Received: 30 May 2019

| Revised: 3 September 2019 | Accepted: 6 September 2019

DOI: 10.1111/cas.14196

ORIGINAL ARTICLE

Generation and evaluation of a chimeric antibody against
coxsackievirus and adenovirus receptor for cancer therapy
Shuichi Sakamoto1 | Hiroyuki Inoue1 | Mika K. Kaneko2 | Satoshi Ogasawara2 |Masunori Kajikawa3 | Sakiko Urano3 | Shun‐ichi Ohba1 | Yukinari Kato2,4|

1,5

Manabu Kawada

1
Institute of Microbial Chemistry(BIKAKEN), Numazu, Japan

Abstract2

Coxsackievirus and adenovirus receptor (CAR) is a single‐pass transmembrane pro‐

Department of Antibody Drug
Development, Tohoku University Graduate
School of Medicine, Sendai, Japan
3
Medical & Biological Laboratories Co., Ltd,
Nagoya, Japan
4tein that is associated with adenoviral infection. CAR is involved in the formation ofepithelial tight junctions and promotes tumor growth in some cancers. Previously,
we developed mouse monoclonal antibodies against human CAR and found that one,

New Industry Creation Hatchery
Center, Tohoku University, Sendai, Japan

mu6G10A, significantly inhibited tumor growth in xenografts of human cancer cells.

5
Laboratory of Oncology, Institute ofMicrobial Chemistry (BIKAKEN), Tokyo,
Japan(ch6G10A) from mu6G10A. ch6G10A had binding activity, inducing antibody‐de‐Herein, we generated and characterized a mouse‐human chimeric anti‐CAR antibody
pendent cellular cytotoxicity and complement‐dependent cytotoxicity, and in vivo

Correspondence
Manabu Kawada, Laboratory of Oncology,
Microbial Chemistry Research Foundation,
Institute of Microbial Chemistry (BIKAKEN),
Tokyo, Japan.
Email: kawadam@bikaken.or.jp

anti‐tumor activity against CAR‐expressing prostate cancer DU‐145 cells. In addi‐

Funding information
Basis for Supporting Innovative Drug
Discovery and Life Science Research[BINDS], Grant/Award Number:
JP18am0101078; Kobayashi Foundation
for Cancer Research; The Japan Society for
the Promotion of Science, Grant/Award
Number: 17K08777 and 26460481

both in vivo orthotopic tumor growth and distant metastatic formation in mouse xen‐tion, cancer tissue array analysis confirmed that CAR is highly expressed in neuroen‐
docrine lung cancers including small cell lung cancer, and treatment with ch6G10A
effectively inhibited in vivo subcutaneous tumor growth of NCI‐H69 small cell lungcancer cells in nude mice. Moreover, treatment with mu6G10A effectively inhibitedograft models of a highly metastatic subline of human small cell lung cancer DMS273
cells. These results suggest that targeting therapy to CAR with a therapeutic anti‐
body might be effective against several cancer types including small cell lung cancer.
KEYWORDS

CAR, mouse‐human chimeric antibody, orthotopic transplantation model, prostate cancer,
small cell lung cancer

1 | I NTRO D U C TI O N

is involved in the formation and/or maintenance of epithelial tightjunctions.1 CAR has an essential role in the development of the heart

Coxsackievirus and adenovirus receptor (CAR) protein that en‐

and lymphatic system in mice. 2,3 Because CAR is the primary cel‐coded by CXADR gene, is a single‐pass transmembrane protein andlular receptor for adenoviruses,1 its expression level is considered

Abbreviations: ADCC, antibody‐dependent cellular cytotoxicity; CAR, coxsackievirus and adenovirus receptor; CDC, complement‐dependent cytotoxicity; CTA, cancer tissue array;
FcγRIIIA, Fcγ receptor IIIA; NK, natural killer; SCLC, small cell lung cancer.
Sakamoto and Inoue contributed equally to this work.

