ceacam7isaneffectivetargetforcart-celltherapyof pancreatic ... · 1/21/2021 · clinical cancer...

CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY

CEACAM7 Is an Effective Target for CART-cell Therapy ofPancreatic Ductal Adenocarcinoma A C

Deepak Raj1, Maria Nikolaidi1, Irene Garces1, Daniela Lorizio1, Natalia M. Castro1, Sabrina G. Caiafa1,Kate Moore1, Nicholas F. Brown1, Hemant M. Kocher2, Xiaobo Duan3,4, Brad H. Nelson3,4,Nicholas R. Lemoine1,5, and John F. Marshall1

ABSTRACT◥

Purpose: To investigate whether CEACAM7 represents a noveltherapeutic target for treating pancreatic ductal adenocarcinoma(PDAC) and to generate CEACAM7-targeting CAR T cells to testthis hypothesis.

Experimental Design: We identified CEACAM7 (CGM2), amember of the CEA family of proteins with expression restrictedto the colon and pancreas, as a potential CAR T-cell target forPDAC. We probed a panel of PDAC tumor sections as well aspatient-derived PDAC cell cultures for CEACAM7 expression. Wegenerated CAR-targeting CEACAM7, and assessed antitumor effi-cacy of CEACAM7 CAR T cells using in vitro and in vivo models.

Results: We show here that CEACAM7 is expressed in a largesubset of PDAC tumors, with low to undetectable expression in allnormal tissues tested. CEACAM7 is also expressed in primaryPDAC cultures isolated from patient-derived tumors, with highexpression within the cancer stem cell-enriched subset. CAR T cellstargeting CEACAM7 are capable of targeting antigen-expressingtumor cells, and mediate remission in patient-derived xenografttumors.

Conclusions:We identify CEACAM7 as a potential therapeutictarget in PDAC and describe the development of CEACAM7-targeted CAR T cells with efficacy against PDAC.

IntroductionPancreatic ductal carcinoma (PDAC) is the fourth most common

cause of deaths due to solid tumors and is characterized by a low 5-yearsurvival rate of <5%. It is generally diagnosed at a late stage, withextensive metastases at presentation (1, 2). PDAC is largely refractoryto conventional therapies, which at best extend survival by a fewmonths and is therefore a disease of unmet need. At a molecular level,PDAC tumors are characterized by considerable genetic heterogene-ity (3, 4) and the presence of a cancer stem cell (CSC) compart-ment (5, 6), that together contribute to disease progression andresistance to chemotherapy.

CAR T cells have shown remarkable efficacy in CD19-expressingB-cell malignancies (7, 8) but limited efficacy in solid tumors (9, 10). Amajor challenge in this respect is the lack of CAR T target antigensstrongly expressed on malignant cells, but with expression on normaltissues sufficiently low as to preclude on-target off-tumor toxicities.The use of CAR T cells against HER2-, CAIX-, and CEACAM5-

expressing tumors has been associated with severe toxicities inclinical trials due to expression of these antigens in lung and bileduct epithelium (11–13). Recent clinical trials using CAR T cellstargeting antigens such as HER2 and mesothelin on PDAC haveshown limited efficacy, with slight to no improvement in overallsurvival (14–16).

Identification of target antigens with more restricted expression innormal tissues is likely to increase the margin of safety of CAR T cellsagainst solid tumors, allowing safe administration of higher doses ofCAR T cells and potentially increasing antitumor efficacy. CEACAM7(CGM2) is a little-studied member of the CEA family, a large group ofproteins with diverse roles, and which are typically dysregulated inmalignancies (17). CEACAM7 is a GPI-anchored protein with nointracellular domain and a relatively small extracellular domain (ascompared with several other CEA members such as CEACAM5;ref. 18). Analysis of the N-terminal region of CEACAM7 suggeststhat it forms a homodimer, with a similar fold to other CEA familymembers, but much higher affinity within the dimer (19). Thephysiologic function of CEACAM7 remains unknown.

Unlike several other members of the CEA family that are expressedwidely, expression of CEACAM7 is restricted to the apical surface ofpancreatic ductal cells and epithelial cells of the adult colon. In fetalcolon tissues, CEACAM7 is localized at the base of epithelial cells, andmoves to the apical surface shortly after birth. No expression ofCEACAM7 has been demonstrated in other areas of the gastrointes-tinal tract such as the esophagus, stomach, and small intestine, nor wasexpression detected in tissues such as lung and biliary system that havebeen previously associated with CAR T-cell toxicity (20, 21). Inter-estingly, CEACAM7 expression is reported to be downregulated incolorectal carcinoma as well as hyperplastic polyps and tubularadenomas, as compared with normal colon (22–24). CEACAM7mRNA expression is, however, detectable in PDAC tumor epithelialcells at higher levels than in normal pancreas, and has been describedas a potential diagnostic marker for PDAC (25); however, muchheterogeneity of expression was observed within the panel of patientswith PDAC examined.

1Centre for Tumor Biology, Barts Cancer Institute, Cancer Research UK Centre ofExcellence, Queen Mary University of London, London, United Kingdom. 2Direc-tor of the Barts Pancreatic Cancer Tissue Bank, Barts Cancer Institute, CancerResearch UK Centre of Excellence, Queen Mary University of London, London,United Kingdom. 3Deeley Research Centre, BC Cancer Agency, Victoria, Canada.4Department of Medical Genetics, University of British Columbia, Vancouver,Canada. 5Director, Barts Cancer Institute, Queen Mary University of London,Cancer Research UK Centre of Excellence.

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

Corresponding Author: John F. Marshall, Barts Cancer Institute, Queen MaryUniversity of London, John Vane Science Centre, Charterhouse Square, LondonEC1M 6BQ, United Kingdom. Phone: 4420-7882-3580; Fax: 4420-7882-3884;E-mail: [email protected]

Clin Cancer Res 2021;XX:XX–XX

doi: 10.1158/1078-0432.CCR-19-2163

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We therefore hypothesized that CEACAM7may be a suitable targetfor CART-cell therapy of PDAC, and describe here the development ofCEACAM7-directed CAR T cells that can efficiently target PDACcultures in vitro and eliminate patient-derived tumor xenografts fromearly as well as patients with late-stage PDAC.

Materials and MethodsPrimary human blood and cancer-derived cells

Blood and tumors from patients with PDAC were supplied by theBarts Pancreas Tissue Bank (www.bartspancreastissuebank.org.uk)and obtained with written consent from all patients. The collectionof human tissue was performed under the Barts Pancreas Tissue BankProtocol, REC reference 13/SC/0592. PDAC tumors were expanded aspatient-derived xenografts (PDXs) in immunocompromised mice asdescribed previously (26).

Several primary patient-derived PDAC cell cultures were used inthis study. PDAC lines 253, 354, 247, 286, 420, A6L, and 185 were giftsfrom James Eshleman and Christine Iacobuzio-Donahue (JohnsHopkins University, Baltimore, MD), PDAC lines 12707, 12556, and12560 were a gift from Aldo Scarpa (University and Hospital Trust ofVerona, Verona, Italy). In addition, circulating tumor cell (CTC)-derived PDAC cell cultures c76, c102, and c139 generated in-housefrompatients with late stage, chemo-na€�ve PDACwere also used. Shorttandem repeat profiling (Public Health England) of all patient-derivedPDAC cell lines was performed and confirmed the uniqueness of eachof these lines.

To culture spheres enriched in CSCs, cells were resuspended in 1�DMEM/F-12 (Gibco) supplemented with 20 ng/mL FGF-2 (CellGS),0.4% amphotericin B, 1% penicillin/streptomycin, 2%B27 supplement(Gibco), and 200 mmol/L of L-glutamine (Gibco). A cell suspension of10,000 cells/mL was then prepared and distributed into ultralowattachment surface flasks (Corning) for 1week. Prior to use, sphereswere filtered using a 40-mmol/L strainer to remove single cells andaggregates.

