Conditions: NSCLC Metastatic Colorectal Carcinoma Refractory Melanoma
Keywords: melanoma PD-1/PD-L1 pathway relapsed metastatic melanoma refractory metastatic melonma MSS Metastatic Colorectal Carcinoma PD-1 R/R NSCLC PD-1 R/R melanoma MSS mCRC
Allocation: NON_RANDOMIZED | Intervention model: PARALLEL
Enrollment: 13
Healthy volunteers: no

Brief summary:
The primary goal of this trial is to assess clinical response to nivolumab and pixatimod, and, nivolumab, pixatimod and cyclophosphamide in three separate patient cohorts. Cohort 1: MSS mCRC in combination with low-dose cyclophosphamide, Cohort 2: PD-1 relapsed/refractory melanoma, and Cohort 3: PD-1 relapsed/refractory NSCLC.

Detailed description:
Nivolumab is approved by the FDA for the treatment of melanoma, non-small cell lung cancer (NSCLC), malignant pleural mesothelioma, renal cell carcinoma, classical Hodgkin lymphoma, squamous cell carcinoma of head/neck, urothelial carcinoma, MSI-H colorectal cancer, hepatocellular carcinoma, gastric carcinoma and gastroesophageal junction (GEJ) cancer.

Pixatimod is an investigational drug that activates the toll-like receptor 9 (TLR9) pathway and is being evaluated in studies involving melanoma, non-small cell lung cancer, and microsatellite stable colorectal cancer.

Cyclophosphamide is approved by the FDA for the treatment of acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloid leukemia, breast cancer, chronic granulocytic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin lymphoma, multiple myeloma, mycosis fungoides, neuroblastoma, non-Hodgkin lymphoma, ovarian cancer, retinoblastoma. Low-dose (immunomodulatory) cyclophosphamide is being studied in combination with various types of immunotherapy including nivolumab and pembrolizumab.

This Phase llA trial hypothesizes that the nivolumab/pixatimod combination in PD-1 relapsed/refractory (R/R) cutaneous melanoma and NSCLC patients will be associated with anti-tumor effects, and that the nivolumab/pixatimod/cyclophosphamide combination in MSS mCRC patients will be associated with anti-tumor effect.

In the 1st stage, 13 patients will be enrolled in cohort 1 while 9 patients each will be enrolled in cohorts 2 and 3. Should the pre-specified efficacy boundary met, further patients will be enrolled to one or more cohorts in 2nd stage. The total enrollment for 1st stage across all 3 cohorts is 31 response-evaluable patients. The total enrollment for both stages across all 3 cohorts is 61 response-evaluable patients.

Arms:
arm 1: Pixatimod : 25 mg IV, weekly

Nivolumab: 480 mg IV, Q4 weeks

Cohort 1 (MSS CRC) arm 2: Pixatimod : 25 mg IV, weekly

Nivolumab: 480 mg IV, Q4 weeks

cyclophosphamide: 50 mg PO twice daily (Day 1-Day 7; Day 15-Day 21) with a 7-day drug free interval (Day 8-Day 14 and Day 22-Day 28)

Cohort 2 (PD-1 R/R melanoma) and Cohort 3 (PD-1 R/R NSCLC)

Interventions: intervention 1: Pixatimod intervention 2: Nivolumab intervention 3: Cyclophosphamide (low dose)
Intervention descriptions: intervention 1: Pixatimod is an investigational drug that is being evaluated in studies involving melanoma, non-small cell lung cancer, and microsatellite stable colorectal cancer. intervention 2: Nivolumab is approved by the FDA for the treatment of melanoma, non-small cell lung cancer (NSCLC), malignant pleural mesothelioma, renal cell carcinoma, classical Hodgkin lymphoma, squamous cell carcinoma of head/neck, urothelial carcinoma, MSI-H colorectal cancer, hepatocellular carcinoma, gastric carcinoma and gastroesophageal junction (GEJ) cancer intervention 3: Cyclophosphamide is approved by the FDA for the treatment of acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloid leukemia, breast cancer, chronic granulocytic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin lymphoma, multiple myeloma, mycosis fungoides, neuroblastoma, non-Hodgkin lymphoma, ovarian cancer, retinoblastoma. Low-dose (immunomodulatory) cyclophosphamide is being studied in combination with various types of immunotherapy including nivolumab and pembrolizumab

Protocol document:
Prot_SAP_000.pdf: 
SUMMARY OF CHANGES – Protocol 
Protocol #: HCC#20-266 
Protocol Date:  02/23/2024 
# 
Section 
Change 
1. 
 
Rationale: The research agreement between the University of Pittsburgh and 
Zucero was terminated as of 2/3/2024 for HCC#20-266. This trial has stopped 
enrolling patients, and the principal investigator has decided to terminate follow up. 
The following language was added to various parts of the protocol. 
 
Added: Note – Patient accrual was terminated early owing to the discontinuation of 
pixatimod supply. Follow-up assessments will no longer be completed upon IRB 
approval of protocol version 02/23/2024. 
 
 
 
 
 


Protocol Number: ZU545201 / HCC 20-266 
IND Number: 155695 
ClinicalTrials.gov number: NCT05061017 
Date: 02/23/2024 
 
 
CLINICAL PROTOCOL ZU545201 
 
Phase IIA Basket Study of Pixatimod (PG545) in Combination with Nivolumab in PD-1 
Relapsed/Refractory Metastatic Melanoma and NSCLC and Pixatimod (PG545) in Combination 
with Nivolumab and low-dose Cyclophosphamide in MSS Metastatic Colorectal Carcinoma 
(mCRC) 
 
 
PRINCIPAL INVESTIGATOR 
Diwakar Davar, MD 
University of Pittsburgh Medical Center and Hillman Cancer Center 
5117 Center Avenue, Suite 1.32d, Pittsburgh, PA 15213 
Telephone (office): 1-412-623-7368 
Pager: 1-412-263-7762 
Fax: 1-412-623-7704 
 
STATISTICIAN 
Hong Wang, PhD 
UPMC Hillman Cancer Center Biostatistics Shared Resource 
Sterling Plaza Suite 325, 201 N. Craig St., Pittsburgh, PA 15213 
Telephone: 412-383-1588 
E-mail: how8@pitt.edu 
 
 
DOCUMENT HISTORY: 
Original 04/26/2021 
First revision 07/19/2021 
Second revision 11/15/2021 
Third revision 03/11/2022 
Fourth revision 04/07/2022 
Fifth revision 04/21/2022 
Sixth revision 07/25/2022 
Seventh version 02/10/2023 
Eighth revision 04/18/2023 
Ninth revision 08/08/2023 
Tenth revision 02/23/2024 
 
 
 
 


Table of Contents 
 
DOCUMENT HISTORY ............................................................................................................... 1 
1. 
SYNOPSIS .......................................................................................................................... 6 
2. 
SCHEDULE OF ACTIVITIES ............................................................................................. 11 
3. 
INTRODUCTION ........................................................................................................... 15 
3.1 
Study Rationale ......................................................................................................... 15 
3.2 
Pharmaceutical and Therapeutic Background: Nivolumab .......................................... 16 
3.3 
Pharmaceutical and Therapeutic Background: Pixatimod (PG545) ............................. 17 
3.4 
Pharmaceutical and Therapeutic Background: Cyclophosphamide ............................. 19 
3.5 
Preclinical and Clinical Trial Data: Nivolumab ............................................................. 20 
3.6 
Preclinical and Clinical Trial Data: Pixatimod (PG545) ................................................ 20 
3.7 
Preclinical and Clinical Trial Data: Cyclophosphamide ................................................ 22 
3.8 
Rationale ................................................................................................................... 22 
3.8.1 
Rationale for Combining Pixatimod (PG545) with Anti-PD(L)1 Antibody ............. 22 
3.8.2 
Rationale for Adding Low-dose Cy in MSS mCRC .............................................. 23 
3.8.3 
Rationale for Dose Selection: Nivolumab ............................................................ 23 
3.8.4 
Rationale for Dose Selection: Pixatimod (PG545) ............................................... 24 
3.8.5 
Rationale for Dose Selection: Cyclophosphamide ............................................... 24 
4. 
OBJECTIVES AND ENDPOINTS ...................................................................................... 25 
4.1 
Primary Objective, Hypothesis, and Endpoint ............................................................. 25 
4.1.1 
Primary Objective ............................................................................................... 25 
4.1.2 
Primary Hypothesis ............................................................................................ 25 
4.1.3 
Primary Endpoint ................................................................................................ 25 
4.2 
Secondary Objectives, Endpoints and Hypotheses .................................................... 25 
4.2.1 
Secondary Objectives......................................................................................... 25 
4.2.2 
Secondary Hypotheses ...................................................................................... 25 
4.2.2.3 
Secondary Hypothesis 3 ..................................................................................... 26 
4.2.3 
Secondary Endpoints ......................................................................................... 26 
4.2.3.3 
Secondary Endpoint 3 ............................................................................................ 26 
4.3 
Exploratory Objectives, Hypotheses, and Endpoints................................................... 26 
4.3.1 
Exploratory Objectives ........................................................................................ 26 
4.3.2 
Exploratory Hypotheses ..................................................................................... 26 
4.3.3 
Exploratory Endpoints ........................................................................................ 27 
5. 
STUDY DESIGN ................................................................................................................ 28 
5.1 
Study Design ............................................................................................................. 28 
5.2 
Trial Procedures ......................................................................................................... 28 
5.3 
Number of Participants ............................................................................................... 29 


6. 
STUDY POPULATION ....................................................................................................... 30 
6.1 
Inclusion Criteria ........................................................................................................ 30 
6.1.1 
General Inclusion Criteria ................................................................................... 30 
6.1.2 
Cohort 1 (MSS mCRC) ....................................................................................... 30 
6.1.3 
Cohort 2 (PD-1 R/R melanoma) ......................................................................... 30 
6.1.4 
Cohort 3 (PD-1 R/R NSCLC) .............................................................................. 31 
6.1.5 
Other Criteria ..................................................................................................... 32 
6.2 
Exclusion Criteria ....................................................................................................... 33 
7. 
TRIAL TREATMENTS ....................................................................................................... 36 
7.1 
Treatment Details ....................................................................................................... 36 
7.2 
Nivolumab Dose Selection, Modification, Timing, and Duration .................................. 36 
7.2.1 
Nivolumab dose selection ................................................................................... 36 
7.2.2 
Nivolumab dose modification .............................................................................. 36 
7.2.3 
Nivolumab timing of administration ..................................................................... 38 
7.2.4 
Nivolumab duration of administration .................................................................. 38 
7.3 
Pixatimod (PG545) Dose Selection, Modification, Timing and Duration ...................... 38 
7.3.1 
Pixatimod (PG545) dose selection ...................................................................... 38 
7.3.2 
Pixatimod (PG545) dose modification ................................................................. 38 
7.3.3 
Pixatimod (PG545) prophylaxis .......................................................................... 38 
7.3.4 
Pixatimod (PG545) timing of administration ........................................................ 39 
7.3.5 
Pixatimod (PG545) duration of administration ..................................................... 39 
7.4 
Cy Dose Selection, Modification, Timing and Duration ............................................... 39 
7.4.1 
Cyclophosphamide dose selection ..................................................................... 39 
7.4.2 
Cyclophosphamide dose modification ................................................................. 39 
7.4.3 
Cy timing of administration ................................................................................. 39 
7.4.4 
Cy duration of administration .............................................................................. 40 
7.5 
Treatment Allocation .................................................................................................. 40 
7.6 
Concomitant Medications ........................................................................................... 40 
7.6.1 
Acceptable Concomitant Medications ................................................................. 40 
7.6.2 
Prohibited Concomitant Medications ................................................................... 40 
7.7 
Rescue Medications and Supportive Care .................................................................. 41 
7.7.1 
Supportive Care Guidelines ................................................................................ 41 
7.8 
Subject Withdrawal/Discontinuation Criteria ............................................................... 44 
7.9 
Subject Replacement Strategy ................................................................................... 44 
7.10 
Clinical Criteria for Early Trial Termination .................................................................. 45 
8. 
STUDY ASSESSMENTS AND PROCEDURES ................................................................. 46 


8.1 
Trial Procedures ......................................................................................................... 46 
8.2 
Treatment Period ....................................................................................................... 46 
8.3 
Administrative Procedures ......................................................................................... 46 
8.4 
Clinical Procedures and Assessments ........................................................................ 47 
8.4.1 
Adverse Events (AE) monitoring ......................................................................... 47 
8.4.2 
Full physical exam .............................................................................................. 47 
8.4.3 
Vital signs........................................................................................................... 47 
8.4.4 
Eastern Cooperative Oncology Group (ECOG) performance scale ..................... 47 
8.5 
Visit Requirements ..................................................................................................... 47 
8.5.1 
Requirements ..................................................................................................... 47 
8.5.2 
Screening ........................................................................................................... 47 
8.5.3 
Thirty-day safety follow-up .................................................................................. 48 
8.5.4 
End of Treatment (EOT) ..................................................................................... 48 
8.5.5 
Post-treatment Surveillance Phase ..................................................................... 48 
8.5.6 
Survival follow up ............................................................................................... 48 
8.6 
Laboratory Assessments ............................................................................................ 48 
8.6.1 
Screening laboratory assessments ..................................................................... 49 
8.6.2 
On-treatment laboratory assessments ................................................................ 49 
8.7 
Efficacy Assessments ................................................................................................ 50 
8.7.1 
Tumor imaging and disease assessment ............................................................ 50 
8.7.2 
Initial tumor imaging ........................................................................................... 51 
8.7.3 
Tumor imaging during the study ......................................................................... 51 
8.7.4 
End of treatment and follow-up tumor imaging .................................................... 51 
8.7.5 
RECIST v1.1 disease assessment ..................................................................... 51 
8.7.6 
iRECIST disease assessment ............................................................................ 52 
8.8 
Adverse Events (AEs) ................................................................................................ 55 
8.8.1 
Definitions .......................................................................................................... 55 
8.8.2 
Evaluation of AEs and SAEs .............................................................................. 56 
8.8.3 
Timeframe for AE/SAE Collection ....................................................................... 58 
8.8.4 
Recording AEs/SAEs ......................................................................................... 59 
8.8.5 
Reporting SAEs and Serious Unexpected AEs (SUAE) ...................................... 59 
8.8.6 
IND safety report ................................................................................................ 61 
8.8.7 
Follow-Up ........................................................................................................... 64 
8.8.8 
Disclaimer .......................................................................................................... 64 
8.8.9 
Reporting AEs to the responsible Institutional Review Board (IRB) ..................... 64 
8.8.10 Follow-up of AEs and SAEs .................................................................................... 65 


8.9 
Tumor Tissue Collection and Correlative Studies ....................................................... 65 
8.9 
Pharmacokinetics Sampling ....................................................................................... 66 
8.10.1 
Pixatimod (PG545) Pharmacokinetics ................................................................ 66 
8.10.2 
Cyclophosphamide Pharmacokinetics ................................................................ 67 
9. 
STATISTICAL CONSIDERATIONS ................................................................................... 69 
9.1 
Study Design ............................................................................................................. 69 
9.2 
Safety Monitoring ....................................................................................................... 69 
9.3 
Efficacy Decision Rule ............................................................................................... 71 
9.4 
Efficacy Analysis ........................................................................................................ 71 
9.5 
Safety Analysis .......................................................................................................... 72 
9.6 
Biomarker Analysis .................................................................................................... 72 
9.7 
Data Safety and Monitoring Plan ................................................................................ 73 
10. 
LABELING, PACKAGING, STORAGE, AND RETURN OF CLINICAL SUPPLIES .........75 
10.1 
Investigational Product: Pixatimod (PG545) ............................................................... 75 
10.1.1 
Packaging and Labeling Information: Pixatimod (PG545) ....................................... 75 
10.1.2 
Handling, Storage and Accountability: Pixatimod (PG545)...................................... 76 
10.1.3 
Dispensing: Pixatimod (PG545).............................................................................. 76 
10.2 
Commercial Product: Nivolumab ................................................................................ 76 
10.3 
Commercial Product: Cyclophosphamide ................................................................... 77 
11. 
REFERENCES .............................................................................................................. 78 
12. 
APPENDICES ............................................................................................................... 87 
12.1 
Appendix 1: ECOG Performance Status115 ......................................................................................................... 87 
12.2 
Appendix 2: Common Terminology Criteria for Adverse Events V5.0 (CTCAE) ..........88 
12.3 
Appendix 3: Contraceptive Guidance and Pregnancy Testing .................................... 89 
12.4 
Appendix 4: Description of the iRECIST Process for Assessment of Disease 
Progression ........................................................................................................................... 90 
12.5 
Appendix 5: Dietary History Questionnaire (DHQ-3) Administration ............................ 95 
 
 
 
 
 
 
 
 
 
 
 
 
5 


1. 
SYNOPSIS 
 
Summary: 
Nivolumab and other agents directed against PD-1/PD-L1 pathway are associated with improved 
response and survival rates in multiple malignancies including advanced melanoma and PD-L1 
expressing NSCLC with objective response rates (ORR) of 35%-45%, the majority of which are 
durable.1-6 However, the majority of treated patients do not respond and not all responses are 
durable. 
Pixatimod (PG545, and hereafter referred to as pixatimod) is a small molecule heparan sulfate 
(HS) mimetic that has natural killer (NK) cell-dependent anti-tumor effects. PG545 is not a TLR9 
agonist that directly activates TLR9, rather, pixatimod results in CpG accumulation in the 
lysosomal compartment of DCs, leading to enhanced production of IL-12, which is essential for 
pixatimod-mediated NK cell activation7,8. In a phase Ia dose-escalation study, single agent 
pixatimod monotherapy at doses up to 150 mg weekly (25-150 mg weekly) demonstrated dose- 
proportional pharmacokinetics and innate immune cell activation.8 In a phase Ib study, 
combination of pixatimod with nivolumab demonstrated promising results in uninflamed tumors 
including microsatellite-stable colorectal carcinoma.9 
Low-dose cyclophosphamide has several immunomodulatory properties including: depletion of 
regulatory T cells; increased IFN production, induction of immunogenic cell death; increased 
CD8+ effector T cells; increased NK cell number and function.10 Pre-clinically, low-dose 
cyclophosphamide diminished immunosuppressive TGF-β and IL-10 and had synergy with intra- 
tumoral type B CpG SD-101 in injected and uninjected tumors.11 Preclinical synergy with PG545 
has also been reported in a murine model of established lymphoma.12 Clinically, low-dose 
metronomic cyclophosphamide has primarily been used in breast cancer, castrate-resistant 
prostate cancer, ovarian cancer and CRC at varying doses (50-100 mg daily) and schedules 
(daily, 1-week-on, 1-week-off). 
We hypothesize that the nivolumab/pixatimod combination in PD-1 relapsed/refractory (R/R) 
cutaneous melanoma and NSCLC patients will be associated with anti-tumor effects. We also 
hypothesize that the nivolumab/pixatimod/cyclophosphamide combination in microsatellite stable 
(MSS) metastatic colorectal carcinoma (mCRC) patients will be associated with anti-tumor 
effects. We further hypothesize that nivolumab/pixatimod/cyclophosphamide combination in MSS 
mCRC patients and nivolumab/pixatimod combination in PD-1 R/R melanoma or NSCLC will be 
associated with increased CD8+ T cells intra-tumorally and PD-1+Ki67+ CD8+ T cells peripherally 
in responders on treatment compared to baseline; and that response will be associated with 
evidence of antigen-specific immunity responses intra-tumorally and peripherally. 
Study 
Population: 
Men or women, ≥18 years of age, with selected solid tumors and an Eastern Cooperative 
Oncology Group (ECOG) performance status of ≤1 and have at least 2 lesions with measurable 
disease as defined by Response Evaluation Criteria in Solid Tumors (RECIST) Version 1.1. 
 
 
 
 
 
 
 
 
 
 
 
 
 
6 


Objectives and 
Endpoints: 
Objective 
Endpoint 
Primary 
• 
To assess efficacy of 
nivolumab/pixatimod combination in 
PD-1 R/R melanoma and NSCLC. 
• 
To assess efficacy of 
nivolumab/pixatimod/cyclophosphamide 
combination in MSS mCRC patients 
• 
Objective response rate by 
RECIST v1.1 to 
nivolumab/pixatimod combination 
in PD-1 R/R melanoma and 
NSCLC. 
• 
Objective response by RECIST 
v1.1 to nivolumab/pixatimod/ 
cyclophosphamide combination 
in MSS mCRC patients 
Secondary 
• 
To assess safety of 
nivolumab/pixatimod combination in 
PD-1 R/R melanoma and NSCLC; and 
nivolumab/pixatimod/cyclophosphamide 
combination in MSS mCRC patients 
• 
To assess efficacy using alternative 
response endpoints. 
• 
To assess CD8+ T cell infiltrate in pre- 
and on- treatment tumor samples by 
multiplex IHC/IF. 
• 
To assess median and landmark 
progression-free survival (PFS) and 
landmark overall survival (OS) in 
participants treated with 
nivolumab/pixatimod or 
nivolumab/pixatimod/ 
cyclophosphamide 
• 
Incidence of all-grade related 
adverse events (AE), serious 
adverse events (SAE) and dose- 
limiting toxicities (DLT) if any in 
each cohort. 
• 
Objective response by iRECIST 
to nivolumab/pixatimod/ 
cyclophosphamide combination 
in MSS mCRC patients and 
nivolumab/pixatimod combination 
in PD-1 R/R melanoma and 
NSCLC. 
• 
Summary measures of change 
(or % change) from baseline in 
the CD8+ T cell or NK cell 
infiltrate in the tumor and TME in 
tumor biopsies at pre- and on- 
(approximately 4 weeks) 
treatment timepoints. 
• 
Median PFS, landmark PFS (6 
months, 1-year and 2-year), 
median OS and landmark OS (1- 
year, and 2-year) in participants 
treated with nivolumab/pixatimod 
or nivolumab/pixatimod/ 
cyclophosphamide 
Exploratory 
• 
To explore potential association 
between biomarkers and clinical 
efficacy of nivolumab/pixatimod or 
nivolumab/pixatimod/ 
cyclophosphamide combination 
• 
Correlation/measures of 
association of biomarkers and 
clinical effect of 
nivolumab/pixatimod or 
nivolumab/pixatimod/ 
cyclophosphamide combination 
 
7 


Overall 
Study 
Design: 
This is a study of nivolumab in combination with pixatimod (PG545, hereafter referred to as 
pixatimod) in 3 separate cohorts: 
• 
Cohort 1: MSS mCRC in combination with low-dose cyclophosphamide 
• 
Cohort 2: PD-1 relapsed/refractory melanoma 
• 
Cohort 3: PD-1 relapsed/refractory NSCLC. 
Each cohort comprises a two-stage Simon phase IIA trial. The goal of this study is to assess 
response of PD-1 R/R melanoma/NSCLC to nivolumab and pixatimod; and MSS mCRC to 
nivolumab, pixatimod and low-dose cyclophosphamide. 
Study 
Evaluation: 
Participants meeting enrollment criteria will be enrolled depending on histology into one of 3 
cohorts (cohort 1: MSS mCRC; cohort 2: PD-1 R/R melanoma; cohort 3: PD-1 R/R NSCLC). 
Number 
of 
Participants: 
In the 1st stage of cohort 1 (MSS mCRC), we will enroll 13 patients. If ≥1 response(s) are seen, 
14 
additional 
patients 
will 
be 
enrolled 
in 
the 
2nd 
stage. 
The 
nivolumab/pixatimod/cyclophosphamide combination will be considered worthy of further 
evaluation if ≥4 responses (ORR 15%) are seen across both stages in this cohort. 
For cohorts 2 (PD-1 R/R melanoma) and 3 (PD-1 R/R NSCLC), we will use the same design, but 
separately, as follows. In the 1st stage of each cohort, will enroll 9 patients. If ≥1 response(s) are 
seen, 8 additional patients will be enrolled in the 2nd stage. The combination will be considered 
worthy of further evaluation if ≥3 responses (18%) are seen across both stages in either (or both) 
cohorts. 
Treatment 
Arms 
and 
Duration: 
There are 3 cohorts in this study: cohort 1: MSS mCRC; cohort 2: PD-1 R/R melanoma; cohort 
3: PD-1 R/R NSCLC. 
Nivolumab has demonstrated clinical activity in participants with melanoma and NSCLC. 
However, single-agent nivolumab has negligible efficacy in PD-1 R/R melanoma/NSCLC and 
MSS mCRC. 
Treatment will be assigned based on histology. Combination therapy will be administered until 
toxicity/discontinuation or completion of 2 years of combination treatment. 
Study 
Assessments 
and Analyses 
Primary Endpoint 
• 
Objective response by RECIST v1.1 to nivolumab/pixatimod/cyclophosphamide 
combination in MSS mCRC (cohort 1) patients and nivolumab/pixatimod combination in 
PD-1 R/R melanoma and NSCLC (cohorts 2 and 3). 
Secondary Endpoints 
• 
Objective response by iRECIST to nivolumab/pixatimod/cyclophosphamide combination 
in MSS mCRC (cohort 1) patients and nivolumab/pixatimod combination in PD-1 R/R 
melanoma and NSCLC (cohorts 2 and 3). 
• 
Summary measures of change (or % change) from baseline in the CD8+ T cell and NK 
cell infiltrate in the tumor and TME in tumor biopsies at pre- and on- (approximately 4 
weeks) treatment timepoints. 
• 
Median PFS, landmark PFS (6 months, 1-year and 2-year), median OS and landmark 
OS (1-year, and 2-year) in participants treated with nivolumab/pixatimod or 
nivolumab/pixatimod/cyclophosphamide. 
Exploratory Endpoints 
• 
Correlation/measures of association of biomarkers and clinical effect of 
nivolumab/pixatimod or nivolumab/pixatimod/cyclophosphamide combination. 
 
