Nonclinical safety of tildrakizumab, a humanized anti–IL-23p19 monoclonal antibody, in nonhuman primates

Journal Pre-proof Nonclinical safety of tildrakizumab, a humanized anti–IL-23p19 monoclonal antibody, in nonhuman primates Michael Santostefano, Danuta Herzyk, Diana Montgomery, Jayanthi Wolf PII:

S0273-2300(19)30240-5

DOI:

https://doi.org/10.1016/j.yrtph.2019.104476

Reference:

YRTPH 104476

To appear in:

Regulatory Toxicology and Pharmacology

Received Date: 1 May 2019 Revised Date:

18 July 2019

Accepted Date: 13 September 2019

Please cite this article as: Santostefano, M., Herzyk, D., Montgomery, D., Wolf, J., Nonclinical safety of tildrakizumab, a humanized anti–IL-23p19 monoclonal antibody, in nonhuman primates, Regulatory Toxicology and Pharmacology (2019), doi: https://doi.org/10.1016/j.yrtph.2019.104476. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.

1

Nonclinical safety of tildrakizumab, a humanized anti–IL-23p19 monoclonal

2

antibody, in nonhuman primates

3

Michael Santostefanoa, Danuta Herzykb, Diana Montgomeryc, Jayanthi Wolfd

4

a

5

Pasteur, Boston, MA, 02115-5727, United States; [email protected]

6

b

7

Pike, West Point, PA, 19486, United States; [email protected]

8

c

9

Pike, West Point, PA, 19486, United States; [email protected]

Safety Assessment and Laboratory Animal Resources, Merck & Co., Inc., 33 Avenue Louis

Safety Assessment and Laboratory Animal Resources, Merck & Co., Inc., 770 Sumneytown

Pharmacokinetics, Predictive and Clinical Immunogenicity, Merck & Co., Inc., 770 Sumneytown

10

d

11

2505, United States; [email protected]

Global Regulatory Affairs, Merck & Co., Inc., 351 N. Sumneytown Pike, North Wales, PA 19454-

12 13

Corresponding author:

14

Michael Santostefano

15

Tel: (617) 992-3030

16

Fax: (617) 992-2487

17 18

Abstract word count: 196

19

Text word count: 4475

20

Reference word count: 1434

21

1

22

Highlights

23

•

24 25

psoriasis in the US, EU, and Australia •

26 27

•

32

Tildrakizumab was well tolerated, with no toxicological findings in repeat-dose toxicity or embryofetal development studies

•

30 31

Safety of subcutaneously administered tildrakizumab was characterized using a pharmacologically relevant cynomolgus monkey

28 29

The anti–IL-23p19 mAb tildrakizumab is approved for moderate-to-severe plaque

No neonatal deaths were noted following in utero exposure at 7x the recommended human dose

•

The results of this comprehensive nonclinical safety program support the safe use of tildrakizumab

33

2

34 35

ABSTRACT Tildrakizumab (also known as MK-3222), is a high-affinity, humanized, immunoglobin

36

G1κ monoclonal antibody targeting the p19 subunit of interleukin-23 recently approved for

37

the treatment of moderate to severe plaque psoriasis in the US, Europe, and Australia. The

38

safety profile of tildrakizumab was characterized in nonclinical studies using a

39

pharmacologically relevant cynomolgus monkey model. In repeat-dose toxicity studies,

40

cynomolgus monkeys were chronically treated with subcutaneous (SC) injections of

41

100 mg/kg of tildrakizumab every 2 weeks up to 9 months. Tildrakizumab was well tolerated,

42

with no toxicological findings (including assessment of reproductive organs; hormonal effects;

43

and cardiovascular, respiratory, and central nervous system function) at systemic exposures

44

approximately 90 times higher than the recommended human dose of 100 mg. An

45

embryofetal developmental study conducted in pregnant monkeys revealed no treatment-

46

related effects to the developing fetus following SC administration of tildrakizumab

47

100 mg/kg. In a pre- and postnatal development study, 2 neonatal deaths due to potential

48

viral infection at 100 mg/kg were considered of uncertain relationship to the treatment based

49

on a lack of historical data on the occurrence of viral infection in neonate cynomolgus

50

monkeys. The results of this comprehensive nonclinical safety program support the safe use

51

of tildrakizumab.

52 53

KEYWORDS

54

Monoclonal antibody; interleukin-23; psoriasis; pre- and postnatal development; animal model

55 56

3

57 58

1. INTRODUCTION Psoriasis is a chronic, noncommunicable, inflammatory skin disorder with a prevalence

59

of 0.09% to 11.4% worldwide (World Health Organization). Plaque psoriasis (psoriasis

60

vulgaris)—the most common form—affects up to 97% of all patients (Kubota et al., 2015) and

61

is characterized by recurrent episodes of inflammatory, sharply demarcated, erythematous,

62

scaly plaques of variable size and confluence (World Health Organization). The primary goal of

63

psoriasis therapies is to maintain control of the lesions by clearing psoriatic plaques, which is

64

often defined in clinical trials by a 75% improvement from baseline based on the Psoriasis Area

65

and Severity Index (Fredriksson and Pettersson, 1978; Mease, 2011).

66

Currently approved biological treatments for psoriasis include tumor necrosis factor -α

67

antagonists (Enbrel® [etanercept]. Full prescribing information, 2017; Humira® [adalimumab]

68

full prescribing information, 2018; Remicade® [infliximab] full prescribing information, 2018); an

69

interleukin (IL)-12/23p40 antagonist (Stelara® [ustekinumab] full prescribing information,

70

2018); IL-17 antagonists (Cosentyx® [secukinumab] full prescribing information, 2018; Siliq™

71

[broadalumab] full prescribing information, 2017; Taltz® [ixekizumab] full prescribing

72

information, 2018); and IL-23p19 antagonists (Ilumya™ [tildrakizumab] full prescribing

73

information, 2018; Tremfya® [guselkumab] full prescribing information, 2017).

74

IL-23 is a naturally occurring heterodimeric cytokine that shares the IL-12/23p40 subunit

75

with IL-12. The IL-12/23p40 subunit couples with either the IL-23p19 or IL-12p35 subunits to

76

form IL-23 and IL-12, respectively (Teng et al., 2015). Until the discovery of IL-23, the IL-

77

12/23p40 subunit was believed to be unique to IL-12, stimulating efforts to understand the

78

relative roles of these 2 cytokines in immune modulation (Teng et al., 2015). IL-23 is a

79

proinflammatory cytokine that plays a role in the pathogenesis of immune diseases including

80

psoriasis, ankylosing spondylitis, and psoriatic arthritis (Teng et al., 2015); a specific

4

81

hypomorphic polymorphism in the gene coding for IL-23 receptor (rs11209026) was identified

82

by genome-wide association studies as protective against these diseases (Abdollahi et al.,

83

2016). IL-23 is overexpressed in psoriasis tissue biopsies (Boutet et al., 2018; Tonel et al.,

84

2010). Sequence variants in the genes for IL-23R and its ligand confer protection against

85

psoriasis (Abdollahi et al., 2016; Capon et al., 2007). The R381Q protective allele attenuates IL-

86

23R signaling in healthy donors and psoriasis patients in in vitro functional studies (Di Meglio et

87

al., 2011).

