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|---|  
| Abbreviated Title:  Metastatic Melanoma Young TIL                                                                   |                                                                                                                       |
|---------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------|
| Version date: 02/15/2019                                                                                            |                                                                                                                       |
| 3.3                                                                                                                 | T Treatment Schedule .                                                                                                |
| 3.4                                                                                                                 | ON Study Evaluation.                                                                                                  |
| 3.4.1                                                                                                               | Within 14 days prior to starting the preparative regimen ......................................................... 19 |
| 3.4.2                                                                                                               | During the preparative regimen: DAILY                                                                                 |
| 3.4.3                                                                                                               | After Pembrolizumab (Retreatment only) and Cell Infusion ............................................. 19             |
| Prior to each Pembrolizumab infusion (days 21, 42, 63) (Retreatment only) ............. 19                          |                                                                                                                       |
| 3.4.4                                                                                                               |                                                                                                                       |
| 3.4.5                                                                                                               | Management of Immune Related Adverse Events (IRAEs) Refer to Appendix 5 ...... 20                                     |
| 3.4.6                                                                                                               | During Hospitalization                                                                                                |
| 3.5                                                                                                                 | Retreatment                                                                                                           |
| 3.6                                                                                                                 | Post Treatment (Follow Up) Evaluation                                                                                 |
| Time period of evaluations:                                                                                         |                                                                                                                       |
| 3.6.1                                                                                                               |                                                                                                                       |
| 3.6.2                                                                                                               | At each scheduled evaluation for response patients will undergo: .................................... 21              |
| 3.7                                                                                                                 | Criteria for Removal from Protocol Therapy and Off Study Criteria ..................... 22                            |
| Criteria for removal from protocol therapy .                                                                        |                                                                                                                       |
| 3.7.1                                                                                                               |                                                                                                                       |
| 3.7.2                                                                                                               | OFF Study Criteria .                                                                                                  |
| 3.7.3                                                                                                               | Off Protocol Therapy and Off-Study Procedure .                                                                        |
| 4                                                                                                                   | CONCOMITANT MEDICATIONS/MEASURES                                                                                      |
| 4.1                                                                                                                 | Infection Prophylaxis                                                                                                 |
| 4.1.1                                                                                                               | Pneumocystis Jirovecii Pneumonia                                                                                      |
| 4.1.2                                                                                                               | Herpes or Varicella Zoster Virus Prophylaxis .                                                                        |
| 4.1.3                                                                                                               | Fungal Prophylaxis (Fluconazole) .                                                                                    |
| 4.1.4                                                                                                               | Empiric Antibiotics                                                                                                   |
| 4.1.5                                                                                                               | Blood Product Support.                                                                                                |
| Other Concomitant medications to control side effects........................................................... 24 |                                                                                                                       |
| 4.2                                                                                                                 |                                                                                                                       |
| s                                                                                                                   | BIOSPECIMEN COLLECTION .                                                                                              |
| 5.1                                                                                                                 | Samples Sent to Dr. Figg's Lab .                                                                                      |
| 5.2                                                                                                                 | Samples Sent to Surgery branch Cell Processing Laboratory.                                                            |
| Prior to chemotherapy administration .                                                                              |                                                                                                                       |
| 5.3                                                                                                                 | Prior to cell infusion                                                                                                |
| 5.4                                                                                                                 |                                                                                                                       |
