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Cancer Letters xxx (2013) xxx–xxx

CAN 11577 No. of Pages 9, Model 5G

11 August 2013

Contents lists available at ScienceDirect

Cancer Letters

journal homepage: www.elsevier .com/locate /canlet

A novel antibody-like TCRcd-Ig fusion protein exhibits antitumoractivity against human ovarian carcinoma

0304-3835/$ - see front matter � 2013 Published by Elsevier Ireland Ltd.http://dx.doi.org/10.1016/j.canlet.2013.07.036

⇑ Corresponding author. Address: Department of Immunology, Institute of BasicMedical Sciences, Chinese Academy of Medical Sciences and Peking Union MedicalCollege, National Key Laboratory of Medical Molecular Biology, 5 Dong Dan SanTiao, Beijing 100005, China. Tel./fax: +86 10 69156474.

E-mail addresses: jingzheng0420@gmail.com (J. Zheng), heweiimu@public.bta.net.cn (W. He).

1 Jing Zheng and Yang Guo contributed equally to this study.

Please cite this article in press as: J. Zheng et al., A novel antibody-like TCRcd-Ig fusion protein exhibits antitumor activity against humancarcinoma, Cancer Lett. (2013), http://dx.doi.org/10.1016/j.canlet.2013.07.036

Jing Zheng 1, Yang Guo 1, Xu Ji, Lianxian Cui, Wei He ⇑Department of Immunology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Key Laboratory of MedicalMolecular Biology, Beijing, China

a r t i c l e i n f o

2526272829303132333435

Article history:Received 13 May 2013Received in revised form 9 July 2013Accepted 28 July 2013Available online xxxx

Keywords:TCR cdADCCTumor-targeted immunotherapyXenograft model

a b s t r a c t

TCRc9d2(OT3) is a tumor-specific TCR with an unique complementarity-determining region 3 (CDR3)sequence, referred to as OT3, in its d2 chain. This region was identified in tumor-infiltrating lymphocytes(TILs) from human ovarian epithelial carcinoma. We demonstrated that TCRc9d2(OT3)-Fc, a fusion pro-tein composed of the complete extracellular domains of the c9 and d2 chains linked to the Fc domains ofhuman IgG1, exhibited successful binding to multiple human carcinoma cell lines. In vitro,TCRc9d2(OT3)-Fc mediated cell killing via antibody-dependent cellular cytotoxicity (ADCC) in adose-dependent manner. In vivo, TCRc9d2(OT3)-Fc significantly inhibited tumor growth and enhancedsurvival in human ovarian carcinoma xenograft models. Our findings suggest that the TCRc9d2(OT3)-Fc fusion protein possesses both the antigen-recognition properties of TCR cd and the Fc-mediatedeffector functions of the antibody.

� 2013 Published by Elsevier Ireland Ltd.

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1. Introduction

Tumor-targeted immunotherapy has significantly developed inrecent years, with T cell-targeted vaccines and adoptive cell trans-fer-based therapies showing promising results in clinical trials[1,2]. For example, Sipuleucel-T, which was approved by the USFood and Drug Administration (FDA) in 2010, showed a survivalbenefit for patients with metastatic prostate cancer [3]. AdoptiveT-cell therapy was also reported to be effective, with an objectiveresponse rate of approximately 50% in patients with metastaticmelanoma [4]. However, these T cell-based therapies encounterseveral considerable obstacles to achieving antitumor responses,including limited infiltration into tumor tissues, functional defectsor anergy and effects of the local immunosuppressive environment[5,6]. Moreover, cell-based therapies require a significant amountof time, money and expertise [7].

In addition to T cell-based strategies, antibody (Ab)-basedtherapies have proven to be successful in the treatment of tumors.Herceptin and rituximab have been used to treat metastatic breastcancer and non-Hodgkin’s lymphoma, respectively, and haveshown great clinical success [8,9]. In Ab-based tumor immunother-

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apy, the identification of tumor-specific antigens for therapeutictargeting is crucial. However, only a limited number of tumor-specific antigens have been discovered, greatly limiting thedevelopment of Ab-centered therapies.

T-cell receptors (TCRs) on tumor-infiltrating lymphocytes (TILs)have evolved to recognize the specific antigens expressed by tumorcells, rendering these receptors ideal candidates for tumor-tar-geted applications. Researchers have attempted to develop novelTCR-based antitumor strategies by fusing tumor-specific TCRs witheffector molecules, such as cytokines, toxins and radionuclides[10,11]. Epel et al. first developed a p53-specific single-chain TCRthat is capable of selectively targeting antigen-presenting cells(APCs) [12]. Wong and his colleagues then reported that asingle-chain TCR-interleukin (IL)-2 fusion protein exhibitedtumor-targeting ability and reduced lung metastases or inhibitedtumor growth in xenograft tumor models [13–15]. Moreover, byfusion to human IgG1, this single-chain TCR-mediated antibody-dependent cellular cytotoxicity (ADCC) displayed potent antitumoreffects in vivo [16]. These reports thus demonstrate the feasibilityof TCR-based antitumor strategies.

