Improved Inhibition of Tumor Growth by Diabody-Drug Conjugates via

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Improved Inhibition of Tumor Growth by DiabodyDrug Conjugates via Half-Life Extension Qing Li, Allison Barrett, Balakumar Vijayakrishnan, Arnaud Charles Tiberghien, Rhiannon Beard, Keith Rickert, Kevin Allen, R. James Christie, Marcello Marelli, Jay Harper, Philip W. Howard, Herren Wu, William Felix Dall'Acqua, Ping Tsui, Changshou Gao, and M. Jack Borrok Bioconjugate Chem., Just Accepted Manuscript • Publication Date (Web): 26 Mar 2019 Downloaded from http://pubs.acs.org on March 27, 2019

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Bioconjugate Chemistry

1

Improved Inhibition of Tumor Growth by Diabody-Drug Conjugates via Half-Life

2

Extension

3 4

Qing Lia,*, Allison Barrettb, Balakumar Vijayakrishnanc, Arnaud Tiberghienc, Rhiannon Beard c,

5

Keith W. Rickerta, Kevin L. Allena, R. Jim Christiea, Marcello Marellia, Jay Harperb, Philip

6

Howardc, Herren Wua, William F. Dall’Acquaa, Ping Tsuia, Changshou Gaoa, M. Jack Borroka

7 8 9

aAntibody

Discovery & Protein Engineering and bOncology Research, AstraZeneca, One

MedImmune Way, Gaithersburg, MD, USA; cSpirogen, 42 New Road, E1 2AX, London, UK

10 11

Corresponding author:

12

Qing Li

13

Department of Antibody Discovery & Protein Engineering

14

AstraZeneca

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One Medimmune Way

16

Gaithersburg, MD 20878

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Email: [email protected]

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ACS Paragon Plus Environment

Bioconjugate Chemistry 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

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Abstract

20 21

Despite some clinical success with antibody-drug conjugates (ADCs) in patients with solid tumors

22

and hematological malignancies, improvements in ADC design are still desirable due to the narrow

23

therapeutic window of these compounds. Tumor-targeting antibody fragments have distinct

24

advantages over monoclonal antibodies, including more rapid tumor accumulation and enhanced

25

penetration, but are subject to rapid clearance. Half-life extension technologies such as PEGylation

26

and albumin-binding domains (ABDs) have been widely used to improve the pharmacokinetics of

27

many different types of biologics. PEGylation improves pharmacokinetics by increasing

28

hydrodynamic size to reduce renal clearance, whereas ABDs extend half-life via FcRn-mediated

29

recycling. In this study, we used an anti-oncofetal antigen 5T4 diabody conjugated with a highly

30

potent cytotoxic pyrrolobenzodiazepine (PBD) warhead to assess and compare the effects of

31

PEGylation and albumin binding on the in vivo efficacy of antibody fragment drug conjugates.

32

Conjugation of 2× PEG20K to a diabody improved half-life from 40 min to 33 h, and an ABD-

33

diabody fusion protein exhibited a half-life of 45 h in mice. In a xenograft model of breast cancer

34

MDA-MB-436, the ABD-diabody-PBD showed greater tumor growth suppression and better

35

tolerability than either PEG-diabody-PBD or diabody-PBD. These results suggest that the

36

mechanism of half-life extension is an important consideration for designing cytotoxic anti-tumor

37

agents.

38 39

Keywords: Antibody fragment, albumin binding domain, polyethylene glycol, pharmacokinetics,

40

antibody-drug conjugate, tumor growth inhibition

41

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INTRODUCTION

43 44

The treatment of solid tumors with antibody-drug conjugates (ADCs) remains challenging, mainly

45

due to the inefficient delivery of these compounds to tumors and their heterogeneous distribution

46

inside tumors 1-5. Antibody distribution in the tumor depends on antibody characteristics such as

47

pharmacokinetics, dose, vascular permeability, binding affinity, size, and hydrophobicity of the

48

drug conjugates, as well as tumor-related factors such as tumor microenvironment and

49

architecture, antigen expression level, and antigen metabolism and internalization

50

studies have shown that antibody fragments tend to have increased vascular permeability, diffuse

51

more rapidly into tumor, and distribute more evenly due to their low molecular weight

52

Nevertheless, because of their small size, serum half-lives of antibody fragments can be short, not

53

allowing enough time to reach and be retained in the tumor. The efficiency of tumor targeting with

54

anticancer agents correlates with their serum half-lives 8, 13-15.

6-9.

Previous

10-12.

55 56

Albumin modification 13, 16-23 and PEGylation 24-30 have been used to extend the serum half-lives

57

of antibody fragments 31 (Fig. 1a). As the most abundant protein in plasma, albumin has neonatal

58

fragment crystallizable receptor (FcRn)–mediated recycling and a size that is above the threshold

59

for renal filtration, leading to a long serum half-life of more than 2 weeks in humans 32. In addition,

60

serum albumin may improve tumor localization of bound proteins or drugs due to its intrinsic

61

capability to extravasate through the gp60 signal pathway and accumulate in solid tumors via

62

binding with SPARC (secreted protein acidic and rich in cysteine)

63

domain (ABD) that is derived from streptococcal protein G and has high affinity to serum albumin

64

has been widely applied to prolong the half-lives of antibody fragments and other small proteins

3


33-35.

An albumin-binding


65

as ABD fusion proteins 36, 37. Considering the potential immunogenicity due to its bacterial origin,

66

efforts have been made to deimmunize ABD by substituting residues in immunogenic regions 37.

