sé. Papot, A. Wagner, Development and evaluation of β-galactosidase-sensitive antibody-drug conjugates, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.08.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Development and Evaluation of β-Galactosidase-Sensitive Antibody-Drug Conjugates.

Sergii Kolodych, Chloé Michel, Sébastien Delacroix, Oleksandr Koniev, Anthony Ehkirch,

AC C

EP

TE D

M AN U

SC

D. Muller, Sébastien Papot* and Alain Wagner.

RI PT

Jitka Eberova, Sarah Cianférani, Brigitte Renoux, Wojciech Krezel, Pauline Poinot, Christian

ACCEPTED MANUSCRIPT

Development and Evaluation of β-Galactosidase-Sensitive Antibody-Drug

RI PT

Conjugates. Sergii Kolodych,a Chloé Michel,a Sébastien Delacroix, a Oleksandr Koniev,a Anthony Ehkirch,a,b Jitka Eberova,c Sarah Cianférani,b Brigitte Renoux,d Wojciech Krezel,e,f,g,h,i Pauline Poinot,j

SC

Christian D. Muller,k Sébastien Papot*, d and Alain Wagnerc. Syndivia SAS, 650 Bd Gonthier d’Andernach, 67400 Illkirch, France.

b

Laboratoire de Spectrométrie de Masse BioOrganique, Université de Strasbourg, CNRS,

M AN U

a

IPHC UMR 7178, 67000 Strasbourg, France. c

Laboratory of Functional ChemoSystems (UMR 7199), Labex Medalis, University of

d

TE D

Strasbourg, France.

Université de Poitiers, UMR-CNRS 7285, Institut de Chimie des Milieux et des Matériaux

de Poitiers, groupe « Systèmes Moléculaires Programmés » (SMP), 4 Rue Michel Brunet,

EP

TSA 51106, 86022 Poitiers, France.

Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France.

f

Institut de la Santé et de la Recherche Médicale, U964, Illkirch, France.

g

Centre National de la Recherche Scientifique, Illkirch, France.

h

Université de Strasbourg, Illkirch, France.

AC C

e

i Fédération de Médecine Translationnelle de Strasbourg, Illkirch, France. j

Institut de Chimie des Milieux et des Matériaux de Poitiers (IC2MP), Université de

Poitiers, CNRS, Equipe Eau, Biomarqueurs, Contaminants Organiques (E.BiCOM) 4 rue Michel Brunet, TSA 51106, F-86073 Poitiers, France.

1

ACCEPTED MANUSCRIPT

k

Chimie Analytique des Molécules Bioactives (CAMBA), Institut Pluridisciplinaire Hubert

Curien, UMR 7178, CNRS, Université de Strasbourg, 67401 Illkirch, France.

RI PT

*Corresponding Author: Phone: +33 549 453 862. E-mail: [email protected]. Keywords: cancer, chemotherapy, drug delivery, self-immolative linker, enzyme-responsive

SC

systems, antibody-drug conjugate

Abstract. The selective destruction of tumour cells while sparing healthy tissues is one of the

M AN U

main challenges in cancer therapy. Antibody-drug conjugates (ADCs) are arguably the most rapidly expanding class of targeted cancer therapies. Efficient drug conjugation and release technologies are essential for the development of these new therapeutic agents. In response to the ever-increasing demand for efficient drug release systems, we have developed a new class of β-

TE D

galactosidase-cleavable linkers for ADCs. Within this framework, novel payloads comprising a galactoside linker, the monomethyl auristatin E (MMAE) and cysteine-reactive groups were synthesized, conjugated with trastuzumab and evaluated both in vitro and in vivo. The ADCs

EP

with galactoside linkers demonstrated superior therapeutic efficacy in mice compared to the

AC C

marketed trastuzumab emtansine used for the treatment of breast cancer.

Introduction

Antibody-Drug Conjugates (ADCs) are efficient cancer therapies that combine the targeting specificity of monoclonal antibodies with the cell-killing potency of small molecular drugs [1–3]. The success of two recently approved ADCs (trastuzumab

2

ACCEPTED MANUSCRIPT

emtansine[4] and brentuximab vedotin[5]) has triggered massive research efforts in the field. More than 60 ADCs are currently undergoing clinical trials for a variety of cancer indications [6].

RI PT

One of the main prerequisites for the activity of ADCs is the efficient release of the cytotoxic drug upon internalization by cancer cells. Hydrazones were among the first cleavable linkers that were investigated for ADC applications [7]. These linkers are

SC

relatively stable in blood circulation and release drugs in the acidic environment of endosomes and lysosomes. Another release strategy consists in the use of disulphide

M AN U

bonds [8], which are cleaved by an exchange with glutathione in the reducing intracellular environment. In order to decrease non-specific release, the disulphide bonds could be stabilised by methyl groups in the α-position [9]. Although they are important steps in ADC development, the stability of these linkers and the specificity of drug release were

TE D

far from being perfect, as in particular illustrated by the withdrawal of gemtuzumab ozogamicin (containing both, hydrazone and disulphide linkers; the reduction of the latter being the triggering step of drug activation) from the market in 2010.

