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.  
Males ≥ 18 years of age with histologically confirmed adenocarcinoma of the prostate.  
.  
Stage D0 disease with documented biochemical progression documented by rising PSA
and no evidence of metastatic disease by physical examination, CT scan or bone scan.
.  
PSADT > 3 months and < 15 months:
Patients must have ≥ 3 PSA measurements over ≥ 3 months.  
.
.  
The interval between PSA measurements must be > 4 weeks.  
.  
Performance Status: ECOG 0-1.  
.  
No other concurrent anticancer therapy or prior prostate cancer vaccines expressing TARP.
untitled h1
study design

.  
Phase II, prospective, single-blinded, randomized, placebo controlled study in men with Stage D0 prostate cancer.  Men with a PSADT ≥ 3 months and ≤ 15 months will be randomized 2:1 to receive ME TARP autologous DC vaccination or a control elutriated monocyte vaccine placebo.
.  
An initial lead-in of 6 patients will be enrolled to allow preliminary assessment of the safety of the ME TARP vaccine platform through 12 weeks before enrollment of prospectively randomized subjects blinded to treatment assignment begins.
.  
All patients will receive a total of 6 doses of vaccine (20 x10 6 viable cells/dose) delivered intradermally at Weeks 3, 6, 9, 12, 15, and 24. All patients will undergo a 15-18L
apheresis at Week 0 and restaging at Weeks 48 and 96 to confirm maintenance of Stage D0 disease.
.  
Sample size:  N = 72 (6 lead-in patients for safety assessment, 2:1 randomization: TARP
N = 44; placebo N =22).  
| TABLE OF CONTENTS                                                                                                                                                   |                                                                                                                                                            |                    |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------|
| PRÉCIS .                                                                                                                                                            |                                                                                                                                                            |                    |
| TABLE OF CONTENTS.................................................................................................................................................4 |                                                                                                                                                            |                    |
| INTRODUCTION                                                                                                                                                        |                                                                                                                                                            |                    |
| l                                                                                                                                                                   | 1.1                                                                                                                                                        | Study Objectives . |
| 1.2                                                                                                                                                                 | Background and Rationale: .................................................................................................................7               |                    |
| 2                                                                                                                                                                   | ELIGIBILITY ASSESSMENT AND ENROLLMENT ...........................................................19                                                        |                    |
| 2.1                                                                                                                                                                 | Eligibility Criteria ............................................................................................................................19        |                    |
| 2.2                                                                                                                                                                 | Pre-Screening and Eligibility Evaluation                                                                                                                   |                    |
| 2.3                                                                                                                                                                 | Baseline Evaluation                                                                                                                                        |                    |
| Registration Procedures                                                                                                                                             |                                                                                                                                                            |                    |
| 2.4                                                                                                                                                                 |                                                                                                                                                            |                    |
| 2.5                                                                                                                                                                 | Treatment Assignment and Randomization Procedures: ...........................................................22                                           |                    |
| 3                                                                                                                                                                   | STUDY IMPLEMENTATION                                                                                                                                       |                    |
| Study Design .                                                                                                                                                      |                                                                                                                                                            |                    |
| 3.1                                                                                                                                                                 | Vaccine Administration                                                                                                                                     |                    |
| 3.2                                                                                                                                                                 |                                                                                                                                                            |                    |
| 3.3                                                                                                                                                                 | Dose Modification and Immunization Stopping Rules .............................................................25                                          |                    |
| Study Calendar .                                                                                                                                                    |                                                                                                                                                            |                    |
| 3.4                                                                                                                                                                 | Criteria for Removal from Protocol Therapy and Off Study Criteria ...............................27                                                        |                    |
| 3.5                                                                                                                                                                 |                                                                                                                                                            |                    |
| 4                                                                                                                                                                   | CONCOMITANT MEDICATIONS/MEASURES ....................................................................28                                                    |                    |
| S                                                                                                                                                                   | BIOSPECIMEN COLLECTION .                                                                                                                                   |                    |
| 5.1                                                                                                                                                                 | Correlative Studies for Research/Pharmacokinetic Studies ................................................29                                                |                    |
| 5.2                                                                                                                                                                 | Sample Storage, Tracking and Disposition .............................................................................30                                   |                    |
| 5.3                                                                                                                                                                 | Samples for Genetic/Genomic Analysis .................................................................................31                                   |                    |
| 6                                                                                                                                                                   | DATA COLLECTION AND EVALUATION ..............................................................................31                                            |                    |
| 6.1                                                                                                                                                                 | DATA COLLECTION                                                                                                                                            |                    |
| 6.2                                                                                                                                                                 | Response Criteria ......................................................................................................................................33 |                    |
| 6.3                                                                                                                                                                 | Toxicity Criteria                                                                                                                                          |                    |
| 7                                                                                                                                                                   | NIH REPORTING REQUIREMENTS / DATA AND SAFETY MONITORING PLAN .33                                                                                           |                    |
| 7.1                                                                                                                                                                 | Definitions                                                                                                                                                |                    |
| 7.2                                                                                                                                                                 | OHSRP Office of Compliance and Training / IRB Reporting...........................................33                                                       |                    |
| 7.3                                                                                                                                                                 | NCI Clinical Director Reporting ......................................................................................................33                   |                    |
| 7.4                                                                                                                                                                 | NIH Required Data and Safety Monitoring Plan ..................................................................34                                          |                    |  
| Version Date: 2/11/2020                                                                                                                         |                                                                                                                                                |                                                                                                         |
|-------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------|
| 8                                                                                                                                               | SPONSOR SAFETY REPORTING ..................................................................................................34                  |                                                                                                         |
| 8.1                                                                                                                                             | DEFINITIONS                                                                                                                                    |                                                                                                         |
| Assessment of Safety Events                                                                                                                     |                                                                                                                                                |                                                                                                         |
| 8.2                                                                                                                                             |                                                                                                                                                |                                                                                                         |
| 8.3                                                                                                                                             | Reporting of Serious Adverse Events ...................................................................................36                      |                                                                                                         |
| Reporting Pregnancy .........................................................................................................................36 |                                                                                                                                                |                                                                                                         |
| 8.4                                                                                                                                             |                                                                                                                                                |                                                                                                         |
| 8.5                                                                                                                                             | Regulatory Reporting for Studies Conducted Under CCR-Sponsored IND ..................36                                                        |                                                                                                         |
| 9                                                                                                                                               | CLINICAL MONITORING .............................................................................................................37            |                                                                                                         |
| 10                                                                                                                                              | STATISTICAL SECTION .....................................................................................................................37    |                                                                                                         |
| COLLABORATIVE AGREEMENTS ..........................................................................................39                           |                                                                                                                                                |                                                                                                         |
| 11                                                                                                                                              | 11.1                                                                                                                                           | Cooperative Research and Development Agreement (CRADA) .........................................39      |
| 12                                                                                                                                              | HUMAN SUBJECTS PROTECTIONS                                                                                                                     |                                                                                                         |
| Rationale For Subject Selection ....................................................................................................39          |                                                                                                                                                |                                                                                                         |
| 12.1                                                                                                                                            |                                                                                                                                                |                                                                                                         |
| 12.2                                                                                                                                            | Participation Of Subjects Unable To Give Consent ..............................................................39                              |                                                                                                         |
| 12.3                                                                                                                                            | Participation Of Children .....................................................................................................40              |                                                                                                         |
| 12.4                                                                                                                                            | Evaluation of Benefits and Risks/Discomforts ...................................................................40                             |                                                                                                         |
| Risks/Benefits Analysis                                                                                                                         |                                                                                                                                                |                                                                                                         |
| 12.5                                                                                                                                            |                                                                                                                                                |                                                                                                         |
| 12.6                                                                                                                                            | Consent Process and Documentation ..........................................................................................41                 |                                                                                                         |
| 13                                                                                                                                              | PHARMACEUTICAL INFORMATION ........................................................................................41                          |                                                                                                         |
| 13.1                                                                                                                                            | Autologous Multi-Epitope (ME) TARP Dendritic Cell Vaccine Description: ..............41                                                        |                                                                                                         |
| Interleukin-4 CellGenix                                                                                                                         |                                                                                                                                                |                                                                                                         |
| 13.2                                                                                                                                            |                                                                                                                                                |                                                                                                         |
| 13.3                                                                                                                                            | KLH (Keyhole Limpet Hemocyanin) .......................................................................................42                      |                                                                                                         |
| 13.4                                                                                                                                            | TARP 27-35 (Wild Type) Peptide NSC#740703                                                                                                      |                                                                                                         |
| TARP 29-37-9V Peptide (Epitope-Enhanced) NSC #740704 .............................................44                                            |                                                                                                                                                |                                                                                                         |
| 13.5                                                                                                                                            |                                                                                                                                                |                                                                                                         |
| 13.6                                                                                                                                            | TARP 1-20 Peptide ..........................................................................................................................45 |                                                                                                         |
| 13.7                                                                                                                                            | TARP 11-30 Peptide ......................................................................................................................46    |                                                                                                         |
| 13.8                                                                                                                                            | TARP 21-40 Peptide ........................................................................................................................47  |                                                                                                         |
| TARP 31-50 Peptide ........................................................................................................................47   |                                                                                                                                                |                                                                                                         |
| 13.9                                                                                                                                            |                                                                                                                                                |                                                                                                         |
| 13.10 TARP 41-58 Peptide ...............................................................................................................48      |                                                                                                                                                |                                                                                                         |
| 13.11 Vitamin D3 Supplements ..........................................................................................................49       |                                                                                                                                                |                                                                                                         |
| REFERENCES                                                                                                                                      |                                                                                                                                                |                                                                                                         |
| 14                                                                                                                                              | APPENDICES                                                                                                                                     |                                                                                                         |
| 15                                                                                                                                              | 15.1                                                                                                                                           | Appendix A: Study Calendar of Clinical and Laboratory Events ........................................54 |
| Appendix B: Performance Status Criteria .........................................................................................57             |                                                                                                                                                |                                                                                                         |
| 15.2                                                                                                                                            |                                                                                                                                                |                                                                                                         |  
Abbreviated Title: TARP Phase II
Version Date: 2/11/2020  
| 15.3 Appendix C: Manufacturing Summary for Dendritic Cell Vaccine and Elutriated                       |                                                                                                                          |
|--------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------|
| Monocyte Placebo                                                                                       |                                                                                                                          |
| 15.4                                                                                                   | Appendix D: Vaccine Branch Vaccine Report Card (VRC) ..............................................61                    |
| Appendix E: Anti-TARP Antibody and Cellular Responses ..............................................62 |                                                                                                                          |
| 15.5                                                                                                   | Appendix F: Exploratory Correlative Studies ..........................................................................63 |
| 15.6                                                                                                   |                                                                                                                          |
| 15.7                                                                                                   | Appendix G: Guidelines and Worksheet for Calculations of PSADT .............................64                           |
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|---------------------------------------|-----------------------------------------------------------------------------------|
| =@7/2E7Q908 BQC890>C                  | HQE7/5 A292 8>C9/>DD7A :2880C7 9/02D ?QFF7?90:7-                                  |
| `11QC>F7C08093                        | =N87DD 2CAr>/ 2C905>A3 /7?E>C?7? 7D08097A 0C 8D0C082D  9/02D?                     |
| 4C8>F7C08093                          | I??>80297A S09@ >C8>F7C08 E/>87?? G0-7- >C8>F7C08  s?7DBt E/>970CJ                |
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| !                                                                 | "# $%& !                                                                               |
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| '() *+,-(+*.                                                      | /(01*2 30-,1*0 4,-5 678-,+8* *+,-(+*. 309 -5*  +(-*0-,38 -( :,09 -( 6(.- ;< 6(8*=78*.                                                                               |
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figure 2

