In Vitro and In Vivo Characterization of a Novel -Like Single-Chain TCR Human IgG1 Fusion

This information is current as Luis A. Mosquera, Kimberlyn F. Card, Shari A. of September 26, 2021. Price-Schiavi, Heather J. Belmont, Bai Liu, Janette Builes, Xiaoyun Zhu, Pierre-Andre Chavaillaz, Hyung-il Lee, Jin-an Jiao, John L. Francis, Ali Amirkhosravi, Richard L. Wong and Hing C. Wong

J Immunol 2005; 174:4381-4388; ; Downloaded from doi: 10.4049/jimmunol.174.7.4381 http://www.jimmunol.org/content/174/7/4381

References This article cites 41 articles, 19 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/174/7/4381.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

In Vitro and In Vivo Characterization of a Novel Antibody-Like Single-Chain TCR Human IgG1 Fusion Protein1

Luis A. Mosquera,* Kimberlyn F. Card,* Shari A. Price-Schiavi,* Heather J. Belmont,* Bai Liu,* Janette Builes,* Xiaoyun Zhu,* Pierre-Andre Chavaillaz,* Hyung-il Lee,* Jin-an Jiao,† John L. Francis,‡ Ali Amirkhosravi,‡ Richard L. Wong,§ and Hing C. Wong2*

We have constructed a protein composed of a soluble single-chain TCR genetically linked to the constant domain of an IgG1 H chain. The Ag recognition portion of the protein binds to an unmutated derived from human p53 (aa 264–272) presented in the context of HLA-A2.1, whereas the IgG1 H chain provides effector functions. The protein is capable of forming dimers, specifically staining tumor cells and promoting target and effector conjugation. The protein also has potent antitumor effects Downloaded from in an in vivo tumor model and can mediate cell killing by Ab-dependent cellular cytotoxicity. Therefore, single-chain TCRs linked to IgG1 H chains behave like Abs but possess the ability to recognize Ags derived from intracellular targets. These fusion represent a novel group of immunotherapeutics that have the potential to expand the range of tumors available for targeted beyond those currently addressed by the conventional Ab-based approach. The Journal of Immunology, 2005, 174: 4381–4388. http://www.jimmunol.org/ argeted for has shown promise as a safe, but not on distant metastases of that tumor. Finally, tumor Ags effective alternative to highly toxic chemotherapeutic ap- shed from the surface of tumor cells can occupy the binding sites T proaches. The clinical and commercial success of human- of antitumor-specific Abs, reducing the number of active mole- ized mAbs Herceptin and , which are used to treat met- cules and resulting in decreased tumor cell death (10). Thus, it astatic breast cancer and B cell lymphoma, respectively, has would be useful to be able to target a broader range of tumor Ags prompted the development of a wide variety of Ab-based therapies that may be derived from either intracellular or extracellular pro- for both solid and blood-borne tumors (1–3). The mechanisms of teins that are not subject to the problems of Ag shedding. action of therapeutic Abs include direct antiproliferative and pro-

The tumor suppressor protein, p53, is mutated and/or overex- by guest on September 26, 2021 apoptotic effects on their targets (4, 5). In addition, through their pressed in a wide variety of human malignancies (11–13), and its Fc domains, Abs may activate Ab-dependent cellular cytotoxicity overexpression can correlate with poor prognosis (14, 15). Thus, 3 (ADCC) (6–8) and phagocytosis (9), which may aid in tumor p53 may be targeted as a general tumor Ag, but because it is an elimination. intracellular protein, it cannot be targeted by Ab-based therapies. Despite the promise of Ab-based immunotherapy, there are a Instead, p53 presented in the context of HLA have been number of potential problems with Ab-based tumor targeting. Abs used as target Ags for -based cancer therapy approaches, only recognize Ags expressed on the cell surface; however, there including tumor vaccination and ex vivo T cell stimulation and are other tumor Ags that are expressed as intracellular proteins. expansion (16, 17). Although these approaches have shown some Moreover, because many tumor-specific Ags are derived from an promise in animal models and some early-stage clinical trials, both aberrant expression of cell type-specific proteins, many tumor Ags of these methods are time consuming, laborious, expensive, and are expressed on only a small number of tumor types, potentially lack standardization, making them unsuitable for treating large pa- limiting the number of tumors that could be treated by any one- tient populations (16, 17). Thus, a therapeutic molecule that can . Furthermore, tumor Ag expression is often het- recognize and bind to Ags derived from intracellular proteins such erogeneous, where the Ag may be expressed on the primary tumor as p53 would significantly broaden the range of tumor Ags that can be targeted. *Altor BioScience Corporation, Miramar, FL 33025; †Hematech LLC, Sioux Falls, We have described the construction, expression, and character- SD 57106; ‡Florida Hospital Cancer Institute, Orlando, FL 32804; and §University of ization of a soluble three-domain mouse single-chain TCR Florida, Gainesville, FL 32612 (scTCR), which recognizes a wild-type human p53 peptide (aa Received for publication October 29, 2004. Accepted for publication January 264–272) presented in the context of HLA-A2.1 (264scTCR) (18). 23, 2005. As an IL-2 fusion protein, the 264scTCR retains its MHC-re- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance stricted, peptide-specific, Ag-binding properties, whereas the IL-2 with 18 U.S.C. Section 1734 solely to indicate this fact. portion binds to IL-2R and retains biological activity. Moreover, 1 This work was supported in part by National Institutes of Health Grant these studies demonstrate that this fusion protein mediates conju- 1R43CA88615-01. gation of target and effector cells, exhibits favorable pharmacoki- 2 Address correspondence and reprint requests to Dr. Hing C. Wong, Altor Bio- netics in mice, binds to target tumor cells, and has antitumor Science Corporation, 2810 North Commerce Parkway, Miramar, FL 33025. E-mail address: [email protected] effects (18). 3 Abbreviations used in this paper: ADCC, Ab-dependent cellular cytotoxicity; In the studies reported here, we constructed a scTCR fusion scTCR, single-chain TCR; HE, dihydroethidium. protein that resembles an Ab. The Ag recognition portion of the