EP

A real breakthrough in the area of specific drug release was achieved through the introduction of enzyme-cleavable linkers into the ADC construct. The pioneering valine-

AC C

citrulline (Val-Cit) linker (Figure 1A) was incorporated in brentuximab vedotin, which was approved by the FDA in 2011 [5]. This dipeptide linker is very stable in blood circulation and is readily cleaved by lysosomal enzymes such as cathepsin B [10,11]. Further research in the area of enzyme-cleavable linkers suitable for ADC engineering led to the development of glucuronide linkers [12,13] cleaved by β-glucuronidase (Figure 1B). Besides the specificity of drug release, the carbohydrate moiety conferred additional

3

ACCEPTED MANUSCRIPT

hydrophilic properties to the payload [14,15]. However, as the concentration of lysosomal enzymes differs from one type of cancer cells to another, it seems worthwhile to widen the scope of enzyme-responsive linkers for ADCs.

RI PT

Over the past two decades, several galactoside prodrugs [16-26] were developed for the selective delivery of potent anticancer drugs in the course of antibody-directed enzyme prodrug therapy (ADEPT) [27-30]. More recently, we reported the first

SC

generation of galactoside prodrugs suitable for the treatment of tumours in prodrug monotherapy (PMT [31,32]). Following selective internalization inside cancer cells

M AN U

overexpressing the folate receptor (FR), these drug delivery systems [33,37] were activated by lysosomal β-galactosidase leading to the release of active compounds. The efficacy of this targeting strategy was assessed in mice using a galactoside prodrug of monomethyl auristatin E (MMAE) which induced a remarkable antitumour effect without

TE D

any detectable side effects [33]. In the course of this study, we demonstrated that the intracellular β-galactosidase-catalysed drug release process was sufficiently efficient to produce a strong by-stander effect. Furthermore, we showed that glucuronide analogues,

EP

while selectively internalized inside FR-positive cancer cells, did not conduct to the intracellular release of the drug. This observation suggested that intracellular activation of

AC C

enzyme-responsive drug delivery systems can be, in some cases, more efficient with galactoside linkers than with glucuronide linkers. These findings urged us to explore the applicability of galactoside linkers in the design of new ADCs. Thus, we report herein the development and evaluation of the first generation of β-galactosidase-sensitive ADCs (Figure 1C).

4

ACCEPTED MANUSCRIPT

Results and Discussion Chemistry. In this pilot study, trastuzumab was chosen as a model antibody in order to

RI PT

compare our new ADCs to the marketed trastuzumab emtansine. The antibody was functionalized on cysteine residues using either payload 5a or 5b (Scheme 1). Payload 5a includes a galactoside trigger, the potent MMAE and a self-immolative linker [39,40]

group.

In

payload

5b,

the

latter

was

SC

bearing a decaethylene glycol side chain terminated by a maleimidocaproyl functional replaced

by

the

previously

reported

M AN U

arylpropiolonitrile (APN) group [40,41] with the aim to increase ADC plasma stability. Indeed, we recently showed that APN linkers, in contrast to maleimide, do not undergo retro-Michael addition when conjugated to cysteine. With this design, the selective internalization of the ADC within HER2+ tumour cells is followed by β-galactosidasecatalysed hydrolysis of the glycosidic bond that triggers the release of MMAE through the

TE D

mechanism depicted in Figure 1C. Payload 5a was prepared in three steps starting from the alkyne 1, which was previously described in the literature [33] (Scheme 1). First, alkyne 1 was placed in CH2Cl2 with commercially available O-(2-aminoethyl)-O’-(2-

EP

azidoethyl)-nonaethylene glycol and Cu(CH3CN)4PF6 in order to form the triazole 2

AC C

through the copper(I)-catalysed azide-alkyne 1,3-cycloaddition (CuAAC) [42]. After 12 hours under these conditions, the reaction mixture was washed with an aqueous solution of EDTA, the solvent was removed and the crude residue was purified by flash column chromatography to yield the protected galactoside 2 in 48 % yield. Full deprotection of the hydroxyl groups with LiOH furnished the derivative 3, which was engaged in the next step without purification. Finally, the reaction of the primary amino group of 3 with the N-hydroxysuccinimide ester 4a afforded the expected product 5a with a 38 % yield.

5

ACCEPTED MANUSCRIPT

Payload 5b was prepared in 71 % yield by reacting the precursor 3 with the APN derivative 4b. In order to compare the efficacy of our galactoside linkers 5a and 5b to that of the enzyme-sensitive linker currently used in clinic, we synthetized MMAE-based

RI PT

payload 5c, containing the Val-Cit linker 9 used in brentuximab vedotin.

ADCs were prepared following the reduction-alkylation procedure. First, trastuzumab was partially reduced by incubation with 2.2 eq of tris(2-carboxyethyl)phosphine (TCEP)

SC

solution for 2 hours at 37°C. The resulting solutions were cooled to 4°C and payloads were added to the mixture. The mixtures were stirred for 2 hours at 4°C for maleimide-

M AN U

containing payloads 5a and 5c, and for 12 hours at 25°C for the APN-containing payload 5b. The resulting conjugates were purified by size-exclusion chromatography. The coupling of trastuzumab with the payloads 5a and 5b provided the β-galactosidasecleavable ADCs T-MC-Gal-MMAE and T-APN-Gal-MMAE, respectively. The coupling

TE D

of trastuzumab with the Val-Cit-containing payload 5c provided the cathepsin B-sensitive ADC T-MC-VC-MMAE (Figure 2A). The drug-to-antibody ratios (DARs), identified using native mass spectrometry [43], were 3.3 for T-MC-Gal-MMAE, 3.3 for T-APN-