| 5.5                                                                                                                 | Post cell infusion evaluations:                                                                                       |
| 5.6                                                                                                                 | Immunological Testing: .                                                                                              |  
5.7 Sample Storage, Tracking and Disposition For SB cell Processing laboratory ....... 26 5.8 Sample Storage, Tracking and Disposition For Dr. Figg's Lab ....................................... 26  
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|-------|-------------------------------------------------------------------------------------------------------------------------------------------|
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| Abbreviated Title:  Metastatic Melanoma Young TIL   |                                                                                       |
|-----------------------------------------------------|---------------------------------------------------------------------------------------|
| Version date: 02/15/2019                            |                                                                                       |
| 7.4                                                 | Data and Safety Monitoring Plan                                                       |
| 7.4.1                                               | Principal Investigator/Research Team:                                                 |
| 7.4.2                                               | Sponsor Monitoring Plan: .                                                            |
| STATISTICAL CONSIDERATIONS .                        |                                                                                       |
| 8                                                   |                                                                                       |
| 9                                                   | COLLABORATIVE AGREEMENTS                                                              |
| HUMAN SUBJECTS PROTECTIONS                          |                                                                                       |
| 10                                                  |                                                                                       |
| 10.1                                                | Rationale for Patient Selection                                                       |
| Participation of Children                           |                                                                                       |
| 10.2                                                | Participation of Subjects Unable to Give consent                                      |
| 10.3                                                |                                                                                       |
| 10.4                                                | Evaluation of Benefits and Risks/Discomforts .                                        |
| 10.5                                                | Risks/Benefits Analysis                                                               |
| 10.6                                                | Consent Process and Documentation                                                     |
| PHARMACEUTICAL INFORMATION                          |                                                                                       |
| 11                                                  | Investigational Regimen                                                               |
| 11.1                                                |                                                                                       |
| 1.1.1                                               | Young TIL                                                                             |
| 11.1.2                                              | Aldesleukin (Interleukin-2, Proleukin, Recombinant Human Interleukin 2) .......... 40 |
| Fludarabine                                         |                                                                                       |
| 11.1.3                                              |                                                                                       |
| 1.1.4                                               | Cyclophosphamide                                                                      |
| 1.1.5                                               | Pembrolizumab                                                                         |
| Support Medications.                                |                                                                                       |
| 1.2                                                 | Mesna (Sodium 2-mercaptoethanesulfonate, Mesnum, Mesnex, NSC-113891)...... 43         |
| 1.2.1                                               |                                                                                       |
| 11.2.2                                              | Filgrastim (Granulocyte Colony-Stimulating Factor, G-CSF, Filgrastim, Neupogen)       |
| 44                                                  |                                                                                       |
| 11.2.3                                              | Trimethoprim and Sulfamethoxazole Double Strength (TMP / SMX DS) .............. 44    |
| 11.2.4                                              | Herpes and Varicella Zoster Virus Prophylaxis                                         |
| 11.2.5                                              | Fluconazole                                                                           |
| Ondansetron hydrochloride.                          |                                                                                       |
| 11.2.6                                              |                                                                                       |
| 11.2.7                                              | Furosemide                                                                            |
| REFERENCE LIST                                      |                                                                                       |
| 12                                                  |                                                                                       |  
13 FIGURES, TABLES & APPENDICES .  
13.1 Table 1  
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Version date: 02/15/2019 For the patients who did not have prior anti-PD1, a single stage design with 21 total evaluable patients will be used. Including the initial 8 patients (with 3 responders), this design will have 80% power to reject a 25% response rate in favor of a 50% response rate, with a one-sided 0.10 alpha level exact binomial test. In practice, the number of patients who respond out of the total nmber evaluated will be determined and presented along with 80% and 95% two-sided confidence intervals. As an example, if there are 8 responses in 21 evaluable patients in this cohort, an exact two-sided 80% confidence interval extends from 25.4% to 52.4%, thus demonstrating results potentially superior to 25% and consistent with 50%.  
For patients who have received prior anti-PD1 therapy, a two-stage Simon MinMax design will be used. This cohort will include the initial 3 patients (with 0 responses) as part of the first stage.  
This portion of the trial will seek to rule out a 10% response rate (p0=0.10) and target a 30%
response rate (p1=0.30). With alpha=0.10 (probability of accepting a poor treatment =0.10) and beta=0.10 (probability of rejecting a good treatment=0.10), this part of the trial will initially enroll 16 evaluable patients (including the 3 from the randomized portion) and if there is only 0 or 1 response out of 16 patients, no further patients will be enrolled. As it may take several weeks to determine if a patient has experienced a response, a temporary pause in the accrual may be necessary to ensure that enrollment to the second stage is warranted. If there are 2 or more responses in the initial 16 patients, then accrual may continue until there are 25 total evaluable patients in this cohort. If there are 2 to 4 responses in 25 patients, this would be an unacceptably low rate, while if there are 5 or more responses in 25 patients, this would be worthy of further evaluation. Under the null hypothesis (10% response rate), the probability of early termination is 51%.  
