CITY OF HOPE NATIONAL MEDICAL CENTER 1500 E. DUARTE · PDF filecity of hope national medical...

of 68

IRB Protocol No. 12022 Version Date: 07/21/2015 Version: 16

CITYOFHOPENATIONALMEDICALCENTER1500E.DUARTEROADDUARTE,CA91010

DEPARTMENT OF HEMATOLOGY & HEMATOPOIETIC CELL TRANSPLANTATION

TITLE: A Phase I Trial To Evaluate Safety And Immunogenicity Of A Cytomegalovirus Peptide Vaccine Co-Injected With PF-03512676 Adjuvant In Recipients Of Allogeneic Hematopoietic Stem Cell Transplant

CITY OF HOPE PROTOCOL NUMBER/VERSION: IRB # 12022 VERSION: 16

Date (S)/ OF AMENDMENT(S)/REVISION(S):

COH Initial Approval Protocol Dated 6/28/2012 Version: 00

COH Amendment 01 Protocol Dated 07/19/2012 Version: 01



COH Amendment 04 Title Page Dated 01/15/2013 Version: 04













SPONSOR/IND NUMBER: BB-13124 DISEASE SITE: Hematopoietic system

of 68


STAGE (if applicable): Hematological malignancies requiring hematopoietic cell transplantation MODALITY: Vaccine Evaluation PHASE/TYPE: Phase I, Therapeutic

PRINCIPAL INVESTIGATOR:

Ryotaro Nakamura M.D. City of Hope National Medical Center

COLLABORATING INVESTIGATOR(S):

City of Hope National Medical Center

Don J. Diamond Ph.D. Stephen J. Forman M.D. Corinna La Rosa Ph.D. John A. Zaia M.D.

PARTICIPATING CLINICIANS:

City of Hope National Medical Center

. Ji-Lian Cai M.D. Robert Chen M.D. Len Farol M.D. Myo Htut M.D. Chatchada Karanes M.D. Samer Khaled M.D. Amrita Krishnan M.D. Auayporn Nademanee M.D. Margaret O’Donnell M.D. Pablo Parker M.D Leslie Popplewell M.D. Firoozeh Sahebi M.D. Tanya Siddiqi M.D. Eileen Smith M.D. David Snyder M.D. George Somlo M.D. Ricardo Spielberger M.D. Anthony Stein M.D. Sean (Shirong) Wang, M.D. Michael Rosenzweig, M.D. Ahmed Aribi, M.D.Nitya Nathwani, M.D

STUDY STATISTICIAN: Jeff Longmate, Ph.D. Department of Information Sciences

PARTICIPATING SITES: City of Hope National Medical Center

of 68


of 68


Experimental Design Schema

*Hematopoietic Cell Stem Transplant

All enrolled patients will undergo HCT standard of care

INTERVENTION

of 68


Protocol Synopsis

Protocol Title:

A Phase I Trial To Evaluate Safety And Immunogenicity Of A Cytomegalovirus Peptide Vaccine Co-Injected With PF-03512676 Adjuvant In Recipients Of Allogeneic Hematopoietic Stem Cell Transplant

Brief Protocol Title for the Lay Public (if applicable):

Evaluation Of A Vaccine To Protect Cancer Patients Receiving Stem Cell Transplant From Life-Threatening Cytomegalovirus Infection

Study Phase:

Phase I, Therapeutic

Participating Sites:

CITY OF HOPE NATIONAL MEDICAL CENTER

Rationale for this Study:

Cytomegalovirus (CMV) infection is the cause of significant complications in CMV+ recipients of allogeneic HCT, a leading therapy for hematological malignancies. Preemptive antiviral chemotherapy limits CMV infection, though its use can have significant toxicity1. A CMV vaccine that confers protective immunity early post-HCT, until normal immunocompetence is re-established in the recipient may reduce CMV morbidity and use of antivirals2. It has been reported that T cells specific for CMV-pp65, a principal target for CD8+ cytotoxic T cells (CTL) can protect HCT recipients from CMV complications3,4. Our group has developed Tet-CMV (IND BB-13124), a peptide vaccine composed of an HLA A*0201 restricted pp65495-503 CTL epitope, covalently linked to a universal tetanus T-helper (TH) epitope5. HLA-restricted peptide epitopes are a promising option to develop a safe, non-infectious subunit CMV vaccine, avoiding CMV immune-evasion mechanisms3. Acceptable safety profiles and vaccine-driven expansion of pp65 T cells in healthy HLA A*0201 adults vaccinated twice with Tet-CMV and PF-03512676 adjuvant support further evaluation of this vaccine formulation in HCT recipients6. Transferring CMV immunity to HCT recipients, by administering Tet-CMV + PF-03512676 to their donors would logically precede direct vaccination of recipients7. However, modern HCT clinical practice makes it impractical to have extended access to the donor, and prior results have shown safety and efficacy of directly immunizing recipients, thereby justifying our evaluation of this approach8. Additionally, in HCT recipients with prolonged CD4+ T cell deficit, stimulation of Toll-like receptor 9 (TLR9) by PF-03512676 could effectively substitute for the normal requirement of TH, as shown in HIV subjects with compromised TH function9. Since PF-03512676 was found to be safe when administered to HCT recipients in a Phase Ib trial (Dr. J. Miller, University of Minnesota, personal communication), there is a precedent for our proposed use in HCT recipients. Importantly, while immune-reconstitution can be limited in allo-HCT population, Tet-CMV has the potential for greater potency when injected into CMV+ HCT recipients, as massive expansion of pp65-specific T cells can occur under the conditions of CMV infection10. Based on these findings, we will perform an evaluation of safety (primary endpoint) and immunogenicity (secondary endpoint) of two injections of Tet-CMV + PF-03512676 in HLA A*0201 CMV+ allogeneic HCT recipients. We plan to extend this Phase I trial if the approach is found to be safe, to assess clinical benefit of Tet-CMV co-injected with PF-03512676 in protecting HCT recipients from CMV-related complications.

of 68


Objectives:

The goal of this Phase I clinical trial is to perform an evaluation of safety and immunogenicity of a HLA A*0201 restricted CMV vaccine named Tet-CMV (IND BB-13124) co-injected with PF-03512676 adjuvant, in HLA A*0201 recipients of allogeneic HCT5,6,11.

The 1o hypothesis is that boosting CMV immunity in HLA A*0201 CMV-positive recipients of allogeneic HCT by vaccination with Tet-CMV peptide + PF-03512676 is safe and leads to expansion of functional CMV-specific T cells1,4,10,12,13. The 2o hypothesis is that CpG DNA will enhance T cell responses, and reduce CMV-specific T cell impairment associated with CMV disease, in vaccinated HCT recipients14-16.

The primary objective is to discover whether two administrations of 2.5 mg Tet-CMV peptide co-injected with 1 mg of PF-03512676, by subcutaneous (SC) route on days 28 and 56 post-HCT are safe and well tolerated in HLA A*0201 CMV-positive recipients of allogeneic HCT.

The secondary objective is to measure levels of CMV-specific T cells in vaccinated compared to unvaccinated HCT recipients (control arm)4.

An additional secondary objective is to assess whether vaccination of HCT recipients with Tet-CMV co-injected with PF-03512676 reduces expression of programmed cell death receptor-1 (PD-1) on CMV-specific T cells17-19.

Study Design:

In this single site, randomized, open label Phase I trial we will perform an evaluation of safety and immunogenicity of Tet-CMV co-injected with PF-035126765,6, in recipients of an HLA-matched allogeneic (related or unrelated donor) HCT, who are at risk for CMV complications11. Tet-CMV consists of the HLA A*0201-restricted CMV pp65495-503 epitope covalently attached to the tetanus toxin tt830-843 epitope (P2), recognized by many HLA DR alleles5,20,21. In healthy HLA A*0201 adults this vaccine has shown a favorable safety profile and led to expansion of pp65 T cells. This Phase I study will have a target enrollment of 36 recipients and a planned duration of 12-18 months. A computer-generated 1:1 randomization, stratified by CMV serostatus will assign each recipient either to the vaccine or to the observational arm. All patients to be enrolled must be recipients of a first HLA-matched allogeneic HCT for hematological malignancies, with the following inclusion criteria; HLA A*0201, CMV-positive, age 18-75, and willing to be monitored for 6 months. Exclusion criteria include receiving T cell depleted HCT, aplastic anemia, autoimmune disease, HIV, HCV and HBV positivity. In the vaccine arm, recipients will be vaccinated SC twice (on days 28 and 56 post-HCT), with 2.5 mg Tet-CMV + 1 mg PF-03512676.

Safety (primary endpoint) will include continuous post-vaccination assessment for the 1st 100 days and as necessary until day 180 for graft versus host disease (GVHD) and adverse events post-vaccination through the 1st 100 days (AEs) in the 18 immunized recipients. GVHD will be monitored till day 180 and AEs will also be monitored day 28 through the 1st 100 days in the observational arm as well. Toxicity of the vaccine formulation is unlikely, based on healthy volunteer studies6. Rigorous stopping rules will be implemented, and will include three major safety outcomes; non-relapse mortality (NRM) at 100 days post HCT, severe (grade 3-4) acute GVHD, and serious AEs (SAE, grade 3-4) related to the vaccination within 2 weeks from each vaccination. CMV-specific immunogenicity (secondary endpoint) will be monitored in all enrolled recipients from both arms (N=36) every 2 weeks from day 28 until day 100 post-HCT, then at days 130, 160 and 180, by measuring levels of CD8+ T cells binding to MHC class I pp65495-503 tetramers4. Levels of pp65-specific T cells associated with protection from CMV

of 68


complications are expected to be detected in a significantly higher proportion among recipients of the vaccine compared to the control arm4. Additional correlative immunogenicity studies will include measuring levels of PD-1 expression on CMV specific-T cells and assessing their proliferative capacity. Since co-injection of PF-03512676 CpG DNA has been reported to up-regulate antiapoptotic molecules15,16, reduction of apoptosis markers and their increased proliferation of CMV-specific T cells is expected in vaccine compared to unvaccinated control recipients.

Endpoints: Endpoints of this trial are safety (primary endpoint) and immunogenicity (secondary endpoint) of Tet-CMV co-injected with PF-03512676, in HLA A*0201 CMV-positive recipients of allogeneic HCT. PRIMARY ENDPOINT. Evaluation of safety will be performed in all recipients, and will be based on assessment of GVHD, graded according to the Keystone Consensus grading system and AEs based on National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE), Version 4.03. SECONDARY ENDPOINT. Immunogenicity will be evaluated by monitoring CMV-specific CD8+ T cells using MHC class I tetramers, cell surface phenotyping, and assessment of CMV-specific proliferative capacity, by multi color flow cytometric analyses.

Sample Size: The target of this study is to enroll and randomize 36 HLA A*0201 CMV-positive recipients of allogeneic HCT to either the vaccine arm (N=18), or to the observational arm.

Estimated Duration of the Study

Based on the yearly volume of HCT patients, we will be able to enroll and randomize into the study 36 HLA A*0201 CMV positive allogeneic HCT recipients in a period of 12-18 months.

Summary of Subject Eligibility Criteria:

Inclusion Criteria: For inclusion in the Phase I study, a male or female recipient must meet ALL of the following criteria:

1. HLA A*0201 subtype

2. Age 18 to 75 years

3. CMV seropositive

4. Able and willing to sign the ICF

5. Willingness to be followed for the planned duration of the trial (6 months post-HCT)

6. Seronegative for HIV, HCV and active HBV (Surface Antigen Negative)

7. Planned related or unrelated HCT, with 8/8 or 7/8 (A,B,C,DRB1) high resolution HLA donor allele matching

8. HCT for the treatment of hematologic cancers including, but not limited to:

Acute lymphoblastic leukemia in first or second remission (For Acute Lymphoblastic Leukemia/Lymphoblastic Lymphoma, the disease status needs to be in hematologic remissionby bone marrow/peripheral blood. Persistent lymphadenopathy on CT or CT/PET scan without progression is allowed.)

Chronic myelogenous leukemia in first chronic or accelerated phase, or in second

of 68


chronic phase

Hodgkin’s and Non-Hodgkin’s lymphoma

Myelodisplastic syndrome

9. Planned HCT with minimal to no-T cell depletion of graft

10. Use of contraception up to 90 days post-HCT

11. Negative pregnancy test for female recipient.

Exclusion Criteria: Recipients will be ineligible under any of the following conditions

1. A poor-risk subject, as defined by any one of the following:

Chronic myelogenous leukemia in blast crisis

Acute myeloid leukemia beyond second remission

Multiple myeloma

Aplastic anemia

2. Planned immunosuppression with alemtuzumab or any equivalent in vivo T-cell depleting agent

3. In vitro T cell depleted graft

4. Planned prophylactic therapy with CMV immunoglobulin

5. Planned CMV prophylactic therapy

6. Experimental anti-CMV chemotherapy in the last 6 months

7. Diagnosed with autoimmune disease

8. Receipt of the following substances:

Any prior investigational CMV vaccine

Live attenuated vaccines, medically indicated subunit or killed vaccines from 30 days prior to participation in the trial and up to 14 days after the second vaccination (day 70 post-HCT)

Investigational research products or allergy treatment with antigens injections from 30 days prior to participation in the trial and up to 14 days after the second vaccination (day 70 post-HCT)

9. Pregnant and/or breast feeding if a female recipient

10. Refusing to use contraception up to 90 days post-HCT.

of 68


Investigational Product Dosage and Administration:

The Phase I trial will consist of two arms, the vaccine and the observational arm. HCT recipients in the vaccine arm will be injected twice on days 28 and 56 post-HCT, with 2.5 mg Tet-CMV + 1 mg PF-03512676 adjuvant in a final 1.0 ml volume, in the upper arm by SC route. A current good manufacturing practice (cGMP)-grade solution of sodium acetate will be used as a diluent for the peptide vaccine. Tet-CMV + PF-03512676 vaccine formulation and dosage were chosen based on the data from the Phase Ib trial, indicating mild reactogenicity and a favorable safety profile for this specific formulation when up to 4 SC injections were administered to healthy volunteers6.

Clinical Observations and Tests to be Performed:

Clinical observations, laboratory tests and blood draws will be performed at the COH “Phase I unit” clinic for outpatients and in-house patients in their respected units, and will include assessment of performance and relapse status, engraftment, GVHD and CTCAE grading, post-HCT toxicities, comprehensive metabolic and chemistry profiles, CMV-specific viral and immunogenicity monitoring. Clinical CMV disease and use of anti-viral drugs will be prospectively monitored.

CMV monitoring will be conducted in a Clinical Laboratory Improvement Amendments (CLIA)-approved laboratory at COH using standard qPCR methods22, to evaluate CMV viral load in plasma. As mandated by the FDA, autoantibodies against dsDNA will be measured in vaccinated recipients using Wampole® DS DNA ELISA II kit system, by Wampole Laboratories (Princeton, NJ). Immunogenicity studies will be performed at the Translational Vaccine Research (TVR) Division and will include monitoring levels of CD8+ T cells binding to pp65 MHC class I tetramers and PD-1 expression on pp65-specific T cells. Additionally CMV-specific T cell growth kinetics and cell death will be studied. All immune-monitoring assays will be performed by flow cytometry techniques. Immunophenotyping will be conducted on freshly thawed peripheral blood mononuclear cells (PBMC) without cultivation or stimulation in vitro. PBMC will be analyzed for levels of CD8+ T cells binding to the pp65 tetramers and PD-1 expression by

FACSCantoTM with FACSDiva software (BD Biosciences, San Jose, CA). Assessment of CMV-specific T cell growth kinetics will be performed using the CFSE dilution method for cell division tracking; cell death will be evaluated by using the ApoAlert Annexin V-FITC Apoptosis kit (Clontech, Mountain View, CA).

Statistical Considerations: SAFETY. Early stopping rules and safety criteria will be based on NRM at 100 days post-HCT and acute GVHD, and related/possibly related SAEs within 2 weeks of injections. These rules and criteria are defined with the objective of maintaining a combined false alarm probability of ~10% under local historical event rates for HCT. Standard probability calculations for two-stage designs will be used, as previously described23. The trial will not reach its safety endpoint if >1 of 18 vaccinated subjects has directly vaccine related NRM at 100 days post-HCT; ≥5 of 18 have any related or unrelated NRM at 100 days post-HCT, or if ≥6 have grade 3-4 acute GVHD. The combined false alarm rate for both two-stage rules is ~10%. It is anticipated that the trial will show that the vaccine does not induce SAEs and/or marked increases in NRM at 100 days post-HCT.

IMMUNOGENICITY. The evaluation of immunogenicity is partly based on data from a prospective multicenter trial, including Dr. Nakamura at COH, in CMV-positive recipients of an allogeneic HCT4. In our study, we will compare levels of CD8+ T cells binding to CMV-specific tetramers in vaccinated and unvaccinated HCT recipients by Wilcoxon rank-sum test, using integrated CMV-specific CD8+ T cells levels over the first 100 days as a numerical outcome. As

of 68


a conservative approach to power, we will assume 4 ordered categories, equally likely under the null hypothesis (consistent with the Gratama et al. investigation4) and with alternative hypothesis probabilities (proportional odds model) of: 0.043, 0.076, 0.170, and 0.71, (89% of vaccinated subjects in the upper two categories). This yields 91% power at the one-sided 0.05 level of significance, with power computed using StatXact/Cytel Studio version 7.0.0. Thus, the study can detect an increase in the underlying probability of rapid recovery of CMV immunity from about 50% to about 90% with good power and conventional significance. Levels of pp65-specific T cells associated with protection from CMV complications are anticipated to be detected in a significantly higher proportion of vaccine recipients compared to those enrolled in the control arm.

Reduction of PD-1 expression and decreased levels of apoptosis markers and increased proliferation of CMV-specific T cells is expected in vaccine recipients compared to unvaccinated control group. These immune-parameters will be compared in both arms using the Kruskall-Wallis rank-sum test, and may provide evidence of the role of PF-03512676 in enhancing CMV-specific T cell activity, in immunosuppressed HCT recipients.