EP

Gal-MMAE, and 2.9 for T-MC-VC-MMAE (Figure 2B). Biological results. Antiproliferative activity of the prepared ADCs was evaluated on both

AC C

HER2+ SK-BR-3 (Figure 2C) and HER2- MDA-MB-231 cell lines. The commercial ADC trastuzumab emtansine (T-DM1) was used to benchmark our approach in this study. As shown in Table 1, all ADCs dramatically affected the viability of HER2+ SK-BR-3 cells, with IC50 values in the picomolar range (Table 1). In these experiments, the three enzyme-responsive ADCs were more potent than T-DM1. On the other hand, no cytotoxicity was observed on MDA-MB-231 cells with any ADC until the highest tested

6

ACCEPTED MANUSCRIPT

dose of 10-9 M. These results demonstrated the selective killing of HER2+ cancer cells by the new enzyme-responsive ADCs. With the aim to evaluate possible toxicity due to payload deconjugation via retro-Micheal addition, the cytotoxicity of maleimide-based

RI PT

payloads 5a and 5c was also measured on both tumour cell lines and compared to that of the free MMAE (Supporting Information). As expected, payloads 5a and 5c showed unspecific antiproliferative activity on both HER2+ and HER2- cell lines. However, they

SC

were significantly less toxic than MMAE indicating that payload deconjugation would induce limited damage.

M AN U

The specificity of β-galactosidase towards the new linker was validated by incubating all the prepared ADCs with 10U/mL of β-galactosidase. The evolution of the DAR over time was monitored by Hydrophobic Interaction Chromatography (HIC). As anticipated, the DARs of T-MC-Gal-MMAE and T-APN-Gal-MMAE decreased rapidly, confirming

TE D

that the linker was cleaved by the enzyme. On the other hand, the DAR of the ADC with the Val-Cit linker (T-MC-VC-MMAE) was unaffected by the presence of β-galactosidase (Figure 2D).

EP

In order to test whether our new linker chemistry affected antigen recognition, the

AC C

affinity of all ADCs was measured using flow cytometry on SK-BR-3 (Figure 2E) and MDA-MB-231 cell lines (Supporting Information). As illustrated by the median fluorescence intensities (MFIs), no significant change in affinity was observed for any ADC compared to the native antibody, trastuzumab (Figure 2E). In vivo therapeutic efficacy of T-MC-Gal-MMAE and T-APN-Gal-MMAE was then evaluated in nude mice bearing subcutaneous BT-474 tumours. The animals (5 per group) received a single 1 mg/kg intravenous dose of either T-MC-Gal-MMAE or T-APN-Gal-

7

ACCEPTED MANUSCRIPT

MMAE. As shown in Figure 3, 31 days post-injection, both ADCs induced a good antitumour response with 57% and 58% reduction of tumour volume, for T-MC-GalMMAE and T-APN-Gal-MMAE respectively, as compared to the control group. In the

RI PT

course of these experiments, the ADCs were well tolerated without any loss in body weight or sign of overt toxicity. Furthermore, no difference in efficacy was observed between the two β-galactosidase-sensitive ADCs T-MC-Gal-MMAE and T-APN-Gal-

SC

MMAE.

T-DM1 was also tested in vivo, following the same protocol. However, the effect of

M AN U

this ADC on tumour growth was not statistically significant at the tested dose of 1 mg/kg (Supporting Information). Nevertheless, the anticancer efficacy of T-DM1 was compared to that of T-MC-Gal-MMAE and T-APN-Gal-MMAE using Principal Components Analysis (PCA) (Figure 4). The results of the PCA showed a higher reduction of tumour

TE D

growth upon treatment with the two β-galactosidase-responsive ADCs than with the clinically used T-DM1. Therefore, the in vivo antitumour efficacy of T-MC-Gal-MMAE and T-APN-Gal-MMAE reveals that β-galactosidase-cleavable linkers are efficient for

EP

the intracellular delivery of anticancer drugs when conjugated to a therapeutic antibody such as trastuzumab. This finding enriches the toolbox of enzyme-responsive linkers

AC C

available for designing ADCs, which could be of great interest. Galactoside linkers could be indeed a valuable alternative to the previously reported valine-citrulline or glucuronide analogues for the therapy of some tumours. For instance, since high level of extracellular β-glucuronidase is present in the microenvironment of solid tumors [31,32,44], ADCs with glucuronide linkers [12,13] can lead to drug release outside of the targeted cancer cells. In this

8

ACCEPTED MANUSCRIPT

context, our β-galactosidase-sensitive linkers may be advantageous in that they release the active compound exclusively within malignant cells.

RI PT

Conclusion In conclusion, we developed the first generation of ADCs bearing β-galactosidasecleavable linkers, designed for the selective delivery of anticancer drugs. These ADCs are

SC

more efficient than trastuzumab emtansine (T-DM1) for the treatment of HER2+ mammary tumours in mice. Thus, we believe that a β-galactosidase-responsive drug

M AN U

delivery system will be of great interest in the field of ADCs and other targeted therapies.

