Clinical trial NCT04684641
Conditions: Cystic Fibrosis
Keywords: Pseudomonas bacteriophage phage
Sponsor: Yale University
Start date: 2021-03-29; Completion date: 2023-06-22
Enrollment: 8 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This is a Phase 2 study with primary objective of looking whether YPT-01 phage therapy reduces sputum bacterial load in cystic fibrosis subjects with Pseudomonas aeruginosa.

In addition, study evaluates the safety profile of phage therapy in this patient population.

Detailed description:
This is a prospective, randomized, placebo-controlled, double-blinded, single-site study of Yale Phage Therapy (YPT) 01 in cystic fibrosis subjects with chronic Pseudomonas aeruginosa airway infections. The study has 2 parallel arms of phage therapy and placebo, with all study materials GMP-manufactured. The purpose of this study is to demonstrate efficacy and safety of inhaled (nebulized) phage therapy YPT-01. Clinically stable subjects who have confirmed diagnosis of CF with PsA in sputum cultures on at least two occasions within past year, and in sputum at screening visit, will be recruited into this study.

An open-label extension is available for subjects in the placebo group to receive YPT-01 following completion of blinded portion of the study.

Nov 2022 study ended enrollment after 8 subjects. The Double-Blind Randomized portion of the study was closed and the Open-Label Extension was opened.

Arms: arm 1: Participants will be randomized to receive 3mL phage therapy, nebulized daily for 7 days. arm 2: Participants will be randomized to receive the 3mL placebo, nebulized daily for 7 days.
Interventions: intervention 1: Standard Dose YPT-01 intervention 2: Placebo
Intervention descriptions: intervention 1: Participants will be randomized to receive the standard dose of phage therapy YPT-01. intervention 2: Participants will be randomized to receive the placebo.

Protocol documents:
Prot_SAP_000.pdf: 
Protocol Number: 2000029160 
November 29, 2022  Version 11 
1 
CYSTIC FIBROSIS PHAGE STUDY AT 
YALE (CYPHY) 
 Protocol Number 
2000029160 
Protocol Version 
November 29, 2022 
 
PRINCIPAL INVESTIGATOR: 
Name: Jon Koff, MD 
Department: Pulmonary, Critical Care, and Sleep Medicine 
Telephone: 203.737.6963 
E-mail Address: jon.koff@yale.edu 
Short Title: 
CYstic fibrosis bacterioPHage study at Yale (CYPHY): A single-site, randomized, double-
blind, placebo-controlled study of bacteriophage therapy YPT-01 for Pseudomonas 
aeruginosa infections in adults with cystic fibrosis 
 
IND Number: 23427 
  
IND Holder: Benjamin Chan, PhD, Research Scientist, Department of Ecology and 
Evolutionary Biology, Yale University 
 
NCT Number: NCT04684641 
Study Phase: 2 
Confidentiality Statement: This document contains confidential information which 
should not be copied, released, or published without written approval from Yale 
University and/or the Principal Investigator and/or the Sponsor. 
 
 
 
 
 
 
 
 


Protocol Number: 2000029160 
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REVISION HISTORY  
Protocol Version And Date 
Submission Description 
IRB Submission 
Number and 
Date  
FDA submission 
Number and 
Date 
1.  June 16, 2020 
Internal Version 
n/a 
n/a 
2.  September 18, 2020 
Initial Submission 
HIC2000029160 
Oct 16, 2020 
SN0001 
Sept 25, 2020 
3.  November 06, 2020 
Internal Version 
n/a 
n/a 
4.  January 06, 2021 
Response to requested 
modifications 
n/a 
n/a 
5.  February 04, 2021 
Amendment #1, 
Remove “low” dose phage 
group  
MOD00038191 
Feb 08, 2021 
SN0002 
Feb 26, 2021 
n/a (no protocol changes) 
CMC info submitted to FDA  
n/a 
SN0003  
June 29, 2021 
6.  June 23, 2021 
Amendment #2, 
Update inclusion criteria and 
travel funds to expand 
enrollment 
MOD00039066 
June 23, 2021 
SN0004  
July 07, 2021 
n/a (no protocol changes) 
 
Provide updated Lab Manual 
and Investigator’s Brochure; 
submitted to FDA July 12, 
2021 & subsequently included 
in IRB amendment 
MOD00039066 
June 23, 2021 
SN0005 
July 12, 2021 
n/a (no protocol changes) 
IND Annual Report submitted 
to FDA 
n/a 
SN0006 
Sept 15, 2021 
7.  Sept 08, 2021 
Amendment #3,  
Response to IRB CR and FDA 
request for information 
MOD00044863 
Sept 23, 2021 
SN0007 
Sept 24, 2021 


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8.  Sept 29, 2021 
Amendment #4, 
Update IRB with the changes 
of SN0007 submitted to FDA 
prior 
MOD00045457 
Sept 29, 2021 
 
n/a (no protocol changes) 
 
IND Annual Review submitted 
to FDA  
n/a 
SN0008 
Sept 02, 2022 
9. October 05, 2022 
Amendment #5, 
End the Double-Blind 
Randomized segment 
including ending enrollment 
Open the Open-Label 
Extension 
MOD00055062 
Nov 01, 2022 
SN0009 
Oct 21, 2022 
10. October 26, 2022 
Amendment #5,  
Additional Open-Label 
changes for IRB review  
MOD00055062 
Nov 01, 2022 
SN0011 
Nov 29, 2022 
11. November 29, 2022 
Amendment #5, response to 
IRB comments. 
Recruitment concluded Oct 21, 
2022 
The study is in the open-label 
stage status upon IRB 
approval of this amendment. 
MOD00055062 
Nov 30, 2022 
SN0011 
Nov 30, 2022 


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Synopsis 
Primary Objective 
The primary objectives of this Phase 2 study are to determine whether YPT-01 phage 
therapy reduces sputum bacterial load in cystic fibrosis subjects with Pseudomonas 
aeruginosa, and to evaluate short-term safety of phage therapy in this patient population. 
Secondary Objectives (if applicable) 
The secondary objectives of this study are to: 
- 
Assess whether YPT-01 induces “trade-offs” between evolved phage resistance 
and decreased virulence factors present in sputum P. aeruginosa; 
- 
Determine whether YPT-01 alters subject sputum microbiome;  
- 
Understand whether YPT-01 changes subject lung inflammation and inflammatory 
markers;  
- 
Describe clinical response in subjects to YPT-01, with respect to change in lung 
function (e.g., FEV1pp), as well as rates of pulmonary exacerbations, acute 
antibiotic use for exacerbations, and hospitalizations;  
- 
Understand whether YPT-01 improves subject-reported quality of life (via CFQ-R); 
- 
Obtain preliminary data on the long-term safety of phage therapy 
Study Duration 
Approximately 3 years from initial IRB approval 
Study Design 
Prospective, randomized, parallel, placebo-controlled, double-blinded with an optional 
open-label extension for subjects who initially received placebo. 
Study Population 
Clinically-stable (e.g., no exacerbations or medication changes within 4 weeks) individuals 
with cystic fibrosis (no solid organ transplant; FEV1pp > 40%) with chronic sputum P. 
aeruginosa. 
Number of Participants  
40 (screened), 36 (enrolled and randomized) 
Number of Study Sites 
Single center study at Yale University 
Primary Outcome Variables 
Change in P. aeruginosa sputum CFU from baseline to day number 14 (7 days after 
completion of phage therapy) 


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Short-term safety profile of inhaled phage therapy during the randomized portion of the 
study (within the first 56 days of the blinded portion of the trial): 
Safety-related outcomes, i.e. frequency and severity of different types of adverse events 
as related to the study intervention, will be actively monitored throughout the study and 
summarized for the following days post-phage treatment during randomized blinded 
(experimental) period: d7, d14, d21, d28, d56. Monthly thereafter until month 6. Safety-
related outcomes will be summarized post-phage treatment on d7, d14, d28, and monthly 
thereafter for a total of 6 months (placebo and open-label time total) during the open-label 
period. 
Secondary and Exploratory Outcome Variables (if applicable) 
Secondary end-points for response profiles at d3, d7, d14, d21, and d28: 
1) Change over time from baseline in PsA sputum load 
2) Change over time from baseline in "trade-offs" of PsA, as measured by change in 
PsA antibiotic sensitivity (after OMKO1 phage), motility and pyocyanin levels (after 
TIVP-H6 phage), and endotoxin levels (after LPS-5 phage); 
3) Changes from baseline in sputum phage presence; 
Secondary end-point at d14: 
1) Change from baseline in lung inflammation as measured by sputum 
transcriptomics; 
Secondary end-points for response profiles at d14 and d28: 
1) Change from baseline in sputum inflammatory markers, as measured by 
inflammatory cytokines; 
Secondary end-points for response profiles at d3, d7, d14, d21, d28, and d56: 
1) Change from baseline in lung function (FEV1pp and absolute FEV1) from baseline; 
2) Change from baseline in quality of life and respiratory symptoms as measured by 
CFQ-R at each clinic visit from baseline; 
3) Rate(s) of pulmonary exacerbations and medication changes from baseline. 
Secondary end-point for long-term safety at month 6: 
1) Frequency and severity of different types of adverse events. 
 
Tertiary end points for response profiles at d7, d14, d21, d28, and d56: 
1) Change from d1 in anti-phage antibody titers. 
 
 
 


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Abbreviations 
Abbreviation  
Explanation 
Ab 
Antibodies 
AE 
Adverse Event 
ANC 
Absolute Neutrophil Count 
ANCOVA 
Analysis of Covariance 
APT 
Adaptive Phage Therapeutics 
CAR 
Carbenicillin 
CF 
Cystic Fibrosis 
CFF 
Cystic Fibrosis Foundation 
CFQ-R 
Cystic Fibrosis Quality-of-Life Revised Survey 
CFR 
Code of Federal Regulations 
CFRSD 
Cystic Fibrosis Respiratory Symptom Diary 
CFTR 
Cystic Fibrosis Transmembrane Receptor 
CFU 
Colony-forming units, a measure of bacterial load 
CI 
Confidence Interval 
CIP 
Ciprofloxacin 
CLIA 
Clinical Laboratory Improvement Amendments 
CONSORT 
Consolidated Standards of Reporting Trials (publishing requirements) 
CRF 
Case Report Form 
CRISS 
Chronic Respiratory Infection Symptom Score 
CS 
Compound Symmetry 
CT 
Computerized Tomography (scan) 
CTCAE 
Common Terminology Criteria for Adverse Events 
CYPHY 
CYstic fibrosis bacterioPHage study at Yale (this study) 
d 
day 


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DMC 
Data Monitoring Committee 
DSMC 
Data Safety Monitoring Committee 
eIND 
emergency Investigational New Drug Application 
EMR 
Electronic Medical Record 
EOP 
Efficiency of Plaquing, an in vitro measure of phage efficacy against a 
pathogen 
FDA 
Food and Drug Administration 
FEV1 
Forced Expiratory Volume over 1 second 
FEV1pp 
Percent predicted of Forced Expiratory Volume over 1 second 
FVC 
Forced Vital Capacity 
GCP 
Good Clinical Practice 
GEE 
Generalized Estimating Equations 
GMP 
Good Manufacturing Practice 
HCl 
Hydrogen Chloride 
HIPAA 
Health Insurance Portability and Accountability Act 
HRPP 
Human Research Protection Program (at Yale) 
ICC 
IntraClass Correlation 
ICF 
Informed Consent Form 
ICH 
International Council for Harmonisation of Technical Requirements for 
Pharmaceuticals for Human Use 
ICOI 
Institutional Conflict of Interest 
ICOIC 
Institutional Conflict of Interest Committee 
IDS 
Investigational Drug Service, a branch of the Yale Pharmacy 
IEC 
Independent Ethics Committee 
IND 
Investigational New Drug Application 
IO 
Institutional Official 
IP 
Intraperitoneal 


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IQR 
Interquartile Range 
IRB 
Institutional Review Board 
ITT 
Intent-To-Treat 
IV 
Intravenous 
IVC 
Inspiratory Vital Capacity 
LB 
Luria-Bertani medium, used to culture bacteria 
LME 
Linear Mixed Effects 
LPS 
Lipopolysaccharide 
LPS-5 
Lipopolysaccharide-targeting phage 5, one of the phages comprising YPT-
01 
LRT 
Likelihood Ratio Test 
LSFI 
Leaders Significant Financial Interests 
MAR 
Missing at Random 
MDR 
Multidrug-resistant, often used in reference to bacteria or pathogens 
MgSO4 
Magnesium Sulfate 
MIC 
Minimum Inhibitory Concentration 
ML 
Maximum Likelihood 
NAL 
Nalidixic acid 
NCFB 
Non-Cystic Fibrosis Bronchiectasis 
OMKO1 
Outer Membrane Knockout-1, an efflux pump targeting phage and one of 
the phages comprising YPT-01 
OR 
Odds Ratio 
PBS 
Phosphate Buffered Saline 
PCR 
Polymerase Chain Reaction 
PEF 
Peak Expiratory Flow 
PFT 
Pulmonary Function Tests (e.g., spirometry) 
PFU 
Plaque-forming units, a measure of phage dosage 


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Phage 
Bacteriophage, a virus of bacteria 
PI 
Principal Investigator 
PsA 
Pseudomonas aeruginosa 
QIC 
Quasi-likelihood under the assumption of independence criterion 
qPCR 
quantitative PCR 
SAE 
Serious Adverse Event 
SN 
Serial Number 
SoA 
Schedule of Activities 
TDN 
Therapeutics Development Network of the Cystic Fibrosis Foundation 
TET 
Tetracycline 
TIVP-H6 
Type IV Pilus Targeting Phage H6, one of the phages comprising YPT-01 
U 
Units, a measure of antibiotic concentration (e.g., meropenem) 
UPIRSO 
Unanticipated Problems Involving Risks to Subjects or Others 
VC 
Vital Capacity 
WHO 
World Health Organization 
YCCI 
Yale Center for Clinical Investigation 
YPT-01 
Yale Phage Therapy 01, a treatment algorithm composed of phages LPS-
5, OMKO1, and TIVP-H6 
 
 


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Table of Contents 
Synopsis ............................................................................................................................... 4 
Abbreviations ........................................................................................................................ 6 
Table of Contents ................................................................................................................ 10 
1 
Introduction .................................................................................................................. 13 
1.1 
Introductory Statement .......................................................................................... 13 
2 
Background .................................................................................................................. 14 
2.1 
Background/prevalence of research topic ............................................................. 14 
2.2 
Preclinical Experience ........................................................................................... 14 
2.3 
Clinical Experience ............................................................................................... 19 
3 
Rationale/Significance.................................................................................................. 22 
3.1 
Problem Statement ............................................................................................... 22 
3.2 
Purpose of Study/Potential Impact ........................................................................ 24 
3.2.1 
Potential Risks ............................................................................................... 24 
3.2.2 
Potential Benefits ........................................................................................... 25 
4 
Study Objectives .......................................................................................................... 27 
4.1 
Hypothesis ............................................................................................................ 27 
4.2 
Primary Objective.................................................................................................. 27 
4.3 
Secondary Objectives ........................................................................................... 27 
5 
Study Design ............................................................................................................... 29 
5.1 
General Design Description .................................................................................. 29 
5.1.1 
Study Date Range and Duration .................................................................... 36 
5.1.2 
Number of Study Sites ................................................................................... 36 
5.2 
Outcome Variables ............................................................................................... 36 
5.2.1 
Primary Outcome Variables ........................................................................... 36 
5.2.2 
Secondary and Exploratory Outcome Variables ............................................. 36 
5.3 
Study Population ................................................................................................... 37 
5.3.1 
Number of Participants .................................................................................. 37 
5.3.2 
Eligibility Criteria/Vulnerable Populations ....................................................... 37 
5.3.3 
Study Specific Tolerance for Inclusion/Exclusion Criteria ............................... 38 
5.3.4 
Screen Fail Criteria ........................................................................................ 38 


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5.3.5 
Change in Medications or Therapies .............................................................. 38 
6 
Methods ....................................................................................................................... 39 
6.1 
Treatment ............................................................................................................. 39 
6.1.1 
Identity of Investigational Product .................................................................. 39 
6.1.2 
Dosage, Administration, Schedule ................................................................. 40 
6.1.3 
Method of Assignment/Randomization ........................................................... 40 
6.1.4 
Blinding and Procedures for Unblinding ......................................................... 41 
6.1.5 
Packaging/Labelling ....................................................................................... 41 
6.1.6 
Storage Conditions ........................................................................................ 43 
6.1.7 
Concomitant therapy ...................................................................................... 43 
6.1.8 
Restrictions .................................................................................................... 43 
6.2 
Assessments ........................................................................................................ 43 
6.2.1 
Efficacy .......................................................................................................... 43 
6.2.2 
Safety ............................................................................................................ 46 
6.2.3 
Biomarkers (if applicable) .............................................................................. 49 
6.3 
Study Procedures ................................................................................................. 50 
6.3.1 
Study Schedule .............................................................................................. 52 
6.3.2 
Informed Consent .......................................................................................... 53 
6.3.3 
Screening ...................................................................................................... 53 
6.3.4 
Enrollment ..................................................................................................... 53 
6.3.5 
On Study Visits .............................................................................................. 53 
6.3.6 
End of Study and Follow-up ........................................................................... 57 
6.3.7 
Removal of subjects ....................................................................................... 57 
6.4 
Statistical Method ................................................................................................. 58 
6.4.1 
Statistical Design ........................................................................................... 58 
6.4.2 
Sample Size Considerations .......................................................................... 60 
6.5 
Planned Analyses ................................................................................................. 61 
6.5.1 
Primary Objective Analysis ............................................................................ 61 
6.5.2 
Secondary Objectives Analyses ..................................................................... 62 
6.5.3 
Safety ............................................................................................................ 63 
6.5.4 
Analysis of Subject Characteristics ................................................................ 64 
6.5.5 
Subsets and Covariates ................................................................................. 64 


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6.5.6 
Handling of Missing Data ............................................................................... 65 
7 
Trial Administration ...................................................................................................... 66 
7.1 
Ethical Considerations: Informed Consent/Assent and HIPAA Authorization ........ 66 
7.2 
Institutional Review Board (IRB) Review ............................................................... 66 
7.3 
Subject Confidentiality .......................................................................................... 67 
7.4 
Deviations/Unanticipated Problems ...................................................................... 67 
7.5 
Data Collection ..................................................................................................... 67 
7.6 
Data Quality Assurance ........................................................................................ 67 
7.7 
Study Records ...................................................................................................... 68 
7.8 
Access to Source Documents ............................................................................... 68 
7.9 
Data or Specimen Storage/Security ...................................................................... 68 
7.10 
Retention of Records ............................................................................................ 68 
7.11 
Study Monitoring ................................................................................................... 69 
7.12 
Data Safety Monitoring Plan ................................................................................. 69 
7.13 
Study Modification................................................................................................. 70 
7.14 
Study Discontinuation ........................................................................................... 70 
7.15 
Study Completion ................................................................................................. 70 
7.16 
Conflict of Interest Policy....................................................................................... 71 
7.17 
Funding Source .................................................................................................... 71 
7.18 
Publication Plan .................................................................................................... 71 
8 
List of Tables ............................................................................................................... 72 
9 
References .................................................................................................................. 73 
10 Appendices .................................................................................................................... 75 
1. 
Cystic Fibrosis Quality-of-Life Questionnaire Revised .................................................. 76 
2. 
Study Drug Diary .......................................................................................................... 84 
3. 
Symptoms Diary ........................................................................................................... 86 
 
 
 
 
 


Protocol Number: 2000029160 
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1 Introduction 
1.1 
Introductory Statement 
This document is a protocol for a human research study. The purpose of this protocol is to 
ensure that this study is to be conducted according to ICH GCP guidelines, CFR 21 Part 
312, and according to other applicable government regulations and institutional research 
policies and procedures. 
 
 


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2 Background 
2.1 
Background/prevalence of research topic 
Cystic fibrosis (CF) is the most common life-limiting genetic disease among caucasians. CF 
is caused by mutation(s) in the cystic fibrosis transmembrane receptor (CFTR), which encodes 
a chloride channel. The majority of individuals with CF develop chronic lung disease 
characterized by recurrent bacterial infections, which cause pulmonary exacerbations that 
result in significant morbidity and mortality. In the lungs, exaggerated mucus production and 
inflammation cause bronchiectasis, which is a change in lung architecture that prevents 
effective bacterial clearance. Pseudomonas aeruginosa (PsA), a Gram-negative bacterium, is 
an important concern because it is the most prevalent bacterial pathogen in CF adults, and 
strongly correlates with lung function decline and increased morbidity and mortality (Parkins 
et al., 2018). Therapies targeting PsA improve clinical outcomes in CF patients, and the 
current focus is on antibiotics to treat chronic lung disease and pulmonary exacerbations 
caused by these bacteria (Mogayzel et al., 2013). Despite this multi-modal approach, PsA 
continues to cause significant disease because it cannot be effectively cleared from the lungs.  
In addition, PsA is one of the emerging multi-drug resistant (MDR) bacteria identified by the 
World Health Organization (WHO). By 2050, MDR pathogens will cause 10 million human 
deaths worldwide (de Kraker et al., 2016; O’Neill, 2014), which will exceed cancer deaths. 
MDR PsA is already increasingly problematic for CF patient care. Therefore, development of 
novel therapeutic approaches that may reduce infection, increase antibiotic sensitivity, or 
decrease inflammation with limited off-target effects are highly desirable for the CF community. 
These goals may be accomplished with bacteriophage (phage) therapy. Phages are lytic 
(virulent) bacteria-specific viruses that may be used as self-amplifying ‘drugs’ to target and kill 
specific bacteria. In addition to their antibacterial effects, phages can also be designed to 
manipulate bacteria to decrease lung inflammation. 
2.2 
Preclinical Experience 
Bacteriophages (phages) are a potential treatment for infections in humans caused by 
bacterial pathogens. Lytic phages are viruses that infect bacteria, generally resulting in 
production of phage progeny, which cause bacterial cell death through lysis (Figure 2-1). 
Given the distinctions in cellular components and molecular machinery of prokaryotic versus 
eukaryotic cells caused by genetic divergence over long spans of evolutionary time, phages 
are restricted to infecting bacteria and cannot infectiously destroy human cells. In addition, a 
majority of phages are restricted in host range to a particular bacterial species (or subset of 
genotypes within the species), or in some cases limited to infecting closely-related species of 
bacteria. 
 