TARP is Expressed in Prostate Tissues with a Range of Gleason Scores Figure 3:
TARP is Expressed in Both Hormone Sensitive and Castrate Resistant Prostate Tissue  
  
Citation: Best CJ, Gillespie JW, Yi Y, Chandramouli GV et al. Molecular alterations in primary prostate
cancer after androgen ablation therapy. Clin Cancer Res 2005 Oct 1;11(19 Pt 1):6823-34. PMID: 16203770 As shown in Figure 1, TARP is expressed both by normal and malignant prostate cancer tissue, with about 95% of prostate cancer specimens reported to be positive for its expression, both primary and metastatic. A recently published study of TARP protein expression in primary prostate cancer specimens from 621 patients who underwent radical prostatectomy (median age 62, median PSA 7.2), documented that TARP was over-expressed in the vast majority (~85%) in comparison to non-neoplastic prostate tissue and its expression was associated with conventional markers of unfavorable and more aggressive tumor behavior. (19)  As shown in Figure 2 and Figure 3, it is also expressed in prostate cancers of all Gleason types and both androgendependent and independent. Therefore, TARP is an ideal tumor antigen target for a vaccine that could be applied to any stage of prostate cancer.  
Oh et al determined two HLA-A2 epitopes that produce cytolytic T cell responses (20).  These sequences map to amino acids 27-35 and 29-37.  TARP27-35 was found to bind with an affinity that was 10 times greater than that of TARP29-37.   These peptides were demonstrated to be immunogenic by immunizing A2K b transgenic mice (expressing human HLA-A*0201) with dendritic cells pulsed with these peptides or with DNA encoding the peptide.  Dendritic cell immunization produced a higher level of immunity than DNA immunization and as expected due to its higher binding affinity, TARP27-35 produced a higher level of CD8+ T cell response than TARP29-37.
untitled h1
1.2.3 epitope enhancement

Modification of the amino acid sequence of epitopes, commonly referred to as epitope enhancement, can improve the efficacy of vaccines through several means: 1) increasing affinity of peptide for MHC molecules (21-23), 2) increasing T cell receptor (TCR) triggering (24-26), or 3) inhibiting proteolysis of the peptide by serum peptidases (21, 27, 28).  Whenever the peptide sequence is altered, it is important to demonstrate that the T cells induced still recognize the ntive peptide sequence.   Epitope-enhanced subdominant peptides can bypass self-tolerance because subdominant epitopes do not generally induce tolerance but can be made more immunogenic by epitope enhancement (29).  
Epitope enhancement of the TARP peptides was performed to increase the level of immunity that could be generated with these peptides.  Amino acid substitutions in the TARP27-35 peptide did not increase binding affinity but two amino acid substitutions in TARP29-37 did produce higher binding affinity peptides.  For TARP29–37, Arg at position 3 and Leu at position 9 were substituted with Ala (TARP29–37-3A) and Val (TARP29–37-9V), respectively.  Substitution at position 3 with Ala in TARP29–37 resulted in the greatest increase in the binding affinity of the peptide.  Although TARP29–37-9V showed a lower binding affinity to HLA-A2 than TARP29– 37-3A did, substitution of Leu at position 9 with Val did enhance the binding affinity compared with the wild-type peptide, TARP29–37.  When the immunogenicity of these peptides was evaluated in A2K b transgenic mice, both of the epitope-enhanced peptides produced a higher percentage of CD8+ T cells specific than the wild type sequence.  It was also shown that T cells generated with the epitope-enhanced TARP29-37 sequences reacted with targets pulsed with the wild type TARP29-37 peptide in the mouse.  
Although immunogenicity of these peptides was demonstrated in the mouse it was necessary to confirm their immunogenicity and cross-reactivity in humans.  Studies of these peptides in human cells showed that TARP29-37, TARP29–37-3A, and TARP29–37-9V were immunogenic in human T cells.  TARP29–37-9V specific T cells recognize targets pulsed with all three peptides equally well whereas TARP29-37-3A specific T cells recognized only targets pulsed with TARP29–37-3A, and TARP29–37 specific T cells recognized targets pulsed with the epitope-enhanced peptides less well.  This to us suggested that the TARP29–37-3A peptide would not be appropriate for immunization in humans whereas the TARP29–37-9V would be more likely to generate T cells that recognize the wild type sequence.  Human T cells specific for TARP27-35 recognized targets pulsed with that sequence as anticipated.  In addition to their ability to kill targets pulsed with TARP peptides, CD8+ T cells specific for TARP peptides were able to kill human tumor targets that were HLA-A2 positive and that expressed TARP sequences, confirming that TARP was endogenously processed and presented in human tumor cells.  The availability of tetramers that react with CD8+ T cells specific for TARP provide a simple means of evaluating the ability to stimulate immunity to the TARP peptides.  In a limited survey tetramer positive cells ranged from 0.66% to 3.9% of the CD8+ T cells in prostate and breast cancer patients compared with .01-.6% in normal controls.  
Clinical Translation: Therapeutic Vaccination Utilizing Wild Type (WT) and Epitope1.2.4 Enhanced (EE) TARP Peptides, NCI 09-C-0139 NCI 09-C-0139 is a prospective, randomized pilot study examining TARP vaccination in HLA-
A*0201 positive men with Stage D0 prostate cancer (PSA biochemical recurrence without evidence of visceral or bony metastatic disease).  Since the optimal method for therapeutic immunization with peptide vaccines in patients with cancer is unclear, patients were randomized to receive vaccination with TARP peptides in Montanide® ISA 51 VG adjuvant plus GM-CSF (Arm A) or as an autologous, TARP peptide-pulsed dendritic cell (DC) vaccine.  The primary objective was to determine the safety and immunogenicity (as measured by IFN- γ ELISPOT, ICS and tetramer assays) of TARP vaccination.  The secondary objectives were to determine the effect of TARP peptide vaccination on PSA doubling time (PSADT) (30) and PSA growth rate and regression rate constants.  All study participants had to have a baseline PSADT (calculated using PSA values within 12 months of study entry) > 3 months and ≤ 15 months.  
| Age                   | GS      | PSA          | Vit D        | ALC        | CD4%        | CD4#        |            |
|-----------------------|---------|--------------|--------------|------------|-------------|-------------|------------|
| Arm A (N = 21)        | 1360    |              |              |            |             |             |            |
| Median                | 64      | 7            | 3.44         | 26         | 40.4        | 584         |            |
| Range                 | 45 - 74 | 6 - 91       | 0.64 - 16.70 | 6 - 79     | 610 - 2160  | 28.5 - 58.4 | 206 - 915  |
| Arm B (N = 20)        |         |              |              |            |             |             |            |
| Median                | 60      | 2.74         | 28.5         | 1230       | 4.7         | 536         |            |
| 7                     |         |              |              |            |             |             |            |
| Range                 | 50 - 82 | 4-9          | 0.48 - 30.70 | 5- 70      | 690 - 3270  | 26.5 - 62.9 | 288 - 1283 |
| All Patients (N = 41) | 62      | 3.44         | 28           | 1270       | 42.5        | 547         |            |
| Median                | 45 - 82 | 0.48 - 30.70 | 5 - 79       | 610 - 3270 | 26.5 - 62.9 | 206 - 1283  |            |
| Range                 | 4-9     |              |              |            |             |             |            |  
Study accrual (N = 41) was completed on 12/19/11, within 28 months of enrollment of the first patient.  Base line demographics for all patients are highlighted in Table 1 :
TARP vaccine was administered by deep subcutaneous injection (Arm A) or intradermally (Arm B, 20x10 6 viable cells/vaccine) at Weeks 3, 6, 9, 12, and 15, with an optional sixth dose of vaccine at Week 36 based on changes in PSADT (≥ 50% increase over pre-vaccine PSADT) or immune parameters (3-fold increase in TARP-specific reactivity as measured by IFN-γ ELISPOT at least two time points) at Week 24.  TARP vaccination was found to be safe and well tolerated, with adverse events limited to injection site reactions < Grade 2.  There were no systemic or immediate hypersensitivity reactions or laboratory abnormalities associated with vaccination. As of 09/02/14, 2 patients currently remain on study for follow up. Patients have gone off study for the following reasons:
.  
| Off Study Reason            | Median Off Study Week 75 (range 9-144 weeks)              |
|-----------------------------|-----------------------------------------------------------|
| Completed Study             | N=18                                                      |
| DC Vaccine Viability Issues | N=1                                                       |
| Lost to Follow-Up           | N=1                                                       |
| Patient Request             | N=4                                                       |
| Progression                 | N=12 (bone metastasis, LAN w/ obstructive uropathy, nodal |
| involvement)                |                                                           |
| PSADT Criteria              | N=2                                                       |
| Secondary Malignancy        | N=3 (superficial MM, invasive SCC of tongue (smoker)      |
| colon adenocarcinoma)       |                                                           |  
Because of responses observed in increases/lengthening of PSADT, the original 48 week study was extended twice to a total of 144 weeks, with additional booster doses of TARP vaccine administered at Weeks 48 and 96.  PSADTs were calculated using the Memorial Sloan Kettering Cancer Center nomogram ( http://nomograms.mskcc.org/Prostate/PsaDoublingTime.aspx ).  The formula takes into account the natural logarithm of 2 divided by the slope obtained from fitting a linear regression of the natural log of PSA over time.  Since the PSADT goes to infinity as the slope approaches 0 and becomes meaningless when the slope is negative, and several patients had dramatic responses to TARP vaccination resulting in negative PSADTs, the slope log (PSA)
parameter was utilized for the formal statistical analysis since the slope log (PSA) is a continuous variable whether positive, zero, or negative.  A representative diagram documenting the relationship of PSADT (in months) to slope log (PSA) is included in Figure 4 below.
untitled h1
figure 4

