Versus-Host Disease (Gvhd) with Amniotic Fluid Eye Drops (Afed)

Abbreviated Title: AFED for the treatment of Ocular GVHD Version Date: 12SEP2017 Principal Investigator: Daniel Couriel, MD

A RANDOMIZED, DOUBLE-BLINDED, PLACEBO-CONTROLLED STUDY FOR THE TREATMENT OF OCULAR CHRONIC GRAFT- VERSUS-HOST DISEASE (GVHD) WITH AMNIOTIC FLUID EYE DROPS (AFED)

IRB # 103515 NCT # HCI-17-HEM-19

Principal Investigator Daniel Couriel, MD, MS Division of Hematology & Hematologic Malignancies Huntsman Cancer Institute - University of Utah 2000 Circle of Hope, Salt Lake City, UT 84112 [email protected]

Sub-Investigators Catherine Lee, MD Division of Hematology & Hematologic Malignancies Huntsman Cancer Institute - University of Utah 2000 Circle of Hope Salt Lake City, UT 84112 [email protected]

Michael Boyer, MD Department of Pediatrics, Primary Children’s Hospital Division of Hematology & Hematologic Malignancies Huntsman Cancer Institute - University of Utah 1950 Circle of Hope Salt Lake City, UT 84112 [email protected] Vedran Radjocic, MD Division of Hematology & Hematologic Malignancies Huntsman Cancer Institute - University of Utah 2000 Circle of Hope, Room 4246 Salt Lake City, UT 84112 [email protected]

Axel Zander, MD Division of Hematology & Hematologic Malignancies Huntsman Cancer Institute - University of Utah 2000 Circle of Hope Salt Lake City, UT 84112 [email protected]

Abbreviated Title: AFED for the treatment of Ocular GVHD Version Date: 12SEP2017

Sub-Investigators John Phillips, PhD Division of Hematology and Hematologic Malignancies University of Utah School of Medicine 5C330 SOM, 30 N. 1900 E. Salt Lake City UT 84132 [email protected]

JoAnna Reems, PhD Cell Therapy & Regenerative Medicine University of Utah Health Sciences 675 Arapeen Drive, Suite 300 Salt Lake City, Utah 84108 [email protected]

Jan Pierce, MBA Cell Therapy & Regenerative Medicine University of Utah Health Sciences 675 Arapeen Drive, Suite 300 Salt Lake City, Utah 84108 [email protected]

Susan Schulman HSEB Clinical Skills Center 26 S 2000 E RM 3550 Salt Lake City, UT 84112 [email protected] Ophthalmology Dix Pettey John A Moran Eye Center 65 N Mario Capecchi Salt Lake City, UT 84132 [email protected]

Statistician Ken Boucher, PhD Huntsman Cancer Institute 2000 Circle of Hope, Room: 4245 Salt Lake City, UT 84112 [email protected]

Investigational agent

IND Number Exempt

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TABLE OF CONTENTS

TABLE OF CONTENTS ...... 3 LIST OF ABBREVIATIONS ...... 5 PROTOCOL SIGNATURE ...... 8 STUDY SUMMARY...... 9 1. OBJECTIVES ...... 11 1.1. Primary Objective ...... 11 1.1.1.2. Changes in the ophthalmologic performance of patients (dry eye symptoms)...... 11 1.2. Secondary Objectives ...... 11 2. BACKGROUND AND RATIONALE ...... 12 2.1. Chronic GVHD ...... 12 2.2. Amniotic Fluid ...... 12 3. DRUG INFORMATION ...... 15 3.1. Description ...... 15 3.2. Potential Side Effects and Hazards ...... 15 3.3. Lot Information ...... 15 4. STUDY DESIGN ...... 16 4.1. Description ...... 16 4.2. Number of Patients ...... 16 4.3. Number of Study Centers ...... 16 4.4. Study Duration ...... 16 5. ELIGIBILITY CRITERIA ...... 17 5.1. Inclusion Criteria ...... 17 5.2. Exclusion Criteria ...... 17 6. TREATMENT PLAN...... 18 6.1. Administration Schedule ...... 18 6.2. Treatment Information ...... 18 6.3. Prohibited Concomitant Medications ...... 19 6.4. Duration of Therapy ...... 19 7. TOXICITIES AND DOSEAGE MODIFICATION...... 20 8. CALENDAR ...... 21

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9. CRITERIA FOR EVALUATION OF ENDPOINTS ...... 21 9.1. Primary Objective: Clinical Effects ...... 21 Parameter d measures the changes in the ophthalmologic performance of patients ...... 22 9.1.2. Definition of Clinical Response ...... 22 9.2. Secondary Objectives ...... 22 10. STATISTICAL CONSIDERATIONS...... 23 11. REGISTRATIONS GUIDELINES ...... 23 12. DATA SUBMISSION SCHEDULE ...... 24 13. ETHICAL AND REGULATORY CONSIDERATIONS ...... 24 13.1. Informed Consent ...... 24 13.2. Institutional Review ...... 24 13.3. Data and Safety Monitoring Plan ...... 24 13.4. Adverse Events / Serious Adverse Events ...... 25 13.5. SAE Reporting Requirements ...... 26 13.6. Protocol Amendments ...... 27 13.7. Protocol Deviations ...... 27 13.8. FDA Annual Reporting ...... 28 13.9. Clinical Trials Data Bank ...... 28 13.10. Record Keeping ...... 28 14. REFERENCES ...... 29 APPENDIX A Patient Instructions and Dosing Diary ...... 31 APPENDIX B FACT-G Quality of Life Questionnaire ...... 33 APPENDIX C Health Care Provider Global Rating of Chronic GVHD ...... 35 APPENDIX D Ocular Rating of GVHD (Eye Score) ...... 36 APPENDIX E Patient Global Rating of GVHD ...... 37 APPENDIX F Dry Eye Severity Grading Scheme ...... 38

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LIST OF ABBREVIATIONS

Abbreviation or Term1 Definition/Explanation AE Adverse event AF Amniotic fluid AFED Amniotic fluid eye drops ALT Alanine aminotransferase ANCOVA Analysis of covariance ANOVA Analysis of variance APTT Activated partial thromboplastin time AST Aspartate aminotransferase AV Atrioventricular β-HCG Beta-human chorionic gonadotropin BID Twice daily BLQ Below limit of quantification BMI Body mass index BP Blood pressure BUN Blood urea nitrogen C-section Cesarean section Ca++ Calcium CBC Complete blood count CFR Code of Federal Regulations CHF Congestive heart failure CI Confidence interval Cl- Chloride

CLcr Creatinine clearance

Cmax Maximum observed concentration

Cmin Trough observed concentration CNS Central nervous system CR Complete response CRF Case report form CT Computed tomography CTCAE Common Toxicity Criteria for Adverse Events CTRM Cell Therapy and Regenerative Medicine Program at the University of Utah CV Coefficient of variation