Flow cytometrySphere-derived pancreatic cancer cells and differentiated cancer

cells were dissociatedwith TrypleE Express (Thermo Fisher Scientific),then diluted with FBS-containing media and washed once with cold

PBS. Spheres were filtered through 40-mmol/L cell strainers. Purified2869 antibody isolated from hybridoma supernatants was used todetect CEACAM7 expression, and cells were incubated for 1 hour at4�C. The cells were washed again with cold PBS and incubated withAlexa Fluor 647–conjugated goat anti-mouse antibody (10337882,Thermo Fisher Scientific) used as a secondary antibody at a 1:200dilution for 30minutes at 4�C in the dark. Following one final washwith cold PBS, cells were resuspended in FACS buffer (1� PBScontaining 3% FBS, 3-mmol/L EDTA, and 0.5 mg/mL DAPI) andanalyzed by flow cytometry on a BD LSR Fortessa (BD Biosciences).

ELISAThe IFNg ELISA was performed on cell-free supernatants from

cytotoxicity cocultures using a kit from eBioscience (88-7316-88)according to the manufacturer's instructions.

Cytotoxicity assaysTarget cells (primary PDAC cultures) and effector cells (T cells

transduced with either 2869 CAR, mutant CAR, or unmodified T cellsas a negative control) were plated at defined effector:target (E:T) ratiosin 96-well cell culture plates. Lysis of target cells was assessed bymicroscopy and quantitated byWST-1 viability assays, using a kit fromRoche (05015944001).

In vivo experimentsAll experiments were approved by the Animal Experimental Ethics

Committee (Home Office Project License PPL P1EE3ECB4) andperformed in accordance with the guidelines for Ethical Conduct inthe Care and Use of Animals. Firefly luciferase-expressing PDAC cellswere established by transducing cells with a PGK-GFP-IRES-Luciferase Lentivector system from Addgene. Cells were sorted forGFP expression with a FACS BD Aria II instrument (BD) andsubsequently expanded in vitro. Then, 50 mL of 1 � 105 PDAC-Luccells were surgically injected either into the pancreas of NSG mice(NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ; Charles River) ages 6–8weeks.To perform surgery, the mice were anesthetized with isofluorane (2%)and an incision was made into the left flank of the skin followingsedation and analgesia. Then, a second incision through the perito-neum was carried out and the spleen with adjacent pancreas wasvisualized for the injection of PDACcells. After closing the peritoneumwith 6/0 sutures (B/Braun; 0022002), the skin was closed by surgicalstaples. An IVIS-200 system (PerkinElmer) was used for weekly in vivoluciferase imaging. Mice were anesthetized with isoflurane (2%) andinjected intraperitoneally with 150mg/kg of luciferin (Caliper LifeSciences) diluted 15mg/mL in PBS. Sequential images were obtainedafter luciferin injection every 30 second (maximum light emission,�20minutes after luciferin injection). Luciferase activity is in photonsper second per square centimeter per steradian (p/s�1 cm�2 sr�1).Living Image software (Caliper Life Sciences) was used for imageanalysis. Once tumors become detectable, mice were randomized tovarious treatments. All animal procedures were conducted in accor-dance to the 3Rs and were approved by the institute's Animal Welfareand Ethics Review Body.

IHCIHC staining was carried out on 2.5-mmol/L sections of PDAC and

normal tissues on a tissuemicroarray, using the VentanaDiscovery XTsystem (Roche). Sections were deparaffinized, hydrated, and loaded onthe Discovery XT. Antigen retrieval was performed using citrate buffersolution. Endogenous peroxidases were quenched using H2O2 reagent(Ventana). The BAC2 mAb has previously been used to specifically

Translational Relevance

Pancreatic ductal adenocarcinoma (PDAC) is characterized by adismal prognosis and poor response to conventional therapies. Ahurdle to CAR T-cell therapy of solid tumors is that commonlytargeted tumor antigens are expressed at low levels in normaltissues, leading to on-target off-tumor toxicity. We here identifyCEACAM7 as a potential target antigen for CAR T-cell therapy ofPDAC. CEACAM7 has low to undetectable expression in allnormal tissues tested, with strong surface expression on a subsetof primary human PDAC tumors. Cell surface expression ofCEACAM7 was maintained in primary cultures from patient-derived PDAC tumors, as well as xenografts generated from thesePDAC cultures. CAR T cells targeting CEACAM7 were generated,and showed significant antitumor activity against patient-derivedPDAC tumor cultures both in vitro and in vivo. Our data suggestthat CAR T-cell therapy targeting CEACAM7 may have potentialfor safe and efficacious therapy of PDAC.

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detect CEACAM7 by immunostaining (20). The BAC2 primaryantibody (Santa Cruz Biotechnology, sc-59946; 1:1,000) was thereforeapplied, incubated for 20minutes, and detected using an anti-mousesecondary antibody and the ChromaMap DAB detection kit (Ven-tana). Tissues were counterstained with hematoxylin, and imageanalysis was performed using Pannoramic Viewer Software(3DHISTECH).

Sequences of 2869 constructsThe protein sequence of the 2869 scFv-Fc is as follows:MALPVTALLLPLALLLHAARPDYKDDDDKGGGGSGGGGSEI-

VLIQSPTTLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLL-IKYASQSIYGTPSRFSGIGSGTDFTLRINSVETEDLGMYFCQQSH-NWPHTFGAGTKLELKGGGGSGGGGSGGGGSQVQLKQSGPGL-VQPSQSLSITCTVSGFSLSSFGIHWVRQSPGKGLEWLGVIWTGG-NTDFNAAFVSRLTITKDNSKSQVFFKMNSLEPDDTAIYYCAKN-HYNSIGHYGMDYWGQGTSVIVSSDKTHTCPPCPAPELLGGPSV-FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE-VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS-NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL-VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV-DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

The protein sequence of the 2869 (with CD8a hinge) CAR is asfollows:

MALPVTALLLPLALLLHAARPDYKDDDDKGGGGSGGGGSQ-VQLKQSGPGLVQPSQSLSITCTVSGFSLSSFGIHWVRQSPGKGLE-WLGVIWTGGNTDFNAAFVSRLTITKDNSKSQVFFKMNSLEPD-DTAIYYCAKNHYNSIGHYGMDYWGQGTSVIVSSGGGGSGGGG-SGGGGSEIVLIQSPTTLSVTPGDSVSLSCRASQSISNNLHWYQQK-SHESPRLLIKYASQSIYGTPSRFSGIGSGTDFTLRINSVETEDLGM-YFCQQSHNWPHTFGAGTKLELKTTTPAPRPPTPAPTIASQPLSL-RPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVI-TLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC-ELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG-RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR-RGKGHDGLYQGLSTATKDTYDALHMQALPPR.

The protein sequence of the 2869 (with IgG4 hinge) CAR is asfollows:

MALPVTALLLPLALLLHAARPDYKDDDDKGGGGSGGGGSEI-VLIQSPTTLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLL-IKYASQSIYGTPSRFSGIGSGTDFTLRINSVETEDLGMYFCQQSH-NWPHTFGAGTKLELKGGGGSGGGGSGGGGSQVQLKQSGPGL-VQPSQSLSITCTVSGFSLSSFGIHWVRQSPGKGLEWLGVIWTGG-NTDFNAAFVSRLTITKDNSKSQVFFKMNSLEPDDTAIYYCAKN-HYNSIGHYGMDYWGQGTSVIVSSESKYGPPCPPCPIYIWAPLA-GTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGC-SCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRR-EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEA-YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

Generation of CAR T cellsLentiviral particles were produced via transfection of the lentiviral

constructs into HEK293FT human embryonic kidney cells. Cells weretransfected with pRRL-SIN-EF1a-WPRE, pMDL, pREV, and pVSVGplasmids using FugeneHD (Promega). Human T cells from healthyvolunteers were obtained by Ficoll-Pacque purification of peripheralblood mononuclear cells (PBMCs) from normal donor whole blood.PBMCs were further activated for 24 hours with CD3/CD28-coatedmagnetic beads (Life Technologies) before infection. Concentratedlentivirus was applied to the activated human T cells in the presence of5mg/mL protamine sulfate and 50 IU/mL IL2 (R&D) and centrifuged

at 1,000� g for 2 hours at 32�C. The 2869 andmutant CAR expressionlevel was determined by flow cytometry with Alexa Fluor 488–labeledanti-FLAG antibody. CAR T cells were then expanded in culture withthe same concentration of IL2, and used for in vitro and in vivo workbetween 5 and 14 days after T-cell isolation.