The response results will primarily be reported separately according to their prior use of anti-
PD1. Survival and progression free survival will also be estimated for the patients, overall and by prior anti-PD1 exposure.  
Toxicity will be evaluated by tabulating the types of toxicity observed and describing the results obtained. This will be done for all patients evaluated as part of the phase II designs as well as the initial patients randomized to receive low dose treatment.  
Patients who are  retreated on arm1P and receive pembrolizumab will be considered to be part of a pilot subset of the main protocol, intended for evaluation for responses separately from patients treated without pembrolizumab, with separate 95% confidence intervals formed around the response fractions of both groups. If the addition of this agent demonstrates the ability to produce at least a small fraction of patients with a clinical response, the combination may be tested in a more definitive subsequent study.  
The initial accrual under the randomized portion of the trial was 24 total patients. Eleven of these 24 who were treated with high dose chemo will be included among the phase II cohorts' maximum accrual of 21+25=46 patients. Thus, up to 13 (randomized to low dose or not treated)
+ 11 (randomized and treated with high dose, and to be included in phase 2) + 35 additional patients in phase 2 may be required (to arrive at 46 total in phase 2). In order to enroll up to these 59 patients, the accrual ceiling will be set at 64. Since 24 patients have already been enrolled, approximately 35 to 40 total additional patients may be enrolled. If the remaining patients are able to be accrued at the rate of approximately one per month, accrual should be completed within approximately 3 to 4 years after approval of amendment D, or 4 to 5 years after the trial initially was open for accrual.  
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Storage: Intact vials are stored in the refrigerator (2° - 8°C) protected from light. Each vial bears an expiration date.  
Dilution/Stability: Reconstituted aldesleukin should be further diluted with 50 mL of 5% Human Serum Albumin (HSA). The HSA should be added to the diluent prior to the addition of RIL-2.  
Dilutions of the reconstituted solution over a 1000-fold range (i.e., 1 mg/mL to 1 mcg/mL) are acceptable in either glass bottles or polyvinyl chloride bags. Aldesleukin is chemically stable for 48 hours at refrigerated and room temperatures, 2° - 30°C.  
Administration: The dosage will be calculated based on total body weight. The final dilution of aldesleukin will be infused over 15 minutes. Aldesleukin will be administered as an inpatient. Toxicities: Expected toxicities of aldesleukin are listed in the product label and in Appendix 3 and 4. Grade 3 toxicities common to aldesleukin include diarrhea, nausea, vomiting, hypotension, skin changes, anorexia, mucositis, dysphagia, or constitutional symptoms and laboratory changes as detailed in Appendix 3. Additional grade 3 and 4 toxicities seen with aldesleukin are detailed in Appendix 4.
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11.1.3 fludarabine

Description: (Please refer to package insert for complete product Information) Fludarabine phosphate is a synthetic purine nucleoside that differs from physiologic nucleosides in that the sugar moiety is arabinose instead of ribose or deoxyribose. Fludarabine is a purine antagonist antimetabolite. How Supplied: It will be purchased by the NIH Clinical Pharmacy Department from commercial sources. Fludarabine is supplied in a 50 mg vial as a fludarabine phosphate powder in the form of a white, lyophilized solid cake.  