8 


Safety Outcome Measures 
• 
Adverse events (AE) will be assessed continuously during the study and for 100 days 
after the last dose of study treatment. 
• 
AEs will be graded using National Cancer Institute (NCI) Common Terminology Criteria 
for Adverse Events (CTCAE) Version 5.0. 
Efficacy Measurements 
• 
Disease assessment with computed tomography (CT) and/or magnetic resonance 
imaging (MRI), as appropriate, will be performed at baseline and every 8 weeks (± 7 
days) until disease progression, at the completion of follow-up, or until participants 
withdraw from the study. Tumor responses will be derived for evaluable participants as 
defined by RECIST Version 1.1 based on recorded tumor measurements. 
• 
Serologic tumor markers will be collected, as appropriate. 
• 
At the Sponsor-Investigator’s discretion, scans and measurements may be collected 
and reviewed by independent radiologists at a later date, or at any time during the 
study. 
Biomarker Measures: 
• 
A broad translational plan to evaluate predictive biomarkers for combination therapy 
(see Section 8.9.1). Analysis of interest may include, but are not limited to: 
• 
Systemic Immunity: 
o Serial peripheral blood samples will be obtained for immuno-monitoring by 
multiplex flow-cytometry and proteomic analysis of immunologically relevant 
soluble factors. 
• 
Tumor Microenvironment: 
o Multiplex IF and IHC to evaluate tumor microenvironment and to assess 
relationships between baseline and on-treatment immune contexture. 
o Transcriptome analysis (to understand gene expression signatures and 
pathways that mediate response, resistance and toxicity). 
o Whole exome sequencing (to understand the relationship between driver 
mutations and response, resistance and toxicity, and to understand the 
relationship between mutational load and neo-antigens to response). 
o Peripheral blood circulating tumor deoxyribonucleic acid (ctDNA) or cell-free 
DNA (cfDNA; to compare this technology to findings derived directly from the 
tumor). 
• 
Host Status 
o Whole blood whole-genome sequencing (WGS) or whole exome sequencing 
(WGS; to assess for the relationship between germline single nucleotide 
polymorphisms with response, resistance and toxicity, and for making mutational 
calls on the tumor DNA sequencing). 
o Stool for microbiome (to assess the impact of fecal metagenomics on IO 
response, resistance and toxicity). 
 
Statistical 
Considerations 
Sample size calculation: 
Stage 1 (cohort 1 – 13 patients; cohorts 2/3 – 9 patients). 
Stage 2 (cohort 1 – 14 patients; cohorts 2/3 – 8 patients). 
Total enrollment for 1st stage across all 3 cohorts = 31. 
Total enrollment for both stages across all 3 cohorts = 61. 
Sample size justification: 
 
9 


In the 1st stage of cohort 1 (MSS mCRC), we will enroll 13 patients. If ≥1 response(s) are seen, 
14 
additional 
patients 
will 
be 
enrolled 
in 
the 
2nd 
stage. 
The 
nivolumab/pixatimod/cyclophosphamide combination will be considered worthy of further 
evaluation if ≥4 responses (ORR 15%) are seen across both stages in this cohort. This study 
design, using Simon’s 2-stage Minimax design, provides 80% power with 5% type I error to detect 
a response rate of 20% against a null hypothesis of 5%. 
For cohorts 2 (PD-1 R/R melanoma) and 3 (PD-1 R/R NSCLC), we will use the same design, 
but separately, as follows. In the 1st stage, will enroll 9 patients. If ≥1 response(s) are seen, 8 
additional patients will be enrolled in the 2nd stage. The combination will be considered worthy of 
further evaluation if ≥3 responses (ORR 18%) are seen across both stages. This study design, 
using Simon’s optimal 2-stage design, provides 80% power with 5% type I error to detect a 
response rate of 25% against a null hypothesis of 5%. 
Statistical plan: 
In the interest of maximizing safety while minimizing exposure to a potentially ineffective 
combination, we have designed parallel two-stage phase II studies in three cohorts. We 
hypothesize that nivolumab/pixatimod combination (and low-dose Cy in MSS mCRC) will 
increase the frequency of responding patients in each cohort. 
Although the nivolumab/pixatimod combination is being evaluated in advanced studies and DLT 
of pixatimod has been established in a prior phase I study,8 the nivolumab/PG545/Cy combination 
has not been evaluated. However, given the Cy dose being studied, no DLTs are expected. To 
mitigate toxicity, we will monitor toxicities continuously (cohort 1) and implement a 4-week DLT 
monitoring period during the enrollment of the first 3 patients across cohorts 2-3. During this 
period, only 1 patient will be enrolled every 4 weeks. If no DLT occurs for the first 3 
patients during the DLT monitoring period, this enrollment cap will be lifted. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
10 


 
2. SCHEDULE OF ACTIVITIES 
 
Procedure/ 
Assessment 
Screening 
(Day -28 to - 
1)A 
Treatment CyclesB 
End of 
Treatment 
C 
30 Day 
Follow- 
UpD 
Post- 
Treatment 
SurveillanceE 
Cycle 1 
Day 1 
Cycle 1 
Day 2 
Cycle 1 
Day 4 
Cycle 1 
Day 8 
Cycle 1 
Day 15 
Cycle 1 
Day 22 
Cycle 2 
Day 1 
Cycle 2 
Day 8 
Cycle 2 
Day 15 
Cycle 2 
Day 22 
Cycle 3+ 
Visit Windows 
None 
None 
None 
± 2 days 
± 2 days 
± 2 days 
± 2 days 
± 2 days 
± 2 days 
± 2 
days 
± 2 days 
± 7 days 
± 7 days 
± 14 days 
NivolumabF 
X 
X (q4 
weeks) 
X (q4 
weeks) 
Pixatimod 
(PG545)G 
X 
X 
X 
X 
X 
X 
X 
X 
X 
(weekly) 
CyH (Cohort 1 
CRC only) 
XH 
XH 
XH 
Informed consent 
X 
Eligibility criteria 
assessment 
X 
Physical exam 
including vital 
signsI 
X 
X 
X 
X (D1) 
XC 
XD 
XE 
Vital signsI 
X 
X 
X 
X 
X 
X 
X (D 
8/15/22) 
Electrocardiogram 
(ECG)J 
X 
X 
Laboratory 
assessments 
XK-S 
XK-B 
XK-T (D15 
only) 
XK-B 
XK-T (D15 
only) 
XK-B (D1 
only) and 
XD 
XE 
including 
XK-T (D15 
screening labsK 
only) 
Serum/urine 
pregnancy testL 
X 
Pixatimod 
pharmacokinetic 
(PK) samplingM 
XM-1 
XM-1 
XM-1 
XM-1 
Cy PK samplingN 
XN-1 
XN-1 
Tumor biopsyO 
XO-S 
XO-O 
XO-P 
Exploratory 
X 
X 
X 
X 
X (C2D15, 
X (q8 
X 
biomarker 
(C2D15, 
C2D22) 
weeks) 
analysesP 
C2D22) 
Stool samplingQ 
X 
 
 
 
X 
X (q8 
X 
 
 
 
weeks) 
 
Dietary history 
questionnaireR 
X 
 
 
11 


 
Disease 
assessmentS 
X 
X (q8 
weeks 
starting 
with 
C3D1) 
XE 
 
Study Calendar Notes: 
AScreening period is 28 days (≤4 weeks) prior to first dose of pixatimod/nivolumab. 
• 
Screening studies including magnetic resonance imaging (MRI)/computed tomography (CT) imaging must be performed ≤4 weeks prior to first dose of 
pixatimod/nivolumab. 
• 
During Screening, MRI brain with contrast (or CT equivalent) should be performed to exclude CNS metastatic disease for melanoma and NSCLC patients. 
• 
CNS imaging is not required for MSS mCRC patients at Screening unless patient is symptomatic. 
• 
CNS imaging need not be repeated on study unless patient is symptomatic. 
• 
CNS imaging should be repeated at investigator’s discretion in patients with a history of treated CNS metastases. 
BTreatment cycle 
• 
Each cycle is 28 days long. 
CEnd-of-treatment (EOT) assessments to be performed within 7 days following removal of subject from pixatimod treatment. Removal of a subject from pixatimod 
treatment is defined as the time in which the Investigator decides to discontinue pixatimod treatment for a subject. 
• 
Management of patients who develop unacceptable toxicity is defined in Section 8.5.4. 
o 
Patients who develop unacceptable toxicity deemed related to pixatimod (or if applicable, cyclophosphamide) but not nivolumab may continue 
Nivolumab (and if applicable cyclophosphamide) after pixatimod discontinuation for duration as defined in Sections 7.3.2 and 7.4.2. 
o 
Patients who develop unacceptable toxicity deemed related to nivolumab but not pixatimod must discontinue both pixatimod and nivolumab. 
D30-day follow-up. 
• 
This begins +30 days following completion of last dose of study treatment or EOT, whichever is earlier. 
• 
Procedures to be followed during this phase are outlined in Section 8.5.3 and will include a visit, labs (CBC, CMP, TSH, free T3, free T4) and any other 
studies deemed necessary at the discretion of the treating physicians and according to established Standard of Care. 
EPost-treatment surveillance. 
• 
This phase begins upon completion of 30 day follow up. 
• 
Procedures to be followed during this phase are outlined in Section 8.8.5 will include imaging (CT or PET/CT), labs (CBC, CMP, TSH, free T3, free T4) 
and any other studies deemed necessary at the discretion of the treating physicians and according to established Standard of Care. 
• 
Note – Patient accrual was terminated early owing to the discontinuation of pixatimod supply.  Follow-up assessments will no longer be completed upon IRB approval of 
protocol version 02/23/2024. 
FNivolumab dosing 
• 
Nivolumab will be dosed at 480 mg q4 weeks IV. 
GPixatimod 
• 
Pixatimod will be dosed 25 mg weekly IV. 
• 
Pixatimod will be administered through either a large-bore IV line placed in the antecubital fossa or central venous catheter. 
• 
Patients in whom pixatimod is administered through a small-bore/distal IV may developed a delayed-type hypersensitivity reaction. 
o 
This should not be treated as an infusion reaction and should recur upon the administration through a proximal large-bore IV line. 
HCyclophosphamide dosing (Cohort 1 only) 
• 
50 mg PO twice daily (D1-D7; D15-D21) with a 7-day drug free interval (D8-D14 and D22-D28). 
• 
Cyclophosphamide dose compliance will be assessed using a pill diary. 
IPhysical exams will be conducted at Screening, each pixatimod treatment visit, EOT, 30-day follow-up and during Post-treatment surveillance. 
• 
Screening physical exam may be performed up to 72 hours prior to the Cycle 1 Day 1 visit. 
• 
Physical exams should be repeated on each D1 visit and should include assessment of ECOG performance status. Requirements are defined in Section 
8.4.2. 
 
12 


• 
On D8/D15/D22 for all cycles, a formal physical exam is not required and only vital signs will be recorded. 
• 
Height will be recorded at Screening visit only. 
JElectrocardiograms (ECGs) should be obtained at Screening and prior to administration of pixatimod C1D1. 
• 
ECG need not be repeated unless clinically indicated at subsequent visits. 
KLaboratory assessments 
• 
Laboratory assessments to be obtained at Screening (XK-S): CBC, renal and liver function, coagulation parameters, infectious parameters (HIV, Hep B/C 
screening), thyroid function (TSH, free T3 and free T4), and urinalysis. 
o 
NOTE: In patients who are currently or have recently (within 6 months) been on heparin, low-molecular weight heparin, fondaparinux or other 
similar agents, anti-heparin antibodies should be obtained during Screening. Per Section 6.2.3, the presence of anti-heparin antibodies is 
exclusionary. 
• 
Laboratory assessments to be obtained on Baseline (Cycle 1 Day 1; XK-B): CBC, renal and liver function, coagulation parameters, fasting lipids, thyroid 
function (TSH , free T3 and free T4) and urinalysis. 
• 
Laboratory assessments to be obtained on On-Treatment (XK-T): CBC, renal and liver function, fasting lipids (D1 of each cycle only starting with C2D1) 
coagulation parameters, and urinalysis. 
• 
There is a 48-hour window to obtain Screening (XK-S), Baseline (XK-B) and/or On-Treatment (XK-T) laboratory assessments. 
LPregnancy testing 
• 
Only required at screening in women of childbearing potential (WOCP). 
MPixatimod PK sampling 
• 
Pixatimod PK timepoints (relative to pixatimod infusion): 
o 
1 – to be performed at pre-dose, 30min (± 5 min), 2h (± 15 min), 4h (± 15 min), 6h (± 30 min), 24h (D2; ± 60 min), 72h (D4; ± 24 hours) and 
168h (prior to C1D8; ± 24 hours) post infusion; 
o 
Windows: 
o 
Pixatimod PK sampling will be collected from 10 subjects in Cohort 1, and a total of 10 subjects from Cohorts 2 and 3. 
• 
Samples to be collected as delineated in Section 8.10.1 and will be processed as delineated in the Laboratory Manual. 
NCyclophosphamide PK sampling 
• 
Cyclophosphamide PK timepoints (relative to pixatimod infusion): 
o 
1 – to be performed at pre-dose, 10min (± 5 min), 30min (± 5 min), 1h (± 10 min), 2h (± 15 min), 4h (± 15 min), 6h (± 30 min), and 24h (D2; ± 
60 min) post cyclophosphamide; 
o 
Cyclophosphamide PK sampling will be collected from 10 subjects in Cohort 1 only. 
• 
Samples to be collected as delineated in Section 8.10.2 and will be processed as delineated in the Laboratory Manual. 
OTumor biopsy must be performed prior to commencing Pixatimod/Nivolumab treatment. 
• 
Punch, core (16 gauge) and/or surgical biopsies are acceptable. 
If core biopsy is performed, pathology confirmation is required to ensure adequate tissue is obtained; and a minimum of 6 cores must be obtained. 
• 
Tumor biopsies will be obtained at Screening (XO-S), on-treatment at C2D1 (XO-O) and at progression (if applicable; XO-P). 
PExploratory biomarker analyses will be obtained as follows: 
• 
Prior to administration of pixatimod/nivolumab at Screening, on C1D1, C1D8, C1D15, C2D1, C2D15, C2D22 in Cycles 1-2. 
• 
Cycles 3 onwards: q8 weekly. 
• 
Samples to be collected as delineated in Section 8.9 and will be processed as delineated in the Laboratory Manual. 
QStool sampling will be obtained as follows: 
• 
Prior to administration of pixatimod/nivolumab on C1D1 and C2D22. 
• 
Cycles 3 onwards: q8 weekly. 
• 
EOT 
• 
Samples will be processed as delineated in the Laboratory Manual. 
RDietary history questionnaire will be on C1D1. Dietary questionnaire is detailed in Appendix 6. 
 
13 


SCT or MRI scans to assess tumor status will be obtained at Screening (baseline value should be performed ≤4 weeks prior to first pixatimod dose). 
• 
Screening: Scans should be performed ≤4 weeks prior to first pixatimod dose. CT (or PET/CT or MRI) can be obtained at Investigator discretion, however, 
contrast-enhanced CT is preferred. 
• 
Cycles 1-2 (+/- 7 days): Contrast-enhanced CT (or PET/CT) at C3D1. 
• 
Cycles 3+ (+/- 7 days): Contrast-enhanced CT (or PET/CT) every 8 weeks. 
• 
Progression: Progression should be confirmed with repeat imaging study done no sooner than 4 weeks after prior study. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
14 


15 
 
 
3. 
INTRODUCTION 
3.1 Study Rationale 
 
PD-1 is a receptor expressed by activated T- and B- cells which binds to two known ligands: PD-L1 (B7- 
H1)13,14 and PD-L2 (B7-DC).15,16 PD-1 negatively regulates T cell functions through the engagement of PD- 
L1, which is expressed by a wide variety of tissues.13-16 PD-L1 is also expressed by human tumors, including 
melanoma, either constitutively or after treatment with IFN-α.17,18 In chronically infected mice, lymphocytic 
choriomeningitis virus-specific CD8+ T-cells exhibit a diminished capability to produce cytokines, lyse infected 
cells and proliferate in a progressive and hierarchical fashion.19 These “exhausted” T-cells have been shown 
to up-regulate PD-1, and blockade of the PD-1/PD-L1 pathway led to increased cytokine production and 
proliferation, resulting in a significant reduction of the viral load.19 
 
We have previously shown that majority of tumor antigen-specific CD8+ T cells upregulate PD-1 expression, 
which is associated with T cell exhaustion/dysfunction in chronic viral infections in animals and humans.20,21 
We observed that PD-1 upregulation on spontaneous tumor antigen-specific CD8+ T cells occurs along with 
T cell activation and is not directly associated with an inability to produce cytokines ex vivo upon stimulation 
with cognate antigen. Blockade of the PD-1/programmed death ligand 1 (PD-L1) pathway in combination with 
prolonged antigen stimulation with PD-L1+ antigen-presenting cells or melanoma cells augmented the 
frequencies of cytokine-producing, proliferating and total tumor antigen-specific NY-ESO-1 CD8+ T cells.20,21 
Collectively, these findings support that PD-1 is a regulator of antigen-specific CD8+ T cell expansion in the 
context of chronic antigen exposure in patients with advanced cancer; and that PD-1 blockade reverses 
tumor-mediated T cell dysfunction. 
 
Immune checkpoint inhibitors (ICI) directed against PD-1/PD-L1 are associated with improved response and 
survival rates in multiple malignancies including melanoma, squamous/non-squamous lung cancer, bladder 
and renal cell carcinoma.4,6,22-25 In advanced melanoma and PD-L1 expressing NSCLC, anti-PD-(L)1 
monotherapy produces overall response probabilities of 42%-45%, the majority of which are durable.1-6 
 
In Checkmate-067, dual anti-PD-1 and anti-CTLA-4 blockade with nivolumab and high-dose ipilimumab 
(3mg/kg every 3 weeks for 4 doses) further improved ORR (58%) in melanoma compared to either nivolumab 
or ipilimumab, albeit with significantly greater toxicity.26-29 Ipilimumab/nivolumab was associated with five year 
progression-free survival (PFS) of 36%, compared with 29% (nivolumab) and 8% (ipilimumab).28 Checkmate- 
227 compared low-dose ipilimumab/nivolumab combination (nivolumab 2mg/kg every 2 weeks; ipilimumab 
1mg/kg every 6 weeks) to nivolumab monotherapy and platinum-doublet chemotherapy in PD-L1≥1% 
NSCLC. Ipilimumab/nivolumab combination produced slightly better response compared to chemotherapy 
(36% vs. 30%), with commensurately improved PFS and overall survival (OS) and led to regulatory approval.30 
 
While anti-PD(L)1 blockade singly and in combination is remarkably efficacious in melanoma and NSCLC, ICI 
therapy is minimally efficacious in microsatellite stable (MSS) CRC. The remarkable efficacy of PD-1/PD- L1 
or PD-1/CTLA-4 blockade in CRC in limited to hyper-mutated subtypes – specifically those with microsatellite 
instability-high (MSI-H)/mismatch repair-deficient (dMMR) CRC or those with POLE mutations.31,32 In these 
patients, the response to ICI is related to increased TIL density secondary to immunogenic neoepitopes and 
increased tumor mutational burden (TMB). However, MSI-H tumors constitute 
~3% of all CRC and ICI therapy is minimally effective in MSS mCRC.33 
 
Biomarkers of response to PD-(L)1 blockade include CD8+ TIL,34,35 interferon gamma (IFN-γ) gene 
expression,36,37 high tumor mutation burden (TMB; 37-39) or PD-L1 expression.40,41 These observations suggest 
that uninflamed tumors represent the bulk of tumors that fail to respond to anti-PD(L)1 ICB; and argue for 
augmenting inflammation as a means to promote T cell responses in PD-1 relapsed/refractory (R/R) 
melanoma and PD-1 R/R NSCLC and MSS mCRC. 


16 
 
Tumor-associated DC are critical regulators of the balance between CD8+ T cell immunity versus tolerance 
to tumor antigens. There are 3 major DC subsets in humans: CD141+ myeloid/conventional DC1 (cDC1), 
CD1c+/CD11b+/BDCA-1+ myeloid/conventional DC2 (cDC2) and CD123+/BDCA-2+/BDCA-4+ plasmacytoid 
DC (pDC). Of these, both CD141+ cDC1 and CD123+/BDCA-2+/BDCA-4+ pDC are critical for CD8+ T cell 
priming and efficacy of PD-1 blockade.42 TLR9 agonists activate pDCs to secrete type I IFN and to express 
increased levels of co-stimulatory molecules such as CD80 (B7.1) and CD86 (B7.2). TLR9 agonism results 
in a range of secondary effects, including secretion of cytokines such as MCP-1, IP-10/CXCL10 that activate 
NK cells and expand T cell subpopulations including CD8+ T cells and TH1 cells.43 However, TLR9 agonists 
are limited as they have to be administered intra-tumorally as systemic administration of TLR9 agonists results 
primarily in liver, spleen, and RES uptake with little specific activation of pDC in draining lymph nodes; and 
immature tumor-associated pDC contribute to tumor growth and an adverse prognosis in patients with 
cancer.44,45 Hence, systemically administered agents may overcome some of limitations of intra-tumoral 
innate immune agonists such as TLR9 and STING agonists, promoting antigen presentation to T cells, 
facilitating cross-priming and augmenting adaptive T cell responses in cancer. 
 
3.2 Pharmaceutical and Therapeutic Background: Nivolumab 
 
The importance of intact immune surveillance in controlling outgrowth of neoplastic transformation has been 
known for decades.46 The inability of the immune system to control tumor growth does not appear to result 
from an inability to recognize the tumor as foreign. Tumor cells have been shown to evade immune destruction 
despite displaying recognizable antigens on their surface and despite the presence of high-avidity T cells that 
are specific for these antigens.47-50 Histologic evaluation of many human cancers show extensive infiltration 
by inflammatory and immune cells,51,52 suggesting that the immune system responds less effectively to 
malignancy. These observations have led to the hypothesis that dominant mechanisms of immune tolerance 
or immune suppression are responsible for the immune system's inability to effectively respond in a way that 
consistently results in rejection. 
 
There are a number of inhibitory mechanisms that have been identified to be involved in tumor-mediated 
immune suppression and include expression of the programmed death ligand-1 (PD-L1), which can engage 
the inhibitory receptor PD-1 on activated T cells; the presence of the tryptophan-catabolizing enzyme IDO1, 
which exploits the exquisite sensitivity of T cells to tryptophan depletion and tryptophan metabolites; and 
infiltration with FoxP3+ regulatory T cells (Treg), which can mediate extrinsic suppression of effector T-cell 
function. Therefore, agents that target these negative regulatory pathways and thereby allow the expansion 
of effector T cells present in the tumor may be beneficial in the clinic. 
 
The PD-1 receptor-ligand interaction is a major pathway stimulated by tumors to suppress immune control. 
The normal function of PD-1, expressed on the cell surface of activated T cells under healthy conditions, is to 
down-modulate unwanted or excessive immune responses, including autoimmune reactions. Programmed 
death receptor-1 (encoded by the gene Pdcd1) is an Ig superfamily member related to CD28 and cytotoxic T- 
lymphocyte-associated antigen-4 (CTLA-4), which has been shown to negatively regulate antigen receptor 
signaling upon engagement of its ligands (PD-L1 and/or PD-L2).14,15,17 The mechanism by which PD-1 down 
modulates T-cell responses is similar to but distinct from that of CTLA-4, as both molecules regulate an 
overlapping set of signaling proteins.53,54 
 
Programmed death (PD) receptor-1 has been shown to be expressed on activated lymphocytes including 
peripheral CD4+ and CD8+ T cells, B cells, Tregs, and natural killer cells.55 Expression has also been shown 
during thymic development on CD4-CD8- (double negative) T cells as well as subsets of macrophages and 
dendritic cells (DCs). The ligands for PD-1 (PD-L1 and PD-L2) are constitutively expressed or can be induced 
in a variety of cell types, including non-hematopoietic tissues as well as in various tumors.15,17,18,41,56,57 Binding 
of either PD-1 ligand to PD-1 inhibits T-cell activation triggered through the T-cell receptor. PD-L1 is expressed 
at low levels on various non-hematopoietic tissues, most notably on vascular endothelium, whereas PD-L2 
protein is only detectably expressed on antigen-presenting cells found in lymphoid tissue or 


17 
 
chronic inflammatory environments. PD-L2 is thought to control immune T-cell activation in lymphoid organs, 
whereas PD-L1 serves to dampen unwarranted T-cell function in peripheral tissues. Although healthy organs 
express little (if any) PD-L1, a variety of cancers were demonstrated to express abundant levels of this T-cell 
inhibitor. PD-1 has been suggested to regulate tumor-specific T cell expansion in subjects with melanoma.20,21 
This suggests that the PD-1/PD-L1 pathway plays a critical role in tumor immune evasion and should be 
considered as an attractive target for therapeutic intervention. 
 
Nivolumab is a fully human Ig G4 antibody that blocks PD-1. Nivolumab is approved for the treatment of 
metastatic melanoma following ipilimumab and, if BRAF V600 mutation positive, a BRAF inhibitor (Checkmate 
037). Nivolumab is also approved for the treatment of advanced metastatic NSCLC of both squamous 
(Checkmate 017) and non-squamous (Checkmate 057) histologies following progression on or after platinum- 
based chemotherapy. 
 
