Clinical trial NCT02298946
Conditions: Colorectal Cancer Colorectal Neoplasms Colorectal Carcinoma
Keywords: Anti-PD1 Therapy Anti-Tumor Immunity Liver Metastatic B7-DC Fc Fusion Protein
Sponsor: National Cancer Institute (NCI)
Start date: 2014-11-21; Completion date: 2017-03-07
Enrollment: 17 participants; Allocation: NON_RANDOMIZED; Intervention model: SINGLE_GROUP

Brief summary:
Background:

\- T-cells are white blood cells that can find and kill germs and tumors. Cancer can keep T-cells from working. Researchers think a new drug called AMP-224 might help the T-cells in people with cancer. They think the drug might work even better when combined with a certain type of radiation therapy.

Objective:

\- To study the safety and effectiveness of AMP-224 together with 1 or 3 days of stereotactic body radiation therapy (SBRT) directed to the liver.

Eligibility:

\- People age 18 and older with metastatic colorectal cancer. Their cancer must have spread to the liver and not be responding to treatment.

Design:

* Participants will be screened with a medical history, physical exam, and blood and urine tests. Their tumors will be measured with computerized tomography (CT) scans or magnetic resonance imaging (MRI) of the chest, stomach, and pelvis. They will have an electrocardiogram (ECG) heart test.
* Participants will have a small part of their tumor removed by needle (biopsy).
* Participants will have 8 study visits over about 10 weeks.
* At 1 visit, they will have another tumor biopsy.
* At 1 visit, they will get a chemotherapy drug through a vein (intravenous (IV)).
* At 6 visits, they will receive AMP-224 through an IV.
* At 1 or 3 visits, they will have SBRT. Computed tomography (CT) scans will map the position of their tumor. Radiation beams of different intensities at different angles will be directed to the tumor.
* At all visits, some screening procedures may be repeated.
* After treatment ends, participants will have 7 follow-up visits over about 5 months. Blood will be drawn. Some screening procedures may be repeated.

Detailed description:
Background:

* Colorectal cancer remains the second leading cause of cancer death in western countries with a median survival of approximately 24 months despite recent advances in systemic treatment.
* Several preclinical studies have documented an increase in peripheral antitumor immunity following radiation, a phenomenon known as the abscopal effect. Tumor PDL1 expression has also been shown to be induced by radiation, which can suppress the anti-tumor immune response. Inhibition of programmed cell death-1 (PD-1)/programmed death ligand-1(PDL-1) axis has been shown to improve anti-tumor immunity by blocking the tumor-mediated suppression of cytotoxic T cells.
* AMP-224, a B7-DC Fc fusion protein, binds to PD-1, an inhibitory receptor that is present on the cell surface of exhausted, activated, effector, and memory T cells. AMP- 224 has a unique mechanism of action in that it binds specifically to PD-1HI T cells (chronically stimulated / exhausted T cells) but not to PD-1LO cells which represent the normal activated T cells population
* The aim of the study is to evaluate whether the anti-tumor immunity of anti-PD1 therapy (with AMP-224) can be enhanced by radiation therapy.

Objectives:

\- To assess safety, tolerability and feasibility of AMP-224 in combination with stereotactic body radiation therapy (SBRT) in patients with metastatic colorectal cancer.

Eligibility:

* Histologically confirmed metastatic colorectal cancer.
* Patient must have progressed on or been intolerant of prior oxaliplatin- and irinotecan containing regimen and have metastatic lesions that are not amenable to curative resection.
* Patient must have one focus of metastatic disease in the liver that is amenable to SBRT.
* Patient must have at least one measurable metastatic lesion by Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 criteria outside the radiation field.
* Patients must be willing to undergo mandatory pre and post treatment tumor biopsy.

Design:

* This is a pilot study whereby all patients will receive SBRT to one liver lesion and concomitant AMP-224. A single treatment of low dose/cyclophosphamide will be administered in conjunction with the SBRT therapy prior to the first AMP-224 treatment.
* Hypofractionated radiation will be administered to a metastatic disease site at a dose and schedule of 8Gy for 1 or 3 days in dose levels (DL)1 or 2 respectively. The day of first administration of AMP-224 will be designated as Day 1. In DL1 the SBRT will be administered on Day 0. In DL2 the SBRT will be administered from D-2 to D0. The study will begin with DL1 and escalate to DL2 once all subjects enrolled at DL1 have remained on study for 4 weeks, which is the DLT period.
* AMP-224 therapy will be given as an intravenous infusion beginning on Day 1 and then every 14 days for a total of 6 treatments only. Optional continuation of treatment q2- weekly until progressive disease (PD) will be considered in responding patients.
* Cyclophosphamide 200 mg/m(2) intravenous will be given on Day 0, prior to the first dose of AMP-224.
* Correlative studies: Peripheral blood will be collected (pre-dose) on days 1, 29, 57 and 93 for immune studies (including immunogenicity, circulating PD plasma samples, immune monitoring for phenotyping and peripheral blood mononuclear cells (PBMC) for T-cell activation). Tumor biopsies (formalin-fixed paraffin-embedded (FFPE) + Frozen) of an irradiated and non-irradiated liver lesion will be collected on day 1 and day 29, which will be analyzed by immunohistochemistry for tumor-infiltrating lymphocytes in addition to ribonucleic acid (RNA) analysis.
* Pharmacokinetic (PK) samples will be collected on Days 1, 15, 29, 43, 57, 71 in addition to up to 5 post treatment dates (if feasible).

Arms: arm 1: Dose Level 1 (DL1) Cyclophosphamide (CTX) 200mg/m(2) intravenous (IV) on day 0. Stereotactic body radiation therapy (SBRT) 8 (gray)Gy x 1 day on day 0, AMP-224 10mg/kg on day 1 then every (q)14 days for a total of 6 doses arm 2: Dose Level 2 (DL2) CTX 200mg/m(2) IV on day 0, SBRT 8Gy x 3 day on days -2, -1, and 0. AMP-224 10mg/kg on day 1 then q14 days.
Interventions: intervention 1: AMP-224 intervention 2: Stereotactic Body Radiation Therapy(SBRT) intervention 3: Cyclophosphamide
Intervention descriptions: intervention 1: 10mg/kg on day 1 then every 14 days for a total of 6 doses. intervention 2: Dose Level 1: 8Gy x 1 day. Dose Level 2: 8Gy x 3 days intervention 3: 200mg/m(2) IV on day 0.

Protocol documents:
Prot_SAP_000.pdf: 
Abbreviated Title: AMP-224 SBRT Met Colorectal Ca 
 
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Abbreviated Title: AMP-224 SBRT Met Colorectal Ca  
NCI Protocol #:  15-C-0021 B 
Version Date:  08/15/2016 
Title: A Pilot Study of AMP-224 – a PD-1 Inhibitor – in Combination with Stereotactic Body 
Radiation Therapy (SBRT) in Patients with Metastatic Colorectal Cancer 
 
Principal Investigator:    
 
Tim Greten, MD A-F 
Thoracic & GI Oncology Branch 
National Cancer Institute  
Building 10, Room 3B43 
9000 Rockville Pike 
Bethesda, MD 20892 
301-451-4723 
FAX: 301-480-8780 
gretentf@mail.nih.gov 
Lead Associate Investigator:      Austin Duffy, MDA-F 
Associate Investigators:    
Deborah Citrin, MD, ROB, CCR, NCIA, B, E  
Brad Wood MD, RAD IS, CC, NIHA, B, E 
William D. Figg, PharmD, GMB, CCR, NCIB, E  
Suzanne Fioravanti, RN, OCD, CCR, NCIA,B  
Melissa Walker RN, OCD, CCR, NCIA, B  
Jennifer Jones MD, PhD, VB, CCR, NCIA, B,E 
Seth Steinberg, PhD, BDMS, OCD, CCR, NCIF 
 
Referral Contact/  
Study Coordinator:   
      Suzanne Fioravanti, RN, OCD, CCR, NCI 
 
10 Center Drive Room 13N220 
 
Bethesda, MD 20982 
 
Phone: (301) 594-6544 
 
Email: fioravas@mail.nih.gov 
Roles of investigators: 
A. Obtain information by intervening or interacting with living individuals for research 
purposes 
B. Obtaining identifiable private information about living individuals 
C. Obtaining the voluntary informed consent of individuals to be subjects 
D. Makes decisions about subject eligibility 
E. Studying, interpreting, or analyzing identifiable private information or data/specimens 
for research purposes 
F. Studying, interpreting, or analyzing de-identified data or specimens for research 
purposes 


Abbreviated Title: AMP-224 SBRT Met Colorectal Ca 
 
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Investigational Agents:     
Drug Name: 
AMP-224 
IND Number: 
123468 
Sponsor: 
Center for Cancer Research, National Cancer Institute 
Manufacturer: 
Amplimmune, Inc. 
 
 
 
 


Abbreviated Title: AMP-224 SBRT Met Colorectal Ca 
 
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PRÉCIS 
Background: 
• Colorectal cancer remains the second leading cause of cancer death in western countries 
with a median survival of approximately 24 months despite recent advances in systemic 
treatment.  
• Several preclinical studies have documented an increase in peripheral antitumor 
immunity following radiation, a phenomenon known as the “abscopal effect”. Tumor PD-
L1 expression has also been shown to be induced by radiation, which can suppress the 
anti-tumor immune response. Inhibition of PD-1/PDL-1 axis has been shown to improve 
anti-tumor immunity by blocking the tumor-mediated suppression of cytotoxic T cells. 
• AMP-224, a B7-DC Fc fusion protein, binds to PD-1, an inhibitory receptor that is 
present on the cell surface of exhausted, activated, effector, and memory T cells. AMP-
224 has a unique mechanism of action in that it binds specifically to PD-1HI T cells 
(chronically stimulated / exhausted T cells) but not to PD-1LOcells which represent the 
normal activated T cells population 
• The aim of the study is to evaluate whether the anti-tumor immunity of anti-PD1 therapy 
(with AMP-224) can be enhanced by radiation therapy.  
Objectives: 
• To assess safety, tolerability and feasibility of AMP-224 in combination with stereotactic 
body radiation therapy (SBRT) in patients with metastatic colorectal cancer. 
Eligibility: 
• Histologically confirmed metastatic colorectal cancer.  
• Patient must have progressed on or been intolerant of prior oxaliplatin- and irinotecan-
containing regimen and have metastatic lesions that are not amenable to curative 
resection. 
• Patient must have one focus of metastatic disease in the liver that is amenable to SBRT. 
• Patient must have at least one measurable metastatic lesion by RECIST 1.1 criteria 
outside the radiation field. 
• Patients must be willing to undergo mandatory pre and post treatment tumor biopsy. 
Design: 
• This is a pilot study whereby all patients will receive SBRT to one liver lesion and 
concomitant AMP-224.  A single treatment of low dose/cyclophosphamide will be 
administered in conjunction with the SBRT therapy prior to the first AMP-224 treatment. 
• Hypofractionated radiation will be administered to a metastatic disease site at a dose and 
schedule of 8Gy for 1 or 3 days in dose levels (DL) 1 or 2 respectively. The day of first 
administration of AMP-224 will be designated as Day 1. In DL1 the SBRT will be 
administered on Day 0. In DL2 the SBRT will be administered from D-2 to D0. The 
study will begin with DL1 and escalate to DL2 once all subjects enrolled at DL1 have 
remained on study for 4 weeks, which is the DLT period. 
• AMP-224 therapy will be given as an intravenous infusion beginning on Day 1 and then 
every 14 days for a total of 6 treatments only. Optional continuation of treatment q2-
weekly until PD will be considered in responding patients. 


Abbreviated Title: AMP-224 SBRT Met Colorectal Ca 
 
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• Cyclophosphamide 200 mg/m2 intravenous will be given on Day 0, prior to the first dose 
of AMP-224. 
• Correlative studies: Peripheral blood will be collected (pre-dose) on days 1, 29, 57 and 93 
for immune studies (including immunogenicity, circulating PD plasma samples, immune-
monitoring for phenotyping and PBMC for T-cell activation). Tumor biopsies (FFPE + 
Frozen) of an irradiated and non-irradiated liver lesion will be collected on day 1 and day 
29, which will be analyzed by immunohistochemistry for tumor-infiltrating lymphocytes 
in addition to RNA analysis.  
• PK samples will be collected on Days 1, 15, 29, 43, 57, 71 in addition to up to 5 post-
treatment dates (if feasible). 
 
 


Abbreviated Title: AMP-224 SBRT Met Colorectal Ca 
 
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SCHEMA 
  
 
  
 
 
Dose Level 1 
Dose Level 2 


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TABLE OF CONTENTS 
PRÉCIS ......................................................................................................................................3 
SCHEMA ...................................................................................................................................5 
TABLE OF CONTENTS ............................................................................................................6 
1 
INTRODUCTION ............................................................................................................. 11 
1.1 
Study Objectives ......................................................................................................... 11 
1.1.1 
Primary Objective ................................................................................................ 11 
1.1.2 
Secondary Objectives ........................................................................................... 11 
1.1.3 
Exploratory .......................................................................................................... 11 
1.2 
Background and Rationale .......................................................................................... 11 
1.2.1 
Metastatic colorectal cancer (CRC) and the current therapeutic paradigm ............ 11 
1.2.2 
The shifting paradigm for the role of immune system in cancer treatment ............ 11 
1.2.3 
The relationship between immune system and colorectal cancer ........................... 12 
1.2.4 
Recent advances in immune-based approaches in solid tumor malignancies ......... 12 
1.2.5 
The PD1/PD-L1 axis ............................................................................................ 13 
1.2.6 
Clinical Studies Evaluating anti-PD-1/anti-PD-L1 therapy ................................... 13 
1.2.7 
Immune responses following radiation, mechanism and rationale for combination 
with anti-PD1 therapy ........................................................................................................ 14 
1.2.8 
AMP-224 (Derived from Investigator’ s Brochure) .............................................. 17 
1.2.9 
Rationale for study population and safety of administering SBRT to hepatic 
metastases ......................................................................................................................... 23 
1.2.10 Justification for dose and schedule ....................................................................... 24 
1.2.11 Justification for Tumor Biopsies .......................................................................... 24 
2 
ELIGIBILITY ASSESSMENT AND ENROLLMENT ..................................................... 25 
2.1 
Eligibility Criteria ....................................................................................................... 25 
2.1.1 
Inclusion Criteria ................................................................................................. 25 
2.1.2 
Exclusion Criteria ................................................................................................ 26 
2.1.3 
Inclusion of Women and Minorities ..................................................................... 27 
2.1.4 
Recruitment Strategies ......................................................................................... 27 
2.2 
Screening Evaluation .................................................................................................. 27 
2.2.1 
History and Physical Evaluation ........................................................................... 27 
2.2.2 
Laboratory Evaluation .......................................................................................... 28 
2.2.3 
Histologic confirmation (at any time point prior to initiation of study therapy) ..... 28 


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2.3 
Registration Procedures............................................................................................... 28 
2.4 
Baseline Evaluation..................................................................................................... 28 
2.4.1 
Imaging studies (baseline – obtained within 28 days prior to enrollment): ............ 28 
2.4.2 
Research Tumor Biopsy (within 16 days of beginning study treatment) ............... 28 
2.4.3 
Laboratory evaluation (obtained within 72 hours prior to first dose of radiation i.e. 
either Day -2 or Day 0.) ..................................................................................................... 28 
2.4.4 
Laboratory evaluation (obtained within 28 days prior to first dose of SBRT)........ 28 
2.4.5 
Laboratory evaluation (obtained within 45 days prior to first dose SBRT) ............ 29 
2.4.6 
History and physical exam with vital signs (obtained within 1 week prior to first 
dose). 
29 
2.4.7 
Electrocardiogram (obtained within 28 days prior to first dose of SBRT) ............. 29 
3 
STUDY IMPLEMENTATION .......................................................................................... 29 
3.1 
Study Design............................................................................................................... 29 
3.1.1 
Protocol Stopping Rules ....................................................................................... 31 
3.1.2 
Dose Limiting Toxicity ........................................................................................ 31 
3.2 
Drug Administration ................................................................................................... 32 
3.2.1 
AMP-224 Drug Administration ............................................................................ 32 
3.2.2 
Cyclophosphamide  Administration ..................................................................... 32 
3.2.3 
Monitoring of Dose Administration...................................................................... 32 
3.3 
Dosing Delays ............................................................................................................. 33 
3.3.1 
Dosing Delays/Dose Modifications: General Guidelines ...................................... 34 
3.4 
Specific Procedures for SBRT ..................................................................................... 34 
3.4.1 
Radiotherapy administration ................................................................................ 34 
3.5 
Study Calendar............................................................................................................ 36 
3.5.1 
Screening/Baseline Calendar ................................................................................ 36 
3.5.2 
On Treatment Calendar ........................................................................................ 38 
3.5.3 
Follow-up Calendar ............................................................................................. 42 
3.6 
Criteria for Removal from Protocol Therapy and Off Study Criteria ............................ 45 
3.6.1 
Criteria for removal from protocol therapy ........................................................... 45 
3.6.2 
Criteria for Removal from Study .......................................................................... 45 
3.6.3 
Off-Study Procedure ............................................................................................ 45 
4 
Concomitant Medications/Measures .................................................................................. 45 
5 
BIOSPECIMEN COLLECTION ....................................................................................... 46 


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5.1 
Correlative Studies for Research/Pharmacokinetic Studies .......................................... 46 
5.1.1 
Immune monitoring ............................................................................................. 47 
5.1.2 
Liver autoantibody panel for autoimmune hepatitis (AIH) .................................... 47 
5.1.3 
Tumor specific responses ..................................................................................... 47 
5.1.4 
Mandatory tumor biopsy ...................................................................................... 48 
5.1.5 
Optional Tumor Biopsy........................................................................................ 50 
5.1.6 
Immunogenicity studies ....................................................................................... 50 
5.1.7 
Pharmacokinetic studies ....................................................................................... 51 
5.2 
Sample Storage, Tracking and Disposition .................................................................. 51 
5.2.1 
Procedures for sample data collection for the Clinical Pharmacology Program: .... 51 
5.2.2 
Procedures for sample storage at the Clinical Pharmacology Program: ................. 51 
5.2.3 
Protocol Completion/Sample Destruction ............................................................. 52 
6 
DATA COLLECTION AND EVALUATION ................................................................... 52 
6.1 
Data Collection ........................................................................................................... 52 
6.2 
Response Criteria ........................................................................................................ 53 
6.2.1 
Definitions ........................................................................................................... 53 
6.2.2 
Disease Parameters .............................................................................................. 53 
6.2.3 
Methods for Evaluation of Measurable Disease .................................................... 54 
6.2.4 
Response Criteria ................................................................................................. 55 
6.2.5 
Duration of Response ........................................................................................... 57 
6.2.6 
Modified immune-related response criteria (irRC) ............................................... 57 
6.3 
Toxicity Criteria .......................................................................................................... 58 
7 
SAFETY REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING PLAN
 
58 
7.1 
Definitions .................................................................................................................. 58 
7.1.1 
Adverse Event...................................................................................................... 58 
7.1.2 
Suspected adverse reaction ................................................................................... 59 
7.1.3 
Unexpected adverse reaction ................................................................................ 59 
7.1.4 
Serious ................................................................................................................. 59 
7.1.5 
Serious Adverse Event ......................................................................................... 59 
7.1.6 
Disability ............................................................................................................. 59 
7.1.7 
Life-threatening adverse drug experience ............................................................. 60 
7.1.8 
Protocol Deviation (NIH Definition) .................................................................... 60 


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7.1.9 
Non-compliance (NIH Definition)........................................................................ 60 
7.1.10 Unanticipated Problem ......................................................................................... 60 
7.2 
NCI-IRB Reporting ..................................................................................................... 60 
7.2.1 
NCI-IRB Expedited Reporting of Unanticipated Problems and Deaths ................. 60 
7.2.2 
NCI-IRB Requirements for PI Reporting at Continuing Review ........................... 60 
7.2.3 
NCI-IRB Reporting of IND Safety Reports .......................................................... 61 
7.3 
IND Sponsor Reporting Criteria .................................................................................. 61 
7.4 
FDA Reporting Criteria ............................................................................................... 61 
7.4.1 
IND Safety Reports to the FDA (Refer to 21 CFR 312.32) ................................... 61 
7.4.2 
FDA Annual Reports (Refer to 21 CFR 312.33) ................................................... 62 
7.4.3 
Safety Reporting to the Manufacturer ................................................................... 62 
7.5 
Data and Safety Monitoring Plan................................................................................. 62 
7.5.1 
Principal Investigator/Research Team .................................................................. 62 
7.5.2 
Sponsor Monitoring Plan ..................................................................................... 62 
8 
STATISTICAL CONSIDERATIONS ............................................................................... 63 
9 
COLLABORATIVE AGREEMENT ................................................................................. 63 
9.1 
Agreement Type ......................................................................................................... 63 
10 HUMAN SUBJECTS PROTECTIONS ............................................................................. 64 
10.1 
Rationale For Subject Selection ............................................................................... 64 
10.2 
Participation of Children .......................................................................................... 64 
10.3 
Evaluation of Benefits and Risks/Discomforts ......................................................... 64 
10.3.1 Risk of Biopsy ..................................................................................................... 64 
10.3.2 Risks of exposure to Ionizing Radiation ............................................................... 64 
10.3.3 Other Risks/Benefits ............................................................................................ 64 
10.4 
Risks/Benefits Analysis ........................................................................................... 64 
10.5 
Consent and Assent Process and Documentation ..................................................... 65 
10.5.1 Re-consent via telephone ..................................................................................... 65 
11 Pharmaceutical Information ............................................................................................... 65 
11.1 
AMP224 (IND# 123468) ......................................................................................... 65 
11.1.1 Source .................................................................................................................. 65 
11.1.2 Toxicity ............................................................................................................... 65 
11.1.3 Formulation and preparation ................................................................................ 65 


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11.1.4 Stability and Storage ............................................................................................ 66 
11.1.5 Administration procedures ................................................................................... 66 
11.2 
Cyclophosphamide .................................................................................................. 66 
11.2.1 Source .................................................................................................................. 66 
11.2.2 Toxicity ............................................................................................................... 66 
11.2.3 Formulation and preparation ................................................................................ 67 
11.2.4 Stability and Storage ............................................................................................ 67 
11.2.5 Administration procedures ................................................................................... 67 
12 REFERENCES .................................................................................................................. 68 
13 APPENDICES .................................................................................................................. 72 
13.1 
Appendix A: Performance Status Criteria ................................................................ 72 
13.2 
Appendix B:  Modified immune-related response criteria (irRC) ............................. 73 
 
