A Safety and Feasibility Trial of Boost Vaccinations of a Lethally Irradiated, Allogeneic Pancreatic Tumor Cell Vaccine Transfected With the GM-CSF Gene Given Alone or in Combination With Either a Single Intravenous Dose or Daily Metronomic Oral Doses of Cyclophosphamide for the Treatment of Surgically Resected Pancreatic Adenocarcinoma
Phase: PHASE2
Conditions: Pancreatic Cancer
Sponsor: Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins

Protocol Version 22, July 24, 2024  
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A safety and feasibility trial of boost vaccinations of a lethally irradiated, allogeneic 
pancreatic tumor cell vaccine transfected with the GM-CSF gene given alone or in 
combination with either a single intravenous dose or daily metronomic oral doses of 
cyclophosphamide for the treatment of surgically resected pancreatic adenocarcinoma 
 
Principal Investigator:  
Daniel Laheru, M.D.. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
IND Sponsor:  
 
Elizabeth Jaffee, M.D.  
 
  
 
 
 
 
 
   
 
 
IND#:  
 
 
BB IND 7136 
 
ClinicalTrials.gov ID:  
NCT01088789 
 
Lead Research Nurse:  
Carol Judkins, R.N., 
 
 
 
 
Statistician: 
 
 
Elizabeth Sugar, Ph.D
 
-
 
 
External Regulatory Affairs: Jennifer Durham, Ph.D., 
 
 
 
Data Management & Regulatory: Jeanne Harrison, 
 
 
IRB: 
 
 
 
Johns Hopkins Medicine- Institutional Review Board 
          
 
 
 
 
 
 
 
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J09100, Protocol Version 22, July 24, 2024 
 
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Table of Contents 
TABLE OF CONTENTS ............................................................................................................. 2 
STUDY SYNOPSIS ...................................................................................................................... 4 
1.0 STUDY SCHEMA .................................................................................................................. 6 
2.0 BACKGROUND AND RATIONALE .................................................................................. 7 
2.1 Introduction ........................................................................................................................ 8 
2.2 Rationale for cell-based immunotherapy of pancreatic adenocarcinoma .......................... 8 
2.3 Vaccine-based Strategies currently undergoing testing in subjects with pancreatic 
adenocarcinoma ................................................................................................................ 9 
2.4 Rationale for the use of a GM-CSF secreting whole cell vaccine approach ..................... 9 
2.5 Rationale of using low dose cyclophosphamide as an immune modulator ..................... 10 
2.5.1 Introduction:  Immune tolerance and regulatory T cells. ......................................... 10 
2.5.2 Evidence from animal studies supporting low dose cyclophosphamide as an 
immune modulator .................................................................................................. 11 
2.5.3 Rationale for using daily metronomic cyclophosphamide for immune modulation 11 
2.6 Phase I study of lethally irradiated allogeneic pancreatic tumor cells transfected with the 
GM-CSF gene. Phase I study at Johns Hopkins ............................................................. 12 
2.6.1 Summary of Study Design ....................................................................................... 12 
2.6.2 Toxicity Events ........................................................................................................ 12 
2.6.3 Disease Free Survival .............................................................................................. 14 
2.6.4 Immunologic Data ................................................................................................... 14 
2.7 Phase II study of lethally irradiated allogeneic pancreatic tumor cells transfected with the 
GM-CSF gene ................................................................................................................. 15 
2.7.1 Summary .................................................................................................................. 15 
2.7.2 Study Design ............................................................................................................ 16 
2.7.3 Safety Data ............................................................................................................... 16 
2.7.4  Survival data ........................................................................................................... 17 
2.7.5 Immunologic Data ................................................................................................... 17 
2.8 Phase II Study of the GM-CSF allogeneic vaccine alone and given in sequence with 
immune modulating doses of Cyclophosphamide in subjects with advanced pancreatic 
cancer .............................................................................................................................. 20 
2.8.1 Summary .................................................................................................................. 20 
2.8.2 Safety and Efficacy Data ......................................................................................... 20 
2.8.3 Immunologic Data ................................................................................................... 21 
2.9 A randomized three-arm neoadjuvant and adjuvant feasibility and toxicity study of a 
GM-CSF secreting allogeneic pancreatic cancer vaccine administered either alone or in 
combination with either a single intravenous dose or daily metronomic oral doses of 
cyclophosphamide for the treatment of patients with surgically resected 
adenocarcinoma of the pancreas ..................................................................................... 22 
2.10 Rationale of boost vaccinations and the long term treatment of immune modulating 
doses of cyclophosphamide ............................................................................................ 24 
2.11 Rationale for Immunobiologic Endpoints ..................................................................... 25 
3.0 STUDY DESIGN AND TREATMENT PLAN .................................................................. 26 
3.1 Study Overview ............................................................................................................... 26 

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3.2 Study Schedule Checklist ................................................................................................ 29 
3.3 Study Population .............................................................................................................. 35 
3.3.1 Eligibility Criteria .................................................................................................... 35 
3.4 Vaccine Production and Administration .......................................................................... 38 
3.5 Study Plan Schedule ........................................................................................................ 39 
3.5.1 Pre-Study Screen ...................................................................................................... 39 
3.5.2 Pre-Study Visit ......................................................................................................... 40 
3.5.3 Study Procedures during the Prime Vaccination Phase (3 monthly vaccination 
cycles) ...................................................................................................................... 40 
3.5.4 Study Procedure during the Boost Vaccination Phase ............................................. 41 
3.5.5 Off Study Visit ......................................................................................................... 43 
3.6 Collection Samples for Correlative Studies ..................................................................... 44 
3.6.1 Leukapheresis .......................................................................................................... 44 
3.6.2 Serum Banking......................................................................................................... 44 
3.6.3 Archival Tumor Tissue ............................................................................................ 44 
3.7 Evaluation for Safety and Anticipated Toxicities ............................................................ 44 
3.7.1 Safety and Anticipated Toxicities of Vaccine ......................................................... 45 
3.7.2 Safety and anticipated toxicities of single injection of low dose cyclophosphamide 
or recurrent administration of metronomic cyclophosphamide .............................. 46 
3.8 Interval Health Care ......................................................................................................... 47 
3.9 Termination of Study Treatment (Off Study) .................................................................. 48 
3.10 Management of Toxicities ............................................................................................. 48 
3.11 Concurrent Medication .................................................................................................. 49 
4.0 STATISTICAL CONSIDERATIONS ................................................................................ 50 
4.1 Sample Size ..................................................................................................................... 50 
4.2 Analysis of Primary Endpoints ........................................................................................ 51 
4.2.1 Safety and Toxicity Measurements .......................................................................... 51 
4.2.2 Measurements of Immune Response ....................................................................... 52 
4.3 Analysis of Secondary Endpoints .................................................................................... 53 
4.3.1 Efficacy Endpoints ................................................................................................... 53 
4.3.2 Relating Immune Response to Clinical Response ................................................... 54 
4.4 Analysis of Exploratory Endpoint ................................................................................... 54 
5.0 RESPONSE CRITERIA ...................................................................................................... 55 
5.1 Evaluation of Clinical Activity ........................................................................................ 55 
5.2 Evaluation of Immune Responses ................................................................................... 55 
6.0 ADVERSE AND PROBLEM EVENT REPORTING ...................................................... 55 
6.1. Responsibilities ............................................................................................................... 55 
6.2 Recording of an Adverse Event ....................................................................................... 56 
6.3 Reporting Guidelines ....................................................................................................... 58 
7.0 CLINICAL TRIAL MONITORING .................................................................................. 58 
8.0 DEVELOPMENTAL METHODS ...................................................................................... 58 
8.1 General Rationale for Banking Patient Materials ............................................................ 58 
8.2 Serum Banking ................................................................................................................ 59 
9.0 REFERENCES ...................................................................................................................... 60 

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Study Synopsis 
 
Title: A safety and feasibility trial of boost vaccinations of a lethally irradiated, allogeneic 
pancreatic tumor cell vaccine transfected with the GM-CSF gene given alone or in combination 
with either a single intravenous dose or daily metronomic oral doses of cyclophosphamide for the 
treatment of surgically resected pancreatic adenocarcinoma 
 
Objectives: 
 
Primary: 
To evaluate the safety and feasibility of long term boost vaccinations of a lethally irradiated, 
allogeneic pancreatic tumor cell vaccine transfected with the GM-CSF gene given alone or in 
combination with either a single intravenous dose or daily metronomic oral doses of 
cyclophosphamide for the treatment of patients with surgically resected adenocarcinoma of the 
head, neck, or uncinate process of the pancreas. 
 
Secondary: 
1. To assess the effect of boost vaccinations and long-term treatment of immune modulating 
doses of cyclophosphamide on the number, repertoire and avidity of peripheral mesothelin-
specific CD8+ T cells. 
2. To estimate disease-free and overall survival of surgically resected pancreatic adenocarcinoma 
patients treated with vaccine boosts with or without low dose cyclophosphamide. 
 
Study Population: 
In order to be considered for this study, patients need to meet the following criteria for inclusion: 
• History of surgically resected pathologic stage 1 (no direct tumor extension beyond pancreas 
and no regional lymph node metastases), 2a (direct extension of tumor beyond pancreas), 
and/or 2b (regional lymph node metastases) adenocarcinoma of the head, neck, tail, or uncinate 
of the pancreas. 
• Cohort 1: Patients who have previously participated in the study titled “A randomized 
three-arm, neoadjuvant and adjuvant, feasibility and toxicity study of a GM-CSF secreting 
allogeneic pancreatic cancer vaccine administered either alone, or in combination with 
either a single intravenous dose, or daily metronomic oral doses of cyclophosphamide for 
the treatment of patients with surgically resected adenocarcinoma of the pancreas” [J0810, 
NA_00015858 (formerly 00-01-58-58)], have no radiographic evidence of pancreatic 
disease recurrence, and have received the GM-CSF secreting allogeneic pancreatic cancer 
vaccine.  
• Cohort 2: Patients who never received any type of pancreatic vaccine or immunotherapy, 
had the Whipple surgery within 18 months and completed the planned adjuvant 
chemotherapy and/or chemoradiation. Have no radiographic evidence of pancreatic cancer 
disease recurrence. Have not received any anti-cancer therapy in the past 28 days. 
• Cohort 3: Patients who have previously participated in the study titled “A Randomized 
Study of a GM-CSF secreting allogeneic pancreatic cancer vaccine with or without a PD-
1 Blockade Antibody (Nivolumab) for the Neoadjuvant and Adjuvant Treatment of 
Patients with Surgically Resectable Adenocarcinoma of the Pancreas” (IRB00050517, 

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J1568), have no radiographic evidence of pancreatic disease recurrence, and received their 
sixth vaccine within 12 months of enrolling in J09100. 
• Cohort 4: Patients who have previously participated in the study titled “A Phase II Study 
of GM-CSF secreting allogeneic pancreatic cancer vaccine in combination with PD-1 
Blockade Antibody (Pembrolizumab) and Stereotactic Body Radiation Therapy (SBRT) 
for the Treatment of Patients with Locally Advanced Adenocarcinoma of the Pancreas” 
(IRB00083132, J15237), have no radiographic evidence of pancreatic disease recurrence, 
and received their last vaccine in the extended treatment phase within 12 months of 
enrolling in J09100. 
• Cohort 5: Patients who have previously participated in the study titled “A Pilot Study of a 
GVAX Pancreas Vaccine (with Cyclophosphamide) in Combination with a PD-1 Blockade 
Antibody (Pembrolizumab) and a Macrophage Targeting Agent (CSF1R inhibitor) for the 
Treatment of Patients with Borderline Resectable Adenocarcinoma of the Pancreas” 
(IRB00130267, J1766), have no radiographic evidence of pancreatic disease recurrence, 
and received their last vaccine in the extended treatment phase within 12 months of 
enrolling in J09100. 
 
Study Design: 
Eligible subjects will receive by intradermal administration the pancreatic tumor vaccine 
consisting of two irradiated, allogeneic pancreatic tumor cell lines transfected with the granulocyte 
macrophage-colony stimulating factor (GM-CSF) gene with or without low dose 
cyclophosphamide. Study participants will be recruited from our prior neoadjuvant vaccination 
with or without low dose cyclophosphamide trial and vaccine naïve patients. The vaccination 
boosts will be offered as a continuation of care.  
 
Patients from the J0810 study (Cohort 1) will remain on the same arm as the J0810 study where 
they have received the parental vaccine (Figure 1). The first vaccine boost will be given no sooner 
than six months (+/- 1 month) after the last prime vaccination. The vaccine will be administered 
for all arms once every six months (+/- 1 month) after the previous vaccine until five years have 
passed and then once every 12 months (+/- 1 month) until 10 years have passed, the subject no 
longer meets the eligibility criteria, no longer wishes to participate in the study, or the vaccine 
supply is exhausted. Arm A participants will receive the pancreatic cancer vaccine alone. Arm B 
participants will be vaccinated and receive a single low-dose of cyclophosphamide (200 mg/m2) 
intravenously one day prior to vaccination (Figure 2). Participants in Arm C will receive 
cyclophosphamide 50 mg once a day starting from 28 days prior to day 1 of vaccination until 28 
days post vaccination. We estimate that approximately 20 patients from the J0810 study will be 
candidates for this boost vaccination study. 
 
Vaccine naïve patients (Cohort 2) will first receive three prime vaccines each one month apart and 
each in combination with a single low-dose of cyclophosphamide (200 mg/m2) intravenously one 
day prior to vaccination (Figure 1). Then, they will receive the boost vaccines as the participant 
in Arm B from the J0810 study (Figure 2). Patients from Cohort 2 may receive treatment until ten 
years have passed, the subject no longer meets the eligibility criteria, no longer wishes to 
participate in the study, or the vaccine supply is exhausted. We estimate the sample size of the 
vaccine-naïve cohort to be 52.  
 

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Participants from clinical trials J1568, J15237 and J1766 (Cohorts 3, 4 and 5) will be vaccinated 
and receive a single low-dose of cyclophosphamide (200 mg/m2) intravenously one day prior to 
vaccination (Figure 2). The first boost vaccine will be given no sooner than six months (+/- 1 
month) and within 12 months after the last prime vaccination on the primary study. The vaccine 
will be administered once every six months (+/- 1 month) after the previous vaccine until five 
years have passed, the subject no longer meets the eligibility criteria, no longer wishes to 
participate in the study, or the vaccine supply is exhausted. We estimate the sample size of Cohort 
3, 4 and 5 to be 68. 
 
Study Drug:  
The vaccine consists of equal numbers (2.5 x 108 each) of Panc 6.03 and Panc 10.05 cells combined 
into a single vaccination. Vaccine cells from each pancreas tumor cell line frozen at 1.25 x108 
cells/vial (2 vials per cell line) in an injectable formulation of hetastarch will be thawed on the day 
of vaccination and taken up into syringes. Each vaccination will consist of six total intradermal 
injections, two each in the right and left thighs, and two in the non-dominant arm. This preparation 
has been used in two completed phase II studies and in two on-going phase II studies. 
Cyclophosphamide (Cytoxan, CTX) is given at 50 mg once daily per oral route or 200 mg/m2 
intravenously as indicated in the study design. 
 
1.0 Study Schema 
 
Figure 1 Vaccination Schedule for Cohorts 1-5 
-6m
0
6m
12m
18m  ……
Last vaccine from 
primary protocol
Consent
Study Entry
Boost Vaccination every
6 months
Combination 
Cohorts
Vaccine-naive 
Cohort
Monthly
IV CTX/Vaccines x 3
Consent
Study Entry
Boost Vaccination every
6 months
 

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Figure 2 Schema for One Boost Vaccination Cycle 
 
 
GVAX Only: Arm A of Cohort 1  
 
 
 
IV Cytoxan with GVAX: Arm B Cohort 1, Cohorts 2-5  
 
 
 
 
Metronomic Cytoxan with GVAX: Arm C Cohort 1  
       -27  -26 . . . . . . .-5  -4  -3  -2  -1    0  1    2    3   4   5. . …. . ..27 28  29  . . . . .2m. . . . . . 5m. . . . . .6m 
        -27 -26. . . . . . …-5  -4  -3  -2  -1    0  1    2    3   4   5. . . . 27 28  29  . . . . . 2m. . . . . . .5m. . . . . . 6m 
        -27  -26 . . . . . . .-5  -4  -3  -2  -1    0  1    2    3   4   5. . . . 27  28  29  . . . . . 2m. . . . . . .5m. . . . . . 6m 
vaccination 
PBL and 
Serum banking 
Metronomic CTX 
IV CTX 
One Boost Vaccination Cycle 

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2.0 Background and Rationale 
 
2.1 Introduction 
 
Pancreatic cancer is the fourth leading cause of all cancer deaths. Although only 37,680 Americans 
are expected be diagnosed with pancreatic cancer, 34,290 will die from pancreatic cancer in 2008 
(American Cancer Society, 2008). Only about 20% of patients diagnosed with pancreatic cancer 
will be eligible for surgical resection with a pancreaticoduodenectomy, the only potentially 
curative treatment. However, even among those patients who undergo surgery and adjuvant 
therapy 79% will eventually die of recurrent disease (Ahlgren, 1996). Pancreatic cancer has the 
most dismal prognosis among 18 cancer diagnoses. The statistics for 1996-2003 for the five-year 
relative survival rates for pancreatic cancer by stage are: 5.0% for all stages, 20.3% for localized, 
8.0% for regional, and 1.7% for distant (American Cancer Society, 2008). Despite significant 
efforts to develop new therapies, locally advanced unresectable disease has a median survival of 
10-12 months and subjects with metastatic pancreatic adenocarcinoma have a median survival of 
3-6 months. While surgical resection is the only curative option, the majority of subjects (80-85%) 
present with advanced unresectable disease. These dismal survival rates require the development 
of novel approaches for the treatment of pancreatic cancer. 
 
