The Journal of Immunology

Retargeting of Human T Cells to Tumor-Associated MUC1: The Evolution of a Chimeric Antigen Receptor1

Scott Wilkie,* Gianfranco Picco,* Julie Foster,§ David M. Davies,* Sylvain Julien,* Lucienne Cooper,* Sefina Arif,† Stephen J. Mather,§ Joyce Taylor-Papadimitriou,* Joy M. Burchell,* and John Maher2*‡

MUC1 is a highly attractive immunotherapeutic target owing to increased expression, altered , and loss of polarity in >80% of human cancers. To exploit this, we have constructed a panel of chimeric Ag receptors (CAR) that bind selectively to tumor-associated MUC1. Two parameters proved crucial in optimizing the CAR ectodomain. First, we observed that the binding of CAR-grafted T cells to anchored MUC1 is subject to steric hindrance, independent of glycosylation status. This was overcome by insertion of the flexible and elongated hinge found in immunoglobulins of the IgD isotype. Second, CAR function was highly dependent upon strong binding capacity across a broad range of tumor-associated MUC1 glyco- forms. This was realized by using an Ab-derived single-chain variable fragment (scFv) cloned from the HMFG2 hybridoma. To optimize CAR signaling, tripartite endodomains were constructed. Ultimately, this iterative design process yielded a potent receptor termed HOX that contains a fused CD28/OX40/CD3␨ endodomain. HOX-expressing T cells proliferate vigorously upon repeated encounter with soluble or membrane-associated MUC1, mediate production of proinflammatory cytokines (IFN-␥ and IL-17), and elicit brisk killing of MUC1؉ tumor cells. To test function in vivo, a tumor xenograft model was derived using MDA-MB-435 cells engineered to coexpress MUC1 and luciferase. Mice bearing an established tumor were treated i.p. with a single dose of engineered T cells. Compared with control mice, this treatment resulted in a significant delay in tumor growth as measured by serial bioluminescence imaging. Together, these data demonstrate for the first time that the near-ubiquitous MUC1 tumor Ag can be targeted using CAR-grafted T cells. The Journal of Immunology, 2008, 180: 4901–4909.