3.3 Pharmaceutical and Therapeutic Background: Pixatimod (PG545) 
 
Pixatimod is a cholestanol-sulfotetrasaccharide conjugated small molecule compound that is a heparan 
sulfate (HS) mimetic with unique NK- and T cell- dependent immunomodulatory properties that is currently 
being evaluated in a variety of different tumors. Beyond anticoagulation, the therapeutic potential of heparin 
derivatives and heparan sulfate (HS) mimetics in oncology is related to their ability to bind and modulate the 
function of a vast array of HS-binding proteins with pivotal roles in cancer growth and progression.7 Defining 
the structural and functional determinants of HS mimetics has clarified the structural requirements for the 
inhibitory effects of HS mimetics on heparanase, selectins, growth factor receptor signaling, 
immunomodulatory activities while limiting potential adverse effects.7 
 
Pixatimod has an oligosaccharide backbone that is derived from starch, and retains the amylose structure of 
α(1 → 4)-linked glucose residues.58 Coupling the sulfated oligosaccharide to a lipophilic cholestanol aglycone 
significantly increased the elimination half-life in vivo, while reducing the unwanted anticoagulant activity 
associated with similar compounds while retaining inhibition of HS-degrading enzyme heparanase-1 
(HPSE).58-61 As such, pixatimod has a half-life of approximately 141 hours (6 days) in humans, considerably 
greater than the 7-8 hours observed with most heparin mimetics - enabling weekly dosing.58 
 
Pixatimod inhibits HPSE in a concentration-dependent manner; a function that is critical to its activity.60,61 
However, it is important to consider that pixatimod has significant immunomodulatory properties as well. In 
multiple orthotopic models, pixatimod inhibited infiltration of tumor-associated macrophages (TAMs).62-65 
TAMs contribute to cancer progression and therapeutic resistance by mediating an immunosuppressive TME 
and facilitating tumor metastases through multiple mechanisms including promotion of tumor cell 
extravasation, invasion, vascularization (see Figure 3.3-1).66 


18 
 
 
 
Figure 3.3-1: Pixatimod inhibits tumor-associated macrophages (TAMs). 
 
Separately, the anti-tumor effects of pixatimod are NK-dependent, which in turn was found to be IL-12 and 
TLR9 dependent (see Figure 3.3-2).12 
 
Figure 3.3-2: Activation of NK cells by pixatimod in tumor-bearing mice. 
 
Pixatimod does not possess CpG ODN motifs and hence does not activate TLR9 directly. Rather, pixatimod 
increases CpG ODN accumulation in endosomal compartment of DCs, leading to enhanced production of IL- 
12, which is essential for pixatimod-mediated NK cell activation (see Figure 3.3-3).12 


19 
 
 
 
Figure 3.3-3: Pixatimod increases human PBMC-derived pDC activation (CD40) in response to 24- hour exposure 
to CpG ODN-2006 in vitro. 
 
Preclinically, pixatimod potently inhibits solid tumor progression and metastasis in a number of syngeneic, 
orthotopic and xenograft murine models of cancer either singly59,63,67-74 or in combination with chemotherapy 
such as paclitaxel or gemcitabine.75,76 Pixatimod has demonstrated synergy with cyclophosphamide, although 
this may be immunodulatory given the effects of low-dose Cy on NK cell activation.12 
 
3.4 Pharmaceutical and Therapeutic Background: Cyclophosphamide 
 
Cy [N,N-bis (2-chloroethyl)-1, 3, 2-oxazaphosphinan-2 amine 2-oxide] is a nitrogen mustard alkylating agent 
from the oxazophorine group. Cyclophosphamide exists as a solid, soluble (in water), and a very weakly acidic 
compound (based on its pKa). It is administered orally or intravenously. Cyclophosphamide is activated in the 
liver, principally activated by cytochrome P450 isoforms, CYP2A6, 2B6, 3A4, 3A5, 2C9, 2C18, and 2C19, of 
which the CYP2B6 isoform has the highest 4-hydroxylase activity. Cy undergoes activation to eventually form 
active metabolites, phosphoramide mustard and acrolein. 
 
As an alkylating agent, cyclophosphamide has three different independent effects: 1) attachment of alkyl 
groups to DNA bases, resulting in DNA being fragmented by repair enzymes as it attempts to replace the 
alkylated bases, preventing DNA synthesis and RNA transcription from the affected DNA; 2) DNA cross- 
linking which inhibits strand separation for replication and/or transcription; 3) induction of nucleotide 
mispairing. As a cytotoxic agent, cyclophosphamide is a key agent in combinations used in the treatment of 
breast cancer, lymphoma, leukemia, AL amyloidosis. 
 
Pioneering work by Dr. Robert North and Dr. Judah Folkmak clarified that low-dose metronomic 
cyclophosphamide had immunostimulatory and antiangiogenic properties, paving the way for combinations 
with vaccines and other agents. While it is unclear how convergent and/or overlapping both these effects are 
with low-dose metronomic cyclophosphamide, what is clear is that these effects are predicated upon the 
following observations: 
• 
Induction of a TH1/TH2 to TH17 skew in cytokine production;77,78 
• 
Depletion of CD4+ Foxp3+ regulatory T cells;79,80 
• 
Enhancement of long-term survival and proliferation of lymphocytes;81,82 
• 
Induction of pro-inflammatory cytokines including IL-1β, IL-7, IL-15, IL-2, IL-21, and IFN- 
γ;83 


20 
 
• 
Augmentation the function of dendritic cells (DC);84-89 and natural killer (NK) cell number 
and function DC function.79,90-92 
 
Low-dose cyclophosphamide has several immunomodulatory properties including: depletion of regulatory T 
cells;79,80 augmentation of DC84-89 and NK79,90-92 cell function. The properties result in TH17 skew in cytokine 
production;77,78 increased IFN production;79,90-92 and induction of immunogenic cell death. 79,90-92 Pre-clinically, 
low-dose cyclophosphamide diminished immunosuppressive TGF-β and IL-1093 and had synergy with intra- 
tumoral type B CpG SD-101 in injected and uninjected tumors.11,93 
 
3.5 Preclinical and Clinical Trial Data: Nivolumab 
 
Refer to the Nivolumab Investigator’s Brochure (IB) for Preclinical and Clinical data. 
 
3.6 Preclinical and Clinical Trial Data: Pixatimod (PG545) 
Refer to the Pixatimod IB for Preclinical and Clinical data. 
Preliminary safety and efficacy in advanced cancer patients 
In early clinical studies, pixatimod was administered subcutaneously (SC), although following local injection 
site reactions, this was switched to intravenous (IV) infusion.76 In the phase I dose-escalation study, escalating 
doses of pixatimod (25 mg, 50 mg, 100 mg, and 150 mg weekly) were administered IV in patients with 
advanced solid tumors.8 Three dose-limiting toxicities (DLTs) were identified in the 150 mg cohort (2 instances 
of hypertension and 1 of epistaxis); and no DLTs were noted in the 100 mg cohort, which was identified as 
the maximum-tolerated dose (MTD).8 Among 16 response-evaluable patients, no objective responses were 
reported; however, disease control rate (DCR) was 38% (6/16).8 Pharmacokinetic exposure was proportional 
up to 100 mg with a mean serum half-life of 141h supporting once weekly administration.8 Pharmacodynamic 
data revealed early increases in CD40+ pDC and NKp46+ NK cells; and increased concentration of IFNγ, 
TNF-α, CXCL10/IP-10 and MCP-1 in plasma.8 
 
Preliminary safety and efficacy in combination with anti-PD(L)1 in advanced cancer patients 
 
Pixatimod has been studied in combination with nivolumab in a phase IB study of advanced PD-1 naive solid 
tumors. In this study, patients with a variety of malignancies (pancreatic cancer, colorectal cancer, uterine 
adenosarcoma, squamous cell carcinoma, endometrial carcinoma, and adrenocortical carcinoma) were 
enrolled to receive escalating doses of pixatimod (25mg up to 50mg) IV weekly in combination with nivolumab 
240mg IV q2 weekly. 
 
A total of 58 patients were enrolled: 55 patients to 25 mg pixatimod/240 mg nivolumab and 3 patients to 50 
mg pixatimod/240 mg nivolumab. Forty-eight (48) subjects withdrew due to disease progression (either 
confirmed per RECIST or unconfirmed clinical progression), seven due to adverse events (two in the 50 mg 
and five in the 25 mg pixatimod cohort), and two withdrew consent Three DLTs occurred. Two DLTs occurred 
in the 50 mg pixatimod cohort: multiorgan failure (assessed as related to pixatimod and nivolumab), and 
pulmonary oedema (assessed as related to pixatimod only). The multiorgan failure resulted in the subject’s 
death. The 50 mg pixatimod/240 mg nivolumab dose was declared a toxic dose. One DLT occurred in the 
25mg cohort: pneumonitis (assessed as related to pixatimod and nivolumab). A summary of treatment- 
emergent adverse events (TEAEs) is presented in Table 3.6-1. Given that only three subjects were recruited 
to the 50 mg pixatimod/240 mg nivolumab, only the 25 mg pixatimod/240 mg nivolumab is presented. 


21 
 
 
Table 3.6-1: 
TEAE Events Possibly, Probably, or Likely related to Pixatimod or P ixatimod/Nivolumab – Occurring in 
Three or More Subjects (Safety Set)1 
Event Type 
25 mg/240 mg2 
N = 55 
General disorders and administration site conditions 
Fatigue 
Pyrexia 
Chills 
Gastrointestinal disorders 
Nausea 
Diarrhoea 
Metabolism and nutrition disorders 
Decreased appetite 
Investigations 
Alanine aminotransferase increased 
Aspartate aminotransferase increased 
Skin and subcutaneous tissue disorders 
Pruritus 
Respiratory, thoracic and mediastinal disorders 
Pneumonitis 
12 (21.8) [17] 
7 (12.7) [8] 
3 (5.5) [3] 
14 (25.5) [19] 
10 (18.2) [16] 
8 (14.5) [9] 
5 (9.1) [6] 
4 (7.3) [6] 
6 (10.9) [6] 
3 (5.5) [3] 
 
1 50 mg Pixatimod/240 mg Nivolumab data not presented as only three subjects were recruited. 
2 For any given adverse event, the results are presented as the number of patients with the event: n, the proportion of patients with 
the event: (%), and the number of events: [e]. 
 
Preliminary data from 26 evaluable patients were previously reported: 3 MSS mCRC patients had objective 
responses, 7 had SD with 12% ORR and 38% disease control rate (DCR).9 Based on this study, the 
recommended phase II dose of pixatimod in combination with anti-PD(L)1 blockade is 25 mg IV weekly. 
 
A summary of severe treatment related adverse drug reactions for both cohorts is presented in Table 3.6-2. 
 
Table 3.6-2: 
Severe Treatment-Emergent Adverse Events, Possibly, Probably, or Likely related 
to Study Drug by MedDRA SOC and Prefered Term (Safety Set) 
 
Event Type 
25 mg/240 mg1 
n (%) 
N = 55 
50 mg/240 mg1 
n (%) 
N = 3 
Causal Agent 
Pixatimod 
Pix/Nivo 
Pixatimod 
Pix/Nivo 
General disorders and administration site 
conditions 
Multiple organ dysfunction syndrome 
Fatigue 
0 (0%) [0] 
0 (0%) [0] 
0 (0%) [0] 
1 (33%) [1] 
0 (0%) [0] 
1 (2%) [1] 
0 (0%) [0] 
0 (0%) [0] 


22 
 
 
Gastrointestinal disorders 
Diarrhoea 
Colitis 
Metabolism and nutrition disorders 
Hyponatremia 
Investigations 
Aspartate aminotransferase increased 
Vascular disorders 
Hypertension 
Nervous system disorders 
Encephalopathy 
Respiratory, thoracic and mediastinal disorders 
Pneumonitis 
Pulmonary oedema 
Hepatobilliary disorders 
Autoimmune hepatitis 
Cardiac disorders 
Cardiomyopathy 
0 (0%) [0] 
2 (4%) [2] 
0 (0%) [0] 
0 (0%) [0] 
0 (0%) [0] 
1 (2%) [1] 
0 (0%) [0] 
0 (0%) [0] 
0 (0%) [0] 
1 (2%) [3] 
0 (0%) [0] 
0 (0%) [0] 
0 (0%) [0] 
1 (2%) [1] 
0 (0%) [0] 
0 (0%) [0] 
2 (4%) [6] 
0 (0%) [0] 
0 (0%) [0] 
0 (0%) [0] 
0 (0%) [0] 
1 (2%) [1] 
0 (0%) [0] 
0 (0%) [0] 
0 (0%) [0] 
1 (2%) [1] 
0 (0%) [0] 
0 (0%) [0] 
0 (0%) [0] 
0 (0%) [0] 
1 (33%) [1] 
0 (0%) [0] 
0 (0%) [0] 
1 (2%) [1] 
0 (0%) [0] 
1 (33%) [1] 
0 (0%) [0] 
0 (0%) [0] 
0 (0%) [0] 
1 (33%) [1] 
1 For any given adverse event, the results are presented as the number of patients with the event: n, the proportion of patients 
with the event: (%), and the number of events: [e]. 
 
Fifty-three (53) serious adverse events were reported in 30 subjects; 13 events were assessed as being possibly/probably, likely, 
or certainly related to the combination. Treatment-related serious adverse events comprised: multiorgan failure, pulmonary 
oedema, cardiomyopathy, and autoimmune hepatitis in the 50 mg pixatimod cohort; autoimmune hepatitis, pneumonitis (three 
events in two subjects), encephalopathy, diarrhea, fever (two events in one subject), and transaminitis in the 25 mg Pixatimod 
cohort. 
 
 
3.7 Preclinical and Clinical Trial Data: Cyclophosphamide 
 
Low-dose cyclophosphamide has several immunomodulatory properties including: depletion of regulatory T 
cells;79,80 augmentation of DC84-89 and NK79,90-92 cell function. The properties result in TH17 skew in cytokine 
production;77,78 increased IFN production;79,90-92 and induction of immunogenic cell death. 79,90-92 Pre-clinically, 
low-dose cyclophosphamide diminished immunosuppressive TGF-β and IL-1093 and had synergy with intra- 
tumoral type B CpG SD-101 in injected and uninjected tumors.11,93 
 
Clinically, low-dose (metronomic) cyclophosphamide has primarily been used in breast cancer, castrate- 
resistant prostate cancer, ovarian cancer and CRC at varying doses (50-100 mg daily) and schedules (daily, 
1-week-on, 1-week-off).94 Two single-arm studies studied cyclophosphamide and PD-1 blockade in sarcomas 
and reported limited activity with unusual toxicity signals.95,96 In these studies, Cy was dosed at 50 mg twice 
daily, 1-week-on, 1-week-off. 
 
3.8 Rationale 
 
3.8.1 Rationale for Combining Pixatimod (PG545) with Anti-PD(L)1 Antibody 
 
The activity of pixatimod is complementary to that of anti-PD(L)1 antibodies in the treatment of malignancies. 
Pixatimod has the dual functions of activating NK cells via DC,12 and inactivating tumor-infiltrating 


23 
 
macrophages by inhibiting heparanase.63,65 In a syngeneic breast cancer model, pixatimod in combination 
with anti-PD-1 improved anti-tumor control compared to anti-PD-1 antibody alone.97 Examination of the 
tumors from this study revealed that the combination significantly increased CD8 and CD4 effector memory 
T cells and NK cell infiltration into the tumors supporting the complementarity of these agents.97 Separately, 
a phase Ib study of the combination of pixatimod with nivolumab showed promising results in MSS mCRC, a 
cancer type that had previously demonstrated negligible response to PD-1 therapies.9 
 
3.8.2 Rationale for Limiting Enrollment by an Immune-inflammatory Biomarker Cutoff 
 
Immune-inflammatory biomarkers (IIBs) showed a prognostic relevance in patients with multiple cancers 
treated with immuno-oncologic agents. Several blood-based, easily obtained, IIBs have demonstrated 
prognostic relevance in the advanced setting in cancer patients across multiple histologies including: 
neutrophil-to-lymphocyte ratio (NLR),98-100 platelet and monocyte counts,101 and systemic immune- 
inflammation index (SII) (based on lymphocyte, neutrophil and platelet counts, but not monocytes).102,103 
 
The pan-immune-inflammation (PIV) is a weighted interaction of both the inflammatory pro-tumor (i.e. 
neutrophils, platelets and monocytes) and anti-tumor (i.e. lymphocytes) cellular populations and is calculated 
as follows: 
 
PIV = [neutrophil count (103/mmc) × platelet count (103/mmc) × monocyte count (103/mmc)]/lymphocyte count 
(103/mmc). 
 
PIV is a strong predictor of survival outcomes with better performance than other well-known immune 
inflammatory biomarkers in patients with 1L mCRC,104 melanoma.105 As such, based on literature data, the 
PIV cutoff utilized in this study is 1200 obtained at any time during Screening in the MSS mCRC cohort 
only (cohort 1). 
 
3.8.3 Rationale for Adding Low-dose Cy in MSS mCRC 
 
The addition of cyclophosphamide to the pixatimod-anti-PD(L)1 combination in MSS mCRC may provide 
benefit via two mechanisms. Firstly, Cy selectively targets suppressive T regs which may alleviate the 
immunosuppressive environment of the liver, the main site to which CRC metastases spread.106-108 Secondly, 
treatment with cyclophosphamide may destroy some tumor cells releasing damage-associated molecular 
patterns (DAMP) and cancer antigens that serve as activating ligands or targets for NK and T cells. Evidence 
in support of this mechanism was seen in a preclinical lymphoma study of the pixatimod/cyclophosphamide 
combination where cyclophosphamide alone led to a transient decrease in A20 tumors which would have 
resulted in the release of tumor associated DAMP and antigens.12 In the pixatimod/cyclophosphamide 
combination, there was complete tumor remission which further analysis indicated was caused by NK cells 
mobilized by DAMP-activated DC in a TLR9 dependent manner.12 
 
3.8.4 Rationale for Dose Selection: Nivolumab 
 
Refer to the eIB and the nivolumab SmPC or USPI for preclinical and clinical study data. 
 
Nivolumab 3 mg/kg has been approved as monotherapy in melanoma and NSCLC patients. Nivolumab 3 
mg/kg has been tested in MMR-deficient colorectal and non-colorectal cancers singly and in combination with 
low-dose ipilimumab.109-111 
 
In the current study, nivolumab will be administered at 480 mg Q4W in all 3 cohorts. The choice of 480mg 
Q4W as an appropriate dose for the switch to fixed dosing is based on simulations performed using the 
population PK model of nivolumab showing that the fixed dose of 240 mg every 2 weeks (and 480 mg Q4W) 
will provide exposures that 1) are optimally consistent with those obtained with the 2 mg/kg dose every 2 
weeks, 2) will maintain individual patient exposures in the exposure range established in melanoma as 


24 
 
associated with maximal efficacy response and 3) will maintain individual patients exposure in the exposure 
range established in melanoma that are well tolerated and safe.112,113 A fixed dose regimen will simplify the 
dosing regimen to be more convenient for physicians and to reduce potential for dosing errors. A fixed dosing 
scheme will also reduce complexity in the logistical chain at treatment facilities and reduce wastage. 
 
3.8.5 Rationale for Dose Selection: Pixatimod (PG545) 
 
The maximum tolerated dose for nivolumab/pixatimod in combination with nivolumab has previously been 
determined to be 25 mg weekly IV administered as an infusion over 1 hour.9 
 
3.8.6 Rationale for Dose Selection: Cyclophosphamide 
 
Rationale for low-dose cyclophosphamide in MSS mCRC is stated in Section 3.8.3. 
 
Low-dose cyclophosphamide is safe, and non-myelosuppressive with an immunomodulatory effect that is 
dependent upon the dose and duration of the drug-free interval.114-116 Given recent data showing rapid anti- 
tumor responses mediated by CD8+ T cells following treatment with a TLR9 agonist and low-dose Cy in a 
colorectal cancer model,11 a clinical investigation of nivolumab/pixatimod/cyclophosphamide is warranted in 
MSS mCRC. 
 
In this context, the ideal immunological dose and schedule of low-dose cyclophosphamide which shows 
sustained natural killer (NK) T cell function and induction of TA- specific memory CD8+ T cells is 50 mg twice 
daily, 1-week-on, 1-week-off.114-116 This dose has been explored in two studies (PEMBROSARC, and 
NCT02406781) in combination with PD-1 inhibitor pembrolizumab in patients with advanced osteosarcoma 
and soft tissue sarcomas.95,96 


25 
 
4. OBJECTIVES AND ENDPOINTS 
 
4.1 Primary Objective, Hypothesis, and Endpoint 
 
4.1.1 Primary Objective 
 
To establish the efficacy of nivolumab/pixatimod combination in PD-1 R/R melanoma and 
NSCLC; and the efficacy of nivolumab/pixatimod/cyclophosphamide combination in MSS 
mCRC. 
 
4.1.2 Primary Hypothesis 
 
That nivolumab/pixatimod combination produces radiographic responses in PD-1 R/R 
melanoma and NSCLC; and that the nivolumab/pixatimod/cyclophosphamide combination 
produces radiographic responses in MSS mCRC. 
 
4.1.3 Primary Endpoint 
 
ORR by RECIST v1.1 to nivolumab/pixatimod combination in PD-1 R/R melanoma and 
NSCLC; and to nivolumab/pixatimod/cyclophosphamide combination in MSS mCRC. 
 
4.2 Secondary Objectives, Endpoints and Hypotheses 
 
4.2.1 Secondary Objectives 
 
4.2.1.1 Secondary Objective 1 
 
To assess safety of nivolumab/pixatimod combination in PD-1 R/R melanoma and 
NSCLC; and nivolumab/pixatimod/cyclophosphamide combination in MSS mCRC 
patients 
 
4.2.1.2 Secondary Objective 2 
 
To assess efficacy using alternative response endpoints. 
 
4.2.1.3 Secondary Objective 3 
 
To assess CD8+ T cell and NK cell infiltrate in pre- and on- treatment tumor samples by 
multiplex IHC/IF. 
 
4.2.1.4 Secondary Objective 4 
 
To assess median and landmark progression-free survival (PFS) and landmark overall 
survival (OS) in participants treated with nivolumab/pixatimod or 
nivolumab/pixatimod/cyclophosphamide. 
 
4.2.2 Secondary Hypotheses 
 
4.2.2.1 Secondary Hypothesis 1 
 
That nivolumab/pixatimod combination augments inflammation by increasing CD8+ T cell 
and/or NK cell infiltrate in on-treatment compared to pre-treatment tumor samples. 


26 
 
4.2.2.2 Secondary Hypothesis 2 
 
That nivolumab/pixatimod combination produces radiographic responses in PD-1 R/R 
melanoma and NSCLC; and that the nivolumab/pixatimod/cyclophosphamide 
combination produces radiographic responses in MSS mCRC as assessed using 
alternative response endpoints. 
 
4.2.2.3 Secondary Hypothesis 3 
 
That nivolumab/pixatimod combination improves PFS and OS in PD-1 R/R melanoma 
and NSCLC and MSS mCRC. 
4.2.3 Secondary Endpoints 
 
4.2.3.1 Secondary Endpoint 1 
 
Incidence of all-grade related adverse events (AE), serious adverse events (SAE) and 
dose-limiting toxicities (DLT) if any in each cohort. 
 
4.2.3.2 Secondary Endpoint 2 
 
ORR by iRECIST to nivolumab/pixatimod combination in PD-1 R/R melanoma and 
NSCLC; and to nivolumab/pixatimod/cyclophosphamide combination in MSS mCRC. 
 
4.2.3.3 Secondary Endpoint 3 
 
Summary measures of change (or % change) from baseline in the CD8+ T cell infiltrate 
and/or NK cell in the tumor and TME in tumor biopsies at pre- and on- (approximately 4 
weeks) treatment timepoints. 
 
4.2.3.4 Secondary Endpoint 4 
 
Median PFS, landmark PFS (6 months, 1-year and 2-year), median OS and landmark OS 
(1-year, and 2-year) in participants treated with nivolumab/pixatimod or 
nivolumab/pixatimod/cyclophosphamide. 
 
4.3 Exploratory Objectives, Hypotheses, and Endpoints 
 
4.3.1 Exploratory Objectives 
 
4.3.1.1 Exploratory Objective 1 
 
To explore potential association between biomarkers and clinical efficacy of 
nivolumab/pixatimod or nivolumab/pixatimod/ cyclophosphamide combination. 
 
4.3.2 Exploratory Hypotheses 
 
4.3.2.1 Exploratory Hypothesis 1 
 
That responders to nivolumab/pixatimod combination demonstrate evidence of immune 
activation in tumor tissue, blood, and plasma by multiparameter flow cytometry, 
transcriptomic, genomic and cytokine analyses. 


27 
 
4.3.3 Exploratory Endpoints 
 
4.3.3.1 Exploratory Endpoint 1 
 
Correlation/measures of association of biomarkers and clinical effect of 
nivolumab/pixatimod or nivolumab/pixatimod/ cyclophosphamide combination. 


28 
 
5. STUDY DESIGN 
 
5.1 Study Design 
 
This is a phase II Simon two-stage study of nivolumab in combination with pixatimod in 3 separate cohorts: 
• 
Cohort 1: MSS mCRC in combination with low-dose cyclophosphamide 
• 
Cohort 2: PD-1 relapsed/refractory melanoma 
• 
Cohort 3: PD-1 relapsed/refractory NSCLC 
The study schema is outlined in Figure 5.1-1 below. 
We hypothesize that the nivolumab/pixatimod combination in PD-1 relapsed/refractory (R/R) cutaneous 
melanoma and NSCLC patients will be associated with anti-tumor effects. We also hypothesize that the 
nivolumab/pixatimod/cyclophosphamide combination in MSS mCRC patients will be associated with anti- 
tumor effect. We further hypothesize that nivolumab/pixatimod/cyclophosphamide combination in MSS mCRC 
patients and nivolumab/pixatimod combination in PD-1 R/R melanoma or NSCLC will be associated with 
increased CD8+ T cells intra-tumorally and PD-1+Ki67+ CD8+ T cells peripherally in responders on treatment 
compared to baseline; and that response will be associated with evidence of antigen-specific immunity 
responses intra-tumorally and peripherally. 
 