Week 24 and Week 48 slope log (PSA) responses to TARP vaccination through 07/31/12 are summarized in Table 2 below:  
| Patients Reaching Week 24*   | Patients Reaching Week 48^   |                       |                 |    |                        |
|------------------------------|------------------------------|-----------------------|-----------------|----|------------------------|
| TOTAL N =                    | 39                           | TOTAL N =             | 31              |    |                        |
| 16                           |                              |                       |                 |    |                        |
| Arm A                        | 21                           | Arm A                 |                 |    |                        |
| Arm B                        | 18                           | Arm B                 | 15              |    |                        |
| Decreased Slope              | 28                           | 71.8% of All Patients | Decreased Slope | 23 | 74.2% of All Patients  |
| Arm A                        | 13                           | 61.9% of Arm A Pts    | Arm A           | 10 | 62.5% of Arm A Pts     |
| 83.3% of Arm B Pts           | 86.7% of Arm B Pts           |                       |                 |    |                        |
| Arm B                        | 15                           | Arm B                 | 11              |    |                        |
| Increased Slope              | 11                           | 2.2% of All Patients  | Increased Slope | 8  | 25.8 % of All Patients |
| Arm A                        | 8                            | Arm A                 | 6               |    |                        |
| Arm B                        | 3                            | Arm B                 | 2               |    |                        |
| Slope Difference             | Slope Difference             |                       |                 |    |                        |
| Slope 3-24 minus             | Slope 3-48 minus             |                       |                 |    |                        |
| Pre-NIH Slope                | Pre-NIH Slope                |                       |                 |    |                        |
| Min -0.175                   | Min                          | -0.178                |                 |    |                        |
| Max 0.144                    | Max                          | 0.066                 |                 |    |                        |  
*At Week 24:Patient 213 excluded due to mixed vaccines Patient 219 off study @ Wk 9 due to ↓PSADT
^At Week 48: 3 patients Off Study- 203, 213 and 219 In September 2012, a final formal analysis was performed on 39 patients with data through 07/31/12 to assess decrease in slope log (PSA) (equivalent to an increase / lengthening in PSADT).  For this analysis:  
.  
Baseline = slope log (PSA) Pre-NIH (outside PSAs from -12 months to entry)
Wk 3-24 = slope log (PSA) @NIH (PSAs from Weeks 3 to 24)
Wk 3-48 = slope log (PSA) @ NIH (PSAs from Weeks 3 to 48)
The primary statistical analysis using a Hochberg adjustment for the pooled cohort of patients from both arms revealed a statistically significant decline in the slope log (PSA):
N = 39 patients at Week 24.  
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28 of 39 patients (71.8%%) exhibited a decrease in slope log (PSA) at Week 24 Among pooled patients in both arms, slope log (PSA) from 3-24 weeks declined significantly compared to baseline, p = 0.0012 .  Median slope decline (range): -0.028 (-0.175 to 0.144)
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Within each arm the decrease in the slope log PSA was statistically significant: p = 0.023 for Arm A and p = 0.026 for Arm B, although there was no statistically significant difference between the two arms.  
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 Figure 6  
  
Waterfall Plot Week 48 Data (through 07/31/12):   TOTAL N = 31  
  
Patients with Decreased Slope Log PSA:
^Three patients off study by Week 48: 203 (patient request), 213 (DC vaccine issues) and 219 (PSADT)
Within each arm the decrease in the slope log (PSA) was not statistically significant, except in Arm B at 48 weeks, which was highly statistically significant (p = 0.0069).  However, there was no formal statistically significant difference observed between Arm A and Arm B. at either 24 or 48 weeks.  Hence, although the arms may not be equal, our analysis does not demonstrate a significant difference between the two.  Importantly, the effect of decreased slope log (PSA) at Weeks 3-24 is similar to that at later time periods i.e. it doesn't wane significantly over time and isn't impacted by an additional vaccine dose at Week 36.  In addition, there were no correlations or associations with change in slope that were strong or statistically significant using any baseline variables including CD4+ T cell percent/absolute count, CD8+ T cell percent/absolute count, 25-OH vitamin D levels, Gleason score, PSA or a baseline PSADT < 6 vs. > 6 months.  
Although TARP vaccination was associated with a decline in the slope log (PSA) in the majority of patients at Week 24 and Week 24, it was associated with an absolute decrease in PSA in only a minority of patients as highlighted in Table 3 below:  
| Patients Reaching Week 24*   | Patients Reaching Week 48^   |                       |                    |    |                       |
|------------------------------|------------------------------|-----------------------|--------------------|----|-----------------------|
| TOTAL N =                    | 39                           | TOTAL N =             | 31                 |    |                       |
| Arm A                        | 21                           | Arm A                 | 16                 |    |                       |
| Arm B                        | 18                           | Arm B                 | 15                 |    |                       |
| Decreased PSA                | 6                            | 15.4% of All Patients | Decreased PSA      | 4  | 12.9% of All Patients |
| Arm A                        | 3                            | 14.3% of Arm A Pts    | Arm A              | 1  | 6.3% of Arm A Pts     |
| Arm B                        | 16.7% of Arm B Pts           | Arm B                 | 20.0% of Arm B Pts |    |                       |
| 3                            | m                            |                       |                    |    |                       |
| Increased or No              | 33                           | Increased or No       | 27                 |    |                       |
| Change in PSA                | Change in PSA                |                       |                    |    |                       |
| Arm A                        | 18                           | Arm A                 | 15                 |    |                       |
| Arm B                        | 15                           | Arm B                 | 12                 |    |                       |
| Greatest                     | Greatest                     |                       |                    |    |                       |
| Decrease in PSA              | Decrease in                  |                       |                    |    |                       |
| PSA                          |                              |                       |                    |    |                       |
| Arm A                        | - 0.48                       | Arm A                 | - 0.36             |    |                       |
| Arm B                        | - 1.60                       | Arm B                 | 0.61               |    |                       |  
*At Week 24:Patient 213 excluded due to mixed vaccines
^At Week 48: Two patients Off Study- 203 and 213 Note: Patient 109 on Arm A, and Patients 201 and 209 had sustained decreases in Week 24 and 48 PSAs when compared to Week 0.  
Immunologic responses to TARP vaccination were examined in ex vivo and 7 day in vitro stimulation (IVS) IFN- γ ELISPOT assays.  While increased TARP-specific reactivity was demonstrated using ex vivo PBMC in a few patients, utilization of the 7 day IVS ELISPOT assay proved to be more sensitive in detecting reactivity to TARP WT27-35 and EE29-37-9V in addition to WT29-37.  However, increases in TARP-specific reactivity were seen in PSADT responders (declining slope log (PSA)) as well as PSADT non-responders (increasing slope log
(PSA)). Additional studies to date examining functional avidity of ELISPOT responses, demonstrated no differences between slope log PSA responders and non-responders.  Studies of tetramer responses and polyfunctional intracellular cytokine staining (ICS) (including Granzyme A and perforin) for assessment of TARP-specific cellular reactivity are in progress. Investigation of anti-TARP antibody responses using microarray platform technology spanning the entire TARP protein is also underway.
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1.2.5 multi-epitope (me) tarp vaccine

The 2 nd generation ME TARP vaccine is based on the amino acid sequence of the entire TARP
protein annotated below.  The vaccine platform includes the original two 9-mer HLA-A*0201 binding TARP peptide epitopes (WT27-35 and EE29-37-9V) utilized in NCI 09-C-0139 as well as an additional proposed five 20-mer TARP peptides overlapping by 10mer for a total of 7 peptides that span the entire TARP sequence as outlined in Figure 7:
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figure 7

Amino Acid Sequence of TARP (58 residues)
MQMFPPSPLFFFLQLLKQSSRRLEHTFVFLRNFSLMLLRGIGKKRRATRFWDPRRGTP
30 31 40 41 11 20 21 58 Original Epitopes in the 1st Generation TARP Vaccine Platform FVFLRNFSL = WT HLA-A*0201-binding peptide TARP 27-35 FLRNFSLMV= EE HLA-A*0201-binding peptide TARP 29-37-9V
Additional Epitopes 2nd Generation TARP Vaccine Platform
- This multi-epitope TARP peptide vaccine consists of 20-mer peptides overlapping by 10-mer spanning the entire 58 amino acid TARP protein as outlined below.  
Amino Acid Sequence of TARP Overlapping Peptides
- TARP 1-20: MOMFPPSPLFFFLQLLKQSS
- TARP 11-30:  FFLOLLKOSSRRLEHTFVFL
- TARP 21-40:  RRLEHTFVFLRNFSLMLLRG - TARP 31-50:  RNFSLMLLRGIGKKRRATRF
TARP 41-58*: IGKKRRATRFWDPRRGTP
.  
*Note: this last peptide is only 18 mer The advantage of this multi-epitope TARP peptide vaccine platform is that the overlapping epitopes cover the entire TARP protein, eliminating the need for HLA restriction in study participants that will greatly facilitate the rate of study accrual.  In addition, these longer synthetic peptides include MHC class II CD4+ T cell helper epitopes that will allow generation of better CD8+ T cell responses with improved functional avidity and longevity (42) as well as humoral anti-TARP antibody responses.
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1.2.6

Rationale for Proposed Phase II Study Design The rationale for the current phase II study design is to definitively establish that the observed declines in the difference in the slope log (PSA) documented in our initial pilot 09-C-0139 TARP study are indeed real and not due to spontaneous or random slowing in the rate of PSA
rise. The observed decreased PSA slope in over 70% of all the patients with a vaccine that has no clinically significant adverse effects other than mild local injection site reactions (< Grade II) is an extremely promising result for any cancer therapy that needs to be confirmed in a prospective, randomized study. Therefore, it is essential to carry out a randomized, placebo-controlled phase I study.  However, to accrue a large enough population for such a randomized phase II study, enrollment cannot be restricted to HLA-A2 positive patients and the excessive screening that entails. We must be able to include patients of all HLA types. We therefore propose to expand the vaccine platform coverage to include the whole TARP protein, as has been done for other tumor antigens like PSA, CEA, and melanoma antigens in the CCR and elsewhere once the value of the antigen target was demonstrated first in HLA-A2 positive patients. (31-38) The whole protein will have all the possible epitopes that can be presented by any HLA molecule and therefore would be suitable for vaccinating the entire population of prostate cancer patients, making adequate accrual feasible.  For the reasons stated earlier, overlapping long peptides such as the 20-mers overlapping by 10 residues have been widely used to represent a whole protein because they contain all the potential epitopes of the whole protein but are more amenable to processing for both class I and class II HLA presentation to CD4 + and CD8 + T cells. (13, 39-41)
We have followed this widespread practice in proposing to include an additional five 20-mer peptides overlapping by 10 residues covering the whole protein.  
Although there was not a formal statistical difference found between the two arms in the pilot study, we have selected the autologous DC vaccine platform in this study because of the statistical trend at both 24 and 48 weeks that suggested it might be better and the logistics of manufacturing a multi-epitope vaccine.  All of the peptide vaccine emulsions (TARP WT27-35 and EE19-37-9V) delivered on Arm A in the 09-C-0139 study were generated manually using a syringe method of emulsification by pharmacists in the Investigational Drug Service.  This approach is not logistically feasible for 7 different peptides and extensive additional studies would be required to determine if peptides could be combined to generate emulsions.  
The proposed phase II clinical trial is a prospective, single-blinded, randomized, placebo controlled study of 96 weeks duration in men with Stage D0 prostate cancer.  Men with a PSADT ≥ 3 months and ≤ 15 months will be randomized 2:1 to receive multi-epitope TARP
autologous DC vaccination or a control elutriated monocyte vaccine placebo. An initial lead-in of 6 patients will be enrolled to allow preliminary assessment of the safety of the multi-epitope TARP vaccine platform through 12 weeks before enrollment of prospectively randomized subjects blinded to treatment assignment begins.
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inclusion criteria