he MUC1 is a large transmembrane in Three properties make MUC1 a highly attractive target for can- which an extracellular (MUC1-N) and a membrane span- cer immunotherapy. First, owing to transcriptional up-regulation T ning subunit (MUC1-C) are held together in a noncova- (2) elevated levels are found in many tumors, notably of the breast lent association. The larger MUC1-N subunit primarily consists of and ovary (3, 4). Second, whereas MUC1 is normally confined to the variable number tandem repeat (VNTR)3 domain copied up to the luminal , polarity of expression is lost upon trans- 125 times per molecule. Because each VNTR contains five poten- formation (5). Third, glycosylation of MUC1 is profoundly dys- tial sites of O-linked glycosylation, MUC1 is normally decorated regulated in cancer. Owing to altered glycosyltransferase expres- with a network of branched “core 2-based” glycans. As a result, sion (6, 7), tumor-associated MUC1 contains a preponderance of the MUC1 ectodomain assumes a rigid structure and may extend shorter glycans including Tn, sialyl Tn (STn), T (Thomsen- up to 500 nm from the cell surface (1). Friedenreich), and ST (6–9). Underglycosylation of MUC1 un- masks cryptic within the VNTR, enabling tumor-selective binding by several Abs (3, 4, 10, 11). *The Biology Group and †Department of Immunobiology, King’s Col- lege London School of Medicine, ‡Department of Clinical Immunology and Allergy, In of these properties, it may seem surprising that MUC1 King’s College Hospital National Health Service Foundation Trust; and §Centre for is not an established target in existing therapeutic regimens for Cancer Imaging, Institute of Cancer and the Cancer Research U.K. Clinical Centre, cancer. However, this mucin presents several obstacles to immu- Barts and The London, Queen Mary’s School of Medicine and Dentistry, Department of Nuclear Medicine, St. Bartholomew’s Hospital, London, United Kingdom notherapy. First, the shedding of soluble MUC1 may inhibit Ab Received for publication August 2, 2007. Accepted for publication January 21, 2008. binding of tumor cells (12). Second, pronounced structural diver- The costs of publication of this article were defrayed in part by the payment of page sity results from alternative splicing, variability in VNTR number, charges. This article must therefore be hereby marked advertisement in accordance and altered glycosylation. Third, steric inhibition by MUC1 may with 18 U.S.C. Section 1734 solely to indicate this fact. compromise Ab binding and recruitment of effector function (13). 1 This work was supported by a Royal College of Pathologists/ Health Foundation Finally, tumor-derived MUC1 can impair growth (14) and Senior Clinician Scientist Research Fellowship (to J.M.), Breast Cancer Campaign Project Grant 2003:552 (to J.M.), and a Cancer Research U.K. Programme Grant (to shield transformed cells from killing by NK and T cells (15). S.J.M.). Recently, Ab- and cell-based immunotherapy of cancer has con- 2 Address correspondence and reprint requests to Dr. John Maher, Breast Cancer verged with the development of chimeric Ag receptor (CAR) tech- Biology Group, Division of Cancer Studies, Third Floor Thomas Guy House, King’s nology. By contrast to TCR, CAR are targeted to native tumor- College London School of Medicine, Guy’s Hospital, St. Thomas Street, London SE1 9RT, U.K. E-mail address: [email protected] associated cell surface molecules (16). Most commonly, these 3 Abbreviations used in this paper: VNTR, variable number tandem repeat; BLI, fusion receptors comprise an Ab-derived single-chain variable bioluminescence imaging; CAR, chimeric antigen receptor; CM, conditioned me- fragment (scFv) coupled via hinge and transmembrane elements to ϩ ϩ dium; ffLUC, firefly luciferase; mIgG, mouse IgG; 435-MUC-LUC, MUC1 ffLUC a signaling domain. Although pioneering clinical studies have MDA-MB-435 cell; scFv, single-chain variable Ab fragment; sTn, sialyl Tn. proven disappointing (17, 18), CAR-based approaches that harness Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 both activating and costimulatory signals have proven much more www.jimmunol.org 4902 MUC1 RETARGETED T CELLS active when tested in vitro (16, 19–21) and in murine models (U-CyTech) in 50 ␮lfor1hat37°C. Plates were developed using activator (20, 22). solutions (U-CyTech), air dried, and analyzed using an ImmunoSpot image To develop a broadly applicable immunotherapy for solid tu- analyzer (Bioreader; Bio-Sys). mors, we set out to engineer a MUC1-specific CAR. We present Production of recombinant MUC1-IgG glycoforms evidence that steric hindrance and glycosylation-related MUC1-IgG fusion carrying T (Thomsen-Friedenreich), ST, and heterogeneity constitute important barriers to this endeavor. None- STn were generated as described (7, 26). To produce unglycosylated theless, by sequential engineering both difficulties have been over- MUC1 and MUC1-Tn, CHO-ldlD cells (27) were stably transfected with a come. We report for the first time that the widely expressed tumor MUC1-mIgG2a cDNA (16 VNTR) and grown in serum-free medium. Cul- marker MUC1 is amenable to targeting by CAR-expressing T ture with 1 mM N-acetylglucosamine (GalNAc) yielded Tn-MUC1-IgG, whereas culture without exogenous sugars yielded unglycosylated MUC1- cells. IgG. Supernatants were concentrated by ultrafiltration using an Amicon YM100 filter. Materials and Methods Comparison of Ab binding to recombinant MUC1 glycoforms Recombinant DNA constructs ELISA plates were coated with 500 ng of MUC1-IgG glycoform and CAR were constructed by overlap extension PCR and sequenced before blocked with 1% BSA. Biotinylated MUC1-specific mAbs were added in cloning in the SFG vector (NcoI site). In S28z and H28z, scFv cloned from serial 2-fold dilutions from 2 ϫ 105 to 2 ng/ml. Following incubation with SM3 (23) or HMFG2, respectively (GenBank accession numbers: SM3 peroxidase-conjugated streptavidin (DakoCytomation) and O-phenylene- VH, AF042142; HMFG2 VH, AM747043; SM3 and HMFG2 VL, diamine (Sigma-Aldrich.), absorbance (A450) was measured using a Dynex AF042143), were substituted