Clinical trial NCT03029988
Conditions: Liver Metastases Colorectal Carcinoma
Keywords: adenocarcinoma, Colorectal Carcinoma, liver metastases
Sponsor: Navidea Biopharmaceuticals
Start date: 2017-07-26; Completion date: 2018-09-24
Enrollment: 3 participants; Allocation: NON_RANDOMIZED; Intervention model: SEQUENTIAL

Brief summary:
Estimation of the concordance of Tc 99m localization in liver metastases from colorectal carcinoma using SPECT/CT imaging and abdominal FDG (PET)/CT imaging per subject.

Detailed description:
This is a prospective, open-label, single-center, comparator study of IV injected Tc 99m Tilmanocept in the localization and detection of liver metastases in subjects with confirmed colorectal carcinoma (CRC). The study will be divided into two cohorts.

If it was determined that additional enrollment would not provide meaningful data, for example no metastatic liver lesions were visualized by Tc 99m tilmanocept for any of the subjects in the cohort, enrollment into Cohort 2 would begin and 3 subjects would be enrolled followed by a review of the imaging and safety data.

This study is designed to evaluate the safety and tolerability of Tc 99m tilmanocept administered intravenously. SPECT/CT imaging of the subject's abdominal cavity will be reviewed to establish concordance with FDG PET imaging.

Arms: arm 1: Subjects will receive 50 µg tilmanocept radiolabeled with 2.0 millicuries (mCi) Tc99m through a single IV injection. arm 2: Subjects will receive 200 µg tilmanocept radiolabeled with 2.0 mCi Tc99m through a single IV injection.

If it was determined that additional enrollment would not provide meaningful data, for example no metastatic liver lesions were visualized by Tc 99m tilmanocept for any of the subjects in the cohort, enrollment into Cohort 2 would begin and 3 subjects would be enrolled followed by a review of the imaging and safety data.
Interventions: intervention 1: Tilmanocept (Technetium Tc 99m tilmanocept Injection) intervention 2: SPECT/CT Imaging
Intervention descriptions: intervention 1: Drug: Technetium Tc 99m tilmanocept intervention 2: 4-6 hours post-injection SPECT/CT will be obtained in the abdominal region.

Protocol documents:
Prot_SAP_001.pdf: 
Clinical Trial Protocol: NAV3-25 
Study Title: 
An Exploratory Evaluation of Technetium Tc 99m Tilmanocept by 
Intravenous (IV) Injection in Subjects with Liver Metastases from 
Colorectal Carcinoma Patients using SPECT/CT Imaging Compared 
to FDG PET/CT Imaging. 
Study Number: 
NAV3-25 
Study Phase: 
1 
Product Name: 
Tilmanocept (Technetium Tc 99m tilmanocept)  
IND Number: 
132943 
Investigator(s): 
University of Alabama Birmingham 
 
 
Sponsor: 
Navidea Biopharmaceuticals 
4995 Bradenton Ave, Suite 240  
Dublin, OH 43017 
 
Sponsor Contact: 
Allison Kissling, CCRP 
Associate Clinical Research Project Manager 
+1 614-973-7551 
 
Medical Monitor: 
Michael Blue, MD 
Senior Medical Director 
+1 614-571-4313 
 
Date 
Original Protocol: 
Amendment 1: 
Amendment 2:  
Amendment 3: 
13 March 2017 
26 April 2017 
08 June 2017 
20 September 2017 
Confidentiality Statement 
This document and its contents are the property of and confidential to Navidea 
Biopharmaceuticals. Any unauthorized copying or use of this document is prohibited  
 
 


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TABLE OF CONTENTS 
SYNOPSIS .................................................................................................................................5 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS ............................................9 
TRIAL ADMINISTRATIVE STRUCTURE ..........................................................................11 
1 
BACKGROUND AND SIGNIFICANCE .......................................................................12 
1.1 
The Unmet Needs: Need for earlier detection of hepatic metastases of 
colorectal primary tumors. .....................................................................................12 
1.2 
The Macrophage Mannose Receptor (CD206) ......................................................12 
1.3 
Current Approval ...................................................................................................13 
1.3.1 
Nonclinical Evaluations – IV AND IP Administration ...............................16 
1.3.2 
Clinical Safety ..............................................................................................18 
1.3.3 
Radiation Dosimetry ....................................................................................19 
1.3.4 
Radiation Exposure ......................................................................................20 
2 
TRIAL OBJECTIVES .....................................................................................................20 
2.1 
Primary Objective(s) ..............................................................................................20 
2.2 
Secondary Objective(s) ..........................................................................................20 
3 
OVERVIEW OF METHODOLOGY AND DESIGN .....................................................21 
3.1 
Overall Trial Design ..............................................................................................21 
3.2 
Justification for Study Design and Population.......................................................21 
3.3 
Protocol Adherence ................................................................................................21 
3.4 
Trial Duration.........................................................................................................21 
4 
STUDY POPULATION ..................................................................................................22 
4.1 
Inclusion Criteria ...................................................................................................22 
4.2 
Exclusion Criteria ..................................................................................................22 
4.3 
Recruitment ............................................................................................................22 
4.4 
Withdrawal .............................................................................................................23 
4.5 
Enrollment and Screen Failures .............................................................................23 
5 
INVESTIGATIONAL PRODUCT ..................................................................................24 
5.1 
Identification of Investigational Product ...............................................................24 
5.2 
Investigational Product Dosage and Administration .............................................24 
5.3 
Treatment Assignment ...........................................................................................24 
5.4 
Packaging and Labeling .........................................................................................24 
6 
THERAPIES OTHER THAN INVESTIGATIONAL PRODUCT .................................26 
6.1 
Prior and Concomitant Therapy .............................................................................26 
6.2 
Post-Study Therapy ................................................................................................26 
7 
STUDY PROCEDURES .................................................................................................27 
7.1 
Screening Visit (Day -29 to Day 0) .......................................................................27 


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7.2 
Day 1 (Injection and Imaging Procedures) ............................................................27 
7.2.1 
Before Injection ...........................................................................................27 
7.2.2 
Injection of Tc 99m tilmanocept ..................................................................27 
7.2.3 
Post-Injection ...............................................................................................28 
7.2.4 
Imaging and Acquisition of Imaging Data ...................................................28 
7.2.5 
Image Acquisition ........................................................................................29 
7.2.6 
Evaluation of SPECT/CT Images ................................................................29 
7.2.7 
End of Study ................................................................................................29 
8 
PROCEDURES AND VARIABLES ..............................................................................30 
8.1 
Population Characteristics .....................................................................................30 
8.1.1 
Demographic and Other Baseline Characteristics .......................................30 
8.1.2 
Medical and Surgical History ......................................................................30 
8.1.3 
Prior and Concomitant Medication ..............................................................30 
8.2 
Safety .....................................................................................................................30 
8.2.1 
Adverse Events ............................................................................................30 
8.2.1.1 
Definition of Adverse Event ..............................................................30 
8.2.1.2 
Categories for Adverse Event Assessment ........................................31 
8.2.1.3 
Assessments and Documentation of Adverse Events ........................32 
8.2.1.4 
Expected Adverse Events ..................................................................32 
8.2.1.5 
Serious Adverse Events .....................................................................33 
8.2.2 
Further Safety Assessments .........................................................................34 
8.2.2.1 
Physical Examination.........................................................................34 
8.2.2.2 
Vital Signs ..........................................................................................35 
8.3 
Other Procedures and Variables.............................................................................35 
8.3.1 
Clinical Laboratory Parameters for Screening and Safety ...........................35 
8.3.2 
Blood Sampling ...........................................................................................36 
9 
STATISTICAL METHODS ............................................................................................37 
10 DATA HANDLING AND QUALITY ASSURANCE ...................................................38 
10.1 
Data Recording ......................................................................................................38 
10.1.1 
CRF Design ..................................................................................................38 
10.2 
Monitoring .............................................................................................................38 
10.3 
Data Processing ......................................................................................................38 
10.4 
Auditing .................................................................................................................38 
10.5 
Archiving ...............................................................................................................39 
10.6 
Premature Termination of the Trial .......................................................................39 
10.6.1 
Trial as a Whole ...........................................................................................39 
10.6.2 
Center ...........................................................................................................40 
10.6.3 
Study Participant ..........................................................................................40 
11 ETHICAL AND LEGAL ASPECTS...............................................................................41 


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11.1 
Ethical and Legal Conduct of the Study ................................................................41 
11.2 
Subject Information and Consent...........................................................................41 
11.3 
Financing/Financial Disclosure .............................................................................42 
11.4 
Publication Policy ..................................................................................................42 
11.5 
Subject Injury .........................................................................................................42 
12 REFERENCE LIST .........................................................................................................43 
 
LIST OF IN-TEXT TABLES 
Table 1.  
The Lymphoseek radiation doses (mGy/MBq) to organs and tissues of 
an average patient (70 kg) with breast cancer or melanoma .......................19 
Table 2.  
Dose Cohort  and Route of Administration Groups.....................................27 
Table 3.  
Clinical Laboratory Parameters ...................................................................36 
Table 4.  
Amount of Blood Withdrawn ......................................................................36 
 
LIST OF IN-TEXT FIGURES 
Figure 1:  
Structure of 99mTc-tilmanocept showing attachment of multiple units 
of DTPA and mannose to a backbone of dextran. The chelating agent, 
DTPA, is shown holding an atom of 99mTechnetium ..................................13 
Figure 2.  
Presurgical lymphoscintigraphy SPECT/CT (3D) image of breast cancer 
patient injected subdermally with 99mTc-tilmanocept. Imaging taken 1 
hour after injection clearly shows localization of 99mTc-tilmanocept in 
a SLN. ..........................................................................................................15 
 
LIST OF APPENDICES 
Appendix 1 
Schedule of Events .............................................................................46 
Appendix 2 
ECOG Performance Status ................................................................47 
Appendix 3 
Sponsor Signatures.............................................................................48 
Appendix 4 
Investigator’s Signature .....................................................................49 
 
 
 
 


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SYNOPSIS 
Study Title 
An Exploratory Evaluation of Technetium Tc 99m Tilmanocept by 
Intravenous (IV) Injection in Subjects with Liver Metastases from 
Colorectal Carcinoma Patients using SPECT/CT Imaging 
Compared to FDG PET/CT Imaging. 
Study Phase 
Phase 1 
Study Objectives 
Primary Objective(s) 
• Estimation of the concordance of Tc 99m localization in liver 
metastases from colorectal carcinoma (CRC) using SPECT/CT 
(single photon emission computed tomography) imaging and 
abdominal FDG Positron Emission Tomography (PET)/CT 
imaging per subject. 
• Evaluation of safety including monitoring the incidence of 
adverse events and changes over time in laboratory tests, vital 
signs, and physical examination findings. 
 
Secondary Objective(s) 
• Estimation of  the optimal dose of Technetium Tc 99m 
tilmanocept. 
• Evaluation of other areas of localization of Technetium Tc 
99m tilmanocept. 
 
