chimeric antigen receptor-glypican-3 t-cell therapy for ... · 6/19/2020 · for advanced...

CLINICAL CANCER RESEARCH | CLINICAL TRIALS: IMMUNOTHERAPY

Chimeric Antigen Receptor-Glypican-3 T-Cell Therapyfor Advanced Hepatocellular Carcinoma: Results ofPhase I Trials A C

Donghua Shi1, Yaoping Shi1, Ahmed O. Kaseb2, Xingxing Qi1, Yuan Zhang1, Jiachang Chi1, Qing Lu3,HuipingGao4, Hua Jiang5, HuamaoWang4,DaijingYuan4, HongMa6, HongyangWang7, Zonghai Li4,5, andBo Zhai1

ABSTRACT◥

Purpose: Our preclinical studies demonstrated the potential ofchimeric antigen receptor (CAR)-glypican-3 (GPC3) T-cell therapyfor hepatocellular carcinoma (HCC). We report herein the firstpublished results of CAR-GPC3 T-cell therapy for HCC.

Materials and Methods: In two prospective phase I studies,adult patients with advanced GPC3þ HCC (Child-Pugh A)received autologous CAR-GPC3 T-cell therapy followingcyclophosphamide- and fludarabine-induced lymphodepletion.The primary objective was to assess the treatment's safety.Adverse events were graded using the Common TerminologyCriteria for Adverse Events (version 4.03). Tumor responses wereevaluated using the RECIST (version 1.1).

Results: A total of 13 patients received a median of 19.9 � 108

CAR-GPC3 T cells by a data cutoff date of July 24, 2019. We

observed pyrexia, decreased lymphocyte count, and cytokinerelease syndrome (CRS) in 13, 12, and nine patients, respectively.CRS (grade 1/2) was reversible in eight patients. One patientexperienced grade 5 CRS. No patients had grade 3/4 neurotox-icity. The overall survival rates at 3 years, 1 year, and 6 monthswere 10.5%, 42.0%, and 50.3%, respectively, according to theKaplan–Meier method. We confirmed two partial responses. Onepatient with sustained stable disease was alive after 44.2 months.CAR T-cell expansion tended to be positively associated withtumor response.

Conclusions: This report demonstrated the initial safety pro-file of CAR-GPC3 T-cell therapy. We observed early signs ofantitumor activity of CAR-GPC3 T cells in patients withadvanced HCC.

IntroductionHepatocellular carcinoma (HCC) is the most common histologic

subtype of liver cancer, which is the sixthmost common cancer and thefourth leading cause of cancer-related death worldwide (1). Half of allliver cancer cases and deaths are estimated to occur in China (2). In theUnited States, the incidence of liver cancer has more than tripled since1980 (3).

Considerable challenges exist in the clinical management of HCC.Only 15% to 20%ofHCC cases are diagnosed at an early stage thatmay

be suitable for curative treatment, such as surgical resection, livertransplantation, local ablation with percutaneous ethanol injection,microwave ablation, and radiofrequency ablation (4–7). Meanwhile,the majority of patients with HCC have underlying chronic liverdisease, and resection in this population is fraught with the potentialfor complications. Patients with intermediate-stage disease may ben-efit from local therapies such as transarterial chemoembolization.However, relapse is a frequent and expected event after systemic andlocal therapies (8). Also, many patients are diagnosed with unresect-able advanced-stage HCC, with macroscopic vascular invasion andextrahepatic spread. Sorafenib, a targeted kinase inhibitor, was the firstsystemic therapeutic approved by the FDA forHCCbased on improve-ment in overall survival (OS) duration by about 3 months (9, 10). Inaddition, regorafenib and two programmed cell death protein 1 (PD-1)inhibitors, pembrolizumab and nivolumab, were recently approved forHCC treatment in patients who experienced progression after takingsorafenib (11–13). Most recently, lenvatinib, cabozantinib, and ramu-cirumab were approved for patients with HCC who had progressionafter sorafenib treatment (14–17). The approval of anti–PD-1 inhi-bitors, pembrolizumab and nivolumab, demonstrates that HCC is animmunosensitive tumor. Furthermore, tremelimumab, an anti-cytotoxic T-lymphocyte–associated protein 4 (CTL-4) antibody, pro-duced response rates of 17% to 26% in patients with HCC (18, 19). Apilot randomized trial of perioperative immunotherapy with nivolu-mab and ipilimumab (anti–CTLA-4) for resectable HCC is ongoing,with preliminary results demonstrating good antitumor activity (20).Despite progress in available therapies, effective systemic treatmentoptions for HCC are still limited. Thus, its 5-year survival rate is adismal 18%; this rate decreases to 11% and 5% if cancer has spread intosurrounding tissues and distant parts of the body, respectively (21).

In recent years, chimeric antigen receptor (CAR) T-cell therapy hasachieved significant efficacy in the treatment of hematologic

1Department of Interventional Oncology, Renji Hospital, Shanghai Jiao TongUniversity School of Medicine, Shanghai, China. 2Department of GastrointestinalMedical Oncology, The University of Texas MD Anderson Cancer Center, Hous-ton, Texas. 3Department of Radiology, Renji Hospital, Shanghai Jiao TongUniversity School of Medicine, Shanghai, China. 4CARsgen Therapeutics Ltd.,Shanghai, China. 5State Key Laboratory of Oncogenes and Related Genes,Shanghai Cancer Institute, Shanghai Jiao Tong University School of Medicine,Shanghai, China. 6CARsgen Therapeutics Corp., Houston, Texas. 7InternationalCooperation Laboratory on Signal Transduction, Eastern Hepatobiliary SurgicalHospital, Second Military Medical University, Shanghai, China.

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

D. Shi and Y. Shi contributed equally to this article.

Clinical Trial registration ID: NCT02395250, NCT03146234

Corresponding Authors: Bo Zhai, Shanghai Jiao Tong University School ofMedicine, Shanghai 200127, China. Phone: 001-7135869741; E-mail:[email protected]; and Zonghai Li, [email protected]

Clin Cancer Res 2020;XX:XX–XX

doi: 10.1158/1078-0432.CCR-19-3259

�2020 American Association for Cancer Research.

AACRJournals.org | OF1

Cancer Research. on December 16, 2020. © 2020 American Association forclincancerres.aacrjournals.org Downloaded from

Published OnlineFirst May 5, 2020; DOI: 10.1158/1078-0432.CCR-19-3259

http://crossmark.crossref.org/dialog/?doi=10.1158/1078-0432.CCR-19-3259&domain=pdf&date_stamp=2020-6-16

http://crossmark.crossref.org/dialog/?doi=10.1158/1078-0432.CCR-19-3259&domain=pdf&date_stamp=2020-6-16

http://clincancerres.aacrjournals.org/

malignancies (22–26). Although a breakthrough in disease controlusing CAR T-cell therapy for solid tumors has yet to be achieved,researchers have observed encouraging antitumor activity in early-phase clinical trials of therapies targeting IL13Ra2 (27), mesothe-lin (28), and claudin18.2 (29, 30), suggesting the feasibility of CART-cell therapy for solid tumors.