This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial License, which permits use, distribution and reproduction
in any medium, provided the original work is properly cited and is not used for commercial purposes.
© 2019 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.
Cancer Science. 2019;110:3595–3602.

﻿ wileyonlinelibrary.com/journal/cas

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an important factor for adenovirus infection and adenoviral vector‐
mediated treatment.

IgG1 were subcloned into pcDNA3.3/Neo or pcDNA3.1/Zeo vectors(Thermo Fisher Scientific Inc.), respectively. Antibody expression

It has been reported that high CAR expression levels occur in var‐

vectors were transfected into CHO‐S cells (Thermo Fisher Scientificious human cancers.4-10 Moreover, CAR promotes tumor growth inInc.) using Lipofectamine 2000 reagent (Thermo Fisher Scientificsome cancer types. CXADR silencing in lung cancer with high CAR ex‐

Inc.). Stable transfectants of CHO‐S/ch6G10A were selected by

11pression suppressed tumor formation ability in xenografts, whereas

culturing the transfectants in medium containing 1 mg/mL G418

CXADR knockdown in oral squamous cell carcinoma resulted in the(Nacalai Tesque, Inc.) and 0.5 mg/mL zeocin (InvivoGen). Stableinhibition of both anchorage‐independent growth and metastatictransfectants were cultured for 14 days in CHO‐S‐SFM II medium

tumor formation in vivo.12 These previous studies suggest that CAR(Thermo Fisher Scientific Inc.), and then ch6G10A immunoglobulin

might be an appropriate target molecule for cancer therapy.
Previously, we found that LNCaP‐CR cells, a highly tumorigenic
subline of the LNCaP human prostate cancer cell line,13 express

was purified from the culture supernatant using Protein G Sepharose(GE Healthcare United Kingdom, Ltd). Purity of ch6G10A was evalu‐
ated by SDS‐PAGE and Coomassie Brilliant Blue staining.

higher levels of CAR than their parental cells.14 We also developed
mouse mAbs against human CAR and found that one of these anti‐
bodies (clone 6G10A) significantly inhibited tumor growth in xeno‐

2.3 | Flow cytometry

grafts of human prostate, pancreatic, and colorectal cancer.14 Based

Cells were fixed in 4% neutral buffered formalin for 10 minutes andon these findings, we proposed that an anti‐CAR antibody might besuspended in PBS containing 0.1% NaN3. Cells were resuspended in14

a feasible candidate for cancer immunotherapy.

PBS containing 0.5% BSA and incubated with anti‐CAR antibodies

Because the immunogenicity of antibodies needs to be reducedfor 1 hour at 4°C and then with PE‐labeled secondary antibodies

for their therapeutic use in humans, mouse‐human chimerization offor 1 hour at room temperature. After washing with PBS containing

mouse mAbs is an important step in the development of therapeutic

0.1% NaN3, cells were analyzed by FACSCalibur (BD Biosciences).

15

antibodies.

In the current study, we generated a mouse‐human chi‐meric anti‐CAR antibody (ch6G10A) from 6G10A mouse anti‐CAR
antibody (mu6G10A), and characterized ch6G10A by flow cytome‐

2.4 | Western blot analysis

try, western blotting, ADCC/CDC analyses, and in vivo anti‐tumor

Western blot analysis was carried out as described previously.19

activity against a prostate cancer cell line. In addition, we carried out

Briefly, cultured cell pellets were lysed with lysis buffer (20 mmol/L

a CTA analysis to investigate CAR expression levels in lung, prostate,

HEPES (pH 7.5), 150 mmol/L NaCl, 1% Triton X‐100, 10% glycerol,and brain tumors, and examined the anti‐tumor activities of anti‐

1 mmol/L EDTA, 50 mmol/L NaF, 50 mmol/L β‐glycerophosphate,

CAR antibodies against SCLC using mouse xenograft models.