88

Treatment of inflammatory diseases with immunosuppressive agents carries a potential

89

unintended consequence of impaired host-defense and/or tumor surveillance (Bongartz et al.,

90

2006; Davies et al., 2013; Langley et al., 2013; Teng et al., 2015). However, selective inhibition

91

of the IL-23p19 pathway is associated with decreased infection liability and decreased risk of

92

tumor incidence or progression compared to broader IL-12/23p40 neutralization (Belladonna et

93

al., 2006; Chackerian et al., 2006; Held et al., 2008; Kapil et al., 2009; Khader et al., 2005;

94

Kortylewski et al., 2009; Langowski et al., 2006; Rudner et al., 2007; Schulz et al., 2008; Teng

95

et al., 2010; Teng et al., 2012; Teng et al., 2011; von Scheidt et al., 2014; Wu et al., 2007; Wu

96

et al., 2009; Zelante et al., 2007). Animal disease model studies point to a critical role of the IL-

97

23 pathway in the pathogenesis of psoriasis, suggesting that IL-23 is an important target for

98

therapeutic intervention in the treatment of inflammatory diseases (Cua et al., 2003; Langrish

99

et al., 2005; Teng et al., 2015; Teng et al., 2011; Tonel et al., 2010) with potential for reduced

100 101

risk for infections and carcinogenesis compared to broader IL-12/23p40 neutralization. This manuscript describes the nonclinical safety program supporting approval of the IL-

102

23p19 antagonist monoclonal antibody (mAb) tildrakizumab (Ilumya™ [tildrakizumab] full

103

prescribing information, 2018), also known as MK-3222, for the treatment of adults with

104

moderate to severe plaque psoriasis who are candidates for systemic therapy or phototherapy.

5

105

Tildrakizumab is a high-affinity (KD – 297 pM; as measured by surface plasmon resonance),

106

humanized IgG1/κ recombinant neutralizing mAb that selectively binds to the IL-23p19 subunit

107

and neutralizes human IL-23 (Kopp et al., 2015). The general expression pattern of IL-23 and

108

its cellular receptor is similar between human, cynomolgus monkey, and mouse; however, the

109

highest IL-23p19 amino acid identity occurs between human and cynomolgus monkey (98%).

110

The safety profile of tildrakizumab was characterized in an extensive nonclinical program

111

conducted in accordance with regulatory guidelines applicable to biotechnology products (ICH

112

M3[R2], 2009; US FDA, 1997) and international regulatory agency feedback throughout

113

development. Using a pharmacologically relevant cynomolgus monkey model system, the

114

toxicity profile was assessed in repeated-dose toxicity studies of up to 9 months in duration.

115

Potential developmental and reproductive effects were characterized in an embryo-fetal

116

development (EFD) study and a pre- and postnatal development (PPND) study in the

117

cynomolgus monkey.

118 119

2. MATERIALS AND METHODS

120

2.1 Ethics statements

121

All study protocols and animal housing were approved by the Institutional Animal Care

122

and Use Committee prior to study initiation, and animals were cared for in accordance with the

123

principles defined in the Guide for the Care and Use of Laboratory Animals (Institute for

124

Laboratory Animal Resources, 2011). All studies were conducted according to site Standard

125

Operating Procedures and the Organization for Economic Co-operation and Development

126

Principles of Good Laboratory Practice in the US.

127

6

128 129

2.2 Physical, chemical, and pharmaceutical properties Tildrakizumab was produced in Chinese Hamster Ovary cells using conventional cell

130

culture and purification processes. The parental mAb of tildrakizumab was produced by

131

immunization of IL 23p19-knockout mice with a recombinant chimeric IL-23 (human p19:

132

mouse p40) as the immunogen. The humanized anti–IL-23 antibody was obtained by grafting

133

the complementarity determining regions from the parental mAb onto the human IgG1

134

framework. Tildrakizumab (50 or 100 mg/mL) was supplied in a frozen liquid formulation and

135

stored at 2℃ to 8℃.

136 137 138

2.3 Animals and husbandry Chinese-origin cynomolgus monkeys (Macaca fascicularis) were housed at an Association

139

for Assessment and Accreditation of Laboratory Animal Care International-accredited facility in

140

species-specific indoor housing. Certified primate diet was provided daily in amounts

141

appropriate for the age and size of the animals, and tap water was available ad libitum to each

142

animal via an automatic watering device. Animals were provided enrichment by additional food

143

supplements and various cage-enrichment devices. Animals were maintained on a 12:12 hour

144

light:dark cycle at 18°C to 29°C with 30% to 70% relative humidity. All animals were negative

145

for simian retrovirus. The cynomolgus monkey was selected as an appropriate test species

146

based on sequence homology to human IL-23 and the similar picomolar binding

147 148

(KD ~ 50 pM) of tildrakizumab; no binding is observed in the rat and mouse. Tildrakizumab also has comparable cellular potency (~125 pM) to human and cynomolgus monkey IL-23.

149

7

150

2.4 Study design

151

2.4.1 Repeat-dose toxicity

152

Assessment of potential toxicity (with evaluation of functional endpoints for

153

cardiovascular, respiratory, and central nervous system [CNS] assessments) of tildrakizumab

154

was evaluated in cynomolgus monkeys in 2 repeat-dose studies of 3 and 9 months in duration.

155

Doses were selected to provide a mean anticipated exposure margin of 10-fold to the clinical

156

exposure and be associated with maximum pharmacology in monkeys based on internal cellular

157

potency. In the 3-month study, 4 groups of cynomolgus monkeys were administered either

158

placebo (Group 1), tildrakizumab 40 or 140 mg/kg by subcutaneous (SC) injection (Groups 2

159

and 3, respectively), or tildrakizumab 140 mg/kg by intravenous (IV) bolus (Group 4) every 2

160

weeks (Q2W) over 15 weeks. In the 9-month study, cynomolgus monkeys were administered a

161

SC dose of placebo or 10, 30, or 100 mg/kg tildrakizumab Q2W for 9 months. In both studies, a

162

4- to 6-month treatment-free postdose period was included. Necropsy was performed at the

163

end of dosing and after the treatment-free period; organ weights were measured, and tissues

164

were sampled for microscopic histopathology. During the study period, assessment of mortality,

165

clinical observations, and qualitative food consumption was made at least daily and body weight

166

was measured weekly. Physical examinations, electrocardiograms (ECG), blood pressure

167

measurements (via the use of cuffs), and ophthalmologic examinations were performed

168

predose, at multiple time points during the dosing period, and once during the treatment-free

169

period. Urine and blood were sampled for urinalysis, hematology, coagulation, and clinical

170

serum chemistry predose, at multiple time points during the dosing period, and once in the

171

treatment-free period. Urine was collected overnight, and standard urinalysis and urine

172

chemistry assessments were performed. Blood samples for toxicokinetic evaluation,

8

173

determination of IL-23 concentration, and immunogenicity analyses were collected as described

174

in the Supplemental Methods.

175

To ensure a sufficient number for fertility assessment, both the 3- and 9-month studies

176

contained sexually mature animals by study termination as determined by morphological

177

examination of the testes in males and daily vaginal swabs for evidence of menses and/or

178

hormone measurements in females. In the 9-month study, blood samples for estradiol analysis

179

were collected on multiple days of an individual female’s cycle and daily progesterone samples

180

were collected for 5 consecutive days during an individual female’s cycle.

181 182

2.4.2 Embryo-fetal development study

183

Pregnant cynomolgus monkeys were administered placebo or tildrakizumab SC at doses

184

of 10, 100, or 300 mg/kg once every 14 days from gestation day 20 (GD20) to GD118. Overall

185

health and food consumption were monitored daily, and pregnancy monitoring was performed

186

regularly via ultrasound to assess embryo-fetal loss (Tarantal and Hendrickx, 1988). Fetal

187

viability, gender, body weight, and placental weight were determined following Cesarean (C-)

188

section at GD140 ± 3 days. Blood samples for toxicokinetic evaluation and immunogenicity

189

analyses were collected as described in the Supplemental Methods. Following euthanasia,

190

external, visceral (including selected organ weights), and skeletal examinations were conducted

191

on all fetuses.

192 193 194

2.4.3 Pre- and postnatal development (PPND) study Pregnant cynomolgus monkeys were administered placebo or tildrakizumab 10 or

195

100 mg/kg SC once Q2W from GD50 until parturition. All dams were first-time mothers.

196

Maternal health and pregnancy progression were monitored as in the embryonic fetal

9

197

development study. Adult females were evaluated for changes in clinical signs, body weight,

198

and other parameters (including nursing behavior and milk let-down) for approximately 6

199

months postpartum. Milk samples were collected from mothers at postpartum day 28 (PPD28)

200

and PPD91 for toxicokinetic analysis. Blood samples for toxicokinetic and antidrug antibody

201

(ADA) analyses were collected from mothers at multiple gestation and postpartum days.

