Clinical trial NCT01740557
Conditions: Metastatic Melanoma Stage III Cutaneous Melanoma AJCC v7 Stage IV Cutaneous Melanoma AJCC v6 and v7
Sponsor: M.D. Anderson Cancer Center
Start date: 2015-01-28; Completion date: 2023-04-21
Enrollment: 10 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
This phase I/II trial studies how well genetically modified therapeutic autologous lymphocytes (patient's own white blood cells) followed by aldesleukin work in treating patients with stage III melanoma or melanoma that has spread to other places in the body (metastatic). Placing chemokine (C-X-C motif) receptor 2 (CXCR2) and nerve growth factor receptor (NGFR) into lymphocytes (white blood cells) may help the body build an immune response to kill melanoma cells. Aldesleukin may enhance this effect by stimulating white blood cells to kill more melanoma cells. Giving genetically modified therapeutic autologous lymphocytes together with aldesleukin may be a better treatment for melanoma.

Detailed description:
PRIMARY OBJECTIVES:

I. To assess the feasibility and safety of CXCR2 and NGFR transduced tumor-infiltrating lymphocytes (TIL) for treating metastatic malignant melanoma.

SECONDARY OBJECTIVES:

I. Determine whether CXCR2 transduction enhances the ability of TIL to migrate to melanoma tumors.

II. Determine the levels of CXCL1 and CXCL8 chemokines produced by melanoma tumors and assess whether this correlates with the tumor localization of CXCR2 transduced TIL.

III. Characterize the clinical response and correlate with migration of CXCR2 transduced TIL to the tumor and levels of CXCL1 and CXCL8 at the tumor site.

OUTLINE:

Patients receive cyclophosphamide intravenously (IV) over 2 hours on days -7 and -6, fludarabine phosphate IV daily over 15-30 minutes on days -5 to -1, and CXCR2-transduced autologous TIL and NGFR-transduced autologous TIL IV over up to 4 hours on day 0. Patients then receive high-dose aldesleukin IV over 15 minutes every 8-16 hours on days 1-5 (up to 15 doses) and 22-26 (up to 15 doses).

After completion of study treatment, patients are followed up at weeks 6 and 12, every 3 months for a year, and then annually for up to 15 years.

Arms: arm 1: Patients receive cyclophosphamide IV over 2 hours on days -7 and -6, fludarabine phosphate IV daily over 15-30 minutes on days -5 to -1, and CXCR2-transduced autologous TIL and NGFR-transduced autologous TIL IV over up to 4 hours on day 0. Patients then receive high-dose aldesleukin IV over 15 minutes every 8-16 hours on days 1-5 (up to 15 doses) and 22-26 (up to 15 doses).
Interventions: intervention 1: Aldesleukin intervention 2: CXCR2-transduced Autologous Tumor Infiltrating Lymphocytes intervention 3: Cyclophosphamide intervention 4: Fludarabine Phosphate intervention 5: Laboratory Biomarker Analysis intervention 6: NGFR-transduced Autologous T Lymphocytes intervention 7: Quality-of-Life Assessment
Intervention descriptions: intervention 1: Given IV intervention 2: Given IV intervention 3: Given IV intervention 4: Given IV intervention 5: Correlative studies intervention 6: Given IV intervention 7: Ancillary studies

Protocol documents:
Prot_SAP_000.pdf: 
Protocol 2009-0471 
September 6, 2017 
Page 1 of 39 
“A Pilot  Study of Lymphodepletion Plus Adoptive Cell Transfer with T-Cell Transduced 
with CXCR2 and NGFR Followed by High Dose Interleukin-2 in Patients with Metastatic 
Melanoma” 
PI:  Rodabe Amaria, MD 


Protocol 2009-0471 
September 6, 2017 
Page 2 of 39 
 
 
Abstract: 
 
This is a pilot study to assess the feasibility and safety of producing sufficient quantities of 
CXCR2 and NGFR transduced tumor infiltrating lymphocytes (TILs) for treatment of metastatic 
melanoma.  A total of 15 patients will be treated. Clinical outcomes that will be observed are 
tumor response by imaging , and possible grade 3 or worse toxicities not normally associated 
with lymphodepletion and high dose IL-2. T-cell based immunotherapy is a promising 
therapeutic modality for cancer. We and others have developed therapies to treat human 
melanomas by isolating and expanding autologous tumor infiltrating lymphocytes (TILs), 
followed by reinfusion into patients.  Although tumor regression can be dramatic in some 
patients, in other patients we see no clinical response.  We hypothesize that inefficient migration 
of T cells to tumors is one of the rate limiting steps in the generation of an effective anti-tumor 
response.  Therefore, we are investigating methods to improve T-cell trafficking to tumors 
through the introduction of specific receptor genes that can enhance migration to tumor sites. 
Melanomas specifically express the chemokines CXCL1 and CXCL8, which are thought to 
promote autocrine growth and angiogenesis.  However, we have found that tumor antigen-
specific T-cells fail to express the chemokine receptors specific for these ligands, including 
CXCR2.  In a strategy designed to direct T cells toward chemokines expressed by tumors, we 
have developed a system to genetically modify T cells using retroviral vectors encoding CXCR2. 
We have demonstrated that murine T-cells transduced to express CXCR2 exhibited enhanced 
trafficking to CXCL1 expressing tumors and this led to improved anti-tumor responses and 
survival.  We now propose to translate our findings from our mouse models to humans by 
treating melanoma patients with autologous tumor-reactive TILs genetically modified to express 
CXCR2.  To compare the ability of CXCR2 transduced TIL to migrate to tumor compared to 
control TIL, each patient will also receive TIL transduced with the truncated NGFR (nerve 
growth factor receptor) marker gene.   
 
Chemokine 
Cell type 
Mea
n SEM 
 
 
 
 
 
RC
C 
FB M71 M73 M72 
 
 
 
 
 
CCL5   (RANTES) 0.25 0.31 0.20 0.29 0.28 0.27 0.02 
 
 
 
 
 
CXCL1  (GRO-α) 0.43 1.12 1.59 0.96 0.50 1.04 0.22 
 
 
 
 
 
CXCL2  (GRO-β) 0.90 0.90 1.04 0.75 0.76 0.86 0.07 
 
 
 
 
 
CXCL8   (IL-8) 
0.47 0.47 0.50 0.50 0.44 0.48 0.01 
 
 
 
 
 
CXCL9   (Mig) 
1.11 1.11 1.20 1.22 1.04 1.14 0.04 
 
 
 
 
 
Chemokine 
Melanoma patient number 
Mea
n SEM 
P1 
P2 P3 P4 P5 
P6 
P7 
P8 
P9 P10 
CCL5   (RANTES) 0.26 0.10 0.24 0.20 0.42 0.57 0.28 0.32 0.24 0.22 0.29 0.04 
CXCL1  (GRO-α) 
0.69 
65.2
6 4.37 0.18 
12.9
8 
1.00 9.89 
23.2
1 
17.7
7 0.22 
13.5
6 5.97 
CXCL2  (GRO-β) 0.46 1.09 2.14 1.12 2.72 0.68 3.57 5.12 1.67 1.27 1.98 0.44 
CXCL8   (IL-8) 
0.54 0.68 0.98 0.36 2.53 0.53 1.03 3.62 0.54 0.38 1.12 0.33 


Protocol 2009-0471 
September 6, 2017 
Page 3 of 39 
 
1.0 
Bac
kground 
and Drug 
Informatio
n: Human 
melanoma 
tumors secrete CXCL1 and CXCL8  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
T-cell migration into tissues is largely dependent on expression of specific chemokines that 
signal through corresponding chemokine receptors expressed by lymphocytes. In order to 
identify chemokines that are expressed constitutively by tumor cells, we first investigated 
chemokine expression in human melanomas freshly isolated  
from patients. Fine needle aspirates of melanomas from ten patients were obtained and 
subjected to comparative cDNA microarray analysis. For controls, we prepared cDNA from 
a renal carcinoma (RCC) cell line, cultured normal fibroblasts and cultured melanocytes. 
Table 1 shows ratios of the signal intensity in the various normal and tumor tissues 
compared to PBMC. Differences are considered significant (in bold, Table 1) when ratios 
are greater than 2 or less than 0.5 and varied from normal tissues and renal cell carcinoma 
(RCC) by a factor of greater than 2 or less than 0.5.  
 
Human melanoma tumors secrete CXCL1 and CXCL8 T-cell migration into tissues is largely 
dependent on expression of specific chemokines that signal through corresponding chemokine 
receptors expressed by lymphocytes. In order to identify chemokines that are expressed 
constitutively by tumor cells, we first investigated chemokine expression in human melanomas 
freshly isolated  
from patients. Fine needle aspirates of melanomas from ten patients were obtained and 
subjected to comparative cDNA microarray analysis. For controls, we prepared cDNA from a 
CXCL9   (Mig) 
1.30 5.27 
15.6
9 1.70 4.89 5.83 4.69 1.45 1.32 2.75 4.49 1.30 
Table 1.  Chemokine expression analysis of human cancers and normal cells. 
(RCC: human renal cell carcinoma; FB: fibroblasts; Mxx: normal melanocytes;P1-
P10: fine needle aspirates from melanoma patient tumors.) 


Protocol 2009-0471 
September 6, 2017 
Page 4 of 39 
 
renal carcinoma (RCC) cell line, cultured normal 
fibroblasts and cultured melanocytes. Table 1 
shows ratios of the signal intensity in the various 
normal and tumor tissues compared to PBMC. 
Differences are considered significant (in bold, 
Table 1) when ratios are greater than 2 or less than 
0.5 and varied from normal tissues and renal cell carcinoma (RCC) by a factor of greater than 2 
or less than 0.5.  
CXCL1 expression, in particular, stood out amongst the other chemokines as being markedly 
upregulated with a high relative signal intensity in a significant proportion of melanomas (6 of 
10) when compared to levels in normal tissues and RCC (mean value 13.56 + 5.97 versus 1.04 
+ 0.22). CXCL9 was also upregulated in a majority of melanomas tested, although the mean 
value of 4.49 + 1.30 was lower than that for CXCL1. The analysis of cDNA from fresh 
melanoma samples suggests that CXCL1 is expressed at the tumor site in vivo.  To confirm that 
melanoma cells, as reported, could produce CXCL1 protein, we next evaluated the production 
of CXCL1 by several human melanoma cell lines. For these purposes, cell lines were plated at 
5x105 cells/ml and, 24 hours later, supernatants were harvested and CXCL1 protein was 
detected using a standard ELISA. With these 
studies, we confirmed that most human 
melanoma cell lines also constitutively produced 
significant amounts of CXCL1. (Table 
2)(Kershaw, Wang et al. 2002).   
CXCL1 binds to the chemokine receptor CXCR2.  
In addition, CXCL8 (IL-8) can bind to CXCR2.  
Because CXCL8 (IL-8) was also seen in some of 
the tumor samples and has been reported to be 
highly expressed in some melanoma cells, we 
evaluated a panel of melanoma cell lines for their 
ability to produce IL-8 (Table 3), and 6 of 8 
melanoma lines produced significant levels of CXCL8.  Moreover, we also analyzed paraffin-
embedded melanoma tissue samples from patients 
for the expression of CXCL1 and CXCL8 by 
immunohistochemistry (Figure 1). Both chemokines 
were found to be present in the majority of lymph 
node and metastatic lesions analyzed, consistent 
with published findings (Richmond, Lawson et al. 
1985; Payne and Cornelius 2002). Thus, the 
majority of human melanomas produce ligands for 
CXCR2, providing a strong rationale for improving tumor homing of T cells through ectopic 
expression of this chemokine receptor. 
 
Table 2.  CXCL1 production by human 
tumor lines 
Cell line 
Human CXCL1 
(pg/ml) 
397 melanoma 
606 
624 melanoma 
1652 
A375 melanoma 
2038 
888 melanoma 
403 
1300 melanoma 
648 
SK23 melanoma 
2801 
SW480 colon CA 
165 
Colo 320 colon CA 
0 
Table 3. IL-8 Production by
human melanoma tumor lines
Tumor Line
IL-8 pg/ml
Mel 624
121
Mel 888
2943
Mel 888-A2
4754
Mel 938-A2
845
A375
9692
WM239
1889
WM2264
684
Mel 526
3080
Medium
0
Figure 1. Human metastatic melanoma 
tumors express ligands for CXCR2. 
Paraffin-embedded, melanoma lymph node 
metastasis analyzed by immunohisto-
chemical staining for (A) chemokine CXCL8 
(B) chemokine CXCL1. Magnification, 40X. 
 


Protocol 2009-0471 
September 6, 2017 
Page 5 of 39 
 
Murine tumors produce CXCL1 
As reported, the expression of CXCL1 appears to be restricted to tumor and inflammatory 
tissues.  This was further confirmed for murine tissues by analysis of the Gene Expression 
Omnibus (GEO) database and the SAGEmap database (both available at 
http://www.ncbi.nlm.nih.gov/).  Evaluation of the murine transcriptome in the GEO database, 
which included the evaluation of transcript levels from 122 samples representing 61 normal 
murine tissues using 31373 distinct probes, revealed that murine CXCL1 RNA levels were not 
significantly higher in any tissue compared to that seen in lung, liver, and kidney.  In addition, in 
our own studies, CXCL1 was undetectable in lysates of minced kidney or lung from normal mice 
(not shown), as determined by ELISA. 
 
We next assessed whether murine tumor cells could produce CXCL1. Fresh tumor explants 
derived from 24JK sarcoma, cultured overnight in medium, produced large amounts of CXCL1, 
as determined by ELISA (not shown).  In addition, cultured MC38 tumor cells expressed high 
levels of CXCL1 (Figure 4B).  While B16 tumor explants expressed CXCL1, cultured B16 tumor 
cells did not.  Therefore, the CXCL1 gene was introduced into B16 for some of our models, as 
discussed below. 
 