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 4382 A SOLUBLE TCR/IgG1 FUSION PROTEIN

fusion protein is composed of a 264scTCR, and a human IgG1 H IgG1 was purified from cell culture supernatant fluid by immunoaffinity chain provides the effector function domain. Fusion of the using the (BF1) 8A3.31 mAb coupled to a Sepharose 4B 264scTCR to the IgG1 H chain allows for dimer formation via the column (Amersham Biosciences). The purified samples were concentrated and buffer-exchanged into PBS using an Ultrafree-15 centrifugal filter with natural disulfide bonding in IgG1 molecules. We evaluate the abil- a 30-kDa molecular mass cutoff membrane (Millipore). The TCR fusion ity of our fusion protein to bind specifically to cells in vitro as well protein samples were stored at 2°C to 8°C (short term) or at Ϫ80°C (long as reduce metastasis in an in vivo experimental metastasis model. term) for biochemical and functional analyses. We also explore the possible mechanisms by which this protein might function. Our data indicate that this molecule exhibits many SDS-PAGE major characteristics of a mAb and that it is possible to create SDS-PAGE was performed under reducing or nonreducing conditions us- scTCR-based, Ab-like molecules that recognize Ags derived from ing 4–20% polyacrylamide gels (NOVEX) and the NOVEX EX-Cell II intracellular proteins such as p53. These novel targeted therapies system. SDS-PAGE gels were stained with Coomassie blue. could prove to be potent immunotherapeutics. ELISA Materials and Methods For determination of 264scTCR/IgG1 concentration from transfected CHO.K1 cells, Maxisorp 96-well plates (Nunc) were coated with anti- Materials Ј human IgG F(ab )2 (Pierce). Purified 264scTCR/IgG1 fusion protein was A2.1 264 CTL clone no. 5 was derived by limiting dilution cloning (19) added and incubated for1hatroom temperature. After washing, bound from a CTL line specific for the human p53 (aa 264–272) peptide gener- fusion protein was detected with HRP-conjugated sheep anti-human IgG1 ated in HLA-A2.1-transgenic mice (11). Expression vectors for producing (The Binding Site) and ABTS substrate (BioFX) and then quenched with 1% SDS (BioFX). Concentration of 264scTCR/IgG1 was determined by peptide-loaded HLA-A2 tetramers were a generous gift from Dr. John Alt- Downloaded from man at Emory University (Atlanta, GA). CHO.K1 Chinese hamster ovary, comparison to a known Ab standard curve. For demonstration of peptide/ T2 human lymphoblast, U937 human promyeloma cells, H57-597, BB7.2, MHC binding, streptavidin plates were coated with p53 (aa 264–272)- loaded HLA-A2 monomers, and purified 264scTCR/IgG1 fusion protein and (BF1) 8A3.31 hybridoma cell lines were purchased from American Type Culture Collection. T2 human lymphoblast cells are positive for was added and incubated for1hatroom temperature. After washing, HLA-A2.1 but deficient in TAP 1 and 2 proteins, which allow them to bound fusion protein was detected with HRP-conjugated sheep anti-human display empty MHC molecules on their surface that can then be loaded IgG1 (The Binding Site) and ABTS substrate (BioFX) and then quenched with exogenous peptide (20). The H57-597 hybridoma produces a mAb with 1% SDS (BioFX). For all ELISAs, absorbance was measured at 405 that recognizes an epitope in the murine TCR-␤ constant region, and the nm using a 96-well plate reader (Bio-Tek Instruments). http://www.jimmunol.org/ BB7.2 hybridoma produces a mAb that specifically recognizes an epitope ␣ on the 2 domain of HLA-A2. The (BF1) 8A3.31 hybridoma produces a Cell staining with 264scTCR/IgG1 mAb that recognizes an epitope in the human TCR-␤ constant region. A T2 cells pulsed with either p53 (aa 149–157) or p53 (aa 264–272) peptide highly metastatic subclone of the human melanoma cell line A375, A375- were incubated with 8.0 pM 264scTCR/IgG1 or 264scTCR/trIgG1 fusion C15N, was used for in vivo metastasis studies and was maintained by Dr. protein in 1% FBS in PBS for 30 min at room temperature. Bound fusion John Francis at the Florida Hospital Cancer Research Institute (Orlando, protein was detected with PE-conjugated anti-TCR C␤ mAb. Samples were FL) (21). Anti-TCR C␤ mAb H57-597, anti-murine TCR V␤3 mAb, and washed with 1% FBS in PBS before analysis on a FACScan flow cytom- FITC-labeled goat anti-mouse IgG were obtained from BD Pharmingen. etry instrument (BD Biosciences). Anti-IgG F(abЈ) and HRP-conjugated anti-IgG were purchased from 2 U937 cells were cultured for 48 h with 62.5 ng/ml human IFN-␥ to