Compared with TCR ab, TCR cd recognizes a broader spectrumof tumor antigens due to the human major histocompatibility com-plex (MHC)-independent antigen recognition of this receptor[17,18]. Activated c9d2 T cells have been reported to exhibit strongcytotoxicity against leukemia, lymphoma and multiple carcino-mas, including ovarian carcinoma [19,20]. We previously identifieda CDR3 sequence, termed OT3, within the d2 chain in TILs inhuman ovarian epithelial carcinoma [21]. A CDR3d(OT3)-grafted

ovarian

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Ab showed specific binding to multiple tumor cell lines and trig-gered an ADCC against tumor cells in vitro [22]. Because all CDRsare reported to influence the antigen recognition of TCR cd [23], fu-sion proteins containing the complete V regions or extracellulardomains of TCR cd may exhibit a better tumor cell-targeting abilitythan the CDR3d(OT3)-grafted Ab. Here, we constructed threeTCRcd-Ig fusion proteins by fusing the complete extracellulardomains or V regions of TCRc9d2(OT3) to the constant domainsof human IgG1. We then compared the tumor binding abilities ofthe three TCRcd-Ig fusion proteins and evaluated the antitumoreffects of the fusion protein with the best tumor binding ability,TCRc9d2(OT3)-Fc, in vitro and in vivo. Our data indicate that theTCRc9d2(OT3)-Fc fusion protein behaves similarly to an Ab butalso possesses the ability of TCR cd to recognize antigens. Withits broad tumor recognition, TCRc9d2(OT3)-Fc may represent anovel agent for tumor immunotherapy.

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2. Materials and methods

2.1. Cell lines

The human tumor cell lines ES-2 (ovarian cancer), SKOV3 (ovarian cancer),HO8910 (ovarian cancer), HeLa (cervical adenocarcinoma), NCI-H520 (pulmonarycancer), A498 (renal cancer), MGc-803 (gastric carcinoma), Raji (Burkitt’s lym-phoma) and K-562 (myelogenous leukemia) were obtained from the Cell CultureCenter of the Institute of Basic Medical Sciences at the Chinese Academy of MedicalSciences (Beijing, China). The ES-2 and SKOV3 cells were cultured in completeMcCoy’s 5A medium (Invitrogen, USA). The HO8910, Raji, K-562 and NCI-H520 cellswere maintained in complete RPMI-1640 medium. The HeLa and MGc-803 cellswere cultured in complete DMEM. The A498 cells were maintained in completeMEM–EBSS. The complete medium mentioned above consisted of medium supple-mented with 10% FBS, 2 mM glutamine, 10 mM HEPES, 50 lg/ml penicillin and100 lg/ml streptomycin. All of the cell lines were cultured at 37 �C and 5% CO2.Additionally, peripheral blood mononuclear cells (PBMCs) were isolated using Fi-coll–Hypaque (TBD, Tianjin, China) centrifugation.

2.2. Mice

Athymic BALB/c (nu/nu) nude mice (female, 4–6 weeks of age) were purchasedfrom the National Rodent Laboratory Animal Resources (Beijing, China). The micewere maintained in specific pathogen-free conditions at the Animal MaintenanceFacility of the Institute of Basic Medical Sciences at the Chinese Academy of MedicalSciences (Beijing, China). All of the mouse experiments were performed in accor-dance with the animal ethics regulations of the Chinese Academy of MedicalSciences.

2.3. Flow cytometry

Tumor cells were incubated with TCRc9d2(OT3)-Fc, TCRVd2(OT3)-Fc,TCRVd2(OT3)H/Vc9Lk or a hIgG1 control (Sigma, USA) diluted in PBS containing1% BSA for 1 h at 4 �C. After three washes, the cells were incubated for 30 min at4 �C with a FITC-labeled goat anti-human IgG (H + L) Ab (Sigma, USA) and thenwashed again. The samples were analyzed with an Accuri C6 Flow Cytometer (BDBiosciences, USA), and the data were analyzed using FlowJo software (Version7.6.4; Tree Star, Inc.).

2.4. Immunofluorescence assay

Cells were fixed on slides using 95% cold ethanol, blocked with PBS containing1% BSA and incubated with the each of the three TCRcd-Ig fusion proteins at 10 lg/ml for 30 min at 37 �C. hIgG1 was used as a control. After three washes, the cellswere incubated for 30 min at room temperature with a FITC-labeled goat anti-hu-man IgG (H + L) Ab and washed again. DAPI (Sigma, USA) was used to counterstainthe nuclei. The slides were then analyzed for fluorescence using a confocal lasermicroscope (LSM 510; Carl Zeiss).

2.5. Immunohistochemistry assay

Paraffin sections of tumor tissues were deparaffinized, rehydrated, treated with0.01 M citrate (pH 6.0) and boiled in a microwave for antigen retrieval. Afterquenching with peroxide, the sections were blocked with 5% goat serum in PBS.The tissues were then incubated with each of the three TCRcd-Ig fusion proteinsor the hIgG1 control for 1 h at room temperature. Primary protein binding wasdetected using a HRP-labeled goat anti-human IgG (Fc) Ab (Sigma, USA) in the

Please cite this article in press as: J. Zheng et al., A novel antibody-like TCRcarcinoma, Cancer Lett. (2013), http://dx.doi.org/10.1016/j.canlet.2013.07.036

presence of the substrate chromagen 3,30-diaminobenzidine (DAB; Zhongshan Jin-qiao Inc., China). Hematoxylin QS (Sigma, USA) was used to counterstain the nuclei,after which the tissue sections were analyzed using light microscopy.

