itacitinib (incb039110), a jak1 inhibitor, reduces cytokines … · 2020. 9. 30. · 1 itacitinib...

Itacitinib (INCB039110), a JAK1 inhibitor, Reduces Cytokines Associated with Cytokine 1

Release Syndrome Induced by CAR T-Cell Therapy 2

3

Eduardo Huarte1, Roddy S. O’Connor

2,3, Michael T. Peel

1, Selene Nunez-Cruz

2,3, John 4

Leferovich2,3

, Ashish Juvekar1, Yan-ou Yang

1, Lisa Truong

1, Taisheng Huang

1, Ahmad Naim

4, 5

Michael C. Milone2,3

, and Paul A. Smith1 6

7 1Incyte Research Institute, Wilmington, Delaware.

2Department of Pathology and Laboratory 8

Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, 9

Pennsylvania. 3Center for Cellular Immunotherapies, Perelman School of Medicine, University 10

of Pennsylvania, Philadelphia, Pennsylvania. 4Incyte Corporation, Wilmington, Delaware. 11

12

13

Running title: Itacitinib (JAK1 Inhibitor) Prevents CAR T-Cell–Induced CRS 14

15

16

Corresponding author: Eduardo Huarte, PhD, Incyte Research Institute, 1801 Augustine Cut-17

off. Wilmington, DE 19810, USA; Phone: 302 304 6332; E-mail: [email protected] 18

19

Disclosure of Potential Conflict of Interests: Eduardo Huarte, Michael T. Peel, Ashish 20

Juvekar, Yan-ou Yang, Lisa Truong, Taisheng Huang, Ahmad Naim and Paul S. Smith are 21

employees and stockholders of Incyte. Michael C. Milone is cofounder and co-chair of Cabaletta 22

Bio and consultant for Novartis. 23

24

on July 6, 2021. © 2020 American Association for Cancer Research.clincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 30, 2020; DOI: 10.1158/1078-0432.CCR-20-1739

http://clincancerres.aacrjournals.org/

Translational relevance 25 The development of chimeric antigen receptor (CAR) technology has dramatically altered the 26

global landscape of cancer cell therapy with unprecedented responses of CAR T-cells targeting 27

CD19 and other B-cell antigens. However, cytokine release syndrome (CRS) and neurotoxicity 28

represent serious, potentially life-threatening side effects often associated with CAR T-cell 29

therapy. In the present study, we demonstrated that JAK1 inhibition with itacitinib reduces levels 30

of cytokines implicated in CRS without affecting CAR T-cell proliferation or cytolytic activity in 31

vitro. Our data show that prophylactic JAK1 inhibition reduces hyperinflammation, and does not 32

affect CAR T-cell antitumor response in preclinical models. This study provides the rationale for 33

a phase II clinical trial of itacitinib for the prevention of CRS induced by CAR T-cell therapy 34

(NCT04071366). 35




Abstract 36 Purpose: T-cells engineered to express a chimeric antigen receptor (CAR T-cells) are a 37

promising cancer immunotherapy. Such targeted therapies have shown long-term relapse-free 38

survival in patients with B-cell leukemia and lymphoma. However, cytokine release syndrome 39

(CRS) represents a serious, potentially life-threatening side effect often associated with CAR T-40

cell therapy. CRS manifests as a rapid (hyper)immune reaction driven by excessive 41

inflammatory cytokine release, including interferon- and interleukin-6. 42

Experimental Design: Many cytokines implicated in CRS are known to signal through the Janus 43

kinase–signal transducers and activators of transcription (JAK-STAT) pathway. Here we study 44

the effect of blocking JAK pathway signaling on CAR T-cell proliferation, anti-tumor activity 45

and cytokine levels in in vitro and in vivo models. 46

Results: We report that itacitinib, a potent, selective JAK1 inhibitor, was able to significantly and 47

dose-dependently reduce levels of multiple cytokines implicated in CRS in several in vitro and in 48

vivo models. Importantly, we also report that at clinically relevant doses that mimic human JAK1 49

pharmacologic inhibition, itacitinib did not significantly inhibit proliferation or antitumor killing 50

capacity of three different human CAR T-cell constructs (GD2, EGFR, and CD19). Finally, in an 51

in vivo model, antitumor activity of CD19-CAR T-cells adoptively transferred into CD19+

tumor 52

bearing immuno-deficient animals was unabated by oral itacitinib treatment. 53

Conclusion: Together, these data suggest that itacitinib has potential as a prophylactic agent for 54

the prevention of CAR T-cell–induced CRS, and a phase II clinical trial of itacitinib for 55

prevention of CRS induced by CAR T-cell therapy has been initiated (NCT04071366). 56




Introduction 57 T-cells engineered to express a chimeric antigen receptor (CAR T-cells) have recently 58

been approved by the US Food and Drug Administration, and commercially available CAR T-59

cell therapies (axicabtagene ciloleucel and tisagenlecleucel) have resulted in an overall response 60

rate of almost 80% (1-4), including long-term relapse-free survival in patients with B-cell 61

leukemia and lymphoma (5). However, an exaggerated immune response by the adoptively 62

transferred CAR T-cells, as well as the host immune system, can lead to a severe cytokine 63

release syndrome (CRS) in up to 40% of treated patients (6, 7). CRS manifests as a rapid 64

immune reaction driven by a massive release of inflammatory cytokines. While individual 65

cytokine levels differs between patients, severe CRS is generally very similar to that seen in 66

hemophagocytic lymphohistiocytosis (HLH) or macrophages activation syndrome (MAS)(8), 67

with elevation of both canonical T-cell cytokines (i.e. IFN-) as well as cytokines compatible 68

with HLH/MAS (i.e. IL-6 and IL-10)(9). Typified by chills and fevers, CRS can also progress to 69

a more severe, even life-threatening reaction (10, 11). 70

To date, low-grade CRS is treated symptomatically with antipyretics and fluids (7). 71