Sponsor/Licensee:

IND NUMBER: BB-13124

Case Report Forms

Eligibility check list at registration Eligibility check list at randomization (day 28) Vaccine day 28; Vaccine day 56 Screening and monitoring for the vaccine arm Screening and monitoring for the observational arm

of 68


Table of Contents

SECTION .............................................................................................................................. PAGE

1.0 Goals and Objectives (Scientific Aims) .......................................................................... 153

2.0 Background ....................................................................................................................... 16

2.1 Introduction/Rationale for Development ............................................................. 16

2.2 Overview of Proposed Study ............................................................................... 16

2.3 Preclinical Studies ................................................................................................ 18

2.3.1 cGMP-grade Tet-CMV± PF-03512676 adjuvant ..................................... 19

2.4 Human Studies ..................................................................................................... 19

2.4.1 cGMP-grade Tet-CMV± PF-03512676 adjuvant ................................... 20

3.0 Patient Eligibility .............................................................................................................. 25

3.1 Inclusion Criteria ................................................................................................. 25

3.1.1 Disease Status ....................................................................................... 24

3.1.2 Age Criteria, Performance Status and Life Expectancy ......................... 25

3.1.3 Child Bearing Potential ........................................................................... 26

3.1.4 Protocol-Specific Criteria ....................................................................... 27

3.1.5 Informed Consent/Assent ....................................................................... 27

3.1.6 Prior Therapy .......................................................................................... 27

3.2 Exclusion Criteria ................................................................................................ 28

3.2.1 Study-Specific Exclusions ...................................................................... 28

3.2.2 Non-Compliance ..................................................................................... 28

3.3 Inclusion of Women and Minorities .................................................................... 29

4.0 Screening and Registration Procedures............................................................................. 29

4.1 Screening Procedures ........................................................................................... 29

4.2 Informed Consent ................................................................................................ 30

4.3 Registration Requirements/Process ..................................................................... 30

4.4 Dose Assignment ................................................................................................. 30

5.0 Treatment Program ........................................................................................................... 30

5.1 Treatment Overview ............................................................................................ 31

5.1.1 Schedule .................................................................................................. 33

5.2 Planned Duration of Therapy ............................................................................... 33

5.3 Criteria for Removal from Treatment .................................................................. 34

5.4 Subject Follow-Up ............................................................................................... 34

of 68


5.5 Supportive Care, Other Concomitant Therapy, Prohibited Medications ............. 34

5.6 Additional Studies ................................................................................................ 36

5.6.1 Laboratory Studies .................................................................................. 33

6.0 Dose Delays/Modifications for Adverse Events ............................................................... 37

7.0 Data and Safety Monitoring .............................................................................................. 38

7.1 Definition of Risk Level ..................................................................................... 35

7.2 Monitoring and Personnel Responsible for Monitoring ...................................... 38

7.3 Definitions ........................................................................................................... 41

7.4 Reporting of Unanticipated Problems and Adverse Events ................................. 36

8.0 Agent Information and Risks ............................................................................................ 44

8.1 Tet-CMV .............................................................................................................. 40

8.1.1 Description .............................................................................................. 44

8.1.2 Toxicology .............................................................................................. 45

8.1.3 Pharmacology – Handling, Storage, Dispensing and Disposal ............... 45

8.2 PF-03512676 ........................................................................................................ 41

8.2.1 Description .............................................................................................. 41

8.2.2 Toxicology .............................................................................................. 42


8.3 Sodium acetate ..................................................................................................... 44

8.3.1 Description .............................................................................................. 44

8.3.2 Toxicology .............................................................................................. 45


8.4 Injectable vaccine formulation ............................................................................. 45

8.4.1 Description .............................................................................................. 45

8.4.2 Toxicology .............................................................................................. 46


9.0 Correlative/Special Studies ............................................................................................... 52

10.0 Study Calendar .................................................................................................................. 53

11.0 Endpoint Evaluation Criteria/Measurement of Effect ...................................................... 55

11.1 Response Criteria ................................................................................................. 55

12.0 Data Reporting/Protocol Deviations ................................................................................. 55

12.1 Data Reporting ..................................................................................................... 55

12.1.1 Confidentiality and Storage of Records and Data ................................... 55

12.1.2 Subject Consent Form ............................................................................. 56

of 68


12.1.3 Data Collection Forms and Submission Schedule .................................. 56

12.2 Protocol Deviations .............................................................................................. 56

12.2.1 Deviation Policy ..................................................................................... 56

12.2.2 Reporting of Deviations .......................................................................... 57

12.2.3 Resolving Disputes ................................................................................. 57

13.0 Statistical Considerations .................................................................................................. 57

13.1 Study Design ........................................................................................................ 57

13.2 Sample Size Accrual Rate .................................................................................... 58

13.3 Statistical Analysis Plan ....................................................................................... 59

14.0 Human Subject Issues ....................................................................................................... 59

14.1 Institutional Review Board .................................................................................. 59

14.2 Recruitment of Subjects ....................................................................................... 59

14.3 Advertisements .................................................................................................... 59

14.4 Study location and Performance Sites ................................................................. 59

14.5 Confidentiality ..................................................................................................... 60

14.6 Financial Obligations and Compensation ............................................................ 60

14.7 Informed Consent Processes ................................................................................ 60

15.0 References ......................................................................................................................... 62

of 68


Abbreviations

Abbreviation Meaning

AE Adverse Event

CFSE Carboxyfluorescein succinimidyl ester

cGMP Current Good Manufacturing Practice

CLIA Clinical Laboratory Improvement Amendments

CMV Cytomegalovirus

COH City of Hope

CRA Clinical Research Associate

CRF Case Report Form

CTCAE Common Terminology Criteria for Adverse Events

CTEP Cancer Therapy Evaluation Program

CTL Cytotoxic T lymphocytes

DSMC Data Safety Monitoring Committee

FDA Food and Drug Administration

FOS Foscarnet

GCP Good Clinical Practice

GCV Ganciclovir

GVHD Graft versus host disease

HBV Hepatitis B virus

HCT Hematopoietic Stem Cell Transplant

HCV Hepatitis C virus

HHV6 Human herpes virus 6

HIV Human immunedeficiency virus

HPV Human papilloma virus

HSV Herpes simplex virus

IB Investigator Brochure

ICF Informed Consent Form

of 68


IM Intramuscular

IND Investigational New Drug

IRB Institutional Review Board

IV Intravenous

NCI National Cancer Institute

NRM Non-relapse mortality

PBMC Peripheral Blood Mononuclear Cells

PD-1 Program death receptor-1

PI Principal Investigator

PMT Protocol Monitoring Team

SAE Serious Adverse Event

SAIC Science Applications International Corporation

SC Subcutaneous

SOC Standard Of Care

Tet-CMV Tetanus-CMV

Tg Transgenic

TH T helper

TLR9 Toll-like receptor 9

TVR Translational Vaccine Research

VAL Valganciclovir

1.0 Goals and Objectives (Scientific Aims)

CMV infection is a leading infectious complication during the first 100 days of the recovery period of HCT, a leading therapy for hematological malignancies24-29. Focus of the clinical trial is to test a vaccine which reduces CMV morbidity in HCT recipients and the use of anti-virals, which impair immune reconstitution and may increase concomitant infections29-31. The goal of the Phase I clinical trial is to perform an evaluation of safety and immunogenicity of a HLA A*0201 restricted CMV vaccine, Tet-CMV co-injected with PF-03512676 adjuvant, in HLA A*0201 recipients of allogeneic HCT5,6,11.

The vaccine formulation has been licensed as an IND (BB-13124) by the FDA, and has been recently used in a dose escalation Phase Ib trial, performed in healthy adults at COH (IRB# 03121)6. Acceptable safety profiles and vaccine-driven expansion of CMV-specific T cells, in volunteers that were vaccinated

of 68


with Tet-CMV and PF-03512676 adjuvant support further evaluation of this vaccine formulation in the setting of HCT.

The 1o hypothesis is that boosting CMV immunity in HLA A*0201 CMV-positive recipients of allogeneic HCT by vaccination with Tet-CMV peptide + PF-03512676 is safe and leads to expansion of functional CMV-specific T cells1,4,10,12,13. The 2o hypothesis is that CpG DNA will enhance T cell responses, and reduce CMV-specific T cell impairment associated with CMV disease, in vaccinated HCT recipients14-16.

This will be an open label Phase I trial consisting of two arms, the vaccine and the observational arm. Randomization will be blocked and stratified by donor CMV status.

It is anticipated that this trial will provide preliminary data on vaccine-induced protective immunity against primary or recurrent CMV infection in vaccinated HCT recipients4.

If the approach is found to be safe, data from this Phase I trial will pave the way for a Phase II trial to achieve sufficient power for assessing clinical benefit of Tet-CMV co-injected with PF-03512676 in reducing primary or recurrent CMV infection during the recovery of immune-compromised HCT recipients4.

1.1 The primary objective of this Phase I trial is to discover whether two administrations of 2.5 mg Tet-CMV peptide co-injected with 1 mg of PF-03512676, by SC route on days 28 and 56 post-HCT are safe and well tolerated in HLA A*0201 CMV-positive recipients of allogeneic HCT.

1.2 The secondary objective is to measure levels of CMV-specific T cells in vaccinated compared to unvaccinated HCT recipients (control arm)4.

An additional secondary objective is to assess whether vaccination of HCT recipients with Tet-CMV co-injected with PF-03512676 reduces expression of PD-1 on CMV-specific T cells17-19.

2.0 Background

2.1 Introduction/Rationale for Development

Human CMV is a highly prevalent, globally-occurring infection that rarely elicits disease in healthy immunocompetent hosts. The human immune system is unable to clear CMV infection and latency, but mounts a spirited immune-defense targeting multiple immune-evasion genes encoded by this double-stranded DNA β-herpes virus. Encoding around 165 genes, CMV is among the largest and most complex of known viruses3. The size of the CMV-specific cellular immune response is the most striking aspect of the dynamic, life-long interaction between the host and CMV. CMV-specific T cells are essential to restrain CMV viral replication and prevent disease, though do not eliminate the virus or preclude transmission.

Significant suppression of host antiviral immunity can alter the life-or-death immune surveillance homeostasis, allowing either CMV reactivation to become detectable or primary infection to cause clinical symptoms. Uncontrolled viral replication and dissemination results in the development of life-threatening end-organ damage (CMV disease)32-34. CMV infection is the cause of major complications and significant morbidity in the recovery of immune-compromised recipients both at early and late times post- HCT26,29,35. HCT patients are vulnerable to herpes virus infections, including CMV, as a result of immunosuppression associated with treatment strategies aimed at preventing rejection or GVHD36-38. Pharmacologic agents used to limit virus replication, such as, GCV or its oral form VAL, and FOS have become the methods of choice for prophylaxis of CMV infection26,39. Despite this, CMV remains an

of 68


important cause of mortality after HCT diminishing the full curative potential this successful cancer therapy4,40. Furthermore, anti-viral chemotherapy has major side effects, including nephrotoxicity, neutropenia, and delayed immune reconstitution which exposes HCT recipients to other opportunistic viral, bacterial and fungal infections1,29. For example, the use of GCV/VAL is associated with a higher proportion of recipients becoming neutropenic and increased numbers of concomitant fatal infections1,29. As GCV/VAL therapy has become ubiquitous in practice, delayed onset of CMV-pneumonia (interstitial pneumonitis, IP) is more frequent which suggests that GCV/VAL impairs immunologic reconstitution41,42. When antivirals are stopped or when virus resistance occurs, the same disease symptoms appear; only frame shifted to ~180 days post-HCT43-46. Thus, different strategies to control CMV are eagerly sought, since antivirals are at best a stop-gap measure, and their use does not address the major risks of late-onset CMV-IP including early CMV reactivation and failure to reconstitute CMV-specific immunity40. Additionally costs associated to the usage of antivirals are significant, with one single course of GCV/VAL reaching $25,000.

Substituting toxic antivirals with a vaccine that harnesses the abundant native immune response to CMV may improve outcomes for HCT recipients. In particular, a CMV vaccine that confers protective immunity early post-transplant, until normal immunocompetence is re-established in the HCT recipient (6 months or earlier post-HCT) may reduce CMV morbidity and the use of antivirals2. It has been shown that control of CMV infection is primarily associated with cellular immune responses47. The abundant tegument pp65 protein is a major contributor to shaping the T cell repertoire in CMV-exposed individuals20,48,49, and is a principal target for HLA Class I-restricted CD8+ CTL3. CMV-infected cells express pp65 both early and late after infection, making it an appropriate vaccine target50-53. Early clinical studies showed that pp65 CTL development is necessary to overcome CMV disease in HCT recipients25,54,55. Adoptive transfer of pp65-specific CTL reduced CMV viremia after HCT in Phase I trials10,13,56-58. In patients who have measurable CMV viremia, expansion of pp65 specific CD8+ T cells occurs, especially after viral reactivation59,60. In agreement with those observations, studies showed that CMV reactivation and disease were detectable only in patients with low level of CMV-tetramer+ CD8+ T cells4,61. Importantly, several reports have associated protection from CMV complications in HCT recipients with CMV-specific CD8+ T cell levels (including pp65-specific T cells) between 7-10/L4,11. A vaccine that induces protective levels of pp65 CTL has the potential to be of therapeutic benefit to an HCT recipient by limiting CMV viremia or disease3,4,10,12,13,51-53.

Our group has identified a repertoire of CTL epitopes within the pp65 protein that can expand human pp65-specific memory CTLs in vitro20,48,62. The pp65495-503 epitope restricted to the high frequency HLA A*0201 allele has been extensively characterized20,48,49. Usage of the pp65495-503 CTL epitope is conserved with limited sequence variation among viral isolates48,62,63. Additionally, using HLA-restricted CTL epitopes to develop a non-infectious subunit CMV vaccine can eliminate the safety concerns for HCT recipients of live-attenuated CMV or recombinant live viral vaccines, while avoiding the many CMV-encoded products involved in immune-evasion51,64,65.

We discovered that covalently linking pp65 CTL and native tetanus or synthetic T fusion epitopes by solid phase synthesis, dramatically enhanced the immunogenicity of epitope vaccines5,6. In HLA A*0201 Tg mice, two candidate vaccine peptides containing the HLA A*0201 pp65495-503 CD8+ T cell epitope fused to universal TH epitopes (either the synthetic PADRE or a natural Tetanus sequence) showed favorable immunogenicity profiles5,6,21,66. The vaccine peptides were named PADRE-CMV and Tet-CMV, respectively6. Co-injection with PF-03512676 adjuvant (a synthetic single stranded CpG DNA with immunostimulatory activity14) further augmented their activity, providing a means to lower vaccine dosage5,67.

After obtaining approval from the IRB (#03121) and FDA (IND, BB-13124), cGMP-grade PADRE-CMV and Tet-CMV, with or without PF-03512676 adjuvant were clinically evaluated for safety and

of 68


immunogenicity. The Phase Ib dose-escalation clinical trial (registry number: [email protected].) was conducted in HLA A*0201 healthy adults and indicated that the CMV peptides co-injected with PF-03512676 were safe and immunogenic. In particular, Tet-CMV + PF-03512676 had a favorable safety profile and led to substantial expansion of pp65495-503 T cells after 2 vaccine injections. These data support further evaluation of Tet-CMV combined with PF-03512676 in the HCT setting6.

Transferring CMV immunity to HCT recipients, by administering Tet-CMV + PF-03512676 to their donors would logically precede direct vaccination of recipients7,68-70. However, modern HCT clinical practice makes it unfeasible to have sufficient access to the donor to conduct immunizations, and prior results have shown safety and efficacy of directly immunizing recipients, thereby justifying our evaluation of this approach8,71. Additionally, in HCT recipients with nascent hematopoietic reconstitution and prolonged CD4+ T cell deficit, stimulation of TLR9 by PF-03512676 may substitute for the requirement of CD4-TH to sustain immune responses9. TLR9 is implicated as an endosomal receptor for bacterial CpG DNA motifs exemplified by PF-0351267614,72. Since PF-03512676 was found to be safe when administered to HCT recipients in a Phase I trial (Dr. J. Miller, University of Minnesota, personal communication), there is a precedent for our proposed use in HCT recipients. While immune-reconstitution can be limited in allo-HCT population, Tet-CMV has the potential for greater potency when injected into CMV+ HCT recipients, since massive expansion of pp65-specific T cells have been shown in HCT patients under the conditions of CMV infection4,10. Based on these findings, we will perform an evaluation of safety and immunogenicity of two injections of Tet-CMV + PF-03512676, in HLA A*0201 CMV-positive adult recipients of an HLA-matched allogeneic HCT for hematologic malignancies. This will be a single site trial to be conducted at COH.

The proposed randomized, open label Phase I trial will be constituted of two arms, the vaccine and the observational arm. HCT recipients in the vaccine arm will be injected twice on days 28 and 56 post-HCT during the critical period in which primary CMV reactivation most predictably occurs (~day 40-100)43,73, with 2.5 mg Tet-CMV + 1 mg PF-03512676 adjuvant in a final 1.0 ml volume, in the upper arm by SC route. A cGMP-grade solution of sodium acetate will be used as a diluent for the peptide vaccine. Tet-CMV + PF-03512676 vaccine formulation and dosage were chosen based on the data from the Phase Ib trial, indicating reduced reactogenicity and a favorable safety profile for this specific immunogen when up to 2 SC injections were administered to healthy volunteers6. The SC route previously evaluated in the Phase Ib trial in healthy volunteers will be also used for the current Phase I trial protocol in HCT recipients. Vaccines co-injected with PF-03512676 are typically administered by IM injections into the deltoid muscle74, however for HCT recipients enrolled in this study only the SC route will be used.

2.2 Overview of Proposed Study

This study will be conducted in compliance with the protocol, GCP and the applicable regulatory requirements.

The objective of the Phase I trial is to perform an evaluation of safety and immunogenicity of Tet-CMV co-injected with PF-035126765,6, in recipients of an HLA-matched allogeneic (related or unrelated donor) HCT, who are at significant risk for CMV complications11. Tet-CMV consists of the HLA A*0201-restricted CMV pp65495-503 epitope covalently attached to the tetanus toxin tt830-843 epitope (P2),

of 68


recognized by many HLA DR alleles5,20,21. The hypothesis to be tested is that boosting CMV immunity in HLA A*0201 CMV-positive recipients by vaccination with Tet-CMV + PF-03512676 is safe and leads to expansion of pp65 T cells, which have been shown to protect HCT recipients from CMV complications1,4,8,10,12,13. Additionally since CpG DNA enhances T cell response, co-injection of PF-03512676 adjuvant (containing CpG DNA motifs with immunostimulatory activity14) may reduce CMV-specific T cell impairment in immunosuppressed HCT recipients15,16. It is anticipated that the trial will provide preliminary data on vaccine-induced protective immunity against primary or recurrent CMV infection, in allogeneic HCT recipients4.

This will be a single site, randomized, open label trial with a target enrollment and randomization of 36 recipients, to be conducted at COH. The planned duration of this Phase I study is 12-18 months. A preliminary eligibility assessment will be performed before HCT. All patients to be enrolled must be recipients of a first 8/8 or 7/8 HLA-matched75-78 allogeneic HCT for hematological malignancies, with the following inclusion criteria; HLA A*0201, CMV-positive, age 18-75, and willing to be monitored for 6 months. Exclusion criteria include receiving T cell depleted HCT, aplastic anemia, autoimmune disease, HIV, HCV and HBV positivity. On day 28 after the HCT procedure, each enrolled patient will be re-evaluated for eligibility, and a computer-generated 1:1 randomization stratified by CMV serostatus will assign each eligible recipient either to the vaccine or to the observational arm. In the vaccine arm, eligible recipients (see Section 5.1) will be vaccinated twice (on days 28 and 56 post-HCT) SC (safest route for patients with incomplete hematopoietic reconstitution), with 2.5 mg Tet-CMV + 1 mg PF-03512676.

Safety (primary endpoint) will include post-vaccination assessment (bi-weekly for 100 days, then at days 130, 160 and 180) of GVHD and AEs (bi-weekly for the 1st 100 days) in the 18 immunized recipients. GVHD will be monitored until day 180 and AEs will also be monitored for the observational arm for the 1st 100 days. Toxicity of the vaccine formulation is unlikely, based on healthy volunteer studies6. Rigorous stopping rules will be implemented, and will include three major safety outcomes; NRM at 100 days post HCT, severe (grade 3-4) acute GVHD, and AEs (grade 3-4) within 2 weeks from each vaccination. CMV-specific immunogenicity (secondary endpoint) will be monitored in all enrolled and randomized recipients from both arms (N=36) every 2 weeks from day 28 until day 100 post-HCT, and afterward on days 130, 160, 180, by measuring levels of CD8+ T cells binding to MHC class I pp65495-503 tetramers4. Levels of pp65-specific T cells associated with protection from CMV complications are anticipated to be detected in a significantly higher proportion of vaccine recipients compared to those enrolled in the control arm4. Additional correlative immunogenicity studies will include measuring levels of PD-1 expression on CMV specific-T cells and assessing their proliferative capacity. Since co-injection of PF-03512676 CpG DNA has been reported to up-regulate antiapoptotic molecules15,16, reduction of apoptosis markers and increased proliferation of CMV-specific T cells is expected in vaccine recipients compared to the unvaccinated control group.

Acceptable safety profiles and enhanced immunogenicity in vaccinated recipients will pave the way for a Phase 2 trial, with enrollment of additional patients to achieve sufficient power for testing clinically significant endpoints.

