Efficient Preparation of Site-Specific AntibodyâDrug Conjugates Using

Subscriber access provided by UNIV OF REGINA

Article

Efficient preparation of site-specific antibody drug conjugates using cysteine-insertion. Nazzareno Dimasi, Ryan Fleming, Haihong Zhong, Binyam Bezabeh, Krista Kinneer, R. James Christie, Christine Fazenbaker, Herren Wu, and Changshou Gao Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00995 • Publication Date (Web): 28 Feb 2017 Downloaded from http://pubs.acs.org on March 1, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Molecular Pharmaceutics is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 54

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

1

Efficient preparation of site-specific antibody drug conjugates

2

using cysteine-insertion.

3 4

Nazzareno Dimasi1,*, Ryan Fleming1, Haihong Zhong2, Binyam Bezabeh1, Krista Kinneer2,

5

Ronald J. Christie1, Christine Fazenbaker2, Herren Wu1, Changshou Gao1,*

6 7 8

1

Antibody Discovery and Protein Engineering and 2Oncology Research MedImmune, Gaithersburg, Maryland, United States of America

9 10

*Corresponding authors’ emails: [email protected] and [email protected]

11 12

Short title: Cysteine-inserted antibody drug conjugates.

13 14

Keywords: cysteine-mutagenesis; cysteine-inserted antibody; antibody drug conjugates,

15

antibody engineering; site-specific conjugation; pyrrolobenzodiazepine dimers; SG3249;

16

tesirine; oncofetal antigen 5T4

17

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

18

ABSTRACT

19 20

Antibody drug conjugates (ADCs) are a class of biopharmaceuticals that combine the

21

specificity of antibodies with the high-potency of cytotoxic drugs. Engineering cysteine residues

22

in the antibodies using mutagenesis is a common method to prepare site-specific ADCs. With

23

this approach, solvent accessible amino acids in the antibody have been selected for substitution

24

with cysteine for conjugating maleimide-bearing cytotoxic drugs, resulting in homogeneous and

25

stable site-specific ADCs. Here we describe a cysteine engineering approach based on the

26

insertion of cysteines before and after selected sites in the antibody, which can be used for site-

27

specific preparation of ADCs. Cysteine-inserted antibodies have expression level and monomeric

28

content similar to the native antibodies. Conjugation to a pyrrolobenzodiazepine dimer (SG3249)

29

resulted in comparable efficiency of site-specific conjugation between cysteine-inserted and

30

cysteine-substituted antibodies. Cysteine-inserted ADCs were shown to have biophysical

31

properties, FcRn and antigen binding affinity similar to the cysteine-substituted ADCs. These

32

ADCs were comparable for serum stability to the ADCs prepared using cysteine-mutagenesis,

33

and had selective and potent cytotoxicity against human prostate cancer cells. Two of the

34

cysteine-inserted variants abolish binding of the resulting ADCs to FcγRs in vitro, thereby

35

potentially preventing non-target mediated uptake of the ADCs by cells of the innate immune

36

system that express FcγRs, which may result in mitigating off-target toxicities. A selected

37

cysteine-inserted ADC demonstrated potent dose-dependent anti-tumor activity in a xenograph

38

tumor mouse model of human breast adenocarcinoma expressing the oncofetal antigen 5T4.

39 40

INTRODUCTION


Page 2 of 54

Page 3 of 54

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


41 42

Antibody drug conjugates (ADCs) are comprised of a tumor-specific monoclonal

43

antibody (mAb) conjugated to highly potent cytotoxic drugs.1 Despite the early potential offered

44

by ADCs due to selective targeting and delivering of highly cytotoxic drugs to tumor cells,2 thus

45

limiting systemic toxicities, FDA approved ADCs are only limited to two examples, Kadcyla3

46

and Adcetris.4 Kadcyla is approved for HER2 positive metastatic breast cancer, while Adcetris is

47

approved for treatment of relapsed Hodgkins Lymphoma. However, in recent years,

48

identification of better therapeutic targets and technological advances in the preparation of ADCs

49

have led to a resurgence of ADCs with currently more than forty ADCs in clinical trials.5,6

50

A central step in the preparation of ADCs is the conjugation of a linker bearing a

51

cytotoxic drug to the mAb.1 ADCs such as Adcetris and Kadcyla are prepared using direct

52

conjugation at solvent-accessible thiols generated by reduction of the mAb interchain disulfide

53

bridges, and at primary amines of lysines, respectively. Conjugation to lysine uses N-

54

hydroxysuccinimide ester, while conjugation to thiols involves N-alkyl maleimide.1 These two

55

methods result in the preparation of heterogeneous conjugates with various drug load, efficacy,

56

pharmacokinetics and therapeutic properties.7

57

One approach to overcome ADCs heterogeneity is to use site-specific conjugation, a

58

method in which drug load and site of conjugation is controlled.8 Moreover, site-specific

59

conjugation at defined sites in the antibody results in stable ADCs in vivo.9-11 Three methods are

60

commonly used to prepare site-specific ADCs: (a) cysteine-mutagenesis at specific residues,9-18

61

(b) replacement of residues with unnatural amino acids with bio-orthogonal reactivity,19-21 and

62

(c) enzymatic ligation approaches.22-25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

63

The substitution of surface exposed residues with cysteines in antibodies, offers a simple

64

method to prepare ADCs, as no cell line engineering and additional production materials (i.e.

65

non-natural amino acids) are required. In addition, maleimide-bearing linkers are highly selective

66

for the sulfhydryl side-chain of the engineered cysteines, and have rapid reaction kinetics in

67

aqueous conditions. Recently, maleimide linkers have been modified to prevent susceptibility to

68

thiol exchange.26-29 These maleimides have been chemically modified to promote

69

thiosuccinimide hydrolysis, therefore precluding premature release of the cytotoxic drug from

70

the antibody. Furthermore, this class of maleimides offers the opportunity to prepare stable site-

71

specific ADCs at any conjugation site.26-30

72

The three most commonly employed sites in the antibodies constant domains, which are

73

used for cysteine-mutagenesis and subsequent preparation of site-specific ADCs, with two

74

cytotoxic drugs conjugated per antibody, are: S239 in the CH2 domain, 10, 15, 16 A114 in the CH1

75

domain,9,11 and V205 in the CL kappa domain.9,11 Cysteine-mutagenesis at these sites don’t have

76

any impact on expression and purification yields, don’t destabilize the structure of the antibody

77

and the resulting sulfhydryls show high reactivity toward maleimides bearing cytotoxic

78

payloads.9-16

79

Guided by the successful use of cysteine-mutagenesis for preparing site-specific ADCs,

80

we sought to explore if inserting cysteines before and after selected sites in the antibody is an

81

efficient method to prepare site-specific ADCs while maintaining IgG-like properties of the

82

ADCs. Herein we describe engineered antibodies that have cysteines inserted before and after the

83

three most employed sites in antibodies used for cysteine-mutagenesis to prepare site-specific

84

ADCs, namely S239, A114 and V205. While technologies for insertion of binding functions in

85

specific regions of the mAb have been reported;31,32 inserting cysteine for conjugation in the


Page 4 of 54

Page 5 of 54

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


86

antibodies has not been reported thus far. Therefore our strategy paves the way to use the amino

87

acid insertion approach to further advance the antibody engineering landscape.

88

The cysteine-inserted antibodies and their correspondent site-specific ADCs, prepared

89

using a maleimide-bearing pyrrolobenzodiazepine dimer (SG3249),33 maintain analytical and

90

functional characteristics similarly to the ADCs prepared by cysteine-mutagenesis at the selected

91

sites. In addition, the cysteine-inserted ADCs before and after position 239, similar to the ADC

92

prepared by mutagenesis at position 239,10 do not bind to FcγRs in vitro, which may abolish

93

antibody-mediated cellular cytotoxicity (ADCC). Loss of FcγRs binding may mitigate off-target

94

toxicities by preventing uptake of the ADCs by cells of the immune system that express FcγRs.

95

One ADC prepared using cysteine-insertion after position 239 showed dose-dependent potent

96

anti-tumor activity in a mouse xenograph model of human breast adenocarcinoma expressing the

97

oncofetal antigen 5T4.34-36

98 99

EXPERIMENTAL SECTION

100 101

General remarks. The antibodies used in this study are an anti-EphA2 antibody 1C110, an anti-

102

oncofetal antigen 5T435,36 and two isotype non-binding antibodies IC10 and ICa (MedImmune),

103

with kappa and lambda light chains, respectively. The maleimide-PEG8 pyrrolobenzodiazepine

104

dimer used for conjugation is SG3249.33 SG3249 was prepared at 10 mM in DMSO and was

105

>95% pure as judged by HPLC. SG3249 was synthetized at Novasep. The structural information

106

for the Fc and the Fab were obtained from the Protein Data Bank using accession code 3AVE37

107

and 3SKJ,38 respectively. Designation of antibodies and ADCs used in this study are listed in

108

Table 1.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

109 110

Materials. Reagents for molecular biology, including cell culture and transfection, were

111

purchased from Thermo Fisher Scientific. E. coli One Shot™ Stbl3™ chemically competent

112

cells, used for DNA amplification, were purchased from Thermo Fisher Scientific. Reagents

113

used for preparing buffers for antibody purification, analytical characterization and conjugation

114

were prepared at MedImmune using reagents from GE Healthcare, Sigma Aldrich, JT Baker,

115

EMD Serono and Thermo Fisher Scientific. Chinese hamster ovary cells (CHO G22) were used

116

for antibody expression; human prostate cancer cell line (PC-3) that express EphA2 and human

117

breast adenocarcinoma (MDA-MB-361) that do not express EphA2 were obtained from the

118

American Type Culture Collection. RPMI medium and HI-FBS used for culturing PC-3 and

119

MDA-MB-361 were purchased from Thermo Fisher Scientific. CellTiter-Glo Luminescent

120

Viability Assay was purchased from Promega. Rat serum was obtained from Jackson

121

Immunoresearch Labs. Anti-human Fc specific antibody agarose beads were purchased from

122

Sigma Aldrich. Syringe filters and vacuum filters were purchased from Nalgene and Millipore,

123

respectively. Materials and buffers used for ProteOn binding were purchased from Bio-Rad. An

124

absorbance of 280 nm was used to monitor antibodies and ADCs during purification and

125

analytical characterization. Figures were prepared using Prism 5, Origin Lab and PyMol.

126 127

Cloning. Selected sites for cysteine-mutagenesis and insertions are in the constant domain of the

128

mAb heavy chain (A114 in the CH1 domain and S239 in the CH2 domain), and the constant

129

domain of the kappa light chain (V205). Cysteine mutations and insertions were carried out

130

using overlapping PCR with synthetic oligonucleotides synthetized at Thermo Fisher Scientific.