Experimental Section

General chemical procedure. 1H and 13C NMR spectra were recorded at 25°C on Bruker 400

TE D

spectrometer. Recorded shifts are reported in parts per million (δ) and calibrated using residual non-deuterated solvent. Data are represented as follow: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, m = multiplet, br = broad), coupling constant

EP

(J, Hz) and integration. High resolution mass spectra (HRMS) were obtained using Agilent QTOF (time of flight) 6520 coupled to Agilent 1200 HPLC with Diode Array Detector; low

AC C

resolution mass spectra, using Agilent MSD 1200 SL (ESI/APCI) coupled to Agilent 1200 HPLC with Diode Array Detector. IR spectra were recorded on a Nicolet 380 FT-IR spectrometer from Thermo Electron Corporation as a DCM solution or solid on a diamond plate. The semi-preparative HPLC system consisted of two Shimadzu LC-8A pumps, an SPD-10A VP detector (Shimadzu), an SCL-10A VP controller (Shimadzu), an SIL-10A autosampler, a 2 mL sample loop and a SunFire C18 column (150 mm × 19 mm i.d., 5 µm, Waters).

9

ACCEPTED MANUSCRIPT

Synthesis of payloads. Compound 5a. To a solution of 1 (28 mg, 0.0218 mmol) and O-(2aminoethyl)-O’-(2-azidoethyl)nonaethylene glycol (12.6 mg, 0.024 mmol, 1.1 eq) in CH2Cl2 (1.8 mL) was added Cu(MeCN)4PF6 (12.2 mg, 0.0327 mmol). The mixture was stirred at room

RI PT

temperature for 12 h. A solution of disodium EDTA (0.13 g) in H2O (2.1 mL) was added. The resulting mixture was stirred for 2 h and extracted with CH2Cl2 (3 x 50 mL). The combined organic layers were dried over MgSO4 and concentrated in vacuo. The crude product was

SC

purified by column chromatography over silica gel (gradient elution 2% to 10% MeOH in CH2Cl2) to give the instable amine 2 (18.9 mg, 0.0104 mmol, 48 %) that was engaged directly in

M AN U

the next step. 2 was dissolved in MeOH (0.8 mL). The mixture was cooled at 0°C and a solution of lithium hydroxide monohydrate (3.9 mg, 0.0915 mmol) in water (0.8 mL) was added dropwise. The mixture was stirred for 15 min, neutralized with IRC-50 acidic resin, filtrated and concentrated in vacuo. The crude product was then dissolved in DMSO (0.5 mL) and the N-

TE D

hydroxysuccinimide ester 4a (1.2 eq, 3.9 mg) was added. The mixture was stirred at room temperature for 12 h and the solvent was removed under reduced pressure. Finally, the payload 5a was purified by preparative-reverse phase HPLC (7.2 mg, 38%, purity > 95% determined by

EP

HPLC). 1H NMR of a mixture of two diastereoisomers of 5a (500 MHz MHz, DMSO-d6): 8.698.46 (m, 1H), 8.32-8.04 (m, 1H), 7.93-7.65 (m, 3H), 7.58-7.43 (m, 1H), 7.31 (m, 6H), 7.00 (s,

AC C

2H), 5.93-5.82 (m, 1H), 5.43-5.38 (m, 1H), 5.19 (m, 1H), 4.99 (m, 2H), 4.68 (m, 2H), 4.43 (m, 6H), 4.14-4.01 (m, 3H), 3.72 (m, 6H), 3.55-3.33 (26H, masked by H2O residual signal), 3.283.17 (m, 18H), 3.07-2.67 (m, 7H), 2.55 (m, 2H), 2.45 (m, 1H), 2.27 (m, 1H), 2.12-2.03 (m, 6H), 1.77 (m, 3H), 1.59-1.46 (m, 5H), 1.35-1.17 (m, 5H), 1.05-0.6 (m, 30H), 0.47 (m, 1H). HRESIMS: m/z 1854.9738 (calcd. for C88H141N11O30Na 1854.9744 [M+Na]+).

10

ACCEPTED MANUSCRIPT

Compound 5b. Compound 5b was obtained using the protocol described for the compound 5a and replacing reagent 4a by the reagent 4b. Compound 5b was obtained in 71% yield after purification by preparative-reverse phase HPLC (purity > 95% determined by HPLC). HRESI-

RI PT

MS: m/z 1814.9204 (calcd. for C88H133N11O28Na 1814.9214 [M+Na]+).

Preparation of the ADCs. To a solution of trastuzumab (5 mg/mL, 1 mL) in PBS (100 mM with 2 mM EDTA, pH 7.4) was added a solution of TCEP (6.88 µL, 2 eq., 10 mM in water). The

SC

resulting mixture was incubated at 37°C for 2h and then cooled to 4°C. To a solution of the partially reduced trastuzumab was added a solution of the payload (20.63 µL, 12 eq., 20 mM in

M AN U

DMSO). The mixture was incubated at 4°C for 2h and then centrifuged at 5000 g for 2 min to remove the insoluble excess of the payload. The supernatant was purified by size-exclusion chromatography on PD-10 columns (GE Healthcare, ref. 17-0851-01) equilibrated with sodium succinate buffer (10 mM, pH 5.0) to give solutions of pure ADC (a purity of >95% was

TE D

confirmed by size exclusion chromatography).