1 mmol/L Na3VO 4, 1× cOmplete Protease Inhibitor Cocktail; Roche).
Lysates were centrifuged at 13 000 g for 15 minutes at 4°C, and the

2 | M ATE R I A L S A N D M E TH O DS
2.1 | Cell lines and reagents
KHYG‐1/FcγRIIIA cells, a human NK cell line stably expressing Fcγ

supernatants were boiled in SDS sample buffer containing 0.5 mol/L
β‐mercaptoethanol. These samples were separated by 12.5% SDS‐
PAGE and transferred to PVDF membranes (Merck KGaA). Protein
levels were detected using the following antibodies: rabbit poly‐clonal antibodies specific for CAR (H‐300/sc‐15405; Santa Cruzreceptor IIIA (FcγRIIIA), were previously established16 and were

Biochemical) and rabbit polyclonal antibodies specific for HSP90

maintained in RPMI‐1640 medium (Nissui Pharmaceutical) supple‐(H‐114/sc‐7947; Santa Cruz Biochemical).

mented with 10% FBS (PAN Biotech GmbH) and 100 units of humaninterleukin (IL)‐2 (FUJIFILM Wako Pure Chemical Corporation).
Human SCLC cell lines NCI‐H69, DMS53, and DMS114 were main‐tained in RPMI‐1640 medium containing 10% FBS. Human prostate

2.5 | Immunohistochemical analysis of cancer
tissue arrays

cancer cell line DU‐145, human SCLC cell line DMS273, GFP‐labeled

Cancer tissue arrays (BC041115c for lung cancer, BC19013 for

subline DMS273‐GFP, highly metastatic subline G3H,17 and C5B (S.

prostate cancer, and GL803b for brain cancer) were purchased

Sakamoto, H. Inoue & M. Kawada, unpubl. data, manuscript in prepa‐

from US Biomax. The arrays were deparaffinized and rehydrated.

ration) were maintained in DMEM (Nissui) containing 10% FBS.

Then, antigens were unmasked using citrate buffer for 10 minutes
at 96°C. Prior to antibody reactions, endogenous peroxidase was

2.2 | Generation of mouse‐human chimeric anti‐
human CAR
A mouse anti‐human CAR mAb, mu6G10A, was developed as de‐inactivated with 3% hydrogen peroxidase for 10 minutes at room
temperature followed by rinsing with distilled water and TBST. The
arrays were blocked with 10% horse serum for 1 hour at 37°C andincubated with primary antibody against CAR (H‐300/sc‐15405,

scribed previously.14 Generation of mouse‐human chimeric anti‐

1:750; Santa Cruz Biochemical) overnight at 4°C. After washinghuman CAR (ch6G10A) was previously described.18 Briefly, thethree times with TBST, the arrays were incubated with second‐

appropriate VH and VL cDNAs of mu6G10A and CH and CL of humanary antibody peroxidase‐conjugated anti‐rabbit IgG (ImmPRESS

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SAKAMOTO et al.

Reagent kit; Vector Laboratories) for 30 minutes at room tempera‐

and ch6G10A, mu6G10A, control human IgG, or control mouse IgG

ture. The arrays were then washed three times with TBST and de‐for 4 hours at 37°C. Cell viability was determined using MTT assay.

veloped using ImmPACT DAB (Vector Laboratories) for 1 minute,

Assays were done in triplicate at least.

and then briefly immersed in hematoxylin for counterstaining andevaluation under light microscopy. Estimated visual intensity of
CAR immunostaining was graded on an arbitrary three‐point scale:
negative (0), positive (1), strongly positive (2).

2.8 | In vivo anti‐tumor activity
Animal experiments were approved by the Institute Committee
for Animal Experiments at the Institute of Microbial Chemistry

2.6 | Antibody‐dependent cellular cytotoxicity

and carried out according to the ethics guidelines of our insti‐
tute. Female BALB/c nude mice and female SCID‐beige mice were

Antibody‐dependent cellular cytotoxicity was measured with aobtained from Charles River Japan (Kanagawa, Japan) and were

calcein AM release assay as described previously.14 Briefly, formaintained in a specific pathogen‐free barrier facility inhouse.

