A T-cell engaging B7-H4/CD3-bispeciﬁc Fab-scFv Antibody ... · B7-H4/CD3 antibody was injected intravenously and imaging was performed at sequential timepoints ranging from 24 hours

Translational Cancer Mechanisms and Therapy

A T-cell–engaging B7-H4/CD3-bispecificFab-scFv Antibody Targets HumanBreast CancerAkira Iizuka1, Chizu Nonomura1, Tadashi Ashizawa1, Ryota Kondou1, Keiichi Ohshima2,Takashi Sugino3, Koichi Mitsuya4, Nakamasa Hayashi4, Yoko Nakasu4, Kouji Maruyama5,Ken Yamaguchi6, and Yasuto Akiyama1,4

Abstract

Purpose: The B7 homolog 4 (B7-H4,VTCN1) is an immunecheckpoint molecule that negatively regulates immuneresponses and is known to be overexpressed in many humancancers. Previously, we generated a mouse anti-human B7-H4mAb that did not have a significant antitumor effect in vivoprobably because of molecule instability. In this study, wedesigned a B7-H4/CD3-bispecific antibody (BsAb) and inves-tigated its antitumor activity in vitro and in vivo using ahumanized mouse model.

Experimental Design: cDNAs of the antibody-bindingfragment (Fab)–single-chain variable fragment (scFv) andscFv-scFv of the anti-B7-H4/CD3 BsAb were synthesized, andthe BsAb antibodies were produced in HEK293 cells. Theantitumor activity against humanbreast cancer cells by humanperipheral blood mononuclear cells (hPBMC) with BsAb was

measured by lactate dehydrogenase release in vitro, and in vivousing hPBMC-transplanted MHC class I- and class II–deficientNOG mice.

Results: hPBMCs with anti-B7-H4/CD3 BsAbs successfullylysed the human breast cancer cell line MDA-MB-468 (EC50:0.2ng/mL) andotherB7-H4þ cell lines in vitro.WhenBsAbwasinjected in a humanized mouse model, there was an imme-diate and strong antitumor activity against MDA-MB-468,HCC-1954, and HCC-1569 tumors and CD8þ and granzymeBþ CTL infiltration into the tumor, and there were no adverseeffects after long-term observation. CD8þ T-cell depletion byan anti-CD8 antibody mostly reduced the antitumor effect ofBsAb in vivo.

Conclusions: An anti-B7-H4/CD3 BsAb may be a goodtherapeutic tool for patients with B7-H4þ breast cancers.

IntroductionBecause of the recent success of immune checkpoint–blocking

antibodies, clinical trials are underway to evaluate their efficacy invarious cancers (1–4).However, amajority of patientswith cancerare unlikely to benefit from anti- programmed death-1 (PD-1)/PD-ligand 1 (PD-L1) antibody treatment because the responserate is approximately 20%–40% even in PD-L1þ cancers.

In addition to these immunomodulatory receptor blockadetherapies, other modulating technologies have been devel-oped (5, 6). MHC and T-cell receptor (TCR)-bypassed T-cellcytotoxicity was first reported in 1985 (7), and over the past 3

decades, CD3 bispecific antibodies (BsAb) and chimeric antigenreceptor (CAR) T cells have been developed (8–10).

Recently, two BsAbs catumaxomab (11) and blinatumo-mab (12, 13) were approved by the FDA, andmore BsAbs directlyengaging immune cells against tumor cells are now in clinicalstudies. Improvements in protein engineering technology haveenabled the creation of various types of artificial antibodies withgreater flexibility in design, size, specificity, half-life, and distri-bution, and dozens of BsAb formats have been proposed (6, 14).

B7 homolog 4 (B7-H4, VTCN1) is considered to be a negativeregulator of immune responses and is overexpressed in manyhuman cancers, which indicates that B7-H4 might be a potentialtarget for cancer therapy. B7-H4 expression is reported to beaffected by the tumor microenvironment (15, 16). Previously,we generated an anti-human B7-H4 mAb that induced T-cellcytotoxicity to a B7-H4þ breast cancer cell line in an in vitroindirect ADCC system, but the antibody did not suppress thetumor growth in a mouse model (17).

In this study, wemanufactured B7-H4/CD3BsAbs based on theantibody-binding fragment (Fab) and single-chain variable frag-ment (scFv) structure of anti-B7-H4 and anti-CD3 mAbs using ahuman cell–based protein expression system. We found that theanti-B7-H4/CD3 Fab-scFv antibody had potent cytotoxic activityagainst a B7-H4þ breast cancer cell line in vitro, and in vivo using aMHC-double knockout (dKO)NOGmousemodel (18).Here, werevealed that the anti-B7-H4/CD3 BsAb might contribute to thedevelopment of novel therapeutic antibodies against solidtumors.