Activated, tumor-reactive T cells do not express CXCR2 
 
We next analyzed the chemokine receptors expressed by tumor-reactive human and murine T 
cells. Using an RNAse protection assay, cultured human T cells appeared to express the 
chemokine receptors CCR1, CCR2, CCR4, CCR5, CXCR3, and CXCR4 (data not shown; 
(Kershaw, Wang et al. 2002)). Notably, human T cells did not express the CXCL1-binding 
chemokine receptor, CXCR2. To confirm this pattern of receptor expression, we evaluated the 
ability of tumor reactive human T cells to respond functionally by measuring calcium flux in 
response to the exogenous addition of various chemokines. We found that the T cells could 
generate a calcium flux in response to the chemokines CCL5 (a ligand for CCR1, 3, and 5), 
CCL2 (ligand for CCR2), and CXCL12 (ligand for CXCR4), but not CXCL1, results that were 
consistent with the expression pattern observed using the RNAse protection assay.  
 
In a similar fashion, we determined whether cultured murine T cells could naturally respond to 
CXCL1.  As with human cells, murine T cells did not naturally migrate towards CXCL1, or exhibit 
calcium flux when exposed to CXCL1. Consistent with this, in an RNAse protection assay, 
murine alloreactive and peptide stimulated T cells were found to express CCR1, CCR2, and 
CCR5, but not CXCR2 (data not shown). 


Protocol 2009-0471 
September 6, 2017 
Page 6 of 39 
 
To test whether expression of CXCR2 would enhance the migration of tumor antigen-specific T 
cells to the tumor and provide better anti-tumor responses, we established a sensitive imaging 
system to track the migration of T cells in a murine tumor model using standard ecotropic 
packaging lines and moloney derived retroviral vectors.  We combined gene transfer with 
imaging vectors and CXCR2 to study the migration and anti-tumor effects of these cells in vivo. 
Visualizing T cell migration in vivo 
To evaluate the effects of CXCR2 gene transfer on T-cell migration in a preclinical study, a 
sensitive and efficient system to trace tumor-specific T cells in vivo was required. Many methods 
have been established to monitor cells in vivo, including bioluminescence imaging (BLI). 
Compared with other technologies, BLI has several advantages that made it a powerful tool for 
the preclinical studies. First, BLI is a 
non-invasive imaging method. It allows 
researchers to continuously monitor the 
same animal for the duration of the 
experiment.  Second, BLI does not 
require 
radioisotopes 
and 
is 
inexpensive. 
Third, 
multiple 
experimental animals can be imaged by 
BLI at the same time, thus saving 
valuable time and resources. 
However, in murine adoptive T-cell 
therapy 
studies, 
the 
number 
of 
transferred T cells migrating to the 
tumor site is typically only a few 
hundred, which was below the level of 
detection 
of 
most 
bioluminescence 
technologies. Therefore, in order to 
monitor the small T cell population in 
vivo, our first priority was to develop a 
sensitive in vivo imaging system based 
on bioluminescence technology. To 
improve the expression of current virus 
based firefly luciferase constructs in 
mouse T cells, we optimized the codon 
usage to the highest frequency of Mus 
musculus, removed the cryptic splice 
sites and added the woodchuck hepatitis virus pre-element to augment export of the viral mRNA 
into the cytosol. These modifications resulted in >80% transduction efficiency in mouse T cells 
and high levels of expression (Figure 2A). After sorting the firefly luciferase transduced T cells, 
we observed a 100 fold increase in signal intensity of T-cells transduced with our enhanced 
luciferase construct compared to those transduced with the standard luciferase construct 
(Figure 2B). By using this modified firefly luciferase construct, as few as three transduced T 
cells could be visualized after subcutaneous injection into the mice (Figure 2C).  
Establishing a new murine tumor model to monitor the migration of transferred T cells in 
vivo  
 
Following optimization of firefly luciferase expression in primary murine T-cells(Rabinovich, Ye 
et al. 2008), we next focused on studying T-cell migration in murine tumor models.  However, 
Figure 2. Comparison of coreporter expression and 
luminescent activity of T cells expressing standard 
versus enhanced retroviral firefly luciferase (A) MurineT 
cells were transduced with the standard firefly luciferase 
construct v-ffLuc (left) or  enhanced firefly luciferase 
construct v-effLuc (Right). The expression of Thy1.1, the 
reporter gene of both luciferase constructs, was assessed 3 
days after transduction by flow cytometry. (B) Murine T cells 
transduced with v-ffLuc or v-effLuc (as indicated) were 
injected s.c. at the indicated numbers into B6 Albino mice, 
and photon emissions were measured on an IVIS 200. (C) 
T cells transduced with v-effLuc were injected s.c. at the 
level of 30, 10, or 3 cells as indicated. 
 


Protocol 2009-0471 
September 6, 2017 
Page 7 of 39 
 
we found that BLI could not be used optimally with the B16 melanoma tumor model, due to the 
blockade of the luciferase signal by the dark skin and fur of the C57BL/6 mice and the black 
pigment of the B16 tumor.  Therefore, we needed to develop a new murine tumor model to 
monitor tumor-specific T cell migration in vivo by BLI. To reduce the absorption of the luciferase 
signal by the dark skin and fur of C57BL/6 mice, the C57BL/6 mouse strain was replaced with 
an albino C57BL/6 mouse strain, which shares the same genetic background with C57BL/6 
mice, but contains a spontaneous mutation of the tyrosinase gene. Additionally, the melanoma 
tumor antigen, gp100, was cloned into a lentiviral vector containing mIL-4R as a reporter gene, 
and transduced into a colon carcinoma derived from C57BL/6 mice, MC38. A stable gp100-
expressing MC38 tumor cell line (MC38/gp100) was generated by sorting transduced tumor 
cells based on the expression of the mIL-4R reporter. We also transduced Pmel T cells with 
retroviral supernatants containing the mCXCR2 and luciferase constructs (Figure 3A). We 
routinely obtained more than 90% transduction efficiency (Figure 3B). Furthermore, 
MC38/gp100 tumor cells could activate Pmel T cells, which express a transgenic TCR 
recognizing an H-2Db restricted gp100 peptide, to exhibit cytolytic activity (Figure 3C) and 
produce effector cytokines such as IFN-γ (Figure 3D). Because MC38/gp100 tumor cells, unlike 
B16 tumor cells, express consistent levels of MHC class I molecules, the activation of Pmel T 
cells by MC38/gp100 does not require IFN-γ pretreatment of tumor to induce class I expression. 
In this respect, the MC38/gp100 tumor cells are more similar to human melanoma than B16, 
since IFN gamma is not required by most human lines for recognition by CD8+ antigen-specific 
T-cells.  The luciferase signal from the transferred T cells in the MC38/gp100 tumor site, could 
be appropriately detected (data not shown).  Therefore, the MC38/gp100 tumor model 
combined with our modified BLI system is a powerful and sensitive tool to study tumor-specific T 
cell migration.    


Protocol 2009-0471 
September 6, 2017 
Page 8 of 39 
 
CXCR2-transduced Pmel T cells migrate towards CXCL1 gradients in vitro 
 
To test whether the exogenous expression of CXCR2 in Pmel T cells could enable these cells to 
respond to CXCL1, Pmel T cells were transduced with either a luciferase construct, OFL-GFP, 
or OFL-GFP and CXCR2. Sorted transduced T cells were used to perform a migration assay 
using a transwell system.  T cell migration to the lower well was measured in vitro by measuring 
luminescent activity after addition of D-luciferin. Higher levels of luciferase intensity were 
observed from Pmel T cells expressing both CXCR2 and luciferase than from Pmel T cells 
expressing luciferase alone. Furthermore,  increased migration of CXCR2-transduced Pmel T 
cells was dependent on CXCL1 as the migration of CXCR2-expressing Pmel T cells was similar 
with control T cells in the absence of CXCL1 (Figure 4A). Moreover, we confirmed the 
migratory ability of CXCR2-expressing Pmel T cells towards CXCL1 produced by murine tumor 
cells. Our results indicated that the concentration of mCXCL1 in overnight cultured medium from 
2x106 MC38 or MC38/gp100 tumor cells was higher than 10ng/ml. Production of mCXCL1 was 
comparable between MC38 and MC38/gp100 tumor cells (Figure 4B). Although mCXCL1 was 
present in B16 tumor explants (data not shown), mCXCL1 produced by in vitro cultured B16 
tumor cells was undetectable by ELISA. Therefore, we also established a stable CXCL1-
expressing B16 tumor cell line (Figure 4B). By using conditioned medium from these tumor 
cells, we investigated the migratory ability of CXCR2-expressing Pmel T cells in vitro.  
Figure 3. Generation and functional characteristics of CXCR2 expressing Pmel T cells. (A) Schematic representation of viral 
vector containing mCXCR2. Long terminal repeat (LTR) from the murine stem cell PCMV virus drives high-level, constitutive 
expression of mCXCR2 in Pmel T cells. (B) FACS analysis of transduced Pmel T cells. Pmel T cells were transduced with modified 
luciferase (OFL) or OFL and mCXCR2. Cells were sorted based on the expression of GFP or CXCR2. Sorted cells were cultured 
for 3 days and used for transfer therapy. Before transfer, the purity of transferred cells was evaluated by flow cytometry. More than 
90% of transferred cells are either GFP positive or GFP and CXCR2 double positive, as expected. (C) Cytotoxic reactivity of 
CXCR2 expressing Pmel T cell against MC38/gp100 tumor. MC38 or MC38/gp100 cells were labeled with Cr51. After labeling, 5000 
tumor cells were cocultured with either CXCR2 expressing Pmel T cells or OFL-expressing Pmel T cells for 6 hours. The 
supernatants were monitored for gamma-activity. (D) IFN-γ secretion by CXCR2 expressing Pmel T cells in response MC38/gp100 
tumor. Similar cell lysis rate and similar amount of IFN-gamma secretion were found between CXCR2-expressing Pmel T cells and 
control Pmel T cell in response to MC38/gp100 tumor.  
C. 


Protocol 2009-0471 
September 6, 2017 
Page 9 of 39 
 
Conditioned medium from either CXCL1-expressing B16 or MC38 tumor cells induced 
enhanced migration of CXCR2 expressing Pmel cells, but not the migration of control T cells 
(Figure 4C). Additionally, the number of migrating T-cells correlated with the concentration of 
CXCL1 in the cell culture supernatants. By contrast, the cell culture medium from B16 tumor 
cells didn’t alter the migration of CXCR2-expressing T cells when compared to medium alone, 
confirming that the migration of CXCR2-expressing T cells was dependent on CXCL1. Taken 
together, these results suggest that the introduction of the CXCR2 gene into Pmel T cells 
enhanced the migratory ability of those T cells towards a CXCL1 gradient in vitro in a dose-
dependent manner, but did not alter the activation of T cells upon antigenic stimulation, in the 
presence or absence of CXCL1 (data now shown).  
 
CXCR2-expressing Pmel T cells possess an enhanced ability to traffic to tumor in vivo 
To analyze the in vivo migratory 
pattern of CXCR2-expressing Pmel 
T cells in tumor-bearing mice, we 
took advantage of our newly 
established MC38/gp100 tumor 
model and modified BLI system. 
1x106 Pmel T cells expressing OFL-
GFP alone or OFL-GFP and CXCR2 
were intravenously injected into 
C57BL/6 albino mice harboring 
MC38/gp100 tumors (average tumor 
size=20 mm2; All the mice receiving 
Pmel T cells 
also received DC vaccination and 
hIL-2 i.p injection as previously 
described (Lou, Wang et al. 2004). 
Luciferase signals from the 
adoptively transferred Pmel T cells were 
monitored on a daily basis. Although the 
transferred Pmel T cells were detected by 
BLI in tumor sites as early as the second 
day after adoptive T cell transfer, most of 
the transferred Pmel T cells were initially 
found in the lung and liver which is 
consistent with adoptive T-cell transfer in 
humans (Figure 5A). Six days after T cell 
transfer, the majority of the transferred 
Pmel T cells were localized at the tumor site. The distribution pattern of transferred tumor-
specific T cells obtained by our BLI technique is similar to the previously reported single-photon 
emission computed tomography (CT)-CT fusion imaging method (Pittet, Grimm et al. 2007). 
Therefore, we used the luciferase results from mice on day six after T cell transfer as 
representative data to characterize the tumor migration of transferred Pmel T cells. As shown in 
Figure 5B, mice receiving CXCR2-expressing T cells displayed a stronger luciferase signal at 
the tumor site than mice receiving T cells expressing luciferase alone on day 6 after T cell 
Figure 4: Enhanced migration ability of CXCR2 
expressing T cells towards CXCL1 gradients (A) 
Migration of CXCR2 expressing T cells towards 
CXCL1 gradients provided by recombinant CXCL1. 
(B) CXCL1 production by murine tumor lines. (C) 
The migration of CXCR2 expressing T cells 
towards CXCL1 gradients provided by murine 
tumor cells. 
 