Pierce and The Binding Site, respectively. All cell culture media and ad- by guest on September 26, 2021 increase expression of FcR on the cell surface. Cells (2 ϫ 105/sample) were ditives were purchased from Mediatech, and all cell culture materials were washed and incubated with or without 4 ␮g of 264scTCR/IgG1 fusion purchased from Nunc unless otherwise noted. All mice were purchased protein for 30 min at room temperature. After washing, cells were incu- from Harlan. Biotinylated peptide/HLA-A2 reagents were generated as de- bated with 4 ␮g of PE-conjugated, HLA-A2/p53 (aa 149–157)- or (aa scribed previously (22). 264–272)-loaded tetramers for 30 min at room temperature. After washing, Cell culture the samples were analyzed by flow cytometry. A375 melanoma cells were stained with either 264scTCR/IgG1 fusion All cell lines were maintained in complete culture medium comprising protein or control fusion protein CMVscTCR/IgG1. A375 melanoma cells IMDM supplemented with 10% FBS plus 2 mM L-glutamine and grown at were cultured on coverslips for 24 h. The cells were fixed with 3.7% form- 37°C with 5% CO2. A375-C15N cells were maintained in RPMI 1640 with aldehyde for 5 min and washed twice with washing buffer (0.5% BSA and 10% heat-inactivated FBS, penicillin, and streptomycin (Invitrogen Life 0.1% sodium azide in PBS). The cells were stained with 10 ␮gof Technologies). For induction of Fc␥R, U937 cells were cultured for 48 h 264scTCR/IgG1 or CMVscTCR/IgG1 fusion protein in 200 ␮lofPBS in IMDM supplemented with 10% FBS, 2 mM L-glutamine, and 62.5 ng/ml containing 5% normal goat serum for 45 min at 23°C. The cells were ␥ ␮ Ј human IFN- (R&D Systems). washed twice and incubated with 6 g of FITC-conjugated F(ab )2 goat anti-human IgG Fc (Jackson ImmunoResearch Laboratories). The cells Constructs were washed three times with equilibration buffer (Molecular Probes). The TCR was cloned from the T cell clone A2.1 264 no. 5 as de- Coverslips were mounted on glass slides with anti-fade reagent in glycerol scribed previously (18). We designated the scTCR derived from this T cell buffer (Molecular Probes) and sealed with nail polish. The slides were clone 264scTCR. To generate the 264scTCR/IgG1 expression construct, documented using a Nikon epifluorescence microscope (Nikon) with a the three-domain single-chain 264scTCR was amplified using the SPOT RT camera and SPOT RT software v3.2 (Diagnostic Instruments). 264scTCR/IL-2 fusion protein construct as a template (18). The scTCR For flow cytometric analysis, A375 cells were detached with 10 mM fragment was ligated into an Ab H chain , replacing the EDTA in PBS (pH 7.4) and washed twice with washing buffer. The cells Ab variable region to yield a scTCR fused to a human IgG1 H chain region. were fixed with 3.7% formaldehyde for 5 min and washed twice. Cell ␮ A control fusion construct, 264scTCR/trIgG1, was created by truncating staining was conducted using 4 g of 264scTCR/IgG1 or CMVscTCR/ ␮ the IgG1 heavy domain before the hinge region, which prevented disulfide IgG1 fusion protein in the presence or absence of 20 g of HLA-A2.1/p53 bonding. The CMV scTCR (CMVscTCR) was cloned from CTLs stimu- (aa 264–272) tetramers for 45 min at 23°C. The cells were washed once ␮ Ј lated with HLA-A2-restricted CMV-pp65 peptides. The IgG1 fragment and incubated with 6 g of FITC-conjugated F(ab )2 goat anti-human IgG ␥ was amplified from 264scTCR/IgG1 DNA to create the CMVscTCR/IgG1 Fc as above. After washing twice, the cells were resuspended and ana- construct. For production of the fusion proteins in mammalian cells, lyzed on a FACScan flow cytometry instrument (BD Biosciences) with CHO.K1 cells were electroporated using a Bio-Rad Gene Pulser, followed CellQuest software (BD Biosciences). by limiting dilution cloning and selection in medium containing 1 mg/ml G418. Affinity determinations Protein purification The affinity of the dimeric 264scTCR/IgG1 fusion protein for HLA-A2.1/ p53 (aa 264–272) was compared with the affinity of the monomeric 264scTCR/IgG1 and 264scTCR/trIgG1 were purified from cell culture su- 264scTCR/trIgG1 fusion protein by surface plasmon resonance (Biacore). pernatant fluid by immunoaffinity chromatography using the H57-597 mAb Approximately 1300 resonance units of biotinylated p53 (aa 264–272)/ coupled to a Sepharose 4B column (Amersham Biosciences). CMVscTCR/ HLA-A2.1 complex were immobilized on a streptavidin sensor chip. Each The Journal of Immunology 4383 surface was used one time only. The average affinity constants were cal- culated based on data from three different scTCR fusion protein concen- trations (5, 10, and 20 ␮M) using the Biacore computer software Biae- valuation 3.1 (Biacore). Cell conjugation T2 cells pulsed with either p53 (aa 264–272) peptide or (aa 149–157) peptides were labeled with 12.6 ␮g/ml dihydroethidium (HE) (Molecular Probes), and U937 cells were labeled with 80 ng/ml calcein AM (Molec- ular Probes). After washing, the two populations of labeled cells were mixed together at a 1:1 ratio in the presence or absence of 2.5 ␮gof 264scTCR/IgG1 fusion protein for 20 min at room temperature. Cells were then analyzed on a FACScan flow cytometry instrument. Pharmacokinetics in mice FIGURE 1. The 264scTCR/IgG1 fusion protein. Schematic representa- For all experiments involving animals, principles of laboratory animal care tion of the domain structure of the 264scTCR/IgG1 fusion protein. were followed, as well as specific national laws where applicable. Female athymic nude mice were injected i.v. via the lateral tail vein with 250 ␮g of 264scTCR/IgG1 fusion protein diluted with PBS to a total volume of 100 ␮l. Serum was collected from one group of mice not injected with constant domain of the human IgG1 H chain (Fig. 1). The cysteine 264scTCR/IgG1 to establish background levels. Serum was collected by residues in the hinge region of the H chain are intact, which allows Downloaded from tail bleed from the injected groups at 15 and 30 min and 1, 2, 4, 8, and 24 h. for appropriate disulfide bonding of the H chain regions and thus Blood samples were centrifuged at 14,000 ϫ g at 4°C for 10 min, and serum was collected and stored at Ϫ80°C until use. 264scTCR/IgG1 con- covalently linked dimer formation of the 264scTCR/IgG1 fusion centrations were determined by ELISA using anti-TCR V␤3 mAb for cap- protein. ture and HRP-conjugated sheep anti-human IgG1 Ab for detection. Expression of TCR/IgG1 fusion protein in mammalian cells Experimental metastasis assay To characterize the soluble 264scTCR/IgG1 fusion protein,