for the scFv in P28z (19). In SD28z and MRX II (Jencons). HD28z, the human IgD hinge (aa 187–289; DNA generously provided by Dr. G. A. Lo¨set, University of Oslo, Norway; Ref. 24) was inserted into the ϩ Binding of recombinant MUC1-IgG glycoforms to CAR-grafted NotI site within S28z and H28z. In SF28z/HF28z, the human IgG1 Fc T cells hinge (aa 238–470; cDNA provided by Dr. M. Stubbs, Cancer Research U.K. (CRUK) Organization) was inserted between the scFv and CD28 (aa -modified T cells (2 ϫ 105) were incubated with MUC1-IgG fusion 153–220) followed by CD3␨ (aa 52–163). In SDF28z/HDF28z the IgD proteins on ice for 30 min. Binding was detected by flow cytometry, fol- hinge was inserted into the NotI site of SF28z/HF28z. In HDF28Tr CD28 lowing incubation with PE-conjugated goat anti-mIgG (DakoCytomation). was truncated by the introduction of a stop codon after aa 182. In HOX/ HBB, the intracellular domain of OX40 (aa 241–277) or 4-1BB (aa 214– Cytotoxicity assays 255) was inserted between the CD28 and CD3␨ sequences in HDF28z. In Four-hour CTL assays were performed using a lactate dehydrogenase re- control CAR lacking scFv, the CD8␣ leader was fused to the indicated lease assay (Boehringer Mannheim) as described (19). hinge. Sequence details of primers are available on request. SFG MUC1 was generated by ligation of SFG (digested with NcoI/ Proliferation of T cells in response to MUC1 XhoI), a 2.9-kb AvaII/XhoI fragment of MUC1 (32 tandem repeats from pcDNA3 MUC1; Ref. 25), and bp 1- 300 of MUC1 PCR amplified to For soluble Ag, T cells were incubated with 2.5 ␮g of a biotinylated MUC1 introduce a 5Ј NcoI restriction site and then digested with NcoI/AvaII. 24-mer peptide or 2.5 ␮g/ml unglycosylated MUC1-IgG. Further cross- Transduced cells were immunoselected using HMFG2 and paramagnetic linking was achieved using paramagnetic beads (Invitrogen Life Technol- beads precoated with goat anti-murine (m)IgG (Invitrogen Life ogies) coated with streptavidin or rabbit anti-mIgG respectively (three Technologies). beads per target cell). For preimmobilized Ag, ELISA plates were coated Firefly luciferase (ffLUC) was expressed using pBabe puro (pBP, cloned with rabbit anti-mIgG (4 ␮g/well; DakoCytomation) followed by recom- as a BglII/BamHI fragment derived from pGL 410[luc2]; Promega. binant MUC1-IgG glycoforms (100 ng/well) and then blocked with mouse serum diluted 1/10 (Sigma-Aldrich). Culture and retroviral transduction of primary human T cells Culture of T cells with tumor cell lines PBMC from anonymous donors were purchased from the U.K. National T cells were cocultivated with confluent MUC1ϩ (lines T47D, BT20, and Blood Service. Gene transfer was performed using PG13 retroviral pack- Ϫ aging cells as described (19) except that PBMC were activated in AIM V MCF7) or MUC1 (line 410.4) tumor cell monolayers in a 24-well dish. medium (Invitrogen Life Technologies). Thereafter, T cells were propa- Where indicated, monolayers were pretreated with inhibitors of O-linked gated in RPMI 1640 plus 10% human AB serum (T cell medium; glycosylation (benzyl-2-acetamido-2-deoxy-␣-D-galactopyranoside at 2 Sigma-Aldrich). mM for 40 h (Sigma Aldrich) and sialylation (Clostridium perfringens neuraminidase) at 50 mU/ml for 2 h (Roche). Effectiveness of inhibitor FACS analysis treatment was confirmed by lectin binding studies (data not shown). FACS analysis was performed on fresh/cultured CARϩ T cells (and un- Demonstration of HMFG2 staining by transduced control cultures) using a Coulter EPICS XL cytometer with Expo32 ADC software. Expression of MUC1-specific CAR was demon- Paraffin sections were dewaxed in xylene and partially rehydrated in alco- strated using the biotinylated peptide (NeoMPS) 24-mer biotinyl-(TAP hol. Endogenous peroxidase was blocked with 0.5% H2O2 in methanol (10 PAHGVTSAPDTRPAPGSTAPP) or 60-mer biotinyl-(VTSAPDTRPAPG min). Subsequent incubations were conducted at 25°C (separated by two washes in PBS): 1) 20% rabbit serum (Sigma-Aldrich) for 15 min; 2) STAPPAHG)3 followed by incubation with PE-conjugated streptavidin (Invitrogen Life Technologies). CD8-PerCP (Becton Dickinson) was used HMFG2 supernatant for 60 min; 3) biotinylated rabbit anti-mIgG (Dako- to assess T cell subset distribution. Cytomation) at 1:200 in PBS for 30 min; 4) streptavidin biotin complex (DakoCytomation) for 30 min; 5) DAB substrate-chromogen solution (Bio- analysis genex) for 5 min. Cell nuclei were lightly counterstained with hematoxylin (Sigma Aldrich). Sections were dehydrated in alcohol, cleared in xylene, Electrophoresis was performed using NuPage 4–12% gradient gels (In- and mounted in Eukit (Fluka). Images were captured using an Olympus vitrogen Life Technologies) under reducing conditions. Western blots were DP50 microscope (UplanFl; ϫ20 objective lens) and processed using Stu- probed with 8D3 (BD Pharmingen) to demonstrate CAR (19) or anti- dio Lite (version 1.0) and Adobe Photoshop (version 9; final magnification mIgG-HRP (DakoCytomation) for MUC1-IgG fusion proteins. ELISA was of ϫ200). used to measure IFN-␥ using paired Ab sets (R&D Systems). Cells pro- ducing IL-17 were quantified by ELISPOT. A Maxisorp plate (Nunc) was Affinity measurement coated with anti-IL-17 capture Ab (R&D Systems) at 1/60 in 100 ␮lof PBS at 4°C overnight and then blocked with 1% BSA at 25°C for 2 h. T Affinity of MUC1 Ab was measured using an IAsys optical biosensor cells (2 ϫ 105 in triplicate wells) were cultured with indicated stimuli or (Fisons). Template material was unglycosylated GST-MUC1 fusion (7 PMA plus ionomycin (Sigma-Aldrich) at 10 ng/ml each as positive control. VNTR). Plates were incubated at 37°C in a 5% CO2 incubator for 72 h and washed Preparation of tumor cell conditioned medium eight times (PBS plus 0.05% Tween 20) before and after the addition of anti-IL-17 detector Ab (R&D Systems) at 1/60 in 100 ␮l of PBS plus 1% Conditioned medium (CM) was harvested from tumor cell lines held at BSA at 4°C overnight and then the addition of gold-labeled anti-biotin Ab confluence for 48 h, filtered (0.44 ␮m), and stored at Ϫ20°C. Depletion of The Journal of Immunology 4903

