Main Text Title. - Cancer Discovery...Aug 22, 2020 · Sakurai. 7 Mailing adress. 2-1-1 Nihonbashi-Muromachi, Chuo-ku, Tokyo, 103-8324, Japan 8 Phone. +81-3-3273-1032 9 FAX. +81-3-3281-0819

p. 1

Main Text 1

2

Title. 3

Antibody to CD137 activated by extracellular adenosine triphosphate is tumor 4

selective and broadly effective in vivo without systemic immune activation 5

6

Running title. 7

Tumor selective anti-CD137 antibody activated by exATP 8

9

Authors. 10

Mika Kamata-Sakurai1,†,

*, Yoshinori Narita2,†

, Yuji Hori3, Takayuki Nemoto

4, Ryo 11

Uchikawa2, Masaki Honda

3, Naoka Hironiwa

5, Kenji Taniguchi

2, Meiri Shida-Kawazoe

3, 12

Shoichi Metsugi2, Taro Miyazaki

6, Naoko A.Wada

3, Yuki Ohte

2, Shun Shimizu

3, 13

Hirofumi Mikami3, Tatsuhiko Tachibana

3, Natsuki Ono

2, Kenji Adachi3, Tetsushi 14

Sakiyama7, Tomochika Matsushita

8, Shojiro Kadono

2, Shun-ichiro Komatsu

2, 3, Akihisa 15

Sakamoto3, Sayuri Horikawa

2, Ayano Hirako

8, Koki Hamada

2, Sotaro Naoi

3, Nasa 16

Savory3, Yasuko Satoh

2, Motohiko Sato

3, Yuki Noguchi

3, Junko Shinozuka

3, Haruka 17

Research. on June 6, 2021. © 2020 American Association for Cancercancerdiscovery.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 August 25, 2020; DOI: 10.1158/2159-8290.CD-20-0328

http://cancerdiscovery.aacrjournals.org/

p. 2

Kuroi3, Ami Ito

3, Tetsuya Wakabayashi

3, Masaki Kamimura

9, Fumihisa Isomura

10, 1

Yasushi Tomii3, Noriaki Sawada

2, Atsuhiko Kato

2, 3, Otoya Ueda

3, Yoshito Nakanishi

11, 2

Mika Endo2, 3

, Kou-ichi Jishage9, 10

, Yoshiki Kawabe2, 3

, Takehisa Kitazawa2, 3

, 3

Tomoyuki Igawa2, 3, 5

4

5

Affiliations. 6

1Translational Research Division, Chugai Pharmaceutical Co., Ltd., 1-1 7

Nihonbashi-Muromachi 2-Chome Chuo-ku, Tokyo 103-8324, Japan. 8

2Research Division, Chugai Pharmaceutical Co., Ltd., 200 Kajiwara, Kamakura, 9

Kanagawa 247-8530, Japan. 10

3Research Division, Chugai Pharmaceutical Co., Ltd., 1-135 Komakado, Gotemba, 11

Shizuoka 412-8513, Japan. 12

4Translational Research Division, Chugai Pharmaceutical Co., Ltd., 200 Kajiwara, 13

Kamakura, Kanagawa 247-8530, Japan. 14

5Chugai Pharmabody Research Pte. Ltd., 3 Biopolis Drive, Synapse 138623, Singapore. 15

6Clinical Development Division, Chugai Pharmaceutical Co., Ltd., 1-1 16





p. 3

7Pharmaceutical Technology Division, Chugai Pharmaceutical Co., Ltd., 5-5-1 Ukima, 1

Kita-ku, Tokyo 115-8543, Japan. 2

8Translational Research Division, Chugai Pharmaceutical Co., Ltd., 1-135 Komakado, 3

Gotemba, Shizuoka 412-8513, Japan. 4

9Chugai Research Institute for Medical Science, Inc., 200 Kajiwara, Kamakura, 5

Kanagawa 247-8530, Japan. 6

10Chugai Research Institute for Medical Science, Inc., 1-135 Komakado, Gotemba, 7

Shizuoka 412-8513, Japan. 8

11Project & Lifecycle Management Unit, Chugai Pharmaceutical Co., Ltd., 1-1 9


†These authors contributed equally to this work. 11

*Corresponding author. 12

13

Keywords. 14

CD137, agonist antibody, extracellular ATP, antigen independent, tumor selective 15

16




p. 4

Additional information. 1

Financial support: 2

This study was funded and supported by Chugai Pharmaceutical Co., Ltd. 3

4

Corresponding author: 5

Mika Kamata-Sakurai 6

Mailing adress. 2-1-1 Nihonbashi-Muromachi, Chuo-ku, Tokyo, 103-8324, Japan 7

Phone. +81-3-3273-1032 8

FAX. +81-3-3281-0819 9

E-mail adress. [email protected] 10

11

A conflict of interest disclosure statement: 12

All authors were employees of Chugai Pharmaceutical Co., Ltd. when this study 13

was conducted. 14

15

Other notes about the manuscript as a whole: 16

Word count. 6000 17

Total number of figures. 6 18




p. 5

Total number of table. 1 1




p. 6

Abstract. 1

Agonistic antibodies targeting CD137 have been clinically unsuccessful due to systemic 2

toxicity. Since conferring tumor selectivity through tumor-associated antigen limits its 3

clinical use to cancers that highly express such antigen, we exploited extracellular 4

adenosine triphosphate (exATP), which is a hallmark of the tumor microenvironment 5

and highly elevated in solid tumors, as a broadly tumor selective switch. We generated a 6

novel anti-CD137 switch antibody, STA551, which exerts agonistic activity only in the 7

presence of exATP. STA551 demonstrated potent and broad anti-tumor efficacy against 8

all mouse and human tumors tested and a wide therapeutic window without systemic 9

immune activation in mice. STA551 was well tolerated even at 150 mg/kg/week in 10

cynomolgus monkeys. These results provide a strong rationale for the clinical testing of 11

STA551 against a broad variety of cancers regardless of antigen expression, and for the 12

further application of this novel platform to other targets in cancer therapy. 13

14

Statement of significance. 15

Reported CD137 agonists suffer from either systemic toxicity or limited efficacy against 16

antigen-specific cancers. STA551, an antibody designed to agonize CD137 only in the 17




p. 7

presence of extracellular ATP, inhibited tumor growth in a broad variety of cancer 1

models without any systemic toxicity or dependence on antigen expression. 2

3

Introduction. 4

Over the past decade, monoclonal antibody therapies delivered significant clinical 5

benefits, including durable responses even in late-stage cancers. Immune checkpoint 6

inhibitors such as anti-CTLA-4 and anti-PD-1/PD-L1 antibodies have achieved 7

impressive clinical outcomes and are changing the paradigm of cancer treatment (1,2). 8

9

CD137 is a co-stimulatory receptor in the tumor necrosis factor receptor superfamily 10

that enhances CD28-independent co-stimulation when T cell receptors recognize 11

antigens, resulting in the proliferation and survival of T cells by upregulating 12

anti-apoptotic signaling, and the production of cytokines such as interferon gamma 13

(IFN-γ) (3,4). The therapeutic potential of agonistic antibody against CD137 was 14

demonstrated in several preclinical models (4,5). The importance of CD137 signaling in 15

T cells has also been demonstrated by the clinical success of second generation chimeric 16

antigen receptor-T (CAR-T) cells incorporating a CD137 co-stimulatory signaling 17

domain (6,7). 18




p. 8

1

Several anti-CD137 agonist antibodies have advanced to clinical stages but have never 2

been clinically successful because of the intolerable toxicity caused by systemic 3

immune activation (8). Urelumab (BMS-663513), an IgG4 antibody, caused severe 4

hepatotoxicity in more than 5% of patients enrolled in phase I and II clinical trials 5

(9,10). In contrast, utomilumab (PF-05082566), an IgG2 antibody, showed fewer grade 6

III–IV adverse effects and no dose-limiting toxicity up to the highest dose evaluated, 7

although it was much less potent (11,12). This on-target off-tumor toxicity seems to be 8

an inevitable problem for all therapeutic antibodies since they cannot discriminate 9

whether the target antigen is in plasma, normal tissue, or tumor (13). 10

11

To overcome the toxicity of anti-CD137 antibody, various bispecific approaches have 12

been developed which induce the CD137 agonistic signal by crosslinking CD137 with 13

tumor-associated antigen (14-16). However, since these bispecific approaches fully rely 14

on expression of specific tumor-associated antigen in the tumor bed to induce CD137 15

agonistic activity, their clinical use is limited to cancer patients that highly express the 16

antigen. This is in sharp contrast to anti-PD-1/PD-L1 antibody and anti-CTLA-4 17

antibody which can be used for patients without considering the expression of specific 18




p. 9

tumor-associated antigen. Thus, a more broadly applicable approach to effectively and 1

safely target CD137 without relying on specific tumor-associated antigen expression is 2

greatly needed. In addition, this bispecific approach is only applicable to agonist targets 3

whose activity depends on its crosslinking. An engineering approach which can also be 4

applied to neutralizing or depleting antibody is desired. 5

6

Elevated extracellular ATP (exATP) is a hallmark of the tumor microenvironment 7

(TME), accumulating in the range of 100 micromolars in the TME (17). The abundance 8

of ATP in the TME is caused by the release of millimolar concentration of intracellular 9