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Abbreviation or Term1 Definition/Explanation CYP Cytochrome P450 D/C Discontinue ECOG Eastern Cooperative Oncology Group eCRF Electronic case report form DLT Dose Limiting Toxicity ECG Electrocardiogram Eg Exempli gratia (for example) FACS Fluorescence Activated Cell Sorting FDA Food and Drug Administration FDG-PET Fluorodeoxyglucose (FDG)-positron emission tomography (PET) GCP Good Clinical Practice GFR Glomerular filtration rate GGT Gamma glutamyl transferase GLP Good laboratory practice HA Hyaluronic acid HBsAg Hepatitis B surface antigen HBV Hepatitis B virus

- HCO3 Bicarbonate HCV Hepatitis C virus HIV Human immunodeficiency virus HR Heart rate hr Hour or hours

IC50 Half maximal inhibitory concentration i.e. Id est (that is) IEC Independent ethics committee INR International normalized ratio IRB Institutional review board IU International unit IV Intravenous, intravenously LDH Lactate dehydrogenase LLQ Lower limit of quantitation MedRA Medical Dictionary for Drug Regulatory Activities MRI Magnetic resonance imaging

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Abbreviation or Term1 Definition/Explanation MRSD Maximum recommended starting dose MTD Maximum tolerated dose NOAEL No-observed-adverse-effect level NOEL No-observed-effect-level PD Pharmacodynamic(s) PFS Progression Free Survival PK Pharmacokinetic(s) PO Per os (administered by mouth) PR Partial response PT Prothrombin time PTT Partial thromboplastin time QC Quality control RBC Red blood cell QD Once daily QTc QT interval corrected QTcF QT interval corrected using Frederichia equation SAE Serious adverse event SD Standard deviation or stable disease

T1/2 Terminal elimination half-life

T3 Triiodothyronine

T4 Thyroxine

Tmax Time of maximum observed concentration TID Three times daily TSH Thyroid-stimulating hormone ULN Upper limit of normal ULQ Upper limit of quantitation UV Ultraviolet WBC White blood cell WOCBP Women of childbearing potential WONCBP Women of nonchildbearing potential

1 All of these abbreviations may or may not be used in protocol

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PROTOCOL SIGNATURE

I confirm that I have read this protocol, and I will conduct the study as outlined herein and according to the ethical principles stated in the latest version of the Declaration of Helsinki, the applicable ICH guidelines for good clinical practice, and the applicable laws and regulations of the federal government. I will promptly submit the protocol to the IRB for review and approval. Once the protocol has been approved by the IRB, I understand that any modifications made during the course of the study must first be approved by the IRB prior to implementation except when such modification is made to remove an immediate hazard to the subject. I will provide copies of the protocol and all pertinent information to all individuals responsible to me who assist in the conduct of this study. I will discuss this material with them to ensure that they are fully informed regarding the study treatment, the conduct of the study, and the obligations of confidentiality. Note: This document is signed electronically through submission and approval by the Principal Investigator in the University of Utah IRB Electronic Research Integrity and Compliance Administration (ERICA) system.

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STUDY SUMMARY

Title A Randomized, Double-Blinded, Placebo-Controlled Study for the Treatment of Ocular Chronic GVHD with Amniotic Fluid Eye Drops (AFED). Short Title AFED for the treatment of Ocular GVHD. IRB Number 103515 IND Exempt Phase Pilot Phase II Design This is a randomized (to each eye, within patient), double-blinded, placebo-controlled study of the efficacy of AFED in patients with hematologic malignancies who have undergone Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) and have been diagnosed with chronic GVHD of the eye. Study Duration Two years Study Center(S) Single Center – Huntsman Cancer Institute Objectives Primary Objective: To determine the clinical effects of AFED in ocular chronic GVHD after 1 month of treatment. Secondary Objectives:  To determine the safety of AFED in patients with ocular chronic GVHD up to 3 months of treatment.  To determine the change in National Institutes of Health (NIH) Consensus Criteria (CC) ocular score of chronic GVHD after up to 3 months of treatment with AFED.  To determine changes in quality of life (measure using FACT-G questionnaires) after 1 and 3 months of treatment with AFED.  To determine changes in visual acuity up to 3 months of treatment with AFED.  To determine the effects up to 3 months of treatment with AFED on the corneal surface.  To determine the changes in dry eye symptoms using the grading provided by the International Dry Eye Workshop (DEWS) 2007 report up to 3 months of treatment with AFED. Number Of Subjects 15 patients (30 eyes). Diagnosis And Main Inclusion: Eligibility Criteria Patients aged 18 years or older with chronic GVHD diagnosed within 3 years after hematopoietic stem cell transplant (HSCT) for any disease, with any graft, and any conditioning regimen with dry eye (patients may be using PROSE lenses at the time of accrual). Exclusion: 1. Patients who have any other reversible cause for dry eye at the time of accrual.

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2. Patients treated with more than 2 lines of therapy beyond corticosteroids with or without calcineurin inhibitors or sirolimus. 3. Patients whose malignancy relapsed after transplantation. 4. Patients with a difference in dryness between both eyes of more than 2 points of the grading provided by the International Dry Eye Workshop (DEWS) 2007 report. Study Product, Dose, AFED will be given in a blinded fashion as one drop (0.25 mL) in one Route, Regimen eye twice daily (the other eye will receive normal saline, the placebo). In patients wearing PROSE lenses, the drops will be applied prior to lens insertion in the morning and immediately after removal at night. Duration of One (1) month. In patients who are responsive to therapy with Administration acceptable toxicity, the treatment period will be extended to 3 months. Statistical This is a randomized (within patient between eyes), double-blinded, Methodology placebo-controlled study. The clinical effects of AFED eye drops will be analyzed using descriptive statistics, t-test, and Fisher’s exact test where applicable. Differences in NIH scores will be analyzed using Wilcoxon signed-rank test. The proportion of subjects who experience adverse events and serious adverse events will be summarized descriptively; no inferential tests will be performed.

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1. OBJECTIVES

1.1. Primary Objective 1.1.1. To determine the clinical effects of AFED in ocular chronic GVHD. Endpoint: the endpoint associated with this objective is clinical response which is a composite of the following four parameters evaluated after 1 month of treatment with AFED: 1.1.1.1. Changes in National Institute of Health Consensus Conference (NIHCC) for assessment of response in chronic GVHD: a. Health Care Provider Global Rating. b. Eye Score. c. Patient Global Rating.

1.1.1.2. Changes in the ophthalmologic performance of patients (dry eye symptoms).

1.2. Secondary Objectives 1.2.1. To determine the safety of AFED in patients with ocular chronic GVHD. Endpoint: frequency and severity of adverse events observed after up to 3 months of treatment with AFED.

1.2.2. To determine the individual changes in NIHCC for assessment of response in chronic GVHD ratings after up to 3 months of treatment with AFED. Endpoints: a. Changes in Health Care Provider Global Rating b. Changes in Eye Score Rating. c. Changes in Patient Global Rating.

1.2.3. To determine the changes in dry eye symptoms after up to 3 months of treatment with AFED. Endpoint: changes in grading according to the International Dry Eye Workshop (DEWS) 2007 report.

1.2.4. To determine the effects of 1 and 3 months of treatment with AFED on the quality of life of patients with ocular chronic GVHD. Endpoint: changes in FACT-G QOL questionnaires scores.