Grade > 2 CRS can be treated with tocilizumab (an anti–IL-6 receptor), recently approved by the 72

US Food and Drug Administration and the European Medicines Agency for treatment of severe 73

and life-threatening CRS (12, 13). Importantly, therapeutic administration of tocilizumab does 74

not influence peak CAR T-cell numbers nor clinical outcomes (14). However, treatment with 75

corticosteroids is also needed in the case of tocilizumab-refractory patients, and concerns remain 76

over the effect of high dose corticosteroids on CAR-T cell expansion and anti-tumor activity 77

(14). To date there are no preventative strategies, and we rely on therapeutic treatments. Thus, 78

the ability to prophylactically inhibit hyper-inflammatory states and the incidence of CRS would 79

represent a new clinical management strategy for patients receiving CAR T therapy. 80

JAKs play an important role in signal transduction following cytokine and growth factor 81

binding to their cognate receptors. Once activated, JAKs phosphorylate signal transducer and 82

activator of transcription proteins (STATs), which results in dimerization and translocation of 83

STATs to the nucleus in order to activate or suppress gene transcription (15). Aberrant 84

production of JAK-mediated cytokines, such as IL-6 and growth factors, has been associated 85

with a number of chronic inflammatory conditions (16, 17). Thus, we reasoned that the blockade 86

of the JAK signaling pathway would attenuate the excessive cytokine response that occurs 87

during CRS without negatively impacting CAR T-cell function. Itacitinib (INCB039110) is a 88

small-molecule inhibitor of the Janus kinase (JAK) family of protein tyrosine kinases (18). As 89

opposed to other JAK inhibitors developed by Incyte (i.e. ruxolitinib) as well as others (i.e. 90

tofacitanib), itacitinib is a JAK1 selective inhibitor, and is therefore expected to minimize the 91

risk of long-term infection or other complications associated with pan-JAK inhibitors (19), while 92

reducing the levels of cytokines that signal trough JAK1, such as IFN-, IL-6, IL-12 or TNF-. 93

In this study, we show that in both in vitro and in vivo preclinical models, itacitinib doses 94

relevant to cellular half maximal inhibitory concentration (IC50; 50–100 nM) were more effective 95

than tocilizumab in reducing CRS-related cytokines produced by CD19-CAR T-cells. Moreover, 96

itacitinib did not significantly inhibit in vitro proliferation or antitumor killing capacity of CAR 97

T-cells, nor, in a lymphoma model, did it affect the antitumor activity of CAR T-cells in a 98

prophylactic setting. Together, these data suggest that itacitinib has potential as a prophylactic 99

agent for the prevention of CAR T-cell–induced CRS, and a phase II clinical trial of itacitinib for 100

the prevention of CRS induced by CAR T-cells is ongoing (NCT04071366). 101

102



https://en.wikipedia.org/wiki/STAT_proteinhttps://en.wikipedia.org/wiki/STAT_proteinhttp://clincancerres.aacrjournals.org/

Methods 103 Animals 104

C57BL/6 and BALB/c animals were purchased from Taconic Biosciences (Rensselaer, 105

New York) and were approximately 8 weeks old at the time experiments were initiated. OT-1 106

TCR-transgenic mice and severely immunodeficient NOD-scid IL2R gamma null (NSG) animals 107

were purchased from the Jackson Laboratory (Bar Harbor, Maine). All mice were used in 108

protocols approved by the Institutional Animal Care and Use Committee. 109

110

T-cell proliferation assay 111

Peripheral blood mononuclear cells (PBMCs) were prepared from human whole blood 112

samples using a Ficoll-Hypaque separation method, and T-cells were then obtained from the 113

PBMCs by centrifugal elutriation. T-cells were maintained in RPMI 1640 medium (Thermo 114

Fisher Scientific, Waltham, Massachusetts) supplemented with 10% fetal bovine serum, 1% 115

HEPES, 2 mM L-glutamine, 0.05 mM 2-mercaptoethanol and 100 μg/mL streptomycin, and 100 116

units/mL penicillin (complete RPMI or CM). T-cells were activated with Dynabeads (Thermo 117

Fisher Scientific; immobilized agonist antibodies against CD3/CD28) at a 3:1 ratio, resuspended 118

at a density of 0.5 × 106 cells/mL in 24-well plates and treated with itacitinib at various 119

concentrations (from 50 to 1000 nM). The plates were incubated at 37°C in 5% CO2 atmosphere 120

for 10 days, and the proliferation was determined every other day by bead-based methods 121

(CountBright™ Absolute Counting Beads, Molecular Probes, ThermoFisher). Cultures were 122

replenished every other day with fresh CM. 123

124

Cytotoxicity assays 125

Luciferase expressing SY5Y neuroblastoma cells (GD-2+) were plated in a 96-well plate 126

at 50,000 cells/well. Twenty-four hours later, 150,000 CAR T-cells were added to corresponding 127

wells in a final volume of 200 L. Target cells alone were seeded in parallel to quantify the 128

maximum luciferase expression (relative luminescent units; RLUmax). Seventeen hours later 129

100 L of luciferin substrate (Bright-Glo™ Luciferase Assay System, Promega, Madison, 130

Wisconsin) was added to the co-culture. Luminescence was measured after a 10-minute 131

incubation using an EnVision (PerkinElmer, Waltham, Massachusetts) plate reader. The percent 132

cell lysis was obtained using the following calculation: [1 − (RLUexperimental)/(RLUmax)] × 133

100. The experiment was performed in triplicates. 134

135

Itacitinib modulation of mouse CRS models 136

CRS was induced in BALB/c animals by intravenous injection (IV) of Concanavalin-A 137

(ConA; 20 mg/kg) or 100 g of an anti-CD3ε antibody (clone 145-2C11). Corresponding 138

animals were orally dosed with 60 or 120 mg/kg of itacitinib 60 minutes prior CRS induction 139