Statistical analysisUnless stated otherwise, results are expressed as the mean � SD.

Statistical analyses were performed using GraphPad Prism 7, twovariables were analyzed using Student t test, and three or morevariables were analyzed using one-way ANOVA with Bonferronicorrection. For statistical analyses of tumor progression (Fig. 5C), alog-rank test was used with x2¼ 4.2 on 1 degree of freedom, with dataplotted � SEM (n ¼ 5).

Patient consentBlood and tumors samples were obtained with written consent

under the Barts Pancreas Tissue Bank Protocol, REC reference 13/SC/0592, and RAS project ID 142061.

Ethics approvalAll experiments were approved by the Animal Experimental Ethics

Committee (Home Office Project License PPL P1EE3ECB4) andperformed in accordance with the guidelines for Ethical Conduct inthe Care and Use of Animals.

ResultsCEACAM7 expression in PDAC tumors and in normal tissues

To assess patterns of CEACAM7 protein expression in PDAC,immunostaining was performed on a panel of human PDAC tumorsections. The BAC2 mAb shows specific binding to CEACAM7 andshows no reactivity against other CEA family members (20), and wastherefore used for all immunostaining work. CEACAM7 expressionwas detectable with high to intermediate expression in 19 of 30 tumorsections, and low to undetectable in 11 of 30 sections. Expression wasclearly visualized in the epithelial component of PDAC tumors, withno staining of the stroma or the blood vessels (Fig. 1A; SupplementaryFig. S1A).

Normal human pancreas and colon are the only tissues whereCEACAM7 expression has been demonstrated (20, 21). To assessCEACAM7 expression in normal tissues, immunostaining was per-formed on a panel of normal tissue sections on the same tissuemicroarray as the PDAC tumor sections. Expression was undetectablein pancreas or colon under the same staining conditions that had beenused for PDAC sections. Expression was also undetectable in a panel ofnormal tissues including tonsil, lung, liver, and prostate, consistentwith previous reports (Fig. 1B; Supplementary Fig. S1B). As previousstudies have reported CEACAM7 transcript expression in pancreasand colon, we performed RT-PCR on RNA isolated from these tissuesfrom normal human donors. CEACAM7 transcript was detectable inthese tissues at levels comparable with a patient-derived primary cellculture (c76) aswell as an established cell line (BxPC-3; SupplementaryFig. S1C). These data do not rule out CEACAM7 expression inpancreas and colon, but suggest significantly lower antigen expressionas compared with PDAC, suggesting that upregulation of CEACAM7protein in PDAC may occur at the posttranscriptional level.

Patient-derived PDAC tumor cultures, unlike cell lines, may retainproperties of the original tumor such as genetic heterogeneity andantigen expression that is not an artefact of long-term culture (27).Wetherefore used qRT-PCR to assess expression of CEACAM7 mRNA

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Figure 1.

Expression of CEACAM7 in PDAC. A panel of PDAC tumor sections (A) and normal human tissues (B) were stained with the CEACAM7-specific BAC2 antibody(scale¼ 50 mm). C, qRT-PCR to determine CEACAM7mRNA expression in adherent and sphere cultures of primary PDAC, as well as non-PDAC cultures and humanpancreas (n¼ 2). b-actin mRNA expression was used as a control. HPDE, human pancreas-derived epithelial cells. WBC, white blood cells. D, qRT-PCR to determineCEACAM7 mRNA expression in a panel of normal tissues from three different human donors (n ¼ 2). b-actin mRNA expression was used as a control.E, Representative flow cytometry using 2869 hybridoma supernatants on adherent and sphere cultures derived from primary PDAC cell lines from stage I–IIpatients (left) or patients with late-stage metastatic disease (right). Values are shown as fold increase in median fluorescence intensity (MFI) compared withunstained controls.

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transcript in a panel of primary cultures from patients with stage I–IIPDAC. CEACAM7 transcript was found to be upregulated in three ofseven primary PDAC cultures tested, with low expression in humanpancreas, and no detectable expression in blood cells nor in non-PDAC cell types (Fig. 1C). The CSC compartment of PDAC has beenshown in several studies to drive chemoresistance, metastasis, diseaseprogression, and relapse and any therapy developed against PDACmust target this subset of cells for long-lasting disease correc-tion (28, 29). We therefore generated nonadherent sphere culturesfrom all the primary PDAC cell cultures to enrich the CSC compo-nent (30). Significantly, CEACAM7 transcript expression was consis-tently raised in CSC-enriched sphere cultures as compared withdifferentiated adherent cultures, suggesting potential to target the keyCSC compartment (Fig. 1C).

To further investigate levels of CEACAM7 expression in normaltissues, CEACAM7 transcript expressionwas analyzed by qRT-PCR ina panel of normal tissues from three human donors, including kidney,lung, skeletal and cardiac muscle, visceral and subcutaneous fat, liver,brain, ovary, and breast. No detectable expression was observed in anyof these tissues, as compared with primary PDAC cultures (Fig. 1D).Expression patterns of CEACAM7 transcript were therefore found tobe consistent with the immunostaining results as well as with previousstudies analyzing patterns of CEACAM7 expression.

We then investigatedwhetherCEACAM7 transcript correlatedwithcell surface expression of CEACAM7 protein in PDAC. Using primarycultures from stage I–II patients with local disease, or blood-derivedCTCs from patients with late-stage metastatic disease, adherent popu-lations and CSC-enriched sphere cultures were generated. The 2869hybridoma was previously raised against CEACAM7, and flow cyto-metry using 2869 hybridoma supernatants showed high CEACAM7expression in one of three metastatic and two of five stage I-II PDACcultures. Consistent with the qRT-PCR data, CEACAM7 expressionwas consistently raised in spheres as compared with adherent cultures,highlighting potential to target the CSC compartment (Fig. 1E; Sup-plementary Fig. S2A). 2869 binding was undetectable in non-PDACcell types such as human PBMCs, human fibroblasts (HFF-1), andembryonic kidney cells (HEK293T; Supplementary Fig. S2B). Likeseveral members of the CEA family, CEACAM7 has no ortholog in themouse. Consistent with this, no 2869 binding was observed on twomouse PDAC lines tested (Supplementary Fig. S2C).

Generation of 2869-derived CAR T cellsOn the basis of these results, we further investigated the specificity of

the 2869 hybridoma against CEACAM7. The 354 and c102 PDACcultures, which showed undetectable CEACAM7 expression whengrown as adherent cultures (Fig. 1E), were modified to express GFPand luciferase (354GL and c102GL) to allow tracking in vitro andin vivo. To assess specificity of 2869 antibody to CEACAM7 protein,354GL and c102GL cells were then further modified to stably expressCEACAM7. Western blotting using 2869 hybridoma supernatantsshowed strong bands of expected size in modified cells, showingsuccessful ectopic expression of CEACAM7, with stronger expressionin c102GL than in 354GL (Fig. 2A). Flow cytometry of both cell typesshowed binding of 2869 hybridoma supernatants specifically in CEA-CAM7-modified cells, suggesting successful membrane localization ofectopic CEACAM7 (Fig. 2B). Consistent with Western blotting data,stronger expression of CEACAM7 was observed on modified c102GLthan on 354GL.

