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table 17 study scenario 1

One combination (G) has true DLT probability more toxic than 33% and the three combinations in Zone 2 have the highest IRR among safe combinations. The true range of optimal combinations is indicated in italicized bold type.  
| Zones                                                                           | 1   | 2   | 3   |     |     |     |     |
|---------------------------------------------------------------------------------|-----|-----|-----|-----|-----|-----|-----|
| Combination                                                                     | A   | B   | C   | D   | E   | F   | G   |
| True DLT probabilities                                                          | 5%  | 6%  | 8%  | 22% | 23% | 25% | 45% |
| True IRR                                                                        | 45% | 50% | 50% | 65% | 65% | 65% | 80% |
| % optimal combination recommendation                                            | 6%  | 10% | 10% | 25% | 22% | 20% | 5%  |
| % participant allocation                                                        | 12% | 14% | 13% | 18% | 18% | 17% | 8%  |
| Average trial size: 45 participants (25th , 50th, 75th percentile = 39, 44, 52) |     |     |     |     |     |     |     |  
9.3.3 Scenario 3:
Combinations in Zones 2 and 3 have true DLT probabilities more toxic than 33% and the tw o combinations in Zone 1 have the highest IRR among safe combinations. The true range of optimal combinations is indicated in italicized bold type.
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table 19 study scenario 3

| Zones                                                                                                                                                                                              | 1   | 2   | 3   |     |     |     |     |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----|-----|-----|-----|-----|-----|-----|
| Combination                                                                                                                                                                                        | A   | B   | C   | D   | E   | F   | G   |
| True DLT probabilities                                                                                                                                                                             | 18% | 20% | 25% | 45% | 48% | 54% | 64% |
| True IRR                                                                                                                                                                                           | 65% | 70% | 70% | 80% | 80% | 80% | 90% |
| % optimal combination recommendation                                                                                                                                                               | 19% | 28% | 25% | 1%  | 1%  | 0%  | 0%  |
| % participant allocation                                                                                                                                                                           | 25% | 29% | 29% | 6%  | 6%  | 5%  | 1%  |
| Average trial size: 29 participants (25th , 50th , 75th percentile = 21, 33, 39) 25.2% of simulated trials were stopped for safety The overall toxicity rate was 25.4%. The overall IRR was 70.6%. |     |     |     |     |     |     |     |  
9.3.4 Scenario 4:
All true DLT probabilities are unsafe (i.e. more toxic than 33%).
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table 20 study scenario 4

| Zones                                                                                                                                                                                       | 1   | 2   | 3   |     |     |     |     |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----|-----|-----|-----|-----|-----|-----|
| Combination                                                                                                                                                                                 | A   | B   | C   | D   | E   | F   | G   |
| True DLT probabilities                                                                                                                                                                      | 50% | 55% | 56% | 65% | 66% | 74% | 80% |
| True IRR                                                                                                                                                                                    | 70% | 70% | 70% | 80% | 80% | 80% | 90% |
| % optimal combination recommendation                                                                                                                                                        | 1%  | 1%  | 3%  | 0%  | 0%  | 0%  | 0%  |
| % participant allocation                                                                                                                                                                    | 32% | 28% | 35% | 2%  | 2%  | 1%  | 0%  |
| Average trial size: 7 participants (25th , 50th, 75th percentile = 2, 3, 6) 96% of simulated trials were stopped for safety The overall toxicity rate was 45.6%. The overall IRR was 70.3%. |     |     |     |     |     |     |     |
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9.4 study design part 2

A fixed sample size design w ill be used in part 2. The vaccine regimen is selected based on available data from Part 1, in w hich the recommended combination w as Arm E. For Part 2, all accruals w ill be to Study Arm E2.
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9.5 sample size/accrual

9.5.1 Part 1 (completed) Maximum sample size is based upon acquiring sufficient information to assess the objective of selecting the arm w ith the highest IRR, assuming at least one optimal combination has been found.  
Data from our prior Mel43 trial in the arms not getting GM-CSF 12 indicated a high T cell response (5-fold increase in reactivity, at least 1200 cells per 100,000 CD8, and no overlap of 2 SD) to the 12 peptide vaccine in PBMC in 30 of 60 participants (50%, 95%CI (37%,
63%)). Using the range as an approximate guideline, maximum sample size is established to ensure that if IRR among all seven arms differ by 30%, w ith the smallest expected rate of 50%, then the arm w ith the higher IRR w ould be selected w ith high probability, P=0.90. The initial maximum target accrual of 20 eligible participants per arm is based upon the ranking and selection procedure, BSH 75, w hich allow s selection of the best treatment w orthy of further investigation in single-factor Bernoulli response experiments. How ever, it is not anticipated that all arms w ill fall in the range of optimal combinations, thus, sample size is estimated from the simulations and w ill be determined by the stopping rules below . Although maximum accrual is set at 140, in the simulation results below , on average a total of betw een 7 and 46 participants w ere required to complete the study.  
Accrual is estimated at 2 participants per month at each site (4 per month overall). The maximum accrual to the study is estimated from the simulations to be approximately 52 participants. Adjusting for 10% ineligibility/dropout/w ithdraw al rate, maximum total target accrual for Part 1 is estimated at 58 participants.
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9.5.2 part 2

Immune response w as observed in 6/16 (37.5%, 90%CI [17.8, 60.9%]) from Part 1. For Part 2 target accrual is set at 16 eligible participants. At study conclusion, if w e observe that at least 12/16 (a doubling of the response rate) eligible participants satisfy the definition of immune response then the low er limit of a one-sided 80%CI around the observed immune response rate for Arm E2 w ould be 61.5% w hich w e w ould conclude supports that the immune response rate is likely to be higher for single site injections. If few er responses are observed then additional studies w ill be needed to better understand the impact of site rotation w ith the use of the tetanus helper peptide.  
Accrual is estimated at 2 participants per month. Adjusting for 10% dropout rate, maximum total target accrual for Part 2 is estimated at 18 participants.
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9.6 stopping rules