(prophylactic) or 30 minutes after (therapeutic) CRS induction. Two hours after ConA or anti-140

CD3 injection, mice were sacrificed and blood was collected into K2EDTA tubes for cytokine 141

measurement. 142

143

Activated macrophage models 144

Spleens were collected from 6- to 8-week-old C57BL-6 female mice and mechanically 145

dissociated. Splenocytes were cultured in RPMI medium supplemented with 10% fetal bovine 146

serum and macrophage colony-stimulating factor. On day 6, cells were treated with itacitinib and 147




then treated with 5 ng/mL lipopolysaccharide (LPS) on day 7. The following day, supernatant 148

was collected and cytokines were measured as described below. Six- to 8-week-old female 149

C57BL/6 mice were prophylactically orally dosed with vehicle, or 60 or 120 mg/kg of itacitinib 150

twice a day (b.i.d.) for 3 days. Mice then received intraperitoneal injections of LPS (5 µg per 151

animal). Two hours after injection, mice were euthanized and 3 mL of sterile saline was injected 152

into the peritoneum to perform a peritoneal lavage. Subsequent cytokine measurements were 153

performed as described below. 154

155

Cytokine measurement 156

Plasma cytokines were measured using multi-spot assay system, pro-inflammatory panel 157

1 (for IFN-, IL-1β, IL-2, IL-4, IL-5, IL-6, KC/GRO, IL-10, IL-1p70, and TNF; Meso Scale 158

Discovery, Meso Scale Diagnostics [MSD], Rockville, Maryland), following manufacturer’s 159

instructions. Briefly, samples, standards, and controls were added at 25 µL per well. The plate 160

was sealed and incubated for 2 hours at room temperature on an orbital shaker (600 rpm). At the 161

end of the incubation, the wells were washed three times using 200 µL phosphate-buffered saline 162

+ 0.05% Tween 20, soaking for 30 seconds and then discarding. Detection antibody was added at 163

25 µL per well, and the plate sealed and incubated for 1 hour at room temperature on an orbital 164

shaker (600 rpm). At the end of the incubation, the plate was washed three times as done 165

previously. 150 µL of the MSD Read Buffer was added to each well, and the MSD plates were 166

measured on a MSD Sector S 600 plate reader. The raw data were measured as an 167

electrochemiluminescence signal detected by photodetectors and analyzed using the MSD 168

Discovery Workbench software. A four-parameter logistic fit curve was generated for each 169

analyte using the standards, and the concentration of each sample was calculated. 170

171

OT-1 T-cell expansion and antitumor activity 172

The OT-1 strain is transgenic for the TCR VV5 specific for the OVA257-264 peptide 173

(SIINFEKL) and restricted to H2-Kb. Naïve OT-1 CD8 cells were isolated from OT-1 spleens by 174

negative selection using Naïve CD8a+ T Cell Isolation Kit (Miltenyi Biotec, Auburn, California). 175

To measure OT-1 CD8+ cell expansion, cells were CFSE-labeled (2 M) and 176

resuspended at 5 × 106 cells/mL in complete RPMI, 20 IU IL-2, 2 g/mL of SIINFEKL peptide. 177

and increasing itacitinib concentrations, from 0 to 1000 nM. Three to five days later, OT-1 cells 178

were collected, stained with an APC-anti-CD8 antibody, and the number of divisions was 179

calculated by measuring the relative carboxyfluorescein succinimidyl ester (CFSE) fluorescence 180

intensity of the different conditions by flow cytometry (20). 181

To study the effect of itacitinib on the antitumor activity of CD8 OT-1 cells, C57BL/6 182

animals received subcutaneous injections of 0.5 × 106 OVA-expressing EG7 tumor cells into the 183

shaved right flank. At day 7, corresponding animals were orally dosed with itacitinib b.i.d. at 60 184

or 120 mg/kg, and at day 10, corresponding animals received an intravenous (tail vein) injection 185

of 5 × 106 naïve OT-1 CD8 cells. Tumor sizes were measured on two perpendicular axes using a 186

digital caliper at least three times a week. Tumor volumes were calculated with the formula: 187

V = (W2 × L)/2 188

Where V is tumor volume, W is tumor width, and L is tumor length. 189

190

Cell Viability Assay 191

Cell proliferation was measured using CellTiter-Glo® Luminescent Cell Viability Assay 192

(Promega, Madison, WI), following manufacturer’s instructions. Briefly, 1000 EG7 cells were 193




seeded in a total volume of 200 l of RPMI + 10% FBS per well in a 96-well plate. The next 194

day, compound plates were reconstituted in complete media with a 10 point dose curve. Cell 195

media of the plate was aspirated and compound with a total volume of 100 l was incubated with 196

the cells for 72 hours. 100 l of CellTiter-Glow reagent was added to each well and protected 197

from the light with a plate seal. Plates were shaken at room temperature for 2 minutes to induce 198

cell lysis and then allowed to sit for approximately 10 minutes. The raw data was measured on a 199

TopCount Luminescence Counter (Perkin Elmer, Waltham, MA) and analyzed in Prism using an 200

[inhibitor] vs. response – variable slope (four parameters) equation. 201

202

Generation of CAR constructs and CAR T-cell preparation 203

The CD19-BBζ CAR consisting of a CD8 hinge, 4-1BB costimulatory domain, and CD3ζ 204

signaling domain was generated as previously described (21). PBMCs were stimulated with 205

Dynabeads (3:1 ratio) and then plated in 24-well plates in X-Vivo medium (Lonza, Morristown, 206

New Jersey) containing IL-2 (200 IU/mL), IL-7 (10 ng/mL), IL-15 (5 ng/mL), and IL-21 (5 207

ng/mL) at 0.5 × 106 cells/mL. Three days later, T-cells were transduced using a lentiviral vector 208

expressing the CAR construct, as described elsewhere (22). 209

Generation of the GD2-E101K CAR lentiviral plasmid was previously described (23). 210