2.2 Rationale for cell-based immunotherapy of pancreatic adenocarcinoma 
 
Adjuvant chemoradiation with a 5-Fluorouracil (5-FU) containing chemotherapy regime has been 
the standard for patients undergoing complete pancreatic cancer resection. However, even the most 
recent studies have demonstrated only modest improvements in disease-free survival (Smeenk et 
al., 2005). Immunotherapy is a potentially therapeutic approach to the treatment of pancreatic 
adenocarcinoma for several reasons. First, immunologic killing of tumor cells acts by a mechanism 
that is distinct from standard chemotherapy and radiation therapy and may represent a non-cross 
resistant treatment modality. Second, the immune system is capable of recognizing a diverse array 
of potential antigens while orchestrating selective and specific cytotoxic responses. This may be 
particularly important in the killing of a heterogeneous tumor population while avoiding normal 
tissue toxicity. Third, preclinical animal models using a vaccine approach for immunotherapy have 
been able to eliminate small burdens of established tumors, a situation that corresponds to the state 
of minimal residual disease commonly found after resection of human tumors (Burris et al., 1997; 
Heineman et al., 2003). Fourth, a completed Phase I trial evaluating an allogeneic, irradiated, 
granulocyte-macrophage colony stimulating factor (GM-CSF) secreting tumor vaccine in patients 
with adenocarcinoma of the pancreas demonstrated both clinical and immunologic responses 
(Jaffee et al., 2001).  In addition, an analysis of 60 patients in a phase II study testing the safety 
and efficacy of the vaccine has shown an 86% one-year survival and a 61% two-year survival 
(Laheru et al., 2007).  There is also a proportion of patients from both the phase I and the phase II 
studies who remain disease-free. These studies, together with the increasing evidence that human 
tumor-specific antigens can be recognized by the immune system suggests that specific immune 
responses can be generated against pancreatic adenocarcinoma if the immune system is sufficiently 
primed. 
Analyses of pre-clinical and human pancreatic tumors have recently demonstrated that immune 
checkpoints such as T regulatory cells are infiltrating pancreatic pre-malignancies (PanIN) and 
early pancreatic tumors (Le, Jaffee, Laheru, et al., unpublished data).  Therefore, it will be 

J09100, Protocol Version 22, July 24, 2024 
 
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important to better understand how to deliver a vaccine together with checkpoint inhibitors even 
to patients with potentially curable cancers. The current J0810 clinical study is designed to test 
the hypothesis that metronomic cyclophosphamide will be more effective than single dose 
cyclophosphamide at inhibiting T regulatory cells, and improve pancreatic cancer immune 
responses induced by this allogeneic paracrine cytokine tumor vaccine when compared with 
vaccine alone.  Unlike the prior clinical trials testing our pancreatic cancer vaccine, in the J0810 
study the first immunization of vaccine occurs in the neo-adjuvant setting, two weeks prior to the 
Whipple procedure.  This will allow us to test whether cyclophosphamide is effective at inhibiting 
T regulatory cells within the tumor and whether the early inhibition of T regulatory cells allows 
for the induction of earlier and more potent anti -tumor immune responses.  
 
2.3 Vaccine-based Strategies currently undergoing testing in subjects with pancreatic 
adenocarcinoma 
 
There are a number of vaccine approaches that have been tested or are undergoing testing in 
pancreatic cancer subjects. Most of these approaches target one candidate tumor antigen. Such 
targets include: MUC-1, CEA, and mutated k-ras. The approaches used have included 
immunization with HLA class I and class II peptides, immunization with the whole protein, or 
delivery by antibody, heat shock protein or dendritic cells (Apostolopos & McKenzie, 1994; 
Kabayashi, Terao, & Kawashima, 1992; Brossart, Heinrich, & Stuhler, 1999; Apostolopous et al., 
1997; Abrams et al., 1996). To date, these studies have demonstrated the induction of T cell 
responses. Significant clinical responses have not yet been observed. This may be due to the lack 
of potency of these approaches, to the existence of host mechanisms of immune tolerance, or both. 
More recent pre-clinical studies suggest that combined vaccine approaches integrating vaccine 
with immunomodulatory agents are significantly more effective than vaccines alone in models of 
tumor tolerance (Reviewed by Prendergast and Jaffee, 2007). 
 
2.4 Rationale for the use of a GM-CSF secreting whole cell vaccine approach 
 
We have developed a cytokine secreting tumor vaccine approach that can cure mice of pre-existing 
tumors. This approach is based on the concept that certain cytokines are required at the site of the 
tumor to effectively prime cancer-specific immunity. In the only study to directly compare a large 
number of immune stimulating cytokines, GM-CSF stood out as the most potent cytokine capable 
of inducing systemic anti-tumor immunity when expressed by the tumor cells for the initial 24-72 
hours of immune priming. GM-CSF is now recognized to be the critical growth and differentiation 
factor for dendritic cells, the most potent professional antigen presenting cell (APC) responsible 
for priming immune responses against infectious agents and tumor antigens. Autologous GM-CSF 
secreting vaccines have been tested in phase I and II trials in subjects with melanoma, renal cell, 
prostate, lung, breast and pancreatic cancers. Most of these studies demonstrated evidence of 
immune activation associated with clinical responses in 10-40% of treated subjects (Berns, et al., 
1995; Simons et al., 1999; Soiffer et al., 2003). 
 
While the use of autologous tumor cells may preserve unique antigens expressed by each subject’s 
cancer, the development of an autologous vaccine requires that extensive processing, in vitro 
expansion, and regulatory testing be performed for each individual subject vaccine. These 
limitations preclude the use of autologous cellular vaccine for most cancers including pancreatic 

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adenocarcinoma. A growing body of evidence supports the immunologic rationale for using 
allogeneic tumor cells rather than autologous cells as the source of antigen used for the vaccination. 
First, studies evaluating human melanoma antigens have demonstrated that most of the human 
tumor antigens identified are shared among at least 50% of known human melanoma tumor cell 
lines, regardless of whether or not they share the same human leukocyte antigen (HLA) type (Cox 
et al., 1994; Kawakami, Eliyahu, & Delago, 1994). In addition, there is now both pre-clinical and 
human data in pancreatic cancer subjects treated with a GM-CSF vaccine to support host derived 
professional APCs as the critical cells required to present immunogen to T cells in the context of 
MHC (Dranoff et al., 1993; Jaffee et al, 2001; Thomas et al., 2004). Therefore, the vaccine cells 
do not need to be HLA compatible with the host’s immune system as long as they can release 
cellular proteins (the tumor antigens) for uptake by professional APCs (macrophages and dendritic 
cells) that are attracted to the vaccine site by GM-CSF. Taken together, the data suggest that 
relevant tumor antigens can be delivered by an allogeneic tumor and still sufficiently mount an 
effective immune response. 
 
Two allogeneic cell lines have been developed from neoplastic tissue harvested from the surgical 
specimens of subjects undergoing pancreaticoduodenectomy at The Johns Hopkins Hospital. 
These cell lines have been characterized as 100% epithelial by cytokeratin staining (Jaffee et al., 
1998). In addition, these cell lines carry the same k-ras mutation as the original tumor specimen 
that supports the conclusion that these lines are derived from malignant pancreatic tumor cells. 
The cell lines Panc 10.05 and Panc 6.03 both contain the most common k-ras mutation at codon 
12 found in greater than 90% of pancreatic cancer. These lines secrete GM-CSF at 80-90 ng/ 106 
cells/ 24 hrs for at least 5 days in culture (Jaffee et al., 1998; Jaffee et al., 1993). These lines have 
undergone extensive regulatory testing and have been shown to maintain GM-CSF secretion, MHC 
class I levels, cytokeratin positive staining and the original K-ras mutation (Jaffee et al., 1998). 
These lines also express 2 new immunogenic pancreatic tumor antigens, mesothelin and PSCA. 
These lines have already been demonstrated to be safe and feasible to produce and administer in 
one phase I and three phase II studies in both the adjuvant and metastatic setting. The clinical lots 
for this long term boost study will be manufactured and released by the GMP Cell Processing and 
Gene Therapy Facility at Johns Hopkins. . 
 
2.5 Rationale of using low dose cyclophosphamide as an immune modulator  
 
2.5.1 Introduction:  Immune tolerance and regulatory T cells. 
 
Efficient immunization against cancer requires a vaccine capable of eliciting potent CD4 and CD8 
T cell immune response.  However, tumors have evolved several mechanisms to escape immune 
surveillance, including immune tolerance involving immunosuppressive T lymphocytes (Drake, 
Jaffee & Pardoll, 2006).  Tumors have been shown to induce rapid expansion of CD4+CD25+ 
regulatory T cells (Treg) in human and mice, leading to delayed rejection of immunogenic tumors 
(Liyanage et al., 2002; Woo et al., 2001; Curiel et al., 2004).  Conversely, elimination of these 
Tregs, which constitute 1-3% of the peripheral CD4+ T cell pool in naïve mice, elicited potent 
antitumor immune responses leading to tumor eradication (Shimizu et al., 1999; Sutmuller et al., 
2001).   
 

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Cyclophosphamide has been studied for its role in breaking the immune tolerance.  For decades, 
it is known that low dose and high dose cyclophosphamide have different biological activities 
when it is used as a chemotherapy agent (Motoyoshi, et al., 2006).  High dose cyclophosphamide 
has a cytotoxic activity; whereas, low dose cyclophosphamide has predominantly an immune 
modulating effect.   Cyclophosphamide, as a cytotoxic chemotherapy agent, is used in treating 
multiple types of cancer including breast cancer, lymphoma, leukemia, sarcoma, etc.  In addition, 
cyclophosphamide is also used as an immune suppressor in autoimmune diseases.   
 
2.5.2 Evidence from animal studies supporting low dose cyclophosphamide as an immune 
modulator 
 
Immune modulating doses of cyclophosphamide-containing chemotherapy have been studied in 
mouse tumor models in conjunct with vaccination.  For example, Dr. Jaffee and colleagues have 
been studying the rodent HER-2/neu transgenic mouse model of mammary tumors (neu-N). These 
mice are a clinically relevant model of breast cancer. They develop spontaneous HER-2/neu 
expressing mammary tumors following over expression of the transgene under the murine 
mammary tumor virus (MMTV) promoter in normal mammary tissue. It has been demonstrated 
that vaccination of these mice induces weak HER-2/neu specific B cell and T cell responses that 
are not capable of controlling mammary tumor growth regardless of the vaccine approach.  Dr. 
Jaffee’s group (Ercolini et al., 2005) reported that treatment of these neu-N mice with such doses 
of cyclophosphamide before vaccination resulted in tumor rejection in 10-30% of mice, which 
were otherwise given vaccination alone and would all become immune tolerant to the tumor 
growth.  Interestingly, it was also shown that this vaccine-enhancing effect of cyclophosphamide 
is mediated through selectively inhibiting the cycling population of CD4+CD25+ Treg cells and 
recruiting high-avidity antitumor effector T cells. In addition, since the cyclophosphamide exerts 
its effect on the regulatory T cells of the immune system rather than on the cancer cell itself, this 
approach can be applied to treat any type of cancer. 
 
2.5.3 Rationale for using daily metronomic cyclophosphamide for immune modulation  
 
In previous clinical trials, a single intravenous dose of cyclophosphamide was given one day prior 
to each vaccination.  Such a schedule was found to be effective in enhancing vaccine induced anti-
tumor immune responses by inhibiting regulatory T cell activity in the neu-N mouse model of 
mammary tumors.  In this animal study, a single dose of cyclophosphamide given by injection one 
day prior to vaccination, consistently led to tumor rejection response rates of no more than 30% of 
treated mice.  Although these responses are statistically better than what is observed in mice treated 
with vaccine alone (0% cures), there is clearly room for improvement.  Evaluation of the kinetics 
of Treg depletion following the one dose of cyclophosphamide revealed that about 50% of Tregs 
are depleted by 48 hours, but the population then rebounds reaching normal peripheral blood and 
lymph node levels by 7-14 days after treatment with cyclophosphamide.  These findings raised the 
possibility that the success of cyclophosphamide modulation depends on the long-term depletion 
or inhibition of Treg cell function since the Treg cell population in neu-N mice recovers within 1-
2 weeks of cyclophosphamide treatment.  Repetitive intravenous cyclophosphamide at these doses 
is not a good option because of the concern that cyclophosphamide would induce lymphopenia 
and inhibit the cycling of antigen-specific effector cells after vaccination, thereby abrogating the 
vaccine-induced antigen-specific T cell response.  

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Recent studies have suggested that metronomic low-dose cyclophosphamide may provide more 
prolonged depletion or inhibition of Treg cell function and more effectively synergize with cancer 
vaccines in mouse models (Taieb et al., 2006; Hermans, et al., 2003; Ghiringhelli et al., 2004; 
Lutsiak, et al., 2005).  Metronomic cyclophosphamide has also been tested for its role in anti-tumor 
angiogenesis in advanced human cancer patients (Orlando et al., 2006a; Orlando et al., 2006b; 
Glode et al., 2003; Bottini et al., 2006; Suvannasankha et al., 2007).  Prolonged treatment with 
metronomic cyclophosphamide results in prolonged clinical benefit, and with minimal acute or 
delayed toxicity.  Ghiringhelli et al. (2007) more recently demonstrated that metronomic 
cyclophosphamide as a single agent treatment selectively depletes CD4+CD25+ regulatory T cells 
and restores T and NK effector function in end stage cancer patients.  This study provides the 
rationale for testing recurrent treatment with metronomic cyclophosphamide in sequence with our 
pancreatic cancer vaccine as a valuable combination for reducing tumor-induced immune tolerance 
and synergizing with the anti-tumor effect of the cancer vaccine.     
 
The above studies provide rationales for the ongoing J0810 (neo)adjuvant pancreatic vaccine trial 
to employ the combinatorial treatment with vaccine and cyclophosphamide given at 50 mg twice 
daily for one week on followed by one week off.  This metronomic cyclophosphamide schedule 
was demonstrated to be safe and effective in modulating immune suppressive activity by 
Ghiringhelli et al. (2007).  
 
2.6 Phase I study of lethally irradiated allogeneic pancreatic tumor cells transfected with the 
GM-CSF gene. Phase I study at Johns Hopkins 
 
2.6.1 Summary of Study Design 
 
This study was the first clinical trial to test the hypothesis that allogeneic GM-CSF secreting 
pancreatic tumor cell lines can prime a systemic immune response in subjects with resected 
pancreatic adenocarcinoma. Fourteen subjects with stage 2 or 3 disease received an initial 
vaccination 8 weeks following resection. This was a dose escalation study in which 3 subjects each 
received 1 X 107, 5 X 107, and 1 X 108 vaccine cells. An additional 5 subjects received 5 X 108 
vaccine cells. Study subjects were jointly enrolled in an adjuvant chemoradiation protocol for 6 
months. Following the completion of adjuvant chemoradiation, subjects were re-assessed and 
those who were still in remission were treated with 3 additional vaccinations given one month 
apart at the same original dose that they received for the first vaccination.   
 
2.6.2 Toxicity Events 
 
Toxicities were limited to grade I/II local reactions at the vaccine site, and self-limited systemic 
rashes, including one documented case of Grover's syndrome (Davis, Dineen, Landa et al., 1999). 
The most frequently occurring toxicities of the vaccine were injection site reactions. See Table 1. 
for a summary of the toxicities. All were grade 1 or 2 by the National Cancer Institute (NCI) 
Common Toxicity Criteria. The total number of injection site reactions in the phase I allogeneic 
pancreatic tumor vaccine study was 28 out of 30 (93%) vaccine treatments. Patients who received 
the second, third, and fourth dose levels of the allogeneic pancreatic tumor vaccine all had grade 
2 injection site reactions. Of these grade 2 injection site reactions all had erythema and induration, 

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with 69% (18/26) of the injection site reactions also having local pruritus at the vaccine sites. One 
patient experienced tenderness at the vaccine sites lasting up to three days after the first and third 
vaccine at dose level four. The patient required no analgesics. All injection site reactions were self-
limiting and no one had any limitations on their activities of daily living related to these local signs 
and symptoms. Sixty-four percent (18/28) of the injection site reactions completely resolved within 
a week. Sixty-nine percent (9/13) of the patients who experienced injection site reactions were free 
of all local toxicities within a week. Thirty-one percent (4/13) of the patients experienced injection 
site reactions lasting more than a week with pruritus at the injection sites being the symptom of 
longest duration, lasting up to 41 days. Two of the four patients with the lengthy local toxicities 
also experienced systemic pruritus and rash. One of which was confirmed by biopsy to be Grover’s 
Syndrome. The other person with the systemic rash and pruritus did not have a skin biopsy at that 
time. The same patient who experienced the Grover’s Syndrome also developed recurrent swelling 
at the vaccine sites at that time which was seven to ten days after the second vaccine at level 4. 
After the third vaccine, this patient also had grade 1 lymphedema with erythema and swelling in 
the lymph drainage areas, particularly in the upper right extremity where two or the six vaccines 
were administered. 
 
Systemic toxicities in these patients receiving Mitomycin-C and vaccine included: Grade 1 
musculoskeletal stiffness and generalized pruritus lasting two hours. One patient on dose level 3 
experienced acute anemia (hemoglobin dropped to 6.3 gm/dl), thrombocytopenia (platelets 
dropped to 5,800/mm3), asymptomatic jaundice (total bilirubin increased to 9.8 mg/dl, direct 
bilirubin to 7.8 gm/dl). This occurred approximately six weeks after completing Mitomycin-C 
containing chemotherapy. There was also asymptomatic anemia with hematocrit nadir of 19.8 %, 
hemoglobin nadir 6.6 g/dl and asymptomatic thrombocytopenia with platelet nadir of 77,000 / mm3 
during the chemotherapy course which included Fluorouracil (5-FU), Leucovorin, Mitomycin-C, 
and Dipyridamole (Persantine) prior to this event. These adverse events were consistent with 
Mitomycin-C associated thrombocytopenic purpura (TTP).  Twenty-four days after the second 
vaccine at dose level three, and seven days after the blood transfusions, the same person 
experienced a grand mal seizure. It is unlikely that this adverse event is related to the vaccine.  
After receiving the vaccine this patient developed symptoms that have since been attributed to 
thrombotic thrombocytopenic purpura (TTP). Although we believe this condition was due to the 
Mitomycin-C treatment completed one month previously, this was reported to the IRB as a 
possible adverse event due to the temporal proximity to immunization with the vaccine. The patient 
was taken off study. The patient’s condition improved, the symptoms of TTP resolved, and the 
patient remains in complete remission at this time. This adverse event has not recurred in the more 
than 200 additional patients who have been treated in follow-up vaccine studies with the same 
vaccine. 
 