2.6. Apoptosis assay

Tumor cells were cultured for 8 h with 50 lg/ml TCRc9d2(OT3)-Fc or hIgG1 at37 �C and 5% CO2. Cell viability was detected using an Alexa Fluor 488 Annexin V/Dead Cell Apoptosis Kit (Invitrogen, USA) according to the manufacturer’s instruc-tions. The stained cells were analyzed with an Accuri C6 Flow Cytometer.

2.7. Cell proliferation assay

Cell Counting Kit-8 (CCK-8; Dojindo, Japan) was used to examine the effects ofTCRc9d2(OT3)-Fc on the proliferation of the human tumor cells in vitro. Briefly, tu-mor cells were cultured for 72 h with various concentrations of TCRc9d2(OT3)-Fc orhIgG1 in 96-well flat-bottom microplates in triplicate. CCK-8 solution (100 ll/ml)was then added to the wells, and the plates were incubated at 37 �C for 1 h. Theabsorbance of the samples at 450 nm was determined using a microplate reader(Thermo Scientific, USA), with a 630 nm read to subtract out the background. Thepercentage of cell proliferation was then calculated as follows:

%Proliferation ¼ As� AbAc� Ab

� 100%

where Ac is the absorbance of the untreated cells, As is the absorbance of theTCRc9d2(OT3)-Fc- or hIgG1-treated cells and Ab is the absorbance of the blank wells.

2.8. ADCC assay

PBMCs were isolated from the whole human blood of healthy donors and usedas effector cells. Target cells (1 � 104/ml) were pretreated with TCRc9d2(OT3)-Fc atvarious concentrations or the hIgG1 control at the maximum concentration of thefusion protein for 40 min at 37 �C. Subsequently, 5000 target cells per well wereco-incubated with effector cells at various effector:target (E:T) ratios (20:1, 40:1and 80:1) in triplicate in 96-well U-bottom microplates for 8 h at 37 �C and 5%CO2. The amount of lactate dehydrogenase (LDH) released into the culturemedium was then measured using a CytoTox 96 Non-Radioactive CytotoxicityAssay (Promega, USA). The percentage of cell lysis was calculated using the follow-ing formula:

%Cytotoxicity ¼ Experimental � Effect spontaneous � Target spontaneousTarget maximum � Target spontaneous

� 100%

2.9. Measurement of antitumor activity of TCRc9d2(OT3)-Fc in tumor xenograft models

Female athymic BALB/c (nu/nu) nude mice (6–8 weeks of age) were subcu-taneously injected in the right armpit with SKOV3 cells (3 � 106/mouse) orES-2 cells (1 � 106/mouse) suspended in PBS. When the resultant tumors grewto an approximate volume of 0.05 cm3, the mice were randomized into groupsof 6–8 mice and treated with the TCRc9d2(OT3)-Fc fusion protein or the equiv-alent volume of PBS every 3 days, resulting in five total treatments. The SKOV3tumor-bearing mice were randomized into two groups: TCRc9d2(OT3)-Fc (n = 6)and PBS (n = 4). These mice were treated with 40 lg/dose of TCRc9d2(OT3)-Fc oran equivalent volume of PBS via intratumoral injection. The ES-2 tumor-bearingmice were randomized into four groups: TCRc9d2(OT3)-Fc (n = 8) via intratu-moral injection, PBS (n = 6) via intratumoral injection, TCRc9d2(OT3)-Fc (n = 8)via intraperitoneal (i.p.) injection and PBS (n = 6) via i.p. injection. These micewere treated with 50 lg/dose of TCRc9d2(OT3)-Fc or the equivalent volume ofPBS via intratumoral injection or i.p. injection. The tumors were measured every3 days using calipers, and the tumor volume was calculated using the followingformula:

V ¼ ðlength�width2Þ=2

2.10. Statistical analysis

For the in vitro experiments, Student’s t-tests and ANOVA (mean ± SD) wereused for statistical analysis where appropriate. The data from the in vivo experi-ments were analyzed using ANOVA and log-rank analysis. All of the data were ana-lyzed using the statistical software SPSS 17.0 (SPSS, Inc., Chicago, IL, USA). Values forwhich P < 0.05 were considered to be statistically significant.

cd-Ig fusion protein exhibits antitumor activity against human ovarian

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Fig. 1. TCRcd-Ig fusion protein formats and SDS–PAGE identification. (a) The formats of TCRc9d2(OT3)-Fc, TCRVd2(OT3)-Fc and TCRVd2(OT3)H/Vc9Lk. (b) The three fusionproteins were separated by SDS–PAGE under reducing and non-reducing conditions, and the SDS–PAGE gels were stained with Coomassie Brilliant Blue.