 
 


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1 
INTRODUCTION 
1.1 
STUDY OBJECTIVES 
1.1.1 Primary Objective 
• To assess safety, tolerability and feasibility of AMP-224 in combination with stereotactic 
body radiation therapy (SBRT) in patients with metastatic colorectal cancer. 
1.1.2 Secondary Objectives 
• To measure changes in CD8+ TIL in hepatic metastases of patients with colorectal cancer 
following treatment with AMP-224 in combination with stereotactic body radiation 
therapy (SBRT). 
• To characterize the pharmacokinetic (PK) parameters of AMP-224 in combination with 
SBRT in patients with metastatic colorectal cancer.  
• To evaluate response rate, progression-free survival and overall survival in patients with 
colorectal cancer during and following treatment with AMP-224 in combination with 
SBRT. 
1.1.3 Exploratory 
• To measure immune parameters in the peripheral blood and tumors in patients with 
metastatic colorectal cancer during and following treatment with AMP-224 in 
combination with SBRT 
1.2 
BACKGROUND AND RATIONALE 
1.2.1 Metastatic colorectal cancer (CRC) and the current therapeutic paradigm 
Colorectal cancer is the second leading cause of cancer death in western countries, with 
approximately 300,000 new cases diagnosed annually in the U.S. and Europe 1.  The cornerstone 
of the systemic treatment of CRC over the past 50 years has been 5-fluorouracil (5FU) and over 
the past decade this has been supplemented by the emergence of new chemotherapeutics 
(oxaliplatin and irinotecan) in addition to so-called targeted agents (bevacizumab and 
cetuximab/panitumumab). These advances have resulted in an increase in the median overall 
survival from 10 to 24 months 2-4.  Whilst questions remain about the exact sequencing and 
specific duration of the biological therapies in particular, the basic paradigm of treatment with 
regard to the chemotherapeutics has been established, with patients and their oncologists having 
the option of either oxaliplatin- or irinotecan-based FU combinations in the first line, followed 
upon disease progression by the regimen not previously given. Once a patient progresses after 
first line treatment the average survival is 12-14 months. 
1.2.2 The shifting paradigm for the role of immune system in cancer treatment 
In general, treatment of cancer has focused on killing tumor cells via cytotoxic doses of either 
chemotherapy or radiotherapy. However, intense research in the past 20 years has revealed the 
partnership between the immune system and various cancer treatment modalities. There are 
several distinct mechanisms by which genotoxic therapies can augment a systemic anti-cancer 
immune response. These mechanisms include, but are not limited to, increased expression of 
antigens that can be recognized either by innate or adoptive immune cells, secretion of 


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chemokines, increased susceptibility to lysis by immune cells and inhibition or elimination of 
immunosuppressive cells, i.e. regulatory T-cells (TREG) or myeloid-derived suppressor cells 
(MDSC) 5. These mechanisms illustrate how cytotoxic therapies may restore 
immunosurveillance against the tumor, which ideally will lead to its elimination.  
Not all cytotoxic therapies result in anti-cancer immunity. In a seminal paper by Obeid et al., 
anthracyclines and irradiation, but not other various classes of chemotherapeutics, were shown to 
yield immunogenic cell death 6. In this paper, the mechanism of immunogenic cell death was 
linked to calreticulin (CRT) exposure on cell membrane, which occurs upstream of apoptosis or 
necrosis, as a part of a specific danger-signaling system. The same group also reported in a 
separate paper that release of high-mobility-group-box-1 (HMGB1) from dying tumor cells was 
central for activating dendritic cells via Toll-like receptor 4 (TLR4) and its adaptor MyD88 7. An 
additional signal to activate the anti-tumor immune system was identified to be ATP released 
from stressed and/or dying cells, which then binds to the purinergic receptor, P2RX7, on the 
dendritic cells 8. The signaling cascade activated by HMGB1 and ATP result in secretion of IL-
1β that in turn polarizes CD8+ T cells towards interferon- γ production 9. Taken together, certain 
chemotherapeutic agents and radiation emit immunogenic signals that are utilized by dendritic 
cells to elicit a productive anti-tumor immune response. In many of these studies colorectal 
cancer models were used  
1.2.3 The relationship between immune system and colorectal cancer 
The correlation with the type of immune infiltrate and survival has been studied extensively in 
colorectal cancer. In a paper by Pages et al., patients who had resected colorectal cancer had 
better survival if the tumor specimen had increased infiltrates of immune cells that were CD8+ T 
cells, ranging from early memory (CD45RO+CCR7–CD28+CD27+) to effector memory 
(CD45RO+CCR7–CD28–CD27–) T cells 10. As a follow up to this study, when genomic and in 
situ immunostaining analyses on these tumor specimens were performed, genes of TH1 
polarization correlated with better survival 11. This correlation was shown to be stronger than the 
classical TNM staging pointing toward the accuracy for the effector immune infiltration to 
predict the prognosis 11. Consistent with these observations, the chemokines that have the most 
favorable impact are CX3CL1, which may attract TH1 cells into the tumor, as well as CXCL10 
and CXCL9, which attract CD45RO+ T cells 12.  
1.2.4 Recent advances in immune-based approaches in solid tumor malignancies 
The past number of years have seen progress for immune-based approaches in solid tumor 
malignancies, with FDA approvals for both dendritic cell vaccination and immune checkpoint 
inhibition in prostate cancer and melanoma respectively 13-15. In melanoma, immune checkpoint 
inhibition with anti-CTLA4 therapy has been shown to demonstrate a median survival benefit in 
two separate phase 3 studies, both of which – more importantly – were associated with long-term 
disease control in approximately one-fifth of patients. Anti-PD1/PDL1 therapy has demonstrated 
a similar degree of clinical activity not only in melanoma and kidney cancer but also in lung 
cancer, a disease type previously thought to be refractory to an immune approach. Appreciation 
of the role in developing tumors of immune-evasion is evidenced by its inclusion as one of the 
(updated) hallmarks of cancer 16.  
Anti-PD1/PDL1 therapy enhances the effector T-cell response by blocking the inhibitory signals, 
which regulate this response. Blocking a single immune checkpoint however is clearly 


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insufficient to induce the necessary antitumor immune response in the majority of patients. Other 
factors such as poor antigenicity or additional negative immune regulators [e.g. myeloid-derived 
suppressor cells (MDSC) or T-regs] may predominate tilting the immune microenvironment 
towards suppression. Combining immune checkpoint inhibition with another modality such as 
radiation or low dose cyclophosphamide – which has been shown to produce a systemic immune 
reaction – has the potential to greatly enhance the anti-tumor immune response.  
The potential for this approach has been demonstrated clinically by Postow et al who reported a 
case of the abscopal effect in a patient with melanoma treated with anti-CTLA4 and 
radiotherapy17. In that case a patient progressed slowly despite treatment with anti-CTLA4 alone, 
but then exhibited a dramatic response after receiving palliative local radiotherapy followed by 
additional treatment with anti-CTLA4. Temporal associations were noted between tumor 
shrinkage and antibody responses to the cancer-testis antigen NY-ESO-1 in addition to changes 
in peripheral-blood immune cells, and increases in antibody responses to other antigens. 
Similarly there is a strong preclinical rationale for the combination of anti-PD1 therapy and 
radiation.  
1.2.5 The PD1/PD-L1 axis 
The PD-L1/PD-1 axis is a potent inhibitor of immune activation, particularly through inhibition 
of effector T cell function 18. PD-L1 (also called B7-H1, the ligand for the PD-1 receptor) is 
undetectable in most normal tissues but is inducible in various cell types by inflammatory 
cytokines and probably plays a tissue-protective role against autoimmunity. Some viruses can 
induce PD-L1/PD-1 signaling in order to escape the host immune response by inducing T cell 
exhaustion, which contributes towards chronic infection 19.  PD-L1 expression has also been 
observed in a wide variety of solid malignancies, suggesting that PD-L1 is a common 
mechanism of immune suppression induced by the tumor 20. Inhibitors of the PD-L1/PD-1 axis 
have been reported to generate potent antitumor activity in murine tumor models and clinical 
trials 20-22. Unlike CTLA4, which is expressed on nearly all T cells and appears to be important 
in controlling T cell proliferation during T cell development, PD-1 is up-regulated on peripheral 
T cells following chronic activation. PD-1 signaling on T cells is induced following binding to 
either PD-L1 (B7-H1, widely expressed, especially on macrophages and some tumors) or PD-L2 
(B7-DC, more limited expression, on antigen presenting cells). PD-1 signaling has been 
associated with chronic T cell activation and T cell exhaustion and it appears to be likely that 
blocking PD-1 may augment responses in the setting of chronic immune activation. The 
differences in the biology between CTLA4 and PD-1 leads to the prediction that PD-1 blockade 
is less likely to induce de novo autoimmunity and more likely to restore responses in the setting 
of chronic antigen exposure.  
1.2.6 Clinical Studies Evaluating anti-PD-1/anti-PD-L1 therapy 
Topalian et al. reported results of a Phase I study of nivolumab, a fully human IgG4 blocking 
antibody against PD-1 22. Doses tested were 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg and 10 
mg/kg using a standard 3+3 design. No MTD was identified. Additional expansion cohorts were 
then studied at 10 mg/kg. Grade 3 or 4 treatment-related adverse events occurred in 14% and 
drug related SAEs occurred in 11%. In general, adverse events were similar in nature, severity 
and reversibility to that seen with ipilimumab, except that the incidence appeared to be less and 
pneumonitis was not observed with a significant frequency as in the ipilimumab studies. 


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Cumulative response rates (all doses) were 18% among patients with non-small-cell lung cancer 
(14 of 76 patients), 28% among patients with melanoma (26 of 94 patients), and 27% among 
patients with renal-cell cancer (9 of 33 patients). Responses were durable; 20 of 31 responses 
lasted 1 year or more in patients with 1 year or more of follow-up. 
In July 2013, Hamid et al. reported results of a Phase I trial of pembrolizumab (previously MK-
3475) a humanized IgG4 mAb that blocks PD-1 23. A total of 135 patients with advanced 
melanoma were treated. Common adverse events attributed to treatment were fatigue, rash, 
pruritus, and diarrhea, the majority of which were low grade. The confirmed response rate across 
all dose cohorts was 38%. The response rate did not differ significantly between patients who 
had received prior ipilimumab treatment and those who had not. Responses were durable in the 
majority of patients.  
Brahmer et al. reported results of anti-PD-L1 blocking antibody therapy in 207 patients with a 
range of cancer 21. Results were similar to that observed with anti-PD1. Objective response rates 
were observed in 6-17% of patient groups including melanoma, renal cell cancer and non-small 
cell lung cancer. Several patients also showed prolonged stabilization of disease and grade 3 or 4 
toxic effects occurred in 9% of patients, and were primarily autoimmune in nature.  
Thus far, anti-PD1 therapy has shown preliminary evidence of clinical activity in also colon 
cancer. A recent article reported that a 71-year-old man affected by CRC who was initially 
treated with surgery plus chemotherapy and, upon disease progression, received anti-PD-1 agent, 
nivolumab. A partial response (PR) was observed on CT scan after only one dose of drug. The 
patient received four more courses of nivolumab over the following 6 months, during which he 
attained a complete response (CR). Therapy was discontinued and radiologic evaluation was 
performed 4 years after the initiation of nivolumab-based therapy, demonstrating no evidence of 
residual disease 24. Moreover, PD-L1 has been shown to be over-expressed in colon cancer but 
conflicting reports have made the prognostic/clinical relevance of this unclear 25,26. 
1.2.7 Immune responses following radiation, mechanism and rationale for combination with 
anti-PD1 therapy 
Several studies have documented an increase in peripheral antitumor immunity following 
radiation. It may even be the case that an intact immune system is critical for RT to exert its 
maximal antitumor effect. This was suggested by a mouse model of melanoma in which single-
fraction RT slowed the growth of small tumors in immune-competent but not immune-deficient 
mice 27.  The same authors also showed that in an animal model of breast cancer ablative RT of a 
primary tumor prevented the growth of metastatic tumor colonies in the lung, an effect that was 
dependent on the presence of CD8+ T cells. Other animal models have likewise displayed 
augmentation of systemic antitumor immunity following local RT 28-30. 
The underlying mechanism appears to be that RT-induced necrosis results in the exposure of 
tumor antigens, thereby increasing the pool of intracellular peptides for cross-presentation 31. 
Radiation has been shown to augment MHC-I expression by tumors, which is critical for antigen 
recognition by cognate CD8+ TCRs and which is known to be diminished in tumors as one of 
their escape mechanisms 31,32. Tumor antigen processing and presentation on MHC-I molecules 
is dependent on expression of a protein called high-mobility group box 1 (HMGB-1), a “danger 
signal” which binds toll-like receptor 4 (TLR4) on dendritic cells. In a pivotal study Apetoh et al. 
demonstrated that RT causes dying tumor cells to release HMGB-1 7. Intriguingly these authors 


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also reported that in breast cancer patients undergoing radiation and chemotherapy the presence 
of a polymorphism in TLR4 – and by implication a less immunogenic drug-induced cell death – 
was associated with an inferior prognosis. Similarly, in esophageal cancer patients preoperative 
chemoradiation has been shown to increase cancer-specific T cell responses and serum levels of 
HMGB-1, the latter of which correlated with overall survival 33. Furthermore, in various in vivo 
and in vitro models a single dose of 10 Gy lead to secretion of ATP and HMGB1 and increased 
expression of calreticulin on the plasma membrane all of which contributed to susceptibility to 
T-cell mediated killing 34. Finally, Camphausen et al. reported inhibition of tumor growth after 
irradiation of the leg of immunocompetent mice bearing a syngeneic tumor injected at a dorsal 
site and this phenomenon required an intact p53 pathway 29.  
Despite this, only a few cases of spontaneous decrease of metastases following radiation – the 
so-called abscopal effect – have been reported 35-38. This is presumably because the immune 
response by itself is too weak to be clinically significant.  
A number of studies have tried to boost this anti-tumor immune response following ablation by 
combining with an immunomodulatory agent:  
- 
A possible abscopal effect was seen in the Phase 1 AMP-224 trial in a patient with ocular 
melanoma who was treated with 9.5 cycles of 10 mg/kg AMP-224 + cyclophosphamide.  The 
subject developed two new brain lesions during the study which required palliative radiation. 
Stereotactic radiation therapy was administered to the brain, neck and abdomen, prior to 
cycle 5.  Per the Cycle 6 CT scan the patient’s target lesion in the liver had shrunk from 7.0 
cm (Cycle 4 scans) to 6.5 cm. The reduction in liver tumor size may be an abscopal effect as 
this lesion was not treated during the subject’s recent radiation therapy. These changes also 
correspond to a drop in the patient’s PD-1H T-cells and an increase in T-cell effector 
function.  
- 
Zeng et al. tested the combination of anti-PD-1 therapy with stereotactic radiosurgery in a 
glioblastoma model. Improved survival was demonstrated with the combination treatment 
compared with either modality alone. Long-term survival was seen only in the combined 
treatment arm, with a fraction (15%-40%) of animals alive at day 180+ after treatment. There 
was also increased tumor infiltration by cytotoxic T cells and decreased regulatory T cells in 
the combined treatment group compared with the single modality arms 39. 
- 
Dewan et al. evaluated RT in combination with anti-CTLA-4 antibody in two separate mouse 
models of breast and colorectal carcinoma 40. The authors founds that the combination of 
anti-CTLA4 and RT achieved enhanced tumor response at the primary site (compared to 
either modality alone) in addition to an abscopal effect (Interestingly this only occurred in 
fractionated versus single-dose RT). The frequency of CD8+ T cells showing tumor-specific 
IFN-gamma production was proportional to the abscopal effect.  
 
 


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Figure 1: (Adopted from 40.) The abscopal effect is induced in MCA38 colorectal cancer tumor-bearing 
mice by fractionated radiation in combination with anti-CTLA4 antibody. 5x105 cells were injected into 
the right and left flanks of C57Bl/6 mice. The left flank was irradiated (while shielding the rest of the 
mouse) with the mentioned doses and anti-CTLA4 was given i.p. every 3 days starting on first day of 
radiation. A: Tumor growth in irradiated (left panel) and non-irradiated flanks (right panel). B: Tumor 
weight in irradiated (left panel) and non-irradiated (right panel) at day 35. C: The non-irradiated tumors 
were excised on day 35 and analyzed by fluorescence microscopy for the presence of CD4+ and CD8+ 
cells. D: Analysis of tumor-specific IFN-γ production by spleen cells harvested at day 35 from treated and 
untreated mice and restimulated in vitro with irradiated MCA38 cells. RMA-S-Ld is an irrelevant target 
used as a negative control. Spleen cells from three mice in each treatment group were pooled. 


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- 
Demaria et al. tested the combination of RT with CTLA-4 blockade in a breast cancer model 
(4T1) known to be poorly immunogenic 41. Anti-CTLA4 alone did not have any effect on 
primary tumor growth or survival. RT was able to delay the growth of the primary irradiated 
tumor, but in the absence of anti-CTLA4 survival was similar to that of control mice. In 
contrast, mice treated with RT + anti-CTLA4 had a statistically significant survival 
advantage. The increased survival correlated with inhibition of lung metastases formation 
and required CD8+. 
- 
Most recently, in the Journal of Clinical Investigation, Deng et al. found that radiation and 
anti–PD-L1 treatment synergistically promoted antitumor immunity in a model of breast 
cancer. 42. Administration of anti–PD-L1 enhanced the efficacy of radiation through a 
cytotoxic T cell–dependent mechanism. Concomitant with radiation-mediated tumor 
regression, they observed that radiation and anti–PD-L1 synergistically reduced the local 
accumulation of tumor-infiltrating MDSCs, which suppress T cells and alter the tumor 
immune microenvironment. Furthermore, activation of cytotoxic T cells with combination 
therapy mediated the reduction of MDSCs in tumors through the cytotoxic actions of TNF. 
These important data provide evidence for a close interaction between external beam 
radiation, T cells, and the PD-L1/PD-1 axis and establish a preclinical basis for the rationale 
of combination therapy with immune modulators and radiotherapy. 
1.2.8 AMP-224 (Derived from Investigator’ s Brochure) 
AMP-224 is a recombinant Fc fusion protein composed of the extracellular domain of human 
B7-DC fused to the hinge and Fc domain of human IgG1. AMP-224 binds to programmed death-
1 (PD-1), an inhibitory receptor that is present on the cell surface of exhausted, activated, 
effector, and memory T cells. AMP-224 has a unique, non-blocking mechanism of action in that 
it binds PD-1HIPD-L1- T cells (chronically stimulated / exhausted T cells) but does not bind PD-
1+PD-L1+ cells (normal activated T cells). 
 
- 
Pre-clinical toxicology studies in cynomolgus monkeys (PD-1 in both species have similar 
affinity to AMP-224) revealed that there were no test article-related effects on clinical 
observations, food consumption, ECG and cardiovascular profiling, ocular exams, clinical 
chemistry, hematology, coagulation and urinalysis indices, organ weights or gross pathology. 
At terminal sacrifice, treatment with >10mg/kg AMP-224 with or without 200mg/m2 
cyclophosphamide (, please see next section for the justification of cyclophosphamide) was 
associated with increased incidence and/or severity of hepatocyte vacuolation, consistent 
with increased glycogen content. In addition, treatment with cyclophosphamide + 10 mg/kg 
AMP-224, and 100 mg/kg AMP-224, with and without cyclophosphamide, was associated 
with minimal to mild thymic atrophy. These findings were not considered adverse and were 
reversible. The no-observed-adverse- effect-level (NOAEL) was 100 mg/kg, the highest dose 
level evaluated, suggesting that saturation of the PD-1 receptor is not associated with 
toxicity. 
- 
In pre-clinical efficacy studies, the CT26 murine colon cancer cell line was used as a model. 
This is a PD-1 negative tumor, in which the anti-tumor immune response is suppressed and 
characterized by tumor expression of PD-L1 and tumor infiltration by CD8+PD-1+ T cells 
and Tregs 43,44. Although AMP-224 as a monotherapy (given 5 mg/kg biweekly for 4 weeks) 


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did not have efficacy in mice subcutaneously injected with CT26, combination with a single 
dose of 100mg/kg intraperitoneal cyclophosphamide resulted in a potent anti-tumor effect.  
 
 
Figure 2: The treatment outcome of AMP-224 given with or without CTX in CT26 murine colon cancer 
model. Tumor volume vs time was plotted for A vehicle, B CTX given only on day 9, C AMP-224 only 
starting on day 3, DAMP-224 starting on day 6, E CTX on day 9 and AMP-224 starting on day 10. Each 
line corresponds to one mouse. 
 
To determine the most effective dose level of AMP-224, the CT25 model was used and dose 
levels of 1.5, 5, 15 and 45 mg/kg following a single dose of cyclophosphamide were tested. 
While there was dose response up to 15 mg/kg, increasing dose to 45 mg/kg did not alter 
efficacy 
 
 


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Figure 3: Tumor eradication and rejection when tumor is reinjected following cyclophosphamide+AMP-
224 treatment in CT26 colon cancer model. Left panel is treatment of primary tumor with indicated doses. 
Cyclophosphamide was given as 100 mg/kg single intraperitoneal injection. Right panel is rejection of 
second tumor injected to the opposite flank. The dose of AMP-224 was 5 mg/kg. Gray line is AMP-224 
alone, blue line is cyclophosphamide alone and red line is the combination.  
 
- 
Immunephenotyping from pre-clinical (CT26-murine studies) conducted on days 15 and 24 
post-inoculation demonstrated that in the group that received cyclophosphamide in 
combination with 5 mg/kg AMP-224, all the tumor-infiltrating lymphocytes (TIL) were 
exclusively PD-1- while in the control tumor, they had high expressions of PD-1. 
Interestingly, the combination treatment resulted in fewer lymphocytes when compared to the 
control group suggesting that AMP-224 can inhibit the proliferation of antigen specific PD-
1+ cells and block the induction of PD-1 on activated T-cells, which most likely contributes 
to the long term anti-tumor effect.  
 