202

Clinical observations; body weight; nursing behavior; and external, morphometric, and

203

neurobehavioral assessments of cynomolgus monkey infants were collected for 6 months

204

postpartum. Blood samples from cynomolgus monkey infants were collected by venipuncture at

205

multiple time points for clinical pathology, toxicokinetic, ADA, and lymphocyte analyses. At

206

postnatal day (PND) 138 (± 2 days) and PND152 (± 2 days), cynomolgus monkey infants were

207

administered intramuscular keyhole limpet hemocyanin (KLH) as a vaccine surrogate. Blood was

208

collected via venipuncture before the first vaccination on PND138 and following the first

209

vaccination at multiple time points, before the second vaccination on PND152 and after the

210

second vaccination at multiple time points, and was analyzed by enzyme-linked immunosorbent

211

assay (ELISA) for the presence of anti-KLH IgG and anti-KLH IgM antibodies to assess T-cell

212

dependent antibody response (TDAR).

213

Mortality data during the study period were compared to the incidence of fetal and

214

neonatal losses in historical controls for untreated animals, as well as published literature of

215

spontaneous/background mortality rates among macaques (Small, 1982). Cynomolgus

216

monkey infants that died during the study period were examined for histomorphology. All

217

surviving cynomolgus monkey infants were euthanized on PND180 (± 2 days), and external and

218

visceral exams and histomorphology were conducted.

219

10

220 221

2.5 Immunophenotyping Quantification of cellular antigens and cell populations from cynomolgus monkey infant

222

blood samples and absolute counts of the subpopulations were generated using specific

223

antibodies against the marker antigens. Initial lymphocyte populations were identified and

224

gated for CD2+/CD20+ to identify all lymphocytes (sample purity/total B-cells.); CD20+ for B-

225

lymphocytes; CD3+ for T-lymphocytes; CD3+/CD4+ for T-helper lymphocytes, and CD3+/CD8+

226

for T-cytotoxic lymphocytes.

227 228

2.6 Bioanalytical, pharmacokinetic, toxicokinetic, pharmacodynamic, and anti-

229

tildrakizumab antibodies analyses

230

2.6.1 Assays for tildrakizumab in monkey serum and milk

231

A validated bridging electrochemiluminescence (ECL) assay using human IL-23 as

232

capture (dynamic range of 3.91–250 ng/mL) was used to quantify tildrakizumab in cynomolgus

233

monkey serum. An exploratory tildrakizumab ECL assay (dynamic range of 3.91–250 ng/mL)

234

was used to quantify tildrakizumab in cynomolgus monkey breast milk in the PPND study.

235 236 237

2.6.2 Assays for anti-tildrakizumab antibodies in monkey serum ADA formation in monkey serum was assessed in the 3- and 9-month repeat-dose

238

toxicity studies in monkeys with an ECL assay that was developed and validated using

239

biotinylated and ruthenylated tildrakizumab as the capture and detection reagents, respectively.

240

A protein G purified rabbit polyclonal (anti-tildrakizumab) antibody was used for the preparation

241

of the positive control. The assay had a dynamic range of 0.781 to 50 µg/mL expressed in

242

undiluted serum. ADA formation in serum samples from the EFD study was assessed using a

243

more sensitive ECL assay with a dynamic range of 100 (lower limit of quantitation [LLOQ]) to

11

244

12800 ng/mL (upper limit of quantitation). Based on the rabbit polyclonal positive control, the

245

sensitivity of this assay was 8.52 ng/mL. This ECL assay was subsequently transferred and

246

validated at Covance Laboratories, Inc. (Chantilly, VA, USA), and used to evaluate ADA in serum

247

samples derived from the monkey PPND study.

248 249 250

2.6.3 Assays for IL-23 in monkey serum In the 3-month toxicity study, IL-23 concentration in monkey serum was determined

251

using a validated ECL assay with an LLOQ of 250 pg/mL expressed in neat serum. The assay

252

used biotin- and SULFO-TAG™-labeled antihuman IL-23 mAbs that recognized an epitope of IL-

253

23 distinct from the one recognized by tildrakizumab. In the 9-month monkey toxicity study, a

254

validated assay with an LLOQ of 16 pg/mL expressed in neat serum was used. Both assays

255

were based on the Meso Scale Discovery platform.

256 257

2.6.4 Pharmacokinetic analysis

258

Serum concentration-time data were analyzed using model-independent methods

259

(Gibaldi and Perrier, 1982). Parameters determined included concentration extrapolated to time

260

0 (Co), maximum observed serum concentration (Cmax), time of maximum observed serum

261

concentration (Tmax), area under the concentration-time curve (AUC), area under the

262

concentration-vs-time curve from zero to time t (AUCt), systemic exposure (AUC[0–∞]),

263

bioavailability (F), accumulation ratio (R), clearance (CL), half-life (t1/2), and steady-state

264

volume of distribution (Vdss).

265

12

266 267

2.6.5 Computer software Pharsight® Knowledgebase Server™ versions 2.0.1 or 4.03 with WinNonlin version 4.0.1

268

(Pharsight Corporation, Cary, NC, USA) or Phoenix Application Suite version 1.3 (Pharsight

269

Corporation, Mountain View, CA, USA) was used to conduct the toxicokinetic analysis. Additional

270

analysis used Microsoft Excel (Redmond, WA, USA).

271 272

3. RESULTS

273

3.1 Chronic toxicity

274

In the 3- and 9-month repeat-dose toxicity studies, exposure to tildrakizumab was

275

independent of gender, increased with increasing dose, and increased with repeated IV or SC

276

administration (Tables 1 and 2). A low incidence (1 of 72 animals) of ADA response was noted

277

in the 3- and 9-month studies with an apparent increase in elimination rate of tildrakizumab

278

(data not shown). In the 9-month study, increases in total serum IL-23 concentrations were

279

observed in monkeys, consistent with binding of tildrakizumab with its target cytokine

280

(Supplemental Figure 1).

281

There was no mortality or effects on clinical observations, body weight, qualitative food

282

consumption, veterinary and ophthalmic evaluations, clinical chemistry, hematology, or

283

urinalysis parameters (data not shown). No toxicological findings were seen in assessment of

284

cardiovascular (including blood pressure and ECGs), respiratory, or CNS functions of

285

cynomolgus monkeys treated with tildrakizumab Q2W for up to 9 months (data not shown). In

286

addition, there were no tildrakizumab-related anatomic pathology (including macroscopic and

287

microscopic observations and organ weights). The no observed adverse effects level (NOAEL)

288

was 140 mg/kg (IV and SC) in the 3-month study and 100 mg/kg (SC) in the 9-month study.

289

13

290

3.2 Developmental and reproductive toxicity (DART)

291

3.2.1 Fertility

292

Communication of potential effects on fertility of new medicines is required in product

293

labels. When nonhuman primates are the only relevant toxicology species, the potential for

294

effects on male and female fertility can be evaluated by examination of reproductive tract

295

(organ weights and histomorphologic evaluation) within repeated-dose toxicity studies using

296

sexually mature monkeys. Therefore, both the 3- and 9-month studies included sexually mature

297

animals to determine potential effects of tildrakizumab on the testes histomorphology and/or

298

testicular weight in males; and vaginal swabs, evidence of menses, and/or hormone

299

measurements (estradiol and progesterone) in females. No tildrakizumab-related effects were

300

observed in any parameter evaluated in these studies.