Stability: Following reconstitution with 2 mL of sterile water for injection to a concentration of 25 mg/ml, the solution has a pH of 7.7. The fludarabine powder is stable for at least 18 months at 2-8°C; when reconstituted, fludarabine is stable for at least 16 days at room temperature. Because no preservative is present, reconstituted fludarabine will typically be administered within 8 hours. Specialized references should be consulted for specific compatibility information. Fludarabine is dephosphorylated in serum, transported intracellularly and converted to the nucleotide fludarabine triphosphate; this 2-fluoro-ara-ATP molecule is thought to be required for the drug's cytotoxic effects. Fludarabine inhibits DNA polymerase, ribonucleotide reductase, DNA primase, and may interfere with chain elongation, and RNA and protein synthesis.  
Storage: Intact vials should be stored refrigerated (2-8°C). Administration: Fludarabine is administered as an IV infusion in 100 ml 0.9% sodium chloride, USP over 15 to 30 minutes. The doses will be based on body surface area (BSA). If patient is obese (BMI > 35) drug dosage will be calculated using practical weight as described in Appendix 2. Toxicities: At doses of 25 mg/m 2 /day for 5 days, the primary side effect is myelosuppression; however, thrombocytopenia is responsible for most cases of severe and life-threatening hematologic toxicity. Serious opportunistic infections have occurred in CLL patients treated with fludarabine. Hemolytic anemia has been reported after one or more courses of fludarabine with  
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| Version date: 02/15/2019   | supportive        |                           |                 |                  |
|----------------------------|-------------------|---------------------------|-----------------|------------------|
| measures                   |                   |                           |                 |                  |
| Edema/Weight               | 3                 | Diuretics prn             | No              | No               |
| gain                       |                   |                           |                 |                  |
| Hypotension                | 3                 | Fluid resuscitation       | If uncontrolled | No               |
| despite all                |                   |                           |                 |                  |
| Vasopressor support        | supportive        |                           |                 |                  |
| measures                   |                   |                           |                 |                  |
| Dyspnea                    | 3 or 4            | Oxygen or ventilatory     | If requires     | No               |
| support                    | ventilatory       |                           |                 |                  |
| support                    |                   |                           |                 |                  |
| Oliguria                   | 3 or 4            | Fluid boluses or dopamine | If uncontrolled | No               |
| at renal doses             | despite all       |                           |                 |                  |
| supportive                 |                   |                           |                 |                  |
| measures                   |                   |                           |                 |                  |
| Increased                  | 3 or 4            | Observation               | Yes (grade 4)   | No               |
| creatinine                 |                   |                           |                 |                  |
| Renal failure              | 3 or 4            | Dialysis                  | Yes             | Yes              |
| Pleural                    | 3                 | Thoracentesis             | If uncontrolled | No               |
| effusion                   | despite all       |                           |                 |                  |
| supportive                 |                   |                           |                 |                  |
| measures                   |                   |                           |                 |                  |
| Bowel                      | 3                 | Surgical intervention     | Yes             | Yes              |
| perforation                |                   |                           |                 |                  |
| Confusion                  | Observation       | Yes                       | No              |                  |
| 3                          |                   |                           |                 |                  |
| Somnolence                 | 3 or 4            | Intubation for airway     | Yes             | Yes              |
| protection                 |                   |                           |                 |                  |
| Arrhythmia                 | 3                 | Correction of fluid and   | If uncontrolled | No               |
| electrolyte imbalances;    | despite all       |                           |                 |                  |
| chemical conversion or     | supportive        |                           |                 |                  |
| electrical conversion      | measures          |                           |                 |                  |
| therapy                    |                   |                           |                 |                  |
| Elevated                   | 3 or 4            | Observation               | Yes             | If changes in LV |
| Troponin                   | function have not |                           |                 |                  |
| levels                     | improved to       |                           |                 |                  |
| baseline by next           |                   |                           |                 |                  |
| dose                       |                   |                           |                 |                  |  
| '()*+,-.+/  012+,*3.)1                               | !           | 4566),3.78 *+,8                        | 98:                                                      | 98:   |
|------------------------------------------------------|-------------|----------------------------------------|----------------------------------------------------------|-------|
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| E853,)681.+                                          | !           | >?:8,7+3.)1                            | E)                                                       | E)    |
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