131

PCR fragments were cloned into a MedImmune proprietary mammalian expression vector.10, 39


Page 6 of 54

Page 7 of 54

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


132

Standard molecular biology methods were used for cloning, vectors preparation and DNA

133

sequencing.

134 135

Expression and purification. Expression vectors containing antibody light and heavy chains for

136

native antibodies and cysteine variants were transfected in CHO-G22 cells. CHO-G22 cells were

137

cultured with 25 µM L-methionine sulfoximine, 100 µg/mL hygromycin in MedImmune’s

138

proprietary CHO-V2 medium at 37°C, 5% CO2 and 80% humidity. 24 hours prior to

139

transfection, CHO-G22 cells were diluted to 1 x 106 cells/mL in CHO-V2 medium without

140

hygromycin and L-methionine sulfoximine and grown overnight. 500 µg of mAb expression

141

vector was diluted into 7.5 mL of 150 mM sodium chloride and 2.5 mL of 1mg/mL PEI-max was

142

mixed with 5 mL of the 150 mM sodium chloride solution. The DNA and PEI-Max solutions

143

were combined and incubated for one minute at room temperature before adding to 500 mL of 2

144

x 106 CHO-G22 cells. The transfected cells were grown for 24 hours as described above.

145

Thereafter the temperature was changed to 34°C and supplemented with 500 mL of CHO-V2

146

medium. The culture medium was collected 14 days after transfection, and all antibody variants

147

were purified using standard protein A affinity chromatography, and were subsequently buffer

148

exchanged using dialysis in 25 mM Histidine–HCl pH 6.0 or PBS pH 7.2, 1 mM EDTA. mAb

149

expression level was determined using an in-house developed protein A binding assay as

150

described previously.39 The purity of the constructs was analyzed using analytical size-exclusion

151

chromatography (SEC-HPLC, method described hereafter). Antibodies had a monomeric content

152

equal or above 90% were used directly for conjugation to SG3249.

153



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

154

Site-specific conjugation. Antibodies variants were reduced using 40 molar equivalents of

155

TCEP (Tris(2-carboxyethyl)phosphine) in PBS pH 7.2, 1 mM EDTA (Ethylenediamine

156

tetraaceticacid) for 3 h at 37°C. Following 2X dialysis in PBS pH 7.2, 1 mM EDTA at 4°C using

157

10,000 MWCO dialysis cassettes, 20 molar equivalents of dehydroascorbic acid were added for

158

4 h at 25°C. The solution was filtered through a 0.2 µm syringe filter and 10% (v/v) DMSO and

159

8 equivalents of SG3249 were sequentially added, followed by incubation at room temperature

160

for 1 h under gentle rotation. The conjugation was quenched by the addition of 4 molar

161

equivalents (over SG3249) of N-acetyl cysteine. The conjugation process resulted in 3 to 5% of

162

aggregate formation. Macromolecular aggregates, conjugation reagents, including cysteine

163

quenched SG3249 were removed using ceramic hydroxyapatite Type II chromatography (CHT)

164

as described previously.10 Site-specific ADCs were formulated at 3 mg/mL in PBS pH 7.2 or 25

165

mM Histidine-HCl pH 6.0.

166 167

Analytical characterization. Before analytical characterization samples were centrifuged at

168

10,000 x g for 5 minutes, and filtered using a 0.22 µm syringe filter. An Agilent HPLC 1260

169

system equipped with an auto sampler and a diode array was used for the analytical

170

characterization. Analytical size-exclusion chromatography (SEC-HPLC), which was used to

171

determine monomeric content, aggregates and fragments, was performed using 100 µg (100 µL

172

volume) of antibodies or ADCs, which were loaded into a TSKgel G3000WXL column (Tosoh).

173

The mobile phase was 0.1 M sodium sulfate, 0.1 M sodium phosphate, 10% isopropanol, pH 6.8.

174

The flow rate was 1 mL/min and each analysis was carried out for 20 minutes at room

175

temperature. Hydrophobic interaction chromatography (HIC-HPLC) was used to assess

176

conjugation and drug load distribution. HIC-HPLC was carried out using a Butyl-non porous


Page 8 of 54

Page 9 of 54

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


177

resin (NPR) column (4.6 µm ID × 3.5 cm, 2.5 µm, Tosoh Bioscience). The mobile phase A was

178

25 mM Tris-HCl, 1.5 M (NH4)2SO4, pH 8.0; and the mobile phase B was and 25 mM Tris-HCl,

179

5% isopropanol, pH 8.0. 100 µL of antibodies or ADCs at a concentration of 1 mg/mL were

180

loaded and eluted at a flow rate of 1 mL/min with a gradient of 5% B to 100% B over 13 min.

181

Reduced reverse phase chromatography (rRP-HPLC) was used to confirm chain-specific

182

conjugation. The antibodies and ADCs were reduced at 37°C for 20 minutes using 42 mM

183

dithiothreitol (DTT) in PBS pH 7.2. 10 µg of reduced antibodies or ADCs were loaded onto a

184

polymeric reverse phase media (PLRP-S), 1000 Å column (2.1 × 50 mm, Agilent) and eluted at

185

80°C at a flow rate of 1 mL/min with a gradient of 5% B to 100% B over 25 min (mobile phase

186

A: 0.1% Trifluoroacetic acid in water; mobile phase B: 0.1% Trifluoroacetic acid in acetonitrile).

187

Reduced liquid chromatography mass spectrometry analysis (rLCMS), which was used to

188

determine conjugation at the light or heavy chain and drug to antibody ratio (DAR), was

189

performed on an Agilent 1290 series uHPLC coupled to an Agilent 6230 TOF. 2 µg of reduced

190

antibodies or ADCs were loaded onto a Zorbax RRHD 300-Diphenyl (2.1 × 50 mm, 1.8 µm,

191

Agilent) and eluted at a flow rate of 0.5 mL/min using a step gradient of 80% B after 2.1 min

192

(mobile phase A: 0.1% Formic acid in water and mobile phase B: 0.1% Formic acid in

193

acetonitrile). A positive time-of-flight MS scan was acquired and data collection and processing

194

was carried out using MassHunter software (Agilent). DAR was calculated using the rLCMS

195

data as described before.10 Efficiency of conjugation was determined using the following

196

equation, where a theoretical DAR of 2 was used:

197

= ( ℎ ) . Thermal

198

denaturation experiments were performed on a MicroCal VP Capillary differential scanning

199

calorimeter (DSC, GE Healthcare). Antibodies and ADCs were formulated at 1 mg/mL and



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

200

extensively dialyzed into 25 mM Histidine-HCl pH 6 before DSC analysis. DSC was performed

201

at a scan rate of 1°C per minute from 20°C to 100°C. The raw data were baseline-subtracted,

202

normalized for concentration and scan rate. Deconvolution analysis was carried out using a non-

203

two-state model and the best fits were obtained using 15 iteration cycles. The denaturation

204

temperatures, Tm, corresponding to the maximum of the transition peaks, were determined for

205

each sample.

206 207

FcRn binding using ProteOn. Neonatal Fc receptor (FcRn) binding was carried out using a

208

ProteOn XRP36 and recombinant human FcRn was prepared at MedImmune.39 Antibody

209

variants and ADCs were immobilized on separate flow cells using a GLC ProteOn sensor chip

210

following manufacturer recommendations. Surface densities were around 2800 RU. A reference

211

cell without protein was prepared using same sensor chip. Three-fold serial dilutions of human

212

FcRn were prepared, ranging from 3000 to 4.1 nM in PBS pH 6 with 0.005% Tween-20 (FcRn

213

buffer). The instrument was primed with the FcRn buffer and then each FcRn dilution was

214

injected over the surface of each antibody variants, ADCs and the reference cell at a flow rate of

215

25 µL/minute. Binding data were collected over 8 minute time frame, followed by 1 minute

216

pulse of a solution of PBS pH 7.4, 150 mM NaCl, 0.005% Tween-20. Data were processed using

217

the ProteOn software.

218 219

FcγγRs binding using ProteOn. The determination of equilibrium binding affinities of the native

220

antibodies and ADCs to human FcγRI, FcγRIIA, FcγRIIIA-158V,and FcγRIIIA-158F, were

221

carried out using ProteOn XPR36 (BioRad). Native antibodies and ADCs were immobilized

222

using amino coupling chemistry on a GLC ProteOn sensor chip. The sensor chip was treated


Page 10 of 54

Page 11 of 54

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


223

with 30 µL of 0.5% SDS, 50 mM NaOH, 100 mM HCl, followed by 30 µL of PBS, 0.005%

224

Tween-20, 3 mM EDTA pH 7.4 (ProteOn running buffer;) at 100 µL/minute. Native antibodies

225

and ADCs were prepared at 10 µM in sodium acetate buffer pH 4 and flowed over the activated

226

sensor chip surface at 30 µL/minute. The surface density for the immobilized proteins ranged

227

from 3230 to 3650 resonance unit (RU). A reference surface was similarly prepared but without

228

immobilized protein. After immobilization, the chip surfaces were inactivated using

229

ethanolamine-HCL (Bio-Rad). Before the binding experiments, the chip surfaces were treated

230

one more time with 30 µL of 0.5% SDS, 50 mM NaOH, 100 mM HCl and 30 µL of ProteOn

231

running buffer at 100 µL/minute. FcγRIIA and FcγRIIIA-158V were prepared at 16 µM in

232

ProteOn running buffer and serially diluted 1:3 to 0.19 nM. FcγRIIIA-158F was prepared in

233

same buffer but at a starting concentration of 32 µM and serially diluted 1:3 to 0.395 nM. FcγRI

234

prepared also in ProteOn running buffer using a starting concentration of 4 µM and serially

235

diluted 1:3 to a final concentration of 5.49 nM. The FcγRs dilutions were injected on the sensor

236

chip with immobilized native antibodies and ADCs at 25 µL/minute for 8 minutes. Between

237

injections 25 µL of 5 mM HCl was used to regenerate the ProteOn chip surface. Data were blank

238

subtracted, reference surface double corrected, and equilibrium binding constant were

239

determined using the ProteOn software (Bio-Rad).

240 241

Flow cytometry. PC-3 and MDA-MB-361 cells were grown in RPMI with 10% FBS

242

(Invitrogen) and DMEM with 10% FBS (Invitrogen), respectively. Cells were detached using

243

enzyme-free dissociation buffer (Invitrogen), washed and suspended in FACS buffer (PBS, 2%

244

FBS). A total of 200,000 cells/well were dispensed into a 96-well microplate and cells were

245

incubated on ice for 30 minutes before binding experiments. Titration binding experiments using



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

246

PC-3 cells with the anti-EphA2 1C1 and the IC native antibodies from 10 µg/mL to 0 µg/mL

247

were carried out to determine the concentration of saturation binding. Binding saturation for the

248

1C1 native antibody was determined to be 5 µg/mL, while no binding at this concentration was

249

observed for the IC native antibody. All antibody variants and ADCs were tested for binding

250

using PC-3 and MDA-MB-361cells at 5 µg/mL. After binding the cells were washed with FACS

251

buffer and stained for 30 minutes on ice with goat anti-human IgG (H+L)-AF488 (Invitrogen) at

252

a concentration of 5 µg/mL. Cells were washed and suspended in FACS buffer containing DAPI

253

(Invitrogen) for live/dead exclusion. Fluorescence of live, single cells was determined via FACS

254

on an LSRII (Becton Dickinson Biosciences) and data were analyzed using FlowJo (Treestar).