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3. Results

3.1. Expression of three TCRcd-Ig fusion proteins

We designed three TCRcd-Ig fusion proteins: TCRc9d2(OT3)-Fc,TCRVd2(OT3)-Fc and TCRVd2(OT3)H/Vc9Lk (Fig. 1a).TCRc9d2(OT3)-Fc is a heterodimer that was constructed by fusingthe extracellular domains of the c9 and d2 chains of TCRc9d2(OT3)with the hinge region and CH2 and CH3 domains of the humanIgG1 H chain. TCRVd2(OT3)-Fc is an Ab fragment homodimer. Each

Fig. 2. The binding of TCRcd-Ig fusion proteins to human ovarian carcinoma cell lines anTCRVd2(OT3)H/Vc9Lk or a hIgG1 control. The bound fusion proteins were identified uindicate the percentage of cells that were positive for the fusion proteins (gray shading)(dark line). (b) SKOV3 cells were incubated with the TCRcd-Ig fusion proteins or a hIgG1analyzed by confocal microscopy. Original magnification, �400. (c) Paraffin sections of huTCRVd2(OT3)H/Vc9Lk or a hIgG1 control and detected using an HRP-labeled goat antrepresentative of at least three independent experiments that yielded similar results. (Foto the web version of this article.)

Please cite this article in press as: J. Zheng et al., A novel antibody-like TCRcarcinoma, Cancer Lett. (2013), http://dx.doi.org/10.1016/j.canlet.2013.07.036

monomer was constructed by fusing the variable region of the d2chain to the hinge region and CH2 and CH3 domains of the humanIgG1 H chain. Lastly, TCRVd2(OT3)H/Vc9Lk is similar to an intactAb, with the variable regions of the H and light chains fused tothe variable regions of the d2 and c9 chains, respectively. The fu-sion proteins were expressed and purified by Sino Biological Inc.(Beijing, China).

The purified proteins were analyzed by SDS–PAGE (Fig. 1b). Underreducing conditions, the major band representing TCRc9d2(OT3)-Fchad a molecular mass of approximately 60 kD, whereas the band

d tissues. (a) SKOV3 cells were incubated with TCRc9d2(OT3)-Fc, TCRVd2(OT3)-Fc,sing a FITC-labeled goat anti-human IgG (H + L) Ab and flow cytometry. The plotscompared with the percentage of the cells that were positive for the hIgG1 controlcontrol as in a. Nuclear staining with DAPI is represented in blue. The samples wereman ovarian cancer tissues were incubated with TCRc9d2(OT3)-Fc, TCRVd2(OT3)-Fc,i-human IgG (Fc) Ab and DAB. Original magnification, �400. The data shown arer interpretation of the references to color in this figure legend, the reader is referred

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corresponding to TCRVd2(OT3)-Fc had a molecular mass of approxi-mately 45 kD. TCRVd2(OT3)H/Vc9Lk yielded two protein bands,which corresponded to the H chains (approximately 50 kD) and lightchains (approximately 30 kD). All of the bands were consistent withthe calculated molecular masses of the deduced amino acid se-quences. There was another visible band (30 kD) for TCRc9d2(OT3)-Fc under reducing conditions, which was shown by western blottingto be the Fc fragment (data not shown). Under non-reducing condi-tions, the molecular masses of TCRc9d2(OT3)-Fc, TCRVd2-Fc andTCRVd2(OT3)H/Vc9Lk were approximately 130 kD, 90 kD and170 kD, respectively. For each fusion protein, the apparent molecularmass of the band in the non-reducing gel was approximately twicethe molecular mass of the band in the reducing gel. This finding indi-cates that the fusion proteins formed dimers under non-reducingconditions. Taken together, these results demonstrate that the fusionproteins were successfully generated and that these proteins formedthe dimers necessary for their biological function.

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3.2. Superior binding of TCRc9d2(OT3)-Fc to tumor cells

Preliminary experiments showed that the three fusion proteinscould bind to SKOV3 cells (data not shown). Flow cytometry, an

Fig. 3. The binding of TCRc9d2(OT3)-Fc to multiple carcinoma cell lines. Human tumoincubated with TCRc9d2(OT3)-Fc (solid line) or a hIgG1 control (dotted line) followed busing flow cytometry. The data shown are representative of at least three independent eHeLa: cervical adenocarcinoma; NCI-H520: pulmonary cancer; A498: renal cancer; MGc-8Paraffin sections of normal human tissues, including the ovary, liver, esophagus and kidnHRP-labeled goat anti-human IgG (Fc) Ab and DAB. Original magnification, �400. The dasimilar results.

Please cite this article in press as: J. Zheng et al., A novel antibody-like TCRcarcinoma, Cancer Lett. (2013), http://dx.doi.org/10.1016/j.canlet.2013.07.036

immunofluorescence assay and an immunohistochemistry assaywere used to determine which fusion protein most effectivelybound to tumor cells and tissues. As shown in Fig. 2a,TCRc9d2(OT3)-Fc, TCRVd2(OT3)-Fc and TCRVd2(OT3)H/Vc9Lkstained 61.2%, 30.4% and 24.8% of the SKOV3 cells, respectively.Consistent with these results, the immunofluorescence assay indi-cated that TCRc9d2(OT3)-Fc bound to SKOV3 better than the othertwo fusion proteins (Fig. 2b). Similar results were observed in bind-ing assays using the human ovarian tumor cell lines ES-2 andHO8910 (data not shown). The immunohistochemistry assayshowed that all three of the fusion proteins could bind to ovariantumor tissues, but TCRc9d2(OT3)-Fc exhibited the best bindingability (Fig. 2c). Based on these data, we concluded that the threefusion proteins are able to recognize several human ovarian carci-noma cell lines and that TCRc9d2(OT3)-Fc has the best bindingability.

