Interleukin-15 Armored Glypican-3-specific Chimeric Antigen Receptor Expressing Autologous T Cells as Immunotherapy for Children With Solid Tumors
Phase: PHASE1
Conditions: Liver Cancer, Rhabdomyosarcoma, Malignant Rhabdoid Tumor, Liposarcoma, Wilms Tumor, Yolk Sac Tumor
Sponsor: Baylor College of Medicine

AGAR Version 1.0 (4/23/2020)                      Version 3.0 (10/23/2020)             Version 6.0 (2/8/2024)       
Version 1.1 (6/16/2020)                      Version 3.1 (5/3/2021)                  Version 6.1 (9/17/2024) 
Version 1.2 (7/13/2020)                      Version 4.0 (12/30/2021)           
Version 2.0 (7/16/2020  
         Version 5.0 (1/19/2023) 
 
 
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Interleukin-15 armored Glypican-3-specific chimeric antigen receptor expressing autologous T 
cells as immunotherapy for children with solid tumors (AGAR)  
 
 
Principal investigators 
Andras Heczey, MD 
David Steffin, MD 
 
 
Co-investigators 
Rajkumar Venkatramani MD, MS 
Helen Heslop, MD 
Valeria Smith MD 
Jennifer Foster MD 
Bilal Omer MD 
Gengwen Tian MD, PhD 
Bambi Grilley, RPh 
Adrian Gee, PhD 
 
 
 
Statistician: 
Tao Wang, PhD 
 
 
 
IND: 26255 
 
 
Center for Cell and Gene Therapy (CAGT)  
 
Texas Children’s Hospital 
6621 Fannin St,  
Houston, TX 77030 
 
Dan L. Duncan Comprehensive Cancer Center 
Baylor College of Medicine   
 
 

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TABLE OF CONTENTS 
 
CHECKLIST FOR PATIENT ELIGIBILITY –PROCUREMENT .................................................. 4 
CHECKLIST FOR PATIENT ELIGIBILITY - TREATMENT ........................................................ 5 
CHECKLIST FOR PATIENT ELIGIBILITY - TREATMENT ........................................................ 6 
1.0 Objectives ........................................................................................................................... 7 
1.1 
Primary Objective ...................................................................................................... 7 
1.2 
Secondary Objective ................................................................................................. 7 
2.0 Background and Rationale ................................................................................................ 7 
2.1 
Glypican 3 (GPC3)-positive tumors of children ....................................................... 7 
2.2 
Glypican-3 .................................................................................................................. 7 
2.3 
Experience targeting glypican-3 with monoclonal antibodies ................................ 8 
2.4 
Chimeric antigen receptors ....................................................................................... 8 
2.5 
Extending the Survival of CAR expressing T Cells ................................................. 8 
2.6 
Generation and preclinical evaluation of GPC3-CAR T cells under study ............. 9 
2.7. 
Inducible caspase 9 (iC9) suicide gene .................................................................... 9 
2.8 Rationale for lymphodepletion before adoptive T-cell transfer .................................10 
2.9 Rationale for the current study ....................................................................................11 
3.0 Risks of this study ............................................................................................................11 
3.1 
Infusion of CAR T cells .............................................................................................11 
3.2 
General Consequences of Retroviral Transduction ...............................................11 
3.3 Specific consequences of transgenes expressed ...........................................................12 
3.3.1 
Targeting GPC3 with CAR T cells ............................................................................12 
3.4 
Cytokine Release Syndrome ....................................................................................13 
3.5 
Lymphodepletion related toxicities .........................................................................14 
4.0 Generation of Transduced Cells ......................................................................................14 
4.1 
Blood collection ........................................................................................................14 
4.2 
Retroviral production ...............................................................................................14 
4.3 
CAR T cell production, transduction, and expansion ............................................14 
4.4 
Testing .......................................................................................................................14 
5.0 Eligibility Criteria ...............................................................................................................14 
5.1 
Procurement Eligibility .............................................................................................15 
5.1.1 Inclusion Criteria: .....................................................................................................15 
5.1.2. Exclusion Criteria .....................................................................................................15 
5.2 
Treatment Eligibility ..................................................................................................15 
5.2.1 Inclusion Criteria.......................................................................................................15 
5.2.2 Exclusion Criteria .....................................................................................................16 
6.0 Treatment Plan ..................................................................................................................16 
6.1 
Pre-infusion lymphodepletion ..................................................................................16 
6.2 
Dose levels and schedule ........................................................................................17 
6.3 
Supportive care .........................................................................................................17 
6.3.1  Venous Access ........................................................................................................17 
6.3.2  Antiviral prophylaxis ...............................................................................................17 
6.3.3  Allergic Reactions ....................................................................................................17 
6.3.4  Fever and Neutropenia ............................................................................................18 
6.3.5  Blood Products ........................................................................................................18 
6.3.6. Concomitant Therapy ...............................................................................................18 
6.3.7. Cytokine Release Syndrome, Neurotoxicity and Other Toxicity ...........................18 
7.0 Patient Evaluation .............................................................................................................19 
7.1 
Study Calendar .........................................................................................................20 
7.2 
Routine Laboratory Investigation ............................................................................21 
7.3 
Diagnostic imaging ...................................................................................................21 
7.4 
Tests of T-cell Function and Persistence ................................................................21 

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7.5 
Safety Testing ...........................................................................................................22 
7.6 
Determination of antitumor responses ...................................................................22 
8.0 Statistical Consideration ..................................................................................................24 
8.1 
Clinical Trial Design ..................................................................................................24 
8.2 
Dose Limiting Toxicity ..............................................................................................24 
8.3 
The Dose Escalation Method and sample size .......................................................24 
8.4. 
Stopping rules ...........................................................................................................26 
8.5  
Data Analysis ............................................................................................................26 
8.5.1. 
Safety Analysis of Adverse Event Data ............................................................26 
8.5.2. 
Clinical response data .......................................................................................26 
8.5.3. 
Analysis of Laboratory Data .............................................................................27 
8.5.4. 
Analysis of Expansion and Persistence of T cells ..........................................27 
9.0 Off Study Criteria: .............................................................................................................27 
10.0 Records to be kept .......................................................................................................28 
11.0 Reporting Requirements ..............................................................................................28 
12.0 Informed Consent .........................................................................................................29 
13.0 Clinical Trial Oversight and Monitoring ......................................................................29 
14.0    References ....................................................................................................................30 
Appendix I – Karnofsky/Lansky Performance Score ............................................................35 
Appendix II - Barcelona Clinic Liver Cancer Stage ...............................................................36 
Appendix III - Child-Pugh-Turcotte Score .............................................................................37 
Appendix IV - Roadmaps ........................................................................................................38 
Appendix V - Potential Side Effects of Lymphodepletion Chemotherapy...........................39 
Appendix VI - Grading of CRS and Neurological Toxicities .................................................42 
 
 

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CHECKLIST FOR PATIENT ELIGIBILITY –PROCUREMENT 
 
PATIENT ID   
 
 
 PATIENT NAME  
 
 
 
 
 
 
YES 
NO 
VALUE/DATE INCLUSION CRITERIA 
Any “NO” answers will make a patient ineligible for study participation. 
 
 
 
Diagnosis of GPC3-positive solid tumor 
 
 
 
Age ≥ 1 year and ≤ 21 years 
 
 
 
Life expectancy of ≥ 16 weeks 
 
 
 
Lansky or Karnofsky score ≥ 60% 
 
 
 
Child-Pugh-Turcotte score < 7 (for patients with 
hepatocellular carcinoma only) 
 
 
 
Barcelona Clinic Liver Cancer Stage A, B or C (for 
patients with hepatocellular carcinoma only) 
 
 
 
Informed consent explained to, understood by and 
signed by patient/guardian. Patient/guardian given 
copy of informed consent 
 
 
 
GPC3 expression (as determined by 
immunohistochemistry) with an extent score of ≥ 
Grade 2 (>25% positive tumor cells) and an intensity 
score of ≥ 2 (scale 0-4). 
 
YES 
NO 
VALUE/DATE 
EXCLUSION CRITERIA 
Any “YES” answers will make a patient ineligible for study participation. 
 
 
 
History of hypersensitivity reactions to murine protein-
containing products or presence of human anti-mouse 
antibody (HAMA) prior to enrollment (only patients 
who have received prior therapy with murine 
antibodies) 
 
 
 
History of organ transplantation 
 
 
 
Known HIV positivity 
 
 
 
Active bacterial, fungal or viral infection (except 
Hepatitis B or Hepatitis C virus infections) 
 
 
 
Actively progressing CNS metastases 
 
 
 
 
Signature of MD ______________________ 
Date ____________________ 
 
 
 

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CHECKLIST FOR PATIENT ELIGIBILITY - TREATMENT 
 
 
 
 
 
 
 
 
 
 
 
 
Page 1 of 2 
 
PATIENT ID  
 
 
 PATIENT NAME  
 
 
 
 
 
 
YES 
NO 
VALUE/DATE INCLUSION CRITERIA 
Any “NO” answers will make a patient ineligible for study participation. 
 
 
 
Age ≥1 year and ≤ 21 years 
 
 
 
Lansky or Karnofsky score ≥ 60% 
 
 
 
Child-Pugh-Turcotte score < 7 (for patients with 
hepatocellular carcinoma only) 
 
 
 
Barcelona Clinic Liver Cancer Stage A, B or C (for 
patients with hepatocellular carcinoma only) 
 
 
 
Creatinine clearance as estimated by Cockcroft Gault 
or Schwartz ≥ 60 ml/min 
 
 
 
Serum AST< 5 times ULN 
 
 
 
Total bilirubin < 3 times ULN for age  
 
 
 
INR ≤ 1.7 (for patients with hepatocellular carcinoma 
only) 
 
 
 
Absolute neutrophil count > 500/μl 
 
 
 
Platelet count > 25,000/μl (can be transfused but must 
be at that level prior to treatment) 
 
 
 
Hgb ≥ 7.0 g/dl (can be transfused but must be at that 
level prior to treatment) 
 
 
 
Pulse oximetry > 90% on room air 
 
 
 
Refractory or relapsed disease after treatment with up- 
front therapy and at least one salvage treatment cycle 
 
 
 
Recovered from acute toxic effects of all prior t and 
investigational agents before entering this study 
 
 
 
Sexually active patients must be willing to utilize one of 
the more effective birth control methods for 3 months 
after the T-cell infusion.  
 
 
 
Informed consent explained to, understood by and 
signed by patient/guardian. Patient/guardian given 
copy of informed consent 
 
Signature of MD ______________________ 
Date ____________________ 
 
 
 
 

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CHECKLIST FOR PATIENT ELIGIBILITY - TREATMENT 
 
 
 
 
 
 
 
 
 
 
 
 
Page 2 of 2 
 
PATIENT ID  
 
 
 PATIENT NAME  
 
 
 
 
 
 
YES 
NO 
VALUE/DATE 
EXCLUSION CRITERIA 
Any “YES” answers will make a patient ineligible for study participation. 
 
