CSPG4-Specific CAR.CIK Lymphocytes As a Novel Therapy for the Treatment of Multiple Soft-Tissue Sarcoma Histotypes

Published OnlineFirst September 8, 2020; DOI: 10.1158/1078-0432.CCR-20-0357

CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY

CSPG4-Speciﬁc CAR.CIK Lymphocytes as a Novel Therapy for the Treatment of Multiple Soft-Tissue Sarcoma Histotypes A C Valeria Leuci1,2, Chiara Donini1,2, Giovanni Grignani1, Ramona Rotolo1, Giulia Mesiano1,2, Erika Fiorino1,2, Loretta Gammaitoni1, Lorenzo D’Ambrosio1, Alessandra Merlini1,2, Elisa Landoni3, Enzo Medico1,2, Sonia Capellero1,2, Lidia Giraudo1, Giulia Cattaneo1,2, Ilenia Iaia1,2, Ymera Pignochino1,2, Marco Basirico1,2, Elisa Vigna1,2, Alberto Pisacane1, Franca Fagioli4, Soldano Ferrone5, Massimo Aglietta1,2, Gianpietro Dotti3,6, and Dario Sangiolo1,2

ABSTRACT ◥ Purpose: No effective therapy is available for unresectable soft- STS histotypes by in silico analysis and on all 16 STS cell tissue sarcomas (STS). This unmet clinical need prompted us to test lines tested by flow cytometry. CSPG4-CAR.CIK displayed whether chondroitin sulfate proteoglycan 4 (CSPG4)-specific chi- superior in vitro cytolytic activity against multiple STS histo- meric antigen receptor (CAR)-redirected cytokine-induced killer typesascomparedwithpairedunmodified control CIK. lymphocytes (CAR.CIK) are effective in eliminating tumor cells CSPG4-CAR.CIK also showed strong antitumor activity against derived from multiple STS histotypes in vitro and in immunode- STS spheroids; this effect was associated with tumor recruitment, ficient mice. infiltration, and matrix penetration. CSPG4-CAR.CIK signifi- Experimental Design: The experimental platform included cantly delayed or reversed tumor growth in vivo in three STS patient-derived CAR.CIK and cell lines established from multiple xenograft models (leiomyosarcoma, undifferentiated pleomor- STS histotypes. CAR.CIK were transduced with a retroviral vector phic sarcoma, and fibrosarcoma). Tumor growth inhibition encoding second-generation CSPG4-specific CAR (CSPG4-CAR) persisted for up to 2 weeks following the last administration of with 4-1BB costimulation. The functional activity of CSPG4-CAR.CIK CSPG4-CAR.CIK. was explored in vitro, in two- and three-dimensional STS cultures, and in Conclusions: This study has shown that CSPG4-CAR.CIK effec- three in vivo STS xenograft models. tively targets multiple STS histotypes in vitro and in immuno- Results: CSPG4-CAR.CIK were efficiently generated from deficient mice. These results provide a strong rationale to translate patients with STS. CSPG4 was highly expressed in multiple the novel strategy we have developed into a clinical setting.

Introduction ularly targeted approaches may offer transient disease control to patients ineligible for radical surgical resection. However, the overall Soft-tissue sarcomas (STS) are rare tumors of mesenchymal origin prognosis remains dismal with a 5-year survival rate of less than that affect both children and adults (1). These peculiar tumors 25% (1–3). STS are also poorly responsive to checkpoint blockade– encompass multiple histotypes, which are characterized by extremely based immunotherapy (3–6). This unmet clinical need prompted variable biological and clinical behaviors. Chemotherapy and molec- us to develop a novel strategy for the treatment of multiple STS histotypes (7). Adoptive immunotherapy with T lymphocytes redirected by 1Candiolo Cancer Institute, FPO-IRCCS, Candiolo, Turin, Italy. 2Department of tumor antigen (TA)-specific chimeric antigen receptors (CAR) is Oncology, University of Torino, Turin, Italy. 3Lineberger Comprehensive Cancer one of the most effective therapies in B-cell malignancies (8–10). Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina. However, application of CAR.T cells to solid tumors remains 4 Pediatric Onco-Hematology, Division of Stem Cell Transplantation and Cellular challenging (11–15). Here, we have tested the ability of cytokine- Therapy, Regina Margherita Children’s Hospital, University of Turin, Turin, Italy. fi 5Division of Surgical Oncology, Department of Surgery, Massachusetts General induced killer lymphocytes (CIK) engineered with a TA-speci c CAR Hospital, Harvard Medical School, Boston, Massachusetts. 6Department of to target tumor cells obtained from multiple STS histotypes in vitro Microbiology and Immunology, University of North Carolina, Chapel Hill, North and in vivo. CIK are patient-derived polyclonal T natural killer (NK) Carolina. lymphocytes endowed with HLA class I–independent antitumor Note: Supplementary data for this article are available at Clinical Cancer activity, mediated mostly by the interaction of their NKG2D receptor Research Online (http://clincancerres.aacrjournals.org/). with stress-inducible targets (MIC A/B and ULBPs 1–6) on tumor – fi V. Leuci and C. Donini contributed equally to this article. cells (16 26). To increase their tumor cell speci city, CIK were engineered with a TA-specific CAR. CAR-engineered CIK have been Corresponding Author: Dario Sangiolo, University of Torino; Candiolo Cancer modeled in hematologic malignancies to target either CD19 or CD33/ Institute FPO-IRCCS, Str Provinciale 142 km 3.95, Candiolo, Turin 10060, Italy. – Phone: 39-011-9933-521; Fax: 39-011-9933-522; E-mail: [email protected] CD23 antigens (27 31), and clinical trials are currently ongoing. We and others studied the use of CAR-expressing CIK in STS (32–35). In Clin Cancer Res 2020;XX:XX–XX our previous study, we demonstrated the preclinical effectiveness of doi: 10.1158/1078-0432.CCR-20-0357 CAR-redirected CIK against CD44v6, which is expressed in about 40% 2020 American Association for Cancer Research. of STS (32). Here, we assessed whether alternative TA could be targeted

