Retargeting T Cells to GD2 Pentasaccharide on Human Tumors ... · T-cell retargeting bispeciﬁc antibodies (BsAb) also haveclinicalpotential,butit is thusfar onlyeffectiveagainst

Research Article

Retargeting T Cells to GD2 Pentasaccharide onHuman Tumors Using Bispecific HumanizedAntibodyHong Xu1, Ming Cheng1, Hongfen Guo1, Yuedan Chen2, Morgan Huse2, andNai-Kong V. Cheung1

Abstract

Anti-disialoganglioside GD2 IgG antibodies have shownclinical efficacy in solid tumors that lack human leukocyte anti-gens (e.g., neuroblastoma) by relying on Fc-dependent cytotox-icity. However, there are pain side effects secondary to comple-ment activation. T-cell retargeting bispecific antibodies (BsAb)also have clinical potential, but it is thus far only effective againstliquid tumors. In this study, a fully humanized hu3F8-BsAb wasdeveloped, in which the anti-CD3 huOKT3 single-chain Fv frag-ment (ScFv)was linked to the carboxyl endof the anti-GD2hu3F8IgG1 light chain, and was aglycosylated at N297 of Fc to preventcomplement activation and cytokine storm. In vitro, hu3F8-BsAbactivated T cells through classic immunologic synapses, inducingGD2-specific tumor cytotoxicity at femtomolar EC50 with >105-fold selectivity over normal tissues, releasing Th1 cytokines

(TNFa, IFNg , and IL2) when GD2(þ) tumors were present. Inseparate murine neuroblastoma and melanoma xenograft mod-els, intravenous hu3F8-BsAb activated T cells in situ and recruitedintravenous T cells for tumor ablation, significantly prolongingsurvival from local recurrence or frommetastatic disease. Hu3F8-BsAb, but not control BsAb, drove T cells and monocytes toinfiltrate tumor stroma. These monocytes were necessary forsustained T-cell proliferation and/or survival and contributedsignificantly to the antitumor effect. The in vitro and in vivoantitumor properties of hu3F8-BsAb and its safety profile supportits further clinical development as a cancer therapeutic, andprovide the rationale for exploring aglycosylated IgG-scFv as astructural platform for retargeting human T cells. Cancer ImmunolRes; 3(3); 266–77. �2014 AACR.

IntroductionNeuroblastoma is a solid tumor model in which monoclonal

antibody (mAb) 3F8 specific for the pentasaccharide on diasia-loganglioside GD2 has been highly successful in controllingchemoresistant microscopic disease (1). These mAbs activateantibody-dependent FcR-mediated cytotoxicity by recruiting nat-ural killer (NK) cells, myeloid cells, and monocyte effectors. TheNCI (Bethesda,MD) program for prioritization of cancer antigenshas ranked GD2 at the 12th position in the list of top 75 cancerantigens (2). Its rank becomes higher when only directly target-able antigens are selected. Malignant solid tumors, such as mel-anoma, soft-tissue sarcomas, osteosarcoma, and small-cell lungcancer (SCLC), express GD2, although with more heterogeneitythan neuroblastoma (3). More recently, GD2 was discovered onneural stem cells, mesenchymal stem cells, and breast cancer stemcells (4).GD2 is rarely expressed innormal tissues except neurons,skin cells, and pain fibers. Among patients treated with anti-GD2

antibodies that were followed for the past two decades, there wereno long-term side effects (5). A recent randomized phase III trialconfirmed the efficacy of anti-GD2 mAb ch14.18, when com-binedwithGM-CSF and IL2, in preventing neuroblastoma relapseamong patients in first complete remission (6). Mouse 3F8(m3F8) was successfully humanized (hu3F8; ref. 7) with a near10-fold slower koff compared with ch14.18. In the phase I trial,hu3F8 showed high tolerability and low immunogenicity (8).Even among patients previously sensitized to m3F8, human anti-human antibody response remained low or undetectable afterrepeated challenges with hu3F8.

T cells can suppress or eradicate human cancers (9). Althoughbothm3F8 and hu3F8 could recruit NK cells andmyeloid cells fortumor cytotoxicity, without FcR, T cells are not involved (1). Theabsence or downregulation of human leukocyte antigen (HLA)expression in neuroblastoma is well known for escaping classic T-cell immunity. The low clonal frequency of cytolytic T cells andtheir inability to home to tumor sites have further emboldenedneuroblastoma to elude T cell–based tumor surveillance (1).Withfew exceptions, T cells are inefficient or incapable of targetingcarbohydrate epitopes.

Bispecific antibodies (BsAb) recruit T cells for tumor cytotox-icity through HLA-nonrestricted CD3-mediated activation. Byengaging polyclonal T cells, BsAb can overcome the low clonalfrequency of classic T cell–mediated antitumor immunity. BsAbspecific for CD3 and tumor antigens, such as CD19, HER2, orEGFR, have successfully retargeted T cells (10, 11). They inducecytotoxic synapse formation in T cells, mobilizing perforinand granzyme to kill tumors (12). CD3 engagement can alsoinduce T-cell proliferation and generation of effector cytokines to

1Department of Pediatrics, Memorial Sloan Kettering Cancer Center,New York, New York. 2Department of Immunology, Memorial SloanKettering Cancer Center, New York, New York.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

Corresponding Author: Nai-Kong V. Cheung, Department of Pediatrics, Memo-rial Sloan Kettering Cancer Center, 1275 York Avenue, Z-1403, NewYork City, NY10065. Phone: 646-888-2313; Fax: 646-422-0452; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-14-0230-T

�2014 American Association for Cancer Research.