Protocol Number: 2000029160 
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Figure 2-1 Lytic phage replication begins when the virus irreversibly binds to a receptor (protein or 
sugar) on the surface of a bacterial cell. The phage delivers its genomic content into the cytoplasm of 
the bacterial cell. Typically, host resources, including proteins and genomes are repurposed to fuel 
phage replication. Replication, transcription and translation of the phage genome begins usually 
through redirecting host metabolism to the production of new phage particles. Upon assembly of new 
phage particles, lysis of the bacterial cell allows newly replicated phage particles to escape the 
cytoplasm and go on to infect other phage-susceptible bacterial cells. 
 
Yale’s Phage Program has identified three environmentally-sourced phages for use in this 
study: OMKO1, TIVP-H6, and LPS-5. Limited preclinical studies have been performed on the 
phages, and pharmacokinetic and toxicology studies have not been completed on them. Each 
phage preparation used in subjects is tested for and meets quality specifications prior to 
clinical use. In addition, extensive use in humans over the last century has suggested that 
phage therapy is generally safe, while demonstrating anecdotal evidence of efficacy (see 
Investigator’s Brochure). The degradation products of bacteriophages are proteins and 
nucleic acids, which are expected to be metabolized in an identical manner to what naturally 
occurs in the body. In addition, it is important to note that CF lung has a distinct microbial 
community that includes phages which may contribute to the spread of virulence or antibiotic-
resistance genes (Brown-Jaque et al., 2018). OMKO1, TIVP-H6, and LPS-5 have been 
confirmed to be lytic, non-lysogenic phages, which minimizes the risk of integration into 
pathogen genomes and passage of virulence and antibiotic-resistance genes. Additionally, 
these three phages provide coverage of approximately 95% of isolates based on analysis of 
96 adult CF sputum culture PsA isolates tested in vitro (see Investigator’s Brochure). 
 
OMKO1 
Phage OMKO1 (family Myoviridae) targets the multidrug efflux pump MexAB-XY of PsA, killing 
susceptible bacterial cells via lysis while exerting selection for the bacterial population to 
evolve phage resistance that can coincide with decreased resistance (i.e., re-sensitivity) to 
certain antibiotics. Prior work demonstrated that phage OMKO1 was generally capable of 
attacking MDR PsA genotypes because it associated with the evolutionarily-conserved oprM 
gene (OprM membrane protein) of MexAB-XY efflux pumps in these bacteria (Chan et al., 
2016). The targeting of OprM results in a tradeoff between efflux pump function and evolved 


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phage resistance, resulting in increased sensitivity of surviving PsA cells to antibiotics. Table 
2-1 shows the change in antibiotic sensitivity between MDR PsA isolates and spontaneous 
phage-resistant mutants that arise in the bacterial population following exposure to phage 
(Chan et al., 2016). Phage-resistant mutants demonstrate a 2- to 12-fold increase in sensitivity 
to antibiotics, which are known or suspected to be exported from the cell via functional MexAB-
XY efflux pumps. 
 
Table 2-1. Mean antibiotic sensitivity of PsA before and after phage-OMKO1-selection in vitro. 
Antibiotic 
Class 
Strain 
Isolate MIC1 
(mg/L) 
Phage Resistant 
Isolate MIC1 
(mg/L) 
Fold-increased 
Antibiotic Sensitivity 
Tetracycline 
Tetracycline 
PA01 
6.333 
1.896 
3.340189873 
  
  
PA14 
10.67 
3.4 
3.138235294 
  
  
PAN 
7.333 
2.8 
2.618928571 
  
  
1845 
6.667 
5.2 
1.282115385 
  
  
1607 
11.33 
2.2 
5.15 
  
  
PAPS 
5.667 
1.95 
2.906153846 
  
  
PASk 
58.67 
14.67 
3.999318337 
  
  
PADFU 
48 
10 
4.8 
Erythromycin 
Macrolide 
PA01 
256 
5.6 
45.71428571 
  
  
PA14 
34.67 
10.8 
3.210185185 
  
  
PAN 
31.33 
4.2 
7.45952381 
  
  
1845 
256 
15.6 
16.41025641 
  
  
1607 
61.33 
7.6 
8.069736842 
  
  
PAPS 
24.67 
4.4 
5.606818182 
  
  
PASk 
149.3 
22 
6.786363636 
  
  
PADFU 
256 
55.67 
4.598527034 
Ciprofloxacin2 
Fluoroquinolone 
PA01 
0.073 
0.057 
1.280701754 
  
  
PA14 
0.21 
0.047 
4.468085106 
  
  
PAN 
0.064 
0.024 
2.666666667 
  
  
1845 
0.136 
0.126 
1.079365079 
  
  
1607 
0.104 
0.045 
2.311111111 
  
  
PAPS 
0.172 
0.014 
12.28571429 
  
  
PASk 
5.333 
1.666 
3.201080432 
  
  
PADFU 
3.667 
1.417 
2.58786168 
Ceftazidime 
Cephalosporin 
PA01 
0.667 
0.663 
1.006033183 
  
  
PA14 
1 
0.8 
1.25 
  
  
PAN 
1.5 
0.6 
2.5 


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1845 
0.917 
0.7 
1.31 
  
  
1607 
1 
0.55 
1.818181818 
  
  
PAPS 
0.667 
0.35 
1.905714286 
  
  
PASk 
1.167 
0.583 
2.001715266 
  
  
PADFU 
1.333 
0.667 
1.99850075 
1 Minimum Inhibitory Concentration (MIC) is the concentration of the drug needed to achieve efficient 
bacterial killing; includes data shown in Chan et al. 2016. 
 
Phage OMKO1 also improved survival in a standardized lethal model of PsA infection in mice 
(Figure 2-2). In this infection model, immunosuppression was induced [cyclophosphamide 
(150 mg/kg IP)] prior to bacterial challenge. Mice were subsequently challenged with PsA 
strain UNC-D instilled intratracheally after isoflurane anesthesia (2-3% in O2). Afterwards, 
OMKO1 phage particles (1x107.5 plaque-forming units, PFU) were administered alone (Figure 
2-2 left), or in combination with meropenem [1250 U (Figure 2-2 center) or 1000 U (Figure 
2-2 right) IV]. These experiments showed that the addition of OMKO1 rescued mice in the 
presence of the sub-therapeutic meropenem concentrations (Figure 2-2 center & right).  
 
 
Figure 2-2. Assessment of phage therapy in a standardized lethal model of PsA 
infection. Mice infected with 1x106 colony-forming units (CFU) PsA were treated with saline (solid line, 
grey circles) or OMKO1 phage (1x107.5 PFU; dashed line, white circles) 3 hours after infection (n=8 
female mice in each group). Mice infected with 1x106 cells of PsA treated with saline (solid line, grey 
circles), meropenem [1250 mg/kg/day; blue line & circles (LEFT), or 1000 mg/kg/day (green line & 
circles; RIGHT)], or OMKO1 phage (1x107.5 PFU; dashed line, white circles) plus meropenem 3 hours 
after infection (n=8 female mice in each group). Phage plus meropenem vs meropenem alone (p = 
0.0006). 
 
TIVP-H6 
Phage TIVP-H6 (family Myoviridae) was discovered by searching for lytic phages that 
specifically impaired the ability for PsA bacteria to produce the virulence factor pyocyanin, 
such that evolved phage resistance resulted in reduced pyocyanin production. To do so, a 
large number of candidate phages were screened for their ability to infect a pyocyanin 
overproducing strain, and it was observed that: 1) phage TIVP-H6 infected this PsA strain, 
and 2) PsA mutants resistant to phage TIVP-H6 failed to produce pyocyanin. The impact of 


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sub-inhibitory concentrations of several antibiotics (ciprofloxacin, tetracycline, nalidixic acid, 
carbenicillin, and erythromycin) on PsA pyocyanin production was also tested in 48-hour 
cultures of bacteria (Figure 2-3). These results showed a significant (p < 0.001) decrease in 
pyocyanin production in PsA relative to controls, when bacteria were exposed to erythromycin 
alone, to phage TIVP-H6 alone, and to erythromycin plus phage TIVP-H6 (Figure 2-3). 
Interestingly, the antibiotic ciprofloxacin was found to increase production of pyocyanin, but 
this effect was reversed when bacteria were exposed to ciprofloxacin plus phage TIVP-H6 
(Figure 2-3, column 6). Analysis of genomes from 10 independently isolated PsA mutant 
strains resistant to phage TIVP-H6 revealed numerous mutations in genes for the type IV pilus. 
Deletion of the type IV pilus in PsA was previously shown to impair pyocyanin production, 
twitching motility and biofilm formation, thus attenuating bacterial virulence (Persat et al., 
2015). 
 
Figure 2-3. Impact of phage and sub-inhibitory concentration of antibiotics on 
production of pyocyanin in PsA. Results are expressed as fold-change in pyocyanin production 
relative to untreated control (y-axis) when PsA is exposed to traditional antibiotics or TIVP-H6 (x-axis). 
Representatives from four major drug classes were tested for their impact on pyocyanin production. 
Ciprofloxacin (CIP), Tetracycline (TET), Nalidixic acid (NAL), Carbenicillin (CAR), and heat-inactivated 
phage (Inactive) TIVP-H6 did not reduce pyocyanin production (left hand bars 1 through 5). In contrast, 
phage TIVP-H6 reduced pyocyanin production and significantly attenuates the increase pyocyanin 
production induced by CIP alone.  (* p < 0.05; ** p < 0.01, significance compared to untreated control) 
 
LPS-5 
Phage LPS-5 (family Podoviridae) was discovered by searching for lytic viruses that 
specifically interacted with lipopolysaccharide (LPS) in PsA, such that evolved phage 
resistance resulted in reduced virulence due to changed LPS production. It was hypothesized 
that phage interaction(s) with LPS could have an impact on the production of outer membrane 
vesicles (Cryz et al., 1984; Kon et al., 1999). Therefore, extracellular endotoxin, measured by 
the Congo-endotoxin assay, would be reduced or absent in bacteria that evolved resistance 
to phage LPS-5. Consistent with this hypothesis, endotoxin production in 48-hour cultures of 


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phage-resistant PsA mutants was observed to be below the limit of detection, indicating that 
bacterial resistance to phage LPS-5 coincided with an evolutionary trade-off evidenced by 
reduced endotoxin production. Ten independently isolated PsA mutants resistant to phage 
LPS-5 were found to have many mutations in LPS synthesis genes, confirming the hypothesis 
that LPS is the primary cellular receptor for phage LPS-5. 
 
2.3 
Clinical Experience 
Phages OMKO1, TIVP-H6, and LPS-5 have been previously used in phage “therapy” for 12 
patients under single-patient emergency INDs (eINDs). Nine were CF patients, two had non-
CF bronchiectasis (NCFB), and one had an infected aortic graft (Chan et al., 2018). The CF 
and NCFB patients tolerated nebulized phage therapy without any adverse side effects. One 
individual had a serious adverse event (SAE); however, it was deemed to be unrelated to 
phage treatment (See Investigator’s Brochure). In all patients, bacterial loads were 
significantly reduced, and in many cases bacterial cultures showed increased antibiotic 
sensitivity or decreased inflammatory products (e.g., pyocyanin) after phage treatment, which 
were the ‘trade offs’ that the Yale investigators intended to leverage.  
 
In addition, several patients’ responses showed evidence of clinical improvement (e.g., 
improved lung function or decreased use of antibiotics, Figure 2-4). Patients FEV1% were 
assessed (Figure 2-4A) and post-phage treatment sputum samples were obtained to 
quantitate PsA titers seven days after completion of phage treatment (Figure 2-4B), to analyze 
antibiotic sensitivity (Figure 2-4D), and virulence factor (e.g., pyocyanin; Figure 2-4C) 
production. These results show log10 -2.66 CFU/mL (±1.17 CFU/mL) decrease in PsA (Figure 
2-4B) seven days after completion of inhaled phage treatment. PsA produces pyocyanin, 
which contributes to lung inflammation. Post TIVP-H6 phage, pyocyanin decreased in patient 
samples 15.67-fold (Figure 2-4C). Treatment with OMKO1 phage resulted in decreased MIC 
for common CF antibiotics by 3.8-fold (Figure 2-1D). CF patients’ pre-phage FEV1% of 35.5% 
(±0.18%) increased to 42.83% (±0.23%) (Figure 2-4B), which is a mean change of 7.3 
percentage points. Narratives of each subject’s experience are presented in the 
Investigator’s Brochure. For these cases, a phage dose of 1e10 PFU provided daily for 7-
10d was used, which was well-tolerated and is thus considered an effective target dose. These 
preliminary data informed the design of a single center trial to compare phage therapy to 
placebo. 
 
 
 


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Table 2-2. Summary of single-patient cases in CF and NCFB treated with OMKO1, 
TIVP-H6, or LPS-5 phages 
Patient 
Background Pathogen Phage(s) Used 
Primary Impact Tradeoff2 Date 
22 yo Female CF 
PDR PsA 
OMKO1, TIVP-H6, 
LPS-5 
CFU Decrease 
Yes 
Dec 2017 
72 yo Male 
NCFB 
MDR PsA TIVP-H6, LPS-5 
CFU Decrease 
Yes 
Sep 2018 
71 yo Male 
NCFB 
MDR PsA TIVP-H6, LPS-5 
Infection 
Resolved1 
Yes 
Oct 2018 
16 yo Female CF 
MDR PsA OMKO1, TIVP-H6, 
LPS-5 
CFU Decrease 
Yes 
Nov 2018 
38 yo Female CF 
PDR PsA 
TIVP-H6, LPS-5 
CFU Decrease 
Yes 
Dec 2018 
26 yo Female CF 
MDR PsA LPS-5 
CFU Decrease 
Yes 
Jan 2019 
38 yo Female CF 
MDR PsA LPS-5 
CFU Decrease 
Yes 
Apr 2019 
46 yo Female CF 
MDR PsA TIVP-H6, LPS-5 
CFU Decrease 
Yes 
May 2019 
27 yo Female CF 
MDR PsA TIVP-H6, LPS-5 
CFU Decrease 
Yes 
May 2019 
26 yo Female CF 
MDR PsA TIVP-H6, LPS-5 
CFU Decrease 
Yes 
Apr 2019 
35 yo Male 
CF 
MDR PsA TIVP-H6, LPS-5 
CFU Decrease 
Yes 
Apr 2019 
CF = cystic fibrosis; NCFB = non-cystic fibrosis bronchiectasis; MDR = multi-drug resistant; PDR = 
pan-drug resistant; PsA = Pseudomonas aeruginosa 
1 Repeat sputum test PsA negative 
2 Sputum evidence for change in PsA virulence factor (e.g., antibiotic resistance, pyocyanin production) 
 
 
 


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Figure 2-4. Comparison of Clinical Measures Using Patient Sputum Pre- vs. Post-Phage 
Therapy.  (A) Average lung function (FEV1%) improvement, (Wilcoxon Signed Rank, Prob>S 
0.0010). (B) Average bacterial count (CFU/mL) reduction, (Wilcoxon Signed Rank, Prob<S 
0.0313). (C) Average inflammatory molecule (Pyocyanin (mg/ml)) reduction (not enough data 
for statistical analysis). (D) Average antibiotic resistance (MIC (mg/ml)) reversion, (Wilcoxon 
Signed Rank, Aminoglycosides and Beta lactams Prob<S 0.0156, Cephalosporins and 
Fluoroquinolones ns). 
 
 
 
 


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3 Rationale/Significance 
3.1 
Problem Statement 
While the emergence of CFTR modulators has the potential to dramatically change CF 
outcomes, the threat of multi-drug resistant pathogens is already a reality for many CF 
patients, and the probability and severity of such infections will only increase with continued 
reliance on traditional antibiotics alone. Therefore, new antibacterial and anti-inflammatory 
approaches to combat these pathogens and their effects are urgently needed. Inhaled phage 
therapy has such potential due to: 1) antibacterial properties that uniquely differ from 
antibiotics; 2) potentially-low side effects given that phages are naturally present (and 
therefore frequently encountered) in the environment, including in the CF lung; and 3) potential 
for administered phage to kill target bacteria while selecting for phage resistance that 
coincides with useful trade-offs, especially reduced lung inflammation and re-sensitivity of 
bacteria to traditional antibiotics. However, carefully designed clinical trials are required to 
develop this therapy.  
Phage therapy is the use of lytic phages to treat bacterial infections. As one of the first 
antibacterial approaches discovered and developed in the early 20th century, phage therapy 
was largely eclipsed by development of chemical antibiotics following the discovery of 
penicillin, and the clinical potential of phage therapy was never fully embraced in Westernized 
countries. However, studies performed in the latter half of the 20th century, recent clinical trials, 
and individual case reports (see Investigator’s Brochure), which demonstrate safety and 
potential efficacy, have renewed interest in phage therapy. As a class of antibacterials, phages 
are distinct from traditional chemical antibiotics in four potentially beneficial ways: 1) phages 
are self-amplifying in the presence of bacteria, and thus 2) are limited in the absence of their 
substrate (i.e., susceptible bacteria); 3) phages are often able to penetrate biofilms to reach 
infectious bacterial cells (Deshpande Kaistha & Devi Umrao, 2016; Pires et al., 2016; 
Szafrański et al., 2017); and 4) phage mechanisms for killing bacteria are distinct from those 
of traditional antibiotics. Exploiting the differences between antibiotics and phage therapy has 
been a driving force for continued research into the potential clinical utility of phage therapy.  
Additionally, knowledge of the cellular-binding targets that phage(s) utilize when attaching to 
pathogenic bacteria may enable prudent choices of phages used in patient treatment. By 
utilizing phages that bind to virulence factors and drug-resistance mechanisms, the phage 
should kill bacteria while selecting for evolution of phage resistance that coincides with 
lowered virulence and drug re-sensitivity (defined as evolutionary “trade-offs”) (Figure 3-1). 
Trade-offs are often observed in biology, where organisms evolve one trait that improves 
fitness (a relative advantage in reproduction or survival), while simultaneously suffering 
reduced performance in another trait (Ferenci, 2016; Goldhill & Turner, 2014). Thus, phage 
therapy could be developed as an ‘evolutionary-based strategy’ that forces a trade-off 
between the evolution of phage resistance, and reduced virulence and/or drug re-sensitivity 
in target bacteria (Figure 3-1). Thus, using trade-off-based approaches in phage therapy can 
be doubly beneficial: 1) success is achieved when the phage kills the target bacterium, and 2) 


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success is also achieved when residual bacteria evolve phage resistance, because this 
causes increased bacterial sensitivity to clinically-approved antibiotics and/or decreased 
bacterial production of inflammatory products (Kortright et al., 2019). 
The Yale Phage Research Team has identified, purified, and sequenced environmentally-
sourced lytic phages that effectively target and kill PsA, while selecting for clinically-useful 
trade-offs. Research at Yale has shown that lytic phage treatment decreases bacterial 
densities in vitro (please see Investigator’s Brochure), and in emergency therapy of a human 
patient (Chan et al., 2016, 2018). These results demonstrated the doubly-beneficial effects of 
using certain phages in therapy, owing to forced trade-offs between evolution of phage 
resistance and reductions in pathogenic traits, especially bacterial re-sensitivity to antibiotics 
(Chan et al., 2018) and decreased bacterial secretion of harmful inflammatory products. In 
particular, the Yale team discovered and characterized phages that utilize three distinct 
receptor binding sites expressed on the surface of PsA cells: multi-drug efflux pumps, type-IV 
pili, and lipopolysaccharide (LPS). When exposed to each of these phages, PsA cells are 
effectively killed, whereas the remaining bacterial population evolves in the predicted manner: 
increased phage resistance evolves at the expense of down-regulation or other functional 
changes in the traits associated with these phage receptors, creating trade-offs that lead to 
drug re-sensitivity and lowered virulence in PsA.  
In unpublished experiments, 96 clinical strains of PsA isolated from adult CF sputum samples 
were examined in vitro for their susceptibility to each of the above-described phages and 
others in the Yale collection and it was observed that 1) PsA bacteria were effectively killed 
and 2) some phages were capable of broad-host-range killing across multiple PsA isolates. 
Using single-patient emergency investigational new drug applications (INDs) [eINDs], the Yale 
Phage Research Team has administered phages OMKO1, TIVP-H6 and LPS-5 via inhalation 
to CF and non-CF bronchiectasis (NCFB) patients to target their multi-drug and pan-drug 
resistant PsA infections. These individuals received nebulized phages for 7 to 10 days, and 
examination of their post-therapy sputum samples showed: 1) decreased bacterial densities; 
2) reduced pathogenic traits in remaining bacteria (i.e., bacteria with increased antibiotic 
sensitivity and decreased pyocyanin production); and 3) evidence for improved clinical 
outcomes (e.g., increased lung function by spirometry measures, and decreased need for 
administered antibiotics). These supportive results prompted the design of the current clinical 
study. 