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(KO Out DMEM, Gibco BRL) or Iscove’s Modified Dulbecco Medium Translational Relevance (Sigma-Aldrich), with 10% or 15% FBS, 25 mmol/L HEPES, 100 U/mL Our study has shown that cytokine-induced killer lymphocytes penicillin, and 100 U/mL streptomycin (Gibco BRL) in a humidified (CIK) engineered with a chondroitin sulfate proteoglycan 4 5% CO2 incubator at 37 C. Patient-derived melanoma cell line, (CSPG4)-specific chimeric antigen receptor (CAR; CSPG4-CAR) M14 (48), which does not express CSPG4, was used as a specificity are effective in eliminating many types of soft-tissue sarcoma control and cultured in RPMI1640 Medium (Sigma-Aldrich), supple- (STS)-derived cells both in vitro and in immunodeficient mice. mented with 10% heat-inactivated FBS, 100 U/mL penicillin, and These preclinical results provide a strong rationale for the clinical 100 U/mL streptomycin (Gibco BRL) at 37 C in a 5% CO2 incubator. translation of the CSPG4-CAR.CIK–based immunotherapeutic The HT1080 cell line used in this study was originally obtained from strategy, which we have developed. Patients with unresectable the ATCC, and was authenticated by genotype analysis with the Cell ID high-grade STS, who respond poorly to checkpoint inhibitor- System (Promega) that compared its profile with those published on based immunotherapy may greatly benefit from this novel the DMSZ database. Adult and neonatal keratinocytes were cultured immunotherapy. with the Lonza KGM Gold Keratinocyte Growth Medium Bullet Kit. Three-dimensional (3D) STS spheroids were generated as a single spheroid per well using ultra-low attachment (ULA) 96-well round bottom plates (Corning) with no additional coating. An STS cell via CAR-engineered immune cells in STS to further expand their suspension of 500–5,000 cells/100 mL was plated into wells and then clinical applicability. centrifuged at 1,000 g for 10 minutes (33). STS spheroids were The TA, chondroitin sulfate proteoglycan 4 (CSPG4), a cell surface assembled in 1–4 days, depending on the target histotypes. We þ proteoglycan that plays an important role in oncogenic pathways generated GFP STS spheroids from cells previously transduced with involved in cancer progression and metastatic spread (36–42), was the pRRL.sin.PPT.hOct4.eGFP.Wpre VSV-G pseudotyped third- selected as the target for the following reasons. CSPG4 is highly generation lentiviral vector. expressed with limited heterogeneity on both differentiated cancer cells and cancer-initiating cells (CIC) in several types of cancer. Generation of CSPG4-CAR.CIK According to the cancer stem cell hypothesis, CIC play a major role Supernatants containing retroviral particles encoding CAR specific in disease recurrence and metastatic spread, the two major causes of for the CSPG4 antigen (CSPG4-CAR) or the control vector encoding patients’ mortality and morbidity. In contrast, CSPG4 is not detectable CAR specific for the CD19 antigen (CD19-CAR), both containing in normal tissues. Furthermore, CSPG4 is expressed by activated 4-1BB costimulatory endodomains, were generated as described pre- pericytes in the tumor microenvironment (43). As a result, CSPG4 viously (41). We generated CSPG4-CAR.CIK and CSPG4-CAR.T cells immunotargeting selectively inhibits neoangiogenesis in the tumor from peripheral blood mononuclear cells (PBMC) isolated from microenvironment, thus contributing to the elimination of the vas- patients diagnosed with STS by density gradient centrifugation using culature that supports tumor growth (44). Lymphosep (Aurogene). Approval was obtained from the IRB per the This article describes the in vitro and in vivo ability of CSPG4- Declaration of Helsinki guidelines for the collection of biological specific CAR-redirected CIK (CSPG4-CAR.CIK) to eliminate STS cells samples (tumors and blood) and for patient informed consent releases following a description of CSPG4 expression on multiple STS cells. (protocol no., 225/2015). For CAR.CIK, PBMCs from 8 patients with STS (Supplementary Table S1) were seeded on day 0 in cell culture flasks at a concentration of 2 106 cells/mL with IFNg (Miltenyi Materials and Methods Biotec; 1,000 U/mL) in RPMI1640 Medium (Gibco BRL), supplemen- Data analysis of CSPG4 RNA expression in The Cancer Genome ted with 10% FBS (Sigma), 100 U/mL penicillin, and 100 U/mL Atlas streptomycin (Gibco BRL). Following a 24-hour incubation at 37C, RNA-sequencing expression data were selected and downloaded PBMCs were activated by anti-biotin MACSiBead particles loaded from the cBioPortal, The Cancer Genome Atlas (TCGA) PanCancer with anti-CD2, -CD3, and -CD28 mAbs (Miltenyi Biotec) and human collections (45, 46). The dataset included 251 STS samples: leiomyo- IL2 IS (Miltenyi Biotec, 300 U/mL). To generate CAR.T cells, PBMCs sarcoma, n ¼ 99; dedifferentiated liposarcoma, n ¼ 58; undifferen- were seeded on day 0 at a concentration of 2 106 cells/mL and tiated pleomorphic sarcoma (UPS)/malignant fibrous histiocytoma/ activated using anti-biotin MACSiBead particles. On day þ1, human high-grade spindle cell sarcoma, n ¼ 50; myxofibrosarcoma, n ¼ 25; IL2 IS (Miltenyi Biotec, 50 U/mL) was added. On day þ2, PBMCs were malignant peripheral nerve sheath tumor (MPNST), n ¼ 9; and transduced with 0.5 mL of retroviral supernatants in retroNectin- synovial sarcoma n ¼ 10. Another 336 melanomas served as a positive coated plates by overnight incubation. Unmodified not transduced expression control and various epithelial tumors (breast cancer, n ¼ (NTD) NTD.CIK and NTD.T cells were used as a paired control. Both 1,082; pancreatic cancer, n ¼ 176; lung adenocarcinoma, n ¼ 510; and CAR.CIK and control NTD.CIK were expanded over 4 weeks, lung squamous cell carcinoma, n ¼ 482) were explored for compar- refreshed with IL2 medium (CIK, 300 U/mL) every 2–3 days as 6 ison. RSEM expression values were plotted after log2 transformation needed, and cultured at 1.8 10 cells/mL. CAR.T cells were cultured with 0.5 jittering on the x-axis using Microsoft Excel. at 1.8 106 cells/mL for 1 week, and the IL2 (50 U/mL) medium was refreshed every 2–3 days as needed. STS cell lines and STS spheroids STS cell lines were generated in our laboratory from patient-derived Flow cytometry surgical biopsies (47). We received approval for collection of patient Conjugated CD3, CD4, CD8, CD56, PD-1, CXCR3, CXCR4, and samples and the associated informed consent document from the CCR7 mAbs (BD Pharmingen) and CD45RO, CD45RA, and CD62L institutional review board (IRB) per Declaration of Helsinki guidelines mAbs (Miltenyi Biotec) were used to characterize lymphocytes. A mAb (protocol no., 225/2015); each patient signed an informed consent. specific for the IgG1/CH2CH3 Spacer (Jackson ImmunoResearch) was Patient-derived STS cell lines were cultured in either KO DMEM F12 used to detect CAR expression. STS cells were stained with conjugated

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mAbs for the expression of CIK NKG2D ligands, MIC A/B (BD ware, and percentage of tumor cell lysis was determined by the þ Pharmingen) and ULBPs (R&D Systems), and for the expression of formula: [100 treated GFP STS spheroid (pixel) 100/untreated þ HLA-ABC, PD-L1, and PD-L2 (BD Pharmingen). STS were stained for GFP STS spheroid (pixel)]. CSPG4 with mAbs 225.28, 763.74, and D2.8.5-C4B8 (49), which recognize distinct and spatially distant epitopes of CSPG4. Cells were Immunofluorescence analysis of CSPG4-CAR.CIK cell first incubated with CSPG4-specific mAbs (1 mg/mL for all mAbs), recruitment and infiltration in STS spheroids þ then washed, and incubated with rabbit anti-mouse IgG-PE Secondary GFP STS spheroids were cocultured with CSPG4-CAR.CIK or Antibody (Miltenyi Biotec). Alternatively, CSPG4 was detected with unmodified NTD.CIK cells stained with red dye PKH26 at E:T ratio 2:1 the conjugated anti-human CSPG4-APC mAb (Miltenyi Biotec). in culture medium (300 U/mL IL2 at 37 C, 5% CO2). Following a 16- CSPG4 molecules expressed on the surface of STS and other cell lines hour coculture at 37C, CIK cells were removed and immunofluo- were measured using a quantitative Immunofluorescence Assay rescence acquisition was conducted on the remaining spheroids. (Bangs Laboratories, Inc.). Briefly, cells of interest and calibration Briefly, STS spheroids were washed twice, centrifuged at 300 g for beads with increasing amounts of antibody capture capability were 3 minutes in PBS, fixed in 4% paraformaldehyde for 1 hour, resus- labeled simultaneously with the anti-human CSPG4-APC mAb. pended with mounting medium, and applied on either glass slides or Labeled cells and calibration beads were analyzed, and a standard glass bottom chamber slide wells. STS spheroids were observed using a regression line was calculated between fluorescence intensity and Leica SP8 AOBS confocal microscope. Next, 80 MHz pulsed white light antigen density, expressed as antibody-binding capacity in molecules laser (470–670 nm) was used to excite the fluorochromes in the per cell. We defined high CSPG4 expression as 2-fold increase as spheroids. Fluorescence channels were scanned sequentially, and compared with normal keratinocytes. Labeled cells were acquired on hybrid Spectral Detectors (HyD SP Leica Microsystems) revealed the FACS Cyan (Cyan ADP, Beckman Coulter SRL) and analyzed using emissions. Image acquisition of the STS spheroids was performed Summit Software. maintaining the same laser power, gain, offset, and magnification (20). We generated maximum intensity projections for each ana- Tumor cell killing assays lyzed spheroid with LAS X Software (Leica) to quantify CIK cell We assessed the tumor-killing ability of patient-derived CSPG4- recruitment and infiltration. Images of the total PKH26 red fluores- CAR.CIK and unmodified NTD.CIK in vitro against STS cell mono- cence area (mm2) present either at the boundary or inside the spheroid layers and STS 3D spheroids. In two cases, CIK and STS cell cultures were analyzed using ImageJ software. were generated from samples collected from the same patient (S1 and S172), while in all other cases, cytotoxicity assays were performed with CIK-cell penetration capability into Matrigel matrix HLA-mismatched effector cells. Cytotoxicity assays against STS cell STS spheroids were collected, washed in PBS, resuspended in 20 mL monolayers were performed using flow cytometry or a biolumines- of liquefied Matrigel (BD Pharmingen) at 4C, and then plated as cence cell viability assay. In the first case, target cells were stained with droplets in well centers of a 24-well tissue culture plate that had been fl either vital dye PKH26 (Sigma-Aldrich) or 5,6-carboxy uorescein prewarmed to 37 C. Plates were incubated at 37 C and 5% CO2 for 15 diacetate succinimidyl ester (CFSE; Molecular Probes), according to minutes to allow the solidification of the Matrigel domes. Domes were the manufacturer’s protocols. Immune-mediated killing was analyzed then overlaid with 500 mL of prewarmed medium with 300 U/mL IL2 by Flow Cytometry (Cyan ADP, Dako) and measured by the DAPI and cocultured with PKH2-stained CSPG4-CAR.CIK or unmodified þ þ permeability of target cells (PKH26 or CFSE gate). For the biolu- NTD.CIK cells (50,000 cells/well) for 5 days. Empty domes were used minescence method, cytotoxicity was measured with the CellTiter-Glo as controls. At the end of the coculture period, each well was washed Luminescent Cell Viability Assay (Promega), in which the number of twice with prewarmed PBS to eliminate any effector cells outside the viable and metabolically active target cells was evaluated by quanti- domes. Fluorescence microscopy (Leica DMI 3000B with Photo- fying the ATP in culture. CIK cells were cocultured at different effector metrics CoolSnap HQ equipped with CCD camera) was used to to target cell (E:T) ratios (10:1, 5:1, 2.5:1, 1:1, 1:2, and 1:4) in visualize CSPG4-CAR.CIK or unmodified NTD.CIK cell migration cytotoxicity assays (300 U/mL IL2 medium at 37 C and 5% CO2) for at the Matrigel boundary and cells that penetrated into the Matrigel 5 hours (short-term assay) and 48 hours (long-term assay). In selected domes. Analysis of the PKH26 red fluorescence dye presence (mm2) experiments, we tested the cytotoxic activity at very low E:T ratio (1:8, was performed with ImageJ software. 1:16, 1:32, and 1:64). Target cells were also tested separately from CIK cells as control to assess their spontaneous mortality. The percentage of In vitro cytokine production STS-specific lysis for each E:T ratio was calculated using the following CSPG4-CAR.CIK and CSPG4-CAR.T cells, and unmanipulated formula: [(experimental spontaneous mortality/100 spontaneous NTD.CIK and NTD.T cells were cocultured alone or with tumor cells mortality) 100]. In selected experiments, growth of residual sarcoma in RPMI1640 medium with 300 U/mL (CIK) or 50 U/mL IL2 (T cells) cells was investigated 48 hours after the treatment with either CSPG4- at a 2:1 E:T ratio and incubated at 37C for 48 hours. Concentrations of CAR.CIK or NTD.CIK. cytokines in culture supernatant were measured using the Bio-Plex Pro In 3D assays, STS spheroids stably expressing GFP were seeded one Human Cytokine 9-plex Assay Kit (Bio-Rad Laboratories Inc.) accord- per well in ULA 96-well round bottom plates. CSPG4-CAR.CIK and ing to the manufacturer’s instructions. Each sample was measured in unmodified NTD.CIK were stained with PKH26 dye and plated at E:T duplicate. Data were acquired and analyzed by Bioclarma (Analysis ratio 2:1 in culture medium with 300 U/mL IL2 at 37 C5%CO2. Service). Granzyme B concentration was measured in supernatants Fluorescence images were acquired at 12-hour intervals over 96 hours from mixed target/effector cell cultures (ELISA Granzyme B Kit, under the same magnification (10). Killing activity was determined Diaclone SAS), as recommended by the manufacturer. as loss of GFP fluorescence spheroid area (pixel) using fluorescence microscopy (Leica DMI 3000B equipped with Photometrics CoolSnap In vivo activity of patient-derived CSPG4-CAR.CIK HQ CCD Camera). Untreated STS spheroids were used to evaluate The antitumor activity of CSPG4-CAR.CIK and unmodified NTD.CIK spontaneous mortality. All images were analyzed with ImageJ soft- was evaluated using STS xenograft models in immunodeficient mice.