Protocol 2009-0471 
September 6, 2017 
Page 10 of 39 
 
transfer. The average luciferase intensity of all mice (N=9) from each group was quantified by 
live imaging software (Figure 5C). By contrast, in peripheral blood, the percentage of  Thy1.1+  
cells, a cell surface marker for transferred Pmel T cells,  among total CD8+ T cells in the 
peripheral blood remained similar between mice receiving CXCR2-expressing Pmel T cells or 
control Pmel T cells (Figure 5D). These results suggest that transduction of T-cells with the 
CXCR2 gene resulted in their preferential accumulation to tumor sites expressing CXCL1. 
Introduction of CXCR2 into human T cells induces calcium flux in response to CXCR2 
ligands 
 
Having demonstrated that CXCR2 transduced murine T-cells were more effective in vivo in 
murine tumor models, we next tested this system with human T-cells in vitro in preparation for 
bringing this concept to the clinic.  We have attempted to utilize knowledge of the expression 
patterns of chemokines and chemokine receptors in tumors and T cells to design strategies to 
enhance T-cell migration to tumors. Our initial approaches have taken advantage of our 
experience in modifying T cells by retroviral gene transduction (Hwu, Shafer et al. 1993; Hwu, 
Yannelli et al. 1993; Hwu and Rosenberg 1994; Treisman, Hwu et al. 1994; Treisman, Hwu et 
al. 1995; Lam, Reeves et al. 1996; Kershaw, Wang et al. 2002; Royal, Kershaw et al. 2002). 
Our strategy thus far has involved 
transduction of human lymphocytes 
with specific chemokine receptor 
genes in order to allow them to 
recognize and respond to the 
chemokines specifically produced by 
tumors. In order to confer 
responsiveness to CXCL1, we cloned 
the human CXCR2 receptor gene into 
a retroviral vector and used it to 
transduce human T cells. Flow 
cytometric analysis demonstrated 
that T cells expressed surface 
CXCR2 following retroviral 
transduction (data not shown).  We 
next evaluated the function of the 
CXCR2 receptor in transduced T 
lymphocytes by measuring calcium 
flux in response to the exogenous 
addition of CXCR2 ligands. T cells 
were transduced with either the 
CXCR2-expressing or control 
retroviral vector were incubated with human 
CXCL8. 
 
Addition of the chemokine CXCL12, which binds 
to the chemokine receptor CXCR4 constitutively  
expressed by mature T cells, was included as a 
positive control. CXCR2-transduced T cells 
responded to CXCL8 and human or mouse 
CXCL1 with a rapid increase in intracellular 
calcium (Figures 6a, 6b, and 6c). The 
Figure 5: Enhanced migration ability of CXCR2 
expressing Pmel T cells to the  tumor sites (A) In 
vivo trafficking of OFL expressing Pmel T cells. 
Imaging was performed at indicated time points after T 
cell transfer. (B) Increased accumulation of CXCR2 
expressing Pmel T cells at the tumor site. Imaging was 
performed at day six after T cell transfer. Data shown 
were from representative mice. (C) Quantitative 
imagng analysis of transferred T cells in tumor bearing 
mice. The intensities of the luciferase signal at the 
tumor sites in all tumor-bearing mice were quantified 
using live imaging software. (D) FACS analysis of the 
percentage of Thy1.1+ in CD8+ cells in peripheral blood 
on day six after T cell transfer. While no differences 
were seen in peripheral blood (D), more CXCR2 
CXCR2 
transduced 
GFP 
transduced 
mCXCL1 
mCXCL1 
hCXCL1 
hCXCL1 


Protocol 2009-0471 
September 6, 2017 
Page 11 of 39 
 
requirement for CXCR2 was demonstrated by the lack of responsiveness to CXCR2 ligands of 
control vector only-transduced T cells (Figures 6e, 6f, and 6g). As expected, both chemokine 
receptor and vector-only transduced T cells were able to respond to CXCL12 (Figures 6d and 
6h). These results clearly demonstrated that introduced human CXCR2 could function in 
transduced primary human T cells and cause mobilization of intracellular calcium in response to 
binding of its chemokine ligands. 
 
CXCR2-transduced human T cells migrate towards CXCL1 produced by tumor cells 
At the time of your death, no matter the cause, we may request permission for an autopsy in 
order to obtain vital information concerning the safety of this experimental therapy approach.  
Please discuss this request with your family to inform them of your wishes. At the time of your 
death, no matter the cause, we may request permission for an autopsy in order to obtain vital 
information concerning the safety of this experimental therapy approach.  Please discuss this 
request with your family to inform them of your wishes. At the time of your death, no matter the 
cause, we may request permission for an autopsy in order to obtain vital information concerning 
the safety of this experimental therapy approach.  Please discuss this request with your family 
to inform them of your wishes. 
We next tested CXCR2-transduced human T cells 
for their ability to migrate specifically toward CXCL1, 
as well as toward CXCL1-producing tumor cell 
supernatants (Figure 7).  Experiments were 
performed using 3 μm pore size transwell plates. As 
shown in Figure 7a, CXCR2-transduced T cells 
migrated 
through 
transwel
l pores 
in 
greater 
numbers 
when 
CXCL1 
was 
present 
in the lower 
chamber 
compared to 
the migration 
in the 
absence of 
CXCL1. This 
specific 
migration was 
evident at 
concentrations of CXCL1 as low as 1 nM. The 
increased migration was dependent upon CXCR2 
expression, as control vector-transduced T cells 
migrated in lower numbers irrespective of the 
CXCL1 concentration (nM)  
CXCL1
Figure 7. Chemotaxis of CXCR2-expressing T cells. 
T cells were assayed for chemotaxis by determining 
their ability to migrate from an upper chamber, through a 
3 μm pore membrane towards a source of chemokine in 
a lower chamber in 24-well plates. (a) CXCR2-
transduced T cells migrated towards CXCL1 in greater 
numbers than control vector-transduced T cells. (b) 
When CXCL1 was included in the upper and lower 
chambers no increase in migration was found, thereby 
indicating that the response to CXCL1 was chemotactic 
in nature and not solely chemokinetic. (c) CXCR2-
transduced T cells migrated towards cell culture 
supernatant derived from the CXCL1- secreting tumor 
cell lines SK23 and HCT/GRO in greater numbers than 
towards supernatants from CXCL1 negative cells or 
media alone. The requirement for CXCR2 for the 
observed effect was evident from the comparatively 
lower migration levels of non-transduced or control 
vector-transduced T cells.  
A
B
Figure 6.  Calcium flux in CXCR2-transduced T 
cells. Human T cells were loaded with the 
fluorescent calcium indicator Fura-2 and assayed 
for calcium flux following the addition of the 
indicated 
chemokines 
(arrows). 
CXCR2-
transduced T cells responded to the CXCR2 
ligands CXCL8, in addition to human and murine 
CXCL1 (a-c), to a similar degree as to the positive 
control 
CXCL12 
(d), 
the 
ligand 
for 
the 
constitutively expressed CXCR4. Control GFP 
vector-transduced T cells did not respond to the 
CXCR2 ligands (e-g), but did respond to the 
positive control CXCL12 (h). 


Protocol 2009-0471 
September 6, 2017 
Page 12 of 39 
 
presence or absence of CXCL1. Migration was demonstrated to be a result of increased specific 
chemotaxis rather than increased chemokinesis since inclusion of CXCL1 in both the upper and 
lower chambers did not result in increased migration of CXCR2-transduced T cells through 
transwells (Figure 7b). Chemotaxis was G-protein-dependent, as it was inhibited by pertussis 
toxin in a dose-dependent manner (data not shown).    
In order to determine whether CXCR2-transduced T cells could respond to CXCL1 derived from 
tumor cells, transwell migration assays were also performed using overnight culture 
supernatants from tumor cell lines as the source of chemokine in the lower chamber. Significant 
numbers of CXCR2-transduced T cells migrated towards supernatant derived from SK23 
melanoma and from a CXCL1-transduced HCT116 colon carcinoma cell line (HCT/GRO), 
approximately 3-fold more than towards media alone (Figure 7c). An approximately 2-fold 
increase in migration was observed in response to CXCL1-expressing tumor cell supernatant 
compared to the CXCL1-negative cell supernatants derived from the colon carcinoma 
COLO205, the ovarian carcinoma line IGROV-1 and non-transduced HCT116. 
 
Interestingly, migration of CXCR2-transduced T cells was greater towards CXCL1-negative 
supernatant derived from ovarian and colon carcinoma cell lines than towards media alone. This 
latter observation may be due to the presence of CXCL8 found in supernatants from these cell 
lines (data not shown), since CXCR2 binds to both CXCL1 and CXCL8. CXCR2 was 
responsible for the increased T-cell migration observed, as demonstrated by the much lower 
level of migration of control vector or non-transduced T cells, irrespective of the source of 
supernatant (Figure 7c). These studies clearly demonstrate the feasibility of altering the 
migratory phenotype of T cells with the introduction of the chemokine receptor CXCR2 
(Kershaw, Wang et al. 2002).   
 
We also demonstrate that tumor infiltrating lymphocytes (TILs) can be readily transduced with 
human CXCR2 expressing vector (Figure 8A). Furthermore,  
transduction of CXCR2 confers enhanced migration capabilities in response to CXCL1 graduent 
compared to NGFR transduced cells (Figure 8B). 
 
 
 
 
 


Protocol 2009-0471 
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Page 13 of 39 
 
Transfer of CXCR2-expressing Pmel T cells further improves T-cell mediated antitumor 
immune responsesSince CXCR2 expression results in increased accumulation of tumor 
antigen specific T cells at the tumor site, we next assessed whether this increased accumulation 
would result in better antitumor immune responses. Seven days after tumor challenge, mice 
were treated with adoptive T-cell transfer (combined with TBI, gp100 peptide pulsed DC 
vaccination and hIL-2 injection as per our routine protocol). The size of MC38/gp100 tumor was 
measured every other day. Compared with untreated mice, transfer of 1x106 Pmel T cells 
resulted in a significant decrease of MC38/gp100 tumor growth, which is consistent with our 
previous studies using the B16 tumor model (Figure 9A).  More importantly, tumor growth in 
mice receiving CXCR2-expressing Pmel T cells was significantly slower than in mice receiving 
control (luciferase only) Pmel T cells (Figure 9A). In another related experiment, tumor growth in 
mice transferred with 1x106 CXCR2-expressing Pmel T cells was comparable to that of mice 
received 5x106 control Pmel T cells (data not shown), suggesting that fewer T-cells need to be 
transferred if migration to the tumor is more efficient. Moreover, the introduction of the CXCR2 
gene into adoptively transferred Pmel T cells also led to a significant increase in the survival of 
MC38/gp100-bearing mice (Figure 9B). Although CXCR2-expressing Pmel T cells did not 
confer better protection in B16 tumor bearing mice than control Pmel T cells (data not shown), 
likely due to the lack of CXCL1 expression by native B16 tumor, the growth of CXCL1-
expressing B16 tumors in vivo was significantly delayed in mice treated with CXCR2-expressing 
Pmel T cells compared with 
control Pmel T cells (Figure 9C). 
Transferring CXCR2-expressing 
Pmel T cells was also found to 
significantly extend the survival 
time of B16-CXCL1 tumor 
bearing mice (Figure 6D). These 
data strongly suggest that 
expression of CXCR2 by 
transferred Pmel T cells 
enhances the in vivo antitumor 
immune response.   
 
Hence, Our results from 
preclinical murine models and 
human TILs have demonstrated 
the feasibility of altering 
lymphocyte migration in vitro and 
in vivo with the use of retroviral 
gene transfer, and also showed 
that improved migration of 
CXCR2-transduced tumor-
specific lymphocytes has 
conferred enhanced tumor 
protection in murine models.  
    
 
 
2.0 Objectives 
 
Figure 9: Regression of established tumors upon adoptive transfer of 
CXCR2 expressing Pmel T cells. Groups of mice were initially implanted with 
5X105 tumor cells in the flank at day 0, subjected to 350cGy TBI at day 6, followed 
by DC vaccination, systemic IL-2 and the adoptive transfer of Pmel T cells either 
expressing OFL alone or OFL and CXCR2. (A) Tumor growth curve of 
MC38/gp100 tumor-bearing mice with adoptive T cell therapy. (B) Kaplan-Meier 
survival curves of MC38/gp100 tumor-bearing mice with adoptive T cell therapy. 
(C) Tumor growth curve of B16-CXCL1 tumor-bearing mice with adoptive T cell 
therapy. (D) Kaplan-Meier survival curves of B16-CXCL1 tumor-bearing mice with 
adoptive T cell therapy.  In both tumor models, CXCR2 transduction enhanced the 
ability of the T-cells to mediate tumor regression and increased survival. 


Protocol 2009-0471 
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Page 14 of 39 
 
Primary Objective 
• 
To  assess the feasibility and safety  of CXCR2 and NGFR Transduced TILs for treating 
metastatic malignant melanoma.  
 
Secondary Objectives 
 
• 
Determine whether CXCR2 transduction enhances the ability of TIL to migrate to 
melanoma tumors  
 
• 
Determine the levels of CXCL1 and CXCL8 chemokines produced by melanoma tumors 
and assess whether this correlates with the tumor localization of CXCR2 transduced TIL 
 
• 
Characterize the clinical response and correlate with migration of CXCR2 transduced 
TIL to the tumor and levels of CXCL1 and CXCL8 at the tumor site. 
 
 
 
3.0 Clinical Pharmacology  
 
3.1 
CXCR2 transduced TIL 
 
CXCR2 is a 67-70 kD seven-transmembrane G-protein coupled chemokine receptor. The 
ligands that bind to CXCR2 include CXCL8 (IL-8) and CXCL1 (GRO-α).  TIL retrovirally 
transduced to express CXCR2 can migrate in vitro and in vivo to CXCL1 and CXCL8 expressing 
tumors.   
 
3.2 
NGFR transduced TIL 
 
Nerve growth factor receptor (NGFR) belongs to the TNF (tumor necrosis factor) superfamily of 
receptors and is one of the receptors for neurotrophins. A non-signaling truncated version of 
human NGFR, lacking the cytoplasmic domain, is routinely used as a marker for gene 
transduction. In this study, a human truncated NGFR will be used as a control for CXCR2 TIL 
transduction. Since it is of human origin and lacks a cytoplasmic domain, it will not elicit any 
immune responses and is incapable of eliciting intracellular signaling cascades. It acts as a 
marker to identify control TIL cells since human T cells normally do not express NGFR.  
 