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Taken together, preclinical and eIND data suggest that phage therapy may be safe and 
efficacious against PsA infections in CF patients. However, these promising clinical data come 
from cases of personalized treatment, and truly convincing results should stem from a blinded, 
randomized, controlled trial. Therefore, CYstic fibrosis bacterioPHage study at Yale (CYPHY) 
proposes to test the safety and efficacy of phage therapy to treat PsA infections in CF patients 
in this investigator-initiated, double-blind, randomized-controlled trial. 
3.2 
Purpose of Study/Potential Impact 
The main purpose of this study is to determine the short-term safety and efficacy of phages 
OMKO1, TIVP-H6, and LPS-5 to treat PsA infections in CF subjects. The use of phages in this 
experimental study may result in reduced bacterial burden. In addition, the strategy of Yale’s 
Phage Program is to use specific phages with targeted ability to exploit cellular receptors 
responsible for bacterial drug-resistance and/or virulence. Therefore, the phage therapy 
approach will kill bacteria, while forcing bacterial evolution in the direction of increased 
sensitivity to traditional antibiotics, and/or decreased inflammation, on average. This research 
may provide data demonstrating that these phages are safe and effective in treating PsA 
infections in CF, building evidence for their general use as an alternative, or combined 
approach, to using conventional antibiotics. This outcome would provide an additional 
therapeutic option for patients. Data from this study may also demonstrate improvement in 
forced expiratory volume over one second (FEV1) or FEV1 percent predicted (FEV1pp) and 
reduction in patient pulmonary exacerbations, which would benefit CF patient outcomes, as 
well as their quality of life.  
The primary outcomes will focus on efficacy, as measured by relative change in CFU of PsA 
in subject sputum, from before to after the intervention, as well as short-term safety as 
determined by number and severity of adverse events. Additional secondary endpoints will be 
clinical outcomes (e.g., FEV1, FEV1pp, CFQ-R), and additional understanding of the effect of 
phage therapy on the human lung (e.g., sputum microbiome, subject transcriptome, and 
phage antibody production). If our hypothesis is supported in this study, these data can be 
used to support further active commercial development of phage as a therapy for PsA in CF 
subjects. 
3.2.1 Potential Risks 
There are multiple AEs that may occur with phage therapy, although an SAE related to 
nebulized phage has not yet been reported. Because phages constitute biologic materials, 
human immunogenic reactions are possible. These responses may include fever, headache, 
and malaise following initial and subsequent phage treatments. Such reactions can be 
addressed with symptomatic treatment, and phage therapy may be continued provided the 
subject and their physician both agree that continuation of the therapy has potential benefit. 
Therefore, phage initial administration occurs under clinical supervision. It is possible, 
although presumed rare, that an allergic or anaphylactic reaction may occur, which would be 
treated in a similar manner as any such patient reaction to a new medication. However, it is 
observed that various largely-uncharacterized phages are commonly found in the CF lung 
(Brown-Jaque et al., 2018), reducing the likelihood that exposure to the characterized 
OMKO1, TIVP-H6, and LPS-5 phages would result in an unfortunate allergic reaction. 


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Additionally, as phages are produced in vitro on bacteria grown in nutrient media, phage 
therapy will not be used in subjects with known allergies to nutrient-media components of soy, 
yeast, egg, or meat. Allergic or anaphylactic reactions will be treated symptomatically (e.g., 
nebulized albuterol, oxygen, and epinephrine, if necessary). In the event of an allergic or 
anaphylactic response, phage therapy will be discontinued.  
Additional potential adverse events of phage therapy administration include increased cough, 
shortness of breath, or wheezing, which may be treated with nebulized albuterol and oxygen 
if necessary. CF is a chronic progressive respiratory disease that is associated with pulmonary 
exacerbations, and potentially hemoptysis and pneumothorax. It is possible that phage 
therapy could exacerbate these conditions. If these conditions occur, they will require 
physician evaluation that may include chest imaging (e.g., chest X-ray or CT scan) or the 
administration of antibiotics, among other interventions.  
One patient treated via eIND (18690), experienced an SAE after the third of three separate 
phage treatments, which resulted in their death. Review at this subject’s institution, and at 
Yale, determined that this event was unrelated to phage therapy (see Investigator’s 
Brochure for details).  
Study drug (phages or placebo), clinical visits, and procedures required for data collection 
(e.g., lung function and blood tests) in this study are provided free of charge. 
Some costs of travel can be covered by the study team as described in section 7.1 of this 
protocol. Administration of this therapy may come with financial risks for subjects because 
funds will not be provided to offset other costs associated with study participation. Such costs 
might include subject travel beyond what the study team can offer, standard-of-care 
treatments for cystic fibrosis, contraception, or if there is a clinical change that requires 
additional treatment. Such costs are not covered by this study.  
Breach of confidentiality is a potential risk of participating in this study. However, all efforts, 
within reason, will be made to keep subject health information private. Because treatment 
involves the use of an investigational drug (phage), Dr. Koff, or his team, may share 
information about subjects, as well as portions of subjects’ medical records, with the federal 
government’s Office of Human Research Protections, the Yale School of Medicine Human 
Investigation Committee, the Food and Drug Administration, and Yale University. Dr. Koff and 
his staff will keep health information in strict confidence and will comply with any and all laws 
regarding the privacy of such information.  
More detailed information about the known and expected risks and reasonably expected 
adverse events of treatments using phages OMKO1, TIVP-H6, and LPS-5 may be found in 
the Investigator’s Brochure. 
3.2.2 Potential Benefits 
Benefit(s) to science: The use of phages in this experimental study may result in data 
supporting the safety and efficacy of phage therapy as a treatment for PsA infections in CF 
patients. These data include the evaluation of phage safety, as well as the provision of 
preliminary efficacy data, such as reduction in sputum bacterial load, and improvement in lung 
function, exacerbations, or symptoms. In addition, the strategy of Yale’s Phage Program is to 


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use phages that target bacterial receptors that function in antibiotic resistance and pathogen 
virulence. Therefore, the phage therapy approach kills bacteria, while selecting for phage-
resistant bacteria with greater sensitivity to traditional antibiotics, and/or decreased ability to 
cause tissue inflammation. Lastly, this study is designed to explore both microbiologic changes 
and human-subject immune changes in response to phages, providing the most detailed 
picture, to date, of mechanisms and outcomes of phage therapy in patients.  
Possible clinical benefits to the subject may include improvement in symptoms, reduction of 
PsA load in sputum, improvement in lung function, improvement in quality of life (as measured 
by CFQ-R), and potentially decreased exacerbation(s) or need for additional medications. 
More detailed information about the known and expected benefits of YPT-01 may be found in 
the Investigator’s Brochure. 
 
 


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4 Study Objectives 
4.1 
Hypothesis 
In this single-center study, randomized double-blind placebo-controlled trial, we will test the 
hypotheses that 7d of inhaled phage therapy will suppress PsA load and will decrease PsA 
antibiotic resistance and virulence factors while safety and potential clinical responses (e.g., 
lung function, respiratory symptoms, and CFQ-R) will be evaluated. More specifically, our 
primary hypothesis for efficacy is that 7d inhaled phage therapy will decrease PsA load from 
baseline to 7 days post completion of phage therapy (14d time on study) in subjects 
randomized to two different doses of phage therapy (active phage treatment arm), as 
compared to subjects randomized to the placebo arm. Our co-primary hypothesis is related to 
the safety profile of phage therapy, as we hypothesize it is safe with respect to the number 
and severity of adverse events attributable to treatment. Our secondary efficacy hypotheses 
are that inhaled phage therapy: (1) will decrease PsA antibiotic resistance and virulence 
factors, (2) will favorably modify sputum phage, host, and microbiome, (3) and will have 
positive impact on clinical and quality of life response in subjects.   
 
4.2 
Primary Objectives 
Our primary objectives are to determine preliminary efficacy of phage therapy in reducing 
sputum bacteria in subjects by comparing changes from baseline to 7 days post completion 
of phage therapy (14d on study) in sputum bacterial load between subjects randomized to 
phage therapy versus placebo, and to obtain short-term safety data on inhaled phage therapy 
by comparing the frequency and severity of adverse events attributable to treatment between 
subjects randomized to phage therapy versus placebo during the blinded portion of the study 
(first 56 days on study). 
 
4.3 
Secondary Objectives  
The secondary objectives for the randomized period of this study are to: 
1) Compare change over time in PsA sputum load between subjects randomized to 
phage therapy versus placebo.  
2) Compare “trade-off” between evolved phage resistance and decreased virulence 
factors, as measured by changes in re-sensitivity to chemical antibiotics (for OMKO1-
treated subjects), PsA motility and production of pyocyanin (for TIVP-H6-treated 
subjects), and production of endotoxin (for LPS-5-treated subjects) in PsA from subject 
sputum prior to, and following, phage therapy, between subjects randomized to phage 
therapy versus placebo. 
3) Compare changes in sputum phage and bacterial microbiome between subjects 
randomized to phage therapy versus placebo.  
4) Compare changes in lung inflammation and inflammatory markers (e.g., sputum 
transcriptomics and inflammatory cytokines) between subjects randomized to phage 
therapy versus placebo. 


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5) Compare clinical response in subjects, with respect to change in lung function (e.g., 
FEV1pp), as well as rates of pulmonary exacerbations, hospitalizations, and acute 
antibiotic use for exacerbations between subjects randomized to phage therapy versus 
placebo. 
6) Compare changes in subject-reported quality of life (via CFQ-R) between subjects 
randomized to phage therapy versus placebo. 
The tertiary exploratory objectives for the randomized period are to:  
- 
Compare change in levels of phage-specific antibodies from baseline to post-treatment 
between subjects randomized to phage therapy versus placebo 
Objectives of Open-label extension (placebo subjects that subsequently receive phage) are 
to: 
- 
Provide subjects who received placebo with phage therapy.  
- 
Continue to gather additional safety data by monitoring for adverse events determined 
to be caused by the investigational product. 
- 
Describe change in PsA sputum load over time 
- 
Describe “trade-off” between evolved phage resistance and reduced virulence factors, 
as measured by changes in sensitivity to chemical antibiotics (for OMKO1-treated 
subjects), PsA motility and production of pyocyanin (for TIVP-H6-treated subjects), and 
production of endotoxin (for LPS-5-treated subjects) in PsA from subject sputum prior 
to, and following, phage therapy. 
- 
Describe differences in FEV1pp and absolute FEV1 (as measured in mL) change prior 
to, and following, phage therapy.  
Objectives of 6-month safety follow-up are to: 
- 
Obtain preliminary data on the long-term safety of phage therapy 
 
 


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5 Study Design 
5.1 
General Design Description 
This is a prospective, randomized, placebo-controlled, double-blinded, single-site study of 
Yale Phage Therapy (YPT) 01 in CF subjects with chronic PsA airway infections. The purpose 
of this study is to demonstrate preliminary efficacy and short-term safety of inhaled phage 
therapy YPT-01. Clinically stable subjects who have confirmed diagnosis of CF with PsA in 
sputum cultures on at least two occasions within past year, and in sputum at screening visit, 
will be recruited into this study.  
 
Experimental design: At Yale, this single-center, parallel trial has been designed to compare 
phage therapy to placebo. This study will screen 40 clinically stable (e.g., no exacerbations or 
medication changes within 4 weeks) CF subjects (no solid organ transplant; FEV1pp > 40% 
to < 100%) with chronic sputum PsA, and enroll 36, of whom 30 are expected to complete the 
study and meet the sample size goal. These 36 subjects will be block randomized to the 
intervention of phage therapy, or placebo, with anticipated enrollment rate of approximately 2 
subjects per week. Per DMC recommendation, the exact size of randomization blocks is not 
listed in the protocol and will only be known to the blinded research team statistician. 
Furthermore, per DMC request, the PI and research team will be blinded to the randomization 
block size during this study. In this study, the research team and subjects will be blinded to 
intervention group. Each subject will receive inhaled, GMP-grade OMKO1, TIVP-H6, or LPS-
5 phages at standard dose ( ≥1x108 PFU/dose) concentrations or placebo for 7d (GMP 
manufactured by Adaptive Phage Therapeuticfs, Gaithersburg, MD). All solutions are 
colorless, odorless, and identical in taste. The first dose of phage is supervised in clinic, and 
effect on lung function is evaluated (≥ 10% decline in absolute FEV1 between pre-phage and 
post-phage will be considered clinically significant, and subject will not continue to receive 
YPT-01 because of “bronchospasm” safety concerns). Subjects will continue daily nebulized 
phage for 7d total. Subjects will return for clinical evaluation on d3, primarily to confirm subject 
has not experienced any SAEs or ≥ 10% decline in absolute FEV1 that is at least probably 
related to YPT-01. Subjects will also visit clinic on d7 for clinical evaluation and spirometry. 
After completion of intervention (7 d), subjects will be evaluated weekly (±3d) until d28, after 
which subjects will be evaluated monthly (i.e., d56, d84, d112, d140, d168, with ±6 days for 
each) through clinic visits or phone calls through month 6 for long-term safety and adverse 
events.  
 
Open-label extension: The open-label extension serves as an opportunity for subjects in the 
placebo group to receive YPT-01 if they are interested. Based upon a survey of cystic fibrosis 
patients at Yale’s Adult Cystic Fibrosis Program and similar discussions that the Cystic 
Fibrosis Foundation has had with cystic fibrosis patients nationally, the addition of the open-
label extension will contribute to improved enrollment. Thus, after completion of visit #7 (d56), 
subjects in the placebo group will be offered open-label phage treatment. For all subjects that 
agree to participate, the open label extension will begin after 3 months (90 days) from dosing 
of first subject in the study. The start date at 90 days was chosen to minimize risk of unblinding 


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the clinical staff to which subjects received placebo. There is a concern that the clinical staff 
might be biased by recognizing subjects that went directly into the open-label extension. 
Therefore, 90 days was chosen to minimize this risk, yet allow for subjects to receive YPT-01, 
if they wished to, without waiting for the entire study to be completed. This would minimize 
subjects waiting to receive the open-label extension without biasing the clinical staff. However, 
DMC will review the relevant safety data and assist with deciding on 90 days for initiation of 
phage therapy. At 90 days after completing placebo, subjects in the placebo group will be 
identified by Yale Clinical Research Pharmacy and the research coordinator will be notified to 
arrange phage treatment As in the randomized trial, the first phage dose will be supervised in 
clinic, and have subsequent clinical follow-up at d7, d14, and d28, and monthly thereafter for 
a total of 6 months starting from initiating therapy in the blinded portion of the study. AE’s and 
SAE’s will be monitored throughout the study to assess the safety of YPT-01. Given that 
approximately 24 of the 36 subjects will be enrolled and have completed at least d3 safety 
visit by 90 days (assuming enrollment of approximately 2 subjects per week), the open-label 
extension will not include a d3 clinic visit. Should any subject exhibit an SAE or ≥ 10% decline 
in absolute FEV1 that is at least probably related to YPT-01 during the 7d of phage treatment, 
the d3 clinic visit will be re-instated for the open-label extension, or at the discretion of the 
DMC. 
 
For both the research and open-label extension, pre- and post-phage sputum samples will be 
collected and processed for PsA bacterial density, antibiotic sensitivity, virulence factors and 
deep sequencing. In addition, clinical responses (e.g., lung function, respiratory symptoms, 
and pulmonary exacerbations) will be evaluated. Should COVID-19 resurge resulting in limited 
research clinic visit availability or subject is unable or unwilling to conduct in person visits, 
monthly follow-up visits for safety and adverse events will be converted to phone calls to 
maximize subject safety. 
 
Phage administration has been carefully reviewed by Yale University Infection Prevention, and 
standard CF infection prevention practices are required for phage administration. 
 
Endpoints for double-blind randomized-controlled study: The primary efficacy endpoint is 
change in sputum PsA load from baseline (pre-phage) to d14 (i.e. 7 days post completion of 
7-day inhaled phage therapy).  
 
The primary short-term safety endpoints: Safety-related outcomes, i.e. frequency and 
severity of different types of adverse events as related to the study intervention, will be actively 
monitored throughout the study and summarized for the following days post-phage treatment 
during randomized blinded (experimental) period: d7, d14, d21, d28, and d56.  
 
Secondary end-points for response profiles at d3, d7, d14, d21 and d28: 
1) Change over time from baseline in PsA sputum load; 
2) Change over time from baseline in "trade-offs" of PsA, as measured by change in PsA 
antibiotic sensitivity (after OMKO1 phage), motility and pyocyanin levels (after TIVP-
H6 phage), and endotoxin levels (after LPS-5 phage); 


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3) Changes from baseline in sputum phage presence; 
 
Secondary end-point at d14: 
1) Change from baseline in lung inflammation as measured by sputum transcriptomics; 
Secondary end-points for response profiles at d14 and d28: 
1) Change from baseline in sputum inflammatory markers, as measured by inflammatory 
cytokines; 
 
Secondary end-points for response profiles at d3, d7, d14, d21, d28, and d56: 
1) Change from baseline in lung function (FEV1pp and absolute FEV1) from baseline; 
2) Rate(s) of pulmonary exacerbations and medication changes from baseline. 
 
Secondary end-points for response profiles at d3, d7, d14, d21, d28, d56, and monthly 
thereafter through month 6: 
1) Change from baseline in quality of life and respiratory symptoms as measured by 
Cystic Fibrosis Questionaire Revised (CFQ-R), Cystic Fibrosis Respiratory Symptom 
Diary (CFRSD) and Chronic Respiratory Infection Symptom Score (CRISS©) at each 
clinic visit from baseline; 
 
Tertiary end points for response profiles at d7, d14, d21, d28, and d56: 
1) Change from d1 in anti-phage antibody titers. 
 