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In vivo experiments received approval by the competent committee slides and visualized with a DM750 Leica Microscope equipped and internal review board (auth. no., 178/2015-PR). STS xenografts with Leica ICC50W CCD Camera (LAS EZ3.4.0 software). were established in 7- to 8-week-old NOD/SCID/gc / (NSG) or NOD/SCID (Charles River Laboratories, SRL) female mice by subcu- Statistical analysis taneous injection with 1 106 cells obtained from three STS All experiments were performed at least twice. Data were analyzed [fibrosarcoma (HT1080), leiomyosarcoma (S172), and UPS (S1)]. using GraphPad Prism 8.0 (GraphPad Software). Descriptive data are Autologous CSPG4-CAR.CIK and unmodified NTD.CIK were presented as mean values SE. To find statistical significance in the available for S172 and S1 xenografts. Allogeneic CSPG4-CAR.CIK comparison of two groups, we used two-tailed Student t tests; for from unrelated patients with STS were generated following the same comparison of three or more groups, the data were analyzed by two- protocol used to generate autologous CIK identical and used in the way ANOVA with Bonferroni multiple comparison post hoc tests. A HT1080 xenograft model. When tumors were approximatively P < 0.05 was considered significant. Significance is represented on 50 mm3 in volume, mice were infused twice a week with 1 graphs as , P ≤ 0.05; , P ≤ 0.01; , P ≤ 0.001; , P ≤ 0.0001. 106 CSPG4-CAR.CIK or unmodified NTD.CIK resuspended in PBS (200 mL), for a total of four infusions. Mice injected with PBS only were used as controls. Treatment and control cohorts included Results 6 mice each for the leiomyosarcoma (S172) group. For the fibro- Generation and characterization of patient-derived CSPG4- sarcoma (HT1080) and UPS (S1) xenografts and control groups, CAR.CIK each group included 3 mice. In the experiments with fibrosarcoma CIK were efficiently generated from PBMCs collected from 8 (HT1080) and leiomyosarcoma (S172), mice were sacrificed at patients diagnosed with STS. We genetically engineered CIK to express the end of treatment. In the experiment with UPS (S1), mice the CSPG4-CAR that includes the 4-1BB costimulatory endodomain. were sacrificed 14 days after the last CIK infusion. Mice were Three weeks following transduction and ex vivo expansion, the mean monitored daily for possible toxicities, while tumor growth was expression of CAR on engineered CIK (CSPG4-CAR.CIK) was 48% measured weekly with manual caliper. Mice were sacrificed at the 6% as assessed by flow cytometry (Supplementary Table S1; Supple- end of treatment or if tumor reached 2 cm along the main diameter. mentary Fig S1A) and was comparable with that obtained using the In additional experiments, mice engrafted with the S172 cell line retroviral vector encoding the CD19-specific CAR (CD19-CAR). (n ¼ 4 mice per treatment) or the HT1080 cell line (n ¼ 5miceper Ex vivo expansion of CSPG4-CAR.CIK was 154-fold (27–348) and was treatment) were infused with CSPG4-CAR.CIK and unmodified comparable with that observed with paired control unmodified NTD.CIK generated from unrelated donors. In these models, when CIK (NTD.CIK). The phenotypic characterization of CSPG4-CAR.CIK the tumor volume was approximatively 20 mm3, mice received two was comparable with that of paired unmodified NTD.CIK þ infusions (on days 0 and þ4) of 3 106 CSPG4-CAR.CIK or showing CD8 cells as the main cellular subset (69% 4%), of which þ þ unmodified NTD.CIK. Mice were sacrificed 11 days after the last 39% 5% of them coexpressed the CD56 molecule (CD3 CD56 ; dose of cells. Tumor volume was calculated by the following Supplementary Table S1). The NKG2D receptor was expressed in 66% þ formula: V ¼ 4/3 p (a/2)2 (b/2), where a is the length and 5%, and 47% 0.1% of the cells were CD62L CD45RA ; the latter b is the width of the tumor. represent the effector memory–like population (Supplementary Fig. S1B). The immune checkpoint receptor, PD-1, was expressed on IHC 12% 2% in both CSPG4-CAR.CIK and NTD.CIK (Supplementary STS xenografts were analyzed by IHC. Samples (5-mmthick) Table S1). were cut from formalin-fixed, paraffin-embedded tissue sections, mounted on slides, and treated as per standard IHC procedures. CSPG4 as a potential CAR target in STS Tissue sections were deparaffinized with 100% xylene and rehy- First, we confirmed that CSPG4 is expressed in multiple STS drated with decreasing concentrations of ethyl alcohol. Antigen histotype cells (leiomyosarcoma; dedifferentiated liposarcoma; retrieval was performed by boiling the sections in 1 mmol/L EDTA UPS, malignant fibrous histiocytoma, and high-grade spindle (pH 9.0) for 60 minutes. Slides were treated with 3% hydrogen cell sarcoma; myxofibrosarcoma; MPNST; and synovial sarcoma) peroxide, 1% BSA (Invitrogen), and 5% normal horse serum in TBS at levels similar to those found in melanoma cells (Fig. 1A)by [25 mmol/L Tris (pH 7.4) and 150 mmol/L NaCl] containing 0.1% in silico analysis with RNA-sequencing expression data from Tween 20 (Sigma-Aldrich, Inc). To confirm CSPG4 expression, TCGA database. Furthermore, using flow cytometry, we observed slides were incubated in a closed humid chamber overnight at 4C CSPG4 expression on the cell surface of 15 of 15 STS cell lines with the CSPG4-specific mAb 225.28, 763.74, and D2.8.5-C4B8 (4 obtained from biopsies of patients affected by different histotypes of mg/mL each) pool. After washing, a secondary anti-mouse IgG advanced STS (UPS, n ¼ 3; gastrointestinal stromal tumor, n ¼ 5; xenoantibody was added. Secondary antibodies conjugated to liposarcoma, n ¼ 4; leiomyosarcoma, n ¼ 2; and MPNST, n ¼ 1) horseradish peroxidase (HRP) were generated and IHC signals and in the HT1080 cell line (fibrosarcoma) from the ATCC were detected with the EnVision1 System-HRP (Dako North (Fig. 1B). America, Inc) and chromogen Diaminobenzidine (DAB) Substrate CSPG4 density on STS cell lines was quantified on per cell basis (DakoCytomation Liquid DABþ Substrate Chromogen System, and found variable among STS samples (mean of 321 47 Dako). Tissue sections were counterstained with Mayer Hematox- molecules/cell; Fig. 1C). STS cell lines were also confirmed to ylin (Bio-Optica). We also explored the presence of infiltrated express variable levels of the main known ligands recognized CSPG4-CAR.CIK, unmodified NTD.CIK cells, and apoptotic tumor by the NKG2D CIK receptor (MIC A/B, 30% 8%; ULBP1, 1% cells in explanted tumors from treated mice. Tissues were stained 0.7%; ULBP2/5/6, 71% 6%; and ULBP3, 25% 9%). HLA according to the manufacturer’s protocols with the primary poly- class I antigens were highly expressed by all STS cell lines tested clonal antibody anti-CD3 (DAKO) and anti-cleaved caspase 3 (Cell (95% 2%), along with varying levels of immune checkpoints, PD- Signaling Technology). Tissue sections were mounted on glass L1 (32% 9%) and PD-L2 (65% 6%; Supplementary Table S2).