  Figure 4: The PD-1 expression in both CD4+ and CD8+ tumor infiltrating cells is inhibited with 
cyclophosphamide +AMP-224 treatment. Tumors were excised on days 15 and 24, followed by digestion 
and analysis of TILs by flow cytometry. In mice that received cyclophosphamide in combination with 5 
mg/kg of murine AMP-224 observe fewer CD8+ TIL on Day 15 were observed. The TIL that were present 
were almost all PD-1-. By Day 24, an expanded population of CD8+PD-1-/LO cells were observed in a 
subset of mice treated with cyclophosphamide + murine AMP-224 and this correlated with elimination of 
the CT26 tumors. The expansion of CD4+ and CD8+ T-cells was not seen in the spleen. 
- 
Human equivalent doses (HED) were calculated based on repeat-dose PK data from mouse 
and cynomolgus monkey studies to provide equivalent levels of drug exposure. AMP-224 
was well-tolerated and an MTD was not identified in preclinical toxicology studies. In 
preclinical murine models, 0.5 mg/kg of AMP-224 was determined to be the lowest dose that 
resulted in a statistically significant improvement in tumor eradication or survival. At this 
dose level, estimated circulating drug levels ranged from approximately 1-3 μg/mL with 
twice weekly dosing. Peak receptor occupancy (on cells expressing high levels of PD-1) was 


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calculated to be 75%, and HED was calculated to be 0.3 mg/kg, the starting dose for Phase 1 
testing. 
 
Human studies from a phase 1, first-in-human, open-label multi-center study were reported at 
ASCO 2013. Twenty-six patients with relapsed or refractory solid tumors were enrolled in 5 
dose cohorts (0.3, 1, 3, 10, 30 mg/kg) in addition to an expansion cohort of melanoma patients 
treated at 10mg/kg. AMP-224 was administered on a 28 day treatment cycle consisting of 200 
mg/m2 of cyclophosphamide given on Day 0 and AMP-224 on Days 1 and 15. To date, (as of 
June 2014) AMP-224 has only been evaluated in combination with cyclophosphamide. The 
number of completed treatment cycles ranged from <1 (6.8%, n=3) to 21 (2.3%, n=1) with 
patients completing 3.3 treatment cycles on average. 
Toxicity/safety: Dose levels up to 30 mg/kg were evaluated without dose-limiting toxicity (DLT).  
AMP-224 was found to have an acceptable safety profile. The most frequently reported 
(occurrence in >10% patients) drug-related Adverse Events (AEs) across all treatment groups 
were as follows: chills (70.5%), fatigue (40.9%), nausea (40.9%), flushing (38.6%), vomiting 
(38.6%), pyrexia (31.8%), back pain (27.3%), headache (25.0%), decreased appetite (18.2%), 
dyspnea (13.6%), non-cardiac chest pain (13.6%), diarrhea (11.4%), feeling cold, (11.4%), 
feeling hot (11.4%), neck pain (11.4%). The majority of AEs considered to be related to AMP-
224 were of mild or moderate severity. Additionally, the following serious adverse events 
(SAEs) were reported:  disease progression (11.4%), neoplasms benign, malignant and 
unspecified (including cysts and polyps) (6.8%), pneumonia (6.8%), back pain (4.5%), bile duct 
obstruction (2.3%), cerebral hemorrhage (2.3%), cerebral ischemia (2.3%), clostridium difficile 
colitis (2.3%), dehydration (2.3%), dyspnea (2.3%), encephalopathy (2.3%), intracranial 
hemorrhage (2.3%), hyperglycemia (2.3%), intestinal perforation (2.3%), malignant neoplasm 
progression (2.3%), metastases to central nervous system (2.3%), metastases to meninges 
(2.3%), pleural effusion (2.3%), pyrexia (2.3%), renal failure (2.3%), spinal cord compression 
(2.3%), vomiting (2.3%). None of the SAEs were considered by the investigators to be related to 
AMP-224.  
10 mg/kg was determined to be the optimal dose level in this study. The most frequently reported 
(occurrence in >10% patients) drug-related AEs in the 10 mg/kg treatment group were as 
follows: chills (87.5%), flushing (45.8%), nausea (41.7%), vomiting (41.7%), pyrexia (37.5%), 
fatigue (33.3%), headache (33.3%), back pain (25.0%), non-cardiac chest pain (20.8%), feeling 
cold (16.7%), feeling hot (16.7%), decreased appetite (16.7%), dyspnea (16.7%), abdominal pain 
(12.5%), upper abdominal pain (12.5%), peripheral edema (12.5%). Additionally, the following 
serious adverse events (SAEs) were reported for the 10 mg/kg treatment group: cerebral 
hemorrhage (4.2%), dehydration (4.2%), intracranial hemorrhage (4.2%), malignant neoplasm 
progression (4.2%), metastases to central nervous system (4.2%), pleural effusion (4.2%), spinal 
cord compression (4.2%), disease progression (8.3%), neoplasms benign, malignant and 
unspecified (including cysts and polyps) (8.3%), pneumonia (8.3%). As previously noted, no 
SAEs were considered to be related to be drug-related. 
Seven patients (16%) did experience drug-related Grade 3 adverse events: One each of 
lymphocytopenia, anemia, hypertension, weight loss, infusion-related reaction, influenza like 
illness, myalgia and thrombocytopenia and three instances of increased blood pressure. There 
was no evidence of pneumonitis, colitis, or autoimmune adverse events (AE). Infusion reactions 


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were the most common AE; observed in 34/44 patients (77.3%) and manageable with pre-
medication and longer (2-3 hour) infusion.  
AMP-224 infusion reactions occurred in all treatment groups with the highest proportion in the 
30 mg/kg treatment group: 
AMP-224 dose 
level 
Number of 
patients enrolled  
Number of 
patients with >1 
infusion reaction 
% Patients with >1 
infusion reaction 
0.3 mg/kg 
6 
2 
33.3% 
1 mg/kg 
4 
2 
50.0% 
3 mg/kg 
4 
2 
50.0% 
10 mg/kg 
24 
22 
91.7% 
30 mg/kg 
6 
6 
100.0% 
Total (all patients) 
44 
34 
77.3% 
 
The most common (occurrence in >10% patients) AMP-224 infusion reactions were as follows: 
chills (72.7%), pyrexia (27.3%), flushing (40.9%), nausea (34.1%), vomiting (34.1%), back pain 
(29.5%), headache (22.7%), feeling hot (13.6%), non-cardiac chest pain (15.9%), feeling cold 
(13.6%), increased blood pressure (11.4%), dyspnea (11.4%) and neck pain (13.6%). The 
majority of the infusion reactions were mild to moderate in severity. There were five instances of 
grade 3 infusion reactions: one instance of hypertension in the 3mg/kg treatment group, 1 
instance of “infusion related reaction” in the 10 mg/kg treatment group and 3 instances of 
increased blood pressure in one patient in the 10 mg/kg treatment group.  
Investigators were permitted to pre-treat patients with prophylactic medication prior to AMP-224 
infusions. The following medications were used for AMP-224 infusion pre-treatment: 
meperidine (12.5, 25 and 50 mg), acetaminophen (650 and 1000 mg), lorazepam (0.5 and 1 mg), 
diphenhydramine (12.5, 25 and 50 mg), ranitidine hydrochloride (50 and 150 mg), promethazine 
(12.5 and 25 mg), loratadine (10 mg), ondansetron (4, 8 and 16 mg) and palonosetron 
hydrochloride (0.25 mg). Dexamethasone (10 mg) was not a permitted pre-treatment medication; 
however, this was also prophylactically administered to two patients in the 10 mg/kg treatment 
group. Patients were not consistently pre-treated for all infusion visits during the escalation phase 
of the study as this was not initially a protocol requirement. Generally subjects in the 0.3, and 1 
mg/kg treatment groups were only pre-treated if an infusion reaction had occurred during the 
prior visit. Subjects in the 10 mg/kg and 30 mg/kg treatment groups were pre-medicated more 
consistently; however, medication combinations generally varied, as they were chosen at the 
discretion of the investigators. 
Pharmacokinetics: AMP-224 is eliminated in a biexponential manner following IV infusion.  In 
the AMP-224-01 trial the mean half-life of AMP-224 in the escalation phase cohorts (0.3 mg/kg, 
n=6; 1 mg/kg, n=4; 3 mg/kg, n=4; 10 mg/kg, n=6; 30 mg/kg, n=6) was 138±23.4 hours and the 
mean half-life in the expansion phase cohort (10mg/kg, n=24) cohort was 111±25.4 hours.  The 
terminal half-life of AMP-224 is approximately 10 days with a linear relationship between dose 


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level and exposure were observed.  Interruptions during the IV infusion did not appear to 
consistently affect PK profile.  
Positive anti-drug antibodies (ADA) were confirmed at one or more time point in 25.6% (n=11) 
of patients tested. (A sample for ADA was not available for 1 subject in cohort 5). 45.5% (n=5) 
of these patients had detectable ADA prior to the first dose of AMP-224. There did not appear to 
be a consistent correlation between titer level and decreased serum AMP-224 concentration 
levels. Exposure values for patients confirmed positive for ADA were not markedly different 
from patients that tested negative. As the number of patients who tested positive for ADA is 
small it cannot be confirmed that there was no effect on the PK of AMP-224. 
Efficacy: Two patients had a clinical response following treatment with AMP-224: one was a 
partial responder (PR) and one had durable stable disease (SD).  Both clinical responders had a 
diagnosis of melanoma, V600K BRAF mutant. All patients with a confirmed clinical response 
had high levels of PD-1+ TIL at baseline as well as normal LDH and ALC values. Additionally, 
patients with the highest pre-treatment levels of PD-1HI cells in peripheral blood also had high 
density of PD-1+ TIL which was associated with clinical response to AMP-224 treatment.  
Seven patients were considered to be “immune responders”, defined as patients assigned to the 
10mg/kg or 30mg/kg dose of AMP-224, who completed a minimum of 4 treatment cycles and 
where emergence of an enhanced effector/functional peripheral T cell response along with 
evidence of tumor reduction in the context of a mixed response was detected post dose.  
Thus, patients’ immune competency at baseline can generally predict whether they will respond 
to AMP-224. It was determined that clinical responders, immune responders and patients with 
progressive disease could be stratified by their baseline ALC counts and expression ofCD8+ TIL 
and PD-1+ TIL within the tumor biopsy. 
Immune-monitoring: Clinical dose-response profile was found to be consistent with the above 
murine CT26 tumor model findings in that sustained reductions in peripheral PD-1HI T cells (the 
so-called ‘exhausted’ T cells which are the specific target of AMP-224) were observed.  PD-1HI 
T-cells were reduced in dose-dependent fashion following the initial dose of AMP-224.  No 
significant difference in the reduction of PD-1HI T-cells was observed between the 10 mg/kg and 
30 mg/kg doses. See Figure 3. 
A sustained reduction in PD-1HI cells was observed in subsequent cycles in most patients who 
received either 10mg/kg or 30 mg/kg of AMP-224; however, failure to obtain sustained 
reduction in PD-1+ T-cells was correlated with rapid disease progression.   


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Figure 5: Reduction in PD-1hi T cells with AMP-224 treatment. 
It was found that failure to control levels of PD-1HI T cells correlated with rapid disease 
progression. AMP-224 treatment did not significantly affect lymphocyte, T cell, CD4+, CD8+, 
or PD-1LO counts. Flow cytometry analysis of PBMC specimens from 10-30 mg/kg cohorts 
showed the following evidence of improved immune function following treatment with AMP-
224: 
- 
Increased numbers of polyfunctional (IFN-γ+TNF-α+IL-2+) CD4+ and CD8+ T cells. 
- 
Increased numbers of effector and EMRA T cells producing the lytic marker Granzyme 
B. 
In addition, IHC analysis of paired tumor biopsy specimens from patients in the 10-30 mg/kg 
cohorts showed increased ratio of CD8+ TIL to PD-1+ TIL in 9/14 cases, including 5/5 
evaluated patients who remained on trial for 4 or more cycles. 
1.2.9 Rationale for study population and safety of administering SBRT to hepatic metastases  
Despite advances in the systemic chemo/biologic therapeutic options, which have become 
available for metastatic colorectal cancer over the past 10-15 years, the majority of patients will 
die from their disease. The majority of patients with metastatic colon cancer develop liver 
metastases so conducting this study in this patient population seeks to address a common 
problem in a common tumor type.  
Administering radiation therapy to the liver is not standard or conventional. With newer 
techniques however this has been shown to be a safe strategy. For example, Dr. Citrin,  one of 
our associate investigators, has previously conducted a study employing hepatic radiation 
combined with vector-based vaccine targeting carcinoembryonic antigen in just this population 
45. In that study patients received a total radiation dose of 32 Gy to sites of metastatic disease in 
the liver, delivered in 8-Gy courses (4 separate 2-Gy fractions) beginning one day after each 
vaccine boost (days 22–25, 36–39, 50–53, 64–67). The treatment was well tolerated, with the 
most common toxicity being a transient dermatologic reaction as a result of the vaccine used in 
the study. There were no ≥ grade 3 toxicities attributable to the treatment and specifically there 
were no ≥ grade 2 hepatic toxicities.  


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Scorsetti et al. evaluated the safety and feasibility of high-dose stereotactic body radiation 
therapy (SBRT) in a phase 2 study of patients with unresectable liver metastases (most frequent 
being colorectal primary) 46. Enrollment was confined to those patients with 1 to 3 liver 
metastases, with maximum individual tumor diameters less than 6 cm. Doses consisted of 75 Gy 
on 3 consecutive days. A total of 61 patients with 76 lesions were treated. Among the patients, 
21 (34.3%) had stable extrahepatic disease at study entry. After a median of 12 months the in-
field local response rate was 94%. None of the patients experienced grade 3 or higher acute 
toxicity. No radiation-induced liver disease was detected. The authors concluded that SBRT for 
unresectable liver metastases can be considered an effective, safe, and noninvasive therapeutic 
option, with excellent rates of local control and a low treatment-related toxicity. 
Other additional reports have documented the safety of SBRT to the liver at doses/schedules of 
60 Gy/10 Fr and 50 Gy/4 Fr 47; 36 to 60 Gy in 6 to 15 Gy per fraction (HCC, cholangio and 
colorectal cancer) 48;  single doses of 17 to 30 Gy (median dose 24 Gy) 49; median dose per 
fraction and total dose of 3.6 Gy (2.0-13.0 Gy) and 55 Gy (30-80 Gy) in 26 patients with 
colorectal cancer 50. 
1.2.10 Justification for dose and schedule 
In the phase 1 trial of AMP-224, although no DLT was identified, immune monitoring studies 
revealed that the dose of 10 mg/kg was equivalent to 30 mg/kg, both given every other week, for 
reducing PD-1HI cells while the latter dose was associated with more infusion reactions. 
Therefore, 10 mg/kg given every 14 days was selected as the dose for the current trial. In the 
Phase 1 AMP-224 study, cyclophosphamide at 200 mg/m2 was administered at the start of each 
treatment cycle (28 days) and no detrimental impact on safety was identified. The rationale for 
including the low dose cyclophosphamide is to reduce Treg T cells and remove any suppressive 
cells that would impede the activation of a robust immune response. However, continued 
repeated infusions of low dose cyclophosphamide may not be necessary.  Firstly, all pre-clinical 
in vivo studies with AMP-224 included a single low dose of cyclophosphamide prior to the first 
AMP-224 treatment.  Secondly, pharmacodyanmic data from the phase 1 clinical trial indicated 
that once an anti-tumor immune response has been activated, it may be detrimental to continue 
dosing with low dose cyclophosphamide. Therefore, in the current trial cyclophosphamide at low 
dose will be given to enhance the immune response by reducing Treg cells only prior to the first 
AMP-224 dose.  
Since in the preclinical model of colon cancer, the most efficacious abscopal effect was 
identified when RT was given in 8 Gy x 3 fractions, this dose schedule was adapted to the 
current study. As mentioned above, doses much higher than 8 Gyx3 were given to liver 
metastases of patients via SBRT without resulting in significant toxicity.  
1.2.11 Justification for Tumor Biopsies 
While the preclinical data suggest important immune-regulatory effects of radiation treatment on 
tumors, with potential for amplification with anti-PD1/PDL1 therapy, the effect on humans is 
really unknown. Given that this is a small pilot study whose next step in development – if safe 
and feasible as per the primary endpoint – will most likely be a larger randomized study, it is 
scientifically important to obtain as much information about the treatment effect. This may lead 
to altered and improved design of the next study. The best strategy for doing this is with tumor 
biopsies. As pointed out by Deng et al.42  administration of anti–PD-L1 enhanced the efficacy of 


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radiation through a cytotoxic T cell–dependent mechanism. Concomitant with radiation-
mediated tumor regression, they observed that radiation and anti–PD-L1 synergistically reduced 
the local accumulation of tumor-infiltrating MDSCs, which suppress T cells and alter the tumor 
immune microenvironment.  It would be important to replicate this observation – or disprove it – 
in humans and would be relevant knowledge whether the clinical data (response, PFS etc.) was 
either positive or negative. Given this and the fact that in order for the biopsy material from any 
single patient to be relevant and worthwhile in the context of N=15 patients we would really 
require participation from all or almost all of the patients. 
2 
ELIGIBILITY ASSESSMENT AND ENROLLMENT 
2.1 
ELIGIBILITY CRITERIA 
2.1.1 Inclusion Criteria 
2.1.1.1 Patients must have histopathological confirmation of Colorectal Carcinoma (CRC) by 
the Laboratory of Pathology of the NCI prior to entering this study. 
2.1.1.2 Patients must have progressed on or been intolerant of prior oxaliplatin and irinotecan-
containing chemotherapeutic regimen and have disease that is not amenable to 
potentially curative resection.  Patients who have a known KRAS wild type tumor must 
have progressed or been intolerant to cetuximab or panitumumab-based chemotherapy. 
2.1.1.3 Patients must have one focus of metastatic disease in the liver that is amenable to SBRT 
in the opinion of radiation oncology. 
2.1.1.4 All patients enrolled will be required to have measurable disease by RECIST 1.1 criteria 
outside the radiation field.  
2.1.1.5 Study patients must have disease that is amenable to pre and post treatment biopsy and 
be willing to undergo this. 
2.1.1.6 Age ≥18 years  
2.1.1.7 Life expectancy of greater than 3 months 
2.1.1.8 ECOG performance status 0-1 (see Appendix A) 
2.1.1.9 Patients must have acceptable organ and marrow function as defined below: 
- 
leukocytes 
>3,000/mcL 
- 
absolute neutrophil count 
> 1,500/mcL 
- 
platelets 
>100,000/mcL 
- 
total bilirubin 
≤1.5X institution upper limit of normal 
- 
Patients are eligible with ALT or AST measuring up to5 x ULN 
given the presence of liver metastasis. 
- 
creatinine 
<1.5X institution upper limit of normal 


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OR 
- 
creatinine clearance 
>45 mL/min/1.73 m2, as calculated 
below, for patients with creatinine levels 
above institutional normal 
Estimated creatinine clearance (mL/min) 
Females 
= 
 
Males = 
 
 
May use a 24 hr. urine collection to determine creatinine clearance. 
Measured creatinine clearance (mL/min) 
 
2.1.1.10 Patients must have recovered from any acute toxicity related to prior therapy, including 
surgery.  Toxicity should be < grade 1 or returned to baseline. 
2.1.1.11 Patients must not have other invasive malignancies within the past 3 years (with the 
exception of non-melanoma skin cancers, localized prostate cancer, carcinoma in situ of 
the cervix and non-invasive bladder cancer that has had successful curative treatment). 
2.1.1.12 Patient must be able to understand and willing to sign a written informed consent 
document. 
2.1.2 Exclusion Criteria 
2.1.2.1 Prior immune checkpoint inhibition with anti-PD1/PD-L1 or anti-CTLA4 therapy or 
other specific T cell targeting agents. 
2.1.2.2 Patients who have had chemotherapy (or so-called ‘targeted’ systemic therapy), large 
field radiotherapy, or major surgery must wait 4 weeks after completing treatment prior 
to entering the study. 
2.1.2.3 Patients with known brain metastases will be excluded from this clinical trial because of 
their poor prognosis and because they often develop progressive neurologic dysfunction 
that would confound the evaluation of neurologic and other adverse events. 
2.1.2.4 Uncontrolled intercurrent illness including, but not limited to, hypertension (systolic BP 
> 160, diastolic BP > 100), ongoing or active systemic infection, symptomatic 
congestive heart failure, unstable angina pectoris, cardiac arrhythmia, uncontrolled 
diabetes or psychiatric illness/social situations that would limit compliance with study 
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2.1.2.5 HIV-positive patients receiving anti-retroviral therapy are excluded from this study due 
to the possibility of pharmacokinetic interactions between antiretroviral medications 
and the investigational agent. 
2.1.2.6 History of chronic autoimmune disease (e.g., systemic lupus erythematosus or 
Wegener’s granulomatosis, Addison’s disease, multiple sclerosis, Graves’ disease, 
Hashimoto’s thyroiditis, hypophysitis, etc.) with symptomatic disease within the 3 years 
before randomization. Note: Active vitiligo or a history of vitiligo will not be a basis for 
exclusion. In addition, a past history of certain automimmunity eg rheumatoid arthritis 
or thyroiditis may be allowed per PI discretion provided it has been quiescent for a 
minimum of three years.  
2.1.2.7 Active or history of inflammatory bowel disease (colitis, Crohn’s), irritable bowel 
disease, celiac disease, or other serious, chronic, gastrointestinal conditions associated 
with diarrhea.  
2.1.2.8 Dementia or significantly altered mental status that would prohibit the understanding or 
rendering of Information and Consent and compliance with the requirements of the 
protocol.  
2.1.2.9 Currently receiving immunosuppressive doses of steroids or other immunosuppressive 
medications (inhaled and topical steroids are permitted) 
2.1.2.10 History of sarcoidosis syndrome 
2.1.2.11 History of hypersensitivity reaction to human or mouse antibody products. 
2.1.2.12 Pregnancy and breast feeding are exclusion factors.  The effects of AMP-224 on the 
developing human fetus are unknown.  Enrolled patients must agree to use adequate 
contraception (hormonal or barrier method of birth control; abstinence) prior to study 
entry, the duration of study participation and 3 months after the end of the treatment. 
Should a woman become pregnant or suspect she is pregnant while she or her partner is 
participating in this study, she should inform her treating physician immediately. 
2.1.2.13 Patients with unhealed surgical wounds for more than 30 days.  
2.1.2.14 Patients with known sensitivity or allergy to any components of AMP-224. 
2.1.3 Inclusion of Women and Minorities 
Men and women of all races and ethnic groups are eligible for this trial. 
2.1.4 Recruitment Strategies 
The study will be posted on the CCR website and on clinicaltrials.gov. 
2.2 
SCREENING EVALUATION 
2.2.1 History and Physical Evaluation 
Complete medical history and physical examination (including height, weight, vital signs, EKG, 
and performance status) will be conducted prior to starting study drug and research biopsy. 