 
Endpoints for Planned Unblinded study extension for subjects randomized to treatment: 
Monthly measurements through month 6 of total follow up for each subject will be collected on 
the following outcomes, i.e. the following secondary endpoints for longer-term efficacy and 
safety will be assessed: 
1) Monthly Frequency and severity of adverse events; 
2) Monthly Medication changes; 
3) Monthly Quality of life and respiratory symptoms as measured by CFQ-R, CFRSD & 
CRISS 
4) M4 and M6 change from baseline lung function (FEV1pp and absolute FEV1), and 
possible additional end-points at M3 and M5 follow up if subject has home spirometry; 
5) M4 and M6 Change from baseline in "trade-off" of PsA virulence characteristics, as 
measured by change in PsA antibiotic sensitivity (after OMKO1 phage), motility and 
pyocyanin levels (after TIVP-H6 phage) and endotoxin levels (after LPS-5 phage). 
6) Frequency and severity of adverse events at month 6 for preliminary long-term safety 
 
Endpoints for Open-label portion for subjects originally randomized to placebo: To minimize 
investigator unblinding, 3 months (90 days) after the first subject in the trial has received the 
first phage therapy dose and following approval from the DMC, subjects who received placebo 
will be offered YPT-01 in an Open-label study extension. Endpoints will comprise assessment 
of subjects at the following days post phage-therapy for: 


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1) Frequency and severity of adverse events and relationship attributable to treatment: 
open-label d1, d7, d14, d28, and monthly thereafter up to 6 months of total follow up 
for each subject from their original randomization to placebo group; 
2) Change in "trade-off" of PsA virulence characteristics, as measured by change in PsA 
antibiotic sensitivity (after OMKO1 phage), motility and pyocyanin levels (after TIVP-
H6 phage) and endotoxin levels (after LPS-5 phage) from open-label d1 to open-label 
d14 and d28; monthly thereafter up to 6 months, if subject agrees and is able to provide 
expectorated or induced sputum. 
3) Change in FEV1pp and absolute FEV1 from open-label d1 to open-label d14 and d28. 
4) Rate(s) of pulmonary exacerbations and medication changes: open-label d1, d7, d14, 
d28, and monthly thereafter up to 6 months of total follow up for each subject from their 
original randomization to placebo group. 
5) Change in Quality of life and respiratory symptoms as measured by CFQ-R, CFRSD 
& CRISS at each clinic visit: open-label d7, d14, d28, and monthly thereafter up to 6 
months of total follow up for each subject from their original randomization to placebo 
group. 
6) Frequency and severity of adverse events at month 6 for preliminary long-term safety 
 
 
 


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5.1.1 CYPHY Trial Design Tables 
Blinded, Randomized, Controlled Study and Unblinded Safety Follow-up for subjects receiving phage (n = 36) 
Screening 
Month 1* 
Month 2* 
Month 3# 
Month 4# 
Month 5# 
Month 6# 
Day 
-7 
1 
3 
7 
14 
21 
28 
56 
84 
112 
140 
168 
Visit 
X 
X 
X 
X 
X 
X 
X 
X 
 
X 
 
X 
Phone Call 
 
 
 
 
 
 
 
 
X 
 
X 
 
Informed Consent 
X 
  
  
  
  
  
  
  
 
 
 
 
Randomization 
X 
  
  
  
  
  
  
  
 
 
 
 
Medical History 
X 
X 
  
  
  
  
  
  
 
 
 
 
Height and Weight 
X 
X 
X 
X 
X 
X 
X 
X 
 
X 
 
X 
Vital Signs 
X 
X 
X 
X 
X 
X 
X 
X 
 
X 
 
X 
Adverse Events 
  
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Medication Regimen 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Physical Exam 
X 
X 
X 
X 
X 
X 
X 
X 
 
X 
 
X 
Daily Temperature 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
Urine Pregnancy Test1 
X 
X 
 
X 
 
 
 
 
 
 
 
 
Pre-treatment PFT2 
  
X 
  
  
  
  
 
 
 
 
Post-dose Observation 
 
X 
 
 
 
 
 
 
 
 
 
 
Post-treatment PFT2 
  
X 
  
  
  
  
 
 
 
 
PFT2 
X 
  
X 
X  
X 
X 
X 
X 
 
X 
 
X 
Sputum 
X 
X 
X 
X 
X 
X 
X5 
 
X5 
 
X5 
Blood for phage Ab3 
X 
X 
X 
X 
X 
X 
 
 
 
 
CFRSD, CRISS & CFQ-R4 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Study intervention 
  
  
  
  
  
  
  
  
 
 
 
 
Dispense diaries 
 
X 
 
 
 
 
 
 
 
 
 
 
Review diaries 
 
 
X 
X 
X 
X 
X 
 
 
 
 
 
* Blinded Portion for Short-term follow up 
3 Ab: antibodies  


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# Unblinded Portion for Longer-term follow up 
1 Administered to females of child-bearing potential 
2 PFT: Pulmonary Function Tests (e.g., spirometry) 
4 CFQ-R: Cystic Fibrosis Quality-of-Life Revised Survey 
5 Expectorated or induced (if the subject agrees) 
 
Open-Label Extension and Monthly Safety Follow-up for placebo-treated Subjects (n = 12) 
Open Label days – if subject enrolled 90 days after study start, they will 
initiate open label in month 3) 
Month 2 
Month 3  
(6 months from Day 1 of Double-Blind 
Arm) 
Day 
Screening 
1* 
7 
14 
28 
56 
84 
Visit 
 
X 
X 
X 
X 
 
 
Visit or Phone Call 
X 
 
 
 
 
X 
X 
Informed Consent 
X 
 
 
 
 
  
 
Medical History 
X 
X 
 
 
 
  
 
Height and Weight 
 
X 
X 
X 
X 
X 
 
Vital Signs 
 
X 
X 
X 
X 
X 
 
Adverse Events 
 
X 
X 
X 
X 
X 
X 
Medication Regimen 
X 
X 
X 
X 
X 
X 
X 
Physical Exam 
 
X 
X 
X 
X 
X 
 
Daily Temperature 
 
X 
X 
X 
X 
 
 
Urine Pregnancy Test1 
 
X 
X 
 
 
 
 
Pre-treatment PFT2 
 
X 
 
 
  
  
  
Post-dose Observation 
 
X 
 
 
 
 
 
Post-treatment PFT2 
 
X 
 
 
  
  
  
PFT2 
 
 
 X 
X 
X 
X 
 
Sputum 
X5 
 
X 
X 
X 
X5 
 
Blood for phage Ab3 
 
 
X 
X 
X 
X 
 
CFRSD, CRISS & CFQ-R4 
 
X 
X 
X 
X 
X 
X 
Study intervention 
 
 
  
  
  
  
  
Dispense diaries 
 
X 
 
 
 
 
 
Review diaries 
 
 
X 
X 
X 
 
 


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* Day numbering restarted for open-label patients (e.g., 
‘open-label d1’) 
1 Administered to females of child-bearing potential 
2 PFT: Pulmonary Function Tests (e.g., spirometry) 
3 Ab: antibodies 
4 CFQ-R: Cystic Fibrosis Quality-of-Life Revised Survey 
5 Expectorated or induced (if the subject agrees) 
 
 
 


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5.1.2 Study Date Range and Duration 
The total duration of the study is expected to be approximately 3 years: 6 months for study 
startup, 27 months for subject recruitment & Double-blind arm procedures, plus 3 months for 
final placebo subject to receive Open-label treatment.  
 
5.1.3 Number of Study Sites 
Single-site study, conducted at Yale University and Yale-New Haven Hospital. 
 
5.2 
Outcome Variables 
5.2.1 Primary Outcome Variables 
The primary efficacy endpoint in the study is the difference from baseline to d14 (7 days after 
completing 7-day intervention period) in PsA sputum load, which will be compared between 
the active treatment and placebo treatment groups. The primary short-term safety endpoints 
are the between-group differences in adverse event frequency and severity between the active 
treatment and placebo treatment groups at d7, d14, d21, d28, and d56. 
 
5.2.2 Secondary and Exploratory Outcome Variables 
Secondary Endpoints: 
• 
Long-term safety as measured by frequency and severity of adverse events at m3, m4, 
m5, and m6 data for subjects randomized to active phage, and monthly measurements 
in the open-label portion for subjects originally randomized to placebo. 
• 
Response profile: change from baseline to d3, d7, d14, d21, and d28 in PsA 
antibiotic sensitivity (if treated with OMKO1 phage), motility and pyocyanin production 
(if treated with TIVP-H6 phage), or endotoxin production (If treated with LPS-5 
phage)  
• 
Response profile: change from baseline to d3, d7, d14, d21, d28 and d56 in FEV1pp 
and absolute FEV1. 
• 
Response profile: change from baseline to d3, d7, d14, d21, d28 and d56 in the rate 
of pulmonary exacerbations. 
• 
Response profile: change from baseline to d3, d7, d14, d21, d28, d56, and monthly 
thereafter through month 6 in quality of life as measured by scales derived from the 
CFQ-R, CFRSD & CRISS. 
• 
Differences in sputum phage, host (e.g., inflammatory cytokines and transcriptomics), 
and microbiome as determined by high-throughput sequencing phage therapy and 
placebo treatment groups compared to each other on d3, d7, d14, d21 and d28 for 
phage presence, on d14 for bacterial microbiome and human lung inflammation 
(transcriptomics), and on d14 and d28 for inflammatory cytokines. 
 
Exploratory Endpoints: 


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• 
Response profile: change from d1 to d7, d14, d21, d28, and d56 in anti-phage 
antibody titers. 
 
5.3 
Study Population 
Subjects diagnosed with cystic fibrosis (CF) who meet all of the inclusion and none of the 
exclusion criteria will be eligible for enrollment in this study. 
 
5.3.1 Number of Participants 
This study will aim to enroll 36 subjects with CF who will have sputum PsA. Pre-phage sputum 
will be screened because, even though phages OMKO1, TIVP-H6, and LPS-5 can infect ~95% 
of clinical isolates assessed in vitro (see Investigator’s Brochure), a small subset of subjects 
may have PsA that cannot be treated with OMKO1, TIVP-H6, and LPS-5 phages. This 
indicates that screening approximately 40 subjects will enable us to enroll 36 subjects for this 
clinical trial. Based on the sample size calculation, we would need only 30 subjects, but 
accounting for attrition (approximately 10%) and equal number of subjects to be randomized 
across the three treatment arms, we propose to enroll 36 subjects.  
5.3.2 Eligibility Criteria/Vulnerable Populations 
Inclusion: 
1. Capable of giving signed informed consent; 
2. Stated willingness to comply with all study procedures and availability for the duration 
of the study; 
3. Age ≥18; 
4. CF diagnosis based upon genetics, sweat chloride testing, or clinical manifestations; 
5. Able to provide repeated induced sputum samples; 
6. Able to use a nebulizer; 
7. PsA culture positive on one occasion within past 2 years and in sputum at screening 
visit; 
8. FEV1 >40%; 
9. Clinically stable lung disease, defined as no decrease in FEV1 >10% or pulmonary 
exacerbations in the 4 weeks prior to screening; 
10. If on CF modulator therapy (e.g., ivacaftor, ivacaftor/elexacaftor/tezacaftor), then 
subject remains on the same modulator therapy for at least 2 months prior to 
enrollment; 
11. For females of reproductive potential: use of effective contraception for at least 1 month 
prior to screening and agreement to use 2 methods of effective contraception during 
study participation, and for an additional 6 weeks after the end of YPT-01 
administration;  
12. Males of non-reproductive potential (e.g., documented congenital bilateral absence of 
vas deferens) or males of reproductive potential (e.g., non-vasectomized males or 


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males vasectomized less than 120 days prior to study start) that agree to use condoms 
with spermicide while engaging in sexual activity or be sexually abstinent. 
Exclusion:  
1. History of solid organ transplant (e.g., lung or liver); 
2. Severe neutropenia, as defined by absolute neutrophil count (ANC) of < 500 per 
microliter;  
3. No YPT-01 phage identified that effectively targets sputum PsA;  
4. Treatment for pulmonary exacerbation within the prior 4 weeks; 
5. Change in pulmonary medications within the prior 4 weeks; 
6. Subjects who are pregnant, who intend to become pregnant, or who do not wish to use 
contraception; 
7. Subjects who are breastfeeding; 
8. Participation in another clinical research study concurrently or within the prior 2 
months; 
9. Known allergy to soy, egg, yeast, or meat. 
10. Any genetic or acquired (including medication-induced) immunocompromised 
condition, beyond the level of immunocompromise typically associated with CF and its 
management. 
5.3.3 Study Specific Tolerance for Inclusion/Exclusion Criteria 
Subjects who fail to meet one or more of the inclusion criteria, or who meet any of the exclusion 
criteria will not be enrolled in this study. Waivers of any of the above study entry criteria will 
not be granted.  
5.3.4 Screen Fail Criteria 
Any consented subject who is excluded from the study before randomization is considered a 
screen failure. All screen failures must be documented with the reason for the screen failure 
adequately stated. If a subject screen fails prior to randomization, they can be rescreened 
once if the site staff feels they meet eligibility criteria, and following confirmation from the 
Sponsor-Investigator or designee. Rescreened subjects are required to complete all screening 
procedures (i.e., test results from previous screenings cannot be used). 
5.3.5 Change in Medications or Therapies 
There are no restrictions on concomitant therapy. However, subjects should not make 
changes to medication unless deemed medically necessary. Subjects will inform the 
investigator of any changes in medication regimen and this will be recorded in the subject’s 
study record. 
 


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6 Methods 
6.1 
Treatment  
6.1.1 Identity of Investigational Product 
YPT-01 is a treatment algorithm comprising phage OMKO1, phage TIVP-H6, and phage 
LPS-5. Phages comprising YPT-01 are provided as a 1 mL solution of a single phage at  
standard dose of ≥1x108 phage particles in phosphate-buffered saline (PBS) and 10 mM 
MgSO4 for administration via inhalation (nebulization), and have been GMP manufactured by 
Adaptive Phage Therapeutics (APT). As YPT-01 phage ages the potency will be monitored 
by APT with a minimum acceptable dose of 1x108 PFU. If the potency drops below 1x108 
PFU/mL the number of vials will be increased to achieve a dose of at least ≥1x108 
PFU/dose. Maximum number of vials is 10 vials. 
Importantly Endotoxin levels will be monitored to ensure that no dose exceeds 5 EU/kg/hr.  
YPT-01 is currently not FDA-approved, although prior administration of YPT-01 phages has 
occurred via emergency INDs for compassionate use (see Investigator’s Brochure).  
YPT-01 is implemented by analyzing subject sputum culture. Phage sensitivity of PsA 
cultured from subject sputum (Figure 6-1) will be used to identify the single phage (Table 6-
1) to that will be administered. 
Placebo control will be GMP-grade phosphate-buffered saline (PBS) and 10 mM MgSO4, 
provided by Adaptive Phage Therapeutics.  
Table 6-1. Phages that comprise YPT-01 phage therapy. 
Bacteriophage 
ID 
Target Pathogen 
Receptor/Target Impact on bacterial 
community 
OMKO1 
Pseudomonas 
aeruginosa 
Efflux pump 
Re-sensitization to small-
molecule antibiotics 
TIVP-H6 
Pseudomonas 
aeruginosa 
Type IV Pilus 
Reduction of pyocyanin, 
impaired twitching motility, 
decreased biofilm formation 
LPS-5 
Pseudomonas 
aeruginosa 
LPS (O-antigen) 
Increased sensitivity to 
hydrophobic antibiotics, 
reduced extracellular 
endotoxin, impaired iron 
uptake 
 
Figure 6-1. Phage therapy algorithm. Briefly, PsA bacteria cultured from subject sputum samples 
are isolated and Efficiency of Plaquing (EOP) assay is performed with each phage (OMKO1, TIVP-H6, 
and LPS-5) as described below. The phage with the greatest EOP will be utilized for treatment. In the 


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event of equal EOP between two or more phages, phage for treatment will be utilized in the following 
order: TIVP-H6, followed by OMKO1, followed by LPS-5.  
 
Determination of Efficiency of Plaquing (EOP) 
10-fold dilutions of each phage will be performed eight times (dilutions -1 through -8) from an 
initial phage stock of approximately 10e9 PFU on both PsA host utilized to amplify phage 
(amplification host) and on PsA isolate from subject (subject host). A 10 µl spot of each dilution 
of phage will be applied to a lawn of amplification host and a lawn of subject host. Following 
incubation for 24 hours at 37°C, plaques will be enumerated and plaquing titer will be 
determined. Titer on subject host will be compared to that on amplification host, the ratio of 
which is the EOP. A value of EOP = 1 would indicate that plaquing titer is identical on the 
amplification and subject hosts; EOP = 10 would indicate that plaquing titer is 10x higher on 
the subject host. Whichever phage has the highest EOP on the subject host will be chosen 
preferentially for deployment. In the event of an equal EOP value between two or more 
phages, administered phage will be chosen based on frequency of use in emergent settings: 
TIVP-H6, followed by OMKO1, followed by LPS-5.  
 
6.1.2 Dosage, Administration, Schedule  
Based on our prior experience in humans (see Investigator’s Brochure), the dose of 1x1010 
PFU/dose has been effective in prior treatment under emergency INDs.  
Subjects will receive 7 containers of YPT-01 to be delivered via nebulization once daily for 7 
days. Subject is provided compressor, nebulizer, and all relevant equipment (InnoSpire 
Elegance Compressor #1099969 with SideStream Reusable neb #HS860, Philips 
Respironics). First dose of nebulized phage or placebo (both are clear odorless suspensions) 
is supervised in clinic. For the first dose of phage therapy, pre- and post-phage spirometry is 
performed to ensure safety of phage administration. If there is >10% change in absolute FEV1, 
then phage administration will be considered “bronchospastic,” and the subject will not 
proceed with additional treatments. Subjects will be observed for 30 minutes following the 
initial nebulization. Subject is provided daily containers for subsequent 6 d of treatment. 
Subjects will be asked to nebulize treatment after standard airway clearance therapies. 
6.1.3 Method of Assignment/Randomization  
Adaptive Phage Therapeutics (APT), Gaithersburg MD, will provide phages (at two 
concentrations) and placebo for the study. Randomization will occur once a subject meets all 
inclusion/exclusion criteria (including once sputum PsA phage sensitivity is obtained), and will 
be performed in block phage (n=24), and placebo (n=12) arms, for a total of 36 subjects. Per 
DMC recommendation, the exact size of randomization blocks will only be known to the 
blinded research team statistician. Furthermore, per DMC request, the PI and research team 
will be blinded to the randomization block size during this study. 


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6.1.4 Blinding and Procedures for Unblinding  
To preserve the blinding of the study, randomization table and treatment assignments are only 
accessible to the Investigational Drug Service (IDS) Pharmacy staff before the study is 
complete. The Investigators, study team managing the study, subjects, and any other 
personnel interacting directly with subjects will remain blinded the final enrolled subject in the 
blinded, randomized portion has completed the d56 study visit. The dispensing procedure will 
be carried out in such a manner that phage and placebo are identical in appearance, labeling, 
preparation time, expiration date and supplies used. 
To provide subjects randomized to placebo a chance to receive YPT-01, subjects in the 
placebo group will be offered open-label phage treatment following all participants’ completion 
of d56 visit. The subjects in the placebo group will be identified by IDS Pharmacy and phage 
will be provided, though the PI will be blinded to the dose of phage subject is receiving. 
In case of an emergency, the PI has the sole responsibility for determining if unblinding of a 
subject’s treatment assignment is warranted for medical management of the event. The 
subject’s safety must always be the first consideration in making such a determination. If the 
investigator decides that unblinding is warranted, investigator will reach out to IDS pharmacy 
to obtain treatment assignment information. It is the responsibility of the PI to promptly 
document the decision and rationale and notify the Sponsor as soon as an unblinding decision 
is made. It is also the PI’s sole discretion to have subject(s) discontinued from the study or 
remain in the study. 
 
6.1.5 Packaging/Labelling 
The following label will be provided by APT on phage doses, which will be identified by serial 
number (SN). A list of serial numbers will be provided to Yale Pharmacy for randomization and 
dispensing: 
 
 
Following administration of initial dose at clinic visit, 6 doses of phage therapy will be given to 
subjects during Visit 1. Subjects will be provided with the following information: 
2-8 °C (36-46 °F) until use 


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Storage and Handling Information on Individual Container: 
 
CAUTION: Investigational New Drug – Limited by 
Federal law to investigational use 
 
Active Ingredient (each container):  
Bacteriophage or Placebo 
SN: 
Storage and Handling Information provided with Outer Container: 
 
CAUTION: Investigational New Drug – Limited by 
Federal law to investigational use 
 
Active Ingredient (each container):  
Bacteriophage or Placebo 
SN: 
Number of containers: 
Expiration Date: 
Subject Name: 
 
Storage 
• Store tubes in the refrigerator (2 – 8 °C) and protect from direct sunlight 
• Do not store in freezer 
• Open each container just prior to use 
• Do not open container when not in use 
 
 
 
 
 


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6.1.6 Storage Conditions 
Single-use phage containers will be stored under refrigeration at 2 °C – 8 °C (36 °F – 46 °F) 
and protected from light until administration. If there is visual evidence for particulate matter 
in the solution, the phage preparation will not be used.  
The investigator or designee must confirm appropriate temperature conditions have been 
maintained during transit for all study intervention received and any discrepancies are reported 
and resolved before use of the study intervention. 
Only participants enrolled in the study may receive study intervention and only authorized site 
staff may supply study intervention. Subjects self-administer study intervention under clinical 
team supervision for the first dose, and administer subsequent doses independently. Prior to 
dispensing to subject, all study intervention must be stored in a secure, environmentally 
controlled, and monitored (manual or automated) area in accordance with the labeled storage 
conditions with access limited to the investigator and authorized site staff. 
The PI, is responsible for study intervention accountability, reconciliation, and record 
maintenance (i.e., receipt, reconciliation, and final disposition records). 
Unused doses and nebulizer provided during the study will be requested from subjects at the 
end of the study and disposed of through appropriate biosafety mechanisms as required by 
Environmental Health and Safety at Yale.  
6.1.7 Concomitant therapy 
There are no restrictions on concomitant therapy, however subjects should not make changes 
to medication unless deemed medically necessary.  Subjects will inform the investigator of 
any changes in medication regimen and this will be recorded in the subject’s study record. 
6.1.8 Restrictions 
No restrictions.  
6.2 
Assessments 
6.2.1 Efficacy 
Primary Objective 
Sputum Bacterial Load 
The parameter for the primary endpoint is change in bacterial load in subject sputum, as 
measured in colony-forming units (CFUs) of PsA, which is determined by serial dilution 
plating assays as described in the Laboratory Manual.  
Induced Sputum Collection 
Subject sputum is collected to assess the primary endpoint of reduction in PsA CFUs, and to 
measure secondary objectives of bacterial re-sensitivity to chemical antibiotics, assaying 
change in bacterial virulence factors, assessing human inflammatory and transcriptomic, and 
genomic changes in sputum microbiome. Sputum is induced as described in the Laboratory 
Manual, with sputum induction protocol repeated until the subject has produced at least 1 mL 
of sputum.  