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Figure 1. A 18 CSPG4 is highly expressed in multiple STS 16 histotypes. CSPG4 mRNA expression in multiple STS histotypes (leiomyosar- 14 coma; dedifferentiated liposarcoma; UPS, RSEM) 2 12 malignant ﬁbrous histiocytoma, and high- grade spindle cell sarcoma; myxoﬁbro- 10 sarcoma; MPNST; and synovial sarcoma). 8 CSPG4 expression in STS was compara- 6 ble with that observed in melanoma. RNA-sequencing expression data were 4 selected and downloaded from the cBio- 2 Portal of TCGA Pan-Cancer Collections. CSPG4 expression (log RSEM expression values were plotted 0 after log2 transformation with 0.5 jittering Lung Lung Breast cancer on the x-axis, using Microsoft Excel. cancer

ﬁ sarcoma

A, CSPG4 expression was con rmed in Melanoma carcinoma Pancreatic Soft-tissue patient-derived STS cell lines of various histologic types by flow cytometry. A squamous cell adenocarcinoma representative flow cytometry histogram is reported for each STS. The M14 mela- B noma cell line that lacks CSPG4 expres- 50 50 S234 S188 S23 50 37 37 sion and normal keratinocytes were used 37 25 25 Counts Counts 25 for comparison. B, Isotype controls 12 Counts 12 12 0 0 are shown in gray. C, Gray histograms 0 0 1 2 3 4 100 101 102 103 104 10 10 10 10 10 100 101 102 103 104 FL 8 Log FL 8 Log FL 8 Log show the number of CSPG4 molecules 50 50 50 S175 S047 S5 expressed on the cell surface of various 37 37 37 fi 25 25 25 Counts patient-derived STS cell lines quanti ed Counts 12 12 12 as the CSPG4-specific mAb-binding 0 0 0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 capacity on a per cell basis. FL 8 Log FL 8 Log FL 8 Log 50 50 50 HT1080 S181 Keranocytes 37 37 37 25 25 25 Counts Counts Counts 12 12 Counts 12 0 0 0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 FL 8 Log FL 8 Log FL 8 Log 50 50 S061 S3 50 S088 37 37 37 25 25 25 Counts Counts

12 12 Counts 12 0 0 0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 FL 8 Log FL 8 Log FL 8 Log 50 50 50 S1 S172 S024 37 37 37 25 25 25 Counts Counts 12 12 Counts 12 0 0 0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 FL 8 Log FL 8 Log FL 8 Log 50 50 50 S309 M14 S25 37 37 37 25 25

25 Counts Counts Counts 12 12 12 0 0 0 0 1 2 3 4 100 101 102 103 104 100 101 102 103 104 10 10 10 10 10 FL 8 Log FL 8 Log FL 8 Log

CSPG4

C 800

600

400

200 CSPG4 expression (molecules/cell)

0 0 1 5 8 3 5 4 4 8 S1 2 S 23 S tes 1 S S y 088 S2 M S234 S175 S06 S18 S047 S181 S172 S309 c S o HT10 n ti ra Ke

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A ** **** 100 **** **** 80 CSPG4-CAR.CIK vs. STS **** **** NTD.CIK vs. STS 60

20 Tumor cell lysis (%)

:1 :1 :1 :2 :4 0:1 5 .5 1 1 1 1 2 Effector/target ratio B C 100 100 NTD.CIK vs. STS

80 **** CSPG4-CAR.CIK vs. STS 80 CSPG4-CAR.CIK vs. STS *** NTD.CIK vs. STS CD19-CAR.CIK vs. STS 60 60

40 40

20 20 Tumor cell lysis (%) Tumor cell lysis (%) 0 0

:8 6 2 4 1 :1 :3 :6 :1 :1 :4 1 1 1 5 1 1 Effector/target ratio Effector/target ratio D E

CSPG4- 100 CSPG4-CAR.CIK vs. M14 100 CSPG4-CAR.CIK vs. STS CSPG4- NTD.CIK vs. M14 NTD.CIK vs. STS 80 80 CSPG4-CAR.CIK vs. keratinocytes NTD.CIK vs. keratinocytes 60 60

40 40

20 vs. keratinocytes 20 Tumor cell lysis (%) Tumor cell lysis (%)

0 0

:1 :1 :1 :2 :1 :2 0:1 5 .5 1 1 1 1 1 2 Effector/target ratio Effector/target ratio F G *** 100 ** 80 CSPG4-CAR.CIK vs. S061NKG2D LIGANDS- ** * NTD.CIK vs. S061NKG2D LIGANDS- 60

40 50

20 Tumor cell lysis (%) CAR-specific lysis (%) 0 0 :1 :1 :1 :1 0 5 .5 1 1 2 Effector/target ratio

CSPG4-high STS CSPG4-low STS H I 150 60 CSPG4-CAR.CIK vs. STS Untreated STS NTD.CIK vs. STS 100 40 ** CSPG4-CAR.T vs. STS NTD.T vs. STS

50 20 * Cell recovery (%) Tumor cell lysis (%)

0 0

K S 1 1 K T : : I 5 .5 R.CI S 2 A TS TD.Cated Effector/target ratio 4-C S N tre PG S C ated tre

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CSPG4 CAR.CIK Effectively Eradicate Soft-Tissue Sarcomas

CSPG4-CAR.CIK effectively and specifically target STS in vitro expressing STS as compared with NTD.CIK were: IFNg (80-fold), We explored the in vitro antitumor activityofCSPG4-CAR.CIK IL1b (4.5-fold), IL6 (1.3-fold), TNFa (7.6-fold), GM-CSF (88.5-fold), against 12 STS cell lines. In two cases (S1 UPS and S172 leiomyo- IL10 (16-fold), IL8 (1.3-fold), IL4 (7-fold), and granzyme B (20-fold). sarcoma), it was possible to obtain peripheral blood from the Complete cytokine values, including CAR.T lymphocytes control, are patient from whom the STS tumor cell line was generated. At reported in Supplementary Fig. S4B. E:T ratios from 10:1 to 1:4, CSPG4-CAR.CIK revealed significantly superior in vitro cytotoxicity against STS (Fig. 2A; Supplementary CSPG4-CAR.CIK effectively target STS cells in 3D spheroids Fig. S2A), as compared with that reported with unmodified NTD. We developed the STS spheroid model that mimics tumor 3D CIK (n ¼ 29; P < 0.0001). Furthermore, the antitumor activity of and allows exploring CAR.CIK migration in a multidimensional CSPG4-CAR.CIK was maintained even at very low E:T ratios (from structure. STS spheroids express GFP, which allows tracking their 1:8 to 1:64; n ¼ 8; P < 0.0001; Fig. 2B). fate by longitudinal imaging. Spheroids were generated for three CSPG4-CAR.CIK antitumor activity was superior to that of control STS (S1, S5, and S172). Spheroids were coincubated with effector CD19-CAR.CIK (n ¼ 5; P < 0.05; Fig. 2C) and was CSPG4 specific cells at E:T ratio 2:1, and loss of fluorescence over time was because CSPG4-CAR.CIK did not eliminate non-expressing CSPG4 considered as a surrogate indication of tumor elimination by target cells (n ¼ 3; P > 0.05; Fig. 2D). Finally, at E:T ratios that CSPG4-CAR.CIK (Fig. 3A and B). CSPG4-CAR.CIK eliminated mediated potent antitumor effects, CSPG4-CAR.CIK had no detect- STS spheroids more effectively than unmodified NTD.CIK (n ¼ 3; able activity against human keratinocytes that show low CSPG4 P < 0.0001; Fig. 4A–C). expression (n ¼ 5; Fig. 2E). CSPG4-CAR.CIK–mediated tumor In selected experiments, we also used live imaging to visualize the elimination was strictly dependent on the CSPG4 expression level on antitumor kinetics of CSPG4-CAR.CIK against STS spheroids tumor cells (n ¼ 7; P < 0.05; Fig. 2F). (Supplementary Videos S1 and S2). Furthermore, measurement of We also measured the antitumor activity of CSPG4-CAR.CIK in an the maximum intensity projections of S172 and S5 spheroids after STS sample expressing CSPG4, but lacking NKG2D ligands, and found incubation with CSPG4-CAR.CIK or unmodified NTD.CIK indi- that CAR expression, per se, promotes antitumor activity of redirected cated that CSPG4-CAR.CIK were present at a higher concentration CIK independently from NKG2D CIK receptor engagement (P < within the STS spheroids as compared with unmodified NTD.CIK 0.001; Fig. 2G). (n ¼ 8; P < 0.05; Fig. 4D–F; Supplementary Videos S3–S6). Finally, Following treatment with CSPG4-CAR.CIK, we evaluated wheth- in selected experiments, we measured the capability of CSPG4- er residual tumor cells spared by the cytotoxic effects of CIK may CAR.CIK to penetrate and migrate toward STS spheroids through regrow. We observed significant delayed in vitro regrowth of STS Matrigel domes, with the intent of mimicking their dynamics cells exposed to CSPG4-CAR.CIK (48 hours after treatment) as through the extracellular matrix (Fig. 5A). Microscopic inspection compared with tumor cells exposed to unmodified NTD.CIK (n ¼ 2; indicated that CSPG4-CAR.CIK readily migrated to the membrane P < 0.05; Fig. 2H). boundary and penetrated the Matrigel domes containing STS Asignificant and superior (3-fold, E:T 5:1) activity level of STS spheroids more efficiently than unmodified NTD.CIK (n ¼ 5; killing was displayed by CSPG4-CAR.CIK compared with CAR.T P < 0.01; Fig. 5B and C). lymphocytes. Both effector cells were generated from PBMCs collected from the same patient and expressed comparable levels CSPG4-CAR.CIK controlled tumor growth in vivo of CSPG4-CAR molecules (n ¼ 2; P < 0.001; Fig. 2I; Supplementary We explored the in vivo antitumor activity of CSPG4-CAR.CIK Fig. S3A and S3B). utilizing three STS xenograft models (HT1080 fibrosarcoma, S172 In selected experiments, we explored the production of Th1- and leiomyosarcoma, and S1 UPS) that were selected because of the Th2-type cytokines and granzyme B by CSPG4-CAR.CIK at baseline different levels of CSPG4 expression. Upon tumor engraftment and following exposure to STS CSPG4-positive targets. Overall, we (50 mm3), mice were treated with intravenous infusions of observed higher baseline productions of IFNg, IL6, IL1b, IL4, IL8, GM- CSPG4-CAR.CIK or unmodified NTD.CIK (Fig. 6A). In two models CSF, TNFa, IL10, and granzyme B by CSPG4-CAR.CIK that markedly (S1 and S172) CIK and tumor cells were autologous, while CAR.CIK increased following engagement with STS CSPG4-positive targets (n ¼ tested in the HT1080 xenograft model were allogeneic. In the S172 4; Supplementary Fig. 4A). The highest cytokine peaks were observed leiomyosarcoma xenograft model characterized by 72% CSPG4 for IFNg, IL6, IL8, TNFa, and GM-CSF. The most differentially expression and a density of 262 molecules/cell, autologous CSPG4- expressed cytokines by CSPG4-CAR.CIK in response to CSPG4- CAR.CIK, but not unmodified NTD.CIK or vehicle, caused significant