1.2.5. To determine the effects of up to 3 months of treatment with AFED on the visual acuity and corneal surface of patients with ocular chronic GVHD. Endpoint: change in visual acuity and corneal surface

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2. BACKGROUND AND RATIONALE

2.1. Chronic GVHD Allogeneic (Allo) Hematopoietic Stem Cell Transplantation (HSCT) is a potentially curative treatment modality for patients with hematologic malignancies who would otherwise have a poor outcome with other conventional treatment approaches alone. Allo HSCT causes donor- derived immune responses that can result in the desired graft-versus-tumor effect as well as an undesired complication, Graft versus Host Disease (GVHD). Chronic GVHD is the main complication for long term survivors of a successful allo HSCT. Chronic GVHD occurs in more than 50% of all patients who undergo an allo HSCT and the majority of patients who develop acute GVHD as a complication of their allo HSCT. Chronic GVHD can involve multiple organs and require prolonged immunosuppressive therapy. Advanced chronic GVHD is typically manifested by significant fibrous tissue deposition in different organs, leading to disabling symptoms, most commonly as a consequence of sicca syndrome and sclerodermatous forms of the disease. Sicca syndrome can lead to severe pain, visual impairment, and mucositis leading to malnutrition. Sclerodermatous chronic GVHD can cause dramatic limitations in the range of motion, with different degrees of immobility, and occasionally restrictive pulmonary disease. At this stage, current treatment strategies are almost always ineffective, at least in part due to the irreversibility of manifestations related to fibrosis. The mainstay of first-line immunosuppressive therapy in patients with chronic GVHD is systemic glucocorticoids and there are no standard second-line therapies. Systemic glucocorticoids have limited efficacy and significant long term complications. Despite the many alternative immunosuppressive agents to systemic glucocorticoids, no single class of immunosuppressive agents has persistently produced a steroid-sparing effect in patients with chronic GVHD.1-7 In conclusion, chronic GVHD and its current standard therapy have a major negative impact on the quality of life (QOL) and survival in patients in whom allo HSCT was able to achieve a cure from their original hematologic malignancy. Therefore, there is a desperate need for more effective agents in treating chronic GVHD. Ocular GVHD in particular affects more than half of the patients within 2 years of the diagnosis of chronic GVHD.8,9 The fibroproliferative nature of ocular chronic GVHD makes it irreversible in its advanced stages, and at this point systemic therapies are ineffective. Thus, the management is supportive, through control of surface inflammation, lubrication, control of drainage and evaporation.10 Despite some advances in the area of supportive care, such as PROSE scleral lenses that effectively control evaporation, the problem of ocular chronic GVHD is far from resolved and it is still associated with worse overall health-related quality of life.9

2.2. Amniotic Fluid 2.2.1. Preclinical Data Early after conception and until the mother’s water breaks for the delivery of their infant, the fetus is bathed in amniotic fluid (AF). AF functions as a supportive cushion to the fetus and provides a protective environment. AF is a rich source of nutrients, cytokines and growth

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factors that are required for fetal development and maturation.11 AF also contains stem cells with the potential to differentiate along multiple cell lineages.12,13 The protective and regenerative properties of AF are achieved via the exchange of water and solutes with surrounding tissues. This is accomplished via the utilization of different pathways during the course of a pregnancy that likely contribute to changes in the composition of the AF with gestational age.11 Among some of the first evidence that AF has protective biological properties is a report describing that concentrates of AF inhibit the development of peritonitis.14 This is followed by a report by Shimberg and co-workers that AF accelerates defense-repair mechanisms within damaged joints.14,15 Since these early publications, more sophisticated evaluations have revealed the presence of antimicrobial, immunomodulatory, and growth-promoting activities in AF.11 Reports of antimicrobial activity in AF differ among investigators.16 Some studies show that AF is inhibitory, while others show no effect against the same microorganisms. Another report provides evidence that AF with low antimicrobial activity is associated with a high incidence of an infectious syndrome in pregnant women.17 Components with antimicrobial, antiviral and antifungal activity that are present in AF include lysozyme, peroxidase, transferrin, β-lysin, immunoglobulins and zinc-peptide complexes.16 Immunomodulatory properties of AF are evident from studies showing that enteral feeding of AF suppresses the pro-inflammatory responses in preterm pigs with necrotizing enterocolitis.18 While growth promoting activities of AF are supported by animal studies and by in vitro culture studies showing that AF can enhance neochondrogenesis,19 regenerate peripheral nerves20 and bone,21 accelerate re- epithelialization in corneas,22 and promote healing of human skin wounds.23 Some of the factors that are found in AF that may contribute to these activities include inflammatory mediators that include, but are not limited to TNF- α, IL-6, IL8, and IL-10,24 trophic factors that include EGF, IGF-1, FGF, HGF and TGF-α,25-29 and HA, an important factor in promoting re-epithelialization in human skin wounds.23 Based on the hypothesis that nutrients, cytokines and growth factors contained in the non- cellular fraction of AF are useful for reparative and regenerative treatments in patients, Pierce et al conducted a study at the Cell Therapy and Regenerative Medicine Program (CTRM) at the University of Utah to address three issues. The first was to determine the feasibility of consenting and screening volunteer donors for the routine collection of AF from full-term pregnant women that were scheduled for caesarean-section (C-sections) and for processing the AF for clinical applications. The second aim was to develop a processing method that resulted in a cell-free AF preparation suitable for clinical applications. The third goal was to gain a better understanding about components of AF procured from full-term pregnancies. Human AF was collected by the staff of the Obstetrical and Gynecological department at the University of Utah hospital. A physician-executed abdominal incision was performed through the abdominal and uterine muscles without cutting into the amnion membrane. Using a sterile soft suction catheter connected to a sterile MediVac Suction Container (Cardinal Health, Waukegan, IL), a blunt end insertion with a catheter was made into the amnion membrane and the AF was aseptically suctioned into a MediVac Container. The container was labelled, wrapped in frozen Insul-ice mats (Fisher Scientific, Hanover Park, IL) and placed in a temperature monitored shipper that is validated for transport between 2 and 8 °C to the Cell Therapy and Regenerative Medicine (CTRM) facility at the University of Utah.

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Upon arrival at the facility, the product was immediately placed in a refrigerator at 2–8 °C until processing occurred. The MediVac container with AF was aseptically placed in a biological safety cabinet and the AF was transferred via aseptic techniques into sterile centrifuge tubes. The total volume and gross appearance of the AF were recorded and samples were removed for sterility testing, cell counts and other relevant testing. The AF was centrifuged at 1400 g for 20 min at 4 °C. Once centrifugation was complete, the supernatant was expressed into a new transfer pack and the remaining cell pellet was characterized and cultured as described below. To produce AFED, the supernatant from the AF was processed using a proprietary filtration technology to sterilize and eliminate cellular debris from the final product. Thirty-six pregnant women consented and passed donor screening to give birth tissue. AF was successfully collected from 17 individuals. Median AF volumes were 70 mL (range 10–815 mL; n = 17). Fluid chemistries were similar, but some differences were noted in HA levels and cytokine profiles. Cytokine arrays revealed that an average of 304 ± 20 of 400 proteins tested were present in AF with a majority of cytokines associated with host defense. AF also supported angiogenesis. Some of the most frequently encountered classified according to function can be found in the table 1. AFs were evaluated for total volume, fluid chemistries, total protein, and hyaluronic acid (HA) levels. AF was also assessed with quantitative antibody arrays, cellular content and for an ability to support angiogenesis. Epithelioid cells were the major cell type in AF with only a minor population of lymphoid cells. Cultures revealed a highly proliferative population of adherent cells capable of producing therapeutic doses of mesenchymal stromal cells (MSCs). Variations noted in components present in different AFs, warrant further investigations to determine their relevance for specific clinical applications.