3.3. Binding of TCRc9d2(OT3)-Fc to a wide variety of carcinoma celllines

Based on the results of the in vitro binding assays, we fo-cused on TCRc9d2(OT3)-Fc in our subsequent studies. The

r cell lines (ES-2, SKOV3, HeLa, MGc-803, NCI-H520, A498, Raji and K-562) werey a FITC-labeled goat anti-human IgG (H + L) Ab. All of the samples were analyzedxperiments that yielded similar results. ES-2, SKOV3 and HO8910: ovarian cancer;03: gastric carcinoma; Raji: Burkitt’s lymphoma; K-562: myelogenous leukemia. (c)ey, were incubated with TCRc9d2(OT3)-Fc or a hIgG1 control and detected using a

ta shown are representative of at least three independent experiments that yielded

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Fig. 4. No effect of TCRc9d2(OT3)-Fc on the viability or proliferation of tumor cellsin vitro. (a) ES-2, SKOV3 and Raji cells were cultured with TCRc9d2(OT3)-Fc (grey)or hIgG1 (black) at 50 lg/ml for 8 h. Cell viability was detected using an Alexa Fluor488 Annexin V/Dead Cell Apoptosis Kit. (b) ES-2, SKOV3 and Raji cells were culturedwith TCRc9d2(OT3)-Fc (box) or hIgG1 (dot) at concentrations ranging from 12.5 to50 lg/ml in triplicate for 72 h in 96-well flat-bottom microplates. Cell proliferationwas detected using CCK-8. Statistical significance was determined by comparisonwith the hIgG1 control. The data represent the means ± SD of three independentexperiments (error bars, SD).

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antigen recognition of TCR cd has been reported to be non-MHCrestricted, and TCR cd-recognized antigens are widely expressedon tumor cells [20,24]. We therefore determined the bindingactivity of TCRc9d2(OT3)-Fc in multiple tumor cell lines. As ob-served for the ovarian carcinoma cell lines, TCRc9d2(OT3)-Fcdisplayed a good binding ability in a wide variety of carcinomacell lines, such as HeLa, MGc-803, NCI-H520 and A498. The per-centage of cells stained with TCRc9d2(OT3)-Fc ranged from97.2% (ES-2 cells) to 42% (A498 cells) (Fig. 3a). However, littleto no binding to the Burkitt’s lymphoma cell line (Raji) or tothe myelogenous leukemia cell line (K-562) was observed(Fig. 3b). Moreover, similar to hIgG1, TCRc9d2(OT3)-Fc eithernever or rarely bound to healthy tissues from various humanorgans, such as the ovary, liver, esophagus and kidney(Fig. 3c). TCRc9d2(OT3)-Fc thus recognized antigens that arespecifically expressed by carcinoma cells at variable levels.Therefore, TCRc9d2(OT3)-Fc was shown to be a broad-spectrumdetector of carcinoma in vitro, which may be a significantadvantage in tumor immunotherapy.

3.4. No effect of TCRc9d2(OT3)-Fc on the viability or proliferation oftumor cells in vitro

According to previous reports, certain therapeutic antibodies,including rituximab and Herceptin, induce tumor cell apoptosisand/or affect tumor cell growth in vitro by blocking receptor signal-ing [25,26]. We therefore tested whether TCRc9d2(OT3)-Fc coulddirectly affect the viability and/or proliferation of ES-2 and SKOV3cells, which are derived from different types of human ovariancancer. The Raji cell line, which was not bound byTCRc9d2(OT3)-Fc, was used as a negative control (Fig. 3b). Thetumor cells were incubated with 50 lg/ml TCRc9d2(OT3)-Fc orhIgG1 for 8 h, and the annexin V+ cells were then identified usingflow cytometry. As shown in Fig. 4a, the apoptosis rates were com-parable for the ES-2, SKOV3 and Raji cells treated withTCRc9d2(OT3)-Fc or hIgG1, indicating that TCRc9d2(OT3)-Fc didnot induce apoptosis in any of these tumor cells. To demonstratethe effects of TCRc9d2(OT3)-Fc on tumor cell proliferation,TCRc9d2(OT3)-Fc or hIgG1 was added to cultured tumor cells atconcentrations ranging from 12.5 to 50 lg/ml for 72 h. Cell prolif-eration was measured using CCK-8, and similar proliferation rateswere observed for cells treated with TCRc9d2(OT3)-Fc or hIgG1,indicating that TCRc9d2(OT3)-Fc did not inhibit the proliferationof tumor cells in vitro (Fig. 4b). Taken together, the data suggestthat TCRc9d2(OT3)-Fc does not have any direct effects on the via-bility or proliferation of human ovarian carcinoma cell linesin vitro.