The CAR contains the 14G2a scFv, as well as an EF1α promoter, CD8 hinge, 4-1BB 211

costimulatory domain, and CD3ζ signaling domain. The anti-EGFR CAR, with 3C10 scFv, was 212

previously described (24). The CAR contains the 3C10 scFV, as well as an EF1α promoter, CD8 213

hinge, 4-1BB costimulatory domain, and CD3ζ signaling domain. 214

215

Antitumor activity (Nalm6 model) 216

To study whether JAK1 inhibition affects the antitumor effect of CAR T-cells, a 217

xenograft model was used as previously reported (25). Briefly, 6- to 10-week-old 218

immunodeficient NOD-SCID γc−/−

(NSG) mice were injected intravenously via tail vein with 2.5 219

× 106 luciferase expressing Nalm6 acute lymphoblastic leukemia cells. Corresponding animals 220

received 120 mg/kg of itacitinib per 10 days (orally, b.i.d.), starting 1 day after the tumor 221

challenge. Mice were then randomized into groups for adoptive transfer of 3 × 106 CAR T-cells, 222

non-transduced cells, or vehicle intravenously at day 4. Anesthetized mice were imaged using a 223

Xenogen IVIS Spectrum system (Caliper Life Sciences, Hopkinton, Massachusetts) once a week. 224

Mice were given an intraperitoneal injection of D-luciferin (150 mg/kg; Caliper Life Sciences). 225

Total flux was quantified using Living Image 4.4 (PerkinElmer) by drawing rectangles of 226

identical area around mice, reaching from head to 50% of the tail length. Background 227

bioluminescence was subtracted for each image individually. Animals were monitored daily, and 228

survival rates were compared between groups. 229

230

Antitumor activity (NAMALWA model) 231

Six- to 8-week-old male NSG mice were subcutaneously inoculated with luciferase-expressing 232

CD19+ NAMALWA lymphoma cells (5 × 10

6). Mice were weighed and monitored two times a 233

week, and bioluminescence signals were measured every 5 days. At day 5 after tumor challenge, 234

animals started a b.i.d. oral treatment of vehicle or INCB039110 (120 mg/kg) for 8 days. At day 235

8 after tumor challenge, animals received an adoptive transfer of 5 × 106 CD19-CAR T-cells. 236

Bioluminiscence was measured as done previously. Animals were monitored daily, and survival 237

rates were compared between groups. 238

239




Data and statistical analysis 240

Data are reported as mean ± SD or mean ± SEM in the relevant figures. Differences between two 241

groups were analyzed by nonparametric Mann-Whitney test. Statistical analysis for multiple 242

groups was performed by Kruskal-Wallis with Dunn’s post hoc test for nonparametric data sets, 243

or ANOVA with Holm-Sidak’s test for parametric results. All tests were performed using 244

GraphPad Prism (GraphPad Software Inc, San Diego, California). 245

246

Results 247 Itacitinib reduces cytokine levels in murine models of acute hyperinflammation 248

As CRS is the most common side effect associated with CAR T-cell treatment, we 249

investigated whether itacitinib is able to reduce the levels of cytokines associated with acute 250

hyperactivation leading to CRS. Therefore, we conducted experiments in which naïve animals 251

were challenged with ConA, a potent T-cell mitogen capable of inducing broad inflammatory 252

cytokine releases and proliferation (26). Similar to individuals experiencing CRS, animals 253

receiving ConA have elevated serum levels of multiple inflammatory cytokines as well as 254

behavioral changes such as fever, malaise, hypotension, hypoxia, capillary leak, multi-organ 255

toxicity, and potentially death. To study the effect of itacitinib in this model, corresponding 256

animals were prophylactically dosed with 60 or 120 mg/kg of itacitinib to achieve JAK1 257

inhibition coverage equivalent to that observed in clinical trials (27-29). When compared with 258

vehicle-dosed animals, itacitinib was able to significantly reduce serum levels of many of the 259

cytokines implicated in CRS (i.e., IL-6, IL-12, and IFN-) in a dose-dependent manner (Fig. 1A). 260

As expected, itacitinib did not have a significant effect on cytokines independent of the JAK1 261

pathway (i.e., IL-5, Fig. 1A). However, not all JAK-mediated cytokines were significantly 262

decreased (i.e. IL-4, Fig 1A). Additionally, itacitinib was also able to dose-dependently reduce 263

CRS-implicated cytokines in a therapeutic mode, where animals were dosed with itacitinib 30 264

minutes after ConA challenge (Fig. 1B). 265

To confirm the capacity of itacitinib to reduce hyperinflammation, naïve animals were 266

challenged with anti-CD3 to induce nonspecific T-cell activation and cytokine response. Once 267

again, corresponding animals were either prophylactically or therapeutically dosed with 120 268

mg/kg of itacitinib. Compared with vehicle-treated mice, itacitinib was able to significantly 269

reduce serum levels of many of the cytokines implicated in CRS, but have no effect on cytokines 270

independent of the JAK1 pathway (Supplementary Fig. S1A). 271

272

Itacitinib reduces IL-6 production by macrophages 273

Recent evidence shows that host macrophages are the major producers of IL-6 after CAR 274

T-cell treatment (30). Therefore, we queried whether itacitinib would prevent IL-6 production by 275

host macrophages. First, murine bone marrow–derived macrophages were expanded in vitro with 276

granulocyte-colony stimulating factor, and itacitinib was added to the cultures at day 6. LPS was 277

added to the culture at day 7 to activate the macrophages (31). Prophylactic treatment with 278

itacitinib reduced IL-6 production in a dose-dependent manner, indicating that the activity of 279

itacitinib in reducing production of inflammatory cytokines is not exclusive to T-cells (Fig. 2A). 280

We also observed a non-significant trend towards reduction on several other cytokines (i.e. IL-281

