Clinical trial NCT02901925
Conditions: Glioma
Keywords: ABY-029 Affibody Molecular Fluorescence-Guided Surgery Epidermal Growth Factor Receptor (EGFR)
Sponsor: Dartmouth-Hitchcock Medical Center
Start date: 2017-02-13; Completion date: 2021-06-29
Enrollment: 14 participants; Allocation: NON_RANDOMIZED; Intervention model: SINGLE_GROUP

Brief summary:
The primary study objective is to determine if microdoses of ABY-029 lead to detectable signals in sampled tissues with an EGFR pathology score ≥ 1 based on histological staining.

The secondary study objective is to assess diagnostic accuracy of ABY-029 detection by iFI and intraoperative probe relative to histopathology diagnosis, and other indicators (e.g. proliferation, infiltration, etc.) as the gold standard, and to measure the molecular uptake and concentration of ABY-029 in resected specimens.

Detailed description:
The investigators plan a sample size of 6-12 patients in this open label, single center, clinical trial of ABY-029. Administration will occur as a single intravenous injection to subjects with recurrent glioma, approximately 1-3 hours prior to surgery.

The protocol is not a safety study since no physiological effects are expected at microdose levels of ABY-029. Rather, doses have been selected to determine if a fluorescence signal can be detected by wide-field imaging technology with a signal-to-noise ratio of 10, which is considered necessary for subsequent assessment of diagnostic performance of ABY-029 as a tumor biomarker sufficient to guide surgical resection in the future. No diagnostic or therapeutic intent is proposed, and administration of the study drug is not intended to alter the extent of planned brain tumor resection during the surgical procedure.

Arms: arm 1: ABY-029 will be administered at the 1X dose level prior to surgery. Probe will be used in vivo to determine if signal is detectable, and ex vivo tissue pathology will measure extent of binding with EGFR positive tumor tissue. arm 2: ABY-029 will be administered at the 3X dose level prior to surgery. Probe will be used in vivo to determine if signal is detectable, and ex vivo tissue pathology will measure extent of binding with EGFR positive tumor tissue. arm 3: ABY-029 will be administered at the 6X dose level prior to surgery. Probe will be used in vivo to determine if signal is detectable, and ex vivo tissue pathology will measure extent of binding with EGFR positive tumor tissue.
Interventions: intervention 1: ABY-029
Intervention descriptions: intervention 1: Using a sample size of 6-12 patients, ABY-029 will be administered via single intravenous injection to subjects with recurrent glioma, approximately 1-3 hours prior to surgery. Microdose levels of ABY-029 have been selected to determine if a fluorescence signal can be detected by wide-field imaging technology with a signal-to-noise ratio of 10, which is considered necessary for subsequent assessment of diagnostic performance of ABY-029 as a tumor biomarker sufficient to guide surgical resection in the future. Administration of the study drug is not intended to alter the extent of planned brain tumor resection during the surgical procedure.

Protocol documents:
Prot_SAP_001.pdf: 
 
CONFIDENTIAL 
CLINICAL RESEARCH PROTOCOL 
 
 
 
 
 
 
 
 
A MICRODOSE EVALUATION STUDY OF ABY-029  
IN RECURRENT GLIOMA 
 
A Phase 0 open label, single-center clinical trial of ABY-029, an Anti-EGFR Fluorescence Imaging Agent 
via single intravenous injection to subjects with recurrent glioma. 
 
 
Principal Investigator: 
 
 
 
 
 
 
David W. Roberts, MD 
Department of Surgery 
Section of Neurosurgery 
Dartmouth-Hitchcock Medical Center 
One Medical Center Drive 
Lebanon, NH 03756 
603-650-8618 
Co-Investigators: 
 
 
Regulatory Sponsor: 
 
 
Funding Sponsor: 
 
Keith Paulsen, PhD 
Nathan Simmons, MD 
 
Keith Paulsen, PhD 
 
NIH/NCI 
Bethesda, MD 20892 
(240) 276-6315 
Study Product: 
ABY-029 (Z03115-Cys-Mal-IRDye800CW) 
Protocol Number: 
D16107         
eIND Number: 
122681 
 
Initial version:  April 19, 2016  
 
 
 
Revised Version:  October 23, 2018 
Revised version:  May 25, 2016 
 
 
 
Revised Version:  February 14, 2019 
Revised version:  June 3, 2016 
Revised version:  June 3, 2016 rev 
Revised version:  June 30, 2016  
Revised version:  November 4, 2016 
Revised version:  November 21, 2016 
Revised version:  December 22, 2016 
Revised Version:  July 7, 2017 
Revised Version:  August 11, 2017 
Revised Version:  November 28, 2017 
Revised Version:  January 29, 2018 
Revised Version:  February 20, 2018 rev 1 
Revised Version:  April 24, 2018 
 


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Table of Contents 
 
LIST OF ABBREVIATIONS......................................................................................................................... IV 
STUDY SUMMARY ...................................................................................................................................... 1 
1 
INTRODUCTION .................................................................................................................................. 3 
1.1 
BACKGROUND INFORMATION .......................................................................................................... 3 
1.2 
INVESTIGATIONAL AGENT ................................................................................................................ 3 
1.3 
PRECLINICAL DATA ........................................................................................................................ 5 
1.3.1 
Pharmacology studies (non-GLP) ........................................................................................... 5 
1.3.2 
Toxicology study (GLP) ........................................................................................................... 6 
1.4 
CLINICAL DATA TO DATE................................................................................................................. 7 
1.4.1 
Related Affibody product ABY-025.......................................................................................... 7 
1.4.2 
IRDye 800CW in Clinical Trials ............................................................................................... 8 
1.5 
DOSE RATIONALE ........................................................................................................................... 9 
1.6 
POTENTIAL RISKS AND BENEFITS .................................................................................................... 9 
1.6.1 
Potential Risks ......................................................................................................................... 9 
1.6.2 
Potential Benefits .................................................................................................................... 9 
2 
STUDY OBJECTIVES ....................................................................................................................... 10 
3 
STUDY DESIGN ................................................................................................................................. 10 
3.1 
GENERAL DESIGN ........................................................................................................................ 10 
3.2 
PRIMARY STUDY ENDPOINTS ........................................................................................................ 10 
3.3 
SECONDARY STUDY ENDPOINTS ................................................................................................... 10 
4 
SUBJECT SELECTION AND WITHDRAWAL .................................................................................. 10 
4.1 
PATIENT SELECTION ..................................................................................................................... 10 
4.2 
INCLUSION CRITERIA .................................................................................................................... 10 
4.3 
EXCLUSION CRITERIA ................................................................................................................... 11 
4.4 
SUBJECT RECRUITMENT AND RETENTION ...................................................................................... 11 
4.5 
EARLY WITHDRAWAL OF SUBJECTS............................................................................................... 11 
4.5.1 
When and How to Withdraw Subjects ................................................................................... 11 
4.5.2 
Data Collection and Follow-up for Withdrawn Subjects ........................................................ 11 
5 
STUDY DRUG .................................................................................................................................... 11 
5.1 
DESCRIPTION ............................................................................................................................... 11 
5.2 
TREATMENT REGIMEN .................................................................................................................. 12 
5.3 
METHOD FOR ASSIGNING SUBJECTS TO TREATMENT GROUPS ....................................................... 12 
5.4 
PREPARATION AND ADMINISTRATION OF STUDY DRUG ................................................................... 12 
5.5 
SUBJECT COMPLIANCE MONITORING ............................................................................................. 12 
5.6 
PRIOR AND CONCOMITANT THERAPY ............................................................................................. 12 
5.7 
PACKAGING ................................................................................................................................. 12 
5.8 
BLINDING OF STUDY DRUG ........................................................................................................... 13 
5.9 
RECEIVING, STORAGE, DISPENSING AND RETURN .......................................................................... 13 
5.9.1 
Receipt of Drug Supplies ....................................................................................................... 13 
5.9.2 
Storage .................................................................................................................................. 13 
5.9.3 
Dispensing of Study Drug ...................................................................................................... 13 
5.9.4 
Drug Accountability ............................................................................................................... 13 
5.9.5 
Return or Destruction of Study Drug ..................................................................................... 13 
6 
STUDY PROCEDURES ..................................................................................................................... 13 
6.1 
SURGICAL VISIT ........................................................................................................................... 14 
6.1.1 
Specimen Preparation and Handling .................................................................................... 14 
6.2 
CLINICAL LABORATORY AND MONITORING DATA ............................................................................ 14 
6.3 
SPECIMEN ANALYSIS .................................................................................................................... 15 


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7 
STATISTICAL PLAN ......................................................................................................................... 15 
7.1 
SAMPLE SIZE DETERMINATION ...................................................................................................... 15 
7.2 
DATA AND STATISTICAL ANALYSIS ................................................................................................. 15 
7.3 
SUBJECT POPULATION(S) FOR ANALYSIS ....................................................................................... 16 
8 
SAFETY AND ADVERSE EVENTS ................................................................................................... 16 
9 
DATA HANDLING AND RECORD KEEPING ................................................................................... 16 
9.1 
CONFIDENTIALITY ......................................................................................................................... 16 
9.2 
CASE REPORT FORMS.................................................................................................................. 17 
10 
CLINICAL SITE MONITORING ......................................................................................................... 17 
10.1 
ON-SITE MONITORING .................................................................................................................. 17 
10.2 
AUDITING AND INSPECTING ........................................................................................................... 17 
11 
ETHICAL CONSIDERATIONS .......................................................................................................... 17 
12 
STUDY FINANCES ............................................................................................................................ 17 
12.1 
FUNDING SOURCE ........................................................................................................................ 17 
12.2 
CONFLICT OF INTEREST ................................................................................................................ 17 
13 
REFERENCES ................................................................................................................................... 18 
14 
ATTACHMENTS ................................................................................................................................ 20 
14.1 
ZEISS PENTERO MICROSCOPE MODIFICATIONS .............................................................................. 20 
14.2 
ZEISS S1 MICROSCOPE MODIFICATIONS......................................................................................... 21 
 
 
 


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List of Abbreviations 
 
ABY-029 
Affibody 
AE 
Adverse Event/Adverse Experience 
CFR 
Code of Federal Regulations 
CPHS 
Committee for the Protection of Human Subjects 
CRF 
Case Report Form 
CSOC 
Clinical Study Oversight Committee 
DCC 
Data Coordinating Center 
DCE 
Dynamic contrast-enhanced 
dFI 
Depth-detected Fluorescence Imaging 
DHHS 
Department of Health and Human Services 
DSMAC 
Data, Safety Monitoring, and Accrual Committee 
FLI 
Fluorescence Imaging 
GCP 
Good Clinical Practice 
HIPAA 
Health Insurance Portability and Accountability Act 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonization 
iFI 
Intraoperative Fluorescence Imaging 
IRB 
Institutional Review Board 
ISM 
Independent Safety Monitor 
FI 
Fluorescence Imaging 
GBM 
Glioblastoma Multiforme 
HGG 
High grade glioma 
MRI 
Magnetic resonance imaging 
NIH 
National Institutes of Health 
OCTOM 
Office of Clinical Trials Operations and Management, NIDCR, NIH 
OHRP 
Office for Human Research Protections 
OHSR 
Office of Human Subjects Research 
PI 
Principal Investigator 
QA 
Quality Assurance 
QC 
Quality Control 
qdFI 
quantitative depth-resolved Fluorescence Imaging 
qFI 
quantitative Fluorescence Imaging 
SAE 
Serious Adverse Event/Serious Adverse Experience 
SOP 
Standard Operating Procedure 
SNR 
Signal-to-Noise  


A Microdose Evaluation Study of ABY-029 in Recurrent Glioma 
 
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Study Summary 
Title 
A Phase 0 open label, single-center clinical trial of ABY-029, an Anti-EGFR 
Fluorescence Imaging Agent, via single intravenous injection in subjects with 
recurrent glioma.  
Short Title 
A Microdose Evaluation Study of ABY-029 in Recurrent Glioma 
Protocol Number 
N/A 
Phase 
Phase 0 microdose 
Methodology 
Patients will be administered a single intravenous dose of ABY-029 1-3 hours 
before surgery. At different times during resection, the coregistered surgical 
microscope (Zeiss Pentero enabled with intraoperative fluorescence imaging, 
iFI) will be focused on a point of interest.  The surgeon will switch to iFI mode 
to capture images of the surgical field. Quantitative measurements of 
fluorophore concentration will be made at the focal point of the operating 
microscope with an intraoperative probe and biopsy samples will be taken at 
the coregistered point of interest for subsequent histological analyses and ex-
vivo assessments of fluorophore concentration.  In addition, an imaging array 
mounted on second commercial, clinical microscope, will be briefly exchanged 
with the primary scope to take additional images of the surgical field.   
Study Duration 
12-18 months 
Study Center(s) 
Single-center, Dartmouth-Hitchcock Medical Center 
Objectives 
The primary study objective is to determine if microdoses of ABY-029 (up to 
6X) lead to detectable signals (defined as signal-to-noise ratio, SNR ≥10, with 
wide-field iFI) in sampled tissues with an EGFR pathology score ≥ 1 based on 
histological staining.  
 