301 302

3.2.2 Embryo-fetal development

303

In the EFD study, peak tildrakizumab serum concentrations were observed in maternal

304

animals from 1 to 7 days after the final dose on GD118 (Supplemental Figure 2). Systemic

305

maternal exposure to tildrakizumab increased with increasing dose (Table 3). Tildrakizumab

306

was detected in fetal serum at the time of C-section on GD140 (Table 5), indicating transfer

307

to the fetus (0.6–0.8 fetal/dam serum ratio). Concentrations of tildrakizumab in the fetus

308

ranged from 21.2 to 69.6, 0.0438 to 458, and 424 to 2180 µg/mL for the 10, 100, and 300

309

mg/kg groups, respectively. Although anti-tildrakizumab antibodies were detected in several

310

animals, including 1 fetus, there was no impact on the animals’ respective serum

311

concentration-time profile (Supplemental Figure 2), with the exception of 1 maternal

312

animal and fetus administered tildrakizumab 100 mg/kg. Anti-tildrakizumab antibodies were

313

present at C-section on GD140 in this dam and fetus; these samples were also positive for

14

314

neutralizing anti-tildrakizumab antibodies. This is consistent with the lower serum drug

315

concentration profile in the dam and fetus as compared with all other animals in the same

316

group.

317

There were no tildrakizumab-related effects on antemortem or postmortem

318

parameters measured, and there were no tildrakizumab-related abnormalities or variations

319

observed in any fetus, including fetal and placental weight at C-section (data not shown). In

320

addition, no toxicity was observed in dams in either the presence or absence of neutralizing

321

anti-tildrakizumab antibodies. The NOAEL was ≥300 mg/kg based on a lack of maternal or

322

developmental toxicity at any dose level.

323 324

3.2.3 Prenatal and postnatal development, including maternal function

325

Systemic exposure to tildrakizumab increased with increasing dose and repeated

326

administration in maternal animals (Table 4); in cynomolgus monkey infants, exposure

327

increased with increasing dose administration to maternal animals (Table 5). Ratios of mean

328

tildrakizumab serum concentrations in cynomolgus monkey infants to dams were

329

approximately 1 between PND7 and PND28 and increased up to 5 on PND180; cynomolgus

330

monkey infants appeared to have slightly reduced clearance relative to the dams from PPD7

331

to PPD180 based on the cynomolgus monkey infant/adult serum concentration ratio (Table

332

5). The mean postbirth serum tildrakizumab concentrations in the cynomolgus monkey infants

333

on PND180 were approximately 0.07 and 0.6 µg/mL at maternal doses of 10 and 100 mg/kg,

334

respectively. Tildrakizumab excretion in breast milk was minimal; mean tildrakizumab

335

concentrations in milk were approximately 0.09% to 0.2% of that in serum on PPD91 for 10 and

336

100 mg/kg, respectively. Some maternal animals in both dosing groups had anti-tildrakizumab

15

337

antibodies, and a subset of these had an apparent increase in tildrakizumab elimination rate

338

(Supplemental Figure 3).

339

Administration of tildrakizumab 10 and 100 mg/kg Q2W to pregnant cynomolgus

340

monkeys was well tolerated. There were no tildrakizumab-related changes in clinical signs,

341

food consumption, or body weights or effects on gestational length or pregnancy/postpartum

342

outcomes that were considered related to maternal administration of tildrakizumab (data not

343

shown).

344

Fetal loss rates (including abortions and stillbirths) were 20% (3/15) in the placebo

345

and 10 mg/kg groups, and 7% (1/15) in the 100 mg/kg group, and all occurred in the third

346

trimester (Table 6). Fetal loss incidence was within the historical control range for the testing

347

facility and in line with published data (Hendrie et al., 1996). Of the fetuses that were aborted

348

or stillborn in all groups, there were no tildrakizumab-related effects observed in the placenta

349

or umbilical cord (when available) or on body weights, morphometric measurements, external

350

or gross/visceral evaluations, or organ weights (data not shown).

351

The number of viable offspring delivered by natural birth was 11 in the placebo group,

352

12 in the 10 mg/kg tildrakizumab group, and 13 in the 100 mg/kg tildrakizumab group. Two

353

neonates (1 in placebo and 1 in the 100 mg/kg tildrakizumab group) were delivered by C-

354

section, resulting in a total of 12, 12, and 14 viable offspring in the control, 10 mg/kg, and

355

100 mg/kg groups, respectively. Of these viable offspring, 7 neonates died or were

356

euthanized within 15 days of birth and were examined by necropsy and histomorphology.

357

Neonatal loss clearly related to maternal neglect occurred in 5 cases within the first week of

358

postnatal life (Table 6); 1/12 (8%) in the placebo group, 2/12 (17%) in the 10 mg/kg group,

359

and 2/14 (14%) in the 100 mg/kg group. In the 10 mg/kg group, 1 moribund neonate was

360

sacrificed because it was unable to adequately grasp the dam to nurse, and another death

16

361

resulted from unsuccessful adoption following maternal death due to extensive blood loss

362

during delivery. Three other neonates were euthanized between PND1 and PND4 due to

363

maternal neglect (1 in the placebo group and 2 in the 100 mg/kg group); histopathological

364

examination showed slight depletion of lymphoid follicles in the spleen and/or moderate

365

lymphoid depletion in the thymus in these animals.

366

The incidence of neonatal loss determined not related to maternal neglect was 0% in

367

the placebo control group, 0% in the tildrakizumab 10 mg/kg group, and 14% (2/14) in the

368

tildrakizumab 100 mg/kg group (Table 6). These values are within the historical control

369

range of the testing facility for untreated animals. The 2 neonates from the 100 mg/kg group

370

that died on PND12 and PND15 appeared icteric with histological findings in the liver and

371

kidneys, suggestive of a possible viral infection.

372

Cynomolgus monkey infants surviving through 6 months postnatal to scheduled

373

necropsy (11, 10, and 10 in the placebo, 10 mg/kg, and 100 mg/kg groups, respectively)

374

showed no tildrakizumab-related changes in clinical signs; body weights; morphometric

375

measurements; external, heart, or gross/visceral assessments; neurobehavioral evaluations

376

(data not shown); clinical serum chemistry; hematology; lymphocyte phenotyping evaluation;

377

immune function competence TDAR test; organ weights; or anatomic pathology, including gross

378

and histopathological assessments (data not shown). In addition, no toxicity was observed in

379

the presence of neutralizing anti-tildrakizumab antibodies.

380

In summary, the no observed effects level (NOEL)/NOAEL in the PPND study was

381

established at 10 mg/kg as there was no difference between the incidences of fetal or neonatal

382

losses (due to maternal neglect or unrelated to maternal neglect) in this group compared to

383

historical controls. At 100 mg/kg, the 2 neonatal deaths due to potential viral infection were

384

considered of uncertain relationship to treatment with tildrakizumab based on the lack of

17

385

historical control histopathology data for occurrence of viral infection in neonate cynomolgus

386

monkeys.

387 388 389

3.3 Exposure multiples Systemic exposure (AUC) multiples for the 3- and 9-month repeat-dose toxicity, EFD,

390

and PPND cynomolgus monkey studies were calculated using clinical exposure derived from a

391

population pharmacokinetic model (AUC0-12 week of 305 µg·day/mL) at the recommended human

392

dose of 100 mg (Jauslin et al., 2019; Jauslin et al., 2018). The monkey-to-human exposure

393

multiple ranged from 7 to 159 times the recommended human dose at the NOAEL in these

394

studies (Table 7).

395 396 397

4. DISCUSSION AND CONCLUSIONS The safety profile of tildrakizumab was characterized in extensive nonclinical safety

398

studies. Tildrakizumab was well tolerated with no toxicological findings in cynomolgus

399

monkeys chronically treated with SC injections of tildrakizumab at 100 mg/kg for up to

400

9 months to maintain high systemic exposure.

401

The repeat-dose toxicity studies in cynomolgus monkeys also included the evaluation

402

of potential impact of tildrakizumab on cardiovascular, respiratory, and central nervous

403

system functions. No tildrakizumab-related changes in any measured safety pharmacology

404

endpoints were detected. These results in monkey studies are consistent with reports that IL-

405

23p19 null mutant mice are viable and breed normally, implying no overt effects in the

406

cardiovascular or other vital systems (Cua et al., 2003; Kurtz et al., 2014).