2.1.1.1 Males ≥ 18 years of age with histologically confirmed adenocarcinoma of the prostate.  
Histology confirmation must be documented with a formal pathology report.  Notes from an outside physician describing the pathologic findings (based on a prior review of the full pathology report) may be used if unable to obtain the original pathology report.  
This will eliminate the need for an additional invasive tissue biopsy.
2.1.1.2  
Must have completed and recovered from all prior definitive therapy (surgery, brachytherapy, cryotherapy or radiotherapy) for the primary tumor, or other definitiveintent local therapy.
2.1.1.3  
Stage D0 disease with documented biochemical progression documented by rising PSA
and no evidence of metastatic disease by physical examination, CT scan or bone scan.  
PSADT > 3 months and < 15 months:
2.1.1.4  
Patients must have > 3 PSA measurements over > 3 months.  
o The interval between PSA measurements must be > 4 weeks.  
2.1.1.5 For patients following definitive radiation therapy or cryotherapy: a rise in PSA of >
2ng/mL above the nadir (per RTOG-ASSTRO consensus criteria).  
2.1.1.6  
For patients following radical prostatectomy: 2 absolute PSA values > 0.2 ng/mL.  
2.1.1.7 Non-castrate level of testosterone: > 50 ng/dL (prior ADT allowed; must be > 6 months since last dose of ADT).  
2.1.1.8 Performance Status: ECOG 0-1 (Refer to Appendix B).  
2.1.1.9 Hemoglobin ≥ 9.0 gm/dL, WBC ≥ 2,500/mm³, ALC ≥ 500/ mm³, ANC ≥ 1,000/mm³,
platelet count > 75,000/mm 3 , and PT/PTT < 1.5X ULN unless receiving clinically indicated anticoagulant therapy; SGPT/SGOT < 3X ULN, total bilirubin < 1.5X ULN;
creatinine < 1.5X ULN and estimated GFR (eGFR) > 60 mL/min.  
2.1.1.10  Hepatitis B and C negative (unless the result is consistent with prior vaccination or prior infection with full recovery); HIV negative.  
2.1.1.11 No use of investigational agents within 4 weeks of study enrollment or use of immunosuppressive or immunomodulating agents within 8 weeks of study entry.  
2.1.1.12 No other concurrent anticancer therapy or prior prostate cancer vaccines expressing TARP.  
2.1.1.13  No alternative medications or nutriceuticals known to alter PSA (e.g. phytoestrogens and saw palmetto).  Note: patients receiving medications for urinary symptoms such as Flomax or 5-alpha reductase inhibitors (finasteride and dutasteride) on a chronic stable dose for at least 3 months prior to study enrollment are allowed .  
2.1.2 Exclusion Criteria 2.1.2.1 Patients with an active second malignancy other than adequately treated squamous or basal cell carcinoma of the skin.  
2.1.2.2 Patients with active infection.  
Patients on immunosuppressive therapy including:
2.1.2.3 Systemic corticosteroid therapy for any reason.  Patients receiving inhaled or o topical corticosteroids may participate.  
Other significant or uncontrolled medical illness. Patients with a remote history of asthma or active mild asthma may participate.  
2.1.2.4 Patients who, in the opinion of the Principal Investigator, have significant medical or psychosocial problems that warrant exclusion.  
2.1.2.5 2.1.3 Recruitment Strategies
We anticipate accrual to this phase II to be brisk given the level of interest in our initial pilot TARP vaccination study NCI 09-C-0139.  Over 220 patients were screened for HLA-A*0201 and PSADT criteria to achieve the final accrual of 41 patients that was accomplished within 28 The multi-epitope (ME) TARP peptide vaccine platform covering the entire TARP
months.  
protein to be used in this study eliminates the need for HLA restriction in study participants and will greatly facilitate the rate of study accrual.  In addition, multiple community-based oncology providers with a substantial number of patients enrolled in the initial 09-C-0139 clinical trial have already begun referring patients in anticipation of this phase II ME TARP DC vaccine study.  
In addition to the standard postings on the CCR website, as the previous Principal Investigator of the ME TARP DC vaccine study, Dr. Wood developed a brief 2-minute video that would provide an informational overview to both patients and health care providers describing the scientific rationale for and design of the clinical study.  
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investigational product. This assessment will include a CBC, acute care panel and liver panel.  
Refer to Appendix C for details concerning the cGMP production of peptide-pulsed dendritic cells and elutriated monocyte placebo by the NIH Clinical Center Department of Transfusion.  
Autologous ME TARP DC and elutriated monocyte placebo vaccine preparations will be
.  
assessed for release standards (nucleated cell content and concentration, appearance, flow cytometric verification of DC validation markers, viability > 60%, and product sterility and safety testing) prior to release for vaccine administration to the patient.  
For both groups, the investigational product will be administered intradermally in two sites
.  
on the forearm with a maximum volume of 0.5 mL per injection.  Administration will be alternated between the left and right forearms with each vaccination, with the exception of patients with a contraindication to using a particular arm.  In these instances, consecutive doses in the same arm will be allowed. All patients will receive a total of 6 doses of vaccine (20 x10 6 viable cells/dose, +/- 10%) delivered at Weeks 3, 6, 9, 12, 15, and 24 and will undergo scans at Weeks 48 and 96 for restaging.  
Patients will be monitored for immediate adverse event vaccine reactions (VS, clinical
.  
assessment) for 1 hour following their first TARP peptide vaccine dose.  If no adverse reactions are observed with the first vaccination, patients will be monitored for 15 minutes for all subsequent vaccinations.  
If an adverse reaction is observed following the first vaccine, the reaction will be
.  
characterized and a determination made as to whether it is considered a dose limiting toxicity (DLT) as outlined in section 3.3.1. If the adverse reaction is determined not to be a DLT, the duration of post-vaccination monitoring for subsequent vaccinations will be determined by the Principal Investigator and Lead Associate Investigator as clinically indicated depending on the severity of the initial vaccine reaction.  
All patients will be given a ME TARP DC Vaccine Report Card (refer to Appendix D) and
.  
instructed on how to complete it, following each ME TARP DC or placebo vaccine dose.
Since this protocol involves multi-epitope TARP vaccination in humans for the first time, enrollment of blinded, randomized subjects will not begin until safety has been established in an initial lead in of 6 patients.  If no adverse events are observed through Week 12 following the first vaccination in these 6 patients, enrollment of additional patients may proceed as quickly as is logistically feasible.
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dose modification and immunization stopping

No dose modifications will be made in patients receiving autologous multi-epitope TARP DC or elutriated monocyte placebo vaccination.  Subjects will cease to receive immunization if they experience dose-limiting toxicity (DLT) as outlined in Section 3.3.1. Toxicity will be assessed according to NCI Common Terminology Criteria for Adverse Events (CTCAE) v4.0 that is available at: http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm
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3.3.1

Immunization-Related Dose Limiting Toxicity (DLT)
The following assessment guidelines for the management of dose limiting toxicity (DLT) are intended to ensure the safety of each patient while on the study.  The descriptions and grading scales found in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE)
version 4.0 will be utilized for AE and DLT reporting.  All appropriate treatment areas should have access to a copy of the CTCAE version 4.0.  A copy of the CTCAE version 4.0 can be downloaded from the CTEP web site
( http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm\#ctc_40 ).  
Immunization-related DLT is defined by the parameters outlined below will be classified by the Principal Investigator as follows for determination of relatedness to ME TARP DC vaccination:
•
Unrelated
.  
Unlikely
.  
Possibly related
.  
Probably related
.  
Definitely related For the purposes of this study, the definitions of dose-limiting toxicities include:
3.3.1.1 Any Grade 2 or Grade 3 or greater allergic/hypersensitivity reaction 3.3.1.2 Any Grade 2 or greater rash consistent with erythema multiforme 3.3.1.3 Grade 3 or greater hematologic or non-hematologic toxicity, excluding lymphopenia. Abnormal laboratory studies will be repeated to verify toxicity 3.3.1.4 Grade 3 or greater acute vascular leak syndrome: respiratory compromise or fluids  
indicated.  
3.3.1.5 The following Grade 3 reactions commonly associated with immunization will be doselimiting:
Injection site reactions: ulceration or necrosis that is severe; operative A
intervention indicated.  
Skin rash/desquamation: severe, generalized erythroderma or macular, papular A
or vesicular eruption; desquamation covering > 50% BSA.  
Urticaria: intervention indicated for > 24 hrs.  
A
3.3.1.6  
The following Grade 3 reactions commonly associated with immunization will not be dose-limiting:
Pruritus/itching: intense or widespread and interfering with ADL lasting < 72 A
hours Fatigue: severe fatigue interfering with ADL lasting ≤ 72 hours A
> Fever: > 40.0° C for < 24hrs Local lymphadenopathy lasting < 1 week A
3.3.2
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delay in vaccination

3.3.3 Patients who are unable to receive their vaccine injection as scheduled due to adverse events, toxicity, failed vaccine release criteria or unforeseen personal or medical circumstances and who are otherwise eligible to continue on protocol, may be continued on vaccine therapy provided that their next vaccine is within 3 weeks of the scheduled target date of vaccine.  Patients will be advised of their options concerning alternative treatments before proceeding with the completion of their study vaccinations.  The reason for the change in vaccination schedule will be documented in the patient's chart. If the delay in the vaccination administration is from cell processing scheduling issues or any other regulatory issues not related to patient's medical condition, delay up to 6 weeks is allowed from the scheduled target date of vaccine administration.  Study weeks will be nmbered to match vaccine dose weeks.Study Stopping Rules The study will be halted pending discussing with the FDA and the NIH Intramural IRB if real time safety assessments in the first six lead-in study subjects include the following:
.  
One or more of the first three subjects (101-103) experiences a Grade II or greater adverse event designated as possibly, probably or definitely related to vaccination with the exception of local injection site reactions.
.  
Two or more of the first six subjects (101-106) experience a Grade II or greater adverse event designated as possibly, probably or definitely related to vaccination with the exception of local injection site reactions.
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study calendar 3.4