Table 1: Common cytokines identified in amniotic fluid, grouped by function. Pro-inflammatory OPN, PAI-I, CD163, RAGE, IL17, IL1R3 Host defense IL-27, LAG-3, GITR, PD1 Innate Immunity hCGb, Galectin-3, TLR-2, Osteoactivin Antimicrobial TSP-1, lactoferrin, CXCL14, Trappin-2, CCL-28, MIG Anti-inflammatory IL1-ra, MBL Embryonic development DKK1, DKK3 Angiogenesis VEGF R1, Transferring, TIMP-2 Wound healing OPN, PAPP-A, FAP

2.2.2. Preliminary Clinical Data Most of the clinical experience at the University of Utah has been in the form of amniotic-fluid embedded membranes applied to patients with second (n=3), third degree (n=1) and electrocution burns (n=1), all of which reveled signs of complete (n=3) or partial healing (n=2), including reduction of pain. An additional 2 patients with chronic ulcers were treated, one related to multiple surgeries and the second one in the setting of sclerodermatous chronic GVHD. In both cases, complete resolution of the ulcer was observed in both patients at 53 and 21 days respectively (unpublished observations).

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AFED were used in a total of 2 patients who were treated at the University of Utah, at a dose of 0.5 ml per eye daily in 2 divided doses, and no toxicities were observed. One patient with severe dry eye had a good partial response with a decrease in the NIH CC eye score and decrease in the need for artificial tears and the use of PROSE lenses. The second patient had stabilization of the disease after discontinuation of all immunosuppression.

3. DRUG INFORMATION

3.1. Description AFED are prepared from birth tissues donated after Cesarean Section delivery from a volunteer donor. The amniotic fluid is retrieved and processed within 72 hours from the time of C- section. The surgical site is prepped according to AORN standards for incision disinfection. Prior to delivery, the amniotic fluid is aseptically aspirated into a sterile container using a soft suction catheter. The donor from whom the allograft is derived must be tested and found to be negative for the following: HBsAg (Hepatitis B Surface Antigen), HBcAb (hepatitis B core antibody), HCV (hepatitis C antibody), HIV I/II-Ab (Antibody to Human Immunodeficiency Virus Types 1 and 2), Syphilis detection test, HIV NAT (HIV Nucleic Acid Test), and HCV NAT (HCV Nucleic Acid Test). Additional tests for other communicable diseases, such as West Nile Virus, T. Cruzi, Cytomegalovirus and Epstein Barr Virus can be performed and their results found to be negative. Donor screening tests are performed by laboratories which are FDA registered for HCT/P, certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA), and use FDA-licensed tests when available, including LABS, Inc., ARUP, Clongen, or VRL Laboratories. Donor selection is based on medical and social history and must meet the Standards of the American Association of Tissue Banks (AATB), United States Public Health Service (USPHS), and the Federal Food and Drug Administration (FDA). Donor suitability is determined by the Manufacturing Facilities’ Medical Directors.

3.2. Potential Side Effects and Hazards Alloimmunization of the recipient to donor histocompatibility antigens may be a consequence of human AFED administration. Side effects and hazards should be considered and weighed against the limitations of alternative eye drops. The manufacturer utilizes strict donor screening procedures to avoid the collection of tissues from donors who may carry infectious agents. Despite rigorous donor screening and laboratory testing, this tissue may transmit infectious agents. Any transmission of disease that is suspected to be caused by the AFED must be reported promptly to Cell Therapy and Regenerative Medicine at the University of Utah (CTRM). Please contact CTRM to report any adverse reactions.

3.3. Lot Information Due to the variable and limited amounts of AF obtained from each donor (200 mL on average), it is not possible for the entire supply of AFED required to treat 15 patients for 3 months to be

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obtained from a single donor. Several lots of AFED will be required to complete the study. Each patients’ supply of AFED for the duration of the study will originate from the same lot.

4. STUDY DESIGN

4.1. Description This is a randomized, double-blinded, placebo-controlled study of AFED used for the treatment of chronic ocular GVHD.

4.2. Number of Patients A total of 15 patients will be enrolled. Treatment with AFED will be randomly assigned for each patient to one eye. The other eye will receive placebo.

4.3. Number of Study Centers This study will be conducted at a single center, the Huntsman Cancer Institute.

4.4. Study Duration Accrual, treatment, and follow up will take approximately 2 years.

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5. ELIGIBILITY CRITERIA

This eligibility checklist is used to determine patient eligibility and filed with the enrolling investigators signature in the patient research chart.

Patient No. ______Patient’s Initials (L,F,M) ______

5.1. Inclusion Criteria Yes/No (Response of “no” = patient ineligible) 5.1.1. _____ Patients with chronic ocular GVHD diagnosed within 3 years after allogeneic hematopoietic stem cell transplant (HSCT) for any hematological malignancy, with any graft and any conditioning regimen, with dry eye (patients may be using PROSE lenses at the time of accrual). 5.1.2. _____ Patients must be ≥ 18 years of age at enrollment. 5.1.3. _____ Patients must be able to provide informed consent and willing to sign an approved consent form that conforms to federal and institutional guidelines.

5.2. Exclusion Criteria Yes/No (Response of “yes” = patient ineligible) 5.2.1. _____ Patients who have any other reversible cause for dry eye at the time of accrual. 5.2.2. _____ Patients treated with more than 2 lines of systemic therapy beyond corticosteroids with or without calcineurin inhibitors or sirolimus. 5.2.3. _____ Patients with a relapsed malignancy after transplantation. 5.2.4. _____ Patients with difference in dryness between both eyes of more than 2 points of the grading provided by the International Dry Eye Workshop (DEWS) 2007 report (see Appendix F, based on the single highest category score in the DEWS grading scale) 5.2.5. _____ Patients with any active ocular infection 5.2.6. _____ Patients with a history or current evidence of any condition, therapy, or laboratory abnormality that might confound the results of the trial, interfere with the subject’s participation for the full duration of the trial, or is not in the best interest of the subject to participate, in the opinion of the treating investigator.

I certify that this patient meets all inclusion and exclusion criteria for enrollment onto this study.

______Investigator Signature Date Time

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6. TREATMENT PLAN

6.1. Administration Schedule Treatment will be given as one drop (0.25 ml) of either AFED or placebo (normal saline) in the appropriate eye twice daily. In patients wearing PROSE lenses, the drops will be applied prior to lens insertion in the morning and immediately after removal at night.