67

In comparison with albumin modification mediated mainly by FcRn recycling, PEGylation

68

improves pharmacokinetics by increasing hydrodynamic size to reduce renal clearance 38-40. In a

69

previous study, we investigated how PEGylation could balance tumor penetration and antibody

70

fragment accumulation with improved serum persistence 14. We found that the pharmacokinetic

71

properties of PEGylated diabodies significantly improved when hydrodynamic radius (Rh)

72

increased to 6 nm, and tumor uptake and biodistribution differed significantly with polyethylene

73

glycol (PEG) size and shape 14. The branched-PEG20K diabody conjugate, which has an Rh of ~6

74

nm, exhibited optimal tumor uptake and retention and a balanced size and pharmacokinetic profile

75

14.

76

PEGylation, it is of great interest to compare the effects of albumin binding and PEGylation on

77

the in vivo efficacy of antibody fragment–drug conjugates.

In light of the different mechanisms that have been used to extend the half-lives of albumin and

78 79

In the current work, we utilized a diabody 41 targeting the oncofetal antigen 5T4 42 as our antibody

80

fragment model format. The ABD was genetically fused to the N-terminus of the diabody as ABD-

81

diabody, and PEG20K was chemically conjugated to the C-terminus of the diabody via a thiol-

82

maleimide conjugation method as PEG-diabody. We evaluated the impact of albumin binding and

83

PEGylation on the in vivo pharmacokinetics of the modified diabodies and found that both ABD

84

fusion and PEGylation prolonged the half-life of the diabody from minutes to days in vivo. To

85

evaluate the in vivo efficacy of diabody-drug conjugates, pyrrolobenzodiazepine (PBD) dimer

86

payloads

87

modified diabodies. PBD was site-specifically conjugated to the C-terminus of ABD-diabody via

43-46,

a potent class of DNA cross-linking agents, were selected for conjugation with

4


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88

a thiol-maleimide conjugation method, and an unnatural amino acid containing an azide moiety

89

[N6-((2-azidoethoxy)carbonyl)-L-lysine]

90

diabody allowed site-specific conjugation of PEG and PBD with the diabody, using click

91

chemistry and thiol-maleimide chemistry to produce a PEG-diabody-PBD. In an in vivo efficacy

92

study, we found that the ABD-diabody-PBD demonstrated higher tumor growth suppression

93

activity and better tolerability than either PEG-diabody-PBD or diabody-PBD.

47

and a cysteine-engineered, dual-functionalized

94 95

a

96 97 98 99 100 101 102

b 1000

Protein concentration (g/mL)

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


Diabody PEG-diabody

100

ABD-diabody 10 1 0.1 0

103

50

100

150

200

Time (h)

104 105 106 107

Fig. 1. (a) Scheme showing different half-life extension mechanisms of diabody through albumin binding and PEGylation. (b) Pharmacokinetics of diabody, PEG-diabody, and ABD-diabody: time course of antibody concentrations in blood after IV injection. 5



108

RESULTS AND DISCUSSION

109 110

Half-life extension of diabody

111

To improve the pharmacokinetics of the diabody, two strategies, PEGylation and albumin binding,

112

were employed (Fig. 1a). To construct an ABD-diabody fusion protein that is capable of binding

113

to mouse serum albumin, ABD 36 was fused to the diabody with a glycine-serine [(G4S)2] linker.

114

SEC-MALS (size exclusion chromatography–multi-angle light scattering) analysis showed that

115

the ABD-diabody eluted as a single species, a monodispersed dimer with an apparent molecular

116

mass of 60 kDa (Supplemental Table S1). For the PEG-diabody, maleimide-PEG20K was

117

covalently conjugated to the C-terminal of the diabody as previously described 14.

118 119

To investigate the ability of PEGylation and ABD fusion to extend the circulation half-life of the

120

diabody, serum pharmacokinetic profiles of diabody, PEG-diabody, and ABD-diabody were

121

examined in mice after a single IV administration at 2.5 mg/kg. Serum concentrations of diabody,

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PEG-diabody, and ABD-diabody in blood samples drawn at different time points after IV injection

123

were determined by ELISA. As shown in Fig. 1b and Table 1, both PEG-diabody and ABD-

124

diabody had significantly higher exposures (area under the curve), with increases of 172-fold (from

125

7 to 1,224 h × µg/mL) and 259-fold (from 7 to 1,816 h × µg/mL), respectively, compared to the

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parental diabody. The unmodified diabody had rapid clearance from blood, with a half-life of 0.6

127

h, and PEGylation or ABD fusion extended the serum half-life of the modified diabody to 32 h or

128

45 h, respectively. Accordingly, the total body clearances of PEG-diabody and ABD-diabody were

129

much lower than that of the diabody. Specifically, the relative decrease in mean total body

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clearance was 0.57% for PEG-diabody and 0.40% for ABD-diabody. In addition, the volume of

6


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distribution at steady state was 132 mL/kg for diabody and ~81 mL/kg for both PEG-diabody and

132

ABD-diabody. This study confirmed that both albumin binding and PEGylation improved the

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pharmacokinetics of the modified diabodies, and ABD-diabody had slightly higher exposures than

134

PEG-diabody.

135 136 137

Table 1. Pharmacokinetic parameters of diabody, PEG-diabody, and ABD-diabody administration Dose Dose AUC0-168 AUCinf CL t1/2 Treatment (mg/kg) (nmol/kg) (h·µg/mL) (h·µg/mL) (mL/h/kg) (h) Diabody 2.5 48.1 NC 7 351 0.64 PEG-diabody 2.5 27.2 1188 1224 2.0 32.70 ABD-diabody

2.5

40.0

1689

1816

1.4

after IV Vss (mL/kg) 131.7 80.6

45.34 81.6

138 139 140 141

AUC0–168 = area under the curve from time 0 to 168 h; AUCinf = area under the curve from time zero to infinity; CL = total body clearance; t1/2 = half-life; VSS = volume of distribution at steady state.