255 256

Determination of in vitro cell viability. PC-3 and MDA-MB-361 were seeded into white

257

polystyrene tissue-culture treated 96-well plates (Costar) in RPMI (Invitrogen) + 10% FBS and

258

DMEM (Invitrogen) + 10% FBS, respectively. Due to differences in growth kinetics, PC-3 cells

259

were seeded at 1500 cells/well and MDA-MB-361were seeded at 5000 cells/well. On the

260

following day, antibodies (native and ADC variants) were spiked into wells in triplicate using an

261

8-point dose curve of 1 to 4 serial dilutions starting from 4 µg/mL. Cell viability was determined

262

5 days later using the Cell Titer-Glo Luminescent Cell Viability Assay kit (Promega) following

263

the manufacturer’s protocol. Luminescence was measured using an EnVision 2104 Multilabel

264

Reader (Perkin Elmer). Cell viability was calculated as a percentage of control untreated cells.

265

EC50 values were determined using logistic non-linear regression analysis with Prism software

266

(GraphPad).

267


Page 12 of 54

Page 13 of 54

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


268

Serum stability of ADCs linker. Before sample preparation, rat serum was centrifuged at

269

12,000 g for 10 minutes followed by filtration through a 0.22 µm syringe filter. 200 µg of ADCs

270

were diluted into 990 µL of serum and incubated at 37°C under gentle rotation for zero, one,

271

three and seven days. Samples were kept frozen after each time point. The time zero samples

272

were immediately frozen after the serum dilution step. ADCs were immune-purified using 50 µL

273

of anti-human Fc agarose beads mixed with 300 µL of PBS and 100 µL of serum sample for 30

274

min at room temperature under continuous and gentle rotation. The beads were washed three

275

times with PBS to remove any unbound serum proteins using centrifugation at 1,000 x g for 1

276

minute at room temperature. ADCs were then eluted from the anti-human Fc agarose beads by

277

using 100 µL IgG elution buffer (Thermo Fisher Scientific), neutralized with 20 µL 1 M Tris pH

278

8, and reduced with 3 µL of 0.5 M DTT at 37°C for 30 min. 40 µL of the reduced samples were

279

analyzed by rLCMS as described above. Raw MS data were deconvoluted and peaks were

280

identified based on their molecular weight. Relative ratio of unconjugated versus conjugated

281

light and heavy chains were calculated and data were plotted as percentage of intact ADC

282

remaining in serum over time.

283 284

In vivo tumor growth inhibition and mice body size. All animal experiments were conducted

285

in accordance with the appropriate regulatory standards under protocols approved by the

286

MedImmune Institutional Animal Care and Use Committee. In vivo experiments were performed

287

using the anti-5T4-C239i-SG3249 and the isotope control antibody ICa-C239i-SG3249. 5- to 6-

288

week-old female athymic nude mice were purchased form Harlan Sprague Dawley Inc. Mice

289

were supplemented with estrogen pellets (0.36 mg, 60-day release; Innovative Research of

290

America), implanted subcutaneously into the dorsal flank the day before tumor cells inoculation.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

291

Ten million of MDA-MB-361 breast adenocarcinoma cells, which express the oncofetal antigen

292

5T4, were prepared in 50% Matrigel (Corning) and injected subcutaneously into the 2nd

293

mammary fat pad of mice. When tumors reached approximately 150 to 200 mm3, mice were

294

randomized based upon tumor volume and assigned into groups (n=10, each group). Anti-5T4-

295

C239i-SG3249 and ICa-C239i-SG3249 at 0.3 and 0.1 mg/kg were administered as a single dose

296

intravenously. Untreated mice were included as positive controls. Tumor volumes were

297

measured twice weekly with calipers, and were calculated using the formula: Tumor volume =

298

(1/2 x Length x Width)2/100. Body weights were monitored twice weekly. Tumor growth and

299

body weight data were plotted using Prism5 software (Prism). Tumor volumes and body weight

300

are expressed as mean ± SEM

301 302

RESULTS

303 304

Cytotoxic drug used for site-specific conjugation. The cytotoxic drug used for site-specific

305

conjugation is the pyrrolobenzodiazepine dimer SG3249.33 PBD dimers are a class of potent and

306

sequence-selective DNA minor groove interstrand cross-linking agents, which have shown

307

activity against both solid tumors and hematological malignancies.40,41 PBD-containing ADCs,

308

including SG3249, are also in clinical development,15,16,42,43 The warhead (i.e. the DNA cross-

309

linking component) of SG3249 is SG3199 (Figure 1A, red), which has at one of the two N10

310

positions a linker composed of maleimide, PEG8, valine-alanine, and a self-immolative PABA

311

group (Figure 1A). The conjugation of SG3249 to the engineered thiols in the mAb occurs

312

through formation of a thiosuccinimide linkage (Figure 1B). SG3199 is released from the ADCs


Page 14 of 54

Page 15 of 54

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


313

in the lysosomal compartment by Cathepsin B, which cleaves the valine-alanine dipeptide in

314

SG3249 (Figure 1A, blue).

315 316

Selection of sites in the antibody for cysteine insertion. The sites selected for inserting

317

cysteines in the mAb were chosen to be before and after positions that have been successfully

318

mutated to cysteine and used for the preparation of site-specific ADCs. 9,10,11,15,16 These sites are

319

A114 in the CH1 domain, V205 in the CL kappa domain of the light chain and S239 in the CH2

320

domain (Figure 2). These three sites are solvent accessible, shown in red in the surface and

321

ribbon representations in Figure 2B, and can be conjugated to high efficiency (>90%) using

322

maleimide functionalized cytotoxic drugs such as auristatins,9,11 tubulysins10 and PBD

323

dimers.15,16 The ADCs prepared using S239C and V205C were stable in serum,9,11,15, 16 while the

324

conjugate prepared at A114C was susceptible to drug loss in serum due to drug exchange

325

associated with the thiosuccinimide linkage.9,11 However, this drug loss propensity can be

326

prevented through selective hydrolysis of the thiosuccinimide linkage,44 by using self-

327

hydrolyzing maleimides,26-28 and other maleimide derivatives.29

328 329

Expression and purification of native, cysteine-substituted and cysteine-inserted antibodies.

330

The native, cysteine-substituted and cysteine-inserted antibodies were transiently expressed in

331

CHO-G22 (1C1 and IC) and HEK293f (5T4 and ICa) cells for 14 and 10 days, respectively. All

332

antibodies have high transient expression levels (Table 2 and Supplementary Figure 2A, 2I),

333

demonstrating that cysteine-substitution (as reported previously)9,10,15,16 and cysteine-insertion at

334

the selected sites do not impair antibody expression. All native antibodies and cysteine variants

335

were purified using protein A chromatography, concentrated to 3 mg/mL and formulated in 25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

336

mM Histidine-HCl pH 6 and PBS pH 7.2. SEC-HPLC analysis showed that native antibodies and

337

antibody cysteine variants have a monomeric content after protein A purification greater than

338

95% (Table 2 and Supplementary Figure 2A-B, 2I-J), except the antibodies with cysteine-

339

inserted before and after position 114, which have a monomeric content between 90 to 92 %

340

(Table 2). Molecular weight of native and antibody cysteine variants was confirmed using

341

rLCMS (Supplementary Figure 1 top panels; Supplementary Figure 2E, 2 M).

342 343

Thermostability of antibodies and antibody cysteine variants. Differential scanning

344

calorimetry (DSC) was used to assess thermostability of the antibody cysteine variants.

345

Transition temperatures (Tm) determined using DSC for each of the cysteine-inserted antibodies

346

were compared to the cysteine mutants at the same site and to the native antibodies (Figure 3).

347

The data showed that cysteine mutation at S239 and V205 result in same Tm as the native anti-

348

EphA2 antibody 1C1, while cysteine mutation at A114 result in an additional Tm of 71°C, which

349

is 2°C lower than the lowest Tm of the native antibody 1C1 (Figure 3). When the cysteine-

350

inserted antibodies before and after S239 were compared to the cysteine mutant at S239, a Tm of

351

63°C is observed in both cysteine-inserted antibodies (Figure 3). When the antibody variants

352

with cysteine-inserted before and after A114 were compared to cysteine mutant at A114, the

353

DSC data showed that cysteine insertions maintain similar Tm as the cysteine mutant, even if

354

deconvolution of the DSC data showed distinct Tm transitions of the antibody domains (Figure

355

3). When comparing the cysteine mutant at position V205 with the cysteine-inserted before and

356

after V205, similar Tm were observed among these variants and the native antibody 1C1 (Figure

357

3). Similar data were also obtained for the IC antibody variants (Supplementary Figure 3).

358


Page 16 of 54

Page 17 of 54

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


359

Site-specific conjugation and analytical characterization of the ADCs. Site-specific

360

conjugation was carried out by reducing the cysteine-mutants and cysteine-inserted antibodies

361

with TCEP followed by oxidation using dehydroascorbic acid to reform the antibody interchain

362

disulfide bonds, which were reduced during TCEP treatment. Antibody concentration used for

363

conjugation ranged from 2 to 8 mg/mL. Site-specific conjugation was achieved by the addition

364

of 8 molar equivalents of SG3249 for one hour at room temperature. CHT chromatography was

365

used to remove the unreacted payload and the macromolecular aggregates that were formed

366

during conjugation. The purified ADCs were > 97% monomer as determined using SEC-HPLC

367

(Figure 4; Supplementary Figure 2B, 2J).