1Immunotherapy Division, Shizuoka Cancer Center Research Institute, Shizuoka,Japan. 2Medical Genetics Division, Shizuoka Cancer Center Research Institute,Shizuoka, Japan. 3Division of Pathology, Shizuoka Cancer Center Hospital,Shizuoka, Japan. 4Division of Neurosurgery, Shizuoka Cancer Center Hospital,Shizuoka, Japan. 5Experimental Animal Facility, Shizuoka Cancer CenterResearch Institute, Shizuoka, Japan. 6Office of the President, Shizuoka CancerCenter Hospital, Shizuoka, Japan.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Yasuto Akiyama, Shizuoka Cancer Center ResearchInstitute, 1007 Shimonagakubo, Sunto-gun, Shizuoka 411-8777, Japan.Phone: 815-5989-5222, ext. 5330; Fax: 815-5989-6085; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-17-3123

�2019 American Association for Cancer Research.

ClinicalCancerResearch

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Published OnlineFirst February 8, 2019; DOI: 10.1158/1078-0432.CCR-17-3123

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Materials and MethodsGene expression profile analysis

A comprehensive gene expression analysis was performed aspreviously described in the HOPE (High-tech Omics-basedPatient Evaluation) project at the Shizuoka Cancer Center(Shizuoka, Japan; ref. 19). Briefly, the tumor tissue samples weredissected from fresh surgical specimens, and the RNA sampleswith an RNA integrity number�6.0 were used for the microarrayanalysis. RNA was amplified, labeled, and hybridized to the SurePrintG3HumanGene Expression 8� 60K v2Microarray (AgilentTechnologies) and Microsoft Excel software. Data analysis wasperformed using GeneSpring GX Software (Agilent Technolo-gies). Ethical approval for the study was obtained from theinstitutional review board at the Shizuoka Cancer Center(Shizuoka, Japan). Written informed consent was obtained fromall the enrolled patients. All the experiments using clinical sam-ples were carried out in accordance with the approved guidelines.

Cell linesHuman breast cancer cell lines (MDA-MB-468, MDA-MB-231,

ZR75, SKBR3, HCC-1954, and HCC-1569) and the lung adeno-carcinoma cell line (NCI-H2170) were purchased from the ATCC,and were maintained in RPMI1640 (Sigma) supplemented with10% FBS (Thermo Fisher Scientific). Humanmammary epithelialcells (HMEC) were purchased from Lonza Ltd., and were culturedin the growth medium MEBM-CC3150 (Lonza Ltd.).

Flow cytometry and antibodiesThe mouse anti-human B7-H4 antibody (clone #25) was

established in-house as described previously (17). Briefly, thehuman B7-H4 isoform 1 extracellular domain was constructedand produced in the Expi293 Expression System (Life Technol-ogies) and was immunized in BALB/cA mice. An antibody secret-ing hybridoma was generated and was screened by a commonmethod using the mouse myeloma cell line P3 � 63ag8.653(ATCC). The flow cytometric analyses were carried out on FACSCanto (BD Biosciences). HMECs and cancer cell lines were incu-bated with anti-B7-H4mAb (clone #25) and were later incubated

with a PE-labeled Polyclonal anti-mouse Ig Ab (BD Biosciences)on ice. The following antibodies were used for flow cytometricanalysis of the in vivo experiments using humanized mice. Forhuman cell labeling, anti-CD3-PerCP (HIT3a), anti-CD4-PE oranti-CD4-PE-Cy5 (RPA-T4), anti-CD8-PE-Cy5 (HIT8a), anti-CD11b-PE-Cy7 (ICRF44), anti-CD14-PerCP (MfP9), anti-CD19-APC (HIB19), anti-CD25-FITC (M-A251), anti-CD33-PE(WM53), anti-CD45-FITC (2D1), anti-CD45RA-FITC (HI100),anti-CD45RO-APC (UCHL1), anti-CD56-PE (B159), and anti-CD127-PE-Cy7 (A019D5) were purchased from BD Pharmingen.The anti-FoxP3-PE (hFOXY) antibody was purchased fromeBioscience, Inc. The anti-TIM3-PE (F38-2E2) and anti-LAG3-FITC (17B4) antibodies were purchased from Miltenyi BiotechandAdipoGen Life Sciences. The anti-mouseCD45 antibody usedto label themouse cells was purchased from BD Pharmingen. Theanti-PD-1-APC (EH12.2H7) and anti-Ki67-PE-Cy7 (Ki-67) anti-bodies were purchased from BioLegend. Splenocyte and periph-eral blood cells were isolated using ACK lysis buffer. Tumor-infiltrating lymphocytes (TIL) were also separated from the con-trol or antibody-treated tumors by anti-humanCD45-microbeads(Miltenyi Biotec) using autoMACS System (Miltenyi Biotec). Thestainingmethodwas described previously (16).Human cellswereidentified by gating human CD45þ fractions.

Production of the B7-H4/CD3 BsAbThemouse anti-human B7H4mAb clone #25–derived VH and

VL genes were cloned, and construct containing Fab (B7-H4)-scFv(CD3) and scFv (B7-H4)-scFv (CD3) linked by a (Gly4Ser)3linker and 6� histidine-tag was designed (Fig. 2A). These cDNAswere chemically synthesized and cloned into the expression vectorpcDNA3.3. The B7-H4/CD3 BsAbs were produced using theExpi293 Expression System (Gibco, Thermo Fisher Scientific) ata ratio of 3:7 (VH-containing long fragment:VL-containing shortfragment) in the Fab-scFv format, purified with a histidine tagaffinity column, and used for experiments.