407 408

Similarly, in extensive developmental and reproductive toxicity studies in cynomolgus monkeys, tildrakizumab was well tolerated during pregnancy and no embryo-fetal toxicity was

18

409

observed in fetuses from pregnant monkeys administered tildrakizumab up to 300 mg/kg SC

410

every other week. In an additional PPND study in cynomolgus monkeys, there were no

411

tildrakizumab-related increases in fetal loss, neonatal loss, or combined fetal and neonatal

412

loss rates, and no effects on morphological or immunological development were observed in

413

offspring exposed to the drug in utero when pregnant monkeys were administered

414

tildrakizumab up to 100 mg/kg SC every other week.

415

As commonly seen in monkey PPND studies (Jarvis et al., 2010), 5 spontaneous

416

neonatal deaths were observed in the PPND study of tildrakizumab and determined to be

417

incidental and associated with maternal neglect. This is a common finding in cynomolgus

418

macaques, particularly in unexperienced, first-time pregnant dams who are less competent

419

than those who have previously given birth (Maestripieri et al., 1997; Prescott et al., 2012;

420

Tsuchida et al., 2008). Maternal neglect and failure to provide food are inducers of stress

421

(Dettling et al., 2007), which is consistent with the observed histopathology of lymphoid

422

tissues from neonates in the PPND study with tildrakizumab (Everds et al., 2013). Of these 5

423

deaths due to maternal neglect, 1 was also associated with failed adoption by a foster

424

mother. Nonhuman primate literature suggests that cross-fostering is less successful than

425

maternal-offspring bonding (Maestripieri, 2001).

426

In the tildrakizumab 100 mg/kg group, 2 additional neonatal deaths were attributed to

427

possible viral infection based on histopathology examination. However, there is a lack of

428

histological control data in nonhuman primate neonates, as neonates found dead typically do

429

not undergo necropsy and histopathology evaluation. Therefore, it is unknown if the observed

430

potential viral infection could be background finding or a drug-related effect. It seems unlikely

431

these observations are a drug-related effect given their low incidence, as well as the good

432

health and confirmed immune competence (ie, normal antibody response to KLH vaccination in

19

433

the TDAR test) of cynomolgus monkey infants monitored through 6 months after birth. Reports

434

of early perinatal and neonatal mortality (PND1−30) caused by infections, such as sepsis,

435

enteritis, and meningitis, in untreated nonhuman primates suggest these 2 neonatal deaths

436

may be coincidental (Price et al., 1973). Furthermore, selective inhibition of the IL-23p19

437

pathway is associated with decreased infection liability compared with broader IL-12/23p40

438

neutralization (Belladonna et al., 2006; Chackerian et al., 2006; Held et al., 2008; Kapil et al.,

439

2009; Khader et al., 2005; Kortylewski et al., 2009; Langowski et al., 2006; Rudner et al.,

440

2007; Schulz et al., 2008; Teng et al., 2010; Teng et al., 2012; Teng et al., 2011; von Scheidt

441

et al., 2014; Wu et al., 2007; Wu et al., 2009; Zelante et al., 2007). Nevertheless, a potential

442

relationship between these 2 neonatal deaths and the treatment of pregnant monkeys with

443

tildrakizumab cannot be ruled out.

444

Immune modulation is sometimes, but not always, associated with a potential for

445

carcinogenicity (Bongartz et al., 2006; Teng et al., 2015). Direct evaluation of carcinogenic

446

potential is not feasible in routine mouse or rat bioassays as tildrakizumab does not bind

447

murine IL-23, most likely due to lower sequence homology. However, multiple literature

448

reports suggest that treatment with a selective IL-23p19 blockade is not associated with an

449

increased tumor risk (Langowski et al., 2006; Teng et al., 2010; Teng et al., 2012; Teng et

450

al., 2011; von Scheidt et al., 2014).

451

In summary, the results of this comprehensive nonclinical safety program evaluating

452

tildrakizumab in cynomolgus monkeys support the safe use of tildrakizumab for the treatment

453

of psoriasis (Ilumya™ [tildrakizumab] full prescribing information, 2018).

454

20

455

5. ACKNOWLEDGEMENTS

456

The authors thank Drs. Kerry Blanchard, Eddie Bowman, Britta Mattson, Judith S. Prescott,

457

Sean Troth, and Gordon K. Wollenberg for their review of this manuscript; Melinda Marian and

458

Robert Venenziale for their roles in planning the early stages of the pharmacokinetic program;

459

and Megan Vogler for her technical input. Editorial support was provided by Kathleen Pieper,

460

PhD, of AlphaBioCom, LLC, and funded by Sun Pharmaceutical Industries, Inc.

461 462

6. AUTHOR CONTRIBUTIONS

463

All authors contributed to the conception, design, interpretation, critical review, and approval of

464

the manuscript to ensure data integrity and accuracy.

465 466

7. DECLARATION OF CONFLICTING INTERESTS

467

All authors are employees of Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc.,

468

Kenilworth, NJ, USA.

469 470

8. FUNDING

471

This work was funded by Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc.,

472

Kenilworth, NJ, USA, and was conducted in the course of the authors’ employment.

21

473

9. REFERENCES

474

Abdollahi, E., et al., 2016. Protective role of R381Q (rs11209026) polymorphism in IL-23R gene

475

in immune-mediated diseases: A comprehensive review. J Immunotoxicol. 13, 286-300,

476

DOI:10.3109/1547691X.2015.1115448.

477 478 479

Belladonna, M. L., et al., 2006. IL-23 neutralization protects mice from Gram-negative endotoxic shock. Cytokine. 34, 161-9, DOI:10.1016/j.cyto.2006.04.011. Bongartz, T., et al., 2006. Anti-TNF antibody therapy in rheumatoid arthritis and the risk of

480

serious infections and malignancies: systematic review and meta-analysis of rare

481

harmful effects in randomized controlled trials. JAMA. 295, 2275-85,

482

DOI:10.1001/jama.295.19.2275.

483

Boutet, M. A., et al., 2018. Role of the IL-23/IL-17 Axis in Psoriasis and Psoriatic Arthritis: The

484

Clinical Importance of Its Divergence in Skin and Joints. Int J Mol Sci. 19,

485

DOI:10.3390/ijms19020530.

486

Capon, F., et al., 2007. Sequence variants in the genes for the interleukin-23 receptor (IL23R)

487

and its ligand (IL12B) confer protection against psoriasis. Hum Genet. 122, 201-6,

488

DOI:10.1007/s00439-007-0397-0.

489

Chackerian, A. A., et al., 2006. Neutralization or absence of the interleukin-23 pathway does not

490

compromise immunity to mycobacterial infection. Infect Immun. 74, 6092-9,

491

DOI:10.1128/IAI.00621-06.

492 493 494 495

Cosentyx® [secukinumab] full prescribing information, 2018. Novartis Pharmaceutical Corporation. East Hanover, NJ. Cua, D. J., et al., 2003. Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain. Nature. 421, 744-8, DOI:10.1038/nature01355.

22

496

Davies, R., et al., 2013. Influence of anti-TNF patient warning regarding avoidance of high risk

497

foods on rates of listeria and salmonella infections in the UK. Ann Rheum Dis. 72, 461-2,

498

DOI:10.1136/annrheumdis-2012-202228.

499

Dettling, A. C., et al., 2007. Behavioral and physiological effects of an infant-neglect

500

manipulation in a bi-parental, twinning primate: impact is dependent on familial factors.

501

Psychoneuroendocrinology. 32, 331-49, DOI:10.1016/j.psyneuen.2007.01.005.

502

Di Meglio, P., et al., 2011. The IL23R R381Q gene variant protects against immune-mediated

503

diseases by impairing IL-23-induced Th17 effector response in humans. PLoS One. 6,

504

e17160, DOI:10.1371/journal.pone.0017160.

505

Enbrel® [etanercept]. Full prescribing information, 2017. Amgen. Thousand Oaks, CA. .

506

Fredriksson, T., Pettersson, U., 1978. Severe psoriasis--oral therapy with a new retinoid.

507

Dermatologica. 157, 238-44.