With dose level four, 50% (3/6) patients experienced a systemic reaction. One patient experienced 
grade 1 constitutional symptom of fatigue and grade 1 musculoskeletal, and achy joints.  Another 
patient on vaccine dose level 4 experienced multiple vaccine related symptomatic toxicities, 
including: a grade 2 rash, systemic pruritus, and Grover’s Syndrome, a grade 1 urticaria, and a 
grade 1 recall induration at the vaccine sites. In conclusion, the main toxicity clearly attributable 
to the vaccine is Grade 1 or Grade 2 and the vaccine seems to be well tolerated by patients 
undergoing their cancer treatment. 

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Table 1. Toxicity Events Associated with Phase I Allogeneic Pancreatic Tumor Vaccine 
 
 
2.6.3 Disease Free Survival 
 
There are three patients who participated in the original study who remain pancreatic cancer 
disease free. The pancreatic cancer disease free survival of patients is defined as the time interval 
from date of diagnosis with adenocarcinoma of the pancreas to the date of radiographic evidence 
of disease recurrence. Only 14 of the 15 patients who participated in this trial were considered for 
the disease free analysis, as one patient had stage 4 disease with liver metastasis prior to entering 
the study. An increase in the disease-free survival was associated with increasing total vaccine 
dose, which is equal to the dose level of cells multiplied by the number of doses received. Using 
the nonparametric correlation of Spearman’s rho the association of total pancreatic tumor vaccine 
dose and disease free survival is statistically significant (p=0.028). Study participants had a 43% 
(6/14) one year disease free survival and 86% (12/14) one year overall survival. Of the fourteen 
patients with resectable disease in this study, three (21%) are still free of pancreatic cancer going 
on 10 years from the date of diagnosis of pancreatic cancer. 
 
2.6.4 Immunologic Data 
 
2.6.4.1 Serum GM-CSF levels 
 
Systemic GM-CSF levels were evaluated as an indirect measure of the longevity of vaccine cells 
at the immunizing site. Serum GM-CSF levels were measured in the patients participating in the 
first pancreatic tumor vaccine study at time 0, 24, 48, 72, 96 and 120 hours following the first 
vaccine. As was observed in pre-clinical studies, GM-CSF levels peaked at 48 hours following 
vaccination.  The peak concentration of serum GM-CSF levels was seen at 48 hours in 83% (5/6) 
of the patients who achieved a measurable serum GM-CSF level. Only one patient who received 
dose level one had a measurable serum GM-CSF level at a minimum level of 1.0 pg/ml at 48 hours. 
There was no measurable serum GM-CSF level in any of the six patients who received dose levels 
two and three at any time point. All five patients who received dose level four had measurable 

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serum GM-CSF levels with the range of 1.2 to 14.0 pg/ml. In addition, serum GM- CSF levels 
could be detected for up to 96 hours following vaccination. GM-CSF levels however became 
undetectable by 120 hours in all patients on study. No side effects other than asymptomatic 
eosinophilia were associated with these low but detectable serum GM-CSF levels.  These data, 
together with data from pre-clinical models, would suggest that detectable serum GM-CSF levels 
may serve as a bio-marker of immune response.  
 
2.6.4.2 Local Immune Reaction of Vaccine Site 
 
The vaccine sites were also evaluated as a measure of the local immune reaction to the vaccine. 
Eleven of 14 subjects demonstrated a similar local inflammatory response to what has been 
observed in pre-clinical models and autologous GM-CSF vaccine clinical trials.  
 
2.6.4.3 Post-Vaccination DTH 
 
Post-vaccination DTH responses to autologous tumor cells have been used in previously reported 
vaccine studies as a surrogate to identify and characterize specific immune responses that are 
associated with vaccination. In this phase I pancreatic cancer vaccine trial, post vaccination DTH 
responses to autologous tumor cells were observed in 1 of 3 subjects receiving 1 X 108 and in 2 of 
5 subjects receiving 5 X 108 vaccine cells (Jaffee et al., 2001). They are the three long-term 
survivors of the initial phase I study and are currently participants in the long-term follow-up study 
(SKCCC J0248/IRB # NA_00036444 [formerly 02-10-14-03]), remain disease-free and 2 subjects 
are receiving additional vaccinations. No additional long-term toxicities have been uncovered in 
this cohort.   
 
2.6.4.4 Mesothelin-Specific T Cell Response 
 
A correlation between the induction of post-vaccination mesothelin-specific T cell responses and 
disease-free survival in these three patients was observed (Thomas et al., 2004).  Consistent with 
their long-term survival and post vaccination DTH responses, CD8+ T cell responses to multiple 
HLA-A2, A3, and A24-restricted mesothelin epitopes were observed exclusively in these three 
patients. Importantly, neither of the vaccinating pancreatic cancer cell lines expressed HLA-A2, 
A3, or A24, therefore providing the first direct evidence that CD8 T cell responses can be generated 
via cross-presentation by an immunotherapy approach designed to recruit APCs to the vaccination 
site.   This result also suggests that the mesothelin-specific T cell response may serve as a better 
surrogate marker for the assessment of vaccine-induced immune response in the future vaccine 
studies.  Thus, in the subsequent phase II study, the role of mesothelin-specific T cell response is 
a primary biomarker of immune response to further validate this marker as a correlate of vaccine 
activity (See Section 2.7 for detail).   
 
2.7 Phase II study of lethally irradiated allogeneic pancreatic tumor cells transfected with 
the GM-CSF gene 
 
2.7.1 Summary 
A follow-up Phase II clinical trial of the pancreatic cancer vaccine in patients who received the 
vaccine after surgical resection of tumor and adjuvant radiation chemotherapy was conducted at 

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Johns Hopkins. All 60 planned subjects on the study completed the treatment phase in June 2006. 
The toxicities associated with the vaccine in this study include: local vaccine site skin reactions 
and systemic rashes similar in severity (grade 1-2) to what was observed in the phase I trial.  There 
were no related serious adverse events observed.  The one-year survival was 86% and the two-
year survival was 61%.  The last vaccination received by patients completing the study was at 18 
months.  The median overall survival was 24.8 months, which compares favorably with a 
previously reported median of 21 months.  A correlation between disease-free survival and the 
induction of mesothelin-specific T cell responses was observed.     
 
2.7.2 Study Design 
 
Sixty research participants received at least one and a maximum of five vaccinations of two 
pancreatic cancer cell lines each delivering 2.5 x 108 cells intradermally distributed among three 
lymph node regions following enrollment into the Johns Hopkins study, IRB # 00-01-13-02 
(SKCCC J9988) titled, A safety and efficacy trial of lethally irradiated allogeneic pancreatic tumor 
cells transfected with the GM-CSF gene in combination with adjuvant chemoradiotherapy for the 
treatment of adenocarcinoma of the pancreas. See Table 2 for the demographic details of this study. 
Enrollment was completed in January of 2005. Vaccine one was administered 8-10 weeks 
following surgical resection. Patients were subsequently treated with 5-fluoruracil (5-FU) 
continuous infusion based chemotherapy integrated with radiotherapy. Research participants who 
remained disease-free one month after completing the chemoradiotherapy received the vaccines 
two through four one month apart. A fifth and final vaccine was administered six months after the 
fourth vaccine to those subjects who remained disease-free. 
 
Table 2. Research Participants’ Demographic Data Characteristic (N=60) 
 
Male  
37  
Female  
23 
Median age  
56.7 
Age range  
41-83 
Node-positive  
52 
Margin-positive  
18 
Node- and Margin-positive  
18 
 
2.7.3 Safety Data 
 
Based on an early analysis, we conclude that the administration of the GM-CSF allogeneic cancer 
vaccine is safe and well tolerated. Treatment related side effects were similar to those side effects 
seen in the phase I study. The most common side effects were vaccine injection site reactions of 
induration and erythema that were transient in all research participants.  In addition, some subjects 
also had transient vaccine injection site reactions of tenderness and pruritus. The systemic 
reactions included transient elevation in eosinophil counts, rashes and flu-like symptoms that have 
included low grade fever, chills, malaise, arthralgias, myalgias, and fatigue. Most patients had a 
transient elevation in their eosinophil count which demonstrates the bioactivitiy of GM-CSF. All 
vaccine related toxicities have been of the same intensity and duration as those observed in the 
phase I study (Jaffee et al., 2001).   

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2.7.4  Survival data 
 
With a median follow up of 24.7 months (Lutz et al., manuscript submitted), median disease-free 
survival is 17.3 months (95% CI: 14.6 – 22.8) with median overall survival of 24.8 months (95% 
CI: 21.2 - 31.6).  While this study was not designed to be directly compared to our surgery 
database, a planned cohort analysis of patients resected at Johns Hopkins who received 
chemoradiation without immunotherapy demonstrated a median overall survival of 20.3 (95% CI: 
18.0 – 23.9) months. The percentage of lymph node-positive patients (most important predictor of 
early recurrence) in this phase II study, 88%,  was higher than those patients enrolled in other 
published PDA adjuvant studies (50-71%).  In addition, 10 patients had a post-resection CA19-9 
> 90 U/ml; whereas similar patients would have been excluded from a recently completed phase 
III (CONKO-1) study comparing 6 months of adjuvant gemcitabine to surgery alone. Nevertheless, 
the median overall survival of 24.8 months compares favorably to two recently published adjuvant 
studies (22 months in the CONKO-1 study and 20.6 months in the RTOG 9704 study)2.  These 
encouraging preliminary clinical data in 60 patients at Johns Hopkins provided the impetus for the 
NCI Gastrointestinal Cancers cooperative group, CALGB, to conduct a follow up multicenter 
study.  The multi-center study of the pancreatic vaccine in the adjuvant setting is under 
development and should begin enrolling by the end of 2009.   
 
2.7.5 Immunologic Data 
 
Peripheral blood lymphocytes (PBL) from two patients demonstrating prolonged disease-free 
survival were used to identify additional mesothelin epitopes by screening 15mer peptides 
overlapping by 10 amino acids covering the entire mesothelin protein sequence.  The PBL used 
for epitope discovery were isolated from an HLA-A0201+ and an HLA-A0101+ patient at 10 and 
22.5 months following treatment completion, respectively.  Both patients were disease free for 
more than 2 years at the time of blood draw.  In total, 8 HLA-A0101-binding and 6 HLA-A0201-
binding mesothelin-derived T cell epitopes have been identified.  IFN ELISPOT analyses were 
performed to measure the frequencies of CD8+ T cells specific for these peptides prior to and 
following each vaccination in the 25 HLA-A0101+ and 23 HLA-A0201+ patients, and to 1 HLA-
A0207+ patient covering 43 of the 60 patients (6 patients expressed both HLA-A1 and HLA-A2).  
These studies showed that CD8+ T cells specific for at least one mesothelin peptide were detected 
following the first vaccine treatment in 38 of the 43 subjects analyzed (Lutz et al. Manuscript 
submitted).  Four of the 5 patients for whom mesothelin peptide-specific T cells were not detected 
following the first treatment recurred before the second scheduled immunotherapy treatment.  In 
addition, when compared to pre-treatment levels, enhanced mesothelin-specific T cell responses 
following the first vaccination were only observed in patients who went on to receive multiple 
treatments.  Furthermore, within the group of patients who received multiple vaccinations, the 
maintenance of enhanced mesothelin peptide-specific responses throughout the course of 
treatment was associated with improved disease-free survival (Figure 3).  We also measured 
responses to negative control peptides (tyrosinase peptides) and a positive control 
CMV/EBV/Influenza A (CEF) peptide pool.  Negative control responses were low or undetectable 
whereas CEF pool responses were detected in all subjects analyzed, but post-vaccination changes 
in CEF pool-specific responses were not detected in any of the groups.  Combined, these data 
suggest that the changes measured were not due to non-specific time point-related differences but 

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reflect actual changes in mesothelin-specific CD8+ T cell responses.  Importantly, these studies 
demonstrated that different patients responded to different epitopes and rarely to the complete 
panel of peptides following the first vaccination.  Surprisingly, enhanced T cell responses were 
observed to an average of approximately 50% of mesothelin peptides following the first 
vaccination in both patients remaining disease-free and patients with progressing disease.  
However, an expansion in the mesothelin epitope-targeted repertoire following boost vaccinations 
was observed almost exclusively in patients who demonstrated longer disease-free survival 
(Figure 4). 
 
Figure 
3. 
Post-immunotherapy 
enhancement of mesothelin-specific 
CD8+ T cell responses correlates with 
disease-free survival.  IFN ELISPOT 
assays were performed to measure the 
frequencies of CD8+ T cells specific for 
mesothelin epitopes in PBL isolated 
from HLA-A1+ and HLA-A2+ patients 
for which pre and post treatment 
lymphocytes were available.  Patients 
were divided into three groups: those 
receiving only one treatment (Single 
Vaccine, 
n=17), 
those 
receiving 
multiple treatments who recurred within 
3 years (DFS < 3yr, n= 18), and those 
receiving multiple treatments who 
remained disease-free for greater than 3 
years (DFS > 3yrs, n=8).  Mesothelin 
responses are reported for the epitope to 
which each individual patient showed a 
maximum response.  Shown for each 
group is the median number of IFN-
secreting CD8+ T cells per 1x106 CD8+ T cells above background measured against irrelevant tumor antigens with the inter-quartile 
range and the maximum value (*).  Responses were measured prior to treatment (Pre-treatment), 14 days following the first 
immunotherapy treatment (Post Vaccine 1) and if given multiple treatments, 28 days following the final immunotherapy treatment 
(Post-treatment). Post-immunotherapy responses were compared to pre-treatment responses using two-tailed Wilcoxon sign-rank 
tests and the calculated p-values are shown. 
Figure 4.  Longer disease-free survival is 
associated with an expansion in the 
CD8+ 
T 
cell 
repertoire 
targeting 
mesothelin epitopes.   IFN ELISPOT 
assays were performed to measure the 
frequencies of CD8+ T cells specific for 
mesothelin epitopes as described in Figure 
1.  Shown are the percentage of mesothelin 
peptides per patient for which a post-
immunotherapy enhancement, defined as a 
2-fold or greater increase in the number of 
IFN-producing peptide-specific T cells, 
was measured following the first (Post 
Vaccine 1) and the final immunotherapy 
treatments (Post treatment, only for 
patients receiving multiple treatments).  
Bars represent the overall percentage of 
peptides for which an enhanced response 
was measured for each group.  Group 
repertoires were compared using logistic  
regression and the calculated p values are 
shown.  
 

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Dilutional tetramer analysis was also performed on PBL isolated prior to and following the first 
and final vaccinations from HLA-A2+ subjects to assess the avidity of T cells specific for each of 
the 6 HLA-A0201-binding mesothelin epitopes.  T cell avidity for each mesothelin peptide was 
plotted against disease-free survival.  Figure 5 shows the relationship between the avidity of 
MesoA2(530-538)-specific T cells and disease-free survival.  For each of the 6 mesothelin peptides 
evaluated, a trend toward improved disease-free survival was observed in patients demonstrating 
higher avidity mesothelin-specific T cells.  In addition, this trend was observed at each time point.  
However, although higher avidity mesothelin-specific T cells were sometimes present in pre-
vaccination PBL, mesothelin-specific IFN responses were frequently not detected until after the 
first vaccination.  Furthermore, in some patients, multiple boosts were required before both high 
avidity T cells and IFN responses could be detected.  These data would suggest that higher avidity 
pre-committed T cells are sometimes already present in resected pancreatic cancer patients that 
can be activated with vaccination.   In addition, the distribution of avidities measured for each 
peptide was used to define cutoffs for distinguishing high from low avidity mesothelin-specific T 
cells.  For this preliminary analysis, DFS>20 months vs. DFS<20 months was chosen as a binary 
clinical outcome parameter.  Figure 6 shows the number of mesothelin peptides for which high 
avidity T cells were detected for each patient evaluated.  Interestingly, a larger repertoire of high 
avidity mesothelin-specific T cells was associated with improved DFS.   
 
Figure 5.  Meso A2(530-538)-specific T cell Avidity vs Disease-
Free Survival.  Dilutional MesoA2(530-538) tetramer analysis was 
performed on PBL isolated a) prior to vaccination, b) 14 days 
following the 1st vaccination or c) 28 days following the final 
vaccination from HLA-A2+ patients.  MesoA2(530-538)-specific T 
cell avidity measured for each patient is shown plotted against 
disease-free survival.  Linear regressions were performed and the 
trendlines are shown. Blue data points were used when 
MesoA2(530-538)-specific IFN responses were also detected.  Red 
data points were used when IFN responses were not detected.  
Dilutions were performed using 1 M tetramer stock solutions.  
 

 
 
 
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Figure 6.  Longer Disease-Free Survival is Associated with 
Larger High Avidity Mesothelin-Specific CD8+ T Cell 
Repertoires.  Dilutional tetramer analysis was performed for 
each of the 6 HLA-A2-binding mesothelin epitopes on PBL 
isolated  prior to vaccination (Pre Vac), 14 days following the 
1st vaccination (Vac 1 Day 14) or 28 days following the final 
vaccination (Post Tx) from HLA-A2+ patients.  Dilutions were 
performed using 1 M tetramer stock solutions.  Patients were 
divided into 2 groups: those who recurred within 20 months 
(< 20 mo DFS) and those who remained disease-free for 
greater than 20 months (> 20 mo DFS). Avidity dilution 
profiles were used to define cutoffs between high and low 
avidity T cells for each peptide.  Shown are the numbers of 
peptides for which high avidity T cells were detected for each 
patient evaluated.  When group sizes were large enough, high 
avidity repertoires were compared between groups using two-
tailed Wilcoxon sign-rank tests and the calculated p-values are 
shown. 
 
2.8 Phase II Study of the GM-CSF 
allogeneic vaccine alone and given in sequence with immune modulating doses of 
Cyclophosphamide in subjects with advanced pancreatic cancer 
 
2.8.1 Summary 
 
As briefly cited above, we have completed a feasibility study of our GM-CSF allogeneic 
vaccine administered alone or in sequence with Cyclophosphamide in subjects with 
advanced pancreatic cancer (Laheru et al., 2008). This study was an open label multi-center 
study sponsored by Cell Genesys, Inc in collaboration with US Oncology.  Subjects were 
enrolled into one of two cohorts: Cohort A- 30 subjects administered a maximum of six 
doses of the same pancreatic cancer vaccine as described above using the two pancreas 
cancer cell lines each delivering 2.5 x 108 cells intradermally administered at 21 day 
intervals; Cohort B- 20 subjects administered cyclophosphamide 250 mg/m2 IV one day 
prior to vaccine as in Cohort A. The primary objective was to evaluate the safety and 
induction of immune responses when treated with either vaccine alone or in sequence with 
cyclophosphamide. Secondary objectives include time to disease progression (TTP) and 
median overall survival (OS).  
 