The secondary study objective is to assess diagnostic accuracy of ABY-029 
detection by iFI and intraoperative probe relative to histopathology diagnosis, 
and other indicators (e.g. proliferation, infiltration, etc.) as the gold standard, 
and to measure the molecular uptake and concentration of ABY-029 in 
resected specimens. 
Number of Subjects 
A minimum of 6 and a maximum of 12 adult patients with a diagnosis of 
recurrent glioma will be enrolled.  
Diagnosis and Main 
Inclusion Criteria 
Operable brain tumor in adults with pre-surgical diagnosis of recurrent glioma. 


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Study Product, Dose, 
Route, Regimen 
 
Criteria for successful iFI are locations of all EGFR positive samples (defined 
as having a pathology score ≥ 1 based on histological staining) having a 
measurable signal (defined as SNR ≥10). A single microdose of ABY-029 at 
30 nanomoles (237µg) will be injected into the first 3 patients. If samples from 
these subjects meet successful iFI criteria, 3 more patients will be enrolled at 
the same dose, concluding the study with a total of 6 subjects. Otherwise, the 
ABY-029 dose in the next 3 patients will be increased to 3X (90 nanomoles, 
711 µg). If samples from these subjects meet successful iFI criteria, 3 more 
patients will be enrolled at the 3X dose, concluding the study with a total of 9 
subjects. If iFI criteria are not met, the next 3 patients will be enrolled at 6X 
dose (180 nanomoles, 1.42 mg).  If iFI criteria are met, 3 more patients will be 
enrolled the 6X dose, concluding the study with a total of 12 subjects. If iFI 
criteria are not met in the first 3 patients at the 6X dose, enrollment will be 
stopped with a total of 9 subjects.  
 
Because of the microdosing levels used, the results of the Dartmouth Toxicity 
Study (at 10X, 100X, 1000X doses), and the safety profile of similar 
compounds (e.g. ABY-025), we do not anticipate any serious adverse events 
(SAEs) related to ABY-029. 
Duration of 
administration 
ABY-029 will be administered by IV injection on day of surgery, approximately 
1-3 hours prior to the procedure. 
Reference methods 
Post-operative histopathology will serve as the gold standard. 
Statistical 
Considerations 
Repeated measures analyses of variance will be performed to account for 
multiple samples from each patient. Student’s t-test will be used to evaluate 
ABY-029 fluorescence signal detection in tumor relative to normal brain with 
iFI and intraoperative probe. Empirical ROC curves and logistic regression 
analysis for clustered binary data will be used to evaluate the accuracy of ABY-
029-specific fluorescence in discriminating between EGFR+ glioma tumor, 
EGFR- glioma tissue, and normal tissue when detected by iFI and/or 
intraoperative probe. 
 


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1 Introduction 
This document is a clinical research protocol and describes a human research study. This study will be 
conducted according to US and international standards of Good Clinical Practice (FDA Title 21 part 312 
and International Conference on Harmonization guidelines), applicable government regulations and 
Institutional research policies and procedures. All personnel involved in the conduct of this study have 
completed human subject protection training. 
1.1 Background Information 
The focus of this exploratory investigational new drug (eIND) development is to realize a fluorescent 
reporter to guide neurosurgical glioma resection using a synthetic peptide that binds to the epidermal growth 
factor receptor (EGFR) and provides an optical fluorescent signal of the concentration.  The combined 
peptide and fluorophore will be called ABY-029, and is the focus of this first-in-human trial in neurosurgery.  
The initial focus of this Phase 0 clinical study is evaluation of drug uptake and concomitant imaging signal.  
 
This research is funded by NIH under grant R01 CA167413.  Following completion of the manufacturing 
and nonclinical plans, we will submit an exploratory IND (eIND) to FDA.  Depending on the findings from 
clinical studies conducted under eIND, we anticipate the submission of a traditional IND. 
 
Several groups have pioneered the use of fluorescence imaging in experimental surgery [1] and a platform 
of targeted compounds for surgical applications [2-5] in which the surgeon’s white light visual field is 
augmented with fluorescent-guided information. Perhaps the largest clinical experience with fluorescence-
guided resection has been gained in neurosurgery with aminolevulinic acid (ALA) induced protoporphyrin 
IX (PpIX). The approach has been evaluated in a prospective randomized Phase III trial [6] with sufficiently 
positive outcomes to alter standard-of-care in Germany for surgical resection of high grade glioma [7] and 
related studies have been completed and are ongoing at Dartmouth under FDA IND #77,751 for oral 
administration of ALA [8-12]. 
 
Clinical motivation exists for pursuing new brain tumor fluorescent probes beyond PpIX for guiding surgical 
resection. First, ALA-induced PpIX fluorescence is enhanced by non-tumor-specific molecular processes 
related to increased metabolism and blood-brain-barrier breakdown. Second, while these processes 
provide reliable enhancement in high-grade glioma tumors, enhancement at the margin and in low-grade 
glioma is relatively poor. Third, the anti-EGFR ABY-029 molecule we are developing has potential to guide 
a range of oncologic surgeries, given the large number of tumor types overexpressing EGFR. Most 
importantly, a wealth of information exists on EGFR fluorophore targeting [13-25] [26-28] which suggests 
our approach is likely to be successful and could benefit patients with malignant glioma and other cancers 
whose prognosis is improved by complete surgical resection. 
1.2 Investigational Agent 
The production of the investigational drug to be used in the clinical study, ABY-029, is being coordinated 
by Dartmouth with project partners Affibody AB, LI-COR Biosciences, and the University of Alabama at 
Birmingham (UAB) Vector Production Facility, with Bachem AG (Switzerland) as the manufacturing 
subcontractor. 
 
The Swedish biotechnology company Affibody AB has developed a group of molecules which are small, 
single domain, non-immunoglobulin proteins based on a 58 amino acid, three-alpha-helical scaffold [29, 
30]. The target binding site is composed of 13 surface-exposed amino acids on helix 1 and helix 2. By 
randomizing these 13 amino acids, the company has created large libraries of Affibody® molecules from 
which ones that bind to a given target protein are selected (examples include EGFR, HER2 or PDGFRß 
[31-33]). Their small size and stability, high affinity and target recognition specificity, and rapid 
biodistribution kinetics and clearance make them ideal for in vivo targeting. Affibody molecules can be 
produced either by recombinant techniques in bacteria or by conventional peptide synthesis and can be 
modified with functional groups for fluorescent or radioactive labels. Site specific modification of Affibody 
molecules is achieved by introducing a single cysteine which can be used for modification of maleimide-
activated functional groups leading to defined and homogenous products [34]. 


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LI-COR is a privately held company that is committed to the development of in vivo fluorescence imaging 
during surgery and has been systematically developing near-infrared (NIR) fluorescent molecules with the 
goal of creating in vivo reporters for targeted contrast. IRDye 800CW is a NIR dye that can be functionalized 
with either an NHS or maleimide reactive group, allowing it to be attached to a number of biomolecules.[35] 
Recently, cetuximab conjugated to IRDye 800CW was used at the University of Alabama in a dose 
escalation study in 12 patients with squamous cell carcinoma in the head and neck.  After passing through 
FDA safety and pharmacokinetic studies, the investigators showed positive correlation between 
fluorescence markers and EGFR levels, and demonstrated that fluorescently labelled molecules can be 
safely administered to humans to identify tumor cells with sub-millimeter resolution [36]. 
  
The ABY-029 conjugate is produced by first incubating the Z03115-Cys peptide with Dithiothreitol (DTT), 
to break up Affibody dimers that naturally form, and then mixing the peptide with IRDye 800 CW maleimide 
dye, at a ratio of one dye molecule per peptide molecule.  
 
Z03115-Cys is a synthetically generated 59 amino acid peptide produced by Bachem (Switzerland). The 
peptide has a molecular formula of C299H468N78092S3 and is composed of (H-Ala-Glu-Ala-Lys-Tyr-Ala-Lys-
Glu-Met-Trp-Lle-Ala-Trp-Glu-Glu-Lle-Arg-Asn-Leu-Pro-Asn-Leu-Asn-Gly-Trp-Gln-Met-Thr-Ala-Phe-Lle-
Ala-Lys-Leu-Leu-Asp-Asp-Pro-Ser-Gln-Ser-Ser-Glu-Leu-Leu-Ser-Glu-Ala-Lys-Lys-Leu-Asn-Asp-Ser-Gln-
Ala-Pro-Lys-Cys-OH).  
 
The dye is coupled to the peptide molecule via maleimide chemistry at a single Cys residue at the C-
terminus of the peptide, as illustrated schematically below.  The conjugation reaction is followed by 
purification from free dye.  
 
 
Figure 1 - Schematic view of ABY-029 (Z03115-Cys-Mal-IRDye800CW) 
The solid phase peptide synthesis production of Z03115-Cys has been subcontracted to Bachem AG 
(Bubendorf, Switzerland). Bachem has automated systems for generation of pharmaceutical grade product, 
and a long history of production for human use.  The maleimide linkage conjugation of Z03115-Cys with 
IRDye800CW has been subcontracted to the University of Alabama at Birmingham Vector Production 
Facility, and will be conducted in their cGMP laboratories. 
 