The goal of this study is to assess response of PD-1 R/R melanoma/NSCLC to nivolumab and pixatimod; and 
MSS mCRC to nivolumab, pixatimod and cyclophosphamide. 
 
Figure 5.1-1: Clinical Trial Schema 
 
5.2 Trial Procedures 
 
The Schedule of Activities in Section 2.0 (Schedule of Activities) summarizes the trial procedures to be 
performed at each visit. Individual trial procedures are described in detail below. It may be necessary to 
perform these procedures at unscheduled time points if deemed clinically necessary by the investigator. 
 
Furthermore, additional evaluations/testing may be deemed necessary by the Sponsor-Investigator and/or 


29 
 
Aculeus Therapeutics for reasons related to subject safety. In some cases, such evaluation/testing may be 
potentially sensitive in nature (e.g., HIV, Hepatitis C, etc.), and thus local regulations may require that additional 
informed consent be obtained from the subject. In these cases, such evaluations/testing will be performed in 
accordance with those regulations. 
 
5.3 Number of Participants 
 
In the 1st stage, 13 patients will be enrolled in cohort 1 while 9 patients each will be enrolled in cohorts 2 and 
3. Should pre-specified efficacy boundary as defined in Section 10.3 be met, further patients will be enrolled 
to one or more cohorts in 2nd stage. The total enrollment for 1st stage across all 3 cohorts is 31 response- 
evaluable patients. The total enrollment for both stages across all 3 cohorts is 61 response-evaluable patients. 


30 
 
6. STUDY POPULATION 
 
6.1 Inclusion Criteria 
 
6.1.1 General Inclusion Criteria 
 
• 
Male/female participants who are at least 18 years of age on the day of signing informed 
consent with advanced/metastatic cutaneous melanoma, NSCLC or MSS mCRC who 
meet the following criteria will be enrolled in this study. 
• 
Male participants: 
o A male participant must agree to use a contraception as detailed in Appendix 3 
of this protocol during the treatment period and for at least 120 days after the last 
dose of study treatment and refrain from donating sperm during this period. 
• 
Female participants: 
o A female participant is eligible to participate if she is not pregnant (see Appendix 
3), not breastfeeding, and at least one of the following conditions applies: 
o Not a woman of childbearing potential (WOCBP) as defined in Appendix 3;OR 
o A WOCBP who agrees to follow the contraceptive guidance in Appendix 3 during 
the treatment period and for at least 120 days after the last dose of study 
treatment. 
 
6.1.2 Cohort 1 (MSS mCRC) 
 
• 
MSS is defined as 0-1 allelic shifts among 3-5 tumor microsatellite loci using a PCR- 
based assay or immunohistochemistry. 
• 
Must have received prior therapy with a fluoropyrimidine, oxaliplatin, and irinotecan. 
• 
Prior treatment with an anti-PD-1/anti-PD-L1 or anti-CTLA-4 antibody is not allowed. 
• 
Prior treatment with BRAF/MEK inhibitor therapy (if BRAF mutated) and/or EGFR 
targeted antibody (if KRAS WT) are allowed. 
• 
No more than 5 prior lines of therapy for metastatic disease. 
• 
Adjuvant therapy will count as 1 prior line of therapy if received within the prior 6 months; 
but if not does not count towards prior line of therapy. 
 
• 
PIV cutoff of 1200 as defined in Section 3.8.2 obtained on labs obtained during 
Screening. 
6.1.3 Cohort 2 (PD-1 R/R melanoma) 
 
• 
PD-1 refractory disease as defined as progression on treatment with anti-PD-(L)1 
inhibitor administered either as monotherapy or in combination with other checkpoint 
inhibitors or other therapies. PD-1 treatment progression is defined by meeting all of the 
following criteria: 
o Receipt of at least 2 doses of an approved or investigational anti-PD-(L)1 inhibitor. 
o Demonstrated PD after anti-PD-(L)1 inhibitor as defined by RECIST v1.1. 
• 
Note: This determination is made by the investigator. Once PD is 
confirmed, the initial date of PD documentation will be considered the 
date of disease progression. 
• 
Note: The initial evidence of PD is to be confirmed by a second 
assessment no less than 4 weeks from the date of the first 
documented PD, in the absence of rapid clinical progression (as 
defined by the criteria in the sub-point below). 


31 
 
• 
Subjects who progressed on/within 3 months of adjuvant therapy with 
anti-PD-(L)1 inhibitor will be allowed; an adjuvant therapy will count as 
1 prior line of therapy if received within the prior 6 months. 
• 
Progressive disease should be documented at least 12 weeks from the 
last dose of anti-PD-(L)1 inhibitor. 
o Primary resistance: Drug exposure for at least 6 weeks with best response of 
PD, or SD for <6 months (including PD at any time after stopping anti-PD-(L)1 for 
any reason unrelated to toxicity if best response while receiving anti-PDx therapy 
was PD or SD <6 months). 
o Secondary resistance: Drug exposure for at least 6 months with best response 
of CR or PR or SD for ≥6 months. If prior anti-PD-(L)1 was stopped prior to PD, 
PD must have occurred at least 12 weeks after the last dose of anti-PD-(L)1. 
• 
Prior treatment with an anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) 
antibody is allowed but not required. 
• 
Prior treatment with BRAF/MEK inhibitor therapy (if BRAF mutated) is allowed but not 
required. 
• 
No more than 5 prior lines of therapy. 
 
6.1.4 Cohort 3 (PD-1 R/R NSCLC) 
 
• 
PD-1 refractory disease as defined as progression on treatment with anti-PD-(L)1 
inhibitor administered either as monotherapy or in combination with other checkpoint 
inhibitors or other therapies. PD-1 treatment progression is defined by meeting all of the 
following criteria: 
• 
Receipt of at least 2 doses of an approved or investigational anti-PD-(L)1 
inhibitor. 
• 
Demonstrated PD after anti-PD-(L)1 inhibitor as defined by RECIST v1.1. 
o Note: This determination is made by the investigator. Once PD is 
confirmed, the initial date of PD documentation will be considered the 
date of disease progression. 
o Note: The initial evidence of PD is to be confirmed by a second 
assessment no less than 4 weeks from the date of the first 
documented PD, in the absence of rapid clinical progression (as 
defined by the criteria in the sub-point below). 
o Subjects who progressed on/within 3 months of adjuvant therapy with 
anti-PD-(L)1 inhibitor will be allowed; an adjuvant therapy will count as 
1 prior line of therapy if received within the prior 6 months. 
o Progressive disease should be documented at least 12 weeks from the 
last dose of anti-PD-(L)1 inhibitor. 
• 
Primary resistance: Drug exposure for at least 6 weeks with best response 
of PD, or SD for <6 months (including PD at any time after stopping anti-PD- 
(L)1 for any reason unrelated to toxicity if best response while receiving anti- 
PDx therapy was PD or SD <6 months). 
• 
Secondary resistance: Drug exposure for at least 6 months with best 
response of CR or PR or SD for ≥6 months. If prior anti-PD-(L)1 was stopped 
prior to PD, PD must have occurred at least 12 weeks after the last dose of 
anti-PD-(L)1. 
• 
Prior treatment with an anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) 
antibody is allowed but not required. 
• 
Prior treatment with BRAF/MEK inhibitor therapy (if BRAF mutated) must have received 
and progressed or have demonstrable intolerance to approved targeted therapy. 


32 
 
• 
Patients with NSCLC with known oncogenic driver (including but not limited to EGFR, 
ALK, ROS, MET alterations) must have received and progressed past driver-specific 
therapy. 
• 
No more than 5 prior lines of therapy. 
 
6.1.5 Other Criteria 
 
• 
The participant (or legally acceptable representative if applicable) provides written 
informed consent for the trial. 
• 
Have measurable disease based on RECIST 1.1. Lesions situated in a previously 
irradiated area are considered measurable if progression has been demonstrated in such 
lesions. 
• 
Have provided newly obtained core or excisional biopsy of a tumor lesion not previously 
irradiated to undergo tumor biopsy (core, punch, incisional or excisional). 
o Biopsy must meet minimal sampling criteria as defined in Schedule of Events 
(Section 2.0). 
• 
Have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 to 1. 
Evaluation of ECOG is to be performed within 28 days prior to the date of enrollment. 
• 
Adequate organ function as defined in Table 6.1.5-1 below performed on screening labs 
obtained within 4 weeks of Cycle 1 day 1 
 
Table 6.1.5–1 Adequate Organ Function Laboratory Values 


33 
 
 
System 
Laboratory Value 
Hematological 
Absolute neutrophil count (ANC) 
≥1500/µL 
Platelets 
≥100 000/µL 
Hemoglobin 
≥9.0 g/dL or ≥5.6 mmol/La 
Renal 
Creatinine OR 
Measured or calculatedb creatinine clearance 
(GFR can also be used in place of creatinine or 
CrCl) 
≤1.5 × ULN OR 
≥30 mL/min for participant with creatinine levels 
>1.5 × institutional ULN 
Hepatic 
Total bilirubin 
≤1.5 ×ULN OR direct bilirubin ≤ULN for participants 
with total bilirubin levels >1.5 × ULN 
AST (SGOT) and ALT (SGPT) 
≤2.5 × ULN (≤5 × ULN for participants with liver 
metastases) 
Coagulation 
International normalized ratio (INR) OR prothrombin 
time (PT) 
Activated partial thromboplastin time (aPTT) 
≤1.5 × ULN unless participant is receiving 
anticoagulant therapy as long as PT or aPTT is 
within therapeutic range of intended use of 
anticoagulants 
ALT (SGPT)=alanine aminotransferase (serum glutamic pyruvic transaminase); AST (SGOT)=aspartate 
aminotransferase (serum glutamic oxaloacetic transaminase); GFR=glomerular filtration rate; ULN=upper limit 
of normal. 
a Criteria must be met without erythropoietin dependency and without packed red blood cell (pRBC) 
transfusion within last 2 weeks. 
b Creatinine clearance (CrCl) should be calculated per institutional standard. 
Note: This table includes eligibility-defining laboratory value requirements for treatment; laboratory value 
requirements should be adapted according to local regulations and guidelines for the administration of specific 
chemotherapies. 
 
• 
Proteinuria exceeding 1gram in a 24 hour period. 
• 
Female subject of childbearing potential should have a negative urine or serum 
pregnancy within 72 hours prior to receiving the first dose of study medication. If the urine 
test is positive or cannot be confirmed as negative, a serum pregnancy test will be 
required. 
• 
Female subjects of childbearing potential should be willing to use 2 methods of birth 
control or be surgically sterile or abstain from heterosexual activity for the course of the 
study through 120 days after the last dose of study medication (Section 5.7.2). Subjects 
of childbearing potential are those who have not been surgically sterilized or have not 
been free from menses for > 1 year. 
• 
Male subjects should agree to use an adequate method of contraception starting with the 
first dose of study therapy through 120 days after the last dose of study therapy. 
• 
Note: Abstinence is acceptable if this is the usual lifestyle and preferred contraception for 
the subject. 
 
6.2 Exclusion Criteria 
 
• 
Is currently participating in or has participated in a study of an investigational agent or using 
an investigational device within 4 weeks of the first dose of treatment. 


34 
 
• 
History of allergy and/or hypersensitivity and/or other clinically significant adverse drug 
reaction to heparin or other anti-coagulant agents, or to any monoclonal antibody. 
• 
Use of heparin (including low-molecular weight heparin and/or fondaparinux) within 2 
weeks prior to enrollment. 
o Patients who are currently receiving low-molecular weight heparin (or fondaparinux 
or other heparin product) for therapeutic anticoagulation may be enrolled if they 
have tested negative for anti-heparin antibodies at Screening. 
• 
Has a diagnosis of immunodeficiency, immunosuppression and/or is receiving systemic 
steroid therapy or any other form of immunosuppressive therapy within 7 days prior to the 
first dose of trial treatment. 
• 
Has had prior chemotherapy, targeted small molecule therapy, or radiation therapy within 
2 weeks prior to study Day 1 or who has not recovered (i.e., ≤ Grade 1 or at baseline) from 
AEs due to a previously administered agent. 
o Note: Subjects with ≤ Grade 2 neuropathy are an exception to this criterion 
and may qualify for the study. 
o Note: If subject received major surgery, they must have recovered adequately 
from the toxicity and/or complications from the intervention prior to starting 
therapy. 
o Note: Subjects with autoimmune disorders of Grade 4 while on prior 
immunotherapy will be excluded. Subjects who developed autoimmune 
disorders of Grade ≤ 3 may enroll if the disorder has resolved to Grade ≤1 and 
the subject has been off systemic steroids at doses >10 mg/d for at least 2 
weeks. 
• 
Active (i.e., symptomatic or growing) central nervous system (CNS) metastases. 
o Note: Subjects with treated and stable CNS metastases are permitted to enroll. 
Stability for prior treated CNS disease should be assessed on a contrast-enhanced 
imaging study obtained no sooner than 14 days from data of definitive radiotherapy 
and/or surgery for CNS disease. 
o Note: Subjects with leptomeningeal carcinomatosis are excluded. 
• 
Has a known additional malignancy that is progressing or requires active treatment. 
Exceptions include basal cell carcinoma of the skin, squamous cell carcinoma of the skin, 
or in situ cervical cancer that has undergone (or is being planned to undergo) potentially 
curative therapy. 
• 
Has a systemic disease that requires systemic pharmacologic doses of corticosteroids 
greater than 10 mg daily prednisone (or equivalent). 
o Note: Subjects who are currently receiving steroids at a dose of ≤10 mg daily do 
not need to discontinue steroids prior to enrollment. 
o Note: Subjects that require topical, ophthalmologic and inhalational steroids are not 
excluded from the study. 
o Note: Subjects with hypothyroidism stable on hormone replacement or Sjogren’s 
syndrome are not excluded from the study. 
o Note: Subjects who require active immunosuppression (greater than steroid dose 
discussed above) for any reason are excluded from the study. 
• 
Has evidence of interstitial lung disease or active, non-infectious pneumonitis. 
• 
Has an active infection requiring systemic therapy. 
• 
Has a history or current evidence of any condition, therapy, or laboratory abnormality that 
might confound the results of the trial, interfere with the subject’s participation for the full 
duration of the trial, or is not in the best interest of the subject to participate, in the opinion 
of the treating investigator. 
• 
Has known psychiatric or substance abuse disorders that would interfere with cooperation 
with the requirements of the trial. 


35 
 
• 
Is pregnant or breastfeeding or expecting to conceive or father children within the projected 
duration of the trial, starting with the pre-screening or screening visit through 26 weeks 
after the last dose of trial treatment. 
• 
Has a known history of Human Immunodeficiency Virus (HIV) (HIV 1/2 antibodies). 
o Note: Subjects with HIV that is well controlled (undetectable viral load and CD4 
count >200 cells/mm3) on anti-retroviral therapy are permitted to enroll. 
• 
Has known active hepatitis B (e.g., HBsAg reactive) or hepatitis C (e.g., HCV RNA 
[qualitative] is detected. 
o Note: Subjects with treated hepatitis B and/or C with no evidence of active infection 
may be enrolled. 
• 
Has a history of significant cardiac disease including but not limited to symptomatic 
congestive heart failure (New York Heart Association Class III/IV), uncontrolled 
hypertension (≥150/90mmHg) despite appropriate anti-hypertensive medication (patients 
with stably controlled hypertension are eligible), unstable angina pectoris or myocardial 
infarction (≤ 6 months prior to screening), uncontrolled cardiac arrhythmia, clinically 
significant cardiac valvopathy requiring treatment. 
• 
Receipt of live vaccine(s) within 30 days prior to the first dose of trial treatment. 
• 
Other uncontrolled intercurrent illness, including but not limited to, ongoing/active infection, 
active interstitial lung disease, serious chronic gastrointestinal conditions associated with 
diarrhea, or psychiatric illness/social situations that would limit compliance with study 
requirement, substantially increase risk of incurring AEs and/or compromise the ability of 
the patient to give written informed consent. 


36 
 
7. TRIAL TREATMENTS 
 
7.1 
Treatment Details 
 
Nivolumab monotherapy (2mg/kg every 3 weeks; 240 mg every 2 weeks; 480 mg every 4 weeks) is approved 
for the treatment of advanced melanoma regardless of BRAF mutation status. Pharmacokinetic analyses 
have established flat exposure-response relationships between these various doses and schedules;112,113 and 
in this study 480 mg q4 will be used. 
 
Pixatimod monotherapy has been demonstrated to be safe and well tolerated in patients with refractory solid 
tumors.8 In a phase Ib study, combination of pixatimod with nivolumab demonstrated promising results in 
uninflamed tumors including pancreatic cancer and microsatellite-stable colorectal carcinoma.9 Based on this 
study, the recommended phase II dose of pixatimod is 25 mg IV weekly. In this study, pixatimod will be dosed 
at 25 mg IV weekly. 
 
Low-dose Cy is safe, and non-myelosuppressive with an immunomodulatory effect that is dependent upon 
the dose and duration of the drug-free interval.114-116 In this context, the ideal immunological dose and 
schedule of low-dose Cy which shows sustained natural killer (NK) T cell function and induction of TA- specific 
memory CD8+ T cells is 50 mg twice daily, 1-week-on, 1-week-off.114-116 This dose has been explored in two 
studies (PEMBROSARC, and NCT02406781) in combination with PD-1 inhibitor pembrolizumab in patients 
with advanced osteosarcoma and soft tissue sarcomas.95,96 
 
In Cohort 1:  
• 
Nivolumab 480 mg Q4W 
• 
Pixatimod 25mg Q1W 
• 
Cyclophosphamide (50 mg twice daily, 1-week-on, 1-week-off). 
 
In Cohorts 2 and 3: 
• 
Nivolumab 480 mg Q4W 
• 
Pixatimod 25 mg Q1W. 
 
7.2 Nivolumab Dose Selection, Modification, Timing, and Duration 
 
7.2.1 Nivolumab dose selection 
 
Rationale for nivolumab dose selection is provided in Section 3.8.3 - Rationale for Dose Selection: 
Nivolumab. 
 
7.2.2 Nivolumab dose modification 
 
No nivolumab dose modification is allowed. 
 
Nivolumab will be withheld for drug-related Grade 4 hematologic toxicities, non-hematological toxicity ≥ 
Grade 3 including laboratory abnormalities, and severe or life-threatening AEs as per Table 7.2.2-1 below. 
 
Table 7.2.2-1: Dose Modification Guidelines for Drug-related Adverse Events (AEs) 
 
Toxicity 
Hold 
Treatment 
For Grade 
Timing for Restarting Treatment 
Treatment Discontinuation 
Diarrhea/Colitis 
2-3 
Toxicity resolves to Grade 0-1 
Toxicity does not resolve within 12 weeks of last 
dose or inability to reduce corticosteroid to 10 mg or 


37 
 
 
Toxicity 
Hold 
Treatment 
For Grade 
Timing for Restarting Treatment 
Treatment Discontinuation 
less of prednisone or equivalent per day within 12 
weeks 
4 
Permanently discontinue 
Permanently discontinue 
AST, ALT, or 
Increased Bilirubin 
2 
Toxicity resolves to Grade 0-1 
Toxicity does not resolve within 12 weeks of last 
dose 
3-4 
Permanently discontinue 
(see exception below)a 
Permanently discontinue 
Type 1 diabetes 
mellitus (if new 
onset) or 
Hyperglycemia 
T1DM or 
3-4 
Hold nivolumab and pixatimod for new 
onset Type 1 diabetes mellitus or Grade 
3-4 hyperglycemia associated with 
evidence of beta cell failure 
Resume nivolumab when patients are clinically and 
metabolically stable 
Hypophysitis 
2-4 
Toxicity resolves to Grade 0-1. Therapy 
with nivolumab and pixatimod can be 
continued while endocrine replacement 
therapy is instituted 
Toxicity does not resolve within 12 weeks of last 
dose or inability to reduce corticosteroid to 10 mg or 
less of prednisone or equivalent per day within 12 
weeks 
Hyperthyroidism 
3 
Toxicity resolves to Grade 0-1 
Toxicity does not resolve within 12 weeks of last 
dose or inability to reduce corticosteroid to 10 mg or 
less of prednisone or equivalent per day within 12 
weeks 
4 
Permanently discontinue 
Permanently discontinue 
Hypothyroidism 
Therapy with nivolumab and pixatimod 
can be continued while thyroid 
replacement therapy is instituted 
Therapy with nivolumab can be continued while 
thyroid replacement therapy is instituted 
Infusion Reaction 
2b 
Toxicity resolves to Grade 0-1 
Permanently discontinue if toxicity develops despite 
adequate premedication 
3-4 
Permanently discontinue 
Permanently discontinue 
Pneumonitis 
2 
Toxicity resolves to Grade 0-1 
Toxicity does not resolve within 12 weeks of last 
dose or inability to reduce corticosteroid to 10 mg or 
less of prednisone or equivalent per day within 12 
weeks 
Permanently discontinue for recurrent Grade 2 
pneumonitis 
3-4 
Permanently discontinue 
Permanently discontinue 
Renal Failure or 
Nephritis 
2 
Toxicity resolves to Grade 0-1 
Toxicity does not resolve within 12 weeks of last 
dose or inability to reduce corticosteroid to 10 mg or 
less of prednisone or equivalent per day within 12 
weeks 
3-4 
Permanently discontinue 
Permanently discontinue 
All Other Drug- 
Related Toxicityc 
3 or Severe 
Toxicity resolves to Grade 0-1 
Toxicity does not resolve within 12 weeks of last 
dose or inability to reduce corticosteroid to 10 mg or 
less of prednisone or equivalent per day within 12 
weeks. Exceptions may be sought for neuropathy or 
other AE following discussion with Principle 
Investigator. 
4 
Permanently discontinue 
Permanently discontinue 
Note: Permanently discontinue for any severe or Grade 3 drug-related AE that recurs or any life-threatening event. 
a For patients with liver metastasis who begin treatment with Grade 2 AST or ALT, if AST or ALT increases by greater than or equal to 
50% relative to baseline and lasts for at least 1 week then patients should be discontinued. 
b If symptoms resolve within one hour of stopping drug infusion, the infusion may be restarted at 50% of the original infusion rate 
(e.g., from 100 mL/h to 50 mL/h). Otherwise dosing will be held until symptoms resolve and the subject should be premedicated for 
the next scheduled dose and the infusion rate reduced by 50% for next scheduled dose. Infusion rate may be subsequently 
increased at investiagtor discretion. 
c Patients with intolerable or persistent Grade 2 drug-related AE may hold study medication at physician discretion. Permanently 
discontinue study drug for persistent Grade 2 adverse reactions for which treatment with study drug has been held, that do not 
recover to Grade 0-1 within 12 weeks of the last dose. 


38 
 
In case toxicity does not resolve to Grade 0-1 within 12 weeks after last infusion, trial treatment should be 
discontinued after consultation with the Sponsor-Investigator. With Sponsor-Investigator’s agreement, 
subjects with a laboratory AE still at Grade 2 after 12 weeks may continue treatment in the trial only if 
asymptomatic and controlled. For information on the management of AEs, see Section 7.7.1. 
 
Subjects who experience a recurrence of the same severe or life-threatening event at the same grade or 
greater with re-challenge of nivolumab should be discontinued from trial treatment. 
 
7.2.3 Nivolumab timing of administration 
 
Trial treatment should be administered after all procedures/assessments have been completed as detailed 
on the Schedule of Activities in Section 2.0. Trial treatment may be administered up to ± 2 days before or 
after the scheduled Day 1 due to administrative reasons. 
 
All trial treatments will be administered on an outpatient basis. 
 
Nivolumab will be administered as a 30-minute IV infusion (treatment intervals may be increased due to 
toxicity as described). Sites should make every effort to target infusion timing to be as close to 30 minutes as 
possible. However, given the variability of infusion pumps from site to site, a window of -5 minutes and +10 
minutes is permitted (i.e., infusion time is 30 minutes: -5 min/+10 min). 
 
7.2.4 Nivolumab duration of administration 
 
In all 3 cohorts, nivolumab will be administered at 480 mg every 4 weeks. Nivolumab 480 mg Q4 will be 
administered till progression and/or unacceptable toxicity. 
 
 
7.3 Pixatimod (PG545) Dose Selection, Modification, Timing and Duration 
 
7.3.1 Pixatimod (PG545) dose selection 
 
Rationale for pixatimod dose selection is provided in Section 3.8.4 - Rationale for Dose Selection: 
Pixatimod (PG545). 
 
7.3.2 Pixatimod (PG545) dose modification 
 
No pixatimod dose modification is allowed. 
 
Pixatimod will be withheld for drug-related Grade 4 hematologic toxicities, non-hematological toxicity ≥ Grade 
3 including laboratory abnormalities (but excluding Grade 3 lipid elevations), and severe or life-threatening 
AEs. 
 
Subjects who experience a recurrence of the same severe or life-threatening event at the same grade or 
greater with re-challenge of pixatimod will have this discontinued. If pixatimod is permanently discontinued 
for, subjects may continue to receive nivolumab. 
 
7.3.3 Pixatimod (PG545) prophylaxis 
 
In the initial 5 patients treated in this study, patients were premedicated with acetaminophen 1000mg prior to 
pixatimod infusion to prophylax against pixatimod-induced cytokine release. However, given the low incidence 
of cytokine-release observed, observed negative impact on vaccination response, and possible deleterious 
effects upon immune therapy observed in retrospective analyses117,118, this was discontinued with protocol 
v6. 