ϫ 5 http://www.jimmunol.org/ Female athymic nude mice were injected with 5.0 10 A375-C15N cells CHO-K1 cells were stably transfected with the 264scTCR/IgG1 via the lateral tail vein on study day 0. The tumors were allowed to estab- lish for 3 days and then the animals were injected i.v. with varying doses expression vector. To determine the concentration of secreted pro- Ј of either 264scTCR/IgG1 (0.1–30 mg/kg in 100 ␮l of total volume), PBS, tein, the fusion protein was captured with an anti-IgG F(ab )2 and or ␣Stx-2 (an irrelevant Ab isotype-matched control at 30 mg/kg in 100 ␮l detected with an anti-IgG1 HRP conjugate. Concentrations of the of total volume) on study days 3, 7, 10, 14, 17, 21, 28, and 35 posttumor 264scTCR/IgG1 fusion protein were estimated based on compar- cell injection. Forty-two days after tumor cell injection, all animals were ison to an IgG1 Ab standard. To determine whether secreted pro- sacrificed humanely, the lungs were removed and fixed in Bouin’s solution, and surface pulmonary tumor nodules were counted. Two observers tein can bind peptide-loaded HLA, the fusion protein was captured counted tumor nodules on each lung, and the average counts were re- with biotinylated HLA-A2/p53 (aa 264–272) monomers on corded. The ␣Stx-2 Ab, a chimeric IgG1 Ab (mouse variable/human H and streptavidin-coated plates and detected with anti-IgG1 HRP con- by guest on September 26, 2021 L chain constant region), was created in our lab and has been described jugate. Positive signals for ELISAs in either format indicate that previously (23). This Ab recognizes an epitope on Escherichia coli 0157 shigatoxin protein and was used for these studies as an irrelevant isotype the transfected cells secrete 264scTCR/IgG1 fusion protein that is control. assembled properly, folded, and secreted from the transfected cells. Moreover, the TCR portion of 264scTCR/IgG1 retains pep- ADCC tide-specific MHC binding ability in this fusion protein format. T2 cells were loaded with p53 (aa 264–272) peptide and used as target The 264scTCR/IgG1 fusion protein was purified from trans- cells. Target cells were labeled with 50 ␮g/ml calcein AM (Molecular fected cell supernatants by immunoaffinity chromatography using Probes) for 30 min at 37°C and washed twice. Effector PBMCs were iso- lated from buffy coats (Community Blood Centers of South Florida) using the one-step gradient method with Histopaque-1077 (Sigma-Aldrich). To measure ADCC activity, 2 ϫ 106 effector cells were incubated with 2 ϫ 104 target cells in 0.25 ml of complete medium with either 264scTCR/IgG1 or CMVscTCR/IgG1 fusion protein at 20 ␮g/ml. The fluorescent intensity of culture supernatant fluid was measured after 4 h with a fluorescent plate reader (excitation ϭ 485 nM; emission ϭ 538 nM; cutoff ϭ 530 nM). ADCC activity was calculated using the following formula: percentage of cytotoxicity ϭ (fluorescent intensity of test sample Ϫ fluorescent intensity of target cells with medium)/(fluorescent intensity of target cells treated with 0.04% Triton X-100 Ϫ fluorescent intensity of target cells with me- dium) ϫ 100. Data are reported as the mean of triplicate determinations Ϯ SEM. Statistical significance was determined by paired t test analysis. Results Generation of TCR fusion protein constructs We have constructed a fusion protein composed of a three-domain scTCR that recognizes an unmutated peptide from human p53 (aa 264–272) (18) fused to the constant domain of the human IgG1 H chain. The scTCR portion of this fusion protein comprises the TCR ␣ ␤ ␤ V region linked to the V /C regions via a flexible linker (G4S)4 FIGURE 2. Production of 264scTCR/IgG1 fusion protein in transfected ␤ ␤ (24). The C domain, which is directly linked to the V domain, Chinese hamster ovary cells. The secreted fusion protein was purified by is truncated at the amino acid residue just before the final cysteine, immunoaffinity chromatography and subjected to SDS-PAGE under either removing the transmembrane and cytoplasmic domains, to gener- reducing (R) or nonreducing (N) conditions. SDS-PAGE gels was stained ate a soluble scTCR molecule. This scTCR is linked directly to the with Coomassie brilliant blue. 4384 A SOLUBLE TCR/IgG1 FUSION PROTEIN an anti-murine TCR C␤ mAb, H57-597. Purified protein was sub- Table I. Affinity determination with 264scTCR/IgG1 fusion proteina jected to SDS-PAGE and Coomassie staining (Fig. 2). Under re- ϳ ducing conditions, the major protein band was 75–80 kDa, kassociation kdissociation ϫ which is consistent with the calculated molecular mass for this Molecule (1/M s) (1/s) KD (M) protein based on its deduced amino acid sequence. Under nonre- 264scTCR/IgG1 1.1 ϫ 104 4.8 ϫ 10Ϫ3 5.6 ϫ 10Ϫ7 ducing conditions, the molecular mass of the predominant protein 264scTCR/trIgG1 5.4 ϫ 104 1.1 ϫ 10Ϫ3 2.2 ϫ 10Ϫ6 ϳ band was 170–180 kDa. The apparent shift in molecular mass of a The affinity of the dimeric 264scTCR/IgG1 fusion protein for HLA-A2.1/p53 (aa the 264scTCR/IgG1 under reducing vs nonreducing conditions is 264– 272) was compared with the affinity of the monomeric 264scTCR/trIgG1 fusion to be expected because there are cysteine residues in the H chain protein by surface plasmon resonance. regions that form disulfide bonds. Thus, as with an IgG1 Ab, the 264scTCR/IgG1 fusion protein forms dimers. Binding of the Fc domain of 264scTCR/IgG1 to FcR MHC/peptide binding of the TCR portion of 264scTCR/IgG1 To demonstrate the ability of the IgG1 Fc portion of the 264scTCR/IgG1 fusion protein to bind to FcR, U937 human pro- The ability of the 264scTCR/IgG1 fusion protein to bind to pep- myeloma cells were stimulated with human IFN-␥ to increase ex- tide-loaded MHC on the surface of APCs was determined by flow pression of the FcR on the cell surface. Stimulated as well as cytometry. T2 lymphoblast cells were loaded with p53 (aa 264– unstimulated cells were incubated with 264scTCR/IgG1 fusion 272) or p53 (aa 149–157) control peptide and incubated with pu- protein, and bound fusion protein was detected with PE-conjugated rified 264scTCR/IgG1 fusion protein. After several wash steps, the HLA-A2.1/p53 (aa 264–272) or HLA-A2.1/p53 (aa 149–157) tet- Downloaded from cells were incubated with PE-labeled anti-TCR mAbs to detect ramers and analyzed by flow cytometry. When the 264scTCR/ cell-bound fusion protein (Fig. 3). T2 cells loaded with p53 (aa IgG1 fusion protein was used, cells stained positively following 264–272) peptide stained positively with 264scTCR/IgG1, incubation with HLA-A2.1/p53 (aa 264–272) tetramers but not whereas cells loaded with p53 (aa 149–157) showed little or no with HLA-A2.1/p53 (aa 149–157) tetramers. Moreover, positive staining, indicating that the 264scTCR/IgG1 could stain cells in a staining with 264scTCR/IgG1 fusion protein was significantly peptide-specific manner. We also attempted to stain peptide-loaded ␥