The protocol study calendar of clinical and laboratory monitoring events is as outlined in Appendix A.  
Research assays for anti-TARP antibody and TARP-specific cellular responses will be conducted as outlined in Appendix E.
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criteria for removal from protocol

A safety visit is required approximately 3-6 weeks following the last dose of study therapy.  A
telephone follow-up will take place should a patient be unable to travel to NIH for a clinic visit.  
3.5.1 3.5.1.1  
Criteria for removal from protocol vaccine therapy Persistent failure of autologous ME TARP DC or elutriated monocyte placebo vaccine to meet DTM release criteria as determined by DTM and the Principal Investigator.  
Delay in vaccination beyond timeframes established in section 3.3.2
3.5.1.2 Patients experiencing a Grade 3 or greater toxicity outlined in sections 3.3.1.3 to 3.3.1.5 attributed as possibly, probably or definitely related to ME TARP DC vaccination.  
3.5.1.3  
Completion of protocol therapy (6 doses), including safety visit.
3.5.2
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off-study criteria

3.5.2.1  
Completion of study.
3.5.2.2 Removal from protocol vaccine therapy as outlined in Section 3.5.1.  
3.5.2.3  
Patient develops evidence of visceral or bony metastatic disease.
3.5.2.4  
Patients with a calculated PSADT of < 3 months after a minimum of 15 weeks on study or 4 doses of vaccine. Prior to removal from study, PSA check and PSADT calculation should be repeated at the next visit in 3 to 6 weeks from the date PSADT < 3 months is identified for an off-study decision.
3.5.2.5  
Patients with a calculated PSADT  that has decreased by > 50% from their calculated Pre-NIH PSADT on or after 24 weeks on study. Prior to removal from study, PSA
check and PSADT calculation should be repeated at the next visit in 3 to 6 weeks from the date PSADT decreased by > 50% is identified for an off-study decision.
3.5.2.6  
Development of a second malignancy other than basal cell or squamous cell carcinoma of the skin that is amendable to local treatment.
3.5.2.7 Patient elects to withdraw from study participation at any time.  
3.5.2.8 Patient is removed from study by the Principal Investigator or Lead Associate Investigator for reasons other than toxicity e.g. failure to adhere to study visits or due to  
the presence of an intercurrent medical condition, etc.
3.5.2.9  
Investigator decision to end the study 3.5.2.10  Death
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once a subject is taken

Authorized staff must notify Central Registration Office (CRO) when a subject is taken off protocol therapy and when a subject is taken off-study. A Participant Status Update Form from the web site (http://home.ccr.cancer.gov/intra/eligibility/welcome.htm) main page must be completed and sent via encrypted email to:  NCI Central Registration Office ncicentralregistration-l@mail.nih.gov .
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concomitant medications/measures 4

Study subjects will be allowed to take multivitamins, analgesics (NSAIDS or
.  
acetaminophen), antipyretics, and antihistamines for symptomatic relief of local or systemic injection site reactions.  
Patients are allowed to continue on medications as clinically indicated for treatment of
.  
chronic medical conditions e.g. hypertension, diabetes, hypercholesterolemia, etc.  
Excluded Therapy:
.  
Chemotherapy: Concomitant use of chemotherapy is not allowed during this trial.  
o Anti-Cancer Radionuclides: Concomitant use of anti-cancer radionuclides is not o allowed during this trial.  Patients will be allowed to co-enroll in studies of novel radionuclide imaging agents e.g. F-18 NaF PET/CT for quantification of metastatic burden in prostate cancer.  
Hormonal Therapies: Concomitant use of anti-androgen hormonal agents is not o alowed.  
Alternative Medications or Nutriceuticals Known to Alter PSA: Concomitant use of o agents known to alter PSA e.g. phytoestrogens and saw palmetto is not allowed.  
Note: Patients receiving medications for urinary symptoms such as Flomax or 5-alpha reductase inhibitors (finasteride and dutasteride) on a chronic stable dose for at least 3 months prior to study enrollment are allowed.  
Corticosteroids: Concomitant, chronic systemic corticosteroids are not allowed o  
during this trial (excepting emergent use for clinical indications).  However, the use of inhaled corticosteroids, intranasal sprays and topical creams on limited body areas is allowed.
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vitamin d3 (cholecalciferol) supplementation

Vitamin D when ingested is metabolized in the liver to 25-OH vitamin D.  Inside cells, it is metabolized further by 1-hydroxylase where it is transformed into a seco-steroid hormone that is important to a host of critical cellular and immune functions within the body.  Within cells is a second enzyme 24-hydroxylase whose function is to decrease vitamin D, thereby maintaining intracellular vitamin D homeostasis.  The classic function of vitamin D is to regulate calcium homeostasis and in turn, bone formation and resorption.  However, additional functions of vitamin D have been demonstrated and include effects on immune response by promoting cellular apoptosis and differentiation.  The exact role of vitamin D deficiency in prostate, breast, colon and other cancers has been controversial, with some laboratory studies suggesting there is a role and other epidemiological studies suggesting that there is no role or even possibly that supplementation should be avoided.  In a recent study by Marshall and colleagues (43) vitamin D
supplementation of 4.000 IU per day for one year was examined in men with low risk prostate cancer (Gleason score of 6, 1-6 cores positive out of 12 possible and a PSA <10) under active surveillance. After one year upon re-biopsy, 60% showed a decrease in the number of positive cores, Gleason score or both and in 6% these factors remained unchanged. In addition PSA
levels did not rise.   In another study reported by Wagner and colleagues at AACR in April 2012
(44), 66 men scheduled to undergo radical prostatectomy were randomly assigned to receive a dily vitamin D dose of 400, 10,000 or 40,000 IU daily for 3 to 8 weeks prior to surgery.  
Calcitriol levels in the prostate increased progressively with increasing vitamin D dosing and corresponded with lower levels of Ki67 as well as higher levels of specific growth-inhibitor microRNAs.  
Several studies have shown that women with low vitamin D levels have an increased risk of breast cancer incidence and mortality, but research is lacking investigating vitamin D levels and prognostic variables e.g. hormone receptor status, Oncotype DX etc. in this patient population.  
In a case control study of 194 women s/p breast cancer surgery and 194 cancer-free controls conducted by Peppone and colleagues at the University of Rochester (45), women with breast cancer were found to have significantly lower 25-OH vitamin levels than controls (32.7ng/mL
vs. 37.4 ng/mL respectively, P=0.02).  Importantly, women with suboptimal 25-OH vitamin D levels (<32 ng/mL) had significantly increased odds of having ER-negative (OR = 2.59, 95%
confidence interval [95% CI] = 1.08-6.23) and triple-negative cancer (OR = 3.15, 95%
CI = 1.05-9.49) than those with optimal 25-OH D concentrations. In addition, women with basallike phenotype had lower 25-OH vitamin D levels than women luminal A phenotype
(24.2 ng/mL vs. 32.8 ng/mL, respectively P = 0.04).  In summary, women with a more aggressive breast cancer molecular phenotype (basal-like) and worse prognostic indicators (ER- and triple-negative) had lower mean 25-OH vitamin D levels. Given its critical role in immune function and possible role in cancer pathophysiology, all patients will have serum 25-OH vitamin D levels obtained at baseline.  Although there is debate about the target level of 25-OH vitamin D for optimum health, many vitamin D experts agree that it should be greater than the 20 ng/mL recommended by the Institute of Medicine (IOM) and U.S. Food and Nutrition Board (46) and that levels of at least 40 ng/mL are necessary for optimum skeletal health as well as the other potential non-skeletal benefits of vitamin D (47).  
All patients with 25-OH vitamin D levels < 40 ng/mL will be initiated on oral supplements of Vitamin D3 (cholecalciferol) per standard clinical care guideline.
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s biospecimen collection 5.1 correlative

This study does not involve any investigation of pharmacokinetic or pharmacodynamic parameters.  
Exploratory analyses will be performed to identify vaccine characteristics using microarrays and TARP-specific cellular or humoral responses. Specific gene expression profiling of autologous ME DC vaccine products will examine genes involved in antigen uptake, apoptosis, cytokine and chemokine expression. We will also examine circulating tumor cells (CTCs) and immune subsets in peripheral blood mononuclear cells including iNKT and myeloid derived suppressor cells
(MDSCs) with Jane Trepel.  This will help to determine if the autologous ME TARP DC
vaccination is associated with a reduction in CTCs as compared to placebo and to assess the impact of vaccination on quantitative and qualitative parameters of iNKT cells. CTCs will also be analyzed in parallel using technology from RareCyte, Inc. to compare the quantitative parameters with those obtained using the standard techniques in the Trepel lab as part as the CRADA \#03048 (see section 11.1.2). In addition, the under the CRADA, molecular characterization of individual CTCs pre and post vaccination will also be performed.  
Preliminary assessment of immune status of subjects enrolled in the first generation 09-C-0139 TARP peptide vaccine study demonstrated normal absolute lymphocyte counts, CD4 and CD8 T-cell subset absolute counts, percentages and ratios as well as excellent immunity to CEF
peptide pooled antigens (CMV, EBV and Flu matrix peptide) utilized as positive controls in IFN-
y ELISPOT assays investigating TARP-specific reactivity.  We will plan to prospectively expand our characterization of immune status and senescence in subjects by exploring investigation of T-cell receptor v-beta repertoire analysis, CD4+CD25+FoxP3+ T-regulatory cell subsets and potentially T-cell receptor excision circles (TREC) as potential correlates of changes in slope log PSA/PSADT and clinical outcomes.
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5.2 sample storage tracking and

For research samples obtained for investigation, the Clinical Support Laboratory, Leidos Biomedical Research processes and cryopreserves samples in support of IRB-approved, NCI clinical trials. The laboratory is located in a controlled-access building and laboratory doors are kept locked at all times. Upon specimen receipt each sample is assigned a unique, sequential laboratory accession I.D. number. All products generated by the laboratory that will be stored either in the laboratory freezers or at a central repository facility are identified by this accession I.D.  An electronic database is used to store information related to patient samples processed by the laboratory. Vial labels do not contain any personal identifier information. Samples are stored inventoried in locked laboratory freezers and are routinely transferred to the NCI-
Frederick repository facilities for long-term storage. These facilities are operated under a subcontract to Leidos Biomedical Research. Access to stored clinical samples is restricted.  
Investigators establish sample collections under "Source Codes" and the investigator responsible for the collections, typically the protocol Principal Investigator and/or Lead Associate Investigator, specifies who has access to the collection.  
When requests are submitted by the NCI investigator for shipment of samples outside of the NIH it is the policy of the laboratory to request documentation that a Material Transfer Agreement
(MTA) is in place that covers the specimen transfer.  The laboratory does not provide patient identifier information as part of the transfer process but may, at the discretion of the NCI
investigator, group samples from individual patients when that is critical to the testing process. Samples will not be sent outside NIH without IRB notification and an executed MTA.  
Once primary research objectives for the protocol are achieved, intramural researchers can request access to remaining samples provided they have an IRB-approved protocol and patient consent.  Access to these samples will only be granted following IRB approval of an additional protocol, granting the rights to use the material.
untitled h1
5.2.2 future use / protocol