142

Generation and characterization of ABD-diabody-PBD

143 144

To test how extended diabody pharmacokinetics affect anti-tumor cytotoxicity, we sought to

145

generate ABD-diabody drug conjugates. For site-specific conjugation of the payload PBD to ABD-

146

diabody, a C-terminal cysteine was introduced in the ABD-diabody to allow for conjugation by

147

thiol chemistry. For thiosuccinimide-linked ADCs, thiosuccinimide hydrolysis stabilizesthe thiol-

148

drug linkage. N-phenyl maleimide PBD was utilized because of its quick hydrolysis, which results

149

in better serum stability than N-alkyl maleimide PBD 48. In addition, ADCs with a noncleavable

150

linker are more stable than ADCs with a dipeptide spacer in the payload due to possible enzymatic

151

cleavage of the drug-linker dipeptides at the exposed drug conjugation position in mouse serum 48,

152

49.

153

site-specific conjugation with the ABD-diabody 48. This was achieved by utilizing thiol-maleimide

SG3683, an N-phenyl maleimide PBD drug with a noncleavable linker, was thus selected for

7



154

chemistry (Fig. 2a) followed by purification with ceramic hydroxyapatite column chromatography.

155

In rLCMS analysis, the reduced ABD-diabody-SG3683 showed a 1,148.69-Da mass increase over

156

the reduced unconjugated ABD-diabody, which corresponded to the addition of a single SG3683

157

molecule and thiosuccinimide hydrolysis with an efficiency of 98% (Fig. 2b). SDS-PAGE

158

confirmed that the ABD-diabody-SG3683 conjugate was homogenous and of the expected size

159

(Fig. 2c). The conjugation efficiency was further confirmed by reversed-phase chromatography

160

(Fig. 2d).

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a

162 163

b

164 165

c

d

166 167 168 169 170 171 172 173 174 175 176 177

Fig. 2. Synthesis and in vitro characterization of ABD-diabody-PBD. (a) Schematic representation of site-specific conjugation of PBD SG3683 to ABD-diabody by thiol-maleimide chemistry. (b) Conjugation of SG3683 to ABD-diabody determined by rLCMS. (c) SDS-PAGE followed by staining with SimpleBlue. Lane M: prestained protein standard; lane 1: ABD-diabody; lane 2: ABD-diabody-SG3683. (d) Analytical characterization of ABD-diabody-SG3683 by reversedphase chromatography.

178 179

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180

Generation and characterization of PEG-diabody-PBD

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For site-specific conjugation of PEG and the PBD payload to the diabody, anti-5T4 azido-diabody

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was generated for orthogonal N- and C-terminal conjugation. Introduction of the unnatural amino

183

acid N6-((2-azidoethoxy)carbonyl)-L-lysine at the N-terminus of a diabody allows conjugation by

184

click chemistry, leaving the engineered the C-terminal cysteine available for conjugation via thiol

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chemistry. An orthogonal pylRS/tRNA pair derived from Methanosarcina mazei was used to

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incorporate the unnatural amino acid into the diabody by transient transfections in CHO cells. The

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expressed azido-diabody was purified by protein-L chromatography. SEC-MALS analysis showed

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that the anti-5T4 azido-diabody was eluted as a single species, as a monodispersed dimer with an

189

apparent molecular mass of 52 kDa, and the incorporation of N6-((2-azidoethoxy)carbonyl)-L-

190

lysine was also verified by electrospray ion mass spectrometry (Supplemental Fig. S1).

191 192

Site-specific drug conjugation of the azido-diabody with SG3658 was achieved by click chemistry

193

(Fig. 3a). SG3658 is an alkyne-functionalized PBD drug-linker analog of SG3683 that allows

194

conjugation with an azide-functionalized diabody to form a stable ADC. The azido-diabody was

195

subjected to a CuAAC reaction with SG3658 armed with a linear alkyne and was purified by

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ceramic hydroxyapatite column chromatography. SDS-PAGE and rLCMS analysis of the click

197

reaction showed an efficient conjugation reaction that resulted in complete consumption of the

198

azido-diabody to a species of higher molecular weight (Fig. 3b and 3c). In the rLCMS analysis,

199

the reduced diabody-SG3658 showed a 958.44-Da mass increase over the unconjugated azido-

200

diabody, which corresponded to the addition of a single SG3658 molecule with an efficiency of

201

90%. The conjugation efficiency was further confirmed by reversed-phase chromatography

202

analysis (Fig. 3d).

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203 204

a

205 206 207 208 209 210 211 212

b

213 214

c

d

215 216 217 218 219 220 221

Fig. 3. Synthesis and in vitro characterization of PEG-diabody-PBD. (a) Schematic representation of site-specific conjugation of PBD SG3658 and PEG to diabody by click chemistry and thiolmaleimide chemistry. (b) Conjugation of SG3658 to diabody determined by rLCMS. (c) SDSPAGE followed by staining with SimpleBlue. Lane M: prestained protein standard; lane 1: unmodified diabody; lane 2: diabody-SG3658; lane 3: PEG-diabody-SG3658. (d) Analytical characterization of PEG-diabody-SG3658 by reversed-phase chromatography.

11



222

Site-specific PEGylation of diabody-PBD was achieved by utilizing thiol-maleimide chemistry.

223

The engineered C-terminal cysteine of the diabody formed inter-VH disulfide bonds during the

224

protein production process. Prior to conjugation, the C-terminal cysteine of diabody-SG3658 was

225

reduced with 10-fold TCEP, and the solvent-exposed free thio group at the C-terminal was

226

available for modification. PEGylation was then conducted with PEG20K-maleimide, which

227

mainly yielded di-PEGylated diabody, followed by purification with ceramic hydroxyapatite

228

column chromatography. Analysis of the final product by SDS-PAGE confirmed the mass

229

differences corresponding to the addition of payload and PEG (Fig. 3c).