39 
 
7.3.4 Pixatimod (PG545) timing of administration 
 
Trial treatment should be administered after all procedures/assessments have been completed as detailed 
on the trial Schedule of Activities in Section 2.0. Trial treatment may be administered up to ± 2 days before 
or after the scheduled Day 1 due to administrative reasons. 
 
All trial treatments will be administered on an outpatient basis. 
 
Pixatimod will be administered as a 60-minute IV infusion (treatment intervals may be increased due to toxicity 
as described). Sites should make every effort to target infusion timing to be as close to 60 minutes as possible. 
However, given the variability of infusion pumps from site to site, a window of -5 minutes and +10 minutes is 
permitted (i.e., infusion time is 30 minutes: -5 min/+10 min). 
 
Pixatimod will be administered after nivolumab. 
 
7.3.5 Pixatimod (PG545) duration of administration 
 
In all 3 cohorts, pixatimod will be administered at 25 mg Q1W. Pixatimod 25 mg Q1W will be administered till 
progression and/or unacceptable toxicity. 
 
7.4 Cy Dose Selection, Modification, Timing and Duration 
 
7.4.1 Cyclophosphamide dose selection 
 
Rationale for pixatimod dose selection is provided in Section 3.8.2. - Rationale for Adding Low-dose Cy 
in MSSm CRC. 
 
7.4.2 Cyclophosphamide dose modification 
 
Cyclophosphamide will be withheld for drug-related Grade 4 hematologic toxicities, non-hematological toxicity 
≥ Grade 3 including laboratory abnormalities, and severe or life-threatening AEs. 
 
Further, in Cohort 1, continuous toxicity monitoring will be done as delineated in Section 9.4. In this context, 
cyclophosphamide dose modification may be permitted for recurrent drug-related G4 hematologic 
 
Subjects who experience a recurrence of the same severe or life-threatening event at the same grade or 
greater with re-challenge of cyclophosphamide will have this discontinued. If cyclophosphamide is 
permanently discontinued for, subjects may continue to receive nivolumab and pixatimod. 
 
7.4.3 Cy timing of administration 
 
Trial treatment should be administered after all procedures/assessments have been completed as detailed 
on the trial Schedule of Activities in Section 2.0. Trial treatment may be administered up to ± 2 days before 
or after the scheduled Day 1 due to administrative reasons. 
 
All trial treatments will be administered on an outpatient basis. 
 
In Cohort 1 only, cyclophosphamide will be administered orally at 50 mg twice daily on D1-D7, and D15-D21 
of a 28-day cycle. Pill compliance will be assessed using a pill diary. 


40 
 
7.4.4 Cy duration of administration 
 
In Cohort 1 only, cyclophosphamide will be administered orally at 50 mg twice daily on D1-D7, and D15- 
D21 of a 28-day cycle. Pill compliance will be assessed using a pill diary. cyclophosphamide will be 
administered till progression and/or unacceptable toxicity. 
 
7.5 Treatment Allocation 
 
Following Enrollment and completion of Screening, patients will enter the Treatment phase. 
 
7.6 Concomitant Medications 
 
Medications or vaccinations specifically prohibited in the exclusion criteria and outlined in Section 7.8.2 are 
not allowed during the ongoing trial. If there is a clinical indication for one of these or other medications or 
vaccinations specifically prohibited during the trial, discontinuation from trial therapy or vaccination may be 
required. The treating Investigator should discuss any questions regarding this with the Principal Investigator. 
The final decision on any supportive therapy or vaccination rests with the investigator and/or the subject's 
treating Investigator. However, the decision to continue the subject on trial therapy or vaccination schedule 
requires the mutual agreement of the treating Investigator, the Principal Investigator, and the subject. 
 
7.6.1 Acceptable Concomitant Medications 
 
All treatments that the investigator considers necessary for a subject’s welfare may be administered at the 
discretion of the investigator in keeping with the community standards of medical care. All concomitant 
medication will be recorded in the research record including all prescription, over-the-counter (OTC), herbal 
supplements, and IV medications and fluids. If changes occur during the trial period, documentation of drug 
dosage, frequency, route, and date may also be included in the research record. 
 
All concomitant medications received within 28 days before the first dose of trial treatment and 30 days after 
the last dose of trial treatment should be recorded. Concomitant medications administered after 30 days after 
the last dose of trial treatment should be recorded for SAEs and ECIs as defined in Section 9.2. 
 
7.6.2 Prohibited Concomitant Medications 
 
Subjects are prohibited from receiving the following therapies during the Screening and Treatment Phase 
(including retreatment for post-complete response relapse) of this trial: 
• 
Anti-cancer systemic chemotherapy or biological therapy; 
• 
Immunotherapy not specified in this protocol; 
• 
Chemotherapy not specified in this protocol; 
• 
Investigational agents other than pixatimod; 
• 
Radiation therapy. 
 
Live vaccines within 30 days prior to the first dose of trial treatment and while participating in the trial. 
Examples of live vaccines include, but are not limited to, the following: measles, mumps, rubella, chicken pox, 
yellow fever, rabies, BCG, and typhoid (oral) vaccine. Seasonal influenza vaccines for injection are generally 
killed virus vaccines and are allowed; however intranasal influenza vaccines (e.g. Flu-Mist®) are live 
attenuated vaccines and are not allowed. 
 
Glucocorticoids for any purpose other than to modulate symptoms from an event of clinical interest of 
suspected immunologic etiology. The use of physiologic doses of corticosteroids may be approved after 
consultation with the Principal Investigator. 


41 
 
 
Subjects who, in the assessment by the investigator, require the use of any of the aforementioned treatments 
for clinical management should be removed from the trial. Subjects may receive other medications that the 
investigator deems to be medically necessary. However, if a patient who is otherwise deriving benefit as 
assessed by the treating investigator may be permitted to receive radiation therapy to a non-target lesion for 
the purposes of symptom control. 
 
The Exclusion Criteria (Section 6.2) describes other medications which are prohibited in this trial. 
There are no prohibited therapies during the Post-Treatment Follow-up Phase (Section 8.5.5). 
Agents known to have TLR9 antagonist activity are prohibited throughout the study. Known antagonists 
include chloroquine, hydroxychloroquine, and quinacrine. 
 
Medications intended solely for supportive care (i.e., antiemetics, analgesics, megestrol acetate for anorexia) 
are allowed. 
 
7.7 Rescue Medications and Supportive Care 
 
7.7.1 Supportive Care Guidelines 
 
Subjects should receive appropriate supportive care measures as deemed necessary by the treating 
investigator. Suggested supportive care measures for the management of AEs with potential immunologic 
etiology are outlined below. Where appropriate, these guidelines include the use of oral or intravenous 
treatment with corticosteroids as well as additional anti-inflammatory agents if symptoms do not improve with 
administration of corticosteroids. Note that several courses of steroid tapering may be necessary as 
symptoms may worsen when the steroid dose is decreased. For each disorder, attempts should be made to 
rule out other causes such as metastatic disease or bacterial or viral infection, which might require additional 
supportive care. The treatment guidelines are intended to be applied when the investigator determines the 
events to be related to nivolumab. 
 
Note: If after the evaluation the event is determined not to be related, the investigator does not need to follow 
the treatment guidance (as outlined below). Refer to Sections 7.2-7.4 for dose modification guidelines for 
nivolumab, pixatimod and cyclophosphamide. 
 
It may be necessary to perform conditional procedures such as bronchoscopy, endoscopy, or skin 
photography as part of evaluation of the event. 
 
• 
Pneumonitis 
 
o For Grade 2 events, treat with systemic corticosteroids. When symptoms improve to Grade 1 or 
less, steroid taper should be started and continued over no less than 4 weeks. 
o For Grade 3-4 events, immediately treat with intravenous steroids. Administer additional anti- 
inflammatory measures, as needed. 
o Add prophylactic antibiotics for opportunistic infections in the case of prolonged steroid 
administration. 
• 
Diarrhea/Colitis 
 
o Subjects should be carefully monitored for signs and symptoms of enterocolitis (such as diarrhea, 
abdominal pain, blood or mucus in stool, with or without fever) and of bowel perforation (such as 
peritoneal signs and ileus). 


42 
 
o All subjects who experience diarrhea/colitis should be advised to drink liberal quantities of clear 
fluids. If sufficient oral fluid intake is not feasible, fluid and electrolytes should be substituted via 
IV infusion. For Grade 2 or higher diarrhea, consider GI consultation and endoscopy to confirm or 
rule out colitis. 
o For Grade 2 diarrhea/colitis, administer oral corticosteroids. 
o For Grade 3 or 4 diarrhea/colitis, treat with intravenous steroids followed by high dose oral 
steroids. 
o When symptoms improve to Grade 1 or less, steroid taper should be started and continued over 
no less than 4 weeks. 
• 
Type 1 diabetes mellitus (if new onset, including diabetic ketoacidosis [DKA]) or ≥ Grade 3 
Hyperglycemia, if associated with ketosis (ketonuria) or metabolic acidosis (DKA) 
o For T1DM or Grade 3-4 Hyperglycemia 
 
Insulin replacement therapy is recommended for Type I diabetes mellitus and for Grade 3-4 
hyperglycemia associated with metabolic acidosis or ketonuria. 
 
Evaluate patients with serum glucose and a metabolic panel, urine ketones, glycosylated 
hemoglobin, and C-peptide. 
• 
Hypophysitis 
 
o For Grade 2 events, treat with corticosteroids. When symptoms improve to Grade 1 or less, steroid 
taper should be started and continued over no less than 4 weeks. Replacement of appropriate 
hormones may be required as the steroid dose is tapered. 
o For Grade 3-4 events, treat with an initial dose of IV corticosteroids followed by oral corticosteroids. 
When symptoms improve to Grade 1 or less, steroid taper should be started and continued over 
no less than 4 weeks. Replacement of appropriate hormones may be required as the steroid dose 
is tapered. 
• 
Hyperthyroidism or Hypothyroidism 
 
o Thyroid disorders can occur at any time during treatment. Monitor patients for changes in thyroid 
function (at the start of treatment, periodically during treatment, and as indicated based on clinical 
evaluation) and for clinical signs and symptoms of thyroid disorders. 
 
o Grade 2 hyperthyroidism events (and Grade 2-4 hypothyroidism): 
 
In hyperthyroidism, non-selective beta-blockers (e.g. propranolol) are suggested as initial 
therapy. 
 
In hypothyroidism, thyroid hormone replacement therapy, with levothyroxine or liothyroinine, is 
indicated per standard of care. 
o Grade 3-4 hyperthyroidism 
 
Treat with an initial dose of IV corticosteroid followed by oral corticosteroids. When symptoms 
improve to Grade 1 or less, steroid taper should be started and continued over no less than 4 
weeks. Replacement of appropriate hormones may be required as the steroid dose is tapered. 
• 
Hepatic 
 
o For Grade 2 events, monitor liver function tests more frequently until returned to baseline values 
(consider weekly). 
 
Treat with IV or oral corticosteroids 


43 
 
o For Grade 3-4 events, treat with intravenous corticosteroids for 24 to 48 hours. 
o When symptoms improve to Grade 1 or less, a steroid taper should be started and continued over 
no less than 4 weeks. 
• 
Nephritis 
 
o For Grade 2 events, treat with corticosteroids. 
o For Grade 3-4 events, treat with systemic corticosteroids. 
o When symptoms improve to Grade 1 or less, steroid taper should be started and continued over 
no less than 4 weeks. 
• 
Management of infusion reactions 
 
o Signs and symptoms usually develop during or shortly after drug infusion and generally resolve 
completely within 24 hours of completion of infusion. 
Table 7.7.1-1 below shows treatment guidelines for subjects who experience an infusion reaction 
associated with administration of nivolumab. 
 
Table 7.7.1-1: Infusion Reaction Management Guidelines 
NCI CTCAE Grade 
Treatment 
Premedication at Subsequent 
Dosing 
Grade 1 
Mild reaction: infusion interruption 
not indicated, intervention not 
indicated 
Increase monitoring of vital signs as medically 
indicated until the subject is deemed medically 
stable in the opinion of the investigator. 
None 
Grade 2 
Requires infusion interruption but 
responds promptly to 
symptomatic treatment (e.g., 
antihistamines, NSAIDS, 
narcotics, IV fluids); prophylactic 
medications indicated for ≤24 h 
Stop Infusion and monitor symptoms. 
Additional appropriate medical therapy may 
include but is not limited to: 
IV fluids 
Antihistamines 
NSAIDS 
Acetaminophen 
Narcotics 
Increase monitoring of vital signs as medically 
indicated until the subject is deemed medically 
stable in the opinion of the investigator. 
If symptoms resolve within one hour of 
stopping drug infusion, the infusion may be 
restarted at 50% of the original infusion rate 
(e.g., from 100 mL/h to 50 mL/h). Otherwise 
dosing will be held until symptoms resolve and 
the subject should be premedicated for the 
next scheduled dose. 
Subjects who develop Grade 2 toxicity despite 
adequate premedication should be 
permanently discontinued from further trial 
treatment administration. 
Subject may be premedicated 1.5 h 
(± 30 min) prior to infusion of 
nivolumab with: 
Diphenhydramine 50 mg po (or 
equivalent dose of antihistamine). 
Acetaminophen 500–1000 mg po 
(or equivalent dose of antipyretic). 
Grades 3 or 4 
Grade 3: 
Prolonged (i.e., not rapidly 
responsive to symptomatic 
medication and/or brief 
interruption of infusion); 
recurrence of symptoms following 
initial improvement; 
hospitalization indicated for other 
clinical sequelae (e.g., renal 
impairment, pulmonary infiltrates) 
Grade 4: 
Stop Infusion. 
Additional appropriate medical therapy may 
include but is not limited to: 
IV fluids 
Antihistamines 
NSAIDS 
Acetaminophen 
Narcotics 
Oxygen 
Pressors 
Corticosteroids 
Epinephrine 
No subsequent dosing 


44 
 
 
Life-threatening; pressor or 
ventilatory support indicated 
Increase monitoring of vital signs as medically 
indicated until the subject is deemed medically 
stable in the opinion of the investigator. 
Hospitalization may be indicated. 
Subject is permanently discontinued from 
further trial treatment administration. 
Appropriate resuscitation equipment should be available in the room and a physician readily available during the period of 
drug administration. 
 
7.8 Subject Withdrawal/Discontinuation Criteria 
 
Subjects are free to withdraw from the study at any time and without penalty or loss of future medical care, or 
any other benefits to which they are otherwise entitled. Subjects may discontinue study medication for any of 
the following conditions: 
• 
Dose-limiting or other unacceptable toxicity considered to be related to either study medication; 
o Patients who develop unacceptable toxicity deemed related to pixatimod but not nivolumab 
may continue nivolumab after pixatimod discontinuation for duration as defined in Section 
7.3.2 if the patients have ongoing benefit in the assessment of treating investigator after 
consultation with Sponsor-Investigator. 
o Patients who develop unacceptable toxicity deemed related to cyclophosphamide but not 
pixatimod or nivolumab may continue pixatimod and nivolumab after cyclophosphamide 
discontinuation for duration as defined in Section 7.4.2. 
o Patients who develop unacceptable toxicity deemed related to nivolumab but not pixatimod 
must discontinue both pixatimod and nivolumab (and if appropriate, cyclophosphamide). 
• 
PD by RECIST (Version 1.1) if accompanied by medically significant clinical deterioration, in the 
judgment of the Investigator. (Continuation of treatment through suspected pseudo-progression is 
permitted.); 
• 
If, in the opinion of the Investigator, it is medically necessary; 
• 
Subject withdraws consent for the study (note that subjects who withdraw consent for additional study 
treatment and procedures will continue to be followed for long-term 
• 
survival unless they explicitly withdraw consent for any follow-up); 
• 
Subject develops an intercurrent illness or AE that precludes further participation, or requires a 
prohibited concomitant treatment; 
• 
Subject becomes pregnant or begins breastfeeding; 
• 
Subject is lost to follow-up. 
 
Patients discontinuing study medication earlier than planned or withdrawing from the study should undergo 
the subsequent End of Treatment (EOT) clinical and laboratory assessments as soon as possible after study 
medication is stopped and the requisite safety follow-up period of 30 days should also be followed. The reason 
for withdrawal will be recorded in the electronic case report form (eCRF) and the subject’s source medical 
record. All subjects will continue to be followed every three months after the last nivolumab/pixatimod 
treatment for long-term survival follow-up until death, loss to follow-up, or withdrawal of consent for follow-up. 
 
7.9 Subject Replacement Strategy 
 
Patients who receive at least 1 dose each of nivolumab and pixatimod (cohorts 2 and 3); and at least 1 dose 
each of nivolumab, pixatimod and low-dose cyclophosphamide (cohort 1) are evaluable for safety. 
 
All patients who met the eligibility criteria; received at least 1 dose each of nivolumab and pixatimod (cohorts 
2 and 3); and at least 1 dose each of nivolumab, pixatimod and low-dose cyclophosphamide (cohort 1); and 
underwent restaging imaging are evaluable for response. 


45 
 
Patients who did not undergo restaging scans for reasons other than disease progression (treatment 
discontinuation, trial withdrawal etc.) will be deemed non-evaluable. Non-evaluable subjects may be replaced. 
 
If in the opinion of the treating investigator and Principal Investigator, the subject’s disease is rapidly 
progressing, restaging scans may be expedited. These patients are evaluable for objective response rate and 
other efficacy endpoints. 
 
7.10 Clinical Criteria for Early Trial Termination 
 
Early trial termination will be the result of the criteria specified below: 
• 
Quality or quantity of data recording is inaccurate or incomplete. 
• 
Poor adherence to protocol and regulatory requirements. 
• 
Incidence or severity of adverse drug reaction in this or other studies indicates a potential health 
hazard to subjects. 
• 
Plans to modify or discontinue the development of the study drugs. In the event of an Aculeus 
Therapeutics decision to no longer supply study drug(s), ample notification will be provided so that 
appropriate adjustments to subject treatment can be made. 


46 
 
8. STUDY ASSESSMENTS AND PROCEDURES 
 
8.1 Trial Procedures 
 
The trial Schedule of Activities in Section 2.0 summarizes the trial procedures to be performed at each visit. 
Individual trial procedures are described in detail below. It may be necessary to perform these procedures at 
unscheduled time points if deemed clinically necessary by the investigator. 
 
Furthermore, additional evaluations/testing may be deemed necessary by the Sponsor-Investigator and/or 
Zuecro for reasons related to subject safety. In some cases, such evaluation/testing may be potentially 
sensitive in nature (e.g., HIV, Hepatitis C, etc.), and thus local regulations may require that additional informed 
consent be obtained from the subject. In these cases, such evaluations/testing will be performed in 
accordance with those regulations. 
 
8.2 Treatment Period 
 
Following Screening, during the Treatment Period, patients will receive treatment delineated below. Each 
cycle is 28 days long. In each cycle, patients will receive pixatimod, nivolumab ± cyclophosphamide (in Cohort 
1). 
 
Cohort 1 (MSS mCRC): 
• 
IV nivolumab 480 mg Q4W 
• 
IV pixatimod 25 mg Q1W 
• 
PO cyclophosphamide at 50 mg twice daily on D1-D7, and D15-D21 of a 28-day cycle 
 
Cohort 2 (PD-1 R/R melanoma): 
• 
IV nivolumab 480 mg Q4W 
• 
IV pixatimod 25 mg Q1W 
 
Cohort 3 (PD-1 R/R NSCLC): 
• 
IV nivolumab 480 mg Q4W 
• 
IV pixatimod 25 mg Q1W 
 
8.3 Administrative Procedures 
 
Informed consent must be documented by the subject’s dated signature or by the subject’s legally acceptable 
representative’s dated signature on a consent form along with the dated signature of the person conducting 
the consent discussion prior to enrollment. 
 
During initial enrollment visit, study investigator (or designee) must discuss the following with patients: 
• 
Inclusion and exclusion criteria 
 
• 
Medical history – pertinently any condition diagnosed within the prior 10 years that are considered to 
be clinically significant by the Investigator. 
 
• 
Prior and concomitant medications. All medications related to reportable SAEs and ECIs should be 
recorded as defined in Section 8.6. 
• 
Disease details and treatments. 


47 
 
8.4 Clinical Procedures and Assessments 
 
8.4.1 Adverse Events (AE) monitoring 
 
The investigator or qualified designee will assess each subject to evaluate for potential new or worsening AEs 
as specified in the trial Schedule of Activities (Section 2.0) and more frequently if clinically indicated. 
Adverse experiences will be graded and recorded throughout the study and during the follow-up period 
according to NCI CTCAE Version 5.0 (see Section 8.6). Toxicities will be characterized in terms regarding 
seriousness,  causality,  toxicity  grading,  and  action  taken  with  regards  to  trial  treatment. 
 
All AEs of unknown etiology associated with nivolumab exposure should be evaluated to determine if it is 
possibly an event of clinical interest (ECI) of a potentially immunologic etiology (irAE). See Section 7.7.1 
regarding the identification, evaluation and management of AEs of a potential immunological etiology. 
 
Related and clinically significant AEs occurring within 30 days after the last dose of study drug should be 
recorded. Patients who are discontinued from the study due to an unacceptable drug-related AE will be 
followed until the resolution of the AE to Grade 0-1 or stabilization or until initiation of a new therapy for their 
cancer, whichever occurs first. Related SAEs that occur within 90 days of the end of treatment or before 
initiation of a new antineoplastic treatment should also be followed and recorded. 
 
Please refer to Section 8.6 for detailed information regarding the assessment and recording of AEs. 
 
8.4.2 Full physical exam 
 
The Investigator or qualified designee will perform a complete physical exam during Screening and as 
otherwise indicated in Schedule of Activities (Section 2.0). Clinically significant abnormal findings should 
be recorded as medical history. 
 
8.4.3 Vital signs 
 
The Investigator or qualified designee will obtain vital signs during Screening and as otherwise indicated in 
trial Schedule of Activities (Section 2.0). Vital signs should include temperature, pulse, respiratory rate, 
weight and blood pressure. Height will be measured at Screening only. 
 
8.4.4 Eastern Cooperative Oncology Group (ECOG) performance scale 
 
The Investigator or qualified designee will assess ECOG status at Screening, prior to the administration of 
each dose of trial treatment and discontinuation of trial treatment as specified in the trial Schedule of 
Activities (Section 2.0). 
 
8.5 Visit Requirements 
 
8.5.1 Requirements 
 
Visit requirements are outlined in trial Schedule of Activities (Section 2.0). Specific procedure-related 
details are provided above in Section 8.1 - Trial Procedures. 
 
 
8.5.2 Screening 
 
Screening starts with the subject’s provision of a written informed consent form (ICF) and should be 
completed within 28 days of initiation of study drug(s). 


48 
 
8.5.3 Thirty-day safety follow-up 
 
The mandatory 30-day Safety Follow-Up Visit should be conducted approximately 30 days after the last dose 
of trial treatment or before the initiation of a new anti-cancer treatment, whichever comes first. All AEs that 
occur prior to the Safety Follow-Up Visit should be recorded. Subjects with an AE of Grade > 1 will be followed 
until the resolution of the AE to Grade 0-1 or until the beginning of a new anti-neoplastic therapy, whichever 
occurs first. SAEs that occur within 90 days of the end of treatment or before initiation of a new anti-cancer 
treatment should also be followed and recorded. 
 
 
8.5.4 End of Treatment (EOT) 
 
Subjects who discontinue trial treatment for disease progression will complete an EOT visit. EOT visit 
requirements include physical exam, medical history, AE assessment and collection of correlative samples 
as indicated in the trial Schedule of Activities (Section 2.0). 
 
8.5.5 Post-treatment Surveillance Phase 
 
Subjects who discontinue trial treatment for a reason other than disease progression (including patients who 
do not progress but discontinue for intolerance/toxicity) will move into the Post-treatment Surveillance 
Phase. These patients will continue to be assessed clinically and radiographically to monitor disease status. 
Post-Treatment Surveillance Phase will start 12 weeks after the last on-treatment time point for patients 
who have no disease progression. 
 
These patients are evaluated per the standard follow-up schedule with imaging [contrast-enhanced CT 
chest/abdomen/pelvis and (if applicable) vs. PET at the discretion of the treating physician] at the following 
intervals. Every 3 months (±2 weeks) if patient is < 2 years from study entry, every 6 months (±4 weeks) if 
patient is 2-5 years from study entry, and every 12 months (±4 weeks) if patient is > 5 years from study entry 
for up to 15 years. 
 
Patients who develop recurrent cancer will be followed for information on survival and for information on 
salvage patterns. The schedule of clinical follow up for these patients will be at the discretion of the treating 
Investigator and according to established guidelines for the relevant disease. AE assessment on the study 
will continue for all patients until 90 days after the last study drug administration. 
 
Every effort should be made to collect information regarding disease status until the start of new anti- 
neoplastic therapy, disease progression, death, end of the study as detailed in Survival Follow Up (Section 
8.5.6). Information regarding post-study anti-neoplastic treatment will be collected if new treatment is initiated. 
 
Note – Patient accrual was terminated early owing to the discontinuation of pixatimod supply. Follow-up 
assessments will no longer be completed upon IRB approval of protocol version 02/23/2024. 
 
8.5.6 Survival follow up 
 
If a subject has confirmed disease progression or starts a new anti-cancer therapy, the subject moves into 
the survival follow-up phase and should be contacted to assess for survival status until death, withdrawal of 
consent, or the end of the study, whichever occurs first. This will be done every 3 months. 
 
