Susan L. Deutscher

Biodistribution of filamentous phage peptide libraries in mice.

In vivo phage display is a new approach to acquire peptide molecules that bind stably to a given target. Phage peptide display libraries have been selected in mice and humans and numerous vasculature-targeting peptides have been reported. However, in vivo phage display has not typically produced molecules that extravasate to target specific organ or tumor antigens. Phage selections in animals have been performed for very short times without optimization for biodistribution or clearance rates to a particular organ. It is hypothesized that peptides that home to a desired antigen/organ can be obtained from in vivo phage experiments by optimization of incubation times, phage extraction and propagation procedures. To accomplish this goal, one must first gain a better understanding of the in vivo biodistribution and rate of clearance of engineered phage peptide display libraries. While the fate of wild type phage in rodents has been reported, the in vivo biodistribution of the commonly used engineered fd-tet M13 phage peptide display libraries (such as in the fUSE5 vector system) have not been well established. Here we report the biodistribution and clearance properties of fd-tet fifteen amino acid random peptide display libraries in fUSE5 phage in three common mouse models employed for drug discovery – CF-1, nude, and SCID mice.

Evaluation of an 111In-Radiolabeled Peptide as a Targeting and Imaging Agent for ErbB-2 Receptor–Expressing Breast Carcinomas

Purpose: The cellular targeting and tumor imaging properties of a novel ErbB-2-avid peptide, discovered from bacteriophage display, were evaluated in human breast carcinoma cells and in breast carcinoma–xenografted mice. Experimental Design: The affinity of the ErbB-2 targeting peptide KCCYSL and its alanine substituted counterparts for the extracellular domain (ECD) of purified recombinant ErbB-2 (ErbB-2-ECD) was assessed by fluorescence titration. Binding of the KCCYSL peptide to breast and prostate carcinoma cells was analyzed by confocal microscopy. A DOTA(GSG)-KCCYSL peptide conjugate was radiolabeled with 111In, and stability, target binding, and internalization were analyzed in vitro. In vivo biodistribution and single-photon emission computed tomography imaging studies were done with the radiolabeled peptide in MDA-MB-435 human breast tumor–bearing severe combined immunodeficient mice. Results: KCCYSL peptide exhibited high affinity (295 ± 56 nmol/L) to ErbB-2-ECD. Substitution of alanine for lysine, tryptophan, and cysteine reduced the peptide affinity ∼ 1- to 2.4-fold, whereas replacing leucine completely abolished binding. Both biotin-KCCYSL and 111In-DOTA(GSG)-KCCYSL were capable of binding ErbB-2–expressing human breast carcinoma cells in vitro. Approximately 11% of the total bound radioactivity was internalized in the carcinoma cells. Competitive binding studies indicated that the radiolabeled peptide exhibited an IC50 value of 42.5 ± 2.76 nmol/L for the breast carcinoma cells. 111In-DOTA(GSG)-KCCYSL was stable in serum and exhibited rapid tumor uptake (2.12 ± 0.32 %ID/g) at 15 min postinjection and extended retention coupled with rapid whole body disappearance, as observed by biodistribution and single-photon emission computed tomography imaging studies, respectively. Conclusions: The DOTA(GSG)-KCCYSL peptide has the potential to be used as a tumor-imaging agent and a vehicle for specific delivery of radionuclide or cytotoxic agents for tumors overexpressing ErbB-2.

Thomsen-Friedenreich and Tn Antigens in Nipple Fluid: Carbohydrate Biomarkers for Breast Cancer Detection