230 231

Competitive binding by ELISA

232 233

To characterize the impact of PEG modification, ABD fusion, and drug conjugation on the binding

234

affinity of the diabody to its target, the antigen-binding affinities of the different diabody formats

235

were determined by competitive ELISA (Fig. 4 and Table 2). In this study, the full-length antibody

236

was labeled with biotin, and the abilities of different diabody formats to inhibit the binding of

237

intact biotinylated immunoglobulin G to 5T4 antigen were compared. When no serum albumin

238

was in the ELISA, the binding activities of the proteins, ranked by the IC50 value, in the competitive

239

binding assay were as follows: diabody (5 nM), diabody-SG3658 (9 nM), PEG-diabody (51 nM),

240

PEG-diabody-SG3658 (58 nM), ABD-diabody (7 nM), and ABD-diabody-SG3683 (10 nM). This

241

suggests that the binding area was partially masked by the PEG modification, whereas ABD fusion

242

and drug conjugation had little impact on binding activity in the assay condition. In addition, when

243

1% mouse serum albumin was added in the binding buffer, the binding activities of the proteins,

244

ranked by IC50 value, were as follows: diabody (12 nM), diabody-SG3658 (20 nM), PEG-diabody

12


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245

(57 nM), PEG-diabody-SG3658 (62 nM), ABD-diabody (138 nM), and ABD-diabody-SG3683

246

(185 nM). In the presence of mouse serum albumin, a ~20-fold increase in IC50 was observed for

247

both ABD-diabody and ABD-diabody-SG3683, suggesting that binding of serum albumin to ABD

248

could partially mask the binding area of the diabody to its target.

249

250 251 252

Fig. 4. Competitive binding assay comparing binding affinities of diabody and modified diabodies with and without the presence of mouse serum albumin.

253 254 255 256

Table 2. Competitive binding ELISA comparing binding affinities of diabody and modified diabodies with and without the presence of mouse serum albumin

257 258

ADC Diabody Diabody-SG3658 PEG-diabody PEG-diabody-SG3658 ABD-diabody ABD-diabody-SG3683 MSA = mouse serum albumin.

IC50 (nM), mean ± SD – MSA 5 ± 0.2 9 ± 0.7 51 ± 6 58 ± 10 7±1 10 ± 2

259 260

13


+ MSA 12 ± 1 20 ± 2 57 ± 5 62 ± 5 138 ± 38 185 ± 45


261

In vitro cytotoxicity assay

262 263

The in vitro cytotoxicities of diabody-SG3658, PEG-diabody-SG3658, and ABD-diabody-

264

SG3683 were determined to evaluate the ability of the ADCs to inhibit tumor cell proliferation,

265

using a CellTiter-Glo luminescent viability assay (Fig. 5). Diabody-SG3658, PEG-diabody-

266

SG3658, and ABD-diabody-SG3683 were able to kill MDA-MB-436 cells and had IC50 values of

267

0.01, 0.78, and 0.74 nM, respectively. The potency of diabody-PBD, PEG-diabody-PBD, and

268

ABD-diabody-PBD were significantly reduced when co-incubated with 100-fold excess

269

unconjugated diabody in the cytotoxicity assay, suggesting specific receptor mediated

270

cytotoxicity. The cytotoxicity activities were confirmed on other 5T4-positive tumor cell lines with

271

different cell surface expression levels of 5T4, namely, MDA-MB-436, MDA-MB-361, DU145,

272

and N87 (Fig. 5). Previously Harper et al50 have shown that MDA-MB-361 breast cancer cells

273

represent high 5T4 expression (~65,000 5T4 molecules/cell), DU145 prostate cancer cells

274

represent moderate 5T4 expression (~30,000 5T4 molecules/cell) and NCI-N87 gastric carcinoma

275

cells represent low 5T4 expression levels (~4,000 5T4 molecules/cell), while MDA-MB-436

276

breast cancer cells were demonstrated to represent high 5T4 expression (~120,000 5T4

277

molecules/cell, Supplemental Table S2). The cytotoxic activities of the ADCs strongly correlated

278

with the level of 5T4 expression50. To further estimate target specificity, the targeting index for

279

each ADC to each cell line was calculated as IC50 (no blocking)/IC50 (with blocking). The targeting

280

index of diabody-PBD for high–5T4-expressing cell lines MDA-MB-436 and MDA-MB-361 were

281

95 and 348, respectively; for DU145, with moderate 5T4 expression, 35; and for N87, with low

282

5T4 expression, 4 (Table 3). The reduced in vitro killing activities of PEG-diabody-SG3658 and

283

ABD-diabody-SG3683, comparing to diabody-SG3658, may be due to potential partial masking

14


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284

of their target binding surfaces by PEGylation and albumin binding. These in vitro cytotoxicity

285

data are consistent with the reduced binding activities of PEG-diabody-SG3658 and ABD-

286

diabody-SG3683 as we observed in the competitive binding assay.

287

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288 289 290 291 292 293 294

Fig. 5. In vitro cytotoxicity activity of modified diabody-PBD against MDA-MB-436, MDA-MB361, DU145, and NCI-N87 cells, as determined by CellTiter-Glo luminescent viability assay. Table 3. In vitro cytotoxicity activity of modified diabody-PBD against MDA-MB-436, MDAMB-361, DU145, and NCI-N87 cells, as determined by CellTiter-Glo luminescent viability assay Cell line MDA-MB-436

ADC Diabody-SG3658 PEG-diabody-SG3658 ABD-diabody-SG3683

IC50, nM No blocking 0.01 0.78 0.74

Targeting index

MDA-MB-361

Diabody-SG3658 PEG-diabody-SG3658 ABD-diabody-SG3683

0.03 0.75 0.22

9.42 >10 >20

348 >13 >90

DU145


0.23 3.78 2.33

8.19 >10 >20

35 >3 >9

N87


5.70 >10 >20

>20 >10 >20

>4 NA NA

+ 100-fold diabody 0.92 1.42 >10

95 2 >13

295 296

16


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In vivo tumor growth inhibition