more intense when the cells were stimulated with human IFN- , http://www.jimmunol.org/ T2 cells with monomeric 264scTCR/trIgG1 fusion protein and which is known to increase the expression of FcR on U937 cells found that no staining was observed on cells loaded with either p53 (Fig. 4). Thus, these data indicate that the IgG1 portion of the (aa 264–272) or p53 (aa 149–157) peptide. These results demon- 264scTCR/IgG1 fusion protein is capable of binding to FcRs. strate that the bivalent nature of 264scTCR/IgG1 greatly increased its ability to stably bind MHC-peptide complexes on the surface of Conjugation of cells mediated by 264scTCR/IgG1 fusion protein cells. In order for the 264scTCR/IgG1 fusion protein to be useful as a therapeutic, it must be able to bring together target and effector Binding affinity of 264scTCR/IgG1 cells through the TCR and Fc components, respectively. To dem- To determine whether the intensity of staining observed with onstrate that the 264scTCR/IgG1 fusion protein can effectively by guest on September 26, 2021 264scTCR/IgG1 could be attributed to a high binding affinity, we conjugate cells, T2 cells were loaded with either p53 (aa 264–272) performed surface plasmon resonance assays. The binding affinity or p53 (aa 149–157) control peptide and then stained with HE.

(KD) of the dimeric 264scTCR/IgG1 with HLA-A2.1/p53 (aa U937 effector cells were differentially stained with calcein AM. 264–272) complexes was estimated to be 5.6 ϫ 10Ϫ7 M, which is The two stained cell populations were mixed, incubated in the significantly higher than the monomeric 264scTCR/trIgG1 (KD of presence or absence of 264scTCR/IgG1, and analyzed by flow 2.2 ϫ 10Ϫ6 M; Table I). The increased affinity of the 264scTCR/ cytometry. When the two cell populations were incubated in the IgG1 fusion protein likely contributes to its ability to bind strongly absence of the 264scTCR/IgG1 fusion protein (Fig. 5, A and B)or to the correct peptide/MHC pair. when the T2 cells were loaded with control peptide p53 (aa 149– 157) and incubated with the U937 cells in the presence of 264scTCR/IgG1 fusion protein (Fig. 5C), the cells remained as