ATP by apoptosis and necrosis of cancer cells, and by exocytosis and channel-mediated 10

release (18,19). Since levels of exATP remain tightly regulated in normal tissues within 11

10 nanomolars (19,20), there is a more than 1000-fold increase of exATP in the TME; 12

this allowed us to exploit exATP as a broadly tumor selective switch to control the 13

activity of antibodies. 14

15

In this study, we developed a novel anti-CD137 agonist switch antibody, STA551, which 16

binds to CD137 only in the presence of ATP and induces strong CD137 agonistic activity 17




p. 10

selectively within the TME. We demonstrated the anti-tumor efficacy of STA551 in a 1

broad variety of tumor models without any dependence on tumor-associated antigen 2

expression. Importantly, while the conventional anti-CD137 antibody showed both 3

intratumor and systemic T cell activation, STA551, the exATP switch antibody, showed 4

potent intratumor T cell activation without affecting normal tissues. A toxicological study 5

of doses up to 150 mg/kg/week in cynomolgus monkeys supported this finding. This 6

paper provides a strong rationale for clinically testing STA551 against a broad variety of 7

cancers, and for the further application of the exATP switch antibody platform to other 8

targets. 9

10

Results. 11

Generation of ATP-dependent anti-human CD137 agonist antibody STA551 12

We obtained the lead ATP-dependent anti-CD137 antibody using a phage-displayed 13

synthetic human Fab library with a built-in ATP-binding motif, D12 library, followed 14

by optimization (Fig. S1A-B). The D12 library was constructed in two steps: first, 15

identifying an anti-ATP antibody from a phage-displayed naïve human Fab library; 16

second, designing and constructing a library with a conserved ATP-binding motif, 17




p. 11

which has a repertoire that maintains the residues involved in D12 binding to ATP and 1

diversifies other residues in the complementarity-determining region (CDR). The lead 2

ATP-dependent anti-CD137 variable region was isolated using phage panning of the 3

D12 library. ATP metabolite is sandwiched between the antibody and CD137 in the 4

ternary complex, and the CDRs and ATP both interact with CD137. After multiple 5

rounds of optimization, including improving CD137 binding, we identified the STA551 6

variable region. For the constant region, several mutations were added, including into 7

the CH2 region of human IgG1 to increase Fc gamma receptor IIb (FcγRIIb)-mediated 8

crosslinking of CD137 by FcγRIIb-expressing cells to enhance CD137 agonistic activity 9

(21) (Fig. 1A). 10

11

The resulting human IgG1/lambda antibody with optimized variable and constant 12

regions, termed STA551, bound to both recombinant human and cynomolgus monkey 13

CD137 extracellular domains in an ATP, adenosine diphosphate (ADP), and adenosine 14

monophosphate (AMP) dependent manner (Fig. 1B-C, Table 1, Fig. S2A-F). Binding 15

affinities of STA551 to FcγRs, C1q, and neonatal Fc receptor (FcRn) are summarized 16

(Fig. S3A-B, S4, Table S1). These results suggest STA551 is unlikely to induce 17




p. 12

antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular 1

phagocytosis (ADCP), or complement-dependent cellular cytotoxicity (CDC). 2

3

In vitro pharmacological profiles of STA551 4

In previous studies, CD137 agonist antibodies were shown to co-stimulate secretion of 5

cytokines such as IFN-γ from human CD8+ T cells (3). To evaluate STA551’s 6

ATP-dependent CD137 agonistic activity, we compared it with Ure-hIgG4, a potent 7

conventional CD137 agonist with an urelumab-like Fab having a human IgG4/kappa 8

isotype (Table S2-3). IFN-γ released from human CD8+ T cells increased in a STA551 9

concentration-dependent manner in the presence of ATP, while almost no increase was 10

observed in its absence (Fig. 2A). In contrast, IFN-γ levels increased in an Ure-hIgG4 11

concentration-dependent manner both in the presence and absence of ATP (Fig. 2A). In 12

addition, the levels of IFN-γ induced by STA551 were at least as strong as Ure-hIgG4 13

and significantly higher than Uto-hIgG2, a conventional CD137 agonist with an 14

utomilumab-like Fab having a human IgG2/lambda isotype (Fig. 2A, S5, Table S2-4). 15

16

17

18

19




p. 13

Next, to further confirm in vitro pharmacological profiles in a more native setting, we 1

mixed STA551 or Ure-hIgG4 with whole human peripheral blood mononuclear cells 2

(PBMCs) from 20 healthy donors in the presence of ATP and evaluated IFN-γ levels. 3

STA551 induced even stronger IFN-γ production than Ure-hIgG4 among PBMCs (Fig. 4

2B, S6), which include immune suppressor cells and FcγR-expressing native myeloid 5

and B cells (22,23). 6

7

Anti-tumor efficacy of STA551 in human CD137 knock-in mice 8

We then examined the in vivo anti-tumor efficacy of STA551 in various immune 9

competent mouse models. Because STA551 does not bind to murine CD137, we 10

established human CD137 knock-in mice (Fig. S7A-D). STA551 specifically bound to 11

membranous human CD137 based on flow cytometry using mouse spleen cells (Fig. 12

S8A). Furthermore, since the FcγR-binding profile in humans is not recapitulated in 13

mice, we generated an engineered constant region of mouse IgG1, MB. Sta-MB, having 14

the same variable region as STA551 but with MB as the constant region, can crosslink 15

human CD137-expressing immune cells with mouse FcγRII-expressing mouse cells to 16

increase agonistic activity in human CD137 knock-in mice (Fig. S8B-C). MB has a 17




p. 14

dissociation constant (KD) against mouse FcγRII-expressing cells comparable to that of 1

STA551 against human FcγRIIb-expressing cells (Fig. S9, Table S5). 2

3

To evaluate the in vivo anti-tumor efficacy of STA551 against various mouse cell lines, 4

five tumors originating from different organs were established in human CD137 5

knock-in mice and treated with Sta-MB or Ure-MB, which have the same variable 6

region as STA551 or urelumab but with MB as the constant region (Table S2). Sta-MB 7

significantly inhibited tumor growth compared to vehicle in all five models and tended 8

to inhibit it more strongly than Ure-MB in the LLC1/OVA/hGPC3 model (Fig. 3A-C). 9

Anti-tumor efficacy lasted over 40 days (Fig. S10). This clearly demonstrates that 10

Sta-MB exerts in vivo agonistic activity in mice regardless of any specific 11

tumor-associated antigen expression, with exATP elevated in all the tumors tested. To 12

support this, we analyzed changes in genes related to exATP such as P2rx7, S100a3 and 13

Rpl38 (Table S6) in tumors and normal tissues using RNA sequencing (RNA-seq). 14

Their mRNA expression was increased in tumors compared to normal tissues (Fig. 15

S11), suggesting elevated exATP in tumors. In addition, tumor cells and 16

tumor-infiltrating lymphocytes (TILs) in these studies expressed CD39, which 17

hydrolyzes ATP to ADP and AMP, and CD73, which hydrolyzes AMP to adenosine 18




p. 15

(ADO) on the cell surface (Fig. S12A-E). These expression levels were consistent with 1

TILs from patients (24). Nevertheless, Sta-MB, which binds to CD137 in the presence 2

of ATP, ADP, or AMP, demonstrated potent anti-tumor efficacy, suggesting that even if 3

CD39 and CD73 are expressed, exATP, exADP, and exAMP remain in the TME at 4

sufficient levels. Furthermore, Sta-MB inhibited lung metastasis of LLC1/OVA as 5

shown by the weight of metastatic nodules (Fig. S13). This suggests that exATP is also 6

enriched in micrometastasis and that Sta-MB was effective therein. 7

8

STA551 response in non-tumor tissues in human CD137 knock-in mice 9

We investigated the pharmacokinetics of Ure-MB and Sta-MB in non-tumor bearing 10

wild-type C57BL/6N mice and human CD137 knock-in mice, which systemically 11

express human CD137, to evaluate the effect of systemic CD137 binding on antibody 12

clearance. Ure-MB showed slow clearance in wild-type mice since it does not bind to 13

mouse CD137, and was rapidly eliminated in human CD137 knock-in mice due to 14

systemic CD137-mediated clearance (Fig. 3D). In contrast, Sta-MB showed slow 15

clearance and similar pharmacokinetics in both wild-type and human CD137 knock-in 16

mice, demonstrating that Sta-MB is not subject to systemic CD137-mediated clearance 17