FIGURE 2. MUC1 retargeted tumor cell cytotoxicity mediated by SM3-derived CAR. A, S28zϩ T cells were established in a 4-h CTL assay with the indicated MUC1ϩ (T47D or RPMI 8226) or MUC1Ϫ (410.4) tumor cell targets. P28z was the control CAR. B, Four-hour CTL assays were performed using SD28zϩ T cells on day 0 (unstim.) or day 28 fol- lowing four cycles (C4) of stimulation on T47D tumor cell monolayers (days 0, 10, 17, and 24). MDA-MB-435, MUC1low control target.

spected daily and sacrificed when symptomatic as a result of tumor pro- gression as specified in the U.K. Home Office Project (license no. PPL 70/5931) that governs this work. Statistical analysis For statistical analysis, the one-tailed homoscedastic Student t test was used. Directional hypothesis testing was performed in accordance with predicted outcomes of individual experiments, thereby minimizing the risk FIGURE 1. MUC1-specific CAR derived from SM3. A, In S28z, an of incurring a type II error in data analysis. SM3 scFv was fused to the hinge, transmembrane, and intracellular se- quence of CD28 followed by the CD3␨ endodomain. In SD28z, the IgD Results hinge was inserted between CD28 and the SM3 scFv. B, Upper panels The IgD hinge improves retargeting capacity of show staining of control (untransduced) or CARϩ T cells with a biotinyl- MUC1-specific CAR ated MUC1 peptide (60-mer) followed by streptavidin-PE (x-axis). Positive The SM3 Ab has renowned selectivity for tumor-associated MUC1 events (%) are indicated. A Western blot (lower panel) was prepared under (1, 4, 5, 11). To generate a MUC1-specific CAR (S28z), an SM3 reducing conditions from the same T cells and probed with anti-CD3␨. ␨ ϩ scFv was fused to sequences derived from CD28 and CD3␨ (Fig. Endogenous CD3 was the loading control. C, CAR or control T cells ϩ were cultured on monolayers of MUC1ϩ T47D tumor cells on the days 1A). However, despite satisfactory expression (Fig. 1B), S28z T ϩ indicated by the arrows. Cells were counted at intervals. D, IFN-␥ produc- cells were poorly activated when cultured with MUC1 tumor tion by indicated T cells (1 ϫ 106/ml) cocultivated for 72 h with T47D or cells such as T47D (proliferation, Fig. 1C; production of IFN-␥, -p Ͻ 0.01). E, S28zϩ T cells were incubated Fig. 1D). Expansion of S28zϩ T cells could be induced with sol ,ء) MUC1Ϫ) tumor cells) 410.4 in the absence of IL-2 with unglycosylated MUC1-IgG or biotinylated uble Ag, either as peptide or unglycosylated MUC1-IgG fusion MUC1 24-mer peptide, cross-linked with beads coated with anti-mIgG (Fig. 1E). By contrast, when MUC1-IgG was immobilized T cell (Anti-mIg) or streptavidin, respectively. Dotted line indicates input cell proliferation was not observed (Fig. 1F). These data indicate that Ͻ ϩ ء number. Columns represent cell number at 8 days ( , p 0.01). F, S28z anchored MUC1 imposes a glycosylation-independent steric bar- and SD28zϩ T cells were incubated in the absence of IL-2 with unglyco- rier upon targeting by S28z. To overcome this, CAR flexibility and sylated MUC1-IgG preimmobilized on a 96-well dish coated with anti- mIgG. To provide a control, mIgG was blocked with mouse serum (Nil). reach was increased by introduction of the IgD hinge (Fig. 1A). Dotted line indicates input cell number. Columns represent cell number at The resultant SD28z CAR consistently expressed at lower levels 8 days. than S28z (Fig. 1B). Nonetheless, SD28z exhibited superior MUC1 retargeting capacity, enabling T cells to proliferate (Fig. 1C) and to produce significantly greater amounts of IFN-␥ in re- MUC1 was achieved by immunoprecipitation with HMFG2 plus protein sponse to T47D cells (Fig. 1D). T cells that express SD28z also A-Sepharose beads (CRUK). proliferate upon stimulation with preimmobilized MUC1-IgG In vivo testing of CAR-grafted T cells (Fig. 1F). MDA-MB-435 tumor cells transduced with SFG MUC1 and pBP ffLUC Inadequate binding to tumor-associated glycoforms is (435 MUC LUC) were inoculated i.p. at the indicated doses in SCID Beige correlated with suboptimal targeting of MUC1 mice (Charles River Laboratories). For therapeutic studies T cells were administered i.p. 4 days after tumor challenge. Bioluminescence imaging Tumor cell killing by S28z-expressing T cells was not reproduc- (BLI) was performed using Xenogen IVIS imaging system with Living ibly detectable in 4-h CTL assays using several tumor targets (Fig. Image software (Xenogen). Mice were injected i.p. with D-luciferin (150 mg/kg; Xenogen) and imaged under 2% isoflurane anesthesia after 10 min. 2A). Similarly, T cells grafted with SD28z did not exhibit robust Image acquisition was conducted on a 15- or 25-cm field of view at me- cytolytic activity in CTL assays conducted using T47D (Fig. 2B) dium binning level for 0.5- to 3-min exposure times. Animals were in- or BT20 cells (data not shown). This deficiency in SD28z function 4904 MUC1 RETARGETED T CELLS

Table I. Production of IFN-␥ by CAR-grafted T cells following cocultivation with O-glycan deficient tumor cellsa

Treated Treated CARa 410.4 410.4 T47D T47D

S28z 67 Ϯ 543Ϯ 4 350 Ϯ 20 911 Ϯ 42* CEA-28z 481 Ϯ 4 467 Ϯ 21 ND ND

a T-cells (1 ϫ 106) expressing S28z (or a control CAR specific for carcinoem- bryonic antigen (CEA)) were plated on the tumor cells listed. Where indicated, tumor cells had been pretreated with inhibitors of O-linked glycosylation and sialylation followed by extensive washing. Supernatants were harvested at 72 h (mean Ϯ SD; IFN-␥ in pg/ml). ND, Not detected. .p Ͻ 0.01 compared to untreated T47D ,ء

panel). These data indicate that tumor-associated MUC1 also imposes glycosylation-related steric constraints on targeting by the SM3-derived CAR, S28z. In agreement with this, MUC1- dependent activation of S28zϩ T cells is enhanced if tumor cells are pretreated with inhibitors of O-linked glycosylation and sia- lylation (Table I).