Glypican-3 (GPC3) is amember of the heparan sulfate proteoglycanfamily and attaches to cell surfaces via a glycosylphosphatidylinositolanchor. Recent studies demonstrated that GPC3 may be a prognosticmarker for HCC, with greater GPC3 expression in tumor cellsassociated with worse prognosis (31). GPC3 is a molecule that is notfully understood in the role of proliferation and suppression of cellgrowth in normal tissues and abnormal or cancerous tissues. Of note,our previous studies demonstrated that GPC3 was highly expressed inHCC and squamous non–small cell lung cancer but showed noexpression in the kidney and gastric glands (32, 33). These findingssuggest that GPC3 is a rational immunotherapeutic target for HCC.Indeed, anti-GPC3 mAbs had good safety profiles in previous stud-ies (34, 35), although significant clinical benefit has yet to be estab-lished in phase II clinical trials. Recently, we demonstrated that GPC3-targeted CAR T cells could eliminate GPC3þ HCC cells in vitro anderadicate GPC3þ HCC tumor xenografts in mice (36–38). Therefore,in the present prospective phase I studies, we explored the safety andpotential efficacy of CAR-GPC3 T-cell therapy in adult Chinesepatients with advanced GPC3þ HCC. We hypothesized thatCAR-GPC3 T cells approach was feasible and could be safely toleratedamong patients with advanced HCC.

Patients and MethodsClinical trial design and patients

Herewe report two sequential phase I studies: NCT02395250 (study1, principal investigator, Z. Li) and NCT03146234 (study 2, principalinvestigator, B. Zhai). Both studies were approved by the EthicsCommittee of Renji Hospital, Shanghai Jiao Tong University Schoolof Medicine. Both trials were performed in accordance with theprinciples of the Declaration of Helsinki. All patients provided writteninformed consent to participate. Pooling the data allowed for a morerobust analysis with an appropriate number of patients to evaluatepharmacology dynamics and clinical responses.

Patients who were screened for GPC3 expression must have hadrelapsed HCC at least twice within 2 years on standard HCC therapy

perGuidelines for Diagnosis and Treatment of Primary Liver Cancer inChina (2011 edition) (39). As described previously (33), their GPC3expression status was determined via IHC staining of fresh tumorsamples or archived samples collected at local laboratories within6 months before enrollment. GPC3 expression was evaluated using a4-point scale: 0, no GPC3 expression; 1þ, weak expression; 2þ,medium expression; 3þ, strong expression (Supplementary Materialsand Methods). Eligibility criteria included one or more measurablelesions per the RECIST (version 1.1), a Child-Pugh score of A or Bwitha score of 7, and an Eastern Cooperative Oncology Group (ECOG)performance score of up to 1. The patients must have had reasonablehematologic cell counts and sufficient renal and hepatic function.Exclusion criteria included active ascites requiring treatment, humanimmunodeficiency virus infection, and the presence of an autoimmunedisease. A full description of the study design and eligibility criteria isprovided in the study protocols (Supplementary Materials andMethods).

Patients who met all screening criteria underwent leukapheresis toobtain peripheral blood mononuclear cells (PBMC) for the generationof autologous CAR-GPC3 T cells. As described previously (36),CAR-GPC3 T cells (product code Y035) consisted of a humanizedanti-GPC3 single-chain variable fragment, CD8a hinge domain,CD8a transmembrane domain, CD28 intracellular domain, andCD3zintracellular signaling domain that were cloned into a lentiviralbackbone (Fig. 1A). After the CAR-GPC3 cells were manufactured,the patients were admitted to the hospital. Two to six days beforeCAR-GPC3 T-cell infusion, patients underwent lymphodepletion(Fig. 1B). In study 1, the lymphodepletion regimen used either (1)cyclophosphamide (Cy) 500 to 1,000 mg/m2/day for 1 to 2 days, or (2)the combination of Cy (500 mg/m2/day for 1–3 days) and fludarabine(Flu, 25–30 mg/m2/day for 2–4 days). In study 2, all patients receivedthe combination regimen of Cy (500 mg/m2/day for 1–2 days) and Flu(20–25mg/m2/day for 3–4 days). The lymphodepletion regimen couldbe adjusted by the treating physician according to patient's diseasecondition (Supplementary Table S1). One subject (patient P5) wasdetermined to be unfit for lymphodepletion due to poor clinicalcondition, with a heavy tumor burden and extremely elevated a-feto-protein (AFP) level of 60,500 ng/mL.

After lymphodepletion, patients received a cycle of Y035CAR-GPC3 T-cell therapy under observation andwere not dischargeduntil their absolute neutrophil counts recovered to at least 500 cells/mm3. In study 1,GPC3-CART cells were administrated froma startingsplit dose (1 � 105 CAR positive cells/kg) and gradually increased bytwo- to threefold until the final total dose of the split infusion wasreached. The maximum dose was 2 � 109 CAR positive cells/kg ifsufficient cells could be manufactured. The maximum dose would bede-escalated if there was a dose-limiting toxicity (DLT) related to theCAR-T treatment. The total dose could be administrated in two to ninesequential split infusions over the treatment cycle. In study 2,manufacturing capabilities were optimized, and all patients (P9-P13) were treated with the fixed dose recommended from study 1(20.0� 108 CAR positive cells). Patient P1 received two cycles of Y035CAR-GPC3 T-cell infusion. The dose of the first cycle was split into sixinfusions given over 13 days to ensure safety. The dose of the first splitinfusion was 0.08� 108 cells (1� 105 cells/kg) and the remaining splitdoses were gradually increased by two to three times until the final splitdose of 20.0 � 108 cells was reached. A total dose of 40.7 � 108 Y035CAR-GPC3 T cells were administered in cycle one. The remaining11.1 � 108 Y035 CAR-GPC3 T cells were administered in cycle two.The interval between the two cycles of Y035 CAR-GPC3 T-cellinfusion in patient P1 was approximately 6 weeks. Patients P2 to

Translational Relevance

Glypican-3 (GPC3) is a member of the heparan sulfate proteo-glycan family that may play a role in the control of cell division andgrowth regulation. GPC3 positive immunostaining can differen-tiate hepatocellular carcinoma (HCC) from dysplastic changes incirrhotic livers. Recent studies demonstrated that greater GPC3expression in tumor cells is associated with a worse prognosis forHCC. This observation provides a rationale for chimeric antigenreceptor (CAR)-GPC3 T-cell therapy in advanced HCC. Thisreport demonstrated that CAR-GPC3 T-cell therapy was feasiblein patients with HCC. Special attention should be given to patientswith rapidly progressive disease and/or heavy tumor burden. Earlysignals of antitumor activity were observed. This publication is thefirst report of CAR-GPC3 T-cell therapy in patients with advancedHCC.

Shi et al.

Clin Cancer Res; 2020 CLINICAL CANCER RESEARCHOF2




P8 received one cycle of Y035 CAR-GPC3 with a mean of 23.2 � 108

cells (median, 9.6 � 108 cells; range, 7.0–92.5 � 108 cells; Table 1).After T-cell infusion, each patient's CAR T-cell DNA copy number

and cytokine levels were monitored. The incidence and severity ofadverse events (AEs) were graded using the Common TerminologyCriteria for Adverse Events (version 4.03). Special attention was givento CAR T-cell–related infusion reactions, cytokine release syndrome(CRS), and preconditioning-related infectious diseases. Early diagno-sis and management of CRS were based on Lee's criteria (40). Tumorresponse was assessed using RECIST (version 1.1). CT or MRI wasperformedduring follow-up. Patientswere evaluated every 4 to 8weeksfor the first 6 months and then about every 3 months per the standardof care.

qRT-PCR analysis of CAR-GPC3 DNA copy numbersReal-time fluorescent qPCR was applied to determine the

CAR-GPC3 DNA copy numbers in peripheral blood as describedpreviously (41). Genomic DNA was extracted from patient PBMCsusing a QIAamp DNA Blood Midi Kit (QIAGEN) before and afterCAR T-cell infusion. The forward primer 50-CCAGCTGCTGATC-TACAAGG-30, reverse primer 50-CAGTAGTACACGCCCACGTC-

30, and TaqMan probe 50-FAM-CGGCACCGACTTCACCCTGA-TAMRA-30 were used in the qPCR assay.