 
3.3  Interleukin-2 (Aldesleukin, Proleukin)  
 
Interleukin-2 (IL-2) will be manufactured and supplied by Norvatis and will be purchased by the 
M.D. Anderson pharmacy from commercial sources. IL-2 is a 133 amino acid-long peptide 
primarily secreted by T-cells in response to various antigenic stimuli.  The cytokine acts through 
a specific IL-2 receptor consisting of α, β, γ subunits.  In addition to T-cell proliferation, IL-2 
leads to activation and proliferation of natural killer (NK) cells, increasing their tumoricidal 
activity.  Other actions of IL-2 include augmentation of B-cell growth and immunoglobulin 
production, enhancement of interferon (IFN)-γ and tumor necrosis factor (TNF)-β production 
from T-cells, IL-6 production by monocytes, modulation of histamine release by basophils, and 
upregulation of IL-2 receptors.  This triggers the release of various other cytokines leading to the 
total immune/ inflammatory reaction and resultant toxicity.   
 


Protocol 2009-0471 
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IL-2 will be administered as an inpatient treatment.  Grade III toxicities common to IL-2 include 
diarrhea, nausea, vomiting, hypotension, skin changes, anorexia, mucositis, dysphagia, 
constitutional symptoms, and laboratory changes.  Additional Grade IV and V toxicities have 
been seen with IL-2 (See Appendix G). 
 
3.4  Fludarabine 
 
Fludarabine phosphate is a fluorinated nucleotide analog of the antiviral agent vidarabine, 9-β-
D-arabinofuranosyladenine (ara-A) that is relatively resistant to deamination.   Fludarabine is a 
purine antagonist antimetabolite.  Fludarabine phosphate is rapidly dephosphorylated to 2-
fluoro-ara-A and then phosphorylated intracellularly by deoxycytidine kinase to the active 
triphosphate, 2-fluoro-ara-ATP.  This metabolite appears to act by inhibiting DNA polymerase 
alpha, ribonucleotide reductase and DNA primase, thus inhibiting DNA synthesis.  The 
mechanism of action of this antimetabolite is not completely characterized and may be multi-
faceted.  
 
It will be purchased by the M.D. Anderson pharmacy from commercial sources. Fludarabine is 
supplied as a fludarabine phosphate powder in the form of a white, lyophilized solid cake.  The 
fludarabine powder is stable for at least 18 months at 2 – 8 degrees C; when reconstituted, 
fludarabine is stable for at least 16 days at room temperature.  Specialized references should be 
consulted for specific compatibility information.  Fludarabine is dephosphorylated in serum, 
transported intracellularly and converted to the nucleotide fludarabine triphosphate; this 2-fluoro-
ara-ATP molecule is thought to be required for the drug’s cytotoxic effects.  Fludarabine inhibits 
DNA polymerase, ribnucleotide reductase, DNA primase, and may interfere with chain 
elongation, and RNA and protein synthesis. 
 
Fludarabine is administered as an I.V. infusion in 100 ml 0.9% sodium chloride, USP over 
approximately 15-30 minutes.  At doses of 25 mg/m2/day for 5 days, the primary side effect is 
myelosuppression.  However, thrombocytopenia is responsible for most cases of severe and 
life-threatening hematologic toxicity.  Hemolytic anemia has been reported after one or more 
courses of fludarabine with or without a prior history of a positive Coomb’s test; fatal hemolytic 
anemia has been reported.  In addition, bone marrow fibrosis has been observed after 
fludarabine therapy.  Other common adverse effects include malaise, fatigue, anorexia, and 
weakness.  Irreversible and potentially fatal central nervous system toxicity in the form of 
progressive encephalopathy, blindness, and coma is rare at the currently administered doses.  
More common neurologic side effects at the current doses of fludarabine include weakness, 
pain, malaise, fatigue, paresthesia, visual or hearing disturbances, and sleep disorders.  
Adverse respiratory effects of fludarabine include cough, dyspnea, and allergic or idiopathic 
interstitial pneumonitis. Tumor lysis syndrome has been rarely observed in fludarabine 
treatment of Chronic Lymphocytic Leukemia (CLL). 
 
3.5  Cyclophosphamide (Cytoxan) 
 
Cyclophosphamide is a synthetic anti-neoplastic drug chemically related to the nitrogen 
mustards.  It is biotransformed principally in the liver to active alkylating metabolites by a mixed 
function microsomal oxidase system.  These metabolites interfere with the growth of susceptible 
rapidly proliferating malignant cells.  The mechanism of action is thought to involve cross-linking 
of tumor cell DNA.   
 


Protocol 2009-0471 
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Cyclophosphamide is well absorbed after oral administration with a bioavailability greater than 
75%.  The unchanged drug has an elimination half-life of 3 to 12 hours.  It is eliminated primarily 
in the form of metabolites, but from 5% to 25% of the dose is excreted in urine as unchanged 
drug.  Several cytotoxic and noncytotoxic metabolites have been identified in urine and in 
plasma.  Concentrations of metabolites reach a maximum in plasma 2 to 3 hours after an 
intravenous dose.  Plasma protein binding of unchanged drug is low but some metabolites are 
bound to an extent greater than 60%.  It has not been demonstrated than any single metabolite 
is responsible for either the therapeutic or toxic effects of cyclophosphamide.  Although elevated 
levels of metabolites of cyclophosphamide have been observed in patients with renal failure, 
increased clinical toxicity in such patients has not been demonstrated. 
 
Following conversion to active metabolites in the liver, cyclophosphamide functions as an 
alkylating agent and possesses potent immunosuppressive activity.   The serum half-life after 
intravenous administration ranges from 3 to 12 hours; the drug and/or its metabolites can be 
detected in the serum for up to 72 hours after administration. 
     
Cyclophosphamide will be obtained from commercially available sources by the M. D. Anderson 
pharmacy.  It will be diluted in 250 ml NS and infused over approximately two hours.  The dose 
will be based on the patient’s body weight, but to prevent undue toxicity, it will not exceed a 
dose greater than 40% of the maximum ideal body weight for drug dosing using MD Anderson 
(MDACC) clinical calculator for Body Surface Area (BSA) and Body Mass Index (BMI). 
Hematologic toxicity occurring with cyclophosphamide usually includes leukopenia and 
thrombocytopenia.  Anorexia, nausea, and vomiting may occur, especially after high-doses.  
Diarrhea, hemorrhagic colitis, and mucosal and oral ulceration have been reported in patients.  
Sterile hemorrhagic cystitis occurs in about 20% of patients; severity can range from 
microscopic hematuria to extensive cystitis with bladder fibrosis.  Although the incidence of 
hemorrhagic cystitis associated with cyclophosphamide appears to be lower than that 
associated with ifosfamide, mesna (sodium 2-mercaptoethanesulfonate) has been used 
prophylactically as an uroprotective agent   Mesna may not be effective in all patients.  Patients 
who receive high dose cyclophosphamide may develop interstitial pulmonary fibrosis, which can 
be fatal.  Hyperuricemia due to rapid cellular destruction may occur, particularly in patients with 
hematologic malignancy.  Hyperuricemia may be minimized by adequate hydration, 
alkalinization of the urine, and/or administration of allopurinol.  If allopurinol is administered, 
patients should be watched closely for cyclophosphamide toxicity due to allopurinal induction of 
hepatic microsomal enzymes.  At high doses, cyclophosphamide can also result in a syndrome 
of inappropriate antidiuretic hormone secretion; hyponatremia with progressive weight gain 
without edema occurs.  Cardiotoxicity has been observed at high doses of cyclophophamide.  
Deaths have occurred from diffuse hemorrhagic myocardial necrosis and from acute 
myopericarditis; in such cases, congestive heart failure may occur within a few days of the first 
dose.  Other consequences of cyclophosphamide cardiotoxicity include arrhythmias, potentially 
irreversible cardiomyopathy, and pericarditis.  Other reported adverse effects of 
cyclophosphamide include headache, dizziness, and myxedema; faintness, facial flushing, and 
diaphoresis.  
  
 
 
 
 
 


Protocol 2009-0471 
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3.6  Mesna (Sodium 2-mercaptoethanesulfonate, Mesnum, Mesnex, NSC-113891) 
 
Mesna (sodium 2-mercaptoethanesulphonate; given by IV injection) is a synthetic sulfhydryl 
compound that can chemically interact with urotoxic metabolites of cyclophosphamide (acrolein 
and 4-hydroxycyclophosphamide) to decrease the incidence and severity of hemorrhagic 
cystitis.   
 
Mesna was developed as a prophylactic agent to reduce the risk of hemorrhagic cystitis.  
Analogous to the physiological cysteine-cystine, mesna is rapidly oxidized to its major 
metabolite, mesna disulfide (dimesna).  Mesna disulfide remains in the intravascular 
compartment and is rapidly eliminated by the kidneys.  In the kidney, the mesna disulfide is 
reduced to the free thiol compound, mesna, which reacts chemically with the urotoxic 
metabolites, resulting in their detoxification.   
 
Mesna will be obtained commercially and is supplied as a 100 mg/ml solution.  Intact ampules 
are stored at room temperature.  Diluted solutions (1 to 20 mg/dl) are physically and chemically 
stable for at least 24 hours under refrigeration.  Mesna is chemically stable at room temperature 
for 48-72 hours in D5W, 48-72 hour in D5W/0.45% normal saline, or 24 hours in normal saline.  
It will be diluted up to 20 mg Mesna/ml fluid in D5W or normal saline and will be administered 
intravenously as a continuous infusion.  Toxicities include nausea, vomiting and diarrhea.  
 
3.7  G-CSF (Granulocyte Colony-Stimulating Factor) 
 
G-CSF may be given in the form of Filgrastim or PEG-Filgrastim at the appropriate doses. The 
side effects of G-CSF are skin rash, myalgia and bone pain, an increase of preexisting 
inflammatory conditions, enlarged spleen with occasional associated low platelet counts, 
alopecia (with prolonged use) elevated blood chemistry levels. 
 
 
3.8 Levofloxacin (Levaquin) and Trimethoprim and Sulfamethoxazole double strength (TMP / 
SMX DS, Bactrim) 
 
Levaquin is used to prevent infections caused by bacteria.  It is a synthetic broad spectrum 
antibacterial agent.  The mechanism of action of levofloxacin involves inhibition of bacterial 
topoisomerase IV and DNA gyrase, enzymes required for DNA replication, transcription, repair, 
and recombination. 
 
TMP/SMX DS will be obtained by the M.D. Anderson pharmacy from commercial sources.  It will 
be used for the prevention of Pneumocystis carinii Pneumonia (PCP).  The oral dose is 1 tablet 
PO bid twice a week. Like other sulfa drugs, Bactrim (sulfamethoxazole-trimethoprim) can 
cause allergies, fever, nausea, and vomiting. Allergies typically develop as a widespread itchy 
red rash with fever eight to fourteen days after beginning the standard dose. Neutropenia, a 
reduction in the number of neutrophils, can also occur.   
 
3.9  Acyclovir (Zovirax) and Valacyclovir Hydrochloride (Valtrex) 
 
Acyclovir and Valtrex will be obtained by the M. D. Anderson pharmacy from commercial 
sources.   
 


Protocol 2009-0471 
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Valtrex is the hydrochloride salt of L-valyl ester of acyclovir.  It is rapidly converted to acyclovir, 
which has demonstrated antiviral activity against herpes simplex virus types 1 (HSV-1) and 2 
(HSV-2) and varicella-zoster virus (VZV).  The inhibitory activity of acyclovir is highly selective 
due to its affinity for the enzyme thymidine kinase (TK) encoded by HSV and VZV.  This viral 
enzyme converts acyclovir into acyclovir monophosphate, a nucleotide analogue.  The 
monophosphate is further converted into diphosphate by cellular guanylate kinase and into 
triphosphate by a number of cellular enzymes.  Acyclovir triphosphate stops replication of 
herpes viral DNA. 
 
Acyclovir will be used to prevent the occurrence of herpes virus infections.  It is supplied as 
powder for injection in 500 mg vials.  Reconstitute in 10 ml of sterile water for injection for 
bacteriostatic water for injection to a concentration of 50 mg/ml. Reconstituted solutions should 
be used within 12 hours.  IV solutions should be diluted to a concentration of 7 mg/ml or less 
and infused over 1 hour to avoid renal damage.  Oral tablets of 200 and 800 mg are available, if 
the patient is able to tolerate medication by mouth.  Reversible renal insufficiency has been 
reported with intravenous but not oral acyclovir.  Neurologic toxicity including delirium, tremors, 
coma, acute psychiatric disturbances, and abnormal EEGs has been reported with higher doses 
of acyclovir.  Should this occur, a dosage adjustment will be made or the drug should be 
discontinued.  Stomach upset, headache, nausea, rash, hives, diaphoresis, hematuria; 
hypotension, and thrombocytosis have been reported.  Hair loss from prolonged use has also 
been documented. Acyclovir will not be used concomitantly with other nucleoside analogs that 
interfere with DNA synthesis, e.g. ganciclovir.  In renal disease, the dose is adjusted as per 
product labeling.  
 