Duration of Study 
Participation 
Approximately 35 days  
Study Drug 
Tilmanocept (Technetium Tc 99m tilmanocept Injection)  
Dose(s) and route of 
administration 
2 Dose Cohorts: 
1) IV injection: 2 mCi, 50 µg 
2) IV injection: 2 mCi, 200 µg 
 
Inclusion Criteria 
1. The subject has provided written informed consent with 
Health Information Portability and Accountability Act 
(HIPAA) authorization before the initiation of any study-
related procedures. 
2. Subjects must be ≥18 years old; 
3. The subject must have a diagnosis of adenocarcinoma of 
the colon and/or rectum with confirmed metastases to the 
liver; 
4. The subjects must have an ECOG performance status of 0-
3; 
5. The subject must be at least 4 weeks past any major 
intraabdominal surgery, including surgery to the liver; 
6. Subjects with prior malignancies other than colon and/or 
rectum cancer are allowed, provided they have been 


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treated with curative intent, and have no evidence of 
recurrence of that malignancy; 
7. If of childbearing potential, the subject has a negative urine 
pregnancy test within 48 hours before administration of Tc 
99m Tilmanocept, has been surgically sterilized, or has 
been postmenopausal for at least 1 year  
Exclusion Criteria 
1. The subject is pregnant or lactating. 
2. The subject has undergone any liver surgery, exclusive of a 
biopsy. 
3. The subject has known sensitivity to dextran. 
4. The subject has had preoperative chemotherapy, 
immunotherapy, or radiation therapy within the 10 days 
prior to Tc 99m Tilmanocept administration 
5. Before the administration of Tc 99m Tilmanocept, has 
received any radiopharmaceutical within 7 radioactive 
half-lives of that radiopharmaceutical 
6. Has received an investigational product within the 30 days 
prior to Tc 99m Tilmanocept administration 
Study design 
Prospective, open-label, single-center, comparator study of IV 
injected Tc 99m Tilmanocept in the localization and detection of 
liver metastases in subjects with confirmed CRC. The study will be 
divided into two cohorts as follows: 
 
Up to 12 subjects (up to 6 per cohort): 
1) 50 µg tilmanocept radiolabeled with 2 mCi Tc99m at 1.0 
mL through IV injection; n = up to 6 
2) 200 µg tilmanocept radiolabeled with 2 mCi Tc99m at 1.0 
mL through IV injection; n = up to 6 
 
Methodology 
The principal investigator (PI) will identify subjects diagnosed  
with CRC with confirmed metastasis to the liver  
 
Subjects will participate in 3 study visits (including screening) over 
the course of the study period. The screening visit will include an 
assessment of eligibility and informed consent. After consent has 
been obtained, subjects will undergo a clinical laboratory 
evaluation.  
Visit 1, Screening (Day -29 to Day 0): The screening visit will 
include informed consent, preliminary review of study eligibility, 
collection of medical history including medications, vital signs,  
physical exam including height and weight, clinical labs, urinalysis, 
and urine pregnancy test for subjects of childbearing potential.  
Subjects will  undergo a FDG PET/CT Scan to identify hepatic 
lesions.   


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Visit 2, Injection and Imaging Procedures (Day 1): 
Pre-Tc 99m Tilmanocept Injection:  
• 
Before injection (Day 1) 
o Urine pregnancy test will be performed for subjects 
of childbearing potential 
o Concomitant medication review 
o Vital Signs - within 15 minutes prior to injection 
 
Tc 99m Tilmanocept Injection: Subjects will receive their open 
label dose assignment in accordance with the 2 dosing groups. 
Post-Tc 99m Tilmanocept Injection:  
• 
Vital signs - within 15 minutes post-injection  
• 
4-6 hours post-injection: 
o Assessment of adverse events 
o SPECT/CT images will be obtained of the 
abdominal cavity (inclusive of entire liver). 
 
5±3 Days Post-injection Follow-Up Visit:  
• 
Vital Signs  
• 
Physical Exam 
• 
Clinical labs and urinalysis 
• 
Concomitant medication review 
• 
Assessment of adverse events 
 
General: Up to 12 subjects (2 cohorts as described above) will be 
injected intravenously with Tc99m (T½ = 6.02 hr).   
 
SPECT/CT Imaging  
Imaging and Acquisition of Imaging Data: Image acquisition of 
patients in this study will be via SPECT/CT scanning. Subjects will 
be imaged by SPECT/CT at 4-6 hours post injection.    
Planned Study Dates 
Start of Recruitment: 
July 2017 
 
End of Recruitment/April 2018 
End of Study/July 2018 
Planned number of study 
centers 
1 center in the United States - University of Alabama at 
Birmingham 
Number of subjects 
Total: Up to 12 evaluable subjects 
Primary Endpoint 
• Estimation of the concordance of Tc 99m SPECT/CT imaging 
localization with FDG PET/CT imaging per subject 


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• Proportion of subjects not experiencing an adverse drug 
reaction (ADR) in each dose group 
Secondary endpoints 
• Estimation of the  concordance of Tc 99m SPECT/CT imaging 
localization with FDG PET/CT imaging per lesion 
• Estimation of the concordance of FDG PET/CT  imaging 
localization with Tc 99m SPECT/CT imaging per lesion 
• Incidence of other areas of localization 
Plan for statistical 
analysis 
Due to the exploratory nature of this study all analyses described in 
this protocol will be descriptive in nature and used in support of 
designing a larger study. 
 


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LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS 
ADR 
adverse drug reaction 
AE 
adverse event 
ALT 
alanine aminotransferase 
AST 
aspartate aminotransferase 
BUN 
blood urea nitrogen 
CD206 
mannose-binding receptor (Ca2+ binding lectin) 
CRC 
colorectal carcinoma 
CRF 
case report form 
CRA 
clinical research associate 
CRO 
contract research organization 
CT 
X-ray computed tomography 
DICOM 
Digital Imaging Communications in Medicine 
DTPA 
pentetic acid 
ECOG 
Eastern Cooperative Oncology Group 
FDA 
Food and Drug Administration 
FDG 
fluorodeoxyglucose 
GCP 
Good Clinical Practice 
Hgb 
Hemoglobin 
HIPAA 
Health Information Portability and Accountability Act 
IB 
Investigator’s Brochure 
ICF 
informed consent form 
ICH 
International Conference on Harmonization  
IEC 
Independent Ethics Committee 
IHC 
Immunohistochemistry 
ILM 
Intraoperative lymphatic mapping  
IND 
Investigational new drug 
IP 
Investigational product  
IRB 
Institutional Review Board 


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ISF 
investigator site file 
IV 
Intravenous 
mCi 
Millicurie 
MedDRA 
Medical Dictionary for Regulatory Activities 
PET 
positron emission tomography 
PI 
principal investigator 
ROI 
region of interest 
SAE 
serious adverse event 
SD 
study day 
SLN 
Sentinel lymph node  
SLNB 
Sentinel lymph node biopsy 
SPECT 
single photon emission computed tomography 
SUSARs 
Suspected, Unexpected, Serious Adverse Reactions 
SUV 
standardized uptake values 
TAMs 
Tumor-associated macrophages 
Tc99m 
technetium-99m metastable isotope; γ-emitting (t½ = 6.02 h) 
Tilmanocept 
DTPA Mannosyl Dextran (the US Adopted Name for the drug substance of 
Lymphoseek) 
TMF 
trial master file 
WBC 
white blood cell 
 
 
 
 
 


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TRIAL ADMINISTRATIVE STRUCTURE 
The principal investigator (PI) must sign the protocol signature sheet before trial participant recruitment 
may start.  Likewise, all protocol amendments must be signed and dated by the PI before coming into 
effect. 
The name and address of the participating center, the investigators, and all required signature 
documents will be maintained in the trial master file (TMF). 
In addition to the PI, there are additional onsite roles that may be performed by other sub-investigators:   
• Subject referral to the trial 
• Review of subject eligibility and medical records  
• Safety assessments 
• Injection and Single Photon Emission Computer Tomography (SPECT) imaging  
 
Trial personnel not listed in this section are identified in a separate personnel list.  This list will be 
updated as needed.  The list of personnel will be available in the center’s investigator site file (ISF). 
 
 


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1 
BACKGROUND AND SIGNIFICANCE 
1.1 
The Unmet Needs: Need for earlier detection of hepatic metastases of 
colorectal primary tumors. 
The liver is a common site of metastases for primary colorectal tumors [1] with between one third and 
one half of all patients diagnosed with colorectal cancer developing hepatic metastases [2].  Perhaps 
fortunately, in 30% to 40% of patients who develop advanced disease, the liver is the only site of 
metastatic disease [3]. As described in the next two sections, it is expected that 99mTc-tilmanocept 
will localize to hepatic metastases of colorectal tumors. If so, it should be possible to image such 
metastatic tumors using SPECT imaging (3D). There are currently available alternative imaging 
technologies, such as fluorodeoxyglucose positron emission tomography/computed tomography 
(FDG PET/CT),  that are used to visualize hepatic tumors. There is a possibility that 99mTc-
tilmanocept enabled imaging may have performance characteristics that would make it preferable to 
the alternatives for imaging of these tumors. If this is demonstrated to be the case, then 99mTc-
tilmanocept would have utility as an imaging agent that could assist physicians to identify hepatic 
metastases, develop surgical plans and monitor patients for the efficacies of non-surgical therapies. 
This study will develop preliminary data relevant to the potential utility of 99mTc-tilmanocept as an 
imaging agent to identify and evaluate metastatic colorectal tumors in the liver.  
1.2 
The Macrophage Mannose Receptor (CD206)  
The Macrophage Mannose Receptor (CD206) is abundantly expressed on tumor associated 
macrophages (TAMs) and on macrophages that reside in regions immediately adjacent to tumors. 
CD206 is a C-type carbohydrate transmembrane, non-circulating binding protein that is expressed on 
the exterior surfaces of macrophages [4-6]. Each CD206 receptor has eight carbohydrate binding 
domains that bind to mannose (3 with mannose specificity) and 5 with recognition of fructose, 
galactose, and N-acetylglucosamine, in addition to mannose. An important feature of the macrophage 
mannose receptor is that multivalent binding is required for high affinity interaction between the 
receptor and its ligands [7]. It is also important to recognize that the level of CD206 expression on 
macrophages can vary greatly between macrophages. Macrophages respond to factors in their 
environments, such as the levels of various cytokines, cellular debris, pathogen derived materials and 
immune complexes, to alter their expression patterns of numerous genes and to acquire various 
activated states or phenotypes [8-10].  The level of CD206 expression is highly dependent on the 
activation state of a macrophage [11]. Our understanding of the full phenotypic plasticity of 
macrophages is still negligible and has as of yet to be fully elucidated.  
TAMs are significant contributors to the cellularity of tumors. In established tumors, the majority of 
TAMs suppress anti-tumor immune responses and secrete various signaling molecules that promote 
tumor growth and progression [11-14]. This common and tumor promoting TAM phenotype includes 
the significant up regulation and abundant expression of CD206 [11, 15-17].  Prior to initiating this 
study, Navidea Biopharmaceuticals, through its academic collaborators, examined histological sections 
of surgical specimens obtained through resections of hepatic metastases of colorectal primary tumors. 
These sections were evaluated by immunohistochemical (IHC) staining using an anti-CD206 antibody. 
These studies confirmed, as has been observed in IHC evaluations using anti-CD206 antibodies of 
similar histological sections derived from a wide variety of cancer types, that macrophages that highly 
express CD206 are common components of the tumors. There were also much lesser but still elevated 
concentrations of CD206 expressing macrophages in anatomically normal tissue that was adjacent to 
the tumor. It is expected that the density of CD206 expressing macrophages will diminish to 
background levels as the distance from the tumor margin increases.  