 
 
Pregnancy or lactation (for women at child-bearing 
age, birth control is required)  
 
 
 
Uncontrolled infection 
 
 
 
Systemic steroid treatment  
( 0.5 mg prednisone equivalent/kg/day, dose 
adjustment or discontinuation of medication must 
occur at least 24hrs prior to CAR T cell infusion) 
 
 
 
Known HIV positivity 
 
 
 
Active bacterial, fungal or viral infection (except 
Hepatitis B or Hepatitis C virus infections) 
 
 
 
History of organ transplantation 
 
 
 
History of hypersensitivity reactions to murine 
protein-containing products or presence of human 
anti-mouse antibody (HAMA) prior to enrollment (only 
patients who have received prior therapy with murine 
antibodies) 
 
 
 
Actively progressing CNS metastases 
 
 
 
Signature of MD ______________________ 
Date ____________________ 
 

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1.0 
Objectives 
The overall goal of this Phase 1 clinical trial is to evaluate the safety of autologous 
glypican-3 specific chimeric antigen expressing T cells co-expressing IL-15 
(15.GPC3-CAR T cells) in children with GPC3-positive solid tumors after 
lymphodepleting chemotherapy. 
1.1 Primary Objective 
1.1.1 
To determine the safety of escalating doses of an intravenous injection 
of 15.GPC3-CAR T cells in children with GPC3-positive solid tumors after 
lymphodepleting chemotherapy. 
 
1.2 Secondary Objective  
1.2.1 
To determine the maximum tolerated dose (MTD) of 15.GPC3-CAR T 
cells in treating patients with GPC3-positive solid tumors after 
lymphodepleting chemotherapy. 
1.2.2 
To assess the anti-tumor effect of infused 15.GPC3-CAR T cells in 
children with GPC3-positive solid tumors. 
 
1.3 Exploratory Objectives:  
1.3.1 
To assess the in vivo persistence, phenotype and functional activity of 
infused 15.GPC3-CAR T cells in children with GPC3-positive solid tumors. 
 
2.0 
Background and Rationale 
2.1 Glypican 3 (GPC3)-positive tumors of children   
Relapsed and refractory solid tumors are challenging to treat. Surgical resection is a 
mainstay of therapy, given conventional cytoreductive treatment with chemo or 
radiotherapy is associated with significant toxicity and often induces limited antitumor 
effect. Thus, safe and effective new therapies are needed. GPC3 is an antigen 
expressed in a heterogeneous group of cancers including hepatoblastoma (HB), 
hepatocellular carcinoma (HCC), Wilm’s tumor, malignant rhabdoid tumor (MRT), yolk 
sac tumor, rhabdomyosarcoma, undifferentiated embryonal sarcoma of the liver, and 
liposarcoma [1-8]. Since GPC3 is selectively expressed on these tumors but not on 
healthy, mature cells, targeting GPC3 may be an effective strategy to eliminate the 
cancer cells. Importantly, two recent Phase 1 clinical studies showed that targeting 
GPC3 is safe, well tolerated and, depending on the density of GPC3 expression on 
tumor cells, can achieve significant antitumor responses in patients with advanced 
HCC [9, 10]. 
 
2.2 Glypican-3 
For safe and effective immunotherapeutic approaches tumor restricted expression of 
the target antigen is necessary. Glypican-3 (GPC3) is one of the six members of the 
membrane-bound proteoglycans found in mammals [11]. It is an attractive 
immunotherapeutic target as it is expressed on the majority of HCCs and not 
expressed on normal hepatocytes after fetal development or on cirrhotic livers [1-8, 
12-14]. In HCC, GPC3 promotes tumor growth by stabilization of Wnt in close physical 
proximity to the cell membrane, thereby increasing Wnt binding to its receptor resulting 
in increased cell proliferation [1, 15, 16]. GPC3 contains heparan sulfate side chains, 
a protein chain containing disulfide bonds and is anchored to the cell surface by 
glycosyl-phosphatidyl-inositol (GPI) [11, 17]. Mutation in GPC3 results in the Simpson-
Golabi-Behmel syndrome as its function is critical for cell proliferation during 
development [18-20].  
 

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2.3 Experience targeting glypican-3 with monoclonal antibodies 
Two recent early phase clinical trials tested a GPC3 targeting monoclonal antibody 
(MAb) “GC33” in 33 patients with advanced HCC. GC33 MAb infusions were well 
tolerated and no dose limiting toxicities were observed. While no patient was cured of 
their disease, significant antitumor activity was observed that correlated with the GPC3 
expression levels in tumors [9, 10]. Thus, GPC3 is a safe target for other means of 
immunotherapy including genetically engineered T cells redirected to GPC3 by 
chimeric antigen receptors (CARs). 
 
2.4 Chimeric antigen receptors 
One potentially effective immunotherapeutic strategy is to generate tumor-specific 
effector cells by genetically engineering T cells to express chimeric antigen receptors 
(CARs), which most commonly consist of an extracellular antigen recognition domain 
(scFv) from a monoclonal antibody (Mab), a transmembrane domain, and intracellular 
signaling domains derived from the T-cell receptor CD3-ζ chain and from costimulatory 
molecules [21]. T cells genetically modified with CARs can combine the specificity of 
monoclonal antibodies with the active biodistribution and cytotoxic function of effector 
cells. CAR T cells have numerous advantages over immunotherapies based on 
monoclonal antibodies or T cells alone [22-24]. 
 
1) CARs are MHC-unrestricted, so that tumor escape by downregulation of HLA 
class I molecules or defects in antigen processing are bypassed.  
2) Both CD4+ and CD8+ T cells can express the same CAR, so that the full 
network of T cell function is directed against tumor cells.  
3) CAR-mediated effector function can be effective against cells that are relatively 
resistant to antibody and complement.  
4) Cytokine secretion upon CAR T cell activation by tumor antigen recruits 
additional components of the immune system, amplifying antitumor immune 
responses.  
5) Unlike intact antibodies, T cells can migrate through microvascular walls, 
extravasate and penetrate the core of solid tumors to exert their cytolytic 
activity.  
6) A single CAR T-cell can sequentially kill a multiplicity of target cells over a 
prolonged period of time.  
 
Clinical trials have shown potent antitumor activity of CAR T cells in patients with 
CD19-positive hematological malignancies, including in patients with significant 
tumor burden [25-28] In regards to targeting GPC3, GPC3-specific CARs (GPC3-
CARs) have been generated by several groups of investigators including ours. 
GPC3-CAR T cells have potent antitumor activity in preclinical models with GPC3-
positive tumors (HCC, lung cancer) [1, 29]. One phase 1 clinical trial with GPC3-CAR 
T cells was recently completed (NCT02395250), and two are currently in progress at 
our institution, including one in in pediatrics (NCT02932956) and one in adults 
(NCT02905188). 
 
This protocol builds on established safety with CAR T cells to redirected to GPC3 by 
co-expressing IL-15 and iC9 to increase expansion, persistence and antitumor 
activity; and enable rapid elimination of IL15 co-expressing cells if necessary. 
 
2.5 Extending the Survival of CAR expressing T Cells 
Full activation of T cells is accomplished after they bind to a target via their native 
receptor and receive stimulation from activating ligands on antigen presenting cells 
(APCs) as well as cytokines secreted in the local microenvironment. Most tumor cells 

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lack the additional costimulatory ligands needed for enhanced T-cell activation; 
therefore, 2nd generation CARs have been engineered to include another stimulatory 
domain [30]. The most common domain added to 2nd generation CARs incorporates 
the intracytoplasmic portion of CD28 that would normally be activated upon binding to 
the activating ligands on APCs and this construct has improved the function of 
adoptively transferred CAR T cells in humans [31]. Other stimulatory domains used 
include OX40, 41BB, inducible T-cell co-stimulator (ICOS), and DNAX activation 
protein 10 (DAP10) [23]. Recently, clinical trials testing CD19-specific CARs with 41BB 
costimulatory endodomain showed breakthrough results in the treatment of CD19-
positive malignancies including 91% complete remission rate of patients with 
relapsed/refractory acute lymphoblastic leukemia [25, 26, 28].  
 
2.6 Generation and preclinical evaluation of GPC3-CAR T cells under study 
We systematically evaluated the anti-tumor properties of T cells expressing GPC3-
CARs containing CD3zeta (Gz) with CD28 (G28z) or 41BB (GBBz) costimulatory 
endodomain. We tested T cells expressing Gz, G28z or GBBz against HB, HCC and 
MRT cell lines in vitro and evaluated their ability to expand, persist and eliminate 
established human HCC and MRT xenografts in vivo. We show that the inclusion of 4-
1BB alone in GPC3-CAR is sufficient to generate T cells with enhanced Th-1-like 
cytokine response, enhanced proliferation and in vivo persistence, and potent 
therapeutic activity in xenogeneic tumor models of GPC3+ positive solid tumors. 
Based on these results, the GBBz construct was selected for further development. 
Next, we evaluated if T cells expressing GBBz and IL-15 in combination have superior 
antitumor properties compared GBBz alone. We determined that GBBz T cells co-
expressing IL-15 specifically and effectively kill GPC3-positive tumor cells in an 
antigen-dependent manner. We also showed that GPC3-CAR T cells co-expressing 
IL-15 exhibit enhanced persistence in vivo and mediate superior tumor control and 
survival of tumor-bearing mice. These results provide a strong rationale to evaluate IL-
15 co-expressing GPC3 CAR T cells in patients with liver tumors in the second cohort 
of this clinical study. 
 
2.7. Inducible caspase 9 (iC9) suicide gene 
 
The use of T cells containing an inducible caspase 9 (iC9) suicide gene have 
the potential to limit toxicities, such as CRS and neurotoxicity that do not 
respond to current standard of care interventions like steroids and IL-1 and IL-
6 inhibitor, and have been evaluated in vitro and in clinical trials. Briefly, iC9-
mediated suicide is based on conditional dimerization of pro-apoptotic molecules, that 
are constructed from human proteins and therefore less likely to be immunogenic [32]  
iC9s generated by joining a drug-binding domain to human caspase 9.  The drug-
binding domain consists of human FK506-binding protein (FKBP12) with an F36V 
mutation. This point mutation increases the binding affinity of FKBP12 to non-toxic 
synthetic homodimerizers, AP20187 or AP1903 (rimiducid) [33, 34].   Administration 
of AP20187 or rimiducid dimerizes and activates caspase 9; this activates downstream 
caspases, leading to apoptosis within 24 hours. In patients receiving stem cell 
transplantation and donor-derived T cells expressing iC9, treatment with rimiducid 
effectively eliminated 99% of T cells and symptoms of graft-versus-host disease after 
a single infusion and repeat infusions could eliminate residual T cells if necessary [35-
37]. These studies provide evidence that the  rimiducid mediated iC9 activation is a 
safe and effective approach to transduced T cells.  
 

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The iC9 genetic modification, unlike other suicide gene systems such as the HSV-TK 
based suicide gene, is human derived and therefore likely to be less immunogenic. 
Previous studies have shown that T-cells genetically modified with iC9 suicide gene 
construct were detected in peripheral blood and increased in number over time, despite 
their constitutive transgene expression. Moreover, pre-clinical and clinical studies show 
that killing occurs with much greater rapidity (within 3 hours) than other suicide gene 
systems. Further advantages of the iC9 system are that killing induced by rimiducid, is 
primarily restricted to activated/proliferating cells, thus preferentially targeting activated 
T cells, and as shown in preclinical studies, furthermore the extent of killing efficacy 
can be modulated through a wide range of Rimiducid concentration eliminating only 
a fraction at lower and close to complete elimination of iC9 expression cells at higher 
concentration [38].   
 