Immunological assays to measure blood concentrations ofcytokines and tumor biomarkers

Both plasma and serum samples were collected from patientsbefore and at different time points after CAR T-cell infusion.Specific circulating cytokines testing was performed on the sametype of samples. The plasma concentrations of IL6, IL12p70, IFNg ,regulated upon activation, normal T cell expressed, and secreted(RANTES), macrophage inflammatory protein (MIP)-1b, andmonocyte chemoattractant protein (MCP)-1 were measured usingcytometric bead array (CBA) according to the manufacturer'sinstructions (BD Biosciences; catalog numbers 558276, 558283,560111, 558324, 558288, and 558287, respectively). The serumconcentration of C-reactive protein (CRP) was measured using ahigh-sensitivity CRP assay with a BN II System (Siemens Healthi-neers). The serum AFP concentrations were measured using achemiluminescent immunoassay according to the manufacturer'sinstructions (ARCHITECT i2000 chemiluminescence immunoas-say analyzer, automated; Abbott Diagnostics).

Figure 1.

CAR-GPC3 construct and study design. A, The modular composition of Y035 CAR-GPC3. scFv, single-chain variable fragment. B, Protocol schedule for screening,manufacturing autologous CAR-GPC3 T cells, and lymphodepletion followed by infusion of CAR-GPC3 T cells and follow-up. The differences in the lymphodepletionregimen and T-cell dose between the two studies are listed.

Phase I Trials of CAR-GPC3 T Cells for Advanced HCC

AACRJournals.org Clin Cancer Res; 2020 OF3




Statistical analysisPatients in study 1 and study 2 were analyzed together. Descrip-

tive statistics consisted of medians with ranges and means with SDsfor continuous variables and counts and percentages for categoricalvariables. AE terms were coded using the Medical Dictionary forRegulatory Activities (version 21.1). Analyses of the association ofDNA copy numbers or cytokine levels with CRS and time points oftumor response were performed using SAS software (version 9.4;SAS Institute). Progression-free survival (PFS) and OS were ana-lyzed using the Kaplan–Meier method. SAS software (version 9.4;SAS Institute) was used to calculate pharmacokinetic parametersusing trapezoidal rule.

ResultsPatient characteristics

A total of 13 patients with HCC received Y035 CAR-GPC3 T-celltherapy from December 2015 to August 2018. P1 to P8 wereenrolled in study 1. P9 to P13 were enrolled in study 2. The patients(11 male and two female) had a median age of 51 years (range, 34–70 years; Table 1). All patient tumors were positive for GPC3according to IHC staining, including 10 patients with a stainingintensity score of 3þ. All patients had Child-Pugh class A diseaseand 3 patients had cirrhosis. Ten patients had extrahepatic disease,9 had longer than a 1-year HCC history, and 10 had BarcelonaClinic Liver Cancer stage C/D disease. Patients had received priorsurgical resection, local therapy, or systemic therapy (Supplemen-tary Table S2). All patients had a history of hepatitis B viralinfection and 5 patients received entecavir antiviral treatment(Supplementary Table S3).

Characteristics of Y035 CAR-GPC3 T cellsWe successfully generated Y035 CAR-GPC3 T cells for all 13

patients. Themedian time tomanufacture the cell products for clinicaluse was 12 days (range, 10–13 days). Amedian of 64.4% T cells (range,41.4–88.4%) expressed CAR-GPC3. Immunophenotypic analysisdemonstrated that more than 95.8% of transduced T cells were CD3þ

[mean, 98.6% (range, 95.8–99.6%)], and both CD8þ andCD4þ subsetswere present in all Y035 CAR T-cell products (data not shown). Y035CAR T-cell products were predominantly terminally differentiatedeffector memory T cells [CD45RAþ/CCR7�; mean, 78.2% (range,42.3–94.1%)], and effector memory T cells [CD45RA�CCR7�; mean,14.1% (range, 0.8–47.4%); Fig. 2A–C].

Adverse effectsY035 CAR-GPC3 T-cell therapy was generally tolerable in

patients who had low tumor burdens and were in good clinicalcondition, even with doses greater than 20.0 � 108 CAR-positivecells. All but one patient experienced an expected transient grade3/4 decrease in lymphocyte count resulting from chemotherapy-induced lymphodepletion. We observed CRS in nine patients(Table 2; Supplementary Table S4). No patients experienced grade3/4 neurotoxicity and no patients experienced CAR T-cell–relatedinfusion reactions.

Persistence of Y035 CAR-GPC3 T cells in peripheral bloodThe CAR-GPC3 vector copy number was closely monitored in

the first 2 weeks after the initial infusion, weekly in the first monthafter the last infusion, and monthly thereafter. The medianCAR-GPC3 DNA copy number in the peripheral blood of allpatients increased rapidly, reaching a peak of 360.4 copies/mgTa

ble

1.Patient

characteristics,Y035

CAR-G

PC3T-celld

oses,an

dclinical

outco

mes.

Subject

IDa

Age

(yea

rs)

Sex

ECOG

score

GPC3IHCstaining

intensityscore

HCChistory

(yea

rs)

BCLC

stag

eMVI

Metastasis

Cirrhosis

Therap

ycycle

CARþT-ce

lldose

(cells)

CARþT-ce

lldose

b

(cells/kg)

Response

OSdurationc

(day

s)PFSduration

(day

s)

P1

54M

03þ

0.6

CYes

Yes

No

140.7

�10

849.6

�10

6SD

1,326

204

211.1�

108

13.5

�10

6

P2

34F

13þ

0.8

CNo

Yes

Yes

117.4

�10

838

.7�

106

NE

95

95

P3

50M

03þ

2.2

CNo

Yes

No

17.8�

108

10.8

�10

6PR

615

111

P4

70M

11þ

10.9

CNo

Yes

No

192.5�

108

146.9

�10

6SD

601

81

P5

59M

13þ

2.0

DNo

Yes

No

19.6

�10

815.3

�10

6PD

2929

P6

39M

03þ

0.4

CNo

No

No

119.7

�10

841.5

�10

6PD

693

33P7

44

F1

1þ1.5

CNo

No

No

18.7

�10

815.3

�10

6NEd

278

18d

P8

46

M1

3þ7.1

CNo

Yes

No

17.0�

108

15.5

�10

6NEd

127

40d

P9

51M

13þ

2.2

CNo

Yes

No

120

.0�

108

26.7

�10

6PD

134

34P10

51M

13þ

0.8

CYes

Yes

No

120

.0�

108

37.7

�10

6PD

48

13P11

68

M1

2þ6.1

BNo

No

Yes

120

.0�

108

30.8

�10

6PD

132

34P12

53M

13þ

2.3

BNo

Yes

Yes

120

.0�

108

44.4

�10

6NE

2424

P13

51M

03þ

3.7

BNo

No

No

120

.0�

108

26.3

�10

6PR

385

99

Abbreviations:BCLC

,Barcelona

ClinicLive

rCan

cer;F,fem

ale;