Blood, urine and tissue specimens collected in the course of this research project may be banked and used in the future to investigate new scientific questions related to this study.  However, this research may only be done if the risks of the new questions were covered in the consent document.  If new risks are associated with the research (e.g., analysis of germ line genetic mutations.) the Principal Investigator must amend the protocol and obtain informed consent from all research subjects. Following completion of this study, samples will remain in storage as detailed above unless a patient has opted out of the future use of specimens and data.  Currently, there is no plan to use these samples outside of the use described in the protocol.  
If the patient withdraws consent, the participant's data will be excluded from future distributions, but data that have already been distributed for approved research use will not be able to be retrieved. The PI will record any loss or unanticipated destruction of samples as a deviation. Reporting will be per the requirements of section 7.2.1.
untitled h1
5.3 s amples for g

No genetic/genomic analyses will be performed as part of this clinical investigation.
untitled h1
data collection and evaluation 6

1.  
Each patient must meet all eligibility requirements and a completed registration must be sent to the NCI Central Registration Office (CRO).  
2.  
The Consent Document must be signed prior to registration with the CRO.
3.  
Treatment will be given according to protocol (on-study and treatment notes, reports of adverse events and documentation of any deviation from the study protocol).
4.  
Data will be entered into a secure software system (C3D Database) produced by Oracle™ Corporation (Redwood Shores, CA).  Data will be collected based on protocol-specific requirements, verified for accuracy and completeness.  Any hard copy data will be kept in locked secure area in the Vaccine Branch Clinical Trials Offices.
5.  
6.  
Toxicity will be assessed according to the protocol using the CTCAE v4.0 that is available at: http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm Response to autologous ME TARP DC or elutriated monocyte placebo vaccination will be assessed by calculation of slope log (PSA)/PSADT at every protocol study time point and specifically used to examine:
o The slope log (PSA for Weeks 3-24 minus that formed for the 12 months prior to enrollment on study (referred to as slope 324 - pre-slope) as well as the slope log
(PSA) for weeks 3-48 versus the same pre-treatment slope log (PSA) (referred to
as slope 348 - preslope) in patients naïve to TARP vaccination receiving active, multi-epitope TARP vaccination vs. placebo.  
7.  
Results of re-staging scans at Weeks 48 and 96 will be documented and verified that the patient remains Stage D0 without evidence of progressive disease unless the scans or clinical findings demonstrate otherwise.  
8.  
Drug accountability records will be maintained for each patient.  
9.  
Vaccine report cards (VRCs) associated with each autologous ME TARP DC or elutriated monocyte placebo vaccination will be obtained for each patient (see Appendix D). If the VRC is not available, investigators may describe the grade and duration of the injection site reaction based on the patient's verbal report. The information recorded on the VRC will also be documented in the medical record.  
10.  
Personal identifiers will not be used when collecting and storing data.  
11.  
An enrollment log will be maintained in the regulatory binder/file that is the only location of personal identifiers with unique subject identification number.  
The PI will be responsible for overseeing entry of data into an in-house password protected electronic system (C3D) and ensuring data accuracy, consistency and timeliness. The principal investigator, associate investigators/research nurses and/or a contracted data manager will assist with the data management efforts.  All data obtained during the conduct of the protocol will be kept in secure network drives or in approved alternative sites that comply with NIH security standards. Primary and final analyzed data will have identifiers so that research data can be attributed to an individual human subject participant. All adverse events, including clinically significant abnormal findings on laboratory evaluations, regardless of severity, will be followed until return to baseline or stabilization of event. Serious adverse events that occur more than 6 weeks  after the last administration of investigational agent/intervention and have an attribution of at least possibly related to the agent/intervention should be recorded and reported as per sections Error! Reference source not found. and 7.4.  
AEs will be documented starting with the first study intervention through 60 days following the last administration of vaccine.  Adverse events that are serious need to be recorded after 60 days following the last intervention, only if they are serious and related to the study intervention.  
An abnormal laboratory value will be considered an AE if the laboratory abnormality is characterized by any of the following:
.  
.  
.  
.  
.  
.  
Results in discontinuation from the study Is associated with clinical signs or symptoms Requires treatment or any other therapeutic intervention Is associated with death or another serious adverse event, including hospitalization.  
Is judged by the Investigator to be of significant clinical impact If any abnormal laboratory result is considered clinically significant, the investigator will provide details about the action taken with respect to the test drug and about the patient's outcome.  
End of study procedures: Data will be stored according to HHS, FDA regulations and NIH
Intramural Records Retention Schedule as applicable.  
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A hospitalization/admission that is pre-planned (i.e., elective or scheduled surgery arranged prior to the start of the study), a planned hospitalization for pre-existing condition, or a procedure required by the protocol, without a serious deterioration in health, is not considered a serious adverse event.
o  
A hospitalization/admission that is solely driven by non-medical reasons (e.g.,
hospitalization for patient convenience) is not considered a serious adverse event.
o  
Emergency room visits or stays in observation units that do not result in admission to the hospital would not be considered a serious adverse event. The reason for seeking medical care should be evaluated for meeting one of the other serious criteria.
.  
Persistent or significant incapacity or substantial disruption of the ability to conduct normal life functions
.  
A congenital anomaly/birth defect
.  
Important medical events that may not result in death, be life-threatening, or require
hospitalization may be considered a serious adverse drug experience when, based upon appropriate medical judgment, they may jeopardize the patient or subject and may require medical or surgical intervention to prevent one of the outcomes listed in this definition.  
8.1.3  
Life-threatening
An adverse event or suspected adverse reaction is considered "life-threatening" if, in the view of either the investigator or sponsor, its occurrence places the patient or subject at immediate risk of death. It does not include an adverse event or suspected adverse reaction that, had it occurred in a more severe form, might have caused death. (21CFR312.32)
8.1.4 Severity The severity of each Adverse Event will be assessed utilizing the CTCAE version 4.0.  
Relationship to Study Product 8.1.5 All AEs will have their relationship to study product assessed using the terms:  related or not related.  
.  
Related - There is a reasonable possibility that the study product caused the adverse event. Reasonable possibility means that there is evidence to suggest a causal relationship between the study product and the adverse event.
.  
Not Related - There is not a reasonable possibility that the administration of the study product caused the event.
untitled h1
8.2 assessment of safety events

AE information collected will include event description, date of onset, assessment of severity and relationship to study product and alternate etiology (if not related to study product), date of resolution of the event, seriousness and outcome. The assessment of severity and relationship to the study product will be done only by those with the training and authority to make a diagnosis and listed on the Form FDA 1572 as the site principal investigator or sub-investigator.  AEs occurring during the collection and reporting period will be documented appropriately regardless of relationship. AEs will be followed through resolution.
untitled h1
ses will be

.  
Assessed for severity and relationship to study product and alternate etiology (if not related to study product) by a licensed study physician listed on the Form FDA 1572 as the site principal investigator or sub-investigator.  
.  
Recorded on the appropriate SAE report form, the medical record and captured in the clinical database.  
.  
Followed through resolution by a licensed study physician listed on the Form FDA 1572 as the site principal investigator or sub-investigator.
For timeframe of recording adverse events, please refer to section 6.1.  All serious adverse events recorded from the time of first investigational product administration must be reported to the sponsor.
untitled h1
reporting of serious adverse events

Any AE that meets protocol-defined serious criteria or meets the definition of Adverse Event of Special Interest that require expedited reporting must be submitted immediately (within 24 hours of awareness) to OSRO Safety using the CCR SAE report form.  
All SAE reporting must include the elements described in section 8.2.  
SAE reports will be submitted to the Center for Cancer Research (CCR) at: OSROSafety@mail.nih.gov and to the CCR PI and study coordinator.  CCR SAE report form and instructions can be found at:
https://ccrod.cancer.gov/confluence/pages/viewpage.action?pageId=157942842 Following the assessment of the SAE by OSRO, other supporting documentation of the event may be requested by the OSRO Safety and should be provided as soon as possible.
untitled h1
8.4 reporting pregnancy 8.4.1 paternal

Male patients should refrain from fathering a child or donating sperm during the study and for 30 after the last vaccine dose. Pregnancy of the patient's partner is not considered to be an AE. However, the outcome of all pregnancies (spontaneous miscarriage, elective termination, ectopic pregnancy, normal birth, or congenital abnormality) occurring from the date of the first dose until 30 days after the last dose should, if possible, be followed up and documented.
untitled h1
8.5 regulatory reporting for studies

Following notification from the investigator, CCR, the IND sponsor, will report any suspected adverse reaction that is both serious and unexpected in an expedited manner to the FDA in accordance to 21 CFR 31.2.32.  CCR will report an AE as a suspected adverse reaction only if there is evidence to suggest a causal relationship between the study product and the adverse
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being given, that the slope 324 - pre-slope may be at least 0.010 or greater, such as 0.015, as it would be expected that the log slope (PSA) would likely very slowly increase, as a minimum, in the absence of treatment. Choosing the smaller difference would suggest that the difference in means for the two groups could be approximately -0.033 vs. +0.015, or a difference of -0.048.  
Assuming a common standard deviation of 0.072 (as was approximately found on the TARP arm B in the prior trial), this would mean that detecting an effect size of 0.67 SD (0.048//0.072=0.67)
could be a reasonable goal. With a 2:1 randomization (2 getting vaccine to 1 getting placebo),
with 44 on the active arm and 22 on the placebo arm, there is 81% power to detect a difference between the two arms on the order of 0.048 slope log PSA units, assuming the parameters from the prior study may be approximately true and a 0.05 one-sided significance level is sought. The analysis is assumed to be done with a two- group t-test, while in practice a Wilcoxon rank sum test will be used if the values in the two arms are not normally distributed (p<0.05 by a Shapiro- Wilks test).   In addition, although using a 0.05 significance level for each test assumes that a Bonferroni correction will be used to adjust the two tests for multiple comparisons, in practice, a less overly conservative Hochberg test may be used to interpret the results. It should also be noted that since the two primary endpoints involve overlapping intervals of time, the results may not be independent of one another and thus this should factor into the interpretation at the conclusion of the study. In addition to the two primary endpoints, the change in log slope (PSA) at later time points, such as weeks 48-96, may also be evaluated as a secondary endpoint. As this is a prospective, singleblinded, randomized placebo controlled trial, the NCI/CCR DSMB will be used to monitor the trial. This monitoring will be done for both futility and for efficacy, as well as for safety.  
The futility monitoring will be performed as follows:  at the first DSMB meeting after which half of the projected patients have been evaluated (33 total patients have been evaluated for both the changes to 24 weeks and the changes to 48 weeks), the slope differences will be calculated on both arms. If at this point, the slope difference for the placebo arm indicates the same or greater benefit compared to that for the active arm, then the trial will not accrue any further patients since the results to date would indicate lack of benefit from using the active vaccine.  
The evaluation for better than expected efficacy will be performed as follows:  at the first DSMB
meeting after which half of the projected patients have been evaluated (33 total patients have been evaluated for both the changes to 24 weeks and the changes to 48 weeks), the slope differences will be calculated on both arms. If at this point, the one tailed p-value is<0.0054, then the trial will stop accrual for better than expected efficacy. By using this interim evaluation for monitoring, the final significance level would need to be <0.0492 to declare the result to be significant at the 0.05 level. Safety and evaluation of other immune parameters will also be included in the analyses, as secondary objectives. These will be explored primarily with descriptive statistics and any comparisons between the two arms will be reported without any formal adjustment for multiple cmparisons.  
With 66 patients as an objective for the randomized portion of the study (N = 44 ME TARP
vaccine and N = 22 elutriated monocyte placebo vaccine) and the initial lead in of 6 patients, a total of 72 patients may be required. In order to allow for a small number of inevaluable subjects, the accrual ceiling will be set at 75. For the data collection and analysis, we will define the evaluable patients as receiving at least 3 doses of vaccines. It is anticipated that up to 2-3 years may be required to enroll 66 patients to the randomized portion of the study.
untitled h1
1 collaborative agreements 11.1 cooperative