3.10 Fluconazole (Diflucan) 
 
Fluconazole will be obtained by the M. D. Anderson pharmacy from commercial sources.  It will 
be used for prophylaxis against fungal infections.  It is available in 200 mg tablets.  It can cause 
headache, nausea, vomiting, diarrhea or abdominal pain, and liver damage that may be 
irreversible.  It can cause rashes and itching, which in rare cases has caused Stevens Johnson 
Syndrome. It has several significant drug interactions.  The package insert should be consulted 
prior to prescribing.  For IV administration in patients who cannot tolerate the oral preparation, 
Fluconazole comes in 2 mg/ml solution for injection, and prepared according to Clinical Center 
Pharmacy standard procedures.  It should be administered at a maximum IV rate of 200 mg/hr. 
 
3.11  Ondansetron hydrochloride (Zofran) 
 
Ondansetron hydrochloride will be obtained by the M. D. Anderson pharmacy from commercial 
sources.  It will be used to control nausea and vomiting during the chemotherapy preparative 
regimen.  It can cause headache, dizziness, myalgias, drowsiness, malaise, and weakness.  
Less common side effects include chest pain, hypotension, pruritis, constipation and urinary 
retention.  Consult the package insert for a complete list of side effects and specific dose 
instructions. 
 
3.12  Furosemide (Lasix) 
 
Furosemide, a loop diuretic, will be obtained by the M. D. Anderson pharmacy from commercial 
sources.  It will be used to enhance urine output during the chemotherapy preparative regimen 
with cyclophosphamide. Adverse effects include dizziness, vertigo, paresthesias, weakness, 


Protocol 2009-0471 
September 6, 2017 
Page 19 of 39 
 
orthostatic hypotension, photosensitivity, rash and pruritis.  Consult the package insert for a 
complete list of side effects and specific dose instructions. 
  
3.13  T Cell Preparation   
 
The procedures and reagents for expanding the human TIL cells and the Certificates of Analysis 
are contained in the CMC located in the IND office. 
 
 
4.0 Eligibility Assessment and Enrollment 
 
 
Patient evaluation for eligibility and registration will occur utilizing a two-turnstile design. 
 
4.1  Turnstile I – Screening – Initiation of TIL Expansion  
 
Patients must fulfill all of the following criteria to be eligible for Turnstile I of the study.  All 
patients entering the MDACC Melanoma Clinic are tested for the BRAF mutation as standard 
practice and are not required to receive treatment with BRAF (and MEK) inhibitors prior to 
therapy with TIL on this study. 
 
 
4.1.1 Turnstile I Inclusion Criteria 
 
a. Patients must have metastatic melanoma or stage III in-transit, subcutaneous, or 
regional nodal disease.  
b. Patients must have a lesion amenable to resection for the generation of TIL. 
c. Patients must receive an MRI/CT/PET of the brain within 6 months of signing informed 
consent. If new lesions are present, patient must have definitive treatment.  PI or his 
designee should make final determination regarding enrollment.   
d. Age greater than or equal to 18 years. 
e. Clinical performance status of ECOG 0 – 2 within 30 days of signing informed consent. 
f. Patients previously treated with immunotherapy, targeted therapy, or no therapy will be 
eligible.  Patients receiving cytotoxic agents will be evaluated by the PI or his designee 
as to suitable eligibility. 
g. Patients with a negative pregnancy test (urine or serum) must be documented within 14 
days of screening for women of childbearing potential (WOCBP).  A WOCBP has not 
undergone a hysterectomy or who has not been naturally postmenopausal for at least 12 
consecutive months (i.e. who has not had menses at any time in the preceding 12 
consecutive months). 
 
4.1.2 Screening Exclusion Criteria 
 
a. Active systemic infections requiring intravenous antibiotics, coagulation disorders or 
other major medical illnesses of the cardiovascular, respiratory or immune system.  PI or 
his designee shall make the final determination regarding appropriateness of enrollment. 
      b. Patients who are pregnant or nursing  
 
 
 


Protocol 2009-0471 
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4.1.3 Screening Procedures 
 
All patients must sign an informed consent form and a negative pregnancy test (urine or serum) 
must be documented for women of childbearing potential before enrollment in Turnstile I and 
being registered in PDMS. Once enrolled into Turnstile I, patients will be screened for Hepatitis 
B Surface Antigen, Hepatitis B Core Antibody, Hepatitis C Virus Antibody, HIV 1/HIV 2 Antibody, 
HTLV I/II Antibody, RPR Qual, CMV Antibody, CMV by PCR, West Nile Virus, Chagas Disease, 
EBV Panel and HLA testing, within 30 days of signing the informed consent. Exception: patient 
does not need infectious disease panel and HLA testing repeated if it was done in Turnstile 1 on 
2004-0069.  An MRI/CT/PET scan of the brain will be performed within 6 months of signing the 
informed consent for Turnstile I.  ECOG assessment will also be done within 30 days of signing 
informed consent. 
 
The primary endpoint is to evaluate the safety and toxicity of CXCR2 and the secondary 
endpoint is to evaluate T-cell migration to tumor. TIL harvests will be obtained on patients to 
obtain 15 treated patients that will be required for the statistical analysis of the study.  The 
samples will be placed in medium in the presence of IL-2.  Tumor Infiltrating lymphocytes (TIL) 
will be transduced with CXCR2 and NGFR and expanded from the patients’ tumor using 
standard, published techniques as described in the CMC. 
 
While T-cells are being grown, patients may be treated with alternative therapies.  However, 
patients must not have received other therapies for 28 days prior to lymphodepletion and T-cell 
infusion.  If the subject is to receive fresh (not frozen and then thawed) TIL, the subject may not 
be treated with any alternative therapy. 
 
Patients must have adequate TIL available. Pre-REP TIL generated in the similar trial 2004-
0069 may also be utilized for Turnstile II. 
 
4.2  Turnstile II  - Treatment – Chemotherapy/Cell Infusion 
 
Patients must sign informed consent document before Turnstile II screening procedures.  Before 
the treatment starts and at each visit, the patient will be asked to complete two quality of life 
questionnaires.  It should take about 15 minutes to complete the questionnaires.  (FACT-G, 
FACT-Melanoma) Appendix D,E. 
Patients must fulfill all of the following criteria to be eligible for Turnstile II of the study. 
 
4.2.1 Chemotherapy/Cell Infusion Inclusion Criteria Turnstile II 
 
a. Patients must have adequate TIL available 
b. Patients must have at least one biopsiable measurable metastatic melanoma, lesion > 
1cm  
c. Patients may have brain lesions which measure < 1cm each. The PI or designee will 
approve the treatment. 
d. Patients of both genders must practice birth control for four months after receiving the 
preparative regimen (lymphodepletion) and continue to practice birth control throughout 
the study.  Patients must have a documented negative pregnancy test (urine or serum) 
for women who have menstruation in the past 12 months and without sterilization 
surgery. 


Protocol 2009-0471 
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Page 21 of 39 
 
e. Unless surgically sterile by bilateral tubal ligation or vasectomy of partner(s), the patient 
agrees to continue to use a barrier method of contraception throughout the study such 
as:  condom, diaphragm, hormonal, IUD, or sponge plus spermicide.  Abstinence is an 
acceptable form of birth control 
f. Patients with a negative pregnancy test (urine or serum) must be documented within 14 
days of screening for women of childbearing potential (WOCBP).  A WOCBP has not 
undergone a hysterectomy or who has not been naturally postmenopausal for at least 12 
consecutive months (i.e. who has not had menses at any time in the preceding 12 
consecutive months). 
 
g. Clinical performance status of ECOG 0-2 within 30 days of signing informed consent..  
h. Absolute neutrophil count greater than or equal to 1000/mm3. 
i. 
Platelet count greater than or equal to 100,000/mm3. 
j. 
Hemoglobin greater than or equal to 8.0 g/dl. 
k. Serum ALT less than three times the upper limit of normal. 
l. 
Serum creatinine less than or equal to 1.6 mg/dl. 
m. Total bilirubin less than or equal to 2.0 mg/dl, except in patients with Gilbert’s Syndrome 
who must have a total bilirubin less than 3.0 mg/dl.  
n. A stress cardiac test (stress thallium, stress MUGA, dobutamine echocardiogram or 
other stress test that will rule out cardiac ischemia) within 6 months of lymphodepletion.   
o. Pulmonary function tests (FEV1>65% or FVC>65%of predicted) within 6 months of 
lymphodepletion. 
p. MRI/CT/PET of the brain within 30 days of lymphodepletion 
 
4.2.2  Chemotherapy/Cell Infusion Exclusion Criteria Turnstile II 
 
a. Has had prior systemic cancer therapy within the past four weeks at the time of the start 
of the lymphodepletion regimen. 
b. Women who are pregnant will be excluded because of the potentially dangerous effects 
of the preparative chemotherapy on the fetus. 
c. Any active systemic infections requiring intravenous antibiotics, coagulation disorders or 
other major medical illnesses of the cardiovascular, respiratory or immune system, such 
as abnormal stress thallium or comparable test, myocardial infarction, cardiac 
arrhythmias, obstructive or restrictive pulmonary disease.  PI or his designee shall make 
the final determination regarding appropriateness of enrollment. 
d. Any form of primary or secondary immunodeficiency.  Must have recovered immune 
competence after chemotherapy or radiation therapy as evidenced by lymphocyte 
counts (> 500/mm3), WBC (> 3,000/mm3) or absence of opportunistic infections.  
e. Require steroid therapy or steroid-containing compounds, or have used systemic 
steroids in the past 4 weeks, or have used topical or inhalational steroids in the past 2 
weeks prior to lymphodepletion; the exception being patients on chronic physiologic 
dose of steroid.   
f. Presence of a significant psychiatric disease, which in the opinion of the principal 
investigator or his designee, would prevent adequate informed consent or render 
immunotherapy unsafe or contraindicated. 
 
5.0 Pretreatment Evaluation Turnstile II 
 


Protocol 2009-0471 
September 6, 2017 
Page 22 of 39 
 
5.1 Lymphodepletion 
 
a. Complete history and physical examination including vital signs, height, weight, noting in 
detail the exact size and location of any lesions that exist will be performed within 14 
days prior to initiation of chemotherapy.   
b. Clinical performance status of ECOG 0-2 within 14 days of lymphodepletion.  
c. Chemistries to include BUN, creatinine, total bilirubin, alkaline phosphatase, LDH, ALT, 
sodium, potassium, chloride, and CO2 will be performed within 14 days prior to 
lymphodepletion.  
d. CBC, differential, PT/PTT, and platelet count will be performed within 14 days prior to 
initiation of chemotherapy. 
e. Urinalysis (micro) within 14 days prior to initiation of chemotherapy. 
f. β-HCG pregnancy test (urine or serum) on all women of childbearing potential will be 
performed within 14 days prior to lymphodepletion. 
g. Baseline radiological studies to evaluate the status of disease must be obtained within 
30 days prior to lymphodepletion (CXR, CT scan of chest, abdomen, and pelvis; MRI/CT 
of brain or a PET/CT may also be used). 
h. EKG within 30 days prior to lymphodepletion. 
i. 
A stress cardiac test (stress thallium, stress MUGA, dobutamine echocardiogram or 
other stress test that will rule out cardiac ischemia) within 6 months of lymphodepletion.   
j. 
Pulmonary function tests (FEV1>65% or FVC>65%of predicted) within 6 months of 
lymphodepletion 
 
6.0 Treatment Plan Turnstile II 
 
6.1  Outline   
 
• 
15 patients will be treated (refer to Section 11.0 Statistical Considerations and Data 
Analysis).  Patients will receive a T cells transduced with CXCR2 as well as T cells 
transduced with NGFR (as a control gene). 
• 
T cells will be expanded in a state-of-the-art GMP facility that will allow compliance with 
all FDA regulations regarding investigational cell transfer products.  We have 
demonstrated that we can successfully expand TIL on the majority of patients and have 
treated patients with up to 150 billion TIL.   
• 
All patients will receive lymphodepleting chemotherapy with cytoxan and fludarabine to 
enhance T cell persistence and effectiveness in vivo.  Cytoxan will be administered at 60 
mg/kg/day I.V. in 250 ml NS over approximately 2 hours on Days –7 and –6.  Mesna 60 
mg/kg with D5W or NS at 125 ml/hr infused intravenously over 24 hours on Days -7 and 
-6.  The dose will be based on the patient’s body weight, but to prevent undue toxicity, it 
will not exceed a dose greater than 40% of the maximum ideal body weight for drug 
dosing using MD Anderson (MDACC) clinical calculator for Body Surface Area (BSA) 
and Body Mass Index (BMI).   Fludarabine will then be infused at 25 mg/m2 IVPB daily 
over approximately 15-30 minutes on Days –5 to –1. 
• 
On day 0, all patients will receive up to 1.5x1011 T cells (including both CXCR2 and 
NGFR transduced TIL in one combined infusion) in NS, depending on the number that 
can be generated in the laboratory. It has been previously shown that infusion of cell 
numbers in this range is well tolerated. Depending on the amount of T-cells and the 
volume, TIL will be infused as an inpatient by I.V. for maximum infusion time of up to 4 
hours, unless ordered otherwise by physician.  Tylenol (acetaminophen) will be given by 