508

Gibaldi, M., Perrier, D., 1982. Pharmacokinetics. 2nd ed Marcel Dekker, Inc., New York.

509

Held, K. S., et al., 2008. Generation of a protective T-cell response following coronavirus

510

infection of the central nervous system is not dependent on IL-12/23 signaling. Viral

511

Immunol. 21, 173-88, DOI:10.1089/vim.2008.0014.

512 513

Hendrie, T. A., et al., 1996. Frequency of prenatal loss in a macaque breeding colony. American Journal of Primatology. 40, 41-53.

514

Humira® [adalimumab] full prescribing information, 2018. AbbVie, Inc. North Chicago, IL.

515

ICH M3[R2], 2009. International Conference on Harmonisation (ICH) Tripartite Guidelines.

516

Guidance on Nonclinical Safety Studies for the Conduct of Human Clinical Trials and

517

Marketing Authorization for Pharmaceuticals.

518 519

Ilumya™ [tildrakizumab] full prescribing information, 2018. Merck & Co, Inc. Whitehouse Station, NJ.

23

520 521 522

Institute for Laboratory Animal Resources, 2011. Guide for the Care and Use of Laboratory Animals, 8th ed. National Academic Press, Washington, DC. Jarvis, P., et al., 2010. The cynomolgus monkey as a model for developmental toxicity studies:

523

variability of pregnancy losses, statistical power estimates, and group size

524

considerations. Birth Defects Res B Dev Reprod Toxicol. 89, 175-87,

525

DOI:10.1002/bdrb.20234.

526

Jauslin, P., et al., 2019. Population-Pharmacokinetic Modeling of Tildrakizumab (MK-3222), an

527

Anti-Interleukin-23-p19 Monoclonal Antibody, in Healthy Volunteers and Subjects with

528

Psoriasis. Clin Pharmacokinet. DOI:10.1007/s40262-019-00743-7.

529

Jauslin, P., et al., 2018. Abstract 6721. Population pharmacokinetic modeling of tildrakizumab

530

(MK-3222), an anti-interleukin-23p19 monoclonal antibody, in healthy volunteers and

531

subjects with psoriasis. J Am Acad Dermatol. 79, AB224.

532

Kapil, P., et al., 2009. Interleukin-12 (IL-12), but not IL-23, deficiency ameliorates viral

533

encephalitis without affecting viral control. J Virol. 83, 5978-86, DOI:10.1128/JVI.00315-

534

09.

535

Khader, S. A., et al., 2005. IL-23 compensates for the absence of IL-12p70 and is essential for

536

the IL-17 response during tuberculosis but is dispensable for protection and antigen-

537

specific IFN-gamma responses if IL-12p70 is available. J Immunol. 175, 788-95.

538

Kopp, T., et al., 2015. Clinical improvement in psoriasis with specific targeting of interleukin-23.

539 540 541

Nature. 521, 222-6, DOI:10.1038/nature14175. Kortylewski, M., et al., 2009. Regulation of the IL-23 and IL-12 balance by Stat3 signaling in the tumor microenvironment. Cancer Cell. 15, 114-23, DOI:10.1016/j.ccr.2008.12.018.

24

542

Kubota, K., et al., 2015. Epidemiology of psoriasis and palmoplantar pustulosis: a nationwide

543

study using the Japanese national claims database. BMJ Open. 5, e006450,

544

DOI:10.1136/bmjopen-2014-006450.

545

Kurtz, S. L., et al., 2014. IL-23 p19 knockout mice exhibit minimal defects in responses to

546

primary and secondary infection with Francisella tularensis LVS. PLoS One. 9, e109898,

547

DOI:10.1371/journal.pone.0109898.

548

Langley, R. G., et al., 2013. Safety results from a pooled analysis of randomized, controlled

549

phase II and III clinical trials and interim data from an open-label extension trial of the

550

interleukin-12/23 monoclonal antibody, briakinumab, in moderate to severe psoriasis. J

551

Eur Acad Dermatol Venereol. 27, 1252-61, DOI:10.1111/j.1468-3083.2012.04705.x.

552 553 554

Langowski, J. L., et al., 2006. IL-23 promotes tumour incidence and growth. Nature. 442, 4615, DOI:10.1038/nature04808. Langrish, C. L., et al., 2005. IL-23 drives a pathogenic T cell population that induces

555

autoimmune inflammation. J Exp Med. 201, 233-40, DOI:10.1084/jem.20041257.

556

Maestripieri, D., 2001. Is there mother–infant bonding in primates? Developmental Review. 21,

557 558 559 560

93-120. Maestripieri, D., et al., 1997. Infant abuse runs in families of group-living pigtail macaques. Child Abuse Negl. 21, 465-71. Mease, P. J., 2011. Measures of psoriatic arthritis: Tender and Swollen Joint Assessment,

561

Psoriasis Area and Severity Index (PASI), Nail Psoriasis Severity Index (NAPSI), Modified

562

Nail Psoriasis Severity Index (mNAPSI), Mander/Newcastle Enthesitis Index (MEI), Leeds

563

Enthesitis Index (LEI), Spondyloarthritis Research Consortium of Canada (SPARCC),

564

Maastricht Ankylosing Spondylitis Enthesis Score (MASES), Leeds Dactylitis Index (LDI),

565

Patient Global for Psoriatic Arthritis, Dermatology Life Quality Index (DLQI), Psoriatic

25

566

Arthritis Quality of Life (PsAQOL), Functional Assessment of Chronic Illness Therapy-

567

Fatigue (FACIT-F), Psoriatic Arthritis Response Criteria (PsARC), Psoriatic Arthritis Joint

568

Activity Index (PsAJAI), Disease Activity in Psoriatic Arthritis (DAPSA), and Composite

569

Psoriatic Disease Activity Index (CPDAI). Arthritis Care Res (Hoboken). 63 Suppl 11,

570

S64-85, DOI:10.1002/acr.20577.

571 572 573 574

Prescott, M. J., et al., 2012. Laboratory macaques: When to wean? Applied Animal Behaviour Science. 137, 194-207. Price, R., et al., 1973. Simian neonatology: III. The causes of neonatal mortality. Veterinary Pathology. 10, 37-44.

575

Remicade® [infliximab] full prescribing information, 2018. Jassen Biotech, Inc. Horsham, PA.

576

Rudner, X. L., et al., 2007. Interleukin-23 (IL-23)-IL-17 cytokine axis in murine Pneumocystis

577 578

carinii infection. Infect Immun. 75, 3055-61, DOI:10.1128/IAI.01329-06. Schulz, S. M., et al., 2008. Protective immunity to systemic infection with attenuated Salmonella

579

enterica serovar enteritidis in the absence of IL-12 is associated with IL-23-dependent

580

IL-22, but not IL-17. J Immunol. 181, 7891-901.

581 582 583 584

Siliq™ [broadalumab] full prescribing information, 2017. Valeant Pharmaceuticals North America, LLC. Bridgewater, NJ. Small, M. F., 1982. Reproductive failure in macaques. American Journal of Primatology. 2, 137147.

585

Stelara® [ustekinumab] full prescribing information, 2018. Janssen Biotech, Inc. Horsham, PA.

586

Taltz® [ixekizumab] full prescribing information, 2018. Eli Lilly and Compnay. Indianapolis, IN. .

587

Tarantal, A. F., Hendrickx, A. G., 1988. Use of ultrasound for early pregnancy detection in the

588

rhesus and cynomolgus macaque (Macaca mulatta and Macaca fascicularis). J Med

589

Primatol. 17, 105-12.

26

590

Teng, M. W., et al., 2010. IL-23 suppresses innate immune response independently of IL-17A

591

during carcinogenesis and metastasis. Proc Natl Acad Sci U S A. 107, 8328-33,

592

DOI:10.1073/pnas.1003251107.

593

Teng, M. W., et al., 2015. IL-12 and IL-23 cytokines: from discovery to targeted therapies for

594

immune-mediated inflammatory diseases. Nat Med. 21, 719-29, DOI:10.1038/nm.3895.