To assess suitability of this hybridoma for CAR development, anscFv was generated on the basis of the variable regions of the heavy andlight chains of the 2869 antibody, and expressed in framewith a human

IgG1-derived Fc fragment to enable detectionwith an anti-Fc antibody(Fig. 2C). The 2869 scFv-Fc fusion was transfected into 293T cells, andsupernatants containing the secreted protein were used for flowcytometry on modified and unmodified 354GL. As expected, the2869 scFv-Fc showed a similar binding pattern to 2869 hybridomasupernatants, with specific binding to cells ectopically expressingCEACAM7, demonstrating the potential of the 2869 scFv for CEA-CAM7-targeted CAR development (Fig. 2D).

Second-generation chimeric antigen receptors were then con-structed by fusing the 2869 scFv to a hinge domain and a CD8atransmembrane domains, in frame with the CD137 (4-1BB) costimu-latory domain and the CD3z activation domain. Several studies haveshown that the distance of the epitope-binding domain from the T-cellsurface can significantly impact CAR T-cell function (31, 32). Wetherefore designed two CARs with either a 45 amino acid CD8a-derived hinge or a 12 amino acid IgG4-derived hinge (Fig. 2E). BothCARs could be efficiently transduced into primary human T cells withexpression ranging between 40% and 90%, though the smaller CAR(IgG4 hinge) consistently showed higher expression, consistent withprevious studies showing that lentiviral transduction efficiencymay beenhanced by smaller constructs (ref. 33; Fig. 2F). T-cell proliferationwas not affected by expression of CAR, nor was there any detectablechange in the phenotypic profile of T cells with either CAR, ascompared with unmodified T cells (Supplementary Fig. S3A and S3B).

Specific activity of 2869 CAR T cells against CEACAM7-expressing targets

The specificity and efficacy of 2869 CAR against CEACAM7-expressing targets was then investigated. Ectopic CEACAM7-expres-sing 354GL and c102GL cells were used as CAR T-cell targets, withunmodified cells as a negative control. T cells that were unmodified, ormodified to express either one of the 2869 CARs, were overlaid on amonolayer of PDAC target cells at a 5:1 E:T ratio. Viability of the targetcells was initially assessed by GFP expression. Lysis of CEACAM7-expressing target PDAC cells, but not unmodified cells, was observedfollowing coculture with either of the 2869 CAR T-cell types (Fig. 3Aand B). Quantitation of target cell viability using WST-1 showed thatneither of the 2869 CAR T-cell types had any effect on unmodifiedPDAC cells, but decreased viability of CEACAM7-expressing targetswhen applied both at 5:1 and 1:1 E:T ratios. Cytotoxic killing appearedto be antigen density dependent, with higher cytotoxicity observedagainst c102GL than 354GL, consistent with higher CEACAM7expression on c102GL cells. The smaller 2869 CAR (IgG4 hinge) hada slight but significantly higher cytotoxic efficacy when CAR T cellswere applied at the 5:1 E:T ratio on either target cell type (Fig. 3CandD). 2869 CAR T cells can therefore specifically target CEACAM7-expressing cells, with no effect on cells that do not express the targetantigen.

Besides enrichment of the CSC compartment, sphere cultures arealso characterized by a dense extracellular matrix that may serve as abarrier to effector cells (34). Cytotoxic assays against sphere culturesmay therefore be informative in (i) CAR T-cell targeting of the CSCcompartment and (ii) ability of T cells to penetrate a barrier thatrecapitulates the physical features of the tumor microenvironment(TME). Sphere cultures of 354GL and c102GL cells with or withoutCEACAM7 expression were therefore seeded and allowed to form for7 days, following which effector T cells were added at a 5:1 E:T ratio.Upon coculture of spheres with T cells expressing either 2869 CAR, noeffect was seen on unmodified spheres, but complete lysis of CEA-CAM7-expressing spheres was observed. As with adherent targets,cytotoxic killing was density dependent, with more efficient clearance






ofCEACAM7-expressing c102GL than 354GL spheres (Fig. 3E andF).2869 CAR T cells can therefore target CEACAM7-expressing PDACsphere cultures, under in vitro conditions that enrich CSC andrecapitulate a stromal barrier.

To compare CAR T-cell activation broadly across both target celltypes under both adherent and sphere conditions, parallel cocultures

were set up with CEACAM7-expressing and unmodified c102GL and354GL target cells cultured as both adherent and spheres, coculturedwith effector T cells at E:T ratios of 5:1 and 1:1. Supernatants from eachindividual coculture were assayed for IFNg secretion. Significantlyhigher secretion of IFNg was observed from T cells expressing either2869 CAR when expressed with CEACAM7-expressing targets, but

Figure 2.

Development of 2869 CAR T cells. A, Lentiviral transduction was used to stably express CEACAM7 in PDAC 354GL and PDAC c102GL adherent cultures. CEACAM7expression was assayed byWestern blotting. B, Ectopic expression of CEACAM7was assessed by flow cytometry using 2869 hybridoma supernatants. C, Sequenceof the 2869 hybridomawas used to generate an scFvwith variable regions of the heavy and light chains connectedwith a 3�(GGGGS) linker, expressed in framewithhuman IgG1 Fc sequence as a tag for detection. D, Flow cytometry to determine binding of 2869 scFv-Fc to CEACAM7-expressing cells. E, Schematic of CAR T cellsbased on the 2869 hybridoma sequence, with either CD8a hinge and transmembrane domain (top), or IgG4 hinge and CD8a transmembrane domain. F, Flowcytometry to detect the N-terminal FLAG tag shows transduction of human T cells to express 2869 CAR.

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Figure 3.

In vitro efficacy of 2869CART cells. Microscopy analysis of GFP-expressing 354GL (A) and c102GL (B), expressing adherent cultures, either unmodified or ectopicallyexpressing CEACAM7, following 24 hours of culture with untransduced T cells or 2869 CAR T cells at a 5:1 E:T ratio (scale ¼ 40 mm). (Continued on the followingpage.)






not with unmodified target cells. Similar patterns of IFNg secretionwere observed upon coculture with either sphere or adherent targets,while unmodified T cells showed no IFNg secretion under anycondition (Fig. 3G and H). These data suggest that 2869 CAR T cellsare activated specifically and effectively by target antigen in the contextof either adherent or sphere culture.

To assess whether CAR T activity was sustained in vivo, weorthotopically implanted 354GL cells (with and without ectopicCEACAM7) to generate a xenograft model of stage I/II PDAC. Thetumors engrafted and expanded rapidly, and were treated with twodoses of 2869 CAR T cells at days 6 and 13 posttumor implantation(Fig. 4A). Rapid tumor progression continued in the 354GL cohort,with all mice reaching endpoints due to tumor burden by day 23.Significant tumor regressionwas seen in the354GLþCEACAM7cohort,with complete tumor clearance in four of five animals (Fig. 4B and C).Nevertheless, all animals in this cohort succumbed to graft vs. hostdisease (GvHD), and were euthanized by day 38 (Fig. 4D).

We wished to investigate the efficacy of 2869 CAR T cells againstlarger, more established tumors. We therefore established a secondxenograft model with 354GL�CEACAM7 cells as before, and mon-itored tumor growth in vivo. When tumors had reached a sizeapproaching the permissible endpoint, we administered two doses of2869 CAR T cells at days 13 and 20 (Fig. 4E). All animals in the 354GLcohort succumbed rapidly to progressive disease, while tumor stabi-lization and significantly extended survival were observed in four offive animals in the 354GLþCEACAM7 cohort (Fig. 4F and G). Thesedata demonstrate the antigen-specific in vivo activity of 2869 CAR Tcells against xenograft models generated from patient-derived primaryPDAC cultures.