2.3 Preclinical Studies

2.3.1 cGMP-grade Tet-CMV ± PF-03512676 adjuvant

Both PADRE-CMV and Tet-CMV ± PF-03512676 adjuvant have been extensively tested in Tg mouse models and the pre-clinical studies have been instrumental to receive both IRB (#03121) and FDA (IND, BB-13124) approval, to perform a Phase Ib trial in healthy adults5. The cGMP-grade CMV vaccine products for the 03121 Phase Ib trial protocol were evaluated in HLA A*0201 HHDII Tg mice, and potency results were confirmed, as illustrated in Figure 16.

of 68


Animal Toxicology

A pre-clinical toxicology study was carried out in rats and has been reported in detail in IRB #03121, and submitted to FDA in IND BB-13124. Briefly, the animal toxicology revealed little to no toxicity of the cGMP-grade CMV peptides at all investigated dosages (from Southern Research Institute, Protocol #11200.11.01).

2.4 Human Studies

2.4.1 cGMP-grade Tet-CMV ± PF-03512676 adjuvant

The proposed evaluation of Tet-CMV + PF-03512676 adjuvant in the HCT setting is supported by the promising safety data and immunogenicity profiles obtained using this vaccine formulation in a clinical study performed in a cohort of healthy volunteers. In particular, based on IRB (#03121) and FDA (IND, BB-13124) approval, cGMP-grade PADRE-CMV and Tet-CMV, with or without PF-03512676 adjuvant were clinically evaluated for safety and immunogenicity (Phase Ib trial) in healthy adults, expressing the HLA A*0201 MHC Class I allele sequence.

Phase Ib Studies in healthy adults

The Phase Ib safety and immunogenicity clinical trial, performed at COH in healthy individuals was a non-randomized, open label, dose escalating study6. Healthy male and female volunteers, 18 and 55 years old, CMV-seropositive or seronegative, and molecularly subtyped as HLA A*0201-positive were enrolled after signing informed consent. Participants were eligible unless they had ≥1 of the following exclusion criteria: abnormal serum chemistry and blood count; hepatitis B or C positive; immunodeficiency, including HIV; taking daily medications for chronic illness, surgery within 6 months of vaccination requiring general anesthesia; known cardiac disease including patients being treated for hypertension and high cholesterol; positive urine pregnancy test/planning to become pregnant within 6 months from vaccination; immunization with other vaccines within 1 month of the study period; participation in a CMV immunotherapy trial in the previous 6 months; history of cancer, depression, allergic diatheses, frequent migraines.

The safety (primary endpoint) and immunogenicity (secondary endpoint) evaluation of PADRE-CMV and Tet-CMV was performed separately in two concomitant stages: Trial A evaluated CMV peptide vaccines alone; whereas Trial B also included co-injected PF-03512676 adjuvant, used at a constant dosage of 1 mg for all research subjects at all dose levels of CMV peptide vaccines74. The vaccine dose escalation (0.5, 2.5, 10 mg vaccine) was based on previous peptide vaccine studies79. A cGMP-grade sodium acetate solution was used as peptide diluent. Trial A formulation was comprised of 1 ml of peptide solution and

of 68


0.1 ml sodium bicarbonate USP to adjust pH; Trial B formulation was comprised of 1.0 ml of peptide solution and 0.1 ml PF-03512676 (1 mg). The final 1.0 ml injection volume was administered SC in the upper arm. To assess safety of multiple injections, booster doses of vaccine ± PF-03512676 (1 mg) were given at day 21, 42, 63. Six volunteers were allotted to each dose level and the vaccine was administered by SC injection in the upper arm.

Between May 2007 and October 2010, 225 volunteers were screened and 63 eligible volunteers were enrolled, vaccinated, and monitored for safety and immunogenicity. However, post-vaccination evaluation of CMV immune responses was not performed for 5 volunteers (1 CMV-seronegative and 4 CMV-seropositive) who received only the first vaccine dose, and withdrew prematurely from the study. Among the 58 evaluable subjects, 3 vaccinated volunteers were lost to follow-up after the d180 visit. The peptide vaccines were safe and tolerated in most subjects, though addition of the PF-03512676 (1 mg) adjuvant increased reactogenicity (Table 1). Most common AEs included mild to moderate cutaneous reactions at the injection site and systemic flu-like symptoms. The duration of related Grade 1 and 2 AEs ranged from 1-2 days. Grade 3 AEs were reported for 4 volunteers: 3 from Trial B and 1 from Trial A (vaccinated with Tet-CMV peptide). These Grade 3 AEs included marked malaise, 39.4–40.5C fever, urticaria and were resolved with non-prescription analgesics (acetaminophen and antihistamine

of 68


respectively), within 8 days. In Trial A, Grade 2 AEs were reported in 2 subjects using both PADRE-CMV (10.5%) and Tet-CMV (16.7%) peptide vaccines. Within Trial B, 6 (31. 6%) and 2 (10.5%) subjects experienced Grade 2 and 3 AEs respectively, following vaccination with PADRE-CMV peptide vaccine + PF-03512676. In contrast, Tet-CMV + PF-03512676 showed a marked favorable safety profile: 2 (15.3%) subjects experienced Grade 2 and 1 (7.7%) Grade 3 AEs. No association was found between severity or grade of AEs and pp65-specific immunity.

Additionally, sera from immunized volunteers were longitudinally evaluated during the whole study observation period (from day -120 to ~1 year after the 1st immunization), for levels of double stranded (ds) DNA autoantibodies. These analyses were requested by FDA, since mouse models of systemic autoimmune diseases have indicated involvement of TLRs in the generation of autoreactive immune responses80. The semi-quantitative detection of human dsDNA IgG autoantibodies in serum was performed using the dsDNA ELISA kit system, by Wampole Laboratories (Princeton, NJ) at COH

of 68


General Clinic Research Centre. As shown in Figure 2 (lowest plot), none of the tested individuals (N=21) vaccinated with the CMV peptides only (Trial A) showed levels of dsDNA autoantibodies above the positivity threshold (>180 AAU/ml). In contrast, for 66.7% (total N=18, upper plots) and 36.4% (total N=11, central plots) of the healthy volunteers vaccinated respectively with PADRE-CMV and Tet-CMV in combination with PF-03512676 CpGDNA adjuvant, levels of dsDNA autoantibodies transiently raised above the positivity threshold, following vaccination. However, by day 360 they returned within the normal range. Our results indicate that co-injection of PF-03512676 CpGDNA adjuvant transiently increases levels of dsDNA autoantibodies. Importantly, none of the immunized volunteers co-injected with PF-03512676 CpGDNA had any evidence of developing autoimmune disease or ever reported autoimmunity-related pathologies, including UPN 228 (2.5 mg Tet-CMV + PF-03512676) who maintained the same levels of dsDNA autoantibodies slightly above positivity threshold, before and after vaccination. The transient and asymptomatic dsDNA autoantibody increases were less frequent and less pronounced in Tet-CMV than in PADRE-CMV peptide immunized subjects (Figure 2, upper and central plots), further supporting the usage of the Tet-CMV formulation for the current study in HCT recipients. The induction of post-vaccination ex vivo immune responses was monitored by flow cytometry analyses, using FACSCantoTM with FACSDiva software (BD Biosciences)5. Positive post-vaccination responses, defined as a >3 fold increase in either MHC class I pp65495-503 tetramer binding or IFN-γ expression by CD8+ T cells compared to baseline were exclusively detected in volunteers who received the vaccine co-administered with PF-03512676 (Figure 3A)52.

Comparing the longitudinal profiles of ex vivo pp65495-503 tetramer+ CD8+ T cell levels, a striking difference was noted in Trial A and B immunized volunteers (Figure 3A; p=0.004, Wilcoxon rank-sum test comparing post-vaccination averages between d14-77). While among Trial A volunteers whose baseline pp65495-503 tetramer levels were minimal-low (<0.2%, referred as CMV+ <0.2%) tetramer levels remained mostly flat during the whole observation period (Figure 3A, left plot), increases in pp65495-503 CD8+ T cells after each vaccination were frequent in Trial B CMV+<0.2% subjects.

A

B

of 68


In all responders, ex vivo CMV responses were still detectable at day 77 (Figure 3A, right plot). These results suggest that PF-03512676 adjuvant contributed to stimulating CMV vaccine responses, though its effect varied in the vaccinated CMV+<0.2% population. Limited variation in pp65495-503 tetramer levels were observed post-vaccination in the CMV-seropositives population with baseline ≥0.2% pp65495-503 tetramer binding from both Trial A and B (Figure 3B). A dose response to the vaccine was not apparent, however data collected from the volunteers immunized with Tet-CMV + PF-03512676 strongly support further evaluation of this vaccine in the HCT setting, due to its satisfactory safety profiles (Table 1) and favorable immunogenicity (Figure 3A, right plot)6. In particular, in the cohort immunized with 2.5 mg Tet-CMV + 1 mg

PF-03512676 adjuvant (the vaccine formulation to be used in HCT recipients) a single Grade 2 AE (38.7–39.3C fever) resolved in 1 day was reported for one vaccinated volunteer (Table 2). Though none of Trial A volunteers showed ex vivo post-vaccination increases in pp65495-503

CD8+ T cells, by using IVS we were able to detect priming of memory T cell responses (>10 fold expansions) in ~30% (4 of 14) of CMV+<0.2% and ~40% (3 of 7) of CMV-seronegatives from Trial A. In addition, pp65495-503 CD8+ T cells could be consistently expanded in all 4 CMV-seronegatives and in 4 CMV-seropositive volunteers from Trial B who failed to demonstrate ex vivo responses. Confirming the ex vivo data, the correlative IVS studies showed that usage of PF-03512676 adjuvant induced significantly higher (p=0.002, two-sided Fisher’s exact test) post-vaccination responses in Trial B compared to Trial A. Stimulation strategies to evaluate vaccine immunogenicity in clinical trials can have limitations, due to

length and conditions of the in vitro culture52,53. However, breadth and consistency of post-IVS responses detected in Trial B subjects (Figure 4), after brief exposure of PBMC to the

of 68


pp65495-503 peptide, are promising. In fact, the vaccine elicited pp65495-503 T cells could be significantly expanded upon exposure to CMV antigens in a viremic individual. In particular, the CMV peptide vaccines may have the potential for greater potency when injected into HCT recipients, as further massive expansion has been reported to occur under the natural stimulation of CMV infection10. The resulting pp65495-503 T cell increase in HCT recipients can be critical to control CMV viremia51-53. In summary, the correlative IVS studies indicate that the CMV peptide vaccines + PF-03512676 are effective in consistently priming pp65-specific T cells.

3.0 Patient Eligibility

3.1 Inclusion Criteria

Patient eligibility will be established pre-HCT, then recipients will be followed for engraftment, GVHD, treatment with corticosteroids, medication/therapies received, relapse, post-HCT toxicities and CMV reactivation. On day 28 post-HCT final eligibility will be decided, and eligible patients will be randomized and stratified by CMV serostatus for initiating (day 28 vaccination and/or continuing the observational/monitoring studies. IRB approved protocol nurses will coordinate the logistics of recipient selection, enrollment, and consenting. Dr. Nakamura, HCT physician and PI of the protocol will determine the eligibility status of the recipient.

For the pre-HCT inclusion in the Phase I study, a male or female recipient must meet ALL of the following criteria:

1. HLA A*0201 subtype 2. Age 18 to 75 years 3. CMV seropositive 4. Able and willing to sign the ICF 5. Willingness to be followed for the planned duration of the trial (6 months post-HCT) 6. Seronegative for HIV, HCV and active HBV (Surface Antigen Negative) 7. Planned related or unrelated HCT, with 8/8 or 7/8 (A,B,C,DRB1) high resolution HLA donor

allele matching 8. HCT for the treatment of hematologic cancers including, but not limited to:

Acute lymphoblastic leukemia in first or second remission (For Acute Lymphoblastic Leukemia/Lymphoblastic Lymphoma, the disease status needs to be in hematologic remissionby bone marrow/peripheral blood. Persistent lymphadenopathy on CT or CT/PET scan without progression is allowed.)

Chronic myelogenous leukemia in first chronic or accelerated phase, or in second chronic phase

Hodgkin’s and Non-Hodgkin’s lymphoma Myelodisplastic syndrome

9. Planned HCT with minimal to no-T cell depletion of graft 10. Use of contraception up to 90 days post-HCT 11. Negative pregnancy test for female recipient.

of 68


3.1.1 Disease Status

Recipients to be enrolled are patients eligible for allogeneic HCT, who were diagnosed with hematologic malignancies including:

Acute Lymphoblastic Leukemia (ALL); B-precursor ALL; T cell ALL Acute Myeloid Leukemia (AML), Acute promyelocytic leukemia; Treatment related AML Chronic Lymphoid Leukemia; Adult T cell leukemia/lymphoma, Chronic lymphocytic leukemia

NOS, Hairy cell leukemia; Prolymphocytic leukemia (B or T); T cell large gran. lymph. leuk; Chronic Myeloproliferative Disease (CML); CEL/Hypereosinophilic syndrome; Chronic

idiopathic myelofibrosis; CML - Philadelphia chromosome; Essential thrombocythemia; Polycythemia vera;

Leukemia, NOS; Myelodysplastic syndrome, NOS; Chronic myelomonocytic leukemia; Hodgkin lymphoma, NOS; Hodgkin lymphoma nodular LP, NOS ; Hodgkin lymphoma - like

PTLD Lymphoma, NOS; Non-Hodgkin Lymphoma (NHL); ALCL, cutaneous, ALCL, systemic ; Burkitt

lymphoma/leukemia; CTCL/Mycosis fungoides; CTC /Sezary syndrome; Diffuse large B-cell lymphoma; MALT-lymphoma; Extranodal NK/T cell lymphoma, Extranodal NK/T lymphoma-nasal; Follicular lymphoma; Lymphoplasmacytic lymphoma Mantle cell lymphoma; Mediastinal large B-cell lymphoma; Nodal marginal zone B-cell lymph.; NHL, aggressive, NOS; NHL, indolent, NOS; NHL, NOS; Peripheral T cell lymphoma, NOS; PTLD (Monoclonal); PTLD (Polyclonal); Precur. B-lymphoblastic lymphoma; Precur. T-lymphoblastic lymphoma; Primary CNS lymphoma; Primary effusion lymphoma; Small lymphocytic lymphoma, NOS

Myeloma, NOS; MGUS; Solitary plasmacytoma.

3.1.2 Age Criteria

Pediatric recipients (children <18 years old of age) are excluded from this study because insufficient data are available in adults to judge potential risks in children. Additionally, vaccine dosage and the blood volume established for immune-monitoring in adults cannot be applicable for both adults and children. Finally, the risk of CMV complications is inversely related to age, and the inclusion of younger children could bias the endpoint observations.

In recent years reduced-intensity conditioning (RIC) regimens allow increased use of allogeneic HCT for elderly patients (≤75 years of age). Since chronologic age itself is no longer being considered a contraindication for this procedure, patients up to 75 years of age, who are fit to undergo allogeneic HCT are included in this study 81,82.

3.1.3 Child Bearing Potential

The effects of Tet-CMV + PF-03512676 on the developing fetus are unknown. However, PF-03512676 had been determined to be embryolethal in rabbits and teratogenic in developing rats and rabbits (see enclosed Pfizer IB). For this reason, women of child-bearing potential and men must agree to use adequate contraception (hormonal or barrier method of birth control or abstinence) prior to study entry and for six months following duration of study participation. Should a woman become pregnant or suspect that she is pregnant while participating on the trial, she should inform her treating physician immediately.

of 68


3.1.4 Protocol-Specific Criteria

All medications, supportive care, blood products or radiation therapy taken or administered during the trial will be documented in the subject’s clinical/hospital and CRF, using COH guidelines. The subject’s clinical information will be recorded on the appropriate CRF.

Concurrent enrollment in other clinical trials using an investigational product is prohibited.

The use of alemtuzumab for immunosuppression is not permitted in this study, because its administration results in in vivo depletion of B, T and dendritic cells, negating any potential positive effect of vaccinating the recipient with Tet-CMV + PF-03512676 adjuvant.

Prophylactic therapy with CMV immunoglobulin or prophylactic antiviral CMV treatment (Antiviral treatment for HSV, HHV6, EBV and adenovirus including the use of GCV/VAL, FOS, Cidofovir, CMX-001) is not allowed.

Medications that might interfere with the evaluation of the investigational product should not be administered, from 30 days prior to participation in the trial and up to 14 days after the second vaccination (day 70 post-HCT). Medications in this category include, but are not limited to:

Live attenuated vaccines Medically indicated subunit (Engerix-B for HBV; Gardasil for HPV) or killed vaccines (e.g.

influenza, pneumococcal, or allergy treatment with antigen injections)

Antiviral treatment for HSV, HHV6, EBV and adenovirus including the use of GCV/VAL, FOS, Cidofovir, CMX-001 may also suppress reactivation of CMV, thus it will not be allowed in this study. Patients requiring such treatment before randomization (day 28) will be removed from the study and replaced. Reasons for removal will be reported in the patient’s CRF

By HCT-SOP as of February 2012, the recommended CMV-PCR value to start anti-CMV therapy is ≥1500 gc/ml, unless there are other risk factors such as GVHD, steroid use, lymphopenia. All enrolled recipients who will require anti-CMV therapy before day 28 will be replaced, treated and monitored as required by COH standard of care. GCV/VAL, FOS, Cidofovir, CMX-001 may be used according to COH SOC for preemptive management of CMV viraemia. Should antiviral treatment be required after day 28, the planned 2nd vaccine injection at day 56 will not be administered (vaccine arm only).

In this study if the treating physician feels that it is in the interest of patient safety to begin treatment at a level lower than 1500 gc/ml, the case will be reported in the analysis as a protocol violation and a 2nd vaccine injection at day 56 will not be administered (vaccine arm only).

For each patients all required medications received after randomization (day 28) will be recorded on the appropriate CRF.

3.1.5 Informed Consent/Assent

All subjects must have the ability to understand and the willingness to sign a written informed consent.

3.1.6 Prior Therapy

See 3.1.4

of 68


3.2 Exclusion Criteria

Recipients will be ineligible under any of the following conditions 1. A poor-risk patient, as defined by any one of the following: Chronic myelogenous leukemia in blast crisis Acute myeloid leukemia beyond second remission Multiple myeloma Aplastic anemia 2. Planned immunosuppression with alemtuzumab or any equivalent in vivo T-cell depleting agent 3. In vitro T cell depleted graft 4. Planned prophylactic therapy with CMV immunoglobulin 5. Planned CMV prophylactic therapy 6. Experimental anti-CMV chemotherapy in the last 6 months 7. Diagnosed with autoimmune disease 8. Receipt of the following substances: Any prior investigational CMV vaccine Live attenuated vaccines, medically indicated subunit or killed vaccines from 30 days prior to

participation in the trial and up to 14 days after the second vaccination (day 70 post-HCT)

Investigational research products or allergy treatment with antigens injections from 30 days prior to participation in the trial and up to 14 days after the second vaccination (day 70 post-HCT)

9. Pregnant and/or breast feeding if a female recipient 10. Refusing to use contraception up to 90 days post-HCT.

3.2.1 Post-HCT Study-Specific Exclusions

On day 28 (immunization day for the vaccine arm) all study recipients (vaccine & observation arm) will be reviewed for eligibility and ruled ineligible to continue the study and receive vaccination (for the vaccine arm) if:

1. Diagnosed with >grade 2 GVHD before day 28 post-HCT, and diagnosed with >grade 2 GVHD between day 28 post-HCT and administration of the 2nd vaccine at day 56..