6.2. Treatment Information

6.2.1. How Supplied, Stored, Packaged and Labeled Patients will receive a monthly supply of study treatment from the CTRM in the form of:  31 bottles containing 500 µl of AFED.  31 bottles containing 500 µl of placebo. Eye drops will be delivered to the patient in a thermosafe box packed with dry ice. They must be transferred to a freezer after transportation and remain frozen until use. Both placebo and AFED will be packaged in identical bottles labelled with the following information:  IRB number and patient study ID  Content: Amniotic Fluid Ey Drops (in order to maintain blinding the placebo bottle will also be labeled as AFED)  Lot number (which pertains to donor ID number, manufacturing batch record, internal identification number, QC/QA review and release, and inventory control system)  Intended eye (right or left)  Storage information (-15 °C) and expiration date  Identification code (to allow cross referencing to the randomization code by CTRM) CTRM will maintain a randomization log based on a randomization code provided by the biostatistician. For each patient the log will capture the following:  Patient study identification in the format IRB number - patient number  Contents of “right eye” bottle (placebo or AFED)  Contents of “left eye” bottle (placebo or AFED) Patient will also receive verbal and written instructions detailing the above (see Appendix A).

6.2.2. Preparation and Administration Each morning, the patient will take one bottle from the “right eye” box and from the “left eye” box and remove them from the freezer. After thawing the eye drops from 5 to 10 minutes, the patient will uncap each bottle and self-administer one drop of fluid in the appropriate eye. Following the morning administration, both bottles will be placed in the refrigerator until the time of the evening administration. The bottles will then be discarded.

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Patients will be provided with a dosing journal (Appendix A) in which they must record the following:  Date  Time bottles were removed from freezer  Time of morning administration (for each eye)  Time bottles were refrigerated  Time bottles were removed from refrigerator  Time of evening administration (for each eye) They will be asked to return their diary as well as any unused bottles (or their empty boxes) at each clinic visit.

6.2.3. Accountability Accountability and compliance should be handled per institutional policies Patients will be provided with a dosing dairy to track ocular treatments. All eye drops will be stored at the CTRM laboratory in accordance with Good Clinical Practice (GCP) and GMP requirements as well as the instructions given by the clinical supplies department of the CTRM, and will be inaccessible to unauthorized personnel. CTRM must maintain a careful record of the inventory and disposition of the agent. An adequate record of receipt, distribution, and return of all study drugs must be kept in the Drug Accountability Form. Subject compliance with the treatment and protocol includes willingness to comply with all aspects of the protocol, and to have blood collected for all safety evaluations. At the discretion of the principal investigator, a subject may be discontinued from the trial for non-compliance with follow-up visits or study drug. Instructions on medication resupply and destruction will be made available to affected parties as applicable. At the conclusion of the study, and, as appropriate, during the course of the study, the Investigator will return all used and unused study drug, packaging, drug labels, and a copy of the completed drug accountability ledger to the CTRM.

6.3. Prohibited Concomitant Medications There are no prohibited concomitant medications on this study.

6.4. Duration of Therapy Patients will self-administer treatment daily for 1 month. In patients who are responsive to therapy with acceptable toxicity (as determined by the investigator), the treatment period will be extended up to 3 months.

6.4.1. Patient Withdrawal from Study Patients can be withdrawn from the study for the following reasons:  Noncompliance with therapy

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 Significant worsening of ocular chronic GVHD after at least 8 weeks of therapy. This decision will be at the discretion of both the treating transplant physician and ophthalmologist  Any other condition that in the opinion of the treating physician and ophthalmologist are not compatible with participation in this study

6.4.2. Discontinuation of Study Significant worsening of ocular chronic GVHD (either disease progression or unacceptable toxicity as determined by the investigator) in the eye treated with AFED during the first month of treatment in > 8 patients will result in the discontinuation of the study.

7. TOXICITIES AND DOSEAGE MODIFICATION

No toxicities are expected and no dosing modifications or interruptions will be allowed.

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8. CALENDAR

Procedure Screening1 End of End of End of Week 122 Week 42 Week 82 End of Study Informed consent X Medical history X Eligibility criteria X Vital signs X X X X Physical examination X X X X NIH CC Health Care Provider Global Rating X X X X NIH CC Health Care Provider Eye Score X X X X NIH CC Patient Global Rating. Visual Exam - dry eye severity level3 X X X X FACT-G QOL questionnaire4 X X X Eye drops X5 Safety Assessment X X X 1. All screening procedures must be completed within 28 days of registration. Treatment must begin within 5 days after registration. 2. Visits will have a ± 3 day window 3. Visual Exam includes visual symptoms, severity and frequency of discomfort, conjunctival injection, conjunctival staining, corneal staining, corneal/tear signs (including filameary keratitis and debris), the eyelids and meibomian glands, and fluorescein tear break up time. Scored 1 to 4 4. http://www.facit.org/FACITOrg/Questionnaires, also listed in Appendix B. 5. Patients will continue to receive treatment after 4 weeks only if they are responsive to therapy with acceptable toxicity in the opinion of the investigator.

9. CRITERIA FOR EVALUATION OF ENDPOINTS

9.1. Primary Objective: Clinical Effects The assessment of clinical effects will be performed after 1 month of therapy with AFED in comparison to baseline for each eye. The associated endpoint is clinical response. Patients will be evaluable for the primary objective if they complete at least 20 full days of treatment (i.e. self-administer ≥ 40 doses of eye drops) during the first 28 days.

9.1.1. Parameters Used to Define Clinical Response Clinical response is defined as a composite of the following four parameters: a. Health Care Provider Global Rating (Appendix C). b. Eye Score (Appendix D). c. Patient Global Rating (Appendix E). d. Dry Eye Score. Parameters a-c measure the changes in a patient’s chronic ocular GVHD in response to treatment and will be assessed using ratings defined by the National Institute of Health

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Consensus Conference (NIHCC) for assessment of response in chronic GVHD, providing three of the four parameters: Parameter d measures the changes in the ophthalmologic performance of patients in response to treatment and will be assessed using the grading scale for signs and symptoms of dry eye provided by the International Dry Eye Workshop (DEWS) 2007 report found in Appendix F.30 Assessment will include visual symptoms, severity and frequency of discomfort, conjunctival injection, conjunctival staining (using NaFl), corneal staining (using NaFl), corneal/tear signs (including filamentary keratitis and debris), the eyelids and meibomian glands, and fluorescein tear break-up time (TFBUT). Each of the signs/symptoms will be graded on a scale of 1 to 4, with 4 being most severe. A total score (the Dry Eye Score) will be obtained at each exam. A cumulative decrease in overall score > 2 will constitute improvement with treatment. Note: Schirmer testing will not be performed or graded as it has no proven value in ocular chronic GVHD.