2869 CAR T cells target endogenously expressed CEACAM7The patient-derived PDAC lines c76 and 253 cells express

moderate levels of CEACAM7 when grown as adherent cultures,though at much lower levels than sphere cultures (Fig. 1E). Adher-ent cultures of c76 and 253 were therefore used to assess activity of2869 CAR against endogenously expressed CEACAM7. As thesmaller 2869 CAR (IgG4 hinge) showed higher activity againstCEACAM7-expressing targets (Fig. 3C), this CAR was used in allsubsequent experiments. 2869 CAR T cells mediated lysis of mono-layers of c76 and 253 cells, while unmodified T cells had no effect(Fig. 5A). Quantitation of target cell viability by WST-1 showedlysis of both target cells types by 2869 CAR T cells in an effector celldose-dependent manner, with higher cytotoxicity observed at E:T5:1 as compared with E:T 1:1 (Fig. 5B). Consistent with target celllysis, raised IFNg secretion was observed upon coculture of 2869CAR T cells with target PDAC (Fig. 5C).

The 2869 CAR contains a 4-1BB costimulatory domain, which hasbeen associated with antigen-independent tonic signaling in previousstudies (35). We wished to investigate whether cytotoxicity observedagainst c76 targets was specifically driven by 2869 CAR engagementwith target antigen, rather than nonspecific stimulation of T cellsdriven by antigen-independent signaling. Amutant CARwas thereforeconstructed with similar intracellular costimulation and activationdomains, but lacking the scFv epitope-binding domain (Fig. 5D).

Mutant and 2869 CAR T cells were generated and used in cyto-toxicity assays with c76 targets at different E:T ratios. In all instances,coculture with 2869 CAR T cells led to significantly diminishedviability of PDAC c76 targets as compared with mutant CAR T cells(Fig. 4F). Finally, cytotoxicity using PDAC 253 targets showedsignificant target lysis by 2869 CAR T cells compared with mutantCAR T cells, demonstrating specific targeting of CEACAM7 by 2869CAR (Fig. 5F).

We next investigated patterns of CEACAM7 expression in moreestablished cell lines. A panel of six PDAC lines showed 2869 bindingin all cell types, at similar levels to PDAC c76 cultures, though at alower level compared with ectopic CEACAM7 expression onmodified354GL cells (Fig. 6A and B). CEACAM7 is therefore expressed inestablished PDAC cell lines as well as in primary PDAC cultures, atsimilar levels on the cell surface.

To further validate the specificity of 2869 CAR, we preparedcocultures of 354GL�CEACAM7 with mutant CAR T cells or 2869CAR T cells at an E:T ratio of 3:1. Microscopy as well as WST-1quantitation of target cell viability confirmed specific targeting ofCEACAM7-expressing targets by 2869 CAR T cells, while mutantCART cells had no effect on antigen-expressing or -nonexpressing celllines (Fig. 6C).We performed cytotoxicity assays on all six PDAC linesusing 2869 CAR T effectors as well as mutant CAR T cells as a control.At E:T ratios of 3:1 and 1:1, significant cytotoxicity was observed on allPDAC lines cocultured with 2869 CAR T cells. Cytotoxicity wascomparable with that observed on primary PDAC cultures, but lowerthan on ectopically expressing 354GLþCEACAM7 cultures (Fig. 6Dand E). These data further demonstrate the specific activity of 2869CAR T cells against CEACAM7-expressing cultures; neverthelesseffector activity appears to be dependent on antigen expression, withlow levels of endogenously expressed CEACAM7 (BxPC-3, PANC-1)being more refractory to cytotoxicity than higher levels of endoge-nously (AsPC-1, CFPAC-1) or ectopically expressed antigen(354GLþCEACAM7).

In vivo activity of 2869 CAR T cells against CEACAM7-expressing xenografts

PDAC c76GL cells, grown as adherent cultures, were implantedorthotopically intoNSGmice. This cell culture, derived from the bloodof a patient with PDAC with metastatic late-stage disease, was chosenfor its aggressiveness, rapid growth, and extensivemetastases (26). Thetumors were monitored until they reached a size of >0.5 cm3, anddetectable metastases had developed, to accurately model late-stagedisease presentation in human patients. At this stage, the animals wereadministered a single dose of 5� 106mutant CART cells or 2869 CART cells (Fig. 7A). In compliance with the 3Rs (UK Home OfficeRegulations), the animals were euthanized under any of the followingconditions: upon tumor size exceeding 1.4 cm3, development of ascites,loss of >20% body weight, or any physical signs of distress or pain.

Continued progression of tumors was observed in five of fiveanimals treated with mutant CAR T cells but only in two of fiveanimals that received 2869 CAR T cells. Complete regression ofprimary tumors and liver metastases was observed in the respondinganimals in the 2869 CAR T-cell cohort (P ¼ 0.04; Fig. 7B and C). Of

(Continued.) The proportion of viable target cells was quantitated using WST-1 assays on 354GL (C) and c102GL (D) targets (n¼ 3; � , P < 0.05; �� , P < 0.01; �� , P <0.001). 354GL (E) and c102GL (F) cells were seeded to form sphere cultures. Seven days later, sphere formation was confirmed by microscopy, and effector T cellswere added to the sphere cultures at a 5:1 E:T ratio. Microscopy was used to visualize sphere integrity following 3 days of coculture with unmodified T cells or 2869CART cells. Apanel of 354GL (G) and c102GL (H) cultureswere seeded as adherent and spheres, andunmodifiedT cells or 2869CART cellswere added at 5:1 and 1:1 E:T ratios. A total of 24 hours later, supernatants were assessed by ELISA for IFNg secretion by effector T cells (n ¼ 3; � , P < 0.05; �� , P < 0.01; �� , P < 0.001).

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Figure 4.

In vivo efficacy of 2869 CAR T cells.A, Early treatment model: PDAC 354GL(with or without ectopic CEACAM7) wereorthotopically engrafted intoNSGmice. Atotal of 5 � 106 2869 CAR T cells wereadministered via tail vein 6 days and13 days later. IVIS bioluminescence imag-ing at various time points (B), IVIS quan-titation of tumors across both cohorts (C),and survival curves (D) are shown (n¼ 5;� , P < 0.05). E, Late treatment model:PDAC 354GL (with or without ectopicCEACAM7) were orthotopically engraf-ted into NSG mice. A total of 5 � 106

2869 CAR T cells were administered viatail vein 13 and 20 days later. IVIS biolu-minescence imaging at various timepoints (F), IVIS quantitation of tumorsacross both cohorts (G), and survivalcurves (H) are shown (n ¼ 5; � , P < 0.05).






note, overall survival of the 2869CART–treated cohort was significantlyimproved over the mutant CAR T–treated cohort (mean survival58.6 days vs. 27.4 days; median survival 61 days vs. 29 days; P < 0.05).

The three surviving mice in the 2869 CAR cohort began to exhibitGvHD symptoms including weight loss, reduced mobility, andhunched posture, and were therefore euthanized 61 days and 84 dayspost–T-cell administration (Fig. 7D). Grossmorphologic examinationconfirmed the absence of tumor. These results therefore demonstratethat 2869 CAR T cells are capable of mediating complete regression oflarge tumors and metastases in an aggressive and metastatic xenograftmodel with presentation similar to late-stage human disease.

DiscussionThe most successful and well-understood example of targeting

tissue-specific antigens is CD19 CAR T-cell therapy of B-cell–derived

hematologic malignancies (36, 37). CAR T-cell therapy in thiscontext generally leads to B-cell aplasia that is generally treated byintravenous immunoglobulin infusions, but is not directly associatedwith toxicity against any other tissue due to B cell–specific expres-sion of CD19 (38). Other B-cell antigens, such as ROR1, are morewidely expressed in tissues throughout the body (39), and targetingthese antigens may place an upper limit on the maximally toleratedCAR T-cell dose.