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2.2.2 Laboratory Evaluation 
• Hematological Profile: CBC with differential and platelet count. 
• Biochemical Profile: Acute, Hepatic, Mineral Panels, amylase and uric acid 
• Serum or urine pregnancy test for female participants of childbearing age and anatomic 
ability (within 72 hours prior to initiating treatment). 
• HLA subtype 
• CT scan (or MRI) 
2.2.3 Histologic confirmation (at any time point prior to initiation of study therapy)  
A block or unstained slides of primary or metastatic tumor tissue will be required from each 
participant to confirm diagnosis with analysis being performed by the Laboratory of Pathology, 
NIH.  
2.3 
REGISTRATION PROCEDURES 
Authorized staff must register an eligible candidate with NCI Central Registration Office (CRO) 
within 24 hours of signing consent.  A registration Eligibility Checklist from the web site 
(http://home.ccr.cancer.gov/intra/eligibility/welcome.htm) must be completed and sent via 
encrypted email to:  NCI Central Registration Office (HOIS) ncicentralregistration-
l@mail.nih.gov.  After confirmation of eligibility at Central Registration Office, CRO staff will 
call pharmacy to advise them of the acceptance of the patient on the protocol prior to the release 
of any investigational agents.  Verification of Registration will be forwarded electronically via e-
mail to the research team.  A recorder is available during non-working hours. 
2.4 
BASELINE EVALUATION 
2.4.1 Imaging studies (baseline – obtained within 28 days prior to enrollment):  
CT scan of chest, abdomen and pelvis or MRI scan if clinically indicated. 
2.4.2 Research Tumor Biopsy (within 16 days of beginning study treatment) 
2.4.3 Laboratory evaluation (obtained within 72 hours prior to first dose of radiation i.e. either 
Day -2 or Day 0.) 
If laboratory tests are done within 72 hours of Cycle 1 Day 1 they do not need to be repeated on 
Day 1. 
• Hematological profile: CBC with differential and platelet count, PT, INR, aPTT, 
fibrinogen. 
• Biochemical profile: Acute, Hepatic, Mineral Panels 
• LDH 
• C-Reactive Protein 
• Tumor marker profile: CEA  
2.4.4 Laboratory evaluation (obtained within 28 days prior to first dose of SBRT) 
• Thyroid function tests (TSH, T3, T4) 


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• ACTH, morning cortisol. 
2.4.5 Laboratory evaluation (obtained within 45 days prior to first dose SBRT) 
• Hepatitis B and/or C viral load and serology 
2.4.6 History and physical exam with vital signs (obtained within 1 week prior to first dose). 
2.4.7 Electrocardiogram (obtained within 28 days prior to first dose of SBRT) 
3 
STUDY IMPLEMENTATION 
3.1 
STUDY DESIGN 
- 
The proposed study is a pilot study of AMP-224 in combination with SBRT of liver at 
two dose levels in patients with metastatic colorectal cancer. 
- 
Hypofractionated radiation will be administered to a metastatic disease site at a dose and 
schedule of 8Gy for 1 (DL 1) or 3 days (DL2) beginning on Day 0 (DL1) or Day -2 
(DL2)  (Total dose 8 - 24Gy).   
 
Dose level 
AMP-224  dose (mg/kg) 
Radiation 
N(total=15) 
1 
10 
8Gy x 1 days 
6 
2 
10 
8Gy x 3 days 
9 
 
- 
AMP-224 therapy will be administered as an intravenous infusion beginning on Day 1 
then q14days for a total of 6 doses only. Optional continuation of treatment on the same 
q2-weekly until disease progression will be considered in patients who appear to be 
responding and who are tolerating treatment.  
- 
Cyclophosphamide 200mg/m2 IV will be given on Day 0.  
- 
Mandatory biopsy of the tumor will be performed at baseline and day 29 of treatment.  
- 
An optional biopsy of a tumor outside the radiation field that meets the criteria for 
response (please see section 6.2 for response criteria) will be performed at day 29 +/- 96 
hours (to allow for schedule challenges).  
- 
Treatment schema is as follows: 
 


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- 
Six patients will be treated on DL1, unless 2 patients develop DLT before the 6th patient 
is enrolled in which case enrollment will stop pending further discussion/amendment.  
- 
If 2 or more out of 6 patients at DL1 encounter DLT we will not proceed to DL2. 
 
 
 
- 
If more than one out of three patients at DL2 cumulatively encounters a DLT, we will not 
investigate this combination further, but may amend the protocol if appropriate. 
Specifically, if 2 or more of 6 patients or 3 or more of 9 patients have a DLT at DL2, then 
we will not proceed with further enrollment or will need to revise the treatment regimen 
before use in a subsequent study, as appropriate. 
- 
Evaluation of DLT will extend for a total of 4 weeks beginning on day 0. 
Dose Level 1 
Dose Level 2 


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3.1.1 Protocol Stopping Rules 
This study will utilize the Common Terminology Criteria for Adverse Events (CTCAE) version 
4.0 for grading systemic toxicity. For safety reasons, the protocol will be temporarily halted 
pending discussions with the NCI IRB and Sponsor regarding necessary amendment for either of 
the following events: 
• One occurrence of grade 5 toxicity by the NCI-CTCAE version 4.0 attributable to the 
treatment regimen. 
• Two occurrences of grade 4 toxicity within a single cohort by the NCI-CTCAE version 
4.0 attributable to the treatment regimen. 
3.1.2 Dose Limiting Toxicity 
3.1.2.1 Definition of Dose-limiting toxicities (DLT) 
A DLT is defined as a ≥ Grade 3 adverse drug reaction (ADR) according to the NCI-CTCAE 
v4.0, that is possibly, probably, or definitely related to the combination of AMP-224 with SBRT, 
occurring during the DLT evaluation period except for any of the following outlined in section 
3.1.2.2. ADRs are defined in this trial as any AEs suspected to be related to AMP-224 by the 
investigator.   
3.1.2.2 Exclusions to Dose Limiting Toxicities 
• Grade 3 infusion-related reactions.  
• Any Grade 3 non-hematologic toxicity that occurs in the first 10 days after SBRT and 
which is an expected toxicity of the procedure in the opinion of the PI. These symptoms 
include, but are not limited to, fevers/rigors, pain, fatigue and nausea. However, if these 
symptoms do not resolve to Grade 1 (or baseline) by 28 days the exception does not 
apply and DLT would be adjudicated. 
• Any grade 3 immune-mediated adverse event (including but not limited to Grade 3 
diarrhea, Grade 3 skin toxicity, or Grade 3 liver enzyme elevation) that improves to ≤ 
Grade 2 within 1 week of onset with supportive care (which may include systemic 
corticosteroids). 
• Nausea and vomiting Grade 3 will only be considered dose-limiting if it is refractory to 
anti-emetic therapy and unable to be corrected to Grade 1 or less within 48 hours. 
• Grade 3 rise in creatinine, not corrected to Grade 1 or less after 2 liters of intravenous 
fluids within 24 hours, will be considered dose limiting.   
• Single laboratory values out of normal range that are unlikely related to trial treatment 
according to the investigator, do not have any clinical correlate, and resolve within 7 days 
with adequate medical management.  
• Transient Grade 3 fatigue (<7days), local reactions, headache, nausea, emesis that 
resolves to ≤ Grade 1.  
• Grade 3 endocrinopathy that is managed with or without systemic corticosteroid therapy 
and/or hormone replacement therapy and the following criteria are met: 
− The subject’s hormone levels are within normal limits 


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− The subject is asymptomatic 
• Grade 3 inflammatory reaction attributed to a local antitumor response 
(e.g., inflammatory reaction at sites of metastatic disease, lymph nodes, etc.). 
3.2 
DRUG ADMINISTRATION 
3.2.1 AMP-224 Drug Administration 
AMP-224 is supplied as a sterile frozen liquid dosage form filled in a glass vial configuration.  The 
product should be stored frozen at ≤-65°C until ready to use.  Each vial contains 300 mg AMP-
224 in 5.0 mL of 60 mg/mL in 10 mM sodium phosphate, 8% sucrose (w/w), 0.01% polysorbate-
80, pH 7.5.  No preservative is used since the vial is designed for single use. 
Once thawed, AMP-224 infusion should begin within 4 hours.  AMP-224 is to be administered 
IV.   
The calculated AMP-224 dose to be administered to the patient (determined by the patient’s body 
weight obtained on the day of dosing) will be removed from the freezer and thawed.  Once thawed, 
each dose will be diluted in 0.9% sodium chloride to equal a total delivery volume of 60 mL.   
15 – 30 minutes prior to the start of the AMP-224 infusion, patients should receive the following 
pre-treatment regimen: 
o Acetaminophen (500-1000 mg) 
o Diphenhydramine (12.5-25mg) 
Dosage of pre-medications depends on the size of the patient and is at the discretion of the 
Investigator.  The same regimen should be used prior to the first AMP-224 infusion, but 
additional medications can be provided at the discretion of the Investigators e.g. Promethazine 
(12.5-25mg), Meperidine (12.5-25mg).  Please see section 11.1.5 as well. 
• A 0.2-0.22 micron filter MUST be added to the IV tubing prior to priming and infusing 
AMP-224 into patients. 
• The final solution will be infused IV via a volumetric infusion pump over a minimum of 2 
hours.  The infusion time may be extended at any time to reduce infusion reactions. 
3.2.2 Cyclophosphamide  Administration 
3.2.2.1 On Day 0 of the study cyclophosphamide will be given as follows:  
The dose of cyclophosphamide will be calculated using the standard CRIS BSA calculator 
(Dubois Formula).  The patient’s body weight and height should be obtained within 3 days prior 
to Day 0. 
- 
Cyclophosphamide will be diluted in 250 mL of 0.9% Sodium Chloride and will be infused 
over 60 minutes.  
3.2.3 Monitoring of Dose Administration 
Vital signs will be collected before investigational product infusion, every 30 minutes during 
infusion, at completion of infusion, and 30 and 60 minutes post infusion.  


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As with any antibody, allergic reactions to dose administration are possible. Therefore, 
appropriate drugs and medical equipment to treat acute anaphylactic reactions must be 
immediately available, and study personnel must be trained to recognize and treat anaphylaxis, 
as per local institutional guidelines. 
3.3 
DOSING DELAYS 
Table 1  
Condition  
Management  
Onset of any toxicity  
 
Rule out alternative etiology  
 
NCI CTCAE Grade 1  
 
Provide symptomatic treatment  
Possible topical steroids if applicable  
 
NCI CTCAE Grade 2  
 
Provide symptomatic treatment  
In the case of an immune-mediated adverse event do 
not give scheduled dose; dosing may be resumed once 
symptoms are resolved.  
Consider oral or IV steroids at the onset of symptoms. 
Taper steroid if symptoms improve.  
NCI CTCAE Grade 3  
 Start high-dose IV steroids at the onset of the 
symptoms  
 Provide symptomatic treatment  
 
 
NCI CTCAE Grade 4  
Start high-dose IV steroids at the onset of the 
symptoms  
Provide symptomatic treatment  
Permanent discontinuation of AMP-224 for all NCI 
CTCAE Grade 4 events (unless specific exemption 
stated elsewhere in protocol) 
 
ALT = alanine transaminase; AST = aspartate transaminase; GI = gastrointestinal; IV = intravenous; NCI CTCAE = 
National Cancer Institute Common Terminology Criteria for Adverse Events.  
a Subjects will not receive any subsequent dose, but will remain on study and follow the other procedures required 
from the study (e.g., follow-up procedures, disease assessment scans, blood sample collections).  
 
During the study, subjects may require immunosuppressive medications such as steroids for 
management of underlying disease, treatment-related toxicity, or unrelated conditions. If 
symptoms resolved to NCI CTCAE Grade ≤ 1, AMP-224 dosing may be resumed during steroid 
taper as long as dose has been reduced to 10mg oral prednisone per day. Subjects with adrenal 


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insufficiency may take daily prednisone or equivalent therapy for their endocrinopathy while 
receiving AMP-224 treatment. Topical and inhaled steroids in standard doses are allowed.  
3.3.1 Dosing Delays/Dose Modifications: General Guidelines  
3.3.1.1 Grade 1 or 2 Toxicity  
Treatment with AMP-224 and cyclophosphamide need not be interrupted, although for chronic 
low grade toxicity causing significant detrimental effect in patient’s well-being the PI may elect 
to delay/omit dosing at his discretion, in particular in the case of a suspected immune-mediated 
adverse event.   
3.3.1.2 Grade 3 Hematologic or Non-Hematologic Toxicity  
Hold AMP-224 and cyclophosphamide and re-evaluate until toxicity improves to ≤ grade 1 or 
pre-treatment baseline within a maximum of 28 days.  Treatment will be discontinued in patients 
who experience grade 3 non-hematologic toxicities felt to be drug-related unless excepted as per 
3.1.2.2. Upon recommencement of AMP-224 patients will continue dosing as at the point of 
discontinuation. 
3.3.1.3 Grade 4 Non-Hematologic Toxicity  
Patients with clinical treatment-related grade 4 non-hematologic toxicity (except pulmonary 
embolism without significant hypoxia and hemodynamic instability) will be taken off treatment 
permanently. Unacceptable toxicities that have not resolved at time of “off treatment” must be 
followed until stabilization or resolution, at which time they will continue in follow up for 
survival.  
3.4 
SPECIFIC PROCEDURES FOR SBRT 
3.4.1 Radiotherapy administration 
3.4.1.1 Modality, Fractionation, and total dose   
Radiation will be delivered with megavoltage external beam radiation with beam energies of 
6MV or higher to one liver lesion. For DL1 treatment will be delivered in 8Gy as a single 
fraction. For DL2 treatment will be delivered in 8 Gy fractions for 3 days.  
3.4.1.2  Simulation 
Patients will be simulated supine with the addition of a 4D CT if appropriate.  A stereotactic 
immobilization device with abdominal compression will be used.  Oral contrast will be delivered 
approximately one hour prior to simulation to allow opacification of small bowel unless 
contraindicated.  IV contrast may be delivered for the simulation if deemed necessary by the 
treating radiation oncologist.   
3.4.1.3 Volume definitions  
GTV:    The gross tumor volume (GTV) will be defined as all gross disease evident on imaging 
and examination at the site of treatment (liver lesion). The GTV will be 6 cm max dimension 
based on clinical information. There will be no CTV margin. 
PTV:    The planning target volume will be a 3-5 mm concentric expansion on the GTV.  An 
additional margin of up to 3 mm may be added as needed if 4D CT reveals extensive respiratory 


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motion of the target. The PTV will be an institution and patient specific margin that will not 
exceed 1 cm. It is recommended that the patient is stimulated and treated with methods to reduce 
or account for organ motion, such as active breathing control or 4 dimensional CT. The PTV 
margin will be at a minimum 3 mm. 
3.4.1.4 Dose specification 
The PTV doses should meet the following criteria: 
1) 93% of the PTV should receive at least 93% of the prescribed dose 
2) < 5% of the PTV should receive more than 110% of the prescribed dose. Efforts will be 
made to reduce heterogeneity if possible. 
Normal Structures The following dose goals will apply for normal tissues: 
− Kidney:  mean dose < 10 Gy (total kidney volume) 
− Small bowel:  maximum 35 Gy, mean <25Gy, V30< 5cc, V35Gy <1cc 
− Duodenum:  maximum 35Gy, mean <25Gy, V30< 5cc, V35Gy <1cc 
− Stomach:  35 Gy, mean <25Gy, V30< 5cc, V35Gy <1cc 
− Large bowel: maximum 35Gy, mean <25Gy, V30< 5cc, V35Gy <1cc 
− Liver: No more than 700 cc of uninvolved liver will receive < 15Gy 
− Spinal cord: maximum 20Gy 
Threshold dose and Max point dose for 1 and 3 fractions will be as follows: 
 
Organ 
One fraction 
Three fractions 
Threshold 
dose (Gy) 
Max point 
dose (Gy) 
Threshold 
dose (Gy) 
Max point 
dose (Gy) 
Ribs 
22 
30 
28.8 (9.6 
Gy/fx) 
36.9 (12.3 
Gy/fx) 
Heart 
16 
22 
24 (8 Gy/fx) 
30 (10 
Gy/fx) 
 
3.4.1.5 Daily treatment delivery 
Treatment will be delivered Monday to Wednesday or Tuesday to Thursday.  In the event that a 
treatment is postponed due to machine malfunction or a federal holiday, the missed treatment 
will be delivered the following Monday. 
Localization will be verified with pretreatment imaging prior to every fraction.  Ideally, this will 
include Tomotherapy localization or cone beam CT, although kV/kV imaging may be used if 
necessary.


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3.5 
STUDY CALENDAR 
3.5.1 Screening/Baseline Calendar 
 
Screening 
Baseline 
Procedure / Study Day 
Within 
28 days of SBRT 
Within 
16 days 
Within 
7 days 
Within 
72 hours 
Physical / Clinical 
 
 
 
 
Written informed consent 
X 
 
 
 
Confirm inclusion / exclusion criteria 
X 
 
X 
 
Demographics, Medical history, cancer history 
X 
 
 
 
Vital signs (T, BP, HR, RR) 
X 
 
X 
 
Physical examination, height, body weight 
X 
 
X 
 
ECOG Performance Status 
X 
 
 
X 
12 lead ECG 
X 
 
 
 
Record concomitant medicationsa 
 
 
 
X 
Disease Assessments 
X 
 
 
 
Histological confirmation of disease 
X 
 
 
 
Tumor Marker profile (CEA) 
 
 
X 
 
CT of chest, abdomen and pelvis OR MRI if clinically indicated  
(same imaging technique must be used throughout study) 
X 
 
 
 
Laboratory Assessments 
X 
 
 
 
CBC/Automated differential/Platelets  
X 
 
 
X 
Coagulation (PT, INR, aPTT, fibrinogen) 
 
 
 
X 
HLA subtype 
x 
 
 
 


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Screening 
Baseline 
Procedure / Study Day 
Within 
28 days of SBRT 
Within 
16 days 
Within 
7 days 
Within 
72 hours 
Biochemical Panel  
Xb 
 
 
X 
Fasting Lipid Panel 
X 
 
 
 
LDH 
 
 
 
X 
C-Reactive Protein 
 
 
 
X 
Thyroid paneld 
X 
 
 
 
Serum pregnancy testf 
 
 
 
X 
Urine Pregnancy test 
 
 
 
X 
Follicle stimulating hormone (FSH)g 
X 
 
 
 
ACTH and morning cortisol 
X 
 
 
 
Viral testinge 
X 
 
 
 
Urinalysis 
X 
 
 
 
Immune Monitoring 
 
 
 
X 
ANA 
 
 
 
X 
AMA and liver microsomal Ab 
 
 
 
X 
Tumor specific T cell responses 
 
 
 
X 
Plasma PD samples  
 
 
 
X 
Tumor biopsy (FFPE + frozen )  
 
X 
 
 
a Document all medications taken within 28 days prior to entry into the study 
b Includes: Acute, Hepatic, Mineral, Uric acid, amylase 
d Thyroid panel includes TSH, T3, Free T4. 
e Viral testing includes HepB SA, HepC Ab (reflex HepCRNA),  
f Serum pregnancy testing obtained from females of child bearing potential only. 
g FSH obtained from females who are post-menopausal for ≥1 year. 


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3.5.2 On Treatment Calendar 


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Radiation 
AMP-224 Treatment 
Procedure / Study Day 
-2 
-1 
0 
1 
1 
8h 
15 
15 
29 
29 
43 
43 
57 
57 
71 
71 
Time point 
 
 
 
Pre 
EOI 
 
Pre 
EOI 
Pre 
EOI 
Pre 
EOI 
Pre 
EOI 
Pre 
EOI 
Window 
 
 
 
 
±1 
ming 
±1 
day 
±2 
day 
 
±2 
day 
±1 
min 
±2 
day 
±1 
min 
±2 
day 
±1 
min 
±2 
day 
±1 
min 
Physical / Clinical 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Vital signsa 
 
 
 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Physical examination 
Xb 
 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Weight 
 
 
 
X 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
Abbreviated symptom-
drive physical exam 
 
 
 
 
 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
ECOG Performance 
Status 
 
 
 
X 
 
 
 
 
 
 
 
 
X 
 
 
 
12 lead ECG 
 
 
 
X 
X 
X 
 
 
 
 
 
 
 
 
 
 
Record adverse eventsc 
Xb Xb 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Record concomitant 
medications 
Xb Xb 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Investigational Agent 
Dosing 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Pre-Medications 
 
 
 
X 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
Cyclophosphamide 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
AMP-224 
 
 
 
X 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
SBRT (DL1 – Day 0) 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
SBRT (DL2 – Days -2-
0) 
X 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Radiation 
AMP-224 Treatment 
Procedure / Study Day 
-2 
-1 
0 
1 
1 
8h 
15 
15 
29 
29 
43 
43 
57 
57 
71 
71 
Time point 
 
 
 
Pre 
EOI 
 
Pre 
EOI 
Pre 
EOI 
Pre 
EOI 
Pre 
EOI 
Pre 
EOI 
Window 
 
 
 
 
±1 
ming 
±1 
day 
±2 
day 
 
±2 
day 
±1 
min 
±2 
day 
±1 
min 
±2 
day 
±1 
min 
±2 
day 
±1 
min 
Local Laboratory 
Assessments 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CBC/Automated 
differential/Plateletse  
Xb 
 
 
 
 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
Biochemical Panele 
Xb 
 
 
 
 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
Coagulation (PT, INR, 
PTT 
Xb 
 
 
 
 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
Thyroid panel (TSH, 
Free T4) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Liver /Kidney 
microsomal antibody 
panel, ANA, AMA 
 
 
 
X 
 
 
 
 
X 
 
 
 
X 
 
 
 
LDH 
 
 
 
X 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
C-Reactive Protein 
 
 
 
X 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
CEA 
 
 
 
X 
 
 
 
 
X 
 
 
 
X 
 
 
 
Urine pregnancy testf 
 
 
 
X 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
Urinalysis 
 
 
 
X 
 
 
 
 
X 
 
 
 
X 
 
 
 
Research Laboratory 
Assessments 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Pharmacokinetic 
samples 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 


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Radiation 
AMP-224 Treatment 
Procedure / Study Day 
-2 
-1 
0 
1 
1 
8h 
15 
15 
29 
29 
43 
43 
57 
57 
71 
71 
Time point 
 
 
 
Pre 
EOI 
 
Pre 
EOI 
Pre 
EOI 
Pre 
EOI 
Pre 
EOI 
Pre 
EOI 
Window 
 
 
 
 
±1 
ming 
±1 
day 
±2 
day 
 
±2 
day 
±1 
min 
±2 
day 
±1 
min 
±2 
day 
±1 
min 
±2 
day 
±1 
min 
Immunogenicity 
samples 
 
 
 
X 
 
 
 
 
X 
 
 
 
X 
 
 
 
Circulating PD samples 
(plasma) 
 
 
 
X 
 
 
 
 
X 
 
 
 
X 
 
 
 
Immune monitoring 
 
 
 
X 
 
X 
 
 
X 
 
 
 
X 
 
 
 
Tumor biopsy (FFPE + 
Frozen) Biopsy to be 
taken from same 
baseline lesion if 
accessible.   
 