368

Drug to antibody ratio (DAR) of ADCs was determined using rLCMS (Supplementary

369

Figure 1 bottom panels; Supplementary Figure 2) and ranged from 1.24 to 1.9, corresponding

370

to an efficiency of conjugation from 62 to 95% (Table 3). The efficiency of site-specific

371

conjugation was high for the S239C, C239i, V205C, C205i and A114C sites. However, less

372

conjugation efficiency was observed at C238i, C113i and C114i sites. rLCMS also confirmed

373

that conjugation was specific to the antibody chain where the engineered cysteines were

374

introduced (Supplementary Figure 1 bottom panels; Supplementary Figure 2). Chain-

375

specific conjugation was further confirmed using rRP-HPLC (Figure 5; Supplementary Figure

376

2). In fact, when conjugation was targeted at engineered cysteines in the heavy chain,

377

conjugation to the light chain was not observed (Figure 5; Supplementary Figure 1 bottom

378

panels). Similarly, when the conjugation was targeted at the engineered cysteines in the light

379

chain, no conjugation was observed to the heavy chain (Figure 5; Supplementary Figure 1

380

bottom panels).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

381

ADCs were further analyzed using HIC, which allows analysis of hydrophobicity change

382

between naked antibodies versus the ADCs, and to compare hydrophobicity of the ADCs

383

prepared at the different sites (Figure 6; Supplementary Figure 2D, 2L). As shown in Figure

384

6, HIC confirmed high efficiency of conjugation for all cysteine variants and showed that

385

conjugation site results in ADCs with different hydrophobicity characteristics. HIC analysis

386

(Figure 6) showed also the ADCs have a high DAR 2 drug load with a minor component of the

387

ADCs having DAR 1. By analyzing the retention time of the HIC main peak that corresponds to

388

the ADCs with DAR 2, the ADCs prepared at S239C, C238i and C239i for both 1C1 and IC

389

have shorter retention time (i.e. less hydrophobic properties) when compared to the other ADCs

390

(Figure 6). Less hydrophobic characteristics for ADCs may result in improved properties in vivo

391

as described previously.45

392 393

Thermostability of ADCs. As carried out for the unconjugated cysteine mutants and cysteine-

394

inserted antibody variants, DSC was used to assess the stability of the ADCs. The Tm for the

395

cysteine mutants ADCs and cysteine-inserted ADCs for 1C1 (Figure 7) and IC (Supplementary

396

Figure 4) are similar to the Tm of the naked cysteine mutants and cysteine-inserted antibodies

397

(Figure 3; Supplementary Figure 3), indicating no negative impact on antibody stability after

398

SG3249 conjugation at the engineered cysteines.

399 400

Binding of cysteine variant ADCs to EphA2 expressed on tumor cells. To confirm the

401

binding of the cysteine variant ADCs we used EphA2-expressing prostate cancer cells (PC-3).10

402

EphA2-negative breast adenocarcinoma cells (MDA-MB-361) were used as negative control.

403

Binding studies were carried out at a saturation concentration of 5 µg/mL using FACS


Page 18 of 54

Page 19 of 54

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


404

(Supplementary Figure 5). All 1C1 cysteine variant ADCs bind to PC-3 cells similarly to the

405

1C1 native antibody (Figure 8A). No binding was detected for the IC cysteine variant ADCs and

406

IC native antibody to PC-3 cells (Figure 8A). In addition, no binding to MDA-MD-361 was

407

observed for the IC and 1C1 cysteine variant ADCs, and the native antibodies (Figure 8B).

408 409

Cytotoxicity evaluation of ADCs. In order to compare functional activity of the cysteine-

410

mutants and cysteine-inserted ADCs, we tested cytotoxicity against EphA2-positive PC-3 cells

411

and EphA2-negative MDA-MD-361 cells as previously described.10 Taking into consideration

412

the differences in DARs among the ADCs and about 2-fold assay variability, the cytotoxic assay

413

showed that cysteine-inserted ADCs have potent cytotoxicity similar to the cysteine mutant

414

ADCs (Figure 9). As expected 1C1 and IC non-conjugated native antibodies and IC ADCs do

415

not kills PC-3 cells (Figure 9). In addition, neither the cysteine-inserted ADCs nor cysteine

416

mutants ADCs killed EphA2-negative MDA-MB-361 cells (data not shown).

417 418

Binding of cysteine variants and ADCs to FcRn and FcγγRs. To confirm that the ADCs have

419

similar binding to human FcRn like the non-conjugated native antibodies, we determined the

420

equilibrium constant KD using ProteOn for the binding to FcRn. Similar equilibrium constant KD

421

for the binding to FcRn between native antibodies and the cysteine-inserted ADCs is important

422

to maintaining similar in vivo half-life.10 As shown in Table 4, equilibrium constant KD for the

423

binding to FcRn at pH 6 is similar, within 2-fold experimental error, among the native anti-

424

EphA2 antibody and ADCs. As expected no binding was detected at pH 7.4 (Table 4). We were

425

also interested in determining the equilibrium constant KD of native antibodies and ADCs to

426

FcγRs. Because the binding of IgG1 to FcγRs involve the Fc domain of the IgG,46 we were



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

427

expecting that the cysteine mutants and cysteine-inserted ADCs at 114 and 205, which are in the

428

Fab domain of the IgG1 (Figure 2), maintain similar binding to FcγRs as the native antibodies.

429

As shown in Table 4, the equilibrium constant KD was similar between the 1C1 native antibody

430

and the ADCs prepared by cysteine-mutagenesis and cysteine-insertions at site 114 and 205. At

431

the same time, we were expecting the ADCs prepared by cysteine-mutagenesis and insertions at

432

239 to have decreased binding to FcγRs since the crystal structure of Fc in complex with FcγRIII

433

showed that S239 in the Fc IgG1 domain makes key contact with both K158 and K117 in

434

FcγRIII.10,47 As shown in Table 4, the ADCs prepared by cysteine-mutagenesis at 239 have

435

much decreased equilibrium constant KD for the FcγRs. Interestingly, complete elimination of

436

binding to the FcγRs resulted from the two ADCs prepared by cysteine-insertion before and after

437

position 239 (Table 4).

438 439

Analysis of ADCs linker stability in rat serum. To study the stability of the ADCs linker in

440

vitro, we incubated the ADCs in rat serum up to seven days followed by affinity capture of the

441

ADCs and determination of ADCs linker remaining over-time using rLCMS. One of the

442

mechanisms of systemic toxicity of ADCs may arise from exchange reactions of the

443

thiosuccinimide drug-linker from the ADCs to thiol containing serum proteins (such as albumin),

444

and from reverse reactions where the thiosuccinimide drug-linker may be released from the

445

ADCs due to competition with thiol-containing serum compounds such as cysteine and

446

gluthathione (Figure 10a).2, 48 ADCs linker remaining over the duration of the study was

447

compared to the ADCs linker at time zero. Remaining ADCs linker was determined after one,

448

three and seven days incubation in rat serum. The ADCs prepared using cysteine insertion before

449

239 (C238i) showed about 33% of drug-linker loss after seven days, while the ADCs prepared


Page 20 of 54

Page 21 of 54

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


450

using cysteine-mutagenesis at 239 (S239C) and cysteine insertion after 239 (C239i) showed 5%

451

of drug-linker loss (Figure 10B). The ADC prepared by cysteine-mutagenesis at 114 (A114C)

452

had 22% of drug-linker loss after seven days, while the cysteine-inserted ADCs before and after

453

114 had 35% (C113i) and 25% (C114i) of drug-linker loss after seven days, respectively (Figure

454

10C). Finally, the ADC prepared by cysteine-mutagenesis at 205 (V205C) had 5% of drug-linker

455

loss after seven days, while the cysteine-inserted ADCs before and after 205 had 25% (C204i)

456

and 17% (C205i) of drug loss after seven days, respectively (Figure 10D).

457 458

In vivo tumor growth inhibition and body weight change. In vivo tumor growth inhibition

459

and body weight change in nude mice was carried out using an anti-5T4 antibody,35,36 and an

460

isotype control antibody ICa with the cysteine-insertion after site 239 (C239i) conjugated to

461

SG3249 (Supplementary Figure 2). The oncofetal protein 5T4 is a cell surface antigen

462

expressed in a variety of solid tumors, whereas expression in normal adult tissues is limited.34-36

463

The restricted tumor expression and the rapid internalization upon antibody binding makes 5T4

464

an attractive target to efficiently deliver cytotoxic drugs into tumor cells.34-36,49 Among the

465

cysteine-inserted variants we chose the C239i for in vivo efficacy and body weight change

466

studies since the ADC prepared at this site is stable in serum (Figure 10), and most important

467

have abolished FcγRs binding (Table 4), which may result in mitigating off-target toxicities by

468

preventing uptake of the ADC by cells of the immune system that express FcγRs.10 As shown in

469

Figure 11A, the anti-5T4-C239i ADC showed potent dose-dependent tumor growth inhibition

470

upon a single i.v. dose in a mouse xenograft model of human breast adenocarcinoma (MDA-MB-

471

361) expressing the 5T4 oncofetal protein. No tumor growth inhibition was observed with the



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

472

ICa ADC (Figure 11A). There were no significant changes in body weight during the studies

473

between each treatment group (Figure 11B).

474 475

DISCUSSION

476 477

During the past few years several methods have been described for preparing site-specific

478

ADCs.1,2,9,10,15,16 Amongst these methods, engineering cysteines in the antibodies has been

479

shown to be a simple and efficient method for the preparation of site-specific ADCs, some of

480

which are in clinical development.16,50 The cysteine-based antibody engineering for preparing

481

ADCs is based on site-specific mutations, where a surface exposed residue in the antibody

482

constant domains is mutated to cysteine.16,50 Cysteine-mutagenesis at solvent accessible residues

483

of antibodies or other proteins is not a straightforward method. Surface exposed cysteines may

484

promote dimer and aggregate through the formation of disulfide bond between the engineered

485

cysteines; may introduce disulfide bond with other cysteines thus rendering the engineered

486

cysteines non-reactive and also contributing to destabilization of the antibody or protein

487

structure; may undergo oxidation that can potentially lead to the loss of function. Despite these

488

challenges, the ADCs based on engineering surface cysteines are advancing the ADCs field from

489

the first generation of non-homogeneous and non-stable ADCs to a new generation of well-

490

defined and stable ADCs, some of which are progressing to the clinic.16,50

491

We sought to investigate if an alternative to cysteine-mutagenesis, consisting of inserting

492

cysteines before and after defined sites in the antibody, would result in homogeneous, stable and

493

differentiated ADCs. We found that inserting cysteines at selected sites in the antibodies is an


Page 22 of 54

Page 23 of 54

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


494

efficient alternative to traditional cysteine-mutagenesis for the preparation of site-specific and

495

stable ADCs.

496

The sites we choose for cysteine-insertion were before and after the sites in the antibodies

497

that were previously reported for the preparation of site-specific ADCs. These sites are A114 and

498

S239 in the heavy chain antibody constant domains and V205 in the antibody kappa light chain

499

constant domain. Insertion of cysteines before and after these sites didn’t impact recombinant

500

antibody expression and purification. This indicates that inserting cysteines at the selected sites

501

result in maintaining IgG1-like folding and structure, which was confirmed by studying the

502

melting temperatures of the cysteine-inserted antibodies using differential scanning calorimetry

503

(DSC). DSC showed that the cysteine-inserted antibodies before and after 114 and 205

504

maintained melting temperatures similar to that of the native antibodies. However, the cysteine-

505

inserted antibodies before and after position 239 resulted in a lower melting temperatures when

506

compared to the cysteine mutants at position 239 and to the native antibodies. Conjugation of

507

SG3249 to the cysteine-inserted antibodies maintained transition temperatures similar to the non-

508

conjugated cysteine-inserted antibodies, demonstrating no structure destabilization after SG3249

509

conjugation.