Protocol 2009-0471 
September 6, 2017 
Page 23 of 39 
 
mouth before the T-cell infusion to decrease the risk of these side effects. Twelve (12) to 
sixteen (16) hours after completing the T cell infusion, all patients will receive high dose 
interleukin-2 (IL-2) on an inpatient basis at the standard dose of 720,000 IU/kg as an 
intravenous bolus over an approximate 15 minute period every 8-16 hours for up to 15 
doses on Days 1 to 5, as tolerated.  Doses will be skipped if patients reach Grade III or 
IV toxicity due to high dose IL-2, except for the reversible Grade III toxicities common to 
high dose IL-2 such as diarrhea, nausea, vomiting, hypotension, skin changes, anorexia, 
mucositis, dysphagia, or constitutional symptoms and laboratory changes (i.e. platelets, 
creatinine, and total bilirubin) as detailed in Appendix G.  If the toxicity is easily reversed 
by supportive measures, then additional doses may be continued.  
• 
If the patient’s platelet count is less than 80K on Day 21 (-2 days/+7 days), the second 
course of high dose IL-2 will be delayed until the platelet count is at least 80K.  The 
second course of IL2 can be omitted at the discretion of PI and/or treating physician.  
For the evaluation of peripheral blood cells following T cell transfer, 70 ml blood samples 
in CPT or heparin tubes will be obtained prior to tumor harvest and/or excision, within 30 
days of lymphodepletion, and again on Days6 (+/- three days),  and day 21(+/- seven 
days)when feasible.  These days were selected based on our previous studies at the 
National Cancer Institute.  During high dose IL-2 administration (Days 1-5 and 22-26), 
peripheral blood mononuclear cell (PBMC) yields are low, possibly due to adhesion of 
immune cells to vasculature.  Therefore, days 6(+/- three days)and day 21(+/- seven 
days)have been selected as the earliest feasible time points to obtain cells and evaluate 
the immediate effects of immunization.  Additional time points to obtain PBMC are week 
6(+/- 7 days) and week 12(+/- 7 days). To evaluate T cells at the tumor site, required 
tumor samples will be obtained by biopsy within 30 days of lymphodepletion and then 
again at approximately day 21 (+/- seven days) when feasible. Additional optional 
biopsies may be done at week 6(+/- 7 days) and week 12(+/- 7 days). 
• 
The retrovirus vector is replication-defective, i.e. will not replicate after infection into the 
host cells. No new viral particles will be formed following infection into the TIL. The 
infection will be performed at the early stages of T-cell expansion. Non infecting vector 
will be washed away during the expansion and harvest process.  The TIL for infusion will 
not contain any retrovirus vector; therefore there will be no danger to the nurses from the 
retrovirus vector. A negative pressure room will not be required; these transduced TIL 
will be administered in the same manner as non-transduced TIL. 
• 
Feeder cells will be CMV negative if pre-treatment testing of the patient reveals absence 
of CMV seropositivity. If a patient is CMV positive at baseline, CMV positive feeder cells 
may be utilized. Pools of feeder cells from CMV seropositive patients will be tested by 
PCR to assess for the presence of CMV in the feeder cell population. Those feeder cells 
that have undetectable levels of CMV by PCR will be deemed eligible to be used as 
feeders in the TIL expansion process. We will monitor all  patients receiving TIL  with 
serum CMV pcr testing at day 3 (+/-24 hours) post TIL infusion and 3 weeks (+/-7 days) 
after TIL infusion.  CMV pcr blood testing is drawn in a single 10mL purple  top tube. The 
test will be performed at MDA through the molecular diagnostics lab. If there is evidence 
of CMV reactivation as evidenced by the CMV pcr, infectious disease specialist will be 
consulted and further management or therapy will be per expert recommendations. 
• 
Administration of the lymphodepletion treatment and concomitant medications including 
supporting prophylactic medication will be documented in the medical record but will not 
be captured in the case report form for this protocol.  TIL administration and IL-2 starting 
at Day 0 will be captured in the case report form. 


Protocol 2009-0471 
September 6, 2017 
Page 24 of 39 
 
• 
Variations from the lymphodepletion (e.g. infusion times; schedule of treatments, etc) 
prior to day 0 will be documented in the medical record but will not be considered 
protocol violations/deviations. 
 
 
 
 
 
 
 
 
 
 
 
 
Treatment schema: 
 
Turnstile 1 
 
Screening 
Within 30 days of signing 
informed consent 
Informed Consent 
X 
 
CT Chest/abd/pelvis or PET1 
X 
 
MRI brain1 
X 
 
Donor Infectious Disease 
Panel2 
 
X 
HLA Testing 
 
X 
Pregnancy Test (serum or 
urine) 
X 
 
Physical exam, Vitals, 
ECOG 
X 
 
CBC with Differential 
X 
 
Serum Chemistries3 
X 
 
Research blood 
 
X 
1: CT chest/abdomen/pelvis or PET and MRI brain within 6 months of screening 
2: Donor infectious disease panel includes: for Hepatitis B Surface Antigen, Hepatitis B Core 
Antibody, Hepatitis C Virus Antibody, HIV 1/HIV 2 Antibody, HTLV I/II Antibody, RPR Qual, 
CMV Antibody, CMV by PCR, West Nile Virus, Chagas Disease, EBV Panel 
3: Serum chemistries: Albumin, alkaline phosphatase, total bilirubin, bicarbonate, BUN, calcium, 
chloride, creatinine, glucose, LDH, magnesium, phosphorus, potassium, total protein, AST, ALT, 
sodium 
4. Pregnancy test (urine or serum) within 14 days of screening and within 14 days of 
lymphodepleting chemotherapy 
 
Turnstile 2 Pretreatment Procedures 
 
Within 6 months 
of 
lymphodepleting 
chemotherapy 
Within 30 days 
of 
lymphodepleting 
chemotherapy 
Within 14 days of 
lymphodepleting 
chemotherapy 
Pulmonary Function 
X 
 
 


Protocol 2009-0471 
September 6, 2017 
Page 25 of 39 
 
Tests 
Cardiac Stress Test 
X 
 
 
Pregnancy Test 
(serum or urine)4 
 
 
X 
Physical exam, Vitals, 
ECOG 
 
 
X 
CBC with Differential 
 
 
X 
Serum Chemistries3 
 
 
X 
PT/PTT 
 
 
X 
Urinalysis 
 
 
X 
EKG 
 
X 
 
CT 
Chest/abdomen/pelvis 
or PET 
 
X 
 
MRI Brain/PET/CT 
 
X 
 
Research Blood 
 
X 
 
Tumor Biopsy 
 
X 
 
Quality of Life 
Questionnaire 
 
X 
 
RCR by PCR 
 
X 
 
 
Turnstile 2 On-Treatment Procedures 
 
During 
Lymphodepletion 
and High Dose 
IL-2 
Day 6 
(+/- 3 
days) 
Day 21 
(+/-7 
days) 
Week 6 
(+/- 7 
days) 
Week 
12 (+/- 
7 days) 
Month 
6, 9, 
12 
Yearly  
Physical 
exam 
Daily 
 
 
X 
X 
X 
X 
CBC with 
Differential 
Every 1-2 days 
 
 
X 
X 
X 
X 
Serum 
Chemistries 
Every 1-2 days 
 
 
X 
X 
X 
X 
CT 
Chest/abd/pel
vis or PET 
 
 
 
X 
X 
X 
X 
MRI 
brain/PET/CT 
 
 
 
X 
X 
X 
X 
Research 
Blood 
 
X 
X 
X 
X 
 
 
Tumor Biopsy 
 
 
X 
Optional 
Optional 
 
 
RCR PCR 
 
 
 
 
X 
Month 
6, 
Month 
12  
X 
 
 


Protocol 2009-0471 
September 6, 2017 
Page 26 of 39 
 
 
Treatment: 
 
Day -7 and –6: 
Cyclophosphamide 60 mg/kg/day I.V. in 250 ml NS over approximately 2 hrs. 
Mesna 60 mg/kg with D5W or NS at 125 ml/hr infused intravenously over 24 hours.  
 
Day -5 to Day -1: 
Fludarabine 25 mg/m2 IVPB daily over approximately 15-30 minutes for 5 days.   
 
Day 0: 
Depending on the amount of T-cells and the volume, T-cells will be infused in NS with maximum 
infusion time of up to 4 hours, unless ordered otherwise by physician. Tylenol (acetaminophen) 
will be given by mouth before the T-cell infusion to decrease the risk of the side effects. 
 
Days 1-5:       
High dose IL-2, 720,000 IU/kg IV bolus (about 15 minutes) every 8-16 hours for up to 15 doses, 
beginning approximately 12-16 hours after T cell infusion. 
 
Day 22-26 (-2 days/ + 7 days): 
High dose IL-2, 720,000 IU/kg IV bolus (about 15 minutes) every 8-16 hours for up to 15 doses.  
If platelet count is greater or equal to 80K. 
 
6.2     Prophylaxes 
 
6.2.1 Infection Prevention and Pneumocystis carinii Pneumonia (PCP) Prophylaxis 
 
Patients will receive Levaquin at 500 mg daily until ANC recovers to greater than 500/mm3 and 
the fixed combination of trimethoprim (TMP) and sulfamethoxazole (SMX) as double strength 
(DS) tablet [ DS tabs = TMP 160 mg/tab and SMX 800 mg/tab ] p.o. b.i.d. twice a week.  
TMP/SMX-DS will be taken by patients beginning on Day -7 and continuing for a minimum of 6 
months post chemotherapy.   For patients with sulfa allergies, Pentamidine will be given (once 
discharged from the hospital) 300 mg IV every 21 days for six months after lymphodepletion.  If 
IV Pentamidine is not feasible after discharge, Pneumocystis Jirovecii Pneumonia (PCP) 
prophylaxis can be substituted with oral antimicrobials such as Atovaquone as per standard of 
care for 6 months after lymphodepletion. 
 
 
 
6.2.2   Herpes Virus Prophylaxis 
 
At the time of the T cell infusion, patients will be administered valtrex 500 mg p.o. daily for 6 
months if the patient is able to take oral medications.  If the patient needs intravenous 
medications give acyclovir 5 mg/kg IVPB every 8 hours, which is continued until absolute 
neutrophil count is greater than 1000/ml.  Reversible renal insufficiency has been reported with 
IV administered acyclovir but not with oral acyclovir.  Neurologic toxicity including delirium, 
tremors, coma, acute psychiatric disturbances, and abnormal EEGs has been reported with 
higher doses of acyclovir.  If symptoms occur, a dosage adjustment will be made or the drug will 
be discontinued.  Acyclovir will not be used concomitantly with other nucleoside analogs (e.g. 


Protocol 2009-0471 
September 6, 2017 
Page 27 of 39 
 
ganciclovir), which interfere with DNA synthesis.  In patients with renal disease, the dose is 
adjusted as per product labeling.  Valtrex/Acyclovir is to be continued for 6 months after 
lymphodepletion. 
 
6.2.3    Fungal Prophylaxis  
 
Patients will begin Fluconazole 200 mg p.o. daily with the T cell infusion (Day 0) and continue 
for 6 months after lymphodepletion. 
 
6.2.4   Ondansetron hydrochloride (Zofran) 
It will be used to control nausea and vomiting during the chemotherapy preparative regimen.  It 
can cause headache, dizziness, myalgias, drowsiness, malaise, and weakness.  Less common 
side effects include chest pain, hypotension, pruritis, constipation and urinary retention.  Consult 
the package insert for a complete list of side effects and specific dose instructions. 
 
6.2.5  Furosemide (Lasix) 
It will be used to enhance urine output during the chemotherapy preparative regimen with 
cyclophosphamide. Adverse effects include dizziness, vertigo, paresthesias, weakness, 
orthostatic hypotension, photosensitivity, rash and pruritis.  Consult the package insert for a 
complete list of side effects and specific dose instructions. 
 
6.2.6   Empiric Antibiotics  
 
Patients will start on broad-spectrum antibiotics, either a 3rd or 4th generation cephalsosporin or 
a quinolone for fevers ≥38.5 C with an ANC less than 500/mm3.  Aminoglycosides should be 
avoided unless clear evidence of sepsis. Infectious disease consultation will be obtained from all 
patients with unexplained fever or any infectious complications. 
 
 
6.2.7   Blood Product Support  
 
In order to reduce neutropenia following chemotherapy and T cell infusion, G-CSF will be given 
at 5 μg/kg/day daily subcutaneously until neutrophil counts reach >500/mm3 Using daily CBC’s 
as a guide, the patient will also receive platelets and packed red blood cells (PRBC’s) as 
needed.  Attempts will be made to keep Hb >8.0 gm/dl, and platelets >20,000/ml.    Leukocyte 
filters will be utilized for all blood and platelet transfusions to decrease sensitization to 
transfused WBC’s and decrease the risk of CMV infection.  Irradiated blood and blood products 
should be used. 
 
7.0    Evaluation During Study 
 
• 
Before the treatment starts and at each visit, the patient will be asked to complete two 
quality of life questionnaires.  It should take about 15 minutes to complete the 
questionnaires.   
• 
During the preparative regimen and high dose IL-2 therapy, patients will have a 
complete blood count (CBC) and electrolytes every 1 to 2 days of treatment.  Patients 
will be monitored with vital signs at baseline, every 15 minutes (+/- 10 minutes) during 
cell infusion and after cell infusion hourly (+/- 30 minutes) for 4 hours after the infusion.   