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Secondary Objectives  
Sensitivity of sputum PsA to chemical antibiotics 
Sputum PsA are assessed for antibiotic sensitivity to determine the “trade-off” of re-
sensitization to antibiotics. Antibiotic sensitivity testing is performed as described in the 
Laboratory Manual using commercially-available antibiotic resistance test kits (e.g., 
eTestSTrip from BioMerieux or similar product) and methods recommended by the 
manufacturer. The following antibiotics may be tested, but will be determined as clinically 
relevant for the strain based on initial PsA isolate resistance profile: cefepime, ceftazidime, 
piperacillin-tazobactam, meropenem, aztreonam, tobramycin, ciprofloxacin, and colistin.  
Levels of bacteria virulence factors (pyocyanin or endotoxin) 
Sputum PsA are assessed for level of pyocyanin and endotoxin production to determine the 
“trade-off” of reduced pyocyanin or endotoxin production. The pyocyanin quantification assay 
is performed as described in the Laboratory Manual and is adapted from (Ingledew & 
Campbell, 1969). The endotoxin quantification assay is performed utilizing a commercially-
available endotoxin detection kit (e.g., Hyglos endoNext endoZyme kit or similar product) using 
methodology recommended by the manufacturer.  
Assess change in PsA motility 
Motility is quantified on PsA isolated from subject sputum using a “Macroscopic Twitching 
Assay” adapted from (Turnbull & Whitchurch, 2014) as described in the Laboratory Manual. 
Briefly, PsA cultures inoculated into the center of a 1% LB-Lennox Agar Plate and allowed to 
grow for 24 hours. The distance in millimeters between the inoculation point and outer halo of 
bacteria is then measured to determine distance traveled by PsA inoculate, informing motility 
function. 
Pulmonary Function tests and Spirometry 
Spirometry (pulmonary function tests, PFT) is assessed to determine change in pulmonary 
function (e.g., FEV1, FEV1pp) and will be measured by a spirometer using standard 
pulmonology methods, as described in the Laboratory Manual. This is a standard test for all 
cystic fibrosis subjects in our practice. 
CF subjects at each clinic visit. 
Spirometry results are provided as raw data, in liters per second, as well as percent predicted. 
Percent predicted is a comparison of the subject’s test results to the predicted values for 
individuals of similar characteristics (e.g., height, age, gender, and ethnicity). Height is 
measured during screening visit per standard protocol. 
Rate of pulmonary exacerbations 
Pulmonary exacerbations will be assessed by collecting medical history data and recent 
changes to symptoms from subjects, and comparing the rate of exacerbations between phage 
treatments and the placebo group during the study, and to historical exacerbation rate from 
prior medical history in the Open-label extension.  


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The presence of a pulmonary exacerbation is established by: 
(1) One major criterion alone, or (2) Two minor signs/symptoms and fulfillment of 
symptom duration 
Major criteria: (One finding alone establishes the presence of a pulmonary 
exacerbation) 
 
 
- Decrease in FEV1 %predicted (pp) of ≥ 10% 
 
 
- Oxygen saturation < 90% on room air or absolute decrease of ≥ 5% 
 
 
- New lobar infiltrate(s) or atelectasis on chest radiograph 
- Hemoptysis (on more than one occasion in past week); hemoptysis is 
defined as 1) mild to moderate (5-240 mL), and 2) massive (>240 mL) 
 
Minor signs/symptoms: (Two minor signs/symptoms are required in the absence of 
major criteria. If at least 2 minor signs/symptoms are present, at least one needs to 
be 3 or more days in duration to meet the pulmonary exacerbation definition) 
 
 
- Increased work of breathing or respiratory rate 
 
 
- New or increased adventitial sounds on lung exam 
 
 
- Weight loss ≥ 5% of body weight 
 
 
- Increased cough 
 
 
- Decreased exercise tolerance or level of activity 
 
 
- Increased chest congestion or change in sputum 
 
Subject Quality of Life 
Quality of life changes will be assessed using the Cystic Fibrosis Questionnaire Revised 
(CFQ-R), a subject-reported outcome report that has been validated to evaluate CF subject’s 
disease-related quality of life (Ronit et al., 2017), as well as the Cystic Fibrosis Respiratory 
Symptom Diary (CFRSD), and the Chronic Respiratory Infection Symptom Score (CRISS). 
These assessments are meant to evaluate subject’s gastrointestinal and respiratory 
symptoms and/or symptom progression, treatment burden, and overall daily functioning. 
These questionnaires will be key secondary efficacy endpoints to the study and can be 
administered as an electronic or written assessment, requiring approximately 20 minutes to 
complete. 
Inflammatory markers in sputum 
An aliquot of subject sputum will be analyzed for inflammation by measuring levels of 
inflammatory proteins (e.g., IL-8, IL-6, TNF-alpha, IL-1beta, MUC5AC, MUC5B, and other 
relevant chemokines and cytokines). Experimental methods for assessment of inflammatory 
markers in the sputum are described in the Laboratory Manual.  
PCR detection of treatment phage 
Polymerase chain reaction (PCR) and quantitative PCR (qPCR) will be performed on subject 
sputum to assess presence of phage used in treatment and will be performed as described 
in the Laboratory Manual.  


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Genomic and transcriptomic changes in sputum microbiome 
Subject sputum will be assessed for genomic and transcriptomic changes in the sputum as 
adapted from (Hunter et al., 2011; Nelson et al., 2019) and (Cobián Güemes et al., 2019) 
and described in the Laboratory Manual. 
Detection of anti-phage antibodies  
Subject blood will be analyzed to assess presence and levels of anti-phage antibodies using 
standard ELISA assays, as described in the Laboratory Manual.  
Detection of Bacterioal Load in Blood 
6.2.2 Subject blood will be analyzed to assess bacterial load, as described in the 
Laboratory Manual.Safety 
This study will collect all adverse events and serious adverse events that occur in subjects 
and record the severity and relationship to investigational product. These data will be 
collected during all clinic visits, through the End of Study Visit (Day 56), and through the end 
of the Open-Label phase for those subjects who enter that portion of the study. 
Given prior experience with YPT-01 in subjects with FEV1 >40% predicted in eINDs (Table 2-
2), incidence of adverse reactions is expected to be extremely low. However, the potential 
always exists for anticipated and/or unanticipated adverse events (serious or otherwise) to 
occur. Since it is not possible to predict with complete certainty the absolute risk in any given 
individual, the following plan is provided for monitoring the data and safety of the proposed 
study as follows:  
 
Plan for Reporting Adverse Events  
For the current study, the following individuals, funding, and/or regulatory agencies will be 
notified:  
1) All co-investigators listed on the protocol 
2) Data and Safety Monitoring Committee (DSMC) 
3) Yale IRB 
4) FDA 
Adverse Events (AE) 
An adverse event (AE) is any untoward medical occurrence in a subject during the study, 
which does not necessarily have a causal relationship with the treatment. This includes any 
newly occurring event or worsening of a pre-existing condition.  
 
The study staff will probe, via discussion with the subject, for the occurrence of AEs during 
each subject visit and record the information in the site’s source documents. Subjects will be 
provided with a diary to record the occurrence of AEs during the study. The diary will be 
reviewed during each study visit. In addition to events spontaneously reported by subjects 
during the visit or in the diary, at each visit subjects will be specifically asked whether they 
have experienced increased sputum production, shortness of breath, hemoptysis, chest pain, 
cough, and fever or chills since their last visit (or after administration of investigational product 
at the Day 1 visit). All subjects will be provided with a thermometer to monitor their temperature 
daily and a diary to record these measurements and subject symptoms.  
 
Adverse events will be recorded in the subject case report form (CRF). Study assessments 
include physical exam(s), vital signs (including pulse oximetry), and spirometry/pulmonary 


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function testing (PFTs). Those deemed to have clinically significant change from baseline 
(e.g., absolute FEV1 10% change) will be documented as an AE. A clinical diagnosis will be 
provided, or if there is no diagnosis, an abnormal study result or assessment will be listed as 
the AE. All subjects will be queried about the occurrence of AEs at each study visit. For each 
AE, documentation will include: event description, classification as “serious” or “non-serious,” 
date of first occurrence and date of resolution (if applicable), severity, causal relationship to 
study intervention, action taken (e.g., diagnostic studies, medication, or treatment given), and 
outcome. 
 
Adverse Events (AE) Severity 
The National Cancer Institute’s Common Terminology Criteria for Adverse Events (CTCAE) 
Version 5.0 will be used to assess and grade AE severity, including laboratory abnormalities 
judged to be clinically significant. It should be pointed out that the term “severe” is a measure 
of intensity and that a severe AE is not necessarily serious.  
 
Table 6-2. AE Severity Grading  
Severity 
(Toxicity Grade)  Description  
Mild (1)  
 
Mild; asymptomatic or mild symptoms; clinical or diagnostic observations 
only; intervention not indicated. 
Moderate (2)  
 
Moderate; minimal, local or noninvasive intervention indicated; limiting 
age-appropriate instrumental activities of daily living (e.g., preparing meals, 
using the telephone, managing money).  
Severe (3)  
Severe or medically significant but not immediately life-threatening; 
hospitalization or prolongation of hospitalization indicated; disabling; 
limiting self-care activities of daily living (e.g., bathing, dressing, feeding 
self, using toilet, taking medications).  
Life-threatening 
(4) 
Life-threatening consequences; urgent intervention indicated.  
Death (5)  
Death related to AE. 
Table 6-3. AE Relationship to Study Drug  
 
Relationship 
to Drug  
Comment 
Definitely  
Previously known toxicity of agent; or an event that follows a reasonable 
temporal sequence from administration of the drug; that follows a known or 
expected response pattern to the suspected drug; that is confirmed by 
stopping or reducing the dosage of the drug; and that is not explained by any 
other reasonable hypothesis.  
Probably 
An event that follows a reasonable temporal sequence from administration of 
the drug; that follows a known or expected response pattern to the suspected 
drug; that is confirmed by stopping or reducing the dosage of the drug; and 
that is unlikely to be explained by the known characteristics of the subject’s 
clinical state or by other interventions.  


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Possibly  
An event that follows a reasonable temporal sequence from administration of 
the drug; that follows a known or expected response pattern to that suspected 
drug; but that could readily have been produced by a number of other factors. 
Unlikely 
There is little evidence to suggest a causal relationship (e.g. the event did not 
occur within a reasonable time after administration of the trial medication) or 
another reasonable explanation for the event (e.g. another clinical condition 
or other concomitant treatment) 
Unrelated  
An event that can be determined with certainty to have no relationship to the 
study drug.  
Serious Adverse Events (SAE)  
An SAE is defined as any AE occurring at any dose that results in any of the following 
outcomes: death, a life-threatening adverse experience, inpatient hospitalization or 
prolongation of existing hospitalization, a persistent or significant disability/incapacity, a 
congenital anomaly/birth defect. Other important medical events may also be considered an 
SAE when, based on appropriate medical judgment, they jeopardize the subject or require 
intervention to prevent one of the outcomes listed.  
 
Serious Adverse Event (SAE) Reporting  
SAEs that occur (whether or not related to study drug) will be reported on an SAE Report 
Form. The collection period for all SAEs will begin after informed consent is obtained, and end 
after procedures for the final study visit have been completed. In accordance with the standard 
operating procedures and policies of the local Institutional Review Board (IRB), the site 
investigator will report SAEs to the IRB.  
 
Assessment of Risks: Subjects will have an opportunity to participate in tests that are routinely 
used in clinical medicine. The risks associated with this study are the following:  
Minimal risk: venipuncture, and sputum collection.  
• 
Minimal risks of venipuncture: Approximately 16 mL of blood will be collected from subjects 
per draw, at each indicated visit in the Study Schedule (Section 5.1), for a total of 
approximately 96 mL of blood for subjects randomized to phage and 160 mL of blood for 
subjects randomized to placebo who choose to subsequently receive phage in the open-
label portion. If a patient weighs less than 110 lbs the blood draw volume will be adjusted 
so as not to exceed the lesser of 50ml or 3ml/kg in an 8-week period. The procedure 
involved in obtaining blood specimens for laboratory analysis is conducted through the use 
of standard phlebotomy methods. The personnel obtaining these samples are highly 
trained nurses or technicians, well versed in this collection method, thereby imposing 
minimal risk. Occasionally, a subject will experience “light-headedness” during the 
phlebotomy procedure. To minimize the risk of this occurrence, all subjects will be placed 
in a supine position on a hospital bed, attended by trained personnel and monitored 
following the procedure. Should a bruise or swelling occur following the phlebotomy 
procedure, ice will be applied to the site, and trained personnel will monitor the site. This 
bruising/swelling is associated with minimal risk.  
• 
Minimal risk of sputum collection. Expectorated sputum is routinely acquired from CF 
subjects at all clinic visits. If unable to spontaneously produce sputum, throat cultures are 
obtained from CF subjects at all clinic visits. No specific intervention occurs outside of 
usual care.  
Moderate risk: nebulization of phage therapy, though no adverse events attributable to therapy 
have been seen in any of the adults who participated in eIND, to date.   


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• 
Risk of nebulization: YPT-01 is an experimental therapy. However, CF subjects nebulize 
several medications and to date there have been no adverse events associated with 
nebulization in CF subjects with mild to severe lung disease. If there is an issue with post-
nebulization wheeze or coughing, nebulized albuterol will be provided. 
For studies conducted under an IND, there are two types of Safety Reports submitted to 
FDA: 
• 
7-Calendar-Day FDA Telephone or Fax Report:  The sponsor-investigator will directly 
notify the FDA, within 7 calendar days after initial receipt of the information, of any 
adverse event that is fatal or life-threatening, unexpected, and considered at least 
possibly related to the investigational product.  
• 
15-Calendar-Day FDA Written Report:  The sponsor-investigator will directly notify the 
FDA within 15 calendar days after initial receipt of the information, of any serious 
adverse event (other than those that are fatal or life-threatening) that is unexpected 
and considered at least possibly related to the investigational product. 
Note:  Serious Adverse Events which do not meet the criteria for expedited reporting will be 
reported to the FDA in the IND Annual Report. 
Stopping rules: 
The following events would result in discontinuation in treatment of an individual subject: 
• 
For first administration of phage in clinic, a 10% decrease in lung function (FEV1) 
• 
For the duration of the trial, a Grade 3 or higher adverse event, or an SAE 
(regardless of grade) deemed at least probably caused by phage therapy  
The following events would result in a pause in study enrollment and a pause in study 
treatment for all enrolled subjects: 
• 
Any subject with an SAE that is at least probably related to phage therapy; 
• 
Two or more subjects with the same SAE (regardless of attributability to phage 
therapy), or two SAEs (Grade 3 or higher) regardless of relatedness; 
o This stopping rule excludes expected hospitalizations for routine CF 
exacerbations (i.e. Pulmonary Exacerbations), which are reported in a 
monthly summary by sponsor to the DMC 
• 
Two or more Grade 3 or higher AEs considered at least possibly related to phage 
therapy. 
Following the imposition of a pause, the Data Monitoring Committee (DMC) created by the 
Cystic Fibrosis Foundation DMC, based at the University of Arizona, will be consulted to 
modify the protocol as needed, and to develop criteria to resume study. Any protocol 
changes would be submitted to the IRB for approval and communicated to the FDA. There 
are no a priori stopping criteria for this trial. 
6.2.3 Biomarkers (if applicable) 
N/A 
 


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6.3 
Study Procedures 
Some subjects may be receiving chronic inhaled antibiotic therapy (e.g., tobramycin, 
aztreonam, or alternating therapy) as part of their treatment regimen. Subjects whose 
treatment regimen includes inhaled tobramycin or alternating therapy between tobramycin and 
aztreonam will initiate nebulization of YPT-01 in the first 14 days of initiating a tobramycin 
treatment cycle. Subjects whose treatment regimen includes inhaled aztreonam will initiate 
nebulization of YPT-01 in the first 14 days of initiating an aztreonam treatment cycle.  
 
Screening visit: Day -7 (±5): 
1. Review the study with the subject and obtain written informed consent and HIPAA 
authorization and assent, if appropriate. 
2. Collect urine for pregnancy test (if female of child-bearing potential). 
3. Perform and record spirometry. 
4. Induce sputum to obtain sputum culture and determine eligibility: 
a. Laboratory assessment of PsA culture susceptibility to YPT-01 as illustrated in 
Figure 6-1, and baseline antibiotic resistance through CLIA laboratory testing. 
b. If sputum culture pathogen meets inclusion criteria, assign the subject a unique 
study number randomized in block format as illustrated in Table 6-4 in a format 
determined by the unblinded DMC statistician and research team statistician, 
which is unknown to the research study team, and schedule subsequent visit 
to complete Day 1 procedures. 
c. If sputum culture pathogen does not meet inclusion criteria, record as Screen 
Failure. 
5. Record demographics. 
6. Record medical history, including a history of CF diagnosis and date. 
7. Record concomitant medications. 
8. Perform a targeted physical examination. 
9. Measure and record height and weight. 
10. Obtain and record vital signs. 
 
Following screening visit, subjects who meet screening criteria will be randomized into one 
of the following treatment arms: 
Table 6-4. Randomization scheme 
Arm Name 
 Phage 
Placebo 
Intervention 
Name 
YPT-01 
Vehicle solution 
Type 
Biologic 
N/A 
Dose 
Formulation 
Liquid for nebulization 
Liquid for nebulization 


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Unit Dose 
Strength(s) 
≥1x108  phage particles 
units/dose 
 
N/A 
Double-Blind 
Arm Dosage 
Level(s) 
One 1mL vial YPT-01 at a dose 
of  >=1.0x108 PFU/mL plus two 
1mL vials placebo, 
administered daily for 7 days 
Three 1mL vials placebo, 
administered daily for 7 days 
Open-Label 
Arm Dosage 
Level(s)  
Up to ten 1mL vials YPT-01 
phage as needed to achieve a 
dose of at least 1x108 PFU/mL 
once daily for 7 days.  
No more than 10 vials will be 
administered at a time to 
minimize the burden of 
administration.  
Placebo vials will be included 
as needed to reach a minimum 
volume of 3mL study drug per 
dose. 
 
N/A 
Route of 
Administration 
Inhalation (nebulization) 
Inhalation (nebulization) 
Use 
Experimental 
Placebo control 
Sourcing 
Adaptive Phage Therapeutics  
Adaptive Phage Therapeutics  
Packaging and 
Labeling 
Each Study Intervention 
container will be labeled as 
required per country 
requirement 
Each Study Intervention 
container will be labeled as 
required per country requirement 
[Current/Former 
Name(s) or 
Alias(es)] 
YPT-01:  OMKO1, TIVP-H6, or 
LPS-5 
N/A 
 
 


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Treatment tracking 
Subjects will be asked to track treatments in a study diary (Appendix 3).  
 
Missed doses  
Subjects are encouraged to administer phage at approximately the same time daily. However, 
missed doses may be made up as long as they are not taken within 8 hours of the next 
scheduled dose. 
 
Voluntary discontinuation 
A subject may, at any time, discontinue participation in the trial for any reason. If a subject 
chooses to discontinue treatment, they will be provided with the option to continue follow-up 
visits to monitor for adverse events. 
 
Discontinuation due to protocol violation 
A protocol violation may lead to discontinuation and occurs when the subject, investigator, or 
the Sponsor fails to adhere to significant protocol requirements affecting the inclusion, 
exclusion, subject safety and primary endpoint criteria. Protocol violations for this study 
include, but are not limited to, the following: 
• 
Failure to meet eligibility criteria following study enrollment 
• 
Failure to obtain induced sputum at visits 
 
Failure to comply with Good Clinical Practice (GCP) guidelines will also result in a protocol 
violation. The Primary Investigator (PI) will determine if a protocol violation should result in 
early discontinuation of study treatment for a subject. 
 
When a protocol violation occurs, it will be discussed with the PI and a Protocol Violation Form 
detailing the violation will be generated. A copy of the form will be filed in the site’s regulatory 
binder. The site will report the violation to their IRB in accordance with their IRB reporting 
requirements. 
 
Pregnancy in subject or subject’s partner 
Should a subject or a subject’s partner become pregnant while subject is on study drug, study 
drug will be discontinued and subject (or subject’s partners’) pregnancy and neonatal 
outcomes will be collected. Should a subject or subject’s partner become pregnant after 
completion of study drug, but during study monitoring (e.g., in the experimental blinded or 
open-label portion of the study), subject pregnancy and neonatal outcomes will also be 
collected.  
 
Subject replacement 
Subjects who withdraw from the study will not be replaced. 
6.3.1 Study Schedule 
Following screening visit, the total number of expected visits is 10 in-person visits and 2 phone 
calls for phage-treated group and 15 in-person visits and 2 phone calls for placebo-treated 
group that continues to open-label, including consent and screening visit, treatment-visits, 
post-treatment follow-up visits, and end of study evaluation. However, should COVID-19 
pandemic result in facility closures that prevent in-person visits as described in Section 5.1, 
the 2 in-person visits for safety follow-ups in month 4 and 6 for both phage-treated and open-
label subjects will be converted to phone calls.  
 


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6.3.2 Informed Consent 
Consent is required and will be acquired by a member of the research team using the Informed 
Consent form, which will be scanned and stored in the EMR. Physical copies will be provided 
to the subject as well as stored in a secure cabinet.  
 
6.3.3 Screening (Day -7±5) 
Screening visit will be performed by PI and/or investigator’s clinical research team and will 
include medical history, physical exam, spirometry (PFT), CFQ-R, sputum collection, and 
informed consent. Sputum is obtained for research laboratory analysis (e.g., phage 
susceptibility) and baseline antibiotic sensitivity testing is assessed from subject medical 
records. Randomization is completed once sputum PsA phage susceptibility is obtained. 
Screen failures are defined as participants who consent to participate in the clinical study but 
are not subsequently entered in the study. A minimal set of screen failure information is 
required to ensure transparent reporting of screen failure participants to meet the 
Consolidated Standards of Reporting Trials (CONSORT) publishing requirements and to 
respond to queries from regulatory authorities. Minimal information includes demography, 
screen failure details, eligibility criteria, and any serious adverse event (SAE). 
Individuals who do not meet the criteria for participation in this study (screen failure) may be 
rescreened. Rescreened participants should be assigned the same participant number as 
identified in the initial screen visit. 
6.3.4 Enrollment  
The investigator will enroll subjects who have consented and met the eligibility criteria during 
screening.  
6.3.5 On Study Visits 
Treatment visit 1: Day 1: 
1. Confirm medical history and any changes in status since screening visit. 
2. Record any changes to concomitant medications. 
3. Administer CFQ-R. 
4. Perform an abbreviated physical examination. 
5. Obtain and record vital signs. 
6. Collect urine for pregnancy test (if female of child-bearing potential). 
7. Blood collection. 
8. Perform and record pre-treatment spirometry. 
9. Dispense study drug, compressor, nebulizer, (i.e., InnoSpire Elegance Compressor 
#1099969 with SideStream Reusable neb #HS860, Philips Respironics), and all 
relevant equipment (e.g., thermometer) to subject. 
10. Review study drug administration with subject. 
11. Supervise subject self-administer first dose of study drug in clinic. 
a. Observe subject for 30 minutes following nebulization for any potential adverse 
events. 
12. Perform and record post-treatment spirometry. If > 10% decline in FEV1 compared to 
pre-treatment spirometry, this will be considered evidence for an adverse response to 
treatment and subject will not continue in the study. 
13. Ask subject about AEs of interest (increased sputum production, shortness of breath, 
hemoptysis, chest pain, cough, and chills or fever). 