Physical Form:   
Clear, green-blue liquid. 
Molecular Formula:    
Cys-((RS)-1-[2-maleimido-ethyl]-IRDye 800CW-OH) 
Molecular Weight:   
 
7914.95 grams/mole 
Solubility:  
Soluble in Phosphate Buffered Saline (PBS)  
pKa Value:   
 
 
6.84E+05 
pH Value: 
 
 
7.4 


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Kd Value: 
1 nanomolar (nM) 
 
The drug will be supplied in vials containing 5 mL of liquid Phosphate Buffered Saline (PBS).  
1.3 Preclinical Data 
1.3.1 
Pharmacology studies (non-GLP) 
The following studies (performed with a non-GMP batch of ABY-029) confirm that fluorescently labeled anti-
EGFR Affibody molecules delineate tumor margins better than a fluorescently labeled anti-EGFR antibody. 
[37,38] 
 
• 
Target binding assay (Biacore) 
Using 3 human tumor models with varying EGFR positive expression, and 1 EGFR negative tumor line for 
rats, Dartmouth determined the EGFR concentration in each tumor model using ABY-029, along with a 
simultaneously delivered reference agent (control affibody-IRDye680RD conjugate). We found that in vivo 
receptor concentration, calculated through receptor concentration imaging (RCI), was strongly correlated 
with ex vivo pathologist-scored immunohistochemistry and computer-quantified ex vivo immuno- 
fluorescence. Weak correlation was observed with ex vivo Western blot or in vitro flow cytometry assays.  
 
• 
Cell binding assays 
o 
Isolated receptor binding assay 
The dissociation constant (KD) and receptor-ligand binding on and off rates (Kon and Koff) were 
determined using surface plasmon resonance (Biacore, GE Life Sciences). Sensograms were 
acquired and evaluated using the Biacore evaluation software to determine Kon and Koff using a 
1:1 binding ratio kinetic fitting scheme. The KD value was subsequently calculated using the 
experimentally determined rate constants. We demonstrated that the binding affinity of anti-EGFR 
Affibody with IRDye 800 CW with human and rat EGFR was maintained as compared with anti-
EGFR Affibody alone. 
 
• 
EGFR phosphorylation assay 
LI-COR Biosciences conducted a study in 2010 in which they labelled an EGFR-specific Affibody molecule 
(Eaff) with IRDye800CW maleimide and tested the molecular binding in cell culture and xenograft mouse 
tumor models. This study showed the dye labelled Affibody was bound and taken up specifically by EGFR-
overexpressing tumor cells. When the fluorescent molecule was intravenously injected into nude mice with 
xenograft tumors, the tumor could be identified 1 hour after injection and was prominent after 1 day. 
Following dissection of tissue sections, they saw the labelled molecule accumulated highest in the liver, 
followed by the tumor and kidney. This study demonstrated that an Affibody labelled with a NIR fluorophore 
holds promise as a molecular imaging agent for EGFR-overexpressing tumors. 
 
• 
In vitro cytotoxicity study  
Phototoxic potential of the infrared light source when cells are exposed to ABY-029 was evaluated, 
compared to a known FDA approved phototoxic agent benzoporphyrin derivative monoacid A (BPD-MA) 
and found to be negligible.   
 
• 
Imaging study for localization in U251 orthotopic xenograft glioma in mice and rats  
Rats or mice were inoculated with orthotopic implantations of a human glioma cell line (U251, F98 or F98-
EGFR) or with PBS (control) in the brain. Tumors were allowed to grow for 2 – 3 weeks before carrying out 
fluorescent tracer experiments. 
 
We demonstrated that ABY-029 penetrated further into the margin of a U251 orthotopic xenograft mouse 
model than did a fluorescently labelled antibody (IRDye 680RD-Erbitux) (Sexton et al, PLoS One 2013). 
Longitudinal imaging of mice bearing U251 xenografts grown orthotopically in the brain was performed by 
tracking the uptake and retention over 0-3 hours in order to mimic in vivo imaging in humans. A reference 
tracer composed of a non-specific Affibody molecule bound to IRDye 680RD (LI-COR Biosciences, Inc) 
was used to quantify blood vessel leakage versus ABY-029 binding to the tumor. We also quantified the 


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tumor to normal brain uptake as a function of time, and compared these results to direct fiber optic probe 
measurements in situ and ex vivo. We validated the concept that sufficiently high signal to noise is 
achievable for ABY-029 uptake in the xenograft tumor model for microdose injection levels. This study 
suggests that microdoses of the compound will be detectable via imaging in human studies.  
 
We used the U251 orthotopic xenograft rat model to demonstrate that distribution and uptake of microdose 
levels of ABY-029 is heterogeneous within a tumor, and that high fluorescing regions closely match with 
highly overexpressing regions of EGFR, as determined by immunohistochemistry (D’Souza et al, submitted 
2016). We evaluated the uptake of the preGMP batch of ABY-029 in glioma after intravenous injection in 
normal rats. Fluorescent imaging of ex vivo brain slices from rats was conducted at different time points 
after ABY-029 administration (1 h – 48 h) to verify which time points provided maximal tumor to normal 
contrast. Although U251 tumor was most clearly visualized 1 hour after ABY-029 injection, tumors could be 
differentiated from the background after 48 hours. These results suggest that ABY-029 shows excellent 
potential to increase surgical visualization of confirmed EGFR positive tumors, with tumor to normal tissue 
contrast levels between 10 and 15. 
 
We compared uptake of pre-GMP ABY-029 and in orthotopic rat model using both an EGFR negative tumor, 
F98, and EGFR positive tumor line, F98-EGFR, which was transfected with EGFR. In addition, we compare 
the uptake of ABY-029 with protoporphyrin IX (PpIX, administered as 5-aminolevulinic acid, ALA), a 
fluorophore currently under investigation for surgical guidance during glioma resection at Dartmouth 
College, and the standard of care for glioma patients in Germany. It was shown that in the EGFR positive 
tumor (F98-EGFR) ABY-029 outperforms PpIX in both the bulk and the margins of the tumors, while they 
performed the same in the bulk EGFR negative tumor (F98) and PpIX showed a slight advantage in the 
margin of the EGFR tumor (Elliott et al, 2016 submitted). These results show a clear indication that ABY-
029 provides a surgical advantage in EGFR positive tumors. 
1.3.2 
Toxicology study (GLP) 
The preclinical toxicology assessment is based on a single-dose toxicity study in one species conducted at 
Dartmouth.  
 
• 
Extended single-dose Safety Study in rats 
We conducted a definitive, extended single-dose toxicity study of ABY-029 in Sprague-Dawley rats, 
consistent with the principles outlined in “Guidance for Industry, Investigators, and Reviewers: Exploratory 
IND Studies” (2006). This study used the non-GMP-manufactured ABY-029 batch created at UAB, which 
was also used to develop the GMP process.  
 
A maximum microdose is 30 nmol per 60 kg human subject (or ~237 µg for a protein drug, or 3.95 µg/kg in 
a human), and this study used the equivalent rat dose of 4.898 µg for a 0.20 kg rat (allowing for a 6.2X rat-
to-human correction for skin surface area). Following review of FDA guidelines and pre-IND meetings with 
FDA, doses of 10X, 100X and 1000X were studied to evaluate whether pharmacologically relevant effects 
exist.  The dose levels tested were 0.049 mg, 0.49 mg and 4.9 mg in the rat toxicity study, along with a 
control group.  
 
Total Study Duration 
1 day and 14 day post-injections 
Recovery 
Control and high dose group, 
sacrifice and analysis on day 15 
Route 
IV (bolus) 
Frequency of dose 
single  
No of doses 
1 
Groups 
Control and 3 dose groups 
No of animals 
6 male + 6 female per group 
Dose levels 
(preliminary) 
0 µg/kg 
244.9 µg/kg  
2449 µg/kg  
24490 µg/kg 


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Main study 
Day 0 - ABY-029 administration  
Recovery period 
1 or 14 days 
Analysis 
All standard parameters: Clinical 
signs, post-dosing observations, 
standard ophthalmology, body 
weight, haematology, bone marrow 
smears, clinical chemistry, 
macroscopic examination and 
organ weights, histopathology 
 
A comprehensive set of protocol tissues was collected during necropsy and sent to a veterinary pathologist 
for microscopic examination. No histopathologic evidence of systemic toxicity was associated with the 
single-dose (10X, 100X, 1000X) intravenous administration of ABY-029 after 24 hours or 14 days in the 
study rats.  Further, no toxic effects were observed among any of the animals during the routine daily 
checks.  
 
• 
Biodistribution Study in normal rats  
Biodistribution of the 100X microdose of ABY-029 was assessed in normal rats and analysis was performed 
at 1 hour after injection. One hour post-intravenous administration of ABY-029 the rats were sacrificed by 
cervical dislocation, and the organs of interest were removed and then imaged on the Pearl Imaging System 
(LI-COR Biosciences). We found a high accumulation of ABY-029 in the liver, skin, lungs, and kidney, which 
are all organs with high endogenous EGFR expression and/or that are involved in the route of excretion. 
No significant difference occurred between ABY-029 accumulation in male and female non-sex organs, 
although males had slightly higher values, possibly due to size of organ versus body weight, as injected 
dose was normalized to body weight. Rats without brain tumors had comparably very low levels of ABY-
029 accumulation in normal brain tissue, due to the blood brain barrier.   
 
• 
Pharmacokinetic Study in normal rats  
Plasma pharmacokinetics of the dye was assessed as function of time by sampling blood from a jugular 
vein catheter at various time points after administration. Pharmacokinetic data was collected from normal 
Sprague Dawley rats at time points from 1 minute to 48 hour) after intravenous injection of 100X microdose 
ABY-029 (244.9 µg/kg). Blood plasma was read on a Fluoromax-3 fluorimeter (Horiba Jobin Yvon), a 
standard curve was created, and all fluorescent data was converted to concentration using the standard 
curve and the bi-exponential plasma curve. The plasma curve created in this study matched closely with 
the previously determined pre-clinical plasma curves, indicating that the physical and biological 
characteristics of ABY-029 were closely retained. The plasma half-life of ABY-029 in normal rats is 
approximately 15-20 minutes and less than 10% remains in the plasma at 24 hours and is undetectable at 
48 hours. In pre-clinical rat and mouse studies, ABY-029 has been detectable in tissues (tumor, liver, 
kidney) for up to 72 hours. 
1.4 Clinical Data to Date 
While no clinical data is available to date on the experimental investigational product, the component 
molecules Z03115-Cys and IRDye800CW have been used in clinical trials, and products similar to ABY-
029 have gone to clinical trial as well. 
1.4.1 
Related Affibody product ABY-025 
ABY-025 (ZHER2:2891-Cys-MMA-DOTA), is a HER2-specific Affibody® molecule that targets a radionuclide to 
HER2-receptor expressing tumors, for in vivo diagnosis of HER2-expressing cancer. ABY-025 is a 61 amino 
acid peptide that is modified site-specifically with one DOTA molecule at the side chain of the C-terminal 
cysteine. DOTA acts as chelator for the radionuclide Indium-111 (111In) or Gallium-68 (68Ga). An 
Investigational Medicinal Product Dossier (IMPD) for ABY-025 labeling with 68Ga or 111In has been filed for 
clinical studies in Sweden and Denmark, as outlined below.   
 
1) ABY-025-MI103 (EudraCT 2010-021078-12, NCT01216033)  


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An exploratory study to evaluate the distribution of [111In]ABY-025 uptake for SPECT imaging in 
subjects with metastatic breast cancer. This study included 7 patients and was completed in Sweden 
in 2011, with results published [39] 
 
2) ABY-025-MI105 (EudraCT 2012-005228-14, NCT01858116).  
An exploratory study to evaluate [68Ga]ABY-025 for PET imaging of HER2 expression in subjects with 
metastatic breast cancer was due to have been completed in December 2014. This study was initiated 
2013 in Swede68n and 16 patients were enrolled to receive at least 2 doses of ABY-025. No adverse 
events with possible or probable relationships to the study drug were reported, and seven non-serious 
adverse events with “unlikely” relationships to the study drug were noted [40]. 
 