Native mass spectrometry experiments. Native MS analyses were carried-out in positive mode, on an ESI-TOF (LCT, Micromass, Altrincham, UK) upgraded by MS Vision (MS Vision,

EP

Almere, Netherlands) coupled with an automated chip-based nanoESI infusion source (Triversa Nanomate, Advion Ithaca, USA). Instrumental parameters were tuned to ensure transmission of

AC C

high molecular weight species and preservation of potential non-covalent interactions disruption. The acceleration voltage was set to 120 V and the pressure in the interface region of the mass spectrometer was 6.0 mbar for T-MC-Gal-MMAE, T-MC-VC-MMAE, T-APN-Gal-MMAE and T-APN-VC-MMAE analysis. Acquisitions were performed during 2 min with a scan time of 4 s after external calibration with cesium iodide 2 mg/mL. MS data interpretations were performed using Mass Lynx V4.1 (Waters, Manchester, UK).

11

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Figure 1. Enzymatically cleavable linkers for traceless drug release. (A). Valine-Citrulline linker cleaved by cathepsin B. (B) Glucuronide linker cleaved by β-glucuronidase. (C) Galactoside

AC C

EP

linker cleaved by β-galactosidase

12

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

Figure 2. Structures and characterization of ADCs. (A) Structures of T-MC-Gal-MMAE, TAPN-Gal-MMAE and T-MC-VC-MMAE. (B) Deconvoluted MS spectra of T-MC-Gal-MMAE, T-APN-Gal-MMAE and T-MC-VC-MMAE. Intensities in percentage and average DAR were calculated according to intensities from mass spectra. (C) In vitro cytotoxic activity of the prepared ADCs and the reference T-DM1 on SK-BR-3 cancer cells. The cell viability was determined with an MTS test after 96 hours of treatment with the corresponding ADC. The

13

ACCEPTED MANUSCRIPT

untreated cells were considered as 100 % viable. The standard deviations are calculated for well triplicates. (D) Specificity of β-galactosidase-induced drug release. All ADCs were diluted to 1 mg/mL in PBS and incubated with β-galactosidase (10 U/mL) at 25°C. Evolution of the DAR

RI PT

was followed by HIC. Both T-MC-Gal-MMAE and T-APN-Gal-MMAE were cleaved by the enzyme at a similar rate. No linker cleavage occurred for T-MC-VC-MMAE in the presence of β-galactosidase. (E) Estimate of antibody affinities of the prepared ADCs, the reference T-DM1

SC

and the native antibody trastuzumab. The affinity of each ADC was established by comparing their median fluorescence intensity (MFI) to that of the native antibody trastuzumab on HER2+

AC C

EP

TE D

at the concentration of 2 µg/mL.

M AN U

SK-BR-3 cells. Rituximab was used as isotype control. All the antibodies and ADCs were tested

14

2500

Vehicle T-MC-Gal-MMAE T-APN-Gal-MMAE

2000

1000 500

* * * * * * *

0 0

10

20

30

40

Day

50

RI PT

1500

SC

Mean tumor volume (mm3)

ACCEPTED MANUSCRIPT

M AN U

Figure 3. In vivo antitumour activity of T-MC-Gal-MMAE and T-APN-Gal-MMAE. BT-474 epithelial ductal carcinoma cell line was subcutaneously implanted into nude female mice. When the tumours reached 250 mm3 a single dose of each indicated ADC (1 mg/kg), or the vehicle, was administered intravenously. Data are represented as mean tumour volumes (n = 5 mice per

TE D

group). The vertical bars depict the SDs. *P<0.05; one-way analysis of variance with Least

AC C

EP

Significant Difference post-test.

15

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Figure 4. PCA plot of T-DM1, T-MC-Gal-MMAE and T-APN-Gal-MMAE according to mice tumour volumes throughout 45 days post-injection of a single 1 mg/kg intravenous

AC C

EP

TE D

dose of antibody-drug conjugates (n = 5 mice per group).

16

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Scheme 1. Synthesis of galactoside payloads 5a and 5b and structure of Val-Cit payloads 5c. a) NH2(CH2CH2O)10CH2CH2N3, Cu(CH3CN)PF6, CH2Cl2, RT, 12 h, 48 %; b) LiOH, MeOH, 0°C,

AC C

EP

TE D

20 min; c) 4a or 4b, DMSO, 16h, 38% and 71% respectively (two steps).

17

ACCEPTED MANUSCRIPT

Table 1. Characterization (DAR) and IC50 (pM) of ADCs on SK-BR-3 and MDA-MB-

231 tumour cell lines, determined by cell viability assays. SK-BR-3

MDA-MB-231

IC50 (pM)

IC50 (pM)

DAR naa

3.3

8.8

T-APN-Gal-MMAE

3.3

14.3

T-MC-VC-MMAE

2.9

15.4

T-DM1

3.5

33.0

naa

naa naa

M AN U

SC

T-MC-Gal-MMAE

EP

TE D

na: no cytotoxic activity

AC C

a

RI PT

ADC

18

ACCEPTED MANUSCRIPT

Appendix A. Supplementary data Supplementary data related to this article can be found at

RI PT

Acknowledgment

This Work was supported by SATT Conectus "BioReLiable" project, CNRS, University of Strasbourg, GIS IBiSA, the Région Alsace, Agence Nationale de la Recherche (ANR,

SC

Programme Blanc – SIMI 7, ProTarget and ANR-10-LABX-0030-INRT, as part of the program Investissements d’Avenir ANR-10-IDEX-0002-02) and La Ligue contre le

M AN U

Cancer (comités Deux-Sèvres et Vienne). The International Center for Frontier Research in Chemistry (icFRC) is also acknowledged for financial support.