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14. Review study drug dose, schedule, and storage conditions with subject. 
a. Subjects are asked to nebulize treatment after standard airway clearance 
therapies at the same time daily. 
15. Provide subject medication and AE diaries and instructions for completion. 
16. Remind subject NOT to discontinue therapy if they experience improvements or feel 
better. 
17. Instruct subject to bring empty study drug containers, and study diary to subsequent 
visit. 
18. Schedule subject for subsequent visit. 
 
Treatment visit: Day 3 (±1): 
1. Record any changes in status since last visit. 
2. Record any changes to concomitant medications. 
3. Administer CFQ-R. 
4. Review subject study diaries and perform container count on returned containers. 
5. Record any AEs. Query for increased sputum production, shortness of breath, 
hemoptysis, chest pain, cough, and chills or fever since their last visit. 
6. Perform an abbreviated physical examination. 
7. Obtain and record vital signs. 
8. Perform and record spirometry. 
9. Induce and collect sputum sample. 
10. Instruct subject to bring empty study drug containers, equipment provided for study 
(e.g., nebulizer), any unused doses of study drug, and study diary to subsequent visit. 
11. Schedule subject for subsequent visit. 
 
Treatment visit: Day 7 (±1): 
1. Record any changes in status since last visit. 
2. Record any changes to concomitant medications. 
3. Administer CFQ-R. 
4. Review subject study diaries and perform container count on returned containers. 
5. Record any AEs. Query for increased sputum production, shortness of breath, 
hemoptysis, chest pain, cough, and chills or fever since their last visit. 
6. Perform an abbreviated physical examination. 
7. Obtain and record vital signs. 
8. Collect urine for pregnancy test (if female of child-bearing potential). 
9. Blood collection. 
10. Perform and record spirometry. 
11. Induce and collect sputum sample. 
12. Request return of equipment provided for study, empty containers of study drug, and 
any unused containers of study drug for disposal. 
13. Schedule subject for subsequent visit. 
 
Follow-up visits: Days 14 (±3), 21 (±3), and 28 (±3):  
1. Record any changes in status since last visit. 
2. Record any changes to concomitant medications. 
3. Administer CFQ-R. 
4. Review subject diary 
5. Record any AEs. (Day 14 only: Query for increased sputum production, shortness of 
breath, hemoptysis, chest pain, cough, and chills or fever since their last visit.) 
6. Perform an abbreviated physical examination. 
7. Obtain and record vital signs. 
8. Blood collection. 
9. Perform and record spirometry. 
10. Induce and collect sputum sample. 


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11. Schedule subject for subsequent visit. 
 
Abbreviated follow-up visit: Day 56 (±7): 
1. Record any changes in status since last visit. 
2. Record any changes to concomitant medications. 
3. Administer CFQ-R. 
4. Record any AEs. 
5. Perform an abbreviated physical examination. 
6. Obtain and record vital signs. 
7. Blood collection. 
8. Perform and record spirometry. 
 
Monthly safety follow-up: M3, M4, M5, M6 (±7 days), alternating in person & phone visit: 
1. Record any changes in status since last visit. 
2. Record any changes to concomitant medications. 
3. Administer CFQ-R. 
4. Record any AEs. 
5. Perform an abbreviated physical examination if subject is in clinic. 
6. Obtain and record vital signs if subject is in clinic.  
7. Perform and record spirometry if subject is in clinic. 
8. Collect sputum if subject is in clinic and can expectorate sputum or agrees to undergo 
induced sputum. 
 
Subjects from the placebo group who continue onto the Open-label portion will undergo the 
following schedule: 
 
Open-Label Treatment Screening Visit (Day -7, ±7 days) 
1. Open Label Consent is required and will be acquired by a member of the research 
team using the Open Label Consent form, which will be scanned and stored in the 
EMR. Photocopies of signed forms will be provided to the subject, and the wet ink 
versions will be stored in a secure cabinet. 
2. Collect sputum sample, either in person or shipped overnight on ice packs. Sputum 
may be induced or produced spontaneously. Sputum is obtained for research 
laboratory analysis (e.g. phage susceptibility and bacterial load), and baseline 
antibiotic sensitivity testing is assessed from subject medical records.  Open Label Day 
1 dosing can occur once PsA phage susceptibility is obtained. 
3. Confirm medical history and any changes in status since last visit. 
4. Record any changes to concomitant medications. 
 
Open-Label Treatment visit 1: Day 1  
1. Confirm medical history and any changes in status since open label screening. 
2. Record any changes to concomitant medications. 
3. Administer CFQ-R and CFRSD. 
4. Perform an abbreviated physical examination. 
5. Obtain and record vital signs. 
6. Collect urine for pregnancy test (if female of child-bearing potential). 
7. Perform and record pre-treatment spirometry. 
8. Review study drug administration with subject. 
9. Supervise subject self-administer first dose of study drug in clinic. 
a. Observe subject for 30 minutes following nebulization for any potential adverse 
events. 
10. Perform and record post-treatment spirometry. If > 10% decline in FEV1 compared to 
pre-treatment spirometry, this will be considered evidence for an adverse response to 
treatment and subject will not continue in the study. 


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11. Ask subject about AEs of interest (increased sputum production, shortness of breath, 
hemoptysis, chest pain, cough, and chills or fever). 
12. Review study drug dose, schedule, and storage conditions with subject. 
a. Subjects are asked to nebulize treatment after standard airway clearance 
therapies at the same time of day. 
13. Provide subject medication and AE diaries and instructions for completion. 
14. Remind subject NOT to discontinue therapy if they experience improvements or feel 
better. 
15. Instruct subject to bring empty study drug containers, and return equipment provided 
for study (e.g., nebulizer), any unused doses of study drug, and study diary to 
subsequent visit. 
16. Schedule subject for subsequent visit. 
 
Open-label Treatment visit: Day 7 (±2): 
1. Record any changes in status since last visit. 
2. Record any changes to concomitant medications. 
3. Administer CFQ-R and CFRSD. 
4. Review subject study diaries and perform container count on returned study drug. 
5. Record any AEs. Query for increased sputum production, shortness of breath, 
hemoptysis, chest pain, cough, and chills or fever since their last visit. 
6. Perform an abbreviated physical examination. 
7. Obtain and record vital signs. 
8. Collect urine for pregnancy test (if female of child-bearing potential). 
9. Blood collection. 
10. Perform and record spirometry. 
11. Induce and collect sputum sample. 
12. Request return of equipment provided for study, empty containers of therapy, and any 
unused containers of therapy for disposal. 
13. Schedule subject for subsequent visit. 
 
Open-Label Follow-up visit: Day 14 (±3):  
1. Record any changes in status since last visit. 
2. Record any changes to concomitant medications. 
3. Administer CFQ-R and CFRSD. 
4. Review subject diary 
5. Record any AEs. Query for increased sputum production, shortness of breath, 
hemoptysis, chest pain, cough, and chills or fever since their last visit. 
6. Perform an abbreviated physical examination. 
7. Obtain and record vital signs. 
8. Blood collection. 
9. Perform and record spirometry. 
10. Induce and collect sputum sample. 
11. Schedule subject for subsequent visit. 
 
Open-Label Follow-up visit: Day 28 (±3):   
1. Record any changes in status since last visit. 
2. Record any changes to concomitant medications. 
3. Administer CFQ-R and CFRSD. 
4. Record any AEs. 
5. Perform an abbreviated physical examination. 
6. Obtain and record vital signs. 
7. Blood collection. 
8. Perform and record spirometry. 
9. Induce and collect sputum sample. 


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Open-Label Monthly safety follow-up: M2, M3(±7 days) in person or on the phone: 
1. Record any changes in status since last visit. 
2. Record any changes to concomitant medications. 
3. Administer CFQ-R and CFRSD. 
4. Record any AEs. 
5. Perform an abbreviated physical examination, if subject is in clinic. 
6. Obtain and record vital signs if subject is in clinic. 
7. Perform and record spirometry if subject is in clinic. 
8. Collect sputum if subject is in clinic and can expectorate sputum or agrees to undergo 
induced sputum. 
 
 
6.3.6 End of Study and Follow-up 
6.3.7 End of blinded study follow-up will occur at the end of day 56 for all randomized 
subjects, following which, subjects randomized to active phage treatment will continue 
being followed monthly up to 6 months of total follow up for each subject, and subjects 
who were originally randomized to placebo, and who will choose to initiate active phage 
therapy, will be followed for 28 days, and then monthly up to a total of 6 months follow 
up from the time of original randomization. Any remaining containers of intervention 
that were not used, and nebulizer used during the study will be collected for disposal.  
Removal of subjects 
In rare instances, it may be necessary for a subject to permanently discontinue (definitive 
discontinuation) study intervention. If study intervention is discontinued in such a manner, the 
participant will remain in the study to be evaluated for adverse events. See the SoA (CYPHY 
Trial Design, pages 32 & 33) for data to be collected at the time of discontinuation of study 
intervention and follow-up, and for any further evaluations that need to be completed. 
Should any serious immune-related adverse event occur (e.g., anaphylactic response), the 
SAE will be reported, and subject will be discontinued from trial. 
A participant may withdraw from the study at any time at his/her own request, or may be 
withdrawn at any time at the discretion of the PI for safety, behavioral, compliance, or 
administrative reasons. This is expected to be uncommon. 
At the time of discontinuation from the study, if possible, an early discontinuation visit should 
be conducted per schedule on day 28 as shown in the SoA (CYPHY Trial Design, pages 32 
& 33). See SoA for data to be collected at the time of study discontinuation and follow-up and 
for any further evaluations that need to be completed (CYPHY Trial Design, pages 32 & 33). 
The participant will be permanently discontinued both from the study intervention and from the 
study at that time. 
If the participant withdraws consent for disclosure of future information, the sponsor may 
retain, and continue to use, any data collected before such a withdrawal of consent. If a 
participant withdraws from the study, he/she may request destruction of any samples taken 
and not tested, and the investigator must document this in the site study records. 
Subjects who are discontinued from the study will not be replaced. 


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6.4 
Statistical Method 
 
6.4.1 Statistical Design 
Hypotheses:  
Primary for efficacy: We hypothesize that subjects randomized to active phage therapy will 
have significantly higher change from baseline in PsA sputum load, as compared to subjects 
randomized to the placebo group during the non-Open-label portion of the study. 
The primary efficacy endpoint is change in PsA sputum load from baseline (pre-phage) to 
d14 on study, i.e. 7 days after completion of phage or placebo treatment in the randomized 
and blinded (non-Open-label) portion of the study.   
Primary for safety: We hypothesize that the short-term safety profile of active phage therapy, 
as measured during the first 2 months of follow up, will show that treatment with phage is safe. 
Primary safety endpoints: frequency and severity of adverse events (AEs and SAEs) on d1, 
d3, d7, d14, d21, d28, d56. 
Secondary: We hypothesize that treatment with active phage : 1) will decrease PsA sputum 
load over time, 2) will decrease PsA antibiotic resistance and virulence factors, 3) will favorably 
modify sputum phage, host, and microbiome, and 4) will have positive impact on clinical and 
quality of life responses in subjects..  
Secondary outcome measures are: 1) monthly tracking for a total of 6-months of individual 
follow up from randomization of AEs and SAEs with active monitoring post d56 measurement 
in subjects randomized to treatment, and monthly measurements in open-label extension 
among subjects originally randomized to placebo who chose active phage therapy;  2) post-
treatment change from baseline in PsA sputum load to d3, d7, d14, d21 and d28; 3) post-
treatment change from baseline in PsA “trade-off” [e.g., decreased resistance to antibiotics, 
inflammatory products (pyocyanin or endotoxin), or motility] to d3, d7, d14, d21 and d28; 4) 
sputum phage presence at d3, d7, d14, d21, d28, host sputum inflammatory markers change 
from baseline to d14 and d28, lung inflammation transcriptomic change from baseline to d14, 
and microbiome changes from baseline to d14; and 5) change from baseline in lung function 
(e.g., FEV1, FEV1pp), pulmonary exacerbations, medication changes, and respiratory 
symptoms (CFQ-R)14 to d3, d7, d14, d21, d28, d56, and monthly thereafter through M6 
following initiation of phage therapy. 
Method: 
Using the intent-to-treat (ITT) approach for all analyses, whereby subjects are analyzed 
according to the group to which they were assigned, data will be examined to ascertain 
whether there is a significant difference in the reduction of PsA sputum load between two 
treatment conditions (phage vs. placebo) on day 14 of study (7 days after completion of 
treatment phase). As this is the only efficacy-related primary outcome, there is no need to 
adjust for either multiple comparisons or false discovery rate, and the significance will be 
established at the two-sided alpha level of 0.05. 
 


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Descriptive 
analyses 
of 
the 
study 
participants, 
as 
well 
as 
all 
primary 
and 
secondary/exploratory endpoints will be tabulated by randomization group, and will include 
mean (standard deviation) or median (IQR: 25th percentile, 75th percentile), for Gaussian 
(Normal) and non-normal distributions of continuous variables, respectively; and using count 
(percent) for discrete/categorical data. Between-group differences will be summarized 
graphically over time (as pertains to the measurement schedule for each outcome of interest) 
using estimated means of change from baseline or Odds Ratios (ORs) and 95% confidence 
intervals (95%CIs).   
 
The primary unadjusted statistical analysis for the between-group change in PsA sputum on 
the log10 scale from baseline to day 14 on study (7 days after completing assigned treatment) 
will be based on the Student’s t-test. In the adjusted analysis, we will use Analysis of 
Covariance (ANCOVA), where we will model the change in PsA on the log10 scale from 
baseline to day 14 on study, with the baseline PsA level as a covariate, as well as the following 
predictors (as the sample size permits): sex, age (a continuous variable), and phage type (a 
3-level categorical variable).  
 
In the additional supportive (secondary) analyses for mean change from baseline to day 3, to 
day 7, to day 14, to day 21, to day 28, and to day 56 (when relevant) in primary and secondary 
continuous outcomes, we will use linear mixed effects (LME) modeling. In the LME models, 
we will approach the effect of time as a continuous variable, thereby allowing the examination 
of the parametric trajectories of change in the outcomes over time and furthermore, we will be 
able to obtain a measure of the within-subject correlation in the response, using the IntraClass 
Correlation (ICC). The latter is particularly useful, as it will allow us to describe the degree of 
heterogeneity in the response due to unmeasured subject characteristics and can be very 
helpful for the future larger trial planning. If parametric trajectories of mean change are not 
feasible, or for continuous outcomes with only 2 time points, we will also consider mean 
response profile (MRP) modeling, with time as a categorical variable, which allows an arbitrary 
change in the means (instead of a parametrically defined change in the mean) of outcomes 
over time. The within-person correlation will be implemented using the covariance pattern 
modeling. Estimation of parameters in the mean response model will be performed using 
maximum likelihood (ML), allowing us to use the Likelihood Ratio Test (LRT) to compare 
nested models. Estimation of covariance parameters in the LME and the covariance pattern 
model will be done with restricted maximum likelihood (REML), in order to use the LRT to 
select the best covariance structure.  We realize that our sample size may not allow us to 
include all potential predictors of interest, and we are also aware of not over-fitting any given 
final model to the observed data; therefore, we will select the most parsimonious final models.  
 
Weekly trade-offs of PsA and phage presence, as well as number of PEs and medication 
changes, these secondary outcomes will be summarized descriptively as numbers (counts) 
and proportions, which will allow us to select optimal statistical tests and models, based on 
the empirical distributions of these outcomes.  As these are discrete outcomes, we propose to 
use marginal models implemented via generalized estimating equations (GEE).  
 
For the analysis of change in lung inflammation transcriptomics change, differentially 
expressed genes (DEGs) related to inflammation at baseline and day 14 on study will be 
compared within each treatment group using paired-samples t-test in the unadjusted analyses, 


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and using ANCOVA (similar to the primary outcome) in the adjusted analyses, which will also 
compare the change in individual DEGs between two groups.  
 
For the microbiome analysis, within each treatment group, active members of microbial 
community will be classified by bacterial taxonomical assignments using KAIJU at the genus 
level (Cobián Güemes et al., 2019) and will be plotted by baseline and day 14 on study, using 
color-coded by phylum bar charts. For each subject, we will output percent with certain top 5 
phyla, and compare the change in these between baseline and day 14 using RPM (as 
described for the primary outcome above), using two time points as a categorical variable, 
treatment group and their interaction. Bacterial rank abundance plots will be generated using 
relative abundance at the genus level and stratified by baseline and day 14, as well as by 
treatment group; and 4 evenness statistics (for treatment group at baseline, for treatment 
group at day 14, for placebo group at baseline and placebo group at day 14) will be calculated 
as Shannon diversity index divided by the natural log of the total number of species (Cobián 
Güemes et al., 2019). Between-group comparisons of the trajectories in relative abundance 
by the bacterial rank will be performed by either using LME (as for the primary outcome above), 
treating the bacterial rank as a polynomial effect.  
Statistical analyses for the blinded portion of the study (through d56 measurement) will be 
done separately from the extended through month 6 of follow up unblinded portion for those 
randomized to active phage, and separately from the data obtained from the open-label 
extension for subjects originally randomized to placebo who chose to receive active phage. 
Statistical analyses will be conducted using SAS 9.4 (Cary, NC). Statistical significance will 
be established at the two-sided alpha=0.05 for non-transcriptomics and non-metagenomics 
outcomes. However, we will also report statistical trends (alpha 0.10) because we are 
interested in exploring associations between the outcomes and other predictors (e.g., subject 
characteristics other than the treatment effect), as well as between secondary outcomes and 
treatment effect, for which we are not specifically powered in this study. 
 
False discovery rate (FDR) using a permutation-based method will be used in transcriptomics 
and metagenomics analyses adjusted for the multiple testing error, so as to preserve the alpha 
level of 0.05. 
 
6.4.2 Sample Size Considerations 
For the primary efficacy objective, based upon preliminary (eIND) data of ~2.5 log10 decrease 
in PsA post-phage (Figure 2-4B), a sample size of 10 subjects in the placebo group, and 20 
subjects in the phage group will have 80% power at the two-sided alpha of 0.05 to reject the 
null hypothesis of equal means when the population mean difference is μphage – μplacebo =2.5-
0.3, with an estimated standard deviation of 2.0, using a two-sided Student’s t-test.  
 


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6.5 
Planned Analyses 
The statistical analysis plan will be finalized prior to unblinding and it will include a more 
technical and detailed description of the statistical analyses described in this section. This 
section is a summary of the planned statistical analyses of the most important endpoints 
including primary and key secondary endpoints. All analyses will be performed using the 
intent-to-treat (ITT) approach. 
6.5.1 Primary Objective Analysis 
The primary unadjusted statistical analysis for the between-group change in PsA sputum 
(continuous variable on the log-10 scale) from baseline to day 14 on study (7 days after 
completing assigned treatment) will be based on the Student’s t-test. In the adjusted analysis, 
we will use Analysis of Covariance (ANCOVA), where we will model the change from baseline 
in PsA at 14 days on study, with the baseline PsA level as a covariate, as well as the following 
predictors (as the sample size permits): sex, age (a continuous variable), and phage type (a 
3-level categorical variable). Results will be summarized as the estimated mean changes in 
PsA sputum from pre-treatment to day 14 on study, with surrounding 95% Confidence 
Intervals.   
 
In the additional supportive analyses for mean change from baseline to day 3, to day 7, to day 
14, to day 21 and to day 28 in primary and secondary continuous outcomes (plus to day 56), 
we will use linear mixed effects (LME) modeling, with a random intercept for subject, and the 
following fixed effects: treatment arm (phage [coded as ‘1’] vs. placebo [coded as ‘0’] in one 
set of models), baseline values of log-10 PsA, time (in days), an interaction of time by 
treatment arm, subject age at randomization, and gender. The joint hypothesis test of the 
effect of treatment arm and treatment arm by time interaction, using the Wald-test, will help us 
ascertain whether there are significant between-treatment arm differences in the adjusted 
changes from baseline in the mean of PsA load across time.  Prior to testing the significance 
of fixed effects, we will also test for the random effect for slope using the LRT (using the 
restricted maximum likelihood, REML) with a mixture of Chi-square distributions. In the LME 
models, we will approach the effect of time as a continuous variable, thereby allowing the 
examination of the parametric trajectories of mean change in the outcomes over time and, 
furthermore, obtain a measure of the within-subject correlation in the outcome variable 
(change from baseline in PsA), using the IntraClass Correlation (ICC). The latter is particularly 
useful, as it will allow us to describe the degree of heterogeneity in the response due to 
unmeasured subject characteristics and can be very helpful for the future larger trial planning.  
 