ADCC activity, target cells were labeled with 10 μg/mL calcein AM

Mice aged 8 weeks were used for the in vivo anti‐tumor assay.

(Dojindo) for 30 minutes at 37°C, washed three times with com‐

To prepare the subcutaneous xenograft model, 1 × 107 DU‐145

plete medium, and further incubated for 1 hour at 37°C. Labeledcells or 1 × 10 6 NCI‐H69 cells in 0.1 mL of 62.5% growth fac‐

4target cells (5 × 10 cells) and effector cells were then cocultured

tor‐reduced Matrigel (BD Biosciences) were injected s.c. into

in 96‐well plates and incubated for 4 hour at 37°C. After centrif‐

the left flanks of the SCID‐beige mice. When the tumor size had

ugation, fluorescence intensity (F) of cell‐free supernatants wasreached approximately 100 mm 3 , i.v. injection of the antibodies

measured (excitation 485 nm, emission 538 nm). Cytolytic activity

was started. To prepare the orthotopic transplanted xenograft

was calculated using the following formula:% of specific lysis = (F experiment − F spontaneous)∕(F maximal − F spontaneous) × 100.
Assays were carried out in triplicate at least.

model of the highly metastatic subline of DMS273 human SCLC
cells, 2.5 × 10 5 C5B cells were injected into the left lungs of nude
mice as described previously.17 An Olympus OV110 Small Animal
Imaging System (Olympus Corp.) was used for imaging orthotopic
and metastatic tumor formation in the orthotopic transplanted
xenograft model. Length (L) and width (W) of the subcutaneous

2.7 | Complement‐dependent cytotoxicity
4and orthotopic tumors were measured by calipers and tumor vol‐ume (TV) was calculated using the formula: TV = (L × W2)/2. All

For CDC activity, target cells (2 × 10 cells) were inoculated into 96‐

the in vivo experiments were carried out at least twice with simi‐

well plates and incubated with 5% rabbit complement (Cedarlane)

lar results.

F I G U R E 1 Generation of a mouse‐human chimeric anti‐coxsackievirus and adenovirus receptor (CAR) mAb, ch6G10A. A, Flowcytometric analysis. DU‐145 human prostate cancer cells were incubated with 10 μg/mL ch6G10A, a mouse‐human chimeric anti‐CAR
mAb and subjected to flow cytometric analysis. B, Assessment of antibody‐dependent cellular cytotoxicity activity. DU‐145 cells wereincubated with human natural killer cell line KHYG‐1/Fcγ receptor IIIA in the presence of the indicated antibodies at 100 μg/mL for 4 h.
Results are expressed as means ± SD of three independent experiments carried out in triplicate. Statistical analysis was done by Student's
t test (**P < .01 vs control). C, Assessment of complement‐dependent cytotoxicity activity. DU‐145 cells were incubated with 5% rabbit
complement in the presence of the indicated antibodies at 10 μg/mL for 4 h. Results are expressed as means ± SD of three independent
experiments carried out in triplicate. Statistical analysis was done by Student's t test (*P < .05 vs control)

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F I G U R E 2 In vivo anti‐tumor effect of chimeric anti‐coxsackievirus and adenovirus receptor mAbs on a subcutaneous transplanted
xenograft model of DU‐145 human prostate cancer cells. Effect of treatment with ch6G10A on the in vivo growth of DU‐145 cells. DU‐145
cells (1 × 107 cells) were implanted s.c. into BALB/c nude mice. Treatment started when average tumor volume had reached 100 mm3 (as day0). A total of 500 μg of ch6G10A, control human IgG or saline was injected i.v. at days 0 and 7 (indicated by arrows). Human natural killer(NK) cells KHYG‐1/Fcγ receptor IIIA (4 × 105 cells) were injected around the tumors at days 0 and 7 (indicated by arrows). A, Tumor volume.
B, Tumor weight at 11 days. Results are expressed as means ± SD (IgG control; n = 7, ch6G10A; n = 5). Statistical analysis was carried out byStudent's t test (*P < .05, **P < .01, n.s., not significant)