FIGURE 3. MHC/peptide-binding ability of the TCR portion of 264scTCR/IgG1 fusion protein. T2 cells were loaded with p53 (aa 264– FIGURE 4. Binding of the Fc domain of 264scTCR/IgG1 to FcR. rIFN- 272) peptide and stained with either 264scTCR/IgG1 (black thin line) or ␥-stimulated U937 cells were stained with 264scTCR/IgG1 fusion protein 264scTCR/trIgG1 fusion protein (gray thick line), followed by anti-TCR followed by HLA-A2/p53 (aa 264–272) (black thin line) or HLA-A2/p53 C␤ mAb. T2 cells loaded with p53 (aa 149–157) control peptide were also (aa 149–157) (black thick line) tetramers. Unstimulated U937 cells were stained with either 264scTCR/IgG1 (dotted black line) or 264scTCR/ also stained with fusion protein and HLA-A2/p53 (aa 264–272) (black trIgG1 fusion protein (black thick line), followed by anti-TCR C␤ mAb. dotted line) or HLA-A2/p53 (aa 149–157) (gray thick line) tetramers. All All samples were analyzed by flow cytometry. samples were analyzed by flow cytometry. The Journal of Immunology 4385

Tumor cell staining To demonstrate the ability of 264TCR/IgG1 fusion protein to bind to tumor cells, the A375 human melanoma cell line, shown in our laboratory to be HLA-A2.1 and p53 positive (data not shown), was used. The 264TCR/IgG1 fusion protein positively stained the A375 cells (Fig. 6A), while staining with CMVscTCR/IgG1 was not detectable above background (Fig. 6B). Staining of A375 cells with 264scTCR/IgG1 was reduced by the addition of HLA-A2.1/ p53 (aa 264–272) tetramers (Fig. 6C), indicating that 264scTCR/ IgG1 specifically binds to tumor cells via the scTCR portion of the fusion protein.

Serum half-life of 264scTCR/IgG1 in mice The pharmacokinetics of the 264scTCR/IgG1 fusion protein was measured in nude mice. All mice were injected with a single i.v. dose of 264scTCR/IgG1 and showed no obvious signs of toxicity. FIGURE 5. Conjugation of cells mediated by 264scTCR/IgG1. T2 cells were loaded with either p53 (aa 264–272) (B and D) or p53 (aa 149–157) Serum samples were collected at various time points, and the 264scTCR/IgG1 fusion protein concentration was determined us-

(A and C) peptides and then labeled with HE. U937 cells were labeled with Downloaded from calcein AM. Labeled cells were mixed and incubated in the presence (C ing ELISA. In these assays, a maximum concentration of ϳ8.8 and D) or absence (A and B) of 264scTCR/IgG1 fusion protein. Single- ␮g/ml 264scTCR/IgG1 was detected with an apparent serum half- stained regions are marked 1 and 3, and the double-stained cell population life of 4 h (Fig. 7). This is consistent with the apparent serum is marked 2. half-life of the 264scTCR/IL-2 fusion protein (18).

Effect of 264scTCR/IgG1 on tumor growth in nude mice two distinct populations on the flow cytometry histograms (Fig. 5, To determine whether the 264scTCR/IgG1 fusion protein exhibits http://www.jimmunol.org/ A–C, regions 1 and 3). However, when the T2 cells were loaded antitumor activity in vivo, an experimental metastasis assay was with p53 (aa 264–272) peptide and incubated with the U937 cells performed. Female athymic nude mice were injected with the in the presence of the 264scTCR/IgG1 fusion protein (Fig. 5D), a highly metastatic A375 human melanoma subclone, A375-C15N, double-stained population of cells appears (Fig. 5D, region 2, con- and tumors were allowed to establish for 3 days. Animals were jugated cells), indicating that T2 cells were conjugated to U937 then treated with varying doses of 264scTCR/IgG1, ␣-Stx-2 (an cells via the 264scTCR/IgG1 fusion protein. These data demon- irrelevant IgG1 isotype control Ab), or vehicle. Forty-two days strate that the 264scTCR/IgG1 fusion proteins can mediate conju- after tumor cell injection, lung nodules were counted. 264scTCR/

gation of effector cells to target cells. IgG1 reduced lung metastasis in a dose-dependent manner reach- by guest on September 26, 2021 ing maximum reduction of metastasis at the 1 mg/kg dose, whereas the isotype control failed to reduce metastasis (Fig. 8). These data indicate that the 264scTCR/IgG1 fusion protein exhibits potent anti-metastatic activities in vivo and has potential use as a cancer therapeutic in humans.

ADCC One possible mechanism of action for therapeutic Abs is ADCC, which is mediated by the FcR-binding portion of the IgG molecule. Because the 264scTCR/IgG1 fusion protein contains a FcR-bind- ing domain, we investigated whether this fusion protein could me- diate ADCC. T2 cells loaded with p53 (aa 264–272) peptide and labeled with calcein AM were used as target cells, and human PBMCs were used as effector cells. Target and effector cells were mixed together at a 1:100 ratio in the presence of 264scTCR/IgG1

FIGURE 6. Tumor cell staining with 264scTCR/IgG1 fusion protein. Formaldehyde-fixed A375 cells were stained with 264scTCR/IgG1 fusion protein (A) or CMVscTCR/IgG1 fusion protein (B). The images were cap- tured at ϫ400. Fixed A375 cells were stained with 264scTCR/IgG1 (black FIGURE 7. Serum half-life of 264scTCR/IgG1 fusion protein. Nude lines) or CMVscTCR/IgG1 (gray lines) fusion protein in the presence (thin mice were injected with 264scTCR/IgG1 fusion protein, and serum sam- or dotted lines) or absence (thick lines) of HLA-A2.1/p53 (aa 264–272) ples were collected at 15 and 30 min and 1, 4, 8, and 24 h postinjection. tetramers (C). Serum concentrations of 264scTCR/IgG1 were determined by ELISA. 4386 A SOLUBLE TCR/IgG1 FUSION PROTEIN