510

ADCs prepared using the cysteine insertion approach maintain binding to target antigen

511

and exhibit potent in vitro cytotoxicity similar to the native non-conjugated antibodies and to the

512

ADCs prepared using cysteine-mutagenesis. Serum stability studies showed that the cysteine-

513

inserted ADC after 239 (C239i) is as stable as the ADC prepared by cysteine-mutagenesis at 239,

514

while drug-linker loss was observed for cysteine-inserted ADC before 239 (C238i). The ADCs

515

prepared by cysteine-insertion before and after the previously reported unstable position 114,9,11

516

also have significaant drug-linker loss after seven days incubation in rat serum. Drug linker loss



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

517

is also observed for the cysteine-inserted ADCs before and after 205 compared to the ADCs

518

prepared by mutagenesis at 205. It has to be noted that drug-linker loss is not a concern for ADcs

519

development since methods for stabilizing the thiossuccinamide-linkage can be employed to

520

stabilize the cysteine-inserted ADCs.26-30

521

Antibody half-life is mediated by the neonatal Fc receptor (FcRn), which protects

522

antibodies from catabolism.51 Therefore, an essential feature of ADCs is to maintain binding to

523

FcRn similar to the naked antibodies. The cysteine-inserted ADCs bind to FcRn similar to the

524

native antibodies, suggesting the ADCs may have an in vivo half-life similar to native antibodies.

525

In addition to binding to FcRn, immune effector functions mediated by the interaction of Fc

526

domain of antibodies with other Fcγ receptors (FcγRs) and complement proteins are important

527

for many therapeutic applications, e.g. elimination of targeted cells via antibody-dependent

528

cellular-cytotoxicity (ADCC), -phagocytosis (ADCP) or complement-dependent cytotoxicity

529

(CDC).52-55 However, in therapeutic applications where the Fc-mediated mechanism of action is

530

not desirable, such as with ADCs,10,56 it may be beneficial to have a silent Fc with no or much

531

reduced binding to FcγRs. A silent Fc could be a desired component of the ADCs since emerging

532

data suggest that potential dose-limiting toxicity of ADCs comes from the non-specific uptake of

533

the ADCs through the binding of the ADCs Fc to cells expressing FcγRs.56 For this reason,

534

developing ADCs with a silent Fc may be beneficial to improve the ADCs therapeutic index.10,50

535

ADCs prepared by cysteine-mutagenesis at S239 have been shown to lack FcγRs binding.10,16,50

536

Therefore, we examined binding of the cysteine-inserted ADCs to FcγRs. Our studies confirmed

537

that the cysteine mutants and insertions before and after 114 and 205 maintain FcγRs binding

538

similar to the native antibodies, which is expected since for these ADCs the cysteine mutations

539

and insertions are in the Fab domain of the antibody. The ADCs prepared by cysteine-


Page 24 of 54

Page 25 of 54

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


540

mutagenesis at 239 have much reduced FcγRs binding as described earlier,10 however the ADCs

541

prepared by cysteine-insertion before and after 239 resulted in complete elimination of FcγRs

542

binding. Therefore, ADCs based on C239i may mitigate the dose limiting toxicities observed

543

with ADCs that have IgG1-like effector functions.55 The potent anti-tumor activity of C239i

544

ADC was demonstrated using a xenograft model of human breast adenocarcinoma expressing the

545

oncofetal protein 5T4. The C239i ADC was also well-tolerated in mice since no significant body

546

weight changes were observed. Furthermore, ADCs prepared at C239i using SG3249 and

547

tubulysin10,57 have been used in combination studies with multiple immunotherapies aimed to

548

increase potential clinical responces.58

549 550

AUTHORS CONTRIBUTIONS

551 552

Conceived and designed the experiments: ND, CG. Performed the experiments: RF, BB,

553

KK, ND, HZ, CF, RJC. Analyzed the data: all authors. Wrote the paper: ND. Provided support:

554

HW. Provided critical review of the paper: all authors.

555 556

ACKNOWOLEDGMENTS

557 558

We are grateful to Jeremy Parker at AstraZeneca for helping procuring SG3249 and our

559

colleagues at Spirogen for the development of pyrrolobenzodiazepine dimers. This study was

560

supported by MedImmune, which is a wholly owned subsidiary of AstraZeneca.

561 562

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

563

The authors are employees of MedImmune and own AstraZeneca stocks.

564 565 566

FIGURE LEGENDS

567 568

Figure 1. Structure of SG3249 and example of an ADC prepared by conjugating SG3249 to

569

engineered cysteines in the mAb. (A) Structure of SG3249. Red denotes the warhead, which is

570

SG3199. The self-immolative spacer para-aminobenzyl alcohol (PABA) is in black. The

571

dipeptide valine-alanine, which is the Cathepsin B recognition site, is in blue. The PEG8 spacer

572

is in magenta; and the maleimide is in green. The molecular weight of SG3249 is 1496 Dalton.

573

(B) Cartoon representation of an ADC prepared by conjugation of SG3249 to engineered

574

cysteines in the mAb. The maleimide in SG3249 reacts with the engineered cysteines in the mAb

575

to form a succinimidyl thioether linkage. The resulting ADC has a drug to antibody ratio (DAR)

576

of two.

577 578

Figure 2. Cysteine-mutagenesis and insertion sites. (A) Sites selected for cysteine-mutagenesis

579

and insertions mapped on a mAb cartoon representation, (B) on the Fc and Fab structures

580

(surface and ribbon representations, left and right, respectively). The cysteine mutation and

581

insertion sites are shown in red in the cartoon and structure representations. (C) Amino acid

582

sequences of the constant domains of the light chain kappa CLκ, CH1 and CH2. Residues

583

selected for cysteine-mutagenesis and insertion before and after the selected sites are shown in

584

red. The sites are V205 in the CLκ, A114 in the CH1 and S239 in the CH2.

585


Page 26 of 54

Page 27 of 54

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


586

Figure 3. Differential scanning calorimetry (DSC) of anti-EphA2 1C1 native antibodies and

587

cysteine variants. The transition temperatures in °C are schematically shown for each antibody

588

variants.

589 590

Figure 4. Size-exclusion chromatography (SEC-HPLC) of unconjugated native antibodies and

591

ADCs. (A) SEC-HPLC of 1C1-Native and 1C1-ADCs. (B) SEC-HPLC of IC-Native and IC-

592

ADCs. The SEC-HPLC for unconjugated native antibodies is shown in red. The ADCs were

593

purified using CHT prior SEC-HPLC analysis. The x-axis is the retention time in minutes and

594

the y-axis is the absorbance at 280 nm. The two native antibodies and all ADCs have a retention

595

time of 8.6 minutes with a monomeric content of 97%.

596 597

Figure 5. Reduced reverse phase liquid chromatography (rRP-HPLC) of unconjugated

598

antibodies and ADCs. Chromatograms are red and blue for unconjugated antibodies and ADCs,

599

respectively. The chromatograms for the two native antibodies, 1C1-Native and IC-Native, are

600

also shown in red for comparative analysis. LC denotes the light chain; LC +1 is the light chain

601

conjugated to one SG3249; HC is the heavy chain; HC +1 is the heavy chain conjugated to one

602

SG3249. The y-axis is the absorbance at 280 nm and the x-axis is the elution time in minutes.

603 604

Figure 6. Hydrophobic interaction chromatography (HIC-HPLC) of unconjugated antibodies and

605

ADCs. Chromatograms are red and blue for unconjugated antibodies and ADCs, respectively.

606

The chromatograms for the two native antibodies, 1C1-Native and IC-Native, are also shown for

607

comparative analysis. The y-axis is the absorbance at 280 nm and the x-axis is the elution time in

608

minutes.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

609 610

Figure 7. Differential scanning calorimetry (DSC) of anti-EphA2 1C1 ADCs. The transition

611

temperatures in °C are schematically shown.

612 613

Figure 8. FACS binding of native antibodies and ADCs to EphA2 positive (A) and EphA2

614

negative (B) cells. The geometric mean fluorescence intensity is shown in the y-axis while the

615

antibodies are listed in the x-axis. All antibodies were tested at 5 µg/mL. The EphA2 positive

616

cell line was human prostate cancer (PC-3), and the EphA2 negative cell line was breast

617

adenocarcinoma (MDA-MB-361). All 1C1 cysteine ADCs bind to EphA2 positive cancer cells

618

similarly to the native 1C1 antibody; while the IC antibodies show no binding.

619 620

Figure 9. In vitro cytotoxicity of ADCs. PC-3 cells were treated with serial dilutions of the

621

ADCs and cell viability is shown as relative percentage to the untreated controls. Error bars

622

indicate SD (n = 3). EC50 (ng/mL) and DAR are shown for each 1C1 ADCs. Negative control

623

antibody (IC) and negative control ADCs (IC-ADCs) have no cytotoxic activity.

624 625

Figure 10. Rat serum stability of ADCs linker. (A) Illustration of the deconjugation mechanism

626

from ADCs in vivo. The retro-Michael reaction regenerates maleimide groups, which may react

627

with endogenous thiols (X-SH) in circulation such as albumin or glutathione to promote

628

deconjugation of the linker-drug from ADCs. (B) ADC linker stability at C238i, S239C and

629

C239i. (C) ADC linker stability at C113i, A114C and C114i. (D) ADC linker stability at C204i,

630

V205C and C205i. The ADCs used in these studies were from antibody IC. In the graphical

631

representations the y-axis is the percentage of intact ADC, and the x-axis represents the different


Page 28 of 54

Page 29 of 54

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


632

ADCs. Time points are schematically shown. These data were determined using rLCMS. The

633

percentage of ADC linker loss over the duration of the study is schematically labelled for each

634

ADCs.

635 636

Figure 11. Anti-tumor activity and body weight change of 5T4-C239i-SG3249 and ICa-C239i-

637

SG3249. (A) Tumor growth curves for MDA-MB-361 breast adenocarcinoma cells after a single

638

i.v. treatment with 5T4-C239i-SG3249 (0.1 and 0.3 mg/kg) and ICa-C239i-SG3249 (0.3 mg/kg).

639

(B) Body weight change for 5T4-C239i-SG3249 (0.1 and 0.3 mg/kg) and ICa-C239i-SG3249

640

(0.3 mg/kg).Untreated mice were used as positive control. Tumor volumes and body weight are

641

expressed as mean ± SEM.

642 643

REFERENCES

644 645 646

(1) Gébleux R, Casi G. Antibody-drug conjugates: Current status and future perspectives. Pharmacol Ther 2016, DOI: 10.1016/j.pharmthera.2016.07.012.

