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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  
- 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.
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7.4.2 fda annual reports (refer

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
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7.5 data and safety monitoring

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 conduct.  
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.
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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.
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10 human subjects protections 10.1

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.
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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.
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10.3 evaluation of benefits and

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.
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10.3.2 risks of exposure to

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.
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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.
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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

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.
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11 pharmaceutical information 11.1 amp224

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 stored at ≤ -65oC. The long-term stability of AMP-224 under the designated storage condition has been demonstrated to exceed 12 months.
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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.
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11.2 cyclophosphamide 11.2.1 source

Cyclophosphamide will be purchased by the NIH Clinical Center Pharmacy Department from commercial sources.
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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.
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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 28° C.
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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 |
| 100                                                                                                                                                                                        | Normal, no complaints, no evidence  of disease.                            |                                                                    |             |
| Normal activity. Fully active, able                                                                                                                                                        |                                                                            |                                                                    |             |
| 0                                                                                                                                                                                          | to carry on all pre-disease  performance without restriction.              | Able to carry on normal activity;                                  |             |
| 90                                                                                                                                                                                         | minor signs or symptoms of  disease.                                       |                                                                    |             |
| 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.   |             |
| 1                                                                                                                                                                                          | Cares for self, unable to carry on                                         |                                                                    |             |
| 70                                                                                                                                                                                         | normal activity or to do active  work. Requires occasional assistance, but |                                                                    |             |
| 60                                                                                                                                                                                         | is able to care for most of his/her  needs.                                |                                                                    |             |
| In bed <50% of the time.  Ambulatory and capable of all selfcare, but unable to carry out any  work activities. Up and about more  than 50% of waking hours.                                                                                                                                                                                            |                                                                            |                                                                    |             |
| 2                                                                                                                                                                                          | 50                                                                         | Requires considerable assistance  and frequent medical care.       |             |
| 40                                                                                                                                                                                         | Disabled, requires special care and  assistance.                           |                                                                    |             |
| In bed >50% of the time. Capable of  only limited self-care, confined to  bed or chair more than 50% of  waking hours.                                                                     |                                                                            |                                                                    |             |
| 3                                                                                                                                                                                          | 30                                                                         | Severely disabled, hospitalization  indicated. Death not imminent. |             |
| 20                                                                                                                                                                                         | Very sick, hospitalization indicated.  Death not imminent.                 |                                                                    |             |
| 100% bedridden. Completely  disabled. Cannot carry on any selfcare. Totally confined to bed or  chair.                                                                                                                                                                                            |                                                                            |                                                                    |             |
| 4                                                                                                                                                                                          | 10                                                                         | Moribund, fatal processes  progressing rapidly.                    |             |
| 5                                                                                                                                                                                          | Dead.                                                                      | 0                                                                  | Dead.       |
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13.2 appendix b modified immune-related

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.  
| Measurable Response                                                                                        | Non-Measurable Response   | Overall Response   |        |
|------------------------------------------------------------------------------------------------------------|---------------------------|--------------------|--------|
| Using Modified irRC                                                                                        |                           |                    |        |
| Index and New, Measurable                                                                                  | Non-Index Lesions         | New, Non-          |        |
| Lesions (Tumor Burden)¹                                                                                    | Measurable Lesions        |                    |        |
| Decrease 100%                                                                                              | Absent                    | Absent             | irCR²  |
| Decrease 100%                                                                                              | Stable                    | irPR²              |        |
| Any                                                                                                        |                           |                    |        |
| Decrease 100%                                                                                              | Unequivocal progression   | irPR 2             |        |
| Any                                                                                                        |                           |                    |        |
| Decrease ≥ 30%                                                                                             | Absent / Stable           | Any                | irPR 2 |
| irPR²                                                                                                      |                           |                    |        |
| Decrease ≥ 30%                                                                                             | Unequivocal progression   | Any                |        |
| Decrease < 30% to increase <                                                                               | Absent / Stable           | Any                | irSD   |
| 20%                                                                                                        |                           |                    |        |
| Decrease < 30% to increase <                                                                               | Unequivocal progression   | Any                | irSD   |
| 20%                                                                                                        |                           |                    |        |
| Increase ≥ 20%                                                                                             | Any                       | Any                | irPD   |
| ' Decreases assessed relative to baseline                                                                  |                           |                    |        |
| 2 Assuming that the response (irCR and irPR) and progression (irPD) are confirmed by a second, consecutive |                           |                    |        |
| assessment at least 4 weeks apart (normally it should be done 6 weeks apart).                              |                           |                    |        |  
Overall Responses Derived from Changes in Index, Non-Index, and New Lesions