2. Received steroid therapy with prednisone >1 mg/kg/day, less than 7 days prior to injection 3. Had relapse 4. Experience graft failure (absolute neutrophil count <500/mm3) 5. Received antiviral treatment with GCV/VAL, FOS, Cidofovir, CMX-001 at any point during the

28 day period 6. There are ongoing post HCT >= grade 3 non-hem AE’s, with exception of grade 3 glucose

intolerance and grade 3 non-hem labs; cholesterol, triglyceride, and hyperglycemia

The recipient will be withdrawn from the trial and will be replaced. Reasons for ineligibility will be detailed in the subject’s CRF and medical record.

On day 56 (immunization day for the vaccine arm) vaccine patients will be reviewed for eligibility. Vaccine patients will be ruled ineligible for the 2nd vaccine injection if:

of 68


1. Diagnosed with >grade 2 GVHD before day 28 post-HCT, and diagnosed with >grade 2 GVHD between day 28 post-HCT and administration of the 2nd vaccine at day 56..

2. Received steroid therapy with prednisone >1 mg/kg/day, less than 7 days prior to injection 3. Had relapse 4. Experience graft failure (absolute neutrophil count <500/mm3) 5. Received antiviral treatment with GCV/VAL, FOS, Cidofovir, CMX-001 at any point during the

56 day period 6. There are ongoing post HCT >= grade 3 non-hem AE’s, with exception of grade 3 glucose

intolerance and grade 3 non-hem labs; cholesterol, triglyceride, and hyperglycemia

Vaccine patients who do not meet eligibility at D+56 will still be monitored and followed per protocol.

3.2.2 Non-Compliance

Subjects, who in the opinion of the PI may not be able to comply with the safety monitoring requirements of the study, can be replaced before day 28 randomization. In contrast, every patient who has been enrolled and randomized (day 28) is considered to be part of the trial.

3.3 Inclusion of Women and Minorities

The study is open anyone regardless of gender or ethnicity. Efforts will be made to extend the accrual to a representative population, but in a trial which will accrue and randomize 36 subjects, a balance must be struck between subject safety considerations and limitations on the number of individuals exposed to potentially toxic or ineffective treatments on the one hand and the need to explore gender, racial, and ethnic aspects of clinical research on the other. If differences in outcome that correlate to gender, racial, or ethnic identity are noted, accrual may be expanded or additional studies may be performed to investigate those differences more fully.

4.0 Screening and Registration Procedures

4.1 Screening Procedures

Diagnostic or laboratory studies performed exclusively to determine pre-eligibility (before HCT) for this trial will be done only after obtaining written informed consent. Studies or procedures that were performed at COH for clinical indications (not exclusively to determine study eligibility) may be used for baseline values and/or to determine pre-eligibility, even if the studies were done before informed consent was obtained. All clinical laboratory tests and blood draw volumes will be performed at a COH CLIA approved lab for both inpatients and outpatients, and as specified in the Study Calendar (Section 10). All test results and assessments required to establish pre-eligibility of a recipient subject (related or unrelated) must be obtained prior to enrollment. Laboratory values obtained at COH CLIA approved labs within days -60 to -9 before HCT window will be considered acceptable. The following “Eligibility Check List” are required at the screening visit:

Signed ICF/disclosure authorization form Medical history Physical exam including vital signs, height and weight HLA molecular typing Hepatitis B surface antigen

of 68


Hepatitis C antibody HIV antibody CMV serostatus CMP + uric acid, phosphorus, cholesterol, & LDH*/CBC with differential** clinical exams Pregnancy test (for female recipients) Review of concomitant medications Assessment of inclusion/exclusion criteria Enrollment and randomization Collection of whole blood for immune-monitoring studies (see Study Calendar, Section 10).

PBMC will be separated from blood specimens, cryopreserved and stored in the vapor phase of centrally monitored liquid nitrogen tanks.

Records will be kept to document screening activities in CRF, and will indicate the reason(s) for any screen failures.

*CMP compete metabolic panel profile performed by Sequential Multiple Analysis at a COH CLIA approved lab, including the following 18 blood chemistry parameters (CMP): glucose, BUN (blood urea nitrogen), creatinine, uric acid, total proteins, albumin, calcium, phosphorous, sodium, potassium, chlorine, total CO2, total bilirubine, alkaline phosphatase, ALT (alanine transaminase), AST (aspartate aminotransferase), LDH (lactate dehydrogenase), total cholesterol.

**CBC: complete blood count with differential performed at the Department of Transfusion Medicine.

4.2 Informed Consent

The investigational nature and objectives of the trial, the procedures and treatments involved and their attendant risks and discomforts, and potential alternative therapies will be carefully explained to the subject and a signed informed consent will be obtained. Documentation of informed consent for screening will be maintained in the subject’s research chart, CRF and medical record.

4.3 Registration Requirements/Process

Once the recipient has been pre-qualified for the trial and all screening activities have been completed (see Section 4.1), the enrolled patient will be registered for the trial by the Study Coordinator. Promptly after completion of the HCT procedure, the study coordinator with the assistance of IRB approved protocol nurses will contact IDS designated COH Pharmacist for the trial, and communicate the possible scheduled dates and acceptable window times for the preparation of the vaccine injections. The study coordinator will also contact IRB approved protocol nurses at the “Phase I Unit” clinic to coordinate the pre-plan of visit scheduling for clinical evaluations/vaccine injections/follows up and blood draws. On day 28 post-HCT, study coordinator will then use a computer-generated randomization program, stratified by CMV serostatus to assign fully eligible recipients to either to the vaccine or to the observational arm.

4.4 Dose Assignment

On days 28 and 56 post-HCT, all eligible recipients assigned to the vaccine arm will receive the identical vaccine formulation at the constant dose of 2.5 mg Tet-CMV + 1 mg PF-03512676 adjuvant, in a final 1.0 ml volume, in the upper arm by SC route.

of 68


5.0 Treatment Program

5.1 Treatment Overview

Only eligible recipients in the interventional arm (N=18) will receive the vaccine treatment, consisting of two injection of 2.5 mg Tet-CMV + 1 mg PF-03512676 adjuvant, in a final 1.0 ml volume . Timing of vaccine administration for HCT recipients will be on days 28 and 56 (±5 days are allowed), during the critical period in which primary CMV reactivation most predictably occurs (~day 40-100)43,73. The “Phase I unit” clinic will be the location where outpatients recipients will receive the treatment. In-house patients will have vaccine administration in their respected units.

In contrast, pre- and post-HCT screening evaluations, assessments of clinical and laboratory profiles, and immune-monitoring analyses will be performed for all enrolled and randomized recipients (N=36), as specified in the Study Calendar (Section 10). All procedures will be carried out in the “Phase I unit” clinic for both inpatients and outpatients recipients.

Specifically, the clinical evaluation will consist of assessing vital signs (blood pressure, heart rate, respiration rate, and temperature) and conducting a comprehensive physical exam.

GVHD and AEs will be monitored for both vaccine recipients and observation arm participants as necessary and no less than bi-weekly from day 28 post-HCT until day 100 post-HCT. Afterwards, GVHD will continue to be monitored as necessary or monthly up until 6 months (Study Calendar, Section 10), and subsequently as per SOC.

Immune-monitoring6,83 will be performed on blood specimens withdrawn biweekly until day 100, and monthly up to 6 months afterward (Study Calendar, Section 10).

Vaccine failure will be defined as a single measurement of ≥1500 gc/ml after 28 and prior to 100 days post-HCT. Patients considered vaccine failures will be still followed for safety, CMV viraemia according to COH SOC, and CMV immune-monitoring, if agreed by the subject. CMV disease will be defined as described84.

The study treatment outline will include the following steps:

1. Pre-HCT eligibility screening/monitoring (Days -60/-9) Please see “Screening Procedures” (Section 4.1)

2. Post-HCT eligibility screening/randomization/monitoring (Day 28 & 56)

On day 28, Dr. Nakamura conferring with the treating physician will decide the full eligibility of enrolled HCT recipients to continue the study based on the criteria listed on Section 3.2.1 (Post-HCT Specific Exclusions). In particular for all recipients will be assessed:

CMP + uric acid, phosphorus, cholesterol, & LDH*/CBC with differential GVHD grading Corticosteroid therapy Relapse status Engraftment CMV viraemia (Patient has NOT had CMV qPCR ≥1,500 gc/ml by standard qPCR methods22

prior to D+28) Usage of antivirals

of 68


No ongoing post HCT >= grade 3 non-hem AE’s, with exception of grade 3 glucose intolerance and grade 3 non-hem labs; cholesterol, triglyceride, and hyperglycemia

For recipients eligible to be randomized and continue the study, and for all who received at least 1 vaccine injection, the following will be performed: Collection of whole blood for immune-monitoring studies (see Study Calendar, Section 10).

PBMC will be separated from blood specimens, cryopreserved and stored in the vapor phase of centrally monitored liquid nitrogen tanks.

3. Treatment Visits (Days 28 and 56: vaccine arm only)

The following activities will occur at each injection visit as indicated in the Study Calendar (Section 10), for those vaccine arm recipients considered eligible as for step 2(:

Pre-injection clinical evaluation (physical exam and vital signs to be performed by the study PI or qualified staff)

Documented negative pregnancy test of female recipients on the day of injection (within 48 hours prior by exception)

qPCR results for CMV viraemia <1500 gc/ml (Patient has NOT had CMV qPCR ≥1,500 gc/ml by standard qPCR methods22 prior to D+28 [to be eligible for D+28 vaccination] and D+56 [to be eligible for Day+56 vaccination])

Review of usage of antivirals Review and recording of concomitant medications Injection of 2.5 mg Tet-CMV + 1 mg PF-03512676 adjuvant, in a final 1.0 ml volume Following injection, subjects will be observed for 30’ The staff will assess vital signs, local and systemic reactogenicity, and AEs at the end of the

30’ observation period

In addition, anaphylactic reactions to the 1st vaccine injection or any grade 3-4 AEs that are definitively related to the vaccine will preclude the administration of a 2nd vaccine injection on day 56.

4. Follow-up evaluations/monitoring visits

All or some of the following activities may occur during follow-up visits as described in the Study Calendar (Section 10)

GVHD grading Engraftment CMV viraemia Review and recording of all AEs (vaccine and observation arm) Review and recording of all concomitant medications Collection of whole blood for immune-monitoring and dsDNA autoantibodies studies PBMC will

be separated from blood specimens, cryopreserved and stored in the vapor phase of centrally monitored liquid nitrogen. Sera for measuring dsDNA autoantibodies will be separated by centrifugation and stored in centrally monitored -20 0C freezers

Additional and/or repeat clinical laboratory tests intended for the purpose of assessing safety and/or to determine outcome of laboratory-related AEs may be ordered by Dr. Nakamura, PI of the study, or the primary care physician in consultation with Dr. Nakamura.

No research blood draws will be made, which duplicate CMP + uric acid, phosphorus, cholesterol, & LDH*/CBC with differential tests for SOC indications.

of 68


5.1.1 Schedule

For a tabular view of the treatment, monitoring, and follow-up schedule, see Study Calendar in Section 10. For both vaccine injections and monitoring blood draws, ± 5 days will be considered an acceptable window in the event that treatment therapy/blood draws cannot be administered/performed exactly on schedule because of unforeseen scheduling problems, weekends and holidays. However, the interval between day 160 and 180 blood draws should be ≥ 20 and ≤ 60 days. Since at this later time points changes in immune response will be unlikely to be detected with short (< 20 days) immune monitoring intervals, the ± 5 day window will be altered to guarantee a minimum of 20 days interval between day 160 and 180 blood draws. Table 3 outlines the study schedule.

5.2 Planned Duration of Therapy

The interventional therapy will consist of 2 vaccine injections which will be administered exclusively to the vaccine arm subjects (N=18), on days 28 and 56 post-HCT. The observational phase of the study will start within 2 months preceding HCT and will be concluded 6 months later for both vaccine and control arms (N=36).

of 68


5.3 Criteria for Removal from Treatment

Any recipient who is removed from treatment prior to randomization on day 28 (see 3.2.1 Post-HCT Specific Exclusions) will be replaced. Reasons for ineligibility will be detailed in the recipient’s CRF and medical record.

Criteria for removal from the study will be applied for recipients who receive specific treatments which may interfere with the vaccine response and the interpretation of the CMV immune-monitoring, and/or are suffering post-HCT serious complications. In particular the following recipients (both from the vaccinations and control arms) will be ruled ineligible to initiate or continue in the study IF:

7. Diagnosed with >grade 2 GVHD before day 28 post-HCT, and diagnosed with >grade 2 GVHD between day 28 post-HCT and administration of the 2nd vaccination at day 56. Received prednisone >1 mg/kg/day, less than 7 days prior to injection

1. Had relapse 2. Experience graft failure (absolute neutrophil count <500/mm3) 3. Have CMV qPCR measurements ≥1500 gc/ml 4. Receiving antiviral treatment with GCV/VAL, FOS, Cidofovir, CMX-001

If there are any ongoing post HCT >= grade 3 non-hem AE’s, with exception of grade 3 glucose intolerance and grade 3 non-hem labs; cholesterol, triglyceride, and hyperglycemia. Patients who relapse after randomization at D+28 through D+180 will be removed from treatment and will be taken off-study and there will be no further follow-up data collected.

5.4 Subject Follow-Up

All randomized recipients will continue to receive follows up and blood draws, as indicated in the Study Calendar (Section 10). Subjects from the vaccine arm who decline a second vaccine injection will be offered to continue immune-monitoring according to the Study Calendar (Section 10). GVHD and CMV monitoring will continue through day 180 and AE and SAE monitoring will be followed from day 28 post-HCT and through the 1st 100 days for vaccine and observation arm participants. Concomitant medications will be followed and recorded at days 28 and 56 for vaccine and observation arm participants. Recipients who are removed or withdraw from the study before randomization (day 28) won’t be further immunologically monitored. Patients who relapse after randomization at D+28 through D+180 will be removed from treatment and will be taken off-study and there will be no further follow-up data collected. As of May 31, 2015,, all of the study-related procedures for the participants have been completed. Our primary analysis will be based on the data up to 180 days post-HCT. However, we have observed an unexpected reduction in the rate of chronic GVHD in patients randomized to the vaccine arm. In order to better assess the incidence of cGVHD after CMVPepVax, the follow up duration is extended from April 17, 2015 to 5/31/15 for all patients. This extended follow up is for data collection from medical records only without requiring additional patient visits or any other interventions. The data to be collected are focused on cGVHD, relapse, and survival.

5.5 Supportive Care, Other Concomitant Therapy, Prohibited Medications

Management of blood draw risks and vaccine reactions

of 68


Blood will be drawn on a maximum of 11 occasions (see Study Calendar, Section 10), and can cause pain and bruising. Though the amount drawn is limited to 30 ml/blood draw, some patients may experience transient hypotension, with symptoms of light-headedness and faintness. Since the patient will be in a reclining chair, the subject will be observed until the blood pressure stabilizes. For light-headedness and/or fainting, fluids by mouth and rest will be recommended.

Tet-CMV + PF-03512676 vaccine injections can cause local pain, redness and swelling, and it is possible that this could last for 1 or 2 days, requiring analgesic agents (Tylenol, 650 mg p.o.) and/or antihistamine (Benadryl, 25-50 mg p.o.). If severe, this could require additional medication, and rarely could result in a sterile abscess that would need to be drained surgically. This has not been reported in the Phase Ib trial in healthy adults (IRB 03121, please see Table 4)6, and it is generally considered unlikely with an s.c. route. Other common side effects of vaccination with Tet-CMV + PF-03512676 (Table 4)6 included injection pain, rash, transient fever, headache, nausea and vomiting, feeling of being generally sick, and tiredness. For pain, headache, and/or malaise, non-prescription analgesics (acetaminophen and antihistamine respectively), will be recommended. A consequence of these is that the HCT recipients may miss time from work. Should serious adverse reactions occur, treatment would be available at City of Hope.

Management of CMV infection and disease in HCT recipients Recipients will be monitored for CMV DNAemia with the schedule indicated in the Study Calendar (Section 10) and as necessary for SOC. Analysis will be performed using LightCycler® PCR. As for COH SOC, a CMV-positive result is defined as ≥500 DNA copies/ml.

Treatment will be initiated per COH institutional protocols.

CMV antiviral drugs including GCV, FOS, VAL, Cidofovir and CMX-001, per package insert instructions are not permitted in this trial. Initiation of CMV antiviral therapy for early reactivation before randomization (day 28 post-HCT) is exclusionary. The recipient will be withdrawn from the trial and additional enrollment will be planned. After day 28, administration of CMV antiviral therapy will be recorded in the patient’s CRF, considered vaccine failure for vaccinated recipients, and will preclude administration of a 2nd injection of vaccine on day 56. Patients will be followed for safety to the greatest extent possible and CMV immune-monitoring will be continued, if agreed by the subject.

Clinically significant CMV viraemia is defined as a level of infection that results in the initiation of CMV antiviral therapy84. By HCT-SOP as of February 2012, the recommended CMV-PCR value to start anti-CMV therapy is ≥1500 gc/ml unless there are other risk factors such as GVHD, steroid use, lymphopenia. CMV disease will be evaluated by the PI Dr. Nakamura, conferring with the treating physician and will be defined as described84.

Management of GVHD and secondary infections Acute and chronic GVHD will be assessed in the recipients with the schedule indicated in the Study Calendar (Section 10) and as necessary for COH institutional SOC. Acute GVHD grade will be assigned according to the Keystone Consensus grading system. Chronic GVHD will be classified by type of onset (progressive, interrupted, de novo, or chronic); basis of diagnosis (histologic/biopsy proven, clinical evidence, both, or unknown); Limited or Extensive chronic GVHD; and overall severity of GVHD (mild, moderate, or severe). Performance status will be evaluated utilizing the Karnofsky Performance Scale.

Diagnosis of >grade 2 GVHD before day 28 post-HCT) is exclusionary, and after day 28 post-HCT will prevent administration of a 2nd vaccination. Secondary infections are defined as bacterial, fungal, viral, protozoal, and helminthic infections that occur with increased frequency among HCT recipients.

of 68


Prophylaxis and treatment associated with GVHD and secondary infections will be administered according to COH institutional protocol and will be recorded in the CRF for each enrolled recipient.

Assessment of engraftment Engraftment will be assessed with the schedule indicated in the Study Calendar (Section 10), by monitoring the recipient’s absolute neutrophil count per COH institutional SOC. The date of engraftment is defined as the first of 3 consecutive days when the peripheral blood absolute neutrophil count is >500/mm3.

Graft failure by day 28 (absolute neutrophil count <500/mm3) is exclusionary. The recipient will be withdrawn from the trial and will be replaced. Subjects in the vaccine arm, who experience graft failure after day 28 (absolute neutrophil count <500/mm3) cannot receive a 2nd vaccine injection. The DSMC will review engraftment and engraftment failure data (which will be recorded in CRF) for all study recipients.

5.6 Additional Studies

Reference is made to Section 9.0 for the correlative studies to be conducted under this protocol.

5.6.1 Laboratory Studies

As for all other procedures described in this protocol, blood for laboratory studies will be drawn at the COH “Phase I unit” clinic for outpatients and in-house patients in their respected units, and with the schedule specified in the Study Calendar (Section 10). One blood draw to be performed 9 to 60 days pre-HCT, and 9 blood draws on days 28, 42, 56, 70, 84, 100, 130, 160, and 180 post-HCT will be required on this study.

Immediately after phlebotomy, a volume of approximately 30 ml (immunogenicity studies) will be handed to authorized personnel of the TVR Division for processing and storage at the TVR. In particular, PBMC will be separated from heparinized blood by standard density gradient centrifugation methods, washed, re-suspended in fetal calf serum (FCS) with 10% DMSO, aliquotted, and cryopreserved in the vapor phase of centrally monitored liquid nitrogen tanks.