References

TE D

[1] Merten, H.; Brandl, F.; Plückthun, A; Zangemeister-Wittke, U. Antibody-Drug Conjugates for Tumor Targeting-Novel Conjugation Chemistries and the Promise of non-IgG Binding

EP

Proteins. Bioconjug. Chem., 2015, 26, 2176–2185. [2] Chari, R. V. J.; Miller, M. L.; Widdison, W. C. Antibody- Drug Conjugates: An Emerging

AC C

Concept in Cancer Therapy. Angew. Chem. Int. Ed., 2014, 53, 3796–3827. [3] Bouchard, H.; Viskov, C.; Garcia-Echeverria, C. Antibody-drug conjugates-A new wave of cancer drugs. Bioorg. Med. Chem. Lett., 2014, 24, 5357–5363. [4] Lambert, J. M.; Chari; R. V. J. Ado-trastuzumab Emtansine (T-DM1): An Antibody-Drug Conjugate (ADC) for HER2-Positive Breast Cancer. J. Med. Chem., 2014, 57, 6949–6964. [6] Senter, P. D.; Sievers; E. L. The discovery and development of brentuximab vedotin for use in relapsed Hodgkin lymphoma and systemic anaplastic large cell lymphoma. Nat. Biotechnol.,

19

ACCEPTED MANUSCRIPT

2012, 30, 631–637. [6] Beck, A.; Goetsch, L.; Dumontet, C.; Corvaïa, N. Strategies and challenges for the next generation of antibody-drug conjugates. Nat. Rev. Drug Discov., 2017, 16, 315-337.

RI PT

[7] Hinman, L. M.; Hamann, P. R.; Wallace, R.; Menendez, A. T.; Durr, F. E.; Upeslacis, J. Preparation and characterization of monoclonal-antibody conjugates of the calicheamicins - A novel and potent family of antitumor antibiotics. Cancer Res., 1993, 53, 3336–3342.

SC

[8] Chari, R. V. J.; Martell, B. A.; Gross, J. L.; Cook, S. B.; Shah, S. A.; Blattler, W. A.; McKenzie, S. J.; Goldmacher, V. S. Immonoconjugates containing novel maytansinoids -

M AN U

Promising anticancer drugs. Cancer Res., 1992, 52, 127–131.

[9] Kellogg, B. A.; Garrett, L.; Kovtun, Y.; Lai, K. C.; Leece, B.; Miller, M.; Payne, G.; Steeves, R.; Whiteman, K. R.; Widdison, W.; Xie, H.; Singh, R.; Chari, R. V. J.; Lambert, J. M.; Lutz, R. J. Disulfide-Linked Antibody-Maytansinoid Conjugates: Optimization of In Vivo

TE D

Activity by Varying the Steric Hindrance at Carbon Atoms Adjacent to the Disulfide Linkage. Bioconjug. Chem., 2011, 22, 717–727.

[10] Doronina, S. O.; Toki, B. E;. Torgov, M. Y.; Mendelsohn, B. A; Cerveny, C. G.; Chace, D.

EP

F.; DeBlanc, R. L.; Gearing, R. P.; Bovee, T. D.; Siegall, C. B.; Francisco, J. A.; Wahl, A. F.; Meyer D. L.; Senter, P. D. Development of potent monoclonal antibody auristatin conjugates for

AC C

cancer therapy. Nat. Biotechnol., 2003, 21, 778–784. [11] Dubowchik, G. M.; Firestone, R. A.; Padilla, L.; Willner, D.; Hofstead, S. J.; Mosure, K.; Knipe, J. O.; Lasch, S. J; Trail, P. A. Cathepsin B-labile dipeptide linkers for lysosomal release of doxorubicin from internalizing immunoconjugates: Model studies of enzymatic drug release and antigen-specific in vitro anticancer activity. Bioconjug. Chem., 2002, 13, 855–869. [12] Jeffrey, S. C.; Andreyka, J. B.; Bernhardt, S. X.; Kissler, K. M.; Kline, T.; Lenox, J. S.;

20

ACCEPTED MANUSCRIPT

Moser, R. F.; Nguyen, M. T.; Okeley, N. M.; Stone, I. J.; Zhang, X.; Senter, P. D. Development and properties of beta-glucuronide linkers for monoclonal antibody-drug conjugates. Bioconjug. Chem., 2006, 17, 831–840.

RI PT

[13] Jeffrey, S. C.; De Brabander, J.; Miyamoto, J.; Senter, P. D. Expanded Utility of the betaGlucuronide Linker: ADCs That Deliver Phenolic Cytotoxic Agents. ACS Med. Chem. Lett., 2010, 1, 277–280.

SC

[14] Lyon, R. P.; Bovee, T. D.; Doronina, S. O.; Burke, P. J.; Hunter, J. H.; Neff-LaFord, H. D.; Jonas, M.; Anderson, M. E.; Setter, J. R.; Senter, P. D. Reducing hydrophobicity of

Biotechnol., 2015, 33, 733–735.

M AN U

homogeneous antibody-drug conjugates improves pharmacokinetics and therapeutic index. Nat.