ID,iden

tification;

M,m

ale;

MVI,macrovascular

invasion;

NE,n

otev

alua

ble;PD,p

rogressivedisea

se.

aP1toP8wereen

rolledinstud

y1a

ndP9to

P13

wereen

rolledinstud

y2.P3an

dP9was

samepatient

who

enrolledtw

ice.After

theinitialenrolm

entinstud

y1(indicated

byP3),the

patient

withd

rewfrom

stud

y1fora

bout

1ye

aran

dthen

enrolledin

stud

y2(ind

icated

byP9).

bCalculatedonthebasisofbodyweight.

c The

datacutoffdatewas

July

24,2

019.T

heOSva

lues

forthepatientswho

werealiveonthat

date(P1an

dP13)werecensored.

dClinical

progressionin

P7an

dP8.

Shi et al.





genomic DNA (range, 28.0–23,358.0 copies/mg genomic DNA) aftera median period of 10.5 days (mean, 13.8 days) and lasting for amedian duration of 19.5 days (mean, 34.4 days; Fig. 3A). The AUCsfor the CAR-GPC3 DNA copy numbers in two patients who hadobjective responses tended to be higher than those in the other 10patients (Fig. 3B), with median AUCs of 98,016.0 and 5,423.1,respectively (P ¼ 0.0606). In the two responders, we observed CART-cell DNA copy number peaks about 2 weeks after Y035 CAR-GPC3 T-cell infusion and before the first partial response(PR; Fig. 3C). The longest CAR T-cell DNA copy number durationwas about 140 days.

Cytokine profile and CRSWe monitored patient serum levels of 12 cytokines during the

studies. The median fold increase in IFNg and IL6 level over baselinewas 68.2 and 34.6, respectively. Themedian peak value of IFNg and IL6level postinfusion was 3.98 and 172.82 pg/mL, respectively (Fig. 3D–E). The levels of CRP, IL6, RANTES, andMCP-1 in patient P3 peakedat the same time as the patient's body temperature increase, which wasconsistent with clinical observations of CRS in patients who receivedY035 T-cell infusion. In addition, we observed IL6, IL10, IL15, andIFNg peaks before the CRS event in patient P13 and significantdecreases in these levels after the event (Fig. 3F).

Figure 2.

Characteristics of Y035 CAR-GPC3 Tcells, which were successfully generat-ed for all patients. A, CAR-GPC3expression in the T cells in the 13patients with HCC. A median of64.4% (range, 41.4–88.4%) of T cellsexpressed CAR-GPC3 according tofluorescence-activated cell sortinganalysis. B, Subset composition ofY035 CAR-GPC3 T-cell productsinfused in all patients. C, Representa-tive histograms anddot plots of infusedY035 CAR-GPC3 T-cell products(patients P6 and P10). CM, centralmemory; EM, effector memory;TEMRA, terminally differentiated effec-tor memory T cells.






Table

2.Treatmen

t-em

ergen

tAEsin

patientswho

received

Y035

CAR-G

PC3T-cellinfusions.

Subject

IDCytopen

ia(A

Egrade)

CRS-relatedev

ents

(AEgrade)

CRS

grade

High-dose

steroids

Use

ofIL-6R

inhibitor

P1

WBCdecreased

(2),plateletco

untdecreased

(2),lympho

cyte

coun

tdecreased

(4)

Pyrexia

(2),APTTincrea

sed(1)

1No

No

P2

Lympho

cyte

coun

tdecreased

(4)

ASTleve

lincreased

(1),APTTincrea

sed(1)

1No

No

P3

WBCdecreased

(2),lympho

cyte

coun

tdecreased

(4)

Pyrexia

(2),bilirubin

leve

lincreased

(2)

1No

No

P4

Lympho

cyte

coun

tdecreased

(3)

Pyrexia

(1),ASTleve

lincreased

(1),ALT

leve

lincreased

(1)

1No

No

P5

Plateletco

untdecreased

(2)

Notap

plicab

le0

No

No

P6

WBCdecreased

(4),lympho

cyte

coun

tdecreased

(4)

Notap

plicab

le0

No

No

P7

Plateletco

untdecreased

(1),lympho

cyte

coun

tdecreased

(4)

Notap

plicab

le0

No

No

P8

Lympho

cyte

coun

tdecreased

(4)

Notap

plicab

le0

No

No

P9

WBCdecreased

(2),plateletco

untdecreased

(3),lympho

cyte

coun

tdecreased

(4)

Pyrexia

(2),APTTincrea

sed(1)

1Yes

No

P10

Plateletco

untdecreased

(3),lympho

cyte

coun

tdecreased

(4)

Pyrexia

(3),rena

lim

pairm

ent(1),ASTleve

lincreased

(2),ALT

leve

lincrea

sed(1),bilirubin

leve

lincreased

(4),APTTincrea

sed(1)

2Yes

No

P11

WBCdecreased

(2),ne

utrophilco

untdecreased

(2),plateletco

unt

decreased

(2),lympho

cyte

coun

tdecreased

(4)

Pyrexia

(2),ASTleve

lincreased

(1),bilirubin

leve

lincreased

(3),APTT

increa

sed(1)

2No

No

P12

Plateletco

untdecreased

(4),lympho

cyte

coun

tdecreased

(4)

ASTleve

lincreased

(2),ALT

leve

lincreased

(1),bilirubin

leve

lincreased

(4),

APTTincrea

sed(1)

5Yes

Yes

P13

Neu

trophilco

untdecreased

(2),plateletco

untdecreased

(3),

lympho

cyte

coun

tdecreased

(4)

Pyrexia(2),hy

potension(2),bilirubin

leve

lincreased

(2),APTTincrea

sed(1)

2Yes

Yes

Note:A

Eterm

swereco

ded

usingtheMed

icalDictiona

ryforReg

ulatory

Activities(version21.1).A

Egradingwas

assessed

usingtheCommonTerminologyCriteriaforAdve

rseEve

nts(version4.03).C

RSgradingwas

assessed

usingLe

e'scriteria

(40).

Abbreviations:ALT

,alanine

tran

saminase;

APTT,a

ctivated

partial

thromboplastin

time;

AST,a

spartate

aminotran

sferase;

ID,iden

tification;

WBC,w

hite

bloodcellco

unt.

Shi et al.





Figure 3.