298 299

To examine the impact of different half-life extension strategies on the in vivo efficacy of modified

300

diabody-drug conjugates, we selected the MDA-MB-436 subcutaneous tumor model in athymic

301

nude mice. The MDA-MB-436 cell line has a high level of 5T4 expression (~120,000 5T4

302

molecules/cell, Supplemental Table S2) and a high targeting index, and PEG-diabody-SG3658 and

303

ABD-diabody-SG3683 showed similar in vitro killing activity in MDA-MB-436 cells. When the

304

average tumor size reached 200 mm3, the mice were treated with a single dose of ADCs as follows:

305

groups 1 and 2 were treated with diabody-SG3658, 6.3 nmol/kg (0.3 mg/kg) or 19.0 nmol/kg (1.0

306

mg/kg) per mouse, groups 3 and 4 were treated with PEG-diabody-SG3658, 6.3 nmol/kg (0.6

307

mg/kg) or 19.0 nmol/kg (1.8 mg/kg) per mouse, and groups 5 and 6 were treated with ABD-

308

diabody-SG3683, 6.3 nmol/kg (0.4 mg/kg) or 19.0 nmol/kg (1.2 mg/kg) per mouse.

309 310

Improved tumor growth inhibition (TGI) was observed for diabody-drug conjugates with half-life

311

extension (Fig. 6a). For example, tumor growth in mice treated with ABD-diabody-SG3683 (6.3

312

nmol/kg) showed a statistically significant difference (P < 0.05) from mice in the control group,

313

with a TGI value of 76%, whereas tumors in mice treated with PEG-diabody-SG3658 (6.3

314

nmol/kg) exhibited a TGI of 45% (P < 0.05). No significant effect on tumor growth inhibition was

315

observed on tumors in mice treated with diabody-SG3658 (6.3 nmol/kg). Furthermore, ABD-

316

diabody-SG3683 exhibited a dose-dependent effect on tumor progression. Tumor growth in mice

317

treated with ABD-diabody-SG3683 at 6.3 nmol/kg was slowed by 76% as compared with tumors

318

in the control group, whereas treatment with ABD-diabody-SG3683 at 19.0 nmol/kg exhibited

319

strong inhibition of tumor growth, with a TGI value of 96%.

17



320

The cytotoxicity activities of the drug conjugates on MDA-MB-436 cells in vitro were not

321

predictive of in vivo activity. Despite the reduced in vitro killing activities due to potential partial

322

masking of their target binding surfaces by PEGylation and albumin binding, both PEG-diabody-

323

SG3658 and ABD-diabody-SG3683 exhibited higher antitumor activities than diabody-SG3658.

324

A relationship was observed between pharmacokinetics and antitumor activity of the drug

325

conjugates in vivo.

326

diabody-SG3658. Slightly higher in vivo exposure via albumin binding and the intrinsic capability

327

of albumin binding to extravasate and accumulate in solid tumors may contribute to the more

328

significant in vivo tumor suppression activity of ABD-diabody-SG3683.

ABD-diabody-SG3683 exhibited more pronounced activity than PEG-

329 330

a

331 332

b 40

Average body weight (g)

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

Untreated Diabody-SG3658 (0.3 mg/kg, 6.3 nmol/kg) Diabody-SG3658 (1.0 mg/kg, 19.0 nmol/kg)

35 30

PEG-diabody-SG3658 (0.6 mg/kg, 6.3 nmol/kg) PEG-diabody-SG3658 (1.8 mg/kg, 19.0 nmol/kg)

25

ABD-diabody-SG3683 (0.4 mg/kg, 6.3 nmol/kg) ABD-diabody-SG3683 (1.2 mg/kg, 19.0 nmol/kg)

20 15 0

333

20

40

60

Days Post Dosage

18


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334 335 336 337 338

Fig. 6. In vivo efficacy of ADCs in the MDA-MB-436 tumor xenograft model. (a) Suppression of MDA-MB-436 tumor growth in mice by treatment with different ADCs, and bracket indicates a significant difference (P < 0.05) between values. (b) Body weight changes of MDA-MB-436– bearing mice in different treatment groups.

339

In addition to exhibiting higher in vivo tumor growth suppression activity, ABD-diabody-SG3683

340

had better tolerability than diabody-SG3658 or PEG-diabody-SG3658. Diabody-SG3658 (19.0

341

nmol/kg) and PEG-diabody-SG3658 (19.0 nmol/kg) treated groups exhibited poor tolerance for

342

the drug conjugates. Four of eight mice treated with diabody-SG3658 (19.0 nmol/kg) and seven

343

of eight mice treated with PEG-diabody-SG3658 (19.0 nmol/kg) had red skin rash at day 3 and

344

died at day 7 (Supplemental Table S3). In comparison, mice treated with ABD-diabody-SG3683

345

(19.0 nmol/kg) showed better tolerance and had no weight loss at the end of the study (Fig 6b).

346 347

Toxicity profiles of ADCs can be impacted by the selection of a cleavable or noncleavable linker.

348

ADCs with noncleavable linker were utilized in the study based on the hypothesis that ADCs with

349

noncleavable linker exhibits little to no bystander activity and the efficacy and toxicity profiles are

350

predominantly due to the internalized ADCs. This could simplify the relationships of ADC formats

351

and in vivo efficacy and toxicity profiles comparing to ADCs with cleavable linker which may

352

generate extracellular warhead catabolite. When administrated at the same dosage, rapidly cleared

353

diabody-SG3658 has poor tolerability and dose limiting toxicity. This is a similar observation as

354

reported in the Hamblett et al51, indicating that the accelerated clearance for noncleavable ADCs

355

led to decreased tolerability at equivalent doses. Interestingly, the slowly cleared PEG-diabody-

356

SG3658 had poor tolerability, despite its improved half-life. Considering similar in vivo

357

pharmacokinetic impact by PEGylation and albumin binding, better tolerability of ABD-diabody-

358

SG3683 may suggest that FcRn recycling of the drug conjugates is a superior approach to

19



359

PEGylation mediated half-life extension. Furthermore, comparing to the intact IgG ADC, ABD-

360

diabody-drug conjugates might not be able to recruit and activate complement components and

361

immune effector cells into the tumor sites for the benefit of anti-tumor activity52. However, ABD-

362

diabody-drug conjugates may avoid the internalization by immune cells resulting in off-target

363

toxicity and avoid sequestering ADCs through immune cells in the circulation52.