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The study described in this protocol will evaluate the ability intravenously (IV) injected 99mTc-
tilmanocept to localize to CD206 expressing TAMs and possibly also to CD206 expressing 
macrophages in tissues immediately adjacent to hepatic metastases of colorectal cancers. 99mTc-
Tilmanocept is, as explained in the next section, a radiopharmaceutical specifically designed to bind to 
CD206. It is envisioned that 99mTc-tilmanocept that has localized to hepatic metastases of colorectal 
tumors can be detected prior to surgery using 3D SPECT imaging.Tilmanocept Structure99mTc-
Tilmanocept is a synthetic radiopharmaceutical molecular construct that was designed to bind 
specifically and with high affinity to CD206 [18-21].  
Figure 1:  
Structure of 99mTc-tilmanocept showing attachment of multiple units of 
DTPA and mannose to a backbone of dextran. The chelating agent, 
DTPA, is shown holding an atom of 99mTechnetium 
 
Formally, 99mTc-tilmanocept is 99mTc-diethylenetriaminepentaacetic acid (DTPA)-mannosyl-
dextran. Tilmanocept is synthesized beginning with a 10 kDa backbone of dextran to which multiple 
amine terminated molecular leashes have been attached. Through these molecular leashes, multiple 
moieties of the chelating agent, DTPA, and the sugar, mannose, are attached to the dextran backbone 
(Figure 1). Each tilmanocept molecule has 3-8 DTPA and 12-20 mannose moieties. DTPA permits 
tilmanocept to be efficiently labeled with 99mTechnetium (Tc99m). The final construct has an average 
molecular weight of 18.7 kDa, is highly water soluble and has an apparent particle diameter of 7 nm. 
The many mannoses on tilmanocept enable high affinity multivalent binding to CD206.  
 
1.3 
Current Approval 
99mTc-Tilmanocept has already been approved by the Food and Drug Administration (FDA) and 
European regulators as a radiopharmaceutical imaging agent to detect sentinel lymph nodes (SLNs) 
before and during surgeries to remove solid tumor cancers [22].  Sentinel lymph nodes are those nodes 
first encountered in the lymphatic flow leading away from a tumor [23, 24]. As such, SLNs are the first 
lymph nodes to which a cancer will metastasize from a primary tumor.  During a surgery to remove a 
primary tumor, it is important to identify and remove all lymph nodes that contain metastatic tumors.  
Lymph node metastases that are left in a patient’s body after surgery can result in disease recurrences. 
In addition, a patient’s lymph node status relative to the presence of nodal metastases provides 
important information that assists physicians in determining cancer patients’ most effective and 
appropriate post-surgical therapies. In the past, cancer patients frequently underwent lymph node 


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dissections (lymphadenectomies) that removed and examined many lymph nodes in the region near a 
tumor. Such lymph node dissections are traumatic, cause surgical recoveries of long durations, often 
lead to lifelong lymphedema [25] and are frequently disfiguring. In addition, in the majority of cases, 
none of a patient’s lymph nodes are found to contain cancer, making the entire lymph node dissection 
unbeneficial and unnecessary. Alternatively and is currently in common practice, if the SLNs can be 
identified, examined and found to be free of cancer, it is extremely unlikely that cancer has spread to 
other lymph nodes further along the lymphatic flow [26]. Therefore, patients with cancer-free SLNs 
can be spared the trauma associated with removal of additional lymph nodes and the sequelae associated 
with lymphadenectomies [27].  
Lymph nodes and especially SLNs are densely populated by macrophages that highly express CD206 
[20]. In current clinical practice, 99mTc-tilmanocept is injected subcutaneously, intradermally or 
peritumorally near a tumor or the location from which a tumor was recently removed. From its injection 
site, 99mTc-tilmanocept rapidly enters the lymphatic drainage and is transported via lymph vessels to 
the first encountered lymph node(s), the SLN(s) where it binds to CD206 and accumulates in 
macrophages through receptor mediated endocytosis. The high density of CD206 expressing 
macrophages in SLNs binds and internalizes all of the 99mTc-tilmanocept delivered via the lymphatic 
flow, thus preventing 99mTc-tilmanocept from further transiting to higher echelon nodes [21, 28]. 
Accumulation of 99mTc-tilmanocept enables identification of SLNs prior to surgery by 
lymphoscintigraphy (Figure 2) or during surgery using a handheld gamma probe [29, 30].  
99mTc-Tilmanocept has been evaluated for its safety and efficacy relative to various lymphatic 
mapping and SLN-related indications in numerous clinical studies including three phase 3 clinical 
studies in patients with melanomas, breast cancers and head and neck squamous cell carcinomas    
[31-33 ]. Based on the results of these trials, 99mTc-tilmanocept was first approved by the FDA for 
lymphatic mapping indications in March, 2013. The label has been expanded since then to 
specifically include SLN-related indications. 99mTc-Tilmanocept has also been approved for 
marketing in Europe. To date, 99mTc-tilmanocept has been used to identify SLNs in over 100,000 
cancer patients.  
 
Throughout this experience, there have been no observed serious adverse events that can be attributed 
to the administration of 99mTc-tilmanocept.  
 
 


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Figure 2.  
Presurgical lymphoscintigraphy SPECT/CT (3D) image of breast cancer 
patient injected subdermally with 99mTc-tilmanocept. Imaging taken 1 
hour after injection clearly shows localization of 99mTc-tilmanocept in a 
SLN. 
 
 
 
 
 


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1.3.1 
Nonclinical Evaluations – IV AND IP Administration 
Dosing in Dogs, Guinea Pigs and Mice: IV dosing in investigational new drug (IND) submitted non-
clinical studies represents, cumulative dosing in male and female beagle dogs [eight dogs (4/sex; wt ♀= 
7.6 kg;  wt ♂ = 10.5 kg ) given an IV injection of saline on Study Day (SD) 1 and 2 and DTPA-
mannosyl-dextran at 0.084 mg/kg on SD 4, 0.42 mg/kg on SD 6, and 0.84 mg/kg on SD 8 and 10] 
1,560X the 50 µg and 195X the 400 µg dose (female dogs) and 1706X the 50 µg dose and 213X the 
400µg dose (male dogs).   Based on the highest single dose administration (0.84 mg/kg) the dogs 
received 600X the 50 µg and 75X the 400 µg dose (female dogs) and 657X the 50 µg and 82X the 400 
µg dose (male dogs). [NOTE: In a separate dog study the dose was 0.56 mg/kg in 2 each ♂ and ♀ dogs 
of similar weight/sex, no effect was noted in that study, consistent with the higher dose dog study]. 
With regard to the sensitization test (IV administration in guinea pigs, wt = 0.415 kg) the maximum 
dose was the equivalent of 96X of the anticipated high dose tilmanocept. 
Lastly, although investigational product (IP) administered in mice, based on the highest single dose 
administration (2000 mg/kg) the mice received 300,0000X the 50 µg and 37,500X the 400 µg dose (♀ 
mice, wt = 22 gm) and 340,000X the 50 µg and 42,500X the 400 µg dose (♂ mice, wt = 29 gm). 
Dosing In Rats: 
Type of Study / 
Description 
Test System 
Method of 
Administration 
Dosing 
Central nervous system 
safety pharmacology  
Rat 
IV 
37, 190, and 380 µg/animal 
or equivalent 490X and 61X 
the anticipated study doses 
of 50 µg and 400 µg in 
humans 
Expanded single-dose 
toxicology (including 
toxicokinetics and local 
tolerance) 
Rat 
IV 
37, 190, and 380 µg/animal 
or equivalent 490X and 61X 
the anticipated study doses 
of 50 µg and 400 µg in 
humans 
Respiratory Safety 
Pharmacology 
Evaluation Using Head-
Out Plethysmography of 
Tilmanocept following 
IV Bolus Injection in 
Male Rats 
Rat 
IV 
60, 120, and 300 µg/animal 
or equivalent 320X and 41X 
the anticipated study doses 
of 50 µg and 400 µg in 
humans 
Pharmacokinetics, 
Excretion, and 
Distribution by 
Quantitative Whole-
Body Autoradiography 
Following IV 
Administration of 
99mTc-Tilmanocept in 
Rats 
Rat 
IV 
25 µg in 0.5 mL with 
collection of  Blood, Urine, 
Feces, and Carcasses for 
QWBA 


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Type of Study / 
Description 
Test System 
Method of 
Administration 
Dosing 
Hemolysis and protein 
flocculation 
Human blood 
samples 
In vitro 
2.5, 25, and 250 µg/mL 
whole human blood 
Target profiling screen 
Ion Channel 
In vitro 
See Individual Tests Below 
K Ion Channel 
Ion Channel 
The cardiac potassium 
channel, hERG, is 
responsible for a rapid 
delayed rectifier current 
(IKr) in human ventricles. 
This channel has been 
selected for evaluation 
because inhibition of IKr 
is the most common cause 
of cardiac action potential 
prolongation by non-
cardiac drugs. In this 
assay, hERG potassium 
channels are expressed in 
a human embryonic 
kidney (HEK293) cell line 
that lacks endogenous 
IKr. 
0.025, 
0.05, 0.25, 
0.5 mg/mL 
Na Ion Channel 
Ion Channel 
Cloned hNav1.5 sodium 
channels (SCN5A gene 
expressed in CHO cells) 
0.025, 
0.05, 0.25, 
0.5 mg/mL 
Ca Ion Channel 
Ion Channel 
1. Cloned L-type calcium 
channels (hCav1.2, 
encoded by the human 
CACNA1C gene and 
coexpressed with the β2 
subunit, encoded by the 
human CACNB2 gene 
and the α2δ1 subunit 
encoded by the human 
CACNA2D1 gene in 
CHO cells), responsible 
for ICa,L, high threshold 
calcium current. 
2. Cloned hNav1.5 
sodium channels (SCN5A 
gene expressed in CHO 
cells). 
0.025-0.5 mg/mL 
 
Conclusions from These Tests: 
CNS: In conclusion, a single IV administration of tilmanocept was well tolerated in rats at levels of 
0.15, 0.75, and 1.50 mg/kg. Brief sedation shortly after dosing was observed at 0.75 and 1.50 mg/kg, 
which resolved by the time of the first functional observational battery assessments and was attributed 
to the mannosyl-dextran content of tilmanocept. There were no tilmanocept-related effects on 
functional observational battery parameters. 