Purpose: Novel biomarkers would facilitate early and accurate diagnosis of breast cancer. The Thomsen-Freidenreich (TF) and Tn antigens are aberrantly glycosylated carbohydrate cancer-associated antigens found in ∼80% of adenocarcinomas. Both TF and Tn are expressed on cell-surface glycoproteins and glycolipids. Nipple aspirate fluid (NAF) is concentrated in secreted proteins and lipids from cells that give rise to cancer. The objective of this study was to determine if NAF from breasts with cancer contains elevated levels of TF and Tn compared with NAF from normal breasts. A sensitive and specific antigen capture immunoassay for TF and Tn detection in NAF was developed for this purpose. Experimental Design: Fifty NAF samples, 25 from breasts with cancer and 25 from normal breasts, were examined. Antigen capture immunoassays were done on the samples using monoclonal antibodies that specifically recognized either TF or Tn antigen in NAF. These antibodies captured serially diluted NAF samples, and the concentration of TF or Tn was determined by comparing absorbance values against a standard curve generated from standard sources of TF or Tn. Results: TF and Tn were detected in 19 of 25 and 20 of 25 NAF samples from breasts with cancer, respectively, compared with 0 of 25 and 1 of 25 NAF samples from breasts without cancer (P < 0.001 for both TF and Tn). In 92% of the cancerous breast NAF samples tested, either TF or Tn was found. Conclusions: Simultaneous measurement of TF and Tn in NAF may facilitate the noninvasive detection of breast cancer and warrants further study.

Identification of peptide sequences that bind the Thomsen-Friedenreich cancer-associated glycoantigen from bacteriophage peptide display libraries

SummaryThe goal of this study was to determine if polypeptides that bind specifically to the carcinoma-associated Thomsen-Friedenreich (T) antigen could be isolated from a random peptide bacteriophage display library. T antigen is a carbohydrate antigen that is exposed and immunoreactive on the surfaces of most primary carcinomas and their metastases, while it is masked on normal cells. Tumor-specific surface carbohydrates are often used as markers of cell differentiation and play a role in cell aggregation, which is an important step in the metastatic process. Therefore, peptides that bind and mask T antigen may yield useful carbohydrate-specific probes and provide insight into carbohydrate-mediated tumor-cell aggregation. A 15-amino acid random peptide bacteriophage display library was screened for polypeptides that exhibited high specificity to two glycoproteins which display T antigen on their surfaces. The results suggest that synthetic peptides identified from the bacteriophage display library have high affinities (Kd ∼ 1 μM) and specificities for proteins and human tumor cells which present T antigen. Thus, random bacteriophage peptide display libraries may be a rich source of sequences that bind to carbohydrate antigen structures.

Phage Peptide Display

Molecular imaging is at the forefront in the advancement of in-vivo diagnosis and monitoring of cancer. New peptide-based molecular probes to facilitate cancer detection are rapidly evolving. Peptide-based molecular probes that target apoptosis, angiogenesis, cell signaling and cell adhesion events are in place. Bacteriophage (phage) display technology, a molecular genetic approach to ligand discovery, is commonly employed to identify peptides as tumor-targeting molecules. The peptide itself may perhaps have functional properties that diminish tumor growth or metastasis. More often, a selected peptide is chemically synthesized, coupled to a radiotracer or fluorescent probe, and utilized in the development of new noninvasive molecular imaging probes. A myriad of peptides that bind cancer cells and cancer-associated antigens have been reported from phage library selections. Phage selections have also been performed in live animals to obtain peptides with optimal stability and targeting properties in vivo. To this point, few in-vitro, in-situ, or in-vivo selected peptides have shown success in the molecular imaging of cancer, the notable exception being vascular targeting peptides identified via in-vivo selections. The success of vasculature targeting peptides, such as those with an RGD motif that bind alpha(v)beta(3)integrin, may be due to the abundance and expression patterns of integrins in tumors and supporting vasculature. The discovery of molecular probes that bind tumor-specific antigens has lagged considerably. One promising means to expedite discovery is through the implementation of selected phage themselves as tumor-imaging agents in animals.

Continuous real time ex vivo epifluorescent video microscopy for the study of metastatic cancer cell interactions with microvascular endothelium