364 365

CONCLUSIONS

366 367

In this study, we showed that ABD fusion and PEGylation prolonged the half-life of a diabody

368

from minutes to days in vivo. ABD-diabody-PBD was engineered by site-specific conjugation of

369

ABD-diabody with PBD, using thiol-maleimide chemistry. By engineering site-specific unnatural

370

amino acid and cysteine dual-functionalized diabody, PEG-diabody-PBD was successfully

371

obtained via site-specific conjugation of PEG and PBD with diabody, using thiol-maleimide

372

chemistry and click chemistry. The in vivo efficacy study in the xenograft model of breast cancer

373

demonstrated that ABD-diabody-PBD exhibited higher in vivo tumor growth suppression activity

374

and better tolerability than diabody-PBD or PEG-diabody-PBD in mice. These results suggest that

375

albumin binding to extend half-life of antibody fragments may be a promising strategy for the

376

development of novel drug delivery systems for cancer treatment.

377 378 379 380 381 382

20


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383


EXPERIMENTAL PROCEDURES

384 385

Materials and cell lines

386 387

Maleimide-PEG20K (catalog no. GL2-200MA) was obtained from NOF America (White Plains,

388

NY). The breast carcinoma cell lines MDA-MB-436 and MDA-MB-361, the prostate carcinoma

389

cell line DU145, and the gastric carcinoma cell line NCI-N87 were obtained from American Type

390

Culture Collection (Rockville, MD). MDA-MB-436 MDA-MB-361 and DU145 cells were

391

cultured in Dulbecco modified Eagle medium, and NCI-N87 cells were grown in Advanced RPMI

392

1640 medium (Gibco, Life Technologies Europe BV, Zug, Switzerland). All media were

393

supplemented with 10% fetal calf serum. Cells were maintained in tissue culture flasks at 37°C in

394

a humidified atmosphere with 5% CO2.

395 396

Cloning, expression, and purification of ABD-diabody and azido-diabody

397 398

We constructed ABD-diabody and azido-diabody plasmids from the diabody plasmid template

399

previously made in our laboratory. Briefly, the diabody was designed with the light-chain variable

400

(VL) and the heavy-chain variable (VH) domains connected by a five–amino-acid GGGGS linker,

401

which allows homodimer formation. An ABD-diabody is constructed by adding an albumin-

402

binding domain, ABD035 36, and a linker (GGGGSGGGGS) to the N-terminus of the VL domain

403

of the diabody. C-terminal GGC residues of the ABD-diabody were designed for site-specific

404

cysteine-maleimide conjugation. Chinese hamster ovary (CHO) cells were transiently transfected

21



405

with the ABD-diabody construct, and the expressed ABD-diabody was purified with protein L

406

affinity chromatography according to standard protocols.

407 408

To construct the azido-diabody, an amber stop codon was introduced into the N-terminus of the

409

diabody to allow the incorporation of an unnatural amino acid containing an azide moiety. C-

410

terminal GGC residues of the diabody were retained in the azido-diabody for site-specific cysteine-

411

maleimide conjugation. A cell-based mammalian expression system was used for site-specific

412

integration of an unnatural amino acid 53. The plasmids used for expression of the azido-diabody

413

were pCEP4-pylRS, the gene encoding for pylRS (reference sequence WP_011033391); pOriP-

414

9×-tRNA, nine tandem copies of a tRNA expression cassette consisting of the U6 snRNP

415

promoter; and the wild-type tRNA-pyl sequence. CHO cells were transiently cotransfected with

416

azido-diabody, pylRS, and tRNA-pyl, using polyethylenimine. For expression of the azido-

417

diabody, cells were grown to a density of 2 × 106 cells per mL, and 2 mM N6-((2-

418

azidoethoxy)carbonyl)-L-lysine 47 was added to the medium and incubated with shaking for 7−14

419

days. Expressed azido-diabody was purified by protein L affinity chromatography according to

420

standard protocols.

421 422

Pharmacokinetics

423 424

Pharmacokinetic studies were conducted in nude mice for diabody, ABD-diabody, and PEGylated

425

diabody. PEGylated diabody was prepared and purified by previously described methods 14.

426

22


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427

Female athymic (nu/nu) mice approximately 5 weeks of age were obtained from Envigo

428

(Indianapolis, IN), housed in individually ventilated cages on hardwood bedding, and fed a

429

commercially available diet (HarlanTeklad 2918 Diet, 18% Global Protein Diet; Harlan,

430

Indianapolis, IN). The mice were routinely tested for rodent pathogens according to guidance from

431

the vendor and quarterly institutional health surveillance programs and were found to be free of

432

these pathogens. All procedures were conducted in accordance with the Guide for the Care and

433

Use of Laboratory Animals in a facility accredited by the Association for Assessment and

434

Accreditation of Laboratory Animal Care and were approved by MedImmune’s Institutional

435

Animal Care and Use Committee. Each animal (n = 9 per group) was injected intravenously (IV)

436

with a dose of 2.5 mg/kg. Blood samples were collected at 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 16 h,

437

1 day, 2 days, 3 days, 4 days, and 7 days post-injection. Concentrations were determined by a

438

protein L enzyme-linked immunosorbent assay (ELISA). Briefly, individual wells of a 96-well

439

immunoplate (half-well) were coated with 1 μg of 5T4 antigen per mL. The plates were blocked

440

with 3% bovine serum albumin (Sigma Chemical, St. Louis, MO) and incubated with samples or

441

standards and then with a protein L–horseradish peroxidase conjugate (Pierce Chemical, Dallas,

442

TX). Peroxidase activity was detected with 3,3',5,5'-tetramethylbenzidine substrate, and

443

absorbance at 450 nm was measured with an EnVision plate reader (Perkin Elmer, Waltham, MA).