Figure 2. CSPG4-CAR.CIK effectively and specifically targets STS cells in vitro. Patient-derived CSPG4-CAR.CIK efficiently targeted STS cells in vitro when using either HLA-mismatched (10 samples) or -matched CIK (two samples). Specific cytotoxicity of CSPG4-CAR.CIK was significantly higher than that obtained with unmodified NTD.CIK. A, Tumor cell–specific cytotoxicity values from 29 experiments are reported (mean SEM). B, CSPG4-CAR.CIK retained their antitumor activity when challenged at very low E:T ratios (n ¼ 8). C, In vitro cytotoxic activity of control CD19-CAR.CIK against STS was comparable with that of unmodified NTD.CIK (n ¼ 5). D, CSPG4-CAR.CIK and paired unmodified NTD.CIK showed similar cytotoxic activity against the M14 control cell line that lacks CSPG4 expression (n ¼ 3). E, Both CSPG4-CAR.CIK and paired unmodified NTD.CIK did not lyse normal keratinocytes (n ¼ 5). F, CSPG4-CAR.CIK exhibited more intense killing of STS cells with high CSPG4 expression as compared with STS cells with low CPSG4 expression (n ¼ 7; , P ≤ 0.05 by t test). G, CSPG4-CAR.CIK, but not paired unmodified NTD.CIK, retained cytotoxic activity against an STS sample expressing CSPG4, but lacking NKG2D ligands (S061; n ¼ 3; two-way ANOVA and Bonferroni post hoc test analysis). H, STS cell growth assessed 48 hours following in vitro treatment was significantly delayed in STS exposed to CSPG4-CAR.CIK, as compared with unmodified NTD.CIK. The dashed line represents the control parallel growth of untreated STS cells (n ¼ 2; , P ≤ 0.05 by unpaired t test). I, CSPG4-CAR.CIK displayed significant and superior STS killing activity compared with paired “non-cytokine induced,” but minimally activated with IL2 (50 U/mL) CAR.T expressing comparable membrane levels of CSPG4-CAR molecules (n ¼ 2; two-way ANOVA and Bonferroni post hoc test analysis). All cytotoxicity assays were analyzed by two-way ANOVA and Bonferroni post hoc test analysis; statistical significance is reported as , P ≤ 0.05; , P ≤ 0.01; , P ≤ 0.001; , P ≤ 0.0001; “n” refers to the number of separate experiments.

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A E:T 2:1 + CSPG4-CAR.CIK

PGK.GFP-LV ULA 96 MW U bottom plate

Plate centrifugation + NTD.CIK

2D monolayer STS cells 2D GFP+ monolayer STS cells 3D GFP+ STS spheroid

B 0 h 24 h 48 h 72 h UNTREATED STS SPHEROIDS NTD.CIK vs. STS SPHEROIDS CSPG4-CAR.CIK vs. STS SPHEROIDS

Figure 3. CSPG4-CAR.CIK effectively target STS cells in 3D spheroids. A, Schematic showing the 3D assay to test the cytotoxic activity of CSPG4-CAR.CIK against GFP- expressing STS spheroids (green). CSPG4-CAR.CIK showed superior tumor elimination, as compared with paired unmodified NTD.CIK. B, Shown are representative microscope fluorescence images and surface plot images of GFP-expressing STS spheroids (green) treated with CSPG4-CAR.CIK (up to 72 hours) and control CIK. Magnification, 10; scale bars, 100 mm.

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A B C **** **** **** **** **** 100 100 **** CSPG4-CAR.CIK vs. S1 CSPG4-CAR.CIK vs. S5 100 CSPG4-CAR.CIK vs. S172 90 90 NTD.CIK vs. S1 NTD.CIK vs. S5 90 **** NTD.CIK vs. S172 80 80 **** 80 70 **** 70 70 60 60 **** 60 50 *** 50 50 40 40 **** 40 30 30 30 Tumor cell lysis (%) Tumor cell lysis (%) 20 20 Tumor cell lysis (%) 20 10 10 10 0 0 0 0 h 0 h Time (h) Time (h) Time (h)

D NTD.CIK vs. STS SPHEROIDS CSPG4-CAR.CIK vs. STS SPHEROIDSE NTD.CIK vs. STS SPHEROIDS CSPG4-CAR.CIK vs. STS SPHEROIDS

F 25,000 * ) 2 20,000 m

15,000

10,000

CIK cell area ( 5,000

IK R.CIK TD.C A N -C 4 PG S C

Figure 4. CSPG4-CAR.CIK infiltrate 3D STS spheroids. CSPG4-CAR.CIK displayed cytolytic activity against 3D STS spheroids. Tumor cell elimination mediated by CSPG4-CAR.CIK was quantified by measuring GFP fluorescence loss over time of S1 (A), S5 (B), and S172 (C) spheroids (pixel) by fluorescence microscopy. Values are reported as mean ( SEM) from three independent wells (E:T ratio, 2:1). Results were analyzed by two-way ANOVA and Bonferroni post hoc test analysis; statistical significance is reported as , P ≤ 0.001; , P ≤ 0.0001. Representative maximum intensity projections of confocal microscopy images for S5 (D) and S172 (E) spheroids (green) treated with unmodified PKH26-stained (red) NTD.CIK and CSPG4-CAR.CIK (E:T ratio, 2:1). Confocal microscopy images taken 16 hours after coincubating CIK and STS spheroids. Magnification, 20 and scale bars, 100 mm are shown. F, CSPG4-CAR.CIK displayed superior infiltration within the STS spheroids (green) as compared with paired unmodified NTD.CIK (n ¼ 8; , P ≤ 0.05 by paired t test). CSPG4-CAR.CIK density was determined as red fluorescence PKH26 area (mm2).

delay in tumor growth (P < 0.05; n ¼ 6 mice/group; Fig. 6B). In the P < 0.0001, S172; Supplementary Fig. S5B–S5E). A complete tumor HT1080 fibrosarcoma and S1 UPS xenograft models, showing 23% regression was observed in 2 of 4 mice bearing leiomyosarcoma and and 95% CSPG4 expression, respectively, but similar densities of 1of5micebearingfibrosarcoma (HT1080). Of note, antitumor target molecules (521 and 499 molecules/cell, respectively), CSPG4- effects were obtained without any macroscopic evidence of toxicity. CAR.CIK also delayed tumor growth as compared with controls (P < 0.001, HT1080; P < 0.0001, S1; n ¼ 3 mice/group; Fig. 6C and D). Tumor infiltration by CSPG4-CAR.CIK was confirmed in Discussion explanted tumors by IHC (Fig. 6E). Cleaved caspase 3 levels were STS are tumors for which the clinical impact of targeted therapies confirmed to be higher in tumors from mice treated with CSPG4- remains modest (3). Here, we report that CSPG4 is a clinically CAR.CIK as compared with those from NTD.CIK-treated or vehi- relevant target in STS and that CSPG4 expression can be efficiently cle-treated mice (Fig. 6F). exploited to eliminate STS tumors by redirecting the specificity of The antitumor activity of CSPG4-CAR.CIK was also verified CIK through CAR engineering. We generated compelling evidence in additional experiments, in which we reduced the initial tumor supporting the antitumor activity of CAPG4-CAR.CIK using cell burden (20 mm3) for both HT1080 and S172 tumors (Supple- lines derived from patients who relapsed after conventional treat- mentary Fig. 5A). In these models, CSPG4-CAR.CIK treatment ments and developing an experimental platform that includes resulted in significant delay of tumor growth up to 11 days after the in vitro bidimensional and 3D assays along with three distinct end of treatment as compared with controls (P < 0.001, HT1080 and in vivo STS xenograft models.