 
 
 
 
 
 
 
Xj 
 
 
 
 
 
 
 
a Vital signs (VS) to be obtained within 30 min prior to infusion & every 30  min during infusion, at the end of infusion, and at 30 min and 1 hr 
post infusion.  If infusion reaction occurs, obtain VS a minimum of every 15 min until symptoms resolve. 
b For subjects enrolled in Dose Level 2  
Weight obtained within 3 days prior to Day 1 & will be used to calculate AMP-224 dose. 
c Adverse event collection begins at the start of  the administration of radiotherapy 
d Same method of disease assessment (CT or MRI) must be used consistently throughout the study. Patients will continue to undergo restaging 
exam every 8 weeks following completion of treatment to assess for PFS. 
e Acute, hepatic and mineral panels, uric acid and amylase. If laboratory tests done within 72 hours of Cycle 1 Day 1 they do not need to be 
repeated on Day 1. For all labs a ‘+/- 24hr’ window applies, with the exception of those needed to determine proceeding with treatment. Labs may 
be performed outside of NIH. Results must be reviewed prior to infusion.  
f Urine pregnancy testing obtained from females of child bearing potential only. 
g+or -1 minute is for guidance only. Leeway is permitted up to 20-30 minutes.  
hthe day 8 visit per PI discretion depending upon logistical issues. 
i This may be a phone call assessment. 
jThe allowed window for tumor biopsy will be +/- 7 days 


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3.5.3 Follow-up Calendar 


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Follow-up 
Procedure / Study Day 
79 
85 
99 
112 
141 
197  
253 
Timepoint 
 
 
 
 
 
 
 
Window 
±2 day 
±2 day 
±4 day 
±4 day 
±4 day 
±7 day 
 
Physical / Clinical 
 
 
 
 
 
 
 
Vital signsa 
 
X 
 
 
 
 
X 
Physical examination 
 
 
 
X 
 
 
X 
Weight 
 
 
 
 
 
 
 
Abbreviated symptom-drive physical exam 
 
X 
X 
 
Xd 
X 
 
ECOG Performance Status 
 
 
 
X 
X 
X 
X 
12 lead ECG 
 
 
 
 
 
 
 
Record adverse eventsb 
X 
X 
X 
X 
X 
X 
X 
Record concomitant medications 
X 
X 
X 
X 
X 
X 
X 
Disease Assessments 
 
 
 
 
 
 
 
Radiologic evaluations (re-staging)e 
 
X 
 
 
X 
X 
X 
Response Assessmentse 
 
X 
 
X 
 
X 
X 
Survival 
 
 
 
 
 
X 
X 
Local Laboratory Assessments 
 
 
 
 
 
 
 
CBC/Automated differential/Plateletsc 
 
 
 
X 
 
 
X 
Biochemical Panelc 
 
 
 
 
 
 
X 
Thyroid panel (TSH, Free T4) 
 
X 
 
 
 
 
 
Liver /Kidney microsomal antibody panel, ANA, AMA 
 
X 
 
 
 
 
 
CEA 
 
X 
 
 
 
 
 


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Follow-up 
Procedure / Study Day 
79 
85 
99 
112 
141 
197  
253 
Central Laboratory Assessments 
 
 
 
 
 
 
 
Pharmacokinetic samples 
X 
X 
X 
X 
 
X 
 
Immunogenicity samples 
 
X 
 
X 
 
X 
 
Circulating PD samples (plasma) 
 
X 
 
 
 
 
 
Immune monitoring 
 
X 
 
 
 
 
 
aVital signs (VS) to be obtained within 30 min prior to infusion & every 30  min during infusion, at the end of infusion, and at 30 min and 1 hr 
post infusion.  If infusion reaction occurs, obtain VS a minimum of every 15 min until symptoms resolve. 
b Adverse event collection begins at the start of  the administration of radiotherapy 
cAcute, hepatic and mineral panels, uric acid and amylase. If laboratory tests done within 72 hours of Cycle 1 Day 1 they do not need to be 
repeated on Day 1. For all labs a ‘+/- 24hr’ window applies, with the exception of those needed to determine proceeding with treatment. Labs may 
be performed outside of NIH. Results must be reviewed prior to infusion.  
d This may be a phone call assessment.e These visits are also at the discretion of the PI  depending upon logistical issues 
eRadiologic assessments/restaging will be performed initially on Day 85 (+/-72hrs) ie post 6 doses of AMP-224 and then q8-weekly subsequently.


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3.6 
CRITERIA FOR REMOVAL FROM PROTOCOL THERAPY AND OFF STUDY CRITERIA 
3.6.1 Criteria for removal from protocol therapy 
In the absence of treatment delays due to adverse events, treatment may continue until one of the 
following criteria applies: 
• Progressive Disease (per immune-related response criteria). 
• Intercurrent illness that prevents further administration of treatment 
• Unacceptable Toxicity  
• Patient decides to withdraw from active therapy 
• Investigator discretion 
• Delayed recovery from toxicity that prevents re-treatment in ≤ 28 days of scheduled 
therapy  
• Completion of protocol therapy including a 30 day safety visit 
3.6.2 Criteria for Removal from Study 
• Completed study follow-up period 
• Patient requests to be withdrawn from study 
• Death 
3.6.3 Off-Study Procedure 
Authorized staff must notify Central Registration Office (CRO) when a subject is taken off-
study. An off-study form from the web site 
(http://home.ccr.cancer.gov/intra/eligibility/welcome.htm) main page must be completed and 
sent via encrypted email to:  NCI Central Registration Office (HOIS) ncicentralregistration-
l@mail.nih.gov. 
4 
CONCOMITANT MEDICATIONS/MEASURES 
All routine and appropriate supportive care (including blood products) will be provided during 
this study, as clinically indicated, and in accordance with the standard of care practices.  Clinical 
judgment should be utilized in the treatment of any AE experienced by the patient. 
Information on all concomitant medications, administered blood products, as well as 
interventions occurring during the study must be recorded on the patient’s eCRF.   


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5 
BIOSPECIMEN COLLECTION 
5.1 
CORRELATIVE STUDIES FOR RESEARCH/PHARMACOKINETIC STUDIES  
The correlative studies which we wish to perform are outlined below and summarized in the 
table. A description of each test including a brief statement of rationale and processing 
information is made below. 
Table 2 
Test/assay 
Volume blood 
(approx) 
Type of 
tube 
Collection point 
(+/- 48hrs) 
Location of 
specimen analysis 
Immune- 
monitoring 
100mls 
(baseline) 
60mls (D1), 
80mls (D8), 
120mls (D29, 
57) (for PBMC) 
EDTA 
Baseline, D1, 8, 
29, D57  
Greten Lab (Deliver 
to Dr. Figg’s lab) 
5-10mls (for 
serum) 
EDTA 
PK (pre & post 
with all six AMP-
224 doses) 
3mls 
SST 
D1 8, 15, 29, 43, 
57, 71 (D79, 85, 
99, 112, 169, 
Final Visit 
Dr. Figg’s lab 
Immunogenicity 
4mls 
SST 
D1 (pre-
infusion), D29 
(pre-infusion), 
D57 (pre-
infusion), D79, 
D85  
90-days post last 
dose (D169) 
Dr. Figg’s Lab 
ANA  
4mls 
SST 
Baseline and 
Day 85 
CC Department of 
Laboratory Medicine 
(DLM) 
AMA & 
Liver/kidney 
microsomal 
antibody 
4 mL 
SST 
Baseline and 
Day 85 
CC DLM  will send 
to Mayo Labs 


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Tumor-specific 
responses  
20mls 
EDTA 
Baseline, D1, 8, 
29, D57 
Greten Lab (Dr. 
Figg’s lab) 
Tumor biopsy 
(All patients) 
NA 
NA 
Baseline and 
D29 +/- 7 days 
Pathology/ Greten 
Lab/Amplimmune 
5.1.1 Immune monitoring 
Immune monitoring will be performed using peripheral blood mononuclear cells (PBMC) for 
immunophenotyping of relevant cell populations including myeloid-derived suppressor cells, 
CD4 and CD8 T cell subsets, T regulatory cells. Patients will undergo blood sampling (c.60-
120mls blood) on the time points outlined in the table (+/- 24hrs). Blood will initially be sent to 
the Figg laboratory for barcoding and processing. On certain occasions the blood may also be 
brought to the Greten lab for processing and analysis. Contact Dr. Figg’s Blood Processing Core 
(BPC) in 10/5A09 at 301-402-3622 or 301-594-6131 with any questions. PBMC will be isolated 
by Ficoll density centrifugation. Aliquots of 1 x 107 PBMC/tube will be individually frozen – 
after initial handling and processing at the Figg laboratory. 
5.1.2 Liver autoantibody panel for autoimmune hepatitis (AIH) 
AIH is a chronic disorder characterized by progressive hepatocellular loss and cell-mediated 
immunologic attack. Histologic inflammation is present and is usually accompanied by fibrosis, 
which can progress to cirrhosis and liver failure. AIH accounts for 11% to 23% of chronic liver 
disease in North America and about 6% of liver transplants in the United States.  
The AIH Diagnostic Panel includes tests for actin (smooth muscle) antibody, antinuclear 
antibodies (ANAs), and liver/kidney microsome antibody (LKM-1). ANAs and actin antibody 
are associated with type 1 AIH, the most common form in the United States, while LKM-1 
antibody is associated with type 2 AIH, more commonly found in Europe and in some South 
American countries. The panel also includes mitochondrial antibody, which can help 
differentiate AIH from PBC. Given that autoimmune hepatitis is a potential complication of 
immune checkpoint inhibition we will perform this panel of autoimmune antibodies to 
investigate this. Samples will be collected at baseline and on D85 approximately. These antibody 
titers are exploratory and will not be used to guide therapy in the absence of clinical correlation. 
5.1.3 Tumor specific responses 
Two oncofetal proteins that are expressed by most of the colorectal cancers are Survivin and 
CEA. The most validated antigenic epitopes for both of these proteins are HLA-A*0201 
restricted and therefore these experiment will be performed in HLA-A*0201-positive subjects. In 
brief, PBMCs will be suspended in medium cultured in duplicate and will be stained with CFSE 
according to manufacturer’s recommendations. Then PBMCs will be cultured with either CEA 
peptide (YLSGANLNL, also known as CAP-1, 52) or Survivin peptide (LMLGEFLKL, also 
known as Sur1M2, 53) or an irrelevant peptide as a negative control (SIINFEKL). To stimulate 
only the CD4 and CD8 T cells that are responsive to the peptide, nanoparticles coated with anti-
CD3 will be used. This method is based on a recent publication in which antigen binding to T 


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cell receptor (TCR) was shown to cause clustering of TCRs that can be recognized by anti-CD3 
that is on a nanoparticle resulting in stimulation of specifically the antigen responsive T cells 54.  
To assess whether a tumor specific response was generated, DCs will be isolated from PBMCs 
using CD14 magnetic beads. Isolated CD14+ cells will be incubated for 5 days and on day 2 rh 
GM-CSF and rh IL-4 will be added to generate immature DCs. On day 5 DCs will be pulsed 
with tumor lysates prepared as explained in section 5.1.4 section 2 at final concentration of 100 
mcg/mL for 12 hours. After washing, cells will be reincubated with rh GM-CSF and TNF-alpha 
to promote DC maturation. To assay T-cell activation, mature DC cells will then be coincubated 
for 3 days with CFSE labeled PBMCs collected on the specified days mentioned in Table 2.  
5.1.4 Mandatory tumor biopsy 
Tumor biopsies will be collected at baseline and day 29+/- 7 days by the Interventional 
Radiology team by a percutaneous approach.  It is preferred that at least two core biopsies ≥18 
gauge in diameter and ≥1 cm in length will be obtained.  
If a site is deemed appropriate for biopsy with minimal risk to the participant by agreement 
between the investigators and Interventional Radiology team, an attempt for biopsy will be 
made. The use of imaging to facilitate biopsies will be decided by members of the Interventional 
Radiology team and may include ultrasound, CT scan, PET scan or MRI. Should a CT scan be 
needed for biopsy, the number of scans for each procedure will be limited to the minimum 
number needed to safely obtain a biopsy. Tumor biopsies and local anesthesia will be 
administered only if they are considered to be of low risk to the participant, as determined by the 
investigators and Interventional Radiology. If an initial attempt at percutaneous biopsy is 
unsuccessful, the participant will be given an option to proceed with a repeated attempt at 
percutaneous biopsy.  
5.1.4.1 Orders and documentation which have to be put in CRIS on the day prior to the 
procedure: 
- 
If an in-patient, full H+P needed. 
- 
Order for non-GYN cytopathology. 
- 
Order for anatomical pathology (Surgical path). 
- 
Order for biopsy procedure under Interventional Radiology menu. 
- 
Note: If patient is on narcotics will need anesthesia consult. 
5.1.4.2 Solid tumor biopsies processing 
Two core biopsies will be attempted in two parts; one for Surgical Pathology and one will be 
split for FFPE, frozen preservation and RNA analysis. If for some reason two cores cannot be 
obtained then the core will be divided into three pieces. These biopsies will be performed using 
ultrasound guidance or CT guided as determined by the radiologist. 
1) Formalin-fixed. 
i. 
The half fixed in 10% formalin will be submitted to Surgical Pathology, 
CCR/NCI (Bldg 10, 2N212).  
ii. 
The specimens will have routine H&E stains made as well as 5 additional 
unstained sections.  


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The unstained sections will be analyzed for phenotyping the immune infiltrates. The tissue 
sections then will be counterstained with hematoxylin. 
2) Frozen-preservation 
i. 
1.5 ml cryogenic vials (obtained from Greten lab) will be labeled with the patients 
name, accession number (HP#) and date using a waterproof sharpie.  
ii. 
The isotherm flask (Greten lab) will be filled with liquid nitrogen on the morning 
of the procedure and will be available together with the cryogenic vials for pick 
up from there when radiology page the contact person to collect the specimens. 
iii. 
Once the biopsy is ready, the core to be cryopreserved will be transferred into an 
empty 1.5-mL cryogenic vial with the use of sterile, pre-chilled (in dry ice) 
disposable tweezers. 
iv. 
The vial with specimen will be immediately dropped into liquid nitrogen 
contained in an isotherm flask.  
v. 
The frozen half will be transferred in the isotherm flask to the protocol-specified 
location for that particular analysis.  
vi. 
The samples will be minced with a scalped under laminar flow conditions 
followed by dissolving in PBS. The samples will then be lysed by five freeze 
thaw cycles. The lysates will be centrifuged 1000 g and supernatant will be 
filtered using 0.45 micrometer pore-size filters. The protein concentration will be 
measured using a validated standard assay. 
        3)  RNA analysis 
i. 
1.5 ml cryogenic vials (obtained from Greten lab) will be labeled with the patients 
name, accession number (HP#) and date using a waterproof sharpie. 
ii. 
Less than 0.5 cm of the tissue to be cryopreserved will be cut using aseptic 
technique. 
iii. 
The cut tissue will be transferred into an empty 1.5-mL cryogenic vial with the 
use of sterile, pre-chilled (in dry ice) disposable tweezers. 
iv. 
1 mL if RNA later (Ambion, Life Technologies) will be added to vial and the 
specimen will be placed into ice and transferred to the protocol-specified location. 
RNA will extracted and sent to Cell Processing Section at DTM for microarray analysis using a 
standard Affymetrix array. Furthermore, based on the correlation between expression of genes 
involved in inflammation, TH1 immunity and immune suppression and survival of patients with 
colorectal cancer 11, we will perform quantative real time PCR for the genes listed in the table 
below. 
Gene 
Encoded protein 
Acc. Number 
IL10 
IL8 
IFNG  
interleukin 10 
interleukin 8 
interferon, gamma IFN 
NM_000572 
NM_000584 
NM_000619 


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TGFB1  
PTGS2  
CEACAM1  
IRF1 
MMP7  
VEGF  
GZMB  
TBX21  
B7H3 
CD8A 
GNLY  
BIRC5  
CD3Z  
TNFRSF10A  
CD4 
transforming growth factor, beta 1  
prostaglandin-endoperoxide synthase 2 (Cox2)  
carcinoembryonic antigen-related cell adhesion 
molecule 1  
interferon regulatory factor 1 
matrix metalloproteinase 7(matrilysin, uterine)  
vascular endothelial growth factor 
granzyme B 
T-box 21 (T-bet) 
B7 homolog 3 
CD8 antigen, alpha polypeptide (p32) 
granulysin 
baculoviral IAP repeat-containing 5 (survivin)   
CD3Z antigen, zeta polypeptide (TiT3 complex)  
tumor necrosis factor receptor superfamily, 10a 
(TRAIL-R)  
CD4 antigen (p55) 
NM_000660 
NM_000963 
NM_001712 
NM_002198 
NM_002423 
NM_003376 
NM_004131 
NM_013351 
NM_025240 
NM_001768 
NM_006433 
NM_001168 
NM_000734 
NM_003844 
NM_000616 
5.1.5 Optional Tumor Biopsy 
If a tumor outside the radiation area meets the response criteria discussed in section 6.2, an 
optional biopsy may be done. The sample will be processed as explained in section 5.1.4. 
5.1.6 Immunogenicity studies 
At each time point outlined in the schedule of assessments, a 5 mL blood sample will be 
collected to assess immunogenicity (Human Anti-Murine Antibodies/HAMA and Human Anti-
Chimeric Antibodies/HACA).  Immunogenicity studies (both HAMA and HACA) will be 
collected at baseline and D1 (pre-infusion), D29 (pre-infusion), D57 (pre-infusion), D79, D85 
and 90-days post last dose (D169).  The date and exact time of each blood draw should be 
recorded on the blood tubes. Sera will be separated and stored at -70°C.  Samples will be batch 
shipped every 3 to 6 months to 3rd party laboratory for analysis.  HAMA and HACA 
concentrations will be measured using validated ELISA methods and evaluated in the context of 
pharmacokinetic parameters and adverse event profiles.   