CMV virology and CMP + uric acid, phosphorus, cholesterol, & LDH*/CBC with differential laboratories will be performed as for COH SOC procedures and processed at COH Clinical Microbiology and Clinical Chemistry Sections, respectively (Department of Transfusion Medicine). Results will be available on OACIS and recorded into the patient CRF for PI revision and data analysis. In particular CMV viraemia will be monitored by standard qPCR methods22 every 2 weeks from days 28 to 100, or more frequently as dictated by SOC. SMA18/CBC laboratories will be performed on days 28 and 56 as for specific protocol criteria (see 3.2.1 Post-HCT Study-Specific Exclusions). Clinical CMV disease and use of anti-viral drugs will be prospectively monitored and recorded in the patient CRF.

As mandated by the FDA, autoantibodies against dsDNA will be measured using Wampole® DS DNA ELISA II kit system, by Wampole Laboratories (Princeton, NJ).

Immunogenicity studies will be performed at the TVR and will include immune-monitoring of levels of CD8+ T cells binding to pp65 MHC class I tetramers and PD-1 expression on pp65-specific T cells. Additionally, we will monitor CMV-specific T cell growth kinetics and early cell death. All immune-monitoring studies will be performed by flow cytometry techniques using the FACS (FACSCantoTM with FACSDiva software; BD Biosciences, San Jose, CA)3.

All immunophenotyping will be conducted on freshly thawed PBMC without cultivation or stimulation in vitro. PBMC will be stained with each fluorochrome-conjugated antibody combination using standard

of 68


methods with commercially fluoresceinated antibodies (BD Biosciences, San Jose, CA), as described in our published studies18,85. Briefly, PBMC will be stained with either CMV pp65495-503 or HIVgag77-85 (as control) APC-conjugated tetramers (Beckman Coulter, Brea, CA) and antibodies against CD3, CD8 and PD-1 labeled with APC-Cy7, FITC and PE, respectively (BD Biosciences, San Jose, CA). PBMC will be analyzed for levels of CD8+ T cells binding to the tetramers and PD-1 expression by FACS (FACSCantoTM with FACSDiva software; BD Biosciences, San Jose, CA)36. In combination with CBC, we will be able to calculate the absolute number of pp65495-503–specific CD8+ T cells/L.

Assessment of CMV-specific T cell growth kinetics will be performed using the carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution method for cell division tracking as previously detailed19. Briefly, proliferation will be analyzed using the CFSE dilution method, according to the manufacturer’s procedure (Molecular Probes, Carlsbad, CA). To assess CD8 T cell proliferation, PBMC will CFSE labeled and incubated for 6 days with 1 µg/ml pp65495-503 peptide or DMSO diluent as control, in the presence of anti-CD49b and CD28 (1 µg/ml). Cells will be then washed and co-stained with anti-CD8 (all antibodies from BD Biosciences) and FACS analyzed for CFSE fluorescence levels.

Apopotic cell death will be determined using the BD ApoAlert annexin V-FITC Apoptosis Kit (BD Biosciences) according to the manufacturer’s instructions, and cells will be analyzed by FACS86.

6.0 Dose Delays/Modifications for Adverse Events

In the interventional vaccine arm, both vaccination dates (days 28 and 56) can be modified ± 5 days from the planned date. This window will allow providing treatment to the recipients when mostly appropriate and specifically only to HCT patients who do not:

experience graft failure (absolute neutrophil count <500/mm3) had relapse Diagnosed with >grade 2 GVHD before day 28 post-HCT, and diagnosed with grade >2

GVHD between day 28 post-HCT and administration of a 2nd vaccination at day 56. receive prednisone >1 mg/kg/day, less than 7 days prior to injection receive antiviral treatment including

the use of GCV/VAL, FOS, Cidofovir, and CMX-001 have viraemia ≥1500 gc/ml by qPCR Ongoing post HCT >= grade 3 non-hem AE’s, with exception of grade 3 glucose

intolerance and grade 3 non-hem labs; cholesterol, triglyceride, and hyperglycemia. .

If any of the conditions listed above are detected and/or reported by day 56 ±5 days, the second vaccine injection will be not administered to the HCT recipient. As for the control arm (prospective immune-monitoring only), the same window time and conditions listed above will be applicable for obtaining blood specimens. All patients from both arms who are ruled not eligible before randomization (day 28) can be replaced. However, every patient who has been enrolled and randomized (day 28) is considered to be part of the trial.

of 68


7.0 Data and Safety Monitoring

7.1 Definition of Risk Level

This is a Risk Level 4 study, as defined in the “City of Hope Data and Safety Monitoring Plan”, http://www.coh.org/dsmc/Pages/forms-and-procedures.aspx involving a COH-held IND.

7.2 Monitoring and Personnel Responsible for Monitoring

The Protocol Management Team (PMT) consisting of PI, collaborating investigator, CRA/protocol nurse, and statistician is responsible for monitoring the data and safety of this study, including the implementation of the stopping rules for safety and efficacy.

Table 1: City of Hope PMT Reporting Timelines for the DSMC

Risk Level Phase Standard Reporting

Requirement

RL 1, RL2, and Compassionate Use Studies

No reports required

3 I Every 3 months from activation date, as indicated in MIDAS

3 Pilot,

Feasibility, II‐IV

Every 6 months from activation date, as indicated in MIDAS

4 Pilot,

Feasibility, I‐IV

Every 3 months from activation date, as indicated in MIDAS

Data and safety will be reported to the COH DSMC using the PMT report and submitted quarterly from the anniversary date of activation. Protocol specific data collection will include the following items: assessment of engraftment, GVHD (graded according to the Keystone Consensus grading system, see table below), AEs based on NCI CTCAE, Version 4.03; relapse status; usage of antivirals or any other infective agent, local and systemic reactogenicity, and toxicity related to the vaccine formulation.

of 68


. CONSENSUS GRADING SYSTEM FOR ACUTE GVHD 1994 Keystone Consensus Criteria

Organ Staging of Clinical Acute GVHD Skin Lower GI Upper GI Liver (Total Bilb)

0- No Rash 1- Maculopapular

rash, <25% of body surface

2- Maculopapular rash, 25-50% of body surface

3- Rash on >50% of body surface, or generalized erythroderma

4- Generalized erythroderma with bullous formation and/or desquamation

0- ≤500 mL/day or <280 mL/m2/day 1- >500 but ≤1000 mL/day or 280-555 mL/m2/day 2- >1000 but ≤1500 mL/day or 556-833 mL/m2/day 3- >1500 mL/day or 833 mL/m2/day 4- Severe abdominal pain with or without ileus, or stool with frank blood or melena

0- No protracted nausea and vomiting 1- Persistent nausea, vomiting, OR biopsy showing acute GVHD of stomach or duodenum

0- <2.0 mg/dL 1- 2.0-3.0 mg/dL 2- 3.1-6.0 mg/dL 3- 6.1-15 mg/dL 4- >15.0 mg/dL

Overall Clinical Grading of Severity of Acute GVHD

Grade Skin Gut Liver

1 Stage 1-2 & None

0

& None

0

11 Stage 3 Or Stage

of 68


1 Or Stage

1

111 Stage

0-4

Or

Stage

2-4

Or

Stage

2-3

1V Stage

4

Or

Stage

0-4

Or

Stage

4

If KPS is ≤ 30%, or decreased ≥ 40% from baseline KPS, the status is Grade IV

Chronic GVHD

Onset of Chronic GVHD

*Karnofsky/Lansky score at time of diagnosis

Progressive (acute GVHD professed directly to chronic)

Interrupted (acute GVHD resolved, then Chronic developed)

De novo (acute GVHD never developed)

Chronic GHVD Flare (symptoms reactivated within 30 days of drug tapering or discontinuation)

Diagnosis of Chronic GVHD based on

Histologic evidence/biopsy proven

Clinical Evidence

Both

Unknown

Maximum Chronic GVHD Limited-localized skin involvement and/or hepatic dysfunction due to chronic GVHD

Extensive-generalized skin involvement; or, liver histology showing chronic aggressive hepatitis, bridging necrosis or cirrhosis; or involvement of eye: Schirmer’s test with <5mm wetting; or, involvement of minor salivary glands or oral mucosa demonstrated on labial biopsy; or, involvement of any other target organ

Overall Severity of Chronic GVHD

Mild-signs and symptoms of chronic GVHD do not interfere substantially with function and do not progress once appropriately treated with local therapy or standard systemic therapy (corticosteroids and/or cyclosporine or FK 506)

Moderate-signs and symptoms of chronic GVHD interfere somewhat with function despite appropriate therapy or are progressive through first line systemic therapy (corticosteroids and/or cyclosporine or FK 506)

Severe-signs and symptoms of chronic GVHD limit function substantially despite appropriate therapy or are progressive through second line therapy

of 68


7.3 Definitions

Adverse event (AE) - An adverse event is any untoward medical experience or change of an existing condition that occurs during or after treatment, whether or not it is considered to be related to the protocol intervention.

Unexpected Adverse Event [21 CFR 312.32 (a) – An adverse event is unexpected if it is not listed in the investigator’s brochure and/or package insert; is not listed at the specificity or severity that has been observed; is not consistent with the risk information described in the protocol and/or consent; is not an expected natural progression of any underlying disease, disorder, condition, or predisposed risk factor of the research participant experiencing the adverse event.

Expected Adverse Event - Any event that does not meet the criteria for an unexpected event OR is an expected natural progression of any underlying disease, disorder, condition, or predisposed risk factor of the research participant experiencing the adverse event

Serious Adverse Event (SAE) [21 CFR 312.32] is defined as any expected or unexpected adverse event that results in any of the following outcomes: Death Is life-threatening event (places the subject at immediate risk of death from the event as it occurred); Requires in-patient hospitalization (not required as part of the treatment) or prolongation of existing

hospitalization; A persistent or significant disability/incapacity; A congenital anomaly/birth defect Secondary Malignancy, or Any other adverse event that, based upon appropriate medical judgment, may jeopardize the subject’s

health and may require medical or surgical intervention to prevent one of the outcomes listed above (examples of such events include allergic bronchospasm requiring intensive treatment in the emergency room or at home, blood dyscrasisas of convulsions that do not result in inpatient hospitalization, or the development of drug dependency or drug abuse).

Unanticipated problem (UP) – Any incident, experience or outcome that meets all three of the following criteria:

1. Unexpected (in term nature, severity, or frequency) given the following: a) the research procedures described in the protocol-related documents such as the IRB approved research protocol, informed consent document or Investigator Brochure (IB); and b) the characteristics of the subject population being studied; AND

2. Related or possibly related to participation in the research (possibly related means there is a reasonable possibility that the incident, experience, or outcomes may have been caused by the drugs, devices or procedures involved in the research); AND

3. Suggests that the research places subjects or others at greater risk of harm (including physical, psychological, economic, or social harm) than previously known or recognized.

Unexpected Adverse Event [21 CFR 312.32 (a) – An adverse event is unexpected if it is not listed in the investigator’s brochure and/or package insert; is not listed at the specificity or severity that has been observed; is not consistent with the risk information described in the protocol and/or consent; is not an expected natural progression of any underlying disease, disorder, condition, or predisposed risk factor of the research participant experiencing the adverse event.

of 68


7.4 Reporting of Unanticipated Problems and Adverse Events

Unanticipated Problems: Unanticipated problems must be reported to the COH DSMC and IRB within 5 calendar days according to definitions and guidelines at http://www.coh.org/hrpp/Pages/hrpp-policies.aspx. Any unanticipated problem that occurs during the study conduct will be reported to the DSMC and IRB by submitting electronically in iRIS (http://iris.coh.org).

Serious Adverse Events - All SAEs occurring during this study, whether observed by the physician, nurse, or reported by the patient, will be reported according to definitions and guidelines at http://www.coh.org/hrpp/Pages/hrpp-policies.aspx and Table 1 below. Those SAEs that require expedited reporting will be submitted electronically in iRIS (http://iris.coh.org/).

Adverse Events - Adverse events will be monitored by the PMT. Adverse events that do not meet the criteria of serious adverse event or are not unanticipated problems will be reported only at the time of protocol continuation reports (see Table 1 below).

City of Hope Adverse Event Reporting Timelines for the IRB

Required Reporting Timelines to DSMC for AE/SAEs Investigator Initiated Studies

Required Reporting Timeframe to DSMC

Attribution UNEXPECTED EXPECTED

Death while on active treatment or within 30 days of last

day of treatment

Possibly, Probably, Definitely 5 calendar days

Unlikely, Unrelated

Death after 30 days of last active treatment/therapy

Possibly, Probably, Definitely

5 calendar days No reporting required

Unlikely, Unrelated No reporting required No reporting required

Grades 3 and 4 AND meeting the definition of "serious"


5 calendar days 10 calendar days

Unlikely, Unrelated 5 calendar days 10 calendar days

Grades 1 and 2 AND resulting in "hospitalization"

of 68



5 calendar days 10 calendar days

Unlikely, Unrelated 10 calendar days 10 calendar days

Externally Sponsored Studies

Required Reporting Timeframe to DSMC

Attribution UNEXPECTED1 EXPECTED

Death while on active treatment or within 30 days of last

day of treatment

Possibly, Probably, Definitely No DSMC reporting required - IRB reporting may be

necessary Unlikely, Unrelated

Death after 30 days of last active treatment/therapy



Grades 3 and 4 AND meeting the definition of "serious"



Grades 1 and 2


No DSMC reporting required - IRB reporting may be necessary

An event determined by the IRB of record to be an Unanticipated Problem (UP) will be communicated to the Investigator and COH DSMC through the COH IRB Operations Director. The DSMC will review the case and make a determination as to whether the study will be suspended, terminated, amended, or allowed to continue without amendment.

of 68


Required Reporting Timeframe to IRB of Record

Attribution UNEXPECTED EXPECTED

Death


5 calendar days Annual

Unlikely, Unrelated Annual Annual

Grades 3 and 4 AND meeting the definition of a UP


5 calendar days Annual


Grade 1 and 2 AND meeting the definition of a UP

Possibly, Probably, Definitely 5 calendar days

Annual


8.0 Agent Information and Risks

8.1 Tet-CMV

8.1.1 Description

cGMP-grade (Bachem Inc, Torrance, CA) peptide vaccine Tet-CMV (=Tetanus-CMV; CODE NAME:

of 68


NSC-721434; IND, BB-13124) consists of the HLA A*0201-restricted CMV pp65495-503 epitope covalently attached to the tetanus toxin tt830-843 epitope (P2), recognized by many HLA DR alleles5,20,21 . The TH and CTL epitope peptide are separated by 3 alanines (Figure 5). The final product contains L-amino acids of the primary structure shown in Figure 5. Tet-CMV vaccine is a 29 amino acid and 3052.6 dalton peptide. Its Molecular Formula is: Tet-CMV = C137H227N35O41S1. Its Amino Acid Sequence is: Tet-CMV: H-Lys-Ser-Ser-Gln-Tyr-Ile-Lys-Ala-Asn-Ser-Lys-Phe-Ile-Gly- Ile-Thr-Glu-Ala-Ala-Asn-Leu-Val-Pro-Met-Val-Ala-Thr-Val-OH. The Pharmacologic Class is: Peptide Vaccine. No preclinical studies have been conducted on Tet-CMV pertaining to its pharmacologic effects or mechanism of action.

8.1.2 Toxicology

In order to obtain IND BB-13124, Tet-CMV was evaluated in toxicology studies in Fisher rats. A study of the effect of the dosages and precise composition of the vaccine formulation given to healthy adults6 was carried out in rats in a GLP setting (Southern Research Institute, Protocol #11200.11.01). The final report revealed no significant toxicity at all dosages. This is the first evaluation of Tet-CMV (IND BB-13124) experimental peptide vaccine in HCT recipients, therefore no Comprehensive Adverse Event and Potential Risks (CAEPR) or toxicology data are available. However, its usage in healthy adults has been studied and evaluated, and the complete panel of safety data from the Phase Ib dose-escalation trial have been published6 and reported in Human Studies (Section 2.4). Briefly, the results indicated that Tet-CMV is safe and well tolerated in most subjects (Table 1 and 2). Most common AEs included mild to moderate cutaneous reactions at the injection site. The duration of related Grade 1 and 2 AEs ranged from 1-2 days. No grade 3 AEs were reported using the vaccine formulation which we are proposing to use in HCT recipients (2.5 mg Tet-CMV peptide co-injected with 1 mg of PF-03512676).

8.1.3 Pharmacology – Handling, Storage, Dispensing and Disposal

There have been no preclinical studies conducted on Tet-CMV pertaining to its pharmacokinetic, absorption, distribution, metabolism and excretion.

Tet-CMV study drug is being provided to COH by the National Cancer Institute sponsored NEXT (NCI-NEXT) program, which contracted with Bachem Incorporated (Torrance, CA) for cGMP-grade production of Tet-CMV. Tet-CMV is supplied frozen in 10 mM sodium acetate (pH 4.2), as a sterile, preservative-free, solution for SC injection at 10 mg/ml in 1.0 ml volume, and packaged in 2-ml USP Type I clear glass vials with gray butyl, FluroTec®-coated stoppers and aluminum flip-off seals. Currently, there are a total of 112 x11 mg/ml vials of Tet-CMV stored in -65 to -80 oC locked and temperature-monitored freezers. Of these vials, 90 are located at COH Pharmacy and 23 at the Biopharmaceutical Development Program (BDP), NCI-Frederick, Frederick, MD. The number of Tet-CMV vials stored at COH Pharmacy exceeds the amount necessary for completing the current Phase I study, since a max of 40 vials are needed to vaccinate the planned 18 HCT recipients, and 3-5 possible replacements. The remaining vials at BDP will be used for stability analyses.

of 68


NCI contracts with SAIC, Frederick, MD to provide interim stability analysis of Tet-CMV every 6 months. The reports by SAIC Biopharmaceutical Quality Control (available upon request) show that the investigational product consistently meets the required specification for stability. Additionally, annual IND (IND, BB-13124) reports have been submitted to FDA, and approval for study continuation has been granted since 2008.

To limit cost and delays, the NCI Pharmaceutical Research Branch has filled vials with vaccine without lyophilization in the formulation solvent of 10mM sodium acetate (pH 4.2) at a concentration of 10 mg/ml. 10mM sodium acetate (pH 4.2) will be used at COH Pharmacy to dilute the peptide, before administration.

As detailed in Section 5.4, concomitant medications that might interfere with the evaluation of the investigational Tet-CMV should not be administered, from 30 days prior to participation in the trial and up to 14 days after the second vaccination (day 70 post-HCT). Medications in this category include, but are not limited to:

live attenuated vaccines medically indicated subunit or killed vaccines (e.g. influenza, pneumococcal, or allergy treatment

with antigen injections).

The use of alemtuzumab for immunosuppression is not permitted in this study (see Section 3.1.4 for additional information).

Antiviral treatment including the use of GCV/VAL, FOS, Cidofovir and CMX-001 are permitted for defined vaccine failure (see Section 5.1) before day 100, and anti-infective agents (e.g. antibiotics, anti-fungals, anti-parasitics) will be administered according to COH recommendation and reported for each enrolled patient in the CRFs.

Recipients in the vaccine arm will be injected twice with 2.5 mg Tet-CMV + 1 mg PF-03512676 adjuvant in a final 1.0 ml volume, in the upper arm by SC route. The dosage and route are based on the Phase Ib trial safety data conducted in healthy volunteers, who received up to 4 injections of the vaccine with adjuvant, and showed substantial expansion of pp65495-503 T cells already after 2 injections6

The vaccine preparation procedure will be performed according to IRB 03121 COH Pharmacy SOP used to perform the Phase Ib trial in healthy adults6:

Thawing and formulation of the vaccine

Put cooling block in hood and set to 4°C. Once the LCD on the cooling block reads 4°C, measure the temperature of the cooling block with the NIST thermometer (take the NIST thermometer out of the freon and lay it on the block). Adjust LCD until NIST thermometer reads 4°C. Only the NIST thermometer should be relied upon for accuracy. Record the LCD and NIST temperature readings on the Vaccine Compounding Record. Obtain frozen Tet-CMV vial from the Pharmacy freezer. Allow vaccine vial to thaw at room temperature (approximately 15-30 minutes). Once thawed, place vial on the cooling block cooled to 4°C (based only on NIST thermometer reading, not the LCD) to keep cold. Record the time at which the vial is completely thawed. This is the start time of the vaccine dose preparation. Administration of the vaccine must occur within 90 minutes after start time of preparation.