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PKH26 stained CIK Wash-out

CSPG4-CAR.CIK CSPG4-CAR.CIK 120-h cocolture

Spheroid embedded in matrigel dome

Wash-out

24 MW plate

PKH26 stained CIK NTD.CIK NTD.CIK 3D STS spheroid

120-h cocolture Wash-out

Empty matrigel dome –NTD.CIK CSPG4-CAR.CIK –NTD.CIK CSPG4-CAR.CIK B EMPTY STS SPHEROID

NTD.CIK CSPG4-CAR.CIK NTD.CIK CSPG4-CAR.CIK

C 80,000 ) 2 ** 60,000

40,000

20,000 CIK cell area ( m

IK K C R.CI TD. A N -C 4 PG S C

Figure 5. CSPG4-CAR.CIK penetrate Matrigel matrix toward the STS spheroids. Schematic showing the Matrigel-based penetration assay. STS spheroids were embedded into Matrigel domes and cocultured with either PKH26-stained CSPG4-CAR.CIK or unmodified NTD.CIK at E:T ratios 10:1. Empty domes served as controls. A, Fluorescence microscope images were acquired at day 5 of coculture. Representative images of CSPG4-CAR.CIK and unmodified NTD.CIK empty or STS spheroids embedded in Matrigel domes are displayed. Dashed lines represent Matrigel boundaries. B, Bright field microscope images (top) and fluorescence images (bottom). Magnification, 4;scalebars,100mm. Summary showing that CSPG4-CAR.CIK displayed superior ability in penetrating Matrigel domes as compared with paired NTD.CIK (n ¼ 5; P ≤ 0.01 by paired t test). C, CSPG4-CAR.CIK density was defined as the fluorescence PKH26 area (mm2). , P ≤ 0.01.

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A B Autologous leiomyosarcoma

VEHICLE 2,000 End of treatment ) 3 1,500

(mm VEHICLE STS xenograft 0yaD 7yaD 41yaD ~50 mm3 NTD.CIK vs. S172 CSPG4-CAR.CIK (1x106) 1,000 CSPG4.CAR.CIK vs. S172 Start volume infusions * 500 STS xenograft Day 0 7yaD 41yaD ~50 mm3 Tumor

NTD.CIK (1x106) 0

Day 0 Day 5 Day 10 Day 15

STS xenograft 0yaD 7yaD 41yaD ~50 mm3

C HT1080 D Autologous UPS

End of treatment

VEHICLE 4,000 VEHICLE

3,000 NTD.CIK vs. HT1080 ) 3 NTD.CIK vs. S1 )

3 2,500 CSPG4.CAR.CIK vs. HT1080 3,000 CSPG4-CAR.CIK vs. S1 2,000 1,500 End of treatment 1,000 ** 2,000 Start *** Start 600 *** *** infusions infusions 400 1,000 200 Tumor volume (mm Tumor volume (mm 0 0

Day 0 Day 5 Day 0 Day 10 Day 15 Day 5 Day 10 Day 15 Day 30 RESCUE

E Vehicle KIC.DTN KIC.RAC-4GPSC CD3

F Vehicle KIC.DTN KIC.RAC-4GPSC Caspase-3

Figure 6. CSPG4-CAR.CIK are active against STS in xenograft models. A, Schematic representation of the STS xenografts and treatment with CSPG4-CAR.CIK (red arrows) and NTD.CIK (blue arrows). Vehicle-treated mice were infused with PBS (gray arrows). CIK were infused intravenously (1 106 cells/infusion) twice a week for 2 weeks. B, Autologous CSPG4-CAR.CIK caused a significant delay of the growth of the S172 leiomyosarcoma (CSPG4 ¼ 72% and CSPG4 density ¼ 262 molecule/cell) as compared with unmodified NTD.CIK or vehicle-treated mice (n ¼ 6; P < 0.05). C, Autologous CSPG4-CAR.CIK caused a significant delay of the growth of the HT1080 fibrosarcoma (CSPG4 ¼ 23% and CSPG4 density ¼ 521 molecule/cell) as compared with unmodified NTD.CIK or vehicle-treated mice (n ¼ 3, P < 0.001). D, Autologous CSPG4-CAR.CIK effectively delayed the growth of S1 UPS (CSPG4 ¼ 95% and CSPG4 density ¼ 499 molecule/cell) as compared with controls (n ¼ 3, P < 0.0001). All results were analyzed by two-way ANOVA and the Bonferroni post hoc test; statistical significance is reported as , P ≤ 0.05; , P ≤ 0.01; , P ≤ 0.001. E, Tumor homing of CSPG4-CAR.CIK and unmodified NTD.CIK was confirmed by IHC in explanted tumors using an anti-human CD3 antibody staining. Magnification, 40; scale bars, 50 mm. F, Apoptotic tumor cells were visualized by detecting cleaved caspase 3 by IHC in explanted tumors.

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The survival rate of adults and children with STS remains extremely further supporting the evidence that NKG2D and CAR engagement poor even in the era of checkpoint inhibitors (3, 50, 51). The latter is not mutually exclusive in CAR-engineered CIK. Our study, disappointing clinical results are likely to be caused at least, in part, by using relatively simple 3D assays to explore the dynamic features of the “cold” or “immunologically ignorant” tumor microenvironment, CAR.CIK ex vivo in a more complex structure compared with liquid low neoantigen load, and defects in HLA class I antigen-presenting culture, also uncovered remarkable migratory and infiltrative abilities machinery in STS (3, 52). of CAR-engineered CIK. These properties further underline the Adoptive transfer of ex vivo–engineered effector T cells, and in potential impact of these cells in solid tumors. It is important to particular CAR.T cells, providing HLA-independent tumor recog- acknowledge that clinical data indicate that CIK are more terminally nition may offer the possibility to elicit immune responses in differentiated as compared with activated and expanded T cells. As otherwise silent tumors, such as STS. However, the identification a result, their longevity in vivo upon adoptive transfer may be of the most appropriate antigen to be recognized by CAR.T cells in limited (32, 57). However, clinical data also indicate that the STS remains to be defined. Here, we show that CSPG4 holds critical manufacturing of CIK both unmanipulated or CAR engineered is biological features to qualify as a valuable target candidate in very robust. This property allows logarithmic expansion of cells in STS (38, 41). Using TCGA dataset we found that CSPG4 mRNA a relatively short period of time and storing of CIK for multiple is highly expressed in STS across multiple histotypes. Furthermore, infusions. Furthermore, the initial evidence of a lower IL1b and IL6 we confirmed CSPG4 protein expression on the cell surface of a wide production by CAR.CIK as compared with paired “conventional” array of STS cell lines spanning multiple STS histotypes and derived CAR.T lymphocytes, may further support a favorable safety profile. from patients who relapsed after conventional treatments. This Overall, our data support CSPG4 as a valuable CAR target for STS information is critical in the clinical setting because STS are intrin- and the use of engineered CIK to express the CSPG4-CAR for the sically highly heterogeneous, especially in relapsed patients (1). development and implementation of a novel and effective immuno- Importantly, CSPG4 expression in all these tumor cell lines therapeutic strategy for the treatment of patients with advanced/ was identified by the antibody that we have used to generate the relapsed high-grade STS. CSPG4-CAR and previously used to show the limited expression of CSPG4 in normal healthy tissues as compared with multiple solid Disclosure of Potential Conflicts of Interest tumors, despite broad CSPG4 mRNA expression in various G. Grignani reports personal fees from Lilly and Merck; grants and personal fees organs (37, 53). In the perspective of a clinical application, a from Bayer, PharmaMar, and Novartis; and personal fees from Eisai outside the systematic confirmation of CSPG4 expression by IHC in STS sec- submitted work. L. D’Ambrosio reports other from GlaxoSmithKline (advisory tions that include tumor microenvironment would be warranted. board), PSI CRO Italy (advisory board), PharmaMar (meeting participation), As for the majority of nonlineage-restricted markers, the density of Celgene (meeting participation), Novartis (editorial activity), and Lilly (meeting participation) outside the submitted work. E. Vigna reports other from METIS CSPG4 expression in STS cell lines was variable, but in general much Precision Medicine B-Corp (company founder) outside the submitted work. G. higher than the expression detected in normal keratinocytes. We found Dotti reports grants from Cell Medica (research sponsor agreement), Bluebird Bio a correlation between CSPG4 expression level and antitumor activity (research sponsor agreement), and Bellicum Pharmaceutical (research sponsor by CSPG4-CAR.CIK with minimal activity against normal keratino- agreement); personal fees from Bellicum Pharmaceutical (scientificadvisory cytes. The latter finding is consistent with previous data underscoring board), MolMed S.p.A. (scientific advisory board), and Tessa Therapeutics the importance of antigen density to define a therapeutic window for (consultant) outside the submitted work; as well as a patent for CSPG4.CAR pending owned by the University of North Carolina and Massachusetts General antigens that are expressed, although at low levels, also by normal Hospital. D. Sangiolo reports grants from AIRC (Associazione Italiana Ricerca sul tissues (54, 55). Cancro), Ministry of Health, FPRC (Fondazione Piemontese per la Ricerca sul While CAR-redirected effector T cells showed potent antitumor Cancro) ONLUS, and University of Torino during the conduct of the study. No effects in B-cell malignancies, it remains to be defined whether other potential conflicts of interest were disclosed by the other authors. cell subtypes such as NK cells, NKT cells, or gdT cells may possess intrinsic biologic characteristics to be exploited in solid tumors to Authors’ Contributions enhance the therapeutic index of engineered immune cells (12). V. Leuci: Conceptualization, formal analysis, supervision, investigation, Here, rather than exploring different immune cell subsets, we visualization, writing-original draft, writing-review and editing. C. Donini: propose that T cells with enhanced cytokine activation during the Conceptualization, formal analysis, supervision, investigation, visualization, ex vivo expansion, such as CAR-engineered CIK, may represent a writing-original draft, writing-review and editing. G. Grignani: Resources, funding acquisition, writing-review and editing. R. Rotolo: Investigation, writing- valid cellular platform for the treatment of STS. Dosing and – original draft, writing-review and editing. G. Mesiano: Resources, investigation, prolonged ex vivo exposure to IL2 induces a mixed T NK pheno- writing-review and editing. E. Fiorino: Resources, investigation, writing-review and type and function in CIK lymphocytes with variable degree of HLA- editing. L. Gammaitoni: Resources, investigation, writing-review and editing. independent (NKG2D-mediated) tumor killing ability (56). We L. D’Ambrosio: Resources, writing-original draft, writing-review and editing. reasoned that CAR-engineered CIK would exploit dual tumor cell A. Merlini: Resources, visualization, writing-review and editing. E. Landoni: killing potential, namely antigen independent via NKG2D receptor Resources, investigation, writing-review and editing. E. Medico: Data curation, formal analysis, investigation, writing-original draft, writing-review and editing. engagement and antigen dependent via CSPG4-CAR engagement. S. Capellero: Investigation, writing-review and editing. L. Giraudo: Investigation, The utilization of these two mechanisms is expected to ultimately visualization, writing-review and editing. G. Cattaneo: Investigation, writing-original amplify the antitumor effects of CSPG4-CAR.CIK and potentially draft. I. Iaia: Investigation, writing-review and editing. Y. Pignochino: Resources, counteract the escape mechanisms utilized by tumor cells with low investigation, writing-original draft. M. Basirico: Investigation. E. Vigna: Resources, CSPG4 expression. We found that CAR.CIK exerted potent anti- writing-original draft, writing-review and editing. A. Pisacane: Resources, validation. tumor effects against a wide array of STS, which was particularly F. Fagioli: Writing-review and editing. S. Ferrone: Conceptualization, resources, writing-original draft, writing-review and editing. M. Aglietta: Resources, writing- evident at very low E:T ratios. fi original draft, writing-review and editing. G. Dotti: Conceptualization, resources, The activity of CSPG4-CAR.CIK was con rmed in three indepen- writing-original draft, writing-review and editing. D. Sangiolo: Conceptualization, dent STS xenograft models and showed clearly gain of function by CIK resources, supervision, funding acquisition, writing-original draft, writing-review cells expressing the CAR as compared with unmodified NTD.CIK, and editing.