2.9 | Statistical analysismice) or saline was injected i.v. at days 0 and 7, and human NK cells
KHYG‐1/FcγRIIIA were also injected around the tumors at days

Results are expressed as mean ± SD. Statistical analysis was carried

0 and 7. ch6G10A significantly inhibited the growth of xenograftout by Student's t test or Mann‐Whitney U test as described in the

DU‐145 tumors in vivo (43% inhibition compared with control

figure legends. P < .05 was considered statistically significant.

human IgG, P < .01) without significant body weight loss in thehost mice (Figure 2, data not shown). These results showed that

3 | R E S U LT S
3.1 | Generation and characterization of a mouse‐
human chimeric anti‐CAR antibody (ch6G10A)
Previously, we developed a mouse mAb against human CAR(mu6G10A) that recognized an extra‐membranous region of CAR
and showed anti‐tumor activities against CAR‐expressing cancer

ch6G10A had ADCC‐/CDC‐inducing activities and in vivo anti‐
tumor activity against CAR‐expressing prostate cancer cells andthat we successfully generated a mouse‐human chimeric anti‐CAR
antibody with potent anti‐tumor activity.

3.2 | Expression of CAR in human lung,
prostate, and brain tumors

cells in xenograft models.14 To reduce the immunogenicity of an‐

Our previous results suggest that CAR is highly expressed in tumor

tibodies in humans, we attempted to generate a mouse‐humantissues such as prostate, lung, and brain.14 Because we used only

chimeric antibody (ch6G10A) by fusing the VH and VL regions ofsmall numbers of tissue sections in the previous study, we further

mu6G10A with the CH and CL regions of human IgG1, respectively.

assessed the expression of CAR in these tumors through immuno‐

As shown in Figure 1A, ch6G10A reacted with the human pros‐

histochemical staining of CTA using commercially available antibody

tate cancer cell line DU‐145, which highly expresses human CAR,

against CAR (H‐300) (Figure 3). CTA analysis showed that CAR was

as strongly as mu6G10A by flow cytometry. Because mu6G10A

considerably expressed in lung squamous cell carcinoma (19/40;

can induce both ADCC and CDC,14 we next examined whether48%), lung adenocarcinoma (14/45; 31%), lung neuroendocrine can‐ch6G10A can also induce both ADCC and CDC. When a humancer (including small cell lung cancer) (7/12; 58%), prostate adenocar‐

NK cell line, KHYG‐1/FcγRIIIA, which stably expresses FcγRIIIA

cinoma (45/56; 80%), and glioblastoma (23/39; 59%). Importantly,

was used as effector cells,16 ADCC by ch6G10A was observed

expression of CAR was significantly higher in all lung cancer types

against DU‐145 cells (Figure 1B). ch6G10A also significantly in‐than in normal lung tissue (lung squamous cell carcinoma and lung

duced CDC activity against DU‐145 cells when using rabbit com‐neuroendocrine cancer, P < .01; lung adenocarcinoma, P < .05), andplement (Figure 1C). Then, we evaluated the anti‐tumor activity of

was significantly higher in glioblastoma than in normal brain tissuech6G10A against DU‐145 cells in vivo using a subcutaneous xeno‐(P < .05). Thus, our results show that CAR was highly expressed ingraft model in nude mice. ch6G10A, control human IgG (500 μg/

these human tumor tissues.

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SAKAMOTO et al.