Obtain diluent and PF-03512676 from Pharmacy refrigerator. Keep cold by placing them on the cooling block. Preparation of vaccine must be done at approximately 4°C to minimize “clouding” of solution.

Using sterile procedures, inject 0.6 ml Normal Saline solution into the PF-03512676 vial (containing 18 mg/1.2 ml of PF-03512676), mix thoroughly and keep on ice. The final concentration of PF-03512676 will be 10 mg/ml in 1.8 ml total volume.

of 68


Vaccine injection preparation Withdraw 0.38 ml of Tet-CMV into a 1 ml sterile syringe. Transfer Tet-CMV into a 5 ml sterile empty vial. Add 0.99 ml (rounded to 1 ml) of 10mM Sodium Acetate, pH 4.2 from diluent vial. Add 0.15 ml of PF-03512676, which will raise the pH of the vaccine formulation above pH 6.0, an

acceptable level for human use. Vortex the vial (containing 1.53 ml of volume) for 30 seconds at highest setting. Withdraw 1 ml and cap syringe. After labeling appropriately, place syringe in sealable plastic amber bag and place in ice bucket. Record the time that the vaccine is placed in ice bucket. Attach a sample label to the green

compounding sheet. The vaccine will be transported per COH standard protocol to “Phase I unit” clinic , in a ice

bucket. Fax Ms. Drake the BLUE compounding form, and also send her the original sheet with the vaccine.

Administration of the vaccine must occur within 90 minutes after start time of preparation, since the potency tests performed in Tg mice to obtain IND BB-13124 showed substantial stability of the vaccine formulation within this time range.

Residual and unused vaccine compounds are to be disposed in the red biohazard bags and incinerated.

8.2 PF-03512676

Pfizer PF0351276 IB is attached to this protocol. The brochure is dated February 2010. We have summarized below its content and added recent findings and studies.

8.2.1 Description

Pfizer PF-03512676 (previously referred as CPG 7909 and CPG ODN 200672) adjuvant is a pure substance named Agatolimod Sodium, and is classified as an investigational agent. PF-03512676 is an immunostimulatory single-stranded, phosphorothioate oligodeoxynucleotide containing four unmethylated deoxycytosine-deoxyguanine dinucleotide (CpG) motifs and synthesized with a nuclease-resistant phosphorothioate backbone. PF-03512676 is 24 nucleotides in length. Its structural formula is: (3'-5')d(P-thio)(T-C-G-T-C-G-T-T-T-T-G-T-C-G-T-T-T-T-G-T-C-G-T-T) tricosasodium salt. Its molecular formula is: C236/H303/N70/O133/P23/S23/Na23. For further details regarding the physical and chemical properties of PF-03512676, please see the enclosed Pfizer IB. The optimal unmethylated CpG motif for interaction with TLR9 is GTC CTT, and in humans this interaction of CpG with TLR9 on selected DCs and on B cells induces the production of TH1-like proinflammatory cytokines, interferons, and chemokines. Activated DCs express increased levels of MHC and costimulatory molecules leading to enhanced antigen presentation and activation of T cells, including cytolytic T cells. There is subsequent secondary activation of macrophages, monocytes, NK cells, other DC subsets, and neutrophils. Activated B cells proliferate, have enhanced antigen presentation, and secrete antibodies and cytokines. PF-03512676 is a potent agonist of the TLR9, and belongs to a group of CpG oligodeoxynucleotides (ODNs) characterized by repeated sequences of unmethylated CpG motifs known to induce strong immunopotentiation14. The natural DNA phosphodiester backbone has been modified with phosphorothioate linkages that provide the molecule with increased stability due to enhanced resistance to enzyme degradation. PF-03512676 mechanism of action is related to direct and indirect stimulation of both innate and adaptive immune responses, as shown in Figure 6.

of 68


This cascade of immune reactions results in increased secretion of antibodies from B cells, cytokines and chemokines from a variety of cells, and increased NK cell activity as well as enhanced antigen presentation and T cell-help that can augment both humoral and T cell-mediated immune responses87. For further details regarding physical and chemical properties, and mechanism of action of PF-03512676, please see enclosed Pfizer IB.

8.2.2 Toxicology

In animal studies, the toxicity of PF-03512676 includes effects of immune stimulation (liver and spleen enlargement and inflammatory infiltrates in rodents), activation of the alternative complement pathway and inhibition of the intrinsic coagulation pathway (shock-like syndrome in monkeys following rapid infusion), and dose-related histopathologic changes in liver and kidney during subacute and chronic administration. All of these effects require dosing significantly higher than 1 mg dose/injection proposed in this trial, e.g., in monkeys the no-effect dose level for complement activation is 2-5 mg/kg.

PF-03512676 had been determined to be embryolethal in rabbits and teratogenic in developing rats and rabbits. Consequently, all women of childbearing potential in clinical studies of PF-03512676 must have a negative pregnancy test before participating in the study and must use effective contraception while on

of 68


study treatment. Use of effective contraception is also required for male patients who participate in clinical trials of PF-03512676.

PF-03512676 has no human genetic (antisense) activity or genotoxicity. Phase I studies in healthy volunteers showed that PF-03512676 was well tolerated at SC doses up to 0.08 mg/kg and IV doses up to 0.32 mg/kg6,88. SC administration caused transient changes in blood leukocyte levels consistent with an immune response and patients had transient injection site reactions and flu like symptoms, but there was no evidence of organ toxicity or autoimmunity88. There were minimal adverse events after IV administration88. Pharmacokinetics studies showed that IV administration resulted in rapid distribution of PF-03512676 to the liver, kidney and spleen, while SC injection resulted in high drug concentrations in the injection site and draining nodes that were maintained for at least 2 weeks88. PF-03512676 has been tested as monotherapy and combination therapy for renal cell cancer, lung cancer, NHL and cutaneous T cell lymphoma, and as an adjuvant for vaccine therapy for breast cancer and melanoma89-95. Monotherapy was well tolerated but had low efficacy, with the best responses seen with direct injection of tumor tissue and SC administration89. Among the most recent Phase I studies96, PF-03512676 administered to patients with early relapsed CLL as a single IV dose was well tolerated with no clinical effects and no significant toxicity up to 1.05 mg/kg. Single dose SC PF-03512676 had a MTD of 0.45 mg/kg with dose limiting toxicity of myalgia and constitutional effects. Multiple weekly SC doses at the MTD were well tolerated. PF-03512676 administration induced immunologic changes in CLL and non-malignanT cells that were dose and route dependent. Further details regarding comprehensive adverse events and potential risks (CAEPR) can be found in the enclosed Pfizer IB.

Unlike the high doses of PF-03512676 given in oncology studies PF-03512676 has only been given at doses of 1 mg as a vaccine adjuvant (as proposed in this trial). A more severe safety profile is associated with IM than SC administration of PF-03512676. As reported in our Phase Ib study6, use of PF-03512676 as a vaccine adjuvant at doses of 1 mg SC/injection results in an AE profile consisting of frequently reported mild to moderate injection site reactions and flu-like symptoms. Adjuvant doses of 1 mg administered one to four times6 were generally well-tolerated and did not result in any related SAEs. Additionally, when PF-03512676 was used as an adjuvant with either Engerix-B hepatitis vaccine, influenza Fluarix vaccine or anthrax vaccine maximum titers (both IgG and neutralizing antibody titers) were greater than with vaccine alone and high titers were achieved earlier9,74,97,98. Induction of cytokines, most notably IP-10 and MCP-1 and a transient leukopenia are additional evidence of the immunomodulatory activity of PF-03512676. In the most recent double-blind study performed in healthy subjects, BioThrax plus 1 mg of PF-03512676, given IM on days 0, 14 and 28 substatnially increased and accelerated the response compared to the response seen in subjects vaccinated with BioThrax alone. No serious AEs related to study agents were reported, and the combination was considered to be reasonably well tolerated.

There is a limited experience regarding the usage of PF-03512676 in transplantation. In a post-bone marrow transplantation AML animal model, PF-03512676 alone had little effect, but substantially improved the efficacy of donor lymphocyte infusion99. In a Phase Ib trial (Dr. Miller, University of Minnesota, personal communication), PF-03512676 was found to be safe and with minimal reactogenicity when administered to HCT recipients.


PF-03512676 is supplied by Pfizer as a sterile, preservative-free, isotonic, phosphate buffered saline solution for SC injection at a concentration of 15 mg/ml, pH 7.4 and packaged in 2-ml USP Type I clear glass vials with gray butyl, FluroTec®-coated stoppers and aluminum flip-off seals. Each vial contains 1.2 ml of extractable volume (18 mg/1.2 ml of PF-03512676). Pfizer is committed to support this Phase I

of 68


trial (please see the enclosed Pfizer letter) with PF-03512676, which will be shipped to COH on ice, and stored under refrigeration (2 to 8°C) at the COH investigation Pharmacy in a locked temperature controlled 4°C. Currently, there are a total of 170 vials of PF-03512676 vials stored in locked and temperature-monitored +4 oC refrigerators, at the COH Pharmacy. It is anticipated that the amount of PF-03512676 is sufficient for completing the current Phase I study, though further material can be obtained by Pfizer upon request. Stability tests on PF-03512676 are routinely performed by Pfizer, and during the Phase I trial will be promptly communicated to the study PI. Residual and unused PF-03512676 vials are to be disposed in the red biohazard bags and incinerated. In the current Phase I trial, PF-03512676 will be administered SC as an adjuvant at the constant dose of 1 mg/vaccine injection74 .

Clinical pharmacokinetics of PF-03512676 were studied following repeated IV or SC administration of PF-03512676. Mean plasma exposure parameters (Cmax and AUC) increased with dose after both IV and SC administrations. Plasma concentrations declined rapidly after Cmax. Tmax shifted to a later time with increase of dose (0.5 hours at 0.0025-mg/kg dose and 2 hours at 0.08-mg/kg dose) following SC administration. There was no apparent plasma PF-03512676 accumulation observed after weekly IV or SC administrations. SC administration of PF-03512676 is associated with greater Cmax levels of selected cytokines, and certain of these cytokines are detectable in plasma for up to seven days. Reflecting this cytokine activation, there is an increase in total white blood cell count approximately 12 hours after dosing followed by a transient fall in total leucocytes, neutrophils, and lymphocytes over the ensuing 24-72 hours. The kinetics of these changes and the absence of immature circulating cell forms suggest that this is due to redistribution of leucocytes, rather than to destruction or altered production. As would be expected from the immunostimulatory activity of PF-03512676, there is at least an additive effect in immune responses when this agent is co-administered with vaccines.

Undesirable pharmacodynamic interactions have not been observed in studies performed to date. At the present time, the effects of PF-03512676 on hepatic drug metabolism, including any effect on P450 enzymes, are not known. The potential for interactions between PF-03512676 and other drugs are also currently not known. Section 5.4 and 8.1.3 provide the list of concomitant medications that might interfere with the evaluation of the investigational Tet-CMV.

All the procedures related to the reconstitution of PF-03512676 with Normal Saline solution, handling precautions, and dispensing instructions for the final preparation of the injectable vaccine formulation are detailed in Section 8.1.3.

8.3 Sodium acetate

8.3.1 Description

A solution of 10 mM sodium acetate (CH3COONa), pH 4.2 will be used as a diluent for Tet-CMV. Formulation studies required to obtain BB-13124 IND indicated that 2.5mg of Tet-CMV + 1 mg of PF-03512676 are soluble in a solution of 10 mM sodium acetate, pH 4.2.

of 68


Sodium is the principal cation of extracellular fluid. It comprises more than 90% of total cations at its normal plasma concentration of approximately 140 mEq/liter. The sodium ion exerts a primary role in controlling total body water and its distribution.

Acetate (CH3COO− ), a source of hydrogen ion acceptors, is an alternate source of bicarbonate (HCO3−) by metabolic conversion in the liver. This has been shown to proceed readily, even in the presence of severe liver disease.

8.3.2 Toxicology

There are no data available on the toxicology of 10 mM sodium acetate, pH 4.2, administered by SC route.

Sterile, nonpyrogenic, 40 mEq (2 mEq/ml) USP solution of sodium acetate are typically administered, after dilution, by the IV route as an electrolyte replenisher. The solution is intended as an alternative to sodium chloride to provide sodium ion (Na+) for addition to large volume infusion fluids for IV use.


This colorless salt is manufactured as a cGMP-grade solution at SAIC (NSC 733084). It is supplied by BDP, NCI-Frederick as a sterile, preservative-free, solution for SC injection at 10mM, 1.0 ml volume, pH 4.2, and packaged in 2-ml USP Type I clear glass vials with gray butyl, FluroTec®-coated stoppers and aluminum flip-off seals. The sodium acetate 10mM solution has passed a safety test, sterility and stability tests, which are performed every 6 months. The reports by SAIC Biopharmaceutical Quality Control (available upon request) show that the solution consistently meets the required specification for stability. Additionally, annual IND (IND, BB-13124) reports have been submitted to FDA, and approval for study continuation has been granted since 2008. Currently, there are a total of 381 vials of sodium acetate vials stored in locked and temperature-monitored +4 oC refrigerators. Of these vials, 71 are located at COH Pharmacy and 310 at BDP, NCI-Frederick. It is anticipated that the amount of sodium acetate vials stored at COH Pharmacy should be sufficient for completing the current Phase I study, though further material can be obtained by BDP upon request.

Pharmacokinetics and pharmacodynamic interactions have not been established for the sodium acetate 10 mM solution. The potential for interactions between sodium acetate 10mM solution and other drugs are also currently not known. Section 5.4 and 8.1.3 provide the list of concomitant medications that might interfere with the evaluation of the investigational Tet-CMV, and should not be administered.

All the procedures related to the handling precautions, and usage of sodium acetate as a diluent for the final preparation of the injectable vaccine formulation are detailed in Section 8.1.3.

Residual and unused sodium acetate vials are to be disposed in the red biohazard bags and incinerated.

of 68


9.0 Correlative/Special Studies

The correlative studies will include CMV-specific viral and immunogenicity monitoring, which will follow the schedule indicated in the Study Calendar (Section 10) and the methodology detailed in Section 5.6.1 (Laboratory Studies).

For CMV monitoring, standard qPCR22 methods will be required to evaluate CMV viral load and possible vaccine failure twice weekly or as required by SOC until day 100. Clinical CMV disease and use of anti-viral drugs will be prospectively monitored.

The correlative immunogenicity studies will include monitoring levels and quality of CMV-specific CD8+ T cells by multi color flow cytometric analyses. Levels of CD8+ T cells ≥7 cells/µL binding to CMV-specific MHC class I tetramers, before day 65 post-HCT were associated with protection from recurrent or persistent CMV infection or disease in a prospective multicenter trial (including Dr. Nakamura at COH), conducted in CMV-positive allogeneic HCT recipients4. Additionally, we and others have reported that up-regulation of PD-1on CMV-specific T cells of immunosuppressed HCT and solid organ transplant (SOT) recipients lead to T cell proliferative exhaustion and accelerated death, and is associated with CMV disease17-19. Co-injection of PF-03512676 CpG DNA in the vaccinated recipients may reduce CMV-specific T cell impairment, since engagement of TLR 9 by CpG DNA enhances T cell response by several mechanisms, including prolonging cell survival through the up-regulation of antiapoptotic molecules15,16.

Based on these findings, immune-monitoring will be performed by MHC class I tetramers, cell surface phenotyping, and assessment of CMV-specific proliferative capacity. Briefly, CMV pp65495-503 and HIVgag77-85 (control) tetramers will be prepared and titrated against reference peripheral blood mononuclear cells (PBMC) at the TVR lab, as previously described5,100. PBMC isolated by standard density gradient centrifugation methods from heparinized blood, will be stained with APC-conjugated tetramers (either CMV pp65495-503 or HIVgag77-85), and antibodies against CD3, CD8 and PD-1 labeled with APC-Cy7, FITC and PE, respectively (BD Biosciences, San Jose, CA). PBMC will be analyzed for levels of CD8+ T cells binding to the tetramers and PD-1 expression by FACS (FACSCantoTM with FACSDiva software; BD Biosciences, San Jose, CA)5. Assessment of CMV-specific T cell growth kinetics will be performed using the CFSE dilution method for cell division tracking; early cell death will be evaluated by using the ApoAlert Annexin V-FITC Apoptosis kit (Clontech, Mountain View, CA)86. Reduction of PD-1 expression and decreased levels of apoptosis markers and increased proliferation of CMV-specific T cells is expected in vaccine recipients compared to unvaccinated control group. Levels of pp65-specific T cells associated with protection from CMV complications are anticipated to be detected in a significantly higher proportion among recipients of the vaccine compared to the control arm.

of 68


10.0 Study Calendar

Daya

-60/ -9

0

HCT

14

28

42

56

70

84

100

130

160

180

IND Vaccine Regimenb R R

Informed consentc R

Inclusion/Exclusionc R R R

Demographicsc X

Medical historyc X

Concurrent medsc X R R

Physical examc R R R

Performance Status R R B-HCG d Rc Rb Rb CMP+4 R R R CBC + differentialI R R R R R R R R R R Engraftment Assessmentf R R R R R R R GVHD Gradingc R R R R R R R R R R CTCAE Reviewc R R R R R R CMV qPCR g R R R R R R Wampole® dsDNA ELISA IIb R R R R CMV immune-monitoring R R R R R R R R R R Blood draw (30 ml)g R R R R R R R R R R

a: All times are relative to the day of HCT, day 0. Visit and relative procedure may be performed +/- 5 days from the time indicated. . The interval between day 160 and 180 blood draws should be not less than 20 days (≥ 20 and ≤ 60 days), thus the ± 5 day window can be altered to fit this time frame requirement (see 5.1.1). No research visit/procedure (R) will be made, which duplicate SOC (X) requirements. Additional assessments/analysis may be performed for SOC indication. b: Vaccine arm only. c: Vaccine and Observation arm d: Serum or urine pregnancy test (women of childbearing potential). e: Sequential Multiple Analysis (CMP+4) comprehensive metabolic panel including phosphorus,uric acid, LDH, and cholesterol. f: Per protocol: Engraftment will be monitored by the recipient’s absolute neutrophil count per COH institutional SOC

of 68


g: Or as necessary based on vaccine failure definition. h: For immunogenicity studies and dsDNA studies at the TVR lab. I: CBC Differential at Pre-HCT, D+28, D+42, D+56, D+70, D+84, D+100, D+130, D+160, & D+180

of 68


11.0 Endpoint Evaluation Criteria/Measurement of Effect

11.1 Response Criteria

Endpoints of this trial are safety (primary endpoint) and immunogenicity (secondary endpoint) of Tet-CMV co-injected with PF-03512676, in HLA A*0201 CMV-positive recipients of allogeneic HCT. PRIMARY ENDPOINT. Evaluation of safety will be performed in all randomized recipients, and will be based on: assessment of GVHD, graded according to theKeystone Consensus grading system and AEs based on NCI CTCAE, Version 4.03, respectively. GVHD, local and systemic reactogenicity, and toxicity related to the vaccine formulation will be evaluated by the study PI, conferring with the treating physician. Specifically, GVHD s will be monitored as necessary and no less than bi-weekly until day 100 post-HCT. GVHD will continue to be monitored as necessary or monthly up until 6 months (see Section 10 for details). AEs and SAEs will be monitored from day 28 post-HCT through the 1st 100 days for both vaccine recipients and observation arm participants. The trial will be suspended for DSMC review if among the first 8 vaccinated recipients any of the following outcomes occur:

≥1 directly vaccine related NRM at 100 days post-HCT; ≥3 NRM at 100 days post-HCT; ≥4 cases of grade 3-4 acute GVHD; ≥3 cases of directly related grade 4 AE within 2 weeks from each

vaccination; ≥3 cases of related/possibly related grade 3 AE within 2 weeks from

each vaccination that do not resolve within 1 week.