9.1.2. Definition of Clinical Response  A complete response (CR) will be defined as resolution of all manifestations of ocular chronic GVHD (i.e. eye score=0), without any worsening in the health care provider or the patient global ratings. Additionally, the ophthalmologic assessment needs to show resolution of manifestations of dry eye in the dry eye grading scale (Appendix F).  A partial response (PR) will be defined as improvement of manifestations of ocular chronic GVHD manifested by a decrease in the eye score by at least 1 point, without any worsening in the health care provider or the patient global ratings. Additionally, there should be a decrease (improvement) of at least 1 point in the dry eye grading scale. Cases with at least 1 point decrease (improvement) in the dry eye grading scale without worsening of NIH CC parameters will also be considered PR.  Progressive Disease (PD) will be defined as increase (worsening) by at least 1 point in the dry eye grading scale irrespective of NIH CC parameters.  Overall response rate (ORR) is defined as CR+PR.

9.2. Secondary Objectives 9.2.1. Safety and Tolerability This endpoint will be evaluated up to 3 months after initiation of therapy based on the results of reported signs and symptoms, scheduled physical examinations, and frequency and severity of adverse events. Toxicities will be reported using Common Terminology Criteria for Adverse Events (CTCAE) version 4. More frequent safety evaluations may be performed if clinically indicated or at the discretion of the investigator.

9.2.2. Changes in Chronic GVHD This endpoint will be evaluated individually (as opposed to being a part of a composite endpoint) after up to 3 months of treatment with AFED. As described in section 9.1.1., changes

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in a patient’s chronic ocular GVHD in response to treatment will be assessed using ratings defined by the National Institute of Health Consensus Conference (NIHCC) for assessment of response in chronic GVHD (found in Appendices C through E).

9.2.3. Changes in Dry Eye Symptoms This endpoint will be evaluated individually (as opposed to being a part of a composite endpoint) after up to 3 months of treatment with AFED. As described in section 9.1.2, signs and symptoms of dry eye will be assessed at each exam using the grading scale provided by the International Dry Eye Workshop (DEWS) 2007 reportError! Bookmark not defined. found in Appendix F.

9.2.4. Changes in Quality of Life Changes in FACT G QOL questionnaires scores will be assessed at 1 and 3 months related to the start of administration of AFED and compared to baseline scores.

9.2.5. Changes in Visual Acuity and Corneal Surface The effects of up to 3 months of treatment with AFED on a patient’s visual acuity and corneal surface will be evaluated by the study ophthalmologist.

10. STATISTICAL CONSIDERATIONS

The primary outcome variable is ORR, defined in 9.1. McNemar’s test for correlated proportions will be used for the primary analysis. The null hypothesis is that the ORR will be equal in the eyes treated with AFED and the eyes treated with placebo. The alternative hypothesis is that the ORR will be higher in the eyes treated with AFED. A sample size of 15 subjects (30 eyes) will provide at least 80% power at the two sided 0.05 significance level for McNemar's test for correlated proportions under the assumptions that the ORR is no more than 5% on placebo and at least 60% on AFT. The impact of different covariates on ORR will be analyzed using descriptive statistics, t-test, and Fisher’s exact test where applicable. Differences in NIH scores will be analyzed using Wilcoxon signed-rank test. For quality of life analysis, the FACT-G survey tool will be used to assess patient-reported outcomes. Scores at 1 month and 3 months post-treatment will be summarized and compared to baseline, utilizing a two-sample t-test with a two-sided alternative. The proportion of subjects who experience adverse events and serious adverse events will be summarized descriptively; no inferential tests will be performed.

11. REGISTRATIONS GUIDELINES

Patients must meet all of the eligibility requirements listed in Section 5 prior to registration. Study related screening procedures can only begin once the patient has signed a consent form. Patients must not begin protocol treatment prior to registration. Treatment should start within five working days after registration.

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To register eligible patients on study, complete a Clinical Trials Office Patient Registration Form and submit to: [email protected].

12. DATA SUBMISSION SCHEDULE

The Case Report Forms (CRFs) are a set of electronic forms for each patient that provides a record of the data generated according to the protocol. CRFs should be created prior to the study being initiated and updated (if applicable) when amendments to the protocol are IRB approved. Data capture should be restricted to endpoints and relevant patient information required for planned manuscripts. These forms will be completed on an on-going basis during the study. The medical records will be source of verification of the data. During the study, the CRFs will be monitored for completeness, accuracy, legibility and attention to detail by a member of the Research Compliance Office. The CRFs will be completed by the Investigator or a member of the study team as listed on the Delegation of Duties Log. The data will be reviewed no less than annually by the Data and Safety Monitoring Committee. The Investigator will allow the Data and Safety Monitoring Committee or Research Compliance Office personnel access to the patient source documents, clinical supplies dispensing and storage area, and study documentation for the above-mentioned purpose. The Investigator further agrees to assist the site visitors in their activities

13. ETHICAL AND REGULATORY CONSIDERATIONS

13.1. Informed Consent Informed consent will be obtained from all research participants prior to performing any study procedures using the most recent IRB approved version.

13.2. Institutional Review This study will be approved by the Institutional Review Board of University of Utah.

13.3. Data and Safety Monitoring Plan A Data and Safety Monitoring Committee (DSMC) is established at Huntsman Cancer Institute (HCI) and approved by the NCI to assure the well-being of patients enrolled on Investigator Initiated Trials that do not have an outside monitoring review. Roles and responsibilities of the DSMC are set forth in the NCI approved plan. The activities of this committee include a quarterly review of adverse events including SAEs, important medical events, significant revisions or amendments to the protocol, and approval of cohort/dose escalations. If the DSMC and/or the PI have concerns about unexpected safety issues, the study will be stopped and will not be resumed until the issues are resolved. The DSMC also reviews and approves audit reports generated by the Research Compliance Office. All phase II studies are reviewed by the full committee at each quarterly DSMC meeting. This includes a review of all serious adverse events (SAEs) occurring in patients treated at HCI or its affiliates as well as all grade 3 or greater toxicities for patients on treatment (only if possibly, probably or definitely related).

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13.4. Adverse Events / Serious Adverse Events This study will utilize the CTCAE (NCI Common Terminology Criteria for Adverse Events) Version 4.0 for AE and SAE reporting.

13.4.1. Adverse Events (AE) An adverse event is the appearance or worsening of any undesirable sign, symptom, or medical condition occurring after starting the study drug even if the event is not considered to be related to study drug. For the purposes of this study, the terms toxicity and adverse event are used interchangeably. Medical conditions/diseases present before starting study drug are only considered adverse events if they worsen after starting study drug. Abnormal laboratory values or test results constitute adverse events only if they induce clinical signs or symptoms, are considered clinically significant, or require therapy. Adverse event will begin at time of first administration of AFED and placebo and continue until the last study visit (M3 follow-up). Information about all adverse events, whether volunteered by the subject, discovered by investigator questioning, or detected through physical examination, laboratory test or other means, will be collected and recorded and followed as appropriate. The occurrence of adverse events should be sought by non-directive questioning of the patient at each visit or phone contact during the study. Adverse events also may be detected when they are volunteered by the patient during or between visits or through physical examination, laboratory test, or other assessments. As far as possible, each adverse event should be evaluated to determine: 1. The severity grade based on CTCAE v.4 (grade 1-5) 2. Its relationship to the study drug(s) (definite, probable, possible, unlikely, not related) For multi-drug regimens, indicate if relationship will be assessed for all drugs, or just study drug. 3. Its duration (start and end dates or if continuing at final exam) 4. Action taken (no action taken; study drug dosage adjusted/temporarily interrupted; study drug permanently discontinued due to this adverse event; concomitant medication taken; non-drug therapy given; hospitalization/prolonged hospitalization) 5. Whether it constitutes an SAE All adverse events will be treated appropriately. Such treatment may include changes in study drug treatment as listed in the dose modification section of this protocol (see section 8 for guidance). Once an adverse event is detected, it should be followed until its resolution, and assessment should be made at each visit (or more frequently, if necessary) of any changes in severity, the suspected relationship to the study drug, the interventions required to treat it, and the outcome. Information about common side effects already known about the investigational product described in the Drug Information (section 3) and the most recent package insert. All adverse events will be immediately recorded in the patient research chart.