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5.1.7 Pharmacokinetic studies 
The data from theAMP-224 Phase 1 study indicates that following IV infusion, median Tmax 
values ranged from 0.533 to 12.7 hours.  After reaching Cmax, serum concentrations of AMP-224 
declined slowly in a biphasic manner, with mean elimination half-life values ranging from 129 to 
160 hours across all dose levels. Further evaluation of the terminal elimination half-life and AUC 
calculations will be performed in this study following a fixed number of AMP-224 doses to 
support the NCA analysis. 
5.2 
SAMPLE STORAGE, TRACKING AND DISPOSITION 
Samples will be ordered and tracked through the CRIS Screens.  Should a CRIS screen not be 
available, the NIH form 2803-1 will be completed and will accompany the specimen and be filed 
in the medical record.  All samples will be sent to Dr. Figg’s lab for processing and storage until 
they are distributed to Dr. Greten’s lab for sample analysis as described in the protocol.   
All specimens obtained in the protocol are used as defined in the protocol. Any specimens that 
are remaining at the completion of the protocol will be stored in the conditions described below. 
The study will remain open so long as sample or data analysis continues.  Samples from 
consenting subjects will be stored until they are no longer of scientific value or if a subject 
withdraws consent for their continued use, at which time they will be destroyed. The PI will 
report any loss or destruction of samples to the NCI IRB as soon as he is made aware of such 
loss. 
If the patient withdraws consent the participants data will be excluded from future distributions, 
but data that have already been distributed for approved research use will not be able to be 
retrieved. 
5.2.1 Procedures for sample data collection for the Clinical Pharmacology Program: 
• All samples sent to the Clinical Pharmacology Program (CPP) will be barcoded, with 
data entered and stored in the Patient Sample Data Management System (PSDMS) 
utilized by the CPP. This is a secure program, with access to PSDMS limited to defined 
CPP personnel, who are issued individual user accounts. Installation of PSDMS is limited 
to computers specified by Dr. Figg. These computers all have a password restricted login 
screen. All CPP personnel with access to patient information annually complete the NIH 
online Protection of Human Subjects course.  
• PSDMS creates a unique barcode ID for every sample and sample box, which cannot be 
traced back to patients without PSDMS access. The data recorded for each sample 
includes the patient ID, name, trial name/protocol number, time drawn, cycle time point, 
dose, material type, as well as box and freezer location. Patient demographics associated 
with the clinical center patient number are provided in the system. For each sample, there 
are notes associated with the processing method (delay in sample processing, storage 
conditions on the ward, etc.). 
5.2.2 Procedures for sample storage at the Clinical Pharmacology Program: 
• Barcoded samples are stored in barcoded boxes in a locked freezer at either -80ºC or 
liquid nitrogen according to stability requirements.  These freezers are located onsite in 
the CPP and offsite at NCI Frederick Central Repository Services (Fisher Bioservices) in 


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Frederick, MD. Visitors to the laboratory are required to be accompanied by laboratory 
staff at all times.  
• Access to stored clinical samples is restricted. Samples will be stored until requested by a 
researcher named on the protocol. All requests are monitored and tracked in PSDMS. All 
researchers are required to sign a form stating that the samples are only to be used for 
research purposes associated with this trial (as per the IRB approved protocol) and that 
any unused samples must be returned to the CPP. It is the responsibility of the NCI 
Principal Investigator to ensure that the samples requested are being used in a manner 
consistent with IRB approval. 
• Following completion of this study, samples will remain in storage as detailed above. 
Access to these samples will only be granted following IRB approval of an additional 
protocol, granting the rights to use the material.  
• If, at any time, a patient withdraws from the study and does not wish for their existing 
samples to be utilized, the individual must provide a written request. Following receipt of 
this request, the samples will be destroyed (or returned to the patient, if so requested), and 
reported as such to the IRB. Any samples lost (in transit or by a researcher) or destroyed 
due to unknown sample integrity (i.e. broken freezer allows for extensive sample 
thawing, etc.) will be reported as such to the IRB. 
• Sample barcodes are linked to patient demographics and limited clinical information. 
This information will only be provided to investigators listed on this protocol, via 
registered use of the PSDMS. It is critical that the sample remains linked to patient 
information such as race, age, dates of diagnosis and death, and histological information 
about the tumor, in order to correlate genotype with these variables. 
5.2.3 Protocol Completion/Sample Destruction 
• Following completion of this study, samples will remain in storage as detailed above only 
for those patients that agreed to future use in the Optional Studies section of the consent 
form.  Access to these samples will only be granted following IRB approval of an 
additional protocol, granting the rights to use the material.  Currently, there is no plan to 
use these samples outside of the use described in the protocol.  
• The PI will report destroyed samples to the IRB if samples become unsalvageable 
because of environmental factors (ex. broken freezer or lack of dry ice in a shipping 
container) or if a patient withdraws consent. Samples will also be reported as lost if they 
are lost in transit between facilities or misplaced by a researcher.  Freezer problems, lost 
samples or other problems associated with samples will also be reported to the IRB, the 
NCI Clinical Director, and the office of the CCR, NCI. 
6 
DATA COLLECTION AND EVALUATION  
6.1 
DATA COLLECTION 
The NCI investigators will be responsible for the collection, maintenance, and quality control of 
the study data. Clinical data will be entered into the NCI C3D electronic database at least once 
every two weeks when patients are enrolled on the trial. Protocol-specific eCRFs will be 
developed for this trial in C3D. All data will be kept secure. Personal identifiers will not be used 
when collecting and storing data. An enrollment log will be maintained in the regulatory 


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binder/file which is the only location of personal identifiers with unique subject identification 
number. 
6.2 
RESPONSE CRITERIA 
For the purposes of this study, patients should be re-evaluated for response every 8 weeks.  In 
addition to a baseline scan, confirmatory scans should also be obtained 4-8 weeks following 
initial documentation of objective response. 
Response and progression will be evaluated in this study using the new international criteria 
proposed by the revised Response Evaluation Criteria in Solid Tumors (RECIST) guideline 
(version 1.1) [Eur J Ca 45:228-247, 2009].  Changes in the largest diameter (unidimensional 
measurement) of the tumor lesions and the shortest diameter in the case of malignant lymph 
nodes are used in the RECIST criteria. 
6.2.1 Definitions 
Evaluable for toxicity:  All patients will be evaluable for toxicity from the time of their first 
treatment with SBRT 
1.1.1.1 Evaluable for objective response  
Only those patients who have measurable disease present at baseline, have received at least one 
dose of therapy, and have had their disease re-evaluated will be considered evaluable for 
response.  These patients will have their response classified according to the definitions stated 
below.  (Note:  Patients who exhibit objective disease progression prior to the end of cycle 1 will 
also be considered evaluable.) 
Evaluable Non-Target Disease Response:  Patients who have lesions present at baseline that are 
evaluable but do not meet the definitions of measurable disease, have received at least one cycle 
of therapy, and have had their disease re-evaluated will be considered evaluable for non-target 
disease.  The response assessment is based on the presence, absence, or unequivocal progression 
of the lesions.  
6.2.2 Disease Parameters 
Measurable disease: Measurable lesions are defined as those that can be accurately measured in 
at least one dimension (longest diameter to be recorded) as >20 mm by chest x-ray, as >10 mm 
with CT scan, or >10 mm with calipers by clinical exam.  All tumor measurements must be 
recorded in millimeters (or decimal fractions of centimeters). 
Malignant lymph nodes: To be considered pathologically enlarged and measurable, a lymph 
node must be >15 mm in short axis when assessed by CT scan (CT scan slice thickness 
recommended to be no greater than 5 mm).  At baseline and in follow-up, only the short axis will 
be measured and followed. 
Non-measurable disease: All other lesions (or sites of disease), including small lesions (longest 
diameter <10 mm or pathological lymph nodes with ≥10 to <15 mm short axis), are considered 
non-measurable disease.  Bone lesions, leptomeningeal disease, ascites, pleural/pericardial 
effusions, lymphangitis cutis/pulmonitis, inflammatory breast disease, and abdominal masses 
(not followed by CT or MRI), are considered as non-measurable. 


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Note:  Cystic lesions that meet the criteria for radiographically defined simple cysts should not 
be considered as malignant lesions (neither measurable nor non-measurable) since they are, by 
definition, simple cysts. 
‘Cystic lesions’ thought to represent cystic metastases can be considered as measurable lesions, 
if they meet the definition of measurability described above. However, if non-cystic lesions are 
present in the same patient, these are preferred for selection as target lesions. 
Target lesions: All measurable lesions up to a maximum of 2 lesions per organ and 5 lesions in 
total, representative of all involved organs, should be identified as target lesions and recorded 
and measured at baseline.  Target lesions should be selected on the basis of their size (lesions 
with the longest diameter), be representative of all involved organs, but in addition should be 
those that lend themselves to reproducible repeated measurements.  It may be the case that, on 
occasion, the largest lesion does not lend itself to reproducible measurement in which 
circumstance the next largest lesion which can be measured reproducibly should be selected.  A 
sum of the diameters (longest for non-nodal lesions, short axis for nodal lesions) for all target 
lesions will be calculated and reported as the baseline sum diameters.  If lymph nodes are to be 
included in the sum, then only the short axis is added into the sum.  The baseline sum diameters 
will be used as reference to further characterize any objective tumor regression in the measurable 
dimension of the disease. 
Non-target lesions: All other lesions (or sites of disease) including any measurable lesions over 
and above the 5 target lesions should be identified as non-target lesions and should also be 
recorded at baseline.  Measurements of these lesions are not required, but the presence, absence, 
or in rare cases unequivocal progression of each should be noted throughout follow-up.  
6.2.3 Methods for Evaluation of Measurable Disease 
All measurements should be taken and recorded in metric notation using a ruler or calipers.  All 
baseline evaluations should be performed as closely as possible to the beginning of treatment and 
never more than 4 weeks before the beginning of the treatment. 
The same method of assessment and the same technique should be used to characterize each 
identified and reported lesion at baseline and during follow-up. Imaging-based evaluation is 
preferred to evaluation by clinical examination unless the lesion(s) being followed cannot be 
imaged but are assessable by clinical exam. 
Clinical lesions: Clinical lesions will only be considered measurable when they are superficial 
(e.g., skin nodules and palpable lymph nodes) and ≥10 mm diameter as assessed using calipers 
(e.g., skin nodules).  In the case of skin lesions, documentation by color photography, including a 
ruler to estimate the size of the lesion, is recommended.  
Chest x-ray: Lesions on chest x-ray are acceptable as measurable lesions when they are clearly 
defined and surrounded by aerated lung.  However, CT is preferable.  
Conventional CT and MRI: This guideline has defined measurability of lesions on CT scan based 
on the assumption that CT slice thickness is 5 mm or less.  If CT scans have slice thickness 
greater than 5 mm, the minimum size for a measurable lesion should be twice the slice thickness.  
MRI is also acceptable in certain situations (e.g. for body scans).   


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Use of MRI remains a complex issue.  MRI has excellent contrast, spatial, and temporal 
resolution; however, there are many image acquisition variables involved in MRI, which greatly 
impact image quality, lesion conspicuity, and measurement.  Furthermore, the availability of 
MRI is variable globally.  As with CT, if an MRI is performed, the technical specifications of the 
scanning sequences used should be optimized for the evaluation of the type and site of disease.  
Furthermore, as with CT, the modality used at follow-up should be the same as was used at 
baseline and the lesions should be measured/assessed on the same pulse sequence.  It is beyond 
the scope of the RECIST guidelines to prescribe specific MRI pulse sequence parameters for all 
scanners, body parts, and diseases.  Ideally, the same type of scanner should be used and the 
image acquisition protocol should be followed as closely as possible to prior scans.  Body scans 
should be performed with breath-hold scanning techniques, if possible. 
Ultrasound:  Ultrasound is not useful in assessment of lesion size and should not be used as a 
method of measurement.  Ultrasound examinations cannot be reproduced in their entirety for 
independent review at a later date and, because they are operator dependent, it cannot be 
guaranteed that the same technique and measurements will be taken from one assessment to the 
next.  If new lesions are identified by ultrasound in the course of the study, confirmation by CT 
or MRI is advised.  If there is concern about radiation exposure at CT, MRI may be used instead 
of CT in selected instances. 
Endoscopy, Laparoscopy:  The utilization of these techniques for objective tumor evaluation is 
not advised.  However, such techniques may be useful to confirm complete pathological 
response when biopsies are obtained or to determine relapse in trials where recurrence following 
complete response (CR) or surgical resection is an endpoint. 
Cytology, Histology: These techniques can be used to differentiate between partial responses 
(PR) and complete responses (CR) in rare cases (e.g., residual lesions in tumor types, such as 
germ cell tumors, where known residual benign tumors can remain). 
The cytological confirmation of the neoplastic origin of any effusion that appears or worsens 
during treatment when the measurable tumor has met criteria for response or stable disease is 
mandatory to differentiate between response or stable disease (an effusion may be a side effect of 
the treatment) and progressive disease. 
6.2.4 Response Criteria 
6.2.4.1 Evaluation of Target Lesions 
Complete Response (CR): Disappearance of all target lesions.  Any pathological lymph nodes 
(whether target or non-target) must have reduction in short axis to <10 mm. 
Partial Response (PR): At least a 30% decrease in the sum of the diameters of target lesions, 
taking as reference the baseline sum of diameters. 
Progressive Disease (PD): At least a 20% increase in the sum of the diameters of target lesions, 
taking as reference the smallest sum on study (this includes the baseline sum if that is the 
smallest on study).  In addition to the relative increase of 20%, the sum must also demonstrate an 
absolute increase of at least 5 mm.  (Note:  the appearance of one or more new lesions is also 
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Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase to 
qualify for PD, taking as reference the smallest sum of diameters while on study. 
6.2.4.2 Evaluation of Non-Target Lesions 
Complete Response (CR): Disappearance of all non-target lesions and normalization of tumor 
marker level.  All lymph nodes must be non-pathological in size (<10 mm short axis). 
Note:  If tumor markers are initially above the upper normal limit, they must normalize for a 
patient to be considered in complete clinical response. 
Non-CR/Non-PD: Persistence of one or more non-target lesion(s) and/or maintenance of tumor 
marker level above the normal limits. 
Progressive Disease (PD): Appearance of one or more new lesions and/or unequivocal 
progression of existing non-target lesions.  Unequivocal progression should not normally trump 
target lesion status.  It must be representative of overall disease status change, not a single lesion 
increase.     
Although a clear progression of “non-target” lesions only is exceptional, the opinion of the 
treating physician should prevail in such circumstances, and the progression status should be 
confirmed at a later time by the review panel (or Principal Investigator). 
6.2.4.3 Evaluation of Best Overall Response 
The best overall response is the best response recorded from the start of the treatment until 
disease progression/recurrence (taking as reference for progressive disease the smallest 
measurements recorded since the treatment started).  The patient's best response assignment will 
depend on the achievement of both measurement and confirmation criteria. 
 
For Patients with Measurable Disease (i.e., Target Disease) 
Target 
Lesions 
Non-Target 
Lesions 
New 
Lesions 
Overall 
Response 
Best Overall Response when 
Confirmation is Required* 
CR 
CR 
No 
CR 
>4 wks. Confirmation** 
CR 
Non-
CR/Non-PD 
No 
PR 
>4 wks. Confirmation** 
CR 
Not 
evaluated 
No 
PR 
PR 
Non-
CR/Non-
PD/not 
evaluated 
No 
PR 
SD 
Non-
CR/Non-
PD/not 
evaluated 
No 
SD 
Documented at least once >4 
wks. from baseline** 
PD 
Any 
Yes or 
No 
PD 
no prior SD, PR or CR 


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Target 
Lesions 
Non-Target 
Lesions 
New 
Lesions 
Overall 
Response 
Best Overall Response when 
Confirmation is Required* 
Any 
PD*** 
Yes or 
No 
PD 
Any 
Any 
Yes 
PD 
* 
See RECIST 1.1 manuscript for further details. 
** 
Only for non-randomized trials with response as primary endpoint. 
*** 
In exceptional circumstances, unequivocal progression in non-target 
lesions may be accepted as disease progression. 
Note: Patients with a global deterioration of health status requiring 
discontinuation of treatment without objective evidence of disease 
progression at that time should be reported as “symptomatic 
deterioration.”  Every effort should be made to document the objective 
progression even after discontinuation of treatment. 
 
For Patients with Non-Measurable Disease (i.e., Non-Target Disease) 
Non-Target Lesions 
New Lesions 
Overall Response 
CR 
No 
CR 
Non-CR/non-PD 
No 
Non-CR/non-PD* 
Not all evaluated 
No 
not evaluated 
Unequivocal PD 
Yes or No 
PD 
Any 
Yes 
PD 
*  ‘Non-CR/non-PD’ is preferred over ‘stable disease’ for non-target disease 
since SD is increasingly used as an endpoint for assessment of efficacy in some 
trials so to assign this category when no lesions can be measured is not advised 
6.2.5 Duration of Response 
Duration of overall response:  The duration of overall response is measured from the time 
measurement criteria are met for CR or PR (whichever is first recorded) until the first date that 
recurrent or progressive disease is objectively documented (taking as reference for progressive 
disease the smallest measurements recorded since the treatment started). 
The duration of overall CR is measured from the time measurement criteria are first met for CR 
until the first date that progressive disease is objectively documented. 
 
Duration of stable disease: Stable disease is measured from the start of the treatment until the 
criteria for progression are met, taking as reference the smallest measurements recorded since the 
treatment started, including the baseline measurements.  
6.2.6 Modified immune-related response criteria (irRC) 
Modified immune-related response criteria (irRC) will also be employed in this study. This new 
classification is based on the recent learning from clinical studies with cancer immunotherapies 
that even if some new lesions appear at the beginning of a treatment or if the total tumor burden 
does not increase substantially, tumor regressions or stabilizations might still occur later. The 


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irRC were created using bi-dimensional measurements (as previously widely used in the World 
Health Organization criteria). For this trial, the concepts of the irRC are combined with RECIST 
1.1 to come up with the modified irRC.  Please refer to Appendix B:  Modified immune-related 
response criteria (irRC) section 13.2 for further details. 
6.3 
TOXICITY CRITERIA 
The following adverse event management guidelines are intended to ensure the safety of each 
patient while on the study.  The descriptions and grading scales found in the revised NCI 
Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 will be utilized for AE 
reporting.  All appropriate treatment areas should have access to a copy of the CTCAE version 
4.0.  A copy of the CTCAE version 4.0 can be downloaded from the CTEP web site 
(http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm#ctc_40).  All 
appropriate treatment areas should have access to a copy of the CTEP Active Version of 
CTCAE. 
7 
SAFETY REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING 
PLAN   
7.1 
DEFINITIONS 
7.1.1 Adverse Event 
An adverse event is defined as any reaction, side effect, or untoward event that occurs during the 
course of the clinical trial associated with the use of a drug in humans, whether or not the event 
is considered related to the treatment or clinically significant. For this study, AEs will include 
events reported by the patient, as well as clinically significant abnormal findings on physical 
examination or laboratory evaluation. A new illness, symptom, sign or clinically significant 
laboratory abnormality or worsening of a pre-existing condition or abnormality is considered an 
AE.   
All AEs, including clinically significant abnormal findings on laboratory evaluations, regardless 
of severity, will be followed until satisfactory resolution. AEs should be reported up to 30 days 
following the last dose of study drug.  Serious adverse events that occur more than 30 days after 
the last administration of investigational agent/intervention and have an attribution of at least 
possibly related to the agent/intervention should be recorded and reported as per sections 7.2, 
7.3, 7.4. 
An abnormal laboratory value will be considered an AE if the laboratory abnormality is 
characterized by any of the following: 
• 
Results in discontinuation from the study 
• 
Is associated with clinical signs or symptoms  
• 
Requires treatment or any other therapeutic intervention 
• 
Is associated with death or another serious adverse event, including hospitalization.  
• 
Is judged by the Investigator to be of significant clinical impact 
• 
If any abnormal laboratory result is considered clinically significant, the investigator will 


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provide details about the action taken with respect to the test drug and about the patient’s 
outcome. 
7.1.2 Suspected adverse reaction 
Suspected adverse reaction means any adverse event for which there is a reasonable possibility 
that the drug caused the adverse event. For the purposes of IND safety reporting, ‘reasonable 
possibility’ means there is evidence to suggest a causal relationship between the drug and the 
adverse event. A suspected adverse reaction implies a lesser degree of certainty about causality 
than adverse reaction, which means any adverse event caused by a drug. 
7.1.3 Unexpected adverse reaction 
An adverse event or suspected adverse reaction is considered “unexpected” if it is not listed in 
the investigator brochure or is not listed at the specificity or severity that has been observed; or, 
if an investigator brochure is not required or available, is not consistent with the risk information 
described in the general investigational plan or elsewhere in the current application. 
"Unexpected”, also refers to adverse events or suspected adverse reactions that are mentioned in 
the investigator brochure as occurring with a class of drugs or as anticipated from the 
pharmacological properties of the drug, but are not specifically mentioned as occurring with the 
particular drug under investigation. 
7.1.4 Serious  
An Unanticipated Problem or Protocol Deviation is serious if it meets the definition of Serious 
Adverse Event or if it compromises the safety, welfare or rights of subjects or others. 
7.1.5 Serious Adverse Event 
An adverse event or suspected adverse reaction is considered serious if in the view of the 
investigator or the sponsor, it results in any of the following: 
• 
Death, 
• 
A life-threatening adverse drug experience 
• 
Inpatient hospitalization or prolongation of existing hospitalization 
• 
Persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions 
• 
A congenital anomaly/birth defect. 
• 
Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered a serious adverse drug experience when, based upon 
appropriate medical judgment, they may jeopardize the patient or subject and may require 
medical or surgical intervention to prevent one of the outcomes listed in this definition. 
7.1.6 Disability 
 A substantial disruption of a person’s ability to conduct normal life functions. 


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7.1.7 Life-threatening adverse drug experience 
Any adverse event or suspected adverse reaction that places the patient or subject, in the view of 
the investigator or sponsor, at immediate risk of death from the reaction as it occurred, i.e., it 
does not include a reaction that had it occurred in a more severe form, might have caused death. 
7.1.8 Protocol Deviation (NIH Definition) 
Any change, divergence, or departure from the IRB approved research protocol. 
7.1.9 Non-compliance (NIH Definition) 
The failure to comply with applicable NIH Human Research Protections Program (HRPP) 
policies, IRB requirements, or regulatory requirements for the protection of human research 
subjects.  
7.1.10 Unanticipated Problem 
Any incident, experience, or outcome that: 
• Is unexpected in terms of nature, severity, or frequency in relation to  
(a) the research risks that are described in the IRB-approved research protocol and 
informed consent document; Investigator’s Brochure or other study documents, and 
(b) the characteristics of the subject population being studied; AND 
• Is related or possibly related to participation in the research; AND 
• Suggests that the research places subjects or others at a greater risk of harm (including 
physical, psychological, economic, or social harm) than was previously known or 
recognized. 
7.2 
NCI-IRB REPORTING 
7.2.1 NCI-IRB Expedited Reporting of Unanticipated Problems and Deaths 
The Protocol PI will report to the NCI-IRB: 
• All deaths, except deaths due to progressive disease  
• All Protocol Deviations 
• All Unanticipated Problems 
• All non-compliance 
Reports must be received by the NCI-IRB within 7 working days of PI awareness via iRIS. 
7.2.2 NCI-IRB Requirements for PI Reporting at Continuing Review 
The protocol PI will report to the NCI-IRB:  
1. A summary of all protocol deviations in a tabular format to include the date the deviation 
occurred, a brief description of the deviation and any corrective action. 


