Molecular specialization of breast vasculature: a breast-homing phage-displayed peptide binds to aminopeptidase P in breast vasculature

Abstract

In vivo phage display identifies peptides that selectively home to the vasculature of individual organs, tissues, and tumors. Here we report the identification of a cyclic nonapeptide, CPGPEGAGC, which homes to normal breast tissue with a 100-fold selectivity over nontargeted phage. The homing of the phage is inhibited by its cognate synthetic peptide. Phage localization in tissue sections showed that the breast-homing phage binds to the blood vessels in the breast, but not in other tissues. The phage also bound to the vasculature of hyperplastic and malignant lesions in transgenic breast cancer mice. Expression cloning with a phage-displayed cDNA library yielded a phage that specifically bound to the breast-homing peptide. The cDNA insert was homologous to a fragment of aminopeptidase P. The homing peptide bound aminopeptidase P from malignant breast tissue in affinity chromatography. Antibodies against aminopeptidase P inhibited the in vitro binding of the phage-displayed cDNA to the peptide and the in vivo homing of phage carrying the peptide. These results indicate that aminopeptidase P is the receptor for the breast-homing peptide. This peptide may be useful in designing drugs for the prevention and treatment of breast cancer.

Figure 1

Isolation of a breast-homing phage by in vivo screening of a phage library. A CX7C library (10⁹ pfu) was injected into the tail vein of mice, and 7 min later the mice were perfused through the heart and phage was rescued from breast tissue. The rescued phage was amplified and reinjected in four consecutive rounds. The number of pfu recovered from breast tissue is shown (black bars). As a control, nonrecombinant T7 phage was injected (white bars). In round 5 (R5), the titer of phage recovered from the pancreas was also determined (gray bar).

Figure 2

Homing specificity of CPGPEGAGC phage. CPGPEGAGC phage (10⁹ pfu) was injected into mice and recovered as in Fig. 1 from the indicated organs. The number of pfu recovered is shown. Selective phage homing to the breast is seen. Coinjection of free CPGPEGAGC peptide (1 mg) blocked the breast homing of the phage.

Figure 3

Localization of CPGPEGAGC phage by immunohistochemistry. CPGPEGAGC phage or nonrecombinant control phage (both at 10⁹ pfu) were injected into the tail veins of mice. After 15 min of circulation, mice were perfused through the heart. The indicated organs were dissected, fixed, and cryo-sections were stained for T7 phage proteins by using a rabbit anti-T7 antibody and a peroxidase-coupled anti-rabbit IgG. CPGPEGAGC phage was detected in the blood vessels and surrounding tissue of the mammary fat pad (A) and early MMTV PyMT breast carcinomas (B). No phage staining occurred in the brain (C) and the pancreas (D). The nonrecombinant phage was not detected in normal breast (E) or in breast carcinomas (F).

Figure 4

Isolation of cDNA clones encoding CPGPEGAGC-binding proteins. (A) The CPGPEGAGC peptide was covalently linked to microtiter wells and a phage-displayed cDNA library was screened for CPGPEGAGC-binding clones by performing four consecutive rounds of selection. The number of pfu recovered from the wells is shown. (B) Clones from the screening shown in A were individually tested for binding to wells coated with the CPGPEGAGC peptide. Phage clone 47 avidly bound to the CPGPEGAGC-coated wells. (C) Alignment of the sequences of clone 47 and human membrane-bound APaseP (XNPEP2; GenBank U90724). The dashes denote gaps in the alignment of the sequences. The signal sequence of ApaseP is shown on the first line, and the N-terminal sequence of the mature protein is shown on the second line.

Figure 5

Anti-APaseP antibody and free CPGPEGAGC peptide block CPGPEGAGC phage binding and homing. (A) The binding of phage displaying the APaseP cDNA fragment (10⁸ pfu) to microtiter wells coated with CPGPEGAGC peptide was tested in the presence of the free CPGPEGAGC peptide (2 mg/ml), purified IgG from an anti-APaseP antiserum, or from normal rabbit serum, each at 10 μg/ml, or buffer. As a control, microtiter wells were coated with an unrelated peptide. (B) CPGPEGAGC-displaying phage was injected into the tail veins of mice together with 2.5 mg of the anti-APaseP IgG or control IgG. Anti-APaseP blocked the breast homing, whereas the control antibody had no effect. (C) Recovery of a phage carrying another breast-homing peptide, CRSS, could not be blocked by the anti-APaseP antiserum (2.5 mg) or by free CPGPEGAGC peptide (1 mg).

Figure 6

APaseP specifically binds to a CPGPEGAGC peptide affinity column. An extract of MMTV PyMT tumor tissue was fractionated on a CPGPEGAGC peptide column. The column was washed and eluted with the CPGPEGAGC peptide. Aliquots from the tumor extract before (extract) and after (unbound) application to the column, the wash fraction, CPGPEGAGC-peptide eluate, and 8 M urea eluate were analyzed by immunoblotting with anti-APaseP.