[15] Jeffrey, S. C.; Nguyen, M. T.; Moser, R. F.; Meyer, D. L.; Miyamoto, J. B.; Senter, P. D. Minor groove binder antibody conjugates employing a water soluble beta-glucuronide linker.

TE D

Bioorg. Med. Chem. Lett., 2007, 17, 2278–2280.

[16] Fernandes, A.; Viterisi, A.; Coutrot, F.; Potok, S.; Leigh, D. A.; Aucagne, V.; Papot, S. Rotaxane-Based Propeptides: Protection and Enzymatic Release of a Bioactive Pentapeptide.

EP

Angew. Chem. Int. Ed., 2009, 48, 6443-6447.

[17] Kamal, A.; Tekumalla, V.; Krishnan, A.; Pal-Bhadra, M.; Bhadra, U. Development of

AC C

pyrrolo[2,1-c][1,4]-benzodiazepine beta-galactoside prodrugs for selective therapy of cancer by ADEPT and PMT. ChemMedChem, 2008, 3, 794-802 [18] Thomas, M.; Rivault, F.; Tranoy-Opalinski, I.; Roche, J.; Gesson, J. P.; Papot, S. Synthesis and biological evaluation of the suberoylanilide hydroxamic acid (SAHA) beta-glucuronide and beta-galactoside for application in selective prodrug chemotherapy. Bioorg. Med. Chem. Lett., 2007, 17, 983-986.

21

ACCEPTED MANUSCRIPT

[19] Bakina, E.; Farquhar, D. Intensely cytotoxic anthracycline prodrugs: galactosides. AntiCancer Drug Des., 1999, 14, 507-515. [20] Barat, R.; Legigan, T.; Tranoy-Opalinski, I.; Renoux, B.; Péraudeau, E.; Clarhaut, J.;

RI PT

Poinot, P.; Fernandes, A. E.; Aucagne, V.; Leigh, D. A.; Papot S. A mechanically interlocked molecular system programmed for the delivery of an anticancer drug. Chem. Sci., 2015, 6, 26082613.

SC

[21] Wirth, T.; Schmuck, K., Tietze, L. F.; Sieber, S. A. Duocarmycin Analogues Target Aldehyde Dehydrogenase 1 in Lung Cancer Cells. Angew. Chem. Int. Ed. 2012, 51, 2874-2877.

M AN U

[22] Tietze, L. F.; von Hof, J. M.; Müller, M.; Krewer, B.; Schuberth, I. Glycosidic Prodrugs of Highly Potent Bifunctional Duocarmycin Derivatives for Selective Treatment of Cancer. Angew. Chem. Int. Ed., 2010, 49, 7336-7339.

[23] Tietze, L. F.; Krewer, B. Antibody-Directed Enzyme Prodrug Therapy: A Promising

TE D

Approach for a Selective Treatment of Cancer Based on Prodrugs and Monoclonal Antibodies. Chem. Biol. Drug Des., 2009, 74, 205-211.

[24] Tietze, L. F.; Major, F.; Schuberth, I.; Spiegl, D. A.; Krewer, B.; Maksimenka, K.;

EP

Bringmann, G.; Magull, J. Selective treatment of cancer: Synthesis, biological evaluation and structural elucidation of novel analogues of the antibiotic CC-1065 and the duocarmycins. Chem.

AC C

Eur. J., 2007, 13, 4396-4409.

[25] Tietze, L. F.; Major, F.; Schuberth, I. Antitumor agents: Development of highly potent glycosidic duocarmycin analogues for selective cancer therapy. Angew. Chem. Int. Ed., 2006, 45, 6574-6577.

[26] Tietze, L. F.; Feuerstein, T.; Fecher, A.; Haunert, F.; Panknin, O.; Borchers, U.; Schuberth, I.; Alves, F. Proof of principle in the selective treatment of cancer by antibody-directed enzyme

22

ACCEPTED MANUSCRIPT

prodrug therapy: The development of a highly potent prodrug. Angew. Chem. Int. Ed., 2002, 41, 759-761.

Cancer, 1987, 56, 531-532.

RI PT

[27] Bagshawe, K. D. Antibody directed enzymes revive anticancer prodrugs concept. Br. J.

[28] Bagshawe, K. D. Antibody-directed enzyme prodrug therapy (ADEPT) for cancer. Expert Rev. Anticancer Ther., 2006, 6, 1421-1431.

SC

[29] Yu, Y.; Fang, L.; Sun, D. Biodistribution of HuCC49 Delta CH2-beta-galactosidase in colorectal cancer xenograft model. Int. J. Pharm., 2010, 386, 208-215.

M AN U

[30] Fang, L.; Battisti, R. F.; Cheng, H.; Reigan, P.; Xin, Y.; Shen, J.; Ross, D.; Chan, K. K.; Martin Jr, E. W.; Wang, P. G.; Sun, D. Enzyme specific activation of benzoquinone ansamycin prodrugs using HuCC49 Delta CH2-beta-galactosidase conjugates. J. Med. Chem., 2006, 49, 6290-6297.

TE D

[31] Bosslet, K.; Czech, J.; Hoffmann, D. A novel one-step tumor-selective prodrug activation system. Tumor Target., 1995, 1, 45-50.