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2. A summary of any instances of non-compliance 
3. A tabular summary of the following adverse events: 
• All Grade 2 unexpected events that are possibly, probably or definitely related to the 
research;  
• All Grade 3 and 4 events that are possibly, probably or definitely related to the 
research;  
• All Grade 5 events regardless of attribution;  
• All Serious Events regardless of attribution.  
NOTE: Grade 1 events are not required to be reported.  
7.2.3 NCI-IRB Reporting of IND Safety Reports 
Only IND Safety Reports that meet the definition of an unanticipated problem will need to be 
reported to the NCI IRB. 
7.3 
IND SPONSOR REPORTING CRITERIA  
An investigator must immediately report to the sponsor using the mandatory MedWatch form 
3500a any serious adverse event, whether or not considered drug related, including those listed in 
the protocol or investigator brochure and must include an assessment of whether there is a 
reasonable possibility that the drug caused the event.  
Study endpoints that are serious adverse events (e.g. all-cause mortality) must be reported in 
accordance with the protocol unless there is evidence suggesting a causal relationship between 
the drug and the event (e.g. death from anaphylaxis).  In that case, the investigator must 
immediately report the death to the sponsor. 
Events will be submitted to Dr. William Dahut, authorized representative for the IND holder 
(CCR) at: 
William Dahut, M.D. 
Bldg 10, Room 3-2571 MSC 1206 
Bethesda, MD 20892 
Telephone: 301-496-4251 
William.Dahut@nih.gov 
Copy all MedWatch reports to nciprotocolsupportoffice@mail.nih.gov. 
7.4 
FDA REPORTING CRITERIA 
7.4.1 IND Safety Reports to the FDA (Refer to 21 CFR 312.32) 
The Sponsor will notify the FDA of any unexpected fatal or life-threatening suspected adverse 
reactions as soon as possible but no later than 7 calendar days of initial receipt of the information 
using the MedWatch Form 3500a.  
The Sponsor is also responsible for reporting any:  
• suspected adverse reaction that is both serious and unexpected 


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• any findings from clinical, epidemiological, or pooled analysis of multiple studies or any 
findings from animal or in vitro testing that suggest a significant risk in humans exposed 
to the drug 
• clinically important increase in the rate of a serious suspected adverse reaction over that 
listed in the protocol or investigator brochure  
to the FDA and to all investigators no later than 15 calendar days after determining that the 
information qualifies for reporting using the MedWatch Form 3500a. If FDA requests any 
additional data or information, the sponsor must submit it to the FDA as soon as possible, but no 
later than 15 calendars days after receiving the request.  
7.4.2 FDA Annual Reports (Refer to 21 CFR 312.33) 
The study Sponsor will submit a brief report annually of the progress of the trial within 60 days 
of the anniversary date that the IND went into effect as indicated in 21CFR 312.33, and any 
associated FDA correspondences regarding the IND annual report.  
7.4.3 Safety Reporting to the Manufacturer 
All safety reports and annual reports that are submitted to the FDA will be submitted to 
AmpImmune at: 
AmpImmune, INC. 
45 West Watkins Mill Road 
Gaithersburg, MD 20878 
Fax: 301-309-9801 
7.5 
DATA AND SAFETY MONITORING PLAN 
7.5.1 Principal Investigator/Research Team  
The clinical research team will meet on a regular basis when patients are being actively treated 
on the trial to discuss each patient. Decisions about dose level enrollment and dose escalation if 
applicable will be made based on the toxicity data from prior patients.  
All data will be collected in a timely manner and reviewed by the principal investigator or a lead 
associate investigator. Adverse events will be reported as required above. Any safety concerns, 
new information that might affect either the ethical and or scientific conduct of the trial, or 
protocol deviations will be immediately reported to the IRB using iRIS and to the Sponsor.  
The principal investigator will review adverse event and response data on each patient to ensure 
safety and data accuracy. The principal investigator will personally conduct or supervise the 
investigation and provide appropriate delegation of responsibilities to other members of the 
research staff.  
7.5.2 Sponsor Monitoring Plan 
This trial will be monitored by personnel employed by Harris Technical Services on contract to 
the NCI, NIH. Monitors are qualified by training and experience to monitor the progress of 
clinical trials. Personnel monitoring this study will not be affiliated in any way with the trial 
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At least 25% of enrolled patients will be randomly selected and monitored at least biannually or 
as needed, based on accrual rate. The patients selected will have 100% source document 
verification done. Additional monitoring activities will include: adherence to protocol specified 
study eligibility, treatment plans, data collection for safety and efficacy, reporting and time 
frames of adverse events to the NCI IRB and FDA, and informed consent requirements. Written 
reports will be generated in response to the monitoring activities and submitted to the Principal 
investigator and Clinical Director or Deputy Clinical Director, CCR, NCI. 
8 
STATISTICAL CONSIDERATIONS 
The primary objective of this pilot study is to determine if it is feasible to safely administer 6 
doses of AMP-224 in combination with radiation to patients with metastatic colorectal cancer 
and examining a small number of immunologic parameters prior to treatment and after treatment 
to determine if there is evidence of patients exhibiting an immune response to treatment. 
Secondary objectives include characterizing PK parameters and evaluating clinical outcomes in a 
preliminary fashion in a small trial. 
Relative to the feasibility endpoint, all patients enrolled on the trial will be considered as one 
group. Fifteen evaluable patients will be enrolled on this trial. It would be desirable if the 
fraction of patients who could receive six doses of AMP-224 were consistent with 80% or higher 
and greater than 50%. With 15 evaluable patients, if there are 11 or more patients who are able to 
safely receive six doses of treatment, then there is a 5.9% probability of this being true if the true 
probability of an individual patient being able to receive this much antibody were 50% and there 
is a 83.6% probability of this being true if the true probability for an individual were 80%. Thus, 
11 or more out of 15 patients receiving six doses of AMP-224 would provide strong evidence 
that it is feasible to administer this treatment in a substantial fraction of patients, consistent with 
80% or more and this would be considered a successful outcome for the trial. 
In addition, the actual levels of changes of immune parameters will be determined, and the 
fraction that are noted to have a change in the parameter values which would be considered 
immune responses will be reported. Focusing on two primary immune parameters, such as CD8+ 
TIL in tumor and in blood, with 15 patients with paired data, there would be 89% power to detect 
a change from baseline of 1 SD or greater magnitude (effect size=1.0) with a two-tailed 0.025 
significance level test for each of the two measures using a paired t-test.  If there are 12 
patients with paired data, there would be 79% power for the same analysis.  Tests of other 
immune parameters will be considered secondary and exploratory analyses. Safety and toxicity 
will also be evaluated and addressed by tabulating and monitoring the grades of toxicity 
experienced by patients in the study. 
It is expected that 1-2 patients per month may be able to enroll onto this trial. The accrual ceiling 
for the protocol will be 25 patients to allow for a small number of inevaluable patients to be 
enrolled. Thus, it is expected that 12 months may be needed to accrue all patients onto this trial. 
9 
COLLABORATIVE AGREEMENT 
9.1 
AGREEMENT TYPE 
The CRADA with AmpImmune for this protocol has been executed. 


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10 HUMAN SUBJECTS PROTECTIONS  
10.1 RATIONALE FOR SUBJECT SELECTION 
Subjects treated on this study, will be individuals with metastatic colorectal cancer, which has 
recurred (or persisted) after appropriate standard treatment.  Individuals of any race or ethnic 
group will be eligible for this study.  Eligibility assessment will be based solely on the patient’s 
medical status.  Recruitment of patients onto this study will be through standard CCR 
mechanisms.  No special recruitment efforts will be conducted. 
10.2 PARTICIPATION OF CHILDREN 
Individuals under the age of 18 will not be eligible to participate in this study because they are 
unlikely to have colorectal carcinoma, and because of unknown toxicities in pediatric patients. 
10.3 EVALUATION OF BENEFITS AND RISKS/DISCOMFORTS 
10.3.1 Risk of Biopsy 
All care will be taken to minimize risks that may be incurred by tumor sampling.  However, 
there are procedure-related risks (such as bleeding, infection and visceral injury) that will be 
explained fully during informed consent. 
10.3.2 Risks of exposure to Ionizing Radiation  
This research study involves exposure to radiation from stereotactic body radiation therapy and 2 
CT guided biopsies. This radiation exposure is not required for medical care and is for research 
purposes only. The amount of radiation received in this study is 309.29 rad. The amount of 
radiation received in this study exceeds the dose guideline established by the NIH Radiation 
Safety Committee for research subjects.  The guideline is an effective dose of 5 rem (or 5,000 
mrem) received per year. More information about radiation is available in the pamphlet, An 
Introduction to Radiation for NIH Research Subjects.  
10.3.3 Other Risks/Benefits 
The potential benefit to a patient that goes onto study is a reduction in the bulk of their tumor 
which may or may not have favorable impact on symptoms and/or survival.  Potential risks 
include the possible occurrence of any of a range of side effects which are listed in the Consent 
Document.  The procedure for protecting against or minimizing risks will be to medically 
evaluate patients on a regular basis as described. 
10.4 RISKS/BENEFITS ANALYSIS 
It is possible that treatment on this protocol may reduce tumor burden or lessen symptoms 
caused by the cancer. While treatment on this protocol may not individually benefit subjects, the 
knowledge gained from this study may help others in the future who have colorectal cancer.   
Potential risks include the possible occurrence of any of a range of side effects listed.  If patients 
suffer any physical injury as a result of the biopsies, immediate medical treatment is available at 
the NIH’s Clinical Center in Bethesda, Maryland.  Although no compensation is available, any 
injury will be fully evaluated and treated in keeping with the benefits or care to which patients 
are entitled under applicable regulations. 


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10.5 CONSENT AND ASSENT PROCESS AND DOCUMENTATION 
Patients will meet with an associate or principal investigator on the trial in the GI oncology 
Clinic, during the initial evaluation for this study.  During that meeting, the investigator will 
inform patients of the purpose, alternatives, treatment plan, research objectives and follow-up of 
this trial.  The investigator will then provide a copy of the IRB-approved informed consent 
document that is included in this protocol.  The patient will be allowed to take as much time as 
he wishes, in deciding whether or not he wishes to participate.  If a prolonged period of time 
expires during the decision making process (several weeks, as an example), it may be necessary 
to reassess the patient for protocol eligibility. The original signed consent goes to Medical 
Records; copy placed in research record (NIH policy). 
All patients must have a signed informed consent form and an on-study (confirmation of 
eligibility) form filled out and signed by a participating investigator before entering on the study. 
10.5.1 Re-consent via telephone 
The informed consent document will be sent to the subject.  An explanation of the study will be 
provided over the telephone after the subject has had the opportunity to read the consent form.  
The subject will sign and date the informed consent. A witness to the subject’s signature will 
sign and date the consent. 
The original informed consent document will be sent back to the consenting investigator who 
will sign and date the consent form with the date the consent was obtained via telephone.   
A fully executed copy will be returned via mail for the subject’s records. 
The informed consent process will be documented on a progress note by the consenting 
investigator and a copy of the informed consent document and note will be kept in the subject’s 
research record. 
 
11 PHARMACEUTICAL INFORMATION 
11.1 AMP224 (IND# 123468) 
11.1.1 Source  
Amplimmune, Inc  
11.1.2 Toxicity  
See section 1.2.6 for complete account of human toxicity. 
11.1.3 Formulation and preparation 
The AMP-224 drug product will be supplied as a sterile frozen liquid dosage form filled in a 5 
mL glass vial configuration (coated stopper and aluminum crimp). Once thawed, the drug is to 
be administered by IV infusion, within 4 hours. The drug product is formulated in a phosphate 
based system that is stabilized against freeze-thaw at a protein concentration of 60 mg/mL. No 
preservative is used since the vial is designed for single use. The AMP-224 drug product is 


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stored at ≤ -65oC. The long-term stability of AMP-224 under the designated storage condition 
has been demonstrated to exceed 12 months. 
11.1.4 Stability and Storage  
AMP-224 is stable at room temperature (after thawing) for at least 8 hours. 
11.1.5 Administration procedures 
Once thawed, AMP-224 infusion should begin within 4 hours.  AMP-224 is to be administered 
IV.  The calculated AMP-224 dose to be administered to the patient (determined by the patient’s 
body weight obtained on the day of dosing) will be removed from the freezer and thawed.  Once 
thawed, each dose will be diluted in 0.9% sodium chloride to equal a total delivery volume of 60 
mL.   
• Regarding pre-medications please refer to Section 3.2.1. 
• A 0.2-0.22 micron filter MUST be added to the IV tubing prior to priming and 
infusing AMP-224 into patients. 
• The final solution will be infused IV via a volumetric infusion pump over a minimum of 2 
hours.  The infusion time may be extended at any time to reduce infusion reactions. 
11.2 CYCLOPHOSPHAMIDE 
11.2.1 Source  
Cyclophosphamide will be purchased by the NIH Clinical Center Pharmacy Department from 
commercial sources. 
11.2.2 Toxicity  
a) Nausea and vomiting - variable; symptomatically improved with standard anti-emetics 
and/or benzodiazepines [e.g., lorazepam]. 
b) Water retention – cyclophosphamide may rarely provoke the syndrome of inappropriate 
antidiuretic hormone secretion and resultant hyponatremia, usually manifested 12-48 hrs 
after IV administration, necessitating frequent accurate assessment [q 1-2 hrs] of intake, 
urine output and urine specific gravity.  This effect can be counteracted by furosemide.  
Fluid restriction is not feasible during administration of high dose cyclophosphamide. 
c) Cardiomyopathy - cyclophosphamide may cause severe, sometimes lethal, hemorrhagic 
myocardial necrosis or congestive cardiomyopathy.  Patients may present with congestive 
cardiomyopathy as late as 2 weeks after the last dose of cyclophosphamide.  The clinical 
syndrome has been observed in patients receiving the dose of cyclophosphamide used in 
this protocol.   
d) Hemorrhagic cystitis – this is a serious, potentially life-threatening complication related 
to injury of the bladder epithelium by cyclophosphamide metabolites. 
e) Sterility 
f) Less common but serious complications include pulmonary fibrosis and secondary 
malignancies. Less common but reversible toxicities include alopecia and skin rash. 


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11.2.3 Formulation and preparation  
Cyclophosphamide is supplied as a lyophilized powder in various vial sizes. It will be 
reconstituted with sterile water for injection to yield a final concentration of 20 mg/ml as 
described in the package insert. 
11.2.4 Stability and Storage  
The vials are stored at room temperature.  Following reconstitution as directed, solutions of 
cyclophosphamide are stable for 24 hours at room temperature, or 6 days when refrigerated at 2-
8° C. 
11.2.5 Administration procedures 
Cyclophosphamide will be diluted in 250 mL of 0.9% Sodium Chloride Injection and will be 
infused over 60 minutes.  Following cyclophosphamide infusion, 500 mL of 0.9% Sodium 
Chloride Injection will be infused at a bolus rate. 
 
 


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13 APPENDICES 
13.1 APPENDIX A: PERFORMANCE STATUS CRITERIA 
ECOG Performance Status Scale 
Karnofsky Performance Scale 
Grade 
Descriptions 
Percent 
Description 
0 
Normal activity.  Fully active, able 
to carry on all pre-disease 
performance without restriction. 
100 
Normal, no complaints, no evidence 
of disease. 
90 
Able to carry on normal activity; 
minor signs or symptoms of 
disease. 
1 
Symptoms, but ambulatory.  
Restricted in physically strenuous 
activity, but ambulatory and able to 
carry out work of a light or sedentary 
nature (e.g., light housework, office 
work). 
80 
Normal activity with effort; some 
signs or symptoms of disease. 
70 
Cares for self, unable to carry on 
normal activity or to do active 
work. 
2 
In bed <50% of the time.  
Ambulatory and capable of all self-
care, but unable to carry out any 
work activities.  Up and about more 
than 50% of waking hours. 
60 
Requires occasional assistance, but 
is able to care for most of his/her 
needs. 
50 
Requires considerable assistance 
and frequent medical care. 
3 
In bed >50% of the time.  Capable of 
only limited self-care, confined to 
bed or chair more than 50% of 
waking hours. 
40 
Disabled, requires special care and 
assistance. 
30 
Severely disabled, hospitalization 
indicated.  Death not imminent. 
4 
100% bedridden.  Completely 
disabled.  Cannot carry on any self-
care.  Totally confined to bed or 
chair. 
20 
Very sick, hospitalization indicated. 
Death not imminent. 
10 
Moribund, fatal processes 
progressing rapidly. 
5 
Dead. 
0 
Dead. 
 
 
 


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13.2 APPENDIX B:  MODIFIED IMMUNE-RELATED RESPONSE CRITERIA (IRRC) 
This new classification is based on the recent learning from clinical studies with cancer 
immunotherapies that even if some new lesions appear at the beginning of a treatment or if the 
total tumor burden does not increase substantially, tumor regressions or stabilizations might still 
occur later. The irRC were created using bi-dimensional measurements (as previously widely 
used in the World Health Organization criteria). For this trial, the concepts of the irRC are 
combined with RECIST 1.1 to come up with the modified irRC.  
For modified irRC, only target and measurable lesions are taken into account. In contrast to the 
RECIST 1.1 criteria, the modified irRC criteria (a) require confirmation of both progression and 
response by imaging at 6 weeks after initial imaging and (b) do not necessarily score the 
appearance of new lesions as progressive disease if the sum of lesion diameters of target lesions 
(minimum of 10 mm per lesion, maximum of 5 target lesions, maximum of 2 per organ) and 
measurable new lesions does not increase by ≥ 20%.  
The same method of assessment and the same technique should be used to characterize each 
identified and reported target lesion(s) at baseline, during the trial, and at the end of trial visit. 
All measurements should be recorded in metric notation. The modified irRC based on RECIST 
1.1 are displayed below.  
 
 
 
 
 
 
 
 


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Overall Responses Derived from Changes in Index, Non-Index, and New Lesions 
 
 
 

ICF_001.pdf: 
MEDICAL RECORD 
CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH STUDY 
• 
Adult Patient or 
• Parent, for Minor Patient 
INSTITUTE: 
National Cancer Institute 
STUDY NUMBER: 
15-C-0021 
PRINCIPAL INVESTIGATOR: 
Tim Greten, M.D. 
STUDY TITLE:  
A Pilot Study of AMP-224, a PD-1 Inhibitor, in Combination with Stereotactic Body 
Radiation Therapy (SBRT) in Patients with Metastatic Colorectal Cancer 
Continuing Review Approved by the IRB on 08/24/16 
Amendment Approved by the IRB on 08/27/16 (B) 
 
Date posted to web:  09/02/16 
Standard 
PATIENT IDENTIFICATION 
CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH STUDY 
• 
Adult Patient or 
• Parent, for Minor Patient 
NIH-2514-1 (07-09) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent (1) 
 
INTRODUCTION 
We invite you to take part in a research study at the National Institutes of Health (NIH). 
First, we want you to know that: 
Taking part in NIH research is entirely voluntary.  
You may choose not to take part, or you may withdraw from the study at any time.  In 
either case, you will not lose any benefits to which you are otherwise entitled.  However, 
to receive care at the NIH, you must be taking part in a study or be under evaluation for 
study participation. 
You may receive no benefit from taking part.  The research may give us knowledge that 
may help people in the future. 
Second, some people have personal, religious or ethical beliefs that may limit the kinds of 
medical or research treatments they would want to receive (such as blood transfusions).  If you 
have such beliefs, please discuss them with your NIH doctors or research team before you agree 
to the study. 
Now we will describe this research study.  Before you decide to take part, please take as much 
time as you need to ask any questions and discuss this study with anyone at NIH, or with family, 
friends or your personal physician or other health professional.   
Why is this study being done? 
Our immune system helps to protect us from germs and can also fight against cancer.  T-cells are 
a type of white blood cell that can find and kill germs and tumors.  T-cells are often present 
inside tumors, but cancers block the activity of these T-cells in many different ways.  In tumors, 
some T-cells become dysfunctional, meaning that they are not able to kill tumor cells.  These 
dysfunctional T-cells in the tumor make a protein called “PD-1” which they express on their 
surface.  Some functional T-cells may also be present, but they are usually outnumbered.  


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 15-C-0021 
CONTINUATION: page 2 of 15 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
AMP-224 is an investigational drug that binds to the PD-1 on the surface of the T-cells. AMP-
224 therefore blocks the dysfunctional cells so that the functional cells can fight the tumor. 
“Investigational” means that AMP-224 has not been approved by the Food and Drug 
Administration (FDA) as either a prescription or over-the-counter drug. It has not been tested in 
combination with stereotactic body radiation therapy (SBRT), which is the main goal of this 
study.   
Stereotactic radiation treatment is a type of radiation therapy in which a few very high doses of 
radiation are delivered to small, well-defined tumors. The goal is to deliver a radiation dose that 
is high enough to kill the cancer while minimizing exposure to surrounding healthy organs. In 
this protocol SBRT is being used for research to see if tumor antigens will be released.  This 
research is being done to study if the safety and effectiveness of the investigational drug AMP-
224 together with either 1 or 3 days of SBRT directed to the liver. 
This is the first study in which AMP-224 will be tested in combination with SBRT.   
In addition to the AMP-224 and radiation treatment you will also receive an additional small 
dose of chemotherapy drug called cyclophosphamide. Cyclophosphamide is a chemotherapy 
drug that has been shown to also have some immune effects at lower doses. 
Why are you being asked to take part in this study? 
You have been invited to take part in this research study because you have been diagnosed with 
metastatic colorectal cancer that has spread to the liver and that has either not responded to prior 
treatment, or there are no curative treatment options available.   
How many people will take part in this study?  
Approximately 25 people will take part in this study. 
Description of Research Study 
What will happen if you take part in this research study?  
Before you begin the study 
Before you begin this study you will have several exams and tests to make sure you are eligible 
for this study.  The exams and tests are part of regular cancer care and may be done even if you 
do not join the study.  If you recently had some of the tests, they may not need to be repeated. 
The research team will explain these exams and tests to you. You will have: 
• History and physical exam 
• Review of current medications and past treatments 
• Routine blood work 
• Viral blood tests 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 15-C-0021 
CONTINUATION: page 3 of 15 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
• Routine urine tests 
• Tumor measurements using special x-rays called computerized tomography (CT or CAT 
scans) or magnetic resonance imaging (MRI) of your chest, stomach, and pelvis areas 
• A heart test called an electrocardiogram or ECG to check your heart 
• Urine or blood pregnancy test 
During the study 
If the exams, tests and procedures show that you can be in the study, and you choose to take part, 
then you will begin treatment.  
Before you begin treatment you will have a mandatory biopsy of your tumor using a needle. You 
will have another mandatory biopsy of your tumor around day 29 after you start treatment unless 
you were unable to tolerate the first biopsy. 
Treatment involves receiving Cyclophosphamide (CTX) through your vein (IV) and SBRT on 
day 0 and AMP-224 through an IV once every 2 weeks for 6 doses. After you have completed 
the first two doses of AMP-224, you will have another biopsy of your tumor.  If your disease is 
responding and you are tolerating the treatment well you will have the option of continuing the 
same every two week dose schedule until your disease shows clear evidence of growth. 
SBRT begins with a treatment planning session, which involves using imaging (4D 
computerized tomography) to precisely map the exact position of the tumor to be treated. These 
images are then used to create customized treatment plans in which sophisticated computerized 
devices direct several radiation beams of different intensities at different angles, so that the 
radiation is directed precisely to the tumor.  
The dose of AMP-224 is 10 milligrams per kilogram (a unit of body weight equal to about 2.2 
pounds).  AMP-224 will be administered over a minimum of 2 hours in the first treatment cycle.  
For subsequent treatment cycles, AMP-224 will be administered over a minimum of 1 hour. The 
AMP-224 infusion duration may be increased for patient safety at any time at the Investigator’s 
discretion. CTX and AMP-224 will be administered on different days as an intravenous (injected 
into a blood vessel through a plastic tube or needle) injection.   
There are two groups of patients on the study. The first group of patients will receive one dose of 
radiation treatment (the SBRT). We plan to treat approximately 6 patients like this and this will 
be the first part of the study. 
Once the first part of the study is complete we will enroll new patients to the second part of the 
study. In this part of the study patients will receive 3 doses of the SBRT. In other words the main 
difference between the first and second parts of the study will the length of time you receive the 
radiation – either one or three doses.  