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Single Bolus Toxicity: Tilmanocept-related clinical pathology changes were limited to minimally 
greater, dose-related, aspartate aminotransferase (AST) values for 0.75 and 1.5 mg/kg males and 
females that were likely caused by muscle or erythrocyte release as changes did not occur in other 
hepatobiliary-related clinical pathology parameters. This change had resolved at the end of the recovery 
phase. 
At the Day 2 necropsy, dark focus in the glandular stomach was considered to be a potential test article-
related gross pathology finding in males at ≥ 0.75 mg/kg. The gross finding of dark focus in the stomach 
correlated microscopically with focal erosion or minimal hemorrhage. Focal erosion in the stomach 
was test article related in males at ≥0.75 mg/kg, but was considered to be of little toxicological 
significance. No test article-related organ weight changes were noted.  At the end of the recovery phase 
(Day 15), there were no test article-related findings in gross pathology, organ weights, or 
histopathology. 
Respiratory: In conclusion, respiratory function was assessed in male Crl:CD(Sprague Dawley) rats 
given a single IV injection dose of vehicle control article or 0.150, 0.300, or 0.750 mg/kg of tilmanocept 
at a dose volume of 3 mL/kg.  Administration of tilmanocept had no effect on mortality, but it was 
associated with severe abnormal clinical observations of hypoactivity, ataxia, labored or irregular 
respiration, and pale skin of entire body for animals given 0.750 mg/kg.  Administration of >0.150 
mg/kg tilmanocept had no direct effect on respiration rate, but it was associated with higher tidal volume 
(up to +26, +14, and +50% in animals given 0.150, 0.300, or 0.750 mg/kg, respectively) and higher 
minute volume (up to +18, +5, and +40% 0.5 through 1 hour post-dose in animals given 0.150, 0.300, 
or 0.750 mg/kg, respectively). 
Hemolysis: No hemolysis and no flocculation were observed following in vitro treatment of human 
whole blood with tilmanocept at final whole blood concentrations of 2.5, 25 or 250 μg/mL. 
Ion Channels (Na+, K+, Ca2+): Although there is a small dose-dependent effect that is fractional to 
the positive control, variability within concentration renders the median value observations not 
significantly different (Kruskal-Wallis). 
Cytochrome P-450 (CYP) inhibition and drug transporter activity as modulated by tilmanocept 
exposure: Navidea has completed in vitro studies on the effects of tilmanocept on human hepatic 
Cytochrome P-450 isozymes and multi-drug transporters derived from human liver biopsies.  
Concentrations of tilmanocept utilized in these studies represent the equivalent of the injection 
2,500,000 µg injected into the total blood pool of reference human (75kg).  There were neither effects 
on any of the CYP isozymes nor on any of the multi-drug transporters.  These data are consistent with, 
to date, the lack of any known drug interactions in >135,000 clinical experiences (administered via any 
route).   
1.3.2 
Clinical Safety 
Non-IV Experience - Well over 150,000 patients in clinical trials and US commercial use for 
intraoperative lymphatic mapping (ILM) with sentinel lymph node biopsy (SLNB) have been exposed 
to tilmanocept, and there have been no safety signals, no deaths due to drug, and no serious adverse 
events (SAEs) due to tilmanocept. There are no known drug interactions leading to contraindications 
with the use of tilmanocept. Post-marketing reports have shown less than 0.09% of subjects 
experiencing adverse events (AEs), with the most common one being lack of node localization. 


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IV Experience – Navidea is currently conducting disease imaging studies in patients with rheumatoid 
arthritis utilizing 99mTc-tilmanocept administered IV. Dosages consist of 50, 200 and 400 µg of 
tilmanocept labeled with 1, 5, or 10 millicurie (mCi) of 99mTc injected IV.  At the time of this writing, 
twenty-two subjects have been administered either 50 µg (9 subjects), 200 µg (7 subjects) or 400 µg (6 
subjects) of tilmanocept with 1, 5, or 10 mCi of  99mTc.  No subject has exhibited any adverse reaction 
to the drug that was related to the drug.  This study, when complete, will provide data on 33 subjects 
with IV administration of  99mTc-tilmanocept ( at each mass dose level with varied 99mTc-tilmanocept 
specific activity). 
1.3.3 
Radiation Dosimetry 
Table 1.  
The Lymphoseek radiation doses (mGy/MBq) to organs and 
tissues of an average patient (70 kg) with breast cancer or 
melanoma  
Target Organ 
Adults With Breast Cancer 
Adults With Melanoma 
brain 
0.0002 
0.0050 
breast (injection site) 
0.0897 
0.0427 
gall bladder wall 
0.0019 
0.0038 
lower large intestine wall 
0.0007 
0.0031 
small intestine 
0.0005 
0.0032 
stomach 
0.0010 
0.0030 
upper large intestine wall 
0.0007 
0.0031 
kidney 
0.0101 
0.0150 
liver 
0.0018 
0.0050 
lungs 
0.0020 
0.0032 
muscle 
0.0005 
0.0024 
ovaries 
0.0101 
0.0162 
red marrow 
0.0007 
0.0027 
bone 
0.0010 
0.0047 
spleen 
0.0015 
0.0032 
testes 
0.0027 
0.0056 
thymus 
0.0063 
0.0031 
thyroid 
0.0048 
0.0025 
urinary bladder 
0.0032 
0.0076 
total body (blood)c 
0.0011 
0.0030 
Effective Dose  
(males, mSv/MBq) 
0.01600 
0.01094 
Effective Dose 
(females, mSv/MBq) 
0.01785 
0.01357 
a Calculated from data of 18 breast cancer patients who received four peritumoural injections of 4, 20, and 100 µg doses of 
Lymphoseek. 
b Calculated from data of 18 melanoma patients who received four intradermal injections of 20, 100, and 200 µg doses of 
Lymphoseek. 
c Blood represents total body exposure segregated from independent measurements of other organs and tissues. 


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1.3.4 
Radiation Exposure 
Participants in this study will be exposed to radiation during both scanning procedures.  The SPECT/CT 
scan will expose the participant to approximately 6.1 millisieverts of radiation.  The FDG PET/CT scan 
will expose the participants to approximately 13-15 millsieverts. 
2 
TRIAL OBJECTIVES 
2.1 
Primary Objective(s)  
• Estimation of the concordance of Tc99m localization in liver metastases from colorectal 
carcinoma (CRC) using SPECT/CT imaging and abdominal FDG PET/CT imaging per subject. 
• Evaluation of safety including monitoring the incidence of adverse events and changes over time 
in laboratory tests, vital signs, and physical examination findings. 
 
 
2.2 
Secondary Objective(s) 
• Estimation of the  the optimal dose of  Technetium Tc 99m tilmanocept 
• Evaluation of other areas of localization of Technetium Tc 99m tilmanocept. 
 


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3 
OVERVIEW OF METHODOLOGY AND DESIGN 
3.1 
Overall Trial Design 
This is a single-center, open-label, within-subject feasibility study of tilmanocept in the localization of 
liver metastasis from CRC. Up to 6 subjects will receive a single IV dose of 50 µg tilmanocept 
radiolabeled with 2 mCi Tc 99m and imaged at 4-6 hours post-injection.  Up to 6 subjects will receive 
a single IV dose of 200 µg tilmanocept radiolabeled with 2 mCi of Tc99m and imaged at 4-6 hours 
post-injection. 
See Appendix 1, Schedule of Events. 
3.2 
Justification for Study Design and Population 
This study is designed to evaluate the safety and tolerability of a single dose of Tc 99m tilmanocept 
administered intravenously. SPECT/CT imaging of the subject’s abdominal cavity (to include the liver) 
will be reviewed to establish concordance with images previously acquired with FDG PET/CT imaging.   
This study is designed to evaluate the use of Tc 99m tilmanocept as an imaging agent in subjects with 
known active liver metastasis from CRC by comparing to FDG PET/CT images.   
No clinical trials have been conducted to evaluate IV administration of Tc 99m tilmanocept 
performance in this population. The rationale for evaluating Tc 99m tilmanocept in this subject 
population is discussed in Section 1.1 and Section 1.2 
3.3 
Protocol Adherence 
Strict adherence to all specifications outlined in this protocol is required for all aspects of the study 
conduct; the investigator may not modify or alter the procedures described in this protocol. If protocol 
modifications are necessary, all alterations that are not solely of an administrative nature require a 
formal protocol amendment for the involvement of Institutional Review Board(s) (IRB(s)). 
If an investigator has deviated from the protocol in order to eliminate an immediate hazard to subjects 
or for other inevitable medical reasons, the investigator shall document all such deviations, including 
the reasons thereof, and submit the document to the sponsor and the head of the medical institution as 
applicable. 
3.4 
Trial Duration 
Subjects will be “on trial” for approximately 35 days. 


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4 
STUDY POPULATION 
The evaluation of tilmanocept in liver metastasis from CRC will involve up to 12 evaluable subjects. 
4.1 
Inclusion Criteria 
Each subject must meet the following criteria to be enrolled in this study. 
1. The subject has provided written informed consent with Health Information Portability and 
Accountability Act (HIPAA) authorization before the initiation of any study-related 
procedures; 
2. The subject must be ≥18 years old; 
3. The subject must have a diagnosis of adenocarcinoma of the colon and/or rectum with 
confirmed metastases to the liver 
4. The subject must have an Eastern Cooperative Oncology Group (ECOG) performance status 
of 0-3; 
5. The subject must be at least 4 weeks past any major intraabdominal surgery, including 
surgery to the liver; 
6. Subjects with prior malignancies other than colon and/or rectal cancer are allowed, provided 
they have been treated with curative intent, and have no evidence of recurrence of that 
malignancy; 
7. If of childbearing potential, the subject has a negative urine pregnancy test within 48 hours 
before administration of Tc 99m Tilmanocept, has been surgically sterilized, or has been 
postmenopausal for at least 1 year  
4.2 
Exclusion Criteria 
Subjects who meet any of the following criteria will be excluded from the study. 
1. The subject is pregnant or lactating; 
2. The subject has undergone any liver surgery, exclusive of a biopsy; 
3. The subject has known sensitivity to dextran; 
4. The subject has had preoperative chemotherapy, immunotherapy, or radiation therapy within 
the 10 days prior to Tc 99m Tilmanocept administration; 
5. Before the administration of Tc 99m Tilmanocept, has received any radiopharmaceutical 
within 7 radioactive half-lives of that radiopharmaceutical; 
6. Has received an investigational product within the 30 days prior to Tc 99m Tilmanocept 
administration 
 
4.3 
Recruitment 
Up to 12 evaluable subjects will be recruited from medical practice in accordance with the inclusion 
and exclusion criteria listed above. Potentially suitable subjects will be asked by the treating specialist 
about their willingness to participate in this trial. After 3 subjects have been enrolled into Cohort 1, the 
data will be reviewed by the PI and Navidea to determine if additional subjects should be enrolled and 
if additional timepoints should be considered.  If it is determined that additional enrollment will not 
provide meaningful data, enrollment into Cohort 2 will begin.  After 3 subjects have been enrolled into 
Cohort 2, the data will be reviewed by the PI and Navidea to determine if additional subjects should be 
enrolled and if additional timepoints should be considered.  


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4.4 
Withdrawal 
In accordance with the Declaration of Helsinki, each subject is free to withdraw from the trial at any 
time and without providing a reason. 
Should a subject withdraw after administration of the IP, all efforts will be made to complete and report 
the observations up to the time of withdrawal as thoroughly as possible.   
The investigator may withdraw a subject from the trial at any time at the discretion of the investigator 
for any of the following reasons:  
• A protocol violation occurs  
• A serious or intolerable AE occurs  
• A clinically significant change in a laboratory parameter occurs  
• At the investigator’s/sponsor’s discretion as long as it is in the best interest of the subject  
• The sponsor or investigator terminates the study  
• The subject requests to be discontinued from the study 
4.5 
Enrollment and Screen Failures 
Subjects who sign an informed consent form (ICF) but are ultimately not injected will be considered 
screen failures. Subjects will be assigned a subject identification number at the time the ICF is signed. A 
subject will be considered enrolled once injected with Technetium Tc 99m on Day 1. Demographic 
data and the reason for the screen failure will be collected for all screen failure subjects.  Enrollment 
will continue until a total of up to 12 subjects have completed SPECT/CT imaging.   
Subject Identification 
After the subject provides written informed consent, the site will assign the subject a 7-digit subject 
number. Subject numbers are to be assigned in a sequential manner using the following format:  
Digits 1 to 2: Trial number (e.g., “25”)  
Digits 3 to 4: Site number (e.g., “01”)  
Digits 5 to 7: Sequential subject number (e.g., “001”, “002”, “003”)  
 
For example, the first subject consented at site 01 is subject number “25-01-001.”  
 