Protocol 2009-0471 
September 6, 2017 
Page 28 of 39 
 
• 
Phlebotomy will be performed prior to tumor harvest and/or excision, or within 30 days of 
lymphodepletion, and again on Day 6 (+/- three days) and day 21 (+/- seven days) after 
the cell infusion when feasible, and again at 6 week (+/- 7 days) and 12 week (+/- 7 
days) after the cell infusion when feasible. These blood samples will be used to evaluate 
T cell persistence and function, using a variety of assays. To assess the secondary 
endpoint of T cell migration to the tumor, required tumor samples will be obtained within 
30 days of lymphodepletion and then again at approximately day 21 (+/-7 days).Optional 
biopsies may be obtained at 6 weeks (+/- 7 days) and 12 weeks (+/- 7 days).   
• 
 The presence of CD4+ T-cells at the tumor site will also be evaluated.  Patients” blood 
samples will be obtained and undergo analysis for detection of RCR by PCR prior to 
lymphodepletion and RCR PCR will be performed approximately at 3 and 6 months and 
at approximately one year post cell administration.  RCR PCR assays detect the GaLV 
envelop gene and are performed by research staff in the Department of Medical and 
Molecular Genetics, Indiana University. Blood samples will be archived annually 
thereafter 10 years if all previous testing has been negative with a brief clinical history.  If 
patients die or develop neoplasms during this trial, efforts will be made to assay a biopsy 
sample for RCR.  If any post-treatment samples are positive, further analysis of the RCR 
and more extensive patient follow-up will be undertaken, in consultation with CBER.  The 
results of these tests are maintained by the Department of Medical and Molecular 
Genetics and by the MD Anderson Cancer Center research team.   
• 
Complete evaluation of evaluable lesions with physical examination and appropriate CT 
scans or PET/CT will be performed approximately at 6 weeks (+/- 7 days) and at 12 
weeks (+/- 7 days) after the cell infusion, at which time patient will be restaged. 
• 
After 12 weeks and for patients who have progression of disease at 6 weeks or at 12 
weeks post cell infusion, if feasible, patients will be evaluated per Melanoma Standard of 
Care and follow-up visit tests and scans will be physician dependent. 
• 
Tumor measurements will not be required for patients who have progression of disease.  
• 
If patients are unable to return for follow-up visits, the patients will be followed for 
survival via phone or clinic visit approximately every 3 months for a year and then yearly 
for 10 years or until patient death, patient withdraws consent, patient is lost-to follow-up 
or the study is completed. 
• 
At the time of the patient’s death, no matter the cause, we may request permission for 
an autopsy in order to obtain vital information concerning the safety of this experimental 
therapy approach.  
 
• 
We will send the patient two questionnaires (FACT-G, FACT-Melanoma)(Appendix D, E) 
yearly to get information regarding their quality of life for 15 years.  For this reason, we 
will ask the patients to continue to provide us with a current address and telephone 
number, even after completion of this research study 
 
 
Blood and tissue specimens collected in the course of this research project may be banked and 
provided in the future to investigators with IRB approved research protocols. 
 
 
 
 
Re-Treatment 
 
If patients have stable disease or a partial response to treatment, they may be re-treated after 
re-evaluation with the same schedule that they had been given safely previously [less than 
Grade III (except for common high dose IL-2 toxicities noted in Appendix) or Grade IV toxicity 


Protocol 2009-0471 
September 6, 2017 
Page 29 of 39 
 
easily reversible by standard measures].  Re-treatment benefits and risks will be carefully 
explained.  A maximum of 1 re-treatment course may be given for each patient. 
 
8.0 Evaluation of Toxicity 
 
This study will utilize the National Cancer Institute Common Terminology Criteria (CTC) for 
Adverse Events version 4.0 for toxicity and Adverse Event reporting.  A copy of the CTCAE 
version 4.0 can be found in Appendix A.  All appropriate treatment areas should have access to 
a copy of the CTCAE version 4.0. 
 
Careful evaluation to ascertain the toxicity, immunologic effects and anti-tumor efficacy of cell 
infusions will be performed.  Toxicities will be monitored and documented on a daily basis 
beginning at day 0 (T-cell infusion) and continuing until discharged from the hospital following 
the initial IL-2 infusion.  The criteria for stopping the study and halting enrollment and the 
administration of study drug will be as follows: 
 
1. A Grade IV non-hematologic toxicity not explained by IL-2 infusion as outlined in 
Appendix G. 
2. Death not due to disease progression within 3 months of the initiation of the conditioning 
regimen. 
 
The principal investigator will monitor the data and toxicities to identify trends.  The principal 
investigator will be responsible for revising the protocol as needed to maintain safety.  The MD 
Anderson IRB will review serious adverse events as they are submitted.  Serious adverse 
events will be submitted to the FDA by the IND Safety Project Manager in the IND Office.  The 
principal investigator will also review serious adverse events and evaluate trends.  Whenever a 
trend is identified, the principal investigator will determine an appropriate follow up plan.  The 
investigator or physician designee is responsible for verifying and providing source 
documentation for all adverse events and assigning the attribution for each event for all subjects 
enrolled on the trial.  
The use of the nonmyeloablative regimen in this protocol is a major procedure which entails 
serious discomforts and hazards for the patient.  Although it is anticipated that this protocol is 
relatively safe because of the expected recovery of the patients’ bone marrow within 2 to 4 
weeks, fatal complications are possible.  It is therefore only appropriate to carry out this 
experimental procedure in the context of life threatening metastatic cancer.  The major hazards 
are infection and disease progression.  The major discomforts are nausea, mucositis, anorexia, 
diarrhea, fever and malaise.  Side effects of common drugs used in this nonmyeloablative 
regimen include: 
 
Cyclophosphamide: Marrow suppression, nausea, mucositis, rash, hemorrhagic cystitis, 
myocardial damage, alopecia, infertility, nausea and vomiting, SIADH.  
  
Fludarabine: Myelosuppression, fever and chills, nausea and vomiting, malaise, fatigue, 
anorexia, weakness, neurologic toxicity, and interstitial pneumonitis.  Serious opportunistic 
infections have occurred in CLL patients treated with fludarabine. 
 
Antimicrobials in general: Allergic reactions, renal impairment, nausea, vomiting, hepatic 
damage, marrow suppression.  
 


Protocol 2009-0471 
September 6, 2017 
Page 30 of 39 
 
High Dose IL-2:  A variety of side effects have been associated with high-dose IL-2 
administration in our experience at the NCI and a listing of these side effects in 652 patients 
who received 1,039 treatment courses are listed in Appendix G.  
 
Serious Adverse Event Reporting (SAE) 
 
A serious adverse event is-any adverse drug experience occurring at any dose that 
results in any of the following outcomes: 
 
 
a.  Death 
b.  A life-threatening adverse drug experience – any adverse experience 
that places the patient, in view of the initial reporter, at immediate risk of 
death from the adverse experience as it occurred, it does not include an 
adverse experience that, had it occurred in a more severe form, might 
have caused death. 
c.  Inpatient hospitalization or prolongation of existing hospitalization 
d. A persistent or significant incapacity or substantial disruption of the 
ability to conduct normal life functions. 
 
 
e.  a congenital anomaly/birth defect. 
 
 
Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered a serious adverse drug experience when,, based 
upon appropriate medical judgment, they may jeopardize the patient or subject and may 
require medical or surgical intervention to prevent one of the outcomes listed in this 
definition.  Examples of such medical events include allergic bronchospasm requiring 
intensive treatment in an emergency room or at home, blood dyscrasias or convulsions 
that do not result in inpatient hospitalization, or development of drug dependency or drug 
abuse (21 CFR 312.32). 
 
a. Important medical events as defined above, may also be  
considered serious adverse events.  Any important medical event can 
and should be reported as an SAE if deemed appropriate by the 
Principal Investigator or the IND Sponsor/ IND office.,  
b. All events occurring during the conduct of a protocol and meeting the 
definition of a SAE must be reported to the IRB in accordance with the 
timeframes and procedures outlined in “University of Texas M.D. 
Anderson Cancer Center Institutional Review Board Policy for 
Investigators on Reporting Serious Unanticipated Adverse Events for 
Drugs and Devices”.  Unless stated otherwise in the protocol, all 
SAEs, expected or unexpected, must be reported to the IND office 
regardless of attribution (within 5 working days of knowledge of the 
event). 
c. All life-threatening or fatal events, expected or unexpected, and  
     regardless of attribution to the study drug, must have a written   
     report submitted within 24 hours 
(next working day) of knowledge of the event to the Safety Project 
Manager in the IND office. 
d.  The MDACC “Internal SAE Report Form for Prompt Reporting” will be 
used for reporting to the IND office. 
e. Unless otherwise noted, the electronic SAE application (eSAE) will be 
utilized for safety reporting to the IND office and MDACC IRB. 


Protocol 2009-0471 
September 6, 2017 
Page 31 of 39 
 
f. Serious adverse events will be captured from the time the patient 
signs consent until 30 days after the last dose of drug unless the 
participant withdraws consent.  Serious adverse events must be 
followed until clinical recovery is complete and laboratory tests have 
returned to baseline, progression of the event has stabilized, or there 
has been acceptable resolution of the event. 
g. Additionally, any serious adverse events that occur after the 30 day 
time period that are related to the study treatment must be report to 
the IND office.  This may include the development of a secondary 
malignancy. 
h. The adverse events will be recorded in CORe according to the 
following guidelines and all other information will be entered into 
PDMS. 
 
 
Recommended Adverse Event Recording Guidelines  
Attribution 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Grade 5 
  
  
  
  
  
  
Unrelated 
Phase I 
Phase I 
Phase I           
Phase II   
  
Phase I         
Phase II  
Phase III 
Phase I                
Phase II  Phase 
III 
Unlikely 
Phase I 
Phase I 
Phase I           
Phase II   
  
Phase I         
Phase II  
Phase III 
Phase I                
Phase II  Phase 
III 
Possible 
Phase I    
Phase II 
Phase I      
Phase II  
Phase III 
Phase I           
Phase II  
Phase III 
  
Phase I         
Phase II  
Phase III 
Phase I                
Phase II  Phase 
III 
Probable 
Phase I    
Phase II 
Phase I      
Phase II  
Phase III 
Phase I           
Phase II  
Phase III 
  
Phase I         
Phase II  
Phase III 
Phase I                
Phase II  Phase 
III 
Definitive 
Phase I    
Phase II 
Phase I      
Phase II  
Phase III 
Phase I           
Phase II  
Phase III 
  
Phase I         
Phase II  
Phase III 
Phase I                
Phase II  Phase 
III 
Exceptions to this rule will include events and laboratory abnormalities that represent 
common symptoms and abnormalities of melanoma and chemotherapy and/or have 
no clinical significance: 
 
• 
Abnormalities in hematologic parameters due to myelosuppressive therapeutic effect: 
 
i. 
Anemia, neutropenia, lymphopenia, thrombocytopenia 
ii. 
Epistaxis or bleeding except for catastrophic CNS or pulmonary     
hemorrhage 
 
• 
Common symptoms of cancer (unless grade >/= 3) including: 
 
i. 
Fatigue 
ii. 
Weakness 
iii. 
Bone, joint or muscle pain 


Protocol 2009-0471 
September 6, 2017 
Page 32 of 39 
 
iv. 
Alopecia 
v. 
Loss of appetite, Nausea, Vomiting 
vi. 
Chemistry abnormalities (phosphorus, calcium, glucose) 
vii. 
Coagulation abnormalities (shortened PT, PTT, increased fibrinogen) 
 
• 
Laboratory abnormalities: 
 
i. 
LDH (increased or decreased) 
ii. 
Alkaline phosphatase (increased or decreased) 
iii. 
Low levels of the following: AST, ALT, creatinine, BUN, uric acid, bilirubin, 
albumin, total protein 
iv. 
Electrolyte abnormalities (sodium, potassium, bicarbonate, CO2, magnesium 
 
• 
General therapy related events 
 
i. 
Catheter related events 
ii. 
Rash related to antibiotic use 
 
Abnormal laboratory test results will be captured if intervention is required. 
 
 
Reporting to FDA 
 
a. Serious adverse events will be forwarded to FDA by the IND Sponsor 
(Safety Project Manager, IND office according to 21 CFR 312.32. 
b. The gene therapy reporting addendum (“Additional Reporting Form for 
Serious Adverse Events on Gene Therapy Trials”) must be included 
with each SAE submitted. 
 
 
It is the responsibility of the PI and the research team to ensure 
serious adverse events are reported according to the Code of Federal 
Regulations, Good Clinical Practices, the protocol guidelines, and Institutional 
Review Board policy. 
 
Careful evaluation to ascertain the toxicity, immunologic effects and anti- tumor efficacy 
of therapy will be performed.   


Protocol 2009-0471 
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9.0 Criteria for Response 
Tumor response to therapy in this study will be done using immune-related response criteria 
(irRC) which is a modified version of WHO criteria. 
 
9.1  
Definition of Measureable and Non-Measurable Lesions 
Measurable Lesions are lesions that can be accurately measured in two 
perpendicular diameters, with at least one diameter > 10 mm.  The area will be 
defined as the product of the largest diameter with its perpendicular.  Skin lesions 
can be considered measurable.  Cutaneous lesions that are > 5 mm in diameter 
can be considered measurable. 
 
Non-Measurable (evaluable) Lesions are all other lesions, including 
unidimensionally measurable disease and small lesions. 
 
Definition of Index/Non-Index Lesions 
 
All measurable lesions, up to a maximum of five lesions per organ and ten 
lesions in total, should be identified as index lesions to be measured and 
recorded on the medical record at baseline.  The index lesions should be 
representative of all involved organs.  In addition, index lesions should be 
selected based on their size (lesions with the longest diameters), their suitability 
for accurate repeat assessment by imaging techniques, and how representative 
they are of the patient’s tumor burden.  A sum of the products of diameters (SPD) 
for all index lesions will be calculated and considered the baseline sum of the 
products of diameters.  Response criteria to be followed are listed below.  The 
baseline sum will be used as the reference point to determine the objective tumor 
response of the index lesions at tumor assessment (TA).  Measurable lesions, 
other than index and all sites of non-measurable disease will be identified as 
non-index lesions.  Non-index lesions will be recorded on the medical record and 
should be evaluated at the same assessment time points as the index lesions.  In 
subsequent assessments, non-index lesions will be recorded as “stable or 
decreased disease,” “absent”, or “progression.” 
 
9.2  
Definition of Tumor Response Using irRC 
The sum of the products of diameters at tumor assessment using the immune-
related response criteria (irRC) for progressive disease incorporates the 
contribution of new measurable lesions.  Each net Percentage Change in Tumor 
Burden per assessment using irRC criteria accounts for the size and growth 
kinetics of both old and new lesions as they appear. 
 