Note – Patient accrual was terminated early owing to the discontinuation of pixatimod supply. Follow-up 
assessments will no longer be completed upon IRB approval of protocol version 02/23/2024. 
 
8.6 Laboratory Assessments 
 
Details regarding specific laboratory assessments to be performed in this trial are provided below. 


49 
 
8.6.1 Screening laboratory assessments 
 
Screening laboratory tests should be performed within 28 days of registration. Requirements are stated 
below in Table 8.6.1-1. Results must be reviewed by the Investigator or qualified designee and found to be 
acceptable prior to each dose of trial treatment. 
 
There is 28-day window to obtain Screening (XK-S) laboratory assessments from signing consent. 
 
Table 8.6.1-1: Screening laboratory assessments (XK-S) 
Blood 
Urine 
Other 
• 
CBC: WBC (total and 
differential including 
absolute neutrophil count), 
hemoglobin and 
hematocrit; platelet count 
• 
Renal function: blood urea 
nitrogen, bicarbonate, 
calcium, chloride, 
creatinine, glucose, 
phosphorus, potassium, 
magnesium and sodium 
• 
Liver function: albumin, 
total protein, alkaline 
phosphatase (ALP), 
alanine aminotransferase 
(ALT), aspartate 
aminotransferase (AST), 
total bilirubin (direct only if 
total is elevated above 
ULN) 
• 
Lactate dehydrogenase 
(LDH) 
• 
Coagulation parameters: 
PT and aPTT 
• 
Thyroid function studies: 
TSH, free T3 and free T4 
• 
$Infectious serologies: HIV, 
hepatitis B surface antigen 
(HBsAg) and hepatitis B 
surface antibody (HBsAb) 
and HCV RNA 
• 
pH 
• 
Specific gravity 
• 
Glucose 
• 
Protein 
• 
Blood 
• 
Nitrites 
• 
WBCs 
• 
Microscopic battery (RBCs, 
WBCs, epithelial cells, 
casts) - only if significant 
positive findings on 
urinalysis 
• 
*Serum or urine pregnancy 
test (β-human chorionic 
gonadotropin) 
• 
&Anti-heparin antibodies 
(patients on heparin, low- 
molecular weight heparin, 
fondaparinux or other 
similar agents) 
*Perform on women of childbearing potential and at Screening only. If urine pregnancy results cannot be 
confirmed as negative, serum β-human chorionic gonadotropin will be required. 
$HIV, hepatitis B and C studies will be obtained at Screening only 
&Anti-heparin antibodies will be obtained at Screening only in patients who are currently or have recently (within 6 
months) been on heparin, low-molecular weight heparin, fondaparinux or other similar agents. 
 
 
8.6.2 On-treatment laboratory assessments 
 
Pre-C1D1 and on-treatment laboratory tests should be performed at the appropriate intervals as indicated in 
the trial Schedule of Activities (Section 2.0). Requirements are stated below in Table 8.6.2-1. Results must 
be reviewed by the Investigator or qualified designee and found to be acceptable prior to each dose of trial 
treatment. 
 
There is a 2-day window to obtain on-treatment (XK-B and XK-T) laboratory assessments. 


50 
 
Table 8.6.2-1: On-treatment laboratory assessments (XK-B and XK-T) 
Blood 
Urine 
Other 
• 
CBC: WBC (total and 
differential including 
absolute neutrophil count), 
hemoglobin and 
hematocrit; platelet count 
• 
Renal function: blood urea 
nitrogen, bicarbonate, 
calcium, chloride, 
creatinine, glucose, 
phosphorus, potassium, 
magnesium and sodium 
• 
Liver function: albumin, 
total protein, alkaline 
phosphatase (ALP), 
alanine aminotransferase 
(ALT), aspartate 
aminotransferase (AST), 
total bilirubin (direct only if 
total is elevated above 
ULN) 
• 
Lactate dehydrogenase 
(LDH) 
• 
Coagulation parameters: 
PT and aPTT 
• 
*Thyroid function studies: 
TSH, free T3 and free T4 
• 
pH 
• 
Specific gravity 
• 
Glucose 
• 
Protein 
• 
Blood 
• 
Nitrites 
• 
WBCs 
• 
Microscopic battery (RBCs, 
WBCs, epithelial cells, 
casts) - only if significant 
positive findings on 
urinalysis 
• 
*Fasting lipids 
(triglycerides, LDL, HDL, 
total cholesterol) 
*Only on D1 of each cycle (XK-B) only 
 
8.7 Efficacy Assessments 
 
8.7.1 Tumor imaging and disease assessment 
 
Tumor imaging is strongly preferred to be acquired by contrast-enhanced computed tomography (CT). Brain 
imaging will be obtained only at Screening in melanoma and NSCLC patients and need not be repeated 
unless prior CNS disease was identified, and previously treated CNS lesions were captured as non-target 
lesions. For CRC patients, brain imaging at screening is not required. For brain imaging a contrast enhanced 
MRI is ideal. If an MRI of the brain cannot be done (or is contraindicated) a contrast enhanced CT of the brain 
is acceptable. 
 
Confirmation that the participant’s imaging shows at least 1 lesion that is appropriate for selection as a target 
lesion per RECIST 1.1 is highly recommended prior to participation. 
 
Participant eligibility will be determined using Investigator assessment based on RECIST 1.1. In addition, 
images (including via other modalities) that are obtained at an unscheduled time point to determine disease 
progression, as well as imaging obtained for other reasons, but which demonstrate radiologic progression, 
should also be used to determine progression. 
 
When the Investigator identifies radiographic progression per RECIST 1.1, efforts should be made to verify 
radiologic PD. Treatment should continue until PD has been verified. Regardless of whether PD is verified, if 
the Investigator considers the participant has progressed, but elects to implement iRECIST, the Investigator 
will assess for confirmation of progression by iRECIST at subsequent time points. 


51 
 
8.7.2 Initial tumor imaging 
 
Initial tumor imaging at Screening must be performed within 28 days prior to the Cycle 1 Day 1 date. The site 
study team must review screening images to confirm the participant has measurable disease per RECIST 
1.1. 
 
8.7.3 Tumor imaging during the study 
 
The first on-study imaging assessment should be performed at 8 weeks (56 days ± 7 days) from the date of 
start of treatment. Subsequent tumor imaging should be performed every 8 weeks (56 days ± 7 days) or more 
frequently if clinically indicated. In patients who complete therapy and enter Post-Treatment Surveillance, 
imaging will be performed as outlined in Section 8.5.5. Imaging timing should follow calendar days and should 
not be adjusted for delays in cycle starts. Imaging should continue to be performed until disease progression 
is identified by the Investigator. 
 
All objective response should be ideally confirmed by a repeat imaging assessment where possible. Tumor 
imaging to confirm PR or CR should be performed at least 4 weeks after the first indication of a response is 
observed. Participants will then return to regular scheduled imaging every 8 weeks (56 days ± 7 days), starting 
with the next scheduled imaging time point. Participants who receive additional imaging for confirmation do 
not need to undergo the next scheduled tumor imaging if it is less than 4 weeks later; tumor imaging may 
resume at the subsequent scheduled imaging time point. 
 
Per iRECIST (Section 8.7.6), disease progression should be confirmed 4 to 8 weeks after first radiologic 
evidence of PD in clinically stable participants. Participants who have unconfirmed disease progression may 
continue on treatment at the discretion of the investigator until progression is confirmed by the site provided, 
they have met the conditions detailed in Section 8.7.6. Participants who receive confirmatory imaging do not 
need to undergo the next scheduled tumor imaging if it is less than 4 weeks later; tumor imaging may resume 
at the subsequent scheduled imaging time point, if clinically stable. Participants who have confirmed disease 
progression by iRECIST (Section 8.7.6), as assessed by the site, will discontinue study treatment. 
 
 
8.7.4 End of treatment and follow-up tumor imaging 
 
In participants who discontinue study treatment, tumor imaging should be performed at the time of treatment 
discontinuation (±4-week window). If previous imaging was obtained within 4 weeks prior to the date of 
discontinuation, then imaging at treatment discontinuation is not mandatory. In participants who discontinue 
study treatment due to documented disease progression and the investigator elects not to implement 
iRECIST, this is the final required tumor imaging. 
 
For participants who discontinue study treatment without documented disease progression, every effort 
should be made to continue monitoring their disease status by tumor imaging using the same imaging 
schedule used while on treatment (as outlined in Section 8.5.5.) to monitor disease status until the start of a 
new anti-cancer treatment, disease progression, pregnancy, death, withdrawal of consent, or the end of the 
study, whichever occurs first. 
 
8.7.5 RECIST v1.1 disease assessment 
 
Response and progression will be evaluated in this study using the international criteria proposed by the 
Response Evaluation Criteria in Solid Tumors (RECIST v.1.1) Committee.119 It is known that patients receiving 
immunotherapy have atypical response patterns that in some cases may lead to incorrect determination of 
the response status. In the case of a measurable lesion increase or detection of a previously occult tumor 
lesion, RECIST 1.1 would fail to recognize the potential pseudo-progression and long-term effectiveness of 


52 
 
immunotherapy. Since significant tumor growth and/or newly detectable tumor lesions will generally be 
classified as progressive disease (PD) based on RECIST 1.1, this could result in an erroneous termination of 
the treatment and unjustified patient exclusion from clinical studies. 
 
Although RECIST 1.1 references a maximum of 5 target lesions in total and 2 per organ, the Sponsor- 
Investigator allows a maximum of 10 target lesions in total and 5 per organ, if clinically relevant to enable a 
broader sampling of tumor burden. 
 
 
8.7.6 iRECIST disease assessment 
 
To address this limitation of RECIST 1.1 in cases of pseudoprogression under immunotherapy, Wolchok et 
al. developed modified ‘immune-related Response Criteria’ (irRC) based on the WHO criteria for the first time 
in 2009.120 Subsequently, bi-dimensional irRC were adapted to the uni-dimensional immune-related RECIST 
(irRECIST) criteria.121,122 In both irRC and irRECIST, new measurable tumor lesions are to be added to the 
sum of the target lesions, while only a significant increase (irRC ≥25%; irRECIST ≥20%) results in 
determination of tumor progression (iPD = ‘immune-related progressive disease’). One point of criticism with 
respect to these criteria, particularly irRC, was that non-measurable tumor lesions (i.e. non-target lesions) did 
not contribute to tumor progression. Moreover, in case of stable or minimal tumor decrease following 
pseudoprogression, iPD was confirmed according to irRC and irRECIST. 
 
To reduce inconsistency between different studies depending on which response assessment protocol was 
utilized, the official RECIST Working Group published the new iRECIST guideline in 2017 to assess response 
to immunotherapy in clinical trials. In iRECIST, the basic principles of defining tumor lesions as measurable 
or non-measurable and assessing tumor responses are unchanged from RECIST 1.1. However, an 
additional confirmatory imaging study must be done to confirm or withdraw an ‘unconfirmed’ tumor 
progression after any initial increase in size. 
 
Given the novel nature of pixatimod and the uncertain nature of tumor kinetics in patients receiving 
nivolumab/pixatimod ± Cy, although RECIST will be primarily used to assess response, a key secondary 
endpoint is response by iRECIST as delineated in Figure 8.7.6-1 and Table 8.7.6-2 below.123 iRECIST will 
be used by the Investigator to assess tumor progression, and make treatment decisions. This is further 
elaborated upon in Appendix 4. 


53 
 
 
Figure 8.7.6-1: iRECIST Response Evaluation 
 


54 
 
 
 
Table 8.7.6-2: Imaging and Treatment after First Radiologic Evidence of Progressive Disease 
 
Clinically Stable 
Clinically Unstable 
Imaging 
Treatment 
Imaging 
Treatment 
First radiologic evidence of 
Repeat imaging at 4 to 
May continue study treatment at 
Repeat imaging at 4 to 
Discontinue treatment 
PD by RECIST 1.1 
8 weeks to confirm PD 
the Investigator’s discretion 
8 weeks to confirm PD per 
while awaiting confirmatory 
Investigator’s discretion 
tumor imaging by site by 
only 
iRECIST 
Repeat tumor imaging 
No additional imaging 
Discontinue treatment 
No additional imaging 
Not applicable 
confirms PD (iCPD) by 
required 
(exception is possible upon 
required. 
iRECIST per Investigator 
consultation with Sponsor- 
assessment 
Investigator) 
Repeat tumor imaging 
Repeat imaging at 4 to 
Continue study treatment at the 
Repeat imaging at 4 to 
Discontinue treatment 
shows iUPD by iRECIST 
8 weeks to confirm PD. 
Principal Investigator’s 
8 weeks to confirm PD per 
per Investigator 
May occur at next 
discretion 
Investigator’s discretion 
assessment 
regularly scheduled 
only 
imaging visit 
Repeat tumor imaging 
Continue regularly 
Continue study treatment at the 
Continue regularly 
May restart study treatment if 
shows iSD, iPR, or iCR by 
scheduled imaging 
Principal Investigator’s 
scheduled imaging 
condition has improved and/or 
iRECIST per Investigator 
assessments 
discretion 
assessments. 
clinically stable per Investigator’s 
assessment. 
discretion. Next tumor imaging 
should occur according to the 
regular imaging schedule. 
iCPD = iRECIST confirmed progressive disease; iCR = iRECIST complete response; iRECIST = modified Response Evaluation Criteria in Solid Tumors 1.1 for 
immune-based therapeutics; iSD = iRECIST stable disease; iUPD = iRECIST unconfirmed progressive disease; PD = progressive disease; RECIST 1.1 = 
Response Evaluation Criteria in Solid Tumors 1.1. 


55 
 
 
8.8 Adverse Events (AEs) 
 
8.8.1 Definitions 
 
8.8.1.1 Definition of AEs 
An AE is an untoward or medical occurrence associated with the use of study drug (active or 
placebo drug, biologic, or device) in clinical investigation subjects, which does not necessarily 
have a causal relationship with the study drug. An AE can therefore be any unfavorable and 
unintended symptom, sign, disease or condition, or test abnormality whether or not considered 
related to study drug. AEs that do not meet the definition for serious AEs (SAEs) are considered 
non-SAEs. 
 
AEs include: 
• 
Changes described by the subject or signs observed by the Investigator or medical staff. 
• 
Test abnormalities (i.e., laboratory tests, ECGs) that result in an alteration in medical care 
(diagnostic or therapeutic). 
 
Disease Progression is not considered an AE in this study. 
 
Abnormalities present at baseline are considered AEs only if they reoccur after resolution or they 
worsen during the study. 
 
8.8.1.2 SAE and serious unexpected adverse reactions (SUSAR) 
An SAE is any AE that fulfills one of the criteria outlined in Table 8.8.1.2-1. 
Table 8.8.1.2-1: Criteria for Determination of Serious Adverse Events (SAEs) 
Death 
An AE that results in death. 
*In this study, deaths that are unequivocally due to Disease Progression 
are not to be reported as SAEs. 
Life-threatening AE 
An AE that places the subject, in the view of the Investigator, at immediate 
risk of death from the AE as it occurred (i.e., does not include an AE that 
had it occurred in a more severe form, might have caused death). 
Required or prolonged 
inpatient hospitalization 
An AE that results in an initial inpatient hospitalization or prolongs an 
existing hospitalization of the subject. If a subject is hospitalized as part of 
the clinical use of the study drug, a period of normal hospitalization will be 
outlined in the protocol or by the judgment of the Investigator. 
Hospitalizations longer than this period will be prolonged hospitalizations. 
Persistent or significant 
disability/incapacity 
An AE that results in a substantial disruption of a subject’s ability to conduct 
normal life functions. 
Congenital 
anomaly/birth defect 
A congenital anomaly/birth defect that occurs in the offspring of a subject 
exposed to the study drug. 
Important medical event 
An AE that may not result in death, be life-threatening, or require 
hospitalization may be considered an SAE when, based upon appropriate 
medical judgment, the event may jeopardize the subject and may require 
medical or surgical intervention to prevent 1 of the outcomes listed above. 


56 
 
Examples of such “important medical events” include allergic bronchospasm requiring intensive 
treatment in an emergency room or at home, blood dyscrasias or convulsions that do not result 
in inpatient hospitalization, or the development of drug dependency or drug abuse. 
 
Medical and scientific judgment should be exercised in deciding whether expedited reporting is 
appropriate in other situations, such as with important medical events described above. 
 
Events that meet SAE criteria must be recorded and reported regardless of expectedness or 
assessed association with study drug. 
 
Note: 
• 
Planned hospital admissions or surgical procedures for elective procedures or for an 
illness or disease that existed before the signing of the ICF or before the subject was 
enrolled in the study will not be captured as SAEs. 
• 
If planned admissions or procedures occur at a time other than what was planned (i.e., 
due to an exacerbation in the preexisting illness or disease), they should be reported as 
SAEs. 
 
8.8.1.3 Unexpected AEs 
 
An unexpected AE is any AE that is not consistent in specificity or severity with the current IB for 
either pixatimod or nivolumab. 
 
 
8.8.2 Evaluation of AEs and SAEs 
 
The Investigator or designee is responsible for making an assessment as to the severity/grade 
(as defined in Section 8.8.2.1, below), causality/relationship (as defined in Section 8.8.2.2, 
below), and outcome of AEs and SAEs (as defined in Section 8.8.2.3, below). In addition, the 
Investigator or designee must report any actions taken as a result of an AE or SAE. 
 
8.8.1.4 AE severity/grade 
 
For each recorded AE or SAE, the Investigator or designee must make an assessment of Grade 
using the Common Terminology Criteria for Adverse Events (CTCAE) version 5. Grade refers to 
the severity of the AE. Note that severity is not the same as “seriousness”. Seriousness (not 
severity) serves as a guide for defining regulatory reporting obligations. 
 
8.8.1.5 Causality and relationship to study drug(s) 
 
For each AE or SAE, the Investigator will determine whether there is a reasonable possibility 
demonstrated by evidence that suggests a causal relationship between the study drug regimens 
– nivolumab and pixatimod (Cohorts 2 and 3) and nivolumab, pixatimod and low-dose 
cyclophosphamide (Cohort 1) - and the AE according to the categories provided in Table 8.8.2.2- 
1. Attribution of adverse events specifically to either pixatimod or nivolumab is challenging, 
therefore, the relationship to study drug should be based on attribution to the combination of the 
two drugs, not a single drug. 


57 
 
The cumulative experience with low-dose cyclophosphamide suggests that AE/SAE related to 
this agent and/or its administration are rare. However, investigators will make every effort to 
evaluate for AE/SAE that may be related to cyclophosphamide. 
 
An AE with causal relationship not initially determined will require follow-up to assign causality 
which must be made by the Investigator prior to completion of the study. The Investigator may 
change his/her opinion of causality in light of follow-up information; if this occurs, the Investigator 
must amend the AE or SAE information accordingly. 
 
Table 8.8.2.2-1: Classifications for Adverse Event (AE) Causality/Relationship 
Classification 
Definition 
Unrelated 
There is no suspicion of a causal relationship between exposure to 
the study drug regimen and the AE; another cause of the AE has 
been identified, no temporal association with study drug has been 
identified, or the study drug cannot be implicated. 
Possibly related 
There is some evidence supporting the possibility of a causal 
relationship between study drug regimen exposure and the AE; an 
alternative explanation (i.e., concomitant drug or concomitant 
disease) is inconclusive, the temporal association with study drug is 
reasonable, and the causal relationship cannot be excluded. 
Probably related 
An adverse event that has a timely relationship to the administration 
of the investigational drug regimen and follows a known pattern of 
response, but for which a potential alternative cause may be 
present. 
Definitely related 
There is strong evidence that there is a causal relationship between 
study drug regimen and the AE; the AE cannot be reasonably 
explained by an alternative explanation (i.e., concomitant drug or 
concomitant disease) and the temporal association with study drug 
is suggestive of a causal relationship. 
 
8.8.1.6 Classification of AE outcome 
 
Adverse event outcome describes the status of the AE at the last observation. The Investigator 
will document the outcome of each AE or SAE using the categories provided in Table 8.8.2.3-1. 
 
Table 8.8.2.3-1: Classifications for Adverse Event (AE) Outcome 
Classification 
Definition 
Fatal 
Termination of life as a result of an AE. 
Not recovered/not resolved 
Subject has not recuperated, or the AE has not improved. 
Recovering/resolving 
Subject is recuperating or the AE is improving. 
Recovered/resolved 
Subject has recuperated, the AE resolved, or returned to baseline 
status / stabilized. 
Recovered/resolved with 
sequelae 
Adverse event has resolved, but the subject has been left with 
symptoms or pathology. 
Unknown 
Not known, not observed, not recorded, or refused. 
 
 
8.8.1.7 Action taken regarding AE 
 
The Investigator will provide the action taken regarding study drug in response to the AE. 
Classifications for each of the potential actions taken are provided in Table 8.8.2.4-1. More than 
one option may apply to a single AE/SAE. For example, study drug may be delayed, and the dose 


58 
 
reduced in response to an AE. Action related to nivolumab, pixatimod and/or low-dose 
cyclophosphamide will be assessed and recorded separately in the EDC. 
 
Table 8.8.2.4-1: Classifications for Actions Taken Regarding an Adverse Event (AE) 
Classification 
Definition 
Dose not changed 
No change in administration of study drug 
Dose reduced1 
Reduction in the amount of study drug administered 
Study drug interrupted 
Temporary interruption (termination) in administration of the study 
drug 
Study drug withdrawn 
Administration of the study drug terminated (no further dosing) 
Not applicable 
Determination of a value is not relevant in the current context 
Unknown 
Not known, not observed, not recorded, or refused 
1Refer to Section 7.2.2. (nivolumab), Section 7.2.3. (pixatimod) and Section 7.2.4. (Cy) 
regarding dose reductions of nivolumab, pixatimod or Cy due to dose-limiting toxicities, 
respectively. 
 
 
8.8.3 Timeframe for AE/SAE Collection 
 
The Investigator is required to record all AEs occurring during the clinical study (21 CFR 312.64[b] 
and ICH E6 [R1]) starting from the date of first dose of study treatment until 30 days after the last 
dose of nivolumab and pixatimod (± cyclophosphamide) on the AE page of the eCRF. SAEs that 
occur within 90 days of the end of treatment or before initiation of a new anti-cancer treatment 
should also be followed and recorded. SAEs as defined in Section 8.8.1.2. must also be reported 
to the Sponsor-Investigator or representative within 24 hours of knowledge of their occurrence, in 
accordance with Section 8.8.5. 
 
8.8.3.1 TRAE and treatment-related SAE (TRSAE) 
 
TRAEs and treatment-related SAEs (TRSAEs) are defined as AEs or SAEs that started or 
worsened in severity on or after the date the study drugs were first administered. 
 
TRAE/TRSAE information will be collected from the time of the subject’s first receipt of nivolumab 
and pixatimod (± cyclophosphamide) until 30 days after the last dose of nivolumab and pixatimod 
(± cyclophosphamide). 
 
 
8.8.3.2 SAEs 
 
SAE information will be collected from the point the subject starts study treatment until 90 days 
after the last dose of nivolumab and pixatimod (± cyclophosphamide). If, at any time after the 
subject has completed participation in the study, the Investigator or study staff becomes aware of 
an SAE that they believe is possibly related or related to nivolumab and pixatimod (± 
cyclophosphamide, see Section 8.8.1.2), then the event and any known details must be reported 
promptly to the Sponsor-Investigator. The reporting instructions described in Section 8.8.5 must 
be followed. 


59 
 
8.8.4 Recording AEs/SAEs 
 
All AEs and SAEs experienced by a subject will be recorded on the appropriate research record. 
Information including a concise description of the event; date and time of event onset and 
resolution; determination of seriousness, severity, corrective treatment, outcome, relationship to 
study drug; and action taken regarding the study drug will be recorded. 
 
Vital signs, laboratory results, and other safety assessments as obtained as detailed in Sections 
8.4, 8.5, and 8.6 will be recorded as AEs if they are determined to be clinically significant findings 
in the opinion of the Investigator. When possible, a diagnosis should be recorded as an AE, rather 
than symptoms or isolated laboratory abnormalities related to that diagnosis. A medical or surgical 
procedure is not an AE; rather the condition leading to the procedure should be recorded as the 
AE. If the condition is not known, the procedure must be recorded as an AE instead. Similarly, 
death is not an AE, but is rather the outcome of the AE(s) that resulted in death. If the AE(s) 
leading to death are not known, then death must be reported as an AE. 
 
All SAEs experienced by the subject will be entered into the CTMA and reported to the Sponsor- 
Investigator or designee, in accordance with Section 8.8.5. 
 
 
8.8.5 Reporting SAEs and Serious Unexpected AEs (SUAE) 
 
8.8.5.1 SAE and SUAE reporting 
 
All events meeting the definition of a serious adverse event, which occur after the date of first 
dose of study treatment and within 30 days after the last dose of study treatment, should be 
reported according to the CRS SAE checklist and SAE form. The initial SAE form should be 
sent to the following within 24 hours of the Principal Investigator becoming aware: 
• 
Sponsor-Investigator 
Email: davard@upmc.edu; 
• 
crssafetysubmissions@upmc.edu 
• 
The Aculeus Therapeutics Drug Safety contact information is available for SAE reporting 
on a 24-hour basis and is reviewed during normal business hours. The contact information 
is as follows: Email: mdevlin@aculeustx.com  
• 
Local Institutional Review Board when reporting requirements are met 
 
In addition to completing appropriate patient demographic and suspect medication 
information, the report should include as applicable the following information that is available 
at the time of report within the CRS departmental SAE form: 
o CTCAE term(s) and grade(s) 
o Current status of study drug 
o Intervention(s) to address the AE (testing and result, treatment and response) 
o Hospitalization and/or discharge dates 
o Event relationship to study drug combination 
 
8.8.5.2 Events of clinical interest (ECI) 


60 
 
Selected non-serious and serious adverse events are also known as Events of Clinical Interest 
(ECI) and must be reported within 24 hours to the Sponsor-Investigator and within 2 working days 
to Aculeus Therapeutics Drug Safety Contact (see Section 8.8.5.1.). 
 