In two previous clinical trials, patients who developed Graft Versus Host Disease 
(GVHD) after infusion of allogeneic iC9 gene-modified T-cells, were given a single 
dose of dimerizing drug,  rimiducid. This led to elimination of more than 90% of the 
iC9-gene modified T cells within 30 minutes after administration and a further log 
reduction within 24 hours without any reports of infusion related toxicity. The residual 
iC9 gene modified T cell population expanded over the next 4-14 days and continued 
to help repopulate the subjects’ immune system without recurrence of GVHD. When 
examined ex vivo, these non-alloreactive iC9 T cells remained susceptible to apoptosis 
following exposure to the dimerizer  rimiducid. The iC9 gene is most highly expressed 
in activated and proliferating T cells and if cells are not activated, expression of the 
transgene is lower and iC9 levels are insufficient to induce apoptosis of the entire 
population after single rimiducid dose exposure. This hypothesis was supported by the 
CASPALLO [35] and DOTTI [37] clinical trials showing activation- dependent 
induction of iC9 in T lymphocytes and enhanced susceptibility to dimerizer drug in 
activated versus resting T cells. 
 
T cells in this protocol will be co-transduced with two retroviral vectors: GPC3-CAR 
(SFG.GPC3.4-1BB.z) and iC9.NGFR.IL15 (SFG.iC9.2A.NGFR.2A.IL-15.). This 
strategy will therefore allow for the preferential killing of the CAR T cells with the 
highest potency, thereby acting as a safety switch in the setting of toxicities. 
 
2.8 Rationale for lymphodepletion before adoptive T-cell transfer 
Lymphodepletion has been shown to improve the anti-tumor efficacy of adoptive cell 
therapies by direct antineoplastic effect of the used chemotherapeutic agents and by 
improving survival and function of adoptively transferred cells [39]. The size of the 
lymphoid compartment is tightly controlled by homeostatic factors, including the 
access to antigen-presenting cells (APCs), major histocompatibility complex (MHC) 
presenting self and antigenic peptides, and direct influence of host T lymphocytes 
including regulatory cells CD4+CD25+FoxP3+ Treg, as well as the competition for 
cytokines such as IL-2, IL-7, IL-15 and IL-21 [40-48]. Administration of certain 
lymphodepleting chemotherapy or total body irradiation, reduces the patient’s 
lymphoid cell pool, improves the adoptively transferred cells access to APCs, peptides 
presented by MHCs and homeostatic cytokines resulting in superior expansion and 
persistence of transferred cells [39, 49, 50]. Most importantly, after lymphodepletion, 
long term complete remissions are achievable in heavily pretreated cancer patients 
with genetically engineered adoptive cell therapies [25, 27, 51]. The most commonly 
used lymphodepleting regimens combine cyclophosphamide and fludarabine (cy/flu) 

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and a fractionated cy/flu regimen was used in the only clinical protocol to date resulting 
in sustained complete remission in a patient with bulky disease [39, 51, 52]. Therefore, 
we will use a fractionated cy/flu regimen for lymphodepletion in this trial. 
 
2.9 Rationale for the current study 
Patients with relapsed / refractory GPC3+ tumors are in urgent need for novel 
therapies. We systematically tested T cells expressing distinct GPC3-CARs in 
preclinical models of GPC3+ solid tumor models and found robust antitumor 
activity against these tumors by GBBz which can be further enhance by IL-15 co-
expression in GBBz T cells [53, 54]. We hypothesize that autologous T cells 
expressing a 15.GPC3-CAR will be safe and we further hypothesize that these 
cells will have antitumor activity in patients with relapsed/ refractory GPC3+ tumors. 
We will conduct a dose escalation Phase 1 clinical trial to test our hypotheses. 
2.10. Rationale for CAR T cell dose:  
Our group has treated three children with GPC3-CAR T cells at the dose level of 
1x107/m2 dose (NCT02932956). The infusions were well tolerated and dose 
limiting toxicities were not detected. We are enrolling on the 3x107/m2 dose level 
currently. A recent publication showed T cells expressing a 3rd generation GPC3-
CAR to be safe when total of 13 adult patients with hepatocellular carcinoma 
received a median of 19.9 × 108 cells [55]. We expect that enrollment on the 
3x107/m2 GPC3-CAR T cells will be safe and we will subsequently start enrollment 
on the 3x107/m2 dose level with 15.GPC3-CAR T cells. If safety is not confirmed 
on this dose level with GPC3-CAR T cells, we will start enrollment on 1x107/m2 
dose with 15.GPC3-CAR T cells. 
3.0 
Risks of this study 
Potential toxicities may be categorized as those related to: i) infusion of T cells; ii) general 
consequences of retroviral transduction; iii) specific consequences of the transgene 
expressed (targeting GPC3, cytokine release syndrome, neurotoxicity); and iv) risks of 
using fludarabine and cyclophosphamide. 
 
3.1 Infusion of CAR T cells 
In this study, we will be administering transduced autologous peripheral blood 
activated T cells modified to express a CAR directed against GPC3 and expectedly 
the infusion product will contain some percentage of non-transduced T cells. Many 
previous studies have infused larger numbers of ex-vivo cultured T cells that have 
been activated ex vivo, with no adverse effects [56, 57]. Thus, the risk from infusing 
autologous T cells will be minimal. 
 
3.2 General Consequences of Retroviral Transduction 
Retroviral transduction results in new, random integrations in host cell DNA, which rarely 
may cause abnormal or uncontrolled proliferation. This effect is much more common with 
replication-competent retrovirus (RCR) where each cell receives multiple integrants. 
Several publications have now reviewed extensive and long-term data on RCR monitoring 
in T cells immediately after gene modification with retroviral and in subsequent follow-up 
samples from patients; no evidence for RCR has yet been found, providing important 
evidence for safety[58-60]. 
Genotoxicity with retroviral vectors was seen in the initial trials transferring retroviral 
vectors to hemopoietic stem cells to correct inherited immunodeficiencies in the late 
1990s and early 2000s. These studies demonstrated clinical activity in patients, but 
several years after treatment, some patients in the SCID trials, as well as patients 
treated for chronic granulomatous disease and Wiskott-Aldrich syndrome, developed 
acute leukemias due to activation of proto-oncogenes adjacent to proviral insertions[61-

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63]. These serious toxicities led to adoption of enhancer-deleted lentiviral or retroviral 
vectors for HSC clinical gene therapies. Up to now, these newer vectors have produced 
disease amelioration without genotoxicity[64]. 
Our proposal should have a very different risk profile as we are transferring retroviral 
vectors into T cells. Although the inclusion of the 41BB endodomain in the CAR may 
enhance proliferation of transgenic T cells in response to the GPC3 antigen, there is no 
reason to believe this effect will lead to uncontrolled T-cell expansion. Ex vivo and 
published in vivo data using several artificial T-cell receptors shows that 41BB sustains 
limited and temporary expansion over approximately 3-6 weeks [65, 66]. Thereafter, the 
cells maintain the expression of the transgene, but proliferate further only in the presence 
of both antigen and exogenous cytokines. Our recent clinical trials using CAR-T cells 
including the 41BB endodomain also indicate that these T cells expand significantly for 
only 2-3 weeks and then they decline without evidence of further expansion [67, 68].  
To date more than 1000 patients have received genetically modified T cells in clinical trials, 
including patients we have treated on our protocols using T cells modified with 
gammaretroviruses [67-72].   In none of these, nor in clinical trials outside our center, has 
malignancy caused by retroviral transduction been reported.  
Patients eligible for the current study will have relapsed / refractory GPC3-positive solid 
tumors. In light of this, the natural history and poor prognosis of these malignancies, and 
given the entire previous experience with retroviral gene therapy, we feel that the small 
risks of retrovirally-induced leukemogenesis are small and are justified in these patients. 
 
3.3 Specific consequences of transgenes expressed 
Specific consequences of the transgene expressed includes targeting GPC3, the 
use of IL-15 and CRS. 
3.3.1 Targeting GPC3 with CAR T cells 
No clinical trial results have been reported in the literature to date from 
testing GPC3-CAR T cells in patients; thus GPC3-specific side effects are 
not known. Expression of GPC3 is highly specific and restricted to 
malignant tissues (1, 2, 5, 12, 13). While Baumhoer et al reported some 
expression of GPC3 on gastric glands (3/7 samples [43%]), kidney tubules 
(9/17 samples [53%]), and testicular germ cells (2/16 samples [13%]) (4), 
this was not confirmed by Gao et al (1). MAb- based GPC3 targeting in 2 
independent Phase 1 clinical trials was safe, and no dose limiting toxicities 
were observed in 33 HCC patients as discussed in Section 2.3. Mild 
gastrointestinal toxicities were described (nausea/vomiting/diarrhea), but 
no nephritis or testicular inflammation was detected. Should 15.GPC3-CAR 
T cells produce direct toxicity against GPC3-expressing normal tissues, a 
number of therapeutic maneuvers are available. We will first use  rimiducid 
(the chemical inducer of dimerization) (see Section 6.3.8). Alternatively, 
corticosteroids in doses used to treat graft-versus host-disease (GvHD) or 
anti-T-cell antibodies such as Campath 1H can be used, as both of which will 
deplete the majority of circulating transduced cells.  
3.3.2 Co-expression of IL-15 
There is prior safety experience using IL-15 in patients. Conlon et al. 
examined the safety of recombinant IL-15 in eighteen patients with 
melanoma and RCC. Doses ranged between 0.3-3 µg/kg/day given on 
twelve consecutive days intravenously. Peak IL-15 concentrations were 
1260 (+/- 348) pg/ml for 0.3 µg/kg/day dosing. DLTs included 
thrombocytopenia, hypotension and transaminase elevations. The maximum 

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tolerated dose was defined at 0.3 µg/kg/day dose. Stable disease was the 
best response per RECIST criteria, five patients had 10-30% decrease in 
marked lesions with two of these patients clearing their respective lung 
lesions [73].  
 
Based on these results, IL-15 can be administered safely to patients with 
cancer. Peak concentrations in peripheral blood of these patients were high. 
In our in vivo animal studies, systemic levels of IL-15 at the time of peak 
CAR T cell expansion were detected at levels similar to control T cells at 3 -
4 log lower concentrations compared to the human studies using IL-15 bolus 
injections [73, 74]. IL-15 produced by GPC3-CAR T cells will likely only 
locally produce effects on the adoptively transferred CAR T cells and 
bystander cells. Should 15.GPC3-CAR T cells induce toxicity of grade 2 
(grade 3 respiratory toxicity) or higher, we will consider rimiducid (≤ 50 kg: 
starting dose of 0.02 mg then escalate to 0.004 mg/kg/dose, 0.04 
mg/kg/dose and 0.4 mg/kg/dose if needed; > 50 kg: starting dose of 0.0004 
mg/kg then escalate to 0.004 mg/kg/dose, 0.04 mg/kg/dose and 0.4 
mg/kg/dose if needed based on response after 6-8 hours of observation 
following each dose) (see below in Section 6.3.8). We will also consider 
using steroids (1-2 mg/kg/day of methylprednisolone or equivalent) if 
suboptimal response to  rimiducid. Alternatively, we will consider the JAK1 
inhibitor, Ruxolitinib (5 mg PO twice daily for children ≥ 25 kg or 2.5 mg 
twice daily if <25 kg) as it disrupts JAK1 mediated downstream signaling of 
IL15; and we found that ruxolitinib can temporarily halt the CAR T cell 
function.  
 