It is anticipated that the trial will show that the vaccine does not induce SAEs and/or marked increases in NRM at 100 days post-HCT.

SECONDARY ENDPOINT. Immunogenicity will be evaluated by monitoring CMV-specific CD8+ T cells by multi color flow cytometric analyses. Levels of CD8+ T cells ≥7 cells/µL binding to CMV-specific MHC class I tetramers, before day 65 post-HCT were associated with protection from recurrent or persistent CMV infection or disease in CMV-positive allogeneic HCT recipients15. Additionally, up-regulation of PD-1 on CMV-specific T cells of immunosuppressed transplant recipients lead to T cell proliferative exhaustion and accelerated death, and is associated with CMV disease33,37,38. Co-injection of PF-03512676 CpG DNA in the vaccinated recipients may reduce CMV-specific T cell impairment39,40. Based on these findings, vaccine immunogenicity will be monitored by MHC class I tetramers, cell surface phenotyping, and assessment of CMV-specific proliferative capacity. Reduction of PD-1 expression and decreased levels of apoptosis markers and increased proliferation of CMV-specific T cells is expected in vaccine recipients compared to unvaccinated control group. Levels of pp65-specific T cells associated with protection from CMV complications are anticipated to be detected in a significantly higher proportion of vaccine recipients compared to those enrolled in the control arm4.

12.0 Data Reporting/Protocol Deviations

12.1 Data Reporting

12.1.1 Confidentiality and Storage of Records and Data

The original data collected in the CRFs will be sent to the COH clinical trial office (CTO) and stored in a locked cabinet. Data management personnel (Ms. Jennifer Drake and Dr. Corinna La Rosa) will enter the data from the CRFs into a database in password protected, secure computers that meet all HIPAA requirements. The principal investigator, co-investigators, and laboratory technicians will have access to

of 68


this information, but all information will be treated confidentially. Data will be analyzed and stored in encrypted, password protected, secure computers that meet all HIPAA requirements. When results of this study are reported in medical journals or at meetings, identification of those taking part will not be disclosed and no identifiers will be used.

Medical records of subjects will be securely maintained in the strictest confidence, according to current legal requirements. They will be made available for review, as required by the FDA, HHS, or other authorized users such as the NCI, under the guidelines established by the Federal Privacy Act and rules for the protection of human subjects.

12.1.2 Subject Consent Form

At the time of registration, the original signed and dated Informed Consent form, HIPAA research authorization form, and the California Experimental Subject’s Bill of Rights (for the medical record) and three copies (for the subject, the research record, and the Coordinating Center) must be available. All Institutional, NCI, Federal, and State of California requirements will be fulfilled.

12.1.3 Data Collection Forms and Submission Schedule

All data will be collected using study designed CRFs. Data will be sent to the location identified in Section 12.1.1 and stored in a secure location.

12.1.3.1 Eligibility Checklist

The Eligibility Checklist must be completed by a protocol nurse or clinical research associate and signed by an authorized investigator prior to registering the subject. See Section 4.3 for the registration procedure.

12.1.3.2 Prior Therapy Forms and On-Study Forms

Within 2 weeks of registration, the clinical research associate will submit study designed CRF’s to the patient chart and kept in a secure location in the CTO offices.

12.2 Protocol Deviations

12.2.1 Deviation Policy

This protocol will be conducted in accordance with COH’s “Clinical Research Protocol Deviation Policy” located at http://www.coh.org/dsmc/Documents/Institutional%20Deviation%20Policy.pdf.

Deviations from the written protocol that could increase patient risk or alter protocol integrity require prior IRB approval of a single subject exception (SSE) request. In addition, if contractually obligated, the sponsor must also approve the deviation. IRB pre-approved SSE protocol modifications are considered an amendment to the protocol and not a deviation. The submission of a deviation report is not required.

Brief interruptions and delays may occasionally be required due to travel delays, airport closure, inclement weather, family responsibilities, security alerts, government holidays, etc. This can also extend to complications of disease or unrelated medical illnesses not related to disease progression. The PI has the discretion to deviate from the protocol when necessary so long as such deviation does not threaten

of 68


patient safety or protocol scientific integrity. Examples include, but are not limited to: a) dose adjustments based on excessive patient weight; b) alteration in treatment schedule due to non-availability of the research participant for treatment; c) laboratory test results which are slightly outside the protocol requirements but at levels that do not affect participant safety. These instances are considered to be deviations from the protocol. A deviation report will be submitted to the DSMC/IRB within five days.

12.2.2 Reporting of Deviations

All deviations will be reported to the COH DSMC within five days. The DSMC will forward to report to the IRB following review.

12.2.3 Resolving Disputes

The COH Investigational Drug Service (IDS) cannot release a research agent that would cause a protocol deviation without approval by the PI. Whenever the protocol is ambiguous on a key point, the IDS should rely on the PI to clarify the issue.

In situations where there is misperception or dispute regarding a protocol deviation among the persons involved in implementing the protocol, it is the responsibility of the PI to resolve the dispute and the PI may consult with the DSMC chair (or designee) to arrive at resolution.

13.0 Statistical Considerations

13.1 Study Design

This is a randomized, open-label trial for evaluating safety (primary endpoint) and immunogenicity (secondary endpoint) of Tet-CMV co-injected with PF-03512676, in HLA A*0201 CMV-positive recipients of allogeneic HCT. The evaluation of safety will focus on the 18 HCT recipients that will receive the vaccine. The evaluation of immunogenicity will involve comparison of the 18 vaccinated subjects to 18 non-vaccinated HCT recipients.

SAFETY. Early stopping rules and safety criteria are based on NRM at 100 days post-HCT, acute GVHD, and related/possibly related SAEs within 2 weeks of injections. These rules and criteria are defined with the objective of maintaining a combined false alarm probability of ~10% under local historical event rates for HCT. Standard probability calculations for two-stage designs will be used, as previously described23. The trial will be suspended for DSMC review if among the first 8 vaccinated recipients any of the following outcomes occur:

≥1 directly vaccine related NRM at 100 days post-HCT; ≥3 NRM at 100 days post-HCT; ≥4 cases of grade 3-4 acute GVHD; ≥3 cases of directly related grade 4 AE within 2 weeks from each

vaccination; ≥3 cases of related/possibly related grade 3 AE within 2 weeks from

each vaccination that do not resolve within 1 week.

For vaccine eligible recipients (see Section 3.2.1 Post-HCT Study-Specific Exclusions), we assume a NRM rate at 100 days post-HCT of 10%, which implies a false alarm probability of 0.038 for the NRM rule. Acute GVHD incidence is assumed to be ~15% for related and 20% for unrelated HCT. The

of 68


combined incidence of GVHD among our HCT population is ~18%, implying a 4% false alarm rate for the GVHD alarm. Together, these rules are estimated to have a 7.5% false alarm rate for early stopping of the Phase I trial. The trial will not reach its final safety endpoint if:

>1 of 15 vaccinated subjects have directly vaccine related NRM at 100 days post-HCT

≥5 of 15 vaccinated subjects have any related or unrelated NRM at 100 days post-HCT

or if ≥6 have grade 3-4 acute GVHD. Stopping for SAEs, which do not include ordinary injection-site reactions, is based on attribution to the vaccine without an alternative explanation, which we consider to be an unlikely source of false alarms. The combined false alarm rate for both two-stage rules is ~10%. It is anticipated that the trial will show that the vaccine does not induce SAEs and/or marked increases in NRM at 100 days post-HCT.

IMMUNOGENICITY. The evaluation of immunogenicity is partly based on data from a prospective multicenter trial, including Dr. Nakamura at COH, in CMV-positive recipients of an allogeneic HCT4. Development of 7 cells/µL of CD8+ T cells binding to CMV-specific tetramers, before day 65 post-HCT (defined as rapid recovery) was associated with protection from recurrent or persistent CMV infection or disease4. In the prospective trial, Gratama et al.4 reported that 34 out of 72 HCT recipients (47%) achieved rapid recovery. In our study, we will compare levels of CD8+ T cells binding to CMV-specific tetramers in vaccinated and unvaccinated HCT recipients by Wilcoxon rank-sum test, using integrated CMV-specific CD8+ T cells levels over the first 100 days as a numerical outcome. As a conservative approach to power, we will assume 4 ordered categories, equally likely under the null hypothesis (consistent with the Gratama et al. investigation4) and with alternative hypothesis probabilities (proportional odds model) of: 0.043, 0.076, 0.170, and 0.71, (89% of vaccinated subjects in the upper two categories). This yields 91% power at the one-sided 0.05 level of significance, with power computed using StatXact/Cytel Studio version 7.0.0. Thus, the study can detect an increase in the underlying probability of rapid recovery of CMV immunity from about 50% to about 90% with good power and conventional significance.

Reduction of PD-1 expression and decreased levels of apoptosis markers and increased proliferation of CMV-specific T cells is expected in vaccine recipients compared to unvaccinated control group. These immune-parameters will be compared in both arms using the Kruskall-Wallis rank-sum test, and may provide evidence of the role of PF-03512676 in enhancing CMV-specific T cell activity, in immunosuppressed HCT recipients.

13.2 Sample Size Accrual Rate

The target of this study is to enroll and randomize 36 HLA A*0201 CMV-positive recipients of allogeneic HCT to either the vaccine arm (N=18), or to the observational arm.. Since in the years 2006-2010 at COH the average incidence of mortality for recipients of all types of allogeneic HCT has been ~7% (Department of Biostatistics, COH: “Yearly mortality rates for HCT performed at COH”), and some patients may require re-transplantation, or may voluntarily withdraw or be lost to follow up, we will enroll 5-7 additional patients who only will participate in replacement of recipients who become ineligible or withdraw before randomization (day 28). None will be replaced after randomization. Randomization will be blocked and stratified by donor CMV status, and will occur at day 28, just prior to vaccination (for the vaccine arm).

Annually over the past five years, COH has performed an average of 254 allogeneic HCT procedures (Department of Biostatistics). Considering that ~40% of the recipients are HLA A*0201, 80-90% are

of 68


CMV positive, , 80-90 HCT recipients will be eligible/year. Therefore, based on the yearly volume of HCT patients, it is feasible to perform enrollment and randomization into the study of 36 HLA A*0201 CMV positive allogeneic HCT recipients in a period of 12-18 months.

13.3 Statistical Analysis Plan

As stated above, the criterion for judging the vaccine to have passed this trial without triggering safety concerns in the HCT setting, is if no more than 1 of 18 vaccinated subjects have directly vaccine related NRM in the first 100 days post-HCT, 4 of 18 have NRM in the first 100 days post-HCT and no more than 5 have grade 3-4 acute GVHD, and there are no SAEs within two weeks of injections that are not attributable to disease or other transplantation risks. In addition, AEs will be summarized for each treatment group by type (MedDRA codes within organ systems), grade, and attribution.

With regard to immunogenicity, as stated above, we will compare levels of CD8+ T cells binding to CMV-specific tetramers in vaccinated and unvaccinated HCT recipients by Wilcoxon rank-sum test, using integrated CMV-specific CD8+ T cells levels over the first 100 days as a numerical outcome. The trial will also provide preliminary estimates of the number of subjects achieving rapid recovery, for which we will use a definition analogous to that of the invstigation by the group of Gratama4 (development of 7 cells/µl of CD8+ T cells binding to CMV-specific tetramers, before day 65 post-HCT). We also will use graphical and longitudinal data methods to produce a quantitative description of the emergence of CMV-specific T cells with and without vaccination.

14.0 Human Subject Issues

14.1 Institutional Review Board

In accordance with City of Hope policies, an Institutional Review Board (IRB) that complies with the federal regulations at 45 CFR 46 and 21 CFR 50, 56 and State of California Health and Safety code, Title 17, must review and approve this protocol and the informed consent form prior to initiation of the study. All institutional, NCI, Federal, and State of California regulations must be fulfilled.

14.2 Recruitment of Subjects

All subjects will be recruited from any of the IRB approved protocol nurses among patients undergoing HCT. Specifically, the nurses with the assistance of Jennifer Drake, Study Coordinator will organize the logistics of recipient selection, enrollment, and consenting of HLA A*0201 CMV-positive recipients of allogeneic HCT. Dr. Nakamura, an HCT physician and PI will determine the eligibility status of the recipient, after conferring with the treating physician.

14.3 Advertisements

Advertisements to include print, media (radio, television, billboards), telephone scripts, lay summary to be posted on City of Hope’s public Clinical Trials On-LineSM website, etc., will be reviewed and approved by the IRB prior to their use to recruit potential study subjects.

14.4 Study location and Performance Sites

This is a single center study to be performed at COH, an NCI-designated Comprehensive Cancer Center since 1998. The COH Comprehensive Cancer Center was funded initially by the NCI as a Clinical Cancer Center in 1981. COH encompasses an extensive organization focused on laboratory research, clinical

of 68


research and clinical care within the COH Medical Center and Beckman Research Institute. COH leads the field of HCT as one of the world’s largest and most successful transplant centers. Currently, the COH Hematological Malignancies Program performs over 500 HCT per year in patients with the diagnosis of hematological malignancies, approximately 50% of whom receive an allogeneic HCT. The center has performed 10,000 bone marrow/HCT transplants as of January, 2011. Patients accessing the City of Hope Comprehensive Cancer Center represent the broad spectrum of racial and ethnic diversity of the greater Los Angeles metropolitan area.

Our extensive knowledge in conducting CMV clinical trials and observational natural history studies of CMV infection in HCT makes our clinical site (ranked 2nd in HCT volume in the US) an excellent choice with proven infrastructure for conducting the Phase I feasibility study in HCT recipients proposed in this application6,18,73,85,101-103. Specifically this study will involve multiple sites at COH, including the Departments of Hematology & Hematopoietic Cell Transplantation, Transfusion Medicine, Inpatient Pharmacy and Virology, and the Clinical Trial Office, the Division of Translation Vaccine Research, and the “Phase I unit” clinic.

14.5 Confidentiality

This research will be conducted in compliance with federal and state of California requirements relating to protected health information (PHI). The study will record individual immunological response to the vaccine and any side effects, and this will be linked to the subject’s identity using a coded study number. In addition, results of screening tests to rule out HIV, HBC, and HCV performed in the Department of Transfusion Medicine will be used with the participant’s permission if performed in the past 4 months. The principal investigator, co-investigators, and laboratory technicians will have access to this information, but all information will be treated confidentially. No identifiers will be used in any subsequent publication of these results.

14.6 Financial Obligations and Compensation

The investigational drug including Tet-CMV diluted in Na acetate and co-injected with PF-03512676 adjuvant, will be provided free of charge by NCI and Pfizer, respectively.

In the event of physical injury to a research participant, resulting from research procedures, appropriate medical treatment will be available at the City of Hope to the injured research participant, however, financial compensation will not be available.

The research participant will not be paid for taking part in this study.

14.7 Informed Consent Processes

The Principal Investigator together with the Study Coordinator will explain the nature, duration, purpose of the study, potential risks, alternatives and potential benefits, and all other information contained in the informed consent document. In addition, they will review the experimental subject’s bill of rights and the HIPAA research authorization form. Research subjects will be informed that they may withdraw from the study at any time and for any reason without prejudice, including as applicable, their current or future care or employment at City of Hope or any relationship they have with City of Hope. Research subjects will be afforded sufficient time to consider whether or not to participate in the research.

Should sufficient doubt be raised regarding the adequacy of comprehension, further clarifications will be made and the questionnaire repeated until a satisfactory result is obtained. Prospective research subjects who cannot adequately comprehend the fundamental aspects of the research study with a reasonable

of 68


amount of discussion, education and proctoring will be ineligible for enrollment. For those subjects who do comprehend the fundamental aspects of the study, consent will be obtained and documented, followed by eligibility testing. The research team will review the results of eligibility testing and determine if the subject is a candidate for study enrollment.

of 68


15.0 References

Reference List

(1) Hakki M, Riddell SR, Storek J et al. Immune reconstitution to cytomegalovirus after allogeneic hematopoietic stem cell transplantation: impact of host factors, drug therapy, and subclinical reactivation. Blood. 2003;102:3060-3067.

(2) Khanna R, Diamond DJ. Human cytomegalovirus vaccine: time to look for alternative options. Trends Mol Med. 2006;12:26-33.

(3) Sylwester AW, Mitchell BL, Edgar JB et al. Broadly targeted human cytomegalovirus-specific CD4+ and CD8+ T cells dominate the memory compartments of exposed subjects. J Exp Med. 2005;202:673-685.

(4) Gratama JW, Boeckh M, Nakamura R et al. Immune monitoring with iTAg MHC Tetramers for prediction of recurrent or persistent cytomegalovirus infection or disease in allogeneic hematopoietic stem cell transplant recipients: a prospective multicenter study. Blood. 2010;116:1655-1662.

(5) La Rosa C, Wang Z, Brewer JC et al. Preclinical development of an adjuvant-free peptide vaccine with activity against CMV pp65 in HLA transgenic mice. Blood. 2002;100:3681-3689.

(6) La Rosa C, Longmate J, Lacey SF et al. Clinical Evaluation of Safety and Immunogenicity of PADRE-Cytomegalovirus (CMV) and Tetanus-CMV Fusion Peptide Vaccines With or Without PF03512676 Adjuvant. J Infect Dis. 2012.

(7) Lindemann M, Barsegian V, Runde V et al. Transfer of humoral and cellular hepatitis B immunity by allogeneic hematopoietic cell transplantation. Transplantation. 2003;75:833-838.

(8) Rolland A. TransVax™, a therapeutic DNA vaccine for vontrol of cytomegalovirus in hematopoietic cell transplant recipients Results of a Phase 2 Clinical Trial [abstract]. ASGCT 14th Annual Meeting - Seattle, WA. 2011.

(9) Cooper CL, Davis HL, Angel JB et al. CPG 7909 adjuvant improves hepatitis B virus vaccine seroprotection in antiretroviral-treated HIV-infected adults. AIDS. 2005;19:1473-1479.

(10) Peggs KS, Verfuerth S, Pizzey A et al. Adoptive cellular therapy for early cytomegalovirus infection after allogeneic stem-cell transplantation with virus-specific T-cell lines. Lancet. 2003;362:1375-1377.

(11) Aubert G, Hassan-Walker AF, Madrigal JA et al. Cytomegalovirus-specific cellular immune responses and viremia in recipients of allogeneic stem cell transplants. J Infect Dis. 2001;184:955-963.

(12) Einsele H, Hebart H. Cellular immunity to viral and fungal antigens after stem cell transplantation. Curr Opin Hematol. 2002;9:485-489.

(13) Cobbold M, Khan N, Pourgheysari B et al. Adoptive transfer of cytomegalovirus-specific CTL to stem cell transplant patients after selection by HLA-peptide tetramers. J Exp Med. 2005;202:379-386.

(14) Krieg AM. CpG motifs in bacterial DNA and their immune effects. Annu Rev Immunol. 2002;20:709-760.

(15) Davila E, Velez MG, Heppelmann CJ, Celis E. Creating space: an antigen-independent, CpG-induced peripheral expansion of naive and memory T lymphocytes in a full T-cell compartment. Blood. 2002;100:2537-2545.

of 68


(16) Kuo CC, Liang SM, Liang CM. CpG-B oligodeoxynucleotide promotes cell survival via up-regulation of Hsp70 to increase Bcl-xL and to decrease apoptosis-inducing factor translocation. J Biol Chem. 2006;281:38200-38207.