(Fig. 3D). Sta-MB showed faster clearance in human CD137 knock-in mice bearing 18




p. 16

LLC1/OVA/hGPC3 and Colon38 than in non-tumor bearing mice (Fig. S14A-B), 1

suggesting that Sta-MB bound to human CD137 within tumors and was eliminated by 2

CD137-mediated clearance there. STA551 was internalized into human 3

CD137-expressing cells (Fig. S14C), consistent with reports (25). These 4

pharmacokinetic studies indicate that, while Ure-MB strongly binds to human CD137 5

systemically, Sta-MB only does so minimally in blood and normal tissues, places where 6

exATP concentration is reported to be very low; thus, Sta-MB is not expected to exert 7

CD137 agonistic activity systemically. 8

9

Next, to confirm that the minimal binding of Sta-MB in non-tumor tissues does not 10

induce systemic immune activation in mice, we evaluated its effect in non-tumor 11

tissues. Human CD137 knock-in mice bearing LLC1/OVA/hGPC3 or Colon38 tumors 12

were given various doses of Sta-MB or Ure-MB. After the treatment, Ure-MB started to 13

elicit systemic response from 0.093 mg/kg, but Sta-MB showed no clear sign of it even 14

at 7.5 mg/kg. In mice treated with a higher dose of Ure-MB, lymphadenopathy and 15

splenomegaly, characterized by a significant increase of draining lymph node (DLN) 16

and spleen weight, and increased PD-1, KLRG-1, or ICOS expression on CD8+ T cells 17

and FoxP3+ regulatory T cells (Tregs) in CD4

+ T cells in liver and DLN were observed 18




p. 17

(Fig. 3E-G). PD-1+

CD8+ T cells were also increased in liver (Fig. S15A-B). In contrast, 1

mice treated with any dose of Sta-MB showed no sign of those phenomena. These 2

results clearly demonstrate that Sta-MB, due to its exATP switch function, only 3

minimally induces CD137 signaling and subsequent immune activation in non-tumor 4

tissues, consistent with its pharmacokinetic profile. Taken together, these data suggest 5

that Sta-MB has a much wider therapeutic window than Ure-MB. 6

7

Pharmacokinetics and toxicology of STA551 in cynomolgus monkeys 8

For the non-clinical pharmacokinetic and toxicology study, cynomolgus monkeys were 9

selected based on species cross reactivity (Fig. 1B-C, S2A-F). The plasma 10

concentration-time profiles and pharmacokinetic parameters of STA551 following a 11

single intravenous administration of 0.5, 5, and 50 mg/kg in male cynomolgus monkeys 12

are shown (Fig. S16, Table S7). Anti-drug antibodies were transiently detected in one 13

animal from each 0.5 and 5 mg/kg group of 4, but this did not influence the 14

pharmacokinetics profile (data not shown). The t1/2 (elimination half-life) was 15

approximately 14 days independent of the dosage, and other parameters were consistent 16

with typical values for IgG antibodies (26). 17




p. 18

1

The 4-week repeat dose toxicity study in cynomolgus monkeys was conducted using 2

intravenous doses of 5, 30, and 150 mg/kg/week. In this study, STA551 was 3

well-tolerated up to 150 mg/kg/week without any abnormalities in general condition, and 4

the highest non-severe toxic dose was above 150 mg/kg/week in cynomolgus monkeys. 5

In contrast, utomilumab, despite having much weaker agonistic activity, was reported to 6

cause dose-limiting toxicity at doses over 5 mg/kg/week (27). We also conducted an 7

exploratory 4-week repeat dose toxicity study of Uto-hIgG2 in cynomolgus monkeys. 8

Uto-hIgG2 elicited systemic toxicity characterized by general condition abnormalities 9

(e.g., decreased food consumption and body weight), hematological changes (e.g., 10

decrease in neutrophils and platelets), and inflammatory changes in lung and liver even at 11

30 mg/kg/week. 12

13

STA551 response in tumor tissues in human CD137 knock-in mice 14

Since STA551 exerts its activity in a novel exATP-dependent and tumor-selective 15

manner, we examined the mechanism behind its anti-tumor efficacy. First, we analyzed 16

changes in gene related to CD8+ effector T cells, immune checkpoint, IFN-γ, and 17




p. 19

antigen presentation (Table S8) after administration using RNA-seq. Their mRNA 1

expression was increased in mice treated with Sta-MB compared to vehicle in 2

LLC1/OVA/hGPC3 or Colon38 tumors (Fig. 4A-B), suggesting that Sta-MB alters 3

immune status in tumors. 4

5

Next, we analyzed changes in expression of the selected immune-related genes in 6

LLC1/OVA/hGPC3 tumors using the nCounter Analysis System by comparing Sta-MB 7

and Ure-MB at various doses. Compared to vehicle, mice treated with Sta-MB showed a 8

significant increase in CD8β1 RNA as a marker of CD8+ T cell infiltration, granzyme B 9

and perforin as a marker of cytolysis, and IFN-γ as a marker of T cell activation in 10

tumors (Fig. 4C). Sta-MB at 0.83 mg/kg upregulated these molecules to levels similar to 11

7.5 mg/kg of Ure-MB. Meanwhile, Ure-MB did not upregulate IFN-γ even at 7.5 mg/kg 12

compared to vehicle. These results demonstrate that Sta-MB, despite its 13

exATP-dependence and tumor selectivity, activated immune cells within tumor tissue 14

more potently than Ure-MB. Consistent with these gene expression data, the ratio of 15

granzyme B, PD-1, KLRG-1, or ICOS in CD8+ T cells was increased in tumors after 16

treatment with Sta-MB or Ure-MB according to flow cytometry (Fig. 4D). Together 17




p. 20

with the increase in CD8β1 RNA, this indicates an increase of activated CD8+ T cells 1

within tumors. 2

3

Furthermore, we examined the distribution of Sta-MB and Ure-MB in human CD137 4

knock-in mice bearing Colon38 tumors. Both Sta-MB and Ure-MB on CD8+ T cells 5

were detected in tumors one day after injection, but in spleen and liver, Sta-MB was 6

markedly lower than Ure-MB (Fig. S17A-B). 7

8

In addition, CD8+ T cell depletion canceled anti-tumor efficacy completely. However, 9

depletion of CD4+ T cells, B cells, NK cells, neutrophils, or macrophages did not 10

influence anti-tumor efficacy in the LLC1/OVA/hGPC3 model (Fig. S18). This 11

demonstrates that STA551’s anti-tumor efficacy was caused by the increase in CD8+ T 12

cells and their higher level of activation and cytolysis, which is also supported by gene 13

expression analysis (Fig. 4), consistent with previous reports on conventional CD137 14

agonist antibodies (28). 15

16

Efficacy of STA551 in combination with anti-PD-L1 antibody 17




p. 21

We next evaluated the in vivo efficacy of Sta-MB in combination with anti-PD-L1. 1

Combination treatment with Sta-MB and anti-PD-L1 monoclonal antibody (mAb) 2

completely inhibited Colon38 tumor growth in human CD137 knock-in mice, while 3

monotherapy with either Sta-MB or anti-PD-L1 mAb showed only moderate efficacy 4

(Fig. 5A). All antibodies were well-tolerated in the monotherapy and combination 5

groups, with mice losing no more than 6.5% body weight and no increase in alanine 6

transaminase (ALT) and aspartate transaminase (AST) (Fig. S19A-B). Analysis of 7

whole gene expression indicated that combination treatment tended to increase the 8

expression of immune-related gene sets compared with monotherapies (Fig. 5B, Table 9

S8). The combination increased CD8+ T cell infiltration and PD-L1 expression in 10

tumors more than monotherapy, according to immunohistochemistry (Fig. 5C-D). These 11

results suggest that the combination of Sta-MB with anti-PD-L1 mAb altered the TME 12

to enhance immune activation, leading to synergistic anti-tumor effects. 13

14

While bispecific antibody against human CD137 and human glypican-3 (GPC3), which 15

exerts CD137 agonistic activity only in the presence of human GPC3, demonstrates 16

efficacy in the LLC1/OVA/hGPC3 model (Fig. S20), both Sta-MB monotherapy and 17




p. 22

combination with anti-PD-L1 mAb inhibited tumor growth in human CD137 knock-in 1

mice with LLC1/OVA tumors which do not express GPC3 (Fig. S21A). 2

3

We compared the in vivo efficacy and response in non-tumor tissues of Sta-MB and 4

Ure-MB when combined with anti-PD-L1 mAb. The Sta-MB combination with 5

anti-PD-L1 mAb showed meaningful potent anti-tumor efficacy compared to the 6

Ure-MB combination (Fig. 5E), inhibiting tumor growth in all mice for over 70 days 7