Improved binding to tumor-associated MUC1 glycoforms using HMFG2-derived CAR The ST glycan is very highly represented on tumor-associated MUC1 (7, 8, 28). Because S28z fails to engage this glycoform, we compared binding properties of SM3 with two other MUC1 Ab, HMFG1 and HMFG2 (Fig. 4A and Table II). This analysis indi- cated that HMFG2 has the broadest capacity for strong binding to tumor-associated MUC1 glycoforms. Compared with SM3, HMFG2 was clearly superior in its ability to bind unglycosylated ϩ and sialylated forms of MUC1. A trend toward improved binding FIGURE 3. Interaction of recombinant MUC1 glycoforms with S28z and H28zϩ T cells. A, Tumor-associated MUC1 carries a preponderance to MUC1-Tn and MUC1-T was also observed. Importantly, the of truncated O-linked glycans, including Tn, STn, T (core 1), and ST. broad reactivity of HMFG2 does not compromise its ability to Purified MUC1-IgG fusion proteins containing these glycans were val- discriminate between malignant cells and normal epithelial coun- idated by Western blotting (nonreducing conditions; probed with anti- terparts (Fig. 4B) (5, 11, 29). forms are indicated. Gal, The H28z CAR was created using an HMFG2 scFv. Similar to (ءء) and dimeric (ء) mIgG-HRP). Monomeric Galactose; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglu- the parental Ab, H28z binds well to all tumor-associated glyco- cosamine. B, FACS plots demonstrate the percentage of binding of forms of MUC1, including MUC1-ST (Fig. 3B, middle panel). MUC1-IgG glycoforms to human T cells expressing S28z and H28z. In Binding curves were generated using T cells that express compa- binding curves shown below, MUC1-IgG glycoforms at the indicated rable levels of H28z and S28z and proved divergent in all cases amounts were incubated with T cells expressing S28z or H28z. To (Fig. 3B, lower panel). Similar findings were obtained using correct for a difference in gene transfer (60% S28z and 78% H28z, ϩ determined using 24-mer MUC1 peptide), data were normalized as the CAR PG13 fibroblasts (data not shown). percentage of bound glycoform/percentage of bound MUC1 24-mer peptide ϫ 100. C, S28z- and H28z-expressing T cells were incubated with MUC1-IgG glycoforms preimmobilized on a 96-well dish coated with anti-mIgG. UG, Unglycosylated; Nil, blocked with mouse serum. Dotted line indicates input cell number. Columns represent cell number after 8 days.

could be overcome by repeated stimulation with T47D monolay- ers, resulting in acquisition of potent cytolytic activity against T47D targets (Fig. 2B). The ability of prestimulated SD28zϩ T cells to mediate brisk destruction of T47D tumor monolayers was inhibited by HMFG2 but not by the isotype control Ab (data not shown). To investigate the suboptimal function of SM3-derived CAR, we examined binding to defined tumor-associated MUC1 gly- FIGURE 4. HMFG2 exhibits broad reactivity with tumor-associated glycoforms of MUC1. A, Binding of biotinylated MUC1 Ab (HMFG1, coforms (MUC1 IgG fusions; Fig. 3A). These studies were per- HMFG2, and SM3) to immobilized MUC1 glycoforms was detected using formed using S28z because it expresses at considerably higher a colorimetric assay following incubation with peroxidase-conjugated levels than SD28z (Fig. 1B). S28z binds well with soluble streptavidin. Similar results were obtained in three separate experiments MUC1 that is unglycosylated or that carries Tn or STn (Fig. 3B, (Table II and data not shown). B, Representative tissue section demon- upper panel). However, binding was less efficient with strating HMFG2 staining of normal breast tissue (top) immediately above MUC1-T and was minimal with MUC1-ST (Fig. 3B, upper primary grade III infiltrating ductal (arrow). Scale bar, 50 ␮m. The Journal of Immunology 4905

Table II. Binding of VNTR-specific antibodies to recombinant MUC1 glycoforms

Recombinant MUC1 Glycoforma Affinity Peptide Unglyc Tn STn T ST (M ϫ 10Ϫ8) Epitope

HMFG2 0.23 Ϯ 0.04* 0.07 Ϯ 0.06 0.09 Ϯ 0.02* 0.07 Ϯ 0.04 0.08 Ϯ 0.05* 2.3 DTR SM3 0.71 Ϯ 0.29 0.19 Ϯ 0.11 0.33 Ϯ 0.02 0.16 Ϯ 0.18 1.06 Ϯ 0.58 17.0 PDTRP

a The Ab concentration at which 50% of maximal binding was determined in three separate experiments. Concentrations were normalized with respect to HMFG1 (1 Ϯ 0 in each case; epitope PDTRP) and are expressed as mean Ϯ SD. Unglyc, Unglycosylated. .p Ͻ 0.05 in comparison with both SM3 and HMFG1 ,ء