595

Teng, M. W., et al., 2012. Opposing roles for IL-23 and IL-12 in maintaining occult cancer in an

596 597

equilibrium state. Cancer Res. 72, 3987-96, DOI:10.1158/0008-5472.CAN-12-1337. Teng, M. W., et al., 2011. Anti-IL-23 monoclonal antibody synergizes in combination with

598

targeted therapies or IL-2 to suppress tumor growth and metastases. Cancer Res. 71,

599

2077-86, DOI:10.1158/0008-5472.CAN-10-3994.

600 601

Tonel, G., et al., 2010. Cutting edge: A critical functional role for IL-23 in psoriasis. J Immunol. 185, 5688-91, DOI:10.4049/jimmunol.1001538.

602

Tremfya® [guselkumab] full prescribing information, 2017. Jassen Biotech, Inc. Hrosham, PA.

603

Tsuchida, J., et al., 2008. Maternal behavior of laboratory-born, individually reared long-tailed

604 605

macaques (Macaca fascicularis). J Am Assoc Lab Anim Sci. 47, 29-34. US FDA, 1997. Points to Consider the Manufacture and Testing of Monoclonal Antibody Products

606

for Human Use. US Department of Health and Human Services, Center for Biologics

607

Evaluation and Research.

608

von Scheidt, B., et al., 2014. Combined anti-CD40 and anti-IL-23 monoclonal antibody therapy

609

effectively suppresses tumor growth and metastases. Cancer Res. 74, 2412-21,

610

DOI:10.1158/0008-5472.CAN-13-1646.

611

World Health Organization, Global Report on Psoriasis. 2016.

612

http://apps.who.int/iris/bitstream/10665/204417/1/9789241565189_eng.pdf Accessed

613

Jan 8, 2019.

27

614

Wu, Q., et al., 2007. IL-23-dependent IL-17 production is essential in neutrophil recruitment

615

and activity in mouse lung defense against respiratory Mycoplasma pneumoniae

616

infection. Microbes Infect. 9, 78-86, DOI:10.1016/j.micinf.2006.10.012.

617 618 619

Wu, S., et al., 2009. A human colonic commensal promotes colon tumorigenesis via activation of T helper type 17 T cell responses. Nat Med. 15, 1016-22, DOI:10.1038/nm.2015. Zelante, T., et al., 2007. IL-23 and the Th17 pathway promote inflammation and impair

620

antifungal immune resistance. Eur J Immunol. 37, 2695-706,

621

DOI:10.1002/eji.200737409.

28

622

10. TABLES AND FIGURES

623

Table 1. Mean toxicokinetic parameters in cynomolgus monkeys (sexes combined) following

624

administration of tildrakizumab in the 3-month repeat-dose toxicity study

Dose route IV

Number of dosesa

Sex (n)

1

F (6) M (6)

7

Nominal AUC(0-14 days) dose µg·day/mL)b (mg/kg) (µ 140

19300 (9)

Cmax (µ µg/mL)b

Tmax (days)c

Mean t½ (days)b,d

Re

3220 (18)

NA

NA

NA f

47200 (37)

6990 (30)

NA

17.7 (16)

2.44

40

6900 (29)

709 (40)

3 (1–14)

NA

NA

140

15900 (18)

1380 (22)

3 (1–14)

NA

NA

40

14100 (59)

1200 (62)

3 (1–7)

21.0 (17)

2.04

140 40500 (53) 3810 (58) 3 (0–3) 17.9 (17) Animals received a SC dose of 40 or 140 mg/kg or an IV dose of 140 mg/kg Q2W for 3

2.56

1

F (6) M (6)

7

F (6) M (6)

SC

625

a

626

months. Data is shown for animals receiving either the first or seventh Q2W dose.

627

b

628

c

629

administered as an IV bolus, as the timing of the bolus can affect the observed Tmax.

630

d

631

per sex per group). The mean elimination t½ was 18.9 days across groups (n = 17).

632

e

633

f

634

antibodies on tildrakizumab serum concentrations.

635

AUC, area under the serum concentration-time curve; Cmax, maximum observed serum

636

concentration; IV, intravenous; NA, not applicable; Q2W, once every 2 weeks; R, accumulation

637

ratio; SC, subcutaneous; t½, terminal phase half-life; Tmax, time of maximum observed serum

638

concentration.

Geometric means (coefficient of variation).

Median (minimum−maximum). Tmax is not listed for studies in which tildrakizumab was

Elimination t½ was determined for the animals assigned to the postdose period (n = 3 animals

Accumulation (R) = AUC(0–14 days)Dosing Interval n (after repeated dosing) ÷ AUC(0–14 days)Dosing Interval 1.

One monkey was excluded from t½ calculations due to the influence from anti-tildrakizumab

29

639

Table 2. Mean toxicokinetic parameters in cynomolgus monkeys (sexes combined) following SC

640

administration of tildrakizumab in the 9-month repeat-dose toxicity study Number of dosesa

Nominal dose (mg/kg)

AUC(0-14 days) (µ µg·day/mL)b

Cmax (µ µg/mL)b

Tmax (day)c

Rd

10 30 100

1190 (50) 3340 (21) 11600 (25)

133 (97) 476 (30) 1080 (29)

1 (0.25–14) 1 (1–1) 3 (0.25–3)

NA NA NA

10 ≥13 30 100 a Animals were dosed Q2W.

3290 (29) 9250 (27) 27500 (39)

295 (31) 893 (29) 2640 (51)

3 (0.0833–7) 3 (1–3) 3 (1–7)

2.95 2.92 2.69

F (6) M (6)

1

e

641

Sex (N)

F (12) M (12)

642

b

643

c

644

d

645

e

646

12 animals/sex assessed).

647

AUC, area under the serum concentration-time curve; Cmax, maximum observed serum

648

concentration; NA, not applicable; Q2W, once every 2 weeks; R, accumulation ratio; SC,

649

subcutaneous; Tmax, time of maximum observed serum concentration.

Mean (coefficient of variation).

Median (range). Accumulation (R) = AUC(0–14 days)Dosing Interval n (after repeated dosing) ÷ AUC(0–14 days)Dosing Interval 1.

Mean of data from dosing intervals 13 and 19 (N = 6 animals/sex per interval for a total of

30

650

Table 3. Mean toxicokinetic parameters following subcutaneous administration of tildrakizumab

651

in pregnant cynomolgus monkeys on gestation day 118

Number of dosesa 8

Nb

Nominal dose (mg/kg)

AUC(0-14 days) (µ µg·day/mL)c

12 11 11

10 100 300

1720 (17) 16300 (27) 48500 (22)

Cmax (µ µg/mL)c 151 (20) 1600 (25) 4820 (21)

Tmax (day)d 3 (3–7) 3 (3–3) 3 (1–3)

652

a

653

b

654

c

655

d

656

AUC, area under the serum concentration-time curve; Cmax, maximum observed serum

657

concentration; GD, gestation day; Tmax, time of maximum observed serum concentration.

Monkeys were administered every 14 days from GD20 to GD118. Number of pregnant dams.

Mean (coefficient of variation). Median (range).

31

658

Table 4. Maternal serum tildrakizumab toxicokinetic parameters on gestation day 120 in the

659

pre- and postnatal development study

Day GD 120 660

Dosea (mg/kg/dose)

AUC0-336 hr (µg/mL·hr)

Cmax (µg/mL)

Tmax (hr)

10

47500 (2820)b

193 (14.7)

54 (5.4)

1760 (107)

61 (4.4)

c

100 434000 (28500) Data are presented as mean (SE).

661

a

662

b

663

after dose administration on GD134.

664

c

665

these animals, the sample at 336 hours postdose on GD120 was collected after dose

666

administration on GD134.

667

AUC, area under the serum concentration-time curve; Cmax, maximum observed serum

668

concentration; GD, gestation day; Q2W, once every 2 weeks; SE, standard error; Tmax, time of

669

maximum observed serum concentration.