10, IL-12p70, KC/GRO, Supplementary Fig. S2) 282 Having determined that itacitinib is able to reduce IL-6 production in vitro, we next 283

expanded our studies to an in vivo context to assess the effect of itacitinib on activated 284




macrophages in mice. Mice were prophylactically treated with itacitinib or vehicle for 3 days to 285

achieve steady state before receiving an intraperitoneal injection of LPS. Two hours after 286

injection, cytokines were measured from intraperitoneal lavages. As seen in Fig. 2B, doses of 287

itacitinib that mimic the JAK1 inhibition coverage achieved in clinical trials significantly 288

reduced IL-6 production by activated macrophages in vivo. Thus, data from both in vitro and in 289

vivo systems indicate that itacitinib is capable of downregulating the major cellular source of 290

inflammatory IL-6 during an experimentally induced model of CRS. 291

292

Itacitinib reduces CAR T-cell cytokine production 293

Whereas cytokine production by the host immune system is a primary cause of CRS, 294

production of inflammatory cytokines by CAR T-cells is also a major contributor (32). Having 295

shown that itacitinib is capable of reducing inflammatory cytokine production by the host 296

immune system, we next wanted to examine whether itacitinib reduces inflammatory cytokine 297

production by CAR T-cells. Human CD19-CAR T-cells were expanded under concentrations of 298

tocilizumab or itacitinib equivalent to human doses achieved in clinical trials (33). After a 3-day 299

expansion, CAR T-cells were co-cultured with CD19 expressing NAMALWA target cells, and 6 300

hours later supernatants were collected to quantify cytokines. Itacitinib, but not tocilizumab, was 301

able to significantly reduce the levels of many inflammatory cytokines (i.e., IL-2, IFN-, IL-6, 302

and IL-8) (Fig. 3). As an internal control (background), we also measured cytokines produced by 303

non-transduced T-cells (Fig. 3). 304

305

Itacitinib at clinically relevant concentrations does not affect PBMC proliferation 306 As T-cells play an important antitumor role, the effect of itacitinib on T-cells was studied 307

to determine if treatment may result in broad immunosuppression potentially interfering with 308

normal T-cell proliferation and function. T-cells were obtained from freshly isolated human 309

PBMCs of healthy adults. Following activation with anti-CD3/anti-CD28 coated beads (23), T-310

cells were expanded in the presence of increasing concentrations of itacitinib, and proliferation 311

was measured by flow cytometry. When compared with the dimethyl sulfoxide (DMSO) control, 312

concentrations of itacitinib relevant to the IC50 (50–100 nM) (34) did not have a significant effect 313

on anti-CD3/anti-CD28–induced expansion of human T-cells (Fig. 4A), demonstrating that 314

itacitinib treatment has no negative impact on the ability of T-cells to proliferate. As an internal 315

control, and consistent with the disruption of the JAK/STAT signaling activity, higher 316

concentrations of itacitinib (1000 nM) blocked T-cell proliferation (Fig. 4A). 317

318

Itacitinib does not affect CAR T-cell proliferation 319

To study the effect of itacitinib specifically on CAR T-cell expansion and cytolytic 320

activity, GD2-CAR T-cells (23) were expanded with anti-CD3/anti-CD28 coated beads in the 321

presence of increasing itacitinib concentrations, ranging from 100 to 500 nM. Once again, when 322

compared with the DMSO control, itacitinib at 100–250 nM did not have a significant effect on 323

CD3/CD28-induced expansion of GD2-CAR T-cells, whereas 500 nM was enough to block 324

CAR T-cell proliferation (Fig. 4B). Importantly, GD2-CAR T-cells expanded in the presence of 325

low (100 and 250 nM) itacitinib concentrations were able to efficiently lyse GD2-expressing 326

tumor cells (Fig. 4C). As a negative (background) control, we also measured target cell lysis by 327

non-transduced (nonspecific) T-cells. As expected, GD2-CAR T-cells expanded in the presence 328

of a high dose of itacitinib (500 nM) and were unable to induce the lysis of target cells beyond 329




background levels (Fig. 4C). Taken together, these data indicate that IC50 relevant doses of 330

itacitinib do not interfere with CAR T-cell proliferation or antitumor activity in vitro. 331

Next, we queried whether itacitinib has an effect on CAR T-cell antigen-specific 332

proliferation. EGFR-CAR T-cells (35) were expanded with anti-CD3/CD28 beads for 2 weeks 333

and then restimulated with EGFR beads in the presence of itacitinib or DMSO controls. Once 334

again, doses of itacitinib relevant to the IC50 (100 or 250 nM) did not affect CAR T-cell antigen-335

specific proliferation (Fig. 4D). 336

337

Itacitinib does not impair T-cell antitumor activity in vivo 338

To further test the effect of itacitinib on antigen-specific T-cell proliferation and in vivo 339

antitumor activity, we isolated splenocytes from OT-1 mice, transgenic for the TCR V2V5 340

specific for the peptide OVA257-264 (SIINFEKL) (36). OT-1 T-cells were expanded with the 341

SIINFEKL peptide in the presence of increasing concentrations of itacitinib, and their expansion 342

was measured by flow cytometry. As expected, itacitinib concentrations relevant to the IC50 have 343

a minimal effect on the expansion rate (Supplementary Fig. S3A). Itacitinib concentrations 344

relevant to maximum free concentrations (189 and 244 nM, in the absence or presence of potent 345

CYP3A4 inhibitors, respectively) induced a modest reduction on T-cell proliferation 346

(Supplementary Fig. S3A). To evaluate the effect of itacitinib on the in vivo antitumor efficacy 347

of OT-1 CD8, we conducted experiments involving adoptive transfer of OVA-specific CD8 cells 348

into C57BL/6 mice previously transplanted with an OVA-expressing tumor cell line (EG7). Five 349

days after tumor challenge, corresponding animals were orally dosed b.i.d. with vehicle or 60 or 350