To compare function of S28z and H28z, T cells were cultured molecular mass (Fig. 5A and data not shown). To compare func- with preimmobilized MUC1-IgG glycoforms. Only H28zϩ T cells tion, engineered T cells were cocultivated with tumor cell proliferated robustly in all cases (Fig. 3C). monolayers. H28z and all IgD hinge-containing CAR enable T cells to proliferate similarly through several rounds of stimu- Combining flexibility and binding power to optimize a lation on MUC1ϩ T47D cells (Fig. 5B). No T cell proliferation MUC1-specific CAR was seen in the absence of stimulation or on MUC1Ϫ 410.4 ϩ Data presented above indicate that an elongated hinge or en- monolayers (data not shown). In all cases, CAR T cells be- hanced Ag binding capacity can improve CAR performance. To came enriched in restimulated cultures (Fig. 5C). Activation explore whether both properties are independently important, was accompanied by production of IFN-␥, which was greatest an HMFG2-based CAR incorporating the IgD hinge was con- for HMFG2 CAR containing an IgD hinge (Fig. 5D). Notably, structed (HD28z). A CAR was also designed containing IgG1 T cells bearing the HF28z (and SF28z) CAR expanded very Fcϩ hinge (HF28z), an element commonly used to distance the poorly, owing to high levels of activation-induced cell death Ag-combining site from the membrane (30). In a third fusion (data not shown). (HDF28z), both elements were included. Matched SM3-based The HDF28z fusion receptor was selected in preference to constructs were also prepared as controls (SF28z and SDF28z). HD28z because it consistently expressed at higher levels on the T All CAR expressed on the T cell surface and were of predicted cell surface (Fig. 5A). Furthermore, HDF28z mediated production of higher levels of IFN-␥ by T cells when incubated with tumor cells that expressed intermediate amounts of MUC1 (e.g., BT20; Fig. 5D). Unlike SM3-derived CAR, newly transduced HDF28zϩ T cells effectively killed MUC1ϩ breast cancer (T47D and BT20 lines) and myeloma cells (RPMI 8226 line), but spared an immor- talized human mammary epithelial line (MTSV1–7) that expresses low levels of the HMFG2 epitope (Fig. 6).

Evaluation of a tri-modular MUC1 CAR Recently, a potent CAR has been described containing a fused CD28 plus OX40 plus CD3␨ endodomain (21). To test applicabil- ity to MUC1 targeting, OX40 or 4-1BB signaling sequences were inserted into HDF28z to create HOX and HBB. To provide con- trols, receptors with a truncated endodomain or lacking scFv were constructed (Fig. 7A). Stable CAR expression was demonstrated in

FIGURE 5. Comparison of HMFG2-derived CAR. A, Expression of the indicated CAR was detected in T cells by flow cytometry (using a biotin- ylated MUC1 24-mer peptide) or Western blotting (reducing conditions; probed with anti-CD3␨). B, CARϩ or control T cells were cocultivated with T47D tumor cells at the time points indicated by the overhead arrows. Cell number was evaluated periodically. C, FACS analysis was performed on days 0, 14 (data not shown), and 28 on the transduced T cell cultures shown in B following staining with CD8 PerCP and a biotinylated MUC1 60-mer peptide plus streptavidin-PE. D, CARϩ T cells (5 ϫ 105/ml) were plated in a 24-well dish on a confluent monolayer of the indicated tumor cell line. FIGURE 6. MUC1 retargeted tumor cell cytotoxicity. CARϩ T cells IFN-␥ was measured in supernatant after 72 h. Owing to consistently lower were established in a 4-h CTL assay with indicated MUC1ϩ tumor cell expression of HD28z, data have been normalized for CAR expression de- targets (T47D, BT20, and RPMI 8226) or a nontransformed breast epithe- termined as (IFN-␥ in pg/ml)/percentage of CARϩ T cells (assessed using lial cell line (MTSV1–7). Dot plots demonstrate CAR expression detected -p Ͻ 0.01 with using PE-conjugated anti-mIgG (P28z) or biotinylated MUC1 24-mer pep ,ء ;24-mer peptide) ϫ 100. P28z, Irrelevant Ag control respect to H28z. tide plus streptavidin-PE (HDF28z). P28z, Irrelevant Ag control. 4906 MUC1 RETARGETED T CELLS

FIGURE 7. Structure of OX40- and 4–1BB-containing “third genera- tion” CAR. A, Western blotting was performed using lysates prepared from CARϩ T cells (reducing conditions) and probed with anti-CD3␨. B, Cell surface CAR expression was demonstrated using a biotinylated MUC1 24-mer peptide plus streptavidin-PE (upper row) or PE-conjugated anti- FIGURE 9. Testing of HOXϩ human T cells in tumor xenograft-bear- human Fc antiserum (lower row). Positive events are indicated (%). ing mice. A, MDA-MB-435 tumor cells were engineered to coexpress MUC1/ffLUC (435-MUC-LUC) and inoculated i.p. at the indicated dose in SCID Beige mice. Serial BLI was performed in two mice per group (mean T cells by Western blotting (Fig. 7A) and flow cytometry (Fig. 7B). data are presented graphically). The third mouse in each panel is a tumor- ϩ To compare function, CAR T cells were cocultivated with free control. B, To test therapeutic efficacy of MUC1 targeted T cells, 20 MUC1ϩ tumor cell lines or 410.4 as control. When activated SCID Beige mice were inoculated i.p. with 2 ϫ 106 435-MUC-LUC tumor cells. After 4 days, mice bearing an established tumor (proven by BLI) were treated with 2 ϫ 107 human T cells (20% CARϩ) or medium (PBS) alone. Serial BLI of representative mice is presented. C, Mean biolumi- ;p Ͻ 0.05 (HOX v DOX ,ء .(nescence is plotted (n ϭ 5 mice per group ;p Ͻ 0.05 (HOX v DOX; HOX vs medium ,ءء ;(HOX vs medium if present HOX vs HDFTr); †, p Ͻ 0.05 (HDFTr vs DOX).