F I G U R E 3 Coxsackievirus and adenovirus receptor (CAR) expression in various human tumor tissues. A, CAR expression in human lung,
prostate, and brain tumors. Immunohistochemical staining was carried out on cancer tissue arrays using CAR antibody H‐300. Estimatedvisual intensity of CAR immunostaining was graded on an arbitrary three‐point scale: negative (0), positive (1), strongly positive (2). CAR,
coxsackievirus and adenovirus receptor; SCLC, small cell lung cancer. Statistical analysis was done by Mann‐Whitney U test (*P < .05,**P < .01, n.s., not significant). B‐D, Representative CAR staining in the lung cancer tissue array. B, SCLC (score 2). C, SCLC (score 1). D,
Normal lung tissue (score 0). All data were documented under a magnification of ×400

3.3 | In vivo anti‐tumor activity of ch6G10A
antibody against NCI‐H69 small cell lung cancer cells
In addition to our CTA results, other studies also showed that CAR

3.4 | In vivo anti‐tumor activity of anti‐CAR
antibody against an orthotopic transplantation
model of SCLC

was highly expressed in SCLC patients.5,8,10 Therefore, we investi‐

We previously developed an orthotopic transplantation model ofgated CAR protein levels in four human SCLC cell lines (DMS114,

SCLC using GFP‐labeled human SCLC cell line DMS273 (DMS273‐

DMS53, DMS273, and NCI‐H69) by western blot analysis, and

GFP) and two highly metastatic sublines (G3H, C5B); this model has

found that CAR was expressed in three of the four lines and wassignificant metastatic activity and comparable metastatic tropism

particularly overexpressed in NCI‐H69 cells (Figure 4A). These ex‐with that of SCLC patients.17 Because CAR expression was clearly ob‐pression data of CAR prompted us to evaluate the in vivo anti‐tumor

served in these cells (Figure 4A), we attempted to elucidate whether

activity of ch6G10A against NCI‐H69 using a subcutaneous xeno‐

anti‐CAR antibody could suppress orthotopic tumor growth and dis‐

graft model in nude mice. ch6G10A or control human IgG (500 μg/

tant metastasis formation in the model using C5B cells. Owing tomice) was injected i.v. at days 0 and 7, and human NK cells KHYG‐1/

the difficulty of giving systemic human NK cells to xenograft mice,FcγRIIIA were injected around the tumors at days 0 and 7. As shown

which is essential for the anti‐tumor activity of ch6G10A, we usedin Figure 4B,C, ch6G10A significantly inhibited the growth of xeno‐

mouse monoclonal CAR antibody mu6G10A in this experiment.

graft NCI‐H69 tumors in vivo (37% inhibition compared with control

Intravenous injection with mu6G10A or control mouse IgG (250 μg/

human IgG, P < .01) without significant body weight loss in the hostmice) was started a day after orthotopic transplantation of C5B cells.

mice (data not shown). These results showed that ch6G10A had anti‐

The injection was carried out once a week for 5 weeks. Orthotopic

tumor activity against CAR‐expressing SCLC cells and suggested

tumor growth was prominently inhibited by mu6G10A (78% inhibi‐that SCLC might be a candidate disease for targeting therapy to CARtion compared with control mouse IgG, P < .005) without signifi‐

with therapeutic antibody.

cant body weight loss in the host mice (Figure 5, data not shown).

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F I G U R E 4 In vivo anti‐tumor effect of chimeric anti‐coxsackievirus and adenovirus receptor (CAR) mAb on a subcutaneous transplanted
xenograft model of NCI‐H69 human small cell lung cancer (SCLC) cells. A, CAR expression in human SCLC cell lines. Whole‐cell lysates(20 μg) of indicated cell lines were separated by 12.5% SDS‐PAGE and analyzed by western blotting using CAR antibody H‐300. HSP90 wasused as a loading control. CAR, coxsackievirus and adenovirus receptor; B‐C, Effect of treatment with ch6G10A on the in vivo growth ofNCI‐H69 cells in a subcutaneous transplanted xenograft model. NCI‐169 cells (1 × 106 cells) were transplanted s.c. into BALB/c nude mice.
Treatment started when average tumor volume had reached 100 mm3 (as day 0). Total of 500 μg of ch6G10A or control human IgG was
injected i.v. at days 0 and 7 (indicated by arrows). Human natural killer cells KHYG‐1/Fcγ receptor IIIA (4 × 105 cells) were injected aroundthe tumors at days 0 and 7 (indicated by arrows). B, Tumor volume. C, Tumor weight at 11 days. Results are expressed as means ± SD (IgG
control; n = 14, ch6G10A; n = 10). Statistical analysis was carried out by Student's t test (*P < .05, **P < .01)
the mu6G10A‐treated group compared with the control mouse IgG‐
treated group (P < .05; Figure 5B). Thus, treatment with anti‐CAR
antibody efficiently inhibited tumor growth in the lung as well as me‐tastasis of SCLC. These results support the notion that SCLC might
be a potential disease for targeting therapy to CAR with therapeutic
antibody.