9.6.1 Part 1 (completed)
The study w ill be stopped according to the follow ing rules:
1. The study w ill stop early for safety if the first three entered participants in Zone 1 experience DLT on combinations in Zone 1 in Stage 1.  
2. If at any point in Stage 2, the set of acceptable combinations is empty, the trial w ill stop for safety.  
3. The study w ill stop if the recommendation is to assign the next participant to a combination that already has 20 participants treated at that combination.
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9.6.2 part 2

Safety w ill be assessed by monitoring the number of participants w ho experience a DLT. In Part 1, 1/16 (6.3%, 95%CI [0.2, 30.2%]) participants treated on Arm E experienced a DLT.  
For Part 2 the upper boundary of a sequential probability ratio test (SPRT) based upon a binomial test of proportions for DLT w ill be used for monitoring to protect against excessive DLT rates. The stopping boundary is for a SPRT contrasting a 5% versus 30% DLT rate, w ith nominal type I and II errors of 5% and 20%, respectively. Safety stopping guidelines for the occurrence of DLTs are detailed in the follow ing table. If a stopping bound is crossed then accrual to the study w ill be suspended until the study PI, co-investigators and the DSMC can review the data, and determine if the study should continue, be amended or be closed to further accrual.  
| Table 21: Incidence of Treatment-Related DLTs  Incidence of treatment related DLTs Number of participants Stopping boundary 2-4 ≥ 2 5-11 ≥ 3 12-18 ≥ 4   |
|----------------------------------------------------------------------------------------------------------------------------------------------------------|
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9.7 analyses 9.7.1 safety

All participants w ho receive any study intervention w ill be monitored for adverse events.  
Adverse events w ill be described and coded based upon the NCI CTCAE v4.03. A DLT is defined as any unexpected adverse event that is possibly, probably or definitely related to treatment and satisfies the criteria in section 7.8. Occurrence of DLTs w ill guide escalation and stopping decisions. At study conclusion frequency, proportion and severity of adverse events, and DLTs by arm w ill be tabulated.
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9.7.2 immunologic response

Subjects are evaluable for immune response if at least one post treatment sample is measurable for response. Peak immunogenicity w ill be evaluated by direct ELIspot assay against the defined nonamer peptides in the peripheral blood. The presence or absence of an immune response w ill be defined by induction of at least a 2-fold increase in IFN-gammasecreting cells over background, and over pre-existing responses, and at least 30 IFNgamma secreting spots per 100,000 CD8 T cells, as described 34. This assessment w ill be performed on blood through w eek 12, although a minimum of 4 w eeks of data w ill be used to guide decisions about the range of optimal dose combinations. IRR is defined as the proportion of participants w here presence (as defined above) of an immune response has been observed. Durable immunogenicity w ill be evaluated w eek 26 by ELIspot assay against the defined nonamer peptides in the peripheral blood. The presence or absence of an immune response at that time w ill be defined by induction of at least a 2-fold increase in IFN-gamma-secreting cells over background, and over pre-existing responses, and at least 30 IFN-gamma secreting spots per 100,000 CD8 T cells, as described 34. This assessment w ill be performed on blood through w eek 26.
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9.7.3 at study conclusion

Frequency and magnitude of adverse events w ill be summarized by arm. Point estimates and confidence intervals w ill be calculated for all dichotomous endpoints. If more than one combination is contained w ithin the range of optimal combinations, estimates and confidence intervals w ill be used to estimate the difference in IRR betw een pairs of combinations among those in the range of optimal combinations.  
Immunogenicity w ill be evaluated by direct ELIspot assay against the defined nonamer peptides in vaccine site biopsies using the above criteria. Disease-free survival is defined as the duration of time from start of treatment to time of recurrence/progression or death from any cause, w hichever occurs first. Participants w ho do not experience an event
(recurrence/progression or death) w ill be censored at date of last contact. Overall survival is defined as the duration of time from start of treatment to time of death from any cause.  
Participants w ho do not experience an event (death) w ill be censored at date of last contact.  
Disease-free survival and overall survival distributions w ill be estimated by the product limit method of Kaplan and Meier.
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10.0 appendices

