Clinical trial NCT00960752
Conditions: Melanoma
Keywords: gp100 peptide MAGE-3 peptide R848 Resiquimod Toll Like Receptor TLR T-cells Tumor Metastatic Lesions Injection Site
Sponsor: M.D. Anderson Cancer Center
Start date: 2010-05-20; Completion date: 2020-07-20
Enrollment: 47 participants; Allocation: RANDOMIZED; Intervention model: FACTORIAL

Brief summary:
The goal of this clinical research study is to learn if the vaccines, gp100(g209-2M), and MAGE-3, when given in combination with resiquimod (R848), can help to stimulate the immune system against melanoma.

Detailed description:
The Study Drug/Vaccines:

Resiquimod (R848) is designed to increase the production of antigen-specific T-cells, to activate immune cells, and to increase T-cells going into the tumor, which may cause the tumor cells to die.

gp100(g209-2M) and MAGE-3 are vaccines designed to stimulate immune cells, also called T-cells, which may help the immune system to recognize and kill melanoma cells.

Study Groups:

If you are found to be eligible to take part in this study, and you have metastatic melanoma, you will be assigned to Group 3. If you have had the melanoma removed and want to be treated to try to keep it from coming back, you will be randomly assigned (as in the flip of a coin) to Group 1 or 2.

* If you are in Group 1, you will receive the vaccines gp100(g209-2M) and MAGE-3 and R848 for 8 weeks. After 8 weeks, if you have melanoma lesions, you will apply R848 to half of the lesions on your body for 16 weeks.
* If you are in Group 2, you will receive only the vaccines (gp100(g209-2M) and MAGE-3) only for 8 weeks. After 8 weeks, if you have melanoma lesions, you will apply R848 to half of the lesions on your body for 16 weeks.
* If you are in Group 3, you will receive the vaccines gp100(g209-2M) and MAGE-3 and R848 for 8 weeks. After 8 weeks, if you have melanoma lesions, you will apply R848 to half of the lesions on your body for 16 weeks.

After 8 weeks, if you no longer have melanoma lesions, you will have completed the study treatment.

Study Drug/Vaccine Administration:

Once a week for 8 weeks, you will receive the vaccines, gp100(g209-2M) and MAGE-3, intradermally (through a needle into your skin) and subcutaneously (through a needle under your skin) at 2 separate sites in separate extremities. Half of each dose will be given one way, and half given the other way. These vaccines will also be given on the same day. You will receive a total of 4 injections each week.

If you are in Group 1, every time you have the vaccine injection, R848 will be applied as a gel over the gp100(g209-2M) injection site. The R848 gel will be allowed to air dry for 30 minutes and then covered with a gauze dressing which remain on overnight.

After 8 weeks, for participants with melanoma lesions, 2 times a week for 16 weeks, you will apply the R848 gel to half of the lesions on your body. You will be given written instructions and a "hands-on" demonstration on how to apply the gel. You will also be given a worksheet to help you with applying the gel.

Study Visits:

One day after your first dose of vaccine (Day 2), you will have a punch biopsy of the vaccine injection site to check your immune system. A punch biopsy is when a hollow instrument that is used to remove a small coin-shaped sample of tissue.

At Week 2, the following tests and procedures will be performed:

* You will have a physical exam, including measurement of your vital signs and weight.
* Your performance status will be recorded.
* Blood (about 1 teaspoon) will be drawn for routine tests.
* Blood (about 4 tablespoons) will be drawn to study your immune response to the vaccine combinations.
* You will have a punch biopsy of the injection site to check your immune system the day after the vaccine is given.

Every 4 weeks, the following tests and procedures will be performed:

* You will have a physical exam, including measurement of your vital signs and weight.
* Your performance status will be recorded.
* Blood (about 1 teaspoon) will be drawn for routine tests.
* Blood (about 4 tablespoons) will be drawn to study your immune response to the vaccine combinations.

Every 8 weeks, for participants with metastatic disease, the following tests and procedures will be performed:

* You will have CT or PET/CT scans of the chest, abdomen, and pelvis to check the status of the disease.
* The tumors will be measured and photographed. Your private areas will be covered (as much as possible), and a picture of your face will not be taken unless there are tumors on your face.
* If the doctor thinks it is necessary, the tumors will be measured by CT scan.
* If the doctor thinks it is needed, you will have an MRI/CT of the brain to check the status of the disease.

The day after you first start the gel (Week 8), you will have 2 punch tumor biopsies to check your immune system. One biopsy will be of a lesion with the R848 and one without, if it is available. For participants without disease, the study will be completed and your follow-up will be decided by your doctor.

At Week 0 (before vaccine is started), after treatment at Week 8 (+/- 1 week)., and Week 16 (+/- 1 week), white blood cells will be collected by leukapheresis to test for antibodies. If you have the leukapheresis, blood (about 2 teaspoons) will be drawn as well. If you do not have leukapheresis, blood (about 4 tablespoons) will be drawn instead.

The leukapheresis procedure is research-related and would not be performed as standard of care if you were not on study. Blood for the leukapheresis will be taken through a needle in a vein in one of your arms. A solution will be added to keep your blood from clotting while it goes through the machine that collects and removes white blood cells from your blood. The remaining blood is returned to you through a needle in a vein of your other arm. This is may take up to 3-4 hours. Although several pints of blood will go through the machine, only about 1/2 a pint (1 cup) will be in the machine at any time. If you cannot have a needle in your vein, this blood will be removed thorough a central venous catheter (CVC). A CVC is a sterile flexible tube that will be placed into a large vein while you are under local anesthesia. You will be given another informed consent document to sign, which describes the risks of the CVC and the care involved.

Length of Study:

You will continue to receive treatment for up to 4 6 months, as long as you may benefit from the study and are responding to the gel. You will be taken off study if the disease gets worse or you experience any intolerable side effects. If you are in the group without disease, you will not receive the study treatment.

Follow-Up:

All three groups will be called 1 time a month for 12 months after coming off the study drugs to check on the status of the disease.

This is an investigational study. The study drugs/vaccines gp100(g209-2M), MAGE-3, and R848 are not FDA approved or commercially available. At this time, they are only being used in research.

Up to 71 participants will take part in this study. All will be enrolled at MD Anderson.

Arms: arm 1: 1 ml of gp100 peptide vaccine and 1 ml of MAGE-3 peptide vaccine daily for 8 weeks with R848 Gel applied to gp100 injection site immediately. Injections given intradermally and subcutaneously at 2 separate sites in separate extremities. Half of each dose given one way, and half given the other way. Vaccines given on the same day. Participant receives a total of 4 injections each week.

After 8 weeks, if participant has melanoma lesions, R848 applied to half of the lesions on body for 16 weeks. arm 2: 1 ml of gp100 peptide vaccine and 1 ml of MAGE-3 peptide vaccine daily for 8 weeks. Injections given intradermally and subcutaneously at 2 separate sites in separate extremities. Half of each dose given one way, and half given the other way. Vaccines given on the same day. Participant receives a total of 4 injections each week.

After 8 weeks, if participant has melanoma lesions, R848 applied to half of the lesions on body for 16 weeks. arm 3: 1 ml of gp100 peptide vaccine and 1 ml of MAGE-3 peptide vaccine daily for 8 weeks with R848 Gel applied to gp100 injection site immediately. Injections given intradermally and subcutaneously at 2 separate sites in separate extremities. Half of each dose given one way, and half given the other way. Vaccines given on the same day. Participant receives a total of 4 injections each week.

After 8 weeks, if participant has melanoma lesions, R848 applied to half of the lesions on body for 16 weeks.
Interventions: intervention 1: gp100 intervention 2: R848 gel intervention 3: MAGE-3
Intervention descriptions: intervention 1: 1 ml of gp100 peptide vaccine: 0.5 ml administered intradermally and the other 0.5 ml subcutaneously in the same extremity weekly (every 7 days +/- 2 days) for 8 weeks. intervention 2: Applied to the gp100 vaccine injection site immediately following the injection, allowed to air dry for 30 minutes then covered with gauze dressing. intervention 3: 1 ml of MAGE-3 peptide vaccine: 0.5 ml administered intradermally and the other 0.5 ml subcutaneously in the same extremity weekly (every 7 days +/- 2 days) for 8 weeks.

Protocol documents:
Prot_SAP_000.pdf: 
2008-0416 
May 7, 2015 
 
 
 
1 
 
 
Activation of pDCs at the Tumor and Vaccine Site with a TLR Agonist 
Abstract 
Although cancer vaccines offer significant promise, clinical effectiveness has yet to be realized.  
Effective vaccines may require inclusion of basic elements of the immune response which allow 
successful elimination of pathogens.  It is now clear that strong adaptive immune responses are 
preceded by a potent innate immune response, triggered by pathogen-associated molecular 
patterns (PAMPs) that are recognized by immune cells expressing Toll-like receptors (TLRs).  In 
the absence of TLR signaling, release of inflammatory cytokines by innate immune cells is sub-
optimal, likely explaining why tumors are poor at eliciting antigen-specific adaptive immunity. 
Harnessing and adapting the mechanisms used by pathogens to induce effective specific 
immunity represents a very promising approach to improving antigen-specific antitumor immune 
responses.    The plasmacytoid dendritic cell, the primary producer of type I interferons in the 
body, is a central mediator of inflammation in response to TLR activation and coordinates 
immune cell interactions which lead to a potent adaptive immune response.  This inflammatory 
response is important not only to trigger strong T-cell priming, but also to induce inflammation at 
the target site which leads to enhanced T-cell migration and effector function.  In our murine 
models, we have found that plasmacytoid dendritic (pDC) cells can lead to enhanced antigen-
specific immune responses, partially through synergy with myeloid dendritic cells (mDC).  In 
addition, we and others have found that pDC can be activated directly in vivo through specific 
toll-like receptor (TLR) ligands.  In this proposal, we will test these concepts in melanoma 
patients and will utilize a vaccine in combination with a TLR agonist capable of activating both 
pDC and mDC in order to model the synergy observed in our murine system.  We will measure 
T-cell priming in patients immunized in the presence or absence of TLR activation.  Subsequent 
to T-cell priming, we will administer the TLR agonist at the tumor site in order to induce 
inflammation at sites of cutaneous metastases and will test the ability of this intervention to 
activate pDC at the tumor site and enhance T-cell migration into the tumor and T-cell effector 
function.  Our goal is to identify principles which may be generalized towards improving cancer 
vaccines against other common cancers. 
1.0  
Objectives 
 
 
1.1  
Primary Objective 
To compare the ability of vaccines in combination with TLR stimulation at the site 
of vaccine (R848; Resiquimod) to vaccines alone in the ability to enhance the 
generation of circulating antigen-specific T-cells (T-cell priming). 
 
 
 
1.2  
Secondary Objectives 
A.  To evaluate the ability of locally administered TLR agonist (R848 gel) to 
activate innate immune cells at the gp100 (g209-2M) vaccine site. 
 
 
 
B.   To evaluate the ability of R848 gel, administered at the tumor 
                             site, to: 
 
i.   Induce inflammation and upregulation of adhesion molecules on tumor 
vasculature 
ii.   Enhance T-cell infiltration into tumor 
iii.  Generate T-cells against additional tumor antigens, not  present in the 
vaccine (i.e., antigen spreading). 
iv. Mediate clinical response with measure of objective tumor response rate and 
progression free survival. 


2008-0416 
May 7, 2015 
 
 
 
2 
 
 
C.   To assess the association between clinical response and laboratory 
parameters of T-cell priming, T-cell migration to tumor, and inflammation at the 
vaccine and tumor sites. 
 