647

(2) Polakis P. Antibody Drug Conjugates for Cancer Therapy. Pharmacol Rev 2016, 68, 3-19.

648

(3) Lambert JM, Chari RV. Ado-trastuzumab Emtansine (T-DM1): an antibody-drug conjugate

649 650

(ADC) for HER2-positive breast cancer. J Med Chem 2014 57, 6949-6964. (4) Senter PD, Sievers EL. The discovery and development of brentuximab vedotin for use in

651

relapsed Hodgkin lymphoma and systemic anaplastic large cell lymphoma. Nat Biotechnol

652

2012, 30, 631-637.

653 654

(5) Jerjian TV, Glode AE, Thompson LA, O'Bryant CL. Antibody-Drug Conjugates: A Clinical Pharmacy Perspective on an Emerging Cancer Therapy. Pharmacotherapy 2016, 36, 99-116.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

655

(6) Bakhtiar R. Antibody drug conjugates. Biotechnol Lett 2016 38, 1655-1664.

656

(7) Hamblett KJ, Senter PD, Chace DF, Sun MM, Lenox J, Cerveny CG, Kissler KM, Bernhardt

657

SX, Kopcha AK, Zabinski RF, Meyer DL, Francisco JA. Effects of drug loading on the

658

antitumor activity of a monoclonal antibody drug conjugate. Clin. Cancer Res 2004, 10,

659

7063−7070.

660

(8) Akkapeddi P, Azizi S-A, Freedy AM, Cal PMSD, Gois PMP, Bernardes GJL. Construction

661

of homogeneous antibody–drug conjugates using site-selective protein chemistry. Chem. Sci

662

2016, 7, 2954-2963.

663

(9) Junutula JR, Raab H, Clark S, Bhakta S, Leipold DD, Weir S, Chen Y, Simpson M, Tsai SP,

664

Dennis MS, Lu Y, Meng YG, Ng C, Yang J, Lee CC, Duenas E, Gorrell J, Katta V, Kim A,

665

McDorman K, Flagella K, Venook R, Ross S, Spencer SD, Lee Wong W, Lowman HB,

666

Vandlen R, Sliwkowski MX, Scheller RH, Polakis P, Mallet W. Site-specific conjugation of

667

a cytotoxic drug to an antibody improves the therapeutic index. Nat Biotechnol 2008, 26,

668

925-932.

669

(10)

Thompson P, Fleming R, Bezabeh B, Huang F, Mao S, Chen C, Harper J, Zhong H, Gao

670

X, Yu XQ, Hinrichs MJ, Reed M, Kamal A, Strout P, Cho S, Woods R, Hollingsworth RE,

671

Dixit R, Wu H, Gao C, Dimasi N. Rational design, biophysical and biological

672

characterization of site-specific antibody-tubulysin conjugates with improved stability,

673

efficacy and pharmacokinetics. J Control Release 2016 236, 100-116.

674

(11)

Shen BQ, Xu K, Liu L, Raab H, Bhakta S, Kenrick M, Parsons-Reponte KL, Tien J, Yu

675

SF, Mai E, Li D, Tibbitts J, Baudys J, Saad OM, Scales SJ, McDonald PJ, Hass PE,

676

Eigenbrot C, Nguyen T, Solis WA, Fuji RN, Flagella KM, Patel D, Spencer SD, Khawli LA,

677

Ebens A, Wong WL, Vandlen R, Kaur S, Sliwkowski MX, Scheller RH, Polakis P, Junutula


Page 30 of 54

Page 31 of 54

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


678

JR. Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug

679

conjugates. Nat Biotechnol 2012 30, 184-189.

680

(12)

Lyons A, King DJ, Owens RJ, Yarranton GT, Millican A, Whittle NR, Adair JR. Site-

681

specific attachment to recombinant antibodies via introduced surface cysteine residues.

682

Protein Eng 1990, 3, 703-708.

683

(13)

Stimmel JB, Merrill BM, Kuyper LF, Moxham CP, Hutchins JT, Fling ME, Kull FC Jr.

684

Site-specific conjugation on serine right-arrow cysteine variant monoclonal antibodies. J Biol

685

Chem 2000, 275, 30445-30450.

686 687 688

(14)

Voynov V, Chennamsetty N, Kayser V, Wallny HJ, Helk B, Trout BL. Design and

application of antibody cysteine variants. Bioconjug Chem 2010 21, 385-392. (15)

Jeffrey SC, Burke PJ, Lyon RP, Meyer DW, Sussman D, Anderson M, Hunter JH, Leiske

689

CI, Miyamoto JB, Nicholas ND, Okeley NM, Sanderson RJ, Stone IJ, Zeng W, Gregson SJ,

690

Masterson L, Tiberghien AC, Howard PW, Thurston DE, Law CL, Senter PD. A potent anti-

691

CD70 antibody-drug conjugate combining a dimeric pyrrolobenzodiazepine drug with site-

692

specific conjugation technology. Bioconjug Chem 2013 24, 1256-1263.

693

(16)

Kung Sutherland MS, Walter RB, Jeffrey SC, Burke PJ, Yu C, Kostner H, Stone I, Ryan

694

MC, Sussman D, Lyon RP, Zeng W, Harrington KH, Klussman K, Westendorf L, Meyer D,

695

Bernstein ID, Senter PD, Benjamin DR, Drachman JG, McEarchern JA. SGN-CD33A: a

696

novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active

697

in models of drug-resistant AML. Blood 2013 122, 1455-1463.

698

(17)

Shiraishi Y, Muramoto T, Nagatomo K, Shinmi D, Honma E, Masuda K, Yamasaki M.

699

Identification of highly reactive cysteine residues at less exposed positions in the Fab

700

constant region for site-specific conjugation. Bioconjug Chem 2015 26, 1032-1040.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

701

(18)

Shinmi D, Taguchi E, Iwano J, Yamaguchi T, Masuda K, Enokizono J, Shiraishi Y. One

702

Step Conjugation Method for Site-Specific Antibody-Drug Conjugates through Reactive

703

Cysteine-Engineered Antibodies. Bioconjug Chem 2016 27, 1324-1331.

704

(19)

Zimmerman ES, Heibeck TH, Gill A, Li X, Murray CJ, Madlansacay MR, Tran C, Uter

705

NT, Yin G, Rivers PJ, Yam AY, Wang WD, Steiner AR, Bajad SU, Penta K, Yang W,

706

Hallam TJ, Thanos CD, Sato AK. Production of site-specific antibody-drug conjugates using

707

optimized non-natural amino acids in a cell-free expression system. Bioconjug Chem 2014

708

25, 351-361.

709

(20)

Tian F, Lu Y, Manibusan A, Sellers A, Tran H, Sun Y, Phuong T, Barnett R, Hehli B,

710

Song F, DeGuzman MJ, Ensari S, Pinkstaff JK, Sullivan LM, Biroc SL, Cho H, Schultz PG,

711

DiJoseph J, Dougher M, Ma D, Dushin R, Leal M, Tchistiakova L, Feyfant E, Gerber HP,

712

Sapra P. A general approach to site-specific antibody drug conjugates. Proc Natl Acad Sci

713

USA 2014, 111 1766-1771.

714

(21)

VanBrunt MP, Shanebeck K, Caldwell Z, Johnson J, Thompson P, Martin T, Dong H, Li

715

G, Xu H, D'Hooge F, Masterson L, Bariola P, Tiberghien A, Ezeadi E, Williams DG, Hartley

716

JA, Howard PW, Grabstein KH, Bowen MA, Marelli M. Genetically Encoded Azide

717

Containing Amino Acid in Mammalian Cells Enables Site-Specific Antibody-Drug

718

Conjugates Using Click Cycloaddition Chemistry. Bioconjug Chem 2015 26, 2249-2260.

719 720 721

(22)

Kornberger P, Skerra A. Sortase-catalyzed in vitro functionalization of a HER2-specific

recombinant Fab for tumor targeting of the plant cytotoxin gelonin. MAbs 2014 6, 354-366. (23)

Beerli RR, Hell T, Merkel AS, Grawunder U. Sortase Enzyme-Mediated Generation of

722

Site-specifically conjugated antibody drug conjugates with high in vitro and in vivo potency.

723

PLoS One 2015 10(7):e0131177.


Page 32 of 54

Page 33 of 54

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

724


(24)

Drake PM, Albers AE, Baker J, Banas S, Barfield RM, Bhat AS, de Hart GW, Garofalo

725

AW, Holder P, Jones LC, Kudirka R, McFarland J, Zmolek W, Rabuka D. Aldehyde tag

726

coupled with HIPS chemistry enables the production of ADCs conjugated site-specifically to

727

different antibody regions with distinct in vivo efficacy and PK outcomes. Bioconjug Chem

728

2014 25, 1331-1341.

729

(25)

Dennler P, Chiotellis A, Fischer E, Brégeon D, Belmant C, Gauthier L, Lhospice F,

730

Romagne F, Schibli R. Transglutaminase-based chemo-enzymatic conjugation approach

731

yields homogeneous antibody-drug conjugates. Bioconjug Chem 2014 25, 569-578.

732

(26)

Lyon RP, Setter JR, Bovee TD, Doronina SO, Hunter JH, Anderson ME,

733

Balasubramanian CL, Duniho SM, Leiske CI, Li F, Senter PD. Self-hydrolyzing maleimides

734

improve the stability and pharmacological properties of antibody-drug conjugates. Nat

735

Biotechnol 2014 32, 1059-1062.

736 737 738

(27)

Fontaine SD, Reid R, Robinson L, Ashley GW, Santi DV. Long-term stabilization of

maleimide-thiol conjugates. Bioconjug Chem 2015 26, 145-152. (28)

Christie RJ, Fleming R, Bezabeh B, Woods R, Mao S, Harper J, Joseph A, Wang Q, Xu

739

ZQ, Wu H, Gao C, Dimasi N. Stabilization of cysteine-linked antibody drug conjugates with

740

N-aryl maleimides. J Control Release 2015 220(Pt B), 660-670.

741

(29)

Kalia D, Malekar PV, Parthasarathy M. Exocyclic Olefinic Maleimides: Synthesis and

742

Application for Stable and Thiol-Selective Bioconjugation. Angew Chem Int Ed Engl 2016

743

55, 1432-1435.

744

(30)

Palanki MSS, Bhat A, Lappe RW, Liu B, Oates B, Rizzo J, Stankovic N, Bradshaw C.

745

Development of novel linkers to conjugate pharmacophores to a carrier antibody. Bioorg.

746

Med. Chem. Lett. 2012 22, 4249–4253.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

747

(31)

Bezabeh B, Fleming F, Fazenbaker C, Zhong H, Yu X-Q, Coffman K, Leow CC, Gibson

748

N, Wilson S, Stover K, Wu H, Gao G. Insertion of scFv into the hinge domain of full-length

749

IgG1 monoclonal antibody results in tetravalent bispecific molecule with robust properties..