1.1.1  CRADA \# 03039 was previously place between the Vaccine Branch, CCR, NCI and PDS
Biotechnology, Newark New Jersey in order to carry out ongoing studies of chemistry, manufacturing, control and optimization of the ME TARP peptide dendritic cell cancer vaccine platform utilizing PDS' proprietary Versamune® immunotherapeutic technology to optimize peptide antigen uptake and processing.  This will be accomplished by studies in the Cell Processing Section in the department of Transfusion Medicine: parallel DC vaccine manufacturing runs and final product parameters will be compared to document whether there is better antigen expression without compromise of the FDA mandated release criteria. The vaccines were manufactured in parallel with Versamune® will NOT be administered to study subjects.
untitled h1
note as of amendment h

11.1.2 CRADA \# 03048 is in place between the Vaccine Branch, CCR, NCI and RareCyte, Inc to investigate the use of RareCyte's technology as described in section 5.1.
untitled h1
12 human subjects protections 12.1

All subjects enrolled in this study will be male as the disease under study does not affect women or children.  We will make every effort to accrue patients from all racial and ethnic groups. Since our referral population is the nation, we should obtain referrals representative of the ntional composition of races.  Since this trial is investigating a multi-epitope TARP peptide vaccine platform, there is no need for HLA restriction as was required in the 09-C-0139 pilot study of HLA-A*0201 restricted WT27-35 and EE29-37-9V TARP peptides Participants will be accrued through web-based advertisements, community-based contacts and referrals of patients from other studies at the Clinical Center.  This protocol will also be advertised on the NCI Clinical Trials Search website as well as through ClinicalTrials.gov.
untitled h1
12.2 participation of subjects unable

Adults unable to give consent are excluded from enrolling in the protocol.  However, re-consent may be necessary and there is a possibility, though unlikely, that subjects could become decisionally impaired. For this reason and because there is a prospect of direct benefit from research participation (section 12.4), all subjects ≥ age 18 will be offered the opportunity to fill in their wishes for research and care, and assign a substitute decision maker on the "NIH Advance Directive for Health Care and Medical Research Participation" form so that another person can make decisions about their medical care in the event that they become incapacitated or cognitively impaired during the course of the study. Note: The PI or AI will contact the NIH
Ability to Consent Assessment Team (ACAT) for evaluation as needed for the following: an independent assessment of whether an individual has the capacity to provide consent; assistance in identifying and assessing an appropriate surrogate when indicated; and/or an assessment of the capacity to appoint a surrogate.  For those subjects that become incapacitated and do not have pre-determined substitute decision maker, the procedures described in NIH HRPP SOP 14E for appointing a surrogate decision maker for adult subjects who are (a) decisionally impaired, and
(b) who do not have a legal guardian or durable power of attorney, will be followed.
untitled h1
12.3 participation of children

Since the safety of Multi-Epitope TARP has not been adequately assessed in children, patients under the age of 18 years will not be eligible to participate.
untitled h1
12.4 e valuation of b

The potential benefit of vaccination with multi-epitope TARP peptides is unknown.  Although a majority of patients (~72%) in our first 09-C-0139 TARP peptide vaccine study exhibited a decline in the slope log PSA compared to their Pre-NIH slope that was sustained out to 48 weeks, these responses were associated with absolute declines in PSA levels in only a minority of patients (~15% of responders).  In addition, it is unknown and unproven whether slowing PSA velocity through vaccination or any other treatment intervention, will in turn impact clinical outcomes.  Ongoing long-term observation out to 144 weeks on the 09-C-0139 study suggests that responses to TARP vaccination may wane.  In addition, there were study subjects who had objective evidence of clinical progression following an initial, favorable slowing in PSA velocity.  We believe that this second generation multi-epitope TARP vaccine comprised of longer synthetic peptides will include MHC class II CD4+ T cell helper epitopes that will allow generation of better CD8+ T cell responses with improved functional avidity and longevity (42) as well as humoral anti-TARP antibody responses.  
TARP peptide vaccination was shown to be safe and very well tolerated in the 09-C-0139 study and we think it is highly likely that the multi-epitope TARP vaccine will have a similar, favorable safety and tolerability profile.  Vaccination was also associated with induction of TARP-specific cellular immune responses in a majority of patients (~75%) as well.  However the clinical significance of these immune responses remains unknown. In this study, we will attempt to definitely establish whether the observed declines in the difference in the slope log (PSA)
documented in the 09-C-0139 study are real and not due to spontaneous or random slowing in the rate of PSA rise.  We will also conduct an exploratory assessment of progression free survival (PFS) at 96 weeks to see if there is any indication of efficacy regarding clinical outcomes in patients receiving autologous ME TARP DC vaccination.  The most significant risk for study subjects randomized to receive the autologous elutriated monocyte vaccine placebo is the apheresis required to collect cells, as autologous, minimally manipulated thawed elutriated monocytes are unlikely to be associated with any adverse reactions other than local injection site discomfort.
untitled h1
12.5 r isks / b

This study offers patients the prospect of direct benefit and the treatment has an acceptable risk profile. Therefore, the benefits outweigh the risks. The risk/benefit analysis is the same for patients unable to consent as it is for less vulnerable patients.
untitled h1
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13.4.4 administration procedures

Autologous peptide-pulsed dendritic cell vaccines will be prepared under GMP conditions from cryopreserved patient monocytes. After thaw, the monocytes will be placed into a 5 day culture with rhIL-4 and rhGM-CSF to generate immature dendritic clls, followed by pulse with KLH and maturation with LPS and IFN-γ.  A fraction of autologous dendritic cells will be pulsed separately with TARP WT27-35 peptide. After removing peptide-pulsing media, dendritic cells, individual fractions will be combined and will be concentrated down at 40 x 10 6 cells/mL in infusion media (Plasma-Lyte A containing 10% autologous heat inactivated plasma). The final peptide-loaded, volumereduced mature dendritic cell product will be prepared in sterile syringes for fresh administration intradermally.
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13.4.5 incompatibilities none known 13.4.6

As the TARP peptide is being delivered as a vaccine, likely adverse events include local injection site reactions commonly associated with vaccination.  TARP is found in normal prostate tissue as well and over expressed prostate cancer. Because the peptide mimics portions of a prostate protein found naturally in the body, there is a chance for development of an autoimmune reaction to it and may result in the possible development of inflammation in the prostate gland (if not removed with radical prostatectomy).  
However, this was not observed in the initial pilot 09-C-0139 study investigating TARP
peptide vaccination.
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13.4.7 special handling

The peptide is NOT a cytotoxic or infectious agent and requires no special handling.
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13.5 tarp 29-37-9v peptide (epitope-enhanced)

TARP 29-37-9V is investigational. TARP 29-37-9V, also called TARP 29-37 (37V) is a synthetic HLA-A2-restricted 9-amino acid epitope of the tumor associated protein TARP, with a single amino acid substitution (valine at position 9 in this peptide or position 37 in full TARP protein, instead of leucine) to increase its binding affinity and immunogenicity. Amino acid sequence: Phenylalanine-Leucine-Arginine-Asparagine-
Phenylalanine-Serine-Leucine-Methionine-Valine (FLRNFSLMV)
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molecular weight 1126.4 13.5.2 formulation

The peptide is vialed in a 5 mL siliconized sterile amber type 1 glass vial with a Teflonlined stopper containing 0.5 mL of a sterile clear solution.  Each mL contains 2.2 mg of TARP:29-37(37V) Peptide and 0.5 mcL of trifluoroacetate 0.05% v/v. Vaccine preparation will occur according to Protocol Specific Instructions (PSI) and established SOPs of The Center for Cellular Engineering, NIH Clinical Center.
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13.5.3 stability and storage

Store the finished injectable dosage forms in the freezer (-70°C or below) for long-term storage. Intact vials are stable for at least 6 months when stored at controlled room temperature (15°C - 30°C), at least 9 months when stored in the refrigerator (2°C - 8°C),
and for at least 36 months when stored in the freezer (-10ºC to -25ºC and -70ºC). The peptide vial contains no preservatives; once the peptide vial is entered, discard unused peptide solution after 3 hours. Stability will be monitored according to a stability program approved by the NIH CC Pharmacy.
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13.5.4 administration procedures

Autologous peptide-pulsed dendritic cell vaccines will be prepared under GMP
conditions from cryopreserved patient monocytes. After thaw, the monocytes will be placed into a 5 day culture with rhIL-4 and rhGM-CSF to generate immature dendritic clls, followed by pulse with KLH and maturation with LPS and IFN-γ.  A fraction of autologous dendritic cells will be pulsed separately with TARP WT27-35 peptide. After removing peptide-pulsing media, dendritic cells, individual fractions will be combined and will be concentrated down at 40 x 10 6 cells/mL in infusion media (Plasma-Lyte A
containing 10% autologous heat inactivated plasma). The final peptide-loaded, volumereduced mature dendritic cell product will be prepared in sterile syringes for fresh administration intradermally.
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13.5.5 incompatibilities none known.