with T47D cells (MUC1 strongly positive; Fig. 8A), HDF28z, HOX, and HBB all mediate high-level production of IFN-␥ (not significantly different). However, upon activation by tumor cells that express lower levels of MUC1 (BT20, Fig. 8A; MCF7, Fig. 8B), HOXϩ T cells produced significantly greater amounts of IFN-␥ than T cells that express HDF28z or HBB. The HOX, HBB, and HDF28z CAR also enable T cells to produce addi- tional cytokines in response to tumor-associated MUC1, includ- ing IL-2 and the proinflammatory cytokine IL-17 (Fig. 8C and data not shown). To examine responsiveness to soluble tumor-derived MUC1, CM was prepared from the MUC1ϩ breast cancer lines T47D and MCF7. MUC1 was demonstrated in CM by immunopre- cipitation (data not shown). In the presence of MUC1ϩ CM, HOX-grafted T cells proliferated in the absence of exogenous cytokines (Fig. 8D, upper panel). The growth-promoting effect ϩ FIGURE 8. Functional comparison of third generation MUC1-specific of MUC1 CM was abrogated by depletion of MUC1 (Fig. 8D, CAR. A, CARϩ T cells (5 ϫ 105/ml) were cocultivated in a 24-well dish lower panel). Enrichment of gene-modified T cells was also with a confluent well of indicated tumor cells for 72h. Supernatants were observed in these cultures, which could be expanded in MUC1- analyzed for IFN-␥. To correct for small differences in gene transfer, data containing CM without exogenous cytokines for up to 7 wk Ͻ ء were normalized as described in Fig. 5D ( , p 0.01). B, Similar to A (data not shown). No differences were observed in proliferation Ͻ ء except that MCF7 tumor cells were used ( , p 0.01). C, CM was prepared between HOXϩ and HDF28zϩ T cells in response to either cell from the MCF7 (MUC1ϩ) and 410.4 (MUC1Ϫ) cell lines. CARϩ T cells were associated- or soluble tumor-derived MUC1 (data not shown). cultured in 50% CM/50% T cell medium for 72 h (PMA/ionomycin was the positive control). IL-17 production was demonstrated by ELISPOT. D, CARϩ To examine MUC1-dependent cytolytic activity, a CTL assay was performed using newly transduced T cells. This confirmed that T cells were plated in 50% T cell medium plus 50% indicated tumor CM ϩ (depleted of MUC1 where indicated). Dotted lines indicate input cell number. HOX T cells are highly cytolytic to T47D targets, achieving Columns represent cell number after 8 days. E, CARϩ T cells were incubated comparable efficiency to HDF28z and HBB (Fig. 8E). Rapid de- ϩ in a 4-h CTL assay with T47D or 410.4 target cells. struction of T47D but not 410.4 monolayers by HOX T cells was The Journal of Immunology 4907 also demonstrated by time-lapse video microscopy (data not Next, a TNF receptor signaling module was incorporated to- shown). gether with CD28 to optimize the CAR endodomain (32). In- clusion of 4-1BB sequences did not improve function. By con- HOX-grafted human T cells exhibit antitumor activity in vivo trast, the OX40-containing CAR (HOX) mediated greater IFN-␥ To test antitumor function in vivo, a model was established in production in response to tumor cells that express intermediate which MDA-MB-435 cells were engineered to coexpress levels of MUC1. No alteration in CD4/CD8 subset distribution MUC1 and ffLUC and then injected i.p. into SCID/Beige mice. was observed as a result of insertion of either OX40 or 4-1BB This model was selected because tumor take is highly repro- sequences (data not shown). In contrast to an earlier study, (21) ducible, dose-dependent, estrogen-independent (unlike T47D or incorporation of OX40 sequences did not increase MUC1-de- MCF7),and can be monitored noninvasively by BLI (Fig. 9A;in pendent cytolytic activity or proliferation. This may reflect the contrast to our experience with BT20). Mice bearing an estab- distinctive nature of CAR cross-linking achieved by complex lished tumor were treated i.p. with a single dose of human T polyvalent Ags such as MUC1. cells that express HOX, DOX, or HDFTr (two control CAR) or An important finding reported in this study is that, upon stim- were treated with medium alone. Administration of HOXϩ T ulation with tumor-associated MUC1, engineered T cells se- cells resulted in a significant delay in tumor growth compared crete proinflammatory cytokines indicative of both type-1 with control mice (medium alone or DOX-expressing T cells; (IFN-␥) and Th17 (IL-17) differentiation. Tissue destruction is Fig. 9, B and C). A small delay in tumor growth was also ob- driven by IL-17 in a number of autoimmune disease models, served following treatment with HDFTr T cells, which achieved most notably experimental autoimmune encephalomyelitis (33). significance at day 6 (Fig. 9, B and C). When compared with Nonetheless, the role played by IL-17 production in antitumor HDFTr, HOX-grafted T cells also confer a significant survival advantage upon estrogen-supplemented mice bearing 1-wk es- immunity is presently unclear with evidence in favor of both tablished i.p. MCF7 breast cancer xenografts (data not shown). antitumor (34) and protumor (35) effects. Development of immunotherapy directed to MUC1 raises Discussion two additional concerns. First, MUC1 is expressed at lower The “holy grail” of cancer immunotherapy is the identification of levels by several normal tissues. Nonetheless, toxicity has not an Ag that is universal in malignancy but not found in normal proven problematic in Ab-based