10.1 Appendix 1: X-page  
| Active Treatment                                   | Follow-up   |     |       |    |    |    |    |    |     |    |     |     |     |
|----------------------------------------------------|-------------|-----|-------|----|----|----|----|----|-----|----|-----|-----|-----|
| Day                                                | Pre         | 1   | 8     | 15 | 22 | 36 | 43 | 57 | 78  | 85 | 183 | 365 | 730 |
| Week                                               | 0           | 1   | 2     | 3  | 5  | 6  | 8  | 11 | 12  | 26 | 52  | 104 |     |
| Vax #                                              | 1           | 2   | 3     | 4  | 5  | 6  |    |    |     |    |     |     |     |
| Informed consent a                                 | X           |     |       |    |    |    |    |    |     |    |     |     |     |
| Class I HLA-typing a                               | X           |     |       |    |    |    |    |    |     |    |     |     |     |
| Pathology review a                                 | X           |     |       |    |    |    |    |    |     |    |     |     |     |
| -HCG c                                            | X           |     |       |    |    |    |    |    |     |    |     |     |     |
| HIV / Hepatitis C d                                | X           |     |       |    |    |    |    |    |     |    |     |     |     |
| HGBA1C b                                           | X           |     |       |    |    |    |    |    |     |    |     |     |     |
| Urinalysis b                                       | X           |     |       |    |    |    |    |    |     |    |     |     |     |
| CBC with differential b                            | X           | X   | X     | X  |    |    |    |    |     |    |     |     |     |
| Comprehensive chemistry b, f                       | X e         | X   | X     | X  |    |    |    |    |     |    |     |     |     |
| LDH b                                              | X           | X   | X     | X  |    |    |    |    |     |    |     |     |     |
| Anti-nuclear antibody / Rf  factor                 | X           | X   | X     | X  |    |    |    |    |     |    |     |     |     |
| Chest x-ray / CT b, g                              | X           | X   | X     |    |    |    |    |    |     |    |     |     |     |
| Abdominal CT b, g                                  | X           |     |       |    |    |    |    |    |     |    |     |     |     |
| Pelvic CT b, g                                     | X           |     |       |    |    |    |    |    |     |    |     |     |     |
| Head MRI / CT b, g                                 | X           |     |       |    |    |    |    |    |     |    |     |     |     |
| History & physical b                               | X           | X   | X     | X  | X  | X  | X  | X  | X   | X  | X   | X   | X   |
| 120cc green top tubes                              | X           |     |       |    |    |    |    |    |     |    |     |     |     |
| 80cc green top tubes                               | X           | X   | X     | X  | X  | X  | X  |    |     |    |     |     |     |
| 20cc red top tubes                                 | X           | X   | X     | X  | X  | X  | X  | X  |     |    |     |     |     |
| Vaccination                                        | X           | X   | X     | X  | X  | X  |    |    |     |    |     |     |     |
| Biopsy normal skin, pts 1-6,  in Part 1            | Xi          | Xi  |       |    |    |    |    |    |     |    |     |     |     |
| Vaccine Site Biopsies                              | X           | X   |       |    |    |    |    |    |     |    |     |     |     |
| Visual                                             | acuity,     | and | color |    |    |    |    |    |     |    |     |     |     |
| acuity examinations                                | X           | X   |       |    |    |    |    |    |     |    |     |     |     |
| Note hair and eye color                            | X           | X   |       |    |    |    |    |    |     |    |     |     |     |
| Note vitiligo                                      | X           | X   | X     | X  |    |    |    |    |     |    |     |     |     |
| Note neurologic function                           | X           | X   | X     | X  |    |    |    |    |     |    |     |     |     |
| Distribute/review participant  toxicity diary      | X           | X   | X     | X  | X  | X  | X  | X  | X h |    |     |     |     |
| Studies & Tests a Any point prior to randomization |             |     |       |    |    |    |    |    |     |    |     |     |     |  
a Any point prior to randomization b Pre-study within 6 weeks of randomization c Within 2 weeks of randomization (f or childbearing women) d Within 6 months of randomization e To include f asting glucose. Fasting blood sugars may be ev aluated 4h or more af ter last eating.  
f Comprehensiv e chemistry panel to include sodium, potassium, creatinine, glucose, calcium, total bilirubin, AST, ALT, and alkaline phosphatase g PET-CT may replace CT of the chest, abdomen, and/or pelv is. New abnormalities of AST, ALT, or LDH lev els, or new nodules on CXR will warrant more complete staging with CT scans of the chest, abdomen and pelv is or PET-CT and with CT or MRI of the brain as appropriate.  
h Rev iew only i Biopsies of normal skin to be done just f or the f irst 6 patients enrolled in Part 1.
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10.2 appendix 2 ajcc staging

| Melanoma TNM Classification T Classification Thickness   | Ulceration Status                                                                                                      |                                                                                                                                             |
|----------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------|
| T1                                                       | ≤ 1.0 mm                                                                                                               | a: without ulceration and level II/III b: with ulceration or mitoses ≥1/mm2                                                                 |
| T2                                                       | 1.01 - 2.0 mm                                                                                                          | a: without ulceration b: with ulceration                                                                                                    |
| T3                                                       | 2.01 - 4.0 mm                                                                                                          | a: without ulceration b: with ulceration                                                                                                    |
| T4                                                       | > 4.0 mm                                                                                                               | a: without ulceration b: with ulceration                                                                                                    |
| N Classification                                         | # of Metastatic Nodes                                                                                                  | Nodal Metastatic Mass                                                                                                                       |
| N1                                                       | 1 node                                                                                                                 | a: micrometastasis* b: macrometastasis† a: micrometastasis* b: macrometastasis† c: in transit met(s)/satellite(s) without  metastatic nodes |
| N2                                                       | 2 - 3 nodes 4 or more metastatic nodes,  or matted nodes, or  in transit met(s)/satellites(s)  with metastatic node(s) |                                                                                                                                             |
| N3 M Classification                                      | Site                                                                                                                   | Serum Lactate Dehydrogenase                                                                                                                 |
| M1a                                                      | Distant skin, subcutaneous or nodal mets                                                                               | Normal                                                                                                                                      |
| M1b                                                      | Lung metastases                                                                                                        | Normal                                                                                                                                      |
| M1c                                                      | All other visceral metastases                                                                                          | Normal                                                                                                                                      |
| Any distant metastasis                                   | Elevated                                                                                                               |                                                                                                                                             |
| *                                                        | Micrometastases are diagnosed after sentinel or elective lymphadenectomy.                                              |                                                                                                                                             |
| †                                                        | Macrometastases are defined as clinically detectable nodal metastases confirmed by therapeutic                         |                                                                                                                                             |  
| Stage Groupings for Cutaneous Melanoma Clinical Staging Pathologic Staging T N M T N M 0 Tis N0 M0 Tis N0 M0 IA T1a N0 M0 T1a N0 M0 IB T1b N0 M0 T1b N0 M0 T2a N0 M0 T2a N0 M0 IIA T2b N0 M0 T2b N0 M0 T3a N0 M0 T3a N0 M0 IIB T3b N0 M0 T3b N0 M0 T4a N0 M0 T4a N0 M0 IIC T4b N0 M0 T4b N0 M0 III‡ Any T N1 N2 N3 M0 IIIA T1-4a N1a M0 T1-4a N2a M0 IIIB T1-4b T1-4b T1-4a T1-4a T1-4a/b M0 M0 M0 M0 M0 IIIC T1-4b T1-4b Any T N1a N2a N1b N2b N2c M0 M0 M0 IV Any T Any N Any M1 Any T Any N Any M1 * Clinical staging includes microstaging of the primary melanoma and clinical/radiologic evaluation  for metastases. By convention, it should be used after complete excision of the primary  N1b N2b N3   |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
M0 M0 M0 IV Any T Any N Any M1 Any T Any N Any M1
* Clinical staging includes microstaging of the primary melanoma and clinical/radiologic evaluation for metastases. By convention, it should be used after complete excision of the primary melanoma with clinical assessment for regional and distant metastases.  
† Pathologic staging includes microstaging of the primary melanoma and pathologic information about the regional lymph nodes after partial or complete lymphadenectomy. Pathology stage 0 or stage 1A patients are the exception; they do not require pathologic evaluation of their lymph nodes.  
‡ There are no stage III subgroups for clinical staging.
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addendum staging for mucosal melanomas