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Published OnlineFirst December 26, 2014; DOI: 10.1158/2326-6066.CIR-14-0230-T

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potentiate the antitumor effect (13). Picogram quantities of BsAbcan exert significant antitumor effects in vitro as well as in vivo inpreclinical animal models and in patients (10). These antitumormechanisms can even recruit na€�ve T cells and stimulate thegeneration of tumor-specific T cells at tumor sites.

Yet, BsAb could overactivate T cells to discharge toxic cytokinestorms, analogous to the overwhelming toxicity from an anti-CD28 superagonist antibody (14). OKT3 (muromonab-CD3,Orthoclone OKT3) is a mouse anti-CD3 antibody with decadeslong safety record in humans (15). It is a proven agent foractivating human T cells for in vitro expansion. It has also beensuccessfully humanized (huOKT3) to reduce immunogenicity(16). OKT3 has been used to build BsAb for a number of tumormodels, many safely tested in the clinic (17). Various forms ofBsAb have been explored; among them are monovalent andbivalent forms made either chemically or genetically. Blinatumo-mab (BiTE AMG103, CD19-CD3 BsAb), an example of a tandemmonovalent scFv, is highly effective at extremely low doses (0.06mg/m2/d) in the treatment of patients with pre-B acute lympho-blastic leukemia and non-Hodgkin lymphoma with mild cyto-kine storm and no autoimmune phenomenon, except for theexpected depletion of B cells (10). However, by bolus injection, itengenders substantial central nervous system (CNS) toxicities,although the underlying mechanism remains unclear. In animalmodels, long-term treatment of mice with BiTE antibody did not

result in T-cell anergy or sustained cytokine release (18). BiTEtechnology has since been applied to other tumor targets, includ-ing chondroitin sulfate proteoglycan 4 (CSPG4) for melanoma,EpCAM for pancreatic carcinoma, carcinoembryonic antigen(CEA) for epithelial cancers, and EGFR for colorectal cancer(11, 19). Thus far, activation of T cells by BiTE is restricted totumors expressing the proper target antigen, and clinical efficacy islimited to tumors of the blood through targeting CD19 (13).Despite these encouraging preclinical and clinical studies, tandemscFvs have unique drawbacks. Their size (�50 kDa; ref. 20) andtheir inability to bind to neonatal FcRn leads to short serum half-lives. Thus, they require continuous infusion over 4 to 8 weeks tobe clinically effective. In addition, asmonovalentmolecules, scFvsdirected at tumor antigens need to have substantially higheraffinity.

We and others have previously shown that IgG-scFv (Fig. 1A) asa tetravalent format for BsAb can penetrate solid tumors (21, 22).Here, the bivalent IgG is derived from a tumor-specific antibody,whereas scFv with a second specificity is attached to the carboxylend of the light chain. For most tumor-selective antibodiesdirected at carbohydrates, bivalency is necessary for optimaltumor targeting, because their Fabs are rarely in the nmol/L orsub-nmol/L range. Similar to IgG (160 kDa), themolecular size ofIgG-scFv (�210 kDa) is in favorable balance between systemicclearance and vascular extravasation to achieve maximal tumor

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Figure 1.A–C, schematic diagram of hu3F8-BsAb in IgG-scFv format (A), reducedSDS-PAGE (B), and SE-HPLCchromatography (C): major peak(16.310 minutes) is the fully pairedBsAb (molecular weight 210 kDa), saltbuffer peak (25 minutes).

Retargeting T Cells to GD2 on Human Tumors

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uptake (20), while being denied entry into the CNS because ofthe blood–brain barrier (BBB). In addition, the human IgGbackbone allows a reproducible and FDA-approved affinitypurification method, as well as binding to FcRn to enhanceserum half-life (23).

In this article, we describe the first humanized anti-GD2BsAb using an IgG-scFv format by attaching the huOKT3-scFv tothe carboxyl end of the hu3F8 IgG1 light chain. The IgGbackbone was aglycosylated to prevent Fc receptor–mediatedcytokine storm (14) as well as pain side effects secondary tocomplement activation (24). We provide evidence that thishu3F8-BsAb has excellent antitumor activity both in vitro andin vivo. The IgG-scFv format may provide a versatile platform forbuilding cell-engaging BsAb.

Materials and MethodsCloning and expression of BsAbs

The hu3F8-BsAb format was designed as a huOKT3 scFvfusion to the C-terminus of the light chain of a human IgG1(21). Nucleotide sequences encoding VH and VL domains fromour hu3F8, and the OKT3 scFv were synthesized by GenScriptwith appropriate flanking restriction enzyme sites, and weresubcloned into a mammalian expression vector. Two controlBsAbs were built on the same platform, Herceptin-huOKT3 andhu3F8-C825 (22). Linearized plasmid DNA was used to trans-fect CHO-S cells (Invitrogen) for the stable production of BsAb,similar to that described in our previous report (25). Hu3F8-BsAb titer was determined by ELISA using antigen GD2 and theCD3(þ) Jurket cell line, and stable clones with highest expres-sion were selected.

TheBsAbproducer linewas cultured inOptiCHOmedium, andthemature supernatantwasharvested. AproteinAaffinity column(GE Healthcare) was used to purify hu3F8-BsAb as previouslydescribed (7), and BsAb was dialyzed into 25 mmol/L of sodiumcitrate, 0.15 mol/L of NaCl, pH 8.2, and frozen in aliquots at�80�C. The purity of hu3F8-BsAb was evaluated by both SDS-PAGE (7) and size-exclusion high-performance liquid chroma-tography (SE-HPLC).