2.0 
Background Drug Information 
2.1  
Natural antitumor immune responses are often weak compared with 
antiviral immune responses 
The notion of whether the immune system is capable of recognizing, responding 
to, and eradicating established tumors was at one time a rather contentious and 
controversial issue 1,2.  However, much evidence has accumulated over the past 
two decades, both in humans and in mouse models, and at the cellular and 
molecular level, to establish that immune cells can play an important role in 
inducing successful cancer regression 3,4.  Multiple studies using different tumor 
models have shown that administration of immune cytokines, specific vaccines, 
and adoptive transfer of immune cells can all lead to effective tumor eradication 
in the appropriate setting 5-7. Nonetheless, it is true that all of these treatments 
rely on specific immune interventions for their success.  The generation of 
effective natural immunity against established tumors is likely to be a very 
infrequent event, as evidenced by the clinical manifestation of tumors in non-
immunocompromised hosts and by the fact that spontaneous regressions are 
very rarely observed.  
 
By contrast, the natural generation of effective immunity against viral infections 
remains the rule rather than the exception. Multiple studies have demonstrated 
that viral infections frequently lead to the spontaneous generation of strong 
immune responses that are often not only capable of inducing viral clearance, but 
also in generating long-lived memory responses capable of protecting the host 
against re-infection 8-10.  In the past ten years, a number of seminal research 
studies have shed much light on why viruses and other pathogens can elicit such 
potent and effective natural immune responses. We believe that harnessing and 
adapting the mechanisms used by pathogens to induce effective specific 
immunity represents a very promising approach to improving specific antitumor 
immune responses.  
 
2.2  
Activation of innate immunity is critical for the generation of effective 
adaptive immune responses 
Effective antiviral immune responses are initiated through activation of innate 
immune cells, including NK cells, conventional myeloid dendritic cells (mDCs), 
and plasmacytoid dendritic cells (pDCs), by specific TLR ligands 11-13.  Activation 
of innate immunity induces the production of proinflammatory cytokines which 
can directly activate cells important for the initiation of adaptive immune 
responses.  Type I interferons (IFNs) and tumor necrosis factor (TNF-β), for 
example, are potent inducers of mDC maturation, inducing upregulation of major 
histocompatibility complex (MHC) and costimulatory molecules as well as 
production of IL-12, both of which are important for the priming of naïve T cells 
14,15.  In addition, the activation of NK cells by pDC, cytokines and TLR ligands 
may lead to increased lysis of virally-infected cells or tumors which, in turn, can 
provide antigen to mDCs for presentation to T cells.  Activation of innate 


2008-0416 
May 7, 2015 
 
 
 
3 
 
 
immunity is important not only for the generation of antigen-specific T-cells, but 
also to induce inflammation at the pathogen site which leads to enhanced 
migration of antigen-specific T-cells to the infected tissue.  
2.3  
Plasmacytoid DCs represent a critical link  
between innate and adaptive immunity  
As the major producer of type I IFNs (represented by IFN- and IFN-), pDCs 
represent one of the most important links between innate and adaptive immunity 
16-20. Upon triggering of TLR7 or TLR9 by virus, pDCs rapidly produce large 
amounts of Type I IFNs, activate a variety of immune cells such as B cells, 
natural killer (NK) cells and macrophages, and differentiate into antigen 
presenting cells (APCs) to induce antigen-specific T cell responses 21. Both 
mDCs and NK cell activation can also be partially mediated by Type I interferons.  
IFNRc -/- mDCs are defective in the ability to adequately respond to viral 
infections 22, suggesting that interferon-producing pDCs may be critical for the 
activation of mDCs and subsequent development of adaptive immunity.  A 
number of lines of evidence suggest that 
pDCs may interact with mDCs to induce an 
enhanced adaptive immune response in the 
development of antiviral immunity 23-25. 
Activation of mDCs by double stranded RNA 
or viral infection has been shown to be 
dependent on exposure to IFN- 22,25-27. In 
addition, HIV was found to be able to activate 
pDC, which could subsequently activate mDC 
upon co-culture 24. In addition, it has been 
recently demonstrated that pDCs may interact 
with lymph node mDCs in the generation of 
anti-HSV CTL 28.   
  
2.4 
Cancer vaccines have significant 
potential, but require considerable 
optimization 
The identification of tumor antigens 
recognized by T-cells has allowed the 
development of rational cancer vaccine 
strategies.  Current evidence suggests that 
cancer vaccines have the ability to increase 
the levels of circulating T-cells capable of 
recognizing tumor antigens.  However, this 
has not led to significant tumor regressions in 
patients.  For example, an analysis of the 
response rate in over 500 patients with 
metastatic melanoma treated with vaccines 
was under 3%.29   Our hypothesis is that this 
is due to inadequate activation of innate 
immunity at the site of immunization as well 
as at the tumor site itself.  Lack of adequate 
inflammation at the vaccine site may lead to suboptimal T-cell priming, while equally 
 
Figure 1. Immunization with a  combination of CpG-
activated pDC plus mDC significantly enhances 
antigen-specific CTL responses. Mice were immunized 
s.c. with purified pDC, mDC, or a combination of pDC 
and mDC (1:1) which were first co-cultured for 4 hours 
with  CpG and OVA257-264 peptide (the total number of 
DCs injected were kept at the same level for all 
groups). Seven days after immunization, mice were 
bled and PBL were isolated. Ag-specific IFN-γ 
production by immune cells was assessed by ELISPOT 
assay upon in vitro restimulation with or without OVA 
peptide. ELISPOT picture represents duplicate wells in 
each immunized group in the presence of OVA peptide 
stimulation. 


2008-0416 
May 7, 2015 
 
 
 
4 
 
 
important; the lack of inflammation at the tumor site may lead to inefficient migration of 
T-cells back to the tumor. 
 
 
2.5  
Plasmacytoid dendritic cells and myeloid dendritic cells synergize in their ability 
to generate antigen specific immune reactions, resulting in an enhanced 
antitumor response in vivo  
We hypothesized that by their capacity to activate innate immunity, pDCs would 
potentiate the function of mDCs when used in combination. Indeed, when purified 
murine pDCs and mDCs were co-cultured in the presence of antigen and a TLR 
activation stimulus, such as CpG, followed by in vivo administration, antigen-specific T-
cell levels were higher than with the administration of either DC subset alone (figure 1).  
Since the total number of DCs was kept constant for all groups, the interaction between 
pDC and mDC in their ability to stimulate T-cells was synergistic and not simply additive.   
Importantly, tumor reduction was also enhanced when mice were treated with the 
combination of activated, antigen-pulsed pDC and mDC compared to either DC 
subset alone (Figure 2).  Treatment of tumors with a combination of pDC and 
mDC resulted in mice with both smaller tumors (Figure 2A) and enhanced 
survival (Figure 2B). 
 
Since pDC and mDC synergize in the generation of antigen-specific T-cells, we 
propose to use a TLR agonist, R848, which is capable of activating both pDC 
and mDC through TLR 7 and 8, respectively, in combination with vaccine in order 
to induce antigen-specific T-cells in melanoma patients. 
 
2.6  
TLR agonists can activate pDC and mDC and can induce inflammation 
 
       
 
a. R848 (Resiquimod) 
Resiquimod (also known as R-848) is a novel heterocyclic amine  that acts as a 
dual TLR7 and TLR8 agonist and therefore is capable of activating both 
plasmacytoid dendritic cells (pDC), which express TLR7, and myeloid dendritic 
cells (mDC), which express TLR8.  Resiquimod is a member of the 
imidazoquinoline family of IRMs, of which imiquimod has been most extensively 
characterized (and is in active clinical use for the treatment of actinic keratosis 
and basal cell cancer).  Resiquimod is consistently more potent than imiquimod 
(50-100 fold) based on median minimum concentration required to elicit a 3-fold 
increase in IFN-α, TNF-α, and IL-12 production by human PBMC.  Resiquimod 
can promote functional maturation of DC subsets as defined by enhanced 
expression of co-stimulatory molecules important for antigen presentation and T-
cell activation. 
Resiquimod is available in a gel formulation for topical use and has been used 
previously in clinical trials of herpetic lesions, actinic keratosis and anogenital 
warts.  These studies revealed that topical resiquimod is well tolerated and 
capable of inducing inflammatory reactions consistent with activation of 
plasmacytoid and myeloid dendritic cells.  As we have shown synergy between 
pDC and mDC in the induction of antigen-specific T-cell priming, we propose to 
utilize topical resiquimod over the vaccine site. 


2008-0416 
May 7, 2015 
 
 
 
5 
 
 
In a study of multiple doses of topical resiquimod applied to intact skin of the 
upper arm (1246-R848), with a maximum exposure of 2.5mg (1gm of 0.25% gel) 
2x/week for 3 weeks, dose-related , but tolerable, systemic adverse effects 
consistent with systemic cytokine induction were observed.  These included 
fever, chills, and transient neutropenia.  Application site reactions including 
erythema, stinging, and pruritis were also observed.  Dose-related increases in 
local cytokine production and T-cell infiltration were also reported. 
     
Because the 0.25% dose provided the highest level of immune cell infiltration, we 
will plan to utilize a dose of 0.2% R848 2 times per week in our study (currently, 
this is the highest clinical grade formulation available), with the option of dose 
reducing to 1 time per week should patients not tolerate the treatment due to 
adverse reactions.  
      
In murine models, we have found that TLR agonists, given at the site of vaccine, 
can enhance the generation of antigen-specific T-cells (T-cell priming).    
 
2.7  
TLR 7/8 agonist R848 enhances T-cell priming against   gp100(g209-2M) 
While activated pDC at the site of immunization resulted in enhanced T-cell 
priming, use of purified activated pDC would be challenging in the clinic due to 
the extensive and personalized nature of the preparation.  Although interferon- 
is a major mediator of the effects of pDC, it is clear from our studies that cell-cell 
contact between pDC and mDC also play an important role and providing 
interferon as an adjuvant will be insufficient 
to obtain the full benefits of pDC activation.  
Therefore, we have focused on the use of 
toll-like receptor agonists capable of 
activating pDC in vivo.  Because our 
preliminary data indicates that activated 
pDC and mDC synergize in the priming of 
specific T-cells, we have selected a novel 
molecule, Resiquimod (R848), which 
stimulates both TLR 7 and 8, thereby 
activating both pDC and mDC, respectively.    
In order to evaluate the ability of R848 to 
enhance antigen specific vaccination, we 
first administered naïve pmel T-cells, which 
are transgenic to express a T-cell receptor 
specific for the gp100(g209-2M) antigen.  
We found that administration of R848 gel 
over a vaccine site using the melanoma 
antigen gp100(g209-2M) enhances the 
proliferation of gp100(g209-2M).  Specific 
pmel T-cells compared to the vaccine alone 
(Figure 3).  
 
Besides enhancing T-cell priming, we have found that the administration of TLR 
agonists at the tumor site induces pDC activation, and subsequent T-cell infiltration, 
tumor regression, and generation of T-cells against other (“de novo”) antigens.  
 
0
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CD8+/Thy1.1+ (% of CD8+)
no treatment
R848                
vaccine              
vaccine + R848


2008-0416 
May 7, 2015 
 
 
 
6 
 
 
 
2.8  
TLR agonist administration results in activation of pDC at the tumor site. 
To determine the ability of TLR agonists 
to activate pDC at the tumor site, 
Resiquimod (R848) or CpG, capable of 
activating pDC through TLR 7 or TLR 9, 
respectively, was injected intratumorally 
and 24 hours later tumor was harvested 
and pDC were evaluated by flow 
cytometry. Intratumoral injection of both 
Resiquimod and CpG resulted in pDC 
activation, as determined by CD86 
expression (Figure 4 ).  Similar results 
were seen using CD40 as a marker of pDC activation (not shown). 
 