Stage IIB: Clinically localized primary melanoma > 4mm thick Stage III: Lymph node metastases Stage IV: Distant metastases
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10.3 appendix 3 ecog performance

| Grade                                  | Performance                                                                                                                                               |
|----------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------|
| 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 house work, 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                                                                      |
| 5                                      | Dead                                                                                                                                                      |
| * As published in Am. J. Clin. Oncol.: | Oken, M.M., Creech, R.H., Tormey, D.C., Horton, J., Davis, T.E.,                                                                                          |
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10.4 appendix 4 new york

| Functional Capacity                                                                                                                                                                                                              | Objective Assessment                                  |           |          |            |                |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------|-----------|----------|------------|----------------|
| Class I.                                                                                                                                                                                                                         | Patients with cardiac disease but                     |           |          |            |                |
| without resulting limitation of physical activity.  Ordinary physical activity does not cause undue  fatigue, palpitation, dyspnea, or anginal pain.                                                                             | No                                                    | objective | evidence | of         | cardiovascular |
| disease.                                                                                                                                                                                                                         |                                                       |           |          |            |                |
| Class II. Patients with cardiac disease resulting  in slight limitation of physical activity. They are  comfortable at rest. Ordinary physical activity  results in fatigue, palpitation, dyspnea, or  anginal pain.             | Objective evidence of minimal cardiovascular  disease |           |          |            |                |
| Class                                                                                                                                                                                                                            | III.                                                  | Patients  | with     | cardiac    | disease        |
| resulting in marked limitation of physical activity.  They are comfortable at rest. Less than ordinary  activity causes fatigue, palpitation, dyspnea, or  anginal pain.                                                         | Objective                                             | evidence  | of       | moderately | severe         |
| cardiovascular disease.                                                                                                                                                                                                          |                                                       |           |          |            |                |
| Class                                                                                                                                                                                                                            | IV.                                                   | Patients  | with     | cardiac    | disease        |
| resulting in inability to carry on any physical  activity without discomfort. Symptoms of heart  failure or the anginal syndrome may be present  even at rest. If any physical activity is  undertaken, discomfort is increased. | Objective                                             | evidence  | of       | severe     | cardiovascular |
| disease.                                                                                                                                                                                                                         |                                                       |           |          |            |                |
| * The Criteria Committee of the New York Heart Association. Nomenclature and Criteria for Diagnosis of                                                                                                                           |                                                       |           |          |            |                |  
* The Criteria Committee of the New York Heart Association. Nomenclature and Criteria for Diagnosis of  
Diseases of the Heart and Great Vessels . 9th ed. Boston, Mass: Little, Brown & Co; 1994:253-256  
69  
10.5 Appendix 5: Lot Testing A. Preparation of the synthetic melanoma and tetanus peptides All peptides have been synthesized under GMP conditions by Polypeptide Group (San Diego, CA) or comparable provider. Certificates of analysis and technical summaries for each of the peptides w ill be included in the chemistry and manufacturing portion of the IND application.
Peptide preparation and vialing has been performed under GMP conditions by Clinalfa/Merck for the tetanus peptide, and Bachem for the NY-ESO-1 long peptide, and w ill be performed under GLP conditions by the UVA Human Immune Therapy Center laboratory for the 6 peptides in the LPV6 long peptide mixture. Documentation relating to the procedures used to prepare and vial the peptides w ill comply w ith the CFR and w ill be included in the Chemistry and Manufacturing Section of the IND application.
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b. lot release quality assurance

1. Identity: Identity w ill be confirmed by structural studies. The individual peptides w ill be tested for identity by mass spectrometry (to define molecular mass and amino acid sequence) and high performance liquid chromatography (HPLC, to confirm purity) in a GMP laboratory (Polypeptide Group). Amino acid analysis w ill also be performed to confirm identity further.  
Before mixing, the amino acid sequence of each individual peptide preparation w ill be reconfirmed by mass spectrometry or co-elution.  
After combining the peptides, the mixture w ill be subjected to HPLC analysis.  
2. Purity: Purity w ill be assessed before and after vialing the peptide mixtures. Before vialing the peptide mixtures, each synthetic peptide w ill be evaluated for the presence of a single dominant species by high performance liquid chromatography
(HPLC). Purity of each peptide component w ill exceed 90% (expected 94%-98%).  
Variants of the original peptide may include incomplete products of synthesis, minor degradation products due to oxidation of methionine residues, and dimerization of cysteine-containing peptides. After vialing the peptide mixture, purity w ill be reconfirmed by HPLC.  
3. Aggregation: Aggregation of peptides w ill be assessed by HPLC. Aggregation is not expected.  
4. Trifluoroacetic acid (TFA): The amount of total fluorine in each peptide preparation w ill be less than 0.5% or 5000 ppm.  
5. Potency: The net peptide content w ill be calculated for each synthesis. The amounts of each peptide (mcg quantities) added to the vaccine vials are calculated based on the net peptide content of the original stock of lyophilized peptides.  
The amount of peptide in each sterile filtered peptide solution w ill be measured by amino acid analysis. The final vials w ill also be tested for quantity of total peptide by amino acid analysis. Lot release criteria are defined in the Chemistry and Manufacturing section of the IND application.
6. Endotoxin: Endotoxin testing w ill be conducted in accordance w ith CFR guidelines.  
7. General Safety: General safety testing w ill be conducted in accordance w ith US
CFR guidelines.
8. Sterility: Sterility testing w ill be conducted in accordance w ith CFR guidelines.  
C. Ongoing Quality Assurance Testing Stability: We w ill follow guidelines for quality assurance studies to assure stability of the peptide preparation over time during the trial76. The peptide preparations w ill be assayed for stability every six months for 2 years and every year thereafter. The follow ing analyses w ill be performed to confirm stability: 1. HPLC: HPLC w ill be performed to confirm purity. A comparison to previous HPLC
data w ill be performed. Ideally, the purity of each peptide component w ill exceed 90% (94%-98%). Variants of the original peptide may include incomplete products of synthesis, minor degradation products due to oxidation of methionine residues, and dimerization of cysteine-containing peptides. Such minor variants w ill be tolerated as long as the intended peptide represents at least 75% of the total peptide species. With liquid formulations of this peptide preparation, w e have observed dimerization of cysteine-containing peptides, yet this peptide retained immunogenicity even w hen as little as 10% remains as monomer. For the present study, dimerization of cysteine-containing peptides w ill be acceptable as long as at least 25% remains as monomer by HPLC. Because measures of peptide quantity are subject to variability, a peptide lot w ill be rejected only if tw o sequential measures fail to meet the criterion stated above. If a second measure is required, the lot w ill be held until the second reading is obtained and show n to be acceptable.
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2. aggregation. aggregation w ill