More recently, development of CAR T cells targeting clau-din18.2 has been described for preclinical therapy of a gastriccarcinoma model. Claudin18.2 is expressed in the stomach epi-thelium, with low to undetectable expression elsewhere in the body.Gastric carcinoma xenografts were also shown to express highlevels of antigen, and CAR-T therapy directed against claudin18.2successfully mediated gastric carcinoma regression. No toxicity toany normal tissue including, surprisingly, the stomach, was

Figure 5.

2869 CAR T cells target endogenouslyexpressed CEACAM7 on primaryPDAC cell lines. A, PDAC c76 andPDAC 253 adherent targets were cul-tured with untransduced T cells or2869 (IgG4 hinge) CAR T cells at E:Tratio of 5:1. Target cells viability wasassessed by light microscopy (scale¼100 mm). B, WST-1 quantitation ofPDAC cell viability of adherent targetsfollowing coculture with unmodifiedT cells or 2869 CAR T cells at E:T ratiosof 5:1 and 1:1 (n ¼ 3; � , P < 0.05; �� , P <0.01). C, IFNg secretion by effectorT cells assessed by ELISA followingcoculture for 24 hours with PDACc76 targets (n ¼ 3; �� , P < 0.001).D, Schematic of mutant CAR, withsimilar costimulatory and activationdomains as well as similar signal pep-tide and FLAG tag, but lacking anti-gen-binding domain. E, WST-1 quan-titation of c76 viability followingcoculture with mutant CAR T cells or2869 CAR T cells at various E:T ratios(n ¼ 3; �� , P < 0.01; ��, P < 0.001).F, WST-1 quantitation of 253 viabilityfollowing coculturewithmutant CARTcells or 2869 CAR T cells at E:T ratio of1:1 (n ¼ 3; � , P < 0.05).

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Figure 6.

2869 CAR T cells target CEACAM7-expressing established PDAC lines. A, Flow cytometry showing 2869 binding on PDAC c76 as well as six established PDAC lines(B) quantified bymean fluorescence intensity (MFI) normalized to the unstained control. C, PDAC 354GL (with orwithout ectopic CEACAM7)were coincubatedwithmutant CAR T cells or 2869 CAR T cells. Microscopy (left) andWST-1 assay (right) show specific targeting of CEACAM7-expressing 354GL by 2869 CAR T cells at E:T::3:1 (n¼ 3; � ,P<0.05).WST-1 quantitation of cytotoxicity of six different established PDAC cell lines following co-culturewith eithermutant or 2869CART cells at E:T ratio of 3:1 (D) or 1:1 (E; n ¼ 3; � , P < 0.05; �� , P < 0.01; �� , P < 0.001).






observed in any of the animals (40). This study highlights theutility of targeting a tumor antigen expressed solely in the tissue oftumor origin.

CEACAM7 is a little-studied protein, not implicated in cancerbesides being downregulated in colorectal carcinomas (22–24).Previous studies have shown CEACAM7 to have a very restrictedtissue expression as compared with other CEA members, withexpression detected only in pancreas and colon (20). In a studythat searched for diagnostic markers for PDAC in cellular materialfrom pancreatic juice, CEACAM7 mRNA expression was found to

be significantly upregulated as compared with healthy controls,suggesting tumor upregulation of CEACAM7 (25). To our knowl-edge, CEACAM7 protein expression in PDAC has not previouslybeen studied.

We therefore analyzed CEACAM7 protein expression on a panelof PDAC tumor sections as well as normal tissues on a tissuemicroarray. We found robust staining of CEACAM7 on themajority of PDAC tumors, with expression restricted to the apicalsurface of epithelial cells, typical of expression patterns of otherCEA proteins (41, 42). CEACAM7 expression was not observed on

Figure 7.

2869 CAR T cells target disseminatedxenograft tumors in vivo. A, MutantCAR T cells and 2869 CAR T cells weregenerated and CAR expression con-firmed by flow cytometry (untrans-duced T cells were used as a control).Cells were expanded in cultureand administered to c76GL tumor-bearing animals in respective cohorts.B, Representative IVIS images show-ing tumor progression ofmice in eithercohort. C, IVIS quantitation of total(primary and metastatic) tumor bur-den over time (� , P¼ 0.04). E, Survivalcurves of animals in both cohorts(� , P < 0.05).

Raj et al.





any normal tissue tested, including pancreas and colon. It isimportant to note that all samples were on a tissue microarraystained in a single run, therefore allowing analysis of comparativeexpression between the samples. Our data therefore suggest thatwhile pancreas and colon may express CEACAM7 at basal levels,expression in PDAC is substantially upregulated. CEACAM7expression was observed on the cell surface of a subset of primaryPDAC cultures, and consistently higher expression was observedon CSC-enriched sphere cultures. These data, showing (i) surfaceexpression of CEACAM7 on PDAC, (ii) enhanced expression onCSC-enriched PDAC cultures, and (iii) lack of expression in allnormal tissues tested, suggest that CEACAM7 would be an idealtarget for CAR T-cell development against PDAC.

We constructed a CAR to target CEACAM7 based on thesequence of a hybridoma raised against this antigen. PDAC culturesectopically expressing CEACAM7 could be specifically targetedusing CAR T cells, with unmodified PDAC cells serving as anegative control. Endogenous CEACAM7 expression in mostPDAC cultures were, however, far lower, but CAR T cell–mediated cytotoxicity was none the less observed. The costimula-tory domain 4-1BB within a CAR has previously been associatedwith nonspecific and antigen-independent signaling in CAR Tcells (35), and we speculated whether this phenomenon may drivenonspecific target cell killing within our in vitro model. To controlfor this effect, we generated a mutant CAR with similar intracellulardomains as the 2869 CAR (4-1BB costimulation and CD3z acti-vation domains), but lacking a tumor antigen-binding domain.Using this negative control, CEACAM7-directed CAR T cells killingof PDAC targets was confirmed both in differentiated PDAC as wellas CSC-enriched sphere cells.

For in vivomodelling of CEACAM7 CAR T-cell activity, we chose amodel that would recapitulate the most aggressive features of humanPDAC disease. We have previously shown that PDAC c76 cells,isolated from a patient with PDAC with late-stage metastatic disease,form extremely aggressive and metastatic tumors in immunodeficientanimals (26). We therefore generated xenograft models of PDAC c76in NSG mice and allowed tumors to progress with extensive metas-tases; thus, recapitulating the common clinical presentation of late-stage PDAC (43). Following a single dose of 2869 CAR T cells,complete tumor regression was observed in three of five animals.CEACAM7 expression in c76 xenografts was previously observed to bemodest, similar to differentiated c76 cells in culture, and considerablylower than CSC-enriched sphere cultures (Supplementary Fig. S4A).This modest expression, as compared with HER2 in similar xenograftmodels (26), may have precluded a more complete response across thecohort.

As in previous studies, we have observed a strong GvHD effect ina subset of animals following CAR T-cell administration (26, 44).NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice are useful modelsfor CAR-T study due to the efficient engraftment of human T cellsas well as primary tumors, the latter also retaining their uniquecharacteristics such as rapid invasiveness and propensity formetastases (26). This strain is therefore commonly used as a modelfor GvHD, which in this system is mediated by human T-cellreceptor (TCR) targeting mouse tissues; consequently NSG ani-mals lacking MHC class I and class II are resistant to GvHDfollowing human T-cell administration (45). Xenogenic GvHDfollowing CAR T-cell administration to NSG is therefore likelyto be a side effect of TCR signaling rather than CAR activity, andmay be an artefact of these animal models, rather than reflect

potentially debilitating clinical phenomena such as cytokine releasesyndrome observed in patients.