120 mg/kg of itacitinib for 2 weeks. Eight days after tumor inoculation, animals received an 351

adoptive transfer of OVA-specific OT-1 naïve CD8 cells. When compared with the control 352

group, adoptive transfer of OT-1 CD8 was able to significantly reduce tumor growth (Fig. 4). 353

Importantly, itacitinib doses equivalent to the JAK1 coverage target doses do not affect the 354

antitumor efficacy of OT-1 cells (Fig. 5). 355

To rule out a direct effect of itacitinib on EG7 tumor cell growth, we expanded EG7 cells 356

in the presence of increasing itacitinib concentration. Even very high concentrations of itacitinib 357

(25 M) were unable to inhibit tumor proliferation (Supplementary Fig. S3B). 358

359

Itacitinib does not affect CD19-CAR T-cell cytolytic activity in vitro 360

To study the effect of itacitinib on human CAR T-cells targeting the CD19 antigen, we 361

treated CD19-CAR T-cells with either itacitinib or anti–IL-6 receptor (tocilizumab) for 3 days 362

and then measured their cytolytic activity against CD19-expressing target cells. Whereas 363

concentrations of tocilizumab that mimic human pharmacologic activity significantly reduced the 364

cytolytic activity of CAR T-cells (37), 100 nM of itacitinib showed no significant effect when 365

compared with control CAR T-cells (Fig. 6A). CAR T-cells expanded with a higher dose of 366

itacitinib (300 nM, approximately five-fold higher that the cellular IC50) showed a modest but 367

statistically significant inhibition on the antitumor activity (Fig. 6A). As internal controls, the 368

antitumor activity of non-transfected PBMCs was also tested. Non-transduced T-cells were 369

unable to specifically lyse the target cells and were unaffected by neither itacitinib nor 370

tocilizumab treatment beyond background levels (Fig. 6B). 371

372

Itacitinib does not affect CD19-CAR T-cell antitumor activity in vivo 373

To test the effect of itacitinib in vivo, we studied the effect of oral itacitinib on the 374

antitumor efficacy of CD19-CAR T-cells. Immunodeficient mice (NSG) were challenged with 375




CD19+ nalm6-luciferase expressing human lymphoma cells. Once the tumor was engrafted (day 376

4), the mice received an adoptive transfer of 3 × 106 human CD19-CAR T-cells. In this model, 377

CAR T-cells were able to control tumor growth, measured by luciferin expression (Fig. 7). 378

Importantly, daily oral itacitinib dosing (120 mg/kg) of the animals did not affect the antitumor 379

activity of adoptively transferred CAR T-cells, thus indicating that, at targeted doses, itacitinib 380

does not have a significant effect on the antitumor activity of CAR T-cells. 381

Finally, to confirm that itacitinib would not affect CAR T-cell activity on a more 382

aggressive model, immunodeficient mice (NSG) were challenged with 5 × 106 CD19

+ 383

NAMALWA-luciferase expressing human lymphoma cells. Once the tumor was engrafted (day 384

7), the mice received an adoptive transfer of human CD19-CAR T-cells. When compared with 385

animals receiving control cells, animals receiving an adoptive transfer of CAR T-cells had a 386

significant tumor growth delay, measured both by luciferin expression (Supplementary Fig. 387

S4B) and expansion of survival (P = 0.0014) (Supplementary Fig. S4C). Even in this 388

aggressive tumor model, daily oral itacitinib dosing of the animals did not affect the antitumor 389

activity of adoptively transferred CAR T-cells (P = 0.1860), thus confirming the safety of 390

prophylactic itacitinib dosing to prevent CRS. 391

392

Discussion 393 CAR T-cell therapy has shown unprecedented remission rates in patients with relapsed or 394

refractory B-cell acute lymphoblastic leukemia. Unfortunately, the high frequency of side effects 395

(mainly CRS and neurotoxicity) that often require extensive critical care support remains a 396

barrier to the further development of CAR T-cell therapy. Current therapeutic options are limited 397

to treating severe cases of CRS with tocilizumab or corticosteroids, but unfortunately severe 398

toxicities and death still occasionally occur (38). Therefore, there is a clear need to develop 399

prophylactic treatments to prevent (rather than treat) CRS without impairing antitumor efficacy. 400

Despite being a very active research field, only recently clear predictors of CAR T-cell 401

associated toxicities have been identified, and several factors, such as ferritin, C-reactive protein 402

or tumor burden can be reliable predictors of severe CRS (11). A recent clinical trial on pediatric 403

leukemia patients suggests that early therapeutic intervention with tocilizumab is effective at 404

reducing CRS rates without affecting CAR T-cell proliferation or antitumor efficacy (39). 405

However, this data should not be generalized to other conditions such as lymphoma as clinical 406

management strategies are different than leukemia and concerns remain over the potential impact 407

of corticosteroids on CAR T-cell activation and proliferation (14). 408

Recent advances in our understanding of the JAK/STAT pathway have highlighted its 409

role in T-cell priming, activation, and expansion. Indeed, several small-molecules targeting the 410

JAK/STAT pathway have shown clinical potential in a number of indications (reviewed in (40)). 411

Biochemical and cellular profiling of itacitinib confirmed it to be a potent JAK1 inhibitor with 412

large fold-selectivity margins over the other family members (JAK2, JAK3, and TYK2) or 413

unrelated kinases (41). Importantly, JAK1 either independently or in cooperation with other 414

members of the JAK family, mediate the signaling of a number of inflammatory cytokines 415

implicated in CRS, including GM-CSF, IFN-, IL-6 and IL-12 (42, 43). Crucially, specific 416 inhibition of JAK1 may prevent some of the side effects associated with JAK1/JAK2 inhibitors 417

such as lymphopenia (42). 418

Consistent with the potent disruption of the JAK1-specific STAT signaling activity, here 419

we show that itacitinib inhibited multiple cytokines involved in CRS (including IL-6, IL-12, and 420




