Development of novel breast cancer-targeted spect imaging peptides by phage display

Breast cancer is the second leading cause of cancer and cancer-related mortality among women. The survival rate of the malignancy declines considerably as the disease progresses, w hich n ecessitates r apid discovery and t reatment [1]. C urrent m ethods of detection including mammograms and breast exams have been successful, however they have limita tions [2]. B oth t echnologies r ely on the de tection o f a l arge mass of c ells, which i s not conducive to identifying aggressive cancer at an early s tage. Additionally, these m ethodologies cannot pr ovide bi ological i nformation t hat m ay dictate t he us e o f specific targeted therapies. As treatment for breast cancer continues to progress towards xv therapies targeted at specific carcinogenic antigens, an ability to detect these antigens will improve p atient c are a nd p revent in effective tr eatment r egimens. T argeted i maging agents not only provide the ability to detect breast cancer, but also allow for simultaneous non-invasive characterization. The c hoice of v ector f or a ta rgeted ima ging a gent d ictates its p robability o f success. P eptides pr ovide s everal p roperties t hat m ake t hem a s trong c andidate f or imaging agents. A peptide is easily synthesized, and can be conjugated in a s ite specific manner with a molecule allowing for in vivo detection. For breast cancer detection, the use of a bi functional chelator allows for binding of a radiometal, which in turn permits clear s ignal d etection at an y d epth o f t issue [3, 4] . A ra diolabeled p eptide’s s mall s ize provides it with rapid pharmacokinetics and enhanced tumor penetration, allowing for the peptide to quickly reach a tumor and bind, followed by clearance of unbound peptide [4]. This is especially important for radiolabeled imaging agents, as circulating radionuclide can deliver an unwanted dose of radiation to radiosensitive organs such as the bone [5]. A major drawback of a peptide is i ts moderate affinity, however the use of bacteriophage (phage) display technology allows for the selection of peptides with suitable affinity for in vivo imaging. Phage display, pioneered by George P. Smith here at the University of Missouri, utilizes the tolerance of phage particles to the insertion of random peptides into i ts coat proteins i n order t o s elect a di splayed peptide w ith a desired function [6]. A l ibrary of phage particles displaying random peptides can be created by altering the phage genome, allowing for production of up t o 10 unique peptide sequences. Phages can be incubated xvi with a t arget of i nterest, a nd onl y t hose bound recovered. R ecovered p hages a re t hen propagated in bacteria, creating an enriched subpopulation of phages displaying peptides with the desired function. Phage display can be used to select peptides with affinity for a purified a ntigen in vitro or a gainst w hole or gans or e ven t umors in vivo [7-9]. It is hypothesized in this dissertation that novel breast cancer targeted peptides can be selected by phage display for the purpose of imaging human breast tumors in vivo. Development of a peptide for imaging purposes begins with the choice of a target. In b reast can cer, E RBB2 i s a h ighly i mportant t arget. E RBB2 i s a m ember o f t he epidermal growth factor re ceptor (E GFR/ERBB) fa mily of t ransmembrane r eceptors, consisting of E GFR, E RBB2, E RBB3 a nd E RBB4. E RBB2 i s ove r-expressed i n up t o 30% of breast cancers, and is expressed in almost 90% of all tumors [10]. Not only is the receptor h ighly p revalent, but its p resence co rrelates w ith i ncreased a ggressiveness and decreased s urvival r ates [11]. S tability and pr omiscuity o f E RBB2 a ccount f or i ts unfavorable phenotype. The receptor does not have a ligand, and is instead constitutively receptive to dimerization and mitogenic signaling [12]. Additionally, a dimer containing ERBB2 is h ighly s table and transmits s ignals lo nger than o ther m embers o f its f amily. Fortunately, h ighly s table cel l s urface r eceptors ar e an i deal t arget f or i maging an d therapy. T wo cl inically approved therapeutic a ntibodies, t rastuzumab a nd pe rtuzumab, are u sed t o t reat b reast can cers w ith E RBB2 over-expression [13, 14] . W hile bot h therapies h ave i ncreased disease f ree survival r ates of pa tients w ith E RBB2 ove rexpressing b reast cancer by 5 m onths, r esistance to th e th erapy is common [15]. Currently, b reast cancers ar e s creened for potential t reatment w ith an ti-ERBB2 t herapy xvii by biopsy. Biopsies m ay not be i deal due t o pa tient pa in a nd us e of ex vivo detection, which m ay not be ph ysiologically r elevant f ollowing r emoval f rom t he pa tient [16]. A non-invasive i maging a gent, s uch as a r adiolabeled pe ptide, w ould pr ovide r eal-time detection of E RBB2, and e nsure t hat t he a ntigen i s bi ologically a ccessible t o t he therapeutic. Previously, a 6 amino acid p eptide ( KCCYSL) was s elected b y in vitro phage display for ERBB2 extracellular domain binding, and was consequently demonstrated to image ERBB2 expressing tumors in mice [8, 17]. While tumor uptake was sufficient for SPECT i maging, non-target or gan upt ake pos ed potential pr oblems f or f urther s tudies. The biodistribution indicated the peptide would be better suited for clinical applications with a d ecreased non-target organ uptake. Here, it was hypothesized that a microlibrary displaying the KCCYSL peptide f lanked by random amino acids at both the Nand Ctermini c ould be us ed t o s elect a pe ptide w ith improved in vivo tumor ta rgeting a nd pharmacokinetics. T his pr ocess, t ermed affinity m aturation, i s a nalogous t o t he development of hi gh affinity antibodies b y the i mmune s ystem. The KC CYSL microlibrary was used for in vivo selections in mice bearing ERBB2-expressing MDAMB-435 hum an br east x enografts. S elected phages a nd c orresponding s ynthesized peptides w ere evaluated f or E RBB2 and c ancer cel l b inding affinity and s pecificity in vitro. Two target specific peptides were DOTA-conjugated and In-labeled for in vivo biodistribution and SPECT imaging analyses for comparison with previously publ ished In-DOTA-KCCYSL.