(data not shown). Even with its greater potency, neither Sta-MB monotherapy nor 8

combination increased DLN and spleen weight, or the absolute number of LAG-3+

9

CD8+ T, PD-1

+ CD8

+ T, KLRG-1

+ CD8

+ T, ICOS

+ CD8

+ T cells, and FoxP3

+ CD4

+ T 10

cells in the DLN, whereas Ure-MB monotherapy and combination increased these 11

populations dramatically (Fig. 5F-H, S21B-C). The Ure-MB therapies significantly 12

increased LAG-3, PD-1, KLRG-1, or ICOS expression on CD8+ T cells and FoxP3

+ 13

Tregs in CD4+ T cells in spleen and liver, but Sta-MB therapies did not (Fig. 5I-J). 14

Those cells in spleen and liver were also increased by Ure-MB, but not by Sta-MB (Fig. 15

S22A-B). In addition, blood cell analysis demonstrated that the density of white blood 16

cells, lymphocytes, and platelets was decreased more by Ure-MB than by Sta-MB (Fig. 17

5K). These results demonstrate that Sta-MB shows potent anti-tumor efficacy against 18




p. 23

broad variety of cancer irrespective of the expression of specific tumor-associated 1

antigen, and the combination of Sta-MB with anti-PD-L1 mAb enhanced anti-tumor 2

activity without causing systemic responses. 3

4

Efficacy of STA551 in combination with T-cell redirecting antibody (TRAB) 5

TRAB is a bispecific antibody targeting tumor-associated antigen and CD3, which 6

activates T cells in an antigen-dependent manner to kill antigen-expressing cancer cells. 7

Because CAR-T cells require CD137 signaling in addition to CD3 signaling for clinical 8

efficacy (6,7), CD137 agonist antibody would be an ideal combination partner with 9

TRAB. Since TRABs targeting various tumor-associated antigens, such as GPC3, Her2, 10

and CEA, are being tested in clinical, it is not realistic to develop a unique CD137 11

bispecific agonist against each and every tumor associated antigen. To test whether 12

efficacy of GPC3-specific TRAB can be enhanced by a CD137 agonist antibody which 13

does not rely on GPC3 expression, we evaluated the efficacy of STA551 in combination 14

with anti-human GPC3/mouse CD3 (anti-hGPC3/mCD3) bispecific antibody. The 15

combination completely inhibited tumor growth in human CD137 knock-in mice with 16

human GPC3-expressing LLC1 (LLC1/hGPC3) tumors, which is a non-inflamed tumor 17

(29), while the efficacy of either monotherapy was limited (Fig. 6A). The combination 18




p. 24

also notably increased RNA levels of CD3, CD8β1, cytolysis markers including 1

granzyme B and perforin, and IFN-γ in tumors (Fig. 6B). 2

3

To further evaluate STA551 combined with TRAB in a setting with human cancer cells 4

and immune cells, we used human hematopoietic stem cell (HSC)-engrafted 5

NOD/Shi-scid, IL-2RγKO Jic (huNOG) mice expressing human CD137 on T cells and 6

human FcγRIIb on B cells. As TRAB, we used anti-human GPC3/human CD3 7

bispecific antibody, GPC3-TRAB. The combination of STA551 with GPC3-TRAB 8

strongly inhibited growth of established NCI-H446, which endogenously expresses 9

human GPC3, and dramatically increased the number of human T cells, while the 10

efficacy of either monotherapy was limited (Fig. 6C-D). This result demonstrates that 11

STA551 also exerts exATP-dependent agonistic activity in human cancer cells and 12

immune cells. 13

14

Discussion 15

Although the importance of CD137 signaling in cancer immunotherapy has been 16

validated both pre-clinically and clinically (4-7), anti-CD137 agonist antibodies have 17




p. 25

not progressed beyond early phase clinical trials. Urelumab was hampered by 1

hepatotoxicity, while utomilumab showed limited efficacy possibly due to its weak 2

agonistic activity (3). 3

4

Addressing these issues, recent publications describe several bispecific tumor-targeted 5

CD137 agonists (14-16). However, their therapeutic efficacy relies fully on the 6

expression of tumor-associated antigen, limiting their clinical application to patients 7

who highly express those antigens. Tumor-associated antigens must be highly tumor 8

specific, and the expression of these rare tumor-associated antigens are often limited to 9

only specific types of cancer. In contrast, the success of anti-PD-1/PD-L1 antibody is 10

partially attributed to its applicability to broad variety of cancers regardless of antigen 11

expression. In addition, EGFR-targeted CD137 bispecific agonist is rapidly eliminated 12

(16), and FAP-targeted CD137 bispecific agonist requires combination partners like 13

anti-PD-L1 antibody or TRAB to strongly inhibit tumor growth (14). Clearly, a novel 14

anti-CD137 agonist antibody, having strong agonistic activity, long half-life, improved 15

safety profile and broad applicability without tumor-associated antigen expression, is 16

greatly needed. 17

18




p. 26

Our exATP switch antibody platform provides a novel tumor selective approach which 1

is not dependent on the expression of tumor-associated antigens, and we believe the 2

exATP switch is superior to other approaches, such as using low pH and hypoxia, which 3

are also recognized hallmarks of TME that have been widely investigated for use in 4

tumor-specific therapeutics (30,31). It has been confirmed that healthy tissues and 5

plasma contain very low levels of exATP (10 to 100 nanomolars) (19,32), while over 6

100 micromolars of exATP was detected in the TME (17). P2X7 is a receptor for 7

exATP with 100 micromolars KD and is activated in the TME (33), indicating that 8

exATP is present in the TME at concentrations over 100 micromolars. Based on these 9

published reports, we speculate there is more than a 1000-fold difference in exATP 10

concentration between normal tissues and tumor. Meanwhile, pH in normal tissues and 11

tumor is reported to be 7.2-7.4 and 6.5-6.9, respectively, with a less than 10-fold 12

difference in proton concentration (34,35), and oxygen levels in arterial blood and the 13

TME are reported to be around 9.5% and 0.3%-4.2%, respectively, with a several-fold 14

difference in oxygen concentration (36,37). Considering the inefficient distribution of 15

antibodies into solid tumor, a less than 10-fold difference in concentration is clearly not 16

sufficient. 17

18




p. 27

We generated STA551, an ATP-dependent agonist switch antibody to CD137 that does 1

not rely on the expression of a specific tumor-associated antigen. It was designed to 2

bind to CD137 strongly in the presence of 100 micromolars of ATP, but not in the 3

presence of one micromolar. STA551 inhibited tumor growth in all the mouse models 4

we tested, indicating that exATP concentration is at least 100 micromolars in the TMEs 5

of a broad range of mouse tumors, which is consistent with a previous estimation of 6

exATP concentration in the TME (17). 7

8

Although CD73 was expressed in those tumors, STA551 activated immune cells and 9

exhibited anti-tumor efficacy, indicating that a high enough concentration of exATP, 10

exADP, and exAMP remains in the tumors for STA551 to work. This is supported by a 11

previous estimation that exADO stays in the micromolar-range in TME (38). Also, in a 12

recent publication, exATP levels were 100-fold higher in tumor tissues than in adjacent 13

non-tumor tissues in human pancreatic ductal adenocarcinoma (PDAC) (39) even 14

though CD73 is expressed in all PDAC (40). This provides more evidence that, even if 15

CD73 is expressed in human tumors, the remaining exATP levels are still high enough 16

for STA551 to achieve efficacy. Furthermore, STA551 did not show CD137 mediated 17




p. 28

systemic clearance and immune activation, indicating that exATP concentration is less 1

than one micromolar in normal tissues, also consistent with previous reports (19). 2

3

Unlike CD137 agonists which failed to show strong efficacy in most tumor models 4

(14,41), STA551 monotherapy achieved potent efficacy in various tumor models. We 5

believe this is because STA551 has an engineered IgG1 Fc with enhanced binding to 6

FcγRIIb on the surface of tumor-associated B and myeloid cells, allowing enhanced 7

cross-linking and multimerization of CD137, which is important for CD137 signal 8

activation (42). The details on Fc engineering and its biological effect will be published 9

elsewhere. 10

11

However, this anti-tumor efficacy may not be as impressive as that of anti-mouse 12

CD137 antibodies such as 1D8-rat IgG2a (43). This is probably because 1D8-rat IgG2a 13

induces ADCC in mice, therefore it depletes Tregs, which expresses CD137 (44), 14

leading to strong anti-tumor efficacy. Sta-MB does not induce ADCC because it does 15

not bind to mouse FcγRIV, therefore it demonstrated anti-tumor efficacy purely through 16

agonistic activity. In addition, treatment started very early in previous reports, before or 17




p. 29

within three days after transplanting of tumor cell lines (45). Such experiments might 1

represent the prevention effect. Sta-MB was injected after tumors were established, 2

therefore the results should reflect the therapeutic effect. 3

4

In the Colon38 tumor model, 0.093 mg/kg of Ure-MB showed no statistically 5

significant efficacy, but did significantly increase T cell activation in the liver. This 6

suggests that the therapeutic window of urelumab is too narrow for efficacy without 7

hepatotoxicity, which is consistent with the clinical results. Even though there are many 8