FIGURE 9. 264scTCR/IgG1 mediation of ADCC. T2 cells loaded with p53 (aa 264–272) peptide were labeled with calcein AM and incubated with human PBMCs (100:1 E:T ratio) in the presence of 264scTCR/IgG1 FIGURE 8. Antitumor effects of 264scTCR/IgG1. Female athymic nude (f) or CMVscTCR/IgG1 ( ) fusion protein. After 4 h, culture supernatants mice were injected with highly metastatic A375-C15N cells and treated were collected, and cell lysis was quantified. with 264scTCR/IgG1 at doses ranging from 0.1 to 30 or 30 mg/kg anti- Stx-2 (an irrelevant IgG1 isotype control Ab) or a dose volume equivalent of PBS. Forty-two days after tumor cell injection, the lungs were removed, vations) have been produced and shown to retain their respective lung nodules were counted, and the mean number of lung nodules was bispecific functions. In another approach, Abs with specificity to plotted. peptide/MHC complexes have been generated and have been used Downloaded from to quantitate peptide/MHC complexes on the surface of APCs and deliver cytotoxic effector toxin to APCs in a peptide-dependent or CMVscTCR/IgG1 fusion protein. Cell killing with 264scTCR/ manner (34, 35). IgG1 ranged from 48 to 89%, while cell killing with CMVscTCR/ TCRs are known to have low affinity for cognate peptide/MHC IgG1 protein was significantly lower ( p ϭ 0.02) (Fig. 9). These complexes due to negative selection in the thymus during the T

data indicate that the 264scTCR/IgG1 protein is capable of medi- cell maturation process (36). In addition, the density of a particular http://www.jimmunol.org/ ating ADCC and that this mechanism may play a role in the an- epitope displayed in the context of MHC on an APC surface is titumor activity of this fusion protein. Given that the population of predictably low (34). These two factors raised significant doubts FcR-positive effector cells in PBMCs used for the ADCC experi- about whether it was feasible to use TCRs as targeting molecules. ment is ϳ15–20% of the population, the high level of killing seen The tumor cell staining results in this study demonstrate that a in these assays suggests that there may be one or more other mech- dimeric form of the scTCR does in fact have sufficient affinity to anisms of action used for cell killing mediated by 264scTCR/IgG1. function as an Ag recognition molecule in vitro. The antitumor effects of the 264scTCR/IgG1 fusion protein in our experimental Discussion metastasis model additionally demonstrate that the Ag-targeting In these studies, we have described the construction and charac- capability of this type of scTCR molecule also functions in vivo. by guest on September 26, 2021 terization of a novel 264scTCR/IgG1 fusion protein, which recog- We are currently performing experiments to determine whether the nizes an unmutated peptide from human p53 (aa 264–272) pre- density of the peptide/MHC complexes on the tumor cell surface sented in the context of HLA-A2.1. The IgG1 H chain domain directly influences the ability of scTCR fusion proteins to target confers FcR binding and allows for dimer formation, and the tumor cells. 264scTCR portion of the molecule confers peptide-specific MHC Affinity plays an important role in tumor retention and penetra- binding. This fusion protein is capable of specifically staining tu- tion by macromolecules such as Abs. It has been shown that in- mor cells and promotes target and effector cell conjugation. The creasing the affinity of Abs increases tumor retention, but at a KD 264scTCR/IgG1 fusion protein also has potent antitumor effects in Ͼ 10Ϫ9 M, tumor retention is not further improved, and penetra- an in vivo tumor model and can mediate cell killing by an ADCC tion is hindered significantly (37, 38). The 264scTCR was derived mechanism of action. Thus, we have demonstrated that it is fea- from a T cell line generated from a HLA-A2.1-transgenic mouse. sible to create scTCR fusion molecules, which are functionally TCRs derived from these mice are CD8 independent and are pos- equivalent to therapeutic mAbs but with the ability to recognize tulated to have high affinity for their cognate peptide/MHC com- target Ags derived from intracellular proteins. plex (11). Our measurements indicate that the affinity of the The development of TCR-based therapeutic agents has been dimeric 264scTCR/IgG1 fusion molecule for peptide/MHC is on ϫ Ϫ7 precluded by the difficulty of producing stable, functionally active the order of 5.6 10 MKD, which is exceptionally high for a TCR molecules in sufficiently large quantities (25–32). We have TCR. Abs with this level of affinity show low levels of tumor overcome this obstacle by constructing soluble, single-chain, retention but very efficient tumor penetration (37, 38). Whether the three-domain TCR molecules comprising the TCR-␣ variable do- unusually high affinity of this scTCR for its cognate peptide/MHC main linked to the TCR-␤ variable and constant regions (18, 33). is the key to successful tumor cell staining and antitumor activity These constructs are active with respect to specific peptide/MHC is unknown but is receiving additional investigation in our labo- binding, easily expressed in large quantities in mammalian cells, ratory. The affinity of scTCR molecules can also be increased sub- and readily purified for use in in vitro and in vivo assays (18). stantially using a variety of in vitro enhancement methods in com- Expression and secretion of scTCR-based proteins in mammalian bination with mutagenesis (33, 39). For example, we have cells offers advantages over bacterial expression systems that re- developed a method wherein scTCRs are expressed quire time-consuming, laborious refolding methods to produce as bacteriophage M13 gene VIII fusions (33). In vitro multimer- TCR-␣/␤ heterodimers (28, 30, 32). In addition, solubly expressed ization methods, such as birA-streptavidin conjugation or cross- scTCRs can be genetically fused with molecules exhibiting effec- linking with liposomes or dendrimers, may also increase the avid- tor functions such as cytokines to create bifunctional-bispecific ity of scTCR molecules. Such multimerization should increase the fusion proteins. For example, scTCR molecules linked to human avidity of a scTCR-based therapeutic agent for its target without IL-2 (18), GM-CSF, and IFN-␣2b (K. Card, unpublished obser- sacrificing Ag recognition specificity. Dimerization using the IgG The Journal of Immunology 4387