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13.4.2. Serious Adverse Event (SAE) Information about all serious adverse events will be collected and recorded. A serious adverse event is an undesirable sign, symptom or medical condition which:  Is fatal or life-threatening  Results in persistent or significant disability/incapacity  Is medically significant, i.e., defined as an event that jeopardizes the patient or may require medical or surgical intervention to prevent one of the outcomes listed above  Causes congenital anomaly or birth defect  Requires inpatient hospitalization or prolongation of existing hospitalization, unless hospitalization is for: o Routine treatment or monitoring of the studied indication, not associated with any deterioration in condition (procedures such as central line placements, paracentesis, pain control) o Elective or pre-planned treatment for a pre-existing condition that is unrelated to the indication under study and has not worsened since the start of study drug o Treatment on an emergency outpatient basis for an event not fulfilling any of the definitions of a SAE given above and not resulting in hospital admission o Social reasons and respite care in the absence of any deterioration in the patient’s general condition Any death from any cause while a patient is receiving treatment on this protocol or up to 30 days after the last dose of protocol treatment, or any death which occurs more than 30 days after protocol treatment has ended but which is felt to be treatment related, must be reported. Toxicities which fall within the definitions listed above must be reported as an SAE regardless if they are felt to be treatment related or not. Toxicities unrelated to treatment that do NOT fall within the definitions above, must simply be documented as AEs in the patient research chart.

13.5. SAE Reporting Requirements SAEs must be reported to the DSMC as well as the IRB and the FDA (when applicable), according to the requirements described below: A MedWatch 3500A form must be completed and submitted to [email protected] as soon as possible and within 24 hours of first knowledge or notification of event.

13.5.1. DSMC Notifications An HCI Research Compliance Officer (RCO) will process and submit the MedWatch form to the proper DSMC member as necessary for each individual study. The RCO will summarize and present all reported SAEs according to the Data and Safety Monitoring Plan at the quarterly DSMC meeting.

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13.5.2. FDA Notifications Adverse events occurring during the course of a clinical study that meet the following criteria will be promptly reported to the FDA:  Serious  Unexpected  Definitely, Probably or Possibly Related to the investigational product Fatal or life-threatening events that meet the criteria above will be reported within 7 calendar days after first knowledge of the event by the investigator; followed by as complete a report as possible within 8 additional calendar days. All other events that meet the criteria above will be reported within 15 calendar days after first knowledge of the event by the investigator. The RCO will review the MedWatch report for completeness, accuracy and applicability to the regulatory reporting requirements. The RCO will ensure the complete, accurate and timely reporting of the event to the FDA. The MedWatch report will be submitted to the FDA through the voluntary reporting method by the Regulatory Coordinator.

13.5.3. IRB Notification Events meeting the University of Utah IRB reporting requirements will be submitted through the IRB’s electronic reporting system within 10 working days.

13.6. Protocol Amendments Any amendments or administrative changes in the research protocol during the period, for which the IRB approval has already been given, will not be initiated without submission of an amendment for IRB review and approval. These requirements for approval will in no way prevent any immediate action from being taken by the investigator in the interests of preserving the safety of all patients included in the trial.

13.7. Protocol Deviations A protocol deviation (or violation) is any departure from the defined procedures and treatment plans as outlined in the protocol version submitted and previously approved by the IRB. Protocol deviations have the potential to place participants at risk and can also undermine the scientific integrity of the study thus jeopardizing the justification for the research. Protocol deviations are unplanned and unintentional events. Because some protocol deviations pose no conceivable threat to participant safety or scientific integrity, reporting is left to the discretion of the PI within the context of the guidelines below. The IRB requires the prompt reporting of protocol deviations which are:  Exceptions to eligibility criteria.  Intended to eliminate apparent immediate hazard to a research participant or,

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 Harmful (caused harm to participants or others, or place them at increased risk of harm, including physical, psychological, economic, or social harm), or  Possible serious or continued noncompliance.

13.8. FDA Annual Reporting This study is IND exempt therefore there are no annual reporting requirements to the FDA.

13.9. Clinical Trials Data Bank The study will be registered on http://clinicaltrials.gov and the NCI CTRP (Clinical Trials Reporting Program) by the Clinical Trials Office.

13.10. Record Keeping Per 21 CFR 312.57, Investigator records shall be maintained for a period of 2 years following the date a marketing application is approved; or, if no application is filed or the application is not approved, until 2 years after the investigation is discontinued and the FDA is notified.