[32] Bosslet, K.; Straub, R.; Blumrich, M.; Czech, J.; Gerken, M.; Sperker, B.; Kroemer, H. K.;

EP

Gesson, J. P.; Koch, M.; Monneret, C. Elucidation of the mechanism enabling tumor selective prodrug monotherapy. Cancer Res., 1998, 58, 1195-1201.

AC C

[33] Legigan, T.; Clarhaut, J.; Tranoy-Opalinski, I.; Monvoisin, A.; Renoux, B.; Thomas, M.; Le Pape, A.; Lerondel S.; Papot, S. The First Generation of β -Galactosidase-Responsive Prodrugs Designed for the Selective Treatment of Solid Tumors in Prodrug Monotherapy. Angew. Chem. Int. Ed. Engl., 2012, 51, 11606–11610. [34] Thomas, M.; Clarhaut, J.; Strale, P.-O.; Tranoy-Opalinski, I.; Roche J.; Papot, S. A Galactosidase-Responsive "Trojan Horse" for the Selective Targeting of Folate Receptor-

23

ACCEPTED MANUSCRIPT

Positive Tumor Cells. ChemMedChem, 2011, 6, 1006–1010. [35] Clarhaut, J.; Fraineau, S.; Guilhot, J.; Peraudeau, E.; Tranoy-Opalinski, I.; Thomas, M.; Renoux, B.; Randriamalala, E.; Bois, P.; Chatelier, A.; Monvoisin, A.; Cronier, L.; Papot, S.;

acute myelogenous leukemia blasts. Leuk. Res., 2013, 37, 948–955.

RI PT

Guilhot, F. A galactosidase-responsive doxorubicin-folate conjugate for selective targeting of

[36] Grinda, M.; Legigan, T.; Clarhaut, J.; Peraudeau, E.; Tranoy-Opalinski, I.; Renoux, B.;

SC

Thomas, M.; Guilhot, F.; Papot, S. An enzyme-responsive system programmed for the double

Biomol. Chem., 2013, 11, 7129–33.

M AN U

release of bioactive molecules through an intracellular chemical amplification process. Org.

[37] Alsarraf, J.; Péraudeau, E.; Poinot, P.; Tranoy-Opalinski, I.; Clarhaut, J.; Renoux, B.; Papot, S. A dendritic beta-galactosidase-responsive folate-monomethylauristatin E conjugate. Chem. Commun., 2015, 51, 15792–15795.

TE D

[38] Papot, S.; Tranoy, I.; Tillequin, F.; Florent, J.; Gesson, J.-P. Design of selectively activated anticancer prodrugs: elimination and cyclization strategies. Curr. Med. Chem. Agents, 2002, 2, 155–185.

EP

[39] Tranoy-Opalinski, I.; Fernandes, A.; Thomas, M.; Gesson; J.-P.; Papot, S. Design of selfimmolative linkers for tumour-activated prodrug therapy. Anti-Cancer Agents Med. Chem., 2008,

AC C

8, 618–637.

[40] Koniev, O.; Leriche, G.; Nothisen, M.; Remy, J.-S.; Strub, J.-M.; Schaeffer-Reiss, C.; Van Dorsselaer, A.; Baati, R.; Wagner, A. Selective Irreversible Chemical Tagging of Cysteine with 3-Arylpropiolonitriles. Bioconjug. Chem., 2014, 25, 202–206. [41] Kolodych, S.; Koniev, O.; Baatarkhuu, Z.; Bonnefoy, J.-Y.; Debaene, F.; Cianférani, S.; Van Dorsselaer, A.; Wagner, A. CBTF: New Amine-to-Thiol Coupling Reagent for Preparation

24

ACCEPTED MANUSCRIPT

of Antibody Conjugates with Increased Plasma Stability. Bioconjug. Chem., 2015, 26, 197–200. [42] Rostovtsev, V. V ; Green, L. G.; Fokin, V. V; Sharpless, K. B. A stepwise Huisgen

alkynes. Angew. Chem. Int. Ed. Engl., 2002, 41, 2596–2599.

RI PT

cycloaddition process: Copper(I)-catalyzed regioselective "ligation" of azides and terminal

[43] Debaene, F.; Bœuf, A.; Wagner-Rousset, E.; Colas, O.; Ayoub, D.; Corvaïa, N.; Van Dorsselaer, A.; Beck, A.; Cianférani, S. Innovative Native MS Methodologies for Antibody Drug

SC

Conjugate Characterization: High Resolution Native MS and IM-MS for Average DAR and DAR Distribution Assessment. Anal. Chem., 2014, 86, 10674–10683.

M AN U

[44] Tranoy-Opalinski, I.; Legigan, T.; Barat, R.; Clarhaut, J.; Thomas, M.; Renoux, B.; Papot, S. beta-Glucuronidase-responsive prodrugs for selective cancer chemotherapy: An update. Eur.

AC C

EP

TE D

J. Med. Chem., 2014, 74, 302-313.

25

ACCEPTED MANUSCRIPT Synthesis of the first β-galactosidase-sensitive antibody-drug conjugates. Efficient release of the drug upon enzymatic activation. Selective cytotoxic activity against HER2+ cancer cell lines. Significant antitumor effect in vivo following the administration of a single 1 mg/kg dose.

AC C

EP

TE D

M AN U

SC

RI PT

The β-galactosidase-sensitive antibody-drug conjugates are more efficient than trastuzumab emtansine (T-DM1) for the treatment of HER2+ mammary tumours.