The efficacy of CAR T-cell targeting of tumor antigen is deter-mined by a number of factors, including (i) antigen density ontumor cells and (ii) binding affinity of CAR to antigen. It has beenobserved that increased antigen density is associated with moreefficient killing (46), but the relationship of CAR-antigen bindingaffinity to CAR efficacy is more complex, with extremes of bothlow- and high-affinity target binding associated with diminishedefficacy (47, 48). It is also important to note that xenograft modelsof human PDAC in immunodeficient animals do not model theimmunosuppressive TME. Clinical use of CAR T cells againstPDAC may therefore need to be complemented with agents thattarget the TME.

In summary, we have identified CEACAM7 as a novel targetantigen for CAR T-cell therapy of a sizeable fraction of PDAC.We have developed a CAR that can specifically and effectivelytarget CEACAM7-expressing PDAC cells, and we demonstratethat CEACAM7-directed CAR T cells can effectively mediateremission of late-stage patient-derived PDAC xenograft tumors.Because of its restricted tissue expression, CEACAM7-directedCAR T cells may offer a higher safety margin than commonlyused targets such as CEACAM5 and HER2 with more broadsystemic expression (11, 13).

Authors’ DisclosuresH.M. Kocher reports grants fromCelgene,Mylan,Medtronic, andOncosil outside

the submitted work. No disclosures were reported by the other authors.

Authors’ ContributionsD. Raj: Data curation, formal analysis, investigation, writing-original draft.M. Nikolaidi: Investigation. I. Garces: Investigation. D. Lorizio: Investigation.N.M. Castro: Investigation. S.G. Caiafa: Investigation. K. Moore: Investigation.N.F. Brown: Investigation. H.M. Kocher: Resources, writing-review and editing.X. Duan: Resources. B.H. Nelson: Resources. N.R. Lemoine: Funding acquisition,writing-review and editing. J.F. Marshall: Supervision, funding acquisition, writing-original draft, project administration, writing-review and editing.

AcknowledgmentsResearch was supported by a Pancreatic Cancer UK Grand Challenge award to

Nicholas Lemoine, John F Marshall, John Maher, and Yaohe Wang. Barts PancreaticTissue Bank (BTPB) is funded by the Pancreatic Cancer Research Fund. Funding forantibody production was generously provided to Brad H. Nelson by the CanaryFoundation of British Columbia Cancer Foundation. Cancer ResearchUK funded theBarts Cancer Institute Core Facilities (C16420/A18066).

We are grateful to patients with pancreatic cancer whodonated tissues to the BPTB(www.bartspancreastissuebank.org.uk). We also thank Claude Chelala, ArchanaAmbily, Thomas Dowe, and Dayem Ullah and the Tissue Access Committee, keymembers of BPTB who contributed to this study We are grateful to James Eshlemanand Christine Iacobuzio-Donahue (Johns Hopkins University, Baltimore, MD) forthe gift of the PDAC lines 253, 354, 247, 286, 420, A6L, and 185, to Aldo Scarpa(University and Hospital Trust of Verona, Verona, Italy) for the gift of PDAC lines12707, 12556, and 12560, and to Irene Esposito (University of D€usseldorf, D€usseldorf,Germany) for tissue samples.We are grateful to George Elia for expert assistance withIHC, and to Cancer Research UK funded Barts Cancer Institute Core Facilities(C16420/A18066). The project concept was started in C. Heeschen's laboratory whenan employee of QMUL.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Received July 1, 2019; revised November 13, 2020; accepted December 21, 2020;published first January 20, 2021.





http://www.bartspancreastissuebank.org.uk


References1. Kleeff J, Korc M, Apte M, La Vecchia C, Johnson CD, Biankin AV, et al.

Pancreatic cancer. Nat Rev Dis Primers 2016;2:16022.2. Kamisawa T, Wood LD, Itoi T, Takaori K. Pancreatic cancer. Lancet 2016;388:

73–85.3. Cancer Genome Atlas Research Network. Integrated genomic characterization

of pancreatic ductal adenocarcinoma. Cancer Cell 2017;32:185–203.4. Lomberk G, Blum Y, Nicolle R, Nair A, Gaonkar KS, Marisa L, et al. Distinct

epigenetic landscapes underlie the pathobiology of pancreatic cancer subtypes.Nat Commun 2018;9:1978.

5. Sancho P, Alcala S, Usachov V, Hermann PC, Sainz B Jr. The ever-changinglandscape of pancreatic cancer stem cells. Pancreatology 2016;16:489–96.

6. Raj D, Aicher A, Heeschen C. Concise review: stem cells in pancreatic cancer:from concept to translation. Stem Cells 2015;33:2893–902.

7. Kalos M, Levine BL, Porter DL, Katz S, Grupp SA, Bagg A, et al. T cells withchimeric antigen receptors have potent antitumor effects and can establishmemory in patients with advanced leukemia. Sci Transl Med 2011;3:95ra73.

8. Maude SL, Frey N, Shaw PA, Aplenc R, Barrett DM, Bunin NJ, et al. Chimericantigen receptor T cells for sustained remissions in leukemia. N Engl JMed 2014;371:1507–17.

9. CastellarinM,Watanabe K, June CH, Kloss CC, Posey AD Jr. Driving cars to theclinic for solid tumors. Gene Ther 2018;25:165–75.

10. Newick K, O'Brien S, Moon E, Albelda SM. CAR T cell therapy for solid tumors.Annu Rev Med 2017;68:139–52.

11. MorganRA,Yang JC, KitanoM,DudleyME, Laurencot CM,Rosenberg SA. Casereport of a serious adverse event following the administration of T cellstransduced with a chimeric antigen receptor recognizing ERBB2. Mol Ther2010;18:843–51.

12. Lamers CH, Sleijfer S, van Steenbergen S, van Elzakker P, van Krimpen B, GrootC, et al. Treatment of metastatic renal cell carcinoma with CAIX CAR-engineered T cells: clinical evaluation and management of on-target toxicity.Mol Ther 2013;21:904–12.

13. Thistlethwaite FC, Gilham DE, Guest RD, Rothwell DG, Pillai M, Burt DJ, et al.The clinical efficacy of first-generation carcinoembryonic antigen (CEACAM5)-specific CAR T cells is limited by poor persistence and transient pre-condition-ing-dependent respiratory toxicity. Cancer Immunol Immunother 2017;66:1425–36.

14. Feng K, Liu Y, Guo Y, Qiu J,Wu Z, Dai H, et al. Phase I study of chimeric antigenreceptor modified T cells in treating HER2-positive advanced biliary tractcancers and pancreatic cancers. Protein Cell 2018;9:838–47.

15. Beatty GL, Haas AR, Maus MV, Torigian DA, Soulen MC, Plesa G, et al.Mesothelin-specific chimeric antigen receptor mRNA-engineered T cells induceanti-tumor activity in solid malignancies. Cancer Immunol Res 2014;2:112–20.

16. Beatty GL, O'Hara MH, Lacey SF, Torigian DA, Nazimuddin F, Chen F, et al.Activity of mesothelin-specific chimeric antigen receptor T cells against pan-creatic carcinoma metastases in a phase 1 trial. Gastroenterology 2018;155:29–32.

17. Hammarstrom S. The carcinoembryonic antigen (CEA) family: structures,suggested functions and expression in normal and malignant tissues.Semin Cancer Biol 1999;9:67–81.

18. Tchoupa AK, Schuhmacher T, Hauck CR. Signaling by epithelial members of theCEACAM family - mucosal docking sites for pathogenic bacteria. Cell CommunSignal 2014;12:27.

19. Bonsor DA, Beckett D, Sundberg EJ. Structure of the N-terminal dimerizationdomain of CEACAM7. Acta Crystallogr F Struct Biol Commun 2015;71:1169–75.

20. Scholzel S, Zimmermann W, Schwarzkopf G, Grunert F, Rogaczewski B,Thompson J. Carcinoembryonic antigen family members CEACAM6 andCEACAM7 are differentially expressed in normal tissues and oppositely deregu-lated in hyperplastic colorectal polyps and early adenomas. Am J Pathol 2000;156:595–605.