Persistence of CAR-GPC3 T cells as determined by DNA copy numbers, and cytokine levels in the patients with HCC. A, Persistence of CAR-GPC3 T cells in patientswith HCC assessed using qRT-PCR to determine the expression of CAR-GPC3 transgenes in peripheral blood genomic DNA. Semi-log CAR-GPC3 copy numberswereplotted over time. One patient (P8) exhibited clinical disease progression after the treatment and withdrew consent for CAR-GPC3 copy-number testing. B, Theassociation of CAR-GPC3DNA copy numberwith tumor response. Copy numbers in the two responders (P3 and P13)were comparedwith those in the other patients.The area under the CAR-GPC3 T-cell copies–time curve from time zero to time of lastmeasurable value (AUClast for the CAR-GPC3 copy number)wasmeasured afterthe first cycle of CAR-GPC3 T-cell infusion. Themedian CAR-GPC3 DNA copy number as calculated according to the AUClast tended to be higher in P3 and P13 than inthe 11 other patients (P¼ 0.0606). Circles indicate individual patients. C, Semi-log CAR-GPC3 DNA copy numbers in patients P3 and P13 plotted with CRS (red) andtime points of tumor responses (blue). Each CAR T-cell infusion is marked by a green triangle. PD, progressive disease. D, The peak levels of three representativecytokines after the first cycle of CAR-GPC3 T-cell infusion in all patients. Hs-CRP, high-sensitivity CRP. E, The peak fold increases in the levels of three representativecytokines after the first cycle of CAR-GPC3 T-cell infusion in all patients. F, Results of cytokine analyses in P3, P12, and P13. The body temperature of both patientsincreased by more than 39�C after CAR-GPC3 T-cell (CART) infusion and was accompanied by CAR T-cell copy expansion. P3 exhibited rapid elevations in Hs-CRP,IL6, RANTES, and MCP-1 levels, accompanied by the peak CAR-GPC3 DNA copy number of 569 copies/mg genomic DNA on day 14. The patient experienced grade 1CRS on day 2 and recovered. P13 exhibited rapid elevation of IFNg , IL10, IL15, and IL6 levels. CRS (grade 2) was diagnosed on day 10 and resolved after tocilizumaband corticosteroid administration. The peak CAR-GPC3 DNA copy number was 23,358 copies/mg genomic DNA on day 15. P12 rapidly exhibited elevation of IL2, IL6,IFNg and IL10 levels. CRS was diagnosed on day 1 and treated with the tocilizumab and high-dose corticosteroid. The peak CAR-GPC3 DNA copy number was10,713 copies/mg genomic DNA on day 7. The patient's condition rapidly deteriorated and the patient died on day 19.






CRS is a major clinical concern for patients receiving CAR T-celltherapy (22, 25, 26). Eight subjects experienced low-grade CRS(grade 1 or 2), which was self-limiting and reversible. All patientsexperienced fever: 11 at grade 1 or 2 and 2 at grade 3. We didnot see a clinically meaningful difference in the incidence andseverity of CRS when comparing patients who received a dose ofY035 CAR-GPC3 T cells greater than 20.0 � 108 (n ¼ 2) with theother patients (data not shown). Four patients received high-dosesteroids, two of whom also received the mAb tocilizumab againstIL6 receptor to manage CRS (Table 2). Surprisingly, one patientexperienced grade 5 CRS when given a dose of 20.0 � 108 cells eventhough 6 patients in the studies had safely received a similar orhigher dose of Y035. To the best of our knowledge, this occurrencewas the first reported case of grade 5 CRS in a patient with HCCafter CAR T-cell therapy.

Patient P12 was a 53-year-old man with metastatic HCC and aGPC3 IHC staining intensity score of 3þ. Prior to enrollment, thepatient underwent partial hepatectomy, transcatheter arterial che-moembolization, radiofrequency ablation, sorafenib administra-tion, and other targeted HCC therapies. His ECOG performancescore was 1, and his body weight was 45 kg. Because of a large tumorburden and high AFP level of 52,096 ng/mL, the patient receivedmicrowave ablation therapy for his cancer after screening. However,MRI showed a rapid progression of the disease. About 1 monthprior to CAR T-cell therapy, the number of intrahepatic lesionsincreased from about 10 to more than 30, and the diameter ofthe largest target lesion increased from 4 to 8 cm. On the day ofCAR T-cell infusion, patient P12 had no significant liver functiondeterioration or progressive weight loss, and the patient's ECOGperformance status remained at 1. After the risk of T-cell treatmentwas described again to the patient and family, the patient wasadministered Y035 CAR-GPC3 T cells at a dose of 20.0 � 108 cells.The next day, the patient experienced severe CRS-related hypoten-sion, fever, and respiratory difficulties indicating pulmonary edema.The patient was then transferred to the intensive care unit for closemonitoring and intensive supportive care, including the adminis-tration of tocilizumab and high-dose steroids. Laboratory testresults demonstrated an IL6 level greater than 18,000 pg/mL andIL2 level greater than 1,000 pg/mL a few hours after T-cell infusion,and elevation of IFNg and IL10 levels in the following days(Fig. 3F). The patient's condition improved initially after treatmentin the intensive care unit. However, the patient P12 died of multipleorgan failure and end-stage HCC on day 19.

Antitumor activity of CAR-GPC3 T-cell therapyWe evaluated all 13 patients for antitumor activity. Two patients

were alive at the time of data analysis. The survival probabilities at3 years, 1 year, and 6 months were 10.5%, 42.0%, and 50.3%, respec-tively, according to the Kaplan–Meier method, with a median OSduration of 278 days (39.7 weeks; 95% confidence interval, 48–615 days; Fig. 4A and B).

Two patients (P3 and P13) had confirmed PRs (Fig. 4C), and one(P1) had a sustainable stable disease (SD). The target lesions in the twopatients with PRs exhibited significant shrinkage. Their OS durationswere 615 and 385 days, respectively, and their PFS durations were 111and 99 days, respectively. Patient P13 was alive at the time of dataanalysis with an AFP level of 1.97 ng/mL (Supplementary Fig. S1B). Inaddition, patient P1, despite having less than a 30% reduction in thesize of target lesions, exhibited a significant clinical benefit of T-celltherapy, with a remarkable reduction in serum AFP level and con-tinued long-term survival. The patient remained alive with a serum

AFP level in the normal range at the time of data analysis (Table 1;Supplementary Fig. S1B). Of note, this patient had vessel invasion(inferior vena cava tumor thrombus and right atrium tumor throm-bus) andmetastatic lymphnodes at baseline, which generally indicate apoor prognosis.

We monitored serum AFP levels throughout the studies. PatientsP1, P3, and P13, who had clinically meaningful antitumor activity ofPD or sustainable SD, had high-percentage reductions in serumAFP levels after Y035 CAR-GPC3 T-cell infusion (SupplementaryFig. S1A).

DiscussionWe conducted the first-in-human clinical trials of GPC3-directed

CAR T cells in patients with advanced GPC3þ HCC. To ourknowledge, this is the first published clinical report of GPC3CAR-T therapy. Because of a lack of expression in normal tissuesand high expression in HCC, GPC3 is considered a promisingimmunotherapeutic target for HCC. Consistent with a good safetyprofile observed in a phase I clinical trial of the anti-GPC3 antibodyGC33 (35), CAR-GPC3 T cells were tolerated in patients with agood performance status and clinical condition. We expected themost common AEs to be hematologic toxic effects, especially grade4 lymphocytopenia, due to the preconditioning regimens used priorto CAR T-cell treatment. These toxic effects were recoverable anddid not result in any serious infectious AEs. In addition, no grade 3/4 neurotoxic effects or CAR T-cell–related infusion reactionsoccurred in the studies. The toxic effects potentially associatedwith the CAR-GPC3 T-cell therapy included CRS and its relatedsymptoms, such as fever and liver function abnormalities. Toprevent severe CRS, investigators should make special considera-tions regarding the eligibility of patients in clinical trials of HCCand pay special attention to the management of AEs in patients withHCC with heavy tumor burdens.