3.5. TCRc9d2(OT3)-Fc-mediated cell killing through ADCC in vitro

Monoclonal antibodies (mAbs) may exert therapeutic effectsthrough ADCC and complement-dependent cytotoxicity (CDC),both of which are mediated by the Fc fragment of IgG [25,26]. AsADCC and CDC mainly differ in their downstream signals, we fo-cused on analyzing whether TCRc9d2(OT3)-Fc could induce tumorcell lysis in vitro through ADCC. To achieve this aim, freshly isolatedPBMCs from healthy donors were used as effector cells, ES-2 andSKOV3 cells were used as target cells and Raji cells were used asa negative control. The tumor cells were incubated with serial dilu-tions of TCRc9d2(OT3)-Fc or hIgG1 and PBMCs at various ratios(20:1, 40:1 and 80:1). As shown in Fig. 5a, TCRc9d2(OT3)-Fc in-creased the killing of ES-2 cells by PBMCs. At an E:T ratio of 80:1,the addition of 32 lg/ml TCRc9d2(OT3)-Fc significantly increasedES-2 cell lysis (27.10 ± 3.51%) compared with the hIgG1 control(9.39 ± 1.97%, P < 0.001). Cell killing was enhanced with increasedconcentrations of TCRc9d2(OT3)-Fc. Similar ADCC assay results

Please cite this article in press as: J. Zheng et al., A novel antibody-like TCRcarcinoma, Cancer Lett. (2013), http://dx.doi.org/10.1016/j.canlet.2013.07.036

were observed for the SKOV3 cells (Fig. 5b). No increase in specificlysis was observed after adding TCRc9d2(OT3)-Fc to the Raji cells(Fig. 5c). These data indicate that TCRc9d2(OT3)-Fc is capable ofmediating ADCC in a dose-dependent manner. ADCC may play animportant role in the antitumor activity of TCRc9d2(OT3)-Fc.

3.6. Antitumor activity of TCRc9d2(OT3)-Fc in tumor xenograft models

After demonstrating that TCRc9d2(OT3)-Fc can mediate ADCCagainst human ovarian carcinoma cell lines, we determined theantitumor efficacy of TCRc9d2(OT3)-Fc in vivo using tumor xeno-graft models. Female BALB/c (nu/nu) nude mice were s.c. injectedwith SKOV3 or ES-2 cells. When the resultant tumors reachedapproximately 0.05 cm3 in volume, the mice were treated withTCRc9d2(OT3)-Fc or an equivalent volume of PBS every 3 days,resulting in five total treatments. In the SKOV3 xenograft model,the mean tumor volume in the mice treated with TCRc9d2(OT3)-Fc was much smaller than the volume in the mice treated withPBS alone (0.33 ± 0.17 and 0.67 ± 0.13 cm3, respectively, P < 0.05)(Fig. 6a).

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Our preliminary tests showed that the ES-2 cells grew muchfaster than the SKOV3 cells both in vitro and in vivo. Thus, to inhibittumor growth effectively, ES-2 xenograft models were treated witha higher dose of TCRc9d2(OT3)-Fc. TCRc9d2(OT3)-Fc showedantitumor effects in the xenograft model in which SKOV3 cellswere introduced via intratumoral injection. Therefore, in the sub-sequent experiments with the ES-2 xenograft model, we testedwhether another route of administration (i.p. injection) affectedthe antitumor efficiency of TCRc9d2(OT3)-Fc.

In the ES-2 xenograft model, the mice treated with PBS alone viaintratumoral or i.p. injection exhibited rapid tumor growth, reach-ing a mean tumor volume of 2.11 ± 0.57 cm3 and 1.97 ± 0.34 cm3,respectively. In contrast, the mice treated with TCRc9d2(OT3)-Fcvia intratumoral or i.p. injection showed greatly suppressed tumorgrowth (0.86 ± 0.28 cm3 and 1.0 ± 0.25 cm3, respectively) after fivetreatments. Compared with the PBS control, the treatment withTCRc9d2(OT3)-Fc significantly inhibited tumor growth in thexenograft models (P < 0.001) (Fig. 6b). Consistent with this finding,the survival of the mice treated with TCRc9d2(OT3)-Fc via intratu-moral or i.p. injection was significantly improved compared withthe survival of the mice treated with PBS alone (Fig. 6c, P < 0.01).These results demonstrate that the TCRc9d2(OT3)-Fc fusion pro-tein significantly inhibits human ovarian tumor growth in xeno-graft models and improves mouse survival, suggesting that thisfusion protein could serve as an agent for ovarian carcinomaimmunotherapy.

Fig. 5. TCRc9d2(OT3)-Fc mediated cell killing through ADCC in vitro. Freshly isolated PBMRaji (c) cells were used as the target cells. The target cells were incubated with various cofor the fusion protein for 40 min. The target cells were then mixed with the PBMCs at E:T rLDH activity using the CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit. Statistical sP < 0.01; ���, P < 0.001). The data represent the means ± SD of three independent experim

Please cite this article in press as: J. Zheng et al., A novel antibody-like TCRcarcinoma, Cancer Lett. (2013), http://dx.doi.org/10.1016/j.canlet.2013.07.036