13.5.6 Reported Adverse Events and Potential Risks:
As the TARP peptide is being delivered as a vaccine, likely adverse events include local injection site reactions commonly associated with vaccination.  TARP is found in normal prostate tissue as well and over expressed prostate cancer. Because the peptide mimics portions of a prostate protein found naturally in the body, there is a chance for development of an autoimmune reaction to it and may result in the possible development of inflammation in the prostate gland (if not removed with radical prostatectomy).  
However, this was not observed in the initial pilot 09-C-0139 study investigating TARP peptide vaccination.
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13.5.7 special handling

The peptide is NOT a cytotoxic or infectious agent and requires no special handling.
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13.6 tarp 1-20 peptide 13.6.1

TARP 1-20 is investigational. Amino Acid Sequence: H-Met-Gln-Met-Phe-Pro-Pro-Ser-
Pro-Leu-Phe-Phe-Leu-Gln-Leu-Leu-Lys-Glyn-Ser-Ser-OH Acetate.  
13.6.2 Formulation and preparation:
The peptide is vialed by in a 2 mL clear type-1, borosilicate glass vial with a 13 mm gray, chlorobutyl, polytetrafluoroethylene (PTFE) "Teflon" lined stopper, and a 13 mm aluminum flip-off seal.  Vial contains 1.2 mL of a 1 mg/mL sterile solution of TARP 1-  
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containing 10% autologous heat inactivated plasma). The final peptide-loaded, volumereduced mature dendritic cell product will be prepared in sterile syringes for fresh administration intradermally.
untitled h1
13.8.2 formulation and preparation

The peptide is vialed in a 2 mL clear type-1, borosilicate glass vial with a 13 mm gray, chlorobutyl, polytetrafluoroethylene (PTFE) "Teflon" lined stopper, and a 13 mm aluminum flip-off seal.  Vial contains 1.2 mL of a 1 mg/mL sterile solution of TARP 2140 Peptide (MPS-481) in sterile water for injection. Vaccine preparation will occur according to Protocol Specific Instructions (PSI) and established SOPs of The Center for Cellular Engineering, NIH Clinical Center.
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13.8.3 stability and storage

Peptide is stored at -70°C or below. Stability will be monitored according to a program approved by the NIH CC Pharmacy. Administration procedures: Autologous peptide-pulsed dendritic cell vaccines will be prepared under GMP
conditions from cryopreserved patient monocytes. After thaw, the monocytes will be placed into a 5 day culture with rhIL-4 and rhGM-CSF to generate immature dendritic clls, followed by pulse with KLH and maturation with LPS and IFN-γ.  A fraction of autologous dendritic cells will be pulsed separately with TARP 21-40 peptide. After removing peptide-pulsing media, dendritic cells, individual fractions will be combined and will be concentrated down at 40 x 10 6 cells/mL in infusion media (Plasma-Lyte A
containing 10% autologous heat inactivated plasma). The final peptide-loaded, volumereduced mature dendritic cell product will be prepared in sterile syringes for fresh administration intradermally.
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13.9.2 formulation and preparation

The peptide is vialed in a 2 mL clear type-1, borosilicate glass vial with a 13 mm gray, chlorobutyl, polytetrafluoroethylene (PTFE) "Teflon" lined stopper, and a 13 mm aluminum flip-off seal.   Vial contains 1.2 mL of a 1 mg/mL sterile solution of TARP 3150 Peptide (MPS-482) in sterile water for injection. Vaccine preparation will occur according to Protocol Specific Instructions (PSI) and established SOPs of The Center for Cellular Engineering, NIH Clinical Center.
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13.9.3 stability and storage

Peptide is stored at -70°C or below. Stability will be monitored according to a program approved by the NIH CC Pharmacy.
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13.9.4 administration procedures

Autologous peptide-pulsed dendritic cell vaccines will be prepared under GMP
conditions from cryopreserved patient monocytes. After thaw, the monocytes will be placed into a 5 day culture with rhIL-4 and rhGM-CSF to generate immature dendritic cells, followed by pulse with KLH and maturation with LPS and IFN-γ.  A fraction of autologous dendritic cells will be pulsed separately with TARP 31-50 peptide. After removing peptide-pulsing media, dendritic cells, individual fractions will be combined and will be concentrated down at 40 x 10 6 cells/mL in infusion media (Plasma-Lyte A containing 10% autologous heat inactivated plasma). The final peptide-loaded, volumereduced mature dendritic cell product will be prepared in sterile syringes for fresh administration intradermally.
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13.10 tarp 41-58 peptide 13.10.1

TARP 41-58 is investigational. Amino Acid Sequence: H-Ile-Gly-Lys-Lys-Arg-Arg-Ala- Thr-Arg-Phe-Trp-Asp-Pro-Arg-Arg-Gly-Thr-Pro-OH Acetate 13.10.2 Formulation and preparation:
The peptide is vialed in a 2 mL clear type-1, borosilicate glass vial with a 13 mm gray, chlorobutyl, polytetrafluoroethylene (PTFE) "Teflon" lined stopper, and a 13 mm aluminum flip-off seal.  Vial contains 1.2 mL of a 1 mg/mL sterile solution of TARP 4158 Peptide (MPS-483) in sterile water for injection.  Vaccine preparation will occur according to Protocol Specific Instructions (PSI) and established SOPs of The Center for Cellular Engineering, NIH Clinical Center.
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13.10.3 stability and storage

Peptide is stored at -70°C or below. Stability will be monitored according to a program approved by the NIH CC Pharmacy. .
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13.10.4 administration procedures

Autologous peptide-pulsed dendritic cell vaccines will be prepared under GMP
conditions from cryopreserved patient monocytes. After thaw, the monocytes will be placed into a 5 day culture with rhIL-4 and rhGM-CSF to generate immature dendritic clls, followed by pulse with KLH and maturation with LPS and IFN-γ.  A fraction of autologous dendritic cells will be pulsed separately with TARP 41-58 peptide. After removing peptide-pulsing media, dendritic cells, individual fractions will be combined and will be concentrated down at 40 x 10 6 cells/mL in infusion media (Plasma-Lyte A containing 10% autologous heat inactivated plasma). The final peptide-loaded, volumereduced mature dendritic cell product will be prepared in sterile syringes for fresh administration intradermally.
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13.11 vitamin d3 supplements 13.11.1

Vitamin D3 (also known as cholecalciferol) is available as an over-the-counter nutritional/vitamin supplement in capsule, tablet and liquid drop form.  For study subjects identified to have 25-OH vitamin D levels < than 40 ng/mL on screening, vitamin D3 supplementation will be recommended rather than vitamin D2 since cholecalciferol is the form of vitamin D naturally produced by the skin in response to sun exposure.
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13.11.2 formulation and preparation

Vitamin D3 supplements are manufactured by multiple suppliers and come in standard dose formulations ranging from 100 to 5000 IU per dose in capsule, tablet or liquid drop form.  
13.11.3 Stability and Storage:
Oral vitamin D3 supplements should be stored according to the manufacturer's specifications.  
13.11.4 Administration Procedures:
Patients will receive Vitamin D3 according to standard of care guidelines. The dose of 2000 IU daily is consistent with the recommended daily dose of 1500 - 2000 IU by the Endocrine Society (48) and is also less than the upper limit of 4000 IU/day set by the U.S. Food and Nutrition Board (46).
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13.11.5 incompatibilities none known. 14

l.
Cheever MA, Allison JP, Ferris AS et al.   The prioritization of cancer antigens: a National Cancer Institute pilot project for the acceleration of translational research.  Clin Canc Res 2009;15:5323-5337.  
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| ?@ABC                           | DCEF@GHIGJKE                                                                                                                                                                            |
| L                               | MJ@NAO AFIGPGIQR STOOQ AFIGPCU AVOC IJ FA@@Q JK AOO H@CWBGECAEC  HC@XJ@NAKFC YGIZJTI @CEI@GFIGJKR                                                                                       |
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|                                  | DCABR                                                                                                                                                                                   |
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loaded, volume-reduced mature dendritic cell product will be prepared in sterile syringes for fresh administration intradermally.  A validated manufacturing process described in the Department of Transfusion Medicine, Clinical Center, NIH standard operating procedures will prepare the dendritic cell vaccine product. Detailed standard operating procedures for processing, labeling, storage, and quality assays are available on site in the Cell Processing Section of the Department of Transfusion Medicine.
untitled h1
standard operating procedure for elutriated

Blood collection shall be by standard lymphapheresis.10 to 15 liters of whole blood will be processed in order to collect peripheral blood mononuclear cells (PBMC).  Lymphocytes will also be cryopreserved. Apheresis will be performed in the Clinical Center (CC) Department of Transfusion Medicine (DTM) using approved standard operating procedures. Bilateral peripheral venous access will be used for apheresis whenever possible. Alternatively, a temporary femoral central venous catheter (CVL) will be placed as an outpatient, if indicated, for collection on the dy of apheresis. The CVL will be inserted by appropriately trained personnel in special procedures with removal of the CVL by 3SE day hospital or DTM clinical staff.  Prophylactic intravenous CaC12 and MgSO4 infusions may be administered during apheresis to treat or prevent citrate toxicity at the discretion of the DTM physician per routine.
untitled h1
patient cell processing

All cell processing will be conducted in accordance with approved DTM policies and procedures.  
Formulation and Preparation Elutriated Monocyte Placebo Vaccine: Autologous elutriated monocyte placebo cell vaccines will be prepared under cGMP conditions from cryopreserved patient monocytes obtained during the original Week 0 apheresis. Autologous monocytes for these placebo vaccines will be enriched from peripheral blood MNC apheresis collections by counter-flow elutriation, aliquoted into at least 8 vials with ~ 333 x 10 6 cells/vial and cryopreserved for future preparation of the elutriated monocyte placebo cell vaccine products. Elutriated monocytes will be thawed the morning of scheduled vaccine delivery.  After thaw, elutriated monocytes will be concentrated down at 40 x 10 6 cells/mL in infusion media (Plasma- Lyte A containing 10% autologous heat inactivated plasma). The final, volume-reduced elutriated monocyte product will be prepared in sterile syringes for fresh administration intradermally.  A validated manufacturing process described in the Department of Transfusion Medicine, Clinical Center, NIH standard operating procedures will prepare the elutriated monocyte placebo vaccine product. Detailed standard operating procedures for processing, labeling, storage, and quality assays are available on site in the Cell Processing Section of the Department of Transfusion Medicine.  
Stability and Storage:  Autologous ME TARP peptide-pulsed dendritic cell vaccines will be harvested from the 5-day culture product and autologous elutriated monocyte placebo vaccine from the single day thaw product.  Both will be packaged for fresh administration on the same dy according to Standard Operating Procedures of the Department of Transfusion Medicine. A fixed autologous ME TARP peptide-pulsed dendritic cell or elutriated monocyte placebo vaccine dose of 20 X 10 6 viable cells/ in 0.25mL or 0.5mL will be administered immediately upon Abbreviated Title: TARP Phase II
Version Date: 2/11/2020 receipt in the clinical setting.  Post packaging tests indicated that the product is stable for at least 2 hours.  
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