2.8.2 Safety and Efficacy Data 
 
We have reported the following findings (Laheru et al., 2008): 
 
1. The administration of a GM-CSF allogeneic pancreatic cancer vaccine is safe both alone 
and when given in sequence with cyclophosphamide. It is well-tolerated by patients with 
advanced pancreatic cancer, and the majority of these patients had received two or more 
prior chemotherapy regimens. The median number of vaccines administered was 2 in 
Cohort A and 3 in Cohort B. Treatment related adverse events reported in > 5% of subjects 
included local vaccine injection site reactions (100%), fever (14%), rigors (10%) and rash 
(6%). Grade 3/4 treatment related events identified in only one JHU subject and included 
leukocytosis, dehydration, and fatigue. 
 

 
 
 
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2. Serum GM-CSF levels peaked at 48 hours post vaccination consistent with published 
results in the adjuvant setting and was seen following repeated vaccination, suggesting that 
vaccine cells are not rapidly cleared by an allogeneic response with repeat administration. 
 
3. Stable disease was noted in 16.7 % of subjects in Cohort A (vaccination alone) and 40% 
of subjects in Cohort B (vaccination plus cytoxan).  Median survival in Cohort A and 
Cohort B were 2.3 months and 4.7 months respectively in a subject population that had 
received ≥ 2 prior chemotherapy in 12/20 subjects for Cohort B and in 30/50 subjects 
overall. 
 
2.8.3 Immunologic Data 
 
Immune analyses in a subset of patients with available lymphocytes isolated at a number 
of time points following multiple immunizations demonstrated a correlation of increased 
mesothelin-specific CD8+ T cell responses with progression-free survival (Table 3). CD8+ 
T cell response to HLA restricted mesothelin epitopes were augmented post cycle 3 and 6 
of the therapy predominantly in patients treated with Cytoxan + vaccination  (9 of 10) as 
compared with patients treated with vaccination alone (4 of 8).  CD8+ T cell responses were 
detected against the positive control CMV/EBV/Influenza A (CEF peptide) pool in all 
patients treated in both Cohorts.  While the analysis to date is limited to HLA- A1+, -A2+ 
and -A3+ patients, median survival from this subset of patients with induction or 
enhancement of mesothelin-specific T cell responses treated with immunotherapy alone is 
7.6 months versus 10.4 months for patients treated with cyclophosphamide + 
immunotherapy.   
  
Table 3. Summary of mesothelin-specific T cell responses in the HLA-A1+/ HLA-A2+/ 
HLA-A3+ patients treated with the vaccine alone or in sequence with Cytoxan 
 
 

 
 
 
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In addition, there was a trend toward prolonged progression-free survival in those patients 
who demonstrated persistent mesothelin-specific T cell responses with therapy.   In this 
same study, mesothelin-specific T cells from a subset of HLA-A2+ subjects were assessed 
for avidity by dilutional HLA-A2/Meso(20-28) and HLA-A2/Meso(530-538) tetramer analysis 
and the data are summarized in Table 4.  As an example, tetramer analysis of cohort B 
subject 7 showed an increase in the frequency and avidity of mesothelin-specific T cells in 
post-treatment PBL compared to PBL isolated prior to treatment.  In contrast, a decrease 
in post-treatment mesothelin-specific T cell frequency and avidity was measured in cohort 
B subject 8.  Whereas changes in frequencies of mesothelin-specific T cells were detected, 
changes in frequencies of T cells specific for tyrosinase, an irrelevant melanoma antigen, 
were not detected in any of the 9 subjects evaluated (Laheru et al. 2008).  This suggests 
that the changes measured were not due to time point-related differences in non-specific 
tetramer staining.   Although the analysis was performed on a small number of subjects, it 
is interesting that the post-treatment MesoA2(530-538) tetramer titration was associated with 
overall survival.  In the same cohort B, subject 7 had a survival of 13.07 months whereas 
subject 8 only had a survival of 3.7 months (Table 4).  Importantly, these tetramer changes 
appear to be antigen specific since time point differences in tyrosinase tetramer titrations 
were not observed.   
 
Table 4.  Summary of HLA-A2 tetramer titrations in a subset of HLA-A2+ patients 
treated with the vaccine alone or in sequence with cyclophosphamide. 
 
 
 
2.9 A randomized three-arm neoadjuvant and adjuvant feasibility and toxicity study 
of a GM-CSF secreting allogeneic pancreatic cancer vaccine administered either 
alone or in combination with either a single intravenous dose or daily metronomic 
oral doses of cyclophosphamide for the treatment of patients with surgically resected 
adenocarcinoma of the pancreas 
 
At present, the JHMI IRB approved J0810 study titled, a randomized three-arm 
neoadjuvant and adjuvant feasibility and toxicity study of a GM-CSF secreting allogeneic 
pancreatic cancer vaccine administered either alone or in combination with either a single 
intravenous dose or daily metronomic oral doses of cyclophosphamide for the treatment of 
patients with surgically resected adenocarcinoma of the pancreas, is recruiting 39 research 
participants, who are randomized into three arms. All participants are receiving the first 

 
 
 
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vaccination of 5 x 108 cells of a equal mixture of two allogeneic GM-CSF secreting 
pancreatic vaccine cell lines (Panc 10.05 and Panc 6.03) two weeks before a 
pancreaticoduodenectomy, a second vaccination between 6 and 10 weeks following the 
pancreaticoduodenectomy (4 weeks prior to adjuvant chemoradiation), and then four 
additional vaccinations once every 28-days beginning 1-2 months following completion of 
chemoradiation. A total of six prime vaccinations will be administrated. For participants in 
Arm B and Arm C, each vaccination is combined with a single low-dose cyclophosphamide 
or repetitive twice-daily metronomic doses of cyclophosphamide, respectively. The 
vaccine dose is the same dose found to be safe and to induce immune responses in the 
above described phase I and II studies.   
 
Since approval in July 2008, we have enrolled and treated 10 patients (between 7/30/08 
and 12/31/08). All ten patients enrolled into the study have received their first (neoadjuvant) 
cycle of treatment and underwent tumor resection (Table 5).  Because a biopsy-proved 
diagnosis is not routinely obtained as part of standard of care prior to the surgery, this study 
does not require a biopsy for the study entry.  With the improvement of pre-operative 
imaging techniques, the misdiagnosis of PDA is uncommon.  Nonetheless, the participants 
were informed that there is a small chance of having a tumor different from PDA. Only 
one patient turned out to have ampullary adenocarcinoma and was subsequently removed 
from the study.  Ampullary cancers are not eligible for this vaccine study because they 
have a different biology and likely different tumor antigens than those expressed by our 
vaccines which derive from PDA of the pancreatic head and neck.  Another three patients 
did not meet eligibility criteria for the study continuation due to disease recurrence 
 
and unresolved post-operative complications at the time of screen visits prior to the second 
vaccination cycle 
  Such a drop-off rate is anticipated.  Therefore, six 
patients remain in the study, and three of them have gone far enough to receive the second 
vaccination.  We estimate that this study needs to recruit an additional 50-54 patients for 
the accrual of 39 evaluable patients during the grant award period.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
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24 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
No serious adverse events were observed in these patients as a result of vaccine therapy 
with or without cyclophosphamide.  Similar to prior vaccine studies, all subjects 
experienced grade I/II local reactions at the vaccine site.  Systemic reactions rarely 
occurred (Table 5) and were self-limiting.  Systemic lymphopenia, which is anticipated to 
result from the cyclophosphamide treatment, occurred in two patients and was resolved 
within one week and prior to the surgery.  No patients’ surgery was delayed because of the 
neoadjuvant treatment.  Thus, the preliminary results suggest that the PDA vaccine and/or 
immune modulating doses of cyclophosphamide is safe and feasible to give prior to 
surgical resection of PDA.   In addition, these preliminary data demonstrate the feasibility 
of enrolling and completing this study in an acceptable time frame.  
 
Building on our prior experience in clinical development of our pancreatic cancer vaccine, 
we anticipate that the J0810 study will demonstrate the safety and feasibility of giving the 
vaccine in the neoadjuvant setting and in combination with immune modulating doses of 
cyclophosphamide.  We also anticipate that vaccine-induced immune response is 
associated with an improved survival of patients.  We are designing this trial as the follow-
up study of the same subjects in the J0810 study and designed to test the hypothesis that 
boosting with the combinatorial treatment of vaccine and immune modulating doses of 
cyclophosphamide results in durable suppression of Tregs and consequently induces more 
durable immune responses that are associated with a continuous pancreatic cancer free 
survival. 
 
2.10 Rationale of boost vaccinations and the long term treatment of immune 
modulating doses of cyclophosphamide 
 
Published studies and our immune analysis with previous vaccine studies suggest that 
antigen-specific T cell response is often not sustained after the last prime vaccination and 
that diminishing T cell response may be associated with early disease recurrence.  We have 
recently completed the accrual of the J0619 study which is testing the safety and efficacy 
of boosting the patients who remain disease free in our phase I and phase II vaccine studies 
and who have received four vaccinations in the J0619 study.   The preliminary safety 
analysis suggests that boost vaccinations are safe with low grade local and systemic 
toxicities that have been observed with prime vaccinations.  The preliminary immune 
analysis also suggests that sustained immune response is observed in patients who are 
receiving boost vaccines.  
 
A more intriguing question is whether sustained Treg suppression is required for sustained 
T cell response.  It is believed that suppression on Treg will diminish after metronomic 
cyclophosphamide is stopped.  Our preclinical model showed that Tregs start to bounce 
back in one week following the treatment of one single intravenous low-dose 
cyclophosphamide (Ercolini et al., 2005); and in patients, peripheral Tregs started to 

 
 
 
J09100, Protocol Version 22, July 24, 2024 
 
25 
bounce back in two months after the completion of daily oral metronomic 
cyclophosphamide (personal communication).   It thus will be important to explore whether 
the avidity and quantity of mesothelin T cell response start to drop after the treatment of 
vaccine and cyclophosphamide is completed through the prime vaccination protocol 
(J0810).  Additionally, we wish to discern whether long-term intermittently administrated 
low-dose cyclophosphamide can reactivate T cells and whether low dose 
cyclophosphamide can further induce T cell response in combination with boost 
vaccinations.   
 
Long term treatment with metronomic cyclophosphamide has been widely tested in clinical 
trials for a variety of metastatic cancer diseases and demonstrated to be safe.  It has been 
tested as a daily maintenance therapy in combination with gleevec in treating patients with 
Gastrointestinal stromal tumor (GISTs) and also demonstrated to be safe and well tolerated 
(Personal communication).  Long-term metronomic cyclophosphamide is also used in the 
rheumatology clinic for patient with certain conditions of vasculitis.   
 
The suppressive effect of one-month metronomic cyclophosphamide on Tregs was 
reported. It is however not known what is the optimal duration of metronomic 
cyclophosphamide treatment for Treg suppression prior to each vaccination.  In this study, 
for Arm C participants, metronomic cyclophosphamide will be given for one month to 
achieve Treg suppression prior to each boost vaccination.  Thus, metronomic 
cyclophosphamide will be continued for one more month post vaccination.   
 
In this boost vaccine study, metronomic cyclophosphamide will be given to Arm C 
participants at 50 mg daily, instead of 50 mg twice daily one week on and one week off.  
The total monthly accumulated dose of cyclophosphamide remains to be same and is 
thought to be equivalent in immune modulation.  The 50 mg daily regimen is recommended 
in the design of immunotherapy studies of other malignant diseases (personal 
communication).  Such a regimen is also the one that was most frequently used in published 
non-immunotherapy cancer treatment studies.   Importantly, its convenience will make 
participants more compliant with the long-term treatment.    
 
2.11 Rationale for Immunobiologic Endpoints  
 
The major limitation to developing cancer vaccines has been the lack of identified 
pancreatic tumor antigens that are the known targets of the immune response. As such, 
current immune based approaches either target a small group of candidate antigens 
expressed by the tumor or rely on whole tumor cells as the immunogen. However, with the 
recent sequencing of the human genome and the development of rapid methods for 
identifying genes that are differentially expressed by tumor cells (Iacobuzio-Donahue, 
Maitra, Shen-Ong et al., 2002), potential candidate immune targets are being discovered 
that may serve as immunogens for treatment as well as prevention. 
 
Mesothelin, a transmembrane glycoprotein member of the mesothelin/megakaryocyte 
potentiating factor (MPF) family was identified by differential gene expression to be over 
expressed by most pancreatic adenocarcinoma (Argani, Iacobuzio-Donahue, Ryu et al., 

 
 
 
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26 
2001).  In the phase I GM-CSF pancreatic cancer vaccine study, we observed a post-
vaccination induction of mesothelin-specific T cell responses in uncultured CD8+ T cells 
isolated from the three subjects who were long-term survivors after vaccination. However, 
the patients who relapsed, we did not observe evidence of mesothelin recognition in post-
treatment lymphocytes (Thomas et al., 2004). These data suggest that mesothelin may serve 
as a biomarker of vaccine-induced T cell responses.  Finally, follow up data from our 
recently completed phase II adjuvant clinical trial provides further support that the post-
vaccination induction of mesothelin specific T cell responses is associated with improved 
disease-free and overall survival in treated patients (see section 2.7.5)  
 
Therefore, in this study, we will further evaluate the mesothelin specific T cell response as 
a biomarker of vaccine induced immune response.  Specifically, we will compare the 
effects of vaccine boosts alone versus vaccine boosts given with either a single dose of 
intravenous cyclophosphamide or daily metronomic cyclophosphamide on changes in the 
number and function of peripheral mesothelin-specific CD8+ T cells.    Three parameters 
of vaccine induced mesothelin-specific T cell function will be assessed in this study as well 
as their association with disease-free and overall survival:  1) T cell number and cytokine 
expression; 2) T cell avidity; and 3) T cell repertoire changes. For exploratory purpose, we 
will assess whether sustained T cell function, measured by the repertoire and avidity of 
mesothelin specific CD8+ T cells, is associated with long-term combinatorial treatment 
with immune modulating doses of cyclophosphamide, and whether it is associated with 
prolonged survival. 
 
3.0 Study Design and Treatment Plan 
 
3.1 Study Overview 
 
This vaccine boost trial will evaluate an equal mixture of two allogeneic GM-CSF secreting 
pancreatic vaccine cell lines, Panc 10.05 and Panc 6.03, in addition to low dose 
cyclophosphamide either as a single intravenous dose or as multiple metronomic oral doses 
for: (1) safety of administration, (2) disease-free and overall survival in patients with 
resected adenocarcinoma of the head, neck, or uncinate of the pancreas, (3) induction of 
mesothelin-specific T cell immune responses, and (4) number and function of Treg cells 
systemically.  
 
Candidates for this study will include the following patient Cohorts who have a history of 
surgically resected pathologic stage 1 (no direct tumor extension beyond pancreas and no 
regional lymph node metastases), 2a (direct extension of tumor beyond pancreas), and/or 
2b (regional lymph node metastases) adenocarcinoma of the head, neck, tail, or uncinate 
of the pancreas: 
• Cohort 1: Patients who have previously participated in the study titled “A 
randomized three-arm, neoadjuvant and adjuvant, feasibility and toxicity study of 
a GM-CSF secreting allogeneic pancreatic cancer vaccine administered either alone, 
or in combination with either a single intravenous dose, or daily metronomic oral 
doses of cyclophosphamide for the treatment of patients with surgically resected 
adenocarcinoma of the pancreas” [J0810, NA_00015858 (formerly 00-01-58-58)], 

 
 
 
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27 
have no radiographic evidence of pancreatic disease recurrence, and have received 
the GM-CSF secreting allogeneic pancreatic cancer vaccine.  
• Cohort 2: Patients who never received any type of pancreatic vaccine or 
immunotherapy, had the Whipple surgery within 18 months and completed the 
planned adjuvant chemotherapy and/or chemoradiation. Have no radiographic 
evidence of pancreatic cancer disease recurrence. Have not received any anti-cancer 
therapy in the past 28 days. 
• Cohort 3: Patients who have previously participated in the study titled “A 
Randomized Study of a GM-CSF secreting allogeneic pancreatic cancer vaccine 
with or without a PD-1 Blockade Antibody (Nivolumab) for the Neoadjuvant and 
Adjuvant Treatment of Patients with Surgically Resectable Adenocarcinoma of the 
Pancreas” (IRB00050517, J1568), have no radiographic evidence of pancreatic 
disease recurrence, and received their sixth vaccine within 12 months of enrolling 
in J09100. 
• Cohort 4: Patients who have previously participated in the study titled “A Phase II 
Study of GM-CSF secreting allogeneic pancreatic cancer vaccine in combination 
with PD-1 Blockade Antibody (Pembrolizumab) and Stereotactic Body Radiation 
Therapy (SBRT) for the Treatment of Patients with Locally Advanced 
Adenocarcinoma of the Pancreas” (IRB00083132, J15237), have no radiographic 
evidence of pancreatic disease recurrence, and received their last vaccine in the 
extended treatment phase within 12 months of enrolling in J09100. 
• Cohort 5: Patients who have previously participated in the study titled “A Pilot 
Study of a GVAX Pancreas Vaccine (with Cyclophosphamide) in Combination 
with a PD-1 Blockade Antibody (Pembrolizumab) and a Macrophage Targeting 
Agent (CSF1R inhibitor) for the Treatment of Patients with Borderline Resectable 
Adenocarcinoma of the Pancreas” (IRB00130267, J1766), have no radiographic 
evidence of pancreatic disease recurrence, and received their last vaccine in the 
extended treatment phase within 12 months of enrolling in J09100. 
 
All patients must meet the eligibility criteria including having no radiographic evidence of 
disease recurrence at the first vaccination and every semi-annual vaccination on this 
protocol. 
 
All participants will receive 5 x 108 cells of an equal mixture of two allogeneic GM-CSF 
secreting pancreatic vaccine cell lines Panc 10.05 and Panc 6.03 divided into six 
intradermal injections. The first vaccine boost on this study will be given no less than six 
months (+/- 1 month) after the last vaccination from the prior study. The vaccine boosts 
will then be administered every six months (+/- 1 month) until 5 years have passed and 
then every 12 months (+/- 1 months) until ten years have passed for Cohorts 1 and 2 or five 
years have passed for Cohorts 3, 4 and 5, the subject no longer meets the eligibility criteria, 
no longer wishes to participate in the study, or the vaccine supply is exhausted. If the last 
vaccination date from the prior trial has occurred more than one year ago, new semi-annual 
dates for vaccine boosting may be established.  
 