H chain illustrates the feasibility of such an approach because the range of tumor or viral Ags for targeted therapies beyond those dimeric 264scTCR/IgG1 fusion protein has a significantly higher currently addressed by the conventional Ab-based approach. affinity for its cognate peptide/HLA complex than the monomeric 264scTCR/trIgG1 protein. Acknowledgments The serum half-life of the 264scTCR/IgG1 molecule is ϳ4h, We thank Dr. Peter Rhode for his thoughtful review of this manuscript. which is consistent with that of our other scTCR fusion proteins (18). Although this apparent serum half-life is short compared with Disclosures other full-length Ab molecules, it is significantly longer that that of The authors have no financial conflict of interest. Fab and single-chain variable fragments. Moreover, having a shorter half-life for a targeted IgG1-based therapeutic may help to References improve on the safety profile of such a targeted therapeutic agent 1. Carter, P., L. Presta, C. M. Gorman, J. B. Ridgway, D. Henner, W. L. Wong, while maintaining the effector function of the FcR-binding portion A. M. Rowland, C. Kotts, M. E. Carver, and H. M. Shepard. 1992. Humanization of an anti-p185HER2 antibody for human cancer therapy. Proc. Natl. Acad. Sci. of the molecule. It is possible that this fusion protein may be USA 89:4285. cleaved in vivo as has been shown for some other Ab-based fusion 2. Leget, G. A., and M. S. Czuczman. 1998. Use of rituximab, the new FDA- proteins (40). Analyzing the blood serum of animals given approved antibody. Curr. Opin. Oncol. 10:548. 3. Pegram, M. D., A. Lipton, D. F. Hayes, B. L. Weber, J. M. Baselga, D. Tripathy, 264scTCR/IgG1 using ELISAs in other formats (different capture D. Baly, S. A. Baughman, T. Twaddell, J. A. Glaspy, and D. J. Slamon. 1998. and detection, i.e., TCR/TCR, TCR/IgG, and IgG/IgG), as well as Phase II study of receptor-enhanced chemosensitivity using recombinant human- ized anti-p185HER2/neu plus cisplatin in patients with SDS-PAGE and immunoblots, may help determine whether this HER2/neu-overexpressing metastatic breast cancer refractory to Downloaded from fusion protein is cleaved in vivo. In addition, it is possible that the treatment. J. Clin. Oncol. 16:2659. fusion protein is taken up rapidly in tissues after administration. 4. Shan, D., J. A. Ledbetter, and O. W. Press. 1998. Apoptosis of malignant human B cells by ligation of CD20 with monoclonal . Blood 91:1644. Biodistribution studies will elucidate whether the fusion protein is 5. Sliwkowski, M. X., J. A. Lofgren, G. D. Lewis, T. E. Hotaling, B. M. Fendly, and taken up in the tissues and, if so, what proportion is taken up J. A. Fox. 1999. Nonclinical studies addressing the mechanism of action of tras- compared with that remaining in circulation. tuzumab (Herceptin). Semin. Oncol. 26:60. 6. van de Winkel, J. G. J., and P. J. Capel. 1993. Human IgG Fc receptor hetero- Mechanisms of action that have been described for therapeutic geneity: molecular aspects and clinical implications. Immunol. Today 14:215. Abs include ADCC, direct blockage of critical survival signals, 7. Wallace, P. K., A. L. Howell, and M. W. Fanger. 1994. Role of Fc ␥ receptors http://www.jimmunol.org/ in cancer and infectious disease. J. Leukocyte Biol. 55:816. and induction of apoptotic signals leading to cell death (4–9). Our 8. Fanger, M. W., L. Shen, R. F. Graziano, and P. M. Guyre. 1989. Cytotoxicity data indicate that the 264scTCR/IgG1 fusion protein is capable of mediated by human Fc receptors for IgG. Immunol. Today 10:92. mounting a potent ADCC-mediated killing of targeted cells, al- 9. Wallace, P. K., P. A. Kaufman, L. D. Lewis, T. Keler, A. L. Givan, J. L. Fisher, M. G. Waugh, A. E. Wahner, P. M. Guyre, M. W. Fanger, and M. S. Ernstoff. though its effect on the unmanipulated tumor cells in vitro is min- 2001. Bispecific antibody-targeted phagocytosis of HER-2/neu expressing tumor imal under similar experimental conditions (data not shown). This cells by myeloid cells activated in vivo. J. Immunol. Methods 248:167. 10. Welch, W. R., J. M. Niloff, D. Anderson, A. Battaile, S. Emery, R. C. Knapp, and result does not eliminate the possibility that ADCC is a possible R. C. Bast, Jr. 1990. Antigenic heterogeneity in human ovarian cancer. Gynecol. mechanism of action for the antitumor activity of the 264scTCR/ Oncol. 38:12. 11. Theobald, M., J. Biggs, D. Dittmer, A. J. Levine, and L. A. Sherman. 1995.

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