3.4 Cytokine Release Syndrome and Neurotoxicity 
 
There have been several reported SAEs associated with cytokine release syndrome 
(CRS) in patients who received T cells [75] or bispecfic T-cell engager molecules [76]. 
The majority of CRS have been reported after the infusion of CAR T cells [27, 28, 77], 
but CRS can also occur after the infusion of conventional antigen-specific T cells [78] 
or tumor infiltrating lymphocytes [79]. Patients will be monitored closely as per study 
calendar and assessed for evidence of incipient CRS (onset of fever, malaise and 
dyspnea) and treated promptly. Management of CRS will follow published guidelines 
[75, 80], and is described in more detail in SOP F 05.11.XX and includes treatment 
options based on the clinical severity of the symptoms, such as oxygen, inotropic 
agents, and IL-6 receptor antibody (4-8 mg/kg). If any grade 2 CRS or above not 
responsive to the aforementioned agents, then rimiducid (see below in 6.3.8) will be 
administered. Alternatively, TNF-α antibody (5-10 mg/kg) may be given. If resistant to 
the aforementioned modalities, steroids (1-2 mg/kg/day of methylprednisolone or 
equivalent) will be administered. The JAK1 inhibitor, Ruxolitinib (5 mg PO twice daily 
for children ≥ 25 kg or 2.5 mg twice daily if <25 kg) may be considered for cases 
resistant to above reagents as it disrupts JAK1 mediated downstream signaling of 
IL15. 
 
There have been several reports of neurotoxicity (e.g. encephalopathy, somnolence, 
aphasia) after the infusion of CD19-specific CAR T cells [27, 28, 77]. Patients will be 
monitored closely as per study calendar and assessed for evidence of incipient 
neurotoxicity (mild somnolence, drowsiness or sleepiness) and treated promptly. 
Management will include IL-6 receptor antibody (4-8 mg/kg),  rimiducid, and if resistant 
to other modalities, steroids (1-2 mg/kg/day of methylprednisolone or equivalent), as 
well as prophylactic anti-epileptics as described in the literature [27, 75, 80]. 
 

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3.5 Lymphodepletion related toxicities 
In this protocol we will use fractionated cyclophosphamide and fludarabine for 
lymphodepletion. This has been a widely used lymphodepletion protocol prior to CAR 
T-cell infusion [39, 49, 81]. Both cyclophosphamide and fludarabine are commercially 
available and a list of their toxicities is attached in Appendix V.  
 
3.6 Safety of the synthetic homodimerizer and the inducible Caspase 9 (iC9) safety 
switch in the IL15-encoding vector (iC9.NGFR.IL-15):  
No known risks have been associated with the synthetic homodimerizer rimiducid, 
which has been evaluated in the United Kingdom and has successfully completed a 
phase I clinical safety study when rimiducid was administered over a 0.01mg/kg to 
1.0mg/kg dose range [34]. The maximal plasma level attained over this dose range 
was 10 to 1275ng/ml  rimiducid (equivalent to 7 to 892nM).  rimiducid will be infused 
as described in Section 6.3.8. 
4.0 
Generation of Transduced Cells  
4.1 Blood collection 
Up to 90 ml (max of 3ml/kg/day) of peripheral blood will be collected over 24 hours 
from a given patient. This may be repeated a second time at least 24 hours later, with 
the limitation not to exceed 180 ml total over the 2 separate collections. The blood is 
used to generate the cellular components listed below, and for infectious disease 
testing and HLA typing. 
 
4.2 Retroviral production 
A producer line for a gamma-retrovirus encoding the GPC3-CAR and the 
iC9.NGFR.IL15 has been generated and vector lots have been prepared under cGMP 
conditions for these patients. The producer cells have been tested for replication 
competent retrovirus and contamination by bacteria, fungi and mycoplasma and by 
other viruses as per our certificates of analysis. The harvested supernatants have 
been filtered, aliquoted and rapidly frozen and stored at –80C. The retroviral 
supernatants are tested for sterility and endotoxin and to exclude replication 
competent retrovirus (RCR). We have issued a certificate of analysis in 
accordance with our SOPs for both vectors. 
4.3 CAR T cell production, transduction, and expansion 
Briefly, isolated PBMCs will be activated with clinical grade CD3 and CD28 MAbs, 
transduced with the GPC3-CAR and the iC9.NGFR.IL15 retroviral vector and 
expanded using cytokines as per SOP D03.53.XX. 
 
4.4 Testing 
Products that meet study specific release criteria, as detailed on the Certificate of 
Analysis (CofA), will be infused as per Section 6.0. 
 
If a positive sterility testing result is reported after the product was infused, the FDA 
and other relevant parties would be notified as per our manufacturing SOP B01.03.XX 
(Product Quality Assurance Program and Release and Return of Clinical GMP/GTP 
Products) and our clinical research SOP J02.06.XX (Serious Adverse Experience and 
Unanticipated Problem Reporting). Our management of such a situation is further 
described in our SOP F05.09.XX (Management of Culture Positive Cell Therapy 
Products).  
 
5.0 
Eligibility Criteria 
 

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5.1 Procurement Eligibility 
5.1.1 Inclusion Criteria:  
 
Diagnosis 
of 
GPC3-positive* 
solid 
tumors 
(as 
determined 
by 
immunohistochemistry with an extent score of >=Grade 2 [>25% positive 
tumor cells] and an intensity score of >= 2 [scale 0-4]). 
 
Age ≥1 year and ≤ 21 years  
 
Lansky or Karnofsky score ≥60% (See Appendix I) 
 
Life expectancy ≥16 weeks 
 
Barcelona Clinic Liver Cancer Stage A, B or C (for patients with 
hepatocellular carcinoma only, See Appendix II) 
 
Child-Pugh-Turcotte score <7 (for patients with hepatocellular carcinoma 
only, See Appendix III) 
 
Informed consent explained to, understood by and signed by 
patient/guardian. Patient/guardian given copy of informed consent 
 
* GPC3 expression will be evaluated by standard immunohistochemistry 
(IHC) at Texas Children’s Hospital/Baylor College of Medicine, Department 
of Pathology for all patients to meet procurement eligibility. All patients will 
send at least 5 unstained slides. 
 
5.1.2. Exclusion Criteria 
 
History of hypersensitivity reactions to murine protein-containing products 
OR presence of human anti-mouse antibody (HAMA) prior to enrollment 
(only patients who have received prior therapy with murine antibodies). 
 
History of organ transplantation 
 
Known HIV positivity 
 
Active bacterial, fungal or viral infection (except Hepatitis B or Hepatitis C 
virus infections) 
 
Actively progressing CNS metastases 
 
5.2 Treatment Eligibility 
5.2.1 Inclusion Criteria 
 
Age ≥ 1 year and ≤ 21 years  
 
Barcelona Clinic Liver Cancer Stage A, B or C (for patients with 
hepatocellular carcinoma only, See Appendix II) 
 
Lansky or Karnofsky score ≥ 60% (See Appendix I) 
 
Child-Pugh-Turcotte score < 7 (for patients with hepatocellular carcinoma 
only, See Appendix III)  
 
Adequate organ function: 
 
Creatinine clearance as estimated by Cockcroft Gault or Schwartz 
≥ 60 ml/min 
 
serum AST< 5 times ULN  
 
total bilirubin < 3 times ULN for age  
 
INR ≤1.7 (for patients with hepatocellular carcinoma only) 
 
absolute neutrophil count > 500/µl 
 
Platelet count > 25,000/μl (can be transfused but must be at that 
level prior to treatment) 
 
Hgb ≥ 7.0 g/dl (can be transfused but must be at that level prior to 
treatment) 

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 
Pulse oximetry >90% on room air 
 
Refractory or relapsed disease after treatment with up- front therapy and 
at least one salvage treatment cycle 
 
Recovered from acute toxic effects of all prior chemotherapy and 
investigational agents before entering this study 
 
Sexually active patients must be willing to utilize one of the more effective 
birth control methods for 3 months after the T-cell infusion.  
 
Informed consent explained to, understood by and signed by 
patient/guardian. Patient/guardian given copy of informed consent  
 
5.2.2 
Exclusion Criteria 
 
Pregnancy or lactation  
 
Uncontrolled infection  
 
Systemic steroid treatment ( 0.5 mg prednisone equivalent/kg/day, dose 
adjustment or discontinuation of medication must occur at least 24hrs prior 
to CAR T cell infusion) 
 
Known HIV positivity 
 
Active bacterial, fungal or viral infection (except Hepatitis B or Hepatitis C 
virus infections) 
 
History of organ transplantation 
 
History of hypersensitivity reactions to murine protein-containing products 
OR presence of human anti-mouse antibody (HAMA) prior to enrollment 
(only patients who have received prior therapy with murine antibodies) 
 
Actively progressing CNS metastases 
 
 
6.0 
Treatment Plan 
In this Phase 1 clinical study, patients will receive lymphodepleting 
chemotherapy (Appendix IV) followed by infusion of 15.GPC3-CAR T cells. 
6.1 Pre-infusion lymphodepletion 
Patients will receive 3 daily doses of cyclophosphamide together with fludarabine to 
induce lymphopenia, finishing at least 48 hours before CAR T cell infusion. 
Cyclophosphamide will be given at a dose of 500 mg/m2/dose followed by Fludarabine 
30 
mg/m2/dose. 
Infusions 
should 
be 
given 
following 
hospital/pharmacy 
recommendations however at a minimum the cyclophosphamide should be infused 
over 1 hour and the fludarabine should be infused over 30 minutes. Mesna, IV 
hydration and anti-emetics should also be provided following local institutional 
guidelines. T-cell infusion will be given 48 to 96 hours post completion of 
chemotherapy. 
 
Day 
Agent 
Dose/Administration 
-4 
Cyclophosphamide/Fludarabine 
500mg/m2/dose IV / 30mg/m2/dose IV 
-3 
Cyclophosphamide/Fludarabine 
500mg/m2/dose IV / 30mg/m2/dose IV 
-2 
Cyclophosphamide/Fludarabine 
500mg/m2/dose IV / 30mg/m2/dose IV 
-1 
 
 
0 to +2 
15.GPC3-CAR T cells 
 Dose level 1, 2, 3, or 4 as specified below. 
 
 

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6.2 Dose levels and schedule 
Four dose levels (DL) will be evaluated. Three to six patients will be assessed on DL 
(see Section 8). 
The following dose levels will be evaluated: 
 
DL1: 3x107/m2 
DL2: 1x108/m2 
DL3: 3x108/m2 
DL4: 1x109/m2 
 
If dose de-escalation is required on DL1, patients will be enrolled on DL0 at 1x107/m2 
dose. If further dose de-escalation is necessary, half log decreases will be used on 
DL-1 and on DL-2.  
 
The first patient on each dose level has to be 14 days post T-cell infusion before the 
second patient can be enrolled.  
 
The doses are calculated according to the actual number of GPC3-CAR transduced T 
cells. 
 
 
Patients may be premedicated with Benadryl 0.1-0.5 mg/kg IV (max 25mg) and 
Tylenol 10mg/kg po (max 650mg). 
 