FcγRIIb-expressing cells in liver (46) to crosslink STA551, STA551 still did not 9

activate T cells in liver, even at dosages far higher than required for efficacy. Hence, 10

STA551 has a therapeutic window wide enough to achieve therapeutic efficacy. 11

12

STA551 was carefully designed so that nonclinical safety assessment in cynomolgus 13

monkeys can be translated into humans. In both species, STA551 binds with 14

comparable affinity to CD137 in the presence of 100 micromolars of ATP, while the 15

binding is not detectable in the absence of ATP. Affinity to both human and 16

cynomolgus monkey FcγRIIb is also enhanced compared to conventional IgG1. 17




p. 30

Systemic toxicity of STA551 was significantly less than conventional CD137 agonist 1

antibody, with no elevation of liver enzymes such as ALT, AST, or glutamate 2

dehydrogenase, no activation of immune cells in peripheral blood, and no abnormalities 3

in the histopathological assessment including lung and liver in cynomolgus monkeys at 4

up to 30 mg/kg. In contrast, all these changes were observed with utomilumab at the 5

same dose. The 14 day half-life of STA551 in cynomolgus monkeys suggests a dosing 6

schedule convenient for patients, such as once every 3 weeks. 7

8

One remaining challenge is in detecting the target binding of switch antibody in vivo. 9

This is technically challenging because switch antibodies would dissociate from the 10

antigen during the process of ex vivo analysis, wherein ATP concentration decreases 11

either by enzymatic and spontaneous hydrolysis as well as by dilution. Thus, observed 12

ex vivo binding may underestimate actual in vivo binding. Given the challenge of 13

quantitatively comparing non-switch and switch antibody biodistribution, we evaluated 14

pharmacodynamic responses and pharmacokinetics in tumor, DLN, spleen and liver 15

which reflect antibody binding to the target. Although we were able to directly detect 16

the binding of Sta-MB to target CD8+ T cells in tumors, the binding was lower than 17

Ure-MB. Since Sta-MB activated immune cells within tumor tissue more potently than 18




p. 31

Ure-MB, lower binding of Sta-MB could be due to its dissociation from the antigen 1

during the ex vivo process. 2

3

STA551 showed a synergistic anti-tumor effect in combination with anti-PD-L1 4

antibody and TRAB by increasing CD8+ T cell proliferation and/or infiltration and 5

expression of immune-related gene sets in tumors. These combinations could further 6

amplify its anti-tumor efficacy in non-inflamed intermediate/immunosuppressed tumors 7

(47). Importantly, STA551 was effective in combination with these reagents, which 8

could affect the TME (i.e. reducing the exATP concentration by inducing the expression 9

of CD39 which hydrolyzes exATP (48)). Combinations with other reagents such as 10

chemotherapy should be tested in the future. 11

12

We have proven preclinically that the exATP switch antibody platform was very effective 13

in expanding the therapeutic window of CD137 agonist antibody. A drug target for which 14

the therapeutic window of conventional antibody is not sufficient could be transformed 15

into a target where no systematic effect was detected even at a dosage much higher than 16

required for maximum efficacy. In the oncology field, there are many drug targets that 17




p. 32

suffer from on-target off-tumor side effects and cannot be safely made into drugs for 1

patients. We believe that the exATP switch antibody platform can reach those previously 2

undruggable targets. Thus, it could be utilized for antibody drug conjugates and other 3

approaches where systemic binding may be too toxic, such as with TRABs, CAR-T, and 4

other antibody therapies. 5

6

In conclusion, we generated a novel anti-CD137 switch antibody, STA551, that exerts 7

agonistic activity selectively in tumors, in which exATP is elevated more than 8

1000-fold compared to normal tissues and plasma, without relying on expression of 9

tumor-associated antigen. STA551 monotherapy demonstrated potent and broad 10

anti-tumor efficacy against all tumors tested without systemic immune activation. These 11

results strongly support the clinical testing of STA551 for the treatment of a wide variety 12

of solid tumors not restricted by antigen expression. Preclinical evidence that the exATP 13

switch antibody platform expands the therapeutic window warrants its application to 14

other targets stymied by on-target toxicity in cancer therapy. STA551 is currently being 15

tested in a phase 1 clinical study. 16

17




p. 33

Methods. 1

Study design 2

The main objective of our study was to evaluate the anti-tumor efficacy and safety of 3

STA551 targeting CD137. Anti-tumor efficacy was assessed in two different 4

tumor-grafted mouse platforms: immunocompetent human CD137 knock-in mice and 5

huNOG mice in which CD34+ human HSCs were transplanted to reconstitute human 6

immune cells. Sample size (n=6 to 15 per group in human CD137 knock-in mouse 7

model and n=5 per group in huNOG mouse model) was determined based on the 8

consistency of tumor growth observed in preliminary experiments to allow for 9

statistically significant differences in tumor size between the various treatment groups. 10

Animals were randomly assigned to groups based on tumor size so that each group had 11

the same average size. Tumor volumes were calculated according to the following 12

formula: [(length × width2)/2]. All tumor volume data (mean tumor volume with SD) 13

were plotted. Animals were sacrificed at the end of the study. The use of human HSCs 14

was approved by an Institutional Review Board. STA551 toxicity was assessed using 15

cynomolgus monkeys. Animals were assigned to each group using a computerized 16

procedure designed to balance body weight equally among groups. The number of 17

animals per group (n=3 per sex per group) was chosen according to ICH-S4A guidelines 18




p. 34

for reaching scientific conclusions on safety with consideration for animal welfare. All 1

animal studies described above were reviewed and approved by the Institutional Animal 2

Care and Use Committee (IACUC) at each facility. 3

4

Cell lines 5

C1498, E.G7-OVA, Hepa1-6, LLC1, and NCI-H446 were purchased from the American 6

Type Culture Collection. Colon38 was obtained from National Institutes of Health. 7

GPC3 and/or chicken ovalbumin (OVA)-overexpressing Hepa1-6/hGPC3 (29), 8

LLC1/OVA/hGPC3, LLC1/hGPC3, and LLC1/OVA cells were established by 9

transfecting GPC3 and/or OVA-expressing plasmids into parental cells to enhance the 10

immunogenicity of each tumor, and also to allow us to test the combination of STA551 11

with a TRAB against GPC3. Human Fc gamma receptor IIb (FcγRIIb)-expressing 12

CHO-k1 cells (CHOk1/human FcγRIIb cells) were purchased from Promega, and 13

CHO-DG44 from Thermo Fisher Scientific. Mouse FcγRIIb-overexpressing CHO, 14

human FcγRIIa-overexpressing CHO, and human FcγRIIb-overexpressing CHO were 15

established by transfecting mouse FcγRIIb-expressing plasmids, H allotype of human 16

FcγRIIa-expressing plasmids, or human FcγRIIb-expressing plasmids into parental 17




p. 35

CHO-DG44 cells. Human CD137 overexpressing CHO was established by transfecting 1

human CD137-expressing plasmids into parental CHO-DG44 cells. 2

3

Measurement of small molecule dependent binding and binding kinetics against 4

human or cynomolgus monkey CD137 5

Binding of anti-CD137 antibodies against recombinant human CD137 extracellular 6

domain (Genbank accession number AAH06196.1) or recombinant cynomolgus 7

monkey CD137 extracellular domain (Genbank accession number ABY47575.1) was 8

assessed in the presence or absence of adenosine-5'-triphosphate (ATP, Nacalai 9

Tesque), adenosine-5'-diphosphate (ADP, Nacalai Tesque), 10

adenosine-5'-monophosphate (AMP, Nacalai Tesque) or adenosine (ADO, Nacalai 11

Tesque) at 37°C using Biacore T200 instrument (GE Healthcare). Antibody was 12

captured onto a Biacore CM4 sensor surface immobilized with protein G (Merck). 13

Biacore sensorgrams were obtained at 37°C by injecting human CD137 or cynomolgus 14

monkey CD137 in the presence of ATP, ADP, AMP, or ADO at 1000, 100, 10, 1, and 0 15

μmol/L, over a sensor surface capturing STA551. Kinetics parameters were determined 16

by fitting sensorgrams with 1:1 binding model using Biacore T200 Evaluation Software 17

Version 2.0 (GE Healthcare). 18




p. 36

1

In vitro CD8+ T cell assay with Fcγ receptor IIb-expressing cells 2

All human CD8+ T cells were purchased from Astarte Biologics, Inc. Human CD8

+ T 3

cells from healthy donors were stimulated with a fixed concentration of immobilized 4

anti-human CD3ε (1 μg/mL, clone SP34, BD Biosciences) and anti-human CD28 5

antibodies (5 μg/mL, clone CD28.2, BD Biosciences) for 6 hours, and then variable 6

concentrations of STA551 or Ure-hIgG4 in the absence or presence of 100 μmol/L ATP 7

with CHOk1/human FcγRIIb cells were added and incubated for 18 hours in AIM-V 8

medium (Thermo Fisher Scientific) supplied with 5% human serum (Sigma-Aldrich). 9