In vitro binding kinetic studies by surface plasmon resonanceIn vitro binding affinity for GD2 was assayed by Biacore T-100

Biosensor aspreviously described (7). Separately, forCD3affinity,CD3 recombinant protein (CD3de-Fc; ref. 26; also produced inCHO-S cells) as the active surface, and blank as the reference, wereimmobilized using the Amino Coupling Kit (GE Healthcare).Purified hu3F8-BsAb and control antibodies, diluted in HBS-EPbuffer (0.01 mol/L HEPES, pH 7.4, 0.15 mol/L NaCl, 3 mmol/LEDTA, 0.05% v/v Surfactant P20), were injected over the sensorsurface. The data were analyzed using the Biacore T-100 evalua-tion software, and the apparent associationon-rate constant (kon),dissociation off-rate constant (koff), and equilibrium dissociationconstant (KD ¼ koff/kon) were calculated.

Cell linesThe cell lines LAN-1 and M14 were obtained from the Univer-

sity of California, Los Angeles (Los Angeles, CA); NMB-7 fromDr.SK Liao ofMcMaster University (Hamilton, ON, Canada). SKNJB,SKNJC2, and SKEAW were developed at the Memorial SloanKettering and all others in Table 1were purchased from the ATCC.They were authenticated by short-tandem repeat profiling using

the PowerPlex 1.2 System (Promega) andwere periodically testedfor Mycoplasma using a commercial kit (Lonza). The luciferase-labeled tumor cell lines IMR-32-Luc andM14-Luc were generatedby retroviral infection with an SFG-GFLuc vector.

Cytokine release assayCytokine release was assayed as previously described (25),

using human peripheral blood mononuclear cells (PBMC) iso-lated from the whole blood of healthy donors (New York BloodCenter).

Cell cytotoxicity (chromium-51 release assay)Cell cytotoxicity was assayed by 51Cr release as previously

described (25), and EC50 was calculated using SigmaPlot soft-ware. Effector T cells were purified from human PBMCs using aPan T-cell isolation kit (Miltenyi Biotec), and then activated andexpanded with CD3/CD28 Dynabeads (Invitrogen) according tothe manufacturer's protocol.

Xenograft studiesAll animal procedures were performed in compliance with

the Institutional Animal Care and Use Committee guidelines.The immunodeficient mice colony BALB-Rag2�/�IL-2R-gc-KO(DKO) was maintained at MSK under sterile conditions, andprovided with Sulfatrim food. In vivo experiments were per-formed with 6- to 10-week-old mice, and each treatment groupincluded 5 mice. Human PBMCs were prepared as previouslydescribed (25). All PBMC samples had similar percentages ofT-cell subpopulations (30%–50% CD3-positive). PBMC deple-tion of subpopulations was done with either CD56 or CD14Microbeads (Miltenyi Biotec), and <0.1% of target populationsremained after depletion.

Subcutaneous tumor plus subcutaneous effector cells (1:1 mixing).Purified PBMCs, activated T cells (ATC), or freshly prepared Tcells were mixed with IMR-32-Luc cells (1:1 ratio, 5 millioneach) in Matrigel (BD Biosciences) and implanted s.c. intoDKO mice in the right flank. Treatment was started accordingto the schedules indicated in the Results and figures. ATCs orfreshly prepared T cells were also given IL2 (1,000 U i.p., 2�/wk� 2 weeks) to maintain T-cell survival in the mice. Tumor sizewas measured by calipers twice per week, and tumor volumeswere calculated using the approximated formula V ¼ 0.5(length � width � width). The percentage of growth was thencalculated.

Subcutaneous tumor plus intravenous effector cells. Five millionM14-Luc cells inMatrigel were implanted s.c. intoDKOmice, andother procedures were similar to those described in the previousparagraph.

Intravenous tumor plus intravenous effector cells. IMR-32-Luc cells(0.5 million) or M14-Luc cells (1 million) were inoculated intoDKOmice i.v. via the lateral tail vein. For IMR-32 xenografts, bothT-cell groups (ATCs) were given IL2 (1,000 U i.p., 2�/wk � 3weeks). For M14 xenografts, all effector cell groups (ATCs orPBMCs) were given IL2 (1,000 U s.c., 2�/wk � 2 weeks). Tumorgrowth was assessed by luciferin bioluminescence once a week.Bioluminescence imaging was conducted using the Xenogen InVivo Imaging System (IVIS) 200 (Caliper LifeSciences). Briefly,

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Cancer Immunol Res; 3(3) March 2015 Cancer Immunology Research268




mice were injected i.v. with 0.1 mL solution of D-luciferin (GoldBiotechnology; 30 mg/mL stock in PBS). Images were collected 1to 2 minutes after injection using the following parameters: a 10-to 60-second exposure time, medium binning, and an 8 f/stop.Bioluminescence image analysis was performed using LivingImage 2.6 (Caliper LifeSciences).

Immunohistochemistry staining. The immunohistochemical(IHC) detection was performed at the MSK Molecular CytologyCore Facility using a Discovery XT processor (Ventana MedicalSystems). Paraffin-embedded tumor sections were deparaffinizedwith EZPrep buffer (Ventana Medical Systems), antigen retrievalwas performed with CC1 buffer (Ventana Medical Systems), andsections were blocked for 30 minutes with Background Bustersolution (Innovex). Anti-CD3 (Dako; cat. no. A0452; 1.2 mg/mL),anti-CD4 (Ventana; cat. no. 790-4423; 0.25 mg/mL), CD8 (Ven-tana; cat. no. 790-4460; 0.35 mg/mL), and anti-CD68 (Dako; cat.no. M0814; 0.02 mg/mL) antibodies were applied and sectionswere incubated for 5 hours, followed by 60-minute incubationwith biotinylated goat anti-rabbit IgG (Vector Labs; cat. no.PK6101) for CD3, CD4, and CD8 antibodies or biotinylatedhorse anti-mouse IgG (Vector Labs; cat. no. MKB-22258) forCD68 antibodies at a 1:200 dilution. The detection was per-formed with a DAB detection kit (Ventana Medical Systems)according to themanufacturer's instructions. Slides were counter-stained with hematoxylin and coverslipped with Permount (Fish-er Scientific). Images were captured from tumor sections using aNikon ECLIPSE Ni-U microscope and NIS-Elements 4.0 imagingsoftware, and counts of IHC-positive cells were averaged from tworandomly selected fields (�200 magnifications).