therapeutic protocols targeted tissues. Against these parameters, MUC1 measures up well be- to this Ag (36, 37). We did not observe killing of a nontumori- ϩ cause it contains several tumor-selective glyco-epitopes and is genic mammary cell line by CAR T cells despite low-level overexpressed in many cancers. To target tumor-associated expression of MUC1. This may reflect the predominance of core MUC1, the S28z CAR was constructed using an SM3 scFv. How- 2 glycans carried by MUC1 on nontransformed cells (9, 10) that ever, S28z exhibited poor activity as a result of steric inhibition inhibit HMFG2 binding (11, 29). Together, this provides reas- and by suboptimal binding to some glyco-epitopes, notably MUC1 surance when considering clinical risks that may be posed by ST. Poor binding to MUC1 ST is particularly noteworthy because MUC1-targeted T cells. it is highly enriched in breast tumors, owing to endosomal re- A second concern with Ab-based immunotherapy is reduced cycling and up-regulation of the ST3Gal I glycosyltransferase efficacy due to binding of soluble target Ag. The polyvalent (7, 28). nature of MUC1 raises concerns that Ab binding might yield To ameliorate steric inhibitory effects, we focused upon im- large circulating immune complexes, resulting in immunopa- provement of the mobility and reach of CAR binding arms. The thology. By contrast, binding of soluble MUC1 may favor a importance of Ab flexibility has become increasingly appreci- CAR-based approach, facilitating tumor-dependent T cell ex- ated in explaining why naive B cells coexpress cell surface IgM pansion and persistence. Such a mechanism has been implicated (which lacks a hinge) and IgD, whose elongated monomeric in the in vivo efficacy of CAR-grafted T cells against hinge is the longest of all Ab isotypes (24). As a consequence, tumors (38). IgD can assume a “T-shape” in which Fab regions can engage To test activity in vivo, a xenograft model was established Ag in virtually any orientation (31). The IgD hinge was inserted using MUC1ϩffLUCϩ MDA-MB-435 (435-MUC-LUC) tumor into S28z, resulting in a marked improvement in MUC1-depen- cells. Tumor growth was significantly delayed by administra- dent T cell proliferation and IFN-␥ production. Tumor cell kill- ϩ tion of a single dose of HOX T cells compared with DOX ing by SD28zϩ T cells was slow. However, this could be mark- ϩ (lacks a MUC1 binding moiety) or an untreated control. Nota- edly enhanced by repeated stimulation with MUC1 tumor ϩ bly, a signaling-defective MUC1 CAR (HDFTr) also mediated cells. In part, this may reflect enrichment of CAR T cells, weak antitumor activity. This suggests that colocalization of T particularly in the CD8 subset, together with Ag-mediated se- cells and tumor can contribute to CAR-independent antitumor lection for increased expression of the SD28z CAR (Fig. 5C). Repeated stimulation in vitro with cell-associated Ag is likely to activity. prove highly cumbersome in clinical protocols. Consequently we Ultimately, tumor growth occurred in all animals, including elected to optimize our MUC1 CAR further. the HOX group, despite persistent expression of the MUC1 tar- To improve binding power for tumor-associated MUC1, an get Ag (data not shown). A recent study has provided evidence HMFG2 scFv was cloned. Compared with SM3, HMFG2 exhibits in support of a role for repeated T cell therapy in this setting a 7.4-fold improvement in affinity for unglycosylated MUC1 (39). However, this approach may impose prohibitive costs (Table II). Nonetheless, this was sufficient to enable T cells upon clinical translation, particularly in resource-limited health grafted with the H28z CAR to proliferate robustly when plated services. We are currently investigating the duration of T cell on anchored glycan-free MUC1. Incorporation of the IgD hinge survival in treated animals with the goal of investigating strat- together with IgG1 Fc yielded a CAR (HDF28z) that expressed egies to prolong this if necessary. at high levels on the cell surface and successfully retargeted the Immune targeting of MUC1 using gene-modified cells has cytolytic activity of newly transduced T cells against diverse been described previously by Finn’s group (40). An MHC un- MUC1ϩ tumor cells. restricted single-chain TCR with SM3-like specificity for 4908 MUC1 RETARGETED T CELLS

MUC1 was fused to CD3␨ and delivered to murine hematopoi- 11. Tarp, M. A., A. L. Sorensen, U. Mandel, H. Paulsen, J. Burchell, etic stem cells. Engraftment primarily occurred in NK and my- J. Taylor-Papadimitriou, and H. Clausen. 2007. Identification of a novel cancer- specific immunodominant epitope in the MUC1 tandem repeat. Gly- eloid cells and protected against subsequent challenge with a cobiology 17: 197–209. MUC1ϩ xenograft. Our study complements these data because 12. Hayes, D. F., H. Sekine, T. Ohno, M. Abe, K. Keefe, and D. W. Kufe. 1985. Use of murine monoclonal antibody for detection of circulating DF3 plasma antigen we show that human T cells may be genetically targeted to levels in breast cancer patients. J. Clin. Invest. 75: 1671–1678. MUC1 found on a range of human tumor types. In support of 13. Ragupathi, G., N. X. Liu, C. Musselli, S. Powell, K. Lloyd, and P. O. Livingston. the cancer stem cell model, insertional mutagenesis has been 2005. 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