After incubation, the supernatants were harvested and the IFN-γ concentrations in the 10

medium were measured by enzyme-linked immunosorbent assay (ELISA). 11

12

In vitro PBMC assay 13

Human PBMCs were purified from the fresh blood of 20 healthy donors using a 14

conventional Ficoll-Paque PLUS gradient (GE healthcare). Human PBMCs were 15

stimulated with a fixed concentration of anti-human CD3ε (0.01 μg/mL) and 16

anti-human CD28 antibodies (5 μg/mL) for 6 hours, and then STA551 or Ure-hIgG4 (5 17




p. 37

μg/mL) were added and incubated for 42 hours in the absence or presence of 250 1

μmol/L ATP in AIM-V medium supplied with 5% human serum. After incubation, 2

supernatants were harvested and IFN-γ concentrations were measured by ELISA. 3

4

In vivo human CD137 knock-in mouse model 5

All mouse studies were performed according to IACUC policies. Human CD137 6

knock-in mice were generated by replacing mouse Cd137 with its human counterpart, 7

CD137 (Fig. S7A-D). The C1498, E.G7-OVA, Hepa1-6/hGPC3, LLC1/OVA/hGPC3, 8

Colon38, LLC1/hGPC3, or LLC1/OVA cells were inoculated subcutaneously into 9

human CD137 knock-in mice. After palpable tumors were established, mice were 10

randomized based on tumor volume and body weight. Subsequently, 2.5 mg/kg of 11

Sta-MB, 7.5 mg/kg of Ure-MB, or vehicle were intravenously administered twice. In 12

the combination models, 10 mg/kg of anti-PD-L1 Ab (clone 10F.9G2, Bio X cell) was 13

intraperitoneally administered, and 1 mg/kg of anti-human GPC3/mouse CD3 bispecific 14

antibody and 2.5 mg/kg Sta-MB was intravenously administered. Tumor size was 15

measured twice per week. 16

17




p. 38

Flow cytometry staining 1

LLC1/OVA/hGPC3, Colon38, or LLC1/OVA cells were inoculated subcutaneously into 2

the flanks of human CD137 knock-in mice. After the tumors were established, tumors 3

or livers were removed from mice treated with anti-human CD137 antibodies on day 4 4

after the first dose. Tumors or livers were digested, processed, and enriched into single 5

cell suspensions with tumor or liver dissociation kit (Miltenyi Biotec) followed by red 6

blood-cell lysis. Both digestions were according to the manufacturer’s protocol. Single 7

cell suspensions from DLN and spleen cells were obtained with nylon mesh followed by 8

red blood-cell lysis. These suspensions were used for the following experiments. 9

Antibodies to CD3ε (clone 145-2C11), CD8α (clone 53-6.7), CD4 (clone RM4-5), 10

CD19 (clone 1D8/CD19), granzyme B (clone QA16A02), ICOS (clone C398.4A), 11

CD279 (clone 29F.A12), CD11b (clone M1/70), CD39 (clone Duha59) and CD73 12

(clone TY/11.8) were purchased from BioLegend. Antibodies to CD45 (clone 30-F11), 13

KLRG-1 (clone 2F1), LAG-3 (clone C9B7W) and FoxP3 (clone MF23) were purchased 14

from BD Biosciences. Antibody to ICOS (clone 7E.17G9) and human IgG (H+L) was 15

purchased from Thermo Fisher Scientific. Antibody to CD8 (clone KT15) was 16

purchased from MBL. Antibody to human GPC3 (clone GC33) was generated in 17

Chugai Pharmaceutical. Dead cells were stained by Zombie Aqua Fixable Viability Kit 18




p. 39

(BioLegend) or Fixable Viability Dye eFluor780 (Thermo Fisher Scientific). These cells 1

were incubated with antibodies for 30 minutes at 4°C with Foxp3/Transcription Factor 2

Staining Buffer Set (Thermo Fisher Scientific) after treatment with mouse Fc receptor 3

blocking reagent (Miltenyi Biotec). Staining was conducted according to the 4

manufacturer’s protocol. Cells were analyzed by FACSLyric (BD Biosciences) or 5

LSRFortessa™ X-20 (BD Biosciences). 6

7

NCI-H446 cells were inoculated subcutaneously into the flanks of huNOG mice. The 8

single cell suspensions were obtained from tumors using the same method as above on 9

day 7. Antibodies to human CD4 (clone RPA-T4), human CD8 (clone SK1), and human 10

CD19 (clone HIB19) were purchased from BioLegend. Antibody to human CD45 11

(clone HI30), human CD3 (clone UCHT1), and mouse CD45 (clone 30-F11) were 12

purchased from BD Biosciences. Dead cells were stained by Fixable Viability Dye 13

eFluor 780. The staining buffer was the same as above. Cell counts by flow cytometry 14

were determined using CountBright Absolute Counting Beads (Thermo Fisher 15

Scientific). Cells were incubated with antibodies for 30 minutes at 4°C after treatment 16

with both human and mouse Fc receptor blocking reagents (Miltenyi Biotec). Cells were 17

analyzed by LSRFortessa™ X-20. 18




p. 40

1

Statistical analysis 2

Data are presented as means ± SD, means + SD, or means only as stated in the figure 3

legends. Statistically significant differences were determined using specific tests as 4

indicated in the figure legends. P < 0.05 was considered statistically significant. 5

6

Author contributions. 7

Writing, review, and/or revision of the manuscript: M.Kamata-Sakurai, Y.Narita, 8

T.Nemoto, M.Honda, N.A.Wada, and T.Igawa 9

Design, production, and characterization of the antibodies: M.Kamata-Sakurai, 10

Y.Hori, N.Hironiwa, M.Shida-Kawazoe, S.Shimizu, T.Sakiyama, S.Kadono, 11

A.Sakamoto, S.Naoi, N.Savory, M.Sato, Y. Noguchi, H.Kuroi, A.Ito, T.Wakabayashi, 12

M.Kamimura, and T.Igawa 13

Facilitation and execution of the pharmacological studies, and analysis of the assay 14

data: M.Kamata-Sakurai, Y.Narita, R.Uchikawa, K.Taniguchi, S.Metsugi, T.Miyazaki, 15

Y.Ohte, H.Mikami, N.Ono, S.Horikawa, K.Hamada, Y.Satoh, F.Isomura, Y.Tomii, 16

N.Sawada, Y.Nakanishi, M.Endo, Y.Kawabe, and T.Kitazawa 17




p. 41

Facilitation and execution of the pharmacokinetics studies, and analysis of the 1

assay data: T.Nemoto and T.Tachibana 2

Facilitation and execution of the toxicological studies, and analysis of the assay 3

data: M.Honda, K.Adachi, T.Matsushita, S.i.Komatsu, A.Hirako, J.Shinozuka, and 4

A.Kato 5

Generation of human CD137 knock-in mice: N.A.Wada, O.Ueda, and K.Jishage 6

Study supervision: M.Kamata-Sakurai, Y.Narita, T.Nemoto, M.Honda, Y.Nakanishi 7

and T.Igawa 8

9

Acknowledgments. 10

We thank the donors who consented to the use of their cells for these studies. We 11

acknowledge the technical and scientific support of teams at Chugai Pharmaceutical Co., 12

Ltd. and Chugai Research Institute for Medical Science Inc. at Kamakura, Gotemba, 13

and Ukima. We also thank Masaaki Goto and Kei Esaki for their help with antibody 14

production and characterization; Suguru Kenmoku, Shohei Kobayashi, Noribumi 15

Tomiyama, Naoki Akiyama, and Ai Shigemasa for large scale antibody production, 16




p. 42

manufacturing method, and formulation; Keiichi Morita for his help with 1

pharmacokinetic studies; and Jacob Davis for review of the manuscript. 2

3

Data and materials availability. 4

All data associated with this study are present in the paper or the Supplementary 5

Materials. All materials and the novel mouse strain generated in this study can be made 6

available on request under a material transfer agreement with Chugai Pharmaceutical 7