Cell Administration: 15.GPC3-CAR T cells at an expected concentration of 
1x107 cells/ml (volume of 2 – 200 ml) will be given by intravenous infusion over 
up to 5-10 minutes through either a peripheral or a central line. 
 
Monitoring will follow institutional standards for administration of blood 
products with the exception that the infusion will be given by a physician. 
 
Patients will receive supportive care for acute or chronic toxicity. Such care will 
include blood components or antibiotics and other interventions as appropriate. 
 
All treatments will be given at the Center for Cell and Gene Therapy at Texas 
Children’s Hospital. 
 
Patients should not receive other treatment for their cancer for at least 4 weeks 
post T-cell infusion (for purposes of evaluation). If a patient receives other 
treatment for their cancer during the first 4 weeks post T-cell infusion, he/she 
is not evaluable and will need to be replaced. 
 
6.3 Supportive care 
6.3.1  Venous Access  
Chemotherapy and T cells can be given through peripheral or central 
intravenous access. 
 
6.3.2  Antiviral prophylaxis 
Patients with chronic hepatitis B virus (HBV) infection must receive antiviral 
prophylaxis (e.g. entecavir) starting 2 weeks prior to CAR T cell infusion until 
at least 6 weeks post-infusion to prevent viral reactivation.  
 
6.3.3  Allergic Reactions 
Standard medications (hydrocortisone, diphenhydramine, and epinephrine) 

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to treat allergic reactions should be available at the bedside prior to T-cell 
infusions.  
 
6.3.4  Fever and Neutropenia  
Standard of care should be administered for complications resulting from 
neutropenia. 
 
6.3.5  Blood Products  
Blood products should be given when appropriate.  
 
6.3.6. Concomitant Therapy 
No other cancer chemotherapy or immunomodulating agents should be 
used.  
 
6.3.7. Cytokine Release Syndrome, Neurotoxicity and Other Toxicity  
 
6.3.7.1  CRS, management will follow published guidelines, and is described in more detail in 
SOP F 05.11.XX which includes treatment options based on the clinical severity of the 
symptoms, such as oxygen, steroids  and IL-6 receptor antibody (4-8 mg/kg). 
 
6.3.7.2 For CRS persistent despite standard therapy or other Grade 2 (grade 3 respiratory 
toxicity) toxicities possibly related to 15.GPC3-CAR T cells, we will plan to administer Rimiducid. 
Dosing will be as follows:  
 
For Weight ≤ 50 kg: 
1) Start at 0.02mg/dose. 
2) Dose may be escalated to 0.004mg/kg/dose and then a maximum dose of 
0.4mg/kg/dose may be given based on the clinical condition of the patient after 6-8 hours of 
monitoring following each administration.  
For weight >50 kg:  
1) Start at dose of 0.0004 mg/kg/dose 
2) Dose may be escalated to 0.004 mg/kg/dose, 0.04 mg/kg/dose and 0.4 mg/kg/dose if 
needed based on the clinical condition of the patient after 6-8 hours of monitoring 
following each administration. 
6.3.7.3 If suboptimal response to rimiducid, we will consider the JAK1 inhibitor, Ruxolitinib (5 mg 
PO twice daily for children ≥ 25 kg or 2.5 mg twice daily if <25 kg) as it disrupts JAK1 mediated 
downstream signaling of IL15; and we found that ruxolitinib can temporarily halt the CAR T cell 
function 
 
 
 
6.3.8. Use of rimiducid, chemical inducer of dimerization 
Packaging and Formulation: The rimiducid for Injection is packaged in a 3 mL Type 
1 clear glass serum vials. The content of each vial is composed of the labeled 
content 10mg (2mL) of rimiducid drug substance dissolved in a sterile, endotoxin 
free, 25% Solutol HS 15/Water for Injection solution at a rimiducid concentration of 
5 mg/mL and at pH 5.0 – 7.5. Each vial is stoppered with a Teflon© coated serum 
stopper and a flip-off seal. Rimiducid is manufactured and provided by Bellicum 
Pharmaceuticals. 
 
Labeling: The primary product label (applied directly to the vial) for the Rimiducid 
for Injection will contain the following information: product name, Rimiducid for 
Injection; the manufacturer’s lot number; product concentration, 5 mg/mL; volume 
of solution available in the vial; total rimiducid contents of the vial (10mg); a 
statement, “For IV Administration, contains no preservatives” and the IND notation, 

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“Caution: New Drug-Limited by Federal Law to Investigational Use”.  
 
Storage: The Rimiducid for Injection vials must be stored at 5°C ± 3°C (41°F ± 5°F) 
in a limited access, qualified refrigerator, preferably without light. 
 
Preparation for Treatment and dosing escalation: Rimiducid will be diluted prior to 
administration. The rimiducid is administered via IV infusion at the target dose 
diluted in normal saline with volume as appropriate for weight. Rimiducid will be 
given at escalating doses starting with 0.0004 mg/kg/dose, 0.04mg/kg/dose and 
0.4mg/kg/dose. Dose increase may be given following 6-8 hours of observation 
based on the indications below: 
 
Indications:  
1) Any Grade 2 or greater CRS and neurotoxicity at least possibly related to IL15 
expressing T cells. 
3) Amy Grade 2 or greater non-CRS related toxicities at least possibly related to 
IL15 expressing T cells that do not respond to standard of care. 
 
Prescribers: Authorized prescribers of rimiducid are Principal Investigators and IND 
holders - Dr. Malcolm Brenner and Dr. Helen Heslop, and the lab medical director. 
 
Laboratory tests after rimiducid administration: Peripheral blood samples will be 
collected immediately prior to administration of rimiducid, 4-8hr, and 72hrs post-
rimiducid dose and will be evaluated with FACS and RT-PCR to confirm elimination 
of iC9 expressing cells. Additional time-points may be collected based on the 
clinical condition of the patient. 
 
 
7.0 
Patient Evaluation 
A complete history, physical examination, and performance status is necessary prior to 
lymphodepletion (week -1) and prior to T-cell administration. A complete history, physical 
exam and performance status will be collected at week 1, 2, 3, and 4. A complete history 
will continue to be collected at week 8, every three months (starting at month 3) for a year, 
every 6 months for 4 years, and then yearly for a total of 15 years. After week 4, follow up 
visits may be conducted at the subject’s local pediatric oncologist’s office with appropriate 
documentation provided to the research team.  
 
 

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7.1 Study Calendar 
The detailed schedules for clinical monitoring are included below.   
Study 
 
 
Wk
-1^ 
Wk 
0 
Wk 
1 
Wk 
2 
 
Wk 
3 
Wk 
4 
Wk 
8 
Mth 
3 
Mth
6 
Mth
9 
Mth 
12 
Every 
6 mths 
x 4 
years 
Annually 
x 15 years 
(total) 
Ctx + Flu 
X 
 
 
 
 
 
 
 
 
 
 
 
 
CAR T-cell 
Infusion 
 
X 
 
 
 
 
 
 
 
 
 
 
 
Hx^ 
X 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
PE^ 
X 
X 
X 
X 
 
X 
 
 
 
 
 
 
 
Performance 
Status 
X 
X 
X 
X 
 
X 
 
 
 
 
 
 
 
CBC with diff 
X 
X 
X 
X 
X 
X 
 
X 
X 
X 
X 
 
 
Lytes,  
BUN, Cr 
X 
X 
X 
X 
 
X 
X 
 
 
 
 
 
 
 
AST, Total Bili, 
Alb 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
 
AFP@ 
X  
X 
X  
X 
X 
X 
 
 
 
 
 
 
 
HBV-PCR@@ 
X 
X 
 
X 
 
X 
 
 
 
 
 
 
 
Pregnancy 
Test (Child 
Bearing 
Potential)  
X 
 
 
 
 
 
 
 
 
 
 
 
 
Function and 
Persistence 
Studies### 
X 
X* 
X** 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
RCR Testing/ 
Archiving### 
 
X 
 
 
 
 
 
X 
X 
 
X 
X*** 
X*** 
Cytokines/ 
Chemokine### 
X 
X 
X**  
X 
X 
X 
X 
X 
 
 
 
 
 
Serum stored 
for HAMA### 
 
X 
 
 
 
X 
 
 
 
 
 
 
 
Imaging 
X 
 
 
 
 
X~ 
 
 
 
 
 
 
 
Tumor biopsy 
 
 
 
X$, 
### 
 
 
 
 
 
 
 
 
 
Ctx + Flu: Cyclophosphamide and fludarabine ^starts day -4; *pre-infusion and 1 to 4 hours post infusion; **one sample will be 
drawn 1 week post infusion, a 2nd sample 3-4 days post infusion is optional; ###study specific tests; ***will be done annually; 
Pregnancy testing is only required in female patients of childbearing potential; ~Post infusion imaging may be performed from 
4-6 weeks post infusion with choice of tests dependent on the previous tests that have best estimated the amount of disease.@: 
Patients with AFP producing tumors only. @@ Patients with HBV infection only.;  $At 2-4 weeks post-CAR T cell infusion tumor 
biopsies will be obtained. ^After week 4, follow up visits may be conducted at the subject’s local physician’s office with 
appropriate documentation provided to the research team. Monthly visits can be done within +/- 1 month; weekly visits can be 
done within +/- one week of the scheduled visit.  Must be completed prior to lymphodepletion and prior to T-cell administration. 
 

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7.2 Routine Laboratory Investigation 
The following routine laboratory investigations will be obtained as outlined in the 
study calendar: 
 CBC and differential: pre-chemotherapy, pre-infusion, at 1, 2, 3 and 4 weeks post 
T-cell infusion (each weekly visit can be within + and then at 3, 6, 9, and 12 months.  
 BUN, Creatinine, Na+, K+, Cl-, CO2: pre-chemotherapy, pre-infusion, at 1, 2, 3 and 
4 weeks  post T-cell infusion 
 Bilirubin, Albumin, AST:  pre-chemotherapy, pre-infusion, at 1, 2, 3 and 4 weeks 
post T-cell infusion 
 AFP (for patients with AFP producing tumors only): pre-chemotherapy, pre-
infusion, at 1, 2, 3 and 4 weeks post T-cell infusion 
 HBV-PCR (for patients with HBV infection only): pre-chemotherapy, pre-infusion, 
at 2, and 4 weeks post T-cell infusion 
 Pregnancy test will be performed within the week preceding starting chemotherapy 
on female patients of childbearing potential unless they have no possibility of 
pregnancy (for example post hysterectomy) 
 Other tests: Infectious disease testing will be done at the time of blood 
procurement for T-cell production; in addition, HLA typing (via blood or buccal 
swab) will be performed to ensure autologous identity of T-cell product. 
 Additional routine laboratory investigations may be performed as per institutional 
guidelines during the administration of cyclophosphamide and fludarabine. 
**for the weekly visits immediately following infusion (1,2,3, and 4 weeks), these can 
be done within +/- one week of the scheduled visit; Monthly visits can be done within 
+/- 1 month from the scheduled visit. 
 