4. Discussion

CDR3d has been identified as a key determinant of the specific-ity of TCR cd antigen recognition [27]. OT3 is a CDR3 sequence thatwas identified on in the d2 chain on TILs from ovarian epithelialcarcinoma [21]. We previously showed that a CDR3d(OT3)-graftedAb maintained its tumor-targeting ability and ADCC [22]. However,the binding ability of this CDR3d(OT3)-grafted Ab was weak. Be-cause all of the CDRs have been reported to play important rolesin the antigen recognition of TCR cd [23], TCRcd-Ig fusion proteinscontaining the complete V regions or extracellular domains of TCRcd may possess a better binding ability than the CDR3d(OT3)-grafted Ab. Thus, in the present study, we constructed threeTCRcd-Ig fusion proteins (Fig. 1). TCRc9d2(OT3)-Fc exhibitedbetter binding to human ovarian carcinoma cells and tissuesthan TCRVd2(OT3)-Fc, TCRVd2(OT3)H/Vc9Lk (Fig. 2) and theCDR3d(OT3)-grafted Ab [22]. In our previous study, Jurkat cells thatexpressed TCRcd(OT3), which were created by gene transfer, werecytotoxic to tumor cell lines, including SKOV3 and HeLa but notRaji [28,29]. This finding was consistent with the tumor recogni-tion of TCRc9d2(OT3)-Fc reported here and indicates thatTCRc9d2(OT3)-Fc retains the specificity of the original TCR. In thepresent investigation, TCRc9d2(OT3)-Fc was also demonstrated tobe capable of binding to multiple carcinoma cell lines, includingcarcinomas of the ovary, cervix, lung, kidney and stomach(Fig. 3), suggesting a broad spectrum of tumor recognition.

Cs from healthy donors were used as the effector cells, and ES-2 (a), SKOV3 (b) andncentrations of TCRc9d2(OT3)-Fc or with hIgG1 at the maximum concentration usedatios of 20:1, 40:1 or 80:1 in triplicate for 8 h. Cell lysis was then measured based onignificance was determined by comparison with the hIgG1 control (�, P < 0.05; ��,ents (error bars, SD).

cd-Ig fusion protein exhibits antitumor activity against human ovarian

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Moreover, TCRc9d2(OT3)-Fc never or rarely bound to healthytissues from various human organs (Fig. 3c), indicating tumor-specific recognition. TCRc9d2(OT3)-Fc was additionally shown tomediate cell killing in vitro by ADCC and, notably, to inhibit tumorgrowth and enhance survival in xenograft models (Fig. 5 and 6).These data suggest that TCRc9d2(OT3)-Fc possesses both theeffector functions of the Ab and the ability of TCR cd to recognizeantigens. Thus, TCRc9d2(OT3)-Fc may be a potential agent forantitumor therapies.

TCRs play a crucial role in tumor-cell killing, as the forcedexpression of tumor-specific TCRs was shown to transfer tumor-cell recognition and cytotoxicity [2,30]. Therefore, the specificityof TCRs is the foundation of T cell-based immunotherapies [2].TCRs on TILs, which are capable of specifically recognizing tumorcells, have been demonstrated to be ideal candidates for tumor-targeted applications. Tumor-specific TCRs can be engineered todeliver toxins, radionuclides, immunomodulatory molecules andother effector agents to tumor sites to mediate antitumor effects[26]. For example, the use of TCR ab for tumor targeting has beenreported in several studies [10–16]. Additionally, Epel et al.reported that a p53-specific single-chain TCR could be used as atargeting agent to selectively deliver a cytotoxic effector moleculeto APCs expressing a specific MHC/peptide complex and thusmediate efficient cell killing [12]. In other studies, Wong et al.

Fig. 6. Antitumor effects of TCRc9d2(OT3)-Fc in xenograft models. (a and b) Female BALBcells (b). When the resultant tumors reached an approximate volume of 0.05 cm3, thtreatments in total. (a) The SKOV3 tumor-bearing mice were treated with 40 lg/dose of T6, respectively). (b) The ES-2 tumor-bearing mice were treated with 50 lg/dose of TCRcrespectively) or i.p. injection (n = 6 and 8, respectively) (points, mean value of six to etreatment. The data were analyzed for significance using ANOVA or log-rank analysis (�

Please cite this article in press as: J. Zheng et al., A novel antibody-like TCRcarcinoma, Cancer Lett. (2013), http://dx.doi.org/10.1016/j.canlet.2013.07.036

reported that a single-chain TCR that is specific for the p53peptide/human leukocyte antigen (HLA)-A2 complex exhibits atumor-targeting ability and antitumor effects following fusionto the effector molecule IL-2 [13–15] or human IgG1 [16]. TCRab-mimicking antibodies were also reported to display tumor-targeting abilities that are similar to that of TCR ab [31]. However,as TCR ab is confined to MHC-restricted, peptide-specific antigenrecognition, TCR ab-based immunotherapies are limited by theHLA type of the patient. Moreover, this type of immunotherapymay be ineffective due to the downregulation of HLA expressionin certain tumors [32]. In contrast, as the antigen recognition ofTCR cd is independent of MHC [17,33], TCR cd-based tumor immu-notherapies should not be disrupted by the downregulation of HLAexpression and should be available to a broader cancer patientpopulation.