If the effect of time in the LME cannot be parameterized as a parametric effect, we will also 
consider mean response profile (MRP) modeling, with time as a categorical variable, which 
allows an arbitrary change in the means of outcomes over time. The model for the mean 
response and the primary hypothesis test will be the same as in the LME. The within-person 
correlation will be implemented using the covariance pattern modeling, testing different within-
person correlation structures, such as Compound Symmetry (CS), Ar(1), Toeplitz, 
Exponential, and unstructured. As this supportive analysis is designed to get better estimates 
for a larger Phase II efficacy trial, we will not apply a correction to our significance level, as we 
would normally do so when comparing multiple means (e.g., change from baseline to day 7 
vs. to day 14 vs. to day 21 vs. to day 28, etc).  
 


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Estimation for both, LME and MRP with covariance pattern modeling, will be performed using 
maximum likelihood (ML and REML), allowing us to use the Likelihood Ratio Test (LRT) to 
compare nested models and Akaike Information Criterion (AIC) for non-nested models. Thus, 
the final LME model can be compared to the final covariance pattern model to choose the best 
fit to the data. We realize that our sample size may not allow us to include all potential 
predictors of interest, and we are also aware of not over-fitting any given final model to the 
observed data; therefore, we will select the most parsimonious final models.  
 
The estimated adjusted mean changes from baseline and 95% Confidence Intervals (95%CIs) 
at day 3, day 7, day 14, day 21 and day 28 will be plotted by treatment arm.  
 
Statistical analysis for the primary safety-related objective is summarized in Section 6.5.3 
Safety – see below. 
 
6.5.2 Secondary Objectives Analyses 
Differences between phage and placebo treatment arms with respect to the change in 
continuous secondary and tertiary endpoints (FEV1, FEV1pp, CFQ-R, and anti-phage 
antibody titers) will be analyzed in the similar fashion as described for the primary endpoint. 
For example, sustained effects of treatment over time will be examined using LME or MRP 
with covariance pattern modeling. Estimated effect sizes and 95%CIs, in particular for FEV1, 
FEV1pp and CFQ-R, will be useful for designing a larger multi-site trial.  
 
Weekly trade-offs in PsA and phage presence, number of PEs and medication changes – 
these secondary outcomes will be summarized descriptively as proportions and numbers 
(counts), which will allow us to select optimal statistical tests and models, based on the 
empirical distributions of these outcomes.  As these are discrete outcomes, we propose to use 
marginal models implemented via generalized estimating equations (GEE), with a separate 
model for the transformed mean response (link=logit for a binary outcome, link=log for a count 
outcome) and a model-based (naïve) or robust (sandwich) estimation of standard errors.  For 
example 
for the 
probability 
of 
a compound 
binary 
outcome 
(e.g., 
1=gaining 
sensitivity/decreased pyocyanin/endotoxin or motility, 0=no improvement) over the course of 
the study follow up in each treatment arm, we propose to separately model the transformed 
mean response using the logit link and the main effects of treatment arm (phage [coded as 
‘1’] vs. placebo [coded as ‘0’] in one set of models), categorical time (e.g., baseline, day 3, day 
7, day 14, day 21 and day 28 – as relevant for a specific secondary outcome), an interaction 
of time by treatment arm, subject age at randomization, and gender. The time by treatment 
arm interaction will be tested using the Wald test, and will allow us to ascertain whether the 
probability of a response differs across the follow up time between the two treatment arms. 
Since our sample size is relatively small to take complete advantage of the robust (sandwich 
estimator) standard errors from GEE, we will implement the Wald Test using both types of 
standard errors, the empirical/robust standard errors and model-based or naïve standard 
errors using the ‘best’ working correlation. The within-subject association will be modeled on 
the log-odds scale, and we will use the Quasi-likelihood under the assumption of 
independence criterion (QIC) to select among the ‘best’ working correlations (Compound 
Symmetry, Unstructured and Toeplitz). Results will be summarized using estimated Odds 
Ratios (ORs) and surrounding 95%CIs. 
 


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For the analysis of change in lung inflammation transcriptomics, differentially expressed genes 
(DEGs) related to inflammation at baseline and day 14 on study will be compared within each 
treatment group using paired-samples t-test in the unadjusted analyses, and using ANCOVA 
(similar to the primary outcome) in the adjusted analyses, which will also compare the change 
in individual DEGs between two groups.  
 
For the analysis of microbiome, within each treatment group, active members of microbial 
community will be classified by bacterial taxonomical assignments using KAIJU at the genus 
level (Cobián Güemes et al., 2019) and will be plotted by baseline and day 14 on study, using 
color-coded by phylum bar charts. For each subject, we will output percent with certain top 5 
phyla, and compare the change in these between baseline and day 14 using RPM (as 
described for the primary outcome above), using two time points as a categorical variable, 
treatment group (phage vs. placebo), and their interaction. Bacterial rank abundance plots will 
be generated using relative abundance at the genus level and stratified by baseline and day 
14, as well as by treatment group; and 4 evenness statistics (for treatment group at baseline, 
for treatment group at day 14, for placebo group at baseline and placebo group at day 14) will 
be calculated as Shannon diversity index divided by the natural log of the total number of 
species (Cobián Güemes et al., 2019). Between-group comparisons of the trajectories in 
relative abundance by the bacterial rank will be performed by either using LME (as for the 
primary outcome above), treating the bacterial rank as a polynomial effect.  
Tertiary/exploratory endpoint analysis of anti-phage antibody titers will follow similar analytical 
plans for continuous outcomes - as for the primary and secondary efficacy endpoints.  
Results from the open-label period of the study and from the extended unblinded portion of the 
study will be summarized using descriptive statistics with surrounding 95%CIs, and will be 
modeled over the follow up time, as the data permit, using similar statistical approaches as 
described for the primary and secondary efficacy endpoints, but we will not conduct between-
treatment comparisons. Furthermore, these data will not be combined with the data obtained 
from the blinded portion of the study. 
 
6.5.3 Safety 
All safety analyses will be made on the Safety Population: all subjects who received any study 
treatment (including placebo), but excluding subjects who drop out prior to receiving any 
treatment. 
Safety will be evaluated with summary of adverse events for the safety population (see 
description under “Section 6.2.2, “Safety”). The summary statistics will be produced in 
accordance with the types and groupings of AE and SAEs of the study protocol. Treatment 
emergent adverse events (AEs) are those events that occur after the baseline assessment.  
A tabular summary of AE will present: Number of subjects with any AE; Number of SAEs with 
outcome death; Number of subjects with SAE; Number of subjects with AEs leading to 
discontinuation of study intervention; Number of subjects with AEs leading to discontinuation 
of study; Total number of AEs; Total number of SAEs. 


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The Adverse Events summary tables will include number of adverse events, the number of 
subjects in each treatment group in whom the event occurred, and the incidence of occurrence 
and should be grouped by system organ class, preferred terms and/or other interested 
variables (e.g., relatedness, intensity and seriousness). 
We will provide tabulated summaries for the short-term and longer-term safety profiles. The 
short-term safety profile from the randomized blinded portion of the study will include data 
from d1, d3, d7, d14, d21, d28, and d56. The longer-term safety profile will come from the 
monthly measurements of the extended unblinded portion for subjects originally randomized 
to active phage. We will also tabulate short-term safety profile, separately, for the open-label 
portion of the study for subjects originally randomized to placebo who chose to receive active 
phage after the blinded portion; and the open-label extension monthly measurements will also 
have additional longer-term safety data. 
All formal testing of adverse effects will be based on the subject as the experimental unit.  
Thus, for comparing incidence of AE within system organ by treatment group, a one-sided 
Fisher’s exact test will be conducted at 0.05 level (higher incidence in experimentally treated 
group is the alternative hypothesis). Also, highest AE grade will be determined for each subject 
and compared by treatment group using a 2 sample Mann-Whitney test (one-sided at 0.05). 
No correction for multiple testing will be employed in order that the statistical power is 
maintained. 
 
6.5.4 Analysis of Subject Characteristics 
Descriptive analyses of the study participants will be tabulated by randomization group, and 
will include mean (standard deviation) or median (IQR: 25th percentile, 75th percentile), for 
Gaussian and non-normal distributions of continuous variables, respectively; and using count 
(percent) for discrete data. We will report the following subject characteristics: age at 
randomization, gender, height, weight, race, FEV1, FEV1 percent predicted, BMI, CF-related 
diabetes (CFRD), pancreatic insufficiency (PI), number of pulmonary exacerbations during 
previous six months, most recent treatment for pulmonary exacerbation prior to first study 
dose, baseline pathogen isolation (e.g., PsA, Staphylococcus, etc.), concomitant medications 
or therapies at study start, use of inhaled antibiotic and which type, type of CFTR mutation, 
use of CFTR modulator, and which CFTR modulator.  
6.5.5 Covariates 
We will include the following covariates in the adjusted models: gender (male vs. female) and 
age, as these are known to be significantly associated with a number of our outcomes of 
interest. For example, age could be a proxy for the length of time diagnosed with CF, and 
hence can indirectly describe CF severity. Gender can also an important predictor of FEV1 
and quality of life outcomes (based on CFQ-R).  
Safety data will be stratified by (1) short-term safety obtained on all randomized subjects from 
the blinded portion of the study (through d56 measurement), which will allow us the between-
treatment group comparison; (2) short-term safety data obtained from the open-label portion 
(through d28), which will give us descriptive information, (3) longer-term safety data obtained 


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from monthly follow up during the unblinded portion of the study on subjects originally 
randomized to the active phage, which will also give us descriptive information, and (4) longer-
term safety data obtained from the monthly follow up data in the open-label portion of the study 
on subjects originally randomized to placebo who chose to receive active phage post the 
randomized portion of the study (also, for descriptive analyses).   
6.5.6 Handling of Missing Data 
The primary approach will be to minimize the occurrence of missing data by implementing 
follow-up procedures that ensure a close to 100% follow-up for all subjects, especially for the 
primary endpoint. In addition, we propose the following two approaches from a methodological 
perspective: (1) we anticipate the attrition to be at most 10%, therefore, we will inflate the 
sample size from 10 subjects per group to 10/0.9=12 subjects per group, and (2) our analytic 
approach for the primary outcome will treat missing outcomes as missing at random (MAR). 
The latter assumption for the missing data mechanism cannot be tested statistically, however 
we will check whether subjects with missing outcomes are significantly different from those 
with complete observations based on important subject characteristics. Even if we find such 
differences, the assumption of MAR will also be reasonable, especially when we incorporate 
variables predictive of the probability of missingness (e.g., available data: outcomes at other 
time points) into the statistical analyses, whereby the inference becomes conditional on what 
we know about our subjects, which generally meets the definition of MAR.  
GEE estimation relies on the assumption that outcomes are missing completely at random 
(MCAR). While this assumption cannot be verified, we can test for departures from this 
assumption by comparing characteristics and available outcome data for subjects with/out 
missing outcomes using standard statistical tests used for between-group comparisons. If we 
find such departures, we will then approach our inference using generalized linear mixed 
effects modeling (GLMM). This approach is similar to the methods described for LME, but we 
will use the transformed means (link=logit or link=log) in the analyses and will report effect 
sizes using the marginalized (integrated over the distributed of random effects) estimates of 
the means on the original scale (e.g., proportions and mean counts). 
 
 


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7  Trial Administration 
7.1 
 Ethical Considerations: Informed Consent/Assent and HIPAA Authorization 
This study will be conducted in accordance with the protocol and with the following: 
o   Consensus ethical principles derived from international guidelines including the Declaration 
of Helsinki and Council for International Organizations of Medical Sciences (CIOMS) 
International Ethical Guidelines 
o   Applicable ICH Good Clinical Practice (GCP) Guidelines 
o   Applicable laws and regulations 
        The protocol, protocol amendments, ICF, Investigator Brochure, and other relevant 
documents (e.g., advertisements) must be submitted to an IRB by the investigator and 
reviewed and approved by the IRB before the study is initiated. 
·        Any amendments to the protocol will require IRB approval before implementation of 
changes made to the study design, except for changes necessary to eliminate an immediate 
hazard to study participants. 
·        The investigator will be responsible for the following: 
o   Providing written summaries of the status of the study to the IRB annually or more frequently 
in accordance with the requirements, policies, and procedures established by the IRB 
o   Notifying the IRB of SAEs or other significant safety findings as required by IRB procedures 
o   Providing oversight of the conduct of the study at the site and adherence to requirements 
of 21 CFR, ICH guidelines, the IRB, European regulation 536/2014 for clinical studies (if 
applicable), and all other applicable local regulations 
Travel Accomodations 
In an effort to make study participation attainable for more patients, the trial will cover some of the 
costs associated with travel & hotel accommodations.  
If patietns are traveling >200 miles they will be compensated up to $5,000. If patients are traveling 
<=200 miles they will be compensated up to $600.  
Costs will be covered either by the study team booking directly through the Yale University portal 
Egencia or otherwise, or via compensation provided in the form of a Bank of America Card loaded 
remotely with the value indicated in travel & hotel receipts and/or bank statements.   
Costs will be covered by the Yale Phage Center. Prior approval will need to be obtained for all costs 
associated with travel & hotel accommodations. Please feel free to discuss this with the study staff. 
7.2 
Institutional Review Board (IRB) Review 
The protocol will be submitted to the IRB for review and approval. Approval of the protocol 
must be obtained before initiating any research activity. Any change to the protocol or study 


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team will require an approved IRB amendment before implementation.  The IRB will determine 
whether informed consent and HIPAA authorization are required.   
The IRB will conduct continuing review at intervals appropriate to the degree of risk, but not 
less than once per year. 
A study closure report will be submitted to the IRB after all research activities have been 
completed.    
Other study events (e.g. data breaches, protocol deviations) will be submitted per Yale 
University’s IRB's policies. 
7.3 
Subject Confidentiality 
Subject confidentiality will be protected in accordance with Yale University IRB Policy 400. 
The investigator will make all efforts, within reason, to keep subject health information private 
and confidential.  
7.4 
Deviations/Unanticipated Problems 
Adverse events, including Serious Adverse Events (SAEs) and Unanticipated Problems 
Involving Risks to Subjects or Others (UPIRSOs) will be handled and reported to the Yale 
University IRB in accordance with IRB Policy 700 and 710. 
7.5 
Data Collection 
Data collection and storage will be conducted in compliance with Yale University IRB Policies 
440 and 445 and HRPP Policy 730. Briefly: 
• 
Participants will be assigned a unique identifier. Any participant records or datasets 
that are transferred to the sponsor will contain this identifier only; participant names or 
any information which would make the participant identifiable will not be transferred. 
• 
The participant will be informed that his/her personal study-related data will be used 
by the sponsor in accordance with local data protection law. The level of disclosure will 
also be explained to the participant who will be required to give consent for their data 
to be used as described in the informed consent 
• 
The participant will be informed that his/her medical records may be examined by 
Clinical Quality Assurance auditors or other authorized personnel appointed by the 
sponsor, by appropriate IRB members, and by inspectors from regulatory authorities. 
• 
When University human research records are no longer deemed useful following the 
proscribed record retention period, the records will be de-identified or destroyed in 
accordance with University media control and/or biological specimen policies. 
7.6 
Data Quality Assurance 
All participant data relating to the study will be recorded on printed or electronic CRF unless 
transmitted to the sponsor or designee electronically (e.g., laboratory data). The investigator 
is responsible for verifying that data entries are accurate and correct by physically or 
electronically signing the CRF. 
The investigator must maintain accurate documentation (source data) that supports the 
information entered in the CRF. 


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7.7 
Study Records 
Study records are defined in the Yale University HRPP Policy 730 as any information 
preserved in a fixed medium, whether on paper, electronically, or otherwise. These include 
but are not limited to regulatory documents, protocols, consent forms, case report forms, 
subject medical records, and surveys.  
7.8 
Access to Source Documents 
·        Source documents provide evidence for the existence of the participant and substantiate 
the integrity of the data collected. Source documents are filed at the investigator’s site. 
·        Data reported on the CRF, or entered in the eCRF, which are transcribed from source 
documents must be consistent with the source documents or the discrepancies must be 
explained. The investigator may need to request previous medical records or transfer records, 
depending on the study. Also, current medical records must be available. 
·        Definition of what constitutes source data can be found in Yale University IRB Policy 440 
under “Materials”: Data, films, biological specimens, or other recorded information that may 
be useful for research. Examples include vials of blood, medical records, tumor specimens, 
scans, and videotapes of interviews. Note that sources of “information” or “data” (e.g., facts 
contained in articles or books) are not affected by this policy because they do not identify any 
human subjects or contain PHI, or otherwise implicate any unique privacy concerns. 
·        Source documents will be available to the Coordinator, DMC, and Data Quality Monitor, 
Investigator, and Investigator’s clinical team (e.g., nurses, clinical fellows). Data will be 
collected from them and entered into the site’s electronic system (e.g., eCRF, ONCOR).  
·        Source documents will be kept at a secure, locked site by the investigator for a minimum 
of three (3) years as required by Yale University IRB Policy 730.1.  
7.9 
Data or Specimen Storage/Security 
Data and specimens will be stored in a secure manner in accordance with Yale University IRB 
Policy 440 and 730. When data or specimens are no longer deemed useful following the 
proscribed record retention period, the records will be de-identified or destroyed in accordance 
with University media control and/or biological specimen policies. 
7.10 Retention of Records 
Study documents will be retained for a minimum of 2 years after the last approval of a 
marketing application in an International Conference on Harmonization (ICH) region and until 
there are no pending or contemplated marketing applications in an ICH region or until at least 
2 years have elapsed since the formal discontinuation of clinical development of the study 
intervention. These documents will be retained for a longer period, however, if required by 
local regulations. No records will be destroyed without the written consent of the sponsor, if 
applicable. It is the responsibility of the sponsor to inform the investigator when these 
documents no longer need to be retained. 


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7.11 Study Monitoring 
Study site monitoring is necessary to assure adequate protection of the rights of human 
subjects and the safety of all subjects involved in clinical investigations and the quality and 
integrity of the resulting data submitted.  
The Study Principal Investigator-designated monitor(s) conducts monitoring visits to ensure 
that clinical investigators and study team members are compliant with the protocol, ICH good 
clinical practice, federal, state and local regulations and institutional policies and procedures, 
that data are of high quality and integrity, and that the facilities and staffing are adequate for 
continued study participation. This will be performed by conducting monitoring visits including 
a site initiation visit, regularly scheduled interim monitoring visits and/or remote interim 
monitoring visits while subjects are on study, and a site close-out visit at the site. Following 
each site visit, a visit report will be generated containing information on site activities and a 
summary of pertinent points and action items. The report will be provided with a follow-up 
letter. Site-specific data status reports will be distributed to the site regularly to outline planned, 
missing or incomplete case report forms and any outstanding data queries. 
During monitoring visits, the following may be reviewed: 
• 
Protection of the rights, safety and welfare of subjects through review of informed 
consent process and documentation, adverse events (AEs) and serious adverse 
events (SAEs) and safety procedures 
• 
Subject eligibility 
• 
Source verification 
• 
Protocol compliance 
• 
Deviations and Non-compliance 
• 
Investigator Site File 
• 
GCP compliance 
• 
If applicable, include: Investigational Drug/ Device Storage and Accountability 
(including quantity and disposal procedures) 
• 
If applicable, include: Laboratory Facilities 
• 
If applicable, include: Equipment maintenance and calibration 
• 
Additional study supplies inventory and assessment 
• 
Study progress and/or follow-up on issues with Site Principal Investigator (PI) and 
relevant members of the study team 
The Study PI and YCCI will define the required study monitoring activities in a Study 
Monitoring Plan. 
7.12 Data Safety Monitoring Plan 
Data Safety Monitoring Plan 
The Investigator has communicated with Miriam Hunt-Bennett, the CFF DMC  Senior Program 
Coordinator at the University of Arizona Health Sciences. This meeting with Miriam and Dr. 
Wayne J. Morgan (Professor of Pediatrics and Physiology at the University of Arizona) initiated 
the process for DMC and completion of a DMC Charter. Since then, the DMC has met to 
review the CYPHY protocol, and their requests and suggestions have been incorporated into 
this protocol. 
A formal data review will occur after the first 5 participants have completed visit 7; A full interim 
data review will occur after 50% completed visit 7, or 6 months after the first randomization. 
Unblinding in the event of an SAE 


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The data monitoring comittee (DMC) will be responsible for subject safety and will have the 
authority to stop or modify this study. The DMC chair has the option to unblind him/herself or 
the entire DMC for SAEs. This will be done on an individual SAE-basis, thus ensuring that the 
DMC is not unblinded for the whole study. 
 