Subjects will maintain the same number given at screening for the entire trial. If a subject is a screen 
failure, the number will not be used for any other subject. 


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5 
INVESTIGATIONAL PRODUCT 
5.1 
Identification of Investigational Product 
Tilmanocept is a radiotracer that accumulates in CD206-expressing tissue by binding to the CD206 
mannose binding receptors that reside on the surface of dendritic cells and macrophages. 
5.2 
Investigational Product Dosage and Administration 
Subjects will receive the administration of Tc 99m tilmanocept through an IV route of administration.  
Cohort 1 will receive an IV injection of 50µg tilmanocept radiolabeled with 2 mCi (74MBq) Tc99m 
in 1.0 mL from a single dose syringe.  Cohort 2 will receive an IV injection of 200µg tilmanocept 
radiolabeled with 2mCi (74MBq) Tc 99m in 1.0mL from a single syringe.  Tc 99m tilmanocept will 
be administered as a slow push through a catheter.  At the completion of the injection, a 10 cc sterile 
normal saline flush will be administered. The preferred site of IV placement will be the left or right 
antecubital vein. 
 
The final administered dose will be ± 20% of the tilmanocept mass dose and radiolabel mCi dose 
assignment.  
Dose levels administered in this study were based on the safety and imaging results of Navidea 
studies NAV3-21, “An Evaluation of the Safety of Escalating Doses of Tc 99m Tilmanocept by 
Intravenous (IV) Injection and Skeletal Joint Imaging with SPECT in Subjects with Active 
Rheumatoid Arthritis (RA) and Healthy Controls” and NAV3-23, “Evaluation of Subcutaneously 
(SC) Injected Tc 99m Tilmanocept Localization in Active Rheumatoid Arthritis (RA) Subjects by 
SPECT and SPECT/CT Imaging.”  The safety and localization in this study is expected to be similar 
to the results collected in these studies. 
 
5.3 
Treatment Assignment 
In this open-label, non-randomized, single center, study all subjects will receive either 50µg or 200µg 
tilmanocept radiolabeled with 2mCi (74MBq) Tc99m in 1.0mL.  The clinical site will send a request 
to Navidea for subject cohort assignment.  The Navidea project manager will confirm treatment group 
assignment and send authorization back to the investigator prior to placement of the dose order. 
The investigator will receive the dose from the supporting Cardinal Health pharmacy. 
5.4 
Packaging and Labeling 
Tilmanocept cartons ready for radiolabeling will be shipped and stored at the region-specific Cardinal 
Health radiopharmacy.  Tilmanocept is provided in a vial. Vials are packaged as a kit. A carton contains 
five vials of tilmanocept. One kit, which is one tilmanocept vial, should be used for no more than one 
subject.  The carton also contains five shield labels and 25 syringe labels. This package has been 
designed specifically for tilmanocept and protects the vials during shipment, handling, and storage.  
Navidea will provide a radiolabeling protocol and Quality Control worksheets. Cardinal Health will 
radiolabel tilmanocept with either 50 µg or 200µg tilmanocept radiolabeled with 2mCi (74MBq) 
Tc99m in 1.0 mL and deliver one syringe to the clinical site radiopharmacy that is ready for 
administration.   


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The investigator (or designated personnel) will confirm receipt of the IP in writing and will use the IP 
only within the framework of this clinical study and in accordance with this study protocol.  For each 
subject, he/she will keep a record of the IP dispensed and store all other forms that accompanied the 
delivery of the radiolabeled product to the clinical site. These documents are to be filed in the ISF. 
Overall drug accountability and reconciliation will be completed by the sponsor or its representative.  
A list of IP vials and other materials that were returned, or destroyed, must be recorded and signed by 
the PI or an appropriately qualified designee as documented in the study site responsibility sheet. An 
overall accountability and reconciliation form of the IP will be prepared and completed. If there are any 
discrepancies, they must be investigated and their resolution documented. All unused study kits will be 
destroyed in accordance with institutional destruction procedures. 


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6 
THERAPIES OTHER THAN INVESTIGATIONAL PRODUCT 
6.1 
Prior and Concomitant Therapy 
All medications taken 30 days prior to Tc 99m tilmanocept injection through the 5 ± 3 days post-
injection safety follow-up will be documented. Subjects must not have chemotherapy, immunotherapy, 
or radiation therapy within the 10 days prior to Tc 99m Tilmanocept administration  
6.2 
Post-Study Therapy  
There are no post-study therapy restrictions. 


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7 
STUDY PROCEDURES 
7.1 
Screening Visit (Day -29 to Day 0) 
• Obtain signed informed consent for study participation 
• Preliminary review of inclusion and exclusion criteria 
• Allocation of unique subject number; this number will be used to document the subject data in the 
case report form (CRF) and enrollment log 
• Interview including the following subject-specific characteristics: 
• Demographic data 
• Medical/surgical history (including concomitant diseases) 
• Medication history 
• Vital signs (body temperature, heart rate, blood pressure, and respiratory rate). 
• Blood draw for routine hematology and chemistry 
• Urine collection for routine urinalysis and for pregnancy testing for women of child-bearing 
potential. Females of child bearing potential are defined as women that are not surgically sterile 
(hysterectomy or bilateral oophorectomy) nor postmenopausal for at least 1 year prior to 
screening. Women who are not of childbearing potential will not require a pregnancy test.  
• Physical examination including height and weight and a review of major body systems; any 
clinically relevant finding is to be documented as baseline finding. 
• Subjects will undergo a FDG PET/CT scan to identify hepatic lesions.   
 
7.2 
Day 1 (Injection and Imaging Procedures) 
7.2.1 
Before Injection 
• Assessment of AEs 
• Concomitant medication review   
• Final check of inclusion/exclusion criteria 
• Vital signs (body temperature, heart rate, blood pressure, and respiratory rate) within 15 minutes  
7.2.2 
Injection of Tc 99m tilmanocept 
Both cohorts will be injected from a single syringe in a single IV injection. 
Table 2.  
Dose Cohort  and Route of Administration Groups 
Dosing Cohorts 
Cohort 1 
IV Injection 
50µg tilmanocept in 1.0 mL 
2 mCi Tc99m 
Cohort 2 
IV Injection 
200µg tilmanocept in 1.0 mL 
2 mCi Tc99m 
Up to 6 subjects 
 Up to 6 subjects 
 


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Up to 12 subjects may be injected and imaged, divided into 2 cohorts of up to 6 subjects each.   
Cohort 1 subjects will receive a Tc 99m tilmanocept IV injection in 1.0 mL and 2 mCi injected as a 
slow push into a catheter.  At the completion of the injection, a 10 cc sterile normal saline flush will be 
administered.  The preferred site of IV placement will be the left or right antecubital vein.  
Cohort 2 subjects will receive a Tc 99m tilmanocept IV injection in 1.0 mL and 2 mCi injected as a 
slow push into a catheter.  At the completion of the injection, a 10 cc sterile normal saline flush will be 
administered.  The preferred site of IV placement will be the left or right antecubital vein.  
Injection and imaging will begin with Cohort 1.  After 3 subjects have been enrolled into Cohort 1, the 
imaging and safety data will be reviewed by the site staff and Navidea personnel to determine if 
additional subjects should be enrolled.  If it is determined that additional enrollment will not provide 
meaningful data, for example no metatstic liver lesions were visualized by Tc 99m tilmanocept for any 
of the subjects in the cohort, enrollment into Cohort 2 will begin.  After 3 subjects have been enrolled 
into Cohort 2, the data will be reviewed by the site staff and Navidea personnel to determine if 
additional subjects should be enrolled. 
7.2.3 
Post-Injection 
The following procedures will be completed at the specified timepoints: 
• 
15 Minutes Post-Injection 
o  Vital signs will be obtained (body temperature, heart rate, blood pressure, and respiratory 
rate) 
• 
4-6 Hours Post-Injection  
o Assessment of AEs  
o SPECT/CT will be obtained in the abdominal region (to include entire liver) 
  
• 5 ± 3 Days Post-Injection 
o Assessment of AEs 
o Vital signs (body temperature, heart rate, blood pressure, and respiratory rate, after at 
least 5 minutes in a resting position)  
o Physical examination including a review of major body systems; any clinically relevant 
changes from screening are to be documented  
o Blood draw for routine hematology and chemistry  
o Urine collection for routine urinalysis  
 
7.2.4 
Imaging and Acquisition of Imaging Data 
The camera used to obtain the images should be a 2 or 3-headed SPECT/CT camera equipped with a 
low-energy, high-resolution collimator with a 15% window (20% can be used if 15% setting not 
available) centered over a 140keV peak.   
The camera must have passed the daily SPECT QC tests as per the manufacturer’s recommendation for 
that day’s scan schedule. 


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Whenever possible subject should be asked to void after injection and prior to the first imaging session 
and again between imaging sessions.  
Image acquisition of subjects in this study will be via SPECT/CT at 4-6 hours post-injection.   The 
abdominal regional will be imaged and will include the entire liver. 
7.2.5 
Image Acquisition 
The investigator will document the manufacturer and type of the SPECT or SPECT/CT scanner used 
in both the subject’s file and CRF. The same scanner should be used during the entire course of the 
study and any updates to hardware or software should be avoided.  If changes occur, they must be 
reported to the sponsor. 
The manufacturer’s standard SPECT acquisition parameters for Hepatobiliary SPECT (HIDA SPECT) 
should be followed. This would typically result in a total acquisition time of 30 ± 10 minutes. For 
SPECT/CT cameras; the CT scan should be set for “low-dose” mode to obtain and use the transmission 
scan for attenuation correction. For non-CT coupled SPECT cameras, attenuation correction would be 
applied using standard ellipse-based Chang methods. Regardless of the number of heads on the SPECT 
camera, the emission data should be acquired with each camera head orbiting the full 360° around the 
patient with typically 90-120 projection views per head. 
7.2.6 
Evaluation of SPECT/CT Images  
Tilmanocept localization in subjects with liver metastasis from a CRC will be evaluated through 
SPECT/CT image analysis by the onsite Nuclear Medicine physician. First, a “routine clinical” 
assessment of the overall technical adequacy of the scan will be made. It is anticipated that this would 
be done by viewing the projection data cine (allowing for an initial visual assessment of patient motion 
during the acquisition). This would be followed by a visual evaluation of the transverse reconstructed 
slices of the liver to assess whether hotspots could be attributed to expected metastases sites. The 
clinician reading the transverse SPECT liver slices will have access to the previously conducted PET 
scans and will report any concordance between uptake of tilmanocept with FDG at the metastases sites. 
The number of sites of concordance (between SPECT and PET) as well as the total number of diagnosed 
metastases sites for each subject imaged will be reported to the sponsor. Following the technical 
assessment, an overall assessment of the scan will be made. A de-identified copy of the Nuclear 
Medicine scan report will be provided by the site to the sponsor. For each subject the SPECT/CT scans 
and FDG PET/CT scans (in Digital Imaging Communications in Medicine [DICOM] format) will be 
forwarded to the sponsor. 
All regions of interest (ROIs), standardized uptake values (SUVs), and quantitative analyses will be 
done by the sponsor. 
Given that the imaging site has electronic transfer capabilities, each subject’s scans will be transferred 
to the sponsors data server. If such an e-transfer is not possible, a standard “patient CD disc” will be 
generated and sent to the sponsor via ground mail. 
7.2.7 
End of Study 
For the entire study, end of study is defined as the last subject visit. 
 