Having demonstrated that administration of TLR agonists resulted in pDC activation at the tumor 
site, we next studied whether this led to enhanced infiltration of T-cells into the tumor.  To study 
this, mice bearing subcutaneous B16-ova melanoma tumors were injected with naïve pmel T-
cells, which express a T-cell receptor specific for the gp100(g209-2M) melanoma antigen, 
expressed by B16 tumor.  Use of the pmel system allows the tracking of vaccine-induced 
lymphocytes in vivo, as well as the simulation of the circulating levels of T-cells we find in 
patients following immunization.  Mice were then immunized with gp100(g209-2M)  peptide in 
IFA.  TLR activation at the tumor site was performed by giving intratumoral CpG every other 
day.  Five days later, tumors were harvested and the percentage of Pmel and ova-specific T-
cells were enumerated by flow cytometry.  Pmel T-cells could be distinguished from 
endogenous T-cells by expression of the congenic marker Thy 1.1.  This experiment allowed us 
to determine whether TLR activation at the tumor site increased vaccine induced gp100(g209-
2M) specific T-cells as well as non-vaccine induced T-cells against a de novo antigen (ova in 
Fig. 5. Intratumoral TLR triggering enhances accumulation and anti-tumor effect of vaccine-induced and de novo induced tumor-
specific T cells. C57BL/6 mice were injected with 3x105 B16.OVA cells on day 0, 10 g Flt3L plasmid i.v. on day 2 (to increase 
circulating pDC), 1x10
7 Thy1.1+ pmel-1 cells and hgp100(g209-2M) /IFA peptide vaccination on day 6 and i.t. injection of 100 g CpG 
on days 13, 15 and 17. On day 18 tumors were excised (n=3) and infiltrating pmel-1 (A) and OVA-specific (B) T cells were counted 
by FACS. (C) Five mice per group continued to receive i.t. CpG every other day and tumor growth was followed. (D) Mice were 
treated as in (C) but instead of i.t. CpG injections, topical imiquimod (Aldara cream) was applied on the s.c. tumors every three days. 
Tumor size on day 22 after tumor injection is shown. 
 
A 
B 
C 
D 
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pmel-1 +
gp100/IFA
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imiquimod
on tumor
tumor size on day 22 (mm2)
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p<0.05
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gp100/IFA
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imiquimod
on tumor
tumor size on day 22 (mm2)
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pmel-1 +
gp100/IFA
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imiquimod
on tumor
tumor size on day 22 (mm2)
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tumor size (mm2)
no tx
i.t. CpG
pmel + gp100/IFA
pmel + gp100/IFA + i.t. CpG
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* p<0.01
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tumor size (mm2)
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i.t. CpG
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** *
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* p<0.01
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pmel-1 +
gp100/IFA
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OVA-spec. T cells/g of tumor (x103)
p<0.05
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*
p<0.05
Figure 4.  B16 tumor bearing mice (3 mice per group) received either CpG (37 ug/mouse) or resiquimod (40 ug/mouse) 
intratumorally. One day later mice were  sacrificed and a single cell suspension was prepared from tumors. The 
percentage of CD86 positive pDC was analyzed using flow cytometry.  
0
20
40
60
80
100
CpG
resiquimod
no tx
Percentage of CD86+ pDC (of total pDC) 


2008-0416 
May 7, 2015 
 
 
 
7 
 
 
 
Figure 6: Activated pDC at the tumor site can lead 
to cross-priming of tumor antigens. C57Bl/6 mice 
were injected subcutaneously with 3x105 B16 
melanoma cells.  10 days later, tumors were 
injected with pDC that were either unactivated or 
activated for 6 hours with the TLR 9 agonist CpG.  
NK cells were depleted in the indicated groups by 
i.p. injection of 200 ug anti-asialo-GM1 Ab on days 
-2, 0 and 2 (in repeat experiments, anti-NK1.1 was 
used with similar results).  Five days after pDC 
injection, splenocytes were harvested, and pulsed 
with an H2-Kb restricted peptide derived from 
p15E, an endogenous retroviral antigen expressed 
by B16.  IFN-γ production was measured by 
intracellular staining and flow cytometry. 
this model) through cross-priming.  High levels of vaccine-induced, antigen-specific Pmel T-cells 
were found to infiltrate tumors only in mice receiving TLR activation at the tumor site (Fig 5a).  
Moreover, using tetramer analysis, endogenously derived ova-specific T-cells were found in 
high numbers in tumors receiving TLR activation compared to those that did not (Fig 5b). This 
suggests that TLR activation at the tumor site results in increased accumulation of vaccine-
induced T-cells, as well as cross-priming of endogenous T-cells against other antigens present 
in the tumor.  Finally, mice receiving a combination 
of vaccine and intratumoral TLR activation 
exhibited tumor regression compared to control 
groups (Fig 5c and 5d). This  
 
 
 
 
 
 
 
was evaluated using both intratumoral CpG administration (TLR 9 agonist, Fig 5c), as well as 
topical imiquimod (TLR7 agonist, Fig 5d).   
These studies suggest that the induction of high numbers of circulating vaccine-induced, 
antigen specific T-cells followed by TLR activation at the tumor site can result in 
migration of the vaccine-induced T-cells to the tumor. In addition, this can result in 
increased cross priming against other antigens expressed by the tumor, and, 
importantly, tumor regression.  
 
2.10 Activated pDC can enhance cross-priming of tumor antigens 
To specifically evaluate the role of activated pDCs at the tumor site, we injected activated 
purified pDC into tumors then evaluated splenic T-cells one week later for their ability to 
specifically recognize tumor antigens.  Following the intratumoral injection of TLR-activated but 
not non-activated pDC, T-cells were found to be reactive against the native tumor antigen 
p15E, an endogenous retroviral antigen which has been found to be upregulated in murine 
tumors (Figure 6). 
 
Cross-primed T-cells were found to infiltrate and induce regression of both pDC injected and 
non-injected tumors.  Interestingly, we found this cross-priming to be NK cell dependent, since 
generation of pDC-induced p15E specific T-cells was not seen following depletion of NK cells 
(Fig 6). In further studies, we found that activated pDC chemoattract NK cell migration into the 


2008-0416 
May 7, 2015 
 
 
 
8 
 
 
tumor site (Fig 7a).  We hypothesize that this leads to NK cell activation and IFN production, 
tumor lysis (Fig 7b), uptake and cross-priming of tumor antigen by mDC, and activation of 
specific T-cells in the draining lymph nodes.  Indeed, antigen-specific T-cells were decreased 
when activated pDC were intratumorally injected in mice deficient for either perforin or IFN.  
Furthermore, mDCs isolated from lymph nodes draining pDC injected, GFP-expressing tumor 
were found to be GFP positive, and capable of activating antigen-specific T-cells.  Therefore, we 
further hypothesize that TLR-mediated activation of pDC at the tumor site will result in NK cell 
infiltration and cross-priming of antigens to generate T-cells with de novo specificities.  
 
Testing our hypothesis in patients, that vaccines in combination with TLR agonists will enhance 
both T-cell priming and migration/effector function, requires vaccines which can be used to elicit 
antigen specific T-cells that can be reliably measured in immune monitoring assays. 
 
2.11   Gp100(g209-2M) peptide vaccine 
 
Gp100(g209-2M) is a melanocyte differentiation antigen with high levels of expression in 
melanomas.  A synthetic peptide of gp100(g209-2M) with the replacement of threonine 
by methionine at the second position of the immunodominant peptide, referred as g209-
2M appeared to be more immunogenic in vitro than the native peptide, due to enhanced 
binding to the HLA-A*0201 molecule. This peptide has been studied in vivo in patients 
with metastatic melanoma. Tumor-specific immune responses to gp100(g209-2M) 
peptide vaccines have been demonstrated. 
     
Previously our group has reported that the immunization of patients with g209-2M 
emulsified in Incomplete Freund’ adjuvant (IFA) could generate T cell responses against 
the native peptide and melanoma cells5.As shown in a characteristic experiment in Table 
1, PBMCs obtained from patients prior to immunization exhibited no in vitro sensitization 
against peptide after 11 days of incubation with g209 or g209-2M peptide, but developed 
0
2
4
6
8
PBS
pDC
pDC/CpG
Percentage of NK1.1+CD3- in 
total live cell gate 
Figure 7: Activated pDC induce NK cell migration and activation.  A. Infiltration of NK cells in tumor induced by intratumoral 
injection of pDC. Mice (3 mice per group) were first inoculated with 3 x 105 B16 tumor cells. Ten days later, mice received intratumoral 
injection of non-activated or CpG-activated pDC. Thirty six hrs after pDC intratumoral injection, mice were sacrificed and the 
percentage of NK cells in tumor were evaluated by staining cells with NK1.1 and CD3. The percentage shown is the percent of NK1.1+ 
CD3- cells in total live cell gate after excluding dead cells by PI staining. 
B. Activation of NK cells in vivo by pDC. Mice (3 mice per group) received i.v. injection of non-activated or CpG-activated pDC. Mice were 
sacrificed 36 hrs later and NK cells were enriched from spleen using DX5 microbeads and then FACS-sorted for NK1.1+/CD3-. Cytotoxicity 
of NK cells was tested in 4hrs Chromium release assay by using 51Cr labeled B16 tumor cells as target cells and with different effector: 
0
5
10
15
20
25
30:1
10:1
3:1
PBS
pDC
pDC+CpG
Specific Lysis (%)
E : T
0
5
10
15
20
25
30:1
10:1
3:1
PBS
pDC
pDC+CpG
Specific Lysis (%)
0
5
10
15
20
25
30:1
10:1
3:1
PBS
pDC
pDC+CpG
Specific Lysis (%)
E : T
A. 
B. 


2008-0416 
May 7, 2015 
 
 
 
9 
 
 
specific anti-influenza reactivity ater incubation with the control flu peptide. However, 
PBMCs obtained following two immunizations with g209-2M in IFA at a 3-week interval 
could be specifically sensitized in vitro to recognize native peptide as well as two HLA-
A*0201+ melanoma cell lines.  Furthermore, administration of g209-2M with concurrent 
IL-2 therapy resulted in tumor regression in 13 of 31 patients (42%) with metastatic 
melanoma.  This response rate is higher than previous studies of IL-2 alone (17%) 31. 
 
Table 1. Specificity of reactivity against g209-2M peptide and HLA-A*0201+ melanomas. 
 
 
In a study designed to identify the optimal schedule for immunization, patients received g209-
2M peptide vaccines in three different schedules: once weekly for three weeks, every day for 4 
days every three weeks, or once every 3 weeks (figure 8).   
 
 
 
Figure 8. Antigen-specific CD8+ T cells 
were detected at similar levels following 
three different vaccination schedules.  
 
1mg of gp209-2M in IFA was injected 
subcutaneously every 3 weeks, once weekly, 
or every day for 4 days every 3 weeks. 
PBMCs from each group of patients were 
collected and analyzed at 3-month (one 
course) following immunization. Antigen-
specific CD8+ T cells were evaluated by 
tetramer assay. 
 
For this trial, we will adopt the weekly peptide vaccination schedule based on its efficacy (Figure 
8) and feasibility. 
 
In a group of patients who received vaccination every 3 weeks for 12 months, antigen-specific 
CD8+ T cells were analyzed at 3, 6, 9, and 12 months by tetramer assay. As shown in Figure 9, 
the number of antigen-reactive CD8+ T cells gradually increased over this period. 
Tetramer 209 Analysis - Adjuvant Patients
                            Post 4
Tetramer positive/104 CD8
1
10
100
1000
10000
q3w
qw
qdx4
 
 
 
Simulator (IFN-γ pg /ml) 
No. of 
immunizations 
In vitro 
sensitization 
with peptide 
 
T2 
T2 (flu) 
T2 
(209) 
501 
mel 
(A2+) 
SK23.mel 
(A2+) 
888 
mel 
(A2-) 
None 
209-2M 
135 
230 
146 
195 
180 
115 
 
209 
118 
238 
84 
148 
231 
261 
 
Flu 
178 
35,570 
124 
290 
284 
282 
 
 
 
 
 
 
 
 
2 
209-2M 
86 
61 
24,150 
72,780 
43,250 
33 
 
209 
86 
165 
9,890 
25,710 
19,480 
98 
 
Flu 
121 
36,460 
132 
313 
448 
362 


2008-0416 
May 7, 2015 
 
 
 10 
 
 
 
 
 
 
 
Figure 9. Levels of antigen-reactive 
CD8+ T cells gradually increased over 
12-month vaccination period.  
 