In this study, research participants from Cohort 1 will remain on the same study arm as the 
prior study. Therefore, Arm A participants will receive the pancreatic cancer vaccine alone. 

 
 
 
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28 
Arm B participants will be vaccinated and receive a single low-dose of cyclophosphamide 
(200 mg/m2) intravenously one day prior to vaccination. Participants in Arm C will receive 
cyclophosphamide 50 mg once a day starting 28 days prior to day 1 of vaccination until 28 
days post vaccination. Research participants from Cohorts 2-5 will be vaccinated and 
receive a single low-dose of cyclophosphamide (200 mg/m2) intravenously one day prior 
to vaccination. The vaccine dose is the same dose found to be safe and to induce immune 
responses in the phase I and II studies. 
 
If patients are also on protocol SKCCC J0248/IRB # NA_00036444 (formerly 02-10-14-
03), titled “Long term follow-up of patients who received lethally irradiated pancreatic 
tumor cells transfected with the GM-CSF gene”, the J0248 protocol will be considered 
inactive for them until they are off this protocol. Thus, assessments and tests included in 
this protocol would not be duplicated.  

 
 
 
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29
3.2 Study Schedule Checklist 
 
Table 6. Prime vaccination phase for Cohort 2 
 
Pre-
study 
Each prime vaccination cycle 
Day 30 
to 
Day 0 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 
Eligibility assessment 
Informed consent 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Inclusion/Exclusion1 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Pregnancy test 
X2 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Performance status 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Safety assessment 
Vital signs 
X 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical exam 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Toxicity assessments 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Laboratory test 
Hematology3 
X 
X5 
 
 
 
 
 
 
 
X6 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X8 
Comprehensive4 
X 
X5 
 
 
 
 
 
 
 
X6 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X8 
CA 19-9 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Amylase 
X 
X5 
 
 
 
 
 
 
 
X6 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X8 
Leukapheresis or blood 
draw (200 cc) for 
immune monitoring 
X 
X5 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X8 
Blood draw for serum 
banking (30 cc) 
X 
X5 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X8 
Efficacy Assessments 
CT chest/abd/pelvis7 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Treatment 
IV cyclophosphamide 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Vaccine9 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
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In order to minimize the need for research-only in-person visits, telemedicine visits may be substituted for in-person clinical trial visits 
or portions of clinical trial visits where determined to be appropriate and where determined by the investigator not to increase the 
participants risks. Prior to initiating telemedicine for study visits the study team will explain to the participant, what a telemedicine visit 
entails and confirm that the study participant is in agreement and able to proceed with this method. Telemedicine acknowledgement will 
be obtained in accordance with the Guidance for Use of Telemedicine in Research. In the event telemedicine is not deemed feasible, the 
study visit will proceed as an in-person visit. Telemedicine visits will be conducted using HIPAA compliant method approved by the 
Health System and within licensing restrictions. 
 
1 Review eligible criteria for continuation. 
2 For women of childbearing potential 
3 Heme-8 with differential including absolute eosinophil count, absolute neutrophils, absolute lymphocytes 
4 Comprehensive chemistry panel including electrolytes, BUN, creatinine, SGOT, SGPT, total bilirubin, alkaline phosphatase.  
5 For prime vaccinations #2-3 only and if not done within 7 days. 
6 Can be done on day 8+/-1.   
7 Can be done within 30 days 
8 Done after prime vaccine 3 only, within +/- 7days. 
9 Prime vaccine can be administered within 28 +/- 4 days. 

 
 
 
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Table 7. Boost vaccination phase for Cohorts 1-5 
 
 
Pre-study1 
Pre-Cycle2 
Arm 
C 
Only 
Each boost vaccination cycle 
Off 
Study 
15 
D-27 
to  
D-1 
0d 1d 2d 3d 4d 5d 6d 7d 8d 9d 10d 11d 12d 13d 14d 15d 16d 17d 18d 19d 20d 21d 22d 23d 24d 25d 26d 27d 28d 29d 
Eligibility assessment 
Informed 
consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Inclusion/ 
Exclusion 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Medical history X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Pregnancy test3 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Performance 
status 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Safety assessment 
Vital signs 
X 
 
 
X8 X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Physical exam 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Review interval 
oncology notes4 X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Toxicity 
assessments 
X X5 
 
X8 X9 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Laboratory test 
Hematology6 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X13 
X 
Comprehensive7 X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X13 
X 
CA 19-9 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 

 
 
 
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32
 
Pre-study1 
Pre-Cycle2 
Arm 
C 
Only 
Each boost vaccination cycle 
Off 
Study 
15 
D-27 
to  
D-1 
0d 1d 2d 3d 4d 5d 6d 7d 8d 9d 10d 11d 12d 13d 14d 15d 16d 17d 18d 19d 20d 21d 22d 23d 24d 25d 26d 27d 28d 29d 
Amylase 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X13 
X 
Efficacy Assessments 
CT chest/ 
abd/pelvis14 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Treatment 
IV CY Cohort 1 
(Arm B) and 
Cohorts 2-511 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Daily 
metronomic CY 
for Arm C of 
Cohort 112 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
Vaccine 
 
 
 
 
X10 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Tetanus toxoid 
boost16 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Archival Tumor 
Tissue17 
X 
 
 
In order to minimize the need for research-only in-person visits, telemedicine visits may be substituted for in-person clinical trial visits 
or portions of clinical trial visits where determined to be appropriate and where determined by the investigator not to increase the 
participants risks. Prior to initiating telemedicine for study visits the study team will explain to the participant, what a telemedicine visit 
entails and confirm that the study participant is in agreement and able to proceed with this method. Telemedicine acknowledgement will 
be obtained in accordance with the Guidance for Use of Telemedicine in Research. In the event telemedicine is not deemed feasible, the 
study visit will proceed as an in-person visit. Telemedicine visits will be conducted using HIPAA compliant method approved by the 
Health System and within licensing restrictions. 
 

 
 
 
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1Pre-Study visit to be done at least one month after candidate’s last cancer treatment and up to thirty days before participants receive their first boost 
treatment (cytoxan or vaccine, whichever is administered first).  The first boost vaccination must be no earlier than 6 months and no later than 18 
months after the last prime vaccine.  
2For Cycle 2 and thereafter, to be done within 30 days before participants receive the next boost treatment.   
3For women of childbearing potential 
4Reviewing pertinent interval local oncology notes including laboratory test and CT scan reports.  
5Clinic visits not required.  Research nurse calls patients to assess the toxicity on the phone. It should be done no less frequently than the following 
occasions: 2 months +/-15 days, 4 months +/- 15 days after the last boost treatment and within 30 days before the next boost treatment. Based on 
toxicity assessment, clinic visits and laboratory/imaging tests may be recommended as per standard of care and as clinically indicated.     
6Heme-8 with differential including absolute eosinophil count, absolute neutrophils, absolute lymphocytes. 
7Comprehensive chemistry panel including electrolytes, BUN, creatinine, AST, ALT, total bilirubin, and alkaline phosphatase. 
8For Cohort 1 (Arm B) and Cohorts 2-5.  Vital signs will be taken before IV cyclophosphamide administration.  For Cycle 2 and thereafter, toxicity 
assessment will be assessed prior to IV cyclophosphamide administration. 
9For all Cohorts and all cycles, toxicity will be assessed after the vaccination.  For Cycle 2 and thereafter and for Cohort 1 (Arm A and C), toxicity 
will also be assessed prior to the vaccination.   
10In general, 6 +/-1 months should be between two boost vaccinations. However, if participants do not meet eligibility criteria due to a reversible 
cause, vaccination can be postponed until they become eligible. Vaccination may also be postponed for other reasons at the discretion of the study 
team.  The interval between two boost vaccinations is not recommended to be shorter than 5 months or more than one year. In the event of persistent 
vaccine-related responses, the semi-annual boost vaccination may be delayed up to one year after the last vaccine-related response.  
11For Cohort 1 (Arm B) and Cohorts 2-5, if a patient has received IV cytoxan, but not received the vaccine on the following day for some reasons, 
IV cytoxan may be repeated again one day prior to the rescheduled vaccination only after the event has been discussed between PI and the study 
team, given that the patient remains eligible. The repeated dose of IV cytoxan must await at least 7 days after the original dose.   
12For Cohort 1 (Arm C), vaccination should be scheduled on the day when the participant has taken 28 +/- 4 days of oral cytoxan.  If the oral cytoxan 
is stopped with intention, it may be resumed only after the event has been discussed between PI and the study team, given that this participant 
remains eligible. The protocol recommends that the numbers of days of oral cytoxan prior to and post each vaccination are added together to be 56.  
However, if a participant misses a dose of cytoxan accidentally and without an intention, the missed dose will not be re-administrated, and a deviation 
should be recorded.  The day of vaccination is always set as Day 1.       
13To be completed on day 29 (+7/- days) after each boost vaccination for Cohort 1 (Arms A and B) and Cohorts 2-5, and within 7 days after 
completing the last dose of cytoxan for Cohort 1 (Arm C). 
14Can be done within one month before participants receive study treatment (cytoxan or vaccine, whichever first). If allergic to the CT scan contrast, 
a non-contrast CT of the chest and a MRI of the abdomen and pelvis will be obtained. Note: Participants who have been for 5 years and more since 
the pancreatic surgery can have either CT scan of chest, abdomen and pelvis with IV contrast or MRI of abdomen and pelvis done at the discretion 
of the PI of the study following the same protocol. If MRI of abdomen and pelvis is chosen, a non-contrast CAT scan of the chest should be done. 

 
 
 
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15Can be done 28 days +/- 3 days of the twentieth boost vaccination cycle or within four weeks of the date when the patient is considered to be off 
study.  Patients are considered off study beginning on 28 days after the last study treatment of the 20th boost vaccination cycle or on the day when 
the patient is considered to be off study prior to the twentieth vaccination. 
16Tetanus toxoid boost if last tetanus vaccination > 10 years. Tetanus/diphtheria (Td) may be substituted if tetanus alone is not available. If both are 
not available, no tetanus vaccine will be administered. 
17See Section 3.6.3. Archival tissue may be collected at any point after consent. 

 
 
 
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35 
3.3 Study Population 
 
One cohort of eligible patients are recruited from those who had a surgically resected and 
pathologically proved AJCC stage I or stage II adenocarcinoma of the head, neck, or 
uncinate of the pancreas (see Section 3.2.1.1 for staging criteria) and who participated in 
the JHMI J0810 three-arm neoadjuvant and adjuvant pancreatic cancer vaccine trial and 
who have no radiographic evidence of pancreatic cancer disease recurrence (Cohort 1).   
 
We will recruit 52 eligible patients to a second cohort from vaccine-naïve patients who had 
a surgically resected and pathologically proved AJCC stage I or stage II adenocarcinoma 
of the head, neck, or uncinate of the pancreas (see Section 3.2.1.1 for staging criteria), and 
who completed adjuvant chemotherapy and/or radiation, and who have no radiographic 
evidence of pancreatic cancer disease recurrence (Cohort 2). 
 
We are also recruiting patients from prior Johns Hopkins SKCCC pancreatic cancer 
vaccine protocols J1568,  J15237 and J1766 (Cohorts 3, 4 and 5). Patients will be eligible 
for this arm of the study if they had a surgically resected adenocarcinoma of the pancreas 
(see Section 3.3.1.1 for staging criteria), have completed the primary vaccines on the initial 
protocol, and have no radiographic evidence of pancreatic cancer disease recurrence. 
 
Participants will need to be off all anti-cancer therapy for at least 28 days. In the event that 
a research participant has evidence of persistent vaccine-related responses (including, but 
not limited to: vaccine site flares such as recurrent erythema, induration, and pruritus at 
previous vaccine administration sites; urticaria) occurring at the frequency of more than 
once in the previous three months, the research participant may choose to delay the semi-
annual boost vaccination for up to one year after the last vaccine-related response. The 
research participant may also choose to continue to receive the semi-annual boost 
vaccinations per protocol with evidence of persistent vaccine-related responses. 
 
3.3.1 Eligibility Criteria 
 
Eligibility to receive a vaccination must be determined with the first vaccination (the first 
prime for the Cohort 2 or the first boost vaccine for Cohorts 1, 3, 4 and 5) and then again 
with each semi-annual vaccination by the Principal Investigators or their designee prior to 
the administration of the research product. No repeated evaluation of eligibility will be 
done with the second and third prime vaccination for Cohort 2. However, the second and 
third prime vaccinations may be held at the discretion of the investigator teams or if the 
patient meets the off study criteria.  If the eligibility criteria for vaccination are not met, 
the research participant may be re-evaluated if the Principal Investigators anticipates that 
the research participant may later meet the eligibility criteria. There is no time limit.  
 
3.3.1.1 Inclusion Criteria for Vaccinations 
 
Research participants must meet the following criteria: 
 

 
 
 
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36 
1. Have a history of surgically resected and pathologically proven adenocarcinoma of the 
pancreas. Please refer to individual protocols for specific resection criteria. 
  
2. Cohorts 1, 3, 4 and 5: Have been a participant in Hopkins IRB protocol J0810, J1568, 
J15237 or J1766.   
 
3. Cohort 2: Have never received any type of pancreatic cancer vaccine/immunotherapy, 
had the Whipple surgery within 18 months and completed the planned adjuvant 
chemotherapy and/or chemoradiation.  
 
4. Cohorts 1, 3, 4 and 5: Received the last irradiated GM-CSF transfected allogeneic 
pancreatic cell lines Panc 10.05 and Panc 6.03 at least 6-12 months prior.  
 
5. Received the last anti-cancer therapy at least 28 days ago.  
 
6. Have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. 
 
7. Have provided informed consent. 
 
8. Have adequate hematologic function (Hemoglobin  9 gm/dl, ANC ≥ 1500/cu mm,  
platelets ≥ 100,000/cu mm, Absolute lymphocyte count  500/ cu mm)  
 
9. Have adequate renal function (Serum creatinine ≤ 2 mg/dl) 
 
10. Have adequate hepatic function (Bilirubin ≤ 2.0 mg/dl, unless known Gilbert's 
Syndrome; AST, ALT and amylase ≤ 2x upper limit of normal:  Alk Phos ≤ 5x upper 
limit of normal.)  
 
11. Agree to use adequate birth control, if of childbearing potential. 
 
3.3.1.2 Exclusion Criteria for Vaccinations 
  
Research participants with any of the following will be excluded from study entry:  
  
1. Radiographic evidence of pancreatic cancer recurrence.  
 
2. Documented history of certain autoimmune diseases such as systemic lupus 
erythematosus, sarcoidosis, rheumatoid arthritis, glomerulonephritis, or vasculitis.  
 
3. Uncontrolled medical problems.  
 
4. Systemic steroid therapy within 28 days before vaccine administration with the 
exception of steroids utilized for the purpose of premedication for contrast CT.  
 
5. Anticipated need for systemic steroid therapy within 28 days after vaccine 
administration.  

 
 
 
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37 
 
6. Evidence of active infections.  
 
7. Pregnant.  
 
8. Have been diagnosed with another cancer or myeloproliferative disorder in the past 5 
years whose natural history or treatment has the potential to interfere with safety or 
efficacy assessment of this study’s investigational drugs. 
 
9. History of noncompliance during previous vaccination cycles with study treatment 
and/or monitoring which is concerning for continued noncompliance.  
 
3.3.1.3 Staging Information 
 
Staging criteria are from the “American Joint Committee on Cancer (AJCC) Staging 
Criteria for Pancreatic Cancer Version 7.” Patients with stage ≤ IIb at time of surgery are 
eligible for this study. 
 
Stage Grouping 
______________________________ 
Stage Ia  T1 N0 M0 
          Ib  T2 N0 M0 
Stage IIa T3 N0 M0 
Stage IIb T1-3 N1 M0 
Stage III T4 Any N M0 
Stage IV Any T Any N M1 
_______________________________ 
 
Primary Tumor (T) 
TX Primary tumor cannot be assessed 
T0 No evidence of primary tumor 
T1 Tumor limited to the pancreas 2 cm or less in greatest dimension 
T2 Tumor limited to the pancreas more than 2 cm in greatest dimension 
T3 Tumor extends beyond the pancreas but without involvement of the celiac axis or the 
superior mesenteric artery. 
T4 Tumor involves the celiac axis or the superior mesenteric artery (unresectable primary 
tumor). 
 
Regional Lymph Nodes (N) 
NX Regional lymph nodes cannot be assessed 
N0 No regional lymph node metastasis 
N1 Regional lymph node metastasis 
 
Distant Metastasis (M) 
MX Presence of distant metastasis cannot be assessed 
M0 No distant metastasis 

 
 
 
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M1 Distant metastasis 
 
3.4 Vaccine Production and Administration 
 
-
-
 
 
 
 
 
 
 
 
 
 
 

 
 
 
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39 
 
 
 
3.5 Study Plan Schedule 
 
Patients in the Cohort 2 (vaccine-naïve cohort) will first enter the Prime Vaccination 
Phase. During this phase, they will receive 3 vaccinations each one month apart and each 
in combination with a single low-dose cyclophosphamide intravenously before they 
proceed to the Boost Vaccination Phase. During the Prime Vaccination Phase, with Day 
1 considered as the day of vaccination and Day 0 as the day of cyclophosphamide 
administration, each vaccination cycle will span from Day 0 to Day 28.  
 
Patients in Cohort 1 (from J0810) will directly enter the Boost Vaccination Phase. During 
this phase, with Day 1 considered as the day of vaccination, each vaccination cycle will 
span from the first day when the patient receives either oral cyclophosphamide or 
intravenous cyclophosphamide prior to each vaccination to Day 28 or their last day of 
taking oral cyclophosphamide, whichever happens later. 
 
Patients in Cohort 3, 4 and 5 (from J1568, J15237 and J1766) will directly enter the Boost 
vaccine phase. During this phase, with Day 1 considered as the day of vaccination, each 
vaccination cycle will span from the first day (Day 0) when the patient receives intravenous 
cyclophosphamide prior to each vaccination. 
 