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8 
PROCEDURES AND VARIABLES 
8.1 
Population Characteristics 
8.1.1 
Demographic and Other Baseline Characteristics 
Up to 12 subjects aged ≥ 18 years, with confirmed CRC with confirmed liver metastases will be 
included. 
8.1.2 
Medical and Surgical History 
Relevant medical and surgical histories are to be recorded for the following body systems: head, ear, 
eye, nose, throat, respiratory, cardiovascular, gastrointestinal, genitourinary, musculoskeletal, 
endocrine, neurological, psychiatric, and skin/dermatological.   
As part of the medical history, the date of the last spontaneous menstruation will be recorded, if 
childbearing potential is not excluded by surgical sterilization. 
8.1.3 
Prior and Concomitant Medication 
All medications taken 30 days prior to injection through the 5 ± 3 day follow-up visit will be 
documented. 
8.2 
Safety 
8.2.1 
Adverse Events 
8.2.1.1 
Definition of Adverse Event 
The definitions below follow International Conference on Harmonization (ICH) – Good Clinical 
Practice (GCP) (see also ICH Guideline for Clinical Safety Data Management:  Definitions and 
Standards for Expedited Reporting). 
Adverse Event (AE) 
An AE is defined as any untoward medical occurrence in a subject administered a pharmaceutical 
product and which does not necessarily have a causal relationship with this treatment.  An AE can 
therefore be any unfavorable and unintended sign (including an abnormal laboratory finding), 
symptom, or disease temporally associated with the use of a medicinal (investigational) product, 
whether or not considered related to the medicinal (investigational) product. 
By definition for this trial, all untoward medical occurrences beginning on the day of injection through 
the assessment 5 days post-injection are to be reported as AEs.  Additionally, untoward medical events 
occurring prior to the day of injection and after the 5 ± 3 day post-injection follow-up will only be 
captured as AEs if they are related to a trial procedure or study drug.  Any clinically significant change 
in condition (worsening) from screening that results in a change in subject management will be 
considered an AE and will be recorded on the AE page of the CRF.  SAEs will be reported from the 
time of consent through the end of participation. 


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8.2.1.2 
Categories for Adverse Event Assessment 
All AEs will be assessed and documented by the investigator according to the categories detailed below 
Severity 
The severity of an AE is classified according to the following categories, taking into account the 
possible range of the intensity of the event: 
• Mild  
 
The AE is transient and easily tolerated by the subject. 
• Moderate  The AE causes the subject discomfort and interrupts the subject’s usual activities 
• Severe  
The AE causes considerable interference with the subject’s usual activities and may 
be incapacitating or life-threatening. 
Specific drug treatment 
Any specific drug treatment will be documented. 
Causal relationship to investigational product 
The investigator will use the following definitions to assess the relationship of the AE to the use of IP:  
Definitely related: 
Event can be fully explained by administration of the IP. 
Probably related: 
Event is most likely to be explained by administration of the IP rather than 
the subject’s clinical state or other agents/therapies. 
Possibly related: 
Event may be explained by administration of the IP or by the subject’s 
clinical state or other agents/therapies. 
Probably not related: 
Event is most likely to be explained by the subject’s clinical state or other 
agents/therapies, rather than the IP. 
Definitely not related: 
Event can be fully explained by the subject’s clinical state or other 
agents/therapies. 
For causality assessments, events meeting the categories of definitely, probably, or possibly related will 
be considered to be related to IP. 
 
Outcome 
The outcome of the AE is to be documented as follows: 
• Resolved 
• Resolved with sequelae 
• Ongoing 


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• Unknown 
• Lost to follow-up 
• Death 
8.2.1.3 
Assessments and Documentation of Adverse Events 
Attention shall be paid to the occurrence of AEs at all stages of the SPECT evaluation.  Thus, the subject 
should be closely observed by the investigator both during and after the evaluation.   
Any AE (observed, volunteered, or elicited) should be recorded in detail in the source documentation. 
The following information is required: 
• The date and time of onset of any AE. 
• The duration (the entire duration of an event or symptom, calculated from date of onset to date 
of end, if not recorded directly). 
• The seriousness of the AE will be assessed by the investigator.  If the investigator deems that an 
AE qualifies as an SAE, a special form provided by the sponsor should be completed and the 
event must be immediately reported to the sponsor.  A definition of SAEs is provided below.   
• The maximum intensity (mild, moderate, or severe). 
• Specific drug treatment 
• The relationship of the AE to the IP and to trial conduct (for definitions, see above). 
• The outcome of the AE (resolved, resolved with sequelae, ongoing, unknown, lost to follow-up, 
death). 
AEs will be coded according to an internationally recognized dictionary (Medical Dictionary for 
Regulatory Activities [MedDRA]). 
8.2.1.4 
Expected Adverse Events 
Expected Adverse Drug Reactions 
The definition below follows ICH-GCP (see also ICH Guideline for Clinical Safety Data Management:  
Definitions and Standards for Expedited Reporting): 
Adverse Drug Reaction (ADR) 
In the pre-approval clinical experience with a new medicinal product or its new usages, particularly as 
the therapeutic dose(s) may not be established, all noxious and unintended responses to a medicinal 
product related to any dose should be considered as adverse drug reaction (ADR).  The phrase 
‘responses to a medicinal product’ means that a causal relationship between a medicinal product and 
an AE is at least a reasonable possibility (definitely, probably, possibly), i.e., the relationship cannot be 
ruled out. 
 
 


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Investigational Product-Related Risks 
In previous clinical trials with tilmanocept (n = 553 subjects injected subcutaneously, intradermally, 
subaereolarly, peritumorally), all individual AEs were experienced by less than ≤ 1% of all subjects 
enrolled in sponsor-initiated trials.  The most commonly reported AEs in all subjects were injection site 
irritation, which occurred in 0.6% (n = 3), and pain, which occurred in 0.2% (n = 1) of all subjects.  All 
other treatment-emergent AEs occurred in 0.2% or less of all subjects AEs from the radioactive dose 
are not expected, since the applied radiation doses are far below doses that can cause acute effects in 
human tissues. 
Precautionary Measures 
Special precautionary measures are not considered to be necessary for this trial.  In case of emergency, 
standard emergency procedures will be employed. 
Unexpected Adverse Drug Reactions 
An unexpected ADR is defined as an adverse reaction that in nature and severity is not consistent with 
the applicable product information (e.g., Investigator’s Brochure [IB]). 
Any adverse experience that is not listed in the current IB or which is, with regard to the specificity or 
severity, not consistent with the risk information shall be regarded as unexpected. 
Hypothetical examples would be (a) acute renal failure listed in the IB with a subsequent new report of 
interstitial nephritis and (b) hepatitis with a first report of fulminant hepatitis.  “Unexpected” as used in 
this definition refers to an adverse drug experience that has not been previously observed and included 
in the product information, rather than from the perspective of such experience not being anticipated 
from the pharmacological properties of the IP. 
8.2.1.5 
Serious Adverse Events 
Definition of Serious Adverse Events 
The following SAE definition is based on ICH guidelines and the final rule issued by the FDA and 
effective 06 Apr 1998.   
An SAE is classified as any untoward medical occurrence that at any dose: 
• results in death, or 
• is life threatening, or 
• requires inpatient hospitalization or prolongation of existing hospitalization, or 
• results in persistent or significant disability/incapacity, or 
• is a congenital anomaly/birth defect, or 
• is an important medical event (see paragraphs below). 
The term ‘life threatening’ in the definition refers to an event in which the subject was at risk of death 
at the time of the event; it does not refer to an event that hypothetically might have caused death if it 
were more severe. 


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Medical and scientific judgment should be exercised in deciding whether it is appropriate to report an 
AE as serious also in other situations, such as important medical events that may not be immediately 
life threatening or result in death or hospitalization but may jeopardize the subject or may require 
intervention to prevent one of the other outcomes listed in the definition above.  These should also 
usually be considered serious.  Examples of such events are intensive treatment in an emergency room 
or at home for allergic bronchospasm or blood dyscrasias or convulsions that do not result in subject 
hospitalization. 
Actions and reporting obligations in case of SAEs 
The investigator should take appropriate diagnostic and therapeutic measures to minimize the risk to 
the subject.   
If any SAE occurs over the course of the trial, investigators or other site personnel will inform Navidea 
Biopharmaceutical representatives within one day (i.e., within 24 hours) of becoming aware of the 
SAE. Written notification on FRM-1084 of the SAE will be emailedto Navidea Biopharmaceuticals 
Pharmacovigilance at safety@navidea.com. 
For fatal or life-threatening adverse events where important or relevant information is missing, active 
follow-up is undertaken immediately. 
All SAEs must also be recorded on the AE CRFs. 
Notification of the IRB(s) 
The sponsor and/or the investigator will notify the IRB(s) about all relevant events (e.g., SAEs and 
suspected, unexpected, serious adverse reactions [SUSARs]) according to all applicable regulations. 
Notification of the authorities 
The sponsor will process and report all relevant events (e.g., SAEs, SUSARs) to the authorities 
according to all applicable regulations. 
Sponsor’s notification of the investigators 
The sponsor will inform the investigator about reported relevant events (e.g., SAEs, SUSARs) 
according to all applicable regulations. 
8.2.2 
Further Safety Assessments 
8.2.2.1 
Physical Examination 
Physical examinations will be performed for the following body systems: 
• General appearance 
• Skin/dermatological 
• Eyes, ears, nose, throat 
• Head and neck (including thyroid) 
• Lungs 


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• Heart 
• Abdomen (liver, kidney, spleen, gastrointestinal) 
• Lymph nodes 
• Musculoskeletal 
8.2.2.2 
Vital Signs 
Vital signs comprise the measurement of body temperature, heart rate, systolic and diastolic blood 
pressure, and respiratory rate All measurements will be taken after the subject has been in a resting 
position for at least 5 minutes.  Measurements will be taken at Screening, Day 1 predose (15 minutes 
before injection), and postdose (within 15 minutes after injection). 
Any clinically significant change from screening (worsening) that results in a change in subject 
management will be considered an AE and will be recorded on the AE page of the CRF. 
8.3 
Other Procedures and Variables 
8.3.1 
Clinical Laboratory Parameters for Screening and Safety 
Clinical laboratory tests to be evaluated in this trial include hematology, serum chemistry and 
urinalysis.  Blood and urine samples for safety will be obtained at screening.  
Subjects will be in a seated or supine position during blood collection. Clinical laboratory tests will 
include the following:  
 
 