PBMCs from patients receiving vaccine 
q3w were collected at 4 different time 
points and analyzed by tetramer assay. 
 
 
To test whether the frequency of antigen-specific T cells analyzed by tetramer assay correlates 
with that of ELISPOT, PBMCs were also analyzed by IFN-γ ELISPOT. Similar to the tetramer 
assay, increased levels of IFN-γ producing CD8+ T cells specific for gp209-2M and melanoma 
cells was detected at 3-months after initiation of vaccine, with higher levels at 12 months (data 
not shown). Antigen-specific T cell reactivity analyzed by tetramer assay correlated well with 
IFN-γ ELISPOT assay as shown in Figure 10. 
 
 
                       Post 3 and 4 Courses
Tetramer positive/104 CD8
1
10
100
1000
10000
Elispot positiv e/10 5 PBMC
1
10
100
1000
10000
 
Figure 10. Comparison of tetramer and 
ELISPOT in patients immunized with 
gp209-2M peptide.  
 
PBMCs were collected at different time 
points, both tetramer and ELISPOT assays 
were performed to test the levels of 
antigen-specific CD8+ T cells.  
2.12 
MAGE-3 peptide vaccines 
MAGE family antigens are also attractive targets for the development of cancer vaccines, 
because they are tumor specific, and are shared by different types of cancers. Among 
these, MAGE-3 is the most frequently expressed in melanomas, with 55% expression in 
melanoma primary lesions, and 74% in metastatic lesions 46. Several MAGE-3 peptides 
were identified that can be recognized by CD4+ T cells that are restricted by MHC class II 
molecules. Specifically, MAGE-3243-258 peptide (KKLLTQHFVQENYLEY) was identified to be 
presented by HLA-DP4 molecules47, a HLA class II allele expressed by approximately 76% 
of Caucasians.  Exposure of MAGE-3243-258 peptide presented by DP4 molecules to specific 
CD4+ T cell clones resulted in a robust production of IFN-γ. Furthermore, this specific CD4+ 
T cell clone exhibited lytic activity against melanoma cells mediated by the recognition of 
MAGE-3243-258 antigen. MAGE-3 antigens have been used in several small-scale therapeutic 
trials. The vaccines consisted of an antigenic peptide, a protein, a pox family recombinant 
Tetramer positive/104 CD8
1
10
100
1000
3m                 6m                 9m         
12m
Tetramer positive/104 CD8
1
10
100
1000
Tetramer positive/104 CD8
1
10
100
1000
3m                 6m                 9m         
12m


2008-0416 
May 7, 2015 
 
 
 11 
 
 
virus carrying a MAGE-3 sequence, or dendritic cells (DCs)-pulsed with an antigenic 
peptide48-51. No significant toxicity was observed. Clinical responses were observed in a 
small number of patients52. 
3.0 
Clinical Pharmacology 
3.1  
gp100(g209-2M): 209-217 amino acid sequence: IMDQVPFSV:  
 
This class I HLA-A2 restricted peptide will be manufactured and purchased from 
a commercial source (Merck Sciences, Clinalfa). This peptide will be provided as 
sterile, pyrogen-free lyophilisate, aliquoted in 3 ml serum vials.  Each vial will 
contain 1mg gp100(g209-2M) peptide with ≥ 95% purity. The vial will be 
individually labeled and store at –20oC. 
 
3. 2     MAGE-3243-258  peptide amino acid sequence: KKLLTQHFVQENYLEY: 
 
This class II HLA-DP4-restricted peptide will also be manufactured and 
purchased from a commercial source (Merck Sciences, Clinalfa). Similarly, this 
peptide will be provided as sterile, pyrogen-free lyophilisate, aliquoted in 3 ml 
serum vials. Each vial will contain 2 mg MAGE-3 243-258 peptide with ≥ 95% purity. 
The vial will be individually labeled and stored at –20oC. 
 
3.3 
Peptide preparation and administration: 
On the day of injection, peptides, I mg of the class I peptide (gp100(g209-2M) 
and 500 mcg of the class II peptide (MAGE-3), will be emulsified individually with 
1mL saline and 1.5 ml Incomplete Freund’s Adjuvant (IFA) formulated as 
Montanide ISA-51.   Each injection will consist of intradermal and subcutaneous 
administration at the same site.   The class I and class II peptides will be injected 
in separate extremities and the same extremity will be used for each peptide 
throughout the 8 weeks of immunizations.  Extremities that have undergone 
regional lymph node dissection will not be used for vaccine administration since 
we believe that T-cell priming occurs in the regional lymph nodes.  In addition, in 
order to obtain a prime-boost effect, the same extremity will be used for serial 
administration of each individual class I and class II peptides. 
 
3.4 
R848 Resiquimod (gel) 
 
Resiquimod is solubilized in a single phase, translucent gel formulation 
composed of propylene glycol, colloidal silicon dioxide, and triacetin. The 0.2% 
Resiquimod gel is packaged in 3.5g multidose tubes.Topical resiquimod gel 
preparations are to be stored between 15C (59F) and 30C (86F) with excursions 
permitted to 4C (39F) and 40C (104F).  R848, 0.2%, will be applied over the 
gp100(g209-2M) vaccine injection site each week. 
 
4.0  
Eligibility Assessment and Enrollment 
      
4.1       Inclusion Criteria 
4.1.1 HLA-A*0201 positive (to enable immunization with the HLA class I 
restricted gp100(g209-2M) peptide). Stage IIB or IIC patients will be 


2008-0416 
May 7, 2015 
 
 
 12 
 
 
enrolled after review and approval by the PI. (a tool to determine the 
projected survival at 5 years, like, but not limited to, the nomogram at 
www.melanomaprognosis.org. If the projected survival is less than 50% at 
5 years, then the patient is considered for enrollment. This is with the 
recognition that the adjuvant, if effective offers a significant impact in that 
group of stage II patients.) 
4.1.2 Patients > 18 years old with histologically documented melanoma with: 
a.(Metastatic disease cohort) Measurable disease, stage IIIB, IIIC 
(in transit lesions with or without nodal metastases) or 
stage IV M1A with disease that includes lesions accessible 
for biopsies or IV M1B 
b. (Adjuvant cohort) subjects who are NED and stage III or IV. 
This includes patients with stage IV disease resected to 
NED.  Stage IIB or IIC patients will be enrolled after review 
and approval by the PI. 
4.1.3    ECOG performance status of 0-1 
4.1.4    At least 2 biopsiable easily accessible cutaneous and subcutaneous 
lesions in patients in the metastatic disease cohort 
4.1.5    WBC > 3000/mm3  (part 1 & 2)  
4.1.6    Platelet count > 90,000/mm3  (part 1 & 2) 
4.1.7    Serum ALT and AST < 3 X upper limit of normal (ULN) (part 1 & 2) 
4.1.8    Total bilirubin < 2 X ULN, except for patients with Gilbert’s syndrome who  
must have a total bilirubin less than 3.0 mg/dl  (part 1 & 2) 
4.1.9   Seronegative for HIV antibody.  (The experimental treatment being  
evaluated in this protocol depends on an intact immune system.  Patients 
who are HIV seropositive can have decreased immune competence and 
thus may be less   responsive to the experimental treatment and more 
susceptible to its toxicities.) 
4.1.10 Negative pregnancy test for women of childbearing potential (WOCBP).  
A WOCBP is a woman who has not undergone a hysterectomy or who 
has not been naturally postmenopausal for at least 12 consecutive 
months (i.e., who has had mense at any time in the preceding 12 
consecutive months) 
4.1.11  Patients of both genders must practice a barrier method of birth control 
while participating in this trial. 
  
     
4.2     Exclusion Criteria 
4.2.1 
Active autoimmune disease requiring active therapy with any form of 
steroid or immunosuppressive therapy or a documented history of any of 
the following:  inflammatory bowel disease; regional enteritis; systemic 
lupus erythematosis; Sjogren's syndrome; inflammatory neurologic 
disorder such as multiple sclerosis; or any immune mediated disease that 
can cause life-threatening symptoms or severe organ/tissue damage in 
the opinion of the principle investigator. 
4.2.2 
Concurrent systemic or inhaled steroid therapy or herbal preparations 
4.2.3 
Any form of active primary or secondary immunodeficiency 
4.2.4 
Prior malignancy except for the following: adequately treated basal cell or 
squamous cell skin cancer, in-situ cervical cancer, thyroid cancer (except 
anaplastic) or any cancer from which the patient has been disease-free 
for 2 years 


2008-0416 
May 7, 2015 
 
 
 13 
 
 
4.2.5 
History of immunization with gp100(g209-2M)  
4.2.6 
Active systemic infections requiring intravenous antibiotics 
4.2.7 
Women who are breastfeeding 
4.2.8 
Prior systemic therapy, radiation therapy or intracavitary surgery (intra-
thoracic, intra-abdominal or intracranial) within 28 days of starting study 
treatment 
4.2.9 
Patients on chronic anticoagulation such as Aspirin, Plavix, or Coumadin 
who cannot have anticoagulation held for procedures are not eligible due 
to the need for leukapheresis 
 
    
4.3    Inclusion of Women and Minorities 
Both men and women and members of all ethnic groups are eligible for this trial.  
Women who are breastfeeding are ineligible. 
5.0 
Pretreatment Evaluation 
 
At the screening visit, patients will be assessed for study eligibility.  All patients 
must sign an informed consent before any protocol specific testing for eligibility 
and be registered in CORE/PDMS. 
The following baseline studies must be completed within 14 days of treatment 
initiation: 
5.1 
Complete history and physical examination 
5.2 
CBC, differential, PT/PTT, platelet count 
5.3 
Chemistries to include serum electrolytes, BUN, creatinine, glucose, albumin, 
alkaline phosphatase, ALT, AST, LDH, calcium, and total bilirubin 
5.4 
β-HCG pregnancy test on all women of child-bearing potential or who have not 
undergone sterilization procedure and have had menstruation in the past 12 
months. 
5.5 
EKG 
5.6 
HIV serology 
 
The following baseline studies must be completed within 28 days of treatment 
initiation: 
5.7 
Radiological studies (CT scans of chest, abdomen, pelvis; or PET/CT scan, MRI/CT 
of brain) to evaluate the status of disease. 
 
The following test will be performed with no time restriction 
5.8 
HLA-A* and HLA-DP4 typing 
 
6.0 
Treatment Plan 
All patients will receive the class I, HLA-A2 restricted gp100(g209-2M) peptide vaccine. 
Patients who are HLA-DP4 positive will also receive the class II restricted MAGE-3 
peptide, in separate extremities.   This will allow us to generate melanoma-specific CD8 
+ and CD4+ T-cells.  The gp100(g209-2M) immune response will be our primary 
endpoint for assessment of T-cell priming, since we have extensive previous experience 
with this vaccine and believe that CD8+ responses are critical to the development of a 
strong antitumor immune response.  The MAGE-3 peptide will be given in order to 
stimulate antigen-specific CD4+ T-cells, which we believe are also important in the 
generation of strong antitumor immunity.  Patients positive for both HLA types will 


2008-0416 
May 7, 2015 
 
 
 14 
 
 
receive both vaccines, gp100(g209-2M) and MAGE-3, for eight weeks Patients who are 
HLA-A2 positive and HLA-DP4 negative will recieve only the gp100 peptide for 8 weeks.  
There will be two arms in Part 1: Arm A is the peptide vaccines plus the TLR7/8 agonist, 
R848 and Arm B is the peptide vaccines only. All patients will be randomized to either 
Arm A or Arm B by CORe during the registration process. The  randomization will be 
stratified by cohort (metastatic disease or adjuvant).  In Arm A, R848 gel will be  applied 
to the gp100(g209-2M) vaccine injection site immediately following the injection.  The 
R848 gel will be allowed to air dry for 30 minutes and then covered with a gauze 
dressing. All vaccines will be administered subcutaneously and intradermally.   The 
gp100(g209-2M) peptide will be administered in the same extremity weekly (every 7 
days +/- 2 days) for 8 weeks.  Similarly, in a separate extremity of eligible patients, the 
MAGE-3 peptide will be administered weekly (every 7 days +/- 2 days) for 8 weeks.  
Each syringe (a total of 4, 2 for gp100 and 2 for MAGE-3) will contain 1ml of peptide 
vaccine; 0.5ml of the vaccine in each syringe will be administered intradermally and the 
other 0.5ml will be administered subcutaneously at each injection site for a total of 4 
sites. 
    
Blood samples  will be obtained serially in order to determine the immune response 
against the gp100(g209-2M) and MAGE-3 vaccines (weeks 0, 2, 4, 8, 12, 16, 20, and 
24: weeks 8 and 16 will not be collected when leukopheresis is performed; blood 
samples will be collected by leukapheresis at weeks 0 and 8 (+/- 1 week), and 16 when 
feasible).  To evaluate the inflammatory infiltrate at the site of immunization, biopsies will 
be obtained of vaccination sites as outlined in section 7.3  
 
Subjects in the adjuvant cohort.  
Vaccination will continue for the designated 8 weeks. Blood draw and pheresis times will 
be the same as subjects in the metastatic disease cohorts. Subjects will be monitored for 
disease recurrence, but if recurrence occurs prior to the completion of blood draws or 
pharesis these tests can still be obtained if deemed valid by the PI.  
 
Subjects in the metastatic disease cohort: 
At the end of 8 weeks, we will allow tumor progression since patients will have only 
received vaccine at this point.   We estimate that of a total of approximately 24 patients 
in this cohort, 17% will drop off at this point, leaving a total of 20 patients to be treated in 
Part 2.  To have enough patients to evaluate the secondary endpoints, 20 additional 
patients may be enrolled in the metastatic disease cohort so that at least 30 patients 
(10/arm) with gel and 10 are available for Part 2. 
 
At the end of 8 weeks of vaccine, cutaneous lesions (at least 2 and up to 10 lesions) will 
be assigned by the physician according to region to receive or not to receive 0.2% R848 
gel.  The CORe randomization system will not be used in Part 2.  The physician will 
designate the regions and which tumors will be treated.  The area of cutaneous lesions 
will be divided into two regions, Region A and Region B, striving to equalize tumor 
burden in each designated region as best as possible. When feasible, Region A will be a 
proximal group of lesions, and B distal. When this proximal – distal differentiation is not 
feasible because of the characteristics of the metastatic pattern, then A and B will be 
assigned to regions in no particular order.  If the two regions coalesce during the 
vaccination phase, the coalesced tumor may be divided into two regions. Each target 
tumor whether it is treated or untreated will be marked, measured and photographed for 
the medical record.  Because this dose provides the highest level of immune cell 


2008-0416 
May 7, 2015 
 
 
 15 
 
 
infiltration, we plan to utilize this dose of R848 two times per week every 3-4 days (+/- 2 
days), with the option of dose reducing from twice to once a week should patients 
experience any grade 3 or greater toxicity. 
 
The physician will designate which of the two regions to receive or not to receive R848.  
(For example, if the cutaneous disease is limited to the thigh, the area of lesions will be 
divided in two with one region proximal and one region distal. Then the proximal region 
is assigned region A and the distal assigned region B. Resiquimod to be applied to 
region A or B.  A 5mm margin will be included in the treatment area around each of the 
lesions at a spread rate of 10mcg/cm2.  The sum of the area of all treated lesions will not 
exceed 100cm2.  The treatment area will be allowed to air dry for 30 minutes and then 
covered with a gauze dressing which can be removed the following day.  The R848 gel 
will be applied in the clinic by the research nurse for the first two weeks of 
administration, and then the subject will apply the gel at home thereafter. The patient will 
be trained for gel self-administration by the research staff. Sachets of gel will be supplied 
for home use and the subject will be instructed on maintaining a home record of 
administration. This home record or a progress note by one of the research team 
reviewing home administration may each act as a source document for all matters 
regarding home administration during this period. The gel may be applied in the clinic for 
longer than the first two weeks at the discretion of the investigators. 
 
Tumor biopsies of treated and untreated lesions will be obtained as outlined in section 
7.3 when feasible in order to assess T-cell infiltration and function at the site.  
 
In order to give patients the opportunity to receive R848 gel on all their lesions, we will 
allow cross-over of patients who do not respond in untreated lesions by week 16 
(8weeks after initiating gel on the tumors) in the untreated lesions to receive R848 gel on 
all of the lesions, but these patients will not be included in the final analysis of clinical 
endpoints that occur following week 24.  Patients will not cross-over until after all blood, 
biopsies, and data are recovered for assessment of the primary endpoints in any 
subject. Treatment with R848 gel will continue for up to 4 months beyond week 24 as 
long as there is no disease progression. 
 
Subjects in either cohort will be followed for 12 months after the completion of therapy 
(vaccination in the adjuvant cohort, Resiquimod in the metastatic cohort) unless they 
meet criteria for withdrawal as per section 10.0. 
 
 


2008-0416 
May 7, 2015 
 
 
 16 
 
 
7.0 
Evaluation During Treatment 
 
7.1 
     Clinical Evaluation 
 
Patients will be evaluated every 2 weeks (+/- 2 days) through week 4, then 
every 4 weeks (+/- 1 week) during the vaccination/treatment. For subjects in 
the adjuvant cohort, this schedule will continue through week 8, for patients in 
the metastatic cohort, this will continue until Resiquimod treatment is 
discontinued. With each patient visit, the evaluation will consist of complete 
interim history and physical examination, including measurement of vital 
signs, performance status, and weight.  Patients will be evaluated for toxicity 
and concommitant medications at each visit.   Toxicities will be recorded on 
the toxicity sheet and the medications will be recorded in the patient’s 
electronic medical record.  Patients will have a complete blood count (CBC), 
with differential, PT/PTT, serum electrolytes, BUN, creatinine, glucose, 
albumin, alkaline phosphatase, ALT, AST, LDH, calcium, and total bilirubin 
tested at baseline and then tested approximately every 2-4 weeks (using 
pediatric blood collection tubes if feasible). 
 
For patients in the adjuvant arm,  
 
CT scans of the chest, abdomen, and pelvis will be done at baseline, week 8, 
(+/- 1 week) and then repeated at 8 week (+/- 1 week) intervals during 
resiquimod treatment.  MRI/CT of the brain will be done at baseline and 
repeated at the time of the CT scans if clinically indicated. 
 
At discontinuance of vaccination in the adjuvant cohort or resiquimod 
treatment in the metastatic cohort, clinical follow-up, labs, and staging studies 
(CT, MRI, PET scans) will be obtained at the discretion of the treating 
physician following standard clinical pathways.  
 
Subjects in either cohort will be followed on study for 12 months after the 
completion of therapy (vaccination in the adjuvant cohort, Resiquimod in the 
metastatic cohort) unless they meet criteria for withdrawal as per section 
10.0. Clinical events will be tracked from the physician determined routine 
clinical follow-up during this period. Clinical events that occur after this period 
on study may be retroactively recorded from the patient’s medical record 
including tumor progression and death. 
 
7.2 
Immunological Evaluations   
 
Sixty mL (4 tablespoons) of peripheral blood will be obtained at weeks 0, 2, 4, 
8, 12, 16, 20, and 24 (+/- 1 week ). Weeks 0, 8 and 16 will collect only ten mL 
(2/3 tablespoon) of peripheral blood by phlebotomy in addition to the 
leukopheresis. If the leukopheresis is not performed for those time points, 
then we will collect Sixty mL (4 tablespoons) of peripheral blood. When 
feasible, biopsies of the vaccine site will be obtained and tumor will be 
obtained as outlined in section 7.3. 
 
 


2008-0416 
May 7, 2015 
 
 
 17 
 
 
 
7.2.1 Flow cytometric analysis 
Flow cytometry will be utilized to determine the percentage of antigen 
specific CD8+ (gp100(g209-2M) or CD4+ (MAGE -3) T-cells and the 
percentage of cells expressing phenotypic markers to assess 
activation status of T-cells in the circulation and at the tumor site.  In 
addition, this technique will be used to determine the nature of the 
inflammatory infiltrate at the tumor site in the presence or absence of 
TLR agonist. 
 
7.2.2 Immunohistochemistry (IHC) 
IHC will be performed on vaccine site and tumor biopsies and will be 
used to assess the nature of the inflammatory infiltrate at these sites 
in the presence or absence of TLR agonists.  Specifically, antibodies 
specific for pDCs, mDCs, NK cells, and T-cells will be used.  
 
7.2.3 ELISPOT/ Luminex 
These assays will be utilized to determine specific cytokine release or 
number of antigen specific spots per 104 CD8+ (for gp100(g209-2M) or 
CD4+ (for MAGE-3) T-cells.  In addition, Luminex assays will be 
utilized to determine the cytokine profile of antigen-specific T-cells. 
 
7.2.4 Quantitative RT-PCR 
This technique will be used to determine the gene expression of 
specific cytokines at the vaccine and tumor sites. 
 
7.2.5 Leukapheresis will be utilized for blood collection together with a ten 
mL phlebotomy to obtain peripheral lymphocytes and plasma at three 
time points when feasible.  Leukapheresis will be performed prior to 
treatment, after treatment at week 8 (+/- 1 week), and at week 16 (+/- 
1 week). This is for both cohorts.  Each leukapheresis will consist of a 
7.5 to 10 liter exchange lasting about three hours. 
 
7.2.6  Plasma collected from phlebotomy will be used to measure reactivity 
of patient’s antibodies against tumor antigens. 
 
7.3 Biopsies: Two areas will be biopsied during the course of this study, the vaccine 
site and the tumor.  
 
Vaccine biopsies: Vaccine sites will be biopsied from all patients in the study when 
feasible. The vaccine site biopsied will be a GP100 vaccine site. The vaccine site will 
be identified and a punch biopsy taken of full thickness skin with standard sterile 
technique and closure. Vaccine sites will be biopsied at 24 hrs (+/- 6 hours) after the 
first gp100(g209-2M) vaccine and at week two of vaccination. These biopsies will 
occur the day following the vaccination for that week. Failure to obtain vaccine 
biopsies due to refusal, discretion of the investigators or infeasibility will not constitute 
a violation of the trial. 
 
Tumor biopsies will be taken from patients in the cohort with metastatic disease. 
There will be no tumor biopsies in patients enrolled in the adjuvant cohort. The first 


2008-0416 
May 7, 2015 
 
 
 18 
 
 
biopsy will be taken as a base line at any time after enrollment onto the trial, but prior 
to the first vaccination. A second tumor biopsy will be taken at week 8 prior to the first 
dose of R848 on the tumors, and at week 12 (4 weeks after initiating R848 therapy on 
the tumors, but at any time during that week) Tumors will be randomly selected when 
biopsied, but when feasible, a biopsy will be taken of both a treated lesion and 
untreated lesion at week 12. Tumor biopsies may be taken using any biopsy method to 
optimize tumor yield using standard sterile techniques. Tumor volume may not allow 
for all biopsies to be obtained in all individuals, in this case pre-vaccine biopsies and 
week 12 will take precedence. Tumor biopsies are at the option of the enrolled subject 
and the investigators, and failure to obtain tumor biopsies due to refusal, inadequate 
remaining tumor volume, discretion of the investigators or infeasibility will not 
constitute a violation of the trial.  
7.4 Samples for Research 
 
Leftover blood, tissue, and T-cells (and possibly genetic material extracted from these 
samples) will be stored for research. These samples may be analyzed by MD 
Anderson and other qualified investigators. These samples will be de-identified. The 
research studies involving these samples will be performed following IRB approved 
protocols. The samples used in these research studies will be to gain further 
information about the disease and will not be used for future treatment decisions. 
 
8.0 
Serious Adverse Event Reporting (SAE) 
Vaccines are usually well-tolerated. They may cause discomfort, swelling, and/or 
redness around the injection site.  While there has not been significant autoimmune 
symptoms caused by gp100(g209-2M) or MAGE-3 peptide vaccines alone, patients who 
experience any Grade III or greater autoimmune reactivity, except for local skin 
reactions, will be taken off the study. The occurrence of 3 patients with Grade III or 
greater toxicity will stop the entry of the study until an assessment of the risks, 
coordination with MD Anderson IRB and appropriate modifications of the informed 
consent can be completed. 
   
The most frequent systemic toxicities associated with Resiquimod are fever, fatigue, 
headache, pain, dizziness, myalgia, chills, and a small increase in absolute lymphocyte 
count.  Localized toxicities reported are erythema, edema, ulceration, weeping, 
scabbing, flaking, pruritis, and numbness/tingling.  Other infrequent toxicities reported 
that may be related are abnormal LFT, abdominal cramping, nausea, vomiting, diarrhea, 
backache, neck pain, rhinitis, and uriticaria.   
 
All adverse events will be recorded according to this table: 
Attribution 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Grade 5 
Unrelated 
 
 
X 
X 
X 
Unlikely 
 
 
X 
X 
X 
Possible 
X 
X 
X 
X 
X 
Probable 
X 
X 
X 
X 
X 
Definitive 
X 
X 
X 
X 
X 


2008-0416 
May 7, 2015 
 
 
 19 
 
 
 
Exceptions to this rule will include events and laboratory abnormalities that represent common 
symptoms and abnormalities of melanoma and chemotherapy and/or have no clinical 
significance: 
 
• 
Abnormalities in hematologic parameters due to myelosuppressive therapeutic effect: 
 
i. 
Anemia, neutropenia, lymphopenia, thrombocytopenia 
ii. 
Epistaxis or bleeding except for catastrophic CNS or pulmonary hemorrhage 
 
• 
Common symptoms of cancer (unless grade >/= 3) including: 
 
i. 
Fatigue 
ii. 
Weakness 
iii. 
Bone, joint or muscle pain 
iv. 
Alopecia 
v. 
Loss of appetite, Nausea, Vomiting 
vi. 
Chemistry abnormalities (phosphorus, calcium, glucose) 
vii. 
Coagulation abnormalities (shortened PT, PTT, increased fibrinogen) 
• 
Laboratory abnormalities: 
• 
 
i. 
LDH (increased or decreased) 
ii. 
Alkaline phosphatase (increased or decreased) 
iii. 
Low levels of the following: AST, ALT, creatinine, BUN, uric acid, bilirubin, 
albumin, total protein 
iv. 
Electrolyte abnormalities (sodium, potassium, bicarbonate, CO2, magnesium 
 
Abnormal laboratory test results will be captured if intervention is required. 
 
Based on the CTCAE v.4, any grade 3 or greater toxicity will result in a dose reduction of 
the application of R848 from twice a week to once a week, however we will still report 
this AE.  In addition, if this is required in 3 patients, we will temporarily halt accrual so 
that we can reassess the study as indicated.  Abnormal lab values that are considered 
clinically significant by the principal investigator will be tracked.  The criteria used is 
based on our experience with this assay in patients, was that biologically significant 
response is considered to be > 0.1% gp100(g209-2M) tetramer + cells in the CD8 + T-
cell population, and an increase of > 2 fold of gp100(g209-2M) tetramer + cells at any 
time point over the pre-vaccine levels.  This is based on the sensitivity of detection using 
our current tetramers and immunologic reagents. 
 
8.1 
Serious Adverse Event Reporting (SAE) 
An adverse event or suspected adverse reaction is considered “serious” if, in the view of either 
the investigator or the sponsor, it results in any of the following outcomes:  
· 
Death 
· 
A life-threatening adverse drug experience – any adverse experience that places the 
patient, in the view of the initial reporter, at immediate risk of death from the adverse 
experience as it occurred. It does not include an adverse experience that, had it occurred 
in a more severe form, might have caused death. 


2008-0416 
May 7, 2015 
 
 
 20 
 
 
• 
Inpatient hospitalization or prolongation of existing hospitalization. 
· 
A persistent or significant incapacity or substantial disruption of the ability to conduct normal life 
functions. 
· 
A congenital anomaly/birth defect. 
Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered a serious adverse drug experience when, based upon 
appropriate medical judgment, they may jeopardize the patient or subject and may require 
medical or surgical intervention to prevent one of the outcomes listed in this definition. Examples 
of such medical events include allergic bronchospasm requiring intensive treatment in an 
emergency room or at home, blood dyscrasias or convulsions that do not result in inpatient 
hospitalization, or the development of drug dependency or drug abuse (21 CFR 312.32). 
· 
Important medical events as defined above, may also be considered serious adverse events.  
Any important medical event can and should be reported as an SAE if deemed appropriate by the 
Principal Investigator or the IND Sponsor, IND Office. 
· 
All events occurring during the conduct of a protocol and meeting the definition of a SAE must be 
reported to the IRB in accordance with the timeframes and procedures outlined in “The University 
of Texas M. D. Anderson Cancer Center Institutional Review Board Policy for Investigators on 
Reporting Unanticipated Adverse Events for Drugs and Devices”.  Unless stated otherwise in the 
protocol, all SAEs, expected or unexpected, must be reported to the IND Office, regardless of 
attribution (within 5 working days of knowledge of the event). 
 
Investigator may include protocol specific reporting criteria here if desired. 
· 
All life-threatening or fatal events, that are unexpected, and related to the study drug, must have 
a written report submitted within 24 hours (next working day) of knowledge of the event to the 
Safety Project Manager in the IND Office.  
· 
Unless otherwise noted, the electronic SAE application (eSAE) will be utilized for safety reporting 
to the IND Office and MDACC IRB.  
· 
Serious adverse events will be captured from the time of the first protocol-specific intervention, 
until 30 days after the last dose of drug, unless the participant withdraws consent. Serious 
adverse events must be followed until clinical recovery is complete and laboratory tests have 
returned to baseline, progression of the event has stabilized, or there has been acceptable 
resolution of the event. 
· 
Additionally, any serious adverse events that occur after the 30 day time period that are related to 
the study treatment must be reported to the IND Office. This may include the development of a 
secondary malignancy.  
Reporting to FDA: 
· 
Serious adverse events will be forwarded to FDA by the IND Sponsor (Safety Project Manager 
IND Office) according to 21 CFR 312.32. 
 


2008-0416 
May 7, 2015 
 
 
 21 
 
 
It is the responsibility of the PI and the research team to ensure serious adverse events are 
reported according to the Code of Federal Regulations, Good Clinical Practices, the protocol 
guidelines, the sponsor’s guidelines, and Institutional Review Board policy 
 
Investigator Communication with Supporting Companies: 
Serious adverse event reporting requirements to supporting companies can be added here if 
needed. 
 
8.2 
Important medical events that may not result in death, be life-threatening, or 
require hospitalization may be considered a serious adverse drug experience 
when,, based upon appropriate medical judgment, they may jeopardize the 
patient or subject and may require medical or surgical intervention to prevent one 
of the outcomes listed in this definition.  Examples of such medical events 
include allergic bronchospasm requiring intensive treatment in an emergency 
room or at home, blood dyscrasias or convulsions that do not result in inpatient 
hospitalization, or development of drug dependency or drug abuse (21 CFR 
312.32). 
a. Important medical events as defined above, may also be considered serious 
adverse events.  Any important medical event can and should be reported as 
an SAE if deemed appropriate by the Principal Investigator or the IND 
Sponsor/the IND office. 
b. All events occurring curing the conduct of a protocol and meeting the definition 
of a SAE must be reported to the IRB in accordance with the timeframes and 
procedures outlined in “University of Texas M.D. Anderson Cancer Center 
Institutional Review Board Policy on Reporting Serious Adverse Events”.  
Unless stated otherwise in the protocol, all SAEs, expected or unexpected, 
must be reported to the IND office regardless of attribution. 
c. All grade 3-5 reactions to the vaccination and all unexpected SAEs will be 
reported in an expedited fashion. 
d. All life-threatening or fatal events, expected or unexpected, and regardless of 
attribution to the study drug, must have a written report submitted within 24 
hours(next working day) of knowledge of the event to the Safety Project 
Manager in the IND office.  
e. The MDACC “Internal SAE Report Form for Prompt Reporting” will be used for  
reporting to the IND office 
f. 
Serious adverse events will be captured from the time the patient signs 
consent until 30 days after the last application of R848.  Serious adverse 
events must be followed until clinical recovery is complete and laboratory tests 
have returned to baseline, progression of the event has stabilized, or there 
has been acceptable resolution of the event. 
g. Additionally, any serious adverse events that occur after the 30 day time 
period that are related to the study treatment must be report to the IND office.  
This may include the development of a secondary malignancy. 
h. The adverse events will be recorded in PDMS according to the following 
guidelines. 
 


2008-0416 
May 7, 2015 
 
 
 22 
 
 
 
 
 
 
8.3.1 Reporting to FDA - Serious adverse events will be forwarded to FDA by 
the IND Sponsor (Safety Project Manager in the IND office) according to 
21 CFR 312.32. 
8.4 
It is the responsibility of the PI and the research team to ensure serious adverse 
events are reported according to the Code of Federal Regulations, Good Clinical 
Practices, the protocol guidelines, and Institutional Review Board policy. 
Recommended Adverse Event Recording Guidelines  
Attribution 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Grade 5 
  
  
  
    
  
Unrelated 
Phase I 
Phase I 
Phase I       
Phase II   
  
Phase I       
Phase II  
Phase III 
Phase I       
Phase II  
Phase III 
Unlikely 
Phase I 
Phase I 
Phase I       
Phase II   
  
Phase I       
Phase II  
Phase III 
Phase I       
Phase II  
Phase III 
Possible 
Phase I       
Phase II 
Phase I       
Phase II  
Phase III 
Phase I       
Phase II  
Phase III 
  
Phase I       
Phase II  
Phase III 
Phase I       
Phase II  
Phase III 
Probable 
Phase I       
Phase II 
Phase I       
Phase II  
Phase III 
Phase I       
Phase II  
Phase III 
  
Phase I       
Phase II  
Phase III 
Phase I       
Phase II  
Phase III 
Definitive 
Phase I       
Phase II 
Phase I       
Phase II  
Phase III 
Phase I       
Phase II  
Phase III 
  
Phase I       
Phase II  
Phase III 
Phase I       
Phase II  
Phase III 


2008-0416 
May 7, 2015 
 
 
 23 
 
 
 
Tumor response to therapy this study will be done using immune-related response 
criteria (irRC) which is a modified version of WHO criteria. 
9.1 
Definition of Measureable and Non-Measurable Lesions Measurable Lesions 
are lesions that can be accurately measured in two perpendicular diameters, with 
at least one diameter > 10 mm.  The area will be defined as the product of the 
largest diameter with its perpendicular.  Skin lesions can be considered 
measurable.  Cutaneous lesions that are > 5 mm in diameter can be considered 
measurable. 
Non-Measurable (evaluable) Lesions are all other lesions, including 
unidimensionally measurable disease and small lesions. 
 
9.2 
Definition of Index/Non-Index Lesions 
In many patients, all measurable disease will be cutaneous, so we will allow all 
10 index lesions to be in a single organ. All measurable lesions, up to a 
maximum of ten lesions in total, should be identified as index lesions to be 
measured and recorded on the medical record at baseline.  The index lesions 
should be representative of all involved organs.  In addition, index lesions should 
be selected based on their size (lesions with the longest diameters), their 
suitability for accurate repeat assessment by imaging techniques, and how 
representative they are of the patient’s tumor burden.  A sum of the products of 
diameters (SPD) for all index lesions will be calculated and considered the 
baseline sum of the products of diameters.  Response criteria to be followed are 
listed below.  The baseline sum will be used as the reference point to determine 
the objective tumor response of the index lesions at tumor assessment (TA).  
Measurable lesions, other than index and all sites of non-measurable disease will 
be identified as non-index lesions.  Non-index lesions will be recorded on the 
medical record and should be evaluated at the same assessment time points as 
the index lesions.  In subsequent assessments, non-index lesions will be 
recorded as “stable or decreased disease,” “absent”, or “progression.” 
 
9.3 
Definition of Tumor Response Using irRC  
The sum of the products of diameters at tumor assessment using the immune-
related response criteria (irRC) for progressive disease incorporates the 
contribution of new measurable lesions.  Each net Percentage Change in Tumor 
Burden per assessment using irRC criteria accounts for the size and growth 
kinetics of both old and new lesions as they appear. 
 
Definition of Index Lesions Response Using irRC 
 
irComplete Response (irCR): Complete disappearance of all index lesions.  
This category encompasses exactly the same subjects as “CR” by the mWHO 
criteria. 
 
irPartial Response (irPR): Decrease, relative to baseline, of 50% or greater in 
the sum of the products of the two largest perpendicular diameters of all index 
and all new measurable lesions (i.e., Percentage Change in Tumor Burden).  
Note: the appearance of new measurable lesions is factored into the overall 


2008-0416 
May 7, 2015 
 
 
 24 
 
 
tumor burden, but does not automatically qualify as progressive disease until the 
SPD increases by >25% when compared to SPD at nadir. 
 
irStable Disease (irSD): Does not meet criteria for irCR or irPR, in the absence 
of progressive disease. 
 
irProgressive Disease (irPD): At least 25% increase Percentage Change in 
Tumor Burden (i.e., taking sum of the products of all index lesions and any new 
lesions) when compared to SPD at nadir. 
 
Definition of Non-Index Lesions Response Using irRC 
 
irComplete Response (irCR): Complete disappearance of all non-index 
lesions.  This category encompasses exactly the same subjects as “CR” 
by the mWHO criteria 
 
irPartial Response (irPR) or irStable Disease (irSD): non-index lesions 
are not considered in the definition of PR, these terms do not apply. 
 
irProgressive Disease (irPD): Increases in number or size of non-index 
lesions does not constitute progressive disease unless/until the 
Percentage Change in Tumor Burden increases by 25% (i.e., the SPD at 
nadir of the index lesions increases by the required amount). 
 
Impact of New Lesions on irRC 
New lesions in and by themselves do not qualify as progressive disease.  
However, their contribution to total tumor burden in included in the SPD 
which in turn feeds into the irRC criteria for tumor response.  Therefore, 
new non-measurable lesions will not discontinue any subject from the 
study. 
 
9.4  
 
Definition of Overall Response Using irRC 
Overall response using irRC will be based on these criteria: 
 
Immune-Related Complete Response (irCR): Complete disappearance 
of all tumor lesions (index and non-index together with no new 
measurable/unmeasurable lesions) for at least 4 weeks from the date of 
documentation of complete response. 
 
Immune-Related Partial Response (irPR): The sum of the products of the two 
largest perpendicular diameters of all index lesions is measured and captured as 
the SPD baseline.  At each subsequent tumor assessment, the sum of the 
products of the two largest perpendicular diameters of all index lesions and of 
new measurable lesions are added together to provide the Immune Response 
Sum of Product Diameters (irSPD).  A decrease, relative to baseline of the irSPD 
compared to the previous SPD baseline, of 50% or greater is considered an 
immune Partial Response (irPR). 
 


2008-0416 
May 7, 2015 
 
 
 25 
 
 
Immune-Related Stable Disease (irSD): irSD is defined as the failure to meet 
criteria for immune complete response or immune partial response, in the 
absence of progressive disease. 
 
Immune-Related Progressive Disease (irPD): It is recommended in difficult 
cases to confirm PD by serial imaging.  Any of the following will constitute 
progressive disease: 
• 
At least 25% increase in the sum of the products of all index lesions over 
baseline SPD calculated for the index lesions. 
• 
At least a 25% increase in the sum of the products of all index lesions and 
new measurable lesions (irSPD) over the baseline SPD calculated for the 
index lesions. 
 
Immune-Related Response Criteria Definitions 
 
Index 
Lesion 
Definition 
 
Non-Index 
Lesion 
Definition 
New 
Measurable 
Lesions 
New 
Unmeasurable 
Lesions 
Percent 
Change in 
Tumor Burden 
Overall irC 
Response 
Complete 
Response 
Complete 
Response 
No 
No 
-100% 
irCR 
Partial 
Response 
Any 
Any 
Any 
>-50% 
<50%to<+25% 
>+25% 
irPR 
irSD 
irPD 
Stable 
Disease 
Any 
Any 
Any 
<50%tp<+25% 
>+25% 
irSD 
irPD 
Progressive 
Disease 
Any 
Any 
Any 
>+25% 
irPD 
 
 
9.5 
Immune-Related Best Overall Response Using irRC (irBOR) 
irBOR is the best confirmed irRC overall response over the study as a 
whole, recorded between the date of first dose until the last tumor 
assessment before subsequent therapy (except for local palliative 
radiotherapy for painful bone lesions) for the individual subjects in the 
study.  For assessment of irBOR, all available assessments per subject 
are considered. 
 
If a lesion is surgically resected or treated with definitive radiosurgery, the 
size of the lesion prior to the definitive local therapy will be included in the 
calculated irBOR. 
Primary evidence of antineoplastic activity will be evaluated as a function 
of objective tumor response following the vaccination period. An overall 
objective assessment of all measurable and non-measurable disease will 
be performed as outlined in 9.1. Tumor assessments should be 
performed by physical exam, ultrasound, MRI or CT scan, throughout the 
study.  The treating physicians will perform tumor measurement. 
Radiological studies must account for all lesions that were present at 
baseline and must use the same techniques as used at baseline. All 


2008-0416 
May 7, 2015 
 
 
 26 
 
 
complete and partial responses must be confirmed by a second 
assessment at least four weeks later. 
 
9.6 
Baseline measurement 
In this trial a period of immunization opens the treatment, and 
immunization alone mediates tumor response rarely.( Cancer 
immunotherapy: moving beyond current vaccines. Rosenberg SA, Yang 
JC, Restifo NP. Nat Med. 2004 Sep;10(9):909-15.) So with this sequence, 
as has been seen with other immunologic mediators, we should expect 
that a period of progression may precede any response from the 
treatment.   
 
Therefore, because subjects are likely to progress during the vaccination 
portion of the study, the baseline will be that measurement during the first 
10 weeks of the study at which the tumors are at their maximum size 
(based on sum of LD). Progression will be allowed during these first 10 
weeks without withdrawal of the subject from treatment or from the study. 
This includes increase in the size of the lesions as well as the 
appearance of new lesions.  
10.0 
Criteria for Removal From The Study 
     
10.1 
Disease progression: 
All patients who develop recurrent disease will be taken off the study. 
Patients in the either cohort may complete their laboratory evaluation 
(blood draws, biopsies and pharesis) if they recurr/progress before week 
16. Patients will be allowed to continue on the study if their disease 
progresses during the first 10 weeks of study, since they will have only 
received vaccine +/- local TLR agonist at this point.  For subsequent 
evaluations, patients with progressive disease will be removed from the 
treatment.     
10.2    Withdrawal of consent:   
The patient’s desire to withdraw from the study may occur at any time.  If 
a patient withdraws consent, the investigator will assess whether the 
reason for withdrawal is actually an adverse event, in which case the 
adverse event should be noted as the reason for withdrawal. 
 
 
10.3 
Adverse event: 
 
Including unacceptable toxicity or exacerbation of underlying disease 
associated with treatment and necessitating discontinuation of treatment, 
usually associated with events that pose a threat to the patient’s life or 
functioning.  For example, any grade III or greater toxicity, attributable to 
the peptide vaccines or R848 (Resiquimod)  
 
 
 
 
 


2008-0416 
May 7, 2015 
 
 
 27 
 
 
 
 
10.4 
Withdrawal by the physician:  
                         
For clinical reasons not related to treatment. 
 
 
 
10.5 
Violation of the study protocol:   
 
Including patient failure to return for required visits.  Every effort should 
be made to contact such patients to determine their reason for 
withdrawal, and to assess any adverse events that may have contributed 
to the patient’s failure to return.  Patients who miss dose(s) because of 
holidays and/or clinic closures will not be withdrawn from the study. 
 
10.6 
End of study 
 
Subjects in either cohort who do not meet any of the above criteria will be 
followed for 12 months after the completion of therapy (vaccination in the 
adjuvant cohort, Resiquimod in the metastatic cohort) and will be 
removed from the study thereafter.  
 
 


2008-0416 
May 7, 2015 
 
 
 28 
 
 
11.0 
Statistical Considerations 
 
Overview 
 
This is a randomized phase II trial enrolling melanoma patients who are HLA-A2+ with or 
without HLA-DP4+.  Patients of two separate types will be enrolled in separate cohorts. 
One cohort includes patients who are NED but at high risk for recurrence. The second 
cohort will include patients with non-visceral metastatic disease and must have at least 
two evaluable and biopsiable lesions to be eligible for the trial.   
 
The trial will have two parts.  In Part 1 (both cohorts), we will fairly randomize patients to 
receive either the gp100(g209-2M) +/-MAGE3 peptide vaccine plus a topical TLR 7 /8 
agonist, R848, (Arm A) or the gp100(g209-2M) +/- MAGE3 vaccine alone Arm B). The 
randomization will be stratified by cohort.  A total of 48 patients will be enrolled, 24 per 
arm.  All patients will receive the gp100(g209-2M) vaccine (those who are HLA-DP4 +  
will also receive the MAGE3 vaccine) weekly (for 8 weeks) and the patients randomized 
to receive R848 gel will have the gel applied to the gp100(g209-2M)  vaccine site 
weekly.   
 
After 8 weeks of treatment on Part 1, Part 2 of the trial begins.  In Part 2 (only patients in 
the metastatic disease cohort), the treating physician will divide the area of cutaneous 
lesions into regions, Region A and Region B and designate the two regions to receive or 
not to receive R848.  At least one lesion will be randomly assigned to receive R848 from 
weeks 8 through 24, and one will not.  One lesion from each region will be chosen for 
biopsy as outlined in section 7.3.  If a patient has more than two evaluable lesions, we 
will stratify by lesion size to assure that the lesions will be comparable. 
As a safety measure, if a patient’s untreated lesions grow while treated lesions are 
stable or shrink, all lesions will be treated with R848.  This will occur after the 
assessment of the primary endpoints of the study, so it will not affect the analysis of 
these endpoints. 
 
In April 2015, the trial is being further modified to add an additional 20 patients with 
metastatic disease, all of whom will receive the the gp100(g209-2M) +/-MAGE3 peptide 
vaccine plus R848 (Arm C).  These patients will not be part of the primary comparative 
study analysis of T-cell responses in the blood.  The purpose of these patients is to 
provide additional information on characterizing tumor response, defined as at least a 
50% shrinkage in tumor size by irRC criteria. 
 
 
Part 1 
The primary objective of Part 1 of the trial is to compare T-cell responses in the blood 
between the vaccine+R848 and vaccine alone treatment arms. This will be assessed in 
the two cohorts separately and combined. The primary outcome of this part of the trial 
will be T-cell response to gp100(g209-2M) +/- MAGE3 measured at 8 weeks using a 
tetramer/multimer assay measured by flow cytometry.  Based on the number of 
gp100(g209-2M) +/- MAGE3 T-cells measured, when the study began, patients were to 
be categorized as either immune responders or immune non-responders.  From 
previous vaccine studies, a ≥ 0.1% response was thought to be a reasonable target.  
Thus, T-cell response to the gp100(g209-2M) + /- MAGE3 peptide in each patient was 


2008-0416 
May 7, 2015 
 
 
 29 
 
 
defined as ≥0.1% gp100(g209-2M) tetramer+ cells in the CD8+ T-cell population or 
≥0.1% MAGE3 multimer cells in the CD4+ T-cell population.  In September 2013, due to 
problems in obtaining the multimers necessary to perform the CD4+ T-cell assays, these 
assays have not yet been run, although more than 24 patients have reached week 24 on 
the trial.  Based upon the aggregate CD8+ responses, no patients have met the 
definition of response according to the above definition.  Therefore, the study team is 
changing the definition of the primary endpoint to be the actual change from baseline for 
gp100(g209-2M) tetramer+ cells in the CD8+ T-cell population.  The study will be 
stratified by cohort (NED/metastatic), but we expect the same immune response in each 
cohort.   
 
For part 1 the 8-week primary immune outcome values CD8+ T-cell response will be 
compared between the two arms by using a Wilcoxon rank-sum test.  Patients who 
discontinue from the study before week 8 will be assigned the lowest rank in this 
analysis.  With one interim analysis, using an O’Brien-Fleming boundary to stop for 
efficacy, a one-sided significance level of 0.05, with 24 patients per arm, we will have 
80% power to be able to detect an effect size of 0.73*SD(CD8+ T-cell response). 
 
Part 2 
The primary outcomes for part 2 of the trial are immune cell infiltration for pDCs, mDCs, 
and NK cells in the tumor biopsy samples.  This can be assessed in the metastatic 
disease cohort only, since those subjects in the adjuvant therapy cohort are free of 
disease. Tumor samples will be taken as outlined in section 7.3 Three different types of 
analyses will be performed on these tumor biopsies: (1) Immunohistochemistry (IHC) for 
the degree of pDC, mDC, and NK cell infiltration, (2) Flow cytometry (FACS) analysis for 
infiltrating melanoma specific CD8+ T cells, and (3) Quantitative RT-PCR (qRT-PCR) 
assays for IFN-γ, IFN-α (and other  type I interferons), IL-12, and TGF-β.  For each of 
these outcomes, we will determine the differences in each of these parameters between 
the pretreatment and post-treatment samples for each of the biopsied lesions for each 
patient.  Mixed-effect models will be fit for each parameter to assess the effects of 
treatment, time, and the initial randomization to gp100(g209-2M) +/- MAGE-3 vs. 
gp100(g209-2M) +/- MAGE-3 +R848.  If there is no evidence of an effect due to the 
initial randomization in Part 1, the changes from baseline for the R848 treated lesion and 
the untreated lesion will be calculated, and these differences will be compared between 
the R848 and non-R848 lesions by using a paired t-test. 
 
A sample size argument for Part 2, based upon a 2-sample t-test  (which represents a 
lower bound on the mixed-effects model given above), is as follows: we assume that a 
total of 24 patients will be enrolled in the metastatic disease cohort, and of these, 17% of 
the patients initially enrolled will discontinue the trial at 8 weeks due to progressive 
disease and that dropout will be equally distributed across the 2 arms, leaving a total of 
20 patients (10/arm) for Part 2.  The 2-sample t-test will assess differences in the 
change between the two treatment groups with respect to the 3 outcomes described 
previously.  With 20 patients, we will have 80% power to detect an increase of 1.2 
standard deviations in the R848 group for each outcome at 8 weeks, assuming a one-
sided 2-sample t-test with a 5% Type I error rate.  One of the benefits of our mixed-
effects modeling approach is that it allows for the analysis of the data using complete 
likelihood methods (Beunckens, Molenberghs, and Kenward, 2005; Clinical Trials).  In 
addition, we will perform 2 types of sensitivity analysis:  1) We will use last-observation-
carried-forward; 2) We will explore how deviations from the Missing at Random (MAR) 


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assumption of the direct likelihood approach may impact inference using methods 
developed in Verbeke and Molenberghs (2000).   
 
Additional Cohort of Non-Randomized Patients 
 
As noted above in the Overview, after the initial 48 patients have been enrolled in the 
randomized portion of the trial, 20 additional patients with metastatic disease will be 
enrolled into Arm C in order to further characterize tumor response of R848.  These 
patients will not be included in the primary analysis.  Tumor response as measured by a 
50% shrinkage in tumor size by irRC criteria will be the primary endpoint for these 
patients.  The response rate associated with the standard of care in these patients is no 
more than 5%.  We expect a total of 10 metastatic disease patients in Arm A from the 
initial randomized portion of the trial.  Combined with the additional 20 patients (Arm A 
and C, with a total of 30 patients, we will have 82% power to detect an increase to 22%, 
assuming a one-sided exact binomial test with a 5% Type I error rate. 
 
Overview of Analysis of Secondary Endpoints 
In addition to the Part 1 and Part 2 primary endpoints described above, several 
additional secondary endpoints will be investigated. Blood will be drawn at baseline, 2, 4, 
8, 12, 16, 20, and 24 weeks, and every 4 weeks after the 24-week draw.  A secondary 
analysis for data generated from Part 1 will measure ex vivo IFN- via ELISPOT assay.  
This outcome is measured as a continouous variable. Our first step will be to plot each 
laboratory parameter over the 24 weeks of follow-up to visually inspect the data for any 
interesting trends between the two clinical trial arms.  Separate graphical displays will be 
created for each treatment arm (gp100(g209-2M)/MAGE vaccine+R848 and 
gp100(g209-2M)/MAGE vaccine alone).  Subsequent to the initial exploratory data 
analysis discussed above, a repeated measures model will be fit for each measured 
parameter in the blood to the data using Proc Mixed in SAS to model the repeated 
laboratory measurements over the 24 weeks of follow-up.  Tumor biopsies will be taken 
as outlined in section 7.3  In addition, a punch biopsy will be taken at the vaccine site for 
all patients as outlined in section 7.3.  To evaluate the ability of locally administered 
TLR agonist (R848 gel) to activate innate immune cells at the vaccine site we will 
evaluate the inflammatory microenvironment at the site of the immunization.  Immune 
cell infiltration for pDCs, mDCs, NK cells, and T-cells will be measured along with 
immune cell function using IHC and quantitative RT-PCR (qRT-PCR) by Core B.  For 
each patient, changes from baseline will be calculated for both the lesions treated with 
R848 and for the non-treated lesions. The 4 immune cell infiltration measures and qRT-
PCR relative expression measures are all continuous variables. We will use Wilcoxon 
signed rank test to compare the measures between the lesions within patient.  
Significance will be assessed at the 5% level.  
 
To evaluate the ability of R848 gel, administered at the tumor site to induce inflammation 
and up-regulation of adhesion molecules on tumor vasculature we will focus on the 
tumor biopsies taken in R848 treated and untreated tumors at pre and post-treatment 
timepoints outlined in section 7.3. These biopsies will be used to investigate the effect of 
R848 treatment on tumors compared to untreated tumors. We will measure inflammation 
and activation of tumor vasculature using Immuno-histochemistry (IHC) and flow 
cytometry (FACS), We will use mixed models to assess differences between treatment 
groups. Our mixed model will include fixed effects for treatments, and random effects for 


2008-0416 
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time, treatment-by-time interaction and an additional random effect to model the within 
patient variability. Significance will be assessed at the 5% level.  
 
To evaluate the ability of R848 gel, administered at the tumor site to enhance immune-
cell infiltration into tumor we will utilize antibodies to PDC, mDC, NK, and T cells.  Since 
these are continuous measures (assessed at baseline, 2, 4, 8, 12, 16, 20, and 24 weeks, 
and every 4 weeks after the 24-week draw) we will use mixed models to assess 
differences between treatment groups. Our mixed model will include fixed effects for 
treatments, and random effects for time, treatment-by-time interaction and an additional 
random effect to model the within patient variability. Significance will be assessed at the 
5% level.  
 
To evaluate the ability of R848 gel, administered at the tumor site to generate T-cells 
against additional tumor antigens, not  present in the vaccine (i.e., antigen spreading) we 
will assess cytokine production against de novo antigens.  Since these are continuous 
measures (assessed at baseline, 2, 4, 8, 12, 16, 20, and 24 weeks, and every 4 weeks 
after the 24-week draw) we will use mixed models to assess differences between 
treatment groups. Our mixed model will include fixed effects for treatments, and random 
effects for time, treatment-by-time interaction and an additional random effect to model 
the within patient variability. Significance will be assessed at the 5% level.  
 
To evaluate the association between clinical response and laboratory parameters ofT-
cell priming, T-cell migration to tumor, and inflammation at the vaccine and tumor sites 
we will explore the relationship between immune cell activation measures at the vaccine 
site (vaccine site measures) and at the tumor site (tumor site measures). Additionally we 
will assess the effect of the TLR agonist R848 upon clinical response. Clinical response 
will be assessed using the immune-related response criteria (irRC) and will be assessed 
at week 8, 16, and every 8 weeks thereafter. Since both vaccine site measures and 
tumor site measures are continuous variables we will assess association between these 
two types of variables using Pearson’s or Spearman’s correlation coefficient. In addition, 
we will use logistic regression to model the relationship between either vaccine site or 
tumor measures and best clinical response assessed over a 16 weeks period after initial 
vaccination.   
 
To assess the association between R848 treatment on lesions and response, the irRC 
will be used to assess response separately by lesion type (lesions receiving or not 
receiving R848 cream). That is, using the irRC, we will, for each patient, assess if the 
lesions not treated with R848 cream responded and also determine if the lesions treated 
with R848 cream responded. Thus, for each patient we will record 4 possible joint 
outcomes ++,+-,-+, and --, where the ++ indicates that both lesions receiving and not 
receiving R848 cream responded; -- indicates neither types of lesions responded. 
Similarly +- and -+ indicate one type of lesion responded while the other did not. We will 
assess differences between treatments (based on within patient differences) using 
McNemar’s test. A statistically significant difference with respect to the McNemar’s test 
will indicate if cream administered to the lesion results in improved outcome (i.e., 
immune-related rsponse criteria) relative to lesions for which cream was not 
administered.  
 
 


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Study Stopping Criteria 
 
We will conduct an interim analysis after half of the patients have been enrolled and 
have reached week 8 on the study.  At the interim analysis, we will test whether there is 
strong evidence that the immune responses differ between the two treatment arms.  We 
will use an O’Brien-Fleming stopping boundary to stop for efficacy, with a nominal 
significance level of 0.0056.  With the alpha spent at the interim look, the nominal 
significance level at the final analysis will be 0.0482. 
12.0  Data Entry and Protocol Management 
For the purposes of this study at M. D. Anderson Cancer Center, the Protocol Data 
Management System (PDMS) will be employed.  All patients will be registered in 
PDMS/CORE.  This study will be monitored by the MDACC Compliance Office.  The 
randomization will be done by the CORE system and the data will be reviewed annually 
by the institution’s Data Safety Monitoring Board (DSMB).  The statisticians will prepare 
the report for review by the DSMB. 
  13.0  Administrative Procedures 
13.1  Changes to the protocol:  
Any change or addition to this protocol requires a written protocol 
amendment that must be approved by the IRB. A copy of the written 
approval of the IRB must be received before implementation of any 
changes.  The IRB must review and approve all amendments to the 
protocol.    
 
13.2  Ethics and good clinical practice:  
This study must be carried out in compliance with the protocol and Good 
Clinical Practice, as described in:  
 
13.2.1.1.1 
ICH Harmonized Tripartite Guidelines for Good Clinical 
Practice 1996. 
 
13.2.1.1.2  US 21 Code of Federal Regulations dealing with clinical 
studies (including parts 50 and 56 concerning informed 
consent and IRB regulations). 
 
13.2.1.1.3   Declaration of Helsinki, concerning medical research in 
humans (Recommendations Guiding Physicians in 
Biomedical Research 
Involving Human Subjects, Helsinki 1964, amended Tokyo 
1975, Venice 1983, Hong Kong 1989, Somerset West 1996). 
The investigator agrees, when signing the protocol, to 
adhere to the instructions and procedures described in it and 
thereby to adhere to the principles of Good Clinical Practice. 
 
 


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14.0 
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