At any time during the study, additional leukapheresis or approximately 200 cc of blood 
may be obtained, and skin biopsies and photos may be taken of the vaccine sites and rashes, 
as clinical indicated, as long as the research participant is in agreement. 
 
All research participants will be seen in the oncology outpatient center for vaccine 
administration and monitoring. Research participants will be monitored for at least 30 
minutes following vaccination for evidence of acute reaction to the injected vaccine cells. 
 
If not previously enrolled on SKCCC J0248/IRB # NA_00036444 (formerly 02-10-14-03) 
titled, “Long term follow-up of patients who received lethally irradiated allogeneic 
pancreatic tumor cells transfected with the GM-CSF gene,” the research participant will be 
encouraged to join this long-term follow-up study. 
 
3.5.1 Pre-Study Screen 
 
All patients on participating in Hopkins clinical protocols J0810, J1568, and J15237 
(Cohorts 1, 3, and 4) who are potentially eligible will be informed of this study. Participants 

 
 
 
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40 
in the vaccine naïve cohort (Cohort 2) will be primarily recruited based on the physician 
referrals and patient self-referrals.  The following will occur prior to study entry for all 
patients: 
 
1. Review eligibility criteria 
2. An initial follow-up appointment will be made to serve as a pre-study visit. 
 
3.5.2 Pre-Study Visit  
 
The Pre-Study visit is to be done at least one month after candidate’s last cancer treatment 
and up to thirty days before participants receive the first boost treatment 
(cyclophosphamide or vaccine, whichever is administered first) for the participants in the 
Cohorts 1, 3, 4 and 5.  The first boost vaccination must be no earlier than 6 months (+/-30 
days) after the last vaccine and within 12 months from the last vaccine for Cohorts 3, 4 and 
5.  For the participants in Cohort 2 (vaccine naïve cohort), the Pre-Study visit is to be done 
at least one month after candidate’s last cancer treatment and up to thirty days before 
participants receive the cyclophosphamide of the first prime vaccination treatment cycle. 
 
For all participants:  
 
1. Obtain informed consent (if not already obtained) 
2. Confirmation of eligibility (see Section 3.3.1) 
3. A clinical evaluation to include history, vital signs, ECOG status, weight, review of 
systems, and physical examination. 
4. CT scans of the abdomen, pelvis, and chest. If the CT scan is not done at Hopkins, the 
research participant must provide the report and a copy of the scans. If the research 
participant is allergic to the CT scan contrast, a non-contrast CT of the chest and a MRI 
of the abdomen and pelvis will be obtained.  
5. A review of pertinent interval local oncology notes. 
6. Blood testing to include CBC/diff, absolute eosinophils, absolute neutrophils, absolute 
lymphocytes, comprehensive chemistry panel including electrolytes, BUN, creatinine, 
AST, ALT, total bilirubin, alkaline phosphatase, amylase, and CA 19-9. 
7. Pregnancy test for women of childbearing potential. Urine pregnancy test preferred. 
8. Research Blood (Cohort 2 only): A leukapheresis for in vitro studies will be encouraged. 
If the research participant refuses a leukapheresis, approximately 200 cc of blood will 
be attempted to be drawn but may not be exact due to unforeseen technical reasons. An 
additional of approximately 30 cc will be drawn for serum banking. These research 
bloods may be drawn within 7 days of vaccination.  
 
3.5.3 Study Procedures during the Prime Vaccination Phase (3 monthly vaccination 
cycles) 
 
This is for the Cohort 2 (vaccine naïve cohort) only: 
 
Day 0 (one day prior to vaccination) 
1. Administration of cyclophosphamide 200 mg/m2 intravenously  

 
 
 
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2. Vital signs before cyclophosphamide administration  
3. For Prime Vaccination Cycle #2-3, a toxicity assessment will be done prior to IV 
cyclophosphamide administration. 
4. Research Blood: A leukapheresis for in vitro studies will be encouraged. If the research 
participant refuses a leukapheresis, approximately 200 cc of blood will be attempted to be 
drawn but may not be exact due to unforeseen technical reasons. An additional of 
approximately 30 cc will be drawn for serum banking. These research bloods may be drawn 
within 7 days of vaccination (for Prime Vaccination Cycle #2-3 only). 
 
Day 1 Vaccination Day 
1. Administration of vaccine (Topical lidocaine-based anesthetic may be placed 
approximately 1-2 hours prior to planned vaccine administration time to the intended 
vaccine sites.) 
2. Vital signs before and after vaccine administration 
3. Assessment for toxicities prior to and after the vaccination.   
4. Monitor for no less than 30 minutes after vaccine administration. 
5. Tetanus toxoid boost if last tetanus vaccination > 10 years. Tetanus/diphtheria (Td) may 
be substituted if tetanus alone is not available. If both are not available, no tetanus vaccine 
will be administered. 
 
Day 8 (+/- 1 day) for All Arms 
1. Heme-8 with differential, including absolute eosinophil count, absolute neutrophils, 
absolute lymphocytes. 
2. Comprehensive chemistry panel including electrolytes, BUN, creatinine, AST, ALT, 
total bilirubin, alkaline phosphatase, and amylase. 
 
Day 28 (+/- 7 days) 
1. Heme-8 with differential, including absolute eosinophil count, absolute neutrophils, 
absolute lymphocytes. 
2. Comprehensive chemistry panel including electrolytes, BUN, creatinine, AST, ALT, 
total bilirubin, alkaline phosphatase, and amylase. 
3. A research blood draw of approximately 200 cc for immune cell analysis will be 
attempted but may not be exact due to unforeseen technical reasons. An additional of 
approximately 30 cc will be drawn for serum banking. 
 
3.5.4 Study Procedure during the Boost Vaccination Phase 
 
This phase is for Cohorts 1, 2, 3, 4 and 5. 
 
Pre-vaccination Assessment 
The following will be done up to thirty days before participants receive each cycle of boost 
treatment (cyclophosphamide or vaccine, whichever is administered first) except for boost 
vaccine cycle #1 of the participants in Cohorts 1, 3, 4 and 5 who should have already had 
their pre-vaccination assessment: 
 
1. Confirmation of eligibility (see Section 3.3.1) 

 
 
 
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2. A review of pertinent interval local oncology notes including laboratory tests and CT 
scan reports. CT scan of the abdomen, pelvis, and chest may be done within one month of 
boost treatment (cyclophosphamide or vaccine, whichever is administered first). If the 
research participant is allergic to the CT scan contrast, a non-contrast CT of the chest and 
a MRI of the abdomen and pelvis will be obtained Note: Participants who have been for 5 
years and more since the pancreatic surgery can have either CT scan of chest, abdomen 
and pelvis with iv contrast or MRI of abdomen and pelvis done at the discretion of the PI 
of the study following the same protocol. If MRI of abdomen and pelvis is chosen, a non-
contrast CAT scan of the chest should be done. 
3. An assessment for toxicity. A clinic visit is not required. This may be done over the 
phone by the research nurse. Based on the toxicity assessment, clinic visits and 
laboratory/imaging tests may be recommended as per standard of care and as clinically 
indicated. 
4. Blood testing to include CBC/diff, absolute eosinophils, absolute neutrophils, absolute 
lymphocytes, comprehensive chemistry panel including electrolytes, BUN, creatinine, 
AST, ALT, total bilirubin, alkaline phosphatase, amylase, and CA 19-9. 
5. Pregnancy test for women of childbearing potential. Urine pregnancy test preferred. 
 
In general, prime vaccines can be administered 28 days +/- 4 days between each 
vaccination while 6 +/-1months should be between two boost vaccinations. After 5 years 
have passed, boost vaccinations will occur every 12 months (+/- 1 month) up through year 
10. However, if participants do not meet eligibility criteria due to a reversible cause, 
vaccination can be postponed until they become eligible.  Vaccination may be postponed 
for other reasons including, but not limited to the occurrence of vaccine site flare-up, at the 
discretion of the study team.  The interval between two boost vaccinations is not 
recommended to be shorter than 5 months or more than one year.  
 
Day -27 to Day +28 for Arm C of Cohort 1 Only 
Participants are taking cyclophosphamide 50 mg daily orally at home starting Day -27 until 
Day 28.  For Cycle #1, prescriptions will be dispensed after meeting eligibility criteria.  For 
Cycles 2-20, prescriptions will be dispensed on Vaccination Day of the previous cycle.    
 
Day 0 (one day prior to vaccination) for Arm B of Cohort 1 and Cohorts 2-5 
1. Administration of cyclophosphamide 200 mg/m2 intravenously  
2. Vital signs before cyclophosphamide administration  
3. For Cycle #2-20, a toxicity assessment will be done prior to IV cyclophosphamide 
administration. 
 
The following is applied to all participants unless indicated: 
 
Day 1 Vaccination Day 
1. Administration of vaccine (Topical lidocaine-based anesthetic may be placed 
approximately 1-2 hours prior to planned vaccine administration time to the intended 
vaccine sites.) 
2. Vital signs before and after vaccine administration 

 
 
 
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43 
3. Assessment for toxicities. For all arms and all cycles, assessment will be performed after 
the vaccination.  For Cycle #2-20 for Arm A and C of Cohort 1, assessment will also be 
done prior to the vaccination.   
4. Monitor for no less than 30 minutes after vaccine administration. 
5. Tetanus toxoid boost if last tetanus vaccination > 10 years. Tetanus/diphtheria (Td) may 
be substituted if tetanus alone is not available. If both are not available, no tetanus vaccine 
will be administered. 
 
Day 8 (+/- 1 day) for Cohort 2 only 
1. Heme-8 with differential, including absolute eosinophil count, absolute neutrophils, 
absolute lymphocytes. 
2. Comprehensive chemistry panel including electrolytes, BUN, creatinine, AST, ALT, 
total bilirubin, alkaline phosphatase, and amylase. 
 
Day 29 (+/- 7 days) 
1. Heme-8 with differential, including absolute eosinophil count, absolute neutrophils, 
absolute lymphocytes. 
2. Comprehensive chemistry panel including electrolytes, BUN, creatinine, AST, ALT, 
total bilirubin, alkaline phosphatase, and amylase. 
 
3.5.5 Off Study Visit 
Patients are considered off study beginning 28 days after their final boost vaccination or 
the date of study termination for patients who go off study prior to their final boost 
vaccination or because the vaccine supply is exhausted.). Follow-up visits will be 
scheduled at the discretion of the patient’s local oncologist and the results sent to us if the 
patient agrees. All attempts will be made to obtain disease-free and overall survival data 
on each patient.  
 
The following evaluations will be performed at the off study visit: 
 
1. History and Physical exam with ECOG performance 
2. Assessment of vaccine sites. This will include: number of sites that have erythema, 
induration, pruritis, and tenderness; and measurement of induration and erythema of largest 
vaccine site. 
3. Heme-8 with differential, including absolute eosinophil count, absolute neutrophils, 
absolute lymphocytes 
4. Comprehensive chemistry panel including electrolytes, BUN, creatinine, AST, ALT, 
total bilirubin, alkaline phosphatase, and amylase. 
5. CA 19-9 
6. CT scan abdomen/pelvis and chest. If done within 30 days the CT do not need to be 
repeated. (If allergic to CT scan contrast, obtain MRI). Note: Participants who have been 
for 5 years and more since the pancreatic surgery can have either a CT scan of the chest, 
abdomen and pelvis with IV contrast or MRI of abdomen and pelvis done at the discretion 
of the PI of the study following the same protocol. If MRI of abdomen and pelvis is chosen, 
a non-contrast CAT scan of the chest should be done. 

 
 
 
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7. Assessment of toxicities (Information may include evaluations made by the local health 
care provider).  
 
3.6 Collection Samples for Correlative Studies 
 
3.6.1 Leukapheresis 
 
All research participants will be encouraged to undergo a standard leukapheresis for 
immune function analysis before each prime vaccination. Any research participant 
demonstrating an interesting immunological response may be asked to undergo additional 
leukapheresis for research purposes.  This may include physical responses thought to be 
related to the vaccine (including, but not limited to vaccine site flares) or interesting 
laboratory responses (including, but not limited to mesothelin-specific CD8+T cell 
responses).  There will be at least one month between additional leukapheresis procedures. 
Prior to the leukapheresis, subjects will be evaluated by the Hematopoietic and Therapeutic 
Support (HATS) Center to determine if their vascular access appears to be adequate for the 
leukapheresis procedure. If the research participant does not agree to the leukapheresis or 
if it is not feasible for leukapheresis, the standard 200 (+/- 10) cc of peripheral blood will 
be obtained. Peripheral blood mononuclear cells (PBMC) will be prepared by Ficoll-
Hypaque density gradient centrifugation by standard protocol and stored at –80°C or below 
until further analysis. The quantity of these samples is necessary for monitoring the 
quantitative change of peripheral lymphocytes including Tregs and functional analysis of 
T cell immune response following each vaccination. 
 
3.6.2 Serum Banking 
 
At the time intervals indicated above, a maximum of 30 cc (and a minimum of 
approximately 20 cc) of peripheral blood will be drawn to collect serum from research 
participants for serology studies described below.  Any research participant demonstrating 
an interesting immunological response may be asked to undergo additional serum banking 
for research purposes.  This may include physical responses thought to be related to the 
vaccine (including, but not limited to vaccine site flares) or interesting laboratory responses 
(including, but not limited to mesothelin-specific CD8+T cell responses). The samples of 
serum should be allowed to set at room temperature for 20-30 minutes to allow clotting, 
and then centrifuged at 3,000 rpm for 10 minutes. The resultant serum will be frozen at -
80°C or below. 
 
3.6.3 Archival Tumor Tissue 
 
Attempts will be made to obtain archived tissue samples. Archived FNA biopsy samples 
do not contain sufficient tissue and will not be collected. The tissue sample should have 
proper size to enable analysis. Formalin-fixed paraffin-embedded tissue blocks or up to 50 
cut slides will be requested.  
 
3.7 Evaluation for Safety and Anticipated Toxicities 
 

 
 
 
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3.7.1 Safety and Anticipated Toxicities of Vaccine 
 
Severe toxicities are unlikely, based on information from previous GM-CSF gene-
transduced whole cell vaccine studies completed here at Johns Hopkins Hospital.  This 
includes over 200 patients treated at JHH in the Phase I and II pancreatic vaccine studies. 
In experiments involving over 400 mice, use of irradiated GM-CSF secreting tumor cells 
caused only reversible lymphadenopathy and reversible subcutaneous swelling; no 
ulcerations were seen. In our first phase I trial in patients with renal cell carcinoma, only 
local erythema and swelling were seen following intradermal injections of cell doses up to 
4 x 107 GM-CSF modified vaccine cells, and up to 4 x 108 unmodified vaccine cells. At the 
highest dose level, we predict that initially 45 mcg of total GM-CSF will be secreted locally 
per 24 hours, a level that will diminish as tumor cells are killed by invading inflammatory 
cells. To support hematologic recovery in oncology patients after intensive chemotherapy, 
subcutaneous or intravenous doses of GM-CSF between 5 and 10 µg/kg/day are commonly 
used (350-700 mcg total for a 70 kg individual). At this dose range, the following side 
effects are commonly seen: local or generalized skin rashes, bone pain (attributed to 
stimulation of hematopoietic progenitors), fever, and malaise. Although patients in the 
initial Phase I study of the allogeneic tumor vaccine had normal bone marrow function, 
leukocytosis and toxic levels of serum GM-CSF did not occur with the 10 fold lower dose 
of GM-CSF. The maximum serum GM-CSF level obtained was 14.0 pg/ml with dose level 
four at 48 hours after the first pancreatic tumor vaccine. The plasmid used to transfect the 
GM-CSF gene is safe. In contrast to retroviral vectors, it lacks the coding sequences that 
would allow replication and the generation of helper virus. This plasmid containing the 
GM-CSF gene has been sequenced following vector construction to confirm its insertion, 
orientation, and the lack of mutations. In addition, this vector has been confirmed to 
produce bioactive GM-CSF. 
 
The risk of generating autoimmune reactions is unknown but is believed to be small. The 
pancreas would be the most likely organ to be involved. Pancreatitis and loss of pancreatic 
function can be supported by the use of exogenous pancreatic enzymes and insulin 
injections if needed. Other organs that may share tissue specific antigens might also be 
involved, such as the salivary glands and other gastrointestinal organs. In the Phase I and 
II studies there were no evidence of autoimmune reactions. Every patient who has received 
the vaccine will be evaluated for toxicity. The research participant will be taken off-study 
if unacceptable adverse events are experienced. Possible toxicities include local swelling, 
induration, or ulceration at the site of the vaccine, systemic toxicities from paracrine 
secretion of GM-CSF, and induction of autoimmunity.  The risk of generating autoimmune 
reactions might be increased by combination of cyclophosphamide; however, no 
autoimmune reactions have been reported in previously published studies of metronomic 
cyclophosphamide.  Therefore, we anticipate that such a risk is still small even if 
cyclophosphamide is combined with the vaccine.  
 
Blood tests to monitor for systemic toxicities will be obtained a week and four weeks after 
all vaccinations. For the first vaccination, blood tests for systemic toxicities will 
additionally be performed two, three, and four months after vaccination All research 
participants will be followed for cancer recurrence every three to six month by their local 

 
 
 
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health care providers or as determined by each provider. The research participant may be 
contacted by phone or e-mail or the information may be obtained from their local health 
care providers. The research nurse will contact patients to assess the toxicity by phone. It 
will be done no less frequently than the following occasions: 2 months +/-15 days, 4 
months +/- 15 days after the last vaccination and within 14 days before the next study 
treatment. Based on toxicity assessment, clinic visits and laboratory/imaging tests may be 
recommended as per standard of care and as clinically indicated. The research participant 
will be advised to call the research nurse and/or the principal investigator if there are any 
new toxicities, concerns or questions. 
 
3.7.2 Safety and anticipated toxicities of single injection of low dose 
cyclophosphamide or recurrent administration of metronomic cyclophosphamide 
 
The dose of cyclophosphamide studied in this trial is below that in common use for the 
adjuvant therapy of e.g. breast cancer (typically 600 mg/m2 of cyclophosphamide) (Sledge, 
1998). Therefore, we anticipate that the risk of toxicity related to the use of single dose of 
cyclophosphamide at 200 mg/m2 or that of daily metronomic cyclophosphamide at 50 mg 
once daily eight weeks is quite small.  Based on the toxicity studies of higher dose of 
cyclophosphamide in adjuvant therapy of breast cancer, fatigue, alopecia, nausea, vomiting, 
and mild cytopenias are likely to be the most common toxicities of cyclophosphamide.  
With standard dose cyclophosphamide, a rare incidence of leukemia or myelodysplastic 
syndrome as a late toxicity was reported. 
 