7.3 Diagnostic imaging  
Imaging studies are required pre-lymphodepleting chemotherapy (within 4 weeks) and 
6 weeks (range 4-6 weeks) following T-cell infusion. Contrast enhanced computer 
tomography (CT) or magnetic resonance imaging (MRI) are acceptable modalities (for 
patients with liver tumors, arterial and portal venous phase sequences are necessary). 
If diagnostic imaging studies are performed at other times either during or after 
treatment on this study, that data will be collected, and information gained will be used 
for this study. The same modality will be used at the time of enrollment for treatment 
and response evaluation.  
 
7.4 Tests of T-cell Function and Persistence 
The following samples will be obtained pre-chemotherapy, pre-T-cell infusion, 1 to 4 
hours post T-cell infusion, 3-4 days post T-cell infusion (optional depending on patient 
preference), at 1, 2, 3, 4 and 8 weeks, then at 3, 6, 9, and 12 months then at least 
every 6 months for the first 5 years and then yearly thereafter for the next 10 years. 
The following analysis will be performed on these peripheral blood samples to monitor 
function, persistence and safety of transduced T-cells at time-points indicated in the 
study calendar. 
 
Quantitative real-time PCR for GPC3-CAR and iC9.NGFR.IL15 construct. 
 
Immunostaining for GPC3-CAR and NGFR (encoded in the iC9.NGFR.IL15) 
if CAR PCR is greater than 0.5% 
 
If detection of CAR T cells is greater than 0.5% and there is persistent CAR 
T-cell expansion 3 months after T-cell infusion, we will perform clonality 
studies (an extra 5 ml of blood). Further evaluations may be done based on 
clinical decisions. 
 
Immunophenotyping 
 
Serum or plasma cytokines/chemokines levels  
 

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All of these tests may not be performed on every specimen as a limited number of 
cells will be available. In general, priority will be given to PCR assays to detect infusion 
of T-cell lines. 
 
Serum from blood drawn for functional studies on week 0 and week 4 will be stored 
for measurement of human anti-mouse antibodies (HAMA) in the event of a suspected 
immunologic reaction. 
 
The lesser of either 60ml or 3ml/kg of patient weight of peripheral blood will be drawn 
on any one day for these assays. If a patient’s hemoglobin is less than 7.0 g/dl on a 
previous evaluation at any of the evaluation times, the amount of blood drawn for the 
evaluation will be reduced and may be obtained over more than one venipuncture.  
 
Percutaneous biopsy of at least 1 tumor lesion is a required component of this study, 
and will be performed at weeks 2-4 post treatment by an interventional radiologist or 
a surgeon. The biopsy costs will be covered by the study. The specimen will be tested 
for the presence of CAR T cells and their potential effect on the microenvironment. If 
patient is unable to complete the biopsy, he/she will still be allowed to remain on study 
for post-treatment follow up (Section 7.1). 
 
7.5 Safety Testing 
7.5.1 RCR testing by PCR: RCR testing will be performed Week 0, months 3, 6, 12. If 
all post treatment samples are negative during the first year the subsequent 
samples will be archived yearly for a total of 15 years. Aliquot of cells and serums 
will also be archived for use in future studies for RCR as required by the.  
 
7.5.2 The PI will designate the choice of assay depending on the mode of vector 
administration and the clinical application. If all post-treatment assays are 
negative during the first year, blood will be obtained yearly for archiving cells and 
serum as per current FDA recommendations for long-term follow up of gene 
therapy patients. If any post treatment samples are positive, further analysis of 
the RCR will be undertaken, in consultation with CBER.  
 
7.5.3 All positive RCR results will be reported immediately to the FDA as an adverse 
experience in the form of an IND safety report. 
 
7.5.4 The following will be obtained yearly for 15 years: Brief clinical history – with 
specific emphasis on clinical outcomes suggestive of retroviral disease such as 
cancer, neurologic disorders or other hematologic disorders. 
 
7.5.5 For any patients who die or develop a second neoplasm, significant hematologic 
or neurologic disorder during the trial, a biopsy sample of the neoplastic tissue 
or pertinent autopsy tissue will be assayed for RCR (if a sample can be obtained). 
 
7.5.6 Other Tissues: Patients will undergo tumor biopsy post-treatment while they are 
on study, and a sample of this will be used to assess the presence/absence of 
the retrovirally transduced cells (if a sample can be obtained). 
 
7.5.7 Long term follow up for patients receiving gene transfer product will be 
conducted as per SOP J02.51.XX. 
 
7.6 Determination of antitumor responses  

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Although response is not the primary endpoint of this trial, patients with measurable 
disease will be assessed by standard criteria. Evaluations of tumor size will be 
performed within 4 weeks of beginning treatment and 4 weeks (range 4 – 6 weeks) 
after 15.GPC3-CAR T-cell infusion. In addition, we will determine AFP as a tumor 
marker in the peripheral blood of patients pre-chemotherapy, pre-infusion, at 1, 2, and 
4 weeks post T-cell infusion in patients with AFP producing tumors. 
 
Definitions  
Response and progression will be evaluated in this study using the international 
criteria proposed by the Response Evaluation Criteria in Solid Tumors (RECIST) 
Committee [82, 83] and by the Immune-related Response Criteria [84, 85].  
 
RECIST criteria: Changes in only the largest diameter (unidimensional measurement) 
of the tumor lesions are used. Measurable lesions are defined as those that can be 
accurately measured in at least one dimension (unidirectional, longest diameter to be 
recorded) as >20 mm with conventional techniques (CT) or as >10 mm with spiral CT 
scan or MRI.  The investigator will identify up to 10 measurable lesions to be followed 
for response. Serial measurements are to be done with CT or MRI.  The same method 
of assessment is to be used to characterize each identified and reported lesion at 
baseline and during follow-up. 
 
Complete Response (CR):  Disappearance of all target lesions 
 
Partial Response (PR): At least a 30% decrease in the sum of the longest diameter 
(LD) of target lesions, taking as reference the baseline sum LD 
 
Progressive Disease (PD): At least a 20% increase in the sum of the LD of target 
lesions, taking as reference the smallest sum LD recorded since the treatment 
started or the appearance of one or more new lesions 
 
Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient 
increase to qualify for PD. 
 
For patients with metastatic disease, who have accessible tumor sites, we will request 
a biopsy to confirm the imaging results and analyze the tumor infiltrating T-cell 
populations. To determine the presence of 15.GPC3-CAR T cells, we will use Q-PCR 
for the transgene. 
 
Immune-related Response Criteria (IrRC): Pseudoprogression corresponds to 
increase of lesion size in response to therapy, which has been observed particular for 
melanoma, brain tumors after receiving immunotherapy and HCC after administration 
of sorafenib a multi-tyrosine-kinase inhibitor [84-87]. Since AFP serum levels correlate 
with disease status in HCC [88, 89], we will use AFP levels to differentiate between 
progression and pseudoprogression in patients with AFP-secreting HCC. For patients 
with AFP-negative HCC additional diagnostic tests will be performed in close 
consultations with the patient’s primary physician to differentiate between both 
possibilities. To fully describe the responses of patients treated on this protocol, we 
will report responses based on the recently described IrRC and all lesions at 
enrollment will be measured bidirectionally as target lesions: 
 
 
Response by decreasing in AFP levels: Only applicable to patients with elevated 
AFP at the time of enrollment. AFP levels will be measured at the time of starting 

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lymphodepletion (Day -4) and at the end of safety assessment (Week 4).  
 
Complete Response (CR):  Disappearance of all lesions. Nodes must regress to < 
10 mm short axis. No new lesions. Normalization of AFP levels. 
 
Partial Response (PR): ≥50% decrease in tumor burden compared with baseline*. At 
least half log decrease in AFP values. 
 
Progressive Disease (PD): ≥25% increase in tumor burden compared with baseline, 
nadir, or reset baseline*. New lesions added to the tumor burden. Greater than 10% 
increase in AFP levels. 
 
Stable Disease (SD): Neither partial response nor progressive disease. Less than 10% 
increase and less than half log decrease of AFP levels. 
 
*If an increase in tumor burden is observed at the first scheduled assessment, the 
baseline is reset to the value observed at the first assessment. 
 
Both RECIST and IrRC will be reported and IrRC will take priority for study related 
questions. AFP based responses will be reported separately. 
 
8.0 Statistical Consideration 
8.1 Clinical Trial Design 
The Primary objective of this phase I clinical study is to determine the safety of 
escalating doses of 15.GPC3-CAR T cells in patients with GPC3-positive solid tumors 
after lymphodepleting chemotherapy. Secondary objectives are to determine the 
maximum tolerated dose (MTD) of 15.GPC3-CAR T cells and the anti-tumor effects in 
these patients as per the roadmaps in Appendix IV.  
 
8.2 Dose Limiting Toxicity 
DLT will be defined as any of the following that may, after consultation with the FDA, 
be considered possibly, probably, or definitely related to the study cellular products. 
 
Any Grade 5 event 
 
Non-hematologic dose-limiting toxicity is any Grade 3 or Grade 4 non-
hematologic toxicity that fails to return to Grade 2 within 72 hours 
 
Grade 2 to 4 allergic reaction to CAR T cell infusion. 
 
Grade 4 hematologic toxicity that persists for 28 days or greater. 
 
Grade 3 and 4 expected reactions due to CRS and neurotoxicity are seen 
with the use of CAR-based immunotherapy. Grade 3 cytokine release 
syndrome (CRS) infusion reactions and neurologic toxicity will only be 
reported to the FDA if they fail to return to Grade 1 within 7 days. Grade 4 
CRS and neurologic toxicities will be reported to the FDA in an expedited 
fashion. 
 
8.3 The Dose Escalation Method and sample size 
Dose escalation is guided by the Bayesian optimal interval (BOIN) design[90, 91] in 
order to determine the maximum tolerated dose (MTD) of 15.GPC3-CAR T cells. Dose 
limiting toxicity (DLT) is defined in Section 8.2. Each treated patient will be followed for 
4 weeks post the 15.GPC3-CAR T cell infusion for the evaluation of DLT at their 
specified dose level. If no dose limiting toxicities are detected, a maximum of three 

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additional doses of 15.GPC3-CAR T cells may be given on the same dose level. 
Patients will need to meet treatment eligibility at the time of each start of 
lymphodepletion for reinfusions. 
 