3. Mass spectrometry. This w ill be performed on each peptide peak to define the molecular sequence and mass if there is any ambiguity about the identity of the peptides based on HPLC. New peaks on HPLC, or significant change in the elution profiles w ill w arrant determination of the peptide identities by mass spectrometry on each peak.  
4. Sterility: One vial of peptide w ill be submitted to the Clinical Microbiology Laboratory at the University of Virginia or Microbiology Research Associates, Inc. (Acton, MA).  
Sterility testing w ill be conducted in accord w ith FDA guidelines in 21 CFR part 610.12.
71 10.6 Appendix 6: NCI Common Terminology Criteria for Adverse Events v4.03 http://evs.nci.nih.gov/ftp1/CTCAE
72
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10.7 appendix 7 summary of

| 2)                                                                                                                                                                                                                                 | Section                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         | 1.1:   | updated   | to   | address   | current   | treatments   | for   | resected   |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------|-----------|------|-----------|-----------|--------------|-------|------------|
| melanoma in the adjuvant setting.                                                                                                                                                                                                  |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 |        |           |      |           |           |              |       |            |
| 3)                                                                                                                                                                                                                                 | Section 1.2.6: updated to address ipilimumab approval for treatment of  resected melanoma in the adjuvant setting.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              |        |           |      |           |           |              |       |            |
| 4)                                                                                                                                                                                                                                 | Section 4.1: removed reference to interferon packet.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            |        |           |      |           |           |              |       |            |
| 5)                                                                                                                                                                                                                                 | Section 5.6.1: added targeted molecular therapy (e.g. vemurafenib, other  inhibitor of mutant BRAF, MEK, or cKit) as nonmerpitted treatments.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   |        |           |      |           |           |              |       |            |
| 6)                                                                                                                                                                                                                                 | Section 5.6.2.6: edited to indicate that any live vaccines (e.g. FluMist) are  permitted, but should be administered at least 2 weeks prior to or at least  2 weeks after a study vaccine.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      |        |           |      |           |           |              |       |            |
| 7)                                                                                                                                                                                                                                 | Updated reference list.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         |        |           |      |           |           |              |       |            |
| 2)                                                                                                                                                                                                                                 | Updated study personnel                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         |        |           |      |           |           |              |       |            |
| 3)                                                                                                                                                                                                                                 | Updated formatting and numbering throughout study document                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      |        |           |      |           |           |              |       |            |
| 4)                                                                                                                                                                                                                                 | Additional administrative changes and typographical errors corrected  throughout the document to improve readability.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           |        |           |      |           |           |              |       |            |
| 5)                                                                                                                                                                                                                                 | Table of Contents: Updated                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      |        |           |      |           |           |              |       |            |
| 6)                                                                                                                                                                                                                                 | Protocol precis  Design: The study has been divided into Part 1 and Part 2. Part 1  required rotating vaccine sites and accrual to this part was completed. Part 2 of the study has been added and the regimen  requires that each vaccine is administered in the same skin site for  all 6 vaccines.  The objectives have been revised to add objectives specific for part  2.  Table 1 has been re-labeled as Table 1A. The title of the table has  been revised to specify that Table 1A is relevant for Part 1 of the  study.  Addition of Table 1B: Study Arm E2 has been specified for Part 2 of  the study.  The rationale for choosing the vaccine regimen from study arm E  has been added.  Regimen: Revised to describe regimen for Parts 1 and 2 of the  study.  Vaccine site biopsies: revised to specify that 3 punch biopsies of  normal skin will be completed in the first 6 patients enrolled in Part 1  of the study.  Leadership: Revised to specify that Part 2 of the study will be  conducted just at the University of Virgina.  Accrual: Adjusted to allow for an additional patients to be enrolled  to part 2 of the study. |        |           |      |           |           |              |       |            |
| 7)                                                                                                                                                                                                                                 | Section 1.0: Updated accrual information.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |        |           |      |           |           |              |       |            |
| 8)                                                                                                                                                                                                                                 | Section 1.1: Updated information describing incidence of melanoma and  deaths from melanoma in 2017 and added a section on vaccine site  selection to provide a rationale for evaluating same site vaccinations.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                |        |           |      |           |           |              |       |            |
| 9)                                                                                                                                                                                                                                 | Section 1.2.6: Updated information on approved adjuvant therapies for melanoma.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 |        |           |      |           |           |              |       |            |