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Acknowledgments DOD grant W81XWH-16-1-0500. V. Leuci has received a fellowship from the This study was supported by fundings from “Associazione Italiana Ricerca sul Fondazione Nicola Ferrari ONLUS and M. Basirico has received a fellowship from Cancro” IG-2017 n. 20259 (to D. Sangiolo), IG-2019 n. 23104 (to G. Grignani); ADISCO ONLUS. The authors sincerely thank Joan Leonard (Leonard Editorial Ricerca corrente Progetto CAR-T RCR-2019-23669115 (to D. Sangiolo and Services, LLC) for the linguistic revision and editorial assistance. E. Medico); FPRC ONLUS 5 1000, Ministero della Salute 2015 Cancer ImGen (to D. Sangiolo, G. Grignani, and E. Medico); FPRC ONLUS 5 1000 MIUR 2014 The costs of publication of this article were defrayed in part by the payment of page (to G. Grignani and L. D’Ambrosio); Ministero della Salute (GR-2011-02349197 to charges. This article must therefore be hereby marked advertisement in accordance D. Sangiolo), University of Torino Fondo Ricerca Locale 2017 (to D. Sangiolo); with 18 U.S.C. Section 1734 solely to indicate this fact. Ricerca Corrente Ministero Salute 2020; and Fondazione per la ricerca sui tumori dell’apparato muscoloscheletrico e rari Onlus CRT RF ¼ 2016–0917 (to G. Grignani). Received January 30, 2020; revised July 14, 2020; accepted August 25, 2020; S. Ferrone was supported by NIH grants R01DE028172 and R03CA216114 and by published ﬁrst September 8, 2020.

References 1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin 2019;69: adoptive immunotherapy after allogeneic stem cell transplantation. Haemato- 7–34. logica 2010;95:1579–86. 2. Pasquali S, Pizzamiglio S, Touati N, Litiere S, Marreaud S, Kasper B, et al. The 21. Rettinger E, Meyer V, Kreyenberg H, Volk A, Kuci¸ S, Willasch A, et al. Cytotoxic impact of chemotherapy on survival of patients with extremity and trunk wall capacity of IL-15-stimulated cytokine-induced killer cells against human acute soft tissue sarcoma: revisiting the results of the EORTC-STBSG 62931 rando- myeloid leukemia and rhabdomyosarcoma in humanized preclinical mouse mised trial. Eur J Cancer 2019;109:51–60. models. Front Oncol 2012;2:32. 3. Pollack SM, Ingham M, Spraker MB, Schwartz GK. Emerging targeted and 22. Introna M, Golay J, Rambaldi A. Cytokine induced killer (CIK) cells for the immune-based therapies in sarcoma. J Clin Oncol 2018;36:125–35. treatment of haematological neoplasms. Immunol Lett 2013;155:27–30. 4. Wisdom AJ, Mowery YM, Riedel RF, Kirsch DG. Rationale and emerging 23. Cappuzzello E, Sommaggio R, Zanovello P, Rosato A. Cytokines for the strategies for immune checkpoint blockade in soft tissue sarcoma. Cancer induction of antitumor effectors: the paradigm of cytokine-induced killer (CIK) 2018;124:3819–29. cells. Cytokine Growth Factor Rev 2017;36:99–105. 5. Alsaab HO, Sau S, Alzhrani R, Tatiparti K, Bhise K, Kashaw SK, et al. PD-1 and 24. Cappuzzello E, Tosi A, Zanovello P, Sommaggio R, Rosato A. Retargeting PD-L1 checkpoint signaling inhibition for cancer immunotherapy: mechanism, cytokine-induced killer cell activity by CD16 engagement with clinical-grade combinations, and clinical outcome. Front Pharmacol 2017;8:561. antibodies. Oncoimmunology 2016;5:e1199311. 6. Donini C, D’Ambrosio L, Grignani G, Aglietta M, Sangiolo D. Next generation 25. Li M, Wang Y, Wei F, An X, Zhang N, Cao S, et al. Efficiency of cytokine-induced immune-checkpoints for cancer therapy. J Thorac Dis 2018;10:S1581–S601. killer cells in combination with chemotherapy for triple-negative breast cancer. 7. Pollack SM, Loggers ET, Rodler ET, Yee C, Jones RL. Immune-based therapies J Breast Cancer 2018;21:150–7. for sarcoma. Sarcoma 2011;2011:438940. 26. Schmeel LC, Schmeel FC, Coch C, Schmidt-Wolf IG. Cytokine-induced killer 8. Jacoby E, Shahani SA, Shah NN. Updates on CAR T-cell therapy in B-cell (CIK) cells in cancer immunotherapy: report of the international registry on CIK malignancies. Immunol Rev 2019;290:39–59. cells (IRCC). J Cancer Res Clin Oncol 2015;141:839–49. 9. Grupp SA, Kalos M, Barrett D, Aplenc R, Porter DL, Rheingold SR, et al. 27. Magnani CF, Mezzanotte C, Cappuzzello C, Bardini M, Tettamanti S, Fazio G, Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl et al. Preclinical efficacy and safety of CD19CAR cytokine-induced killer cells J Med 2013;368:1509–18. transfected with sleeping beauty transposon for the treatment of acute lympho- 10. Vera J, Savoldo B, Vigouroux S, Biagi E, Pule M, Rossig C, et al. T lymphocytes blastic leukemia. Hum Gene Ther 2018;29:602–13. redirected against the kappa light chain of human immunoglobulin efficiently 28. Marin V, Kakuda H, Dander E, Imai C, Campana D, Biondi A, et al. Enhance- kill mature B lymphocyte-derived malignant cells. Blood 2006;108:3890–7. ment of the anti-leukemic activity of cytokine induced killer cells with an anti- 11. Gauthier J, Yakoub-Agha I. Chimeric antigen-receptor T-cell therapy for CD19 chimeric receptor delivering a 4–1BB-zeta activating signal. Exp Hematol hematological malignancies and solid tumors: clinical data to date, current 2007;35:1388–97. limitations and perspectives. Curr Res Transl Med 2017;65:93–102. 29. Marin V, Pizzitola I, Agostoni V, Attianese GM, Finney H, Lawson A, et al. 12. Rotolo R, Leuci V, Donini C, Cykowska A, Gammaitoni L, Medico G, et al. CAR- Cytokine-induced killer cells for cell therapy of acute myeloid leukemia: based strategies beyond T lymphocytes: Integrative opportunities for cancer improvement of their immune activity by expression of CD33-specific chimeric adoptive immunotherapy. Int J Mol Sci 2019;20:2839. receptors. Haematologica 2010;95:2144–52. 13. Klebanoff CA, Rosenberg SA, Restifo NP. Prospects for gene-engineered T cell 30. Pizzitola I, Anjos-Afonso F, Rouault-Pierre K, Lassailly F, Tettamanti S, immunotherapy for solid cancers. Nat Med 2016;22:26–36. Spinelli O, et al. Chimeric antigen receptors against CD33/CD123 antigens 14. Leuci V, Mesiano G, Gammaitoni L, Aglietta M, Sangiolo D. Genetically efficiently target primary acute myeloid leukemia cells in vivo.Leukemia redirected T lymphocytes for adoptive immunotherapy of solid tumors. 2014;28:1596–605. Curr Gene Ther 2014;14:52–62. 31. Tettamanti S, Marin V, Pizzitola I, Magnani CF, Giordano Attianese GM, 15. Newick K, O’Brien S, Moon E, Albelda SM. CAR T cell therapy for solid tumors. Cribioli E, et al. Targeting of acute myeloid leukaemia by cytokine-induced Annu Rev Med 2017;68:139–52. killer cells redirected with a novel CD123-specific chimeric antigen receptor. Br J 16. Schmidt-Wolf IG, Lefterova P, Mehta BA, Fernandez LP, Huhn D, Blume KG, Haematol 2013;161:389–401. et al. Phenotypic characterization and identification of effector cells involved in 32. Leuci V, Casucci GM, Grignani G, Rotolo R, Rossotti U, Vigna E, et al. CD44v6 as tumor cell recognition of cytokine-induced killer cells. Exp Hematol 1993;21: innovative sarcoma target for CAR-redirected CIK cells. Oncoimmunology 1673–9. 2018;7:e1423167. 17. Giraudo L, Gammaitoni L, Cangemi M, Rotolo R, Aglietta M, Sangiolo D. 33. Merker M, Pfirrmann V, Oelsner S, Fulda S, Klingebiel T, Wels WS, et al. Cytokine-induced killer cells as immunotherapy for solid tumors: current Generation and characterization of ErbB2-CAR-engineered cytokine-induced evidences and perspectives. Immunotherapy 2015;7:999–1010. killer cells for the treatment of high-risk soft tissue sarcoma in children. 18. Gammaitoni L, Giraudo L, Macagno M, Leuci V, Mesiano G, Rotolo R, et al. Oncotarget 2017;8:66137–53. Cytokine-induced killer cells kill chemo-surviving melanoma cancer stem cells. 34. Zuo S, Wen Y, Panha H, Dai G, Wang L, Ren X, et al. Modification of cytokine- Clin Cancer Res 2017;23:2277–88. induced killer cells with folate receptor alpha (FRa)-specific chimeric antigen 19. Mesiano G, Grignani G, Fiorino E, Leuci V, Rotolo R, D’Ambrosio L, et al. receptors enhances their antitumor immunity toward FRa-positive ovarian Cytokine induced killer cells are effective against sarcoma cancer stem cells cancers. Mol Immunol 2017;85:293–304. spared by chemotherapy and target therapy. Oncoimmunology 2018;7: 35. Ren X, Ma W, Lu H, Yuan L, An L, Wang X, et al. Modification of cytokine- e1465161. induced killer cells with chimeric antigen receptors (CARs) enhances antitumor 20. Kuci S, Rettinger E, Voss B, Weber G, Stais M, Kreyenberg H, et al. Efficient lysis immunity to epidermal growth factor receptor (EGFR)-positive malignancies. of rhabdomyosarcoma cells by cytokine-induced killer cells: implications for Cancer Immunol Immunother 2015;64:1517–29.