In vitro BsAb/hPBMC cytotoxicity assayhPBMCs were isolated from the peripheral blood of healthy

volunteers or patients with glioma as effector cells using Ficoll-PaquePLUS (GEHealthcareUKLtd). Effector cellswere incubatedwith cancer cell lines or HMEC at an effector/target (E/T) ratioranging from 1.25 to 40 in the presence of various concentrationsof Fab-scFv or scFv-scFv B7H4/CD3 BsAb at 37�C for 16 or 24hours in 5% CO2 atmosphere. The supernatant from the cultureswere collected and measured using a Lactate DehydrogenaseCytotoxicity Assay Kit (Takara Bio Inc.). The percentage of specificlysis was determined by the following formula: percentage ofspecific lysis ¼ [(effector cells and target cells and agent release –effector cells release) – spontaneous target cell release]/(maximaltarget cells release� spontaneous target cells release)� 100. EC50

was calculated using a 4-parameter logistic curve fitting usingImageJ (ver. 1.51J8, NIH, Bethesda, MD). The effector T-cellsubsets were isolated or depleted from the healthy volunteerPBMCs by an autoMACSMagnetic Cell Isolator (Miltenyi Biotec),and secreted Granzyme B and IFNg in culture supernatants weremeasured by an ELISA Kit (Mabtech AB, BioLegend).

Animal experimentsThe MHC-dKO NOG mice were kindly supplied by Dr.

Mamoru Ito at the Central Institute for Experimental Animals(Kawasaki, Japan). All the animals were cared for and treated

Translational Relevance

Despite the clinical success of immune checkpoint blockadeantibodies against advanced cancers, the response rate isapproximately 20%–40% even for PD-L1þ cancers and mostpatients with cancer are unlikely to benefit from treatment.Other therapeutic antibodies have been developed and eval-uated in clinical trials in combination with anti-PD-1 anti-bodies. In this study, we manufactured anti-B7-H4/CD3 bis-pecific antibodies (BsAb) based on the Fab and scFv structure.We found that the B7-H4/CD3 BsAb had potent cytotoxicactivity against B7-H4þ breast cancer cell lines in vitro, andin vivo using a MHC-double knockout NOG mouse model.Because B7-H4 is highly expressed independently of HER2 orPD-L1 expression in breast cancers obtained by the High-techOmics-based Patient Evaluation for Cancer Therapy project,the B7-H4/CD3 BsAb may be a good therapeutic tool forimmune checkpoint blockade or anti-HER2 antibody unre-sponsive patients with cancer.

Iizuka et al.

Clin Cancer Res; 25(9) May 1, 2019 Clinical Cancer Research2926




according to the Guidelines for the welfare and use of animals incancer research, and the experimental procedures were approvedby the Animal Care and Use Committee of Shizuoka CancerCenter Research Institute (Shizuoka, Japan). Clinical experimentsusing the PBMCs derived from patients with glioma and healthyvolunteers were approved by the Institutional Review Board ofShizuoka Cancer Center (Shizuoka, Japan). All the patients pro-vided written informed consent.

In vivo imaging in the tumor-transplantedNOG-MHCdKOmiceFor in vivo imaging, all the tumor-transplanted MHC-dKO

NOGmice were supplied with a low fluorescence feed for morethan 1 week. Cy5.5 labeling of the Fab anti-B7-H4/CD3 anti-body was performed using a Cy5.5 Labeling Kit (GE HealthcareUK Ltd). Cy5.5-labeled Fab-scFv B7-H4/CD3 BsAb localizationwas performed using the Optix MX2 Laser Scanner System(Advanced Research and Technologies) with excitation at670 nm and emission at �700 nm. The Cy5.5-labeledB7-H4/CD3 antibody was injected intravenously and imagingwas performed at sequential timepoints ranging from 24 hoursto 28 days.

BsAb pharmacokinetics in the BALB/cA miceIn the pharmacokinetic study of anti-B7-H4/CD3 BsAb,

5 9-week-old BALB/cA mice were injected with 100 mg of

Cy5.5-labeled Fab-scFv anti-B7-H4/CD3 BsAb via tail vein, andthen blood was drawn at timepoints ranging from 2 minutes to48 hours after the antibody injection. Serum samples werestored at �80�C until the BsAb concentrations were measuredby a sandwich ELISA or fluorescence intensity. Sandwich ELISAwas performed using the recombinant B7-H4 extracellularregion and horseradish peroxide–labeled polyclonal anti-human Ig antibody (GE Healthcare). Serum BsAb concentra-tion was also determined by fluorescence intensity levels usingthe Optix MX2 imager and was performed using the ART OptixOptiview Software (Advanced Research and Technologies).