We observed two objective responses to the Y035CAR-GPC3T-celltherapy. In addition, one patient with SD also experienced long-termsurvival (44.2 months). Although a higher GPC3 expression in tumorcells is associated with a worse prognosis for HCC, we did not find asignificant correlation between the staining intensity and clinicalefficacy in the studies. This correlation will be intensively investigatedin the ongoing studies. The antitumor activity of CAR-GPC3T cells wereported here was more promising than the efficacy reported in thephase I study of mAb GC33. In the GC33 mAb study, the median OSduration was 16.1 weeks (39.7 weeks in our studies), and no patientshad objective responses (35). Several advantages of our CAR-T cellsmay contribute to better clinical outcomes. First, CAR-T cells elim-inate the tumor cells by directly lysing the target cells upon engagingthe antigen via physiologic cytotoxic lymphocytes and natural killercells, a mechanism different with mAbs even if they share the sameantigen specificity. CAR-T cells are superior to mAbs especially forthose target cells with the low expression level of antigen. Second,CAR-T cells have a longer half-life than mAbs. The combination ofCAR T cells with local treatment such as thermal ablation may releasethe tumor antigen and enhance the CAR-T activity. In our studies,CAR DNA copy numbers in the peripheral blood increased rapidlyafter Y035CAR-GPC3T-cell infusion, indicating successful expansionof the T cells. As described previously, the AUClast values for the copynumbers in the two therapy responders tended to be higher than thosein the other patients, suggesting a relationship between CAR T-cellpersistence and antitumor activity of Y035 CAR-GPC3 T cells. Serumlevels of cytokines such as IL6 and IFNg increased significantly after

Shi et al.





Figure 4.

Clinical responses and exploratory subgroupanalysis in patientswithHCCadministeredCAR-GPC3T cells.A, Swimmer's plot of the time after CAR-T for each patient.A total of 13 patients with HCC received CAR-GPC3 T-cell infusions. Eligibility criteria included an expected survival duration of 12 weeks. At the data cutoff point,two patients were still alive. P5 was lost to follow-up after 1 month. PD, progressive disease. B, Kaplan–Meier curve of OS for patients. Censored patients (those aliveor lost to follow-up at the time of data analysis) are indicated by plus signs. C, Tumor responses in two patients with advanced HCC (P3 and P13). CT scans of onetarget lesion (yellowarrow) in the liver of P3 takenbefore andafter CAR-GPC3T-cell infusion. Thepatientwas a50-yearmanwith amultifocal HCChistory of 2.2 yearsand a GPC3 IHC staining intensity score of 3þ prior to enrollment. He experienced grade 1 CRS and grade 2 fever. CT demonstrated a progressive reduction in lesionsize from 31.0 � 27.8 mm to 12.2 � 9.5 mm. The patient had a confirmed PR and a PFS duration of 111 days. MRI scans of P13 before and after CAR-GPC3 T-celltreatment. The patientwas a 52-year-oldmalewith amultifocal HCChistory of 3.7 years. The patient had aGPC3 IHC staining intensity score of 3þ. His prior therapiesincluded five transcatheter arterial chemoembolization (TACE) procedures and 11 thermal ablations. He hadgrade 2CRS and an IL6 level greater than 1,000pg/mLonday 15. After receiving multiple doses of tocilizumab, the patient gradually recovered from CRS. MRI scans of multiple lesions in the liver are shown (a, b, and c withyellowarrows). Starting 4weeks after CART-cell infusion,MRI revealed significant reductions in lesion sizes,with lesions a and cdisappearing completely and lesionbshrinking slightly from 14.2 � 13.4 mm to 13.9 � 13.0 mm. The patient had confirmed PR and a PFS duration of 99 days. He was alive at the time of data analysis.






CAR T-cell treatment. Such increases may be correlated with clinicalsymptoms such as elevated body temperature andCRS, confirming theimportance of close cytokine level monitoring during CAR T-celltherapy.

The serum AFP level is an important biomarker of tumor responseand disease relapse in patients with HCC (42, 43). Normalization ofserum AFP levels in patients with high baseline AFP levels is a usefulindicator of the success of surgical resection. Furthermore, decreasedserum AFP level is associated with improved OS after treatment withsorafenib (44). Indeed, in this study, the three patients with clinicallymeaningful benefits of the CAR T-cell treatment had greater reduc-tions in serum AFP levels than did the other patients. This finding isconsistent with the favorable antitumor activity of the treatmentobserved in the imaging of these patients.

This report also confirmed that dose modification in patients withHCC with heavy tumor burden is important, particularly when thosepatients are included in clinical trials of CAR-GPC3 T-cell therapy. Toprevent severe CRS, investigators should make special considerationsregarding the eligibility of patients in clinical trials of HCC and payspecial attention to themanagement of AEs in patients withHCCwithheavy tumor burdens. Optimization of CAR T-cell infusion doses andschedules, as well as pretreatment regimens, is warranted to improveCAR T-cell persistence and tumor penetration. We did not observeany difference in clinical outcomes between patients who receivedmultiple split infusions versus those who received single infusions ofCAR-GPC3 T cells. From the point of clinical practice, a single dose ofCAR-GPC3 T cells infusion is more likely to be implemented in futureclinical trials. Dose-modification strategies such as the reduction of theCAR-GPC3 T-cell dose or a single preconditioning chemotherapeuticagent in patients with HCC with heavy tumor burdens shouldbe explored in future clinical trials. This pilot program of the firstever reported experience with CAR T in HCC will inform futurestudies to use a unified regimen and dose to validate the signal ofactivity in HCC. We are currently conducting a CAR-GPC3 clinicaltrial that is currently the first and only investigational new drug clearedfor CAR T research in solid tumors in China.

This report confirms that new CAR T-cell approaches with GPC3should be considered for HCC. For example, in our previous study ofimmunocompetent and immunodeficient HCC mouse models (36),we demonstrated the combined antitumor effects of sorafenib andCAR-GPC3 T cells, suggesting that this combination therapy can beapplied clinically to HCC. We are investigating whether a differentlymphodepletion regimen in the combination of CAR-T cells and atyrosine kinase inhibitor can be an alternative approach to replace thestandard lymphodepletion regimen for patients with HCC, whocommonly develop compromised hepatic function. Further improve-ments of the CAR-GPC3 with next-generation technology are alsopromising and may not require lymphodepletion pretreatment. Forinstance, our recent study demonstrated that IL12 armored GPC3-redirected CAR T cells could greatly improve the antitumor activitiesin mouse model even with large tumor burdens (41). We also showed

that introducing an IL4/21 inverted cytokine receptor into the CAR-GPC3 construct improved the CAR T-cell potency in vitro andin vivo (37). In another recent study, our group disrupted PD-1 geneexpression in CAR-GPC3 T cells using the CRISPR/Cas9 gene-editingsystem. This disruption enhanced the in vivo activity of CAR T cellsagainst HCC and improved the persistence and infiltration of CAR Tcells in mice bearing HCC (45). These new CAR-GPC3 T-cell ther-apeutic approaches are promising and should be explored in futureHCC clinical trials.

In summary, we report herein the initial safety profile of Y035CAR-GPC3 T-cell therapy in patients with advanced HCC. Earlysigns of antitumor activities were observed. Future studies arewarranted to further confirm the safety and efficacy of CAR-GPC3T cells.