For the time period beginning when the consent form is signed until treatment initiation, any ECI, 
or follow up to an ECI, that occurs to any subject must be reported within 24 hours to the Sponsor- 
Investigator and within 2 working days to Aculeus Therapeutics Drug Safety Contact if it causes 
the subject to be excluded from the trial, or is the result of a protocol-specified intervention, 
including but not limited to washout or discontinuation of usual therapy, diet, placebo treatment or 
a procedure. 
 
For the time period beginning at treatment initiation through 90 days following cessation of 
treatment, or 30 days following cessation of treatment if the subject initiates new anticancer 
therapy, whichever is earlier, any ECI, or follow up to an ECI, whether or not related to Checkmate 
product, must be reported within 24 hours to the Sponsor-Investigator and within 24 hours to 
Aculeus Therapeutics Drug Safety Contact. 
 
ECI for this trial include: 
• 
An overdose of Aculeus Therapeutics product 
 
8.8.5.3 Pregnancies 
 
Female subjects or the partners of male subjects who discover they are pregnant within a year of 
their last nivolumab and pixatimod (± cyclophosphamide) dose will be instructed to notify the 
Investigator immediately. 
 
If the Investigator learns of a report of pregnancy at any time after signing the ICF, the Investigator 
must report the pregnancy to Aculeus Therapeutics Pharmaceuticals Drug Safety within 24 hours 
(following the same reporting process outlined in Section 8.8.5.1). 
 
The Investigator will inform the subject that the Sponsor-Investigator or its designee is required to 
gather information regarding the course and outcome of a pregnancy that has occurred after 
exposure to a study drug. The progress of the pregnancy must be followed until the outcome of 
the pregnancy is known (i.e., delivery, elective termination, or spontaneous abortion). If the 
pregnancy results in the birth of a child, additional follow-up information may be requested. 
 
The Investigator will be asked to obtain follow-up information no later than 2 months after the 
gestational period to obtain maternal/fetal/neonatal outcome and any other relevant information. 
Follow-up information may be requested at additional time points. All study-related contacts 
involving a known pregnancy should include pregnancy status assessment until pregnancy 
outcome is known. 
 
Please note that pregnancy in and of itself is not an AE or an SAE. Pregnancy should not be 
entered into the eCRF as an AE unless the Investigator suspects an interaction between the study 
drug and the contraceptive method. Additionally, all information received will be assessed for any 
AEs and SAEs and processed per study guidelines. If the subject is discontinued because of 
pregnancy, pregnancy will be documented as the reason for study discontinuation. Spontaneous 
abortions and stillbirths will be reported as SAEs. 


61 
 
8.8.6 IND safety report 
 
The Sponsor-Investigator must notify FDA and all participating investigators (i.e., all investigators 
to whom the Sponsor-Investigator is providing drug under its INDs or under any investigator's 
IND) in an IND safety report of potential serious risks, from clinical trials or any other source, as 
soon as possible, but in no case later than 15 calendar days after the Sponsor-Investigator 
determines that the information qualifies for reporting under Sections 8.8.6.1. to 8.8.6.3. below. 
In each IND safety report, the Sponsor-Investigator must identify all IND safety reports previously 
submitted to FDA concerning a similar suspected adverse reaction, and must analyze the 
significance of the suspected adverse reaction in light of previous, similar reports or any other 
relevant information. 
 
8.8.6.1 Serious and unexpected adverse reactions 
 
The Sponsor-Investigator must report any suspected adverse reaction that is both serious and 
unexpected. The Sponsor-Investigator must report an adverse event as a suspected adverse 
reaction only if there is evidence to suggest a causal relationship between the drug and the 
adverse event, such as: 
• 
A single occurrence of an event that is uncommon and known to be strongly associated 
with drug exposure (e.g., angioedema, hepatic injury, Stevens-Johnson Syndrome); 
 
• 
One or more occurrences of an event that is not commonly associated with drug exposure, 
but is otherwise uncommon in the population exposed to the drug (e.g., tendon rupture); 
 
• 
An aggregate analysis of specific events observed in a clinical trial (such as known 
consequences of the underlying disease or condition under investigation or other events 
that commonly occur in the study population independent of drug therapy) that indicates 
those events occur more frequently in the drug treatment group than in a concurrent or 
historical control group. 
 
8.8.6.2 Findings from other studies 
 
The Sponsor-Investigator must report any findings from epidemiological studies, pooled analysis 
of multiple studies, or clinical studies (other than those reported under Section 8.8.6.1.), whether 
or not conducted under an IND, and whether or not conducted by the Sponsor-Investigator, that 
suggest a significant risk in humans exposed to the drug. Ordinarily, such a finding would result 
in a safety-related change in the protocol, informed consent, investigator brochure (excluding 
routine updates of these documents), or other aspects of the overall conduct of the clinical 
investigation. 
 
8.8.6.3 Findings from animal and/or in vitro studies 
 
The Sponsor-Investigator must report any findings from animal or in vitro testing, whether or not 
conducted by the Sponsor-Investigator, that suggest a significant risk in humans exposed to the 
drug, such as reports of mutagenicity, teratogenicity, or carcinogenicity, or reports of significant 
organ toxicity at or near the expected human exposure. Ordinarily, any such findings would result 
in a safety-related change in the protocol, informed consent, investigator brochure (excluding 
routine updates of these documents), or other aspects of the overall conduct of the clinical 
investigation. 


62 
 
8.8.6.4 Increase rate of occurrence of serious suspected adverse reactions 
 
The Sponsor-Investigator must report any clinically important increase in the rate of a serious 
suspected adverse reaction over that listed in the protocol or investigator brochure. 
 
8.8.6.5 Submission of IND safety reports 
 
The Sponsor-Investigator must submit each IND safety report in a narrative format or on FDA 
Form 3500A or in an electronic format that FDA can process, review, and archive. FDA will 
periodically issue guidance on how to provide the electronic submission (e.g., method of 
transmission, media, file formats, preparation and organization of files). The Sponsor-Investigator 
may submit foreign suspected adverse reactions on a Council for International Organizations of 
Medical Sciences (CIOMS) I Form instead of a FDA Form 3500A. Reports of overall findings or 
pooled analyses from published and unpublished in vitro, animal, epidemiological, or clinical 
studies must be submitted in a narrative format. Each notification to FDA must bear prominent 
identification of its contents, i.e., "IND Safety Report," and must be transmitted to the review 
division in the Center for Drug Evaluation and Research or in the Center for Biologics Evaluation 
and Research that has responsibility for review of the IND. Upon request from FDA, the Sponsor- 
Investigator must submit to FDA any additional data or information that the agency deems 
necessary, as soon as possible, but in no case later than 15 calendar days after receiving the 
request. 
 
8.8.6.5.1 
Unexpected fatal or life-threatening suspected adverse reaction 
reports 
 
The Sponsor-Investigator must also notify FDA of any unexpected fatal or life-threatening 
suspected adverse reaction as soon as possible but in no case later than 7 calendar days after 
the sponsor's initial receipt of the information. 
 
8.8.6.5.2 
Reporting format or frequency 
 
FDA may require the Sponsor to submit IND safety reports in a format or at a frequency different 
than that required under Sponsor-Investigator paragraph. The Sponsor may also propose and 
adopt a different reporting format or frequency if the change is agreed to in advance by the director 
of the FDA review division that has responsibility for review of the IND. 
 
8.8.6.5.3 
Investigations of marketed drugs 
 
A Sponsor of a clinical study of a drug marketed or approved in the United States that is conducted 
under an IND is required to submit IND safety reports for suspected adverse reactions that are 
observed in the clinical study, at domestic or foreign study sites. The Sponsor must also submit 
safety information from the clinical study as prescribed by the post marketing safety reporting 
requirements. 
 
8.8.6.5.4 
Reporting study endpoints 
 
Study endpoints (e.g., mortality or major morbidity) must be reported to FDA by the Sponsor- 
Investigator as described in the protocol and ordinarily would not be reported under Section 9.6. 
However, if a serious and unexpected adverse event occurs for which there is evidence 
suggesting a causal relationship between the drug and the event (e.g., death from anaphylaxis), 
the event must be reported under Serious and unexpected suspected adverse reaction as a 
serious and unexpected suspected adverse reaction even if it is a component of the study 
endpoint (e.g., all-cause mortality). 


63 
 
8.8.7 Follow-Up 
 
• 
The sponsor must promptly investigate all safety information it receives. 
• 
Relevant follow-up information to an IND safety report must be submitted as soon as the 
information is available and must be identified as such, i.e., "Follow-up IND Safety Report." 
• 
If the results of a sponsor's investigation show that an adverse event not initially 
determined to be reportable under section IND safety reports of this section is so 
reportable, the sponsor must report such suspected adverse reaction in an IND safety 
report as soon as possible, but in no case later than 15 calendar days after the 
determination is made. 
 
8.8.8 Disclaimer 
 
A safety report or other information submitted by a sponsor under this part (and any release by 
FDA of that report or information) does not necessarily reflect a conclusion by the sponsor or FDA 
that the report or information constitutes an admission that the drug caused or contributed to an 
adverse event. A sponsor need not admit, and may deny, that the report or information submitted 
by the sponsor constitutes an admission that the drug caused or contributed to an adverse event. 
 
The principal investigator must promptly review all information relevant to the safety of the drug 
obtained or otherwise received from foreign or domestic sources, including information derived 
from any clinical or epidemiological investigations, animal or in vitro studies, reports in the 
scientific literature, and unpublished scientific papers, as well as reports from foreign regulatory 
authorities and reports of foreign commercial marketing experience for drugs that are not 
marketed in the United States. The study sponsor must notify all participating investigators of 
potential serious risks, from clinical trials or any other source, as soon as possible. 
 
 
8.8.9 Reporting AEs to the responsible Institutional Review Board (IRB) 
 
In accordance with applicable policies of the University of Pittsburgh Institutional Review Board 
(IRB), the Sponsor-Investigator will report, to the IRB, any observed or volunteered adverse event 
that is determined to be 1) associated with the investigational drug or study treatment(s); 2) 
serious; and 3) unexpected. Adverse event reports will be submitted to the IRB in accordance 
with the respective IRB procedures. 
 
Applicable adverse events will be reported to the IRB as soon as possible and, in no event, later 
than 10 calendar days following the sponsor-investigator’s receipt of the respective information. 
Adverse events which are 1) associated with the investigational drug or study treatment(s); 2) 
fatal or life-threatening; and 3) unexpected will be reported to the IRB within 24 hours of the 
Sponsor-Investigator’s receipt of the respective information. 
 
Follow-up information to a reported adverse event will be submitted to the IRB as soon as the 
relevant information is available. If the results of the Sponsor-Investigator’s follow-up investigation 
show that an adverse event that was initially determined to not require reporting to the IRB does, 
in fact, meet the requirements for reporting; the Sponsor-Investigator will report the adverse event 
to the IRB as soon as possible, but in no event later than 10 calendar days, after the determination 
was made. 


64 
 
8.8.10 Follow-up of AEs and SAEs 
 
All AEs and SAEs documented at a previous visit that are designated as either 
recovering/resolving or not recovered/resolved, will be reviewed by the Investigator at subsequent 
visits. 
 
All AEs will be followed until resolution of AE, completion of the subject’s participation, or study 
termination, whichever occurs first. 
 
Serious AEs and AEs resulting in discontinuation will be followed until one of the following occurs: 
• 
The event resolves. 
• 
The event stabilizes. 
• 
The event returns to a baseline value, if a baseline value is available. 
• 
The event can be attributed to agents other than the study drug or to factors unrelated to 
study conduct. 
• 
The Investigator agrees that follow-up is no longer necessary. 
 
Follow-up reports from the Investigator must be provided as indicated using the SAE report form 
within 24 hours of the Investigator’s first knowledge of the new information. Additional information 
(i.e., hospital records, laboratory, or other diagnostic test results) should be provided if requested 
and/or indicated. 
 
Rules for AE/SAE follow up apply to all subjects, including those who withdraw consent prior to 
study completion (to the extent allowed). The Investigator will ensure that follow up includes 
further investigations to elucidate the nature and/or causality of the AE/SAE. These investigations 
must be consistent with appropriate medical management and subject consent. 
 
Investigators are not obligated to actively seek AEs or SAEs in former study subjects that occur 
pursuant to the follow-up period. However, if the Investigator or designee learns of any AE or SAE 
at any time after a subject has been discharged from the study and the event is considered as 
reasonably related to the study drug, the Sponsor-Investigator will notify Aculeus Therapeutics. 
 
 
8.9 Tumor Tissue Collection and Correlative Studies 
 
The total amount of blood/tissue to be drawn/collected over the course of the trial (from pre-trial 
to post-trial visits), including approximate blood/tissue volumes drawn/collected by visit and by 
sample type per subject can be found in the Laboratory Manual and is reproduced below. 
 
Planned correlative analyses will include flow cytometric analyses of tumor samples, TIL and 
peripheral blood mononuclear cells (PBMC), IHC analyses of pre-/post- treatment tumor samples, 
tumor whole exome/RNA sequencing. Single-cell RNA sequencing will be performed on a subset 
of treated patients. Studies will be performed under the direction of the Principal Investigator along 
with co-Investigator Dr. Hassane Zarour in Dr. Zarour’s lab. 
 
Tumor tissue 
Patients must undergo protocol mandated research biopsies during Screening and on Cycle 2 
Day 1. Biopsies may be on any suitable previously non-radiated representative lesion. Biopsy site 
must be sized suitably to permit investigator to obtain minimum quantity of tissue as specified in 


65 
 
the Schedule of Activities (Section 2.0), which is 6 core biopsies of minimum 16gauge (or 
greater). Tumor tissue will be processed as described in the Laboratory Manual. 
 
Primary tumor (if available) 
FFPE tissue block(s) or 10 unstained slides (air dried) from patients’ originally resected primary 
tumor (melanoma, NSCLC or CRC) will be requested as well. 
 
Blood biospecimens 
Blood biospecimens will be collected at baseline, and per the schedule outlined in the trial 
Schedule of Activities (Section 2.0). 
 
At EACH time point please submit the following: 
• 
Six (6) 10mL sodium heparin tubes (Fisher Scientific catalog number 02-685-114A) 
• 
Two (2) 5mL SST tubes (Fisher Scientific catalog number 02-683-145) 
• 
One (1) 10mL Streck cf DNA tube 
• 
One (1) 4mL K2 EDTA tube (SCREENING ONLY) (Fisher Scientific catalog number 02- 
689-4) 
 
Each tube must be clearly labeled to include: 
• 
Protocol number: HCC 20-266 
• 
Patient number: UOP-XXX 
• 
Patient initials: 
• 
Originating institution/investigator name: 
• 
Date and time drawn: 
• 
Collection time point: 
 
Stool specimens 
Stool biospecimens will be collected at baseline, and per the schedule outlined in the trial 
Schedule of Activities (Section 2.0). 
 
Dietary questionnaire 
DHQ-3 questionnaire may be administered electronically or on paper. Electronic administration is 
preferred. Data will be entered into DHQ-3 online per instructions as outlined in Appendix 1. 
 
 
8.9 Pharmacokinetics Sampling 
 
8.9.1 Pixatimod (PG545) Pharmacokinetics 
 
Pixatimod pharmacokinetic sampling will be collected from 10 subjects in Cohort 1, and a total of 
10 subjects from Cohorts 2 and 3. Samples will be collected at the following time-points relative 
to the pixatimod infusion (Cycle 1 Day 1): 
 
Pre-dosing 
Post-dosing: 
• 
30 minutes post-infusion (± 5 minutes) 
• 
2 hours post-infusion (± 15 minutes) 
• 
4 hours post-infusion (± 15 minutes) 
• 
6 hours post-infusion (± 30 minutes) 


66 
 
• 
24 hours post-infusion (± 60 minutes) 
• 
72 hours post-infusion (± 24 hours) 
• 
168 hours post-infusion (± 24 hours) (i.e. prior to dosing on Cycle 1 Day 8). 
 
Post-dose samples cannot be taken from the same vein as used for dosing. At each of the 
sampling periods, samples will be collected into 2.7 mL sodium citrate tubes. At each of the 
collection time points each subject will have 1 x 2.7 mL collection, with the exception of the Cycle 
1 Day 1 pre-dose sample where 2 x 2.7 mL collections will be required. Pharmacokinetic analysis 
will be performed using an LC-MS/MS method validated for pixatimod in human plasma. Samples 
will be clearly labelled, frozen, and forwarded to the contracted laboratory for analysis. The 
laboratory will measure pixatimod concentrations on each sample. The exact date and time of 
infusion state and finish, and the exact time of each sample, will be recorded on a supplementary 
page of the CRF. 
 
The individual pixatimod pharmacokinetic profile and parameters of the AUC (area under the 
curve), Cmax (maximum concentration), Tmax (time to maximum concentration), and t1/2, 
clearance (Cl) and volume of distribution (Vd) will be estimated provided the ‘fit’ of the time versus 
concentration curves permit such analysis. Summary statistics will be used to describe 
pharmacokinetic results. 
 
8.9.2 Cyclophosphamide Pharmacokinetics 
 
Cyclophosphamide pharmacokinetic sampling will be collected from 10 subjects in Cohort 1 only. 
Samples will be collected at the following time-points relative to the pixatimod infusion (Cycle 1 
Day 1): 
 
Pre-dosing 
Post-dosing: 
• 
10 minutes (± 5 minute) 
• 
30 minutes post-dose (± 5 minutes) 
• 
1 hour post-dose (± 10 minutes) 
• 
2 hours post-dose (± 15 minutes) 
• 
4 hours post-dose (± 15 minutes) 
• 
6 hours post-dose (± 30 minutes) 
• 
24 hours post-dose (± 1 hour) 
 
At each of the sampling periods, samples will be collected into 2.0 mL sodium heparin tubes. At 
each of the collection time points each subject will have 1 x 2.0 mL collection, with the exception 
of the Cycle 1 Day 1 pre-dose sample where 2 x 2.0 mL collections will be required. 
Pharmacokinetic analysis will be performed using an LC-MS/MS method validated for 
cyclophosphamide in human plasma. Samples will be clearly labelled, frozen, and forwarded to 
the contracted laboratory for analysis. The laboratory will measure cyclophosphamide 
concentrations on each sample. The exact date and time of infusion state and finish, and the 
exact time of each sample, will be recorded on a supplementary page of the CRF. 
 
The individual cyclophosphamide pharmacokinetic profile and parameters of the AUC (area under 
the curve), Cmax (maximum concentration), Tmax (time to maximum concentration), and t1/2, 
clearance (Cl) and volume of distribution (Vd) will be estimated provided the ‘fit’ of the time versus 


67 
 
concentration curves permit such analysis. Summary statistics will be used to describe 
pharmacokinetic results. 
 
9. STATISTICAL CONSIDERATIONS 
 
9.1 Study Design 
 
This is a phase II study of nivolumab in combination with pixatimod in 3 separate cohorts: 
• 
Cohort 1: nivolumab, pixatimod and low-dose cyclophosphamide in MSS mCRC 
• 
Cohort 2: nivolumab and pixatimod in PD-1 relapsed/refractory melanoma 
• 
Cohort 3: nivolumab and pixatimod in PD-1 relapsed/refractory NSCLC. 
 
The goal of this study is to assess response of PD-1 R/R melanoma/NSCLC to nivolumab and 
pixatimod; and MSS mCRC to nivolumab, pixatimod and low-dose cyclophosphamide. Simon’s 
two-stage design will be used for each cohort separately. 
 
9.2 Safety Monitoring 
 
Anti-PD-1 therapy including nivolumab has been tested in PD-1 R/R melanoma, NSCLC and PD- 
1 naive MSS mCRC in multiple studies. 
 
The safety profile observed thus far suggests that the combination of nivolumab and pixatimod is 
unlikely to pose a high risk of toxicity in the setting of PD-1 R/R melanoma and PD-1 R/R NSCLC. 
Hence, in this study, we will monitor dose limiting toxicities (DLTs, defined below at the end of this 
section) for 4 weeks for the 1st 3 patients accrued in Cohorts 2 and 3. If we see at least 1 DLT 
out of the 3 patients, we will hold the accrual in this cohort, and the study committee and the 
Principal Investigator (Sponsor-Investigator) will decide whether to modify or discontinue the 
study. 
 
The combination of nivolumab and pixatimod has been studied in MSS mCRC in a small phase 
IB study wherein no untoward safety signals were observed. PD-1 inhibitor pembrolizumab and 
low-dose cyclophosphamide (50 mg twice daily, 1-week-on, 1-week-off) has been explored in two 
studies in patients with advanced osteosarcoma and soft tissue sarcomas with a low incidence of 
grade 3/4 adverse events.95,96 Based on the above, it is unlikely that the combination of nivolumab, 
pixatimod and low-dose cyclophosphamide has overlapping toxicities, nor is there a plausible 
basis for pharmacodynamic or pharmacokinetic interactions leading to a DLT. As such, it is not 
felt that this combination requires a formal phase I study. However, the combination of nivolumab, 
pixatimod and low-dose cyclophosphamide has hitherto not been studied in advanced cancer 
patients in general and MSS mCRC patients in particular. To mitigate toxicity, in Cohort 1, we will 
utilize a safety run-in and monitor toxicities continuously using the following method and decision 
rule. 
 
In the safety run-in (Cohort 1 only), 6 patients will be enrolled at a rate of no more than 1 patient 
per month. Toxicities will be monitored as delineated below. At the end of the safety run-in period 
(defined as 1 month following the enrollment of the 6th patient in Cohort 1), the formal DLT rate 
will be calculated. If the DLT rate is ≤25%, the study will continue to proceed. Should the DLT rate 
exceed 25% (i.e. at least 2 DLTs in the first 6 enrolled patients), the study will be stopped. 
 
To further mitigate toxicity, we will use a Bayesian monitoring scheme to continuously monitor the 
DLT rate of the study combination at 25%. A non-informative prior of Beta(1, 1) for the DLT rate 
will be used. We will hold the accrual if the posterior probability Pr (DLT rate>25%) ≥ 0.7 and the 
study committee and the PI will decide whether to modify or discontinue the study. The stopping 
boundary for toxicity is given in the following table. 


68 
 
 
Number of DLTs≥ 
in Number of Patients = 
4 
7-9 
5 
10-12 
6 
13-15 
7 
16-18 
8 
19-22 
9 
23-25 
10 
26-27 
 
 
We simulate the operating characteristics under various assumed true toxicity rate (see the 
following table): 
Scenario 
Prob.Of.Tox 
Prob.Early.Stop 
1 
0.05 
0.001 
2 
0.15 
0.05 
3 
0.25 
0.30 
4 
0.35 
0.67 
5 
0.45 
0.92 
 
A DLT is defined as any adverse event(s) (AEs) considered related to nivolumab and pixatimod 
that occurs during the first 28 days of therapy in Cohorts 2 and 3; and related to nivolumab and 
pixatimod and cyclophosphamide that occurs during the safety run-in period (defined as 1 month 
following the enrollment of the 6th patient) in Cohort 1. During DLT monitoring period, no further 
accrual will be permitted. Any patient who has started the study treatment will be evaluable for 
safety. 
 
The following events will be considered DLTs if deemed related to study therapy: 
• 
Hematologic 
 
o Grade 4 neutropenia 
 
o Febrile neutropenia, defined as absolute neutrophil count (ANC) ≤1000/mm3 with 
a temperature of ≥ 38.3 degrees °C 
 
o Grade ≥3 neutropenic infection 
o Grade ≥3 thrombocytopenia with bleeding 
o Grade 4 thrombocytopenia 
• 
Non-hematologic 
 
o Grade >3 fatigue lasting ≥1 week 
o Grade ≥3 nausea, vomiting or diarrhea despite maximal medical intervention 
o Grade ≥3 toxicities (non-laboratory) 


69 
 
o Grade >3 electrolyte abnormality that last >72 hours, unless the patient has 
clinical symptoms, in which case all grade >3 electrolyte abnormalities regardless 
of duration should count as a DLT. 
o Grade ≥4 hepatic toxicity [excluding isolated grade ≥3 gamma- 
glutamyltransferase (GGT) abnormalities] according to Hy's law.124 
• 
Any death (grade 5 event) not clearly due to the underlying disease and/or extraneous 
cause(s). 
 
• 
Other (non-AST/non-ALT) non-hematologic Grade ≥3 laboratory value if the abnormality 
leads to overnight hospitalization. 
 
An AE believed to be caused by tumor flare or pseudoprogression is not considered a DLT. Any 
such cases that would otherwise meet DLT criteria must be discussed immediately with the 
Principal Investigator. If the Principal Investigator agrees that tumor flare is a likely explanation 
for the AE, treatment with nivolumab/pixatimod may continue so long as the subject is closely 
monitored by the Principal Investigator or study staff while on study. 
 
 
9.3 Efficacy Decision Rule 
 
We hypothesize that the nivolumab/pixatimod combination in PD-1 relapsed/refractory (R/R) 
cutaneous melanoma and NSCLC patients will be associated with anti-tumor effects. We also 
hypothesize that the nivolumab/pixatimod/cyclophosphamide combination in MSS mCRC 
patients will be associated with anti-tumor effects. 
 
We further hypothesize that nivolumab/pixatimod/cyclophosphamide combination in MSS mCRC 
patients and nivolumab/pixatimod combination in PD-1 R/R melanoma or NSCLC will be 
associated with increased CD8+ T cells intra-tumorally and PD-1+Ki67+ CD8+ T cells peripherally 
in responders on treatment compared to baseline; and that response will be associated with 
evidence of antigen-specific immunity responses intra-tumorally and peripherally. 
 