3) MA 1021 (EudraCT 2010-024353-36, NCT02095210) 
An exploratory study of HER2 PET imaging in breast cancer patients using [68Ga]ABY-025 was 
completed. This study was conducted in Denmark from October 2010 to April 2013 with 8 patients 
enrolled. Results have been published [41] 
1.4.2 
IRDye 800CW in Clinical Trials 
As of March 31, 2016, seven trials are listed on http://clinicaltrials.gov/ involving the use of IRDye 800CW, 
two that were completed and five are ongoing with patients being recruited. 
 
1) A Phase 1 study was completed (NCT01508572) (VEGF-targeted Fluorescent Tracer Imaging in 
Breast Cancer) (Oct 2011 – Sept 2012), using a VEGF targeted fluorescent tracer to breast cancer, 
imaging with diffuse tomography, opto-acoustic tomography and fluorescence reflectance imaging, with 
administration of 4.5 mg of bevacizumab-IRDye800 at 1-3 days prior to imaging.  The trial enrolled 20 
subjects total beginning in October 2011 and was completed in January 2015, but no results have been 
published. 
 
2) A Phase 1 study was completed (NCT 02113202) (Molecular Fluorescence Endoscopy in Patients 
with Familial Adenomatous Polyposis, Using Bevacizumab-IRDye800CW) between March 2014 
and January 2016. Seventeen patients with Familial Adenomatous Polyposis were administered 
Bevacizumab-IRDye800 at 4.5mg, 10mg or 25mg at 1-3 days prior to endoscopy.  The study’s primary 
objective was to determine the sensitivity of the fluorescent tracer bevacizumab-IRDye800CW, a NIR 
fluorescence endoscopy platform, in identifying adenomatous polyps. Results have not been published. 
 
3) A Phase 1 study is recruiting patients (NCT 01972373) (Visualization of Rectal Cancer During 
Endoscopy, Using a Fluorescent Tracer), as of 2014, imaging rectal cancer with endoscopy and 
optical tomography methods, using two imaging sessions, each with a dose of 4.5mg of Bevacizumab-
IRDye800, imaged before and then again 3 weeks after the initiation of chemotherapy.  In these cases 
fluorescence imaging is performed 2 days after administration of the conjugate.  30 patients were 
estimated for enrolment.  
 
4) Another Groninigen study is a Phase 1 trial for esophageal imaging (NCT 02129933) (VEGF-targeted 
Fluorescence Near-Infrared (NIR) Endoscopy in (Pre)Malignant Esophageal Lesions), approved 
for April 2014 – June 2016.  In this study, 4.5 mg of Bevacizumab-IRDye800 is given by IV at 2 days 
prior to fluorescence endoscopy imaging in 10 patients.   
 
5) An ongoing Phase 1 trial in the United States with IRDye800CW targeted to the EGF receptor, (NCT 
01987375) (Cetuximab IRDye800 Study as an Optical Imaging Agent to Detect Cancer during 
Surgical Procedures) is a dose escalation trial at the University of Alabama Birmingham. Head and 
Neck cancer is imaged during surgery, with Cetuximab-IRDye800CW conjugate as the administered 
agent. This study is directed by Dr Eben Rosenthal, planned for November 2015 – December 2016. 
Fifteen patients must be shown to tolerate a test dose of unlabelled Cetuximab of 100 mg, in the week 
prior to entering this study.  The second phase of this study will reduce the test dose to 10 mg. 
 


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6) Radboud University in the Netherlands is conducting a Phase 1 trail (NCT 02497599) (Intraoperative 
Dual-modality Imaging in Renal Cell Carcinoma) with Indium-111-DOTA-girentuximab-
IRDye800CW in renal cell carcinoma patients.  The study will assess the feasibility and safety of 
intraoperative dual-modality imaging with Indium-111-DOTA-girentuximab-IRDye800CW in 22 patients 
between June 2015 and December 2016.  Patients will receive a single IV dose of the drug, followed 
by a whole body planar scan and SPECT/CT scan and a partial nephrectomy. The study will be 
extended by use of dual-modality imaging. 
 
7) A Phase 1 and 2 study at the University Medical Center Groningen (NCT 02583568) (Fluorescence 
Guided Surgery in Breast Cancer) is recruiting patients.  This study will enrol 26 patients from October 
2015 to December 2016 to define the optimal dose of the fluorescent tracer Bevacizumab-
IRDye800CW for intraoperative delineation of breast cancer tissue using an improved and optimized 
fluorescent tracer and camera system. 
1.5 Dose Rationale  
We plan a sample size of 6-12 patients in this open label, single center, clinical trial of ABY-029.  
Administration will occur as a single intravenous injection to subjects with recurrent glioma, approximately 
1-3 hours prior to surgery. The protocol is not a safety study since no physiological effects are expected at 
microdose levels of ABY-029. Rather, doses have been selected to determine if a fluorescence signal can 
be detected by wide-field imaging technology with a signal-to-noise ratio of 10, which is considered 
necessary for subsequent assessment of diagnostic performance of ABY-029 as a tumor biomarker 
sufficient to guide surgical resection in the future.  No diagnostic or therapeutic intent is proposed, and 
administration of the study drug is not intended to alter the extent of planned brain tumor resection during 
the surgical procedure. 
 
Dosing Scheme: A single microdose of ABY-029 at 30 nanomoles (237µg) will be injected into the first 3 
patients. If samples from these subjects meet successful iFI criteria (defined as SNR ≥10 at locations of 
samples with EGFR pathology score ≥ 1 based on histological staining), 3 more patients will be enrolled at 
the same dose, concluding the study with a total of 6 subjects. Otherwise, the ABY-029 dose in the next 3 
patients will be increased to 3X (90 nanomoles, 711 µg). If samples from these subjects meet successful 
iFI criteria, 3 more patients will be enrolled at the 3X dose, concluding the study with a total of 9 subjects. 
If iFI criteria are not met, the next 3 patients will be enrolled at 6X dose (180 nanomoles, 1.42 mg).  If iFI 
criteria are met, 3 more patients will be enrolled the 6X dose, concluding the study with a total of 12 subjects. 
If iFI criteria are not met in the first 3 patients at the 6X dose, enrollment will be stopped with a total of 9 
subjects.  
1.6 Potential Risks and Benefits  
The risks to subjects are reasonable in relation to anticipated benefits and/or knowledge that might 
reasonably be expected from the results of this clinical trial. 
1.6.1 
Potential Risks 
Because of the microdose levels to be used, the results of the Dartmouth Toxicity Study, and the safety 
profile of similar compounds (e.g. ABY-025), serious adverse events (SAEs) related to ABY-029 are not 
expected to occur.   
1.6.2 
Potential Benefits 
Patients enrolling in this study will not benefit directly because no diagnostic or therapeutic decisions will 
occur based on the study drug, and thus, administration of the study drug is not intended to alter the extent 
of planned brain tumor resection during the surgical procedure. However, future patients may benefit from 
the knowledge gained from the study. Current literature shows that maximum cytoreductive surgery is of 
significant benefit to patients with malignant gliomas. Patients with complete resections also experience 
survival benefits from post-operative radiation and/or chemotherapy. Unfortunately, achieving a high 
degree of tumor removal is often limited by the surgeon’s ability to distinguish residual disease from 
intervening parenchyma under conventional white light illumination, and rates of complete resection have 


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been reported to be as low as 20% in high grade glioma. iFI augments the surgeon’s white light visual field 
with fluorescent information, and in the case of ALA-induced PpIX, improved patient outcomes in a 
prospective randomized Phase III trial sufficiently to alter standard-of-care in Germany for surgical resection 
of high grade glioma. While ALA appears to be effective, motivation exists for pursuing new fluorescent 
probes beyond PpIX for guiding surgical resection. First, ALA-induced PpIX fluorescence is not tumor-
specific. Second, fluorescent enhancement at the margin and in low-grade glioma is relatively poor. Third, 
ABY-029 has potential to guide a range of oncologic surgeries, given the large number of tumor types that 
overexpress EGFR. Fourth, a wealth of information exists on EGFR fluorophore targeting which suggests 
our approach is likely to be successful and could benefit patients with malignant glioma and other cancers 
whose prognosis is improved by complete surgical resection. 
2 Study Objectives 
The primary study objective is to determine if microdoses of ABY-029 (up to 6X) lead to detectable signals 
(defined as signal-to-noise ratio, SNR ≥10, with wide-field iFI) in sampled tissues with an EGFR pathology 
score ≥ 1 based on histological staining.  
 
The secondary study objectives are to assess the diagnostic accuracy of ABY-029 detection by iFI and an 
intraoperative probe relative to histopathology as the gold standard, and to measure the molecular uptake 
and concentration of ABY-029 in resected specimens.  
3 Study Design 
3.1 General Design 
This study is a Phase 0 open label, single-center clinical trial of ABY-029, an Anti-EGFR Fluorescence 
Imaging Agent, via single intravenous injection to subjects with recurrent glioma. The expected duration for 
enrolling study participants is 12 to 18 months, and up to 12 subjects may be enrolled. Doses of 1X (30 
nanomoles or 237µg), 3X, and 6X may be considered depending on strength of signal detection with iFI in 
tissues within the surgical field, subsequently sampled and determined post-operatively to have positive 
EGFR scores based on histological staining. 
3.2 Primary Study Endpoints 
The primary study endpoint is signal detection (defined as SNR with wide-field iFI) in vivo in brain tissues 
within the surgical field intended for resection that are subsequently sampled during surgery and assigned 
an EGFR pathology score based on histological staining.  
3.3 Secondary Study Endpoints 
Secondary endpoints of the study are diagnostic accuracy of ABY-029 detection by iFI and intraoperative 
probe relative to histopathology tissue diagnosis, and other indicators (e.g. proliferation, infiltration, etc.) as 
the gold standard, and measurement of molecular uptake and concentration of ABY-029 in resected 
specimens.  
4 Subject Selection and Withdrawal 
4.1 Patient Selection 
Patients with recurrence of presumed high grade glioma having EGFR positive tissue (score ≥ 1 based on 
histological staining or molecular pathology analysis) obtained from prior surgery will be eligible to 
participate.  
4.2 Inclusion Criteria 
The following criteria must be met by study subjects to be eligible for study enrollment: 
 
1) 
Preoperative diagnosis of recurrent high-grade glioma having EGFR positive tissue from prior surgery. 