Co., Ltd. The following restrictions apply to this mouse strain: 8

1. Cross-breeding with other mouse strains is prohibited. 9

2. Additional gene manipulations to the mouse strain are prohibited. 10

3. Use of the mouse strain for commercial purposes is prohibited. 11

4. Further transfer of the mouse strain to third parties is prohibited. 12

13

References. 14

1. Postow MA, Callahan MK, Wolchok JD. Immune Checkpoint Blockade in Cancer Therapy. J 15

Clin Oncol 2015;33(17):1974-82. 16

2. Márquez-Rodas I, Cerezuela P, Soria A, Berrocal A, Riso A, González-Cao M, et al. Immune 17

checkpoint inhibitors: therapeutic advances in melanoma. Ann Transl Med 2015;3(18):267. 18




p. 43

3. Chester C, Sanmamed MF, Wang J, Melero I. Immunotherapy targeting 4-1BB: mechanistic 1

rationale, clinical results, and future strategies. Blood 2018;131(1):49-57. 2

4. Yonezawa A, Dutt S, Chester C, Kim J, Kohrt HE. Boosting Cancer Immunotherapy with 3

Anti-CD137 Antibody Therapy. Clin Cancer Res 2015;21(14):3113-20. 4

5. Vinay DS, Kwon BS. Immunotherapy of Cancer with 4-1BB. Mol Cancer Ther 5

2012;11(5):1062-70. 6

6. Park JH, Geyer MB, Brentjens RJ. CD19-targeted CAR T-cell therapeutics for hematologic 7

malignancies: interpreting clinical outcomes to date. Blood 2016;127(26):3312-20. 8

7. van der Stegen SJC, Hamieh M, Sadelain M. The pharmacology of second-generation chimeric 9

antigen receptors. Nature reviews Drug discovery 2015;14(7):499-509. 10

8. Gelao L, Criscitiello C, Esposito A, Goldhirsch A, Curigliano G. Immune checkpoint blockade 11

in cancer treatment: a double-edged sword cross-targeting the host as an "innocent bystander". 12

Toxins 2014;6(3):914-33. 13

9. Segal NH, Logan TF, Hodi FS, McDermott D, Melero I, Hamid O, et al. Results from an 14

Integrated Safety Analysis of Urelumab, an Agonist Anti-CD137 Monoclonal Antibody. Clin 15

Cancer Res 2017;23(8):1929-36. 16

10. Dubrot J, Milheiro F, Alfaro C, Palazón A, Martinez-Forero I, Perez-Gracia JL, et al. Treatment 17

with anti-CD137 mAbs causes intense accumulations of liver T cells without selective antitumor 18

immunotherapeutic effects in this organ. Cancer Immunol Immunother 2010;59(8):1223-33. 19

11. Segal NH, He AR, Doi T, Levy R, Bhatia S, Pishvaian MJ, et al. Phase I Study of Single-Agent 20

Utomilumab (PF-05082566), a 4-1BB/CD137 Agonist, in Patients with Advanced Cancer. Clin 21

Cancer Res 2018;24(8):1816-23. 22

12. Chin SM, Kimberlin CR, Roe-Zurz Z, Zhang P, Xu A, Liao-Chan S, et al. Structure of the 23

4-1BB/4-1BBL complex and distinct binding and functional properties of utomilumab and 24

urelumab. Nat Commun 2018;9(1):4679. 25

13. Sela-Culang I, Kunik V, Ofran Y. The structural basis of antibody-antigen recognition. Front 26

Immunol 2013;4:302. 27

14. Claus C, Ferrara C, Xu W, Sam J, Lang S, Uhlenbrock F, et al. Tumor-targeted 4-1BB agonists 28

for combination with T cell bispecific antibodies as off-the-shelf therapy. Sci Transl Med 29

2019;11(496):eaav5989. 30

15. Hinner MJ, Aiba RSB, Jaquin TJ, Berger S, Dürr MC, Schlosser C, et al. Tumor-Localized 31

Costimulatory T-Cell Engagement by the 4-1BB/HER2 Bispecific Antibody-Anticalin Fusion 32

PRS-343. Clin Cancer Res 2019;25(19):5878-89. 33

16. Compte M, Harwood SL, Munoz IG, Navarro R, Zonca M, Perez-Chacon G, et al. A 34

tumor-targeted trimeric 4-1BB-agonistic antibody induces potent anti-tumor immunity without 35

systemic toxicity. Nat Commun 2018;9(1):4809. 36




p. 44

17. Pellegatti P, Raffaghello L, Bianchi G, Piccardi F, Pistoia V, Di Virgilio F. Increased level of 1

extracellular ATP at tumor sites: in vivo imaging with plasma membrane luciferase. PLoS One 2

2008;3(7):e2599. 3

18. Di Virgilio F, Adinolfi E. Extracellular purines, purinergic receptors and tumor growth. 4

Oncogene 2017;36(3):293-303. 5

19. Gorman MW, Feigl EO, Buffington CW. Human plasma ATP concentration. Clin Chem 6

2007;53(2):318-25. 7

20. Fuentes E, Palomo I. Extracellular ATP metabolism on vascular endothelial cells: A pathway 8

with pro-thrombotic and anti-thrombotic molecules. Vascular pharmacology 2015;75:1-6. 9

21. Mimoto F, Katada H, Kadono S, Igawa T, Kuramochi T, Muraoka M, et al. Engineered antibody 10

Fc variant with selectively enhanced FcγRIIb binding over both FcγRIIa(R131) and 11

FcγRIIa(H131). Protein Eng Des Sel 2013;26(10):589-98. 12

22. Kleiveland CR. Peripheral Blood Mononuclear Cells. In: Verhoeckx K, Cotter P, 13

López-Expósito I, Kleiveland C, Lea T, Mackie A, et al., editors. The Impact of Food Bioactives 14

on Health: in vitro and ex vivo models. Cham: Springer International Publishing; 2015;161-7. 15

23. Kotsakis A, Harasymczuk M, Schilling B, Georgoulias V, Argiris A, Whiteside TL. 16

Myeloid-derived suppressor cell measurements in fresh and cryopreserved blood samples. J 17

Immunol Methods 2012;381(1-2):14-22. 18

24. Gourdin N, Bossennec M, Rodriguez C, Vigano S, Machon C, Jandus C, et al. Autocrine 19

Adenosine Regulates Tumor Polyfunctional CD73+ CD4+ Effector T Cells Devoid of Immune 20

Checkpoints. Cancer Res 2018;78(13):3604. 21

25. Martinez-Forero I, Azpilikueta A, Bolaños-Mateo E, Nistal-Villan E, Palazon A, Teijeira A, et 22

al. T Cell Costimulation with Anti-CD137 Monoclonal Antibodies Is Mediated by K63–23

Polyubiquitin-Dependent Signals from Endosomes. J Immunol 2013;190(12):6694-706. 24

26. Haraya K, Tachibana T, Nezu J. Predicting pharmacokinetic profile of therapeutic antibodies 25

after iv injection from only the data after sc injection in cynomolgus monkey. Xenobiotica; the 26

fate of foreign compounds in biological systems 2017;47(3):194-201. 27

27. Fisher TS, Kamperschroer C, Oliphant T, Love VA, Lira PD, Doyonnas R, et al. Targeting of 28

4-1BB by monoclonal antibody PF-05082566 enhances T-cell function and promotes anti-tumor 29

activity. Cancer Immunol Immunother 2012;61(10):1721-33. 30

28. Weigelin B, Bolanos E, Teijeira A, Martinez-Forero I, Labiano S, Azpilikueta A, et al. Focusing 31

and sustaining the antitumor CTL effector killer response by agonist anti-CD137 mAb. Proc Natl 32

Acad Sci U S A 2015;112(24):7551-6. 33

29. Ishiguro T, Sano Y, Komatsu S-i, Kamata-Sakurai M, Kaneko A, Kinoshita Y, et al. An 34

anti-glypican 3/CD3 bispecific T cell-redirecting antibody for treatment of solid tumors. Sci 35

Transl Med 2017;9(410):eaal4291. 36




p. 45

30. Joyce JA. Therapeutic targeting of the tumor microenvironment. Cancer Cell 2005;7(6):513-20. 1

31. Junttila MR, de Sauvage FJ. Influence of tumour micro-environment heterogeneity on 2

therapeutic response. Nature 2013;501(7467):346-54. 3

32. de Andrade Mello P, Coutinho-Silva R, Savio LEB. Multifaceted Effects of Extracellular 4

Adenosine Triphosphate and Adenosine in the Tumor-Host Interaction and Therapeutic 5

Perspectives. Front Immunol 2017;8:1526. 6

33. Adinolfi E, Raffaghello L, Giuliani AL, Cavazzini L, Capece M, Chiozzi P, et al. Expression of 7

P2X7 receptor increases in vivo tumor growth. Cancer Res 2012;72(12):2957-69. 8

34. Estrella V, Chen T, Lloyd M, Wojtkowiak J, Cornnell HH, Ibrahim-Hashim A, et al. Acidity 9

generated by the tumor microenvironment drives local invasion. Cancer Res 10

2013;73(5):1524-35. 11

35. Stubbs M, McSheehy PMJ, Griffiths JR, Bashford CL. Causes and consequences of tumour 12

acidity and implications for treatment. Mol Med Today 2000;6(1):15-9. 13

36. Vaupel P, Mayer A. Hypoxia in cancer: significance and impact on clinical outcome. Cancer 14

Metastasis Rev 2007;26(2):225-39. 15

37. McKeown SR. Defining normoxia, physoxia and hypoxia in tumours-implications for treatment 16

response. Brit J Radiol 2014;87(1035):20130676. 17

38. Hatfield SM, Kjaergaard J, Lukashev D, Belikoff B, Schreiber TH, Sethumadhavan S, et al. 18

Systemic oxygenation weakens the hypoxia and hypoxia inducible factor 1α-dependent and 19

extracellular adenosine-mediated tumor protection. J Mol Med (Berl) 2014;92(12):1283-92. 20