In vivo study using humanized mouse modelHumanized MHC-dKO NOG mice production method was

reported previously (20). Briefly, 8-week-old MHC-dKO NOGmice were irradiated with X-rays and 1 � 107 hPBMCs frompatients with glioma were intravenously administered to eachmouse via tail vein. The study design for the experiment evalu-ating the mice treated with Fab-scFv B7H4/CD3 BsAb is shownin Figs. 5 and 6 and Supplementary Fig. S5. Four in vivo experi-ments were performed (dose-response, short- and long-termantitumor effect evaluation, and T-cell subset depletion). Specif-ically, we set the starting day of the antibody injection as day 0.As shown in Fig. 5A (the short-term antitumor effect experiment),on day -14, 1 � 106 MDA-MB-468 human breast cancer cells

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Expression of the B7-H4 gene inbreast and gynecologic cancertissues. A, A comprehensive DNAmicroarray analysis was performedusing 2,527 surgically resectedcancer tissue samples, and the majorcancer categories are indicated. Thebox plots of log2-normalized valuesof VTCN1 (B7-H4) expression aredisplayed. The data analysis wasperformed using Microsoft Excel2016 software. B, The B7-H4expression in the HMECs and variouscancer cell lines was determined byflow cytometry. An in-houseanti-B7-H4mAb (clone #25)was used.

B7-H4/CD3-bispecific Antibody Targeting Breast Cancer

www.aacrjournals.org Clin Cancer Res; 25(9) May 1, 2019 2927




(B7-H4þ) or NCI-H2170 lung adenocarcinoma cells (B7-H4�)were subcutaneously injected into the fat pad or flank region ofthe mice. Starting on day 0, each antibody was administeredintravenously. The antitumor activity was evaluated bymeasuringthe tumor volume. Tumor volume was calculated based on theNCI formula as follows: tumor volume (mm3)¼ length (mm)�[width (mm)]2 � 1/2.

One week after the antibody injection, tumors, spleens, andblood were harvested from the groups. The tumors from one setof 3 mice were used for TIL flow cytometry, IHC analysis, andqRT-PCR of the immune response–associated genes. The schemafor the long-term antitumor effect evaluation and T-cell subsetdepletion experiment are shown in Fig. 5B andC. For T-cell subsetdepletion in vivo, anti-CD4 and anti-CD8 mAbs were purchasedfrom Bio X Cell.

In the dose–response experiment, as shown in Fig. 6, human-ized MDA-MB-468 tumor-transplanted mice were administeredwith BsAb sequentially in a dose-escalatingmanner (0.2–200 mg).Two weeks after the start of antibody injection, the tumors wereresected and used for IHC analysis.

Additional in vivo experiment targeting B7-H4þ breast cancercell lines, such as HCC-1954 and HCC-1569, using the human-izedMHC-dKONOGmice was performed in the samemethod asshown in Supplementary Fig. S5C.

IHCThe xenografts were harvested 1 or 2 weeks after the injection

of anti-B7-H4/CD3 BsAb. Formalin-fixed, paraffin-embeddedtissue blocks and sections were made. anti-B7-H4 antibody(clone #25 in-house made; ref. 17), anti-CD8 (C8/144B), andanti-CD4 (4B12) antibodies (Thermo Fisher Scientific), anti-granzyme B antibody (GrB-7, Dako), anti-FoxP3 antibody(236A/E7, Abcam), anti-CD204 antibody (SRA-C6, TransGenicInc.), and anti-PD-L1 antibody (28–8, Abcam) were purchased

and used for IHC analysis. Positively stained cell frequency wascounted using the image-analyzing software, ImageJ (NIH,Bethesda, MD) in randomly selected 1/3 areas of a tumorsection whole image at 200 � magnification. The necrotic areawas excluded.

Human breast cancer paraffin-embedded tissue arrays werepurchased from US Biomax, Inc., (catalog no BR1503f) andhuman normal and tumor paraffin tissue arrays were purchasedfrom BioChain Institute Inc., and were used for IHC study usinganti-B7-H4 mAb #25.

Statistical analysisFor in vivo studies, the intergroup differences were assessed by

two-way ANOVA with Shirley–Williams test. Significant differ-ence in the positive cell frequency by IHC was assessed usingtwo-tailed unpaired Student t test. The correlation betweendifferent gene expression levels was analyzed using a Spearmancoefficiency test. P ¼ <0.05 was considered significant.

ResultsB7-H4 expression in cancer tissues or cell lines

High expression of B7-H4 was frequently observed in breastand ovarian cancer and was partially observed in lung cancersfrom the HOPE project using 2,527 surgically resected tumortissues (Fig. 1A; Supplementary Fig. S1A). The positive rate ofB7-H4 mRNA expression in breast cancers (more than 2-foldupregulation in tumors compared with normal tissues) was 56%.In contrast, PD-L1 expressionwas low inbreast andovarian cancertissues. B7-H4 mRNA expression was detected in various types ofcell lines, especially in breast cancer cell lines (Supplementary Fig.S2A) by qPCR, and B7-H4 protein expression was detected in43.5% of breast cancer tissues by IHC using breast tumor tissuearray (Supplementary Fig. S7). B7-H4 cell surface expression was