The BOIN design has algorithmic escalation/de-escalation rules like the traditional 3+3 
design but allows for specification of various target toxicity rate, cohort size, and 
expanded accrual. With continued consistent toxicity monitoring, this design seeks to 
minimize incorrect escalation/de-escalation decision, thereby optimizing dose 
assignment for each patient.  For a broad range of conditions, the BOIN design 
performs as well as the model-based continual reassessment method (CRM) designs 
in selecting the MTD, but has a lower risk of overdosing or under dosing[92].  
In this trial, three patients will be initially enrolled to the lowest dose level. There will 
be a gap of 2 weeks between the first and second patient on each dose level. When 
these three patients are evaluable for DLT after their 4-week toxicity follow-up, we will 
assign the next cohort of three patients according to the decision rule of the BOIN 
design. The BOIN design is a type of ‘interval design’, in which dose choices are made 
according to pre-specified toxicity tolerance intervals. In general, with a target toxicity 
rate of 0.30 for this trial, we will escalate the dose if the observed toxicity rate at the 
current dose is <= 0.2365, or de-escalate the dose if the observed toxicity rate at the 
current dose is >=0.3585. If the observed toxicity rate is between 0.2365 and 0.3585, 
another cohort of patients will be treated at the current dose. For safety, no dose level 
will be skipped during dose escalation. Enrollment on the next dose level will wait until 
the completion of toxicity evaluation period following the 15.GPC3-CAR T cell infusion 
(4 weeks post infusion) for all treated patients enrolled at the current dose level. Dose 
escalation/de-escalation will continue until the recommendation is to assign the next 
cohort to a dose level that already has 6 patients treated. Dose escalation/de-
escalation is executed according to the rules in the table below:  
 
Dose escalation/de-escalation rule for the BOIN design 
Number of patients treated at the current dose 
3 
4
5 
6 
7 
8
9 
Escalate if # of DLT ≤ 
0 
0
1 
1 
1 
1
2 
Treat additional 3 patients on current dose if number of DLT = 
1 
1
NA
2 
2 
2
3 
De-escalate if # of DLT≥* 
2 
2
2 
3 
3 
3
4 
Eliminate dose level and all higher dose from further use if 
number of DLT≥ 
3 
3
4 
4 
5 
5
5 
*At the lowest dose level, if the recommendation is to de-escalate AND the dose level has not been eliminated 
from consideration, additional patients will be accrued at the same dose level; otherwise the trial will stop. 
“Eliminate” in the table means that we eliminate the current and any higher dose levels 
from the trial because they are overly toxic. Therefore, we will prevent treating any 
future patients at these toxic dose levels. If the current dose is DL1 and the rule 
indicates dose de-escalation, we will treat the next cohort at DL0. If the number of 
DLTs at DL0 reaches the elimination boundary, we will  suspend the trial for safety 
and report to FDA. If FDA approves and further dose de-escalation is necessary, half 
log decreases will be used on DL-1 and on DL-2.If the current dose is the highest dose 
and the rule indicates dose escalation, we will treat additional patients at the highest 
dose until 6 patients are already treated at the current (i.e. highest) dose.  
Once dose escalation is finished and all patients are evaluable for DLT, we will 

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determine the MTD based on isotonic regression as specified in Liu and Yuan[90] 
specifically, the dose for which the isotonic estimate of the toxicity rate is closest to the 
target toxicity rate. If there is no DLT that determine a MTD, the maximum dose level 
will be declared as the MTD. In case the lowest dose level is determined to be too 
toxic and the elimination boundary is reached, no MTD will be defined for the trial.  
Hence, upon completion of the dose escalation, we anticipate that 15 patients will be 
treated with a total 6 of patients accrued at the MTD level. Depending on observed 
toxicities, we expect that at most 24 patients will be infused in the phase-I trial.  
The first three patients on this study will be treated at DL1.  
 
DLTs will be evaluated from start of lymphodepleting chemotherapy to Day 28 (4 
weeks) post-CAR T-cell infusion. We anticipate that we will need to procure no more 
than 26 patients to enroll 15-24 evaluable patients. 
 
8.4. Stopping rules 
The enrollment to the study will be held if DLT0 is overly toxic that the number of DLTs 
at this dose level reaches the elimination boundary. This enrollment hold rule does not 
mandate automatic closure of the study. It serves a rule to trigger the oversight by 
FDA. The FDA will review all available data on any SAE where the relationship to CTL 
is suspected. Based on the evaluations, the FDA may recommend modifications to the 
study in order to ensure subject safety or recommend stopping the trial. 
 
8.5 Data Analysis 
8.5.1. Safety Analysis of Adverse Event Data 
All patients who received CAR T-cell infusions will be included in the safety 
analysis. All analyses will be performed separately for each dose level. Safety 
and toxicity outcomes will be summarized for the overall patient group and by 
dose levels. Adverse event data and corresponding toxicity grades four weeks 
after the T-cell infusion and during long-term follow-up will be summarized in 
the form of tables. Incidence tables will be generated to summarize incidence 
of patients reporting at least one episode of each specific adverse event, 
incidence of adverse events causing withdrawal and incidence of serious 
adverse events. The total number of episodes for each event reported 
(Frequency Table), the severity and attribution to study therapy of each 
episode reported (Severity Table and Attribution Table) will also be displayed. 
 
Listings of adverse events by patients will include the time to onset, the 
duration of each event, the severity of each event, and the relationship of the 
event to study therapy, whether it was a serious event, and whether it caused 
withdrawal. Safety data will be summarized.  
 
8.5.2. Clinical response data 
Response rates will be estimated as the percent of patients whose best 
response is either complete response or partial response (see Section 7.6) by 
combining the data from lesions recorded via imaging or by AFP for each 
patient pre- and post-infusion. These data will be evaluated separately by both 
RECIST and IrRC. To compare with historical data, a 95% confidence interval 
will be calculated for the response rate. 
 
The analysis of time to progression will include all patients who are considered 
evaluable for response. This will include patients that have had pre- and post-

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therapy imaging and that are off-treatment but continue to be enrolled on the 
study protocol and that have been previously evaluated by RECIST and/or IrRc 
criteria. The probability of time to progression at 6 months will be summarized. 
Time to progression will be estimated using the product-limit method of Kaplan 
and Meier, and their associations with biologic biomarkers will be studied by 
the Cox regression in an exploratory way. Time to progression is defined as 
the number of days from enrollment to: (1) disease progression; (2) death 
because of treatment complications; or (3) last patient follow-up whichever is 
first. Patients will be considered to have experienced a progression event if (1) 
or (2) occurs. Otherwise, the patient will be considered censored for time to 
progression. 
 
8.5.3. Analysis of Laboratory Data 
Descriptive statistics (means, standard deviations, medians and ranges) at 
pre-infusion and at 1, 2, and 4 weeks post T-cell infusion will be calculated.  
Laboratory data collected at 3-month intervals for the first year will also be 
summarized. Scatter plots depicting laboratory values at each time point for 
each patient will also be generated. In order to analyze changes in laboratory 
values, a shift table with Stuart-Maxwell chi-square analysis of the change in 
the normal range from pre-infusion to post infusion time points (using high, 
normal, low) will be performed.  When appropriate, these tables are collapsed 
and the McNemar’s test applied in place of the Stuart-Maxwell test. 
 
8.5.4. Analysis of Expansion and Persistence of T cells 
Despite the small patient numbers, a data-dense study will be generated due 
to the repeat measurements on proliferation, immune function, etc. on each 
patient. Descriptive statistics using mean ± SD, medians and ranges will be 
calculated to summarize repeated measurements of T-cell expansion 
(increasing T-cell levels in the peripheral blood over time), and persistence 
(infused T cells continuing at detectable levels over time) following infusion as 
measured by PCR. Plots of growth curves will be generated to graphically 
illustrate patterns of survival and expansion of T cells as well as immune 
response. The validity of the normality assumption on these data will be tested 
and appropriate transformations will be considered whenever indicated. 
 
To compare the persistence of 15.GPC3-CAR T cells among the four dose 
levels, we will calculate the area under the growth curves (AUC) over time for 
T-cell frequencies.  After appropriate log-transformation, ANOVA and t-test will 
be used to compare the AUCs with Bonferroni adjustment for multiple 
comparisons. Alternative strategies such as repeated measurement models 
will be applied to compare immunological and virological efficacy across dose 
levels. We will use a type-I error of 10% in the analysis because of the small 
sample size.  
  
The modeling strategies proposed here are amenable to these types of data 
but will however be considered exploratory and interpreted with caution due to 
limited study power and high type-I error. The results of this study will not be 
definitive but only exploratory. 
 
9.0 Off Study Criteria 
Patients are considered to be off study if they meet the following criteria:  
9.1 
Completion of study specified procedures  

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9.2 
Refusal of further study follow up by patient or legal guardian 
 
9.3 
Lost to follow up 
 
9.4 
Death 
 
  
Any questions regarding patients on this study should be addressed to Dr. Heczey at 
832- 824-4233 Or Dr. Steffin at 832-824-4745. 
 
10.0 
Records to be kept 
The criteria listed in the NCI Common Toxicity Criteria Scale will be used in grading toxicity. 
(Version 4.X located at http://ctep.cancer.gov) with the exception of CRS and neurological 
toxicities that are related to T-cell infusions. CRS and neurological toxicities will be graded 
according to Appendix VI. 
 
The CAGT research nurse/coordinator will maintain a database documenting on study 
information, adverse events, off study notification and death information. The dates and 
doses of therapy as well as clinical chemistries, hematologic parameters, the clinical 
status and occurrence of any adverse events and subsequent interventions are to be kept 
on all patients. 
 
Imaging reports 
Biopsy and surgical summaries 
Autopsy summaries, where appropriate 
Informed consent documents 
 
All required clinical evaluation records will be the responsibility of Dr. Heczey and Dr 
Steffin who will also be responsible for analysis of the clinical outcome and toxicity. 
 
11.0 
Reporting Requirements 
11.1 
Register all patients with Cell and Gene Therapy Research Coordinator. 
 
11.2 
Enter all patients by contacting Drs. Heczey or Steffin. The following data will be 
captured: 
 
Eligibility  
On study  
Concomitant medication 
Off study  
Flow sheets 
Adverse event  
CRS Adverse event (if applicable) 
Neurotoxicity Adverse event (if applicable) 
Death  
 
11.3. Drug Toxicity and/or Adverse Reactions 
11.3.1 Adverse events will be collected as per SOP J 02.05.XX and SOP J 
02.75.XX. 
Data 
on 
adverse 
experiences/toxicities 
regardless 
of 
seriousness, must be collected for documentation purposes only for 4 
weeks after the study drug/biologic. 

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11.3.2 Serious 
adverse 
events 
will 
be 
collected 
as 
per 
SOP 
J 
02.06.XX.   Reporting of serious adverse events related to gene transfer 
will continue for 15 years per SOP J 02.51.XX.  
11.3.3 Grade 3 cytokine release syndrome (CRS) infusion reactions and 
neurologic toxicity will be reported to the FDA if they fail to return to Grade 
1 within 7 days. Grade 4 CRS and neurologic toxicities will be reported to 
the FDA in an expedited fashion. 
 
12.0 Informed Consent 
All patents and/or their legal guardian must sign a document of informed consent 
consistent with local institutional and Federal guidelines stating that they are aware 
of the investigational nature of this protocol and of the possible side effects of 
treatment, including tumor biopsy (Section 7.5). Further, patients must be informed 
that no efficacy of this therapy is guaranteed, and that unforeseen toxicities may 
occur. Patients have the right to withdraw from this protocol at any time. No patient 
will be accepted for treatment without such a document signed by him or his legal 
guardian. Full confidentiality of patients and patient records will be provided 
according to institutional guidelines 
 
13.0 Clinical Trial Oversight and Monitoring 
This protocol will be conducted in accordance with the Cell and Gene Therapy 
Monitoring Plan on file with the FDA.   
 
This protocol will be monitored in accordance with the current Data Safety 
Monitoring Plan. of the Dan L Duncan Cancer Center at Baylor College of Medicine 
 
The conduct of this clinical trial will be evaluated in accordance with the Texas 
Children's Cancer Center and Center for Cell and Gene Therapy Quality 
Assurance Policy and Procedure Plan. 
 