IFN-) in two different models of systemic inflammation in a dose-dependent manner. 421

Importantly, only cytokines dependent upon JAK1 were affected, as levels of IL-5 and other 422

JAK1-independent cytokines were unaffected by itacitinib treatment. It is also important to note 423

that while the effect on inflammatory cytokine levels was profound and statistically significant, 424

plasma levels of such cytokines were still elevated when compared with naïve animals, thus 425

potentially dampening the hyper-immune activation state, but avoiding broad 426

immunosuppression. 427

In addition to the effect of itacitinib on the production of cytokines by T-cells and CAR 428

T-cells, our findings show that itacitinib is also able to reduce the secretion of inflammatory 429

cytokines by macrophages, both in vitro and in vivo. Recent studies have shown that IL-1 and IL-430

6 produced by host monocytes/macrophages play a key role in the pathogenesis of CRS and 431

neurotoxicity (30, 44). Our data demonstrate that itacitinib is able to inhibit IL-6 production by 432

murine macrophages in vivo, demonstrating its potential utility in treating the most prominent 433

source of inflammatory cytokines during CRS. The ability of itacitinib to inhibit the production 434

of CRS-associated cytokines produced by a range of cell types (both of murine and human 435

origin, an important distinction considering the differences between mouse and human biology 436

(45)) is particularly noteworthy, as such a strategy may be necessary for the effective treatment 437

of CRS. 438

To achieve clinical efficacy while preventing CRS, it is imperative that clinical doses of 439

itacitinib do not interfere with CAR T-cell proliferation and antitumor activity. Our findings 440

confirm that itacitinib concentrations relevant for cellular IC50 (in vitro) and drug 441

pharmacokinetic exposures that mimic JAK1 target coverage in vivo have no significant effect 442

on the proliferation rate of PBMCs or four different CAR T-cell constructs. More importantly, 443

our data show that in preclinical models, where animals receive an oral dose of itacitinib that 444

mimic human JAK1 pharmacologic inhibition, prophylactic treatment with itacitinib does not 445

affect the in vivo antitumor efficacy of CAR T-cells. 446

In summary, we have demonstrated that itacitinib at clinically relevant doses can prevent 447

CAR T-cell associated CRS in preclinical models without negatively affecting antitumor 448

efficacy. Reducing the incidence and severity of CRS has the potential to benefit clinical 449

practice, including the use of CAR T-cell therapy in patients in previously high-risk populations. 450

Based on these results, a phase II clinical trial of itacitinib for the prevention of CRS induced by 451

CD19-CAR T-cell therapy was initiated (NCT04071366). Results from our preclinical study may 452

have implications in other settings; future studies are required to further investigate the role of 453

JAK inhibition beyond CAR T-cells induced CRS (i.e., bispecific antibodies (46)) or conditions 454

(including HLH (9, 47) and respiratory infections such as SARS (48), MERS (49), or Covid-19 455

(50-52)) where CSR may play a role. 456

457

Acknowledgments 458 This study was funded by Incyte Corporation, and supported in part by an NIH grant 459

RO1CA226983-03 awarded to R.O’C. 460

We are thankful to April Barbour, Michael Howell, and Lea Burke for useful discussions about 461

pharmacokinetics and translational relevance of the manuscript. Editorial assistance was 462




provided by Envision Pharma Group, Inc. (Philadelphia, PA, USA), and funded by Incyte 463

Corporation. 464

465

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613




Figures Legends 614

615

Figure 1. 616

Itacitinib reduces cytokine levels in murine models of acute inflammation. (A) BALB/c mice 617

were orally dosed with vehicle control, or 60 or 120 mg/kg of itacitinib. One hour later, animals 618

were challenged with Concanavalin-A (ConA), and 120 minutes later sacrificed and plasma 619

collected (prophylactic). (B) BALB/c mice were challenged with ConA, 30 minutes later were 620

orally dosed with vehicle control, or 60 or 120 mg/kg of itacitinib and 2 hours after ConA 621

challenge sacrificed (therapeutic). N = 5 animals per group. MSD analysis was performed to 622

detect the levels of pro-inflammatory cytokines. Data represent mean ± SEM, and P values were 623

calculated by two-way ANOVA. *P < 0.05, ** P < 0.01, ***P < 0.001, **** P < 0.0001. Data 624

are representative of four independent experiments. 625

626

Figure 2. 627

Itacitinib reduces IL-6 production by macrophages. (A) Macrophages harvested from 628

C57BL-6 mice were treated with increasing doses of itacitinib 1 day in advance of being 629

activated with 5 ng/mL LPS. Twenty-four hours after LPS treatment, supernatant was harvested 630

and cytokine levels measured. (B) C57BL/6 mice were prophylactically orally dosed with 631

vehicle, or 60 or 120 mg/kg of itacitinib b.i.d. for 3 days. Mice then received intraperitoneal 632

injections of LPS (5 µg per animal). Two hours after injection, mice were euthanized and IL-6 633

levels were measured in the peritoneal lavage. Data represent mean ± SEM, and P values were 634

calculated by two-way ANOVA. *P < 0.05, ***P < 0.001. Data are representative of two 635

independent experiments. 636

637

Figure 3. 638

Itacitinib reduces CAR T-cell cytokine production. CD19-CAR T-cells were expanded with 639

either itacitinib or the anti–IL-6 receptor tocilizumab. Three days later, they were co-cultured 640

with CD19+ lymphoma cells (E:T ratio = 2.5:1) for 6 hours and cytokines were measured in the 641

supernatant by MSD. (A) Experiment scheme. (B) Data represent mean ± SEM, and P values 642

were calculated by two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. Data are 643

representative of two independent experiments. 644

645

Figure 4. 646

Itacitinib does not affect PBMC proliferation nor cytolytic activity. (A) Flow cytometry 647

quantification of T-cell expansion of human PBMCs expanded with DMSO (positive control) or 648