Definition of Index Lesions Response Using irRC 
  
irComplete Response (irCR): Complete disappearance of all index lesions.  
This category encompasses exactly the same subjects as “CR” by the mWHO 
criteria. 
 
irPartial Response (irPR): Decrease, relative to baseline, of 50% or greater in 
the sum of the products of the two largest perpendicular diameters of all index 


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and all new measurable lesions (i.e., Percentage Change in Tumor Burden).  
Note: the appearance of new measurable lesions is factored into the overall 
tumor burden, but does not automatically qualify as progressive disease until the 
SPD increases by >25% when compared to SPD at nadir. 
 
irStable Disease (irSD): Does not meet criteria for irCR or irPR, in the absence 
of progressive disease. 
 
irProgressive Disease (irPD): At least 25% increase Percentage Change in 
Tumor Burden (i.e., taking sum of the products of all index lesions and any new 
lesions) when compared to SPD at nadir. 
 
Definition of Non-Index Lesions Response Using irRC 
 
irComplete Response (irCR): Complete disappearance of all non-index lesions.  
This category encompasses exactly the same subjects as “CR” by the mWHO 
criteria 
 
irPartial Response (irPR) or irStable Disease (irSD): non-index lesions are not 
considered in the definition of PR, these terms do not apply. 
irProgressive Disease (irPD): Increases in number or size of non-index lesions 
does not constitute progressive disease unless/until the Percentage Change in 
Tumor Burden increases by 25% (i.e., the SPD at nadir of the index lesions 
increases by the required amount). 
 
Impact of New Lesions on irRC 
New lesions in and by themselves do not qualify as progressive disease.  
However, their contribution to total tumor burden in included in the SPD which in 
turn feeds into the irRC criteria for tumor response.  Therefore, new non-
measurable lesions will not discontinue any subject from the study. 
 
9.3  
Definition of Overall Response Using irRC 
Overall response using irRC will be based on these criteria: 
 
Immune-Related Complete Response (irCR): Complete disappearance of all 
tumor 
lesions 
(index 
and 
non-index 
together 
with 
no 
new 
measurable/unmeasurable lesions) for at least 4 weeks from the date of 
documentation of complete response. 
 
Immune-Related Partial Response (irPR): The sum of the products of the two 
largest perpendicular diameters of all index lesions is measured and captured as 
the SPD baseline.  At each subsequent tumor assessment, the sum of the 
products of the two largest perpendicular diameters of all index lesions and of 
new measurable lesions are added together to provide the Immune Response 
Sum of Product Diameters (irSPD).  A decrease, relative to baseline of the irSPD 
compared to the previous SPD baseline, of 50% or greater is considered an 
immune Partial Response (irPR). 
 


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Immune-Related Stable Disease (irSD): irSD is  defined as the failure to meet 
criteria for immune complete response or immune partial response, in the 
absence of progressive disease. 
 
Immune-Related Progressive Disease (irPD): It is recommended in difficult 
cases to confirm PD by serial imaging.  Any of the following will constitute 
progressive disease: 
• 
At least 25% increase in the sum of the products of all index lesions over 
baseline SPD calculated for the index lesions. 
• 
At least a 25% increase in the sum of the products of all index lesions and 
new measurable lesions (irSPD) over the baseline SPD calculated for the 
index lesion. 
 
 
Immune-Related Response Criteria Definitions 
 
Index 
Lesion 
Definition 
 
Non-Index 
Lesion 
Definition 
New 
Measurable 
Lesions 
New 
Unmeasurable 
Lesions 
Percent 
Change 
in 
Tumor Burden 
Overall irC 
Response 
Complete 
Response 
Complete 
Response 
No 
No 
-100% 
irCR 
Partial 
Response 
Any 
Any 
Any 
>-50% 
<50%to<+25% 
>+25% 
irPR 
irSD 
irPD 
Stable 
Disease 
Any 
Any 
Any 
<50%tp<+25% 
>+25% 
irSD 
irPD 
Progressive 
Disease 
Any 
Any 
Any 
>+25% 
irPD 
 
 
 
9.4  
Immune-Related Best Overall Response Using irRC (irBOR) 
irBOR is the best confirmed irRC overall response over the study as a whole, 
recorded between the date of first dose until the last tumor assessment before 
subsequent therapy (except for local palliative radiotherapy for painful bone 
lesions) for the individual subjects in the study.  For assessment of irBOR, all 
available assessments per subject are considered. 
 
If a lesion is surgically resected or treated with definitive radiosurgery, the size of 
the lesion prior to the definitive local therapy will be included in the calculated 
irBOR. 
 
10.0  Criteria for Removal from the Study 
 
Patients will be taken off the study if:  (a) the patient voluntarily withdraws, (b) there is significant 
noncompliance, or (c) there is progression of disease in those patients after being treated by 


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lymphodepletion, T-cell infusion, and IL-2 in Turnstile II.  Any patient who develops Grade IV 
toxicity due to cell infusion will be taken off protocol.   
 
11.0  Statistical Considerations and Data Analysis 
 
This is a pilot study to assess the feasibility and safety of producing sufficient quantities of 
CXCR2 and NGFR transduced tumor infiltrating lymphocytes (TILs) for treatment of metastatic 
melanoma.  A total of 15 patients will be treated. Clinical outcomes that will be observed are 
tumor response by imaging, and possible grade 3 or worse toxicities not normally associated 
with lymphodepletion and high dose IL-2. 
Based on our current experience with generation of gene modified TILs, we are not able to 
adequately fulfill the parameters for a phase I study due to technical challenges with generating 
enough T cells to allow for dose escalation. In our 3 patients treated thus far, we have not been 
able to reach the nominal dose level 2 due to the inherent variability in quantities of T cells able 
to be generated for  individual patients. Thus, this trial is being redesigned as a feasibility study 
with the primary objectives to assess the feasibility of generating TILs and safety of  and  
treating patients with genetically modified T cells.   
 
Secondary objectives include  response, defined following immune-related response criteria.  
Immunological endpoints include number of CXCR2 transduced cells and number of NGFR 
transduced cells at infusion, number of CXCR2 transduced cells and number of NGFR 
transduced cells based on tumor biopsy, and the amounts of CXCR2 and CXCL8 cytokines.  
Regression analysis will be used to estimate the ability of the genetically transduced T-cells to 
differentially recognize the cancer cell cytokines. Logistic regression will be used to assess the 
effects of  immunological variables on the probability of clinical response.  A sample size of 15 
patients was determined primarily by resource limitations. 
 
12.0 
Statistical Considerations. A clinical response/safety summary will be submitted to the 
IND medical monitor after the first five participants are treated and every five thereafter. 
 
12.1 
Outcomes.  The primary clinical endpoint will be response, defined following immune-
related response criteria as a 50% or greater decrease in the tumor’s linear dimension post 
treatment, compared to baseline, evaluated by PET or CT imaging at week 6, week 12 and then 
every 3 month intervals for up to one year of follow up. 
 
 Immunological endpoints will include (i) at infusion, Xc = number of CXCR2 transduced cells 
and Xs = number of NGFR transduced (control) cells, (ii) post treatment, based on tumor 
biopsy, Yc = number of CXCR2 transduced cells and Ys = number of NGFR transduced cells, 
and (iii) post treatment, ZCXCR2  = amount of CXCR2 cytokine and ZCXCR8  = amount of  CXCL8 
(IL-8) cytokine.   
 
 
12.2.  Estimation of Immunological Effects. Denote the pre-treatment and post-treatment 
ratios by RX = Xc/Xs  and RY = Yc/Ys.  In order to test the hypothesis that the CXCR2 cells are 
more likely to find the tumor than the NGFR (control) cells, the linear regression model RY = b0 + 
b1 RX + error will be fit to the (RX , RY) pairs and the hypothesis H0: b1 = 1 will be tested against 
the alternative HA: b1 > 1. [1]  The alternative HA says that the CXCR2 cells are more likely to 
enter and be present in the tumor than the control cells.  This is a statistical version of the 
intuitive idea that, if the TIL methodology works as designed and the genetically transduced T-


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cells differentially recognize the cancer cell cytokines, then the ratio of post- and pre-treatment 
ratios RR = RY/RX should be larger than 1.  
 
12.3.  Estimation of Clinical Response Rate. Additional regression analyses will include 
logistic regression to assess the effects of Yc,  RY and RR on the probability of clinical 
response, as well as regression fits of  ZCXCR2  and ZCXCR8 as functions of Yc,  RY and RR.   
 
12.4.  Sample Size. A total of 15 patients will be treated as determined primarily by resource 
limitations.  
 
13.0  Data Entry and Protocol Management 
 
For the purposes of this study at M. D. Anderson Cancer Center, the Protocol Data 
Management System (PDMS) will be employed.  All patients will be registered in CORe utilizing 
a two-turnstile registration before any study specific tests are performed. The continual 
reassessment method (CRM) used in phase I will be implemented using the Biostatistics 
Department Clinical Trial Conduct Website. 
 
14.0  Administrative Procedures 
 
14.1  Changes to the Protocol:  
 
Any change or addition to this protocol requires a written protocol amendment that must 
be approved by the IND Office and the IRB. A copy of the written approval of the IRB 
must be received  by the IND Office and the principal investigator before implementation 
of any changes.  The IRB must review and approve all amendments to the protocol.    
 
 
14.2  Ethics and Good Clinical Practice:  
 
This study must be carried out in compliance with the protocol and Good Clinical 
Practice, as described in:  
 
14.2.1  ICH Harmonized Tripartite Guidelines for Good Clinical Practice 1996. 
 
14.2.2   US 21 Code of Federal Regulations dealing with clinical studies    
(including parts 50 and 56 concerning informed consent and IRB regulations). 
 
14.2.3   Declaration of Helsinki, concerning medical research in humans  
(Recommendations Guiding Physicians in Biomedical Research Involving Human 
Subjects, Helsinki 1964, amended Tokyo 1975, Venice 1983, Hong Kong 1989, 
Somerset West 1996). 
 
The investigator agrees, when signing the protocol, to adhere to the instructions and 
procedures described in it and thereby to adhere to the principles of Good Clinical 
Practice. 
 
 
 


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15.0 References 
 
 
Hwu, P. and S. A. Rosenberg (1994). "The use of gene-modified tumor-infiltrating lymphocytes 
for cancer therapy." Ann N Y Acad Sci 716: 188-97; discussion 197-203. 
 
 
Hwu, P., G. E. Shafer, et al. (1993). "Lysis of ovarian cancer cells by human lymphocytes 
redirected with a chimeric gene composed of an antibody variable region and the Fc receptor 
gamma chain." J Exp Med 178(1): 361-6. 
 
 
Hwu, P., J. Yannelli, et al. (1993). "Functional and molecular characterization of tumor-infiltrating 
lymphocytes transduced with tumor necrosis factor-alpha cDNA for the gene therapy of cancer 
in humans." J Immunol 150(9): 4104-15. 
 
 
Kershaw, M. H., G. Wang, et al. (2002). "Redirecting migration of T cells to chemokine secreted 
from tumors by genetic modification with CXCR2." Hum Gene Ther 13(16): 1971-80. 
 
 
Lam, J. S., M. E. Reeves, et al. (1996). "Improved gene transfer into human lymphocytes using 
retroviruses with the gibbon ape leukemia virus envelope." Hum Gene Ther 7(12): 1415-22. 
 
 
Lou, Y., G. Wang, et al. (2004). "Dendritic cells strongly boost the antitumor activity of adoptively 
transferred T cells in vivo." Cancer Res 64(18): 6783-90. 
 
 
Payne, A. S. and L. A. Cornelius (2002). "The role of chemokines in melanoma tumor growth 
and metastasis." J Invest Dermatol 118(6): 915-22. 
 
 
Pittet, M. J., J. Grimm, et al. (2007). "In vivo imaging of T cell delivery to tumors after adoptive 
transfer therapy." Proc Natl Acad Sci U S A 104(30): 12457-61. 
 
 
Rabinovich, B. A., Y. Ye, et al. (2008). "Visualizing fewer than 10 mouse T cells with an 
enhanced firefly luciferase in immunocompetent mouse models of cancer." Proc Natl Acad Sci 
U S A 105(38): 14342-6. 
 
 
Richmond, A., D. H. Lawson, et al. (1985). "Characterization of autostimulatory and 
transforming growth factors from human melanoma cells." Cancer Res 45(12 Pt 1): 6390-4. 
 
 
Royal, R. E., M. H. Kershaw, et al. (2002). "Increased functional expression of transgene in 
primary human lymphocytes using retroviral vectors modified with IRES and splicing motifs." 
Gene Ther 9(16): 1085-92. 
 
 


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Treisman, J., P. Hwu, et al. (1995). "Interleukin-2-transduced lymphocytes grow in an autocrine 
fashion and remain responsive to antigen." Blood 85(1): 139-45. 
 
 
Treisman, J., P. Hwu, et al. (1994). "Upregulation of tumor necrosis factor-alpha production by 
retrovirally transduced human tumor-infiltrating lymphocytes using trans-retinoic acid." Cell 
Immunol 156(2): 448-57. 
 
 
Venables, W. and B. Ripley (2002). "Modern Applied Statistics with S." Springer 4th Edition. 
 
 
O’Quigley J, Pepe M, Fisher, L: Continual reassessment method: A practical design for phase I 
clinical trials in cancer. Biometrics 46:33-48, 1990.  
 
Thall PF, Sung H-G, Choudhury A.  Dose-finding based on feasibility and toxicity in T- cell 
infusion trials.  Biometrics, 57:914-921, 2001. 
 
Venables WN and Ripley BD. Modern Applied Statistics with S, 4th Edition. Springer, 2002