Disclosure of Potential Conflicts of InterestD. Yuan and H. Ma are employees/paid consultants for CARsgen. H. Wang and

Z. Li report a patent on the antibody against glypican-3 (CN201610626384.7) issuedto CARsgen Therapeutics. No potential conflicts of interest were disclosed by theother authors.

Authors’ ContributionsConception and design: D. Shi, D. Yuan, H. Wang, Z. Li, B. ZhaiDevelopment of methodology: D. Shi, H. Gao, B. ZhaiAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): D. Shi, Y. Shi, X. Qi, Y. Zhang, J. Chi, Q. Lu, H. Jiang, Z. LiAnalysis and interpretation of data (eg, statistical analysis, biostatistics,computational analysis): D. Shi, Y. Shi, Q. Lu, H. MaWriting, review, and/or revision of themanuscript:D. Shi, A.O. Kaseb, H.Ma, Z. LiAdministrative, technical, or material support (ie, reporting or organizing data,constructing databases): Y. Shi, Q. Lu, H. Wang, H. Ma, H. WangStudy supervision: D. Shi, H. Ma, H. Wang, Z. Li, B. Zhai

AcknowledgmentsWe thank the patients and their families for study participation.We also thank the

study staffs of Dr. Jun Xiao, Ms. Jie Zhang, Ms. Chunyan He, Ms. Huaying Ruan,Mr. Maogen Chen, and Dr. Zhen Liu (CARsgen Therapeutics, Shanghai) as well asDrs. Xiaoou Zhou and Cheryl Askew (CARsgen Therapeutics, Houston) for helpingwith the manuscript preparation. We also thank Donald R Norwood, ScientificPublication Services at The University of Texas MD Anderson Cancer Center formanuscript's editing and revision. Finally, we thank Jianming Yang and Baohua Qianfor providing assistance with autologous T-cell collection This report was supportedby National Science and Technology Major Projects (No. 2017ZX10203206006),National Science and Technology Major Projects (No. 2018ZX09733001-003-001),National Natural Science Foundation of China (81872483 to Z. Li), Shanghai Scienceand Technology Talents Program (No. 16XD1402600), and Collaborative InnovationCenter Grant for Translational Medicine at Shanghai Jiao Tong University School ofMedicine (No. TM201601).

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 October 8, 2019; revised February 17, 2020; accepted April 27, 2020;published first May 5, 2020.

References1. Ferlay J, Ervik M, Lam F, Colombet M, Mery L, Pi~neros M, et al. Global Cancer

Observatory: Cancer Today. Lyon, France: International Agency for Research onCancer; 2018. https://gco.iarc.fr/today/data/factsheets/cancers/11-Liver-fact-sheet.pdf. Accessed August 29, 2019.

2. ChenW, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, et al. Cancer statistics inChina, 2015. CA Cancer J Clin 2016;66:115–32.

3. American Cancer Society; 2019. https://www.cancer.org/cancer/liver-cancer/about/what-is-key-statistics.html. Accessed August 29, 2019.

4. Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma. Lancet 2003;362:1907–17.

5. Cabrera R, Nelson DR. The management of hepatocellular carcinoma.Aliment Pharmacol Ther 2010;31:461–76.

Shi et al.




https://gco.iarc.fr/today/data/factsheets/cancers/11-Liver-fact-sheet.pdf



https://www.cancer.org/cancer/liver-cancer/about/what-is-key-statistics.html




6. Ma S, Ding M, Li J, Wang T, Qi X, Shi Y, et al. Ultrasound-guidedpercutaneous microwave ablation for hepatocellular carcinoma: clinicaloutcomes and prognostic factors. J Cancer Res Clin Oncol 2017;143:131–42.

7. Wang T, Zhang XY, Lu X, Zhai B. Laparoscopic microwave ablation ofhepatocellular carcinoma at liver surface: technique effectiveness and long-term outcomes. Technol Cancer Res Treat 2019;18:1–9.

8. Tabrizian P, Jibara G, Shrager B, Schwartz M, Roayaie S. Recurrence ofhepatocelluar cancer after resection: patterns, treatments, and prognosis.Ann Surg 2015;261:947–55.

9. Cheng AL, Kang YK, Chen Z, Tsao CJ, Qin S, Kim JS, et al. Efficacy and safety ofsorafenib in patients in the Asia-Pacific region with advanced hepatocellularcarcinoma: a phase III randomised, double-blind, placebo-controlled trial.Lancet Oncol 2009;10:25–34.

10. Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, et al. Sorafenib inadvanced hepatocellular carcinoma. New Engl J Med 2008;359:378–90.

11. Bruix J, Qin S, Merle P, Granito A, Huang YH, Bodoky G, et al. Regorafenib forpatients with hepatocellular carcinoma who progressed on sorafenib treatment(RESORCE): a randomised, double-blind, placebo-controlled, phase 3 trial.Lancet 2017;389:56–66.

12. El-Khoueiry AB, Sangro B, YauT, Crocenzi TS, KudoM,HsuC, et al. Nivolumabin patients with advanced hepatocellular carcinoma (CheckMate 040): an open-label, non-comparative, phase 1/2 dose escalation and expansion trial. Lancet2017;389:2492–502.

13. Zhu AX, Finn RS, Edeline J, Cattan S, Ogasawara S, Palmer D, et al. Pembro-lizumab in patients with advanced hepatocellular carcinoma previously treatedwith sorafenib (KEYNOTE-224): a non-randomised, openlabel phase 2 trial.Lancet Oncol 2018;19:940–52.

14. Hiraoka A, Kumada T, Atsukawa M, Hirooka M, Tsuji K, Ishikawa T, et al.Important clinical factors in sequential therapy including lenvatinib againstunresectable hepatocellular carcinoma. Oncology 2019;97:277–85.

15. Llovet JM,Montal R, Sia D, Finn RS.Molecular therapies and precisionmedicinefor hepatocellular carcinoma. Nat Rev Clin Oncol 2018;15:599–616.

16. Abou-Alfa GK, Meyer T, Cheng A-L, El-Khoueiry AB, Rimassa L, Ryoo B-Y,et al. Cabozantinib in patients with advanced and progressing hepatocellularcarcinoma. N Engl J Med 2018;379:54–63.

17. Zhu AX, Kang YK, Yen CJ, Finn RS, Galle PR, Llovett JM, et al. Ramucirumabafter sorafenib in patients with advanced hepatocellular carcinoma and increaseda-fetoprotein concentrations (REACH-2): a randomised, double-blind, placebo-controlled, phase 3 trial. Lancet Oncol 2019;20:282–96.

18. Duffy AG, Ulahannan SV, Makorova-Rusher O, Rahma O,Wedemeyer H, PrattD, et al. Tremelimumab in combination with ablation in patients with advancedhepatocellular carcinoma. J Hepatol 2017;66:545–51.

19. Sangro B, Gomez-Martin C, de la Mata M, Inarrairaegui M, Garralda E, BarreraP, et al. A clinical trial of CTLA-4 blockade with tremelimumab in patients withhepatocellular carcinoma and chronic hepatitis C. J Hepatol 2013;59:81–8.

20. Kaseb AO, Pestana RC, Vence LM, Blando JM, Singh S, Ikoma N, et al.Randomized, open-label, perioperative phase II study evaluating nivolumabalone or nivolumab plus ipilimumab in patients with resectable HCC. J. Clin.Oncol 2019;37: 4098.