Long-term daily use of cyclophosphamide may have different toxicity profiles from higher 
dose of cyclophosphamide given intravenous every 2-3 weeks.  Nonetheless, animal 
studies still suggest that continuous administration of metronomic dose of 
cyclophosphamide is significantly less toxic than the maximum tolerated dose of 
cyclophosphamide given intermittently for 3 doses (Emmenegger et al., 2004).  The 
toxicities of metronomic cyclophosphamide in humans can be anticipated based on the 
previously published studies.  Orlando et al. (2006a) reported in patients with metastatic 
breast cancer that prolonged treatment of metronomic cyclophosphamide at 50 mg/day for 
a median duration of treatment of 20.4 months was well tolerated and side-effects were 
mild.  The most frequently encountered toxicity was grade 1-2 leukopenia, which was 
observed in 54% of the 63 cases.  Increases in transaminase values were registered in 12 
cases, and one patient had grade 3 toxicity.  Other side effects included one patient with 
grade-3 thrombocytopenia, five with grade 1-2 anemia, ten with grade-1 and one with 
grade-2 nausea/vomiting, and small percentages of patients with grade 1-2 mucositis, 
gastric pain, diarrhea, fever, infection, asthenia, etc.  It should be noted that  patients in this 
study were also given metronomic dose of methotrexate which likely resulted in some of 
the side effect profiles  Other studies reported similar toxicity profiles with prolonged use 
of metronomic cyclophosphamide (Orlando et al., 2006b; Glode et al., 2003; Bottini et al., 
2006; Suvannasankha et al., 2007).  The dose and schedule of metronomic 
cyclophosphamide in this study is adopted from Ghiringhelli et al. (2007), where the short-
term and long-term safety was shown.  
 

 
 
 
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Long-term use of metronomic cyclophosphamide has the potential to be associated with 
leucopenia, thrombocytopenia, infection, transaminitis, and hemorrhagic cystitis. The most 
commonly anticipated toxicity due to cyclophosphamide is leukopenia. However, most 
cases of myelosuppression induced by metronomic cyclophosphamide are reversible with 
dose reduction or temporary discontinuation (Austen et al., 2001, O. de Weerdt et al., 2001, 
Lord et al., 2007). For intravenous administration, the nadir in the white blood cell count 
occurs between 8-14 days after starting treatment with a full recovery 21 days after 
administration (Austen et al., 2001). The degree of lymphopenia and the length of time to 
white blood cell count nadir are dose-dependent. The time course of hematologic effect in 
daily oral cyclophosphamide is less predictable but based on other metronomic 
cyclophosphamide studies, anemia, neutropenia and thrombocytopenia occur less 
commonly and to a lesser degree than leukopenia (Lord et al., 2007). The development of 
opportunistic infections is associated with the degree and duration of leucopenia. The risk 
of infection increases when the white blood cell nadir decreases to below 3000 cells/muL 
or the absolute neutrophil count falls below 1000 cells/muL (Fox and Pandya, 2000). The 
most common opportunistic infection is Pneumocystis carinni pneumonia, which can be 
prevented with low-dose trimethoprim-sulfamethoxazole (Beimler et al., 2004). 
 
Hepatotoxicity is known to occur with high dose cyclophosphamide, but is rare at low 
doses. In the cases that hepatotoxicity did occur, liver injury occurs within 2-8 weeks of 
treatment and liver function normalized after discontinuation of cyclophosphamide (Mok 
et al., 2000, Akay et al., 2006). The risk of hemorrhagic cystitis is dose dependent and due 
to accumulation acrolein, a cyclophosphamide metabolite. It may occur during treatment 
or months after treatment. The risk of bladder toxicity is higher with oral metronomic doses 
than intermittent intravenous doses based on studies with lupus nephritis, vasculitis, and 
Wegener’s granulomatosis patients.  However, we expect rare, if any, cases of hemorrhagic 
cystitis since it is associated with higher cumulative doses of 57 to 100 g (Fox and Pandya, 
2000).  As reported, essentially all the cases of hemorrhagic cystitis induced by long-term 
low dose cyclophosphamide treatment were reversible with appropriate treatments.   
 
3.8 Interval Health Care 
 
The research participant disease status may be evaluated more frequently than twice a year. 
Patients may undergo additional abdomen, chest and pelvis CT scans and blood tests, 
including CA 19-9 levels, as recommended by their local oncologist, or by the study team 
as standard of care for possible disease recurrence. The results of the CT scans, blood tests, 
and progress notes will be requested from the research participant’s local oncologist. 
Medical records obtained will be added to the oncology outpatient medical records, 
including electronic forms, when possible. Data related to the toxicity and/or disease status 
assessments will be recorded in the case report form binders and electronic database.  If 
the research participant is followed at Johns Hopkins, the medical records will be accessed 
at least quarterly, and more frequently, if needed for disease recurrence. 
 
If patient is taken off study, the patient’s medical records will be requested from the health 
care provider providing further care for 28 days after the last research product was 
administered, or longer for resolution of any adverse events related to the research product. 

 
 
 
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After off-study, the research participant will be followed annually via the protocol SKCCC 
J0248/IRB # NA_00036444 (formerly 02-10-14-03) titled, “Long-term follow-up of 
patients who receive lethally irradiated allogeneic pancreatic tumor cells transfected with 
the GM-CSF gene”, if consent is granted. 
 
3.9 Termination of Study Treatment (Off Study) 
 
Patients in Cohort 1 and 2 will be considered to have completed the study when they have 
received 10 years of treatment. Patients in Cohort 3, 4 and 5 will be considered to have 
completed the study if they have received all ten boost vaccinations. Every attempt will be 
made to schedule an off-study visit within 4 weeks of the date of study termination for 
patients who were unable to complete the 10 years of treatment for Cohort 1 and 2 or ten 
vaccinations for Cohort 3, 4 and 5. If determined to be off-study, the research participant 
will be followed for 28 days after the administration of the last vaccine for toxicities related 
to the vaccine, or longer, if vaccine-related toxicities occur.  
 
The patient may be discontinued from treatment or participation in the study in the 
following instances: 
1. Patient withdraws consent and refuses future vaccinations 
2. Patient is repeatedly noncompliant with study treatment 
3. Concurrent illness develops that would preclude objective clinical assessments 
4. The incidence or severity of adverse events in this study denotes potential untoward 
health risk to the patient. 
5. If the patient is diagnosed with autoimmune diseases such as systemic lupus 
erythematosus, sarcoidosis, rheumatoid arthritis, glomerulonephritis, or vasculitis.  
6. Patient receives non-study immunotherapy, chemotherapy, radiotherapy, gene therapy, 
biologic therapy, or other investigational therapy for the treatment of pancreatic cancer. 
7. Patient experiences a dose-limiting toxicity attributed to study vaccine. Dose-limiting 
toxicity (DLT) is defined as any grade 3 or 4 non-hematological toxicity excluding alopecia, 
grade 3 hematologic toxicity, and grade 4 hematological toxicity that does not resolve in 
less than 5 days. If a DLT (except alopecia) occurs, treatment will be stopped. Treatment 
may be restarted if the DLT resolves to < grade 2 or to baseline levels. If the toxicity 
continues at > grade 2 for four weeks then the patient will be removed from further 
treatment in the study. 
 
Per the FDA guidance for patients treated with genetically modified products, all research 
participants will be encouraged to enroll in a long-term follow-up protocol, following their 
completion of all interventional studies. These patients will be followed for disease 
progression, survival and potential long term toxicity of gene therapy in an existing 
protocol titled “Long term follow-up of patients who received lethally irradiated allogeneic 
pancreatic tumor cells transfected with the GM-CSF gene (SKCCC J0248/IRB # 
NA_00036444 [formerly 02-10-14-03])”. 
 
3.10 Management of Toxicities 
 

 
 
 
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Local vaccine site reaction may be treated with topical applications of aloe vera or vitamin 
E gel or lotion. Significant local inflammation that is causing the research participant 
severe pain or is interfering with the activities of daily living may be treated with cold 
packs and oral analgesics. Local toxicities of pruritus at the vaccine sites and systemic 
pruritus may be treated with topical or oral diphenhydramine hydrochloride (Benadryl) or 
topical aloe vera. If oral diphenhydramine hydrochloride is used the recommended dose 
shall be 25-50 mg every four to six hours as needed for pruritus, not to exceed 300 mg/day. 
Cases of local ulceration should be manageable with local wound care, with or without 
antibiotics. Severe local inflammation or significant clinical autoimmunity will be 
managed on a case-by-case basis. 
 
For patients with grades 3 and 4 leukopenia, neutropenia or thrombocytopenia who are due 
to receive IV cyclophosphamide , both the IV cyclophosphamide and vaccine will be held 
until the patient's counts return to grade 2 or above.  The patients will be taken off the study 
if their counts do not return to grade 2 or above within 6 months during the prime 
vaccination phase or within 6 months during the first five years or within 12 months in 
after five years during the boost vaccination phase. For patients with Grade 3 or 4 
leukopenia, 
neutropenia 
or 
thrombocytopenia 
who 
are 
still 
receiving 
oral 
cyclophosphamide treatment, treatment can be restarted once the myelosuppression has 
returned to grade 2 or above. If the myelosuppression has not recovered to grade 2 by the 
time of the next scheduled vaccination, both the oral cyclophosphamide and vaccine will 
be held until the patient’s count return to grade 2 or above. The patients will be taken off 
the study if their counts do not return to grade 2 or above within 6 months during the prime 
vaccination phase or within 6 months during the first five years or within 12 months in 
after five years during the boost vaccination phase. Prophylactic doses of trimethoprim-
sulfamethoxazole will be recommended to all patients with Grade 3 or 4 lymphopenia or 
neutropenia lasting more than a month at the discretion of the treating physician. If the 
patient is allergic to trimethoprim-sulfamethoxazole, dapsone will be recommended as an 
alternative. 
 
Patients receiving cyclophosphamide who develop hematuria or polyuria will undergo 
urinalysis and cytology. If hemorrhagic cystitis is identified, oral or IV cyclophosphamide 
will be discontinued and appropriate treatments will be initiated as per standard of care.  
Such patients may receive their scheduled vaccination at the discretion of the study team 
and appropriate surveillance will be performed for transitional cell carcinoma of the 
bladder.  
 
3.11 Concurrent Medication 
 
Concurrent medications will be collected as a part of “Medical History” when it is taken 
as indicated in Section 3.2 - Checklists and Section 3.5 - Study Visit Procedure. In addition, 
concomitant medications will be recorded during each vaccination cycle from Day 0 to 
Day 28 for Cohort 1 (Arm A and Arm B) and Cohorts 2-5 and from Day -28 to Day 28 for 
Cohort 1 (Arm C). No other chemotherapy, immunotherapy, radiotherapy, gene therapy, 
biologic therapy, or other investigational therapy for the treatment of pancreatic cancer is 
allowed during the course of this study. Systemic corticosteroids or other 

 
 
 
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immunosuppressive drugs should be avoided for 28 days before and after each vaccination.  
Local steroid treatments are allowed. 
 
4.0 Statistical Considerations 
 
4.1 Sample Size 
 
The primary statistical endpoint of this study is to determine the safety and feasibility of 
vaccine boosting with lethally irradiated, allogeneic pancreatic tumor cells transfected with 
the GM-CSF gene given alone or in combination with either a single intravenous dose or 
long term daily metronomic oral doses of cyclophosphamide. The sample size of Cohorts 
1, 3, 4 and 5 is predetermined by the prior parent studies J0810, J1568, J15237 and J1766. 
The J0810 study will have enrolled approximately 60 participants. Among them, 39 are 
evaluable subjects who have received at least 3 vaccines.  For patients who receive all six 
vaccinations under the J0810 protocol, the first boost vaccination will be scheduled around 
20 months following surgery. Based on analysis from the phase II adjuvant study (Laheru 
et al., 2007), we anticipate the disease free survival rate at 20 months to be approximately 
40%-50% respectively.  Therefore, we estimate that up to 50% of these 39 evaluable 
patients, equal to approximately 20 patients, will be candidates for this boost vaccination 
study.  It should be noted that some participants from the J0810 may have completed less 
than three vaccination cycles and then been taken off the study due to reversible conditions, 
and may again become eligible for this boost vaccination study. Nonetheless, we estimate 
that such subjects only account for less than 5% of the total participants in the J0810 study.  
 
We have recognized the power of this study would be limited by the sample size that is 
predetermined by the original J0810 study size. To adequately power this study to evaluate 
the immunologic endpoint, we add a vaccine-naïve cohort.  Based upon the previous J0619 
boost vaccine trial, 70% of the patients remained disease-free and completed their first 
follow-up boost.  In addition, in the previous J9988 study, 50% of patients who were tested 
showed a positive response (defined as CD8+ T cells of two high-avidity mesothelin 
epitopes with enhanced functional activity) following their first 6-month boost vaccine.   A 
sample size of 26 would provide 90% power to detect an increase from the null rate of 50% 
to 80% with a two-sided type I error rate of 5%.   Based upon the previous J0619 boost 
vaccine trial, 70% of the patients remained disease-free and completed their first follow-
up boost.  Therefore, we initially estimated that we needed to enroll a total of 38 individuals 
to allow for the 30% loss to follow-up.  In addition to the 20 patients that were estimated 
for the above J0810 cohort, we originally estimated to enroll a total of 58 patients to this 
study.  However, based on the preliminary enrollment data of this current study, 
approximately 50% patients went off the study prior to receiving the first boost vaccine.  
More patients went off the study due to disease recurrence was because of more frequent 
use of CT scan as a surveillance in today’s practice than when the previous J0619 study 
was conducted.  Another reason is that all the patients enrolled in this study were within 
18 months out of the surgery and thus had a high risk of recurrence than those enrolled in 
the J0619 study. After considering the preliminary enrollment data from this current study. 
We re-estimated the sample size of the vaccine-naïve cohort to be 52.  Therefore, we 

 
 
 
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estimate the total sample size of this study including both the J0810 cohort and the vaccine-
naïve cohort to be 72.  
 
We still lack data to distinguish the immune modulatory effects of these two different 
routes of cyclophosphamide administration. Since the single IV dose of cyclophosphamide 
has been used more commonly in the past, we have chosen to add a new cohorts with the 
single IV dose of cyclophosphamide (Cohorts 3-5), which will allow for a better 
comparison to historical cohorts.  It should be noted that the sample size of the new cohorts 
is powered separately, but this new cohorts will not be analyzed separately. Therefore, the 
data analysis part of the protocol remains unchanged. All cohorts will receive the boost 
vaccinations in the same way, which is the main study procedure of the protocol.  The main 
objective is also the same, to study the effect of the vaccine boosting. Nonetheless, we have 
to calculate the sample size of the vaccine cohort separately because the sample number in 
Cohorts 1, 3, 4 and 5 cannot be estimated precisely. The sample number in Cohorts 1, 3, 4 
and 5 depends on how many patients remain disease-free after they complete the primary 
studies and how many are interested in the boost vaccination. When we calculate the 
sample size of the new cohort separately, we can ensure that the sample size of this study 
has the sufficient power to compare with the historical control patients, those treated in the 
J0619 study – the previous boost vaccine study. In addition, there is a small difference 
between how each cohort received the prime vaccinations. Cohort 2 will receive the prime 
vaccination in a similar way to the patients in the J0619 study (historical comparison 
cohort). By calculating the sample size of the new cohort separately, we can perform a 
more formal comparison between patients in this study and those in the J0619 study. 
Certainly, we will also combine the patients in all cohorts for comparison and combine 
Arm B of the J0810 cohort (Cohort 1), and vaccine-naïve cohort (Cohort 2) for comparison.  
We estimate the sample size of Cohort 3, 4 and 5 to be 68. 
 
4.2 Analysis of Primary Endpoints 
 
4.2.1 Safety and Toxicity Measurements 
 
The primary endpoint of this study is safety as measured by local and systemic toxicity. 
For this endpoint, all the patients who are enrolled in the study will be evaluated.  These 
toxicities will be characterized according to the National Cancer Institute Common 
Terminology Criteria for Adverse Events, Version 3.0, and can be accessed and 
downloaded via the website: http://ctep.cancer.gov/reporting. Local dermatologic reactions 
anticipated according to the previous studies of the same vaccine are known to be 
appropriate vaccine-induced immune responses (Jaffee et al. 2001), therefore, are not 
considered toxicities, adverse events or problems, and will no longer be reported.  Plenty 
of scientific information has been collected by the previous studies; thus, measurements of 
the vaccine sites and routine skin biopsy will not be performed.  Nonetheless, unusual types 
of vaccine site reaction including but not limit to ulceration will be recorded.  An optional 
skin biopsy and/or photographs may be obtained if the subject has a systemic rash, or an 
unusual vaccine reaction. In addition to sending this to pathology for diagnosis we will use 
additional material from the biopsy for research purposes to understand the effects of the 
vaccine. For each participant arm, we will tabulate the number, type and degree of toxicities 

 
 
 
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for each round of vaccination. In addition, we will estimate the proportion of individuals 
who have a DLT within the first 28 days of vaccination for each cycle with an exact 95% 
confidence interval.  We will estimate the frequency of each toxicity per total number of 
patients treated (total number of treated patients will be included in denominator), as well 
as per total number of vaccines that are administered (total number of vaccines will be 
included in denominator). 
 
We expect to be able to estimate the frequency of the toxicities that were initially identified 
in the phase I and II studies that are vaccine related. Based on the phase I data, we do not 
expect to detect significant local or systemic toxicities.   The toxicities of low-dose 
cyclophosphamide have also been well characterized.  The toxicity profile of the 
combination of low-dose cyclophosphamide and vaccination can be estimated from the 
above-described phase II study in patients with metastatic pancreatic cancers. Because 
pancreatic adenocarcinoma has a very poor prognosis and there are few available treatment 
options, a <20% non-life threatening toxicity rate would be considered acceptable in this 
study (NCI Common Toxicity Criteria grade 3 or less).   
 
In addition, although it is unlikely that life-threatening toxicities will be uncovered in this 
study, a single life-threatening adverse event would suspend accrual.  Then, a complete 
evaluation of all other observed adverse events to define their severity and to determine the 
degree to which each event can be definitively attributed to the vaccine treatment will be 
warranted.  Based on the results of this evaluation, modifications in the form of dose 
reduction, schedule change, or vaccine lot replacement would be made in order to resume 
clinical testing. 
 