750

MAbs 2016, In press.

751

(32)

Wozniak-Knopp G1, Bartl S, Bauer A, Mostageer M, Woisetschläger M, Antes B, Ettl K,

752

Kainer M, Weberhofer G, Wiederkum S, Himmler G, Mudde GC, Ruker. Introducing

753

antigen-binding sites in structural loops of immunoglobulin constant domains: Fc fragments

754

with engineered HER2/neu-binding sites and antibody properties. Protein Eng Des Sel 2010

755

23, 289-297.

756

(33)

Arnaud C. Tiberghien, Jean-Noel Levy, Luke A. Masterson, Neki V. Patel, Lauren R.

757

Adams, Simon Corbett, David G. Williams, John A. Hartley, and Philip W. Howard. Design

758

and Synthesis of Tesirine, a Clinical Antibody–Drug Conjugate Pyrrolobenzodiazepine

759

Dimer Payload. ACS Med Chem Lett 2016 7, 983-987..

760

(34)

Sapra P, Damelin M, Dijoseph J, Marquette K, Geles KG, Golas J, Dougher M,

761

Narayanan B, Giannakou A, Khandke K, Dushin R, Ernstoff E, Lucas J, Leal M, Hu G,

762

O'Donnell CJ, Tchistiakova L, Abraham RT, Gerber HP. Long-term tumor regression

763

induced by an antibody-drug conjugate that targets 5T4, an oncofetal antigen expressed on

764

tumor-initiating cells. Mol Cancer Ther 2013 12, 38-47.

765

(35)

Kerk S, Finkel K, Pearson AT, Warner K, Nor F, Zhang Z, Wagner VP, Vargas PA,

766

Wicha MS, Hurt E, Hollingsworth RE, Tice DA, Nor JE. 5T4-targeted therapy ablates cancer

767

stem cells and prevents recurrence of head and neck squamous cell carcinoma. Clin Cancer

768

Res. 2016 Oct 25. DOI: 10.1158/1078-0432.CCR-16-1834.


Page 34 of 54

Page 35 of 54

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

769


(36)

Harper H, Lloyd C, Dimasi N, Toader D, Marwood R, Lewis L, Bannister D, Jovanovic

770

J, Fleming R, Mao S, Marrero A, Korade III M, Strout P, Chen T, Wetzel L, Breen S,

771

Rebelatto M, Zhong H, Hurt E, Huang K, Tice DA, Hollingsworth A, Herbst R, Kamal A.

772

Evaluation of antibody-drug conjugate (ADC) payloads with different mechanisms of action

773

reveals a pyrrolobenzodiazepine-conjugated ADC targeting 5T4 with potent anti-cancer stem

774

cell activity. Under Revision

775

(37)

Matsumiya S, Yamaguchi Y, Saito J, Nagano M, Sasakawa H, Otaki S, Satoh M, Shitara

776

K, Kato K. Structural comparison of fucosylated and nonfucosylated Fc fragments of human

777

immunoglobulin G1. J Mol Biol 2007 368, 767-779.

778

(38)

Peng L, Oganesyan V, Damschroder MM, Wu H, Dall'Acqua WF. Structural and

779

functional characterization of an agonistic anti-human EphA2 monoclonal antibody. J Mol

780

Biol 2011 413, 90-405.

781

(39)

Dimasi N, Gao C, Fleming R, Woods RM, Yao XT, Shirinian L, Kiener PA, Wu H. The

782

design and characterization of oligospecific antibodies for simultaneous targeting of multiple

783

disease mediators. J Mol Biol 2009 393, 672-692.

784

(40)

Puzanov I, Lee W, Chen AP, Calcutt MW, Hachey DL, Vermeulen WL, Spanswick VJ,

785

Liao CY, Hartley JA, Berlin JD, Rothenberg ML. Phase I pharmacokinetic and

786

pharmacodynamic study of SJG-136, a novel DNA sequence selective minor groove cross-

787

linking agent, in advanced solid tumors. Clin Cancer Res 2011 17, 794-802.

788

(41)

Janjigian YY, Lee W, Kris MG, Miller VA, Krug LM, Azzoli CG, Senturk E, Calcutt

789

MW, Rizvi NA. A phase I trial of SJG-136 (NSC#694501) in advanced solid tumors. Cancer

790

Chemother Pharmacol 2010 65, 833-838.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

791

(42)

Flynn M, Zammarchi F, Tyrer PC, Akarca AU, Janghra N, Britten CE, Havenith CE,

792

Levy JN, Tiberghien A, Masterson LA, Barry C, d'Hooge F, Marafioti T, Parren PW,

793

Williams DG, Howard PW, van Berkel PH, Hartley JA. ADCT-301, a pyrrolobenzodiazepine

794

(PBD) dimer-containing antibody drug conjugate (ADC) targeting CD25-expressing

795

hematological malignancies. Mol Cancer Ther 2016 15, 2709-2721.

796

(43)

Saunders LR, Bankovich AJ, Anderson WC, Aujay MA, Bheddah S, Black K, Desai R,

797

Escarpe PA, Hampl J, Laysang A, Liu D, Lopez-Molina J, Milton M, Park A, Pysz MA,

798

Shao H, Slingerland B, Torgov M, Williams SA, Foord O, Howard P, Jassem J, Badzio A,

799

Czapiewski P, Harpole DH, Dowlati A, Massion PP, Travis WD, Pietanza MC, Poirier JT,

800

Rudin CM, Stull RA, Dylla SJ. A DLL3-targeted antibody-drug conjugate eradicates high-

801

grade pulmonary neuroendocrine tumor-initiating cells in vivo. Sci Transl Med 2015 302,

802

302ra136.

803

(44)

Tumey LN, Charati M, He T, Sousa E, Ma D, Han X, Clark T, Casavant J, Loganzo F,

804

Barletta F, Lucas J, Graziani EI. Mild method for succinimide hydrolysis on ADCs: impact

805

on ADC potency, stability, exposure, and efficacy. Bioconjug Chem 2014 25, 1871-1880.

806

(45)

Lyon RP, Bovee TD, Doronina SO, Burke PJ, Hunter JH, Neff-LaFord HD, Jonas M,

807

Anderson ME, Setter JR, Senter PD. Reducing hydrophobicity of homogeneous antibody-

808

drug conjugates improves pharmacokinetics and therapeutic index. Nat Biotechnol. 2015 33,

809

733-735.

810 811 812 813

(46)

Nimmerjahn F, Ravetch JV. Fcgamma receptors: old friends and new family members.

Immunity 2006 24, 19-28. (47)

Sondermann P, Huber R, Oosthuizen V, Jacob U. The 3.2-A crystal structure of the

human IgG1 Fc fragment-Fc gammaRIII complex. Nature 2000 406, 267-273.


Page 36 of 54

Page 37 of 54

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

814 815 816


(48)

Baldwin AD, Kiick KL. Tunable degradation of maleimide-thiol adducts in reducing

environments. Bioconjug Chem 2011 22, 1946-1953. (49)

Shapiro GI, Vaishampayan UN, LoRusso P, Barton J, Hua S, Reich SD, Shazer R, Taylor

817

CT, Xuan D, Borghaei H. First-in-human trial of an anti-5T4 antibody-monomethylauristatin

818

conjugate, PF-06263507, in patients with advanced solid tumors. Invest New Drugs 2017 Jan

819

9 In Press

820

(50)

Li JY, Perry SR, Muniz-Medina V, Wang X, Wetzel LK, Rebelatto MC, Hinrichs MJ,

821

Bezabeh BZ, Fleming RL, Dimasi N, Feng H, Toader D, Yuan AQ, Xu L, Lin J, Gao C, Wu

822

H, Dixit R, Osbourn JK, Coats SR. A Biparatopic HER2-Targeting Antibody-Drug

823

Conjugate Induces Tumor Regression in Primary Models Refractory to or Ineligible for

824

HER2-Targeted Therapy. Cancer Cell 2016 29, 117-129.

825 826 827

(51)

Simister NE, Mostov KE. An Fc receptor structurally related to MHC class I antigens.

Nature 1989 337, 184–187. (52)

Abès R, Dutertre CA, Agnelli L, Teillaud JL. Activating and inhibitory Fcgamma

828

receptors in immunotherapy: being the actor or being the target. Expert Rev Clin Immunol.

829

2009 5, 735-747.

830 831 832 833 834

(53)

Desjarlais JR, Lazar GA. Modulation of antibody effector function. Exp Cell Res. 2011

317, 1278-1285. (54)

Gessner JE, Heiken H, Tamm A, Schmidt RE. The IgG Fc receptor family. Ann Hematol.

1998 6, 231-248. (55)

Jiang XR, Song A, Bergelson S, Arroll T, Parekh B, May K, Chung S, Strouse R, Mire-

835

Sluis A, Schenerman M. Advances in the assessment and control of the effector functions of

836

therapeutic antibodies. Nat Rev Drug Discov. 2011 10, 101-111.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

837

(56)

Uppal H,Doudement E, Mahapatra K, Darbonne WC, Bumbaca D, Shen BQ, Du X, Saad

838

O, Bowles K, Olsen S, Lewis Phillips GD, Hartley D, Sliwkowski MX, Girish S, Dambach

839

D, Ramakrishnan V. Potential mechanisms for thrombocytopenia development with

840

trastuzumab emtansine (T-DM1). Clin. Cancer Res. 2015 21, 123–133.

841

(57)

Toader D, Wang F, Gingipalli L, Vasbinder M, Roth M, Mao S, Block M, Harper J,

842

Thota S, Su M, Ma J, Bedian V, Kamal A.Structure-Cytotoxicity Relationships of Analogues

843

of N14-Desacetoxytubulysin H. J Med Chem. 2016 59, 10781-10787.