4 | D I S CU S S I O N
In the present study, we successfully generated a mouse‐human chi‐meric anti‐CAR monoclonal antibody, ch6G10A, which has ADCC‐
and CDC‐inducing activities and in vivo anti‐tumor activity against
F I G U R E 5 In vivo anti‐tumor effect of mouse coxsackievirus
and adenovirus receptor (CAR) mAb on an orthotopic transplanted
xenograft model of the highly metastatic subline of DMS273 humansmall cell lung cancer (SCLC) cells. A‐B, Effect of treatment with
mu6G10A on the in vivo growth of C5B cells, the highly metastatic
subline of DMS273, in an orthotopic transplanted xenograft model.
C5B cells (2.5 × 105 cells) were orthotopically transplanted into thelungs of BALB/c nude mice (day 0). Total of 250 μg of mu6G10A
or control mouse IgG was injected i.v. once a week from day 1 for
5 weeks. Mice were killed and analyzed on days 30‐39. A, Orthotopic
tumor volume at the time of death. B, Distant metastatic formation.
Dotted lines show the means of the number of metastasis‐positive
organs of each group. Percentages show distant metastasis incidence.
Results are expressed as means ± SD (n = 9). Statistical analysis was
carried out by Student's t test (*P < .05, ***P < .005 vs control)

CAR‐expressing cancer cells. In addition, CTA analysis showed that
CAR is highly expressed in neuroendocrine lung cancer including
SCLC, and treatment with anti‐CAR antibody inhibited both subcu‐
taneous and orthotopic tumor growth in mouse xenograft models ofhuman SCLC cell lines. These results suggest that targeting therapy
to CAR with therapeutic antibodies might be effective against sev‐
eral cancers including SCLC.
Our CTA analysis showed significantly higher expression of
CAR in neuroendocrine lung cancers including SCLC compared withnormal lung tissues (Figure 3), consistent with the findings of pre‐vious studies.5,8,10 Furthermore, treatment with anti‐CAR antibody
effectively inhibited both in vivo tumor growth (subcutaneous andorthotopic) and distant metastasis formation in mouse xenograftmodels of human SCLC cell lines (Figures 4 and 5). SCLC accounts for

Incidence of distant metastases formation was 67% in the control

10%‐20% of all cases of lung cancer worldwide and shows aggres‐mouse IgG‐treated group and 33% in the mu6G10A‐treated group,

sive growth and early metastatic spread. The current main treatment

and significantly fewer metastasis‐positive organs were observed inagainst SCLC is chemotherapy but the high chance of disease relapse

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SAKAMOTO et al.

after chemotherapy results in poor median survival. 20 Therefore,

evaluations of the 6G10A‐based antibody using animal models suchnovel therapeutic strategies against SCLC are urgently needed. CAR

as primates or human CAR knock‐in mice are required. If the reac‐

expression data in SCLC and our current results with anti‐CAR anti‐

tivity of 6G10A to normal tissues causes severe problems, cancer

body collectively suggested that therapeutic antibodies against CAR

specificity of the antibody should be improved.

might be an alternative strategy for SCLC treatment. Our CTA analy‐

In conclusion, our studies and other reports collectively suggest

sis also showed significantly higher expression of CAR in lung squa‐that CAR should be considered a candidate target for cancer im‐

mous cell carcinoma, lung adenocarcinoma, and glioblastoma than inmune therapy against several cancers including SCLC.

matched adjacent normal tissues. Because there are many cases ofthese diseases, validation of the anti‐tumor activity of our anti‐CAR
antibody will be meaningful for future investigations.