39. Hu L-P, Zhang X-X, Jiang S-H, Tao L-Y, Li Q, Zhu L-L, et al. Targeting Purinergic Receptor 21

P2Y2 Prevents the Growth of Pancreatic Ductal Adenocarcinoma by Inhibiting Cancer Cell 22

Glycolysis. Clin Cancer Res 2019;25(4):1318. 23

40. Sciarra A, Monteiro I, Ménétrier-Caux C, Caux C, Gilbert B, Halkic N, et al. CD73 expression 24

in normal and pathological human hepatobiliopancreatic tissues. Cancer Immunol Immunother 25

2019;68(3):467-78. 26

41. Wei H, Zhao L, Li W, Fan K, Qian W, Hou S, et al. Combinatorial PD-1 Blockade and CD137 27

Activation Has Therapeutic Efficacy in Murine Cancer Models and Synergizes with Cisplatin. 28

PLoS One 2013;8(12):e84927. 29

42. Vanamee ES, Faustman DL. Structural principles of tumor necrosis factor superfamily signaling. 30

Sci Signal 2018;11(511): eaao4910. 31

43. Melero I, Shuford WW, Newby SA, Aruffo A, Ledbetter JA, Hellström KE, et al. Monoclonal 32

antibodies against the 4-1BB T-cell activation molecule eradicate established tumors. Nat Med 33

1997;3(6):682-5. 34




p. 46

44. Buchan SL, Dou L, Remer M, Booth SG, Dunn SN, Lai C, et al. Antibodies to Costimulatory 1

Receptor 4-1BB Enhance Anti-tumor Immunity via T Regulatory Cell Depletion and Promotion 2

of CD8 T Cell Effector Function. Immunity 2018;49(5):958-70.e7. 3

45. Shuford WW, Klussman K, Tritchler DD, Loo DT, Chalupny J, Siadak AW, et al. 4-1BB 4

costimulatory signals preferentially induce CD8+ T cell proliferation and lead to the 5

amplification in vivo of cytotoxic T cell responses. J Exp Med 1997;186(1):47-55. 6

46. Sorensen KK, Simon-Santamaria J, McCuskey RS, Smedsrod B. Liver Sinusoidal Endothelial 7

Cells. Compr Physiol 2015;5(4):1751-74. 8

47. Galon J, Bruni D. Approaches to treat immune hot, altered and cold tumours with combination 9

immunotherapies. Nat Rev Drug Discov 2019;18(3):197-218. 10

48. Raczkowski F, Rissiek A, Ricklefs I, Heiss K, Schumacher V, Wundenberg K, et al. CD39 is 11

upregulated during activation of mouse and human T cells and attenuates the immune response 12

to Listeria monocytogenes. PLoS One 2018;13(5):e0197151. 13




p. 47

Display Items 1

2

Tables. 3

Table 1. 4

A. Human CD137 5

Antibody

ATP 0 μmol/L ATP 100 μmol/L

KD (mol/L)

STA551 N. D. 9.82 (±0.13) × 10−9

Ure-hIgG4 1.66 (±0.03) × 10−8 1.62 (±0.03) × 10−8

Uto-hIgG2 7.12 (±0.02) × 10−8 7.10 (±0.03) × 10−8

6

7

8

9

10




p. 48

B. Cynomolgus monkey CD137 1

Antibody

ATP 0 μmol/L ATP 100 μmol/L

KD (mol/L)

STA551 N. D. 2.51 (±0.03) × 10−8

Ure-hIgG4 N. D. N. D.

Uto-hIgG2 13.5 (±0.13) × 10−8 13.0 (±0.10) × 10−8

2

KD values for the binding of STA551, Ure-hIgG4, and Uto-hIgG2 to human and 3

cynomolgus monkey CD137. (A)This table shows the mean ± SD (n = 3) of the calculated 4

KD of STA551, Ure-hIgG4, and Uto-hIgG2 binding to human CD137 and (B) 5

cynomolgus monkey CD137 in the absence and presence of 100 μmol/L ATP. Each 6

antibody was measured in independent experiments both with and without ATP. KD: 7

dissociation constant, N.D.: not detectable, ATP: adenosine triphosphate. 8

9

10

11




p. 49

Figures. 1

Figure 1. 2

Generation of STA551. (A) Schematic illustration of STA551. The Fab arm, depicted in 3

purple, ATP dependently binds to CD137. (B) Biacore sensorgram showing binding of 4

STA551 to human CD137 and (C) cynomolgus monkey CD137 at 37°C in the presence 5

of ATP at 1000 μmol/L (shown in red), 100 μmol/L (yellow), 10 μmol/L (blue), 1 μmol/L 6

(green), 0 μmol/L (black). 7

8

Fig. 2. 9

IFN-γ release induced by STA551 or Ure-hIgG4 in the absence or presence of ATP. (A) 10

Human CD8+ T cells derived from three different donors were co-cultured with 11

CHOk1/human FcγRIIb cells and STA551 or Ure-hIgG4 in the absence or presence of 12

100 μmol/L ATP. IFN-γ concentrations were measured by ELISA. The blue circle 13

indicates STA551 in the absence of ATP, the blue triangle STA551 in the presence of 100 14

μM ATP, the pink circle Ure-hIgG4 in the absence of ATP, and the pink triangle 15

Ure-hIgG4 in the presence of 100 μM ATP. Each point represents mean ± SD (n=3). *: 16

Statistical differences in IFN-γ levels between 1 μg/mL STA551 in the absence of ATP 17

and in the presence of 100 μmol/L ATP (*P < 0.0001 by t-test). (B) Human PBMCs 18




p. 50

from the fresh blood of 20 healthy donors were incubated with STA551 or Ure-hIgG4 1

in the presence of 250 μmol/L ATP. IFN-γ concentrations were measured by ELISA. 2

The symbols indicate mean of IFN-γ levels from different 20 donors. **: Statistical 3

differences in IFN-γ levels between the STA551 and Ure-hIgG4 or anti-KLH-hIgG4 4

and Ure-hIgG4 (**P < 0.01 by paired t-test). IFN-γ: Interferon gamma, FcγRIIb: Fc 5

gamma receptor IIb, PBMCs: peripheral blood mononuclear cells, ATP: adenosine 6

triphosphate, ELISA: enzyme-linked immunosorbent assay. 7

8

Fig. 3. 9

Anti-tumor efficacy and systemic responses to Sta-MB or Ure-MB in human CD137 10

knock-in mice. (A) Human CD137 knock-in mice bearing C1498, E.G7-OVA, and 11

Hepa1-6/hGPC3, (B) LLC1/OVA/hGPC3, and (C) Colon38 were administered vehicle 12

(black/gray), Sta-MB (blue), or Ure-MB (pink). Black dashed lines indicate average 13

tumor volume for vehicle (A, n=10, B, n=7, C, n=14). *, **: Statistical analysis between 14

vehicle and Sta-MB on (B) day 21 (*P < 0.01 by Steel’s test) or (C) day 28 (*P < 0.025 by 15

Williams’ test) or Ure-MB (**P < 0.05 by Steel’s test). Non-parametric Steel’s test or 16

parametric Williams’ test was selected by Bartlett's test for assessing tumor volumes of 17

each treatment. †: Euthanasia due to dissemination of tumor cells in the peritoneal cavity, 18




p. 51

#: Euthanasia due to false administration. (D) Human CD137 knock-in mice (bold line) 1

and wild type C57BL/6N mice (dashed line) were administered Sta-MB (blue) and 2

Ure-MB (pink) at 7.5 mg/kg. Antibody concentration in plasma was determined. Human 3

CD137 knock-in mice bearing (E) LLC1/OVA/hGPC3 and (F) Colon38 were 4

administered by vehicle, Sta-MB (blue) or Ure-MB (pink). Weights of draining lymph 5

nodes (DLN) and spleen, and PD-1 expression on liver-derived CD8+ T cells were

Documents

Main Text Title. - Cancer Discovery...Aug 22, 2020 · Sakurai. 7 Mailing adress. 2-1-1 Nihonbashi-Muromachi, Chuo-ku, Tokyo, 103-8324, Japan 8 Phone. +81-3-3273-1032 9 FAX. +81-3-3281-0819