In the 1st stage of Cohort 1 (MSS mCRC), we will enroll 13 patients. If ≥1 response(s) are seen, 
14 
additional 
patients 
will 
be 
enrolled 
in 
the 
2nd 
stage. 
The 
nivolumab/pixatimod/cyclophosphamide combination will be considered worthy of further 
evaluation if ≥4 responses (ORR 15%) are seen across both stages in this cohort. This study 
design, using Simon’s 2-stage Minimax design, provides 80% power with 5% type I error to detect 
a response rate of 20% against a null hypothesis of 5%. 
 
For Cohorts 2 (PD-1 R/R melanoma) and 3 (PD-1 R/R NSCLC), we will use the same design, 
but separately, as follows. In the 1st stage, will enroll 9 patients. If ≥1 response(s) are seen, 8 
additional patients will be enrolled in the 2nd stage. The combination will be considered worthy of 
further evaluation if ≥3 responses (ORR 18%) are seen across both stages. This study design, 
using Simon’s optimal 2-stage design, provides 81% power with 5% type I error to detect a 
response rate of 25% against a null hypothesis of 5%. 
 
9.4 Efficacy Analysis 
 
Definition of response-evaluable patient 
Each patient will be assigned one of the following categories: 1) complete response, 2) partial 
response, 3) stable disease, 4) progressive disease, 5) early death from malignant disease, 6) 


70 
 
early death from toxicity, 7) early death because of other cause, or 9) unknown (not assessable, 
insufficient data). All patients who met the eligibility criteria; received at least 1 dose each of 
nivolumab and pixatimod (cohorts 2 and 3); and at least 1 dose each of nivolumab, pixatimod 
and low-dose Cy (cohort 1); and underwent restaging imaging are evaluable for response. 
 
Definition of survival endpoints 
PFS is defined as the time from initiation of treatment till cancer relapse or death. All suspected 
relapses should be biopsied to confirm relapse. In the event where this is difficult and/or 
dangerous, imaging may be used as a surrogate. 
 
OS is defined as the time from initiation of treatment till death. Where possible, investigators 
should endeavor to confirm if death is related to disease or other concomitant illness. In the event 
where this is impossible to confirm conclusively, it will be assumed that death is related to disease 
progression. 
 
Kaplan-Meier estimates of PFS and OS will be provided. The corresponding median survival time 
(with 95% confidence intervals) will be determined, along with survival estimates at selected time 
points (e.g. 6 months, 1 years, and 2 years). 
 
9.5 Safety Analysis 
 
Definition of safety-evaluable patient 
Any patient who has received at least 1 dose each of nivolumab and pixatimod (cohorts 2 and 
3); and at least 1 dose each of nivolumab, pixatimod and cyclophosphamide (cohort 1) is 
evaluable for safety. 
 
As per NCI CTCAE Version 5.0, the term toxicity is defined as adverse events that are classified 
as either possibly, probably, or definitely related to study treatment. The maximum grade for each 
type of toxicity will be recorded for each patient, and frequency tables will be reviewed to 
determine toxicity patterns. 
 
As these agents singly and in combination have previously been studied in the diseases in 
question and the relevant biologically active doses determined, no “dose-escalation” will be 
performed in this study. However, to accurately capture toxicities for the use of this agent in this 
space, we will be monitoring toxicities closely. The detailed data and safety monitoring plan is in 
Section 9.7. 
 
9.6 Biomarker Analysis 
 
To search for potential prognostic biomarkers (and toxicity marker) for the regimen, logistic 
regression will be used to assess the association between each marker and objective 
radiographic response. 
 
The Cox proportional hazards model will be used to assess the association of each marker and 
survival endpoints (i.e. PFS and OS). 


71 
 
9.7 Data Safety and Monitoring Plan 
 
All enrolled patients will be reviewed weekly to discuss AEs, in particular during DLT period. 
 
Principle Investigator/Sub-investigators, regulatory, CRS management, clinical research 
coordinators, clinical research associates, data managers, and clinic staff meet monthly in 
disease center Data Safety Monitoring Boards (DSMB) to review and discuss study data to 
include, but not limited to, the following: 
 
• 
SAEs 
 
• 
Subject safety issues 
 
• 
Recruitment issues 
 
• 
Accrual 
 
• 
Protocol deviations 
 
• 
Unanticipated problems 
 
• 
Breaches of confidentiality 
 
Minutes from the disease center DSMB meetings are sent to those who are unable to 
participate during the scheduled meeting time. 
 
All toxicities encountered during the study will be evaluated on an ongoing basis according to the 
NCI Common Toxicity Criteria version 5.0. All study treatment associated adverse events that are 
serious, at least possibly related and unexpected will be reported to the IRB. Any modifications 
necessary to ensure subject safety and decisions to continue or close the trial to accrual are also 
discussed during these meetings. If any literature becomes available which changes the 
risk/benefit ratio or suggests that conducting the trial is no longer ethical, the IRB will be notified 
in the form of an Unanticipated Problem submission and the study may be terminated. 
 
All study data reviewed and discussed during these meetings will be kept confidential. Any breach 
in subject confidentiality will be reported to the IRB in the form of an Unanticipated Problem 
submission. The summaries of these meetings are forwarded to the UPCI DSMC which also 
meets monthly following a designated format. 
 
For all research protocols, there will be a commitment to comply with the IRB’s policies for 
reporting unanticipated problems involving risk to subjects or others (including adverse events). 
DSMC progress reports, to include a summary of all serious adverse events and modifications, 
and approval will be submitted to the IRB at the time of renewal. 
 
Protocols with subjects in long-term (survival) follow-up or protocols in data analysis only, will be 
reviewed twice a year rather than monthly by the disease center DSMB. 
 
Both the UPMC Hillman Cancer Center DSMC as well as the individual disease center DSMB 
have the authority to suspend accrual or further investigate treatment on any trial based on 
information discussed at these meetings. 


72 
 
All records related to this research study will be stored in a double locked environment. Only the 
researchers affiliated with the research study and their staff will have access to the research 
records. 
 
10. 
LABELING, PACKAGING, STORAGE, AND RETURN OF CLINICAL SUPPLIES 
 
10.1 Investigational Product: Pixatimod (PG545) 
 
The investigator shall take responsibility for and shall take all steps to maintain appropriate 
records and ensure appropriate supply, storage, handling, distribution and usage of 
investigational product in accordance with the protocol and any applicable laws and regulations. 
 
Clinical supplies provided by Aculeus Therapeutics are summarized in Table 10.1-1. 
 
Table 10.1-1: Product Description 
Product Name 
Dosage Form 
Pixatimod (PG545) 
Pixatimod is a white to off-white powder with no odor. It is manufactured using 
maltotetraose derived from hydrolysis of starch. The compound also features 
a cholestanol moiety conjugated to the synthetic, sulfated tetrasaccharide 
component. This lipophilic moiety is considered a critical structural feature of 
the molecule which leads to novel immunomodulatory activity and enhanced 
pharmacodynamic and pharmacokinetic properties compared to similarly 
designed compounds (known as heparan sulfate mimetics) lacking the 
aglycone. Pixatimod is presented in an anomerically pure form. 
Pixatimod drug product is presented as a frozen aqueous solution for infusion 
containing 60 mg or 100 mg pixatimod per vial (25 mg/mL). Prior to 
administration the appropriate dose of pixatimod is diluted in 250 mL 0.9% 
saline infusion solution and infused over one hour. 
 
 
10.1.1 Packaging and Labeling Information: Pixatimod (PG545) 
 
The pixatimod drug product vials will be labeled with the following information: 
• 
Hillman Cancer Center (HCC) protocol number 
• 
The batch number of the drug 
• 
The drug name, concentration, and nominal volume per vial 
• 
The recommended storage conditions of the secondary container 
• 
Cautionary statement to keep away from children 
• 
The route of administration 
• 
Cautionary statement indicating that the drug is for investigational use only 
• 
The name and address of Aculeus Therapeutics (Aculeus Therapeutics Pty Ltd; Bio 21 
Incubator, 30 Flemington Rd, Parkville VIC 3052, Australia). 
 
The pixatimod kit carton will be labeled with the following information: 
• 
Hillman Cancer Center (HCC) protocol number 
• 
Number of vials per kit 
• 
The batch number of the drug 
• 
The drug name, concentration, and nominal volume per vial 
• 
The recommended storage conditions of the drug 
• 
Cautionary statement to keep away from children 
• 
The route of administration 
• 
Cautionary statement indicating that the drug is for investigational use only; 
• 
The name and address of Aculeus Therapeutics (Aculeus Therapeutics Pty Ltd; Bio 21 
Incubator, 30 Flemington Rd, Parkville VIC 3052, Australia) 


73 
 
10.1.2 Handling, Storage and Accountability: Pixatimod (PG545) 
 
All pixatimod drug product vials will be transported, received, stored, and handled in accordance 
with the container or drug product kit/vial label, the instructions supplied to the site and its 
designated pharmacy personnel, the site’s standard operating procedures (SOPs), and applicable 
regulations. 
 
Appropriate storage and transportation conditions will be maintained for the pixatimod drug 
product vials from the point of manufacture up to delivery of pixatimod. All shipments of pixatimod 
drug product vials will include a temperature monitoring device that records required storage 
conditions for the vials, at regular intervals for the entire time the shipment is in transit. 
 
Upon receipt by the site, the designated site personnel will examine the shipment and temperature 
monitoring devices to verify the pixatimod drug product vials were received in acceptable 
condition. If not received in acceptable condition, the site must notify Aculeus Therapeutics and 
the site should quarantine the drug until a decision has been made by Aculeus Therapeutics. 
Once inspected, vials should be stored at the specified temperature (-20°C) in a locked area 
accessible only to designated site personnel until dispensing. Once dispensed, pixatimod drug 
product vials will be stored in a limited access area under appropriate environmental conditions. 
 
The designated site personnel will be responsible for maintaining accurate records of the quantity 
and dates of all study drug supplies received, dispensed, and returned, in accordance with 
applicable regulations and the site’s SOPs. The quantity of study drug lost, destroyed, etc. must 
also be accounted for and documented. 
 
All original vials, whether empty or containing pixatimod will be kept at the site and destroyed 
according to the site’s drug destruction standard operating procedures. Used pixatimod vials will 
not be dispensed again (even to the same subject) nor will they be relabeled or reassigned for 
use by other subjects. Contents of the pixatimod drug product vials will not be combined. At the 
termination of the study, a final drug accountability review and reconciliation must be completed, 
and any discrepancies must be investigated and their resolution documented. 
 
All pixatimod drug product vials will be destroyed onsite as per institutional standard operating 
procedures, after site close out has been completed. 
10.1.3 Dispensing: Pixatimod (PG545) 
 
The pixatimod drug product vials will be dispensed and pixatimod drug product will be 
administered according to applicable site SOPs. Details regarding the preparation and 
administration of the study drug will be outlined in a study-specific pharmacy manual. Only eligible 
subjects participating in the study will receive pixatimod. Only authorized and qualified site staff 
may supply or administer pixatimod. 
 
 
10.2 Commercial Product: Nivolumab 
 
Pharmaceutical and therapeutic background of nivolumab is detailed in Section 3.2. Preclinical 
and clinical trial data pertaining to nivolumab is summarized in Nivolumab IB. For the purposes 
of this trial, commercial supply of nivolumab will be used. Nivolumab dose, schedule and 
administration is detailed in Section 3.8.3. 


74 
 
10.3 Commercial Product: Cyclophosphamide 
 
Pharmaceutical and therapeutic background of cyclophosphamide is detailed in Section 3.4. 
Preclinical and clinical trial data pertaining to cyclophosphamide is summarized in Section 3.4. 
For the purposes of this trial, commercial supply of cyclophosphamide will be used. 
Cyclophosphamide dose, schedule and administration is detailed in Section 3.8.6. 


75 
 
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84 
 
12. 
APPENDICES 
 
12.1 Appendix 1: ECOG Performance Status125 
 
Grade 
Description 
0 
Normal activity. Fully active, able to carry on all pre-disease performance without 
restriction. 
1 
Symptoms, but ambulatory. Restricted in physically strenuous activity, but 
ambulatory and able to carry out work of a light or sedentary nature (e.g., light 
housework, office work). 
2 
In bed <50% of the time. Ambulatory and capable of all self-care, but unable to carry 
out any work activities. Up and about more than 50% of waking hours. 
3 
In bed >50% of the time. Capable of only limited self-care, confined to bed or chair 
more than 50% of waking hours. 
4 
100% bedridden. Completely disabled. Cannot carry on any self-care. Totally 
confined to bed or chair. 
5 
Dead. 
*As published in Oken MM et al, Am J Clin Oncol 1982.125 


85 
 
12.2 
Appendix 2: Common Terminology Criteria for Adverse Events V5.0 
(CTCAE) 
 
The descriptions and grading scales found in the revised NCI Common Terminology 
Criteria for Adverse Events (CTCAE) version 5.0 will be utilized for adverse event 
reporting. (http://ctep.cancer.gov/reporting/ctc.html) 


86 
 
12.3 
Appendix 3: Contraceptive Guidance and Pregnancy Testing 
Woman of Childbearing Potential (WOCBP) 
• 
A woman is considered fertile following menarche and until becoming post-menopausal 
unless permanently sterile (see below) 
• 
Women in the following categories are not considered WOCBP: 
• 
Premenarchal 
• 
Premenopausal female with 1 of the following: 
o Documented hysterectomy 
o Documented bilateral salpingectomy 
o Documented bilateral oophorectomy 
o Note: Documentation can come from the site personnel’s review of the 
participant’s medical records, medical examination, or medical history 
interview. 
• 
Postmenopausal female 
o A postmenopausal state is defined as no menses for 12 months without an 
alternative medical cause. 
o A high follicle stimulating hormone (FSH) level in the postmenopausal range 
may be used to confirm a postmenopausal state in women not using hormonal 
contraception or hormonal replacement therapy (HRT). However, in the 
absence of 12 months of amenorrhea, confirmation with two FSH 
measurements in the postmenopausal range is required. 
o Females on HRT and whose menopausal status is in doubt will be required to 
use one of the non-hormonal highly effective contraception methods if they 
wish to continue their HRT during the study. Otherwise, they must discontinue 
HRT to allow confirmation of postmenopausal status before study enrollment. 
Contraception Requirements 
Male Participants: 
Male participants with female partners of childbearing potential are eligible to participate if they 
agree to one of the following during the protocol defined time frame in Section 6.1. 
 
● Be abstinent from penile-vaginal intercourse as their usual and preferred lifestyle (abstinent 
on a long term and persistent basis) and agree to remain abstinent. 
● Use a male condom plus partner use of a contraceptive method with a failure rate of <1% per 
year as described in Table A3-1 when having penile-vaginal intercourse with a woman of 
childbearing potential who is not currently pregnant. 
○ Note: Men with a pregnant or breastfeeding partner must agree to remain abstinent from 
penile-vaginal intercourse or use a male condom during each episode of penile 
penetration. 
 
Female Participants: 


87 
 
Female participants of childbearing potential are eligible to participate if they agree to use a highly 
effective method of contraception consistently and correctly as described in Table A3-1 during 
the protocol-defined time frame in Section 6.1. 
 
Table A3-1 Highly Effective Contraception Methods 
Highly Effective Contraceptive Methods That Are User Dependent a 
Failure rate of <1% per year when used consistently and correctly. 
● 
Combined (estrogen- and progestogen- containing) hormonal contraception b, c 
○ 
Oral 
○ 
Intravaginal 
○ 
Transdermal 
○ 
Injectable 
● 
Progestogen-only hormonal contraception b, c 
○ 
Oral 
○ 
Injectable 
Highly 
Effective 
Methods 
That 
Have 
Low 
User 
Dependency 
Failure rate of <1% per year when used consistently and correctly. 
● 
Progestogen- only contraceptive implant b, c 
● 
Intrauterine hormone-releasing system (IUS) b 
● 
Intrauterine device (IUD) 
● 
Bilateral tubal occlusion 
● 
Vasectomized partner 
A vasectomized partner is a highly effective contraception method provided that the partner is the sole male sexual
partner of the WOCBP and the absence of sperm has been confirmed. If not, an additional highly effective method
of contraception should be used. 
● 
Sexual abstinence 
Sexual abstinence is considered a highly effective method only if defined as refraining from heterosexual intercourse
during the entire period of risk associated with the study treatment. The reliability of sexual abstinence needs to be
evaluated in relation to the duration of the study and the preferred and usual lifestyle of the participant.) 
Notes: 
Use should be consistent with local regulations regarding the use of contraceptive methods for participants of clinical 
studies. 
a) Typical use failure rates are lower than perfect-use failure rates (i.e. when used consistently and correctly). 
b) If locally required, in accordance with Clinical Trial Facilitation Group (CTFG) guidelines, acceptable hormonal 
contraceptives are limited to those which inhibit ovulation. 
 
 
Pregnancy Testing 
WOCBP should only be included after a negative highly sensitive urine or serum pregnancy test. 
Pregnancy testing will be performed whenever an expected menstrual cycle is missed or when 
pregnancy is otherwise suspected. 
12.4 Appendix 4: Description of the iRECIST Process for Assessment of Disease 
Progression 
 
Assessment at Screening and Prior to RECIST 1.1 Progression 
Until radiographic progression based on RECIST 1.1, there is no distinct iRECIST 
assessment. 
 
Assessment and Decision at RECIST 1.1 Progression 


88 
 
In participants who show evidence of radiological PD by RECIST 1.1 the Investigator will 
decide whether to continue a participant on study treatment until repeat imaging is 
obtained using iRECIST for participant management (see Section 8.7.6, Figure 8.7.6-1 
and Table 8.7.6-2). This decision by the Investigator should be based on the participant’s 
overall clinical condition. 
Clinical stability is defined as the following: 
• Absence of symptoms and signs indicating clinically significant progression of 
disease 
• No decline in ECOG performance status 
• No requirements for intensified management, including increased analgesia, or 
other palliative care 
Any participant deemed clinically unstable should be discontinued from study treatment 
at site-assessed first radiologic evidence of PD and is not required to have repeat tumor 
imaging 
for 
confirmation 
of 
PD 
by 
iRECIST. 
 
If the Investigator decides to continue treatment, the participant may continue to receive 
study treatment and the tumor assessment should be repeated 4 to 8 weeks later to 
confirm    PD    by    iRECIST,    per    Investigator    assessment. 
 
Tumor flare may manifest as any factor causing radiographic progression per RECIST 
1.1, including: 
• Increase in the sum of diameters of target lesion(s) identified at baseline to ≥ 20% 
and ≥ 5 mm from nadir 
o Please note: the iRECIST publication uses the terminology “sum of 
measurements”, but “sum of diameters” will be used in this protocol, consistent 
with the original RECIST 1.1 terminology. 
• Unequivocal progression of non-target lesion(s) identified at baseline 
• Development of new lesion(s) 
iRECIST defines new response categories, including iUPD (unconfirmed progressive 
disease) and iCPD (confirmed progressive disease). For purposes of iRECIST 
assessment, the first visit showing progression according to RECIST 1.1 will be assigned 
a visit (overall) response of iUPD, regardless of which factors caused the progression. 
 
At this visit, target and non-target lesions identified at baseline by RECIST 1.1 will be 
assessed as usual. 
 
New lesions will be classified as measurable or non-measurable, using the same size 
thresholds and rules as for baseline lesion assessment in RECIST 1.1. From measurable 
new lesions, up to 5 lesions total (up to 2 per organ), may be selected as New Lesions – 
Target. The sum of diameters of these lesions will be calculated and kept distinct from 
the sum of diameters for target lesions at baseline. All other new lesions will be followed 
qualitatively as New Lesions – Non-target. 


89 
 
Assessment at the Confirmatory Imaging 
 
On the confirmatory imaging, the participant will be classified as progression confirmed 
(with an overall response of iCPD), or as showing persistent unconfirmed progression 
(with an overall response of iUPD), or as showing disease stability or response 
(iSD/iPR/iCR). 
 
Confirmation of Progression 
 
Progression is considered confirmed, and the overall response will be iCPD, if ANY of the 
following occurs: 
 
• Any of the factors that were the basis for the initial iUPD show worsening 
o For target lesions, worsening is a further increase in the sum of diameters of 
≥ 5 mm, compared to any prior iUPD time point 
o For non-target lesions, worsening is any significant growth in lesions overall, 
compared to a prior iUPD time point; this does not have to meet the 
“unequivocal” standard of RECIST 1.1 
o For new lesions, worsening is any of these: 
 An increase in the new lesion sum of diameters by ≥ 5 mm from a prior iUPD 
time point 
 Visible growth of new non-target lesions 
 The appearance of additional new lesions 
• Any new factor appears that would have triggered PD by RECIST 1.1 
Persistent iUPD 
 
Progression is considered not confirmed, and the overall response remains iUPD, if: 
 
• None of the progression-confirming factors identified above occurs AND 
• The target lesion sum of diameters (initial target lesions) remains above the initial 
PD threshold (by RECIST 1.1) 
 
Additional imaging for confirmation should be scheduled 4 to 8 weeks from the scan on 
which iUPD is seen. This may correspond to the next visit in the original visit schedule. 
The assessment of the subsequent confirmation scan proceeds in an identical manner, 
with possible outcomes of iCPD, iUPD, and iSD/iPR/iCR. 
 
Resolution of iUPD 
 
Progression is considered not confirmed, and the overall response becomes iSD/iPR/iCR, 
if: 


90 
 
• None of the progression-confirming factors identified above occurs, AND 
• The target lesion sum of diameters (initial target lesions) is not above the initial PD 
threshold. 
 
The response is classified as iSD or iPR (depending on the sum of diameters of the target 
lesions), or iCR if all lesions resolve. 
 
In this case, the initial iUPD is considered to be pseudo-progression, and the level of 
suspicion for progression is “reset”. This means that the next visit that shows radiographic 
progression, whenever it occurs, is again classified as iUPD by iRECIST, and the 
confirmation process is repeated before a response of iCPD can be assigned. 
 
Management Following the Confirmatory Imaging 
 
If repeat imaging does not confirm PD per iRECIST, as assessed by the Investigator, and 
the participant continues to be clinically stable, study treatment may continue and follow 
the regular imaging schedule. If PD is confirmed, participants will be discontinued from 
study treatment. 
 
NOTE: If a participant has confirmed radiographic progression (iCPD) as defined above, 
but the participant is achieving a clinically meaningful benefit, an exception to continue 
study treatment may be considered. In this case, if study treatment is continued, tumor 
imaging should continue to be performed following the intervals as outlined in Section 6. 
 
Detection of Progression at Visits After Pseudo-progression Resolves 
 
After resolution of pseudo-progression (ie, achievement of iSD/iPR/iCR), iUPD is 
indicated by any of the following events: 
 
• Target lesions 
o Sum of diameters reaches the PD threshold (≥ 20% and ≥ 5 mm increase from 
nadir) either for the first time, or after resolution of previous pseudo- 
progression. The nadir is always the smallest sum of diameters seen during 
the entire trial, either before or after an instance of pseudo-progression. 
• Non-target lesions 
o If non-target lesions have never shown unequivocal progression, their doing so 
for the first time results in iUPD. 
o If non-target lesions had shown previous unequivocal progression, and this 
progression has not resolved, iUPD results from any significant further growth 
of non-target lesions, taken as a whole. 
• New lesions 
o New lesions appear for the first time 
o Additional new lesions appear 


91 
 
o Previously identified new target lesions show an increase of ≥ 5 mm in the new 
lesion sum of diameters, from the nadir value of that sum 
o Previously identified non-target lesions show any significant growth 
If any of the events above occur, the overall response for that visit is iUPD, and the iUPD 
evaluation process (see Assessment at the Confirmatory Imaging above) is repeated. 
Progression must be confirmed before iCPD can occur. 
 
The decision process is identical to the iUPD confirmation process for the initial PD, 
except in one respect. If new lesions occurred at a prior instance of iUPD, and at the 
confirmatory scan the burden of new lesions has increased from its smallest value (for 
new target lesions, their sum of diameters is ≥5 mm increased from its nadir), then iUPD 
cannot resolve to iSD or iPR. It will remain iUPD until either a decrease in the new lesion 
burden allows resolution to iSD or iPR, or until a confirmatory factor causes iCPD. 
 
Additional details about iRECIST are provided in the iRECIST publication.123 


92 
 
12.5 Appendix 5: Dietary History Questionnaire (DHQ-3) Administration 
 
Dietary history questionnaire will be administered either on paper or electronically and data entered 
and stored securely electronically. Instrument will be administered by PI or designee. 
 
Detailed information for study staff: 
1. 
Please obtain patient’s username and password from study PI. 
2. 
Please provide instructions (see below) to patients. 
3. 
Patients are to be instructed to provide detailed dietary information either during visit (preferred) or 
at home. 
4. 
Please instruct the patients to report intake as averaged over the preceding 4 weeks. 
 
Detailed information for patients: 
• 
Thank you for participating in this study. We are interested in evaluating your dietary history. To do 
this we are using a validated questionnaire termed the “Diet History Questionnaire (DHQ-3)”. 
• 
This questionnaire takes approximately 30 minutes to complete. You can do this either while 
receiving your therapy or at home. 
• 
After completing the questionnaire, you will receive a Respondent Nutrition Report. This report 
shows estimated daily nutrient and food group intakes based on questionnaire responses. Recommended 
values are only available for some nutrients and food groups. 
• 
Please feel free to discuss this with your study doctor. 
 
Dietary study login URL: 
Your username (case sensitive):  
 
Your study password (case sensitive):