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2) 
Tumor judged to be suitable for open cranial resection based on preoperative imaging studies. 
3) 
Valid informed consent by subject. 
4) 
Age ≥ 18 years old. 
4.3 Exclusion Criteria 
The following criteria would exclude a subject from study enrollment: 
 
1) 
Pregnant women or women who are breast feeding. 
2) 
Patients on any experimental anti-EGFR targeted therapies 
4.4 Subject Recruitment and Retention 
Participation in this research requires informed consent according to Institutional Review Board (IRB) 
guidelines and a signed IRB-approved Consent Form as the means of documenting this understanding. 
Potential recruits are instructed that their participation is completely voluntary and that their medical care 
will not be altered in any way should they elect not to participate at any time prior to surgery. Subjects are 
recruited from patients presenting or referred to the Section of Neurosurgery at Dartmouth-Hitchcock 
Medical Center for surgical resection of intracranial tumor of the types meeting protocol inclusion criteria. 
Potential subjects may learn about the study through its posting on clinicaltrials.gov and contact Dr. Roberts 
or another surgeon in the Neurosurgery Section about participation. Subjects will be invited to participate 
in this study by a member of the Neurosurgery Section which will occur either at the time of consultation 
with the surgeon about the candidate’s standard-of-care procedures or at another time agreed to by the 
potential participant, the candidate’s surgeon and/or Dr. Roberts or Dr. Roberts’s designee. No 
advertisements or other promotional material will be used. No finder fees or recruitment incentives will be 
offered. Women of child bearing potential are eligible for enrollment into this study because ABY-029 
administration is not considered to present any additional risk for these women. The study will exclude 
women who are pregnant or breast-feeding as indicated in the exclusion criteria. Women of child-bearing 
potential, if asked to participate, will be given a pregnancy test at no cost to confirm pregnancy status before 
administration of ABY-029. 
4.5 Early Withdrawal of Subjects 
4.5.1 
When and How to Withdraw Subjects 
Subjects participate in a “one-time” event (i.e., surgery) as part of this study and are not expected to be 
withdrawn because of subsequent post-surgical conditions and/or scenarios (e.g. the need for alternative 
and/or additional treatment). Subjects may withdraw their consent at any time prior to surgery. Subject data 
collected per protocol may subsequently not be included in the final analysis, if IV administration of ABY-
029 is found not to have occurred at the proper dose (237µg, 711µg or 1.42mg, depending on the dose 
group) approximately 1-3 hours prior to surgery or the definitive post-operative diagnosis does not meet the 
presumed entry criteria. 
4.5.2 
Data Collection and Follow-up for Withdrawn Subjects 
No post-operative end-points or follow-up information is needed to meet study objectives; hence, 
consenting subjects who complete surgery will not be lost to follow-up. 
5 Study Drug  
5.1 Description 
 
Patients will be administered ABY-029 by intravenous injection approximately 1-3 hours prior to induction 
of anesthesia. The range of approximately 1-3 hours is acceptable given pre-clinical data indicating that 
imaging signal levels appear to be stable over this time period. Time from ABY-029 administration to 
surgery is collected. Subjects are considered enrolled in the study once ABY-029 has been injected. 


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5.2 Treatment Regimen 
Groups of up to 6 patients (as described in Section 1.5) will receive a single injection of ABY-029 at 1X or 
3X or 6X of the microdose of 30 nanomoles (237µg). The dose will be administered by intravenous injection 
on the day of surgery, approximately 1-3 hours prior to the procedure.  
5.3 Method for Assigning Subjects to Treatment Groups 
No separate treatment groups are part of the study design. 
5.4 Preparation and Administration of Study Drug 
ABY-029 used in this study is stored, prepared, and dispensed by the Investigational Pharmacy at 
Dartmouth-Hitchcock Medical Center. (Address: Dartmouth Hitchcock Medical Center, Investigational 
Pharmacy, Rubin-463N3, One Medical Center Drive, Lebanon, NH 03756; email address: 
investigational.pharmacy@hitchcock.org; Tel: 603-650-7890).   
 
ABY-029 Preparation and Administration Procedures: 
1) ABY-029 is prepared by the Investigational Pharmacy at the appropriate dose, typically on the day 
of or day before surgery, depending on the scheduled surgery date and time.   
2) From the vial containing ABY-029, the required dose is withdrawn into a syringe, labeled, and 
placed into a light-impermeable bag.  
3) Once prepared at the requested dose, the drug in the light-impermeable bag is either held by the 
Investigational Pharmacy until picked up by a member of the study team, or stored in a refrigerated 
and locked box in the Same Day Surgery patient holding area. The key to the lock-box is placed in 
an associated Acudose Unit. 
4) The dose of ABY-029 is administered intravenously through an IV port followed by a saline flush. 
 
Most study subjects will check in to the Same Day Surgery Program on the day of their surgery, though 
some may be admitted inpatients. Study subjects generally have a pre-surgical MRI.  ABY-029 will be 
administered to patients approximately 1-3 hours prior to surgery, in the Same Day Surgery Program 
area, the MRI area, or in an inpatient unit. Patients will be under the care and observation of a research 
nurse or Same Day Surgery nurse, or by an inpatient nurse if ABY-029 is administered in the inpatient 
area, during the time between ABY-029 administration and the start of surgery, and will be monitored 
accordingly and appropriately. If ABY-029 is administered prior to the pre-surgical MRI, a research nurse 
will accompany the patient to the MRI and monitor the patient until he or she is in the Same Day Program 
holding area.  
5.5 Subject Compliance Monitoring 
Subjects participate in a “one-time” event (i.e., surgery) as part of this study and are monitored through the 
in-patient surgical service during the time of their participation.  
5.6 Prior and Concomitant Therapy 
This study is a Phase 0 diagnostic trial of ABY-029 fluorescence as a possible intraoperative imaging 
biomarker during open cranial surgery for tumor resection. Prior and/or concurrent medical therapies do 
not alter the diagnostic intent and/or endpoints associated with the study. 
5.7 Packaging 
ABY-029 is packaged as a transparent, green-blue liquid in 5mL glass vials at a concentration of 0.27 
mg/mL, in PBS (phosphate-buffered saline). It is stored at the Investigational Pharmacy at Dartmouth-
Hitchcock Medical Center (Address: Dartmouth Hitchcock Medical Center, Investigational Pharmacy, 
Rubin-463N3, One Medical Center Drive, Lebanon, NH 03756).  ABY-029 is shipped in clear glass vials, 
each with a rubber stopper in boxes containing 10 mL units. 


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5.8 Blinding of Study Drug 
This study is a Phase 0 diagnostic trial of ABY-029 fluorescence as a potential intraoperative imaging 
biomarker during open cranial surgery for tumor resection and does not include comparison treatment 
groups. Because all subjects receive ABY-029, blinding of the surgeon to the study drug is not practical or 
appropriate. 
5.9 Receiving, Storage, Dispensing and Return 
5.9.1 
Receipt of Drug Supplies 
ABY-029 will be shipped to the Investigational Pharmacy at Dartmouth-Hitchcock Medical Center by UAB. 
Investigational pharmacy staff will count and verify that the shipment contains all the items noted in the 
shipment inventory, providing temperature monitoring data to UAB if necessary. Investigational pharmacy 
staff will document receipt into their electronic investigational drug management database that keeps a 
perpetual inventory.  Any damaged or unusable study drug in a given shipment will be documented in the 
study files, and destroyed on site following standard procedures.   
5.9.2 
Storage 
ABY-029 will be stored as a transparent, green-blue liquid in its shipment vials and outer carton in a freezer 
at temperatures between -15oC and -25oC.  Vials will not be removed from the outer carton until drug is 
prepared as ABY-029, which must be stored under dark conditions. 
5.9.3 
Dispensing of Study Drug 
Once drug assignment is performed, a study authorized staff member (e.g., Dr. Roberts, his designee) will 
order ABY-029 through the institution’s electronic medical record. This order will specify the dose of 1X 
(237 µg), 3X (711 µg) or 6X (1.42mg) for ABY-029. Since the concentration in each vial of ABY-029 is 1.35 
mg or 5.7X, in order to conserve our supply of ABY-029 we will administer a 5.7X dose to the 6X dose 
group in this study, and will call this the “6X dose” throughout this protocol. One dose of ABY-029 will be 
administered to each subject. The investigational pharmacy pharmacist will process this order and a 
pharmacy technician will prepare the ABY-029 dose as described in Section 5.4 (above) 
5.9.4 
Drug Accountability 
When processing an order for ABY-029, an investigational pharmacy pharmacist will make an entry into 
the electronic investigational drug management database to remove vial(s) used to prepare a dose from 
ABY-029 drug inventory. A member of the study team will document the amount of ABY-029 administered 
and any amount remaining in the institution’s electronic medical record.  
5.9.5 
Return or Destruction of Study Drug 
At the completion of the study, a final reconciliation of drug shipped, drug consumed, and drug remaining 
will occur. This reconciliation will be logged on the drug reconciliation form, signed and dated. Any 
discrepancies noted will be investigated, resolved, and documented prior to return or destruction of unused 
study drug. Site policy (i.e., Investigational Pharmacy) is to destroy any used, open drug containers on site. 
Drug destroyed on site will be documented in the study files.  
6 Study Procedures 
This study is a Phase 0 clinical investigation of ABY-029 concentration measured/detected in brain tissue 
through its fluorescence emissions as an intraoperative biomarker of tumor during open cranial surgery for 
tumor resection. Subjects participate in a “one-time” event/visit (i.e., surgery) as part of this protocol. Images 
and data from the patient’s pre- and post-operative MR scans are used for analysis in the study, but these 
acquisitions are part of the patient’s standard-of-care, would occur independently of whether the participant 
is enrolled, and thus, are not considered to be research study visits. No post-surgical follow-up visit is part 
of the protocol data collection or analysis. Patients are monitored for possible adverse events for 
approximately 30 days through the routine follow-up under the care of the operating surgeon involved in 


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the study procedures, post-operatively, and subsequently through the medical record. Hence, this 
information is already being collected as part of standard-of-care and is available for adverse event 
surveillance. 
6.1 Surgical Visit 
After the study drug is administered (~1-3 hours prior to surgery), the patient will be prepared and 
transported to surgery as per routine at Dartmouth-Hitchcock. In the OR, patient positioning, registration of 
the MR image-guidance system, preparation of the surgical field, and draping will follow standard practice, 
as will scalp incision and craniotomy. Upon opening of the dura, the coregistered and tracked Zeiss Pentero 
operating microscope adapted for intraoperative fluorescence imaging (iFI) at 800 nm will be brought in to 
begin conventional image-guided microsurgical technique for tumor resection. At approximately 4 time 
points during each surgery and/or at the discretion of the surgeon, data/image and specimen acquisitions 
will occur. Three (3) will typically occur at first exposure of tumor, at the approximate mid-point of tumor 
resection (when a significant tumor tissue is in the field), and at a point nearing but prior to completion of 
tumor resection (when small amount of tumor tissue remains), and a fourth may occur at presumed end of 
resection. At each time point, iFI acquisitions will be recorded. The surgeon will then focus the coregistered 
microscope at discrete points in the operative field which exhibit non-, weakly, moderately, and strongly 
fluorescent emissions, if evident on the iFI scan. At each point, a second iFI will be acquired, optical probe 
measurements will be recorded and a surgical biopsy specimen will be obtained (except at presumed end 
of resection when no more tissue is planned for excision). Since the 2nd iFI, probe data and biopsy sample 
will occur each time at the focal point of surgical microscope which is coregistered, all intraoperative images, 
data and specimens will also be coregistered. If no signal is evident on the iFI scan, the surgeon will sample 
several locations in the surgical field by focusing the operating microscope on points of interest intended 
for resection, performing the probe measurements and biopsying the positions for later analysis. iFI and 
optical probe readings will also be acquired from normal brain to establish baseline/background signal 
levels for the case. Additionally, an imaging module mounted on a second commercial, clinical microscope, 
will be used to acquire images during surgery.  Acquiring these images will involve moving the primary 
clinical scope out of the field, positioning the new scope to acquire images, and moving the primary scope 
back into place. This process will take approximately three minutes or less and poses no additional risk to 
the patient. When the case is performed in the Center for Surgical Innovation, and when indicated clinically, 
iMR (intraoperative MRI) may be acquired and resection may continue based on the findings. In these 
cases, iFI, optical probe and biopsy may occur at locations planned for resection. We have used these 
same coregistered image, data, and biopsy procedures in our current studies of ALA-induced PpIX 
fluorescence and have collected high quality information from up to 12-16 surgical specimens per case 
(~10 on average). 
6.1.1 
Specimen Preparation and Handling 
Resected specimens used for standard clinical care will be analyzed according to practice. Tissues taken 
as part of the study will be bisected immediately in the OR into nearly equivalent pieces in terms of size 
and gross appearance with one part going into 10% buffered formalin and the other snap frozen in liquid 
nitrogen. If the specimen cannot be divided, the sample will be formalin-fixed. 
6.2 Clinical Laboratory and Monitoring Data  
Since the microdoses that will be administered are presumed to fall below levels needed to cause 
pharmacological and/or physiological responses/effects that increase subject risks, and the preclinical 
(rodent) results from the Dartmouth Toxicity Study showed no evidence of systemic toxicity, either 
histologically or in terms of blood chemistry (in the latter, some clinical chemistry values in dosed animals 
were statistically different from un-dosed controls at 24 hours, but all levels were well within the normal 
range) associated with much higher single-doses of ABY-029 (10X, 100X, 1000X), no protocol-specific 
clinical laboratory or monitoring data associated, for example, with renal or liver function will be collected. 
Data obtained in this patient group as part of standard-of-care laboratory studies and clinical monitoring 
procedures includes hemoglobin, hematocrit, white blood cell count, platelet count, electrolytes (sodium, 
potassium, chlorate, bicarbonate), glucose, BUN and creatinine prior to administration of ABY-029. 
Similarly, vital signs (heart rate, blood pressure, respiratory rate) are monitored pre-operatively, during 