50, 150, or 1000 nM of itacitinib. T-cells were expanded using anti-CD3/CD28-coated beads, 649

and proliferation was measured every other day by flow cytometry. (B) GD2-CAR T-cells were 650

expanded in the presence of different itacitinib concentrations and proliferation was measured 651

every other day by flow cytometry. (C) Lytic activity of GD2-CAR T-cells against SY5Y 652

neuroblastoma cells (GD-2+) was determined by measuring luciferase activity. As an internal 653

control, lytic activity of non-transduced T cells was also measured (background dotted line). (D) 654




EGFR-CAR T-cells were expanded with anti-CD3/CD28 beads for 2 weeks and then 655

restimulated with EGFR beads in the presence of itacitinib (100 or 250 nM). Proliferation was 656

measured every other day by flow cytometry. 657

658

Figure 5. 659

Itacitinib does not impair T-cell antitumor activity in vivo. C57BL/6 animals were 660

subcutaneously inoculated with an OVA-expressing EG-7 tumor cell line. Starting at day 5, 661

corresponding groups received oral doses of itacitinib or vehicle for 2 weeks, and at day 8 they 662

received an adoptive transfer (A.T.) of naïve OT-1 CD8 T-cells. (A) Experiment scheme. (B) 663

Tumor growth was monitored and compared between groups. N = 10 animals per group. Data 664

represent mean ± SEM, and P values were calculated by two-way ANOVA. * P < 0.05, n.s., not 665

significant. Data are representative of two independent experiments. 666

667

Figure 6. 668

Itacitinib does not affect CD19-CAR T-cell cytolytic activity in vitro. (A) CD19-CAR T-cells 669

or (B) nontransduced PBMC were expanded with either itacitinib or anti–IL-6 receptor 670

(tocilizumab) at the indicated concentrations. Three days later, they were co-cultured with 671

luciferin expressing CD19+ lymphoma cells (E:T ratio = 2.5:1) for 6 hours, luciferase substrate 672

was added and luminescence was recorded by a luminometer. Data represent mean ± SEM, and 673

P values were calculated by two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not 674

significant. 675

676

Figure 7. 677

Itacitinib does not affect CD19-CAR T-cell antitumor activity in vivo. Immunodeficient NSG 678

mice were inoculated with 2.5 × 106 CD19

+ human lymphoma Nalm6 luciferase-expressing 679

Nalm6 cell line. Starting 1 day later, animals received b.i.d. doses of itacitinib or vehicle for 10 680

days. At day 4, post-tumor injection, corresponding animals received an adoptive transfer of 3 × 681

106 CD19-CAR T-cells. (A) Experiment scheme. (B) Anesthetized mice were imaged using a 682

Xenogen IVIS Spectrum system to measure bioluminescence once a week. N = 5–10 animals per 683

group. 684




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M

T-c

ells

10

0 μ

g/m

L


DM

SO

20

μg

/mL

20

0 μ

g/m

L

10

0 n

M

30

0 n

M

T-c

ells

10

0 μ

g/m

L


**

***

**

******

*

*

***

*** ***

A

B

CAR T-cell transduction

Itacitinib or tocilizumab

0 1 2 3 days

CD19+ NAWALMA

Supernatant collection

and cytokine quantification




A

8

6

4

2

0Po

pu

lati

on

do

ub

lin

gs

0 2 4 6 8 10

Days of culture

B

4

3

2

1

0

Po

pu

lati

on

do

ub

lin

gs

0 2 4 6 8 10

Days of culture

C

80

60

40

20

10

Tu

mo

r c

ell ly

sis

, %

Non-

transduced

0 100 250 500

D

8

6

4

2

0Po

pu

lati

on

do

ub

lin

gs

0 2 4 6 8 10 12 14 16 18 20

Days of cultureItacitinib, nM

DMSOItacitinib 50 nM

Itacitinib 100 nM

Itacitinib 1000 nM

DMSOItacitinib 100 nM

Itacitinib 250 nM

Itacitinib 500 nM

Non-transducedDMSO

Itacitinib 100 nM

Itacitinib 250 nM




2500

2000

1500

1000

500

0Tu

mo

r v

olu

me

, m

ea

n ±

SE

M

7 9 11 13 16 20 22

Days

*n.s.n.s.

Vehicle

A.T. + vehicle

A.T. + itacitinib 60 mg/kg

A.T. + itacitinib 120 mg/kg

A

B

EG7 (OVA+) cells

Itacitinib

OT-I transfer

0 5 8 2218 days

Tumor monitoring




100

80

60

40

20

0

Cy

toto

xic

ity

, %

DMSO 20 100 200 100 300

Tocilizumab, μg/mL Itacitinib, nM

**** **

***

A B

n.s.

100

80

60

40

20

0

Cy

toto

xic

ity

, %

DMSO 20 100 200 100 300

Tocilizumab, μg/mL Itacitinib, nM

CAR T-cells Nontransduced T-cells




1000

100

10

1

0.1

0.01

0.001

To

tal fl

ux

, P

/S ×

10

9

0 10 20 30 40

Days elapsed

Vehicle

Non-transduced + vehicle

CAR T-cells + vehicle

CAR T-cells + itacitinib 120 mg/kg

A

B

CD19+ Nalm6

Itacitinib

CD19+ CAR T-cells transfer

0 1 4 3511 days

Survival




Published OnlineFirst September 30, 2020.Clin Cancer Res Eduardo Huarte, Roddy S. O'Conner, Michael T. Peel, et al. T-Cell TherapyAssociated with Cytokine Release Syndrome Induced by CAR Itacitinib (INCB039110), a JAK1 inhibitor, Reduces Cytokines

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itacitinib (incb039110), a jak1 inhibitor, reduces cytokines … · 2020. 9. 30. · 1 itacitinib...

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