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 15-C-0021 
CONTINUATION: page 4 of 15 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
When you are finished taking the drugs (treatment) 
You will be seen weekly for three weeks, then every two weeks for one month, then month two, 
and month five after that after completion of the study treatment to assess for possible delayed or 
ongoing side effects and overall clinical status. At these visit, you will have basic blood tests as 
well as blood collected for research purposes. 
Study Chart 
Day 0 
• History and physical exam 
• Review of current medications and treatments 
• Routine blood tests 
• Stereotactic body radiation therapy 
• CTX infusion 
Day 1 
• Physical exam 
• Vital Signs 
• Review of current medications and treatments 
• Routine and research blood tests 
• Routine urine tests 
• ECG 
• Report any side effects 
• Pre-medications 
• AMP-224 Infusion 
Day 8 
• Physical exam 
• ECG 
• Report any side effects 
• Review of current medications and treatments 
• Routine and research blood tests 
Day 15 
• Physical exam 
• Vital Signs 
• Review of current medications and treatments 
• Routine and research blood tests 
• Routine urine tests 
• Report any side effects 
• Pre-medications 
• AMP-224 Infusion 
Day 29 
• Physical exam 
• Vital Signs 
• Review of current medications and treatments 
• Routine and research blood tests 
• Routine urine tests 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 15-C-0021 
CONTINUATION: page 5 of 15 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
• Tumor Biopsy 
• Report any side effects 
• Pre-medications 
• AMP-224 Infusion 
Day 43 
• Physical exam 
• Vital Signs 
• Review of current medications and treatments 
• Routine and research blood tests 
• Routine urine tests 
• Report any side effects 
• Pre-medications 
• AMP-224 Infusion 
Day 57 
• Physical exam 
• CT Scan or MRI 
• Vital Signs 
• Review of current medications and treatments 
• Routine and research blood tests 
• Routine urine tests 
• Report any side effects 
• Pre-medications 
• AMP-224 Infusion 
Day 71 
• Physical exam 
• Vital Signs 
• Review of current medications and treatments 
• Routine and research blood tests. 
• Routine urine tests 
• Report any side effects 
• Pre-medications 
• AMP-224 Infusion 
 
Birth Control 
If you are a woman who is breast feeding or pregnant, you may not take part in the study because 
we don’t know how this medicine would affect your baby or your unborn child. If you are a 
woman who can become pregnant, or are the partner of a woman who can become pregnant, you 
will need to practice an effective form of birth control before starting study treatment, during 
study treatment, and for 3 months after you finish study treatment.  If you think that you or your 
partner is pregnant, you should tell your study doctor or nurse at once.   
Effective forms of birth control include:  


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 15-C-0021 
CONTINUATION: page 6 of 15 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
• abstinence  
• intrauterine device (IUD) 
• hormonal [birth control pills, injections, or implants] 
• tubal ligation 
• vasectomy 
Risks or Discomforts of Participation 
Cyclophosphamide (CTX) 
Specific symptoms that have been associated with cyclophosphamide as listed below. However, 
we will be using cyclophosphamide at a much lower doses at which the side effects listed are 
unlikely: 
• Changes in blood counts including: low red cell count (causing fatigue and shortness of 
breath), low platelet count (increasing the risk of bleeding and bruising), decrease in 
white blood cells (increasing the risk of infection and the need for treatment with 
antibiotics or other treatment)  
• Loss of appetite, nausea, vomiting  
• Diarrhea, stomach pain  
• Mouth sores  
• Hair loss  
• Fatigue  
• Muscle or joint aches  
• Bladder damage which can cause blood in your urine 
Stereotactic body radiation therapy (SBRT)  
In general, radiation can cause the following side effects regardless of the site that is being 
treated.  
• 
Tiredness  
• 
Lowered blood counts  
• 
Skin reddening  
• 
Mild ache at the site that received radiation  
These side effects tend to go away after the radiation therapy is completed. However, there are 
some long-term or chronic side effects that primarily affect the small bowel, liver, kidneys, and 
spinal cord. Many of these side effects take months to years to develop. Rarely, treatment with 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 15-C-0021 
CONTINUATION: page 7 of 15 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
radiation may also lead to developing other types of cancer, usually years after receiving the 
treatment. It is possible that you may experience some, all, or none of the side effects described 
above. It is also possible that your specific treatment may cause some side effects that we cannot 
anticipate. For that reason, you will be watched closely while you are receiving treatment for any 
signs that might signal the earliest stage of toxicity so that we can treat them early.  
Specific symptoms that have been associated with SBRT to the liver area are the following:  
• Nausea, vomiting 
• Diarrhea 
• Abdominal pain 
• Feelings of claustrophobia  
• Skin irritation  
• Fatigue  
• Sore throat and trouble swallowing  
• Late or delayed side effects (the side effects noted above but they occur after the 
radiation has completed) 
AMP-224  
While you are taking part in this study, you are at risk for side effects.  You should talk to your 
doctor about possible side effects.  Other drugs (pre-medications) will be given to make side 
effects less serious and less uncomfortable.  Many side effects may go away shortly after  
AMP-224 is stopped, but in some cases side effects can be serious and long lasting.  It is not 
known how long the side effects may last.  The unforeseen side effects may range from mild to 
life-threatening.  During and after your participation in the study, it will be very important for 
you to report any possible side effects or symptoms you develop to your study doctor.  
Certain side effects can follow the administration of any protein such as AMP-224; these 
include: 
• fever (high body temperature) 
• chills (shaking)  
• skin rash (redness or bumps on the skin)  
• itching  
• joint pain  
• swelling   
If any of these side effects appear, it is anticipated that they will be mild and easy to control.  
There is also a possibility that a more severe response may occur, resulting in a fall in blood 
pressure (causing dizziness, light-headedness and fainting), shortness of breath, and pain in the 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 15-C-0021 
CONTINUATION: page 8 of 15 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
chest, low back and side. Some side effects may be serious and may even result in death.  You 
will be closely observed for all of these symptoms.  Should side effects occur, appropriate 
treatment will be started as determined by your study doctor. 
AMP-224 is developed to play a role in immune function.  There is a risk that AMP-224 could 
affect this function.  There are cases where patients who received other drugs that affect the 
immune system developed illnesses such as dermatitis (inflammation of the skin), enterocolitis 
(inflammation of the bowel), autoimmune hepatitis (inflammation of the liver), lymphopenia 
(low white blood cells that fight infection), fatigue (tiredness), and hypophysitis (inflammation 
of the pituitary gland).  Patients receiving AMP-224 will be carefully monitored for diarrhea, 
abdominal cramping (pain in the abdomen), fatigue (tiredness), headaches, arthralgias (joint 
pain), changes in sight, and any skin changes such as rash (redness or bumps on the skin), 
itching, or discoloration.  If any of these symptoms appear, you should notify your study doctor 
right away.  In addition, blood tests will be done at regular interval to look for changes in liver 
function, thyroid function (a gland important in regulating metabolism), and adrenal gland 
function (a gland important in responding to stress and regulating salt and water levels). 
Side effects associated with other drugs that affect the immune system occurred anywhere from 1 
week to a few months after starting the drug.   
Your body may react to AMP-224 by thinking that the drug is a foreign substance and does not 
belong to your body.  If this happens, your body will develop specific proteins (antibodies 
[substances in the body that normally fight infection]) that block AMP-224.  The effects of 
developing these proteins are not known.  However, if your body makes these proteins, it could 
limit your ability to respond to AMP-224 or related drugs if you were to receive these drugs in 
the future.  There is also the possibility of developing an allergic reaction. 
Because AMP-224 study drug is investigational, its use may involve other risks that are not 
known or cannot be predicted at the present time.  These risks may affect you during the study 
and/or at some point in the future.  During the study, it is important that you report any new or 
unusual symptoms to your doctor immediately.   
To date, AMP-224 has been tested in 44 patients; 6 at 0.3 mg/kg, 4 at 1 mg/kg and 4 at 3 mg/kg, 
24 at 10 mg/kg, and 6 at 30 mg/kg.  Patients enrolled in that study have received between 1 and 
42 doses of AMP-224.  Side effects considered related to AMP-224 have been generally mild to 
moderate in intensity.  Side effects that have occurred in ≥10% of patients that were considered 
at least possibly related to AMP-224 are: chills (shivers), rigors (muscle spasms), fatigue (feeling 
tired), decreased appetite, facial redness, nausea, back pain, diarrhea, and fever.  
One patient who was not pre-medicated had a severe infusion reaction to AMP-224 which 
included chills (shivers), rigors (muscle spasms), and elevated blood pressure.  The patient 
recovered after the infusion was stopped and after receiving treatment medications.   


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 15-C-0021 
CONTINUATION: page 9 of 15 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
Tumor Biopsy 
Before you begin treatment on this protocol we will obtain a piece of your tumor (biopsy) using 
a needle with minimal risk to you.   You will have this biopsy again on day 29. You will be 
given local anesthesia (numbing medicine) and a sedative prior to the biopsy.  The biopsy will be 
taken through a needle put through the skin into your tumor.  After the procedure the nurses will 
watch your blood pressure and other vital signs.  This biopsy is not optional and you cannot 
participate in this study if you do not agree to the biopsies. If the first biopsy is too difficult or if 
you experience too much discomfort as a result of it you will be able to continue on the protocol 
without undergoing the second biopsy. However, an attempt at the first biopsy is needed to enter 
this study. There are other studies at NIH which may also be options for you and which do not 
involve biopsies. 
Risk of Radiation 
This research study involves exposure to radiation from stereotactic body radiation therapy and 
the CT used during your biopsies.   Please note that this radiation exposure is not necessary for 
your medical care and is for research purposes only.   The total amount of radiation you will 
receive in this study is from up to 3 treatments of stereotactic body radiation therapy and 2 CT 
guided biopsies.  The NIH Radiation Safety Committee has reviewed the use of radiation in this 
research study and has approved this use as involving acceptable risk and necessary to obtain the 
research information desired. 
Using the standard way of describing radiation dose, from participating in this study, you will 
receive a total of 2400 rem to your gallbladder wall and liver and 600 rem to your adrenals. The 
total effective dose will be 309 rem.  All other organs will receive smaller amounts of radiation.  
The amount of radiation received in this study exceeds the dose guideline established by the NIH 
Radiation Safety Committee for research subjects.  The guideline is an effective dose of 5 rem 
(or 5,000 mrem) received per year. 
The specific side effects related to the effects of stereotactic body radiation therapy are described 
above. 
If you would like more information about radiation and examples of exposure levels from other 
sources, please ask the investigator for a copy of the pamphlet called, An Introduction to 
Radiation for NIH Research Subjects. 
Please tell your doctor if you have taken part in other research studies or received any medical 
care at the NIH or other places/hospitals that used radiation.  This way we can make sure that 
you will not receive too much radiation.  Consider x-rays taken in radiology departments, cardiac 
catheterization, and fluoroscopy as well as nuclear medicine scans in which radioactive materials 
were injected into your body. 
If you are pregnant, you will not be permitted to participate in this research study.  It is best to 
avoid radiation exposure to unborn or nursing children since they are more sensitive to radiation 
than adults. 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 15-C-0021 
CONTINUATION: page 10 of 15 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
 
Blood Sampling  
Risks of blood sampling include bruising or bleeding at the needle site; rarely infection. This is 
treated with bandages, pressure and, if infection, antibiotic medicines. For more information 
about risks and side effects, ask your study team. 
Potential Benefits of Participation 
Are there benefits to taking part in this study? 
We hope that you will get personal medical benefit from taking part in this study, but we cannot 
be certain.  These potential benefits could include shrinking of your tumor or lessening of your 
symptoms, such as pain, that are caused by the cancer.  Because there is not much information 
about the drug’s effect on your cancer, we do not know if you will benefit from taking part in 
this study, although the knowledge gained from this study may help others in the future who 
have cancer.   
Alternative Approaches or Treatments 
What other choices do I have if I do not take part in this study?  
 
Instead of being in this study, you have these options: 
• Getting treatment or care for your cancer without being in a study 
• Taking part in another study 
• Getting comfort care, also called palliative care.  This type of care helps reduce pain, 
tiredness, appetite problems and other problems caused by the cancer.  It does not treat 
the cancer directly.  Instead, it tries to improve how you feel.  Comfort care tries to keep 
you as active and comfortable as possible. 
Please talk to your doctor about these and other options. 
 
Research Subject’s Rights 
What are the costs of taking part in this study?  
If you choose to take part in the study, the following will apply, in keeping with the NIH policy: 
• 
You will receive study treatment at no charge to you. This may include 
surgery, medicines, laboratory testing, x-rays or scans done at the 
Clinical Center, National Institutes of Health (NIH), or arranged for you 
by the research team to be done outside the Clinical Center, NIH if the 
study related treatment is not available at the NIH.  


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 15-C-0021 
CONTINUATION: page 11 of 15 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
• 
There are limited funds available to cover the cost of some tests and 
procedures performed outside the Clinical Center, NIH.  You may have 
to pay for these costs if they are not covered by your insurance company. 
• 
Medicines that are not part of the study treatment will not be provided or 
paid for by the Clinical Center, NIH. 
• 
Once you have completed taking part in the study, medical care will no 
longer be provided by the Clinical Center, NIH. 
 
Will your medical information be kept private?  
We will do our best to make sure that the personal information in your medical record will be 
kept private.  However, we cannot guarantee total privacy.  Organizations that may look at 
and/or copy your medical records for research, quality assurance, and data analysis include: 
 
• The National Cancer Institute (NCI) and other government agencies, like the Food and 
Drug Administration (FDA), which are involved in keeping research safe for people. 
• National Cancer Institute Institutional Review Board 
• The study Sponsor, Center for Cancer Research or their agent(s). 
• Qualified representatives from Amplimmune, Inc, the pharmaceutical company who 
produces AMP-224. 
Stopping Therapy  
Your doctor may decide to stop your therapy for the following reasons: 
• if he/she believes that it is in your best interest 
• if your disease comes back during treatment 
• if you have side effects from the treatment that your doctor thinks are too severe 
• if new information shows that another treatment would be better for you 
 
In this case, you will be informed of the reason therapy is being stopped. 
You can stop taking part in the study at any time. However, if you decide to stop taking part in 
the study, we would like you to talk to the study doctor and your regular doctor first. 
If you decide at any time to withdraw your consent to participate in the trial, we will not collect 
any additional medical information about you. However, according to FDA guidelines, 
information collected on you up to that point may still be provided to The Center for Cancer 
Research, Amplimmune, Inc. or designated representatives. If you withdraw your consent and 
leave the trial, any samples of yours that have been obtained for the study and stored at the 
NCI can be destroyed upon request. However, any samples and data generated from the samples 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 15-C-0021 
CONTINUATION: page 12 of 15 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
that have already been distributed to other researchers or placed in the research databases cannot 
be recalled and destroyed. 
 
Conflict of Interest 
The National Institutes of Health (NIH) reviews NIH staff researchers at least yearly for conflicts 
of interest.  This process is detailed in a Protocol Review Guide.   You may ask your research 
team for a copy of the Protocol Review Guide or for more information.  Members of the research 
team who do not work for NIH are expected to follow these guidelines but they do not need to 
report their personal finances to the NIH. 
 
Members of the research team working on this study may have up to $15,000 of stock in the 
companies that make products used in this study.  This is allowed under federal rules and is not a 
conflict of interest.  
The National Institutes of Health and the research team for this study are using AMP-224 
developed by Amplimmune, Inc. through a joint study with your researchers and the company.  
The company also provides financial support for this study.  
 
Optional Biopsy 
If you have a tumor outside of the radiation area that shrinks, we may ask you if you will 
undergo a needle biopsy of that particular tumor.  The biopsy to be performed is exclusively for 
research purposes and will not benefit you.  It might help other people in the future.  Even if you 
sign "yes" to have the biopsy you can change your mind at any time.  Please read the sentence 
below and think about your choice. After reading the sentence, circle and initial the answer that 
is right for you. The decision to participate in this part of the research is optional, and no matter 
what you decide to do, it will not affect your care.  
 
I agree to have the tumor biopsy for the research tests in this study.  
 
Yes              No               Initials_________ 
 
Use of Specimens and Data for Future Research 
To advance science, it is helpful for researchers to share information they get from studying 
human samples.  They do this by putting it into one or more scientific databases, where it is 
stored along with information from other studies. A researcher who wants to study the 
information must apply to the database and be approved.  Researchers use specimens and data 
stored in scientific databases to advance science and learn about health and disease.   


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 15-C-0021 
CONTINUATION: page 13 of 15 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
We plan to keep some of your specimens and data that we collect and use them for future 
research and share them with other researchers. We will not contact you to ask about each of 
these future uses.  These specimens and data will be stripped of identifiers such as name, address 
or account number, so that they may be used for future research on any topic and shared broadly 
for research purposes. Your specimens and data will be used for research purposes only and will 
not benefit you.  It is also possible that the stored specimens and data may never be used.  
Results of research done on your specimens and data will not be available to you or your doctor.  
It might help people who have cancer and other diseases in the future.   
If you do not want your stored specimens and data used for future research, please contact us in 
writing and let us know that you do not want us to use your specimens and/or data.  Then any 
specimens that have not already been used or shared will be destroyed and your data will not be 
used for future research. However, it may not be possible to withdraw or delete materials or data 
once they have been shared with other researchers. 


MEDICAL RECORD 
CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH STUDY 
• 
Adult Patient or 
• Parent, for Minor Patient 
STUDY NUMBER: 15-C-0021 
CONTINUATION: page 14 of 15 pages 
 
PATIENT IDENTIFICATION 
CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH 
STUDY (Continuation Sheet) 
• 
Adult Patient or 
• Parent, for Minor Patient 
NIH-2514-1 (07-09) 
P.A.:  09-25-0099 
 
File in Section 4:  Protocol Consent 
 
 
 
OTHER PERTINENT INFORMATION 
1. 
Confidentiality.  When results of an NIH research study are reported in medical journals 
or at scientific meetings, the people who take part are not named and identified.  In most cases, 
the NIH will not release any information about your research involvement without your written 
permission.  However, if you sign a release of information form, for example, for an insurance 
company, the NIH will give the insurance company information from your medical record.  This 
information might affect (either favorably or unfavorably) the willingness of the insurance 
company to sell you insurance. 
The Federal Privacy Act protects the confidentiality of your NIH medical records.  However, you 
should know that the Act allows release of some information from your medical record without 
your permission, for example, if it is required by the Food and Drug Administration (FDA), 
members of Congress, law enforcement officials, or authorized hospital accreditation 
organizations. 
2. 
Policy Regarding Research-Related Injuries.  The Clinical Center will provide short-
term medical care for any injury resulting from your participation in research here.  In general, 
no long-term medical care or financial compensation for research-related injuries will be 
provided by the National Institutes of Health, the Clinical Center, or the Federal Government.  
However, you have the right to pursue legal remedy if you believe that your injury justifies such 
action. 
3. 
Payments.  The amount of payment to research volunteers is guided by the National 
Institutes of Health policies.  In general, patients are not paid for taking part in research studies at 
the National Institutes of Health.  Reimbursement of travel and subsistence will be offered 
consistent with NIH guidelines. 
4. 
Problems or Questions.  If you have any problems or questions about this study, or 
about your rights as a research participant, or about any research-related injury, contact the 
Principal Investigator, Tim Greten, M.D., Building 10, Room 3B43, Telephone:  301-451-4723. 
You may also call the Clinical Center Patient Representative at 301-496-2626. If you have any 
questions about the use of your specimens or data for future research studies, you may also 
contact the Office of the Clinical Director, Telephone:  301-496-4251.  
5. 
Consent Document.  Please keep a copy of this document in case you want to read it 
again. 


MEDICAL RECORD 
CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH STUDY 
• 
Adult Patient or 
• Parent, for Minor Patient 
STUDY NUMBER: 15-C-0021 
CONTINUATION: page 15 of 15 pages 
 
PATIENT IDENTIFICATION 
CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH 
STUDY (Continuation Sheet) 
• 
Adult Patient or 
• Parent, for Minor Patient 
NIH-2514-1 (07-09) 
P.A.:  09-25-0099 
 
File in Section 4:  Protocol Consent 
 
 
 
 
 
COMPLETE APPROPRIATE ITEM(S) BELOW: 
A. Adult Patient’s Consent 
B. Parent’s Permission for Minor Patient. 
I have read the explanation about this study 
and have been given the opportunity to discuss 
it and to ask questions.  I hereby consent to 
take part in this study. 
I have read the explanation about this study 
and have been given the opportunity to discuss 
it and to ask questions.  I hereby give 
permission for my child to take part in this 
study.   
(Attach NIH 2514-2, Minor’s Assent, if 
applicable.) 
 
  
  
 
 
 
Signature of Adult Patient/ 
Legal Representative 
Date 
Signature of Parent(s)/ Guardian Date 
 
 
Print Name 
 
 
Print Name 
C. Child’s Verbal Assent (If Applicable) 
The information in the above consent was described to my child and my child agrees to 
participate in the study. 
 
  
  
 
Signature of Parent(s)/Guardian 
Date 
Print Name 
THIS CONSENT DOCUMENT HAS BEEN APPROVED FOR USE  
FROM AUGUST 24, 2016 THROUGH AUGUST 23, 2017.  
 
 
 
 
 
 
 
 
Signature of Investigator 
Date 
Signature of Witness 
Date 
 
 
Print Name 
 
 
Print Name