| 10) Section 1.2.10: Added prior experience with same site vaccinations. 11) Added Table 5 to describe vaccine site reactions for prior Mel44 and  Mel58 studies. 12) Section 2.0: Revised objectives to add objectives for Part 2. |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 |        |           |      |           |           |              |       |            |  
| 13) Section 3.1: Regimen-added the regimen for Part 2, which includes  same site vaccinations. 14) Section 4.2.7(1): Corrected section reference. 15) Section 4.4.2: Revised randomization section and specified that Part 2 of  the study will not include randomization. 16) Section 5.2.1: Added a description for preparing drug for Study Arm E2. 17) Section 5.4.2: Deleted prior text describing the vaccine regimen and  replaced with a description for the administration of vaccines for Part 1  and Part 2 and a description of expected vaccine site toxicities. 18) Section 5.4.3: A section describing doses for part 2 of the study has  been added. 19) Section 5.4.6 , 6.2.2, 6.2.4, Appendix 1: Clarified that biopsies of normal  skin will be completed in the first 6 participants who are enrolled in Part 1. 20) Section 5.6: Clarified that patients will be randomized in Part 1 and  assigned to an arm in Part 2 of the study. 21) Section 7.4.3: Clarified that grade 3 injection site reactions with significant ulceration of the skin (> 2 cm) will be considered DLTs.  Clarified dermatologic toxicities for resiquimod. 22) Section 7.8: Added grade 3 injection site reaction with ulceration ≤ 2 cm as an exception to the DLT rule 23) Section 9.0: Revised introduction to statistical considerations to add  information about Part 2 of the study. 24) Section 9.1-removed the DLT rule from this section and revised to refer to  section 7.8 of the protocol. 25) Sections 9.2, 9.3, 9.4, 9.5, 9.6: clarified which sections pertain to Part 1  and added statistical language for Part 2. 26) Reformatted headings for the appendices 27) Appendix 7: added a summary of changes list for prior versions of the  protocol   |                                                   |                                                                                          |                           |
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| 06-17-14                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                | 1)                                                | Sapna Patel's role was changed from co-investigator to PI at MD  Anderson Cancer Center. |                           |
| 2)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      | Table of Contents page numbers were updated.      |                                                                                          |                           |
| 06-06-14                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                | 1)                                                | Section 1.2.3: Editorial change for clarification                                        |                           |
| 2)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      | Section 1.2.4: Editorial change for clarification |                                                                                          |                           |
| 3)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      | Section 1.2.5:                                    | The dosing for resiquimod                                                                | was revised to include an |
| acceptable dose range of 400-600 mg. Instructions about coverage and  washing the affected area have been incorporated into the study protocol.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         |                                                   |                                                                                          |                           |
| 4)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      | Section 1.2.8: Reference added                    |                                                                                          |                           |
| 5)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      | Table 3: Reformatted                              |                                                                                          |                           |
| 6)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      | Table 4: Updated with new data                    |                                                                                          |                           |
| 7)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      | Section 4.1.4:                                    | Clarification of inclusion criterion and whether repeat scans                            |                           |
| are needed after stereotactic radiotherapy.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             |                                                   |                                                                                          |                           |
| 8)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      | Section 4.1.5:                                    | Clarification of inclusion criterion and the description of                              |                           |
| required lymph node basins.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             |                                                   |                                                                                          |                           |
| 9)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      | Section 4.1.8:                                    | Clarification of inclusion criterion a) specifications for patients                      |                           |
| with Gilbert's disease and b) re-defined acceptable limit for Hgb-A1C level.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            |                                                   |                                                                                          |                           |
| 10) Section 4.2.11: Addition of the following to allowable autoimmune disorders:  Hypothyroidism of any etiology on stable thyroid hormone replacement  therapy. 11) Section 5.2.1: Arms E and G: Change in mixing instructions for PolyICLC.  PolyICLC will be incorporated into the vaccine mixture rather than  administered as a separate injection.  12) Section 5.2.1: Clarification of vaccine components for Study Arms C, D, F  and G. The dose of resiquimod was adjusted from 500 mg to approximately  500 mg. Instructions aoubt coverage and washing the affected area have                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                |                                                   |                                                                                          |                           |  
| been incorporated into the study protocol.   |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |
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| 13) Reference List: Updated.                 |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |
| 03-25-14                                     | 1) Dr. Geoffrey Weiss and Dr. Elizabeth Gaughan have been added to the list of  co-investigators. 2) The Table of Contents was updated. 3) The dose of resiquimod has been corrected throughout the study document.  The correct dose is 1000 mcg and the drug will be supplied in multi-dose tubes.  Section 1.2.6 also includes information on prior usage with this dose. 4) Section 4.1.4: An editorial change was made and also a clarification that the  brain metastases in this section refer to treated brain metastases. 5) Section 4.1.8: The lab parameters inclusion criteria were reformatted. HIV  and HCV requirements were moved to the exclusion criteria (Section 4.2.6). 6) Section 4.2.3: The reference to Section 4.2.3 within this section was corrected  to Section 4.2.4. 7) Section 4.4.1: Information about the interferon education packets was  changed to include a statement that participants mus t convice the study team  that the answered the questions accurately or understand the correct answers. 8) Section 5.4.1: A clarification was made to this section to specify that chronic  inflammation is expected in subjects who receive Montanide ISA-51. Also,  editorial changes were made to this section. 9) Section 6.2.2: "Vaccine site biopsy (3)" was removed from the Day 8 schedule  of events as the biopsy procedures were listed twice for this day. 10) Sections 6.2.1, 6.2.2, 6.2.3, 6.2.4, 6.2.5, 6.2.6, 6.2.7, 6.2.8: Clarfication was  made to the language pertaining to the interval history and physical exam.  Examination of vaccine sites was added to each of the study visit days, when  applicable.  11) Section 7: Editorial correction was made to change "or" to "for". 12) Section 7.4.3: A redundant section related to AEs that do not require  expedited reporting. Reporting guidelines are provided in Table 12. 13) Section 7.4.3: A clarification was made to the dosing regimen that was used  for resiquimod in prior studies. 14) Section 7.6.1: A clarification was made to the process of reporting AEs. AEs  determined to require expedited reporting to the sponsor, must also be reported  to a site's institution according to local policy and procedures. 15) Section 7.6.3: A clarification was made regarding reporting AEs to the sites.  Serious and unexpected suspected adverse reactions will be reported to the sites  no later than 15 calendar days after the Sponsor determines that the the  requirements for an IND safety report have been met. 16) Section 7.7.2: The phrase "and at UVA by the UVA melanoma team" was  removed from this section as it did not apply to the outside sites. Individual AEs  will be reviewed at each site. 17) Section 7.7.3: The term "weekly" was removed when describing the  melanoma team meetings. The melanoma team meets 20 times a year, as  specified in section 7.7.4 of the DSMP. The term "may" was included in the  description of the events that are reviewed at each meeting as the agenda varies  depending upon study accruals. 18) Section 7.7.4 : The DSMP was modified to specify that month conference  calls will be held between the Sponsor and the sites. 19) Section 9.2.2: "R-package, pocrm" was added for clarification. Additional  statistical methods were added to clarify the model of DLT probabilities. |  
| 20) 9.6.3: The endpoints of disease-free survival and overall survival have been  defined. 21) Appendix 1: editorial change made to subscript "i". 22) Appendix 5: Clarifications were made to the descriptions of the peptide  preparation and vialing.   |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        |
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| 10-10-13                                                                                                                                                                                                                                                   | 1) Cover sheet has been updated  2. New study staff members have been added and the formatting for contact  information has been corrected.  3. Zone information has been added to the précis (Table 1) and Table 5 and the  description of the regimen has been modified to reflect changes in the statistical  section.  4. Table of Contents has been updated.  5. List of abbreviations has been updated.  6. The timing of the negative control biopsies has been clarified throughout the  protocol. The negative control biopsies will be completed on days 8 and 22 in the  first 6 patients.  7. Table 3: An explanation of the grading criteria are provided.  8. Section 1.2.6 and 5.2.1: additional directions for applying the resiquimod have  been added.  9. Section 4.4.1: Replaced the section that participants will have to complete the  interferon packet questions satisfactorily with a statement that answers to  questions in the packet will be reviewed with the participant.  10. Section 4.4.2: A description of when randomization is to take place in relation  to registration and the start of treatment has been added.  11. Section 4.4.3: "suture removal" was removed from the evalution following the  biopsies as punch biopsies will not require sutures.  12. Section 5.2 and 5.4.3: The dosage of and preparation of the vaccines were  clarified.  13. Section 5.4.6.4: Information on specimen collection delays and the associated  documentation are provided.  14. Section 5.6.2.6: clarification made to permitted influenza vaccines  15. Section 6.2 Treatment: The schedules for the immunologic bloods, vaccines,  and biopsies have been moved to this section.  16. Section 7.0 Regulatory and Reporting Requirements: This entire section has  been revised and reformatted to clarify the reporting requirments for the study.  The original reporting tables have been replaced with a template table from the  UVA Cancer Center DSMP and an IRB reporting table has been added. In  addition, reporting criteria for reporting to the FDA have been replaced.  17. Section 7.1.3: A clarification for the definition of hospitalization for expedited  AE reporting has been added.  18. Section 7.5: Adverse event classifications have been updated and the  hospitalization category has been moved to the description for an SAE.  19. Section 7.5: AE classifications have been updated to include  Hematologic/Metabolic and Non-Hematologic/Non-metabolic categories.  Autoimmune has been added to the allergic category.  20. Section 7.7.1: Replaced section stating that someone from the research team  would be available 24 hours a day 7 days a week for questions with the statement  that subjects would be given information on how to contact their providers if they  have questions or problems.  21. Section 9.1: The table has been modified to correct the description of the  adjuvant preparation for each arm/combination.  22. Section 9.2: A description of the randomization has been added. |  
| 23. Appendix 1: Administrative changes have been made to the X-page to  improve readability.  24. Additional administrative changes and typographical errors corrected  throughout the document to improve readability.   |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               |
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| 08-06-13                                                                                                                                                                                                                  | 1) An additional study group has been added in response to the  recommendations of the FDA and of our collaborators at MD Anderson Cancer  Cente 2) The study design has been changed to incorporate an adaptive design in order  to minimize data with the least number of patients. This reduces the target  sample size by half. 3) Section 4.4 of the study protocol has been modified to include additional details  regarding the registration and randomization of study patients. 4) A section for study drug accountability and distribution has been added  (section 5.3) 5) Table of Contents has been updated. 6) The source of Hiltonol (polyICLC) has been updated, in section 1.2.5. 7) Updated section 1.2.10 to specify that the peptides for the vaccines have been  synthesized under GMP conditions, rather than GLP. 8) Appendices about vaccine preparation, immunologic assays, and details of  tissue handling with the biopsies has been removed from the protocol and will be  included ina Study Manual and Investigator Brochure. 9) Numerous small administrative changes have been made to improve clarity,  consistency and readability and to provide appropriate citations. 10) Details about the quality assurance studies to be done on the peptides have  been added to respond to the FDA after our preIND meeting. 11) Skin biopsies have been amended: Patients 1-6 will have 3 skin punch  biopsies rather than 2, on day 1. The 3rd sample from skin on day 1 in those  patients and from all patients on days 8 and 22 will be stored in RNA later rather  than be collected for viable cell studies. 12) Signature page and study staff contact information has been added. 13) X-page has been corrected with regards to the timing of the normal skin  biopsies. |
| 12/05/11                                                                                                                                                                                                                  | 1)Section 4.4 of the study protocol has been modified to include additional  details regarding the registration and randomization of study patients. 2) A section for study drug accountability and distribution has been added  (section 5.3) 3) Table of Contents has been updated.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         |
university of virginia human immune
11.0 reference list