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Table 3.  
Clinical Laboratory Parameters 
Hematology 
hemoglobin (Hgb), platelets, white blood cell (WBC) 
Serum chemistry 
 
creatinine, chloride, potassium, sodium, carbon dioxide /bicarbonate, 
blood urea nitrogen (BUN), alanine aminotransferase (ALT), aspartate 
aminotransferase (AST) 
Urinalysis 
pH, specific gravity 
 
The investigator will provide a list of normal ranges of the local laboratory prior to the start of the study.  
The University of Alabama Birhmingham laboratory will provide the necessary supplies to collect the 
blood and urine samples.   
All laboratory reports must be promptly reviewed by the investigator, and upon review, initialed and 
dated by the investigator.  Change(s) in postdose test values considered clinically significant, which 
would require either additional control or therapy, must be specified in the source documentation and, 
in case of disturbing or influencing factor(s) on values/samples, details of the appropriate value(s) and 
the source of disturbance or influence (e.g., quality of sample, co-medication) are to be recorded.  GCP 
would suggest that a copy of the laboratory results also be provided to the subject’s referring physician.   
Any change in laboratory value which results in a change in subject management (additional controls 
or treatment required) will be reported as a clinically significant change. Clinically significant changes 
in laboratory parameters which are not the result of laboratory error are to be recorded as AEs. 
Any clinically significant changes in laboratory values are to be followed up with repeated tests at 
appropriate intervals (as determined by the investigator and the Medical Monitor) until the values return 
to baseline level or until the abnormality is explained by the investigator.   
8.3.2 
Blood Sampling 
The total amount of blood withdrawn during the trial will be approximately 20 mL over a period of 
approximately 2 weeks as shown in Table 4.  
Table 4.  
Amount of Blood Withdrawn 
Time point of examination 
Amount of blood taken 
Laboratory examination (Screening) 
10 mL 
Follow-up 
10 mL 
Total 
20 mL 
 


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9 
STATISTICAL METHODS 
Statistics will be descriptive in nature. The sample size was chosen in order to provide a reasonable 
amount of data to assess the localization performance of Tc 99m tilmanocept in this population across 
the administered doses. 
Demographics and baseline characteristics will be summarized for all subjects injected with 
tilmanocept using either descriptive statistics (sample size, mean, standard deviation, minimum, 
median, maximum) or frequency counts and percentages. 
Primary and secondary endpoint will be determined as follows: 
• The count and percentage of subjects that had at least one liver lesion identified by Tc99m 
tilmanocept that was also identified by FDG PET/CT. 
• The count and percentage of subjects that had at least one liver lesion identified by FDG PET/CT 
that was also identified by Tc99m tilmanocept. 
• The count and percentage of the number of liver lesions identified by Tc99m tilmanocept that were 
also identified by FDG PET/CT. 
• The count and percentage of the number of liver lesions identified by FDG PET/CT that were also 
identified by Tc99m tilmanocept. 
• Incidence of AEs reported for each cohort 
• Other lesion localization (count and percentage of subjects with ≥ 1 localized lesion by 
tilmanocept). 
• Exploratory statistics may be conducted for other localization of tilmanocept and quantitative 
uptake (radioisotope) in lesion(s) or lymph node(s). 


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10 DATA HANDLING AND QUALITY ASSURANCE 
10.1 
Data Recording 
Data required according to this protocol are to be entered onto the CRFs (provided by the sponsor) as 
soon as possible. 
10.1.1 
CRF Design 
CRFs will be used for collecting all data generated during the trial.  CRF completion details will be 
documented in a separate document that will be provided by the sponsor and maintained in the TMF. 
10.2 
Monitoring 
This study will be monitored regularly by a clinical research associate (CRA) from the sponsor or a 
contract research organization (CRO). Monitoring procedures include one or more visits designed to 
clarify all prerequisites before the study starts. Interim monitoring visits will take place on a regular 
basis according to a schedule fixed by mutual agreement. During these visits, the CRA will check for 
completion of the entries on the CRFs, their compliance with the protocol and with GCP, and will 
compare the CRF entries with the source data. 
 
All data recorded in the CRF will be captured in the source documentation. 
 
The CRA will verify the correct use of the IP. The IP will not be supplied to the investigator site prior 
to a favorable opinion from the IRB/IEC and the regulatory authority and, if appropriate, from the 
radiation protection authorities. In addition, the CRA will determine whether all AEs and SAEs have 
been appropriately reported (including adherence to the time periods required for SAEs). 
 
10.3 
Data Processing 
Trial data documentation will be maintained specifying all relevant aspects of data processing for the 
trial (including data validation, cleaning, correcting, releasing).  This documentation will be stored in 
the TMF. 
For data coding (e.g., AEs, medication, medical/surgical history), internationally recognized and 
accepted dictionaries will be used. 
10.4 
Auditing 
A member of the sponsor’s (or a designated CRO) quality assurance unit may arrange to visit the 
investigator in order to audit the performance of the study at the trial site and the trial documents 
originating there.  The auditor(s) will usually be accompanied by a CRA or the trial team lead.  The 
investigator will be informed about the outcome of the audit. 
In addition, inspections by health authority representatives and independent ethics committee(s) 
(IEC(s)/ IRB(s) are possible at any time.  The investigator is to notify the sponsor of any such inspection 
immediately. 


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10.5 
Archiving 
Essential documents shall be archived safely and securely in such a way that ensures that they are 
readily available upon authorities’ request. 
Patient (hospital) files will be archived according to local regulations and in accordance with the 
maximum period of time permitted by the hospital, institution, or private practice.  Where the archiving 
procedures do not meet the minimum timelines required by the sponsor, alternative arrangements must 
be made to ensure the availability of the source documents for the required period. 
The investigator/institution notifies the sponsor if the archival arrangements change (e.g., relocation or 
transfer of ownership). 
The ISF is not to be destroyed without the sponsor’s approval. 
The investigator’s contract will contain all regulations relevant for the trial center. 
10.6 
Premature Termination of the Trial 
Termination by the Sponsor 
The Sponsor may terminate the trial at any time for any of the following reasons: 
1. Failure to enroll subjects 
2. Protocol violations 
3. Inaccurate or incomplete data 
4. Unsafe or unethical practices 
5. Questionable safety of the IP 
6. Suspected lack of efficacy of the IP 
7. Administrative decision 
Termination by the Investigator 
If the investigator terminates the trial prematurely, the investigator must do the following: 
• Return all unused IP and related trial materials to the Sponsor. 
• Provide the IRB and the sponsor with a written statement describing why the trial was terminated 
prematurely. Prompt compliance with this requirement is essential so that the sponsor may 
comply with its regulatory obligations. 
10.6.1 
Trial as a Whole 
The sponsor retains the right to prematurely terminate the trial as a whole at any time.   
At the discretion of the sponsor, the entire trial may be canceled for medical reasons.  In addition, the 
sponsor retains the right to end the trial at any time if the study cannot be carried out as agreed upon in 
the protocol.  


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In case of premature termination or suspension of the trial, the PI/sponsor will promptly inform the 
investigator/institutions, regulatory authorities, and IRB of the termination or suspension and the reason 
for that. 
10.6.2 
Center 
At any time, the trial may be terminated at an individual center if:  
• The center cannot comply with the requirements of the protocol. 
• It is not possible for the center to comply with GCP standards. 
10.6.3 
Study Participant 
Individual subjects may be withdrawn from the trial according to the criteria specified in Section 4.4. 
 


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11 ETHICAL AND LEGAL ASPECTS 
11.1 
Ethical and Legal Conduct of the Study 
The planning and conduct of this clinical trial are subject to national laws. Only when all of the 
requirements of the appropriate regulatory authority have been fulfilled will the trial begin. The trial 
will be conducted in accordance with the ethical principles that have their origin in the Declaration of 
Helsinki and the ICH-GCP Guidelines of 17 Jan 1997. At the discretion of the PI, the entire trial may 
be canceled for medical reasons. In addition, the sponsor retains the right to end the trial for medical-
scientific or GCP-relevant reasons. In case of premature termination the investigators, IRB/IECs and 
Regulatory Authorities will be informed by the Study Manager. As required by local law, current 
safety-relevant information will be provided to the IEC/IRB and the regulatory authorities by the 
sponsor. The sponsor will also inform all investigators about relevant safety events according to the 
applicable regulations. 
11.2 
Subject Information and Consent 
All relevant information on the trial will be summarized in the subject consent form and additionally 
as required by the investigator’s institution in an integrated subject information and consent sheet. A 
sample subject information and informed consent form is provided as a document separate to this 
protocol. 
Based on this subject consent form and any required information sheet, the investigator will explain all 
relevant aspects of the trial to each subject, before his/her entry into the trial (i.e., before examinations 
and procedures associated with selection for the trial are performed).  
The investigator will also mention that written approval of the IRB/IEC has been obtained.  
Each subject will have ample time and opportunity to ask questions and will be informed about the 
right to withdraw from the trial at any time without any disadvantage and without having to provide 
reasons for this decision.  
Following this informative discussion, the subject will be asked if he/she is willing to sign and 
personally date a statement of informed consent. Only if the subject voluntarily agrees to sign the ICF 
and has done so, may he/she enter the trial. Additionally, the investigator or his nominated designee 
will personally sign and date the form, too. The subject will receive a duplicate of the signed and dated 
form.  
The investigator will record in the source documentation the time and date of obtaining informed 
consent. 
In the event that informed consent is obtained on the date that screening trial procedures are performed, 
the trial record or subject's clinical record must clearly show that informed consent was obtained prior 
to these procedures.  
The ICF and any other written information provided to subjects will be revised whenever important 
new information becomes available that may be relevant to the subject’s consent, or there is an 
amendment to the protocol which necessitates a change to the content of the subject information and/or 
the written ICF. The investigator will inform the subject of changes in a timely manner and will ask the 
subject to confirm his/her participation in the trial by signing the revised ICF form. Any revised written 
ICF and written information must receive the IRB/IEC's approval/favorable opinion in advance of use. 


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11.3 
Financing/Financial Disclosure 
Each investigator (including principal and/or any sub-investigators; as well as their spouses and 
dependent children) who is directly involved in the treatment or evaluation of research subjects has to 
provide a financial disclosure according to all applicable legal requirements. All relevant 
documentation will be filed in the TMF and/or ISF, as appropriate. 
11.4 
Publication Policy 
The Sponsor will be responsible for determining when any trial results should be published. The 
Sponsor will work jointly with the investigator(s) to publish information in a timely manner. The 
investigator(s) shall not submit any information gleaned under the direct support or sponsorship of the 
Sponsor to journals or professional societies without the prior written approval of the Sponsor.  A 
"publication" is meant to include any abstract, letter, manuscript or public announcement in any form 
or length that contains information gleaned under the direct support or sponsorship of the Sponsor. 
11.5 
Subject Injury 
In general, if a subject is injured as a direct result of the IP but not due to medical negligence on the 
part of the PI or trial staff, the Sponsor will pay for reasonable and necessary medical treatment for the 
injury, to the extent the expenses are not covered by the subject’s medical insurance, a government 
program, or other responsible third party.  If laws or regulations of the locality in which the trial is 
taking place require additional payment of expenses, the sponsor shall comply with such law or 
regulation.  Where applicable, the Sponsor has taken specific national insurance. 