7.13 Study Modification 
Study modifications will be made as study amendments to the protocol in accordance with 
Yale University IRB Policy 100 PR.1. The change will be implemented in the study upon 
approval by the IRB and submission to the IND.  
7.14 Study Discontinuation 
The sponsor designee reserves the right to terminate the study at any time for any reason at 
the sole discretion of the sponsor. Reasons for the early termination of the study by the 
sponsor or investigator may include but are not limited to: 
·        Failure of the investigator to comply with the protocol, the requirements of the IRB or local 
health authorities, the sponsor's procedures, or GCP guidelines 
·        Inadequate recruitment of participants by the investigator 
·        Discontinuation of further study intervention development 
If the study is prematurely terminated or suspended, the sponsor shall promptly inform the 
Investigators, the IECs/IRBs, the regulatory authorities, and any contract research 
organization(s) used in the study of the reason for termination or suspension, as specified by 
the applicable regulatory requirements. The Investigator shall promptly inform the subject and 
should assure appropriate subject therapy and/or follow-up. 
Circumstances that may warrant termination or suspension include, but are not limited to: 
• 
Determination of unexpected, significant, or unacceptable risk to participants 
• 
Demonstration of efficacy that would warrant stopping    
• 
Insufficient compliance to protocol requirements 
• 
Data that are not sufficiently complete and/or evaluable 
• 
Determination that the primary endpoint has been met 
• 
Determination of futility 
7.15 Study Completion 
Study sites will be closed upon study completion. A study site is considered closed when all 
required documents and study supplies have been collected and a study-site closure visit has 
been performed. 
The primary date of completion will be defined as “the date the final subject was examined or 
received an intervention for purposes of final collection of data for the primary and secondary 
outcome measures and adverse events (e.g., last subject’s last visit), whether the clinical trial 
concluded according to the pre-specified protocol or was terminated.” Data analysis will occur 
following primary date of completion, after which the study will be complete. The IRB and FDA 
will be notified of study completion through annual reporting. 


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7.16 Conflict of Interest Policy 
Institutional conflict of interest will be handled according to Yale IRB Policy 501, and will be 
defined as “a significant external relationship that creates the opportunity and incentive to 
directly and significantly bias decision-making or compromise the professional judgment of the 
IRB, the HRPP, the researchers, or others in the conduct, review, or oversight of human 
subjects research.” 
The Institutional Conflict of Interest Committee (ICOIC) is responsible for the implementation 
of this policy. The ICOIC establishes and oversees procedures to collect information relating 
to SERs and relevant significant financial interests of University leaders (LSFIs). Additionally, 
any individual employed by the University or engaged in the review or conduct of University 
research who becomes aware of an SER is obligated to bring it to the attention of the ICOIC, 
or to the attention of the Institutional Official (IO) or an Institutional Review Board (IRB) Chair, 
who will in turn forward it to the ICOIC. The ICOIC reviews SERs and LSFIs to determine 
whether they pose the risk of creating ICOIs. If the ICOIC determines that a risk of ICOI exists 
in relation to human-subjects research, it will decide upon actions to eliminate or manage the 
ICOI, and it will report the ICOI along with those decisions to the Human Research Protections 
Program (“HRPP”). The HRPP and the IRBs may conduct additional reviews specific to 
human-subjects research and may impose measures, in addition to those determined by the 
ICOIC, to protect human research subjects and ensure the integrity of research. 
7.17 Funding Source 
The study will be funded through donations from non-profit entities, the Blavatnik Fund at Yale 
University and the Cystic Fibrosis Foundation. Salary support for this study is provided by Yale 
University’s Department of Pulmonology, Critical Care, and Sleep Medicine and the 
Department of Ecology and Evolutionary Biology.  
7.18 Publication Plan 
·        The results of this study may be published or presented at scientific meetings. If this is 
foreseen, the investigator agrees to submit all manuscripts or abstracts to the sponsor before 
submission. This allows the sponsor to protect proprietary information and to provide 
comments. 
·        The sponsor will comply with the requirements for publication of study results. In 
accordance with standard editorial and ethical practice, the sponsor will generally support 
publication of multicenter studies only in their entirety and not as individual site data. In this 
case, a coordinating investigator will be designated by mutual agreement. 
·        Authorship will be determined by mutual agreement and in line with International 
Committee of Medical Journal Editors authorship requirements. 
 


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8 List of Tables 
Table 2-1. Mean antibiotic sensitivity of PsA before and after phage-OMKO1-selection 
in vitro. 
Table 2-2. Summary of single-patient cases in CF and NCFB treated with OMKO1, 
TIVP-H6, or LPS-5 phages 
Table 6-2. AE Severity Grading  
Table 6-3. AE Relationship to Study Drug  
Table 6-4. Randomization scheme 
 
 


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9 References 
Brown-Jaque, M., Rodriguez Oyarzun, L., Cornejo-Sánchez, T., Martín-Gómez, M. T., 
Gartner, S., de Gracia, J., Rovira, S., Alvarez, A., Jofre, J., González-López, J. J., & 
Muniesa, M. (2018). Detection of Bacteriophage Particles Containing Antibiotic 
Resistance Genes in the Sputum of Cystic Fibrosis Patients. Frontiers in 
Microbiology, 9. https://doi.org/10.3389/fmicb.2018.00856 
Chan, B. K., Sistrom, M., Wertz, J. E., Kortright, K. E., Narayan, D., & Turner, P. E. (2016). 
Phage selection restores antibiotic sensitivity in MDR Pseudomonas aeruginosa. 
Scientific Reports, 6(1), 26717. https://doi.org/10.1038/srep26717 
Chan, B. K., Turner, P. E., Kim, S., Mojibian, H. R., Elefteriades, J. A., & Narayan, D. (2018). 
Phage treatment of an aortic graft infected with Pseudomonas aeruginosa. Evolution, 
Medicine, and Public Health, 2018(1), 60–66. https://doi.org/10.1093/emph/eoy005 
Cobián Güemes, A. G., Lim, Y. W., Quinn, R. A., Conrad, D. J., Benler, S., Maughan, H., 
Edwards, R., Brettin, T., Cantú, V. A., Cuevas, D., Hamidi, R., Dorrestein, P., & 
Rohwer, F. (2019). Cystic Fibrosis Rapid Response: Translating Multi-omics Data 
into Clinically Relevant Information. MBio, 10(2), e00431-19, /mbio/10/2/mBio.00431-
19.atom. https://doi.org/10.1128/mBio.00431-19 
Cryz, S. J., Pitt, T. L., Fürer, E., & Germanier, R. (1984). Role of lipopolysaccharide in 
virulence of Pseudomonas aeruginosa. Infection and Immunity, 44(2), 508–513. 
de Kraker, M. E. A., Stewardson, A. J., & Harbarth, S. (2016). Will 10 Million People Die a 
Year due to Antimicrobial Resistance by 2050? PLOS Medicine, 13(11), e1002184. 
https://doi.org/10.1371/journal.pmed.1002184 
Deshpande Kaistha, S., & Devi Umrao, P. (2016). Bacteriophage for Mitigation of Multiple 
Drug Resistant Biofilm Forming Pathogens. Recent Patents on Biotechnology, 10(2), 
184–194. https://doi.org/10.2174/1872208310666160919122155 
Ferenci, T. (2016). Trade-off Mechanisms Shaping the Diversity of Bacteria. Trends in 
Microbiology, 24(3), 209–223. https://doi.org/10.1016/j.tim.2015.11.009 
Goldhill, D. H., & Turner, P. E. (2014). The evolution of life history trade-offs in viruses. 
Current Opinion in Virology, 8, 79–84. https://doi.org/10.1016/j.coviro.2014.07.005 
Hunter, S. J., Easton, S., Booth, V., Henderson, B., Wade, W. G., & Ward, J. M. (2011). 
Selective removal of human DNA from metagenomic DNA samples extracted from 
dental plaque. Journal of Basic Microbiology, 51(4), 442–446. 
https://doi.org/10.1002/jobm.201000372 
Ingledew, W. M., & Campbell, J. J. (1969). A new resuspension medium for pyocyanine 
production. Canadian Journal of Microbiology, 15(6), 595–598. 
https://doi.org/10.1139/m69-101 
Kon, Y., Tsukada, H., Hasegawa, T., Igarashi, K., Wada, K., Suzuki, E., Arakawa, M., & 
Gejyo, F. (1999). The role of Pseudomonas aeruginosa elastase as a potent 
inflammatory factor in a rat air pouch inflammation model. FEMS Immunology & 
Medical Microbiology, 25(3), 313–321. https://doi.org/10.1111/j.1574-
695X.1999.tb01356.x 


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Kortright, K. E., Chan, B. K., Koff, J. L., & Turner, P. E. (2019). Phage Therapy: A Renewed 
Approach to Combat Antibiotic-Resistant Bacteria. Cell Host & Microbe, 25(2), 219–
232. https://doi.org/10.1016/j.chom.2019.01.014 
Mogayzel, P. J., Naureckas, E. T., Robinson, K. A., Mueller, G., Hadjiliadis, D., Hoag, J. B., 
Lubsch, L., Hazle, L., Sabadosa, K., Marshall, B., & the Pulmonary Clinical Practice 
Guidelines Committee. (2013). Cystic Fibrosis Pulmonary Guidelines: Chronic 
Medications for Maintenance of Lung Health. American Journal of Respiratory and 
Critical Care Medicine, 187(7), 680–689. https://doi.org/10.1164/rccm.201207-
1160OE 
Nelson, M. T., Pope, C. E., Marsh, R. L., Wolter, D. J., Weiss, E. J., Hager, K. R., Vo, A. T., 
Brittnacher, M. J., Radey, M. C., Hayden, H. S., Eng, A., Miller, S. I., Borenstein, E., 
& Hoffman, L. R. (2019). Human and Extracellular DNA Depletion for Metagenomic 
Analysis of Complex Clinical Infection Samples Yields Optimized Viable Microbiome 
Profiles. Cell Reports, 26(8), 2227-2240.e5. 
https://doi.org/10.1016/j.celrep.2019.01.091 
O’Neill, J. (2014). Antimicrobial Resistance: Tackling a Crisis for the Health and Wealth of 
Nations: December 2014. Review on Antimicrobial Resistance. 
https://books.google.com/books?id=b1EOkAEACAAJ 
Parkins, M. D., Somayaji, R., & Waters, V. J. (2018). Epidemiology, Biology, and Impact of 
Clonal Pseudomonas aeruginosa Infections in Cystic Fibrosis. Clinical Microbiology 
Reviews, 31(4), e00019-18, /cmr/31/4/e00019-18.atom. 
https://doi.org/10.1128/CMR.00019-18 
Persat, A., Inclan, Y. F., Engel, J. N., Stone, H. A., & Gitai, Z. (2015). Type IV pili 
mechanochemically regulate virulence factors in Pseudomonas aeruginosa. 
Proceedings of the National Academy of Sciences, 112(24), 7563–7568. 
https://doi.org/10.1073/pnas.1502025112 
Pires, D. P., Cleto, S., Sillankorva, S., Azeredo, J., & Lu, T. K. (2016). Genetically 
Engineered Phages: A Review of Advances over the Last Decade. Microbiology and 
Molecular Biology Reviews, 80(3), 523–543. https://doi.org/10.1128/MMBR.00069-15 
Quittner, A. L., Buu, A., Messer, M. A., Modi, A. C., & Watrous, M. (2005). Development and 
Validation of the Cystic Fibrosis Questionnaire in the United States: A Health-Related 
Quality-of-Life Measure for Cystic Fibrosis. Chest, 128(4), 2347–2354. 
https://doi.org/10.1378/chest.128.4.2347 
Ronit, A., Gelpi, M., Argentiero, J., Mathiesen, I., Nielsen, S. D., Pressler, T., & Quittner, A. 
L. (2017). Electronic applications for the CFQ-R scoring. Respiratory Research, 
18(1), 108. https://doi.org/10.1186/s12931-017-0592-z 
Szafrański, S. P., Winkel, A., & Stiesch, M. (2017). The use of bacteriophages to biocontrol 
oral biofilms. Journal of Biotechnology, 250, 29–44. 
https://doi.org/10.1016/j.jbiotec.2017.01.002 
Turnbull, L., & Whitchurch, C. B. (2014). Motility Assay: Twitching Motility. In A. Filloux & J.-
L. Ramos (Eds.), Pseudomonas Methods and Protocols (Vol. 1149, pp. 73–86). 
Springer New York. https://doi.org/10.1007/978-1-4939-0473-0_9 
 


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10 Appendices 
Appendix # 
Title 
1 
Cystic Fibrosis Quality-of-Life Questionnaire Revised 
2 
Study Drug Diary 
3 
Symptoms Diary 
 
 
 
 
 
 
 


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1. Cystic Fibrosis Quality-of-Life 
Questionnaire Revised 
Source:(Quittner et al., 2005), adapted from https://cfqr.github.io/ 
Understanding the impact of your illness and treatments on your everyday life can help your 
healthcare team keep track of your health and adjust your treatments. For this reason, this 
questionnaire was specifically developed for people who have cystic fibrosis. Thank you for 
your willingness to complete this form. 
The following questions are about the current state of your health, as you perceive it. This 
information will allow us to better understand how you feel in your everyday life. Please 
answer all the questions. There are no right or wrong answers! If you are not sure how to 
answer, choose the response that seems closest to your situation. 
 
Please fill-in the information or check the box indicating your answer. 
What's your birth date? 
Enter a date... 
 
 
 
What's your gender? 
- 
Male 
- 
Female 
 
During the past two weeks, have you been on vacation or out of school for reasons NOT 
related to your health? 
- 
Yes  
- 
No 
 
What is your current marital status? 
- 
Single/never married 
- 
Married 
- 
Widowed 
- 
Divorced 
- 
Separated 
- 
Remarried 
- 
With a partner 
 
Which of the following best describes your racial background? 
- 
Caucasian 
- 
African American 
- 
Hispanic 
- 
Asian/Oriental or Pacific Islander 
- 
Native American or Native Alaskan 
- 
Other 


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- 
Prefer not to answer this question 
 
What is the highest grade of school you have completed? 
- 
Some high school or less 
- 
High school diploma/GED 
- 
Vocational school 
- 
Some college 
- 
College degree 
- 
Professional or graduate degree 
 
Which of the following best described your current work or school status? 
- 
Attending school outside the home 
- 
Taking educational courses at home  
- 
Seeking work 
- 
Working full or part time (either outside the home or at a home-based business) 
- 
Full time homemaker 
- 
Not attending school or working due to my health 
- 
Professional or graduate degree 
 
Please check the box indicating your answer. 
During the past two weeks, to what extent have you had difficulty 
Performing vigorous activities such as running or playing sports 
- 
A lot of difficulty 
- 
Some difficulty 
- 
A little difficulty 
- 
No difficulty 
 
Walking as fast as others 
- 
A lot of difficulty 
- 
Some difficulty 
- 
A little difficulty 
- 
No difficulty 
 
Carrying or lifting heavy things such as books, groceries, or school bags 
- 
A lot of difficulty 
- 
Some difficulty 
- 
A little difficulty 
- 
No difficulty 
 
Climbing one flight of stairs 
- 
A lot of difficulty 
- 
Some difficulty 
- 
A little difficulty 
- 
No difficulty 
 
Climbing stairs as fast as others 
- 
A lot of difficulty 


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- 
Some difficulty 
- 
A little difficulty 
- 
No difficulty 
 
During the past two weeks, indicate how often: 
You felt well 
- 
Always 
- 
Often 
- 
Sometimes 
- 
Never 
 
You felt worried 
- 
Always 
- 
Often 
- 
Sometimes 
- 
Never 
 
You felt useless 
- 
Always 
- 
Often 
- 
Sometimes 
- 
Never 
 
You felt tired 
- 
Always 
- 
Often 
- 
Sometimes 
- 
Never 
 
You felt energetic 
- 
Always 
- 
Often 
- 
Sometimes 
- 
Never 
 
You felt exhausted 
- 
Always 
- 
Often 
- 
Sometimes 
- 
Never 
 
You felt sad 
- 
Always 
- 
Often 
- 
Sometimes 
- 
Never 
 
Thinking about the state of your health over the last two weeks: 


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To what extent do you have difficulty walking? 
- 
You can walk a long time without getting tired 
- 
You can walk a long time but you get tired 
- 
You cannot walk a long time because you get tired quickly 
- 
You avoid walking whenever possible because it’s too tiring for you 
 
How do you feel about eating? 
- 
Just thinking about food makes you feel sick 
- 
You never enjoy eating 
- 
You are sometimes able to enjoy eating 
- 
You are always able to enjoy eating 
 
To what extent do your treatments make your daily life more difficult? 
- 
Not at all 
- 
A little 
- 
Moderately 
- 
A lot 
 
How much time do you currently spend each day on your treatments? 
- 
A lot 
- 
Some 
- 
A little 
- 
Not very much 
 
How difficult is it for you to do your treatments (including medications) each day? 
- 
Not at all 
- 
A little 
- 
Moderately 
- 
Very 
 
How do you think your health is now? 
- 
Excellent 
- 
Good 
- 
Fair 
- 
Poor 
 
Thinking about your health during the past two weeks, indicate the extent to which each 
sentence is true or false for you. 
I have trouble recovering after physical effort 
- 
Very true 
- 
Somewhat true 
- 
Somewhat false 
- 
Very false 
 
I have to limit vigorous activities such as running or playing sports 
- 
Very true 
- 
Somewhat true 
- 
Somewhat false 
- 
Very false 
 


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I have to force myself to eat 
- 
Very true 
- 
Somewhat true 
- 
Somewhat false 
- 
Very false 
 
I have to stay at home more than I want to 
- 
Very true 
- 
Somewhat true 
- 
Somewhat false 
- 
Very false 
 
I feel comfortable discussing my illness with others 
- 
Very true 
- 
Somewhat true 
- 
Somewhat false 
- 
Very false 
 
I think I am too thin 
- 
Very true 
- 
Somewhat true 
- 
Somewhat false 
- 
Very false 
 
I think I look different from others my age 
- 
Very true 
- 
Somewhat true 
- 
Somewhat false 
- 
Very false 
 
I feel bad about my physical appearance 
- 
Very true 
- 
Somewhat true 
- 
Somewhat false 
- 
Very false 
 
People are afraid that I may be contagious 
- 
Very true 
- 
Somewhat true 
- 
Somewhat false 
- 
Very false 
 
I get together with my friends a lot 
- 
Very true 
- 
Somewhat true 
- 
Somewhat false 
- 
Very false 
 
I think my coughing bothers others 
- 
Very true 


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- 
Somewhat true 
- 
Somewhat false 
- 
Very false 
 
I feel comfortable going out at night 
- 
Very true 
- 
Somewhat true 
- 
Somewhat false 
- 
Very false 
 
I often feel lonely 
- 
Very true 
- 
Somewhat true 
- 
Somewhat false 
- 
Very false 
 
I feel healthy 
- 
Very true 
- 
Somewhat true 
- 
Somewhat false 
- 
Very false 
 
It is difficult to make plans for the future (for example, going to college, getting married, 
advancing in a job, etc.) 
- 
Very true 
- 
Somewhat true 
- 
Somewhat false 
- 
Very false 
 
I lead a normal life 
- 
Very true 
- 
Somewhat true 
- 
Somewhat false 
- 
Very false 
 
The next questions are about school, work, or other daily tasks. 
To what extent did you have trouble keeping up with your schoolwork, professional work, or 
other daily activities during the past two weeks? 
- 
You have had no trouble keeping up 
- 
You have managed to keep up but it’s been difficult 
- 
You have been behind 
- 
You have not been able to do these activities at all 
 
How often were you absent from school, work, or unable to complete daily activities during 
the last two weeks because of your illness or treatments? 
- 
Always 
- 
Often 


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- 
Sometimes 
- 
Never 
 
How often does CF get in the way of meeting your school, work, or personal goals? 
- 
Always 
- 
Often 
- 
Sometimes 
- 
Never 
 
How often does CF interfere with getting out of the house to run errands such as shopping or 
going to the bank? 
- 
Always 
- 
Often 
- 
Sometimes 
- 
Never 
 
Indicate how you have been feeling during the past two weeks. 
Have you had trouble gaining weight? 
- 
A great deal 
- 
Somewhat 
- 
A little 
- 
Not at all 
 
Have you been congested in your chest? 
- 
A great deal 
- 
Somewhat 
- 
A little 
- 
Not at all 
 
Have you been coughing during the day? 
- 
A great deal 
- 
Somewhat 
- 
A little 
- 
Not at all 
 
Have you had to cough up mucus? 
- 
A great deal 
- 
Somewhat 
- 
A little 
- 
Not at all 
 
Has your mucus been mostly: 
- 
Clear 
- 
Clear to yellow 
- 
Yellowish-green 
- 
Green with traces of blood 
- 
Don’t know 
- 
I don’t cough up mucus 


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How often during the past two weeks: 
Have you been wheezing? 
- 
Always 
- 
Often 
- 
Sometimes 
- 
Never 
 
Have you had trouble breathing? 
- 
Always 
- 
Often 
- 
Sometimes 
- 
Never 
 
Have you woken up during the night because you were coughing? 
- 
Always 
- 
Often 
- 
Sometimes 
- 
Never 
 
Have you had problems with gas? 
- 
Always 
- 
Often 
- 
Sometimes 
- 
Never 
 
Have you had diarrhea? 
- 
Always 
- 
Often 
- 
Sometimes 
- 
Never 
 
Have you had abdominal pain? 
- 
Always 
- 
Often 
- 
Sometimes 
- 
Never 
 
Have you had eating problems? 
- 
Always 
- 
Often 
- 
Sometimes 
- 
Never 
 
 
 


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2. Study Drug Diary 
 
 
 
 


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3. Symptoms Diary 


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4. Daily Temperature Diary