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Design and production of theanti-B7-H4/CD3 BsAbs inFab-scFv and scFv-scFv formats.A,Molecular design of the BsAbs;Fab (anti-B7-H4)-scFv (anti-CD3)construct consisting of mouse anti-human B7H4mAb clone #25-derived VH and VL genes andhuman immunoglobulin constantregion domain sequences linked by(Gly4Ser)3 linker sequence tothe anti-human CD3 antibody VHand VL genes. B, Coomassieblue–stained SDS-PAGE of purifiedanti-B7-H4/CD3 BsAbs, Fab-scFvcontaining a single-short chain(30 kDa) and single-long chain(60 kDa), and scFv-scFvcontaining a single chain (60 kDa).C, Characterization of the Fab-scFvanti-B7-H4/CD3 BsAb. The bindingactivity of Cy5.5-labeled Fab-scFvanti-B7-H4/CD3 BsAb to theB7-H4þbreast cancer cell line wasevaluated by flow cytometry.Gray-dotted line, no antibody;black thick line, Cy5.5-labeledanti-B7-H4/CD3 BsAb.

Iizuka et al.





observed on breast cancer cells and not on HMEC (Fig. 1B;Supplementary Fig. S2C and S2D).

Unstable B7-H4 surface expression was suggested in somereports (21, 22). For example, breast cancer cell line SKBR3changed B7-H4 surface expression under confluent culture con-ditions (Supplementary Fig. S2B).

Generation of anti-B7-H4/anti-CD3 BsAbsTwo types of recombinant BsAbs were constructed from the

novel anti-human B7-H4 mAb clone #25 (17) and classicalanti-CD3 antibody (clone: OKT3). The anti-B7-H4/CD3Fab-scFv format that connected the antigen-binding fragment(Fab), including the human IgG4 consensus region 1, with thescFv was constructed by a single-short chain and a single-longchain (Fig. 2A and B). Therefore, 90 kDa molecular size was

larger than albumin, which contributes to the prevention ofrenal leakage and to the stabilization of anti-B7-H4 affinity. Theanti-B7-H4/CD3 scFv-scFv single-chain bispecific format wasconstructed in a manner similar to BiTE (23). The Fab-scFvanti-B7-H4/CD3 BsAb labeled with Cy5.5 showed positivestaining for B7-H4 in positive breast cancer cell lines by flowcytometry (Fig. 2C).

PBMCs with anti-B7-H4/CD3 BsAb show cytotoxic activityagainst a B7-H4þ breast cancer cell line in vitro

BsAb-mediated crosslinking of B7-H4 on the target cell surfacewith CD3 on T cell causes effector T-cell–dependent lysis ofthe target. The cytotoxic activity of the B7-H4/CD3 BsAbs withhuman PBMCs against MDA-MB-468 cells for scFv-scFv was67 ng/mL (EC50). For Fab-scFv, it was 12 ng/mL after 16 hours,

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Cytotoxic activity of the anti-B7-H4/CD3 BsAb against breast cancer cell lines.A, EC50 of each BsAb for the cytotoxic activity against breast cancerMDA-MB-468 cells is shown. Open circle, scFv-scFv 67 ng/mL; closed circle, Fab-scFv 13 ng/mL; and closed triangle, Fab-scFv 0.2 ng/mL. Effector humanPBMCs were derived from one representative case of 3 healthy volunteers and were used at an E/T 40. Human PBMCs from a patient with glioma, which werealso used for in vivo experiment, were used for a cytotoxicity assay (data not shown). The EC50 values were calculated using ImageJ. B, Cytotoxic activity of anti-B7-H4/CD3 BsAb against B7-H4-positive or -negative cancer cell lines. The E/T ratio was set at 40. Square, ZR75; cross, MDA-MB-231; triangle, SKBR3; diamond,NCI-H2170. C, Cytotoxic effect of the anti-B7-H4/CD3 BsAb against HMECs. PBMCs derived from 3 different healthy volunteers were used as effector cells.Closed marker, MDA-MB-468; open marker, HMEC. Each point shows the average of two experiments from 1 volunteer. The cytotoxic activity of anti-B7/CD3BsAb in various E/T ratios using Fab-scFv (D) and scFv-scFv (E) against MDA-MB-468. From the top line, E/T ratios of 20, 10, 5, 2.5, 1.25, and target cells alone areshown. F, Cytotoxicity assay against MDA-MB-468 using positively or negatively MACS-isolated T-cell subsets. The data are representative of three independentexperiments with each volunteer T-cell subsets at an E/T of 10 for 16 hours.






and for Fab-scFv it was 0.23 ng/mL after 24 hours in vitro (Fig. 3A).The Fab-scFv anti-B7-H4/CD3 antibody showed antibody-depen-dent cytotoxicity on B7-H4þ cells, whereas no cytotoxic activitywas seen against B7-H4� cancer cell lines (Fig. 3B; SupplementaryFig. S5B).