The antigens recognized by TCR cd have not been fully explored[18]. The potential ligands are mainly stress-induced molecules inmalignant cells, including MHC class I chain-related A/B, UL16-binding proteins (ULBPs) and heat shock proteins [24,34], or abnor-mal metabolites, such as isopentenyl pyrophosphate (IPP) andadenosine triphosphate analogue (ApppI) [35,36]. c9d2 T cellswere also reported to be activated by phosphoantigens and theF1-ATPase-ApoA1 complex. IPP and ApppI were observed to abnor-mally accumulate in Daudi cells [36,37], whereas F1-ATPase is

/c (nu/nu) nude mice were s.c. injected with 3 � 106 SKOV3 cells (a) or 1 � 106 ES-2e mice were treated with TCRc9d2(OT3)-Fc or PBS every 3 days, resulting in fiveCRc9d2(OT3)-Fc or an equivalent volume of PBS via intratumoral injection (n = 4 and9d2(OT3)-Fc or an equivalent volume of PBS via intratumoral injection (n = 6 and 8,ight animals; error bars, SD). (c) Survival of the ES-2 xenograft model mice after

, P < 0.05; ��, P < 0.01; ���, P < 0.001).

cd-Ig fusion protein exhibits antitumor activity against human ovarian

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ectopically expressed on the surface of tumor cells; furthermore,data have suggested that F1-ATPase may directly interact withTCR c9d2 [38,39]. Additionally, we previously demonstrated thatULBP4s [34] and the hMSH2 protein [40] are potential ligands forTCR c9d2. ULBP4s are overexpressed in transformed tumor cellsand herpes simplex virus (HSV)-infected cells [34], and hMSH2 isectopically expressed on the cell membrane of human ovariancarcinoma cells [40]. Thus, these ligands, which are abundantlyor ectopically expressed by tumor cells, may play a role inTCRcd-based tumor-targeting therapies. However, further studiesare necessary to determine which antigen is involved inTCRc9d2(OT3)-Fc-mediated ADCC.

The effects of therapeutic mAbs include the inhibition of tumorgrowth and the induction of apoptosis, ADCC or CDC [25,26]. Ourdata show that TCRc9d2(OT3)-Fc could not induce the apoptosisor inhibit the proliferation of ES-2 and SKOV3 cellsin vitro but med-iated cell killing through ADCC (Figs. 4 and 5). The importance ofFc-Fc receptor (FcR) interactions for the in vivo antitumor effectsof mAbs has been demonstrated in mouse models and clinical trials[26,41]. In ADCC, Ab-coated tumor cells are lysed or phagocytosedby effector cells that express FcR, including natural killer (NK) cells,neutrophils, monocytes and macrophages [25]. These effector cellshave been shown to infiltrate into tumor sites and be activated bythe interaction of FccR and the Fc fragment of an Ab. Antibodiesthat induce ADCC are typically of the IgG1 or IgG3 subclass [26].Here, in the case of TCRc9d2(OT3)-Fc, the IgG1 H chain binds tothe FccR, and the TCR portion provides tumor targeting. In additionto the Fc fragment of IgG, TCRc9d2(OT3) can also be engineered todeliver other effector molecules, such as IL-2, IFN-c, toxins orradionuclides, as is performed in certain successful mAb-basedstrategies [26].

Several strategies can be used to enhance the antitumor effectsof TCRc9d2(OT3)-Fc if its ligands are defined. The affinity of an Abis important for its tumor retention and penetration and ADCC[42], but TCRs typically exhibit a low affinity for TCR ligands[43]. The affinity of TCR molecules can be increased substantiallyusing several enhancement methods [44,45]. In one case, selectedmutants of Va CDR3 resulted in more than 100-fold higher affinityfor the cognate peptide/MHC ligand [44]. However, increasing theaffinity of TCRs without sacrificing antigen recognition specificityis a significant challenge [45]. Another option is modification ofthe Fc fragment, such as an alteration of the amino acid sequenceor glyco-engineering, which could enhance the effects of ADCC[46,47]. Amino acid alteration or removal of the core fucose ofthe Fc fragment of therapeutic mAbs was reported to enhancethe binding affinity of CD16A (FccRIII), resulting in increased ADCC[46,47]. Further work is in progress to increase the affinity ofTCRc9d2(OT3)-Fc and to optimize the Fc fragment to enhance theantitumor effects of TCR.

In conclusion, we constructed a TCRc9d2(OT3)-Fc fusion proteinthat possessed both the antigen recognition properties of TCR cdand the effector functions of an Ab. TCRc9d2(OT3)-Fc was capableof binding to multiple human carcinoma cell lines. In vitro,TCRc9d2(OT3)-Fc mediated cell killing by ADCC in a dose-depen-dent manner. In vivo, TCRc9d2(OT3)-Fc significantly inhibited tu-mor growth and improved survival in human ovarian carcinomaxenograft models. These data demonstrate the feasibility of usingTCRc9d2(OT3)-Fc in ovarian tumor-targeted therapy. Moreover,due to its broad spectrum of tumor recognition, TCRc9d2(OT3)-Fchas the potential to expand the range of tumors targeted byimmunotherapies.

585586587588589590

5. Conflict of Interest

The authors declare that they have no conflict of interest.

Please cite this article in press as: J. Zheng et al., A novel antibody-like TCRcarcinoma, Cancer Lett. (2013), http://dx.doi.org/10.1016/j.canlet.2013.07.036

Acknowledgments

This work was supported by grants from the National Programfor Biomedical Hightech, the Ministry of Science and Technology(China PR; 2006AA02A245) and the National Major Science & Tech-nology Specific Projects of the Twelfth Five-Year Plan (China PR;2012ZX10002015-008).

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cd-Ig fusion protein exhibits antitumor activity against human ovarian