844

(58)

Rios-Doria J, Harper J, Rothstein R, Wetzel L, Chesebrough J, Marrero A, Chen C,

845

Strout P, Mulgrew K, McGlinchey K, Fleming R, Bezabeh B, Meekin J, Stewart D, Kennedy

846

M, Martin P, Buchanan A, Dimasi N, Michelotti E, Hollingsworth R. Antibody-drug

847

conjugates bearing pyrrolobenzodiazepine or tubulysin payloads are immunomodulatory and

848

synergize with multiple immunotherapies. Cancer Research 2017, In Press

849


Page 38 of 54

Page 39 of 54

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

850


Table 1: Designation of antibody used herein. Name 1C1-native 1C1-S239C 1C1-C238i 1C1-C239i 1C1-A114C 1C1-C113i 1C1-C114i 1C1-V205C 1C1-C204i 1C1-C205i

Description anti-EphA2 mAb with native sequence anti-EphA2 mAb with mutation S239C in the CH2 domain anti-EphA2 mAb with a cysteine-inserted after position 238 in the CH2 domain anti-EphA2 mAb with a cysteine-inserted after position 239 in the CH2 domain anti-EphA2 mAb with mutation A114C in the CH1 domain anti-EphA2 mAb with a cysteine-inserted after position 113 in the CH1 domain anti-EphA2 mAb with a cysteine-inserted after position 114 in the CH1 domain anti-EphA2 mAb with mutation V205C in the CLκ domain anti-EphA2 mAb with a cysteine-inserted after position 204 in the CLκ domain anti-EphA2 mAb with a cysteine-inserted after position 205 in the CLκ domain

Name IC-native IC-S239C IC-C238i IC-C239i IC A114C IC-C113i IC-C114i IC V205C IC-C204i IC-C205i

Description Isotype control mAb with native sequence Isotype control mAb with mutation S239C in the CH2 domain Isotype control mAb with a cysteine-inserted after position 238 in the CH2 domain Isotype control mAb with a cysteine-inserted after position 239 in the CH2 domain Isotype control mAb with mutation A114C in the CH1 domain Isotype control mAb with a cysteine-inserted after position 113 in the CH1 domain Isotype control mAb with a cysteine-inserted after position 114 in the CH1 domain Isotype control mAb with mutation V205C in the CLκ domain Isotype control mAb with a cysteine-inserted after position 204 in the CLκ domain Isotype control mAb a with cysteine-inserted after position 205 in the CLκ domain

851 852

When the above antibody variants are conjugated to SG3249 a designation ADC is used (e.g. 1C1-S239C-ADC). 1C1 is an anti-

853

EphA2 human IgG1 antibody with kappa light chain; IC is an isotype non-binding human IgG1 antibody with kappa light chain.

854



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Page 40 of 54

855

Table 2: Transient expression in CHO cells after 14 days of culture and monomeric content of native, cysteine-substituted and

856

cysteine-inserted antibodies. Name 1C1-Native 1C1-S239C 1C1-C238i 1C1-C239i 1C1-A114C 1C1-C113i 1C1-C114i 1C1-V205C 1C1-C204i 1C1-C205i

Expression (mg/L) 425 350 303 410 260 400 375 338 232 230

Monomer (%) 99 99 99 99 99 92 92 99 99 99

Name IC-Native IC-S239C IC-C238i IC-C239i IC A114C IC-C113i IC-C114i IC V205C IC-C204i IC-C205i

Expression (mg/L) 780 780 721 721 555 760 750 493 384 335

Monomer (%) 96 95 96 96 97 91 90 98 98 96

857 858

Transient expression level was determined using a protein A binding method and monomeric content was determined using SEC-

859

HPLC. Native represents the wild-type antibody.

860


Page 41 of 54

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

861


Table 3: Drug to antibody ratio (DAR) and efficiency of conjugation of ADCs. Name 1C1-S239C 1C1-C238i 1C1-C239i 1C1-A114C 1C1-C113i 1C1-C114i 1C1-V205C 1C1-C204i 1C1-C205i

DAR 1.90 1.44 1.80 1.86 1.36 1.24 1.96 1.84 1.80

Efficiency of conjugation (%) 95 72 90 93 68 62 98 92 90

Name IC-S239C IC-C238i IC-C239i IC A114C IC-C113i IC-C114i IC V205C IC-C204i IC-C205i

DAR 1.86 1.48 1.80 1.88 1.44 1.28 1.92 1.84 1.80

862 863

DAR was determined using rLCMS. Efficiency of conjugation determined as described in Methods.

864


Efficiency of conjugation (%) 93 74 90 94 72 64 96 92 90


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

865

Page 42 of 54

Table 4: Equilibrium KD (nM) determined using ProteOn for 1C1 native antibody and 1C1-ADCs binding to human FcγRs. FcγRs

Native

FcγRI FcγRIIA FcγRIIIA-158V FcγRIIIA-158F FcRn (pH 6) FcRn (pH 7.4)

2.67 649 136 1635 2580 NB

C204i ADC 1.17 667 226 2130 2340 NB

V205C ADC 1.82 599 219 1965 3660 NB

C205i ADC 1.65 591 220 1995 3240 NB

C113i ADC 2.15 665 204 1905 3920 NB

A114C ADC 3.98 645 196 1920 3770 NB

C114i ADC 5.8 650 119 1830 3630 NB

C238i ADC 58 NB NB NB 3810 NB

866 867

NB = No binding was detected. Similar data were obtained with the IC native and IC ADCs (data not shown).

868


S239C ADC 14.3 5140 3400 8340 3640 NB

C239i ADC 67 NB NB NB 2830 NB

Page 43 of 54

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Structure of SG3249 and example of an ADC prepared by conjugating SG3249 to engineered cysteines in the mAb. (A) Structure of SG3249. Red denotes the warhead, which is SG3199. The self-immolative spacer para-aminobenzyl alcohol (PABA) is in black. The dipeptide valine-alanine, which is the Cathepsin B recognition site, is in blue. The PEG8 spacer is in magenta; and the maleimide is in green. The molecular weight of SG3249 is 1496 Dalton. (B) Cartoon representation of an ADC prepared by conjugation of SG3249 to engineered cysteines in the mAb. The maleimide in SG3249 reacts with the engineered cysteines in the mAb to form a succinimidyl thioether linkage. The resulting ADC has a drug to antibody ratio (DAR) of two. 190x254mm (96 x 96 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Cysteine-mutagenesis and insertion sites. (A) Sites selected for cysteine-mutagenesis and insertions mapped on a mAb cartoon representation, (B) on the Fc and Fab structures (surface and ribbon representations, left and right, respectively). The cysteine mutation and insertion sites are shown in red in the cartoon and structure representations. (C) Amino acid sequences of the constant domains of the light chain kappa CLκ, CH1 and CH2. Residues selected for cysteine-mutagenesis and insertion before and after the selected sites are shown in red. The sites are V205 in the CLκ, A114 in the CH1 and S239 in the CH2. 190x254mm (96 x 96 DPI)


Page 44 of 54

Page 45 of 54

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Differential scanning calorimetry (DSC) of anti-EphA2 1C1 native antibodies and cysteine variants. The transition temperatures in °C are schematically shown for each antibody variants. 190x254mm (96 x 96 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Size-exclusion chromatography (SEC-HPLC) of unconjugated native antibodies and ADCs. (A) SEC-HPLC of 1C1-Native and 1C1-ADCs. (B) SEC-HPLC of IC-Native and IC-ADCs. The SEC-HPLC for unconjugated native antibodies is shown in red. The ADCs were purified using CHT prior SEC-HPLC analysis. The x-axis is the retention time in minutes and the y-axis is the absorbance at 280 nm. The two native antibodies and all ADCs have a retention time of 8.6 minutes with a monomeric content of 97%. 190x254mm (96 x 96 DPI)


Page 46 of 54

Page 47 of 54

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Reduced reverse phase liquid chromatography (rRP-HPLC) of unconjugated antibodies and ADCs. Chromatograms are red and blue for unconjugated antibodies and ADCs, respectively. The chromatograms for the two native antibodies, 1C1-Native and IC-Native, are also shown in red for comparative analysis. LC denotes the light chain; LC +1 is the light chain conjugated to one SG3249; HC is the heavy chain; HC +1 is the heavy chain conjugated to one SG3249. The y-axis is the absorbance at 280 nm and the x-axis is the elution time in minutes. 190x254mm (96 x 96 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Hydrophobic interaction chromatography (HIC-HPLC) of unconjugated antibodies and ADCs. Chromatograms are red and blue for unconjugated antibodies and ADCs, respectively. The chromatograms for the two native antibodies, 1C1-Native and IC-Native, are also shown for comparative analysis. The y-axis is the absorbance at 280 nm and the x-axis is the elution time in minutes. 190x254mm (96 x 96 DPI)


Page 48 of 54

Page 49 of 54

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Differential scanning calorimetry (DSC) of anti-EphA2 1C1 ADCs. The transition temperatures in °C are schematically shown. 190x254mm (96 x 96 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

FACS binding of native antibodies and ADCs to EphA2 positive (A) and EphA2 negative (B) cells. The geometric mean fluorescence intensity is shown in the y-axis while the antibodies are listed in the x-axis. All antibodies were tested at 5 µg/mL. The EphA2 positive cell line was human prostate cancer (PC-3), and the EphA2 negative cell line was breast adenocarcinoma (MDA-MB-361). All 1C1 cysteine ADCs bind to EphA2 positive cancer cells similarly to the native 1C1 antibody; while the IC antibodies show no binding. 190x254mm (96 x 96 DPI)


Page 50 of 54

Page 51 of 54

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


In vitro cytotoxicity of ADCs. PC-3 cells were treated with serial dilutions of the ADCs and cell viability is shown as relative percentage to the untreated controls. Error bars indicate SD (n = 3). EC50 (ng/mL) and DAR are shown for each 1C1 ADCs. Negative control antibody (IC) and negative control ADCs (IC-ADCs) have no cytotoxic activity. 254x190mm (96 x 96 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Rat serum stability of ADCs linker. (A) Illustration of the deconjugation mechanism from ADCs in vivo. The retro-Michael reaction regenerates maleimide groups, which may react with endogenous thiols (X-SH) in circulation such as albumin or glutathione to promote deconjugation of the linker-drug from ADCs. (B) ADC linker stability at C238i, S239C and C239i. (C) ADC linker stability at C113i, A114C and C114i. (D) ADC linker stability at C204i, V205C and C205i. The ADCs used in these studies were from antibody IC. In the graphical representations the y-axis is the percentage of intact ADC, and the x-axis represents the different ADCs. Time points are schematically shown. These data were determined using rLCMS. The percentage of ADC linker loss over the duration of the study is schematically labelled for each ADCs. 190x254mm (96 x 96 DPI)


Page 52 of 54

Page 53 of 54

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Anti-tumor activity and body weight change of 5T4-C239i-SG3249 and ICa-C239i-SG3249. (A) Tumor growth curves for MDA-MB-361 breast adenocarcinoma cells after a single i.v. treatment with 5T4-C239iSG3249 (0.1 and 0.3 mg/kg) and ICa-C239i-SG3249 (0.3 mg/kg). (B) Body weight change for 5T4-C239iSG3249 (0.1 and 0.3 mg/kg) and ICa-C239i-SG3249 (0.3 mg/kg).Untreated mice were used as positive control. Tumor volumes and body weight are expressed as mean ± SEM. 190x254mm (96 x 96 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table of Content Graphic 254x190mm (96 x 96 DPI)


Page 54 of 54

Efficient Preparation of Site-Specific AntibodyâDrug Conjugates Using

Recommend Documents

Efficient Preparation of Site-Specific AntibodyâDrug Conjugates Using