There was no significant difference in cytotoxic activity(EC50 value) against MDA-MB-468 cells between healthyvolunteer–derived PBMCs and glioma patient–derived PBMCs(data not shown). The anti-B7-H4/CD3 BsAb and volunteerPBMCs showed no cytotoxic effect on normal HMEC(Fig. 3C). The BsAb did not kill target cells without PBMCs(Fig. 3D and E). The effector cell killing activity was elicited byBsAb at a dose above 1 ng/mL against MDA-MB-468 cells after16 hours in vitro, and a decrease in the killing activity wasobserved under high concentrations of BsAb (Fig. 3C–E),which might be caused by a decrease in crosslinking of thetarget molecules because of BsAb saturation. Unexpectedly,positively isolated CD4þ as well as CD8þ T-cell subsets

showed strong cytotoxicity against B7-H4þ tumor cell lineswith granzyme B and IFNg secretion by the stimulation ofBsAb. Interestingly, even CD4 and CD8 double–negativeT cells showed a weak cytotoxicity. However, granzyme Band IFNg were not produced (Fig. 3F; Supplementary Fig.S3A and S3B).

Anti-B7-H4/CD3 BsAb pharmacokinetics in vivoBsAb accumulation at the MDA-MB-468 tumor site occurred

within 24 hours after the antibody injection, and the signalwas detected even 28 days after the antibody injection.In contrast, specific antibody accumulation was not recognizedin B7-H4� NCI-H2170 tumor (Fig. 4A and B; SupplementaryFig. S4). The BsAb concentration in the serum was determinedby a recombinant B7-H4 and an anti-human-IgG-Ab sandwichELISA, and the T1/2-beta was 8.5 hours. Twenty-fourhours after the 100 mg/body BsAb injection, the serum con-centration was estimated at 0.1 mg/mL (Fig. 4C), but the serum

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Cy5.5-labeled anti-B7-H4 BsAb (Fab-scFv) localization in tumor-bearing mice and BsAb half-life in mouse blood. A, Sequential fluorescence imaging of theMDA-MB-468 tumor-bearing MHC-dKO NOGmouse after Cy5.5-labeled BsAb intravenous injection from 24 hours to 28 days.B, Fluorescence imaging of theNCI-H2170 tumor-bearing MHC-dKO NOGmouse at 24 hours after the BsAb injection. C, Fluorescence imaging in the resected organs, including theMDA-MB-468 tumor on day 28 after Cy5.5-labeled BsAb injection (200 mg/body). D, Serum BsAb concentration after a 100 mg injection into 5 BALB/cAmice.Blood was sequentially collected for 2 minutes to 48 hours after the injection to measure serum BsAb concentration. Each point represents the mean� SD of5 mice (i.v., intravenous injection).

Iizuka et al.





concentration by fluorescent imaging of the Cy5.5-labeledBsAb was approximately 1 mg/mL after 24 hours (Supplemen-tary Fig. S4), which was higher than the antibody levelobtained by the sandwich ELISA method. The BsAb in thesera may partially exist in complex with other proteins.

Antitumor effect of the anti-B7-H4/CD3 BsAb againstMDA-MB-468 and other breast tumors

A single injection of Fab-scFv B7-H4/CD3 BsAb, triple-neg-ative breast cancer (TNBC) MDA-MB-468 xenograft tumorsdecreased in size by about 70% in hPBMC-transplantedhumanized NOG mouse model in a week (Fig. 5A). In thelong-term experiment, the reduction in tumor size was main-

tained until 2 weeks after the BsAb injection (Fig. 5B). Incontrast, the growth of the tumors treated with full bodyanti-B7-H4 antibody (clone #25) was not inhibited. In addi-tion, no antitumor effect was observed in B7-H4� NCI-H2170tumors treated with BsAb (Fig. 5A). The BsAb also inducedT-cell cytotoxicity to HER2þ and B7-H4þ breast cancer celllines, HCC-1954 and HCC-1569 in vitro (Supplementary Fig.S5A and S5B), and BsAb injection suppressed HCC-1954 andHCC-1956 tumor growth by more than 50% in the humanizedmouse model (Supplementary Fig. S5C).

A relatively high dose of anti-CD3 antibody (clone: OKT3),formerly used in the clinic as an immunosuppressive agent,resulted in no inhibitory effect and obvious weight loss. The

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Antitumor effect of the anti-B7-H4/CD3 BsAb in vivo using a humanized MHC-dKO NOGmouse model. A, Short-term antitumor effect of the antibodies onMDA-MB-468 tumors. Two weeks after the transplantation of PBMCs and cancer cells, each antibody was administered by a 40 mg/body single injection via tailvein. Diamond, control group; closed square, Fab-scFv anti-B7-H4/CD3 BsAb; triangle, anti-B7-H4mouse mAb (clone #25); and cross, anti-human CD3mAb(OKT3). � , P < 0.05. B, Long-term antitumor effect of the antibodies on MDA-MB-468 tumors. Twelve days after the transplantation of PBMCs and cancer cells,the BsAb was administered at 40 mg/body via tail vein two times weekly. Diamond, control group; closed square, Fab-scFv anti-B7-H4/CD3 BsAb. Each pointshows the mean� SD value of 7 mice. � , P < 0.05. C, The effect of T-cell depletion on the antitumor effect of anti-B7-H4/CD3 BsAb. Human T-cell subsets weredepleted by intraperitoneal injection of 100 mg/body/day anti-CD4 or anti-CD8mAb (Bio X Cell) from day 7 to day 9. Diamond, control group (no antibody);closed square, isotype antibodyþ BsAb; closed circle, anti-CD4 antibodyþ BsAb; and closed triangle, anti-CD8 antibodyþ BsAb. Each point shows themean� SE value of 4 mice (i.p., intraperitoneal; i.v., intravenous; s.c., subcutaneous).