Recent studies suggest that only endothelium-attached malignant cells are capable of giving rise to hematogenous cancer metastases. Moreover, tumor cell adhesion to microvascular endothelium could be crucial in metastasis predilection to specific organs or tissues. However, the existing in vitro and in vivo techniques do not provide for sufficient delineation of distinct stages of a dynamic multi-step intravascular adhesion process. Here we report the development of an experimental system allowing for prolonged continuous ex vivo real-time observation of malignant cell adhesive interactions with perfused microvessels of a target organ in the context of its original tissue. Specifically, the vasculature of excised dura mater perfused with prostate cancer cells is described. An advantage of this technique is that selected fluorescently labeled tumor cells can be followed along identified vascular trees across the entire tissue specimen. The techniques provide for superior microvessel visualization and allow for uninterrupted monitoring and video recording of subsequent adhesion events such as rolling, docking (initial reversible adhesion), locking (irreversible adhesion), and flattening of metastatic cancer cells within perfused microvasculature on a single cell level. The results of our experiments demonstrate that intravascular adhesion of cancer cells differs dramatically from such of the leukocytes. Within dura microvessels perfused at physiological rate, non-interacting, floating, tumor cells move at velocities averaging 7.2×103 μm/s. Some tumor cells, similarly to leukocytes, exhibit rolling-like motion patterns prior to engaging into more stable adhesive interactions. In contrast, other neoplastic cells became stably adhered without rolling showing a rapid reduction in velocity from 2×103 to 0 μm/s within fractions of a second. The experimental system described herein, while developed originally for studying prostate cancer cell interactions with porcine dura mater microvasculature, offers great flexibility in adhesion experiments design and is easily adapted for use with a variety of other tissues including human.

111In-Labeled Galectin-3–Targeting Peptide as a SPECT Agent for Imaging Breast Tumors

Galectin-3 is a member of the galectin family of β-galactoside–binding animal lectins. Galectin-3 is overexpressed in a wide range of neoplasms and is associated with tumor growth and metastases. Given this fact, radiolabeled galectin-3–targeting molecules may be useful for the noninvasive imaging of tumors expressing galectin-3, as well as for targeted radionuclide therapy. In this study, the tumor cell–targeting and SPECT properties of a galectin-3–avid peptide identified from bacteriophage display were evaluated in human breast carcinoma cells and in human breast tumor–bearing mice. Methods: The galectin-3–avid peptide G3-C12 (ANTPCGPYTHDCPVKR) was synthesized with a Gly-Ser-Gly (GSG) linker at the amino terminus. After conjugation with 1,4,7,10-tetra-azacyclododecane-N,N′,N″N″′-tetraacetic acid (DOTA), the peptide was labeled with 111In. The radiochemical purity and stability of the compound was assessed by high-performance liquid chromatography. MDA-MB-435 human breast carcinoma cells expressing galectin-3 were used to characterize the in vitro binding properties of the radiolabeled compound. SCID mice bearing MDA-MB-435 xenografts were used as an in vivo model for biodistribution and imaging studies with the 111In-labeled peptide. Results: 111In-DOTA(GSG)-G3-C12 bound specifically to galectin-3–expressing MDA-MB-435 cells. The radiolabeled peptide was stable in serum and was found intact in excreted urine for at least 1 h. Competitive binding experiments indicated that the radiolabeled peptide exhibited an inhibitory concentration of 50% of 200.00 ± 6.70 nM for cultured breast carcinoma cells. In vivo biodistribution studies revealed that tumor uptake was 1.2 ± 0.24, 0.75 ± 0.05, and 0.6 ± 0.04 (mean ± SD) percentage injected dose per gram at 30 min, 1.0 h, and 2.0 h after injection of the radiotracer, respectively. SPECT/CT studies with 111In-DOTA(GSG)-G3-C12 showed excellent tumor uptake and contrast in the tumor-bearing mice. Specificity of peptide binding was demonstrated by successful blocking (52%) of in vivo tumor uptake of 111In-DOTA(GSG)-G3-C12 in the presence of its nonradiolabeled counterpart at 2 h after injection. Conclusion: This study demonstrated the successful use of a new radiolabeled peptide for the noninvasive imaging of galectin-3–positive breast tumors. This peptide may be a promising candidate for future clinical applications.

Evaluation of 99mTc-glucarate as a breast cancer imaging agent in a xenograft animal model.