444 445

Conjugation and purification of ABD-diabody-PBD

446 447

ABD-diabody-PBD was prepared by site-specific conjugation of the PBD payload SG3683 to the

448

C-terminal engineered cysteine of the ABD-diabody. ABD-diabody solution was first treated with

449

the reducing agent tris(2-carboxyethyl)phosphine (TCEP) at a molar ratio of 1:10 at 37°C for 2 h

23



450

to reduce C-terminal cysteine residue. After the initial reducing step, TCEP was removed from the

451

reaction with a desalting column (Zeba; Thermo Fisher Scientific, Waltham, MA). The freshly

452

prepared diabody with a free C-terminal thiol group was immediately combined with dimethyl

453

sulfoxide (final concentration 20%, vol/vol), followed by addition of the N-phenylmaleimide–

454

functionalized PBD payload SG3683 at a molar ratio of 1:5. The reaction proceeded at 37°C with

455

mixing for 1 h and was followed by the addition of N-acetyl-L-cysteine to quench the unreacted

456

maleimide.

457 458

The crude ADC reaction mixtures were diluted five-fold with water and purified with type II

459

ceramic hydroxyapatite column chromatography (Bio-Rad Laboratories, Hercules, CA). Buffer A

460

consisted of 10 mM sodium phosphate, pH 7, and was used as loading buffer, and buffer B

461

consisted of 10 mM sodium phosphate 2 M sodium chloride, pH 7, and was used as elution buffer.

462

ADC was eluted with a 0–100% buffer B linear gradient over 20 min at a flow rate of 5 mL/min.

463

The purified ADC was then buffer exchanged into 1× phosphate-buffered saline (PBS), pH 7.4.

464 465

Conjugation and purification of PEG-diabody-PBD

466 467

For the conjugation of diabody-PBD with the copper(I)-catalyzed azide alkyne cycloaddition

468

(CuAAC) 53, a solution of the azide-containing antibody (azido-diabody) and the cytotoxic alkyne

469

SG3658 (supplemental methods) were combined. In a separate tube, a solution containing copper

470

sulfate (CuSO4), tris(hydroxypropyltriazolylmethyl)amine (THPTA) ligand, amino guanidine, and

471

sodium ascorbate was made. The THPTA-CuSO4 complex was capped, vortexed, and allowed to

472

stand for 10 min. A portion of this complex was added to the antibody-alkyne mixture. The final

24


Page 24 of 37

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473

mixture was capped, vortexed, and allowed to incubate for 1–2 h at room temperature. The crude

474

diabody-SG3658 was then buffer exchanged into 1× PBS, pH 7.4.

475 476

For PEGylation, dialyzed diabody-SG3658 was first treated with 10-fold-molar TCEP to reduce

477

C-terminal cysteine residue in PBS, pH 7.4, at room temperature for 2 h. After the initial reducing

478

step, TCEP was removed from the reaction with a desalting column (Zeba; Thermo Fisher). The

479

freshly prepared diabody with a free C-terminal thiol group was immediately reacted with

480

maleimide-PEG20K in PBS and 1 mM ethylenediaminetetraacedic acid, pH 7.4. The PEGylation

481

reactions were then incubated at 4oC overnight, and 10-fold-molar N-acetyl-L-cysteine was added

482

to quench the reaction. The reactions were then purified with type II ceramic hydroxyapatite

483

column chromatography (Bio-Rad) as previously described. The purified ADC was then buffer

484

exchanged into 1× PBS, pH 7.4.

485 486

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

487 488

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was performed under

489

reducing conditions, using NuPAGE Novex 4–20% Bis-Tris gel (Thermo Fisher) in MOPS [3-(N-

490

morpholino)propanesulfonic acid] buffer (Thermo Fisher) according to the manufacturer’s

491

instructions. The gels were visualized by staining with SimpleBlue protein staining solution

492

(Thermo Fisher).

493 494

Liquid chromatography–mass spectrometry

495

25



496

Conjugation efficiency was determined by using reduced liquid chromatography–mass

497

spectrometry (rLCMS), using a 1200 series high-performance liquid chromatograph coupled to a

498

6520 Accurate-Mass Q-TOF LCMS (Agilent, Santa Clara, CA) with an electrospray ionization

499

source. Approximately 2–10 μg of reduced antibody or ADC was loaded onto a Poroshell 300SB-

500

C3 column (2.1 × 75 mm; Agilent) and eluted at a flow rate of 0.4 mL/min, using a step gradient

501

of 60% B after 6 min (mobile phase A, 0.1% formic acid in water; mobile phase B, 0.1% formic

502

acid in acetonitrile) (JT Baker, Phillipsburg, NJ). MassHunter software (Agilent) was used for data

503

acquisition and chromatogram processing.

504 505

Reversed-phase chromatography

506

Diabody and modified diabodies were reduced at 37°C for 20 min with 42 mM dithiothreitol in

507

PBS, pH 7.2. Approximately 5–20 μg (20 μL by volume) of reduced samples was loaded onto a

508

PLRP-S 1000 Å column (2.1 × 50 mm; Agilent) and eluted at 80°C at a flow rate of 1 mL/min and

509

a gradient of 5–100% B over 25 min (solvent A, 0.1% trifluoroacetic acid in water; solvent B,

510

0.1% trifluoroacetic acid in acetonitrile).