BsAb-treated mice showed no adverse effects such as weight loss.An escalating BsAb dose (0.2–200 mg/body) was administeredwithout harm (Fig. 6A).

In the T-cell subset depletion in vivo study, CD8þ T-cell deple-tion blocked the growth inhibition induced by B7-H4 BsAb, andCD4þ T-cell depletion showed only a weak blocking effect ongrowth inhibition (Fig. 5C). These results demonstrated that theantitumor effect of anti-B7-H4 BsAb was mediated partially byCD4þ T cells and mostly by CD8þ T cells.

Effector immune cell analysis of MDA-MB-468 tumor–bearingmice treated with the anti-B7-H4/CD3 BsAb

From the flow cytometry analysis of TIL and splenocytes fromthe antibody-treatedmice, the total cell number andCD3þCD45þ

T-cell number from spleens showed a tendency to increase in theBsAb-treated mice compared with the controls (SupplementaryTable S1), but therewere no significant differences in the TIL T-cellsubset (data not shown).

In the Fab-scFv anti-B7-H4/CD3 BsAb-administered group,hematoxylin and eosin (H&E)- stained tumor specimens showedremarkable infiltration of lymphoid cells inside the tumor coresand resulted in almost no viable cancer cells at the tumor site(Fig. 6B). Immunostaining revealed that CD8þ and granzyme Bþ

lymphocytes were more frequently observed in the BsAb-treatedgroup (Fig. 6B and C), but CD4þ and FoxP3þ lymphocytes andCD204þ immune cells did not significantly differ (data notshown).

IHC study of B7-H4 in human normal and tumor tissue arraysIn 28 human normal and tumor tissues array analysis, seven

B7-H4þ tumors (pharynx, esophagus, stomach, lung, kidney,

fallopian tube, and kidney) were identified (Supplementary Fig.S6). In the normal tissues, only the tonsil (epithelial cell) waspositively stained with the anti-B7-H4 antibody clone #25. TheB7-H4 IHC study of human breast cancer tissue array showed thata B7H4þ rate of 69 breast cancer tissues was 43.5%, and there wasa tendency of high B7H4 stain in triple-negative tumor comparedwith PD-L1 (Supplementary Fig. S7). However, it was not definitebecause of small number of cases.

DiscussionBecause of the recent success of immune checkpoint blocking

antibodies, such as ipilimumab and nivolumab, in patients withmetastatic melanoma and other malignancies, clinical trials areunderway to evaluate their efficacy in various solid cancers (1–4).These studies show that the modulation of the suppressedimmune system is an effective way to combat cancers and thatimmunotherapy can be used to treat cancer.

In addition to immune checkpoint antibodies, BsAbs thatdirectly engage immune cells (24) are becoming another prom-ising strategy in cancer antibody therapy. Advances in recombi-nant protein technology allow for the construction of BsAbs ina variety of formats with great flexibility. A number of formatshave been proposed including bispecific T-cell engager (BiTE;refs. 25, 26), tandem diabody (27), dual affinity retargeting(DART; ref. 28), and antibody-TCR format (ImmTac; ref. 29).Catumaxomab-targeting EpCAM and blinatumomab-targetingCD19 are approved for clinical use and others are in variousstages of clinical development (30–33).

New T-cell–engaging BsAbs, including BiTE and DART, work ata much lower dose in xenograft models and clinical use (23, 28)

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Breast cancer cell eradication andeffector T-cell infiltration inside thetumor after BsAb treatment. A,PBMCs and MDA-MB-468 tumor–transplanted mice wereadministered BsAb sequentially ina dose-escalating manner (0.2–200 mg) with 3- or 4-day intervalsbetween injections. Two weeksafter the initial antibody injection,the resected control tumorspecimens and BsAb treated–tumor specimens with about70% size reduction were used forIHC analysis. B, Images of theanti-B7-H4 BsAb–treated mousetumors stained with H&E or withanti-B7-H4 (clone #25) and anti-CD8 antibodies. Magnification,200�. C, Infiltrating CD8þ orgranzyme Bþ T-cell counts at thetumor site. Each histogramrepresents the mean� SD of morethan 10 areas of the tumor section(� , P < 0.05; i.v., intravenous; s.c.,subcutaneous).

Iizuka et al.





compared with conventional antibody therapies. Our BsAb,which connects the anti-B7-H4 antibody and anti-CD3 antibody,immediately elicited a strong B7-H4þ target tumor lysis at verylowdosebyCD8þ effector T cells or other T-cell subsets, bypassingpeptide-MHC/TCR recognition (Figs. 3 and 5).