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14. REFERENCES

1 Pidala, J., C. Anasetti, and H. Jim, Quality of life after allogeneic hematopoietic cell transplantation. Blood, 2009. 114(1): p. 7-19. 2 Lee, S.J., G. Vogelsang, and M.E.D. Flowers, Chronic graft-versus-host disease. Biol Blood Marrow Transpl, 2003. 9(4): p. 215-33. 3 Lee, S.J., et al., Severity of chronic graft-versus-host disease: association with treatment-related mortality and relapse. Blood, 2002. 100(2): p. 406-414. 4 Couriel, D., et al., Acute graft-versus-host disease: pathophysiology, clinical manifestations, and management. Cancer, 2004. 101(9): p. 1936-46. 5 Inamoto, Y., et al., Incidence, risk factors, and outcomes of sclerosis in patients with chronic graft- versus-host disease. Blood, 2013. 121(25): p. 5098-103. 6 Ingham, P.W., Y. Nakano, and C. Seger, Mechanisms and functions of Hedgehog signalling across the metazoa. Nat Rev Genet, 2011. 12(6): p. 393-406. 7 Bhatia, S., et al., Late mortality after allogeneic hematopoietic cell transplantation and functional status of long-term survivors: report from the Bone Marrow Transplant Survivor Study. Blood, 2007. 110(10): p. 3784-92. 8 Inamoto, Y., et al., Validation of measurement scales in ocular graft-versus-host disease. Ophthalmology, 2012. 119(3): p. 487-93. 9 Sun, Y.C., et al., Impact of Ocular Chronic Graft-versus-Host Disease on Quality of Life. Biol Blood Marrow Transplant, 2015. 21(9): p. 1687-91. 10 Carpenter, P.A., et al., National Institutes of Health Consensus Development Project on Criteria for Clinical Trials in Chronic Graft-versus-Host Disease: V. The 2014 Ancillary Therapy and Supportive Care Working Group Report. Biol Blood Marrow Transplant, 2015. 21(7): p. 1167-87. 11 Underwood, M.A., W.M. Gilbert, and M.P. Sherman, Amniotic fluid: not just fetal urine anymore. J Perinatol, 2005. 25(5): p. 341-8. 12 Prusa, A.R., et al., Oct-4-expressing cells in human amniotic fluid: a new source for stem cell research? Hum Reprod, 2003. 18(7): p. 1489-93. 13 Bottai, D., et al., Third trimester amniotic fluid cells with the capacity to develop neural phenotypes and with heterogeneity among sub-populations. Restor Neurol Neurosci, 2012. 30(1): p. 55-68. 14 Johnson, H.L., Peritoneal Immunization. The American Journal of Surgery, 1936. 34(2): p. 266-271. 15 Shimberg, M., The Use of Amniotic Fluid Concentrate in Orthopedic Conditions. The Journal of Bone and Joint Surgery, 1938(20): p. 167-177. 16 Ismail, M.A., G.I. Salti, and A.H. Moawad, Effect of amniotic fluid on bacterial recovery and growth: clinical implications. Obstet Gynecol Surv, 1989. 44(8): p. 571-7. 17 Ojo, V.A., E.E. Okpere, and E.E. Obaseiki-Ebor, Antimicrobial properties of amniotic fluid from some Nigerian women. Int J Gynaecol Obstet, 1986. 24(2): p. 97-101. 18 Siggers, J., et al., Postnatal amniotic fluid intake reduces gut inflammatory responses and necrotizing enterocolitis in preterm neonates. Am J Physiol Gastrointest Liver Physiol, 2013. 304(10): p. G864- 75. 19 Ozgenel, G.Y., G. Filiz, and M. Ozcan, Effects of human amniotic fluid on cartilage regeneration from free perichondrial grafts in rabbits. Br J Plast Surg, 2004. 57(5): p. 423-8. 20 Ozgenel, G.Y. and G. Filiz, Combined application of human amniotic membrane wrapping and hyaluronic acid injection in epineurectomized rat sciatic nerve. J Reconstr Microsurg, 2004. 20(2): p. 153-7. 21 Karacal, N., et al., Effect of human amniotic fluid on bone healing. J Surg Res, 2005. 129(2): p. 283-7.

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22 Castro-Combs, J., et al., Corneal wound healing is modulated by topical application of amniotic fluid in an ex vivo organ culture model. Exp Eye Res, 2008. 87(1): p. 56-63. 23 Nyman, E., et al., Hyaluronic acid, an important factor in the wound healing properties of amniotic fluid: in vitro studies of re-epithelialisation in human skin wounds. J Plast Surg Hand Surg, 2013. 47(2): p. 89-92. 24 Weissenbacher, T., et al., Influence of maternal age, gestational age and fetal gender on expression of immune mediators in amniotic fluid. BMC Res Notes, 2012. 5: p. 375. 25 Merimee, T.J., M. Grant, and J.E. Tyson, Insulin-like growth factors in amniotic fluid. J Clin Endocrinol Metab, 1984. 59(4): p. 752-5. 26 Watanabe, H., Epidermal growth factor in urine of pregnant women and in amniotic fluid throughout pregnancy. Gynecol Endocrinol, 1990. 4(1): p. 43-50. 27 Lang, A.K. and R.F. Searle, The immunomodulatory activity of human amniotic fluid can be correlated with transforming growth factor-beta 1 (TGF-beta 1) and beta 2 activity. Clin Exp Immunol, 1994. 97(1): p. 158-63. 28 Kurauchi, O., et al., The concentration of hepatocyte growth factor (HGF) in human amniotic fluid at second trimester: relation to fetal birth weight. Horm Metab Res, 1995. 27(7): p. 335-8. 29 Hirai, C., et al., Trophic effect of multiple growth factors in amniotic fluid or human milk on cultured human fetal small intestinal cells. J Pediatr Gastroenterol Nutr, 2002. 34(5): p. 524-8. 30 The definition and classification of dry eye disease: report of the Definition and Classification Subcommittee of the International Dry Eye WorkShop (2007). Ocul Surf, 2007. 5(2): p. 75-92.

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APPENDIX A Patient Instructions and Dosing Diary

1. You will receive a thermosafe box containing dry ice and the eyedrops that you will use for the study. Use it to transport the eyedrops to your home. 2. Upon arrival at your home, transfer the boxes containing the eyedrops to the freezer. EYEDROPS MUST REMAIN FROZEN UNTIL USE. 3. On the morning on the 1st day of use, removed a single bottle from the box labelled “left eye” and a single bottle from the box labelled “right eye” from the freezer. 4. Record the time that the bottles are removed from the freezer on the patient diary. 5. Allow the eyedrops from both bottles to thaw at room temperature for 5 to 10 minutes. 6. Remove the cap from the bottle designated for the right eye and administer 1 drop from this bottle into the right eye. Recap the bottle. 7. Remove the cap from the bottle designated for the left eye and administer 1 drop from this bottle into the left eye. Recap the bottle. 8. Record the time the drops were added to your eyes on the patient diary. 9. Place both bottles in the refrigerator for later use. DO NOT RETURN THE BOTTLES TO THE FREEZER. 10. Record the time the drops were placed in the refrigerator on the patient calendar/diary. 11. On the evening of the 1st day of use, remove the eyedrop bottles from the refrigerator. 12. Record the time that the bottles are removed from the refrigerator on the patient diary. 13. Remove the cap from the bottle designated for the right eye and administer 1 drop from this bottle into the right eye. Recap the bottle and toss the bottle in the trash. 14. Remove the cap from the bottle designated for the left eye and administer 1 drop from this bottle into the left eye. Recap the bottle and toss the bottle in the trash. 15. Record the time the drops were added to your eyes on the patient diary. 16. Repeat steps 4 through 16 for each subsequent day as directed by study coordinator.

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Patient Name: ______Patient Study ID: ______

Treatment period: ______(First/second/third month)

MORNING EVENING DAY Date Bottles thaw AM dose Bottles refri- Bottles out of PM dose sd mm/dd/yy time time gerated time fridge time time hh:mm hh:mm hh:mm hh:mm hh:mm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

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APPENDIX B FACT-G Quality of Life Questionnaire (Version 4) – Page 1/2

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APPENDIX B FACT-G Quality of Life Questionnaire (Version 4) – Page 2/2

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APPENDIX C Health Care Provider Global Rating of Chronic GVHD According to the National Institute of Health Consensus Conference for assessment of response in chronic GVHD

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APPENDIX D Ocular Rating of GVHD (Eye Score) According to the National Institute of Health Consensus Conference for assessment of response in chronic GVHD

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APPENDIX E Patient Global Rating of GVHD According to the National Institute of Health Consensus Conference for assessment of response in chronic GVHD

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APPENDIX F Dry Eye Severity Grading Scheme (from International Dry Eye Workshop 2007 Report)

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