AC K N OW L E D G M E N T S

Our previous study showed that mu6G10A exerts anti‐tumor

We thank T. Ohishi, A. Harakawa, and Y. Kohda (BIKAKEN) for

activity primarily through both ADCC and CDC activities.14 In thishelpful discussions and their technical assistance. This work wasstudy, we generated ch6G10A with the human IgG1 constant region

supported by grants from the Japan Society for the Promotion of

because human IgG1 exerts higher ADCC/CDC activities among theScience (26460481 and 17K08777) (S.S.) and from the Kobayashi

IgG1 subclass. Therefore, it is presumed that ch6G10A also exerts its

Foundation for Cancer Research (M.K.). This work was also sup‐anti‐tumor activity primarily through both ADCC and CDC. If this isported by the Platform Project for Supporting Drug Discovery

the case, the anti‐tumor activity of ch6G10A could be enhanced by

and Life Science Research (Basis for Supporting Innovative Drug

ADCC‐enhancing technologies such as afucosylation of the Fc do‐

Discovery and Life Science Research [BINDS]) from AMED undermain. 21 Thus, ch6G10A could be a potent lead antibody for cancer

Grant Number JP18am0101078 (Y.K.). We also thank H. Nikki

immunotherapy.
We showed that the treatment with mu6G10A efficiently inhib‐

March, PhD, from Edanz Group (www.edanz​editi​ng.com/ac) for ed‐iting a draft of this manuscript.

ited tumor growth in the lung as well as metastasis in the SCLC met‐
astatic model (Figure 5). Although it is very important as to whether
ch6G10A also inhibits SCLC metastasis, there are no suitable met‐
astatic models for the evaluation of chimeric mAb at this point.

D I S C LO S U R E
Authors declare no conflicts of interest for this article.

However, there is an interesting humanized mouse strain, NOG‐IL‐2
Tg, which can induce human NK cells from hematopoietic stem cells(HSC). 22 The report suggested that chimeric anti‐human CCR4 mAb

ORCID(Poteligeo, Kyowa‐Kirin, Tokyo, Japa) suppressed tumor growth of

Yukinari Katohuman lymphoma cells in human HSC‐transferred NOG‐IL‐2 Tg mice

Manabu Kawada

through ADCC. If SCLC metastatic models can be generated using

https://orcid.org/0000-0001-5385-8201
https://orcid.org/0000-0001-9348-4240

mice, evaluation of the effects of ch6G10A in SCLC metastasis might
be possible.
Another important point about the result of Figure 5 is themechanism by which anti‐CAR mAb inhibited SCLC metastasis. It
is still unclear whether anti‐CAR mAb directly reacts with tumor
cells in blood/lymph vessels and metastatic sites. However, as aresult of technical limitations of our model, it is very difficult toanswer this question. Because we use fluorescence imaging tochase the metastatic tumor in our model, killing and dissection ofmice are needed for quantitative detection of metastatic tumors.
This makes it difficult to start treatment with antibody after con‐firming formation of metastatic tumors in the model. In addition,
enucleation of the orthotopic tumor in lung will result in deathof the model mouse. This also makes it difficult to examine thedirect effect of anti‐CAR mAb on tumor cells in metastatic sitesof the model.
One of the major points for the clinical safety of therapeutic an‐tibodies is reactivity with normal tissues. We previously showed thatmu6G10A reacts with normal skin, prostate, and kidney tissue.14 At
this point, we could not avoid the possibility that the reactivity of
6G10A to normal tissues could cause some adverse events when
the 6G10A‐based antibody is given to humans. Thus, further safety

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How to cite this article: Sakamoto S, Inoue H, Kaneko MK,
et al. Generation and evaluation of a chimeric antibody against
coxsackievirus and adenovirus receptor for cancer therapy.
Cancer Sci. 2019;110:3595–3602. https​://doi.org/10.1111/
cas.14196​