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surgery and post-operatively in the Neurosurgical Special Care Unit for a minimum of 24 hours following 
surgery as part of standard-of-care, and these data are available for adverse event monitoring. EKGs, when 
collected as part of standard-of-care operating procedures on surgical patients at Dartmouth-Hitchcock, are 
available pre-operatively, monitored continuously during surgery and again post-operatively (typically in the 
evening of the day of surgery). These data will not be collected as part of this clinical protocol per se, but 
will be available for adverse event monitoring, as needed. 
6.3 Specimen Analysis  
Biopsy samples taken at the time of surgery for research purposes and processed for IHC will be scored 
by a pathologist according to the following criteria: 0 = no staining; 1 = low membranous staining around 
cells; 2 = medium membranous staining completely around cells; and 3 = high membranous staining 
completely around cells. Standard formalin-fixation with paraffin embedding will be used for histopathologic 
type and grade to confirm diagnosis of astrocytoma or glioblastoma (following WHO criteria) by the study 
neuropathologist. As these tumors are recurrences and patients have received radiation and 
chemotherapy, an assessment of reactive gliosis vs. residual/recurrent glioma will also be scored as well 
as tumor burden and necrosis using methods previously described. Here, histopathology will be classified 
as non-tumor tissue, solid tumor, infiltrating tumor, or indeterminate. Infiltrating tumor will be further graded 
into <33% tumor, >33% but <67% tumor, and >67% tumor). Proliferation will be assessed by quantitatively 
scoring KI-67 immunopositive cells. ABY-029 binding to tumor cells in the specimen will be evaluated by 
performing IHC with secondary antibodies directed against the anti-EGFR Affibody following the protocol. 
EGFR protein expression often corresponds to gene amplification, thus, we will assess gene-protein 
correlations on a cell-by-cell basis by performing both IHC for EGFR protein and CISH for EGFR gene 
amplifications using a multi-spectral analysis microscope (Nuance) to distinguish in individual tumor cells. 
Frozen tissue will be solubilized and evaluated for quantitative fluorescence using fluorimetry. Western 
and/or ELISA will be used if the signal is not sufficiently strong. 
 
7 Statistical Plan 
7.1 Sample Size Determination 
Sample size determination is based on the primary objective of the study as a pilot assessment of 
fluorescence detection of ABY-029 localization in EGFR+ tumors with iFI. We estimate a minimum of 6, 
and more typically 12-16 specimens per case will be collected, and assume an average of 10 is available 
consistent with numbers in similar studies of ALA-induced PpIX fluorescence that have enrolled over 100 
subjects. To assess the adequacy of this sample size, if only 3 patients are enrolled at a specific dose, the 
study will have 80% power to detect a difference in fluorescence signal mean in samples relative to normal 
brain as small as 3.3 standard deviations with a two-sided significance level of 0.05, and a difference in 
fluorescence signal mean in samples between to two EGFR pathology scoring levels of 2.7 standard 
deviation with the same power and significance level (assuming samples are equally distributed between 
EGFR pathology scoring levels). If 6 patients are enrolled a one dose, the detectable difference in 
fluorescence signal mean in samples relative to normal brain drops to 1.4 standard deviations (at the same 
power and significance level) and to 1.8 standard deviations for differences in signal mean between two 
EGFR pathology scoring levels. 
7.2 Data and Statistical Analysis 
The primary study objective is to relate ABY-029 fluorescence (resulting from a specific pre-surgical dose) 
measured by wide-field iFI to EGFR pathology score (based on IHC staining) of coregistered tissue 
specimens sampled at the same time points during surgery. Similar but more sensitive fluorescence data 
will be available from the intraoperative optical probe. A minimum of 3 patients will be enrolled at a given 
dose, and up to 6 patients may be enrolled at one dose, if iFI SNR requirements are met. Assuming 10 
specimens are available for analysis on average from the surgical cases at a given dose (this value is 
consistent with average specimen counts in related ALA-induced PpIX studies which have enrolled more 
than 100 subjects), 30 specimens with corresponding probe signals and IHC pathological scoring will be 
available for analysis, and as many as 60 specimens may exist, if iFI SNR goals are achieved. Clustered 


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data analyses of variance will be performed to account for multiple samples from each patient. Student’s t-
test evaluation will be used to determine signal detectability relative to measurements in normal brain, and 
relative to the noise floor of the iFI system determined from laboratory measurements in phantoms. ANOVA 
will also be used assess differences in signal means when stratified by EGFR pathology score (0 = no 
expression, 1 = low expression, 2 = medium expression, 3 = high expression). Pearson’s and Spearman’s-
rank correlations between iFI signal strength and EGFR pathology score will be computed. These analyses 
will be repeated for the optical probe data as well. 
 
Secondary study objectives will be assessed by performing analyses on the iFI and probe data as a 
diagnostic test with the corresponding specimen histopathological diagnoses as the gold standard. 
Predictive models (for each pathology) of the odds of tumor positivity for biopsies given concentration of 
ABY-029 will be constructed. Histologically confirmed tumor status from biopsies will be treated as a binary 
outcome with a logit link function in a generalized linear mixed model (SAS GLIMMIX) with a subject-specific 
random effect to account for intra-individual correlations among biopsies. The iFI and/or probe fluorescence 
measures will be introduced first as linear terms. Interactions and polynomial fits will also be assessed via 
goodness of fit statistics. The predictive accuracy of the model fit will be summarized through a concordance 
index, i.e., area under the ROC curve formed with the fitted probabilities of tumor. Analyses will be repeated 
for other histopathological indicators (beyond tumor/non-tumor) such as degree of proliferation, infiltration, 
ABY-029 binding, among others. 
7.3 Subject Population(s) for Analysis 
This study will involve patients with recurrent intra-cranial malignant glioma judged to be operable. Subjects 
eligible for enrollment in the trial will be patients with a previously resected high grade glioma with tissue 
positive for EGFR expression (pathology score ≥ 1 based on histological staining). 
8 Safety and Adverse Events 
The study will be monitored by the Data, Safety Monitoring, and Accrual Committee (DSMAC) of the Norris 
Cotton Cancer Center at Dartmouth-Hitchcock Medical Center. All unexpected and/or serious adverse 
events will be reported to committee by the protocol PI or the designee, along with a determination of their 
relationship to study procedures. Given that brain tumor patients undergoing open surgical resection 
procedures present with post-operative complications and medical events that are expected, we will log 
and review all adverse events, but evaluate and report on only those with outcomes that would not routinely 
be expected in this surgical patient population to the NCCC DSMAC, along with a determination of their 
probable relationship to study procedures. Summaries of study enrollments, clinical status, and data will be 
submitted for annual review (or more frequently as required depending on enrollment patterns). In the case 
of any unexpected adverse events, the patient’s medical record will be reviewed by a study physician, and 
if necessary, the patient will be examined by a study physician and appropriate medical action taken. The 
event will be recorded in the patient's study file with the date of occurrence, date of resolution, severity and 
the actions taken. Any unexpected or serious adverse events, or drug reaction beyond those expected from 
open-cranial brain tumor surgery that are determined likely to be related to the study procedures, will be 
reported to the Dartmouth IRB and the FDA as required. 
9 Data Handling and Record Keeping 
9.1 Confidentiality 
Information about study subjects will be kept confidential and managed according to the requirements of 
the Health Insurance Portability and Accountability Act of 1996 (HIPAA). Risk of breach of confidentiality of 
the medical records and status of participants will be minimized. Databases which are used to store subject-
sensitive information are password-protected and encrypted during file/data transfers from viewing 
terminals. Access will be limited to research team members who have undergone CPHS training at 
Dartmouth. Whenever possible and practical standard-of-care clinical data used in the research will be de-
identified when under analysis. 


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9.2 Case Report Forms 
Study case report forms (CRFs) will be the primary data collection instruments for the study.  All data 
requested on CRFs will be recorded.  Any missing data will be explained.  If a space on the CRF is left 
blank because the procedure was not performed or the question was not asked, a written notation will be 
made.  If an item is not applicable to an individual case, written notation will be made.  Changes to the 
CRFs will be initialed and dated. 
  
10 Clinical Site Monitoring 
10.1 On-Site Monitoring  
Clinical research monitoring for regulatory compliance and data integrity will be conducted according to the 
NCI-approved NCCC Data and Safety Monitoring Plan. Internal monitoring is conducted by appropriately 
trained staff of the NCCC Office of Clinical Research and Dartmouth-Hitchcock Medical Center Clinical 
Trials Office who are not involved in the study. This monitoring will include periodic assessment of the 
regulatory compliance, data quality, and study integrity. Study records will be reviewed and directly 
compared to source documents and the conduct of the study will be discussed with the investigator. 
Monitors may request access to all regulatory documents, source documents, CRFs, and other study 
documentation for on-site inspection.  Direct access to these documents is guaranteed by the investigator, 
who must provide support at all times for these activities. 
10.2 Auditing and Inspecting 
The study investigators will permit study-related monitoring, audits, and inspections by the IRB, the sponsor, 
government regulatory bodies, and university compliance and quality assurance groups of all study related 
documents (e.g. source documents, regulatory documents, data collection instruments, study data, etc.). 
The study investigators will ensure the capability for inspections of applicable study-related facilities (e.g. 
pharmacy, diagnostic laboratory, etc.). 
 
Participation as an investigator in this study implies acceptance of potential inspection by government 
regulatory authorities and applicable university compliance and quality assurance offices. 
11 Ethical Considerations 
This study is to be conducted according to US and international standards of Good Clinical Practice (FDA 
Title 21 part 312 and International Conference on Harmonization guidelines), applicable government 
regulations and Institutional research policies and procedures. 
 