Animals were dosed Q2W from GD50 until parturition. One animal was excluded because the sample at 336 hours postdose on GD120 was collected

Two animals were excluded due to insufficient quantifiable time points; additionally, in 1 of

32

670

Table 5. Cynomolgus monkey infant/adult female serum tildrakizumab concentration ratios

671

following tildrakizumab administration to adult females prebirth in the pre- and postnatal

672

development study

Daya 7 28 91 180 ± 2 673

Doseb (mg/kg/dose)

Infant monkey serum concentration (µg/mL)

Adult female monkey serum concentration (µg/mL)

Infant monkey/adult female monkey ratio

10

49.3 (5.5)

51.2 (7.9)

1.0

100

466 (26.1)

468 (56.6)

1.0

10

22.7 (2.6)

19.7 (3.7)

1.2

100

213 (18.2)

157 (22.7)

1.4

10

2.53 (0.46)

0.86 (0.3)

2.9

100

21.9 (2.9)

8.3 (3.1)

2.6

10

0.075 (0.02)

0.015 (0.01)

4.9

0.60 (0.15)

0.28 (0.15)

2.2

100 Data are presented as mean (SE).

674

a

675

b

676

GD, gestation day; PND, postnatal day; PPD, postpartum day; SE, standard error.

PND for cynomolgus monkey infants and PPD for adult females. Maternal animals were dosed once every 2 weeks from GD50 until parturition.

33

677

Table 6. Summary of fetal and neonatal losses in pre- and postpartum developement study Treatment group (number of animals)

Fetal loss (abortion and stillbirths)

Neonatal loss due to maternal neglecta

Neonatal loss not due to maternal neglect

Combined fetal and neonatal loss

Placebo (N = 15)

3/15 (20)

1/12 (8)

0/12

4/15 (27)

Tildrakizumab at 10 mg/kg (N = 15)

3/15 (20)

2/12 (17)

0/12

5/15 (33)

Tildrakizumab at 100 mg/kg (N = 15)

1/15 (7)

2/14 (14a)

2/14 (14)

5/15 (33)

Historical controlsb

7%–29%

0%–17%

0%–20%

17%–43%

678

Data are presented as fetal or neonatal loss per total number of animals (%).

679

a

680

b

Includes 1 neonatal death at 100 mg/kg unable to nurse. Data on file.

34

681

Table 7. Systemic tildrakizumab exposure multiples Dose levels a (mg/kg/day) 40 140 (NOAEL) 140 via IV (NOAEL)

AUCt (µg·day/mL) 14100 40500 47200

Monkey to human b exposure multiple NA 133 155

9 months

10 30 100 (NOAEL)

3290 9250 27500

NA NA 90

EFD

10 100 300 (NOAEL)

1720 16300 48500

NA NA 159

10 1979c 100 (NOAEL) 18083c a Subcutaneous route of administration, except where noted.

7 59

Study in monkeys 3 months

PPND 682 683

b

Exposure multiples calculated from a population pharmacokinetic model derived from the

684

human AUC0-12week of 305 ug·day/mL for 100 mg (MRHD) (Jauslin et al., 2019; Jauslin et al.,

685

2018).

686

c

AUC (µg·hr/mL)/24 hours to convert to AUC (µg/day/mL).

687

AUCt, area under the concentration-vs-time curve from zero to time t; EFD, embryo-fetal

688

development; MRHD, recommended human dose; NA, not applicable; NOAEL, no observed

689

adverse effect level; PPND, pre- and postnatal development; IV, intravenous.

35

690

11. SUPPLEMENTAL MATERIAL

691

Supplemental Methods

692

Three-month repeat-dose toxicity study collection time points

693

Blood samples for toxicokinetic evaluation and determination of interleukin (IL)-23

694

concentration were collected prior to dosing on the first day and at multiple time points within

695

the first 24 hours postdose, then at 24-hour intervals up to 96 hours, and at 168 hours

696

following the first dose. Thereafter, blood samples for toxicokinetic evaluation, IL-23

697

concentration measures, and antidrug antibody (ADA) analyses were collected at day 10; prior

698

to dosing every 2 weeks on days 14 through 70; on days 98, 84, and 112; and every other

699

week after until sacrifice. On day 84, additional samples were taken for toxicokinetic analyses

700

predose and at multiple time points up to 168 hours postdose.

701 702 703

Nine-month repeat-dose toxicity study collection time points Blood samples for toxicokinetic evaluation were collected on days 1, 169, and 253 (at

704

predose and multiple time points up to 336 hours postdose on each of those days); predose on

705

days 43, 85, 127, and 211; at week 8 of the treatment-free period; and on the day of sacrifice.

706

Blood was collected for ADA analysis and IL-23 concentration measures at predose; on days 1,

707

15, 43, 85, 127, 169, and 211; week 8 of the treatment-free period; and on the day of sacrifice.

708

Additional samples for IL-23 concentration were taken on days 3 and 7.

709 710 711 712

Embryo-fetal development study collection time points Maternal blood was sampled predose on gestation day (GD)20, GD34, GD62, and GD90; at multiple time points up to 336 hours postdose on GD118; and once on the day of Cesarean-

36

713

section (GD140) for toxicokinetic and ADA analyses, including a neutralization assay. Fetal

714

umbilical cord blood samples were collected at C-section for toxicokinetic and ADA analyses.

37

715

Supplemental Figure 1. Individual serum IL-23 levels over time during the 9-month repeat-

716

dose toxicity study in males

717 718

N = 6 for all doses. Each line represents an individual male; female data was similar. Day 0 was

719

the first day of tildrakizumab administration. Day 318 and 395 measurements occurred during

720

the recovery phase.

721

a

722

increase in IL-23 in a single monkey at the day 169 time point is unknown but did not affect the

723

toxicity profile.

724

IL, interleukin.

Tildrakizumab exposure in the 30 mg/kg group showed low variability. The reason for the

725

38

726

Supplemental Figure 2. Maternal tildrakizumab serum concentration and antidrug antibody

727

presence during the embryofetal development study

728 729

a

730

b

731

concentrations were measured prior to dosing (trough concentration) with robust sampling after

732

the final dose.

733

Day 0 was the first day of drug administration. Each line represents an individual animal. Time

734

points where ADA was detected are marked with a red X. All dose groups, but not the placebo

735

group, are presented. One animal was positive on day 140 and 160; this animal and the animal

736

positive on day 34 were different from the 5 that were positive on day 20.

737

ADA, antidrug antibody; GD, gestation day; TIL, tildrakizumab.

Five animals were ADA-positive at day 20. Monkeys were administered every 14 days from GD20 to GD118. Tildrakizumab serum

738

39

Supplemental Figure 3. Maternal tildrakizumab serum concentration and antidrug antibody presence during the pre- and postnatal development study for A) the 10 mg/kg group and B) the 100 mg/kg group A)

a

Three animals were ADA-positive at GD50, and 5 animals were ADA-positive at postpartum day 91.

b

Animals were dosed Q2W from GD50 until parturition. Tildrakizumab serum concentrations were measured prior to dosing (trough

concentration) with robust sampling after the final dose. c

Four animals were ADA-positive on postpartum day 180; all animals except 1 that were ADA-positive on postpartum day 91 were

also ADA-positive on postpartum day 180.

40

Each line represents an individual maternal animal’s tildrakizumab serum concentration. Time points where ADA were detected are marked with a red X. ADA, antidrug antibody; GD, gestation day; Q2W, every 2 weeks; TIL, tildrakizumab.

B)

a

Three animals were ADA-positive at gestation day 50.

41

b

Animals were dosed Q2W from GD50 until parturition. Tildrakizumab serum concentrations were measured prior to dosing (trough

concentration) with robust sampling after the final dose. Each line represents an individual maternal animal’s tildrakizumab serum concentration. Time points where ADA were detected are marked with a red X. ADA, antidrug antibody; GD, gestation day; Q2W, every 2 weeks; TIL, tildrakizumab.

42

FUNDING This work was funded by Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA and was conducted in the course of the authors’ employment.

Declaration of interests ☐ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☒The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

All authors are employees of Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA.