Conditions: Melanoma
Allocation: RANDOMIZED | Intervention model: SINGLE_GROUP
Enrollment: 187
Healthy volunteers: no

Brief summary:
The majority of melanoma vaccines tested to date have been antigen-specific vaccines targeting melanoma-specific or associated antigens and utilizing a variety of delivery systems and immune-adjuvants. As opposed to testing an "off the shelf" vaccine that might be able to treat a subset of patients, our approach has been personalized to the patient and applicable to all patients. Our vaccine approach consists of harnessing the most potent antigen presenting cell in the body - the dendritic cell (DC) - together with the full repertoire of tumor antigens from an individual's cancer. We have conducted phase I and II studies using an autologous DC-tumor cell fusion technique that has now been simplified into a DC-tumor cell lysate vaccine. The autologous tumor lysate (TL) is loaded into yeast cell wall particles (YCWP) that are naturally and efficiently taken up into the patient's DC. These autologous tumor lysate, particle-loaded, DC (TLPLDC) are injected intradermally (ID) monthly x 3 followed by boosters at 6, 12, and 18 months.

Detailed description:
Stage III and Stage IV (resected) melanoma patients will be identified prior to definitive surgery and screened for inclusion/exclusion criteria. Eligible patients will be counseled and consented for tissue procurement. Enrolled patients will have their disease surgically resected and a portion 1mg minimum of their melanoma sterilely frozen in provided freezing vials and storage tubes. This tissue will be shipped in liquid nitrogen shippers through FedEx to our central facility in Greenville SC and stored frozen until vaccine preparation. If patients cannot be rendered disease-free, they will be considered screen failures for this study. If melanoma is being resected from multiple locations primary and nodes two different metastatic sites then samples of each would be preferred but not mandatory.

As indicated by SoC per the National Comprehensive Cancer Network (NCCN) guidelines and determined by the treating team, if a patient is to receive systemic therapy (chemotherapy or IFN-aguidelines) and determined by the treating team, if a patient is to receive systemic therapy (chemotherapy or IFN-central facility in Greenville, SC) and stored frozen until vaccine preparation. If patients cannot be rendered disease-free, they will receive a single injection of Neupogen (G-CSF) 300 mod (or its equivalent) SQ 24-48 hrs. prior to having 70 mL of blood collected and sent to our central facility for DC isolation and preparation. Patients who cannot tolerate Neupogen, or its equivalent or refuse it, will have 120 mL of blood drawn and sent. Additional blood may be drawn if additional vaccine doses need to be made or re-made for any reason. Vaccines will be prepared by producing TL through freeze/thaw cycling and then loaded into pre-prepared YCWP. The TL-loaded YCWP will be introduced to the DC for phagocytosis thus creating the TLPLDC vaccine which will be frozen in single dose vials. Each vial will contain 1-1.5 x 106 TLPLDC and will be labeled with the patient's unique study number.

Based on their randomization, autologous TLPLDC (active vaccine) or unloaded YCWP + autologous DC (control) will be sent back to the site in a blinded fashion. Regardless of assigned group, the site will receive 6 single dose vials to be injected intradermal monthly x 3 followed by boosters at 6, 12, and 18 months in the same lymph node draining area (preferably the anterior thigh). Patients must begin vaccinations between 3 weeks and 3 months from completion of (SoC). Frozen tumor will be maintained for active vaccines for all patients to include the control patients. The latter will be offered their active vaccine at time of recurrence in a crossover fashion. Additionally, control patients who do not recur will be offered active vaccine at the completion of the trial.

Safety data will be collected on local and systemic toxicities and graded and reported per the Common Terminology Criteria for Adverse Events (CTCAE) v4.03.

Disease-free status will be monitored per SoC as outlined by NCCN. Suspected recurrences will be documented with biopsy and pathologic confirmation. Time to recurrence will be based on date of randomization to time of confirmed recurrence.

Recurrent patients will be offered participation in the open label portion of the study. New active vaccine will be made for all patients, and they will be inoculated at 0, 1, 2, 3, 6, and 9 mos. Patients will be treated per SoC for their recurrence. Safety and tumor response will be assessed per RECIST and irRC on their SoC follow-up scans.

Blood (50 mL) will be collected from all patients prior to each inoculation and at 24 months from enrollment for a total of 7 time points or a total of 350 mL of blood over 2 years. The collected blood will be sent to our central facility for immunologic testing of the T-cell response.

Arms:
arm 1: autologous TLPLDC (active vaccine) arm 2: unloaded YCWP + autologous DC (control)

Interventions: intervention 1: TLPLDC intervention 2: Placebo
Intervention descriptions: intervention 1: Autologous tumor lysate, particle-loaded dendritic cell vaccine intervention 2: None

Protocol document:
Prot_000.pdf: 
Phase IIb TLPLDC & TLPO vaccine trial 
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Protocol Title: 
A prospective, randomized, blinded, placebo-
controlled, phase IIb trial of an autologous 
tumor lysate (TL) + yeast cell wall particles 
(YCWP) + dendritic cells (DC) vaccine vs 
unloaded YCWP + DC and embedded phase 
I/IIa trial with tumor lysate particle only (TLPO) 
vaccine in stage III and stage IV (resected) 
melanoma to prevent recurrence. 
Protocol Number: 
20141932 
Study Drugs: 
Autologous TLPLDC vaccine (tumor lysate,  
particle-loaded, dendritic cells) 
and 
Autologous TLPO (tumor lysate, particle only) 
Sponsor: 
Elios Therapeutics, LLC 
Director: 
George E. Peoples, MD, FACS 
Cancer Insight, LLC 
1305 East Houston Street 
San Antonio, Texas 78205 
IND:  
TLPLDC IND#16101 & TLPO IND#17274 
Principal Investigator: 
Mark B. Faries, MD, FACS 
Head, Division of Surgical Oncology 
Co-Director, Melanoma Program 
Surgical Director, Experimental Therapeutics 
The Angeles Clinic and Research Institute, a 
Cedars-Sinai Affiliate 
11818 Wilshire Blvd., Suite 220 
Los Angeles, CA 90025 
Medical Monitor:   
Vance Sohn, MD 
Protocol Version/Date: 
2.4/ 15 February 2021 


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Table of Contents 
1.0 
PROTOCOL SYNOPSIS ....................................................................................... 4 
2.0 
INTRODUCTION ............................................................................................. 10 
2.1 
Immunotherapy in Melanoma .......................................................................................................... 10 
2.2 
Dendritic Cell Vaccines ........................................................................................................................ 12 
2.3 
New Vaccine Technology .................................................................................................................... 13 
2.4 
Prior/Ongoing Clinical Trials with Autologous Tumor/DC Vaccine ................................ 16 
3.0 
OBJECTIVES ................................................................................................... 21 
3.1 
Primary Objective .................................................................................................................................. 21 
3.2 
Secondary Objectives ........................................................................................................................... 21 
3.3 
Exploratory Objectives ........................................................................................................................ 21 
4.0 
STUDY DESIGN ............................................................................................... 22 
4.1 
Description of Study ............................................................................................................................. 22 
4.2 
Rationale for Study Design ................................................................................................................. 24 
4.3 
Outcome Measures ................................................................................................................................ 25 
4.3.1 
Primary outcome measures .................................................................................................................. 25 
4.3.2 
Secondary outcome measures ............................................................................................................. 26 
4.3.3 
Exploratory outcome measures .......................................................................................................... 26 
4.4   
Safety Considerations........................................................................................................................... 27 
4.5   
Compliance with Laws and Regulations ....................................................................................... 28 
5.0   
MATERIALS AND METHODS ........................................................................... 28 
5.1 
Subjects ...................................................................................................................................................... 28 
5.1.1 
Subject selection ........................................................................................................................................ 28 
5.1.2 
Inclusion criteria ....................................................................................................................................... 29 
5.1.3 
Exclusion criteria....................................................................................................................................... 30 
5.2 
Method of Treatment Assignment .................................................................................................. 31 
5.3 
Study Treatment..................................................................................................................................... 31 
5.3.1 
Vaccine production ................................................................................................................................... 31 
5.3.2 
Storage ........................................................................................................................................................... 32 
5.3.3 
Inoculation series – administration .................................................................................................. 33 
5.3.4 
Dose reduction for TLPO ........................................................................................................................ 33 
5.4 
Study Assessments ................................................................................................................................ 33 
5.4.1 
Pretreatment assessments .................................................................................................................... 34 
5.4.2 
Assessments during treatment ............................................................................................................ 34 
5.4.3 
Follow-up assessments ............................................................................................................................ 35 
5.4.4  
Blood collection and processing ......................................................................................................... 36 
5.4.5  
Phenotypic assay (for example, dextramer assay) for melanoma-specific antigens . 36 
5.4.6  
Functional assay (for example, ELISPOT assay) for melanoma-specific antigens ..... 37 
5.5 
Discontinuation of Protocol-Specific Therapy ........................................................................... 37 
5.6 
Subject Discontinuation ...................................................................................................................... 37 
5.7 
Study Discontinuation.......................................................................................................................... 38 
5.8 
Data Collection ........................................................................................................................................ 39 
5.9 
Statistical Methods ................................................................................................................................ 39 
5.9.1  
Rationale for study design .................................................................................................................... 39 
5.9.2 
Sample size determination ................................................................................................................... 40 
5.9.3 
Data analysis ............................................................................................................................................... 40 
5.9.4 
Withdrawal .................................................................................................................................................. 43 


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5.9.5 
Missing data ................................................................................................................................................. 43 
6.0 
ADVERSE EVENTS ........................................................................................... 43 
6.1 
Adverse Event and Reporting Definitions ................................................................................... 43 
6.2  
Reporting of Adverse Events Associated with This Study .................................................... 44 
6.3 
Reporting Requirements for IND Sponsor .................................................................................. 45 
6.3.1  
MedWatch 3500A reporting guidelines .......................................................................................... 45 
7.0 
INVESTIGATOR REQUIREMENTS ..................................................................... 46 
7.1  
Study Initiation ....................................................................................................................................... 46 
7.2  
Study Completion................................................................................................................................... 47 
7.3  
Informed Consent .................................................................................................................................. 47 
7.4 
Institutional Review Board or Ethics Committee Approval ................................................. 47 
7.5 
Study Monitoring Requirements ..................................................................................................... 48 
7.6 
Data Safety Monitoring Plan .............................................................................................................. 49 
7.7  
Study Medication Accountability .................................................................................................... 49 
7.8  
Disclosure of Data .................................................................................................................................. 49 
7.9  
Retention of Records ............................................................................................................................ 49 
7.10  
Publications .............................................................................................................................................. 49 
7.11  
Changes to Protocol .............................................................................................................................. 50 
REFERENCES ............................................................................................................. 51 
Appendix A ............................................................................................................... 57 
Appendix B ............................................................................................................... 58 
Appendix C ............................................................................................................... 59 
Appendix D ............................................................................................................... 61 
Appendix E ............................................................................................................... 62 
Appendix F................................................................................................................ 63 
Appendix G ............................................................................................................... 69 
Appendix H ............................................................................................................... 71 
Appendix I ................................................................................................................ 72 
Appendix J ................................................................................................................ 73 
Appendix K ............................................................................................................... 74 
 
 
 
 
 
 
 


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1.0 PROTOCOL SYNOPSIS 
Brief Title 
A prospective, randomized, blinded, placebo-controlled, phase IIb trial of an 
autologous tumor lysate (TL) + yeast cell wall particles (YCWP) + dendritic cells 
(DC) vaccine vs unloaded YCWP + DC and embedded phase I/IIa trial in tumor 
lysate particle only (TLPO) vaccine in stage III and stage IV (resected) melanoma 
to prevent recurrence. 
Test Article 
Name 
Autologous TLPLDC vaccine (tumor lysate, particle loaded, 
dendritic cells) 
Autologous TLPO vaccine (tumor lysate, particle only) 
Activity 
Induction of anti-cancer cell-mediated immune response 
Dosage form 
Intradermal injection of 1-1.5x106 autologous TLPLDC or 1.0x108 
TLPO monthly x 3 followed by boosters at 6, 12, and 18 months 
Development 
Phase 
Phase IIb (with embedded phase I/IIa) 
Indication 
To prevent recurrences in disease-free stage III and stage IV (resected) 
melanoma patients 
Rationale 
Setting:  
Melanoma is a potentially lethal skin malignancy.  Patients with stage III and IV 
(resected) melanoma have a significant risk of recurrence in the 50-90% range.  
Once recurrent, these patients will likely die of disease due to the paucity of 
effective systemic therapies. 
Melanoma is an immunogenic cancer often eliciting an endogenous immune 
response; however, if this protective mechanism fails then the disease progresses.  
Multiple immunotherapies have been tested in melanoma attempting to reinstate 
this immune protection.  Some of the immunotherapies which have shown promise 
have been cytokine therapies (IFN-α, IL-2) as melanoma is an immunogenic cancer 
often eliciting an endogenous immune response; however, if this protective 
mechanism fails then the disease progresses.  Multiple immunotherapies have 
been tested in melanoma-PD-L1 antibodies have shown significant promise with 
the FDA approval of the former in the metastatic setting. 
While these FDA-approved immunotherapies are effective in a small percentage of 
melanoma patients, they are non-specific, often toxic, and poorly tolerated.  
Vaccines are meant to raise a more specific, tumor-targeted immune response with 
minimal toxicity.  Unfortunately, the majority of melanoma vaccines have been 
tested in late-stage patients with substantial tumor burden and have shown minimal 
benefit.  Current data suggests that the best time to intervene with a vaccine 
strategy is in the adjuvant setting after patients have been rendered disease-free 
by standard of care (SoC) therapies.  In this setting, the vaccine-induced immune 
response can attack any minimal residual disease and hopefully provide long-term 
protection through immunologic memory with minimal toxicity.   


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Rationale 
(continued) 
Vaccine:    
The majority of melanoma vaccines tested to date have been antigen-specific 
vaccines targeting melanoma-specific or associated antigens and utilizing a variety 
of delivery systems and immune-adjuvants.  As opposed to testing an “off the shelf” 
vaccine that might be able to treat a subset of patients, our approach has been 
personalized to the patient and applicable to all patients.  Our vaccine approach 
consists of harnessing the most potent antigen presenting cell in the body – the 
dendritic cell (DC) – together with the full repertoire of tumor antigens from an 
individual’s cancer.  We have conducted phase I and II studies using an autologous 
DC-tumor cell fusion technique that has now been simplified into a DC-tumor cell 
lysate vaccine.  The autologous tumor lysate (TL) is loaded into yeast cell wall 
particles (YCWP) that are naturally and efficiently taken up into the patient’s DC.  
These autologous tumor lysate, particle-loaded, DC (TLPLDC) are injected 
intradermally (ID) monthly x 3 followed by boosters at 6, 12, and 18 months. 
The embedded phase I/IIa will introduce the next version of the vaccine, which 
simplifies the production process. It utilizes the autologous tumor to create the 
tumor lysate, which is then loaded into the yeast cell wall particles and then capped 
with silicate for stability. This tumor lysate, particle only (TLPO) vaccine, is then 
injected intradermally and taken up in vivo by dendritic cells and stimulates the 
patient’s immune system to recognize the tumor. These autologous TLPO vaccines 
are injected in the same manner as the TLPLDC, monthly x3 followed by booster 
at 6, 12, and 18 months. 
Prior Results: 
To date, we have vaccinated a total of 36 patients with varying malignancies, and 
25 of these have been late-stage melanoma patients.  These personalized vaccines 
have a very favorable toxicity profile with only flu-like symptoms immediately after 
inoculations, and no related serious adverse events have been observed in any 
patient vaccinated to date.  In the completed phase I/IIa melanoma trial, 25 stage 
IV patients were vaccinated.  The median overall survival (OS) was 14.5 months 
(compared to a historic rate of 8.5 months), and there was a 27% OS at 3 yrs.  
There was a dose response with an OS improvement in patients receiving ≥3 
inoculations compared to patients receiving less (log rank p=0.025).  Additionally, 
among the small group of stage IV (resected) patients (n=4), the OS was 100% at 
20 months median follow-up.  
Primary 
Endpoint 
For the TLPLDC phase IIb: 
To determine 24-month disease-free survival (DFS) in vaccinated (TLPLDC) vs 
control patients. 
For the TLPO phase I/IIa: 
To assess the safety of the vaccine per CTCAEv4.03 
Secondary 
Endpoints 
For the TLPLDC phase IIb: 
1) To determine DFS and OS at 36 months in vaccinated (TLPLDC) vs control 
patients. 
2) To document the safety of the vaccine. 


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Exploratory 
Endpoints 
For both trials: 
 1) To document immunologic response to the vaccines through T-cell assays 
recognizing known melanoma-specific antigens. 
2) To correlate vaccine immune response to clinical outcome. 
3) To assess safety and tumor response of continued vaccination in combination 
with SoC therapies in patients after recurrence. 
Patient 
Population 
Stage IIIA-C, Stage IV (resected) melanoma patients identified prior to definitive 
surgery and anticipated to be clinically disease-free after surgery.   
Treatment Arms 
For the TLPLDC phase IIb:  
1) Vaccinated group = autologous tumor cell lysate + yeast cell wall particles + 
autologous DC (TLPLDC) 
2) Control group = empty yeast cell wall particles + autologous DC 
For the TLPO phase I/IIa: 
1) Vaccinated group = autologous tumor cell lysate + yeast cell wall particles 
(TLPO) 
Stratification, 
Randomization, 
and Crossover 
Patients will be stratified based on stage III vs stage IV. 
Randomization will be 2:1 (vaccine:control). 
All patients will be offered open label active vaccine at first sign of recurrence. 
Non-recurrent control patients will be given the option of receiving active vaccine 
upon their completion of the trial. 
Upon randomization of the 120th patient in the TLPLDC phase IIb, the TLPO 
phase I/IIa trial will be initiated and 60 additional patients will be randomized 2:1 
(TLPO phase I/IIa:TLPLDC).   
Number of 
Patients  
At least N= 180 
Sample Size 
Justification 
Assuming a baseline recurrence rate of 60% (corresponding to a DFS of 40%) at 2 
years in this mixed group of stage III and stage IV (resected) melanoma patients, a 
sample size of approximately 120 will have 80% power to detect a statistical 
difference between treatment proportions controlling the type I error at alpha = 0.05 
(two-sided).   
A blinded evaluation of the first 75 patients enrolled discovered an early recurrence 
rate of 12% prior to completion of primary vaccine series. To compensate for this 
early recurrence rate and preserve the power of the trial, enrollment will be 
extended by at least an additional 20 patients, now totaling at least 140. 
The TLPO phase I/IIa will be assessed primarily for the tolerability of the TLPO 
vaccine as determined by CTCAEv4.03.  Additionally, immunologic assays will be 
assessed for vaccine induced T cell recognition of common melanoma-related 
antigens.  Patients will be followed clinically per standard of care for disease status.  
Number of Sites 
10-12 sites will be established. 
Duration of Trial 
With 10-12 sites enrolling at 0.8 pts/site/month, we anticipate enrollment to be 
complete in 12-18 months.  With a primary endpoint of 2 year DFS and secondary 
endpoints of 3 year DFS/OS, the trial duration is expected to be 4-4.5 years. 


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Trial Design and 
Conduct 
Stage III and Stage IV (resected) melanoma patients will be identified prior to definitive 
surgery and screened for inclusion/exclusion criteria.  Eligible patients will be counseled 
and consented for tissue procurement.  Enrolled patients will have their disease 
surgically resected and a portion (1mg minimum) of their melanoma sterilely frozen in 
provided freezing vials and storage tubes.  This tissue will be shipped in liquid nitrogen 
shippers through FedEx to our central facility in Greenville, SC and stored frozen until 
vaccine preparation.  If patients cannot be rendered disease-free, they will be 
considered screen failures for this study.  If melanoma is being resected from multiple 
locations (primary and nodes, two different metastatic sites), then samples of each 
would be preferred but not mandatory.  
As indicated by SoC per the National Comprehensive Cancer Network (NCCN) 
guidelines and determined by the treating team, if a patient is to receive systemic 
therapy (chemotherapy or IFNα and/or radiation therapy), then the vaccinations will not 
begin until SoC therapy is completed.  Although, patients on adjuvant check point 
inhibitors (CPI) may start the vaccine trial after showing tolerance of the CPI after 3 
doses, given that there may a synergistic benefit and no added toxicity when adding the 
vaccine to CPI. Once SoC therapies are complete (or demonstrating tolerance on CPI 
after 3 doses) and the patient deemed clinically disease-free, they will be consented for 
treatment and randomized.  Once consented, patients will receive a single injection of 
Neupogen (G-CSF) 300 µg (or its equivalent) SQ 24-48 hrs prior to having 70 mL of 
blood collected and sent to our central facility for DC isolation and preparation. Patients 
who cannot tolerate Neupogen, or its equivalent or refuse it, will have 120 mL of blood 
drawn and sent.  Additional blood may be drawn if additional vaccine doses need to be 
made or re-made for any reason. Vaccines will be prepared by producing TL through 
freeze/thaw cycling and then loaded into pre-prepared YCWP.  The TL-loaded YCWP 
will be introduced to the DC for phagocytosis thus creating the TLPLDC vaccine, which 
will be frozen in single dose vials.   Each vial will contain 1-1.5 x 106 TLPLDC and will 
be labeled with the patient’s unique study number. The TLPO is created in a similar 
manner with the freeze/thaw cycling, loaded into pre-prepared YCWP, and then capped 
with silicate for stability.  The TLPO vaccine is then frozen in single dose vials of 1.0 x 
108 TLPO and will be labeled with the patient’s unique study number. 
Based on their randomization, autologous TLPLDC (active vaccine) or unloaded YCWP 
+ autologous DC (control) or autologous TLPO will be sent back to the site in a blinded 
fashion.  Upon randomization of the 120th patient the randomization will transition to 2:1 
(TLPO phase I/IIa:TLPLDC). Regardless of assigned group, the site will receive 6 single 
dose vials to be injected intradermal monthly x 3 followed by boosters at 6, 12, and 18 
months in the same lymph node draining area (preferably the anterior thigh).  Patients 
must begin vaccinations between 3 weeks and 3 months from completion of SoC (or 
demonstrating tolerance on CPI after 3 doses).  Frozen tumor will be maintained for 
active vaccines for all patients to include the control patients.  The latter will be offered 
their active vaccine at time of recurrence in a crossover fashion.  Additionally, control 
patients who do not recur will be offered active vaccine at the completion of the trial. 
Safety data will be collected on local and systemic toxicities and graded and reported 
per the Common Terminology Criteria for Adverse Events (CTCAE) v4.03. 
Disease-free status will be monitored per SoC as outlined by NCCN.  Suspected 
recurrences will be documented with biopsy and pathologic confirmation.  Time to 
recurrence will be based on date of randomization to time of confirmed recurrence.  
Recurrent patients will be offered participation in the open label portion of the study.  
New active vaccine will be made for all patients, and they will be inoculated at 0, 1, 2, 3, 
6, and 9 mos.  Patients will be treated per SoC for their recurrence.  Safety and tumor 
response will be assessed per RECIST and iRECIST on their SoC follow-up scans. 
Blood (50 mL) will be collected from all patients prior to each inoculation and at 24 
months from enrollment for a total of 7 time points or a total of 350 mL of blood over 2 
years.  The collected blood will be sent to our central facility for immunologic testing of 
the T-cell response. 


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Inclusion 
Criteria 
1) Stage IIIA-C patients identified prior to their definitive surgery 
2) Stage IV patients rendered clinically disease-free with surgery 
3) Approximately 1 cm3 is preferred (but 1 mg minimum) of accessible and 
dispensable tumor that will not interfere with pathologic staging 
4) Clinically disease-free after SoC therapies OR clinically disease-free after 3 
doses of CPI therapy, demonstrating tolerance of CPI, and completion of other 
SoC therapies 
5) ECOG 0-1 performance 
6) Not involved in other clinical trials 
7) Capable of giving informed consent 
Exclusion 
Criteria 
1) Evidence of residual disease after surgery and SoC therapies 
2) Insufficient tumor available to produce vaccine 
3) Immune deficiency disease or HIV, active HBV, or active HCV 
4) Steroids or other immunosuppressants 
5) ECOG >2 
Statistical 
Analysis 
The primary endpoint for analysis of clinical efficacy is 24 months DFS when 
analyzed as a proportion using Pearson chi-square test. The intention-to-treat 
(ITT) population analyses and the per protocol (PP) population analyses will be co-
primary given the high early recurrence rate. 
In the time -to-event analysis, Kaplan-Meier curves will be generated to display 
time to events for each treatment group. 
The null and alternative hypotheses are: 
H0: hV / hC = 1   
Ha: hV / hC ≠ 1 
where hV is the constant autologous TLPLDC vaccine hazard rate and hC is the 
constant unloaded YCWP + autologous DC control hazard rate. The active vaccine 
efficacy is demonstrated when hV /hC < 1; the alternative hypothesis is a 0.50 
hazard ratio. 
The overall type 1 error rate of 5% will be used for the rejection of the null 
hypothesis in favor of the alternative hypothesis that autologous TLPLDC or TLPO 
vaccine will prolong DFS over that of unloaded YCWP + autologous DC. 
The stratification factors are the following: 
Stage (III vs IV) 
The time-to-event analysis efficacy analysis will be based on a proportional hazard 
model including the stratification factors to estimate the hazard ratio. Hazard ratios 
and 95% 2-sided confidence intervals will be presented. The model will also 
examine 2-way treatment interactions with the baseline stratification factors. 
Confirmatory analyses will be performed using a stratified log rank test for each of 
the stratification factors. 
Subgroup analyses will be displayed; any p-values for individual strata will be 
regarded as descriptive statistics. 
To explore the possibility of a treatment by investigative site interaction, the 
primary analysis will also be conducted by site for the ITT and PP populations. 
Sites that have enrolled large numbers of subjects relative to other trial centers will 
be identified to see if the results obtained at those investigative sites are influential. 
Investigative sites that individually represent fewer than 10 subjects will be 
combined for this exploratory analysis; geographic region will be substituted if the 
average number of subjects per site is fewer than 10. 


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Planned 
Survival 
Analyses 
1) The primary analysis will be conducted at the completion of two years of 
follow-up on the last enrolled patient and will compare 2-year DFS between the 
two treatment arms. 
2) An interim analysis is planned six months after enrollment of the 120th patient.  
This analysis will compare median DFS between treatment groups.  
3) The secondary analysis will be conducted at the completion of 3 yrs. of follow-
up on the final enrolled patient and will compare 3-year DFS and OS between 
treatment groups.   
 
 
 


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2.0 
INTRODUCTION  
2.1 
Immunotherapy in Melanoma 
Although melanoma is capable of eliciting an endogenous immunogenic response, 
the disease progresses when such protective mechanism fails.  After standard of 
care (SoC) therapy of stage III and IV melanoma, clinically disease-free patients 
have a 50-90% risk of recurrence.  Once recurrent these patients will likely die from 
their disease.  Currently, chemotherapy, radiation therapy and immunotherapy are 
the main choices for metastatic disease with the hope of prolonging survival1.  The 
majority of chemotherapy trials have been disappointing with no consensus 
standard therapy for care.  Overall, immunotherapy appears to be the most 
promising.  Even though immunotherapy has been studied for more than half a 
century as a treatment for cancer, recent advances in molecular immunology now 
make this strategy a viable option for the treatment of patients with advanced 
cancers2.   
Several immunotherapeutic strategies have been studied as methods to re-instate 
the innate and adaptive immune responses in melanoma.  IFN-α and IL-2 
immunotherapies are promising cytokine therapies having gained U.S. Food and 
Drug Administration (FDA) approval for treatment of metastatic melanoma.  More 
recently, ipilimumab, a monoclonal antibody (mAb) targeting CTLA-4, was 
approved by the FDA for use in the metastatic setting in 2011. 
IL-2 therapy is a common agent utilized in community practice despite its 
association with significant toxicities3-5.   Multiple phase II trials have evaluated IL-
2 therapy in conjunction with chemotherapy (cisplatin, vinblastine, dacarbazine, 
interferon-alpha, and IL-2), a treatment strategy with complete response rates of 
15-21% and overall response rates as high as 64%6-8 prompting FDA approval in 
1998.  A phase III trial evaluating biochemotherapy (cisplatin, vinblastine, 
dacarbazine, IL-2 and interferon alpha-2b) showed an increased response rate 
and progression-free survival in comparison to chemotherapy alone, but this was 
not associated with increased overall survival (OS) or improved quality of 
response9.  Subsequent meta-analysis also showed no survival benefit of 
biochemotherapy in metastatic melanoma despite an improved overall response 
rate10.  With toxicities of biochemotherapy substantially higher than chemotherapy 
alone, attempts to decrease this toxicity by administration of lower doses of IL-2 in 
the outpatient setting failed to show benefit of this approach vs chemotherapy 
alone11-13.  Additional synergistic toxicities between IL-2 and ipilimumab (discussed 
below) therapies are also being brought into question14. 
Interferon therapy has been shown to increase DFS but the impact on OS remains 
unclear14.  The World Health Organization conducted the initial major randomized 
trial examining low dose adjuvant interferon in resected stage III melanoma without 
an improvement in OS15.    


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Two subsequent randomized studies in resected stage IIb/III melanoma also failed 
to show improvement in OS or recurrence-free survival16.   
However, in another prospective trial in stage II melanoma patients, DFS was 
increased by 41 months in the adjuvant interferon group17.  In an adjuvant trial by 
the French Cooperative Group, a significant relapse-free survival benefit and trend 
towards increased OS was noted in clinically node-negative patients with 
melanoma > 1.5 mm18.   A randomized trial by the Eastern Cooperative Oncology 
Group (ECOG 1684) examining high-dose interferon-α -2b has also shown 
significant improvement in relapse-free and OS at 6.9 months median follow-up in 
stage IIb and stage III melanoma; although, this effect later waned at 12.6 months 
median follow-up19 with a larger follow-up trial confirming the lack of OS survival 
advantage20.  A pooled analysis of the three randomized trials examining high dose 
interferon-α (E1684, E1690, and E1694) confirmed an improvement in relapse-fee 
survival in the high-risk melanoma population without improvement in OS21.  The 
FDA has since approved the adjuvant use of pegylated interferon-α in node-
positive melanoma patients based upon a significant 4-year relapse-free survival 
benefit (45.6% vs 38.9%) seen in a randomized trial of 1,256 patients with 
completely resected stage III melanoma22.   Yet, despite FDA approval the high 
toxicity of adjuvant high-dose interferon is causing decreased use in most 
institutions although most National Comprehensive Cancer Network (NCCN) 
panelists agree on its role in specialized situations14.   
Ipilimumab gained FDA approval in the metastatic setting after a randomized 
phase III trial revealed a significant OS advantage from gp100 and ipilimumab 
combination 
therapy 
or 
ipilimumab 
monotherapy 
compared 
to 
gp100 
monotherapy23.  A significant OS benefit has been shown in a phase III trial of 
ipilimumab plus dacarbazine as well24.  Unfortunately, ipilimumab’s stimulation of 
T-cells may be associated with significant toxicity with immune-related events 
occurring in 60% of patients from the gp100 and ipilimumab combination trial.  In 
this trial, 10-15% of the ipilimumab-associated immune-related adverse events 
were severe (grade 3 or 4) with immune-related toxicity attributed as the cause of 
seven deaths.  As a T-cell stimulator, ipilimumab is specifically associated with 
significant risk of autoimmune reactions, and with the overall response rate 
occurring in less than 20% of patients, clinical effects often take months to become 
apparent.  However, when therapeutic responses occur, they are often sustainable 
raising questions of application in the adjuvant setting.  ECOG is currently 
conducting a prospective randomized trial of ipilimumab vs IFN-α in the adjuvant 
setting to prevent recurrence in stage III melanoma25. 
Although ipilimumab represents the only FDA approved immunostimulatory mAb, 
promising results are being obtained from clinical trials examining the safety and 
efficacy of the new programmed cell death protein 1 (PD-1)/programmed cell death 
ligand 1 (PD-L1) pathway blocking antibodies.  The PD-1/PD-L1 pathway has been 
increasingly utilized after being found to play an integral role in tumor-induced 
immunosuppression26, 27. Anti-PD-1 and anti-PD-L1 agents are currently being 
tested in early clinical trials28.   


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A recent phase I trial boasts rapid response with 31% of advanced melanoma 
patients reported to have an objective response at 8 weeks with a median OS of 
16.8 months for the total study population29. Noting the possible complementary 
role between CTLA-4 and PD-1, a phase I clinical trial cohort study examined this 
combination therapy with evidence of tumor regression in a substantial number of 
patients30. Given these results, a phase 3 trial comparing CTLA-4/PD-1 to either 
agent alone is currently underway (NCT01844505)31. 
Although promising, setbacks of current therapies include the small percentage of 
melanoma patients in which efficacy is shown, lack of specificity, and high 
toxicities.  Although efficacious against some tumors, the potency of the above 
approaches is limited by their non-specific engagement of the host immune 
system.  An ideal strategy will utilize the full potential of the host immune system 
but in a specific/targeted manner.  As such, a tumor-targeted vaccine strategy is 
an appealing adjuvant strategy. 
2.2 
Dendritic Cell Vaccines  
Sipuleucel-T (Provenge), an autologous dendritic cell-based vaccine, is the only 
FDA-approved cancer vaccine.  The phase III trial examining Provenge’s three-
shot inoculation series improved OS by 4.1 months over placebo32 without toxicity 
leading to regulatory approval for use in patients with metastatic castration-
resistant prostate cancer in 2010.   
Alone or in combination with cytokines, vaccines hold promise of eliciting a 
sustainable, more specific, tumor-targeted immune response with minimal toxicity.  
Several laboratories have demonstrated that tumor cells persist in part because 
they have one or more mutations that allow escape of immune surveillance33-36.  
As exemplified by sipuleucel-T, dendritic cells can be used as professional antigen-
presenting cells (APC) to present tumor antigens to the immune system in an 
attempt to overcome this escape.   
In order to stimulate robust anti-tumor immunity with activation of CD8+ T-cells, 
tumor antigens must be presented by APC in the context of MHC Class I 
molecules.  However, endocytosis of tumor antigens exposed to dendritic cells 
directs presentation by MHC Class II eliciting helper T-cells only, thus failing to 
engage the full power of the immune system with activation of a cytotoxic T-
lymphocyte (CTL) response.   
Laboratories have attempted to use gene transfer methods to introduce specific 
tumor antigens into dendritic cells from which they will be presented via MHC Class 
I37-41.  Unfortunately, numerous challenges include: 1) the inability to identify and 
map all integral tumor specific antigens; 2) the limited number of known tumor 
antigen genes that can be introduced into the dendritic cell; and 3) the complicated, 
time-consuming process required of this strategy.   


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Given the potential disadvantages of specific antigen identification, an ideal 
strategy involves inclusion of the entire content of tumor cells within the dendritic 
cells.  As a result, the entire portfolio of potential tumor antigens will be included 
within the APC.   Formation of a dendritic and tumor cell hybrid cell is one method 
to harness the DC with the full repertoire of tumor antigens from an individual’s 
cancer37, 42.  As such, it is an approach personalized to the patient while potentially 
available to all patients.   Both peptide and tumor lysate-loaded dendritic cells have 
been previously utilized to vaccinate patients43-49.  A preferred method to 
manufacture a tumor cell/dendritic cell hybrid uses un-cultured, post-irradiation 
tumor cells to produce a non-proliferative hybrid cell or dendritoma that retains 
both the character of the tumor cells in addition to the ability to act as an effective 
APC.  Animal studies have shown these hybrid cells capable of producing an anti-
tumor specific immune response50-58.  Results from in vitro studies 37, 59, 60 and 
human clinical trials42, 60 are also encouraging.  Scientists have observed that DC 
pulsed with tumor proteins can be administered repeatedly without side effects, 
suggesting the safety of DC-based vaccines.   
Drawbacks of this approach include the time consuming production process that 
makes application of a dendritoma vaccine within the clinical setting difficult.  
Therefore, we developed a novel technique by which hybrid cells can be easily 
purified from a fusion mixture, instantly and without culture61.  Hybrid cells are 
instantly purified from these fusions between DC and tumor cells.  This process 
allows these dendritomas to retain the characteristics of the tumor cell as well as 
the ability of the DC to act as an effective APC. Animal studies have confirmed that 
the purified dendritomas are better activators than fusion mixtures in stimulating 
specific anti-tumor immunity62.  With the hypothesis that the use of highly purified 
hybrid cells from DC-tumor cell fusion would be more effective than the entire 
fusion mixture in stimulating tumor cell-specific antitumor immunity, we examined 
this instant dendritoma purification technique in a pilot study of ten stage IV renal 
cell carcinoma patients and phase I and IIa trials in stage IV melanoma patients.  
In combination with IL-2 therapy, dendritomas prepared from autologous DC and 
tumor cells were administered by subcutaneous injection.  With toxicities related 
to IL-2 therapy, these studies showed both clinical and immunologic disease-
specific responses63. 
2.3 
New Vaccine Technology 
Unfortunately, although injection of these dendritomas allows for effective 
presentation of tumor epitopes to patients’ immune systems to stimulate a CD8+ 
CTL anti-tumor response61, 62 sufficient tumor tissue is often unavailable to create 
a dendritoma vaccine for most solid tumor patients.  
New technology whereby patients’ tumor cell lysate is loaded into micro particles 
to be phagocytized by DC offers an alternative when sufficient tumor tissue in not 
available.   


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Reproducing the strong CD8+ immune response observed previously with the 
dendritoma vaccines, this technology takes advantage of an immature DC’s avid 
phagocytosis of small particles in the range of 1-5 microns.  Using a minimum 250-
500 microgram sample of tumor tissue, it is repeatedly subjected to freeze-thaw 
cycling to create a tumor lysate suspension that is then loaded within the inner 
space of denatured yeast cell wall particles (YCWP).  These YCWP are then 
incubated with and are engulfed by immature DC.  These DC now contain tumor 
lysate within their cytoplasm, and tumor antigens are processed through the 
endogenous pathway.  These tumor antigen-loaded professional APC are the 
vaccine.  More specifically, the YCWP allow for the tumor proteins to escape the 
lysosome and thus digestion, so epitope products are not shuttled to the MHC 
Class II pathway.  Instead, these particles deliver protein tumor antigens into the 
cytoplasm where they are digested by the proteasome into epitopes that are then 
processed by TAP (transporter associated with antigen processing) and bound to 
MHC class I molecules in the endoplasmic reticulum.  In short, tumor lysate from 
the patient is loaded into YCWP, which are then phagocytized by patient dendritic 
cells.  These tumor lysate, particle-loaded DC (TLPLDC) are then administered as 
the vaccine. 
Theoretically, injection of this solid tumor vaccine intradermally near a lymph node 
allows the TLPLDC to effectively "present" the tumor epitopes to the patient's T-
cells and to stimulate the generation of antitumor immune responses.   
The next iteration of the vaccine production, TLPO, has removed the DC loading 
ex vivo and allows the DC to phagocytize the tumor lysate in vivo. The tumor lysate 
is produced in the same manner as TLPLDC with the autologous tumor tissue 
undergoing the freeze/thaw cycles and then loaded into the yeast cell wall 
particles. The TL-loaded YCWP are then capped with silicate to generate the 
TLPO vaccine. The TLPO vaccine removes the steps of harvesting the DC, 
maturing them, and loading the DC ex vivo. The TLPO method allows the DC to 
be loaded in vivo and present the tumor epitopes. 
The concept of using TLPLDC as tumor-antigen-presenting cells in a therapeutic 
vaccine was initially tested in a murine metastatic melanoma model.  In these 
studies, dendritic cells were prepared from mouse bone marrow and an 
established murine metastatic melanoma cell line was used as the source of the 
tumor cells.  The dendritic cells were loaded with YCWP containing B16F0 tumor 
lysate. Three days prior to the TLPLDC preparation, female C57BL/6J mice were 
challenged with B16F0 melanoma cells by intravenous injection.  Once the 
TLPLDC were prepared, each mouse in the treatment group was injected 
subcutaneously with TLPLDCs and this vaccination repeated for three weekly 
doses.  The mice were monitored up to four weeks for pulmonary metastasis. At 
the end of four weeks, the mice were sacrificed and the metastases were counted.  
All four control animals that were not treated with TLPLDC had more than 50 
tumors.  On the other hand, none of the treated animals had measurable 
metastases.  This experiment has been replicated multiple times, and these data 


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indicate that TLPLDC are effective in treating cancer in a proven animal model 
system.   
The TLPO Investigator’s Brochure details the specifics of the preclinical testing, 
but in summary, to test the TLPO concept, first YCWP were loaded with 
fluorescence labeled albumin and then half capped with silicate and half left 
uncapped. These particles were shaken in solution and tested for fluorescence 
leak at 1 and 2 hours. The fluorescence leak experiments demonstrated decreased 
leak from capped YCWP at 1 and 2 hours (15.8% vs 24.7% and 6.7% vs 16.6%, 
respectively).  Next, the capped and uncapped YCWP were added to cultured 
mouse macrophage cells to evaluate DC uptake at 20 minutes, 1 hour and 2 hours; 
cells were then lysed and centrifuged to evaluate the amount of fluorescent 
material in the cytoplasm versus organelles. There was increased uptake of 
capped YCWP in DC cells compared to uncapped, particularly at 2 hours after 
introduction (fluorescence readings of 11065 vs. 3928). Lysed DC loaded with 
capped YCWP demonstrated delivery of 68.9% of contents to the cytoplasm vs. 
48.8% in uncapped YCWP. Next, C57B mice were injected with 100ul saline 
(group #1), 106 empty capped YCWP (group #2) or 1mcg murine GMCSF (group 
#3). Five hours post-injection, 100ul saline was injected into the same site, then 
withdrawn, placed on a slide and examined under the microscope at 160X to 
evaluate the number of monocytic cells. The in vivo murine model showed greatly 
increased recruitment of monocytic cells in the mice receiving empty capped 
YCWP vs. GMCSF or saline alone. Then the TLPLDC vs TLPO was compared in 
a B16 melanoma model with mice followed to 80 days.  In this murine survival 
model, there was improved survival in those treated with TLPLDC or TLPO vs. 
controls.  
 
 


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Figure 1: Murine Survival Model TLPLDC vs TLPO vs Control 
 
Squares=TLPLDC, n=10 
Triangles=TLPO, n=20 
Circles=controls, n=5 
 
Finally, in a large animal equine model, 4 grey horses with melanoma were treated 
with autologous TLPO created from a biopsy of their individual lesions, injected 
every 2 weeks for a total of 4 vaccinations.  The target lesions were assessed over 
6 months by standard RECIST criteria. The equine model revealed one horse to 
be a complete responder (100% resolution of the target lesion) and the remaining 
3 as partial responders (50%, 68%, and 45% reduction in tumor size). 
Through this series of results, the silicate capped TLPO particles have proven to 
be effective at retaining loaded contents, have improved uptake by DC and, 
specifically, effective delivery to the cytoplasm of DC. In early animal studies, 
TLPO appears to have equivalent efficacy compared to the TLPLDC vaccine, but 
eliminates the need for ex vivo DC loading.  The TLPO vaccine has not been used 
in humans previously, and as such, the TLPO portion of this study is considered a 
first in man study and will be conducted as a phase I/IIa trial with safety, immune 
response and early clinical efficacy endpoints. 
2.4 
Prior/Ongoing Clinical Trials with Autologous Tumor/DC Vaccine 
We have conducted phase I/IIa clinical trials examining DC + autologous tumor 
vaccination of solid tumor patients in combination with IL-2.   To date, we have 
vaccinated 35 late-stage melanoma and renal cell carcinoma patients.  
In a phase I trial (IND# 8851), ten patients with stage IV melanoma received a 
vaccination every three months at a dose ranging from 500,000-1,000,000 or more 
dendritomas.  IL-2 dose was increased from 3 MIU/m2/day if tolerated to a 
maximum dose of 9 MIU/m2/day for five days (administered one day after the first 


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dendritoma vaccine but not after subsequent vaccinations).  There were two 
unrelated serious adverse events: one patient died due to progressive disease 19 
days after the first vaccination, and another patient experienced thrombocytopenia 
that led to a splenectomy 73 days after the last vaccination.  
The maximum level of toxicity experienced by the patients in the phase I trial is 
depicted in Figure 1.  The most common adverse events experienced by patients 
included fever (100%), chills (50%), hypotension (40%), nausea (40%), anemia 
(40%), arthralgia/myalgia (30%), weight gain (30%), stomatitis (30%), and edema 
(30%).   The average survival of nine evaluable patients was 198 days, with one 
patient alive and disease-free 12 years after initial vaccination64. 
Figure 2: Adverse Events by Grade 
 
 
 
 
 
The phase IIa trial (IND# 8851) vaccinated 15 patients with stage IV melanoma. 
Each patient received a vaccination every six weeks at a dose ranging from 
250,000-1,000,000 or more dendritomas up to six times depending on the 
availability of dendritomas and tumor cells.  IL-2 dose was administered from 3 
MIU/m2/day on days 1, 3, and 5 after the first vaccination.  There were two 
unrelated serious adverse events: one patient (on Coumadin) experienced gastric 
bleeding and a second experience hypoxia after IL-2 administration and required 
blood transfusions for treatment of anemia.  Erythema (33%), fever (33%), 
headache (27%), and arthralgia/myalgia (27%) were the most common adverse 
events experienced by patients.  


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Other commonly experienced adverse events included rash (20%), pain (20%), 
chills (20%), fatigue (20%), and nausea (20%).  The average survival of the ten 
deceased patients was 590 days.  Five patients remain alive.  
With 23 evaluable patients out of the 25 enrolled in the completed phase I/IIa 
melanoma trials, the median overall survival (OS) was 12.5 months in comparison 
to the median OS of 8.5 months (4.7 to 11.5) 65 seen in historical controls (Table 1 
and Figure 2).  One patient with stage IV melanoma had a partial response 12 
weeks after initial vaccination and received five vaccinations with a complete 
response (Figure 3).   
At interim follow-up assessment, 13 patients had died, three subjects had 
progressive disease, two subjects had stable disease, one subject had progressive 
disease, and four subjects had complete response (Figure 4).  Of note, OS of all 
four stage IV patients with completely resected disease was 100% at 20 months.  
Although difficult to draw a dose response conclusion in a limited number of 
patients, the four subjects who had a complete response received three to six 
vaccinations and the one patient who had a partial response had five vaccinations.   
Table 1: Evaluable Patients 
  
      
      
 
  
 
 
 


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Figure 3: Overall Survival  
 
 
 
 
 
 
 
 
Figure 4: Number of Inoculations  
 
Figure 5: No Evidence of Disease 
 
 
 
 
 
 
 
 
Overall, data from the above trials in late-stage melanoma patients showed the 
safety of a dendritoma vaccine itself as well as increased survival when compared 
to conventional treatments.  
The adverse events listed above were felt to be related the administration of IL-2 
and not the vaccine itself.  In addition, better survival rates were seen when all 
visible tumor was removed at the time of surgery.   


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A similar vaccination strategy was also utilized in a phase I, open label, study of 
ten patients with stage IV renal cell carcinoma (IND# 9519).  Each patient received 
a vaccination every six weeks at a dose ranging from 500,000-1,000,000 or more 
dendritomas.  IL-2 dose was increased from 3 MIU/m2/day if tolerated to a 
maximum dose of 9 MIU/m2/day for five days (administered one day after first 
dendritoma vaccine but not after subsequent vaccinations).   Twelve weeks after 
initial vaccination, one patient died, four patients had progressive disease, three 
had stable disease, and one had a partial response.  At final follow-up, nine 
patients are deceased.  The remaining patient was withdrawn due to medical 
problems requiring steroid treatment, a prohibited concomitant medication. This 
patient is being followed for survival. The cumulative summary of adverse events 
from the clinical study showed that fever (90%), nausea (70%), vomiting (60%), 
anemia (60%) and chills (60%) were the most common AEs experienced by the 
patients. Other commonly experienced AEs included unspecified pain (50%), 
stomatitis (40%) and weight loss (40%).  Average survival of the first six deceased 
patients in the phase I trial was 274 days with three patients living greater than 18 
months and one patient still alive with no evidence of disease at two years.  These 
results suggest a possible clinical benefit although a larger, controlled trial is 
needed to draw meaningful conclusions. 
As discussed above, the concept of an autologous tumor/DC vaccine has been 
made simpler with the use of the YCWP loading system, which allows the efficient 
and effective delivery of autologous tumor lysate to the patient’s DC.  In addition 
to being simpler, the TLPLDC vaccine has been shown to be more effective in 
animal models compared to the dendritoma approach although both systems 
utilize autologous tumor and DC.  We are currently utilizing the new technology of 
TLPLDC in an ongoing treatment registry study of multiple solid tumors in Cayman.  
This non-IND foreign clinical trial is being conducted by GCP and meets the criteria 
of 21 CFR 312.120.  Thus far, 10 patients have completed the vaccination series 
of 1x106 TLPLDC intradermally per monthly inoculation x 4 with very limited toxicity 
(no grade >2).  Clinical activity has been documented in 60% of the wide variety of 
patients vaccinated to date (2 complete responses, 2 partial responses, and 2 with 
stable disease).  Thus far, 40% have shown progressive disease, but none of these 
received all four inoculations.  One partial responder subsequently developed 
progressive disease.  Initial clinical response in stage IV melanoma patients has 
been impressive but in small numbers.  One of three vaccinated stage IV 
melanoma patients has achieved a complete pathologic response (see detailed 
information in the Investigator’s Brochure).  Based on this initial safety data using 
the new technology and our prior promising clinical responses with autologous 
tumor/DC vaccination in stage IV melanoma patients, especially resected stage 
IV, we are moving forward with a phase IIb trial in the adjuvant setting. 
 
 


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3.0 OBJECTIVES 
In this combined study, we intend to assess the ability of the tumor lysate, particle-
loaded, dendritic cell (TLPLDC) vaccine given in the adjuvant setting to prevent 
recurrences in patients with stage III and IV (resected) melanoma.  Also, the tumor 
lysate, particle only (TLPO) vaccine is being studied in an embedded phase I/IIa 
trial with safety, immune response and early efficacy endpoints as described 
below.       
3.1 
Primary Objective 
For the TLPLDC phase IIb, the primary objective is to compare DFS at 24 months 
between the vaccinated (TLPLDC and TLPO) and control groups analyzed as a 
proportion. 
For the TLPO phase I/IIa, the primary objective is to assess the tolerability of the 
TLPO vaccine per CTCAEv4.03. 
3.2 
Secondary Objectives 
For the TLPLDC phase IIb: 
• Compare DFS and overall survival (OS) at 36 months between the 
vaccinated (TLPLDC and TLPO) and control groups from time of 
randomization.   
• Compare safety between vaccinated (TLPLDC and TLPO) and control 
groups by assessing local and systemic toxicities using standard National 
Cancer Institute (NCI) Common Terminology Criteria for Adverse Events 
(CTCAE) v4.03. 
3.3 
Exploratory Objectives 
For both trials: 
• Evaluate immune responses among the vaccinated patients and 
compare to the control patients. 
• Correlate immunologic responses to clinical outcome.  
• Assess ongoing vaccination after recurrence for safety and tumor 
response in combination with SoC therapies to include checkpoint 
inhibitors. 
 
 


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4.0 STUDY DESIGN 
4.1 
Description of Study 
This will be a multi-center, prospective, randomized, blinded, placebo-controlled 
phase IIb trial of the autologous TLPLDC vaccine and a phase I/IIa trial of the 
TLPO vaccine (Appendixes A and B).  The vaccines to be used in this study are 
investigational and will be used under Investigational New Drug (IND) applications 
(IND#16101 for TLPLDC and IND#17274 for TLPO).  The Sponsor of these INDs 
is Elios Therapeutics, LLC. 
The target study population is melanoma patients after complete surgical excision 
with high risk for recurrence.  High-risk patients are defined as: 
• Stage III resected patients.  Stage IIIA, IIIB, and IIIC will be allowed; 
however, it is unlikely that many stage IIIA patients will be enrolled 
given the requirement for tumor to produce the vaccine (detailed later).   
• Stage IV resected patients.  All clinical/radiographic disease must be 
resected.   
Stage III and Stage IV (resected) melanoma patients will be identified prior to 
definitive surgery and screened for inclusion/exclusion criteria.   
Eligible patients will be counseled and consented for tissue procurement (consent 
#1).  Enrolled patients will have their disease surgically resected per SoC (need 
not occur at the study site), and a portion (approx. 1 cm3 preferred but 1 mg 
minimum of their melanoma sterilely frozen in provided freezing vials and storage 
tubes.   
This tissue will be shipped in liquid nitrogen shippers through FedEx to our central 
facility in Greenville, SC and stored frozen until vaccine preparation.  If patients 
cannot be rendered disease-free, they will be considered screen failures for this 
study.  If melanoma is being resected from multiple locations (i.e., primary and 
nodes or two different metastatic sites), then samples from all locations would be 
preferred but is not mandatory.   
As indicated by SoC per the NCCN guidelines and determined by the treating 
team, if a patient is to receive systemic therapy (chemotherapy or IFN-α) and/or 
radiation therapy, then the vaccinations will not begin until SoC therapy is 
completed. Although, patients on adjuvant check point inhibitors (CPI) may start 
the vaccine trial after showing tolerance of the CPI after 3 doses, given that there 
may be a synergistic benefit and no added toxicity when adding the vaccine to CPI.  
Once the patient is deemed clinically disease-free, they will be consented (consent 
#2) for treatment and randomized.  Consenting should occur as close to 
completion of SoC therapies as possible (or after demonstrating tolerance to CPI 
after 3 doses), but may overlap completion of systemic and/or radiation therapies 
by no more than one month.  Once consented, patients will receive a single 


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injection of Neupogen (G-CSF) 300 µg (or its equivalent) subcutaneously 24-48 
hrs prior to having 70 mL of blood collected from the patient and sent to our central 
facility for DC isolation and preparation.  Patients who cannot tolerate Neupogen 
(or its equivalent) or refuse it, will have 120 mL of blood drawn and sent to our 
central facility for DC isolation and preparation.  Additional blood may be drawn if 
additional vaccine doses need to be made or remade for any reason.  
Vaccines will be prepared by producing tumor lysate (TL) through freeze/thaw 
cycling of the autologous tumor and then loaded into pre-prepared yeast cell wall 
particles (YCWP).  The TL-loaded YCWP will be introduced to the DC for 
phagocytosis; thus creating the TLPLDC vaccine which will be frozen in single 
dose vials.   Each vial will contain 1-1.5 x 106 TLPLDC and will be labeled with the 
patient’s unique study number.  The TLPO will be created by taking the TL-loaded 
YCWP and capping them with silicate. The TLPO vaccine will be frozen in single 
dose vials containing 1 x 108 TLPO and will be labeled with the patient’s unique 
study number. Vaccine production and QA testing for vaccine release and shipping 
will take approximately three weeks.  
Based on their randomization, either autologous TLPLDC or TLPO (active vaccine) 
or unloaded YCWP + autologous DC (control) will be sent back to the site in a 
blinded fashion.  The patients, referring physicians, site PIs, and site pharmacists 
will be blinded to group assignment.  Regardless of assigned group, the site will 
receive six single dose vials to be injected intradermal monthly x 3 followed by 
boosters at 6, 12, and 18 months (Appendix C) from initial inoculation in the same 
lymph node draining area (preferably the anterior thigh).  Patients must begin 
vaccinations between 3 weeks and 3 months from completion of Standard of Care 
(SoC) or patients on adjuvant check point inhibitors (CPI) may start the vaccine 
trial after showing tolerance of the CPI after 3 doses. 
Frozen tumor will be maintained for all patients.  Patients who recur will have the 
option to participate in the open label portion of the trial. At the time of confirmed 
recurrence, the study site can request to have the patient unblinded, and the 
patient will sign the open label consent form (consent #3). In the open label study, 
all patients will have new vaccine doses produced from stored tumor and a new 
blood draw.    If the patient is having surgery, additional tumor tissue may be 
collected or may be required if insufficient tumor is available in the freezer. 
Once open label consent has been signed, patients will receive a single injection 
of Neupogen (G-CSF) 300 µg (or its equivalent) subcutaneously 24-48 hrs. prior 
to having 70 mL of blood collected from the patient and sent to our central facility 
for DC isolation and preparation.  Patients who cannot tolerate Neupogen (or its 
equivalent) or refuse it, will have 120 mL of blood drawn and sent to our central 
facility for DC isolation and preparation.  Additional blood may be drawn if 
additional vaccine doses need to be made or remade for any reason. After vaccine 
is produced and shipped to the study site, patients will begin receiving the 
inoculations on the following schedule: 0, 1, 2 and 3 months ± 1 week followed by 
booster inoculations at 6 and 9 months ± 2 weeks from initial inoculation for a total 


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of six doses (Appendix C).  These vaccinations will be occurring in the background 
and not interfere with any SoC therapies indicated and initiated to treat the patient’s 
recurrence per NCCN guidelines.  For the open label study, patients will be 
followed for a total of 12 months with tumor assessments by iRECIST and RECIST 
(Appendix F) on SoC follow-up imaging 
Control patients who do not recur will be offered active vaccine at the completion 
of the trial.  It will be their choice whether to proceed with active vaccination. 
Safety data will be collected on local and systemic toxicities, graded and reported 
per the NCI Common Terminology Criteria for Adverse Events (CTCAE) v4.03 
(Appendix D). 
Disease-free status will be monitored per SoC as outlined by NCCN for three 
years.  Suspected recurrences will be documented with biopsy and pathologic 
confirmation whenever possible.  Time to recurrence will be based on date of 
randomization to time of confirmed recurrence. 
In addition to the initial blood collection for vaccine preparation, 50 mL of blood will 
be collected from all patients prior to each of the six inoculations and at 24 months 
from enrollment for a total of seven time points or a total of 350 mL of blood over 
two years.  The collected blood will be sent to our central facility for immunologic 
testing of the T-cell response.  The immunologic responses will be correlated with 
the clinical outcomes.  Patients will follow a similar pattern if they recur and 
participate in the open label portion of the study. 
We plan to enroll at least 180 patients (randomized 2:1, vaccine:control) up to 120 
patients; after enrollment of the 120th patient the randomization will transition to 2:1 
(TLPO phase I/IIa:TLPLDC) for the remaining 60 patients.  The TLPO portion of 
the study is being conducted as an embedded phase I/IIa trial.  With 10-12 sites 
enrolling a 0.8 pts/site/month, we anticipate enrollment to be complete in 12-18 
months.  With a 2-year DFS primary endpoint and 3-year DFS/OS secondary 
endpoints, the trial duration is expected to be 4-4.5 years.  Open label patients will 
be followed for a year after recurrence. 
4.2 
Rationale for Study Design 
Based on our prior phase I/IIa clinical trials in stage IV melanoma, clinically 
disease-free patients realized the greatest benefit from vaccination.  As such, 
stage III and IV (resected) patients are the target population for this adjuvant trial. 
Prevention of recurrence is the primary objective.   
Assuming a baseline recurrence rate of 60% (corresponding to a DFS of 40%) at 
two years based on published data in this mixed group of stage III (predominantly 
IIIB and IIIC) and stage IV (resected) melanoma patients, a sample of size of 120 
will have 80% power to detect a statistical difference in proportions between 
treatment arms controlling the type I error at alpha = 0.05 (two-sided). 


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A blinded evaluation of the first 75 patients enrolled discovered an early recurrence 
rate of 12% prior to completion of primary vaccine series. To compensate for this 
early recurrence rate and to preserve the power of the trial, enrollment will be 
extended by at least an additional 20 patients, now totaling at least 140. After the 
randomization of the 120th patient, then randomization will transition to a 2:1 (TLPO 
phase I/IIa:TLPLDC) for an additional 60 patients resulting in an additional 20 
patients receiving the TLPLDC vaccine (therefore 140 as stated above). The total 
expected enrollment after the initiation of the TLPO phase I/IIa trial is at least 60 
patients (40 TLPO phase I/IIa and 20 additional TLPLDC; Appendix B). In the 
TLPO phase I/IIa, the tolerability of the TLPO vaccine will be assessed primarily.  
Given the embedded nature of the trial and the randomization of the patients to the 
TLPO phase I/IIa vs TLPLDC, there will be an opportunity to compare the toxicity 
profiles of the two vaccines. 
In an attempt to allow all patients to benefit from receiving an autologous TLPLDC 
or TLPO vaccine and to assess the potential for synergistic benefit of active 
vaccination added to SoC therapies, all patients regardless of randomization will 
be offered the opportunity to participate in the open label portion of the study if they 
should recur.  These open label secondarily treated patients will be followed for 
documentation of treatment response.  Additionally, control patients who do not 
recur can request active vaccination after they complete the trial. 
4.3 
Outcome Measures 
4.3.1 Primary outcome measures 
Disease state will be determined by the patients’ own physicians at the individual 
study sites during their routine follow-up screening.  This will occur for all enrolled 
patients, regardless of randomization, approximately every three to six months for 
three years from randomization with clinical exam, laboratory and radiographic 
surveillance as indicated.  This follow-up strategy is SoC for these patients per 
NCCN guidelines.  If records are not available, patients, or their referring 
physicians, will be contacted to discern their disease status.  Recurrences will be 
confirmed pathologically, whenever possible, per standard of care.   
For the TLPLDC phase IIb, the primary outcome measure of the trial is DFS at 24 
months compared between the vaccinated (TLPLDC) and control groups after the 
final enrolled patient completes two years of follow-up. 
An interim analysis will be performed six months after the 120th patient is enrolled.  
This analysis will compare median DFS between vaccinated (TLPLDC) and control 
groups. 
For the TLPO phase I/IIa, the primary outcome measure is safety as assessed by 
CTCAE v4.03 graded toxicity scale (Appendix D). 


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4.3.2 Secondary outcome measures 
For the TLPLDC phase IIb, the secondary outcome measures include 36 months 
DFS and OS and assessing local and systemic toxicities. 
DFS and OS at 36 months will be assessed and compared between vaccinated 
(TLPLDC) and control groups.  This assessment will be conducted after the last 
enrolled patient has been followed for three years.  
For both studies, standard local and systemic toxicities will be collected and graded 
per the NCI CTCAE v4.03 graded toxicity scale (Appendix D).   
For both the primary and the booster inoculations, patients will be monitored for 30 
minutes after the inoculation with vital signs taken as needed. Local or systemic 
toxicities will be collected and graded by the research staff at the patient’s next 
visit. Serious AEs will be reported as described in Section 6.0. 
4.3.3 Exploratory outcome measures 
For both trials, immune responses will be primarily documented using the 
assessment of local reactions and future CTL assays on stored blood and 
assessment of stored serum pre- and post-vaccination.  Each of these 
measurements will be performed regardless of randomization.  Detailed 
descriptions of these assays/tests are described in Sections 5.4.4, 5.4.5, and 5.4.6.   
Phenotypic assays (dimer/tetramer) for clonal CTL expansion against common 
melanoma-associated antigens such as gp-100 and MART-1 as well as functional 
assays like ELISPOT against the same antigens will be performed on 
banked/stored blood.  Antibody and cytokine responses may be assessed on the 
stored serum.  Blood/serum will be collected prior to each inoculation (0, 1, 2, 6, 
12, 18 months) and at study completion at 24 months from randomization.  
Comparisons will be made between the immunologic responses in the vaccinated 
(TLPLDC and TLPO) patients vs the control patients.  
In a series of exploratory analyses, the results of these immunologic measures will 
be correlated with clinical outcome in the vaccinated (TLPLDC and TLPO) patients. 
Additional exploratory outcomes will compare clinical and immunological 
outcomes based on the dose of vaccine (TLPLDC and TLPO) and number of 
inoculations received. 
If enrolled patients recur, they will be offered participation in the open label portion 
of the study regardless of their randomization.  All patients will have new vaccine 
produced, and they will receive inoculations at 0, 1, 2, 3, 6, and 9 mos (Appendix 
C).  They will receive the vaccinations in the background of their SoC therapies 
initiated to treat their recurrence.  These patients will be followed for safety and 
tumor response in combination with their SoC therapies and assessed per 
iRECIST and RECIST (Appendix F) of their SoC follow-up scans.  


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4.4   Safety Considerations 
In our previous trials, the safety of the autologous tumor/DC vaccines has been 
excellent.  In the completed melanoma phase I where the autologous tumor/DC 
vaccine was given with IL-2, the most common adverse events experienced by 
patients included fever (100%), chills (50%), hypotension (40%), nausea (40%), 
anemia (40%), arthralgia/myalgia (30%), weight gain (30%), stomatitis (30%), and 
edema (30%).  There were no SAE’s related to the vaccine/IL-2. 
In the completed melanoma phase IIa where the IL-2 dose and frequency was 
reduced, the most common adverse events were erythema (33%), fever (33%), 
headache (27%), and arthralgia/myalgia (27%).  Other commonly experienced 
adverse events included rash (20%), pain (20%), chills (20%), fatigue (20%), and 
nausea (20%).   
Many of these toxicities are attributable to IL-2, which will not be used in the current 
study.  We have seen minor toxicities (grade 1 and 2) transient flu-like symptoms 
in the 12 patients vaccinated without IL-2 in the treatment registry with the new 
technology, TLPLDC vaccine.  We anticipate a similar favorable safety profile in 
this trial, but all local and systemic toxicities will be documented, graded by the 
NCI CTCAE v4.03 and reported as indicated below in Section 6.0. 
Additionally, there is a small risk associated with the protocol-dictated single dose 
of Neupogen, or its equivalent, prior to the initial blood draw for DC isolation.  
Neupogen is a FDA-approved drug for increasing the white blood count, is 
commonly used in oncology patients, and has a well-tolerated toxicity profile. 
Thus far, in this study, we have seen no significant local reactions in any of the 
blinded patients vaccinated with the TLPLDC vaccine or placebo nor has there 
been any systemic safety signal noted by the DSMB.  However, for the TLPO 
vaccine phase I/IIa component, this is a first in human study; therefore, the safety 
of this vaccine is unknown.  As such, the primary endpoint of this embedded trial 
is safety.  Since the silicate-capped, tumor lysate-loaded YCWP are being given 
alone (not contained within autologous DC), then the vaccinated patients may form 
a different reaction to this version of the vaccine.  While the phase I/IIa TLPO trial 
is not a dose escalation trial, we will modify the dose based on local and systemic 
toxicities.   
Dose Reductions of Vaccine.  For patients experiencing robust local reactions 
(>100mm of erythema/induration and/or threatened skin disruption), or >/= grade 
3 local and/or systemic reactions, or >/= grade 2 hypersensitivity reactions, the 
dose of the vaccine will be reduced by 50% for subsequent inoculations.  If 
reactions/toxicities recur with subsequent inoculations, the vaccine dose will 
continue to be reduced serially by 50%.  If dose reduction does not control toxicities 
or they worsen despite reductions, then all further inoculations will be halted.   


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4.5   Compliance with Laws and Regulations 
This study will be conducted in accordance with current FDA regulations and 
Good Clinical Practices (GCPs), and local ethical and legal requirements. 
5.0   MATERIALS AND METHODS 
5.1 
Subjects 
Patients over the age of 18 years with a diagnosis of stage III or IV melanoma 
capable of being rendered clinically disease-free after surgery will be targeted.  
Patients will be recruited from medical and surgical oncology and/or 
hematology/oncology clinics at the individual study sites.   All patients will be 
properly counseled and consented. 
5.1.1 Subject selection 
Potentially eligible patients, specifically patients with clinical stage III and stage IV 
resectable melanoma will be identified by staff in the medical and surgical and/or 
the hematology/oncology clinics at the individual study sites.   
A research nurse and/or study coordinator will approach these patients about 
being in the trial and will introduce the trial to the prospective volunteer.  If the 
volunteer is interested and appears eligible, the nurse will arrange to counsel the 
patient.  The nurse will thoroughly screen the patient for inclusion and exclusion 
eligibility criteria.  If the patient remains interested and eligible, informed consent 
will be obtained for tissue procurement (consent #1). 
Written informed consent will be obtained from all study participants.  Prospective 
participants will be provided with a copy of the consent form #1 to read.   
The research nurse coordinator and/or study coordinator, or Principal Investigator 
(PI), will explain the study and review the consent form with the patient.  Subjects 
will be given ample time to ask and have all questions answered prior to signing 
the consent form.   
After SoC surgery (which need not occur at the study site), if the patient is rendered 
clinically disease-free, sufficient tumor is available for vaccine production (1 cm3 
preferred, 1mg minimum), and all other eligibility criteria are met, then the patient 
can be counseled and consented for randomization and treatment (consent #2).  
This consenting should occur as close to completion of indicated SoC therapies as 
possible (or after demonstrating tolerance to CPI after 3 doses), but may overlap 
completion of systemic and/or radiation therapy by no more than one month.  Once 
the second written informed consent is obtained, the patient will receive a single 
injection of Neupogen (G-CSF) 300 µg (or its equivalent) subcutaneously 24-48 
hours prior to having 70 mL of blood collected from the patient and sent to our 
central facility for DC isolation and preparation.  Patients who cannot tolerate 
Neupogen (or its equivalent) or refuse it, will have 120 mL of blood drawn and sent. 


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Additional blood may be drawn if additional vaccine doses need to be made or 
remade for any reason.  The full course (six doses) of the TLPLDC or TLPO 
vaccine vs placebo vaccine (empty YCWP + DC) will be produced at one time after 
randomization.  The production and QA testing for vaccine release will take 
approximately three weeks.  Vaccination cannot begin until after all SoC therapies 
are completed, but the inoculation series must begin within 3 weeks and 3 months 
from completion of SoC therapies.   
Tumor will be maintained on all patients to include the control patients so that 
active vaccine can be reproduced if necessary, in the case of the vaccine patients 
or be made for the control patients in the case of recurrence or upon request at the 
end of the primary endpoint.     
5.1.2 Inclusion criteria 
Patients will be included in the study based on the following criteria: 
• 18 years or older 
• Eastern Cooperative Oncology Group (ECOG) performance status 0,1 
(Appendix E) 
• AJCC stage III or IV completely resectable melanoma identified before 
surgery 
• Approximately 1 cm3 preferred but 1 mg minimum of accessible and 
dispensable tumor that will not interfere with pathologic staging 
• Clinically disease-free after surgery 
• Completing SoC adjuvant therapy per NCCN guidelines to include 
chemotherapy, radiation therapy, and/or biologic therapy as clinically 
indicated.  (Consent #2 should be signed as close to completion of 
SoC as possible but may overlap completion by up to one month.)  
ONLY for patients on CPI therapy: The patient has completed all other 
SoC therapy per NCCN and has demonstrated tolerance of treatment 
after 3 doses of CPI therapy without serious adverse events and 
remains clinically disease-free. (Consent #2 should be signed after the 
3rd dose of CPI therapy.) 
• Vaccinations initiated between 3 weeks and 3 months from completion 
of SoC multi-modality cancer care or after tolerance to CPI therapy has 
been determined. 
• Adequate organ function as determined by the following laboratory 
values: 


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• ANC ≥ 1,000/μL 
• Platelets ≥ 75,000/μL 
• Hgb ≥ 9 g/dL 
• Creatinine ≤ 1.5 x upper limit of normal (ULN) or Creatinine 
clearance ≥ 50% of lower limit of normal (LLN) 
• Total bilirubin ≤ 1.5 ULN 
• ALT and AST ≤ 1.5 ULN 
• For women of child-bearing potential, agreement to use adequate birth 
control (abstinence, hysterectomy, bilateral oophorectomy, bilateral 
tubal ligation, oral contraception, IUD, or use of condoms or 
diaphragms) 
• Signed informed consent 
5.1.3 Exclusion criteria 
Patients will be excluded from the study based on the following criteria: 
• 
Evidence of residual disease after surgery and SoC adjuvant 
therapies 
• 
Insufficient tumor available to produce vaccine 
• 
ECOG >2 performance status (Appendix E) 
• 
Immune deficiency disease or known history of HIV, HBV, HCV 
• 
Receiving immunosuppressive therapy including chronic steroids, 
methotrexate, or other known immunosuppressive agents 
• 
Pregnancy (assessed by urine HCG) 
• 
Breast feeding 
• 
Active pulmonary disease requiring medication to include multiple 
inhalers (>2 inhalers and one containing steroids) 
• 
Involved in other experimental protocols (except with permission of 
the other study PI) 


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5.2 
Method of Treatment Assignment 
If volunteers meet all inclusion criteria and none of the exclusion criteria and agree 
to participate, they will continue in the study.  After tissue procurement, if adequate 
tumor is available for vaccine production (1 cm3 preferred, 1 mg minimum) and 
patients are rendered clinically disease-free, then they will be consented and 
randomized for treatment assignment.   
Patients will be randomly assigned by the CRO using a computer-generated 
randomization table to receive the autologous TLPLDC vaccine or placebo (empty 
YCWP + autologous DC).  Randomization between the two treatment groups will 
occur in a 2:1 allocation ratio using an institutional balancing algorithm.  After the 
120th patient is randomized, the TLPO phase I/IIa trial will commence with a 2:1 
(TLPO phase I/IIa:TLPLDC) randomization for an additional 60 patients (refer to 
Appendix B). This randomization will use the same computer-generated 
institutional balancing algorithm and the patients will be randomly assigned by the 
CRO.  Patients, treating physicians, and site PIs will be blinded as to the treatment 
assignment.  Active vaccine will be made available to those patients assigned to 
the control group at time of recurrence or after they have completed the trial, 
including all follow-ups and primary and secondary endpoints, if they have not 
recurred and wish to receive the active vaccine.   
5.3 
Study Treatment 
5.3.1 Vaccine production  
Vaccines will be produced by Orbis Health Solutions (Greenville, SC).  Tumor 
lysate from the patient’s tumor is loaded into YCWP which are phagocytized by the 
patient’s dendritic cells in vitro to generate TLPLDC. The TLPO vaccine is 
produced from the patient’s tumor lysate loaded into YCWP and capped with 
silicate.   
The final vaccine contains 1-1.5 x106 TLPLDC or 1.0 x 108 TLPO per dose and will 
be provided in frozen single dose vials in 250 µl of freezing media for TLPLDC and 
saline for TLPO.  The final vaccine is prepared for injection by thawing the single 
dose vial and diluting the vaccine (TLPLDC or TLPO) by adding 500 µl of sterile 
saline for injection.  
For the open label portion of the study, new vaccine will be made for all patients 
from frozen tumor and a new blood draw. 
5.3.1.1 
Preparation of dendritic cells 
Dendritic cells will be generated from the patient's peripheral blood monocytes 
(PBMC) obtained from the post-Neupogen (or its equivalent) 70 mL blood draw or 
from 120 mL of blood drawn if Neupogen (or its equivalent) not used after consent 
#2.  The PBMCs are initially diluted and placed in a plastic culture flask for 2 hours.  


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Non-adherent cells are washed away and collected.  These cells are rich in 
lymphocytes and will be frozen for future assays.   
The adherent cells will be incubated at 37°C in 5% CO2 for two days in serum-free 
cell culture medium with the appropriate cytokines added to generate immature 
monocyte-derived dendritic cells. 
5.3.1.2 
Tumor tissue preparation 
A minimum of 1 mg sample of viable tumor tissue is required to be collected at the 
time of primary surgery.  The specimens will be collected and placed in sterile 
freezing vials that will be provided and then shipped in specialized FedEx shippers 
designed to keep the specimens frozen (also provided).  The cells of the tumor 
tissue will be lysed by multiple freeze/thaw cycles in lysis buffer to produce tumor 
lysate. 
5.3.1.3 
YCWP preparation 
YCWP are prepared by NaOH digestion of all non-cell wall material from 
Fleishman’s Bakers’ yeast, Saccharomyces cerevisiae, followed by thorough 
aqueous, ethanol, and acetone washings. 
5.3.1.4 
Loading of YCWP with tumor lysate 
TLPLDC are generated by incubating tumor lysate loaded YCWP in the presence 
of 
dendritic 
cells 
at 
a 
specific 
time 
during 
day 
2 
of 
the 
DC 
isolation/incubation/maturation process.  The timing is essential in that the 
immature DC are given the YCWP at their most phagocytic phase.  For control 
patients, empty YCWP will be incubated in the presence of DC at the same point 
on day 2 of the DC incubation. 
5.3.1.5 
Capping of TL-Loaded of YCWP with Silicate 
TLPO vaccine is generated by capping tumor lysate-loaded YCWP with silicate. 
The fully loaded, dried YCWP are added to a solution of ammonium hydroxide, 
tetraethylorthosilicate and 100% ethanol and shaken vigorously for 15 minutes in 
order to “cap” the particles with a silicate coating.  Following “capping,” the loaded 
and “capped” YCWP are washed multiple times in PBS with sonication to remove 
all “capping” reagents.  After washing, the suspension of “capped” tumor lysate-
loaded YCWP (TLPO) is aliquoted into individual doses each containing 1x108 
TLPO in 250 uL of sterile saline and frozen. 
5.3.2 Storage 
Six doses of 1-1.5 x 106 autologous TLPLDC or 1.0 x 108 TLPO will be 
cryopreserved in patient-specific labeled single dose vials and maintained at the 
central production facility at -80◦C until shipped. Once approved for release, the 
entire vaccine series (all six doses) will be shipped to the site frozen in specialized 


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LN shippers.  The patient-specific dose series will be stored at the site at least -
70°C in the research pharmacy until serially thawed for use. Any temperature 
excursion for more than 30 minutes at -55◦C or warmer needs to be reported. 
Outside of preparation for patient injection, the vaccine may not, at any time, be 
allowed to thaw. The isolated 250ul vial will thaw in less than one minute at room 
temperature.   
5.3.3 Inoculation series – administration 
Patients will receive six intradermal inoculations on the anterior or medial side of 
the same thigh or arm. The general area of inoculation will be at a location midway 
between the inguinal ligament and the knee. 
Before injection, this cellular vaccine will either be thawed slowly in a 370 C water 
bath or the vaccine vial can be held in a gloved hand until thawed if water bath is 
not available. The vial should not be left lying out to thaw at ambient temperature. 
The 250 µl of thawed TLPLDC or TLPO will be diluted with 500 µl of sterile saline 
for injection.  The final 750 µl volume will be drawn up into a 1 mL syringe and 
should be injected within 4 hours.  The entire contents of the syringe will be injected 
intradermally in two approximately equal inoculums at two different sites 5 cm from 
each other on the anterior or medial thigh or arm. Inoculations will be administered 
in the same lymph node draining area (same arm or leg) (see also Appendix C).   
The research nurse coordinator will administer the inoculations sterilely in the clinic 
facility located at each study site. For female patients with childbearing potential, 
a urine pregnancy test will be performed before each inoculation. If this test is 
positive at any time, the patient will be discontinued from the study. 
Initial inoculations will be performed at 0, 1, and 2 months ± 1 week followed by 
booster inoculations at 6, 12, and 18 months ± 2 weeks from initial inoculation for 
a total of six doses.  
For the open label portion of the study, inoculations will be performed at 0, 1, 2, 
and 3 months, ± 1 week and at 6 and 9 months, ± 2 weeks for a total of six doses. 
5.3.4 Dose reduction for TLPO 
Patients experiencing robust local reactions or unacceptable toxicities (see section 
4.4 for definitions) will have their TLPO dose reduced by 50%.  This will be 
accomplished by doubling the sterile saline diluent and maintaining the same 
inoculation volume.  
5.4 
Study Assessments 
Signed, IRB approved informed consent must be obtained from patients prior to 
any pretreatment assessments.   


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5.4.1 Pretreatment assessments 
This trial requires two consents: one for tissue procurement and one for 
randomization/treatment.  The second consent will be signed once a patient has 
been rendered clinically disease-free after surgery, adequate tumor tissue has been 
acquired for vaccine production, and all eligibility criteria are met.  Consent #2 will 
be signed as close to completion of SoC therapies as possible but may overlap 
completion of systemic and/or radiation therapy by up to a month. For those patients 
on CPI therapy, they must have completed 3 doses of therapy and demonstrated 
clinical tolerance before continuing with consent #2.  Initiation of vaccination cannot 
begin until SoC therapy has been completed (and/or demonstration of 3 doses of 
CPI tolerance, as applicable), but must begin 3 weeks to 3 months from completion 
of SoC (or after 3 doses of CPI therapy demonstrating tolerance). 
All patients must have a CBC with differential, CMP, and LDH within two months of 
trial initiation and, for female patients, a urine pregnancy test (upon consent to 
study), for screening and immediately prior to any vaccine inoculations.  If the 
pregnancy test is positive the patient will be excluded from the study.  Women who 
have had a hysterectomy, bilateral oophorectomy, tubal ligation, documented 
absence of menses for two years, or FSH hormonal laboratory results that verify 
menopause, will not be required to have pregnancy testing.   
For patients who have a complete metastatic evaluation (CBC, LFTs, CXR, chest 
and abdomen CT, bone scan, and PET scan) available, all studies will be screened 
but these studies are not required for enrollment.  Disease-free status will be assured 
by the patients’ primary treating/referring physician.  Overall health screen will be 
assessed utilizing the ECOG performance status grading system (Appendix E). 
All patients regardless of randomization will be assessed for baseline and post-
vaccination immunologic responses.  Blood samples will be obtained prior to each 
inoculation (0, 1, 2, 6, 12, 18 months) and at study completion at 24 months from 
randomization.  The protocol schedule of events is listed in Appendix G.   
Specimen handling, processing, and assays are described in Sections 5.4.4, 5.4.5, 
and 5.4.6. 
5.4.2 Assessments during treatment 
Any laboratory evaluations or imaging studies ordered by the treating oncologist 
as part of standard practice will be reviewed by the research nurses or study 
coordinators and any abnormal results will be reviewed by the study 
investigators. 
Research nurses or study coordinators will assess the patients prior to and during 
a 30 minute observation after vaccine inoculation, and will assess the patient on 
an “as needed” basis. 


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For both the primary inoculations and booster series, the patients will be monitored 
for 30 minutes after the inoculation with vital signs taken as needed.    Local or 
systemic toxicities will be collected and graded by the research staff at the patient’s 
next visit. The NCI CTCAE v4.03 graded toxicity scale (Appendix C) will be utilized 
to assess local and systemic toxicity.   
Blood samples will be obtained from all patients, regardless of randomization, to 
determine induction of anticancer immune responses as described in Sections 
5.4.4, 5.4.5, and 5.4.6. 
5.4.3 Follow-up assessments 
The clinical endpoint of recurrence will be determined by the patients’ own 
physicians at the individual study sites during routine follow-up.  This will include 
history and physical examination every 3-6 months for all patients.  Additional 
laboratory and radiographic surveillance will be performed as indicated and 
directed by the patients treating physicians.  The determination of recurrence will 
be per standard practice by the treating physicians and communicated to the study 
investigators on a routine basis.  Documentation of any recurrence will be 
obtained.  Whenever possible, pathologic confirmation of recurrence will be 
obtained.   
However, a patient will also be considered to have a recurrence if highly suggested 
on radiographic evaluation and oncologic treatment for the recurrence is initiated 
by the treating physician.  Documentation of the latter will be obtained.    For the 
purposes of this trial, DFS will be calculated from the date of randomization until 
date of last follow-up or recurrence.   If records are not available, patients, or their 
referring physicians, will be contacted to discern their disease status and every 
effort will be made to obtain documentation.   
Once a patient recurs, they will be offered the opportunity to participate in the open 
label portion of the study.  The patients will be vaccinated at 0, 1, 2, 3, 6, and 9 
months.  Assessments during inoculations and blood draws will be the same as 
above.  These patients will be treated per SoC for their recurrence.  The 
vaccinations will be performed in the background and will not interfere with SoC 
treatments.  Additional laboratory and radiographic surveillance will be performed 
as indicated and directed by the patients treating physicians and per standard of 
care.  The determination of tumor response will be assessed on these SoC scans 
per RECIST and iRECIST (Guidelines for the Evaluation of Immune Therapy 
Activity in Solid Tumors) (Appendix F).  These results will be communicated by the 
primary study investigators on a routine basis.   
All enrolled patients, regardless of randomization, will be followed for clinical 
recurrence and overall survival for a minimum of three years from randomization.  
The open label patients will be followed for 12 months. 


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5.4.4  Blood collection and processing 
Multiple blood draws will be required for this trial. All blood tubes will be labeled 
only with the patients’ unique study number.  Approximately 50 mL will be collected 
for immunologic assessments prior to each inoculation (0, 1, 2, 6, 12, and 18 
months) and at 24 months from randomization.  Thus, a total of approximately 350 
mL of blood will be drawn over the two-year course of the study.  De-identified 
patient blood samples showing only the unique study ID number will be sent from 
study sites via overnight delivery to our central lab facility in Greenville, SC where 
they will be stored until used for immunologic assays (Section 5.4.6).  At no point 
will laboratory personnel have access to patient identifiers.  Blood will be frozen 
and stored under unique study numbers for up to five years for additional 
immunologic studies related to this protocol (for example, to repeat assays or 
perform new immunologic assays that do not exist at present but may become 
available) as needed and then destroyed.  No genetic testing will be performed on 
this material.  Study participants will not be contacted in the future for additional 
use of these stored blood specimens.  If study participants want their blood 
specimens removed from storage and destroyed, they may do so by contacting 
the PI or research nurse at any time.  Additionally, any stored blood may also be 
utilized to assess new generations of vaccines. A similar schedule will be followed 
for the open label portion of the study.      
The 50 mL of blood will be collected as 10 mL into a BD Vacutainer Rapid Serum 
tube (BD, Franklin Lakes, NJ) which contain a clot activator and silicone coated 
interior.  After receipt and centrifugation, serum will be collected, aliquoted in vials 
and frozen.  The remaining 40 mL will be collected into four BD Vacutainer Heparin 
tubes (green tops), which contain an anticoagulant (sodium heparin).  Once 
received, the heparinized blood will be pooled, diluted, and added to FICOLL 
HYPAQUE density gradient fluid containing tubes to allow for the separation of 
PBMC from the red blood cells by a single step centrifugation process.  The PBMC 
fraction will be collected, aliquoted, and frozen for future use in immunologic 
assays. 
5.4.5  Phenotypic assay (for example, dextramer assay) for melanoma-specific 
antigens 
 
Thawed and cultured PBMC will be stained with aqua live/dead stain (Invitrogen) 
and the following antibodies:  CD8 APC-H7 (BD Biosciences), CD3 PE Cy7 (BD 
Biosciences), gp100-APC-conjugated dextramer (Immudex), and the following 
pacific blue conjugated lineage antibodies:  CD14 (BD Biosciences), CD16 (BD 
Biosciences), and CD19 (Biolegend).  Cells will be analyzed on a Canto flow 
cytometer (BD Biosciences).   
The frequency of gp100-specific CD8+ T-cells will be determined as the percentage 
of cells that are alive, lineage-, CD3+ CD8+ and gp100-dextramer+.  This assay 
may be performed for other known CD8+ epitopes from known melanoma-specific 
antigens.  


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5.4.6  Functional assay (for example, ELISPOT assay) for melanoma-specific 
antigens 
Thawed and isolated PBMC are cultured/stimulated overnight in complete medium 
(RPMI + 5 %FCS + PSG) supplemented with IL-7 (20 ng/mL) with the individual 
peptides at 25 μg/mL (gp100, MART-1, etc.) or PMA + Ionomycin in flat-bottom 
anti-human IFN-γ ELISPOT plates (BD PharMingen) at 5 x 105 cells/well/200 μL in 
duplicate wells.  The plate is incubated at 37ºC overnight after which the wells will 
be washed and incubated with the biotinylated-anti-IFN-γ mAb for two hours.  The 
wells will be washed again and incubated with streptavidin-conjugated HRP for 
one hour.  After a final wash the AEC-substrate solution will be added to the wells 
and allowed to develop for approximately 5-10 minutes at which time the wells will 
be washed with deionized water to stop the reaction.  The number of spots present 
in each well will be enumerated using the CTL ELISPOT analyzer (CTL Analyzers 
LLC, Cleveland, OH). 
5.5 
Discontinuation of Protocol-Specific Therapy 
Protocol-specific therapy may be discontinued for any of the following reasons: 
• Progressive disease 
• Unacceptable toxicity as described in the protocol:  
o Please refer to Section 4.4 (Safety Considerations).  Adverse 
events that do not respond to clinical management of the reaction 
will result in discontinuation from the study. 
o Please refer to Section 5.6 (Subject Discontinuation) for a list of 
severe adverse reactions warranting discontinuation from the study.  
• Patient election to discontinue therapy (for any reason) 
• Physician’s judgment 
5.6 
Subject Discontinuation 
Those patients who display significant reactions (e.g., anaphylactic reaction 
immediately after vaccine administration) or serious toxicities will be discontinued 
from the study as discussed below and as determined by the PI and/or Sponsor.  
They will be followed by the study investigator until resolution of the adverse event. 
Inoculations will be immediately halted if any serious adverse reactions occur to 
include: death, life-threatening adverse drug experience (e.g.,, severe 
anaphylactic reaction immediately after vaccine administration), inpatient 
hospitalization or prolongation of existing hospitalization, a persistent or significant 
disability/incapacity, a congenital anomaly/birth defect, or other important medical 
events that may not result in death, be life-threatening, or require hospitalization 


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but which, when based upon appropriate judgment of the PI, be determined to 
jeopardize the patient or require medical or surgical intervention to prevent an 
outcome listed above.  Any death or grade 4 adverse drug experience found to be 
directly related to the experimental vaccine will result in suspension of patient 
enrollment to the study. 
Patients may withdraw from the study at any time and for any reason.  A patient 
may be asked to withdraw from the study by the PI if they are not compliant with 
the timing of the inoculation series, observation period, or return visits to monitor 
for study-associated toxicities.   
Additionally, if the PI determines that it is no longer safe for a patient to continue  
in the trial for any reason, he/she may be withdrawn.   
Because it is not known whether these inoculations might harm an unborn child, 
patients who are pregnant, plan on becoming pregnant, or who are breast-feeding 
will not be enrolled into the study.  Women of childbearing potential will take a urine 
pregnancy test before starting this study and prior to each inoculation; a positive 
test result will terminate the patient’s participation in the study.  Patients will be 
counseled to avoid becoming pregnant while participating in this study, and that in 
order to prevent pregnancy they should either have no sexual relations or use a 
reliable type of birth control.  They will be counseled that with the exception of 
hysterectomy, bilateral oophorectomy, or tubal ligation, birth control methods are 
not totally effective in preventing pregnancy, and that the only ways to completely 
avoid the risk of the vaccine or immunoadjuvant alone to an unborn baby are (1) 
avoid becoming pregnant, or (2) do not receive these inoculations.  Patients will 
be counseled to avoid becoming pregnant for at least six months after receiving 
the inoculations, as pregnancy within this time after inoculation administration may 
be a risk to an unborn baby.   
If a patient is discontinued from the study for an adverse event or pregnancy, 
they will continue to be followed for resolution of adverse event and clinical 
recurrences unless the patient withdraws consent for further study evaluation.  
The reason for any premature discontinuation of a patient from the study will be 
recorded on the appropriate Case Report Form.  
5.7 
Study Discontinuation 
The IND sponsor, the Data Safety Monitoring Board, and the overall study Principal 
Investigator have the right to suspend or terminate this study entirely or at a 
specific site at any time, and for any reason.  Reasons for suspending or 
terminating the study may include, but are not limited to, the following: 
• The incidence or severity of AEs in this or other studies indicates a 
potential health hazard to subjects 
• Subject enrollment is unsatisfactory 


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• Data recording are inaccurate or incomplete 
• Study protocol not followed 
5.8 
Data Collection 
Basic demographic, pathologic, and relevant clinical information will be gathered 
on each patient and entered into an Electronic Data Capture (EDC) database.  The 
URL for the EDC system is https://cancerinsight.eclinicalhosting.com/OpenClinica. 
The EDC system is accessible from any major web browser.  User name and 
password will be generated by the Data Manager of Cancer Insight, LLC and sent 
via email to appropriate site personnel prior to enrolling their first patient. Clinical 
nurses at the site will be provided with Source Document Flow Sheets to capture 
data at enrollment and for each study visit.  Although some data fields on the Flow 
Sheets will not be entered into EDC, they must be captured on the Flow Sheets 
for monitoring purposes.  
All data must be submitted within 72 hours of the data collection visit.  Data entry 
will begin with the patient’s randomization with enrollment date to be date of signed 
informed consent.  
Edit checks will fire in real time as data is being entered in the EDC system to 
ensure quality data is provided.  In addition to edit checks, queries will be 
generated as Discrepancy Notes from the Data Manager and Monitors.  Sites will 
have ten business days to update a query.  It is the responsibility of the coordinator 
at each site to ensure that data has been submitted.  Cancer Insight personnel or 
the Sponsor’s representative will perform an audit of the site-specific Flow Sheets 
and will match them against source documents to ensure the quality of data 
coming to the Data Manager in the EDC per the internal monitoring plan. 
The database, hosted by OpenClinica, resides in a SAS 70 Type II data center and 
meets ISO 17799 standards for information security. The EDC system is HIPAA 
and 21 CFR Part 11 compliant with robust audit logs, controlled user access, and 
electronic signature/password management.  In addition to the site user, the Data 
Manager, the Monitors, the study PI and Sponsor/Sponsor representative have 
access to the database.  Each user is assigned a role, which grants limited access 
and functionalities dependent upon that specific role.  
5.9 
Statistical Methods 
5.9.1  Rationale for study design 
Based on our prior phase I/IIa clinical trials in stage IV melanoma, the resected 
stage IV patients enjoyed the greatest benefit from vaccination.  As such, stage III 
and IV (resected) patients are the target population for this adjuvant trial. 
Prevention of recurrence is the primary objective.   


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5.9.2 Sample size determination 
Assuming a baseline recurrence rate of 60% (corresponding to a DFS of 40%) at 
two years based on published data in this mixed group of stage III (predominantly 
IIIB and IIIC) and stage IV (resected) melanoma patients, a sample of size of 120 
will have 80% power to detect a statistical difference between treatment 
proportions controlling the type I error at alpha = 0.05 (two-sided). 
A blinded evaluation of the first 75 patients enrolled discovered an early recurrence 
rate of 12% prior to completion of primary vaccine series. To compensate for this 
early recurrence rate and preserve the power of the trial, the enrollment will be 
extended by at least an additional 20 patients, now totaling at least 140. 
After randomization of the 120th patient, the randomization will transition to 2:1 
(TLPO phase I/IIa:TLPLDC) for an additional 60 patients.  The TLPO vaccine group 
(n=40) will be assessed primarily for tolerability of the TLPO vaccine based on 
CTCAE v4.03 graded local and systemic toxicities.  Given that the patients will be 
randomized between the TLPO phase I/IIa and TLPLDC, there will be an 
opportunity to directly compare the toxicity profile of these two vaccines. 
Also, the 20 additional TLPLDC vaccine patients will be assessed with the original 
120 randomized patients to account for the 140 patients mentioned above 
(Appendix B). 
5.9.3 Data analysis 
For the TLPLDC phase IIb, the intention-to-treat (ITT) population will include all 
subjects who were randomly assigned.  ITT analysis will be performed with each 
randomized patient evaluated in the treatment arm to which they are randomized 
regardless of actual treatment received.  The per-protocol (PP) population is a 
subset of the ITT population. 
Subjects may be excluded from the PP population for the following reasons and 
others as determined prior to database lock:  violations of eligibility criteria, 
development of second malignancies, early recurrences occurring prior to 
completion of the first four inoculations, receiving alternative disease-directed 
therapy without evidence of recurrence, or major deviation from prescribed 
vaccination schedule (to include boosters). 
5.9.3.1 
Study patient characteristics 
Demographic characteristics of all patients in the ITT and PP populations will be 
summarized including age, race, disease histology, tumor location, tumor depth, 
ulceration, number of mitoses, nodal status (number and micro- vs macro-
metastasis), AJCC clinical stage, location and number of distant metastases (if 
present), surgery extent, and other disease-directed therapies to include 
chemotherapy, radiation therapy, and biologic therapy.  Continuous variables will 
be summarized using the number of patients, mean, standard deviation, median, 


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minimum, and maximum; and categorical variables will be summarized using the 
frequency count and the percentage of patients in each category.  Differences 
between treatment groups will be determined using a chi-square test for 
categorical variables and a t-test for continuous variables. 
5.9.3.2 
Primary efficacy analysis 
For the TLPLDC phase IIb, the primary efficacy endpoint of the trial is 24 month 
DFS when analyzed as a proportion using Pearson chi-square test. The primary 
efficacy analysis will be performed on the ITT and the PP population as equally 
important analyses given the high early recurrence rate. 
The primary efficacy analysis will also be conducted by site for the ITT and PP 
populations in order to explore the possibility of a treatment by investigative site 
interaction.  Investigative sites that individually represent fewer than five subjects 
will be combined for this exploratory analysis; geographic region will be substituted 
if the average number of subjects per site is fewer than ten. 
The primary analysis will be conducted once the 120th patient enrolled completes 
24 months of follow-up.   
An interim analysis of DFS and compared between treatment groups will be 
performed 6 months after the 120th patient is enrolled.  No early stopping rule is 
included. 
5.9.3.3 
Secondary efficacy analysis 
For the TLPLDC phase IIb, DFS and overall survival (OS) will be compared 
between the vaccinated (TLPLDC and TLPO) and control groups at 36 months 
from the 120th patient enrolled.   Both DFS and OS will be measured from time of 
randomization to date of recurrence and death, respectively.  DFS and OS will be 
determined using the Kaplan-Meier method and differences between treatment 
groups will be assessed using the log-rank test.   
In addition, Cox proportional hazards regression models will be fit to determine the 
association between DFS and OS and treatment group. The secondary efficacy 
analysis will be performed on the ITT and the PP population as equally important 
analyses given the high early recurrence rate. 
Due to changes in standard of practice, specifically the FDA approval of checkpoint 
inhibitors (CPI) in the adjuvant setting, the protocol was modified to allow for the 
inclusion of patients who had demonstrated initial tolerability to CPI.  These 
patients were randomized and received concurrent CPI + either TLPLDC or 
placebo.  Therefore, as a part of the overall secondary efficacy analysis, an 
additional analysis will specifically assess DFS and OS in this subgroup of patients 
who received concurrent therapy. 


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During the course of the trial, it has been noted that the production method of the 
TLPLDC vaccine may impact clinical outcome.  As such, as a part of the overall 
secondary efficacy analysis, an analysis will be performed for DFS and OS 
comparing patients who received TLPLDC produced from 120 mL of blood vs 
TLPLDC produced after G-CSF.  Additionally, these groups will be compared to 
the placebo group in three arm analyses as well as exploratory two arm analyses 
by grouping like arms.  
5.9.3.4 
Safety analysis 
For both trials, the incidence of treatment-emergent AEs, SAEs, severe AEs, AEs 
related to study drug, (defined as not, unlikely, possibly, probably, or definitely 
related to study drug), and AEs which lead to study discontinuation will be 
summarized by treatment group.  Differences between treatment groups will be 
determined using Fisher’s exact tests.  
Time to AE onset will be estimated using the Kaplan-Meier method and treatment 
differences will be determined using the log-rank test. In addition, duration of any 
AE with >10% incidence, total number of AEs, SAEs, and related AEs will be 
summarized and treatment group differences assessed using a t-test or Wilcoxon 
rank sum test, depending on the distribution of the data.  
5.9.3.5 
Immunologic analysis 
For both trials, immunologic responses will be determined using phenotypic and 
functional assays (ex vivo).  
Expression percentages will be compared using a chi-square test while continuous 
outcomes will be compared using a paired t-test (within treatment group) and a 
two-sample t-test (between treatment groups).  For the ex vivo response evaluated 
by the phenotypic and functional assays, the number of peptide-specific CTL will 
be determined at multiple time points (see Sections 5.4.4, 5.4.5, and 5.4.6) during 
the trial.  
Changes from pre-vaccination levels to each time point will be compared, and 
differences within treatment groups will be evaluated using a paired t-test while 
between treatment group differences will be evaluated using two-sample t-tests. 
In a series of exploratory analyses, the different immunologic response indicators 
(in vivo and ex vivo) at different time points will be correlated with clinical outcome 
in order to assess for any predictors of outcome. Additional exploratory outcomes 
will compare clinical and immunological outcomes based on the dose of vaccine 
(TLPLDC and TLPO) and number of inoculations received. 
5.9.3.6 
Open Label Study 
At the time of recurrence, all patients will be offered the opportunity to participate 
in the open label portion of the study regardless of their randomization.  All patients 


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will have new vaccine produced, and they will receive inoculations at 0, 1, 2, 3, 6, 
and 9 months.  They will receive the vaccinations in the background of their SoC 
therapies initiated to treat their recurrence.  These patients will be followed for 
safety and tumor response in combination with their SoC therapies and assessed 
per iRECIST and RECIST of their SoC follow-up scans. 
5.9.4 Withdrawal 
Subjects may withdraw or be discontinued from the study as discussed in Section 
5.6.  Subjects who do withdraw or are discontinued will be included in the efficacy 
analyses unless a subject withdraws consent to participate.  In the instance of a 
subject withdrawing consent, any data collected will be excluded from analysis. 
5.9.5 Missing data 
Every reasonable attempt will be made to recover any missing data.  If any data 
remains missing that data point will be excluded from analysis for that patient.  A 
sensitivity analysis using the tipping point approach is the imputation strategy for 
missing data as described in the SAP, Section 4.7. 
6.0 ADVERSE EVENTS  
Reporting of adverse events (AEs) will be performed in accordance with the Data 
Safety Monitoring Plan (DSMP) (Appendix H), this protocol, and federal 
regulations. 
6.1 
Adverse Event and Reporting Definitions 
With the occurrence of an AE, the first concern will be for the safety of the subject. 
An adverse event (AE) is any untoward medical occurrence in a subject 
participating in an investigational study or protocol regardless of causality 
assessment.  
An adverse event can be an unfavorable and unintended sign (including an 
abnormal laboratory finding), symptom, syndrome, or disease associated with or 
occurring during the use of an investigational product whether or not considered 
related to the investigational product. 
Serious Determination: 
Serious adverse events (SAEs) are AEs occurring at any dose which meet one 
or more of the following serious criteria: 
• Results in death (i.e., the AE caused or led to death) 
• Is life-threatening (i.e. the AE placed the subject at immediate risk of 
death; it does not apply to an AE which hypothetically might have caused 
the death if it were more severe) 


P h ase IIb T L P L D C & I/II a T L P O v acci ne tri al  
 Versi o n 2. 4/ 1 5  Fe br u ary 2 0 2 1  
C o nfi de nti al  
4 4  
•  R e q uir e s or pr ol o n g s i n p ati e nt h o s pit ali z ati o n (i. e., t h e A E r e q uir e d at l e a st 
a 2 4 -h o ur h o s pit ali z ati o n. (I n p ati e nt h o s pit ali z ati o n or pr ol o n g e d a 
h o s pit ali z ati o n b e y o n d t h e e x p e ct e d l e n gt h of st a y; h o s pit ali z ati o n s f or 
el e cti v e m e di c al/ s ur gi c al pr o c e d ur e s, s c h e d ul e d tr e at m e nt s, or r o uti n e 
c h e c k -u p s ar e n ot S A E s b y t hi s crit eri o n.)  
•  I s di s a bli n g (i. e., t h e A E r e s ult e d i n a s u b st a nti al di sr u pti o n of t h e s u bj e ct’ s 
a bilit y t o c arr y o ut n or m al lif e f u n cti o n s)  
•  I s a c o n g e nit al a n o m al y/ birt h d ef e ct (i. e., a n a d v er s e o ut c o m e i n a c hil d or 
f et u s of a s u bj e ct e x p o s e d t o t h e st u d y dr u g pri or t o c o n c e pti o n or d uri n g 
pr e g n a n c y)  
•  D o e s n ot m e et a n y of t h e a b o v e s eri o u s crit eri a b ut m a y j e o p ar di z e t h e 
s u bj e ct a n d m a y r e q uir e m e di c al or s ur gi c al i nt er v e nt i o n t o pr e v e nt o n e of 
t h e o ut c o m e s li st e d a b o v e 
E x p e ct e d/  U n e x p e ct e d  D et er mi n ati o n:  
 
E x p e ct e d  A E s  ar e  t h o s e A E s  t h at ar e  li st e d or  c h ar a ct eri z e d  i n t h e c urr e nt  
I n v e sti g at or Br o c h ur e  (I B) a n d/ or  ar e  li st e d i n t h e i nf or m e d c o n s e nt  d o c u m e nt.  
U n e x p e ct e d  A E s  ar e  t h o s e n ot  li st e d i n t h e c urr e nt  I B a n d/ or  i nf or m e d c o n s e nt  
d o c u m e nt . T hi s  i n cl u d e s A E s  f or w hi c h  t h e s p e cifi cit y  or  s e v erit y  i s n ot  c o n si st e nt  
wit h  t h e d e s cri pti o n  i n t h e I B. F or  e x a m pl e,  u n d er  t hi s d efi niti o n,  h e p ati c  n e cr o si s  
w o ul d  b e  u n e x p e ct e d  if t h e I n v e sti g at or Br o c h ur e  o nl y  r ef err e d t o el e v at e d  h e p ati c  
e n z y m e s  or  h e p atiti s.  
 
St u d y  Dr u g  R el ati o n s hi p:  
 
T h e  Pri n ci p al  I n v e sti g at or will  d et er mi n e  if t h er e i s a  p o s si bilit y  t h at t h e st u d y  dr u g  
c a u s e d  t h e a d v er s e  e v e nt.  I n t h e r e p ort t o t h e s p o n s or,  t h e i n v e sti g at or m u st  
i n cl u d e a n  a s s e s s m e nt  of  c a u s alit y,  i. e., w h et h er  t h er e i s a  p o s si bilit y  t h at t h e dr u g  
c a u s e d  t h e e v e nt.  S o m e  fa ct or s  t h at m a y  b e  c o n si d er e d  i n d e t er mi ni n g if a n  A E  
s h o ul d  b e  r e g ar d e d a s  p o s si bl y  r el at e d t o t h e u s e  of  t h e dr u g  c o m p o n e nt s  ar e  a s  
f oll o w s: 
 T h er e i s a cli ni c all y pl a u si bl e ti m e s e q u e n c e b et w e e n o n s et of t h e A E a n d 
a d mi ni str ati o n of dr u g c o m p o n e nt s;  
 T h er e i s a bi ol o gi c all y pl a u si bl e m e c h a ni s m f or dr u g c o m p o n e nt s t o c a u s e 
or c o ntri b ut e t o t h e A E;  
T h e A E c a n n ot b e attri b ut e d s ol el y t o c o n c urr e nt/ u n d erl yi n g ill n e s s, ot h er 
dr u g s, or pr o c e d ur e s.  
6 . 2  
R e p orti n g of A d v er s e E v e nt s A s s o ci at e d wit h T hi s S t u d y 
I n v e sti g at or s ar e  r e q uir e d t o r e p ort t o t h e s p o n s or  a n d/ or  s p o n s or’ s  
d e si g n at e d  r e pr e s e nt ati v e: 
 


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• Any adverse event that may be regarded as caused by, or possibly 
caused by, the study drug  
• All serious adverse events  
• All unanticipated problems involving risks to human subjects or others 
(UPIRSOs), including any adverse event that falls into this category 
• All serious and unexpected adverse events for which there is a reasonable 
possibility that the adverse event was caused by the study drug (SUSARs) 
 
Investigators are required to report to the Institutional Review Board (IRB):  
 
• All unanticipated problems involving risks to human subjects or others 
(UPIRSOs) 
• All adverse events that falls into the category of UPIRSO.  An adverse 
event is a UPIRSO when it is unexpected, possibly related to participation 
in the research, and places the subject or others at a greater risk of harm 
than was previously known.  
• All serious and unexpected adverse events for which there is a reasonable 
possibility that the adverse event was caused by the study drug (SUSARs) 
 
If an event is reportable under 21 CFR §312.32, i.e., if the event is a SUSAR, the 
IND Sponsor or sponsor’s representative will report these to the FDA per the 
requirements of 21 CFR § 312.32.   
 
All events meeting the outlined criteria will be reported for the time period 
beginning with any amount of exposure to vaccine components through the 
protocol-defined follow-up period. 
6.3 
Reporting Requirements for IND Sponsor 
6.3.1  MedWatch 3500A reporting guidelines 
For sponsors of IND Studies, there are additional reporting requirements for the 
FDA in accordance with the regulations set forth in 21 CFR § 312.32.  If an event 
is reportable under the U.S. Code of Federal Regulations, the sponsor will submit 
a Form FDA 3500A (MedWatch), as required by regulation.  Form FDA 3500A 
Supplement provides general instructions for filling out the Form FDA 3500A, and 
should be used as guidance. Events meeting the following criteria need to be 
submitted to the FDA as expedited IND Safety Reports according to the following 
guidance and timelines: 
7 Calendar-Day Telephone or Fax Report: The Sponsor is required to notify the 
FDA of any unexpected fatal or life-threatening suspected adverse reactions as 
soon as possible, but no later than 7 calendar days after the sponsor’s initial receipt 
of the information. 
15 Calendar-Day Written Report: The Sponsor is required to notify the FDA and 
all participating investigators, in a written IND Safety Report, of any suspected 


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adverse reaction that is both serious and unexpected.  The sponsor must report 
an adverse event as a suspected adverse reaction only if there is evidence to 
suggest a causal relationship between the drug and the adverse event.  
• Written IND Safety Reports should include an Analysis of Similar 
Events in accordance with regulation 21 CFR § 312.32. All safety 
reports previously filed with the IND concerning similar events 
should be analyzed and the significance of the new report in light of 
the previous, similar reports commented on. 
• Written IND safety reports with Analysis of Similar Events are to be 
submitted to the FDA and all participating investigators within 15 
calendar days of first learning of the event. The FDA prefers these 
reports on a MedWatch 3500A Form but alternative formats are 
acceptable (e.g., summary letter). 
Follow-up information: 
If any information necessary to evaluate the suspected unexpected serious 
adverse reaction is missing or unknown, or if new information becomes available, 
that information should be submitted as a Follow Up IND Safety Report as soon 
as the information is available, but no later than 15 calendar days after the sponsor 
receives the information.   
Study Drug Relationship Determination for Reporting Purposes: 
 
For reporting purposes, suspected adverse reactions are those where there is a 
reasonable possibility that the study drug caused the adverse event. For reporting 
purposes, “reasonable possibility” means there is evidence to suggest a causal 
relationship between the drug and the adverse event.  See 21 CFR 312.32.   
7.0 INVESTIGATOR REQUIREMENTS 
7.1  
Study Initiation 
Before the start of this study, the following documents must be on file with the 
Sponsor or Sponsor’s representative: 
• Original U.S. Form FDA 1572 for each Principal Investigator (for all 
studies conducted under U.S. Investigational New Drug [IND] 
regulations), signed by the Principal Investigator. The names of any 
sub-investigators must appear on this form, as well as all other 
information required by the form. 
• Current curriculum vitae of the Principal Investigator 
• Written documentation of IRB approval of protocol and informed 
consent document 


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• A copy of the IRB-approved informed consent document 
• A signed Clinical Research Agreement 
• A signed investigator’s agreement (Appendix K) 
• Laboratory Documentation 
• All other essential documents required to be obtained under the 
CFR and ICH GCP, as applicable to this study  
7.2  
Study Completion 
The following materials are requested by the sponsor when the study is 
considered complete or terminated: 
• A summary, prepared by the Principal Investigator, of the study, 
and/or a study manuscript, and/or a study abstract submitted to 
scientific conferences. 
7.3  
Informed Consent 
Informed consent form templates will be provided, and the final IRB-approved 
document must be provided to the Sponsor or Sponsor’s representative for 
regulatory purposes. The informed consent document must be signed by the 
subject or the subject’s legally authorized representative before his or her 
participation in the study. The case history for each subject shall document that 
informed consent was obtained prior to participation in the study.  
Copies of the informed consent form documents must be provided to the subject 
or the subject's legally authorized representative. If applicable, it will be provided 
in a certified translation of the local language. 
Signed consent forms must remain in each subject’s study file and must be 
available for verification by study monitors at any time. 
7.4 
Institutional Review Board or Ethics Committee Approval 
This protocol, the informed consent document, and relevant supporting information 
must be submitted to the IRB for review and must be approved before the study is 
initiated. The study will be conducted in accordance with U.S. FDA, applicable 
national and local health authorities, and IRB requirements. 
The Principal Investigator is responsible for keeping the IRB apprised of the 
progress of the study and of any changes made to the protocol as deemed 
appropriate, but in any case the IRB must be updated at least once a year. 
Investigators are required to promptly notify their respective IRB of events as 
described in this protocol in Sections 6.0, 6.1, and 6.2.  Some IRBs may have other 


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specific adverse event requirements to which investigators are expected to adhere. 
Investigators must immediately forward to their IRB any written safety report or 
update provided by the Sponsor (e.g., IND safety report, IB, safety amendments 
and updates, etc.). 
Ethics and Regulatory Considerations 
The protocol will be reviewed and approved by the IRB or Independent Ethics 
Committee (IEC) of each participating center prior to study initiation. A list of 
IRB/IEC members should be obtained by the investigator and provided to the 
sponsor and sponsor representative. Any documents that the IRB/IEC may need 
to fulfill its responsibilities, such as protocol amendments and/or information from 
the sponsor will be submitted to the IRB/IEC. The IRB/IEC’s written unconditional 
approval of the study protocol and the informed consent form will be in the 
possession of the PI and the Sponsor before the study is initiated. The IRB/IEC’s 
unconditional approval statement will be transmitted by the investigator or 
designee to the sponsor/sponsor representative prior to shipment of study drug 
supplies to the site. This approval document must refer to the study by exact 
protocol title and protocol version number/date and must identify the documents 
reviewed and the date of review. 
Protocol modifications or changes may not be initiated without prior written 
IRB/IEC approval except when necessary to eliminate immediate hazard to the 
patients. Such modifications will be submitted to the IRB/IEC and written 
verification 
that 
the 
modification 
was 
submitted 
and 
IRB/IEC 
acknowledgement/approval should be obtained and transmitted to the 
Sponsor/Investigator or Sponsor/Investigator’s representative. The IRB/IEC must 
be informed by the principal investigator of any changes or revisions of informed 
consent forms or other documents originally submitted for review; adverse events 
and other reportable events as described in this protocol in Sections 6.0, 6.1, and 
6.2; any new information that may affect adversely the safety of the patients or the 
conduct of the study; annual updates and/or request for re-approval; and when the 
study has been completed. 
7.5 
Study Monitoring Requirements 
Site visits may be conducted by authorized Sponsor representative or CRO 
representatives to inspect study data, subjects’ medical records, and CRFs in 
accordance with current U.S. GCPs and the respective local and national 
government regulations and guidelines (if applicable). 
The Principal Investigator will permit authorized representatives of Sponsor, CRO, 
the FDA, and the respective national or local health authorities to inspect facilities 
and records relevant to this study. 


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7.6 
Data Safety Monitoring Plan 
A DSMP (Appendix H) describing the CRO internal monitoring plan includes data 
safety and integrity and site initiation/QA monitoring, as well as external monitoring 
plan, the Data Safety Monitoring Board (DSMB) charter and responsibilities. 
7.7  
Study Medication Accountability 
The study drug will be provided by the Sponsor.  The recipient will acknowledge 
receipt of the drug by returning the INDRR-1 form indicating shipment content and 
condition. Damaged supplies will be replaced. Accurate records of all study drug 
dispensed from and returned to the study site should be recorded by using the 
institution’s drug inventory log or the NCI drug accountability log.  All partially used 
or empty containers should be disposed of at the study site according to 
institutional standard operating procedure. Return unopened, expired, or unused 
study drug with the Inventory of Returned Clinical Material form as directed by the 
Sponsor. 
7.8  
Disclosure of Data  
Subject medical information obtained by this study is confidential, and disclosure 
to third parties other than those noted above is prohibited. Upon the subject’s 
permission, medical information may be given to his or her personal physician or 
other appropriate medical personnel responsible for his or her welfare. Data 
generated by this study must be available for inspection upon request by 
representatives of the FDA, national and local health authorities, the Sponsor, and 
the IRB for each study site, if appropriate. 
7.9  
Retention of Records  
U.S. FDA regulations (21 CFR §312.62[c]) require that records and documents 
pertaining to the conduct of this study and the distribution of investigational drug, 
including CRFs, consent forms, laboratory test results, and medication inventory 
records, must be retained by the Principal Investigator for two years after 
marketing application approval. If no application is filed, these records must be 
kept two years after the investigation is discontinued and the FDA and the 
applicable national and local health authorities are notified. The Sponsor will notify 
the Principal Investigator of these events.  
For studies conducted outside the United States under a U.S. IND, the Principal 
Investigator must comply with U.S. FDA IND regulations and with the record 
retention policies of the relevant national and local health authorities. 
7.10  Publications  
The investigator must agree to send to the Sponsor or Sponsor’s representative, 
for review all manuscripts, abstracts and presentations using data from this study 
prior to their submission. The Sponsor or Sponsor’s representative reserves the 


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right to delete from such materials any part or parts deemed to be confidential or 
proprietary. 
7.11  Changes to Protocol  
The protocol may not be modified without written approval of the Sponsor or 
Sponsor’s representative, or the Study Director. All changes to the protocol must 
be submitted to the FDA, the overseeing IRB, and local IRB/IEC. Additionally, 
changes must be approved by overseeing IRB prior to their implementation. 
Documentation of IRB/IEC approval must be sent to the Sponsor or Sponsor’s 
representative, and the Study Director immediately upon receipt.  Any changes 
and modifications to the informed consent language must be reviewed and 
approved by the Sponsor or Sponsor’s representative, and the Study Director prior 
to submission to the local IRB. 


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Appendix A 
 
Trial Schema 
 
Study Population 
Adjuvant Melanoma 
patients, n=140, 
randomized 2:1 
(vaccine:control) 
• Stage III and IV 
(resected) 
• NED after standard of 
care (SOC) 
• Sufficient tumor (1 mg/1 
cm3) available to 
produce vaccine 
• Stratified by Stage 
• Crossover – open label 
treatment of recurred 
controls 
0 
1 
2 
  Endpoints 
  1o            2o 
DFS     DFS/OS 
Tumor Lysate 
Loaded YCWP 
+ DC 
+ DC 
Empty YCWP 
Dosing  
by Month 
6 
A prospective, randomized, blinded, placebo-controlled, phase IIb 
trial of an autologous tumor lysate (TL)+yeast cell wall particles 
(YCWP)+dendritic cells (DC) vaccine vs unloaded YCWP+DC in 
stage III and stage IV (resected) melanoma to prevent recurrence 
12 
18 
24 
36 


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Appendix B 
 
Combined Trial Schema 
 
 
CombinedTrialsSchema
Melanoma
StageIII
StageIV
(resected)
R
A
N
D
O
M
I
Z
A
T
I
O
N
Controls
(n=40)
TLPLDC
(n=80)
TLPLDC
(n=20)
TLPO
PhaseI/IIa
(n=40)
TLPLDCPhaseIIb Trial
n=120
CombinedTrials
n=60
1
2
1
2
Primary
Analysis
(n=120)
TLPLDC
(n=100)
Per
Treatment
Analysis
(n=140)


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Appendix C 
 
Vaccination Timeline 
 
1
2
6
12
18
24
36
0
Vax1
Vax2
Vax3
Booster
x1
Booster
x2
Booster
x3
RO
R1
R2
R3
RB1
RB2
RB3
Before 
1st
vaccine
Serum & 
Blood 
sample
Serum & 
Blood 
sample
Serum & 
Blood 
sample
Serum & 
Blood 
sample
Serum & 
Blood 
sample
Serum & 
Blood 
sample
Serum & 
Blood 
sample
MONTHS 
Phase IIb Clinical Schedule
Consent #1
Tumor Acquisition
Consent #2
Blood for DC
Randomization
First vaccination
3wks-3mos from
Completion of SoC
DFS
DFS/OS
Primary
Endpoint
Secondary
Endpoint
Vax2-3 given +1wk
Booster 1-3 given +2wk


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Appendix C 
 
Open Label Vaccination Timeline 
 
 


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Appendix D 
 
NCI Common Terminology Criteria for Adverse Events, v4.03 
 
obtained from http://ctep.cancer.gov/forms/CTCAEv4.03.pdf 
http://evs.nci.nih.gov/ftp1/CTCAE/CTCAE_4.03_2010-06 14_QuickReference_8.5x11.pdf 
 
 


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Appendix E 
 
ECOG Performance Status Criteria 
 
Score 
Description 
0 
Fully active, able to carry on all pre-disease performance 
without restriction 
1 
Restricted in physically strenuous activity but ambulatory 
and able to carry out work of a light or sedentary nature, 
e.g., light housework, office work 
2 
Ambulatory and capable of all self-care but unable to carry 
out any work activities.  Up and about more than 50% of 
waking hours. 
3 
Capable of only limited self-care, confined to bed or chair 
more than 50% of waking hours 
4 
Completely disabled.  Cannot carry on any self-care.  Totally 
confined to bed or chair. 


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Appendix F 
 
1. Quick Reference for the Response Evaluation Criteria in Solid Tumors 
(RECIST Criteria) 
 
 
A. Indications for use of RECIST Criteria 
- 
The RECIST criteria is useful in all trials where objective response is the 
primary study endpoint, as well as in trials where assessment of stable 
disease, tumor progression or time to progression analyses are 
undertaken, since all of these outcome measures are based on an 
assessment of anatomical tumor burden and its change on the study. 
 
B. Identification of Lesions 
I. 
Measurable disease 
- 
All patients must have measurable disease to be enrolled into this trial. 
- 
All baseline imaging for determining these target lesions must be 
completed no more than 4 weeks prior to beginning treatment 
- 
Measurable disease includes: 
▪ Tumors that can be accurately measured along the 
longest diameter  
▪ Tumors must be >10mm by CT (CT must use 5mm cuts or 
smaller) 
▪ If tumor lesions are measured on clinical exam, the lesion 
should be superficial, >10mm in diameter when measured 
by a caliber 
▪ Tumors must be >20mm by chest xray.  
 
II. 
Lymph nodes must be measured along its shortest axis  
▪ Nodes must be >15mm by CT scan (CT must use 5mm 
cuts or smaller) 
 
III. 
Target lesions 
- 
Target lesions must be determined by each primary investigator 
- 
Baseline imaging must be completed no more than 4 weeks before 
initiation of trial 
- 
All target lesions must be measurable tumors that are easily monitored 
with reproducible repeated measurements. 
▪ Maximum of 5 target lesions 
▪ Maximum of 2 lesions per organ   
▪ A sum of all the diameters for each target lesion is 
recorded and monitored throughout the study forming a 
baseline sum of diameters. 
- 
All other lesions will be deemed non-target lesions.  They may be 
followed throughout the trial, but will not be incorporated into the 
baseline sum of diameters.  These non-target tumors must be recorded 


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separately. 
IV. 
Imaging Modalities 
- 
All lesions must be measurable per iRECIST 1.1 Criteria.  It is preferred 
to monitor each lesion with an imaging study if able.   
▪ CT scans must be completed as IV contrast and a 5mm 
cuts. 
▪ MRI may also be used. 
▪ FDG-PET useful in monitoring lesions.  It should be 
combined with a CT scan for complete evaluation of 
tumors. 
C. Evaluation of Tumor Response  
- 
Tumor response of target lesions include: 
▪ Complete response (CR): Disappearance of all target 
lesions.  Any pathologic lymph nodes must be <10mm 
(target or non-target nodes).  
▪ Partial Response (PR): At least a 30% decrease in the 
sum of diameters for target lesions in comparison to 
baseline sum of diameters. 
▪ Progressive Disease (PD): At least a 20% increase in the 
sum of diameters for target lesions.  In addition to this 
increase, the sum of diameters must increase by at least 
5mm.  The development of new lesions is also evidence 
of PD. 
▪ Stable Disease (SD): Tumors neither fit criteria for PR or 
PD. 
- 
Tumor response of non-target lesions include: 
▪ CR - Disappearance of all target lesions.  Any pathologic 
lymph nodes must be <10mm (target or non-target nodes). 
▪ Non-CR/Non-PD - Persistence of one or more non-target 
lesions and maintenance of tumor marker level above the 
normal limits. 
▪ PD – Unequivocal progression of existing non-target 
tumors, or the appearance of new lesions.  
- 
The primary investigator at each site will verify the presence of changes 
in target/non-target lesions.   
- 
Target lesions which shrink, and are deemed too small to measure 
should be given a default measurement of 5mm.   
- 
If target lesions have completely disappeared is should be measured as 
0mm.   
- 
Lymph nodes may regress to <10mm but because they are still 
measurable, the sum of diameters may never equal 0mm in the setting 
of CR when they are included as target lesions. 
- 
Please see table below regarding the Best overall response 
 
 
Target lesions 
Non-Target lesions 
New Lesions 
Overall response 


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CR 
CR 
No 
CR 
CR 
Incomplete 
response/SD 
No 
PR 
PR 
Non-PD 
No 
PR 
SD 
Non-PD 
No 
SD 
PD 
Any 
Yes or No 
PD 
Any 
PD 
Yes or No 
PD 
Any 
Any 
Yes 
PD 
 
 
* Please refer to the complete New response evaluation criteria in solid tumors: 
Revised RECIST guidelines – version 1.1 for further details74.  
 
2. Quick Reference for iRECIST Criteria 
 
A. 
Indications: 
- The guidelines continue to recommend use of RECIST 1.1 to define 
whether tumor lesions are measurable or non-measurable and the 
principles used to determine objective tumor response are largely 
unchanged from RECIST 1.1. The concept change for iRECIST is the 
resetting the bar if RECIST 1.1 progression is followed at the next 
assessment by tumor shrinkage. 
-iRECIST defines unconfirmed progress (iUPD) on the basis of RECIST 1.1 
principles; however, iUPD requires confirmation, which is done on the basis 
of observing either a further increase in size (or in the number of new 
lesions) in the lesion category in which progression was first identified in (ie, 
target or non-target disease), or progression (defined by RECIST 1.1) in 
lesion categories that had not previously met RECIST 1.1 progression 
criteria. However, if progression is not confirmed, but instead tumor 
shrinkage occurs (compared with baseline), which meets the criteria of iCR, 
iPR, or iSD, then the bar is reset so that iUPD needs to occur again 
(compared with nadir values) and then be confirmed (by further growth) at 
the next assessment for iCPD to be assigned. If no change in tumor size or 
extent from iUPD occurs, then the time point response would again be 
iUPD. This approach allows atypical responses, such as delayed responses 
that occur after pseudoprogression, to be identified, further understood, and 
better characterized. 
-However, many aspects of new lesion assessment are unique to iRECIST. 
If a new lesion is identified (thus meeting the criteria for iUPD) and the 
patient is clinically stable, treatment should be continued. New lesions 
should be assessed and categorized as measurable or non-measurable 
using RECIST 1.1 principles.  
-The algorithm for patients with no previous iUPD is identical to RECIST 
1.1. For patients with iUPD at the last timepoint response, the next timepoint 


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response is dependent on the status of all lesions, including target, non-
target, new lesion target, and new lesion non-target; on whether any 
increase in size has occurred (either a further increase in size or a sufficient 
increase to assign a new iUPD if the criteria were not previously met); or 
the appearance of additional new lesions. 
 
B. 
Identification of tumors, refer to RECIST 1.1 
 
C. 
Assignment of time point response using iRECIST26 
 
Time 
point 
response 
with no 
previous 
iUPD in 
any 
category 
Time 
point 
response 
with 
previous 
iUPD in 
any 
category * 
Target lesions: iCR; non-target 
lesions: iCR; new lesions: no  
iCR 
iCR 
Target lesions: iCR; non-target 
lesions: non-iCR/non-iUPD; 
new lesions: no  
iPR 
iPR 
Target lesions: iPR; non-target 
lesions: non-iCR/non-iUPD; 
new lesions: no  
iPR 
iPR 
Target lesions: iSD; non-target 
lesions: non-iCR/non-iUPD; 
new lesions: no  
iSD 
iSD 
Target lesions: iUPD with no 
change, or with a decrease 
from last timepoint; non-target 
lesions: iUPD with no change, 
or decrease from last 
timepoint; new lesions: yes  
Not 
Applicable 
New lesions 
confirm iCPD 
if new 
lesions were 
previously 
identified and 
they have 
increased in 
size (≥5 mm 
in sum of 
measures for 
new lesion 
target or any 
increase for 
new lesion 
non-target) 
or number; if 
no change is 
seen in new 
lesions (size 
or number) 
from last 
timepoint, 
assignment 


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remains 
iUPD  
Target lesions: iSD, iPR, iCR; 
non-target lesions: iUPD; new 
lesions: no  
iUPD 
Remains 
iUPD unless 
iCPD is 
confirmed on 
the basis of a 
further 
increase in 
the size of 
non-target 
disease 
(does not 
need to meet 
RECIST 1.1 
criteria for 
unequivocal 
progression)  
Target lesions: iUPD; non-
target lesions: non-iCR/non-
iUPD, or iCR; new lesions: no  
 
iUPD 
Remains 
iUPD unless 
iCPD is 
confirmed on 
the basis of a 
further 
increase in 
sum of 
measures ≥5 
mm; 
otherwise, 
assignment 
remains 
iUPD  
Target lesions: iUPD; non-
target lesions: iUPD; new 
lesions: no  
 
iUPD 
Remains 
iUPD unless 
iCPD is 
confirmed 
based on a 
further 
increase in 
previously 
identified 
target lesion 
iUPD in sum 
of measures 
≥5 mm or 
non-target 
lesion iUPD 
(previous 
assessment 
need not 
have shown 
unequivocal 
progression)  


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Target lesions: iUPD; non-
target lesions: iUPD; new 
lesions: yes  
 
iUPD 
Remains 
iUPD unless 
iCPD is 
confirmed on 
the basis of a 
further 
increase in 
previously 
identified 
target lesion 
iUPD sum of 
measures ≥5 
mm, 
previously 
identified 
non-target 
lesion iUPD 
(does not 
need to be 
unequivocal), 
or an 
increase in 
the size or 
number of 
new lesions 
previously 
identified  
Target lesions: non-iUPD or 
progression; non-target 
lesions: non-iUPD or 
progression; new lesions: yes  
iUPD 
Remains 
iUPD unless 
iCPD is 
confirmed on 
the basis of 
an increase 
in the size or 
number of 
new lesions 
previously 
identified  
Target lesions, non-target lesions, and new lesions defined 
according to RECIST 1.1 principles; if no pseudoprogression 
occurs, RECIST 1.1 and iRECIST categories for complete 
response, partial response, and stable disease would be the 
same. *Previously identified in assessment immediately 
before this timepoint. “i” indicates immune responses 
assigned using iRECIST. iCR=complete response. 
iPR=partial response. iSD=stable disease. 
iUPD=unconfirmed progression. non-iCR/non-iUPD=criteria 
for neither CR nor PD have been met. iCPD=confirmed 
progression. RECIST=Response Evaluation Criteria in Solid 
Tumors. 
 
 
* Please refer to the complete Guidelines for iRECIST.75   
 
 


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Appendix G 
 
Schedule of Events 
 


Phase IIb TLPLDC & TLPO vaccine trial 
 
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70 
 
Open Label Protocol Schedule of Events 
 
 
CLINIC VISIT 
 
PROCEDURES 
 
TOTAL 
Visit # 
Time Req   
(hr) 
 
Time Point 
 
Purpose 
 
Screening 
 
History 
Nursing 
Visit 
 
UPT 
Blood 
Draws 
Inoculation 
# of Shots 
This Visit 
Time for 
Inoc + 
Follow-Ups 
1 
2 
Pre-treatment 
Consent/Tissue Procurement 
Gather Baseline Data 
x 
x 
x 
x 
 
 
0 
N/A 
2 
2 
Baseline 
Consent/Randomization & Treatment 
Neupogen Injection 
Gather Current Baseline Data 
x 
x 
x 
x 
 
X 
(Neupogen 
injection) 
1 
N/A 
3 
1 
Baseline 
Draw Blood to Obtain Dendritic Cells 
 
x 
x 
 
x 
 
0 
N/A 
 
START PRE-INOCULATION BLOOD DRAWS AND ADMINISTRATION OF INOCULATIONS 
4 
1 
Starting Time 
Point 
Draw blood for Immunologic Sample R0 
Administer Inoculation #1 
 
x 
x 
x 
x 
x 
2 
 
1 hr  
5 
1 
Week 4 
+ 1 week 
Draw blood to record immunologic levels pre-inoculation #2  
Administer Inoculation #2 
 
x 
x 
x 
x 
x 
2 
 
1 hr  
6 
1 
Week 8 
+ 1 week 
Draw blood to record immunologic levels pre-inoculation #3 
Administer Inoculation #3 
 
x 
x 
x 
x 
x 
2 
 
1 hr  
7 
1 
Week 12 
+ 1 week 
Draw blood to record immunologic levels pre-inoculation #4 
Administer Inoculation #4 
 
x 
x 
x 
x 
x 
2 
1 hr 
 
START PRE-BOOSTER BLOOD DRAWS AND ADMINISTRATION OF BOOSTERS 
8 
1 
Month 6 from 
Starting Time 
Point (+2 
weeks) 
Draw blood to record immunologic levels pre-booster #1 
Administer Booster #1 
 
x 
x 
x 
x 
x 
2 
 
1 hr 
9 
1 
Month 9 
(+ 2 weeks) 
Draw blood to record immunologic levels pre-booster #2 
Administer Booster #2 
 
x 
x 
x 
x 
x 
2 
1 hr 
10 
1 
Month 12 
(+2 weeks) 
Draw Blood for final Immunologic Sample 
Final Study Assessment 
 
x 
x 
x 
x 
 
0 
1 hr 
 


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Appendix H 
 
Data Safety Monitoring Plan 
 
 
This is a free-standing document 


Phase IIb TLPLDC & TLPO vaccine trial 
 
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Appendix I 
 
Adverse Events Reporting Algorithm 
 
 
 


Phase IIb TLPLDC & TLPO vaccine trial 
 
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Appendix J 
 
MedWatch FDA Form 3500A Link 
 
 
Form FDA 3500A Link 
 
 
 
 


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74
Appendix K 
 
PROTOCOL TITLE:  
A prospective, randomized, blinded, placebo-
controlled, phase IIb trial of an autologous tumor 
lysate (TL) + yeast cell wall particles (YCWP) + 
dendritic cells (DC) vaccine vs unloaded YCWP + 
DC and embedded phase I/IIa trial with tumor 
lysate particle only (TLPO) vaccine in stage III and 
stage IV (resected) melanoma to prevent 
recurrence 
 
PROTOCOL NUMBER: 
 
20141932 
 
STUDY DRUG:  
Autologous tumor lysate (TL) + yeast cell wall   
particles (YCWP) + dendritic cells (DC) 
 
and 
 
Autologous tumor lysate (TL) + yeast cell wall 
particles only (PO) 
 
PRINCIPAL INVESTIGATOR:  
Mark B. Faries, MD, FACS 
 
PROGRAM DIRECTOR: 
 
George E. Peoples, MD, FACS 
 
PROTOCOL VERSION/DATE:  
2.4/ 15 February 2021 
 
INVESTIGATOR’S AGREEMENT / INVESTIGATOR’S SIGNATURE PAGE 
I have read the protocol described above. I have fully discussed the objectives of this 
trial and the content of this protocol with the sponsor or sponsor’s representative. I 
understand that the information in this protocol is confidential and should not be 
disclosed, other than to those directly involved in the execution or the ethical review of 
the trial, without written authorization from Cancer Insight. It is, however, permissible to 
provide information to a patient in order to obtain consent.  I agree to conduct this trial 
according to the protocol and to comply with its requirements, subject to ethical and 
safety considerations and guidelines, and to conduct the trial in accordance with all 
applicable regulations, and guidelines as stated in the protocol and other information 
supplied to me. I understand that the sponsor may decide to suspend or prematurely 
terminate the trial at any time, for whatever reason. Such a decision will be 
communicated to me in writing. Conversely, should I decide to withdraw from execution 
of the trial, I will communicate my intention immediately, in writing to the Sponsor. 
 
 
Signed:  
 
 
 
 
 Date:   
 
 
 
                                          
 
Investigator’s Name and Address:  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

SAP_001.pdf: 
Page 1 of 15 
Statistical Analysis Plan for Protocol 20141932 
Confidential 
 
 
STATISTICAL ANALYSIS PLAN v2.5 
(replaces SAP v2.4, 15 December 2020) 
 
 
 
SPONSOR:   
 
Elios Therapeutics, Inc. 
PROTOCOL TITLE:   
A prospective, randomized, blinded, placebo-controlled, phase IIb 
trial of an autologous tumor lysate (TL) + yeast cell wall particles 
(YCWP) + dendritic cells (DC) vaccine vs unloaded YCWP + DC 
and embedded phase I/IIa trial with tumor lysate particle only 
(TLPO) vaccine in stage III and stage IV (resected) melanoma to 
prevent recurrence. 
PROTOCOL NUMBER: 
20141932 
PROTOCOL VERSION: 
2.4 
PROTOCOL DATE: 
15 February 2021 
SAP VERSION:  
v2.5 
SAP DATE: 
15 February 2021 
 
 


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Table of Contents 
1 
Rationale for study design ....................................................................................................... 4 
2 
Study Endpoints ....................................................................................................................... 4 
2.1 
Primary Endpoint ............................................................................................................. 4 
2.2 
Secondary Endpoints ........................................................................................................ 4 
3 
Trial Overview ......................................................................................................................... 4 
3.1 
Trial Design and Conduct ................................................................................................ 5 
3.2 
Patient Population ............................................................................................................ 6 
3.3 
Treatment Arms................................................................................................................ 6 
3.4 
Sample Size Justification ................................................................................................. 6 
3.5 
Randomization and Unblinding ....................................................................................... 7 
3.5.1 
Randomization Methodology.................................................................................... 7 
3.5.2 
Unblinding procedures .............................................................................................. 7 
3.6 
Schedule of Events ........................................................................................................... 8 
4 
Statistical Methodology .......................................................................................................... 8 
4.1 
General Considerations .................................................................................................... 8 
4.2 
Study Populations for Analysis ........................................................................................ 8 
4.2.1 
Intention to Treat Population .................................................................................... 8 
4.2.2 
Per Protocol Population ............................................................................................ 9 
4.2.3 
Safety Population ...................................................................................................... 9 
4.3 
Subject Disposition .......................................................................................................... 9 
4.4 
Demographics and other Baseline Characteristics ........................................................... 9 
4.4.1 
Demographics ........................................................................................................... 9 
4.4.2 
Baseline Characteristics and Disease History ........................................................... 9 
4.4.3 
Medical history ....................................................................................................... 10 
4.4.4 
Urine Pregnancy Test .............................................................................................. 10 
4.5 
Protocol Deviations ........................................................................................................ 10 
4.6 
Extent of Exposure ......................................................................................................... 10 
4.7 
Methods for Handling Missing Data .............................................................................. 10 
4.8 
Analysis of Primary Endpoint ........................................................................................ 11 
4.9 
Analysis of Secondary Endpoints .................................................................................. 12 
4.9.1 
Efficacy ................................................................................................................... 12 


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Statistical Analysis Plan for Protocol 20141932 
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4.9.2 
Safety ...................................................................................................................... 13 
4.10 Subgroup Analyses ......................................................................................................... 13 
4.10.1 Stage ........................................................................................................................ 13 
4.10.2 Other subgroup analyses ......................................................................................... 13 
4.11 Interim Analysis and Early Stopping Guidelines ........................................................... 14 
4.12 Safety Evaluation ........................................................................................................... 14 
4.12.1 Adverse Events ....................................................................................................... 14 
4.12.2 Deaths ..................................................................................................................... 15 
4.12.3 Laboratory Data ...................................................................................................... 15 
4.12.4 Vital Signs ............................................................................................................... 15 
4.12.5 Physical Examination.............................................................................................. 15 
4.12.6 Concomitant Medications ....................................................................................... 15 
 


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Statistical Analysis Plan for Protocol 20141932 
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1 
Rationale for study design 
Based on our prior phase I/IIa clinical trials in stage IV melanoma, the resected stage IV 
patients enjoyed the greatest benefit from vaccination. As such, stage III and IV (resected) 
patients are the target population for this adjuvant trial. Prevention of recurrence is the 
primary objective. 
2 
Study Endpoints 
2.1 
Primary Endpoint 
• To determine 24-month disease-free survival (DFS) in vaccinated vs. control patients in 
the intention to treat (ITT) and per protocol (PP) population as defined in section 4.2.2. 
2.2 
Secondary Endpoints 
• To determine DFS and overall survival (OS) at 36 months in vaccinated vs. control 
patients in the ITT and PP population. 
• To determine DFS and OS at 36 months in vaccinated vs. control patients who received 
concurrent CPI. 
• To determine DFS and OS at 36 months comparing patients who received TLPLDC 
produced from 120 mL of blood vs TLPLDC produced after G-CSF.   
• To determine DFS and OS at 24 months in TLPO vaccinated vs. TLPLDC (randomized 
and pooled) vs. control patients 
• To determine DFS and OS at 36 months in TLPO vaccinated vs. TLPLDC (randomized 
and pooled) vs. control patients 
• To determine DFS and OS at 36 months in TLPO vaccinated vs. TLPLDC (randomized 
and pooled) vs. control patients comparing patients who received concurrent CPI. 
• To determine DFS and OS at 36 months in TLPO vaccinated vs. TLPLDC (randomized 
and pooled) vs. control patients comparing patients who received TLPLDC produced 
from 120 mL of blood vs TLPLDC produced after G-CSF.   
• To document the safety of the vaccine, assessed by CTCAEv4.03. 
3 
Trial Overview 
The primary aim of the present trial is to evaluate the efficacy of TLPLDC as an adjuvant 
treatment for resected stage III and IV melanoma. The primary endpoint of this trial will be DFS 
at 24 months in the ITT and PP population. Secondary endpoints include DFS and OS at 36 
months in the ITT and PP populations and safety of the vaccine. Endpoints will also be analyzed 
to compare vaccine manufacturing method and/or presence of adjuvant check point inhibitors. 
Approximately 120 patients will be randomized in a 2:1 ratio to receive vaccine or placebo. The 
embedded phase I/IIa trial of TLPO vs. TLPLDC will randomize patients 2:1 to TLPO or 
TLPLDC. Given that the patients will be randomized between the TLPO phase I/IIa and 
TLPLDC, there will be an opportunity to directly compare the toxicity profile of these two 
vaccines. Also, the 20 additional TLPLDC vaccine patients will be assessed with the original 120 
randomized patients to account for the 140 patients mentioned below in section 3.4.  


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3.1 
Trial Design and Conduct 
Stage III and Stage IV (resected) melanoma patients will be identified prior to definitive surgery 
and screened for inclusion/exclusion criteria. Eligible patients will be counseled and consented 
for tissue procurement. Enrolled patients will have their disease surgically resected and a portion 
(1mg minimum) of their melanoma sterilely frozen in provided freezing vials and storage tubes. 
This tissue will be shipped in liquid nitrogen shippers through FedEx to our central facility in 
Greenville, SC and stored frozen until vaccine preparation. If patients cannot be rendered 
disease-free, they will be considered screen failures for this study. If melanoma is being resected 
from multiple locations (primary and nodes, two different metastatic sites), then samples of each 
would be preferred but not mandatory.  
As indicated by SoC per the National Comprehensive Cancer Network (NCCN) guidelines and 
determined by the treating team, if a patient is to receive systemic therapy (chemotherapy or 
IFNα and/or radiation therapy), then the vaccinations will not begin until SoC therapy is 
completed. Although, patients on adjuvant check point inhibitors (CPI) may start the vaccine 
trial after showing tolerance of the CPI for at least 3 months given that there may a synergistic 
benefit and no added toxicity when adding the vaccine to CPI. Once SoC therapies are complete 
(or demonstrating 3 months of tolerance on CPI) and the patient deemed clinically disease-free, 
they will be consented for treatment and randomized. Once consented, patients will receive a 
single injection of Neupogen (G-CSF) 300 μg (or its equivalent) SQ 24-48 hrs prior to having 70 
mL of blood collected and sent to our central facility for DC isolation and preparation. Patients 
who cannot tolerate Neupogen, or its equivalent or refuse it, will have 120 mL of blood drawn 
and sent. Additional blood may be drawn if additional vaccine doses need to be made or re-made 
for any reason. Vaccines will be prepared by producing TL through freeze/thaw cycling and then 
loaded into pre-prepared YCWP. The TL-loaded YCWP will be introduced to the DC for 
phagocytosis thus creating the TLPLDC vaccine which will be frozen in single dose vials. Each 
vial will contain 1-1.5 x 106 TLPLDC and will be labeled with the patient’s unique study 
number. The TLPO is created in a similar manner with the freeze/thaw cycling, loaded into pre-
prepared YCWP, and then capped with silicate for stability. The TLPO vaccine is then frozen in 
single dose vials of 1.0 x 108 TLPO and will be labeled with the patient’s unique study number.  
Based on their randomization, autologous TLPLDC (active vaccine) or unloaded YCWP + 
autologous DC (control) or autologous TLPO will be sent back to the site in a blinded fashion. 
Upon randomization of the 120th patient the randomization will transition to 2:1 (TLPO phase 
I/IIa : TLPLDC). Regardless of assigned group, the site will receive 6 single dose vials to be 
injected intradermal monthly x 3 followed by boosters at 6, 12, and 18 months in the same lymph 
node draining area (preferably the anterior thigh). Patients must begin vaccinations between 3 
weeks and 3 months from completion of SoC (or demonstrating 3 months of tolerance on CPI). 
Frozen tumor will be maintained for active vaccines for all patients to include the control 
patients. The latter will be offered their active vaccine at time of recurrence in a crossover 
fashion. Additionally, control patients who do not recur will be offered active vaccine at the 
completion of the trial. 


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Safety data will be collected on local and systemic toxicities and graded and reported per the 
Common Terminology Criteria for Adverse Events (CTCAE) v4.03. Disease-free status will be 
monitored per SoC as outlined by NCCN. Suspected recurrences will be documented with biopsy 
and pathologic confirmation. Time to recurrence will be based on date of randomization to time 
of confirmed recurrence. Recurrent patients will be offered participation in the open label portion 
of the study. New active vaccine will be made for all patients, and they will be inoculated at 0, 1, 
2,3, 6, and 9 mos. Patients will be treated per SoC for their recurrence. Safety and tumor 
response will be assessed per RECIST and iRECIST on their SoC follow-up scans. 
3.2 
Patient Population 
Patients with stage III melanoma whose disease is determined to be resectable, and stage IV 
melanoma patients who are rendered disease-free by surgery, will be randomized 2:1 to receive 
TLPLDC or placebo. Following the completion of the phase IIb portion of the study, patients 
with stage III melanoma whose disease is determined to be resectable, and stage IV melanoma 
patients who are rendered disease-free by surgery will be randomized 2:1 to receive TLPO or 
TLPLDC. 
3.3 
Treatment Arms 
There will be two treatment arms for the phase IIb portion of the study evaluating TLPLDC 
versus placebo: 
1) Vaccinated group = autologous tumor cell lysate (TL) + yeast cell wall particles (YCWP) 
+ autologous DC (TLPLDC) 
2) Control group = empty YCWP + autologous DC 
There will be two treatment arms for the phase I/IIa portion of the study evaluating TLPO versus 
TLPLDC: 
1) Vaccinated group = autologous tumor cell lysate + yeast cell wall particles (TLPO) 
2) Vaccinated group = autologous tumor cell lysate (TL) + yeast cell wall particles (YCWP) 
+ autologous DC (TLPLDC) 
3.4 
Sample Size Justification 
Assuming a baseline recurrence rate of 60% (corresponding to a DFS of 40%) at 2 years in this 
mixed group of stage III and stage IV (resected) melanoma patients, a sample size of 
approximately 120 will have 80% power to detect a statistical difference between treatment 
proportions controlling the type I error at α = 0.05 (two-sided). 
A blinded evaluation of the first 75 patients enrolled discovered an early recurrence rate of 12% 
prior to completion of the primary vaccine series. To compensate for this early recurrence rate 
and preserve the power of the trial, enrollment will be extended by at least an additional 20 
patients, now totaling at least 140. 
After randomization of the 120th patient, the randomization will transition to 2:1 (TLPO phase 
I/IIa : TLPLDC) for an additional 60 patients. The TLPO vaccine group (n=40) will be assessed 
primarily for tolerability of the TLPO vaccine based on CTCAE v4.03 graded local and systemic 
toxicities. Given that the patients will be randomized between the TLPO phase I/IIa and 


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TLPLDC, there will be an opportunity to directly compare the toxicity profile of these two 
vaccines. Also, the 20 additional TLPLDC vaccine patients will be assessed with the original 120 
randomized patients to account for the 140 patients mentioned above (see protocol Appendix B, 
Combined Trials Schema). 
3.5 
Randomization and Unblinding 
3.5.1 Randomization Methodology 
Patients will be randomized 2:1 to either the vaccine or placebo. A central, permuted 
block randomization scheme will be used with a selected blocking factor (block size) 
determined based on the proposed allocation ratio and number of subjects. The block size 
will be specified in the randomization plan. An Interactive Web Response System 
(IWRS) will be used for the randomization to either the vaccinated vaccine group or the 
control group.  
Study staff will log into the IWRS using unique credentials and input subject-specific 
screening and baseline information. In accordance with the randomization schedule, the 
IWRS will assign the randomization number to each subject. Each subject must be given 
only the investigational product assigned by IWRS. The study staff will document the 
randomization number in the electronic Case Report Form (eCRF). Subjects are to be 
randomized in the order in which they qualify from the screening phase for inclusion in 
the study. 
Upon randomization of the 120th patient in the TLPLDC phase IIb, the TLPO phase I/IIa 
trial will be initiated and 60 additional patients will be randomized 2:1 (TLPO phase I/IIa 
: TLPLDC). 
3.5.2 Unblinding procedures 
The investigators, study personnel, subjects, medical monitor, and clinical monitors will 
remain blinded throughout the study, unless safety concerns necessitate unblinding.  
If a medical emergency occurs and a decision regarding the subject’s clinical treatment 
requires knowledge of the treatment assignment, the study blind may be broken for the 
specific subject. Unless the medical emergency is deemed to be life-threatening, the 
medical monitor must first be consulted before unblinding. The Investigator would then 
utilize the IWRS for unblinding and the unblinding procedure will be provided in the 
eCRF Completion Guidelines. The date, time, and reason for unblinding must be 
documented in the source documents and on the applicable unblinding form in the IWRS. 
Investigators should note that the occurrence of a SAE should not routinely trigger 
immediate unblinding. If the medical monitor was not notified prior to breaking the blind, 
the investigator must notify the medical monitor of any and all blinds broken within 24 
hours of each occurrence. 
All patients will be offered open label vaccine at first sign of recurrence. Non-recurrent 
control patients will be given the option of receiving active vaccine upon their 
completion of the trial.  


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3.6 
Schedule of Events 
The Schedule of Events is defined in Appendix G of the study protocol. 
4 
Statistical Methodology 
4.1 
General Considerations 
All statistical analyses will be performed using SAS® Version 9.3 or higher, unless otherwise 
noted. The analyses of efficacy and safety endpoints will be based on the study populations for 
analysis defined in section 4.2. Sensitivity analyses for primary and key secondary efficacy 
endpoints may be performed to test the robustness of the treatment effects observed, including 
comparisons of vaccine manufacturing method and/or presence of adjuvant check point 
inhibitors. Two-sided p-values will be presented for all statistical testing, unless otherwise noted. 
Continuous variables will be summarized using descriptive statistics, which includes n (number 
of non-missing observations), the mean, median, standard deviation, minimum, and maximum. 
The minimum and maximum will be reported using the same precision as the original 
measurement. The mean, median, other selected percentiles, and standard deviation will be 
reported to one decimal place more than the precision of the original measurement. For 
categorical variables, descriptive statistics include frequency and percentage will be presented. 
Rates of 24- and 36-month DFS will be compared using the Pearson chi-square test.  
For time to event analyses, the Kaplan-Meier method will be used to estimate the quartiles (25th 
percentile, median and 75th percentile) of time to event variable for each treatment group, along 
with 95% confidence intervals (Kaplan and Meier, 1958). Kaplan-Meier event rates with 2-sided 
95% confidence intervals at year 1, year 2 and year 3 will also be estimated. The hazard ratio and 
its 2-sided 95% CI will be estimated using Cox proportional hazards model (Cox, 1972) with 
treatment group as the independent variable and stratified by the randomization stratification 
factors. 
In general, all summary tables will be supported by a relevant patient data listing including all 
patients enrolled into the study. The listings will include all data collected and will be sorted by 
patient identifier and actual visit date, as applicable, unless otherwise noted. Baseline is defined 
as the last non-missing valid value prior to first study treatment. Change from baseline, if 
presented will be the post-treatment value minus baseline value, unless otherwise noted. 
Concomitant medications will be coded by the WHO Drug Dictionary and adverse events will be 
coded using the Medical Dictionary for Regulatory Activities (MedDRA). The dictionary 
versions will be documented in the Data Management Plan for the study. 
4.2 
Study Populations for Analysis 
4.2.1 Intention to Treat Population 
Analyses of the ITT population will include all patients who undergo randomization. ITT 
analysis will be performed with each randomized patient evaluated in the treatment arm 
to which they are randomized regardless of actual treatment received. 
 


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4.2.2 Per Protocol Population 
The per protocol (PP) population is a subset of the ITT population. Per the protocol, 
subjects may be excluded from the PP population for the following reasons and other as 
determined prior to database lock: violations of eligibility criteria, development of second 
malignancies, early recurrences occurring prior to completion of the first four 
inoculations, receiving alternate disease-directed therapy without evidence of recurrence, 
or major deviation from prescribed vaccination schedule (to include boosters). For the 
purposes of the primary statistical analysis, the PP population will include all subjects 
except those who experience early recurrence prior to completion of the fourth 
inoculation timepoint at 6 months. 
4.2.3 Safety Population 
The Safety Population will consist of all patients who received at least 1 dose of 
investigational product. In the event of study drug administration errors, analyses of the 
Safety Population will be performed according to the treatment the subject actually 
received. 
4.3 
Subject Disposition 
All patients enrolled into the study (sign informed consent) will be accounted for in the Patient 
Disposition Table.  Disposition information, such as the following will be summarized by the 
number and percentages of patients in each treatment group and overall: 
• Patients who comprise the ITT, safety and PP populations  
• Patients who discontinue treatment and the reason for treatment discontinuation 
• Patients who discontinue the study and reason for discontinuation 
• Patients who die during the study  
4.4 
Demographics and other Baseline Characteristics 
4.4.1 Demographics 
Demographics such as gender, age, race and ethnicity will be summarized descriptively 
by treatment group and overall for the ITT population. Demographic characteristics of all 
patients in the ITT and PP populations will be summarized including age, race, disease 
histology, tumor location, tumor depth, ulceration, number of mitoses, nodal status 
(number and micro- vs macrometastasis), AJCC clinical stage, location and number of 
distant metastases (if present), surgery extent, and other disease-directed therapies to 
include chemotherapy, radiation therapy, and biologic therapy. 
4.4.2 Baseline Characteristics 
Baseline characteristics such as the following will be summarized descriptively by 
treatment group and overall for the ITT population: 
• Age 
• Race 
• Disease histology 
• Tumor location 
• Tumor depth 


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• Ulceration 
• Number of mitoses 
• Nodal status (number and micro- vs macro-metastasis) 
• AJCC clinical stage 
• Location and number of distant metastases (if present) 
• Surgery extent 
• Other disease-directed therapies to include chemotherapy, radiation therapy, and 
biologic therapy 
4.4.3 Medical history 
Medical History will be coded using the MedDRA dictionary terms and will be 
summarized descriptively by SOC and PT by treatment group and overall for the ITT 
population. 
4.4.4 Urine Pregnancy Test 
Data on urine pregnancy test (UPT) will not be summarized but will be provided as a 
listing. 
4.5 
Protocol Deviations 
Protocol deviations (major and minor) will be defined prospectively prior to database lock by a 
review committee or medical monitor who is blinded to treatment assignment. Major protocol 
deviations as well as those deviations that could possibly affect the interpretation of the primary 
endpoint (subset of major deviations) will be tabulated by treatment group and overall. All 
deviations will be presented in listings. 
4.6 
Extent of Exposure 
The number of months on study, number of injections received, and cumulative investigational 
product exposure will be summarized using descriptive statistics by treatment group and overall 
for the ITT population.   
4.7 
Methods for Handling Missing Data 
Missing data for the binary landmark endpoint will be handled by a tipping point analysis 
including the following steps: 
1. The data are filled in m times to generate m complete data sets. 
2. The m complete data sets are analyzed by using standard procedures. 
3. The results from the m complete data sets are combined for the inference. 
4. Repeat step #1 to generate multiple imputed data sets, with a specified shift parameter 
that adjusts the imputed values for observations in the treatment group (not the placebo 
group). 
5. Repeat step 2 for the imputed data sets with shift parameter applied. 
6. Repeat step 3 to obtain the p-value to see if the p-value is still<=0.05. 
7. Repeat steps 4-6 with more stringent shift parameter applied until the p-value >0.05. 
 


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Censoring rules will be applied to the estimation of the distribution of the time-to-event 
endpoints. 
4.8 
Analysis of Primary Endpoint 
The primary endpoint of this study is to determine 24-month disease-free survival (DFS) in 
vaccinated vs control patients analyzed as a proportion. The primary efficacy analysis will be 
performed on the ITT and the PP population as equally important analyses given the high early 
recurrence rate. The Bonferroni method will be used to control the overall type 1 error rate, and 
the study will be considered successful if analysis in either analysis population (ITT or PP) if the 
two-sided p-value is less than or equal to 0.025. All stratified analyses will utilize the 
stratification factors as entered at the time of randomization. Analyses may also be performed 
using the subgroups listed in Section 4.10. 
The primary efficacy analysis will also be conducted by site for the ITT and PP populations in 
order to explore the possibility of a treatment by investigative site interaction. Investigative sites 
that individually represent fewer than five subjects will be combined for this exploratory 
analysis; geographic region will be substituted if the average number of subjects per site is fewer 
than ten. 
The primary analysis will be conducted 24 months after the 120th patient is enrolled. 
DFS will be analyzed both as a proportion and time-to-event outcome. Rates of 24- and 36-
month DFS will be compared using the Pearson chi-square test. The overall type 1 error rate of 
2.5% will be used for rejection of the null hypothesis in favor of the alternative hypothesis that 
autologous TLPLDC vaccine will have a different 24-month DFS event rate over that of 
unloaded YCWP + autologous DC in the ITT and PP populations. The study will be considered 
successful if the two-sided p-value is less than or equal to 0.025 in either analysis population 
(ITT or PP). 
In the time-to-event analysis, DFS is defined as the time interval (months, calculated as 
days/30.4) from the randomization date to the date of first observation of disease recurrence (i.e., 
first documented evidence per radiographic analysis or per the investigator via confirmation 
procedure of biopsy or cytology) or death from any cause, whichever comes first.  
Subjects last known to be alive, who have not experienced recurrence of disease, are censored at 
their last assessment date (using standard of care (SoC) assessment) for disease recurrence 
defined above, or at the primary analysis data cut-off date, whichever comes first. Subjects who 
had first disease recurrence before randomization or have no radiographic post-baseline tumor 
assessment, will be censored at their randomization date. 
The Hazard ratio of vaccine compared with control and its 2-sided 95% confidence interval will 
be estimated using a Cox proportional hazards model with treatment groups as the independent 
variable and adjusted for the randomization stratification factors. 
Kaplan-Meier curves will be used to assess DFS for each treatment group. A log-rank test 
stratified by the randomization stratification factors will be used to compare the treatment groups 
in the ITT and PP populations. Any subgroup analyses will be displayed; any p-values for 
individual strata will be regarded as descriptive statistics. 


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To explore the possibility of a treatment by investigative site interaction, the primary analysis 
will also be conducted by site for the ITT and PP populations. Sites that have enrolled large 
numbers of subjects relative to other trial centers will be identified to see if the results obtained 
at those investigative sites are influential. Investigative sites that individually represent fewer 
than 10 subjects will be combined for this exploratory analysis; geographic region will be 
substituted if the average number of subjects per site is fewer than 5. 
4.9 
Analysis of Secondary Endpoints 
4.9.1 Efficacy 
• To determine DFS and OS at 36 months in vaccinated vs. control patients  
• To determine DFS and OS at 36 months in vaccinated vs. control patients who 
received concurrent CPI. 
• To determine DFS and OS at 36 months comparing patients who received 
TLPLDC produced from 120 mL of blood vs TLPLDC produced after G-CSF.   
• To determine DFS and OS at 24 months in TLPO vaccinated vs. TLPLDC 
(randomized and pooled) vs. control patients 
• To determine DFS and OS at 36 months in TLPO vaccinated vs. TLPLDC 
(randomized and pooled) vs. control patients 
• To determine DFS and OS at 36 months in TLPO vaccinated vs. TLPLDC 
(randomized and pooled) vs. control patients comparing patients who received 
concurrent CPI. 
• To determine DFS and OS at 36 months in TLPO vaccinated vs. TLPLDC 
(randomized and pooled) vs. control patients comparing patients who received 
TLPLDC produced from 120 mL of blood vs TLPLDC produced after G-CSF.   
DFS at 36 months will be analyzed using the same methodology as the primary endpoint, both as 
a proportion and time-to-event outcome.   Overall Survival (OS) at 36 months will be calculated 
as the duration of survival from randomization until the date of death from any cause. If the date 
of death is not available, OS will be censored at the date the patient was last known to be alive.  
OS will be summarized by treatment group using median, Q1 and Q3 from the KM estimate of 
survival.  The KM survival curve will be provided as a figure. 
Due to changes in standard of practice, specifically the FDA approval of checkpoint inhibitors 
(CPI) in the adjuvant setting, the clinical protocol was modified to allow for the inclusion of 
patients who had demonstrated initial tolerability to CPI.  These patients were randomized and 
received concurrent CPI + either TLPLDC or placebo.  Therefore, an additional secondary analysis 
will be performed specifically to assess DFS and OS in this subgroup of patients who received 
concurrent therapy.  This analysis will be conducted using the same methodology as the primary 
endpoint.   
DFS and OS comparing vaccinated patients who received TLPLDC produced from 120 mL of 
blood vs vaccinated patients who received TLPLDC produced after G-CSF to determine if there 
is any difference in clinical outcomes due to the different production methods.  Additionally, each 
vaccinated group with the different production methods will be compared separately to the placebo 
group using the same methodology as the primary endpoint.  Finally, these groups will be 


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compared to the placebo group in three arm analyses as well as exploratory two arm analyses by 
grouping like arms to generate assumptions for phase 3 planning as appropriate. 
Similarly, DFS and OS comparing vaccinated patients who received TLPO in the phase I/IIa 
portion of the study will be compared to patients who were randomized to TLPLDC as well as 
placebo. The comparison to TLPLDC will be first evaluated in patients randomized to TLPLDC 
in the phase I/IIa portion of the study and then in a pooled manner to all patients who received 
TLPLDC in both portions of the study. Following this, patients who received concurrent CPI 
versus no concurrent CPI will be compared. To compare patients who received TLPO to TLPLDC 
produced from 120 mL of blood vs vaccinated patients who received TLPLDC produced after G-
CSF, the following four-arm comparison will be used:  
1) Vaccinated group = autologous tumor cell lysate + yeast cell wall particles (TLPO) 
2) Vaccinated group = autologous tumor cell lysate (TL) + yeast cell wall particles (YCWP) 
+ autologous DC (TLPLDC) produced from 120 mL of blood   
3) Vaccinated group = autologous tumor cell lysate (TL) + yeast cell wall particles (YCWP) 
+ autologous DC (TLPLDC) produced after G-CSF 
4) Control group = empty YCWP + autologous DC 
This four-arm comparison will be conducted similarly to the primary endpoint analysis with 24- 
and 36-month DFS and OS rates being calculated in each arm and KM estimates and figures 
generated. The hazard ratio of TLPO vaccine compared with each of the other three arms and the 
associated 2-sided 95% confidence intervals will be estimated using a Cox proportional hazards 
model with treatment groups as the independent variable and adjusted for the randomization 
stratification factors. TLPO will serve as the referent group to permit the calculation of hazard 
ratios among each of the other three arms.  
4.9.2 Safety 
To determine the safety of the vaccine, assessed by CTCAEv4.03. Safety will be 
determined by adverse events, physical examinations, vital signs, laboratory parameters 
and ECOG performance status (described in section 4.12). 
4.10 
Subgroup Analyses 
4.10.1 Stage 
Subgroup analyses will be conducted by stratifying for stage (III vs. IV). This subgroup 
analysis may include 24-month DFS and OS as well as 36-month DFS and OS. 
4.10.2 Other subgroup analyses 
Subgroup analyses may be conducted for other selected endpoints. These subgroup 
analyses will be descriptive in nature and may include but are not limited to the 
following: 
• Age (18-64 years, ≥65 years old) 
• Age (<60 years, ≥60 years old) 
• Gender (Male, Female) 
• Race 


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• Geographic Region 
In addition, Cox proportional hazards regression models will be fit to determine the 
association between DFS and OS and treatment group. The secondary efficacy analysis 
will be performed on the ITT and PP population as co-primary analyses given the high 
early recurrence rate. 
4.11 
Interim Analysis and Early Stopping Guidelines 
A Data Safety Monitoring Board (DSMB) external to the sponsor will be formed with members 
chosen for their expertise in oncology, melanoma, and statistical methods in clinical trials. 
The primary role of the external DSMB is to protect the safety of the study participants 
throughout the study duration. They will accomplish this by periodically reviewing safety and 
efficacy analysis results in order to assess the risk-benefit profile for subjects enrolled in this 
study. Access to both safety and efficacy data will provide the DSMB a broad perspective with 
which to generate recommendations for appropriate actions. The external individuals serving on 
the DSMB will have access to subjects’ individual treatment assignments. To minimize the 
potential introduction of bias, these individuals will not have direct contact with the study site 
personnel or subjects.  
Selected sponsor staff may serve as liaisons to the external DSMB, but will not be voting 
members, and will not be unblinded to the results.  
The data for review are outlined in the DSMB charter and agreed to in advance by the DSMB 
members. Unblinded reports for review will be generated by an independent CRO that is external 
to the sponsor as well. The external DSMB will convene periodically, as outlined in the DSMB 
Charter, and the start date will depend on subject accrual rates.  
An interim analysis is planned 6 months after the 120th patient is enrolled. No early stopping rule 
is included. There are no plans to stop the study early for efficacy, thus there are no adjustments 
for multiplicity. 
At other interim evaluations, the DSMB will focus on safety, and will review efficacy data only 
to balance the risk-benefit assessment. Any specific guidelines for study stopping for safety 
reasons will be developed by the DSMB and documented in the DSMB Charter. 
4.12 
Safety Evaluation 
4.12.1 Adverse Events 
Treatment emergent adverse events (TEAEs) reported during the study will be listed, 
documenting onset, action taken, change in dosing, severity, relationship to 
Investigational product, severity and outcome.  A treatment emergent adverse event is 
defined as an AE that occurs on or after the date of the first study treatment.  
Verbatim terms on the eCRFs will be linked to preferred term (PT) and system organ 
class (SOC) using the Medical Dictionary for Regulatory Activities (MedDRA).  
A summary of all TEAEs by the number and percentages of subjects who experienced 
any of the following will be provided by treatment group and overall:  


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• Any TEAE, any TEAE with CTCAE severity grade > 3, any TEAEs definitely, 
probably or possibly related to the Investigational Product, any serious TEAEs, any 
AEs leading to dose reductions and drug interruptions or drug withdrawal, and AEs 
leading to study discontinuation. 
• Any fatal TEAEs, including relationship to Investigational Product (Definite, 
Probable, Possible) 
Additionally, the number and percent of unique subjects reporting any TEAE will also be 
summarized by treatment group and overall, SOC and PT, seriousness, maximum 
CTCAE grade, relationship to Investigational Product (Definite, Probable, Possible) and 
AEs leading to study discontinuation or drug withdrawal.  AE summaries will utilize the 
Safety population.  For all AE summaries, if a subject has more than one AE within a PT, 
the subject is counted once in that PT. If a subject has more than one AE within a SOC, 
the subject is similarly counted once in that SOC. 
4.12.2 Deaths 
The number of percent of subjects experiencing death will be summarized by each 
treatment group and overall along with a listing of deaths and the number of days of the 
death since the last injection of Investigational Product.   
4.12.3 Laboratory Data 
Clinical laboratory tests will be evaluated for any clinically significant changes during the 
study period.  All laboratory data (hematology and chemistry) will be listed and reported 
by treatment group and overall in the units recorded on the eCRFs. Shift tables by analyte 
and by out of range flag (high, low, normal) may be presented to facilitate the evaluation 
of change from baseline to each subsequent visit, in the safety population.  A listing of 
the laboratory data will be presented including the clinical significance flag. 
4.12.4 Vital Signs 
Vital signs, including body mass index (BMI), weight (standardized to kg), blood 
pressure (mmHg), heart rate (beats/min) and body temperature (standardized to F), will 
be summarized by treatment group and overall. Observed values will be tabulated at 
baseline, and at each scheduled post-baseline scheduled visit. Change from baseline at 
each scheduled post-baseline time point will also be summarized. A listing of vital signs 
will be provided. 
4.12.5 Physical Examination 
Physical examination findings, including ECOG Performance Score will be summarized 
by treatment group and overall.  Observed values will be tabulated at baseline, and at 
each scheduled post-baseline scheduled visit.   
4.12.6 Concomitant Medications 
Concomitant Medications will be coded per the WHO Drug dictionary.  Concomitant 
Medications will be tabulated by treatment group, Drug Class (pharmacological level, 
ATC3) and Drug Name (chemical substance level, ATC5).  These data will be provided 
in subject data listings along with the verbatim drug term and usage details.