4.2.2 Measurements of Immune Response 
 
We will measure the number, repertoire and avidity of peripheral mesothelin-specific CD8+ 
T cells.  When these parameters of immune response are measured, continuous variables 
will be summarized with means and standard deviations. Dichotomous and categorical 
variables will be summarized using proportions with exact 95% confidence intervals and 
counts, respectively. These summaries will be computed for each vaccinated patient at 
multiple time points before and after vaccine administration: pre-vaccination and four 
weeks post vaccination (also as indicated in Study Plan). Plots will be used to show the 
changes in immune response over time both for each individual and for each arm. For each 
vaccination, comparisons in the pre and post-vaccine responses will be compared using 
paired t-tests (or Wilxcoxon signed rank tests if appropriate) for continuous variables and 
McNemar’s test for dichotomous or categorical variables. Associations between immune 
responses will be explored graphically (e.g. scatterplots, boxplots) and numerically (e.g. 
correlations,  χ² tests).  
 
Since the initial therapies differ between the four cohorts, we will consider comparing each 
cohort against a historical control, respectively. Therefore, while the sample size of 
Cohorts 1, 3, and 4 is small and predetermined, the sample size of Cohort 2 (the vaccine-
naive cohort) is powered separately. We will also consider merging the following cohorts 
to compare against the historical control:  Cohort 1 (Arm B), Cohort 2, Cohort 3 (Arm A), 

 
 
 
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Cohort 4 and Cohort 5. We will also compare between the two cohorts although it is limited 
by the small sample sizes.  
 
One of the main goals of this study is to determine whether or not the boost vaccination 
scheme is safe and feasible. If the study treatment is not feasible, we would never be able 
to answer the immunologic questions.  In addition, till today, none of long-term immune 
response parameters have been accepted as the primary endpoints. Therefore, we will still 
keep the safety endpoint as the primary endpoint.   
   
4.3 Analysis of Secondary Endpoints 
 
4.3.1 Efficacy Endpoints 
 
The secondary efficacy endpoint of interest is the overall survival (OS) of patients treated 
with vaccine. The overall survival is defined as the time from initial randomization for the 
J0810 study until death. If a patient is lost to follow-up or the study is ended prior to death, 
the patient will be considered censored at their last recorded follow-up. For each arm, 
Kaplan Meier curves will be constructed after combining all patients from three arms and 
the median survival estimates will be calculated with 95% confidence intervals using 
Greenwood’s formula.  All participants will be evaluable for efficacy endpoints. 
 
We are also interested in investigating the effect of vaccine on disease/progression free 
survival (PFS). Patients who are treated at Johns Hopkins will be followed every 3 months 
and evaluated for recurrence with the CA19-9 level and the CT scan, as recommended by 
the NCCN guideline.  However, the standard of care follow-up, such as the interval of 
follow-up and the type of follow-up imaging, varies from institution to institution. PET 
scan is not typically done as a follow-up image for resected pancreatic cancer.  However, 
if a PET scan has been done and its result is available, we may use the results of PET scan 
to help determining the recurrence by following the guidance of RECIST 1.1.  In order to 
prevent biasing our results, it is necessary to coarsen the follow-up interval and the 
selection of imaging type so that every individual is evaluated in the same manner 
regardless of the institution. This coarsening of the data may cause an overestimation of 
PFS. In order to take into account both potential forms of bias, we will perform our analysis 
of PFS on the entire cohort as well as the subpopulation of patients from Johns Hopkins 
(estimated to be approximately 50% of the research participants). PFS will be defined as 
the time from the first vaccine until evidence of disease recurrence. If a patient withdraws 
from the study prior to being diagnosed with progressive disease, they will be censored at 
the date of their last follow-up visit. If an individual dies prior to being diagnosed with 
progressive disease, they will be considered to have progressed at the date of their last 
follow-up visit. This is a conservative estimate if we count the disease recurrence at the 
next planned visit then this will bias the PFS time upward. The PFS will be summarized 
using Kaplan-Meier curves and estimates of median PFS with 95% confidence intervals 
based upon Greenwood’s formula. Due to potential causes of bias discussed earlier, the 
results will be considered extremely preliminary. If the PFS estimates for patients treated 
at Johns Hopkins appear to be lower than that of the population as a whole and the Johns 
Hopkins patients are followed at more frequent intervals than the other patients, then this 

 
 
 
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indicate that an overestimation of PFS may exist. No formal comparison is planned due to 
the small sample size. Such results could affect the future planning of follow-up treatment 
for patients in subsequent trials.  
 
Although cyclophosphamide administrated in two different ways will be tested in different 
arms, the primary endpoint is to evaluate their safety, feasibility and immune responses 
including mesothelin-specific T cell responses and Treg quantity and function.  Our second 
endpoint is to evaluate whether cyclophosphamide administrated as in Arm B and Arm C 
would have significant impacts on clinical efficacy.  However, even if cyclophosphamide 
administration would have a significant impact on clinical efficacy, we do not anticipate 
that the sample sizes will be adequately powered to compare the clinical efficacy between 
arms.  The purpose of different design is to discern the difference in anti-tumor immune 
responses when cyclophosphamide is administrated in a different way.  The data will serve 
as the rationale for the choice of cyclophosphamide administration schedule/dose in the 
future clinical studies.  For instance, if one modality of cyclophosphamide administration 
results in a more favorable immune response, we will postulate that such a schedule of 
cyclophosphamide administration would also result in a more favorable clinical response.   
We will also combine all three arms and compare them with data from our prior phase II 
adjuvant study or historical data of non-vaccinated patients.  
 
4.3.2 Relating Immune Response to Clinical Response 
 
The relationships between immune and clinical responses will be assessed using a variety 
of statistical techniques. Preliminary explorations will be graphical in nature (e.g. boxplots, 
scatterplots). Univariate and multivariate modeling will be used quantify the associations. 
In the case of a binary clinical outcome (e.g. toxicity), logistic regression will be used. In 
the case of a time-to-event clinical outcome (e.g. OS), the Cox proportional hazards model 
will be used. 
 
4.4 Analysis of Exploratory Endpoint 
 
Genomic sequencing library construction, whole genome/exome sequencing, whole 
transcriptome sequencing, microbial sequencing, neoepitope prediction, mutation burden, 
and bioinformatic analysis may be performed either at an on-campus laboratory or at an 
off-campus sequencing service. All the samples will be de-identified before sending to any 
laboratory for sequencing.  The FASTQ files, BAM files and VCF files will be generated 
and analyzed. Other sequencing assays may be performed on a subset of samples according 
to specific requirements of collaboration projects.  
 
Results from the sequencing studies will not be released to the patients. These studies are 
for research purposes only and are not using a clinically validated platform. 
 
Genomic sequencing data will be either destroyed or stored on a JHU managed, HIPAA-
compliant, password protected hard drive or Johns Hopkins University School of Medicine 
PMAP. 
 

 
 
 
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Genomic sequencing data will be either destroyed after the research or stored and 
computations in a HIPAA-compliance space.  
 
5.0 Response Criteria 
 
5.1 Evaluation of Clinical Activity 
 
Most patients will be expected to have only minimal residual disease if they remain to be 
eligible for this study following the surgery. Therefore, there will be no disease to measure 
at baseline. Patients will be monitored for disease-free and overall survival. The results 
from this trial will be compared among different arms, to the results of our prior phase II 
adjuvant study, to historical controls seen at the Johns Hopkins Hospital. Patients recently 
seen at the Johns Hopkins Hospital who can be matched for pathologic stage, surgical 
intervention, and adjuvant combination chemotherapy and radiation therapy, are the most 
accurate group of historical controls since our institution has the largest reported 
experience treating patients with this disease and has recently reported the best survival 
statistics for current interventions. Patients may undergo standard of care evaluations 
consisting of abdominal, chest, and pelvis CT scans at regular intervals to evaluate for local 
recurrence and distant metastases. In addition, any patient presenting with symptoms will 
undergo evaluation for metastases. Recurrent disease is defined as evidence of either local 
or metastatic recurrence by CT scan. The serum tumor marker CA19-9 lacks a sufficient 
sensitivity and specificity to serve as reliable indicators of response. The CA 19-9 levels 
will be followed to evaluate whether large and persistent changes might correlate with 
either in vitro immune responses or with time to clinical recurrence. 
 
5.2 Evaluation of Immune Responses 
 
A central goal of the clinical trial is to identify immunologic changes associated with the 
vaccine therapy that may be markers of potential clinical responses. Studies include, but 
are not limited to, the analysis of mesothelin-specific antitumor immune responses. All the 
techniques involved are well established at Johns Hopkins, and these studies will be 
performed in close collaboration with appropriate CORE facilities. 
 
The leukapheresis product or peripheral blood will be obtained from each research 
participant at periodic protocol-specified intervals for the in vitro assays. Serum will also 
be obtained at the same intervals for humoral immune response.  In addition, research 
participants may be asked to donate additional lymphocytes by leukapheresis and 
additional serum.  Informed consent will be obtained for this procedure using the 
Leukapheresis Informed Consent Form, which is a separate document than the main 
vaccine study informed consent form. 
 
6.0 Adverse and Problem Event Reporting 
 
6.1. Responsibilities 
 

 
 
 
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It is the responsibility of the principal investigator to notify the IND sponsor of the vaccine 
research product, Elizabeth Jaffee, M.D., Hopkins Medicine Institutional Review Board, 
and Hopkins Institutional Biosafety Committee of any serious adverse event due to any 
cause, which occurs during the course of this investigation, and is believed to be in any 
way related to study drug. The sponsor will notify the appropriate federal regulatory 
agencies, including but not limited to the Food and Drug Administration. The Principal 
Investigator or their designees must notify the IND sponsor, Elizabeth Jaffee, M.D. of any 
Serious Adverse Event (SAE) within 24 hours of the investigator learning that the adverse 
event has occurred. Events must be documented on the Johns Hopkins Medicine IRB form 
titled, “Problem/Event Report Form” available at http://irb.jhmi.edu. See section 6.3 for 
IRB reporting guidelines. 
 
All adverse and problem events as well as serious adverse events occurring from day 0 to 
28 of each vaccine cycle will be recorded.  
 
A serious adverse event (SAE) is one that: 
• Results in death 
• Is life-threatening 
• Requires inpatient hospitalization or prolongation of an existing hospitalization > 
24 hours 
• Results in persistent or significant disability/incapacity 
• Is a congenital anomaly/birth defect in the offspring of an exposed patient 
 
An important medical event 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, it jeopardizes the subject and may require medical or 
surgical intervention to prevent one of the outcomes listed in this definition. 
A life threatening adverse event is defined as any adverse experience that places the patient 
or subject, in the view of the investigator, at immediate risk of death from the reaction as 
it occurred, i.e., it does not include a reaction that, had it occurred in a more severe form, 
might have caused death. 
An adverse drug reaction (ADR) is a noxious and unintended response to a medicinal 
product in which a causal relationship between a medicinal product and an adverse event 
is at least a reasonable possibility, i.e. that the relationship cannot be ruled out. 
A planned hospitalization however is not considered a SAE. 
An elective surgery will be considered an AE but not an SAE unless the hospitalization is 
prolonged due to complications. 
 
6.2 Recording of an Adverse Event 
 
The principal investigator is responsible for evaluating all adverse events, obtaining 
supporting documents, and determining that documentation of the event is adequate. The 
principal investigator is responsible for determining the severity and relationship of the 
adverse event to the investigational drug. The principal investigator may delegate these 
duties to sub-investigators and must assure that these sub-investigators are qualified to 
perform these duties under the supervision of the principal investigator. 

 
 
 
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All adverse events will be recorded in the subject’s Case Report Form and in the study 
database. The detailed description of the event will include appropriately graded severity 
of the adverse event and its relationship to the study drug. 
 
Severity will be categorized by toxicity grade according to the NCI Common Terminology 
Criteria 
for 
Adverse 
Events 
version 
3.0 
available 
at 
http://ctep.cancer.gov/reporting/ctc.html  
 
Adverse events not listed in the NCI Common Terminology Criteria for Adverse Events 
will be evaluated using the following criteria: 
• Grade 1, Mild: Awareness of symptom, but easily tolerated; usually transient 
requiring no special treatment; does not interfere with usual status or activities 
• Grade 2, Moderate: May be ameliorated by simple therapeutic measures; may 
interfere with usual activities 
• Grade 3, Severe: Incapacitating, inability to perform usual activities 
• Grade 4, Life-threatening/Disabling: Subject was at risk of death or significant 
disability at the time of the event 
• Grade 5, Death related to AE  
 
Relationship of the adverse event to the investigational drug will be determined by the 
principal investigator, and will be categorized as: 
• Not Related: The adverse event is clearly related to other factors such as the 
subject’s clinical state, environmental factors, or other modes of therapy or 
concomitant drugs administered to the subject. 
• Possible: The adverse event follows a reasonable temporal sequence from 
administration of the study drug, and/or follows a known response pattern to the 
study drug, but could readily have been produced by the subject’s clinical state, 
environmental factors, or other modes of therapy or concomitant drugs 
administered to the subject. 
• Probable: The adverse event follows a reasonable temporal sequence from 
administration of the study drug and follows a known response pattern to the study 
drug, and cannot readily have been produced by the subject’s clinical state, 
environmental factors, or other modes of therapy or concomitant drugs 
administered to the subject. 
 
All grade 3 and 4 clinical laboratory results that represent an increase in severity from 
baseline will be reported as adverse events. A grade 1 or 2 clinical laboratory abnormality, 
except for lymphopenia, neutropenia and leukopenia should be reported as an adverse event 
only if it is considered clinically significant by the investigator. Regular vaccine site 
reactions such as erythema, swelling, indurations, pruritus and tenderness will be noted by 
the RN in each visit note but will not be reported as an AE. However, reactions outside the 
vaccination site such as hives and rashes or any significant reaction due to vaccination will 
be reported as an AE. 
 

 
 
 
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In the event of death, the cause of death should be recorded as the adverse event. An attempt 
will be made to obtain a copy of the death certificate. Because the long-term effects of gene 
therapy are not known, the National Institutes of Health (NIH) would like an autopsy, in 
the event of death. If an autopsy is performed, a copy of the autopsy report should be 
obtained. 
 
6.3 Reporting Guidelines 
 
We will use the current JHM IRB, Institutional Bio-safety Committee (IBC), NIH 
Recombinant DNA Advisory Committee (RAC), FDA guidelines and SAE Reporting 
Criteria and Safety Reporting Requirements for IND Holders for reporting relevant 
problems, events, adverse events, and adverse drug reactions. 
 
7.0 Clinical Trial Monitoring 
 
On a regular basis, the protocol will be internally monitored by the principal investigator, 
and the study’s sponsor, Dr. Elizabeth Jaffee.  
 
The SKCCC Compliance Monitoring Program will provide external monitoring for JHU-
affiliated sites in accordance with SKCCC DSMP (Version 6.0, 02/21/2019). The SMC 
Subcommittee will determine the level of patient safety risk and level/frequency of 
monitoring. 
 
8.0 Developmental Methods 
 
The following projects have been developed with the goal of improving the specificity and 
efficacy of this GM-CSF transfected whole tumor pancreatic cell vaccine. These methods 
should be considered developmental.   
 
8.1 General Rationale for Banking Patient Materials 
 
Recent data from human melanoma studies have revealed that the majority of tumor-
specific antigens identified so far are shared among MHC-matched melanoma tumors. In 
addition, the cellular proteins from which they derive are often shared among non-MHC 
matched tumors.  Preliminary studies of human renal cancer have demonstrated similar 
findings (Zhou et al., 2005). We have begun to address this question in patients with 
pancreatic adenocarcinoma using patient materials from the phase I and phase II studies.  
The demonstration of shared antigens will drive an intensified effort to identify these 
antigens for the purpose of developing future, antigen-specific vaccines, thereby 
eliminating whole cells as a source of antigen. In particular, these antigens could then be 
employed in novel strategies that take advantage of our more recent understanding of tumor 
antigen processing and presentation mechanisms, hopefully leading to enhanced immune 
priming beyond that which can be achieved with cellular vaccines. The following materials 
will be collected for these developmental goals. 
 

 
 
 
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8.2 Serum Banking 
 
Serological analyses (SEREX or SERPA) are commonly used to identify tumor-associated 
antigens or antigens targeted by T cells (Nishikawa et al., 2005; Zhou et al., 2005).  To 
establish a both reliable and feasible way to predict anti-tumor immune response and also 
to identify novel pancreatic tumor antigens, Dr. Jaffee’s laboratory has used the serum from 
patients who have shown anti-tumor immune response in the previous phase II clinical 
trials to develop a functional proteomic approach to screen the antigens targeted by the 
antibody responses induced by vaccination (unpublished data).  The whole cell extract from 
Panc 6.03 and Panc 10.05 cells were used as the proteome and fractioned by 
chromatography and chromatographic fractions of proteins were further separated by 2D 
protein electrophoresis.  They were then subjected to immunoblotting analysis by using 
patients’ sera to identify the antigens that can be recognized by the antibodies in the sera.  
The immunoblots with the serum from patients prior to vaccination was compared with 
those with the post-vaccination serum.  The proteins that were unique in the immunoblots 
with the serum from post-vaccination patients were sequenced by mass spectrometry and 
their entities were identified.  These proteins, representing the antigens that are targeted by 
vaccine-induced humoral immune response, comprise a proteomic array that can be used 
to predict anti-tumor immune responses.   
 
This trial will provide us with an opportunity to begin to validate the vaccine-induced 
antibody responses to these antigens and the values of the proteomic array comprised of 
these antigens in predicting anti-tumor immune responses.  In this study, we will bank the 
serum from each participant prior to and following each vaccination.  As noted above, 
approximately 20-30 ml of blood will be required at these time points as well as at other 
indicated time points for serum banking.   They will be used in the immunoblot analyses 
of individual vaccine-specific antigens or in the ELISAs to assess whether antibodies to 
these antigens are specifically induced by vaccination.  Once the analysis of clinical 
efficacy is completed, the induction of vaccine-specific humoral immune response, 
reflected by the presence of vaccine-specific antibody production in serum, will be 
correlated with patients’ clinical response and T cell immune response.  
  

 
 
 
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