AACRJournals.org Clin Cancer Res; 2020 OF13

Leuci et al.

36. Wang X, Wang Y, Yu L, Sakakura K, Visus C, Schwab JH, et al. CSPG4 in cancer: 48. Luo W, Hsu JC, Tsao CY, Ko E, Wang X, Ferrone S. Differential immunogenicity multiple roles. Curr Mol Med 2010;10:419–29. of two peptides isolated by high molecular weight-melanoma-associated anti- 37. Wang Y, Geldres C, Ferrone S, Dotti G. Chondroitin sulfate proteoglycan 4 as a gen-specific monoclonal antibodies with different affinities. J Immunol 2005; target for chimeric antigen receptor-based T-cell immunotherapy of solid 174:7104–10. tumors. Expert Opin Ther Targets 2015;19:1339–50. 49. Wang Y, Sabbatino F, Wang X, Ferrone S. Detection of chondroitin sulfate 38. Ilieva KM, Cheung A, Mele S, Chiaruttini G, Crescioli S, Griffin M, et al. proteoglycan 4 (CSPG4) in melanoma. Methods Mol Biol 2014;1102:523–35. Chondroitin sulfate proteoglycan 4 and its potential as an antibody immuno- 50. D’Angelo SP, Mahoney MR, Van Tine BA, Atkins J, Milhem MM, Jahagirdar BN, therapy target across different tumor types. Front Immunol 2017;8:1911. et al. Nivolumab with or without ipilimumab treatment for metastatic sarcoma 39. Geldres C, Savoldo B, Hoyos V, Caruana I, Zhang M, Yvon E, et al. T lymphocytes (Alliance A091401): two open-label, non-comparative, randomised, phase 2 redirected against the chondroitin sulfate proteoglycan-4 control the growth of trials. Lancet Oncol 2018;19:416–26. multiple solid tumors both in vitro and in vivo. Clin Cancer Res 2014;20:962–71. 51. Tawbi HA, Burgess M, Bolejack V, Van Tine BA, Schuetze SM, Hu J, et al. 40. Harrer DC, Dorrie€ J, Schaft N. CSPG4 as target for CAR-T-cell therapy of various Pembrolizumab in advanced soft-tissue sarcoma and bone sarcoma (SARC028): tumor entities-merits and challenges. Int J Mol Sci 2019;20:5942. a multicentre, two-cohort, single-arm, open-label, phase 2 trial. Lancet Oncol 41. Pellegatta S, Savoldo B, Di Ianni N, Corbetta C, Chen Y, Patane M, et al. 2017;18:1493–501. Constitutive and TNFa-inducible expression of chondroitin sulfate proteogly- 52. Pollack SM, He Q, Yearley JH, Emerson R, Vignali M, Zhang Y, et al. T-cell can 4 in glioblastoma and neurospheres: implications for CAR-T cell therapy. infiltration and clonality correlate with programmed cell death protein 1 and Sci Transl Med 2018;10:eaao2731. programmed death-ligand 1 expression in patients with soft tissue sarcomas. 42. Chekenya M, Krakstad C, Svendsen A, Netland IA, Staalesen V, Tysnes BB, et al. Cancer 2017;123:3291–304. The progenitor cell marker NG2/MPG promotes chemoresistance by activation 53. Beard RE, Zheng Z, Lagisetty KH, Burns WR, Tran E, Hewitt SM, et al. Multiple of integrin-dependent PI3K/Akt signaling. Oncogene 2008;27:5182–94. chimeric antigen receptors successfully target chondroitin sulfate proteoglycan 4 43. Ozerdem U, Stallcup WB. Pathological angiogenesis is reduced by targeting in several different cancer histologies and cancer stem cells. J Immunother pericytes via the NG2 proteoglycan. Angiogenesis 2004;7:269–76. Cancer 2014;2:25. 44. Wang J, Svendsen A, Kmiecik J, Immervoll H, Skaftnesmo KO, PlanagumaJ, 54. Majzner RG, Theruvath JL, Nellan A, Heitzeneder S, Cui Y, Mount CW, et al. et al. Targeting the NG2/CSPG4 proteoglycan retards tumour growth and CAR T cells targeting B7-H3, a pan-cancer antigen, demonstrate potent pre- angiogenesis in preclinical models of GBM and melanoma. PLoS One 2011;6: clinical activity against pediatric solid tumors and brain tumors. Clin Cancer Res e23062. 2019;25:2560–74. 45. Gao J, Aksoy BA, Dogrusoz U, Dresdner G, Gross B, Sumer SO, et al. Integrative 55. Du H, Hirabayashi K, Ahn S, Kren NP, Montgomery SA, Wang X, et al. analysis of complex cancer genomics and clinical profiles using the cBioPortal. Antitumor responses in the absence of toxicity in solid tumors by targeting Sci Signal 2013;6:pl1. B7-H3 via chimeric antigen receptor T cells. Cancer Cell 2019;35:221–37. 46. Cerami E, Gao J, Dogrusoz U, Gross BE, Sumer SO, Aksoy BA, et al. The cBio 56. Nishimura R, Baker J, Beilhack A, Zeiser R, Olson JA, Sega EI, et al. In vivo Cancer Genomics Portal: an open platform for exploring multidimensional trafficking and survival of cytokine-induced killer cells resulting in minimal cancer genomics data. Cancer Discov 2012;2:401–4. GVHD with retention of antitumor activity. Blood 2008;112:2563–74. 47. Sangiolo D, Mesiano G, Gammaitoni L, Leuci V, Todorovic M, Giraudo L, et al. 57. Franceschetti M, Pievani A, Borleri G, Vago L, Fleischhauer K, Golay J, et al. Cytokine-induced killer cells eradicate bone and soft-tissue sarcomas. Cytokine-induced killer cells are terminally differentiated activated CD8 cyto- Cancer Res 2014;74:119–29. toxic T-EMRA lymphocytes. Exp Hematol 2009;37:616–28.

OF14 Clin Cancer Res; 2020 CLINICAL CANCER RESEARCH

CSPG4-Specific CAR.CIK Lymphocytes as a Novel Therapy for the Treatment of Multiple Soft-Tissue Sarcoma Histotypes

Valeria Leuci, Chiara Donini, Giovanni Grignani, et al.

Clin Cancer Res Published OnlineFirst September 8, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-20-0357

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