21. Frangsmyr L, Baranov V, Hammarstrom S. Four carcinoembryonic antigensubfamily members, CEA, NCA, BGP and CGM2, selectively expressed in thenormal human colonic epithelium, are integral components of the fuzzy coat.Tumor Biol 1999;20:277–92.

22. Thompson J, ZimmermannW,Nollau P, NeumaierM,Weber-Arden J, SchreweH, et al. CGM2, amember of the carcinoembryonic antigen gene family is down-regulated in colorectal carcinomas. J Biol Chem 1994;269:32924–31.

23. Nollau P, Prall F, Helmchen U, Wagener C, Neumaier M. Dysregulation ofcarcinoembryonic antigen group members CGM2, CD66a (biliary glycopro-tein), and nonspecific cross-reacting antigen in colorectal carcinomas. Com-

parative analysis by northern blot and in situ hybridization. Am J Pathol 1997;151:521–30.

24. Nollau P, Scheller H, Kona-HorstmannM, Rohde S, Hagenmuller F,Wagener C,et al. Expression of CD66a (human C-CAM) and other members of thecarcinoembryonic antigen gene family of adhesion molecules in human colo-rectal adenomas. Cancer Res 1997;57:2354–7.

25. Yoshida K, Ueno S, Iwao T, Yamasaki S, Tsuchida A, Ohmine K, et al. Screeningof genes specifically activated in the pancreatic juice ductal cells from the patientswith pancreatic ductal carcinoma. Cancer Sci 2003;94:263–70.

26. RajD, YangMH, Rodgers D,Hampton EN, Begum J,Mustafa A, et al. SwitchableCAR-T cells mediate remission in metastatic pancreatic ductal adenocarcinoma.Gut 2019;68:1052–64.

27. Knudsen ES, BalajiU,Mannakee B,Vail P, Eslinger C,MoxomC, et al. Pancreaticcancer cell lines as patient-derived avatars: genetic characterisation and func-tional utility. Gut 2018;67:508–20.

28. Valle S, Martin-Hijano L, Alcala S, Alonso-Nocelo M, Sainz B Jr. The ever-evolving concept of the cancer stem cell in pancreatic cancer. Cancers 2018;10:33.

29. Prieto-Vila M, Takahashi RU, Usuba W, Kohama I, Ochiya T. Drug resistancedriven by cancer stem cells and their niche. Int J Mol Sci 2017;18:2574.

30. Bahmad HF, Cheaito K, Chalhoub RM, Hadadeh O, Monzer A, Ballout F, et al.Sphere-formation assay: three-dimensional in vitro culturing of prostate cancerstem/progenitor sphere-forming cells. Front Oncol 2018;8:347.

31. James SE, Greenberg PD, Jensen MC, Lin Y, Wang J, Till BG, et al. Antigensensitivity of CD22-specific chimeric TCR is modulated by target epitopedistance from the cell membrane. J Immunol 2008;180:7028–38.

32. Hudecek M, Lupo-Stanghellini MT, Kosasih PL, Sommermeyer D, Jensen MC,Rader C, et al. Receptor affinity and extracellular domain modifications affecttumor recognition by ROR1-specific chimeric antigen receptor T cells.Clin Cancer Res 2013;19:3153–64.

33. Cante-Barrett K, Mendes RD, Smits WK, van Helsdingen-vanWijk YM, PietersR, Meijerink JP. Lentiviral gene transfer into human and murine hematopoieticstem cells: size matters. BMC Res Notes 2016;9:312.

34. Olejniczak A, SzarynskaM, Kmiec Z. In vitro characterization of spheres derivedfrom colorectal cancer cell lines. Int J Oncol 2018;52:599–612.

35. Gomes-Silva D,MukherjeeM, SrinivasanM, Krenciute G, Dakhova O, Zheng Y,et al. Tonic 4–1BB costimulation in chimeric antigen receptors impedes T cellsurvival and is vector-dependent. Cell Rep 2017;21:17–26.

36. Park JH, Riviere I, Gonen M, Wang X, Senechal B, Curran KJ, et al. Long-termfollow-up of CD19 CAR therapy in acute lymphoblastic leukemia. N Engl J Med2018;378:449–59.

37. Schuster SJ, Svoboda J, Chong EA, Nasta SD, Mato AR, Anak O, et al. Chimericantigen receptor T cells in refractory B-cell lymphomas. N Engl J Med 2017;377:2545–54.

38. Porter DL, HwangWT, Frey NV, Lacey SF, Shaw PA, Loren AW, et al. Chimericantigen receptor T cells persist and induce sustained remissions in relapsedrefractory chronic lymphocytic leukemia. Sci Transl Med 2015;7:303ra139.

39. Balakrishnan A, Goodpaster T, Randolph-Habecker J, Hoffstrom BG, Jalikis FG,Koch LK, et al. Analysis of ROR1 protein expression in human cancer andnormal tissues. Clin Cancer Res 2017;23:3061–71.

40. Jiang H, Shi Z, Wang P, Wang C, Yang L, Du G, et al. Claudin18.2-specificchimeric antigen receptor engineered T cells for the treatment of gastric cancer.J Natl Cancer Inst 2019;111:409–18.

41. Bamberger AM, Riethdorf L, Nollau P, Naumann M, Erdmann I, Gotze J, et al.Dysregulated expression of CD66a (BGP, C-CAM), an adhesion molecule of theCEA family, in endometrial cancer. Am J Pathol 1998;152:1401–6.

42. Ogura E, Senzaki H, Yoshizawa K, Hioki K, Tsubura A. Immunohistochem-ical localization of epithelial glycoprotein EGP-2 and carcinoembryonicantigen in normal colonic mucosa and colorectal tumors. Anticancer Res1998;18:3669–75.

43. McGuigan A, Kelly P, Turkington RC, Jones C, Coleman HG, McCain RS.Pancreatic cancer: a review of clinical diagnosis, epidemiology, treatment andoutcomes. World J Gastroenterol 2018;24:4846–61.

44. Rodgers DT, Mazagova M, Hampton EN, Cao Y, Ramadoss NS, Hardy IR, et al.Switch-mediated activation and retargeting of CAR-T cells for B-cell malignan-cies. Proc Natl Acad Sci U S A 2016;113:E459–68.

45. BrehmMA,Kenney LL,WilesMV, LowBE, Tisch RM, Burzenski L, et al. Lack ofacute xenogeneic graft- versus-host disease, but retention of T-cell functionfollowing engraftment of human peripheral blood mononuclear cells in NSGmice deficient in MHC class I and II expression. FASEB J 2019;33:3137–51.

Raj et al.





46. Walker AJ, Majzner RG, Zhang L, Wanhainen K, Long AH, Nguyen SM, et al.Tumor antigen and receptor densities regulate efficacy of a chimeric antigenreceptor targeting anaplastic lymphoma kinase. Mol Ther 2017;25:2189–201.

47. Schlenker R, Olguin-Contreras LF, Leisegang M, Schnappinger J, Disovic A,Ruhland S, et al. Chimeric PD-1:28 receptor upgrades low-avidity T cells and

restores effector function of tumor-infiltrating lymphocytes for adoptive celltherapy. Cancer Res 2017;77:3577–90.

48. Park S, Shevlin E,Vedvyas Y, ZamanM, Park S,HsuYS, et al.Micromolar affinityCAR T cells to ICAM-1 achieves rapid tumor elimination while avoidingsystemic toxicity. Sci Rep 2017;7:14366.






Published OnlineFirst January 21, 2021.Clin Cancer Res Deepak Raj, Maria Nikolaidi, Irene Garces, et al. Pancreatic Ductal AdenocarcinomaCEACAM7 Is an Effective Target for CAR T-cell Therapy of

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