Statistical analysisDifferences between samples indicated in the figures were

tested for statistical significance by the Student t test, and P <0.05 was considered statistically significant.

Additional experimental details are described in Supplemen-tary Materials and Methods.

ResultsHu3F8-BsAb design

We designed hu3F8-BsAb using the IgG-scFv format (Fig. 1A).The heavy chain was identical to that of a hu3F8 IgG1 (7), exceptfor an N297A mutation to remove glycosylation. The light chainwas constructed by extending a hu3F8 IgG1 light chain with a C-terminal (G4S)3 linker followed by huOKT3 scFv. The DNAencoding both heavy chain and light chain was inserted into amammalian expression vector, transfected into CHO-S cells, andstable clones of high expression were selected. Supernatants werecollected from shaker flasks and purified by protein A affinitychromatography. Under reducing conditions, hu3F8-BsAbshowed two bands of around 50 kDa; besides the heavy chain,huOKT3 scFv fusion increased hu3F8 light chain molecularweight to approximately 50 kDa (Fig. 1B). By SEC-HPLC, a majorpeak (85%) showed amolecular weight of 210 kDa, the rest beingaggregates removable by gel filtration (Fig. 1C). Hu3F8-BsAbremained stable after multiple freeze and thaw cycles whenassayed by SDS-PAGE and SEC-HPLC (data not shown).

Hu3F8-BsAb binding to both tumor cells and T cellsBy FACS, hu3F8-BsAb was equally efficient as parental hu3F8

IgG1 in binding to the GD2(þ) neuroblastoma cell line LAN1

Table 1. Potency of hu3F8-BsAb

Tumor type Cell line GD2 expression (MFI)a EC50 (ng/mL)b EC50

Neuroblastoma LAN-1 1,467 0.0009 4.5 fmol/LNeuroblastoma NMB7 1,476 0.001 5 fmol/LNeuroblastoma IMR32 1,229 0.005 25 fmol/LMelanoma HT-144 1,227 0.016 80 fmol/LSCLC NCI-H524 2,437 0.02 100 fmol/LMelanoma SKMEL1 576 0.05 250 fmol/LMelanoma M14 745 0.08 400 fmol/LNeuroblastoma SKNBE(1)N 509 0.08 400 fmol/LOsteosarcoma U2OS 283 0.3 1.5 pmol/LNeuroblastoma SKNBE(2)C 478 0.5 2.5 pmol/LOsteosarcoma CRL1427 371 0.8 4 pmol/LSCLC NCI-H69 232 0.8 4 pmol/LNeuroblastoma SKNJB 196 2 10 pmol/LMelanoma SKMEL28 24 4 20 pmol/LNeuroblastoma SKNJC2 28 20 100 pmol/LBreast carcinoma MCF7 6 21 105 pmol/LEwing SKEAW 102 150 750 pmol/LSCLC NCI-H345 38 320 1.6 nmol/LColon carcinoma SW620 6 700 3.5 nmol/LBreast carcinoma AU565 8 >1,000 >5 nmol/LBreast carcinoma MDA-MB-361 6 >1,000 >5 nmol/LBreast carcinoma SKBR3 7 >1,000 >5 nmol/LBreast carcinoma MDA-MB-231 5 >1,000 >5 nmol/LBreast carcinoma MDA-MB-468 6 >1,000 >5 nmol/LEwing SKES-1 24 >1,000 >5 nmol/LOvarian carcinoma OVCAR3 4 >1,000 >5 nmol/LOvarian carcinoma SKOV3 5 >1,000 >5 nmol/LRhabdomyosarcoma HTB82 8 >1,000 >5 nmol/L

Abbreviation: MFI, mean fluorescence intensity.aFACS analysis using hu3F8 IgG1, with rituximab as negative control (MFI set at 5).bFour-hour 51Cr release assay at 10:1 E:T ratio. Maximum antibody concentration at 1 mg/mL. EC50 (concentration of antibody at half maximal killing) was calculatedusing SigmaPlot.






(Fig. 2A), although 100-fold less in binding to CD3(þ) T cellswhen compared with parental huOKT3 IgG1(N297A; Fig. 2B).This is consistent with our observation that light chain–anchoredscFv has lower avidity for T cells than regular huOKT3 IgG1, tominimize nonspecific cytokine release (see below).

The binding affinity by Biacore of hu3F8-BsAb to GD2 (7)showed a kon of 1.57 � 105/(mol/L)�s, a koff of 9.12 � 10�4/s,an overall KD of 5.8 nmol/L, comparable with that of parentalhu3F8 (1.79 � 105/(mol/L)�s, 2.91 � 10�3/s, and 16.3 nmol/L,respectively). For CD3 (26), hu3F8-BsAb had a kon of 5.43 � 105/(mol/L)�s, a koff of 1.05� 10�1/s, an overallKDof 194nmol/L, lessavid than parental huOKT3 IgG1(N297A; 1.68 � 106/(mol/L)�s,1.09 � 10�1/s, and 64.6 nmol/L, respectively). In summary,hu3F8-BsAb gained in GD2 avidity but displayed a decreased konfor CD3 binding compared with huOKT3.