21. American Society of Clinical Oncology, 2019. https://www.cancer.net/cancer-types/liver-cancer/statistics. Accessed August 29, 2019.

22. Brentjens RJ, Davila ML, Riviere I, Park J, Wang X, Cowell LG, et al.CD19-targeted T cells rapidly induce molecular remissions in adults withchemotherapy-refractory acute lymphoblastic leukemia. Sci Transl Med2013;5:177ra38.

23. Brentjens RJ, Riviere I, Park JH, Davila ML,Wang X, Stefanski J, et al. Safety andpersistence of adoptively transferred autologous CD19-targeted T cells inpatients with relapsed or chemotherapy refractory B-cell leukemias. Blood2011;118:4817–28.

24. Garfall AL, Maus MV, Hwang WT, Lacey SF, Mahnke YD, Melenhorst JJ, et al.Chimeric antigen receptor T cells against CD19 formultiple myeloma. New EnglJ Med 2015;373:1040–7.

25. Locke FL, Neelapu SS, Bartlett NL, Siddiqi T, Chavez JC, Hosing CM, et al.Phase 1 results of ZUMA-1: a multicenter study of KTE-C19 anti-CD19

CAR T cell therapy in refractory aggressive lymphoma. Mol Ther 2017;25:285–95.

26. Maude SL, Frey N, Shaw PA, Aplenc R, Barrett DM, Bunin NJ, et al. Chimericantigen receptor T cells for sustained remissions in leukemia. New Engl J Med2014;371:1507–17.

27. BrownCE, AlizadehD, Starr R,Weng L,Wagner JR, Naranjo A, et al. Regressionof glioblastoma after chimeric antigen receptor T-cell therapy. New Engl J Med2016;375:2561–9.

28. Beatty GL, O'Hara MH, Lacey SF, Torigian DA, Nazimuddin F, Chen F, et al.Activity of mesothelin-specific chimeric antigen receptor T cells againstpancreatic carcinoma metastases in a phase 1 trial. Gastroenterology2018;155:29–32.

29. 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.

30. Zhan X,Wang B, Li Z, Li Jie, Wang H, Chen L, et al. Phase I trial of Claudin 18.2-specific chimeric antigen receptor T cells for advanced gastric and pancreaticadenocarcinoma. J. Clin. Oncol 2019;37(15_suppl):2509.

31. Kaseb AO, Hassan M, Lacin S, Abdel-Wahab R, Amin HM, Shalaby A, et al.Evaluating clinical and prognostic implications of glypican-3 in hepatocellularcarcinoma. Oncotarget 2016;7:69916–26.

32. Bi Y, Jiang H, Wang P, Song B, Wang H, Kong X, et al. Treatment ofhepatocellular carcinoma with a GPC3-targeted bispecific T cell engager.Oncotarget 2017;8:52866–76.

33. Gao H, Li K, Tu H, Pan X, Jiang H, Shi B, et al. Development of T cells redirectedto glypican-3 for the treatment of hepatocellular carcinoma. Clin Cancer Res2014;20:6418–28.

34. Abou-Alfa GK, Puig O, Daniele B, KudoM,Merle P, Park JW, et al. Randomizedphase II placebo controlled study of codrituzumab in previously treated patientswith advanced hepatocellular carcinoma. J Hepatol 2016;65:289–95.

35. Zhu AX, Gold PJ, El-Khoueiry AB, Abrams TA, Morikawa H, Ohishi N, et al.First-in-man phase I study of GC33, a novel recombinant humanized antibodyagainst glypican-3, in patients with advanced hepatocellular carcinoma.Clin Cancer Res 2013;19:920–8.

36. Wu X, Luo H, Shi B, Di S, Sun R, Su J, et al. Combined antitumor effects ofsorafenib and GPC3-CAR T cells in mouse models of hepatocellular carcinoma.Mol Ther 2019. 27:1483–94.

37. Wang Y, Jiang H, Luo H, Sun Y, Shi B, Sun R, et al. An IL 4/21 inverted cytokinereceptor improving CAR-T cell potency in immunosuppressive solid-tumormicroenvironment. Front Immunol 2019;10:1691.

38. Guo X, Jiang H, Shi B, Zhou M, Zhang H, Shi Z, et al. Disruption of PD-1enhanced the anti-tumor activity of chimeric antigen receptor T cells againsthepatocellular carcinoma. Front Pharmacol 2018;9:1118.

39. Qin S. Primary Liver Cancer Diagnosis and Treatment Expert Panel of theChinese Ministry of Health. Guidelines on the diagnosis and treatment ofprimary liver cancer (2011 edition). Chin Clin Oncol 2012;1:1–10.

40. Lee DW, Gardner R, Porter DL, Louis CU, Ahmed N, Jensen M, et al. Currentconcepts in the diagnosis and management of cytokine release syndrome.Blood 2014;124:188–95.

41. Liu Y, Di S, Shi B, Zhang H,Wang Y, Wu X, et al. Armored inducible expressionof IL-12 enhances antitumor activity of glypican-3-targeted chimeric antigenreceptor-engineered T cells in hepatocellular carcinoma. J Immunol 2019;203:198–207.

42. Sauzay C, Petit A, Bourgeois AM, Barbare JC, Chauffert B, Galmiche A, et al.Alpha-foetoprotein (AFP): A multi-purpose marker in hepatocellular carcino-ma. Clin Chim Acta 2016;463:39–44.

43. Bruix J, Reig M, Sherman M. Evidence-based diagnosis, staging, and treat-ment of patients with hepatocellular carcinoma. Gastroenterology 2016;150:835–53.

44. Personeni N, Bozzarelli S, Pressiani T, Rimassa L, TronconiMC, Sclafani F, et al.Usefulness of alpha-fetoprotein response in patients treated with sorafenib foradvanced hepatocellular carcinoma. J Hepatol 2012;57:101–7.

45. Sun L, Guo H, Jiang R, Lu L, Liu T, He X. Engineered cytotoxic T lymphocyteswith AFP-specific TCR gene for adoptive immunotherapy in hepatocellularcarcinoma. Tumour Biol 2016;37:799–806.





https://www.cancer.net/cancer-types/liver-cancer/statistics




Published OnlineFirst May 5, 2020.Clin Cancer Res Donghua Shi, Yaoping Shi, Ahmed O. Kaseb, et al. TrialsAdvanced Hepatocellular Carcinoma: Results of Phase I Chimeric Antigen Receptor-Glypican-3 T-Cell Therapy for

Updated version

10.1158/1078-0432.CCR-19-3259doi:

Access the most recent version of this article at:

Material

Supplementary

http://clincancerres.aacrjournals.org/content/suppl/2020/05/05/1078-0432.CCR-19-3259.DC1Access the most recent supplemental material at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. (CCC)Click on "Request Permissions" which will take you to the Copyright Clearance Center's

.http://clincancerres.aacrjournals.org/content/early/2020/06/19/1078-0432.CCR-19-3259To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-19-3259

http://clincancerres.aacrjournals.org/content/suppl/2020/05/05/1078-0432.CCR-19-3259.DC1

http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://clincancerres.aacrjournals.org/content/early/2020/06/19/1078-0432.CCR-19-3259


chimeric antigen receptor-glypican-3 t-cell therapy for ... · 6/19/2020 · for advanced...

Documents