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12 REFERENCE LIST 
1. 
Millikan, K.W., E.D. Staren, and A. Doolas, Invasive therapy of metastatic 
colorectal cancer to the liver. Surg Clin North Am, 1997. 77(1): p. 27-48. 
2. 
Mayo, S.C. and T.M. Pawlik, Current management of colorectal hepatic 
metastasis. Expert Rev Gastroenterol Hepatol, 2009. 3(2): p. 131-44. 
3. 
Hugh, T.J., A.R. Kinsella, and G.J. Poston, Management strategies for colorectal 
liver metastases--Part I. Surg Oncol, 1997. 6(1): p. 19-30. 
4. 
Gazi, U. and L. Martinez-Pomares, Influence of the mannose receptor in host 
immune responses. Immunobiology, 2009. 214(7): p. 554-61. 
5. 
Apostolopoulos, V. and I.F. McKenzie, Role of the mannose receptor in the 
immune response. Curr Mol Med, 2001. 1(4): p. 469-74. 
6. 
Ezekowitz, R.A., et al., Molecular characterization of the human macrophage 
mannose receptor: demonstration of multiple carbohydrate recognition-like 
domains and phagocytosis of yeasts in Cos-1 cells. J Exp Med, 1990. 172(6): p. 
1785-94. 
7. 
Taylor, M.E. and K. Drickamer, Structural requirements for high affinity binding 
of complex ligands by the macrophage mannose receptor. J Biol Chem, 1993. 
268(1): p. 399-404. 
8. 
Murray, P.J., et al., Macrophage activation and polarization: nomenclature and 
experimental guidelines. Immunity, 2014. 41(1): p. 14-20. 
9. 
Mantovani, A., et al., The chemokine system in diverse forms of macrophage 
activation and polarization. Trends Immunol, 2004. 25(12): p. 677-86. 
10. 
Chavez-Galan, L., et al., Much More than M1 and M2 Macrophages, There are also 
CD169(+) and TCR(+) Macrophages. Front Immunol, 2015. 6: p. 263. 
11. 
Dong, P., et al., CD86(+)/CD206(+), Diametrically Polarized Tumor-Associated 
Macrophages, Predict Hepatocellular Carcinoma Patient Prognosis. Int J Mol Sci, 
2016. 17(3). 
12. 
Martinez, F.O., et al., Macrophage activation and polarization. Front Biosci, 2008. 
13: p. 453-61. 
13. 
Chanmee, T., et al., Tumor-associated macrophages as major players in the 
tumor microenvironment. Cancers (Basel), 2014. 6(3): p. 1670-90. 
14. 
Ruffell, B., N.I. Affara, and L.M. Coussens, Differential macrophage programming 
in the tumor microenvironment. Trends Immunol, 2012. 33(3): p. 119-26. 


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15. 
Laoui, D., et al., Tumor-associated macrophages in breast cancer: distinct 
subsets, distinct functions. Int J Dev Biol, 2011. 55(7-9): p. 861-7. 
16. 
Zhang, Y., et al., Crosstalk between colon cancer cells and macrophages via 
inflammatory mediators and CD47 promotes tumour cell migration. Eur J 
Cancer, 2013. 49(15): p. 3320-34. 
17. 
Hu, W., et al., Alternatively activated macrophages are associated with 
metastasis and poor prognosis in prostate adenocarcinoma. Oncol Lett, 2015. 
10(3): p. 1390-1396. 
18. 
Wallace, A.M., et al., Lymphoseek: a molecular radiopharmaceutical for sentinel 
node detection. Ann Surg Oncol, 2003. 10(5): p. 531-8. 
19. 
Hoh, C.K., A.M. Wallace, and D.R. Vera, Preclinical studies of [(99m)Tc]DTPA-
mannosyl-dextran. Nucl Med Biol, 2003. 30(5): p. 457-64. 
20. 
Azad, A.K., et al., gamma-Tilmanocept, a New Radiopharmaceutical Tracer for 
Cancer Sentinel Lymph Nodes, Binds to the Mannose Receptor (CD206). J 
Immunol, 2015. 
21. 
Cope, F.O., et al., The inextricable axis of targeted diagnostic imaging and 
therapy: An immunological natural history approach. Nucl Med Biol, 2016. 
43(3): p. 215-25. 
22. 
Surasi, D.S., J. O'Malley, and P. Bhambhvani, 99mTc-Tilmanocept: A Novel 
Molecular Agent for Lymphatic Mapping and Sentinel Lymph Node Localization. 
J Nucl Med Technol, 2015. 43(2): p. 87-91. 
23. 
Alex, J.C., et al., Gamma-probe-guided lymph node localization in malignant 
melanoma. Surg Oncol, 1993. 2(5): p. 303-8. 
24. 
Krag, D.N., et al., Surgical resection and radiolocalization of the sentinel lymph 
node in breast cancer using a gamma probe. Surg Oncol, 1993. 2(6): p. 335-9; 
discussion 340. 
25. 
Paskett, E.D., et al., Cancer-related lymphedema risk factors, diagnosis, 
treatment, and impact: a review. J Clin Oncol, 2012. 30(30): p. 3726-33. 
26. 
Mansel, R.E., N.I. Khonji, and D. Clarke, History, present status and future of 
sentinel node biopsy in breast cancer. The Mary Beves Lecture. Acta Oncol, 2000. 
39(3): p. 265-8. 
27. 
Essner, R. and A.J. Cochran, Sentinel node biopsy: not only a staging tool? Recent 
Results Cancer Res, 2002. 160: p. 133-48. 


Tc 99m tilmanocept 
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28. 
Wallace, A.M., et al., Sentinel lymph node accumulation of Lymphoseek and Tc-
99m-sulfur colloid using a "2-day" protocol. Nucl Med Biol, 2009. 36(6): p. 687-
92. 
29. 
Tokin, C.A., et al., The efficacy of Tilmanocept in sentinel lymph mode mapping 
and identification in breast cancer patients: a comparative review and meta-
analysis of the (9)(9)mTc-labeled nanocolloid human serum albumin standard of 
care. Clin Exp Metastasis, 2012. 29(7): p. 681-6. 
30. 
Marcinow, A.M., et al., Use of a novel receptor-targeted (CD206) radiotracer, 
99mTc-tilmanocept, and SPECT/CT for sentinel lymph node detection in oral 
cavity squamous cell carcinoma: initial institutional report in an ongoing phase 3 
study. JAMA Otolaryngol Head Neck Surg, 2013. 139(9): p. 895-902. 
31. 
Sondak, V.K., et al., Combined analysis of phase III trials evaluating 
[(9)(9)mTc]tilmanocept and vital blue dye for identification of sentinel lymph 
nodes in clinically node-negative cutaneous melanoma. Ann Surg Oncol, 2013. 
20(2): p. 680-8. 
32. 
Wallace, A.M., et al., Comparative evaluation of [(99m)tc]tilmanocept for sentinel 
lymph node mapping in breast cancer patients: results of two phase 3 trials. Ann 
Surg Oncol, 2013. 20(8): p. 2590-9. 
33. 
Agrawal, A., et al., [(99m)Tc]Tilmanocept Accurately Detects Sentinel Lymph 
Nodes and Predicts Node Pathology Status in Patients with Oral Squamous Cell 
Carcinoma of the Head and Neck: Results of a Phase III Multi-institutional Trial. 
Ann Surg Oncol, 2015. 22(11): p. 3708-15. 
 
 


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Appendix 1 
Schedule of Events 
Evaluation 
VISIT 1 
Screening 
Days 
 -29 to 0 
VISIT 2 
Injection and Imaging 
Procedures  
Day 1 
VISIT 3 
Follow-up 
Day 5±3 
0:00 
4-6 hrs 
 
Informed 
Consent 
x 
 
 
 
Entry Criteria  
x 
 
 
 
Medical History 
x 
 
 
 
Vital Sign 
Assessment 
x 
xbd 
x 
x 
Physical 
Examination 
xc 
 
 
x 
Review of 
Medications 
x 
x 
 
x 
Clinical 
Laboratory 
Evaluation 
x 
 
 
x 
Urine Pregnancy 
Test 
x 
xa 
 
 
Tc99m 
tilmanocept 
Administration 
 
x 
 
 
FDG PET/CT 
Imaging 
x 
 
 
 
 SPECT/CT 
Imaging 
 
 
x 
 
Adverse event 
Monitoring 
x 
x 
x 
x 
a. Urine dipstick pregnancy test must be completed and determined to be negative in women of 
childbearing potential within 48 hours of injection. 
b. Time point 0:00 is before tilmanocept injection (a 15 minute pre-injection window is 
permitted). 
c. Physical examinations done within 30 days of injection may be used even if conducted prior to 
obtaining informed consent if they are ordered and conducted at the direction of the Investigator 
as part of his/her standard of care. 
d. Timepoint is within 15 minutes post-injection. 
 


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Appendix 2 
ECOG Performance Status 
Grade 
Eastern Cooperative Oncology Group 
0 
Fully active, able to carry on all pre-disease performance without restriction 
1 
Restricted in physically strenuous activity but ambulatory and able to carry out work of 
a light or sedentary nature, e.g., light house work, office work 
2 
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 
Capable of only limited self-care, confined to bed or chair more than 50% of waking 
hours 
4 
Completely disabled.  Cannot carry on any self-care.  Totally confined to bed or chair 
5 
Dead 
 
 
 


Tc 99m tilmanocept 
Clinical Trial Protocol:NA V3-25 
Sponsor Signatures 
Navidea Biopharmaceuticals 
20 September 2017 
Appendix 3 
Study Title: 
An Exploratory Evaluation of Technetium Tc 99m Tilmanocept 
by Intravenous (IV) Injection in Subjects with Liver Metastases 
from Colorectal Carcinoma Patients using SPECT Imaging 
Compared to FDG PET/CT Imaging. 
Study Number: 
Original Protocol Date: 
Amendment 1 Date: 
Amendment 2 Date: 
NAV3-25 
13 March 2017 
26 April 2017 
08 June 2017 
Amendment 3 Date: 
20 September 2017 
following personnel c 
, ..... --, 
/ 
Signed: 
.,.... ~ 
Michael Blue, MD 
Senior Medical Director 
Confidential 
Page 48 of49 
Date:_q_,,_L-_~_J_t_/_ 
' 
Date: 9 · 2 ~ ' I 7 


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Navidea Biopharmaceuticals 
Clinical Trial Protocol:NAV3-25 
 
20 September 2017  
 
 
 
 
Confidential 
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Appendix 4 
Investigator’s Signature 
Study Title: 
An Exploratory Evaluation of Technetium Tc 99m Tilmanocept 
by Intravenous (IV) Injection in Subjects with Liver Metastases 
from Colorectal Carcinoma Patients using SPECT Imaging 
Compared to FDG PET/CT Imaging. 
Study Number: 
NAV3-25 
Original Protocol Date: 
Amendment 1 Date: 
Amendment 2 Date: 
Amendment 3 Date: 
13 March 2017 
26 April 2017 
08 June 2017 
20 September 2017 
I have read the protocol described above. I agree to comply with all applicable regulations and to 
conduct the study as described in the protocol.  
 
 
Signed: 
 
 
Date: 
 
 
             Pradeep Bhambhvani, MD               
             Associate Professor  
             Division of Molecular Imaging and Therapeutics 
             The University of Alabama at Birmingham