Hu3F8-BsAb redirected T-cell killing of human tumor cell linesIn a standard 4-hour 51Cr release assay, hu3F8-BsAb demon-

strated potent cytotoxicity against the MYCN-amplified neuro-blastoma cell line IMR32 (EC50, 25 fmol/L; Fig. 3A). ATCs plustwo negative control BsAbs of the IgG-scFv format, hu3F8-C825[ref. 22; anti-GD2-DOTA (metal)] and HER2-BsAb (Herceptin-huOKT3), showed negligible cytotoxicity (Fig. 3A). When anextensive panel of human tumor cell lines (including neuroblas-toma, melanoma, SCLC, osteosarcoma, breast carcinoma, Ewingsarcoma, colon carcinoma, ovarian carcinoma, and rhabdomyo-sarcoma) were tested, hu3F8-BsAb killing potency (nmol/L tofmol/L range) correlated with tumor GD2 expression by FACS(Table 1). On the basis of the >105 difference in EC50 on tumorcells versus normal human cardiacmyocytes, hepatocytes, adrenalcortical cells, renal mesangial cells, or pulmonary alveolar epi-thelial cells, the safety margin for hu3F8-BsAb–redirected T-cellcytotoxicity was quite wide (Fig. 3B).

GD2-specific activation of T cells by Hu3F8-BsAbTo further investigate the mechanism of Hu3F8-BsAb–medi-

ated cytotoxicity, we imaged primary human T cells together with

IMR32 targets in the presence of various BsAb reagents. Hu3F8-BsAb induced stable T cell–target cell conjugate formation,accompanied by a rapid and robust Ca2þ flux within the T cell(Fig. 4A and B). This phenomenon, which is characteristic of

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cells) and Rituxan was used asnegative control (mean fluorescenceintensity set at 5).

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cytolytic synapse formation, was not observed in the presenceof either hu3F8-C825 or HER2-BsAb, indicating that recognitionof bothCD3andGD2by theBsAbwas aprerequisite. To assess thestructure of Hu3F8-induced contacts in more detail, we imaged Tcells on supported lipid bilayers containing purified GD2 andthe adhesion molecule ICAM-1, which binds to the aLb2integrin LFA-1. These surfaces elicited T-cell Ca2þ flux in astrictly Hu3F8-BsAb–dependent manner, validating their utilityas a GD2(þ) target-cell proxy (Fig. 4C). Total internal reflectionfluorescence microscopy revealed that the contacts formed byHu3F8-BsAb–treated T cells on the GD2 surfaces contained acentral accumulation of T-cell receptors surrounded by aperipheral ring of filamentous actin (Fig. 4D and E). This radiallysymmetric architecture was also observed on positive controlbilayers containing directly coated huOKT3, and is characteristicof mature immunologic synapses. In contrast, T cells treated withhu3F8-C825 or HER2-BsAb exhibited disorganized contacts withGD2bilayers similar to that of no antibody treatment (Fig. 4DandE). Taken together with the killing assays described above, thesedata strongly suggest that Hu3F8-BsAb induces the formation ofbona fide cytolytic synapses.

PBMCs, after 24 hours of hu3F8-BsAb activation, releasedprotumoricidal Th1 cytokines (TNFa, IFNg , and IL2) only in thepresence of GD2(þ) tumor cells, whereas the Th2 cytokine (IL10)release was less intense (Fig. 4F). Similar results were obtainedwith freshly isolated peripheral T cells instead of PBMCs (data notshown).

Efficacy of hu3F8-BsAb in humanized miceFor in vivo therapy studies, DKO mice were used for human

xenografts (27). In three different humanized mouse xenograftmodels (s.c. tumor plus s.c. effector cells, s.c. tumor plus i.v.effector cells, and intravenous i.v. tumor plus i.v. effector cells), i.v.hu3F8-BsAb showed high activity against established tumors,with either early (starting on day 4–5) or late (starting on day10) treatment. No clinical neurotoxicities were observed.

Subcutaneous effector cells plus subcutaneous tumors (1:1mixing) tosimulate T cells residing within tumor. IMR-32-Luc (luc, luciferasereporter) cells were mixed (1:1) with either T cells (freshlyisolated or ATC), or PBMCs (unactivated from the buffy coat)and planted s.c. On day 4, treatment with BsAb (5 mg i.v., 2�/wk � 2 weeks) was started and tumor size was measured.Although control BsAb (hu3F8-C825) plus PBMCs had mini-mal effect, hu3F8-BsAb plus PBMCs was curative (see survivalcurves; Supplementary Fig. S1). As effectors, PBMCs were super-ior to purified fresh T cells, and more effective than ATC in thistumor model (Fig. 5A).

Intravenous effector cells plus subcutaneous tumor to simulatehoming of T cells to soft-tissue tumor. M14-Luc melanoma cells(BRAF-mutated) were planted s.c., and hu3F8-BsAb (40 mg i.v.,2�/wk � 3 weeks) was started on day 5, while fresh PBMCs (1 �107 cells i.v., onceweekly� 3weeks) started onday 7. In thismorestringent model, hu3F8-BsAb without effector cells or controlBsAb with PBMCs was ineffective. Hu3F8-BsAb with PBMCs wascurative (Fig. 5B; Supplementary Fig. S1).

Intravenous effector cells plus intravenous tumor to simulate circu-lating T cells againstmetastatic disease. In Fig. 5C, IMR-32-Luc cellswere inoculated i.v. to mimic metastatic model, hu3F8-BsAb

treatment was started on day 4 (40 mg i.v., 2�/wk � 3 weeks),1� 107 ATCs (half i.v. and half i.p.) started on day 6 (once weekly� 3 weeks) and tumor luciferin bioluminescence signal wasrecorded and quantified weekly. ATC in combination withhu3F8-BsAb suppressed tumor progression.