Trastuzumab and other HER2-targeted agents improvedpatient outcomes in breast cancer therapy, but initially theresponsive tumors develop resistance. HER2-targeted BsAbshowed an antitumor potency against trastuzumab-resistanttumor cells in in vitro and in vivo models (32). Its EC50 wasinversely correlated with surface HER2 expression and could beeffective for tumors that express a lower level of HER2. Giventhat B7-H4 is frequently expressed on breast cancer cells irre-spective of HER2 expression (Supplementary Fig. S8) and thatB7-H4 expression has a tendency to be higher in TNBCcells (34), B7-H4 is potentially a novel substitute target forprimary and recurrent breast cancers, including triple-negativecancers that are nonresponsive to HER2-targeting therapies.Interestingly, our anti-B7-H4 BsAb showed a potent antitumoreffect in vivo against B7-H4þ breast cancer cell lines, HCC-1954and HCC-1569, which was demonstrated to be basal typeHER2þ and trastuzumab-resistant by some researchers (35, 36).This observationmight suggest that this type of breast cancerswithpositive B7-H4 expression could be the target for B7-H4-targetingtherapy.

In addition, based on B7-H4 gene expression and the clinicaldata from the TCGA tumors samples, high B7-H4 gene expressioncan be a poor prognostic factor in patients with breast cancer(Supplementary Fig. S1B).

B7-H4 is widely detected in cancers and normal tissues at themRNA level, but its cell surface expression is limited and tends tobe unstable (21, 22). Our IHC study showed a limited expressionin normal tissues and positive expressions in several tumor tissuesother than breast cancer (Supplementary Fig. S6).

The BsAb eliminated breast cancer cells in a short amount oftime (Fig. 5A, 6B), and the immediate antitumor response mayhelp overcome cancers with continuous genomic evolution andimmune evasions in contrast to the immune checkpoint blockadeantibody therapy, which takes several weeks to induce an ade-quate amount of tumor antigen–specific effector T cells at thetumor site.

Tumor-specific antigens, such as carcinoembryonic antigens,cancer/testis antigens, or differentiation antigens, are alsoexpressed in normal tissues to varying degrees, except formutation neoantigens. Therefore, the therapeutic approachestargeting these antigens require antibody dose control forharnessing cytotoxic effector cells. B7-H4-targeted CAR-T–celltreatment showed lethal toxicity including a delayed GVHD ina mouse model (37), and a clinical trial using trastuzumab-based CAR-T cells resulted in patient death and wasaborted (38). These reported adverse events suggest that the

strict regulation of effector function is important for clinicaluse. A shorter in vivo half-life of the smaller size BsAb isdescribed as a negative property, but this characteristic enableseasier control of effector cells for clinical use compared withCAR-T–cell therapy.

Immune check point modulators are not effective for themajority of patients with cancer, and positive outcome is likelyto require a high mutation burden and effector T-cell accumula-tion againstmutation-derived neoantigens (39, 40). BsAb therapymay be an alternative anticancer immunotherapeutic strategy tobypass neoantigen-MHC/TCR recognition.

In this study, we demonstrated that the B7-H4-targeted BsAbhad potent antitumor activity in vitro and in vivo, which wasspecific for B7-H4þ tumors and was not cytotoxic on a normalmammary duct epithelial cell line. More importantly, in vivoimaging showed a long-lasting retention of the injectedantibody at the tumor site. These results suggest that the Fab-scFvanti-B7-H4/CD3 BsAb might be a therapeutic agent for PD-L1�

B7-H4-expressing tumors or anti-HER2 antibody nonresponsivebreast tumors.

Disclosure of Potential Conflicts of InterestY. Nakasu reports receiving speakers bureau honoraria from Eisai Co.,

Chugai Co., and Dai-ichi Pharmaceutical Co., and is a consultant/advisoryboard member for Ono Pharmaceutical Co. No potential conflicts of interestwere disclosed by the other authors.

Authors' ContributionsConception and design: A. Iizuka, Y. AkiyamaDevelopment of methodology: A. IizukaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A. Iizuka, T. Ashizawa, K. Mitsuya, N. Hayashi,Y. NakasuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. Iizuka, C. Nonomura, R. Kondou, K. Ohshima,T. SuginoWriting, review, and/or revision of the manuscript: A. Iizuka, K. Yamaguchi,Y. AkiyamaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C. Nonomura, K. Maruyama, Y. AkiyamaStudy supervision: A. Iizuka

AcknowledgmentsWe thank Koji Takahashi for his excellent assistance in maintaining the

NOG-dKO mouse in the animal facility. This study was supported by JSPSKAKENHI, Japan (grant no. 17K07235; to A. Iizuka).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received October 23, 2017; revised February 24, 2018; accepted February 6,2019; published first February 8, 2019.

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2019;25:2925-2934. Published OnlineFirst February 8, 2019.Clin Cancer Res Akira Iizuka, Chizu Nonomura, Tadashi Ashizawa, et al. Human Breast Cancer

engaging B7-H4/CD3-bispecific Fab-scFv Antibody Targets−A T-cell

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A T-cell engaging B7-H4/CD3-bispeciﬁc Fab-scFv Antibody ... · B7-H4/CD3 antibody was injected intravenously and imaging was performed at sequential timepoints ranging from 24 hours