(17) Gallez-Hawkins GM, Thao L, Palmer J et al. Increased programmed death-1 molecule expression in cytomegalovirus disease and acute graft-versus-host disease after allogeneic hematopoietic cell transplantation. Biol Blood Marrow Transplant. 2009;15:872-880.

(18) La Rosa C, Krishnan A, Longmate J et al. Programmed death-1 expression in liver transplant recipients as a prognostic indicator of cytomegalovirus disease. J Infect Dis. 2008;197:25-33.

(19) Krishnan A, Zhou W, Lacey SF et al. Programmed death-1 receptor and interleukin-10 in liver transplant recipients at high risk for late cytomegalovirus disease. Transpl Infect Dis. 2010;12:363-370.

(20) Diamond DJ, York J, Sun J, Wright CL, Forman SJ. Development of a candidate HLA A*0201 restricted peptide-based vaccine against human cytomegalovirus infection. Blood. 1997;90:1751-1767.

(21) Panina-Bordignon P, Tan A, Termijtelen A et al. Universally immunogenic T cell epitopes: promiscuous binding to human MHC class II and promiscuous recognition by T cells. Eur J Immunol. 1989;19:2237-2242.

(22) Boeckh M, Huang M, Ferrenberg J et al. Optimization of quantitative detection of cytomegalovirus DNA in plasma by real-time PCR. J Clin Microbiol. 2004;42:1142-1148.

(23) Simon R. Optimal two-stage designs for phase II clinical trials. Control Clin Trials. 1989;10:1-10.

(24) Applebaum FR, Meyers JD, Fefer A et al. Nonbacterial nonfungal pneumonia following marrow transplantation in 100 identical twins. Transplantation. 1982;33:265-268.

(25) Quinnan GV, Jr., Kirmani N, Rook AH et al. Cytotoxic T cells in cytomegalovirus infection: HLA-restricted T- lymphocyte and non-T-lymphocyte cytotoxic responses correlate with recovery from cytomegalovirus infection in bone-marrow- transplant recipients. N Engl J Med. 1982;307:7-13.

(26) Reusser P, Einsele H, Lee J et al. Randomized multicenter trial of foscarnet versus ganciclovir for preemptive therapy of cytomegalovirus infection after allogeneic stem cell transplantation. Blood. 2002;99:1159-1164.

(27) Winston DJ, Gale RP, Meyer DV, Young LS. Infectious complications of human bone marrow transplantation. Medicine (Baltimore). 1979;58:1-31.

(28) Boeckh M, Nichols WG. The impact of cytomegalovirus serostatus of donor and recipient before hematopoietic stem cell transplantation in the era of antiviral prophylaxis and preemptive therapy. Blood. 2004;103:2003-2008.

(29) Nichols WG, Corey L, Gooley T, Davis C, Boeckh M. High Risk of Death Due to Bacterial and Fungal Infection among Cytomegalovirus (CMV)-Seronegative Recipients of Stem Cell Transplants from Seropositive Donors: Evidence for Indirect Effects of Primary CMV Infection. J Infect Dis. 2002;185:273-282.

(30) Hakki M, Riddell SR, Storek J et al. Immune reconstitution to cytomegalovirus after allogeneic hematopoietic stem cell transplantation: impact of host factors, drug therapy, and subclinical reactivation. Blood. 2003;102:3060-3067.

of 68


(31) Salzberger B, Bowden RA, Hackman RC, Davis C, Boeckh M. Neutropenia in allogeneic marrow transplant recipients receiving ganciclovir for prevention of cytomegalovirus disease: risk factors and outcome. Blood. 1997;90:2502-2508.

(32) Steininger C. Clinical relevance of cytomegalovirus infection in patients with disorders of the immune system. Clin Microbiol Infect. 2007;13:953-963.

(33) Limaye AP, Bakthavatsalam R, Kim HW et al. Impact of cytomegalovirus in organ transplant recipients in the era of antiviral prophylaxis. Transplantation. 2006;81:1645-1652.

(34) Griffiths P, Whitley R, Snydman DR, Singh N, Boeckh M. Contemporary management of cytomegalovirus infection in transplant recipients: guidelines from an IHMF workshop, 2007. Herpes. 2008;15:4-12.


(36) Boeckh M, Nichols WG, Papanicolaou G et al. Cytomegalovirus in hematopoietic stem cell transplant recipients: Current status, known challenges, and future strategies. Biol Blood Marrow Transplant. 2003;9:543-558.

(37) Tomblyn M, Chiller T, Einsele H et al. Guidelines for preventing infectious complications among hematopoietic cell transplantation recipients: a global perspective. Biol Blood Marrow Transplant. 2009;15:1143-1238.

(38) Zerr DM, Fann JR, Breiger D et al. HHV-6 reactivation and its effect on delirium and cognitive functioning in hematopoietic cell transplantation recipients. Blood. 2011;117:5243-5249.

(39) Reusser P. Oral valganciclovir: a new option for treatment of cytomegalovirus infection and disease in immunocompromised hosts. Expert Opin Investig Drugs. 2001;10:1745-1753.

(40) Ljungman P, Hakki M, Boeckh M. Cytomegalovirus in hematopoietic stem cell transplant recipients. Infect Dis Clin North Am. 2010;24:319-337.

(41) Nguyen Q, Champlin R, Giralt S et al. Late cytomegalovirus pneumonia in adult allogeneic blood and marrow transplant recipients. Clin Infect Dis. 1999;28:618-623.

(42) Razonable RR, Rivero A, Rodriguez A et al. Allograft rejection predicts the occurrence of late-onset cytomegalovirus (CMV) disease among CMV-mismatched solid organ transplant patients receiving prophylaxis with oral ganciclovir. J Infect Dis. 2001;184:1461-1464.

(43) Boeckh M, Leisenring W, Riddell SR et al. Late cytomegalovirus disease and mortality in recipients of allogeneic hematopoietic stem cell transplants: importance of viral load and T-cell immunity. Blood. 2003;101:407-414.


(45) Peggs KS, Mackinnon S. Immune reconstitution following haematopoietic stem cell transplantation. Br J Haematol. 2004;124:407-420.

(46) Singh N. Delayed occurrence of cytomegalovirus disease in organ transplant recipients receiving antiviral prophylaxis: are we winning the battle only to lose the war? Eur J Clin Microbiol Infect Dis. 2002;21:902.

of 68


(47) Gamadia LE, Remmerswaal EB, Weel JF et al. Primary immune responses to human CMV: a critical role for IFN-gamma -producing CD4+ T cells in protection against CMV disease. Blood. 2003;101:2686-2692.

(48) La Rosa C, Krishnan R, Markel S et al. Enhanced immune activity of cytotoxic T-lymphocyte epitope analogs derived from positional scanning synthetic combinatorial libraries. Blood. 2001;97:1776-1786.

(49) Wills MR, Carmichael AJ, Mynard K et al. The human CTL response to cytomegalovirus is dominanted by structural protein pp65: Frequency, specificity, and T Cell Receptor usage of pp65-Specific CTL. J Virol. 1996;70:7569-7579.

(50) Riddell SR, Rabin M, Geballe AP, Britt WJ, Greenberg PD. Class I MHC-restricted cytotoxic T lymphocyte recognition of cells infected with human cytomegalovirus does not require endogenous viral gene expression. J Immunol. 1991;146:2795-2804.

(51) Bernstein DI, Reap EA, Katen K et al. Randomized, double-blind, Phase 1 trial of an alphavirus replicon vaccine for cytomegalovirus in CMV seronegative adult volunteers. Vaccine. 2009;28:484-493.

(52) Wloch MK, Smith LR, Boutsaboualoy S et al. Safety and immunogenicity of a bivalent cytomegalovirus DNA vaccine in healthy adult subjects. J Infect Dis. 2008;197:1634-1642.

(53) Berencsi K, Gyulai Z, Gonczol E et al. A canarypox vector-expressing cytomegalovirus (cmv) phosphoprotein 65 induces long-lasting cytotoxic t cell responses in human cmv- seronegative subjects. J Infect Dis. 2001;183:1171-1179.

(54) Reusser P, Riddell SR, Meyers JD, Greenberg PD. Cytotoxic T-lymphocyte response to cytomegalovirus after human allogeneic bone marrow transplantation: pattern of recovery and correlation with cytomegalovirus infection and disease. Blood. 1991;78:1373-1380.

(55) Rook AH, Quinnan GV, Jr., Frederick WJ et al. Importance of cytotoxic lymphocytes during cytomegalovirus infection in renal transplant recipients. Am J Med. 1984;76:385-392.

(56) Walter EA, Greenberg PD, Gilbert MJ et al. Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor. N Engl J Med. 1995;333:1038-1044.

(57) Feuchtinger T, Opherk K, Bethge WA et al. Adoptive transfer of pp65-specific T cells for the treatment of chemorefractory cytomegalovirus disease or reactivation after haploidentical and matched unrelated stem cell transplantation. Blood. 2010;116:4360-4367.

(58) Einsele H, Roosnek E, Rufer N et al. Infusion of cytomegalovirus (CMV)-specific T cells for the treatment of CMV infection not responding to antiviral chemotherapy. Blood. 2002;99:3916-3922.

(59) Cwynarski K, Ainsworth J, Cobbold M et al. Direct visualization of cytomegalovirus-specific T-cell reconstitution after allogeneic stem cell transplantation. Blood. 2001;97:1232-1240.

(60) Gallez-Hawkins G, Thao L, Lacey SF et al. Cytomegalovirus immune reconstitution occurs in recipients of allogeneic hematopoietic cell transplants irrespective of detectable cytomegalovirus infection. Biol Blood Marrow Transplant. 2005;11:890-902.

(61) Gratama JW, van Esser JW, Lamers CH et al. Tetramer-based quantification of cytomegalovirus (CMV)-specific CD8+ T lymphocytes in T-cell-depleted stem cell grafts and after transplantation may identify patients at risk for progressive CMV infection. Blood. 2001;98:1358-1364.

of 68


(62) Longmate J, York J, La Rosa C et al. Population coverage by HLA class-I restricted cytotoxic T-lymphocyte epitopes. Immunogenetics. 2001;52:165-173.

(63) Zaia JA, Gallez-Hawkins G, Li X et al. Infrequent Occurrence of Natural Mutations in the pp65(495-503) Epitope Sequence Presented by the HLA A*0201 Allele among Human Cytomegalovirus Isolates. J Virol. 2001;75:2472-2474.

(64) Mocarski ES, Jr. Immunomodulation by cytomegaloviruses: manipulative strategies beyond evasion. Trends Microbiol. 2002;10:332-339.

(65) Adler SP, Hempfling SH, Starr SE, Plotkin SA, Riddell S. Safety and immunogenicity of the Towne strain cytomegalovirus vaccine. Pediatr Infect Dis J. 1998;17:200-206.

(66) Alexander J, Sidney J, Southwood S et al. Development of high potency universal DR-restricted helper epitopes by modification of high affinity DR-blocking peptides. Immunity. 1994;1:751-761.

(67) Rothenfusser S, Hornung V, Ayyoub M et al. CpG-A and CpG-B oligonucleotides differentially enhance human peptide-specific primary and memory CD8+ T-cell responses in vitro. Blood. 2004;103:2162-2169.

(68) Horn B, Bao L, Dunham K et al. Infusion of cytomegalovirus specific cytotoxic T lymphocytes from a sero-negative donor can facilitate resolution of infection and immune reconstitution. Pediatr Infect Dis J. 2009;28:65-67.

(69) Shenoy AG, Solomon SR, Pichon S et al. Protecting stem cell transplant recipients against CMV reactivation by vaccinating their donors with a Canarypox pp65 Vaccine (ALVAC) [abstract]. Blood. 2006;108.

(70) Leung AY, Chow HC, Kwok JS et al. Safety of vaccinating sibling donors with live-attenuated varicella zoster vaccine before hematopoietic stem cell transplantation. Bone Marrow Transplant. 2007;39:661-665.

(71) Storek J, Dawson MA, Lim LC et al. Efficacy of donor vaccination before hematopoietic cell transplantation and recipient vaccination both before and early after transplantation. Bone Marrow Transplant. 2004;33:337-346.

(72) Hartmann G, Krieg AM. Mechanism and function of a newly identified CpG DNA motif in human primary B cells. J Immunol. 2000;164:944-953.

(73) Zhou W, Longmate J, Lacey SF et al. Impact of donor CMV status on viral infection and reconstitution of multifunction CMV-specific T cells in CMV-positive transplant recipients. Blood. 2009;113:6465-6476.

(74) Cooper CL, Davis HL, Morris ML et al. CPG 7909, an immunostimulatory TLR9 agonist oligodeoxynucleotide, as adjuvant to Engerix-B HBV vaccine in healthy adults: a double-blind phase I/II study. J Clin Immunol. 2004;24:693-701.

(75) Woolfrey A, Klein JP, Haagenson M et al. HLA-C Antigen Mismatch Is Associated with Worse Outcome in Unrelated Donor Peripheral Blood Stem Cell Transplantation. Biol Blood Marrow Transplant. 2010.

(76) Saber W, Opie S, Rizzo JD et al. Outcomes after matched unrelated donor versus identical sibling hematopoietic cell transplantation in adults with acute myelogenous leukemia. Blood. 2012;119:3908-3916.

(77) Bacigalupo A. Matched and mismatched unrelated donor transplantation: is the outcome the same as for matched sibling donor transplantation? Hematology Am Soc Hematol Educ Program. 2012;2012:223-229.

of 68


(78) Anasetti C. What are the most important donor and recipient factors affecting the outcome of related and unrelated allogeneic transplantation? Best Pract Res Clin Haematol. 2008;21:691-697.

(79) Vitiello A, Ishioka G, Grey HM et al. Development of a lipopeptide-based therapeutic vaccine to treat chronic HBV infection. I. Induction of a primary cytotoxic T lymphocyte response in humans. J Clin Invest. 1995;95:341-349.

(80) Waldner H. The role of innate immune responses in autoimmune disease development. Autoimmun Rev. 2009;8:400-404.

(81) Sorror ML, Sandmaier BM, Storer BE et al. Long-term outcomes among older patients following nonmyeloablative conditioning and allogeneic hematopoietic cell transplantation for advanced hematologic malignancies. JAMA. 2011;306:1874-1883.

(82) Warlick E, Ahn KW, Pedersen TL et al. Reduced intensity conditioning is superior to nonmyeloablative conditioning for older chronic myelogenous leukemia patients undergoing hematopoietic cell transplant during the tyrosine kinase inhibitor era. Blood. 2012;119:4083-4090.

(83) Brooimans RA, Boyce CS, Popma J et al. Analytical performance of a standardized single-platform MHC tetramer assay for the identification and enumeration of CMV-specific CD8+ T lymphocytes. Cytometry A. 2008;73:992-1000.

(84) Ljungman P, Griffiths P, Paya C. Definitions of cytomegalovirus infection and disease in transplant recipients. Clin Infect Dis. 2002;34:1094-1097.

(85) La Rosa C, Limaye AP, Krishnan A, Longmate J, Diamond DJ. Longitudinal assessment of cytomegalovirus (CMV)-specific immune responses in liver transplant recipients at high risk for late CMV disease. J Infect Dis. 2007;195:633-644.

(86) DeYoung MP, Johannessen CM, Leong CO et al. Tumor-specific p73 up-regulation mediates p63 dependence in squamous cell carcinoma. Cancer Res. 2006;66:9362-9368.

(87) Krieg AM. Therapeutic potential of Toll-like receptor 9 activation. Nat Rev Drug Discov. 2006;5:471-484.

(88) Krieg AM, Efler SM, Wittpoth M, Al Adhami MJ, Davis HL. Induction of systemic TH1-like innate immunity in normal volunteers following subcutaneous but not intravenous administration of CPG 7909, a synthetic B-class CpG oligodeoxynucleotide TLR9 agonist. J Immunother. 2004;27:460-471.

(89) Krieg AM. Development of TLR9 agonists for cancer therapy. J Clin Invest. 2007;117:1184-1194.

(90) Leonard JP, Link BK, Emmanouilides C et al. Phase I trial of toll-like receptor 9 agonist PF-3512676 with and following rituximab in patients with recurrent indolent and aggressive non Hodgkin's lymphoma. Clin Cancer Res. 2007;13:6168-6174.

(91) Manegold C, Gravenor D, Woytowitz D et al. Randomized phase II trial of a toll-like receptor 9 agonist oligodeoxynucleotide, PF-3512676, in combination with first-line taxane plus platinum chemotherapy for advanced-stage non-small-cell lung cancer. J Clin Oncol. 2008;26:3979-3986.

(92) Link BK, Ballas ZK, Weisdorf D et al. Oligodeoxynucleotide CpG 7909 delivered as intravenous infusion demonstrates immunologic modulation in patients with previously treated non-Hodgkin lymphoma. J Immunother. 2006;29:558-568.

of 68


(93) Yamada K, Nakao M, Fukuyama C et al. Phase I study of TLR9 agonist PF-3512676 in combination with carboplatin and paclitaxel in patients with advanced non-small-cell lung cancer. Cancer Sci. 2010;101:188-195.

(94) Liang X, Moseman EA, Farrar MA et al. Toll-like receptor 9 signaling by CpG-B oligodeoxynucleotides induces an apoptotic pathway in human chronic lymphocytic leukemia B cells. Blood. 2010;115:5041-5052.

(95) Kim YH, Girardi M, Duvic M et al. Phase I trial of a Toll-like receptor 9 agonist, PF-3512676 (CPG 7909), in patients with treatment-refractory, cutaneous T-cell lymphoma. J Am Acad Dermatol. 2010;63:975-983.

(96) Zent CS, Smith BJ, Ballas ZK et al. Phase I clinical trial of CpG oligonucleotide 7909 (PF-03512676) in patients with previously treated chronic lymphocytic leukemia. Leuk Lymphoma. 2012;53:211-217.

(97) Rynkiewicz D, Rathkopf M, Sim I et al. Marked enhancement of the immune response to BioThrax(R) (Anthrax Vaccine Adsorbed) by the TLR9 agonist CPG 7909 in healthy volunteers. Vaccine. 2011;29:6313-6320.

(98) Cooper CL, Davis HL, Morris ML et al. Safety and immunogenicity of CPG 7909 injection as an adjuvant to Fluarix influenza vaccine. Vaccine. 2004;22:3136-3143.

(99) Blazar BR, Krieg AM, Taylor PA. Synthetic unmethylated cytosine-phosphate-guanosine oligodeoxynucleotides are potent stimulators of antileukemia responses in naive and bone marrow transplant recipients. Blood. 2001;98:1217-1225.

(100) Lacey SF, Gallez-Hawkins G, Crooks M et al. Characterization of cytotoxic function of CMV-pp65-specific CD8+ T- lymphocytes identified by HLA tetramers in recipients and donors of stem-cell transplants. Transplantation. 2002;74:722-732.

(101) Lacey SF, Diamond DJ, Zaia JA. Assessment of cellular immunity to human cytomegalovirus in recipients of allogeneic stem cell transplants. Biol Blood Marrow Transplant. 2004;10:433-447.

(102) Wang Z, La Rosa C, Mekhoubad S et al. Attenuated poxviruses generate clinically relevant frequencies of CMV-specific T cells. Blood. 2004;104:847-856.

(103) Wang Z, Zhou W, Srivastava T et al. A fusion protein of HCMV IE1 exon4 and IE2 exon5 stimulates potent cellular immunity in an MVA vaccine vector. Virology. 2008;377:379-390.

CITY OF HOPE NATIONAL MEDICAL CENTER 1500 E. DUARTE · PDF filecity of hope national medical...

Documents

Transcript of CITY OF HOPE NATIONAL MEDICAL CENTER 1500 E. DUARTE · PDF filecity of hope national medical...