All subjects for this study will be provided a consent form describing this study and providing sufficient 
information for subjects to make an informed decision about their participation in this study.    This consent 
form will be submitted with the protocol for review and approval by the IRB.  The formal consent of a subject, 
using the IRB-approved consent form, will be obtained before that subject undergoes any study procedure. 
The consent form will be signed by the subject and the investigator-designated research professional 
obtaining the consent.  
12 Study Finances 
12.1 Funding Source 
This study is financed through a grant from the US Department of Health and Human Services, National 
Institutes of Health, National Cancer Institute, grant # 5R01CA167413. 
12.2 Conflict of Interest 
All Dartmouth investigators will follow the Dartmouth conflict of interest policy, and will have disclosed 
potential conflicts-of-interest related to the study. 


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13 References 
 
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radionuclides for pretargeted radioimmunotherapy and imaging. Nucl Med Biol, 2011. 38(2): p. 223-33. 
4. Gibbs-Strauss, S.L., et al., Nerve-highlighting fluorescent contrast agents for image-guided surgery. 
Mol Imaging, 2011. 10(2): p. 91-101. 
5. Orcutt, K.D., et al., A modular IgG-scFv bispecific antibody topology. Protein Eng Des Sel, 2010. 23(4): 
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6. Stummer, W., et al., Fluorescence-guided surgery with 5-aminolevulinic acid for resection of malignant 
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9. Valdes, P.A., et al., Gadolinium- and 5-aminolevulinic acid-induced protoporphyrin IX levels in human 
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11. Valdes, P.A., et al., Quantitative fluorescence in intracranial tumor: implications for ALA-induced PpIX 
as an intraoperative biomarker. J Neurosurg, 2011. 
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13. Leung, K., IRDye 800-albumin-binding domain-fused-ZHER2:342 Affibody. 2004. 
14. Chopra, A., Cy5.5 conjugated anti-epidermal growth factor receptor monoclonal antibody. 2004. 
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16. Helman, E.E., et al., Optical imaging predicts tumor response to anti-EGFR therapy. Cancer Biol Ther, 
2010. 10(2): p. 166-71. 
17. Wang, K., et al., Characterizing breast cancer xenograft epidermal growth factor receptor expression 
by using near-infrared optical imaging. Acta Radiol, 2009. 50(10): p. 1095-103. 
18. Gleysteen, J.P., et al., Fluorescent labeled anti-EGFR antibody for identification of regional and distant 
metastasis in a preclinical xenograft model. Head Neck, 2008. 30(6): p. 782-9. 
19. Koyama, Y., et al., In vivo molecular imaging to diagnose and subtype tumors through receptor-targeted 
optically labeled monoclonal antibodies. Neoplasia, 2007. 9(12): p. 1021-9. 
20. Barrett, T., et al., In vivo diagnosis of epidermal growth factor receptor expression using molecular 
imaging with a cocktail of optically labeled monoclonal antibodies. Clin Cancer Res, 2007. 13(22 Pt 1): 
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21. Kulbersh, B.D., et al., Sensitivity and specificity of fluorescent immunoguided neoplasm detection in 
head and neck cancer xenografts. Arch Otolaryngol Head Neck Surg, 2007. 133(5): p. 511-5. 
22. Rosenthal, E.L., et al., In vivo detection of head and neck cancer orthotopic xenografts by 
immunofluorescence. Laryngoscope, 2006. 116(9): p. 1636-41. 
23. Verbeek, F.P., et al., Near-Infrared Fluorescence Imaging of Both Colorectal Cancer and Ureters Using 
a Low-Dose Integrin Targeted Probe. Ann Surg Oncol, 2014. 
24. Ryu, J.H., et al., In vivo fluorescence imaging for cancer diagnosis using receptor-targeted epidermal 
growth factor-based nanoprobe. Biomaterials, 2013. 34(36): p. 9149-59. 
25. Tafreshi, N.K., et al., Synthesis and characterization of a melanoma-targeted fluorescence imaging 
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26. Huang, C.W., Z. Li, and P.S. Conti, In vivo near-infrared fluorescence imaging of integrin alpha2beta1 
in prostate cancer with cell-penetrating-peptide-conjugated DGEA probe. J Nucl Med, 2011. 52(12): p. 
1979-86. 
27. Terwisscha van Scheltinga, A.G., et al., Intraoperative near-infrared fluorescence tumor imaging with 
vascular endothelial growth factor and human epidermal growth factor receptor 2 targeting antibodies. 
J Nucl Med, 2011. 52(11): p. 1778-85. 
28. Miller, S.J., et al., In vivo fluorescence-based endoscopic detection of colon dysplasia in the mouse 
using a novel peptide probe. PLoS One, 2011. 6(3): p. e17384. 
29. Nord, K., A combinatorial library of an alpha-helical bacterial receptor domain. Protein Eng, 1995. 8(6): 
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30. Lofblom, J., et al., Affibody molecules: engineered proteins for therapeutic, diagnostic and 
biotechnological applications. FEBS Lett, 2010. 584(12): p. 2670-80. 
31. Friedman, M., et al., Directed evolution to low nanomolar affinity of a tumor-targeting epidermal growth 
factor receptor-binding affibody molecule. J Mol Biol, 2008. 376(5): p. 1388-402. 
32. Orlova, A., et al., Tumor imaging using a picomolar affinity HER2 binding affibody molecule. Cancer 
Res, 2006. 66(8): p. 4339-48. 
33. Lindborg, M., et al., Engineered high-affinity affibody molecules targeting platelet-derived growth factor 
receptor beta in vivo. J Mol Biol, 2011. 407(2): p. 298-315. 
34. Orlova, A., et al., Synthetic affibody molecules: a novel class of affinity ligands for molecular imaging 
of HER2-expressing malignant tumors. Cancer Res, 2007. 67(5): p. 2178-86. 
35. Sampath, L., et al., Dual-labeled trastuzumab-based imaging agent for the detection of human 
epidermal growth factor receptor 2 overexpression in breast cancer. J Nucl Med, 2007. 48(9): p. 1501-
10. 
36. Rosenthal, E., et al., Safety and Tumor-specificity of Cetuximab-IRDye800 for Surgical Navigation in 
Head and Neck Cancer. Clin Cancer Res, 2015 21:3658-3666. 
37. Gong, H., et al., In vivo imaging of xenograft tumors using an epidermal growth factor receptor-specific 
affibody molecule labeled with a near-infrared fluorophore. Neoplasia, 2010. 12(2): p. 139-49. 
38. Sexton, K., et al., Fluorescent affibody peptide penetration in glioma margin is superior to full antibody. 
PLoS One, 2013. 8(4): p. e60390. 
39. Sorensen, J, et al., First-in-Human Molecular Imaging of HER2 Expression in Breast Cancer 
Metastases Using the 111IN-ABY-025 Affibody Molecule. J Nucl Med. 2014 May;55(5):730-5. 
40. Sorensen, J, et al., Measuring HER2-Receptor Expression In Metastatic Breast Cancer Using 
[68Ga]ABY-025 Affibody PET/CT, Theranostics 2016, Vol 6, issue 2, p 262-271. 
41. Sandstrom, M, et al., Biodistribution and radiation dosimetry of the anti-HER2 Affibody molecule 68Ga-
ABY-025 in breast cancer patients, J Nucl Med. 2016 Feb [epub ahead of print]. 
 


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14 Attachments 
14.1 Zeiss Pentero microscope modifications 
The figure below illustrates modifications made to the Zeiss Pentero surgical microscope. The device as 
modified presents a non-significant risk to patients. It will not be in contact with the subject in any way, 
including as an implant. The device will not be used in any way to direct or manage patient care, nor to 
collect data, diagnose or treat the patient in any way. It is used purely as part of an observational study.
 


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14.2 Zeiss S1 microscope modifications 
The figure below illustrates modifications made to the Zeiss S1 surgical microscope. The device as modified 
presents a non-significant risk to patients. It will not be in contact with the subject in any way, including as 
an implant. The device will not be used in any way to direct or manage patient care, nor to collect data, 
diagnose or treat the patient in any way. It is used purely as part of an observational study. 
 

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CONSENT TO TAKE PART IN RESEARCH
Dartmouth-Hitchcock Medical Center 
A Microdose Evaluation Study of ABY-029 In Recurrent Glioma
David W. Roberts, MD, Nathan Simmons, MD, and Keith Paulsen, PhD
Introduction:  You are being asked to take part in a research study.  Taking part in research 
is voluntary.  
You are being asked to take part in this study because you have been diagnosed with a brain 
tumor that will be removed by surgery as part of your care. 
Taking part in a research study is not the same as getting regular medical care.  The purpose of 
regular medical care is to improve your health.  The purpose of a research study is to gather 
information to help patients in the future.  
Your decision whether or not to take part in this study will not influence your future medical 
care. Please ask questions if there is anything about this study you do not understand.
Background:
ABY-029 is an experimental drug that has not been tested in humans yet.  Our previous animal 
research showed that ABY-029 can light up portions of many brain tumors, making them glow 
a green color.  The purpose of this study is to determine if surgeons can see ABY-029 in human 
brain tumors by using a special microscope.  
ABY-029 is given by IV to patients a few hours before surgery.  Giving patients ABY-029 
before brain surgery has not been approved by the U.S. Food and Drug Administration (FDA).  
We have received permission from the FDA to use ABY-029 in one limited research study.  In 
an earlier research study, we showed that ABY-029 has the potential to help the surgeon to 
identify brain tumor tissue during surgery because the tumor cells glow a green color.  This 
approach could benefit patients with malignant brain tumors that need to be more completely 
removed through surgery.
As part of our previous research, we developed a probe that measures the concentration of 
ABY-029 in tissue.  We have also developed a sensitive imaging system that is able to detect 
smaller amounts of fluorescence (glow) from ABY-029 than the naked human eye can see.  
Both of these devices are experimental and have not been approved by the FDA.  In a previous 
research study, they have been determined to pose no significant risk to you or to your 
surgical procedure, according to the FDA guidelines.
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What is the purpose of this study?
The purpose of the study is to measure the amount of ABY-029 that reaches your brain tumor 
with the help of the devices we have made. 
We will use a probe and imaging system during your surgery to record the amount of ABY-
029 in your tumor and in the surrounding tissue.  We will use these instruments during your 
surgery to figure out where and how much of the ABY-029 is distributed in your tumor and 
the surrounding tissue.
Will you benefit from taking part in this study?
This study is not intended to provide a personal benefit to you. The MRI of your tumor is 
helpful in guiding the surgeon to remove it, and is part of standard practice for your condition. 
This information will be available and used during your surgery as it would be, even if you 
chose not to participate in this study. 
This study is the first time that ABY-029 will be used in humans to determine if it can help 
surgeons safely remove as much tumor as possible. As a result, predicting whether you will 
benefit from your participation in this study is not possible at this time. We hope to gather 
information that may help people with brain tumors similar to yours in the future.
What does this study involve?
In many cases, pathology reports from your previous surgery contain information that can 
help us determine your eligibility. If your eligibility for this study could not be determined 
from information on file, we have run an additional test on those samples. The study will pay 
for this additional testing and you will and/or your insurance will not be charged.  
We will analyze your MRI brain scans, usually taken in the first few days after surgery, and 
we may examine your health records during the first 30 days after your surgery to satisfy FDA 
monitoring and reporting requirements associated with the experimental use of ABY-029. You 
will have post-operative MRI and clinic follow-up visits as part of the routine care that you 
would undergo regardless of whether you enroll in this study or not. 
Your surgery will be very similar to what would happen if you decide not to participate, 
except that the surgeon will use an operating microscope to visualize ABY-029 on the surface 
of your brain. The operating microscope is FDA-approved and color images of your brain 
surface will be captured. A second imaging system, which is attached to the operating 
microscope, will be used to measure the ABY-029 fluorescence light signal coming from the 
tissue. This device is not FDA-approved, but is not considered to be of significant risk to your 
health, safety and welfare during surgery. Images will also be captured with this system. 
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After the images are captured, the surgeon will place a small probe on the exposed surface of 
your brain in different spots, depending on whether the ABY-029 fluorescence is present or 
not, in order to make accurate measurements of the ABY-029 concentration at each location. 
The probe is not FDA-approved, but is considered not to be of significant risk to your health, 
safety and welfare during surgery. The surgeon may then decide to remove a small piece of 
brain tissue that will be analyzed after to surgery so we know exactly the diagnosis of the 
tissue that was measured by the probe and visualized with the images. The surgeon will only 
take the sample if this piece of tissue would generally be removed during the course of 
surgery. The entire process of using the probe and removing a tissue sample will be repeated a 
number of times during your procedure. Depending on the size and location of your tumor, 
the surgeon may record information from as many as 20 different locations during your 
surgery. 
What are the options if you do not want to take part in this study?
In order to be considered for this study, your doctor has already recommended that your brain 
tumor be surgically removed as part of your care, and you will most likely have surgery even 
if you decide not to participate in this study. Other research studies may be available for 
patients with your condition, and you can discuss those options with your doctor. 
If you take part in this study, what activities will be done only for research purposes?
If you take part in this study, the following activities will be done only for research purposes:
 

Being given a very small amount of ABY-029 dissolved in about one teaspoon of saline 
in a syringe approximately 3 hours before surgery.  The amount of ABY-029 we will 
give is called a microdose, a tiny dose weighing about 0.25 mg, which is about 1/10th 
the weight of a paper clip.