 

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Appendix I – Karnofsky/Lansky Performance Score 
 
 
Performance Status Criteria 
 
Karnofsky and Lansky performance scores are intended to be multiples of 10 
Karnofsky (age ≥16) 
Lansky (age<16) 
Score 
Description 
Score 
Description 
100 
Normal, no complaints, no evidence 
of disease 
100 
Fully active, normal. 
90 
Able to carry on normal activity, 
minor signs or symptoms of 
disease 
90 
Minor restrictions in physically 
strenuous activity 
80 
Normal activity with effort; some 
signs or symptoms of disease. 
80 
Active, but tires more quickly 
70 
Cares for self, unable to carry on 
normal activity or do active work. 
70 
Both greater restriction of and 
less time spent in play activity 
60 
Required occasional assistance, 
but is able to care for most of 
his/her needs. 
60 
Up and around, but minimal 
active play; keeps busy with 
quieter activities. 
50 
Requires considerable assistance 
and frequent medical care. 
 
50 
Gets dressed, but lies around 
much of the day; no active 
play, able to participate in all 
quiet play and activities. 
40 
Disabled, requires special care and 
assistance. 
40 
Mostly in bed; participates in 
quiet activities 
30 
Severely disabled, hospitalization 
indicated. Death not imminent. 
30 
In bed; needs assistance even 
for quiet play. 
20 
Very sick, hospitalization indicated. 
Death not imminent. 
20 
Often sleeping; play entirely 
limited to very passive activities 
10 
Moribund, fatal processes 
progressing rapidly. 
10 
No play; does not get out of 
bed
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Appendix II - Barcelona Clinic Liver Cancer Stage 
 
The BCLC algorithm classifies HCC into five stages—based on the extent of disease, Child-Pugh 
score, and ECOG performance status—that enables prognostication and informs allocation of 
first-line treatment. If the proposed first-line treatment is contraindicated owing to a patient's 
clinical status, the treatment approach recommended for the subsequent disease stage should 
be considered. For example, a patient with early stage HCC (BCLC stage A) might benefit from 
TACE. Abbreviations: BCLC, Barcelona Clinic Liver Cancer (group); HCC, hepatocellular 
carcinoma; PS, performance status 
 
 
 

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Appendix III - Child-Pugh-Turcotte Score 
 
 
Measure 
1 point 
2 points 
3 points
units
Bilirubin (total) 
<2 
2-3 
>3 
mg/dl 
Serum albumin 
>35 
28-35 
<28 
g/L 
INR 
<1.7 
1.71-2.20 
>2.2 
No unit 
Ascites 
None 
Suppressed 
with medication 
Refractory 
No unit 
Hepatic 
encephalopathy
None 
Grade I-II (or 
suppressed 
with medication 
Grade III-IV 
(refractory) 
No unit 
Interpretation: The score employs five clinical measures of liver disease. Each measure is 
scored 1-3, with 3 indicating most severe derangement. Chronic liver disease is classified into 
Child-Pugh Turcotte class A (5-6), B (7-9), and C (10-15), employing the added score from above. 
Hepatic encephalopathy: Grade 1 - Trivial lack of awareness; euphoria or anxiety; shortened 
attention span; impaired performance of addition or subtraction. Grade 2 - Lethargy or apathy; 
minimal disorientation for time or place; subtle personality change; inappropriate behavior. Grade 
3 - Somnolence to semistupor, but responsive to verbal stimuli; confusion; gross disorientation. 
Grade 4 – Coma. 
 
 
 
 
 

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Appendix IV – Roadmaps 
 
 
Lymphodepletion with Cyclophosphamide and Fludarabine  
 
 
 
 
 
DAY 
SCHEDULED 
START 
DATE 
DATE 
STARTED
FLUDARABINE 
(30MG/M2/DOSE) 
CYCLOPHOSPHAMIDE
(500MG/M2/DOSE) 
CAR T-
CELL 
INFUSION 
-4 
 
 
_____ mg 
_____ mg 
 
-3 
 
 
_____ mg 
_____ mg 
 
-2 
 
 
_____ mg 
_____ mg 
 
-1 
 
 
 
 
 
0 to +2 
 
 
 
 
X 
 
Infusions should be given following hospital/pharmacy recommendations however at a minimum 
cyclophosphamide should be infused over 1 hour and fludarabine should be infused over 30 
minutes. Mesna, IV hydration and anti-emetics should also be provided following local institutional 
guidelines. T cell infusion will be given 2 to 4 days post completion of chemotherapy. For follow 
up post T-cell infusion please refer to patient-specific follow up schedule as outlined in Section 7 
of the complete protocol, 
 
 
 
 

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Appendix V - 
Potential Side Effects of Lymphodepletion Chemotherapy 
 
 
Risks and side effects related to CYCLOPHOSPHAMIDE include those which are: 
Likely
Less likely
Rare but serious
 
Loss of appetite 
 
Nausea 
 
Vomiting 
 
Fewer white blood cells in 
the blood. 
 
A low number of white 
blood cells may make it 
easier to get infections. 
 
Hair loss 
 
Decreased ability of the 
body to fight infection 
 
Absence or decrease in 
the number of sperm 
which may be temporary 
or permanent which may 
decrease the ability to 
have children 
 
Abnormal hormone 
function which may lower 
the level of salt in the 
blood 
 
Abdominal pain 
 
Diarrhea 
 
Fewer red blood cells and 
platelets in the blood 
 
A low number of red 
blood cells may make you 
feel tired and weak. 
 
A low number of platelets 
may cause you to bruise 
and bleed more easily. 
 
Bleeding and 
inflammation of the 
urinary bladder 
 
Absence or decrease 
monthly periods which 
may be temporary or 
permanent and which 
may decrease the ability 
to have children 
 
Temporary blurred vision 
 
Nasal stuffiness with IV 
infusions 
 
Skin rash 
 
Darkening of areas of the 
skin and finger nails 
 
Slow healing of wounds 
 
Infections 
 
Heart muscle damage 
which may occur with 
very high doses and 
which may be fatal. 
 
Abnormal heart rhythms 
 
Damage and scarring of 
lung tissue which may 
make you short of breath 
 
A new cancer or leukemia 
resulting from this 
treatment. 
 
Damage or scarring of 
urinary bladder tissue 
 
Severe allergic reaction 
which can be life 
threatening with 
shortness of breath, low 
blood pressure, rapid 
heart rate chills and fever  
 
Infertility which is the 
inability to have children 
 
 
 
 

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Risks and side effects related to FLUDARABINE (IV) include those which are: 
Likely 
Less likely 
Rare but serious 
 
Loss of appetite 
 
Nausea or the urge to 
vomit 
 
Decreased number of red 
blood cells, white blood 
cells 
(neutrophil/granulocyte), 
and/or platelets (a blood 
cell that helps clot blood) 
 
Muscle weakness of the 
whole body 
 
Cough 
 
Shortness of breath 
 
Fatigue or tiredness 
 
Fever 
 
Infection 
 
Pain 
 
Increased risk of unusual 
infections 
lasting 
more 
than 6 months 
 
Skin 
rash 
with 
the 
presence of macules (flat 
discolored 
area) 
and 
papules (raised bumps) 
 
Diarrhea 
 
Irritation or sores in the 
lining of the mouth, voice 
box, throat, and windpipe 
 
Vomiting 
 
Peripheral Neuropathy - 
Commonly 
known 
as 
"pins and needles," where 
part of the body (typically 
a foot or hand) begins to 
tingle 
and 
becomes 
numb, or "falls asleep" 
 
Blurred 
vision, 
double 
vision and/or loss of vision 
(blindness) 
 
Fear of light 
 
Inflammation of the lungs 
that may cause difficulty 
breathing and can be life-
threatening 
 
Chills  
 
An increase in the number 
of a type of white blood 
cell (called eosinophils) in 
the blood 
 
Agitation or restlessness 
 
Confusion 
 
Weakness or paralysis 
(loss of muscle function) 
caused by damage to 
peripheral nerves (those 
nerves outside of brain 
and spinal cord) 
 
Inflammation 
(swelling 
and 
redness) 
or 
degeneration 
of 
the 
peripheral nerves (those 
nerves outside of brain 
and spinal cord) causing 
numbness, 
tingling, 
burning  
 
Pain of the urinary tract  
 
Inflammation 
(swelling 
and 
redness) 
of 
the 
paranasal sinuses which 
 
Severe 
rash 
with 
redness, 
pain 
and/or 
blisters.  When pressure 
is applied to an area, the 
skin will detach from the 
lower layers. 
 
A 
rare 
autoimmune 
disorder called Evan’s 
syndrome in which the 
body makes antibodies 
that destroy the red blood 
cells, platelets and white 
blood cells  
 
Sudden damage to the 
red blood cells (hemolytic 
anemia) 
which 
could 
cause a rapid decrease in 
the number of red blood 
cells such that you may 
be tired, weak, feel short 
of 
breath, 
and 
may 
require 
a 
blood 
transfusion 
 
Coma and/or abnormal 
brain function 
 
Convulsion or seizure 
 
Blindness 
 
A 
rare 
disorder 
that 
damages 
the 
material 
that covers and protects 
nerves in the white matter 
of the brain. The disorder 
may cause headaches, 
loss 
of 
coordination, 
clumsiness, 
loss 
of 
language ability, memory 
loss, 
vision 
problems, 
and weakness of the legs 
and arms that gets worse. 
 
Inflammation 
(swelling 
and 
redness) 
of 
the 
bladder not due to urinary 
tract infection 
 
Severe potentially life-
threatening damage to 
the lungs which can lead 
to fluid in the lungs 
 
Bleeding from the lungs 
 
Kidney damage which 
may require dialysis 

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may or may not be a 
result of infection 
 
 
 
 
 

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Appendix VI - 
Grading of CRS and Neurological Toxicities 
CRS Grading Scale 
Grade 
Symptoms 
1 
 Symptoms are not life threatening and require symptomatic treatment only 
(e.g. fever, nausea, fatigue, headache, myalgia, malaise)  
2 
 Symptoms require and respond to moderate intervention  
 Oxygen requirement <40% or hypotension responsive to fluids or  
 low dose of one vasopressor or Grade 2 organ toxicity  
3 
 Symptoms require and respond to aggressive intervention  
 Oxygen requirement ≥ 40% or  
hypotension requiring high dose or multiple vasopressors or  
 Grade 3 organ toxicity or Grade 4 transaminitis  
4 
 Life-threatening symptoms 
 Requirements for ventilator support or  
Grade 4 organ toxicity (excluding transaminitis)  
5 
 Death 
 
 
 

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Neurological Toxicity Grading Scale 
Grade 
Symptoms 
1 
 Somnolence-mild drowsiness or sleepiness  
 Confusion-mild disorientation  
 Encephalopathy-mild symptoms  
 Dysphasia-not impairing ability to communicate  
2 
 Somnolence-moderate, limiting instrumental ADL*  
 Confusion-moderate disorientation, limiting instrumental ADL*  
 Encephalopathy-limiting instrumental ADL*  
 Dysphasia-moderate impairing ability to communicate spontaneously  
3 
 Somnolence – obtundation or stupor  
 Confusion-severe disorientation, limiting self-care-ADL*  
 Encephalopathy-limiting self- care ADL*  
 Dysphasia-severe receptive or expressive characteristics, impairing ability 
to read, write or communicate intelligibly  
4 
 Life-threatening Consequences of Grade 3 Toxicities 
 Urgent Intervention Indicated  
 Mechanical Ventilation  
5 
 Death 
 
*ADL: activities of daily living