511 512

Competitive binding assay

513 514

The antibody-binding activities of diabody and modified diabodies were determined by

515

competition ELISA. Ninety-six–well immunoplates (half-well; Corning, Corning, NY) were

516

coated with 1 µg of 5T4 antigen per mL in PBS at 4oC overnight and blocked with blocker casein

517

(Thermo Fisher) at room temperature for 2 h. Serially decreasing dilutions of diabody or

518

PEGylated diabodies were mixed with a constant optimal binding concentration of biotin-labeled

26


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519

5T4 immunoglobulin G, with or without the presence of 1% mouse serum albumin (Sigma), and

520

were then added to the plate and incubated at room temperature for 1 h, followed by incubation

521

with a streptavidin–horseradish peroxidase conjugate (Pierce). Peroxidase activity was detected

522

with tetramethylbenzidine substrate (Thermo Fisher), and the absorbance at 450 nm was measured

523

with an EnVision plate reader (Perkin Elmer).

524 525

In vitro killing assay

526 527

MDA-MB-436, MDA-MB-361, DU145, and NCI-N87 cells were plated in culture media at a

528

density of 2,000–5,000 cells per well (depending on the growth kinetics of each cell line) of tissue

529

culture–treated, 96-well plates in a volume of 50 µL and allowed to adhere overnight. A 4×

530

concentration of each dose of diabody-PBD to be tested was prepared by diluting the test articles

531

in culture medium. In addition, a 400× concentration of naked diabody was prepared for the block

532

wells. First, 25 µL of either medium alone or the 400× naked diabody was added to cells in

533

triplicate such that the final dose curve ranged from 2,500 nM down to 0.38 nM in a stepwise 1:3

534

serial dilution series. Next, 25 µL of each test article was added to cells in triplicate such that the

535

final dose curve ranged from 25 nM down to 0.004 nM in a stepwise 1:3 serial dilution series. The

536

treated cells were cultured at 37°C with 5% CO2 for 144 h. The CellTiter-Glo Luminescent

537

Viability Assay (Promega, Madison, WI) was used to determine relative cytotoxicity. Briefly, 100

538

µL of CellTiter-Glo reagent was added to each well and allowed to incubate for 10 min at room

539

temperature with mild shaking, and then the absorbance of each sample at 560 nM was read with

540

an EnVision luminometer (Perkin Elmer). The percent cell viability was calculated by the

541

following formula: (average luminescence of treated samples/average luminescence of control

27



542

samples) × 100. Half-maximal inhibitory concentrations (IC50 values) were determined by logistic

543

nonlinear regression analysis with Prism v.7.04 (GraphPad Software, La Jolla, CA). Results shown

544

are representative of at least three individual experiments.

545 546

In vivo tumor growth inhibition

547 548

Tumor growth inhibition studies were performed at HD biosciences in accordance with all

549

appropriate regulatory standards under protocols approved by the Institutional Animal Care and

550

Use Committee. In vivo efficacy of ADCs were evaluated with 6- to 8-week-old female athymic

551

(nu/nu) mice (Charles River Laboratories, Wilmington, MA), each weighing approximately 18–

552

20 g. A total of 5 × 106 MDA-MB-436 cells were implanted into the right flank of each mouse to

553

establish a subcutaneous tumor disease model. When the mean tumor volume reached 185–225

554

mm3, the tumor-bearing mice were randomly divided into groups (n = 8 per group) and treated

555

with a single IV dose of diabody-SG3658 (0.3 mg/kg, 6.3 nmol/kg), diabody-SG3658 (1.0 mg/kg,

556

19.0 nmol/kg), PEG-diabody-SG3658 (0.6 mg/kg, 6.3 nmol/kg), PEG-diabody-SG3658 (1.8

557

mg/kg, 19.0 nmol/kg), ABD-diabody-SG3683 (0.4 mg/kg, 6.3 nmol/kg), or ABD-diabody-

558

SG3683 (1.2 mg/kg, 19.0 nmol/kg). Untreated mice were included as controls. Mice were

559

monitored daily and tumors were measured twice weekly with calipers. Tumor volumes were

560

calculated with the formula 1/2 × (length × width)2. Body weights were measured daily to assess

561

treatment tolerability. The study was terminated 53 days after tumor implantation or when the

562

tumor volumes reached ∼1,000 mm3, whichever occurred first. Tumor growth inhibition was

563

plotted with Prism v.7.04 (GraphPad Software). Tumor volumes are expressed as mean ± standard

564

error of the mean. Tumor growth inhibition (TGI) was calculated according to the following

28


Page 28 of 37

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565

equation: TGI (%) = (Vc – Vt)/(Vc – V0) × 100, where Vc and Vt indicate the mean tumor volume

566

in the control and the treated groups, respectively, at the end of the study and V0 is the mean tumor

567

volume in the groups at the start of the study.

568 569

Statistical analysis

570 571

Data were analyzed with Prism v.7.04 (GraphPad Software). Results are presented as mean ±

572

standard deviation. Two-way ANOVA and Bonferroni post hoc analyses were performed to

573

determine statistical significance (defined as P < 0.05).

574 575 576

ACKNOWLEDGMENTS

577

We would like to acknowledge HD biosciences and Haihong Zhong for in vivo study assistance,

578

and Pamela Thompson for discussion on drug conjugation methods. Editorial support was

579

provided by Deborah Shuman of AstraZeneca.

580 581

SUPPORTING INFORMATION

582

The Supporting Information is available free of charge on the ACS Publications website.

583 584

DECLARATION OF INTEREST

585

All authors are employees of AstraZeneca and have stock ownership and/or stock interests or

586

options in AstraZeneca.

587 588

FUNDING SOURCE

589

This study was supported by AstraZeneca. 29



590

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591 592

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