INTRODUCTION The use of [(99m)Tc]glucarate has been reported as an infarct-avid agent with the potential for very early detection of myocardial infarction. [(99m)Tc]Glucarate has also been postulated as an agent for non-invasive detection of tumors. The aim of our study was to develop a Glucarate kit and evaluate [(99m)Tc]glucarate as a potential cancer imaging agent in female SCID mice bearing human MDA-MB-435 breast tumors. METHODS Glucarate in a kit formulation was labeled with (99m)Tc and evaluated for radiolabelling efficiency and radiochemical purity. The Glucarate kit stability was assessed by monthly quality controls. The pharmacokinetics of [(99m)Tc]glucarate were determined in female SCID mice bearing MDA-MB-435 human breast carcinoma tumors at 0.5, 1, 2, 4 and 24 h. Nuclear imaging studies were performed with a micro-single photon emission tomography (SPECT)/computed tomography (CT) system at 2 h post injection, while magnetic resonance imaging (MRI) was employed for tumor morphology analysis and metastatic deposit localization. RESULTS The Glucarate kits exhibited a stable shelf life of 6 months. [(99m)Tc]Glucarate was obtained with radiochemical purity greater than 95%. Biodistribution studies demonstrated moderate tumor uptake coupled with high renal clearance. Tumor-to-muscle ratios were 4.85 and 5.14 at 1 and 4 h post injection. MRI analysis showed tumors with dense cellular growth and moderate central necrosis. [(99m)Tc]Glucarate uptake in the primary MDA-MB-435 shoulder tumors and metastatic lesions were clearly visualized with micro-SPECT/CT imaging. CONCLUSIONS Selective tumor uptake and rapid clearance from nontarget organs makes [(99m)Tc]glucarate a potential agent for breast cancer imaging that awaits validation in a clinical trial.

Combinatorial evolution of high-affinity peptides that bind to the Thomsen-Friedenreich carcinoma antigen.

Thomsen-Friedenreich (TF) antigen occurs on approximately 90% of human carcinomas, is likely involved in carcinoma cell homotypic aggregation, and has clinical value as a prognostic indicator and marker of metastasized cells. Previously, we isolated anti-TF antigen peptides from bacteriophage display libraries. These bound to TF antigen on carcinoma cells but were of low affinity and solubility. We hypothesized that peptide amino acid sequence changes would result in increased affinity and solubility, which would translate into improved carcinoma cell binding and increased inhibition of aggregation. The new peptides were more soluble and exhibited up to fivefold increase in affinity (Kd ≅ 60 nM). They bound cultured human breast and prostate carcinoma cells at low concentrations, whereas the earlier peptides did not. Moreover, the new peptides were potent inhibitors of homotypic aggregation. The maturated peptides will have expanded applications in basic studies of the TF antigen and particular utility as clinical carcinoma-targeting agents.

Equilibrium Binding Studies of Recombinant Anti-single-stranded DNA Fab ROLE OF HEAVY CHAIN COMPLEMENTARITY-DETERMINING REGIONS

We previously isolated nucleic acid-binding antibody fragments (Fab) from bacteriophage display libraries representing the immunoglobulin repertoire of autoimmune mice to expedite the analysis of antibody-DNA recognition. In the present study, the binding properties of one such anti-DNA Fab, high affinity single-stranded (ss) DNA-binding Fab (DNA-1), were defined using equilibrium gel filtration and fluorescence titration. Results demonstrated that DNA-1 had a marked preference for oligo(dT) (100 nM dissociation constant) and required oligo(dT) >5 nucleotides in length. A detailed analysis of the involvement of the individual heavy chain (H) complementarity-determining regions (CDR) ensued using previously constructed HCDR transplantation mutants between DNA-1 and low affinity ssDNA-binding Fab (D5), a Fab that binds poorly to DNA (Calcutt, M. J. Komissarov, A. A., Marchbank, M. T., and Deutscher, S. L.(1996) Gene (Amst.) 168, 9-14). Circular dichroism studies indicated that the wild type and mutant Fab studied were of similar overall secondary structure and may contain similar combining site shapes. The conversion of D5 to a high affinity oligo(dT)-binding Fab occurred only in the presence of DNA-1 HCDR3. Results with site-specific mutants in HCDR1 further suggested a role of residue 33 in interaction with nucleic acid. The results of these studies are compared with previously published data on DNA-antibody recognition.