Measurements of your brain tissue with an intraoperative optical probe.

Recording of fluorescence images of your brain during the course of your surgery.

Specimens of your tumor will be taken from locations measured with the intraoperative 
optical probe that would normally be removed during surgery, in addition to the 
biopsy specimens used to confirm your clinical diagnosis as part of standard-of-care for 
your condition.

Additional lab work that is not part of the standard of care:
o
A pregnancy test (urine or blood) will be performed on all women of child-
bearing potential prior to your surgery.
o
If additional testing on previous tissue collected from your prior surgery is 
needed to verify eligibility, the study will pay for this.
What are the risks involved with taking part in this study? 
We cannot be sure how your body may respond to the ABY-029 used in this study.  However, 
from our previous work with this drug we do not believe there are any safety risks with giving 
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you a very small amount of ABY-029.  The research team will discuss possible problems and 
the chances that they will happen.  Unknown problems may happen.  Problems may be small, 
such as a minor side effect, or they may be so serious that they result in death. Or they may be 
somewhere in between.  You should report any problems to your neurosurgeon by calling 
(603-650-5109 or to the director of this study, David W. Roberts, MD by calling 603-653-9920.
Risks/Side Effects of ABY-029
None known at this time from ABY-029.
Because this drug is given by IV, there may be some pain or mild bruising from the IV.
Reproductive Risks and Risks to Pregnant Women:
The risks of ABY-029 to a pregnancy or fetus are unknown.  These may be a minor 
inconvenience or may be so severe as to cause death. Women should not be or become 
pregnant while on this research study. Pregnant women or women who are breast-feeding 
may not take part in this research study.  All women who could become pregnant will have a 
pregnancy test before surgery. The sponsor of this study will pay for a pregnancy test.  
All women who could become pregnant need to use a medically approved method of birth 
control in order to take part in this research study.  Please discuss options with the researcher.
If a female study subject becomes pregnant, they should let us know right away. Call David 
W. Roberts, MD or his study staff at 603-653-9920.
Other important items you should know:
• Leaving the study: You may choose to stop taking part in this study at any time for any 
reason. If you decide to stop taking part, it will have no effect on the quality of your medical 
care.
• A description of this clinical trial will be available on www.clinicaltrials.gov, as required by 
U.S. Law.  This Web site will not include information that can identify you.  At most, the Web 
site will include a summary of the results.  You can search this Web site at any time. 
• This research involves the use of new technology that could lead to a product sold for profit 
in the future. If the results of this research are used to develop a product sold for a profit, you 
will not share in the profit.  You will not receive money from the profits.
• New Information: New information related to this research will be made known to you 
when it becomes available.  This may affect your decision to stay in this study. 
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• Funding:  The National Institutes of Health is the sponsor of this research. Some of the 
technology being used in the study is being provided by Carl Zeiss, Inc., at no cost to the 
project. The ABY-029 is being provided by Dartmouth College through federal funding for this 
study.  The investigators involved in this research are inventors of some of the technology 
used in this study, and hold intellectual property rights to these ideas jointly with the Trustees 
of Dartmouth College.
• Number of people in this study:   It is expected that between 6 and 12 people will be 
enrolled at this institution.
How will your privacy be protected?
The information collected for this study will be used only for the purposes of research as 
stated earlier in this form. It includes: your pre-operative and first post-operative brain scans, 
optical images and signals collected in the operating room during your surgical procedure, 
and image and histological analyses of the tissue specimens removed during your surgery. 
Information on your gender, age, brain tumor diagnosis, etc., will be collected in order to 
characterize some basic demographics of the subject population enrolled in the study. The data 
collected for this will be maintained indefinitely.
We are careful to protect the identities of the people in this study.  We also keep the 
information collected for this study secure and confidential.  Specifically, your identity is 
coded into a study number and most of the data and analysis occurs with information that is 
archived only by study number. Information which identifies you as participating in the study 
is maintained in binders as required by the FDA, and these binders will be kept in the office of 
research team. The office is kept locked when not in use, and the study team is trained in the 
importance of maintaining your confidentiality. Research data, if sent electronically to 
members of the research team, is de-identified and only secure file transfer methods are used. 
Pathology information may be sent to a pathologist working outside the institution for 
evaluation purposes. This data and information is de-identified and is referenced only by the 
study number of the participant.
Who may use or see your health information?
Federal law provides additional protections of your medical records and related health 
information. By signing this form, you allow the research team to use your health information 
and give it to others involved in the research.  The research team includes the study director 
plus others working on this study at Dartmouth-Hitchcock Medical Center and elsewhere.  
You also permit any health care provider holding health information needed for this study to 
give copies of your information to the research team.
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The information collected for this study may be used by researchers or officials of the 
following institutions.

Dartmouth College

Mary Hitchcock Memorial Hospital

Dartmouth-Hitchcock Clinic

Dartmouth-Hitchcock Medical Center 

National Institutes of Health (NIH)

Federal Drug Administration (FDA)

Carl Zeiss, Inc.

Affibody AB (developer of the study drug)

LI-COR Biosciences (manufacturer of the glowing dye in ABY-029)

University of Alabama (research facility where ABY-029 was made)

The Dartmouth-Hitchcock Health Institutional Review Board (D-HH IRB) 
In order to conduct this study, researchers need to use your health care information.  This 
data is called Protected Health Information ("PHI”).  PHI is protected by federal privacy laws 
(HIPAA).  By signing this consent form, you give your permission to have your PHI collected, 
used and disclosed for purposes of this study.  There is no intention to disclose your PHI to 
others outside of the study. There are protections in place to keep your PHI and research data 
confidential.  However, HIPAA requires notification so you are aware that if your PHI is 
disclosed to others, it may no longer be protected by federal privacy laws.
No publication or public presentation about the research described above will reveal your 
identity without another authorization from you.
Identifiable data collected for this study will be used for research purposes which are 
determined to be reasonable and in line with expectations by a review committee.
Once data collected for this research study is no longer identifiable, the data may be used or 
disclosed for other purposes.
Your permission to use your health information for this study will not end until the study is 
completed. During this study, you and others who take part in the study may not have access 
to the study data.  You may ask for study data once the study is over.  You have a right to 
receive a copy of the information in your medical record at any time.
Whom should you call about this study?
If you have questions about this study or need to report a study related injury, you can call 
your doctor or the research office for this study at 603-653-9920 during normal business hours 
and they will be able to contact Dr. Roberts for you. If Dr. Roberts is not available, other 
                   D-HH IRB Approved                    Effective Date: 6/17/2021                    Expiration Date: 6/16/2022


v. 07/20/2020
Page 7
members of the section of Neurosurgery will be available to answer your questions during 
normal business hours.   
This research has been reviewed and approved by the Dartmouth-Hitchcock Health 
Institutional Review Board (D-HH IRB). You may talk to them at (603) 650-1846 or 
irb@hitchcock.org if:
Your questions, concerns, or complaints are not being answered by the research team.
You cannot reach the research team.
You want to talk to someone besides the research team.
You have questions about your rights as a research subject.
You want to get information or provide input about this research.
What about the costs of this study?
ABY-029 will be supplied free of charge.  All additional tests/visits/procedures as described in 
the “If you take part in this study, what activities will be done only for research purposes?” 
section will be paid for by the sponsors of the study.
Insurance plans will not be billed for research procedures that are not the usual care for your 
condition. Some of the medical care that you will receive during this study is the usual care a 
doctor would recommend for your condition. You or your insurance plan are expected to pay 
for the costs of this usual medical care. 
Will you be paid to take part in this study?
You will not be paid for participating in this study.
What happens if you get sick or hurt from taking part in this study?
Sponsor Information: The sponsor of this research is the National Institutes of Health. Carl 
Zeiss, Inc., provides equipment for use in the study at no cost and ABY-029 was made for use 
in the study with funds from the sponsor. If you develop an illness or have an injury because 
you are in this research study, the sponsor will not pay for:

Treatment of illness or injury that results from the negligence of a health care provider, 
or

Treatment of a condition that you had before you were in the study.
The sponsor will not offer any other payments for your study-related illness or injury such as 
lost wages, expenses other than medical care, or pain and suffering.
This study is federally funded by NIH and compensation for a research-related injury or 
illness is not provided by federal law.
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v. 07/20/2020
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If you are injured or become ill as a result of research procedures, you will be provided with 
medical treatment but the following organizations do not plan to pay for this treatment.

Mary Hitchcock Memorial Hospital

Dartmouth-Hitchcock Clinic

Dartmouth-Hitchcock Medical Center 

Trustees of Dartmouth College

The sponsor of this study
If you have any questions or concerns about the legal responsibility of these organizations, 
please call the Mary Hitchcock Memorial Hospital Office of Risk Management at 603-650-1250 
during normal business hours.
If you agree to take part in this study and you sign this consent form, you are not giving up 
any of your legal rights.
Your responsibilities as a person taking part in this study
(1) Be aware it is important for your safety that the research team knows about your 
medical history and current condition.
(2) Notify the research team in advance if you plan to undergo any other medical 
treatment during this study, or are taking or plan to start taking any medications.
(3) Notify the research team immediately if you suffer any injury or unexpected reaction 
to the study medication or procedures.
(4) Seek treatment with the help of the research team if you suffer any injury or 
unexpected reaction to the study medication or procedures.
(5) Make reasonable efforts to cooperate with the instructions of the research team.
CONSENT  
I have read the above information about “A Microdose Optimization Study of ABY-029 in 
Recurrent Glioma” and have been given time to ask questions.  I agree to take part in this 
study and understand I will be given a copy of this signed consent form.
Participant's Signature                                      Date
PRINTED NAME 
Researcher or Designee Signature                   Date
PRINTED NAME 
                   D-HH IRB Approved                    Effective Date: 6/17/2021                    Expiration Date: 6/16/2022