(1) Kenter GG, Welters MJ, Valentijn AR et al. Vaccination against HPV-16 oncoproteins for vulvar intraepithelial neoplasia. *N Engl J Med* 2009;361:1838-1847.  
(2) Sabbatini P, Tsuji T, Ferran L et al. Phase I Trial of Overlapping Long Peptides from a Tumor Self-Antigen and Poly-ICLC Show s Rapid Induction of Integrated Immune Response in Ovarian Cancer Patients. *Clin Cancer Res* 2012;18:6497-6508.  
(3) Gnjatic S, Saw hney NB, Bhardw aj N. Toll-like receptor agonists: are they good adjuvants? *Cancer J* 2010;16:382-391.  
(4) Speiser DE, Lienard D, Rufer N et al. Rapid and strong human CD8+ T cell responses to vaccination w ith peptide, IFA, and CpG oligodeoxynucleotide 7909. J Clin Invest 2005;115:739-746.  
(5) Schw artzentruber DJ, Law son DH, Richards JM et al. gp100 peptide vaccine and interleukin-2 in patients w ith advanced melanoma. *N Engl J Med* 2011;364:21192127.  
(6) Ascierto PA, Gogas HJ, Grob JJ et al. Adjuvant interferon alfa in malignant melanoma: an interdisciplinary and multinational expert review . *Crit Rev Oncol* Hematol 2013;85:149-161.  
(7) Agarw ala SS, Lee SJ, Flaherty LE et al. Randomized phase III trial of high-dose interferon alfa-2b (HDI) for 4 w eeks induction only in patients w ith intermediate- and high-risk melanoma (Intergroup trial E 1697) . [abstract]Agarw ala SS, Lee SJ,
Flaherty LE et al. *J Clin Oncol* 2011;29 suppl:Abstract 8505
(8) Slingluff CL, Jr., Petroni GR, Chianese-Bullock KA et al. Immunologic and clinical outcomes of a randomized phase II trial of tw o multipeptide vaccines for melanoma in the adjuvant setting. *Clin Cancer Res* 2007;13:6386-6395.  
(9) Slingluff CL, Jr., Chianese-Bullock KA, Bullock TN et al. Immunity to melanoma antigens: from self-tolerance to immunotherapy. *Adv Immunol* 2006;90:243-95.:243295.  
(10) Kirkw ood JM, Lee S, Land S et al. E1696: Final analysis of the clinical and immunological results of a multicenter ECOG phase II trial of multi-epitope peptide vaccination for stage IV melanoma w ith MART-1 (27-35), gp100 (209-217, 210M),
and tyrosinase (368-376, 370D) (MGT) +/- IFNa2b and GM-CSF. [abstract]Kirkw ood JM, Lee S, Land S et al. *J Clin Oncol (Meeting Abstracts)* 2004;22:7502
(11) Rosenberg SA, Sherry RM, Morton KE et al. Tumor progression can occur despite the induction of very high levels of self/tumor antigen-specific CD8+ T cells in patients w ith melanoma. JI 2005;175:6169-6176.  
(12) Slingluff CL, Jr., Petroni GR, Olson WC et al. Effect of GM-CSF on circulating CD8+
and CD4+ T cell responses to a multipeptide melanoma vaccine: Outcome of a multicenter randomized trial. *Clin Cancer Res* 2009;15:7036-7044.  
(13) Slingluff CL, Jr., Petroni GR, Yamshchikov GV et al. Immunologic and clinical outcomes of vaccination w ith a multiepitope melanoma peptide vaccine plus low -
dose interleukin-2 administered either concurrently or on a delayed schedule. *J Clin* Oncol 2004;22:4474-4485.  
(14) Slingluff CL, Jr., Petroni GR, Olson W et al. Helper T cell responses and clinical activity of a melanoma vaccine w ith multiple peptides from MAGE and melanocytic differentiation antigens. *J Clin Oncol* 2008;26:4973-4980.  
(15) Rosenberg SA, Yang JC, Restifo NP. Cancer immunotherapy: moving beyond current vaccines. *Nat Med* 2004;10:909-915.  
(16) Rosenberg SA, Yang JC, Kammula US et al. Different adjuvanticity of incomplete freund's adjuvant derived from beef or vegetable components in melanoma patients immunized w ith a peptide vaccine. *J Immunother* 2010;33:626-629.  
(17) Slingluff CL, Jr., Petroni GR, Smolkin ME et al. Immunogenicity for CD8+ and CD4+ T
cells of tw o formulations of an incomplete Freund's adjuvant for multipeptide melanoma vaccines. *Journal of Immunotherapy* 2010;33:630-638.  
(18) Hailemichael Y, Dail Z, Jaffarzad N et al. Persistent antigen at vaccination sites induces tumor-specific CD8+ T cell sequestration, dysfunction and deletion. *J Exp* Med 2013;in press.  
(19) Salerno EP, Shea SM, Olson WC et al. Activation, dysfunction and retention of T
cells in vaccine sites after injection of incomplete Freund's adjuvant, w ith or w ithout peptide. *Cancer Immunol Immunother* 2013.  
(20) Law son DH, Lee SJ, Tarhini AA, Margolin KA, Ernstoff MS, Kirkw ood JM. E4697:
Phase III cooperative group study of yeast-derived granulocyte macrophage colonystimulating factor (GM-CSF) versus placebo as adjuvant treatment of patients w ith completely resected stage III-IV melanoma. [abstract]Law son DH, Lee SJ, Tarhini AA, Margolin KA, Ernstoff MS, Kirkw ood JM. *J Clin Oncol* 2010;28:abstract 8504
(21) Song XT, Turnis ME, Zhou X et al. A Th1-inducing adenoviral vaccine for boosting adoptively transferred T cells. *Mol Ther* 2011;19:211-217.  
(22) Ly LV, Sluijter M, Versluis M et al. Peptide vaccination after T-cell transfer causes massive clonal expansion, tumor eradication, and manageable cytokine storm.  
Cancer Res 2010;70:8339-8346.  
(23) Song S, Zhang K, You H et al. Significant anti-tumour activity of adoptively transferred T cells elicited by intratumoral dendritic cell vaccine injection through enhancing the ratio of CD8(+) T cell/regulatory T cells in tumour. *Clin Exp Immunol* 2010;162:75-83.  
(24) Lou Y, Wang G, Lizee G et al. Dendritic cells strongly boost the antitumor activity of adoptively transferred T cells in vivo. *Cancer Res* 2004;64:6783-6790.  
(25) Pilon-Thomas S, Mackay A, Vohra N, Mule JJ. Blockade of programmed death ligand 1 enhances the therapeutic efficacy of combination immunotherapy against melanoma. *J Immunol* 2010;184:3442-3449.  
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