In Fig. 5D and E, DKO mice implanted i.v. with M14-Lucmelanoma cells were treated with BsAbs started on day 10 (5 mg,2�/wk � 2 weeks), in combination with either PBMCs or ATCsstarted on day 12 (1� 107 cells i.v., once weekly� 2weeks). Micetreated with saline (Control), BsAb only, or effector cells pluscontrol BsAb, had equally rapid tumor progression. Hu3F8-BsAbplus effector cells suppressed tumor progression, regardless ifeffector cells were PBMCs or ATCs; survival was significantlyimproved (Supplementary Fig. S1).

In summary, using freshly isolated T cells or ATCs, we dem-onstrated that hu3F8-BsAb tumor cytotoxicity indeed functionedthrough T cells in vivo. Fresh PBMCs from healthy donors, whichmimicked a more realistic clinical situation, showed even betterefficacy with hu3F8-BsAb.

The importance of monocytes for antitumor activityTo address the cellularmechanism as towhy PBMCsweremore

effective than T cells alone, we tested whether monocytes couldparticipate in T-cell tumor infiltration using the s.c. tumor modeldescribed in Fig. 5B. Tumorswere collected 5 days after i.v. PBMCsand IHC was performed (Fig. 6A). In the PBMCs-plus-controlBsAb tumors, only circulating T cells in the blood vessels andnoneinside the tumor were detected. In contrast, PBMCs plus hu3F8-BsAb tumor clearly demonstrated T-cell tumor infiltration byCD3(þ) staining, including both CD4(þ) and CD8(þ) populations.Furthermore, CD68(þ)monocytes in the PBMCpopulation werealso found infiltrating the tumor stroma, but only when hu3F8-BsAb was given.

The importance ofmonocytes in the antitumor effect in vivowastested using the tumor model described in Fig. 5A, by comparingPBMCs and purified T cells to NK-depleted PMBCs, and mono-cyte-depleted PBMCs. Although depleting NK cells (CD56þ pop-ulation) showed comparable antitumor effect as PBMCs, deplet-ing monocytes (CD14þ population) resulted in a substantial lossin tumor suppression (Fig. 6B). In vitro, depleting monocytesmarkedly decreased Th1 cytokine release (TNFa and IFNg ; Fig.6C) and tumor cytotoxicity (Fig. 6D).When T cells were identifiedin the monocyte-depleted PMBC group by IHC, their numberswere reduced substantially in tumors harvested 9 days afterstarting BsAb treatment (Fig. 6E and Supplementary Fig. S2). Weinterpret these results to suggest a critical role of monocyte tumorinfiltration in sustaining T-cell infiltration, survival, or prolifera-tion, directly or indirectly through cytokines, and contributingsignificantly to the exceptional antitumor effect of BsAb (28).

DiscussionIn this study, we described the successful engineering of

hu3F8-BsAb to engage polyclonal T cells to target the penta-saccharide of GD2 on tumors not recognizable by classic T cellsbecause of low or absent HLA expression. Using an IgG-scFvplatform with proven ability to penetrate solid tumors, a fullyhumanized BsAb was built, riding on decades of safety recordsof OKT3 and m3F8, carrying the N297A mutation to prevent Fcreceptor–mediated cytokine storm and pain side effects fromcomplement activation. The potency of this BsAb, as well as its






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Figure 4.Hu3F8-BsAb activation of T cells. A and B, human T cells loadedwith Fura2-AMwere preincubatedwith BsAb and added towells containing GD2(þ) IMR32 target cells. A,representative time-lapse montages [time (m:ss) below the last montage] showing T cells contacting individual IMR32 cells (IMR) in the presence of BsAb reagents.Ca2þ concentrationwithin the T cells is displayed in pseudocolor, blue indicating lowand red indicating high concentrations. B, average single-cell Ca2þ responses in T cellsattached to IMR32 targets plotted against time. Each curve represents the average of 10 aligned responses. C, human T cells loaded with Fura2-AM were preincubatedwith BsAb reagents and plated on bilayers containing ICAM-1 and either GD2 or huOKT3. Ca2þ responses were quantified by calculating the average Fura ratio overfour imaging fields (�120 cells) during the plateau phase of the response.D andE, humanT cells preincubatedwithBsAb reagents andplated onbilayers containing ICAM-1and either GD2 or huOKT3, were fixed and stained for CD3 and F-actin and then imaged using TIRFmicroscopy. D, representative images of CD3 accumulation and F-actinring formation. Scale bars, 10 mm. E, quantification of F-actin ring formation by clearance ratio (n¼ 20 cells/sample). Error bars denote standard error of the mean (SEM).�� , P < 0.001, extremely statistically significant; ns, not statistically significant, between the indicated groups, respectively. F, cytokine release from PBMCs activated byhu3F8-BsAb in the absence or presence of IMR-32 neuroblastoma cells. Three whole IgGs (huOKT3, huOKT3-aGlyco, and hu3F8) were used as controls. Mean þ SD.

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tumor selectivity and in vivo efficacy in preclinical models, wasexceptional.

To exploit the Fc-independent T cell–mediated effectors, weadopted an IgG-scFv BsAb platform to develop the hu3F8-BsAb.We surveyed a number of uniquely different bivalent formats,

including chemical conjugation (29), dual-variable-domain(DVD), or attaching huOKT3 scFv to different positions in thehu3F8 IgG (C-terminal of heavy chain or C-terminal of lightchain; ref. 30), and found that the last option gave the bestfunctionality. Although this format has been previously described

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Figure 5.Efficacy of hu3F8-BsAb in humanized DKO mice. Treatment schedules, doses of BsAbs (hu3F8-BsAb or control hu3F8-C825), and effector cells (PBMC, ATC,or fresh T cells) are detailed in Materials and Methods and Results. Data shown as mean þ SEM (n ¼ 5); � , P < 0.01 determined by the Student t test whentreatment groups (ATC/PBMC þ hu3F8-BsAb) were compared with the corresponding control groups, respectively. A, s.c. tumor plus subcutaneous effectorcells (1:1 mixing) model: % tumor growth of IMR-32 neuroblastoma. B, s.c. tumor plus i.v. effector cells model: % tumor growth of M14 melanoma. C–E, i.v. tumorplus i.v. effector cells model: C, bioluminescence changes of IMR-32 neuroblastoma during treatment; D, bioluminescence changes of M14 melanoma duringtreatment; E, representative images at day 31.






(21), it has never been used for engagement of T cells forimmunotherapy. This hu3F8-BsAb represents a log-fold improve-ment over the monovalent anti-GD2 5HLDS(15)BA(Y) BsAb(BiTE format; ref. 25), primarily due to the far superior innatebinding affinity (KD) to GD2 (5.8 vs. 250 nmol/L), resulting in

substantially higher in vitro killing potency (EC50 in fmol/L rangevs. pmol/L range). Because hu3F8-BsAb is four times the size ofthe monovalent BiTE format and retains the binding to FcRn (seebelow), its half-life is measured in days instead of minutes (datanot shown). This should obviate the need for continuous infusion

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Figure 6.The role of monocytes in antitumor activity. A, M14 s.c. tumormodel as detailed in Fig. 5B, with treatments of one dose of PBMCs (2� 107 cells i.v.) at day 15, and twodoses of BsAbs (40 mg i.v.) at days 13 and 16. Representative images of IHC staining of tumor sections collected 5 days after i.v. PBMCs were shown. B, s.c.tumor model as detailed in Fig. 5A, with 5 million each of IMR-32 mixed with PBMCs, or subpopulations processed from 5 million PBMCs. PBMCs plus hu3F8-C825as control group, while all other four groups plus hu3F8-BsAb. Treatment schedule of BsAbs (5 mg i.v.) were indicated in the figure. C, cytokine release fromthe same PBMC subpopulations as described in Fig. 6B, activated by hu3F8-BsAb in the presence of M14 cells. D, 51Cr release of the same PBMC subpopulationswith M14 cells (E:T ¼ 50:1) in the presence of hu3F8-BsAb. PBMC subpopulations were first incubated with BsAb for 18 hours before 51Cr-labeled target cellswere added for 4 hours. E, tumors (from groups in Fig. 6B) were harvested 9 days after starting BsAb treatment, and CD3(þ) T cells by IHC staining of tumorsections were counted from two randomly selected fields (�200 magnifications).

Xu et al.





to be clinically effective. Indeed, hu3F8-BsAb had much better invivo efficacy than 5HLDS(15)BA(Y), despite dosing only two tothree times versus six to seven times weekly (Supplementary Fig.S3AandS3B). Thebigger size of thehu3F8-BsAb could also reducethe likelihood of leakage into the CNS and neurotoxicity, which isa major adverse side effect of blinatumomab.

The study of cellular mechanism behind how hu3F8-BsAbworked in vivo, especially in the presence of PBMCs, revealed animportant role of monocytes during this T cell–mediated antitu-mor effect. In the presence of hu3F8-BsAb, T cells extravasated toinfiltrate solid tumors (Fig. 6A). Monocytes followed the track ofT cells. Once inside the tumor, these monocytes/macrophagessustained T-cell proliferation and/or survival and also contributedto its antitumor effect (Fig. 6B and E). The co-migration of T cellsand monocytes into the tumor stroma was highly suggestive of across-talk between the different infiltrating white-cell popula-tions, possibly through chemokines or cytokines. Because mono-cytes or macrophages could not by themselves exploit the tumorspecificity of the BsAb (because this BsAb was devoid of all Fcreceptor binding), this cross-talk was most likely orchestrated bytumor-infiltrating T cells. The role of auxiliary leukocytes insupporting T cell–mediated tumor ablation has been observedrecently by other groups,who showed thatmacrophages and IFNgwere critical for eradicating large solid tumors by destroyingstroma cells that support tumor growth (31).

OKT3 can cause cytokine release syndrome, although manage-able and rarely reaching the life-threatening cytokine stormsexperienced by superagonist anti-CD28 TGN1412 (14). Cytokinestorm is known to be FcgR-dependent, with severity mainlycorrelating with IL2 and/or IL6 release (14, 32, 33). The IgG-scFvplatformwas purposely adopted to reduce CD3 binding to T cells(Fig. 2B), andN297A (aglycosylation) introduced to eliminate allFcgR binding to accessory cells, thereby minimizing spontaneousT-cell activation and cytokine release in the absence of tumortargets (Fig. 4F). In fact, IL2 and IL6 released by hu3F8-BsAb werecomparable with the equivalent monovalent BiTE format, andless than the chemical conjugate of hu3F8-IgG � OKT3 (Supple-mentary Fig. S4). Although the singleN297Amutation eliminatedFcgRbinding, it did not affect protein A or FcRn affinities (34, 35),or in vivo pharmacokinetics (36). Indeed, hu3F8-BsAb had noFcgR binding by Biacore, no antibody-dependent cell-mediatedcytotoxicity (ADCC), and minimal complement-mediated cyto-toxicity (CMC) functions in vitro, while retaining Biacore bindingto FcRn (data not shown). Because aglycosylated OKT3 is knowntobemuch lessmitogenic, triggering only partial signaling both invitro and in vivo (15), and aglycosylated humanized anti-CD3antibody (otelixizumab; ref. 35) was safe in humans (37), weexpect hu3F8-BsAb to have an acceptable safety profile. Anothercritical consideration is the pain side effects observed during anti-GD2 therapy (5, 6), generally ascribed to complement activation(24). Removing CMC in hu3F8-BsAb should reduce this sideeffect. The absence of pain side effect when T cells were driven byGD2-chimeric antigen receptors (CAR) further supported explo-ration of this T-cell approach (38). Moreover, aglycosylated BsAbcan be produced in nonmammalian or cell-free systems providingsignificant advantages in speed and cost during manufacture,bypassing problems associated with glycan heterogeneity of con-ventional antibodies (39).

Onehighlight of hu3F8-BsAb is the exceptional potency againsta wide range of different types of GD2(þ) tumor cells (Table 1).With in vitro EC50 at femtomolar concentrations (<50 molecules

of hu3F8-BsAb per T cell), its potency is more than 1,000-foldhigher than that of the parental hu3F8 IgG in a typical ADCCassayusing CD16-transfected NK92 cells (7). This in vitro potencytranslated into a robust and enduring antitumor effect in xeno-graftmodels, prolonging tumor-free survival (Supplementary Fig.S1). It is reassuring that T cells in the presence of hu3F8-BsAbdisplayed >105 safety margin (based on EC50) against normaltissue cells including cardiac myocytes, hepatocytes, adrenal cor-tical cells, renal mesangial cells, or pulmonary alveolar epithelialcells (Fig. 3B). Similar to m3F8 (5) and hu3F8 IgG1 (40),although hu3F8-BsAb plus T cells showed cytotoxicity to GD2(þ) neurons (data not shown), the absence of acute or long-termneuronal toxicity in anti-GD2 clinical trials was likely due to theBBB.

Although many different platforms of T cell–engaging BsAbhave been described, only one (catumaxomab) has beenapproved for malignant ascites by the European Medicines Agen-cy, and none so far has been proven to be superior when onebalances potency and toxicity (19).When comparedwith the onlyanti-GD2 BsAb (ektomab), built using the catumaxomab plat-form and now in preclinical testing, hu3F8-BsAb has substantialdifferences. Ektomab was built from mouse ME361, with lowaffinity for GD2 compared with 3F8 (41) and cross-reactive withGD3 (42). Its anti-CD3 was derived from a rat IgG2b antibody.These murine and rat components are expected to induce neu-tralizing antibodies in humans, and unlikely to allow repeatedadministrations. Ektomab was built using a quadroma-basedformat known to be relatively inefficient (19). Its in vitro potency(43) was substantially (>500-fold based on EC50) lower than thatof hu3F8-BsAb. More importantly, it was made with high Fcaffinity for human FcR to enhance binding to dendritic cells(44) to produce its vaccination effect (45). With this intact Fcfunction, it runs a higher risk of cytokine storm syndrome. Forexample, for catumaxomab, doses above 150mg over 6 hours haveencountered dose-limiting toxicities consistent with cytokinestorm (46).

In summary, this study shows the first demonstration of asuccessful IgG-scFv BsAb platform for engaging T cells to targetcarbohydrates for cancer immunotherapy. This BsAb for retarget-ing T cells is built with structural considerations for bivalencytoward the target, no Fc function, minimal spontaneous cytokinerelease, and long serum half-life. With the excellent antitumoractivity both in vitro and in vivo, and the large safety margin,hu3F8-BsAb has considerable clinical potential. This platformmay be relevant for other tumor or antigen systems, and likely hasbroad implications for therapeutic lymphocyte-redirecting BsAbin general.

Disclosure of Potential Conflicts of InterestNK. V. Cheung has ownership interest (including patents) in scfv constructs

of anti-GD2 antibodies, therapy-enhancing glucan, use of mAb 8H9, methodsfor preparing and using scFv, GD2 peptidemimics,methods for detectingMRD,anti-GD2 antibodies, generation and use of HLA-A2–restricted peptide-specificmAbs and CARs, high-affinity anti-GD2 antibodies, and multimerization tech-nologies. No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: H. Xu, NK. V. CheungDevelopment of methodology: H. Xu, M. Cheng, M. Huse, NK. V. CheungAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): H. Xu, M. Cheng, Y. Chen, M. HuseAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): H. Xu, M. Cheng, Y. Chen, M. Huse, NK. V. Cheung






Writing, review, and/or revision of themanuscript:H. Xu,M. Cheng,M.Huse,NK. V. CheungAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): H. Xu, H. Guo, NK. V. CheungStudy supervision: H. Xu, NK. V. Cheung

AcknowledgmentsThe authors thank Dr. Mamoru Ito of the Central Institute for Experimental

Animals (Kawasaki, Japan) for providing the DKO mice, Dr. Gloria Koo,Hospital for Special Surgery (New York, NY), for her advice in handling DKOmice, MSK Small-Animal Imaging Core Facility and Molecular Cytology CoreFacility for providing technical services, Dr. Mahiuddin Ahmed for his expertisein antibody structural design, and Dr. Irene Cheung for reviewing the article.

Grant SupportThis study was supported, in part, by grants from the Band of Parents, Kids

Walk for Kids with Cancer NYC, Cookies for Kids' Cancer, Robert SteelFoundation, Geoffrey Beene Cancer Research Center of MSK, and the Technol-ogy Development Fund of MSK. Technical service provided by the MSK Small-Animal Imaging Core Facility and Molecular Cytology Core Facility weresupported, in part, by the NIH Cancer Center Support Grant P30 CA008748.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received December 8, 2014; accepted December 10, 2014; publishedOnlineFirst December 26, 2014.

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2015;3:266-277. Published OnlineFirst December 26, 2014.Cancer Immunol Res Hong Xu, Ming Cheng, Hongfen Guo, et al. Using Bispecific Humanized AntibodyRetargeting T Cells to GD2 Pentasaccharide on Human Tumors

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Retargeting T Cells to GD2 Pentasaccharide on Human Tumors ... · T-cell retargeting bispeciﬁc antibodies (BsAb) also haveclinicalpotential,butit is thusfar onlyeffectiveagainst