64Cu-1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid-Arg-rich Cys knot scaffold grafted with integrin αvβ6-binding peptide RSLARTDLDHLRGR

64Cu-DOTA-R02

Leung K.

Publication Details

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In vitro Rodents

Background

[PubMed]

Integrins are a family of heterodimeric glycoproteins on cell surfaces that mediate diverse biological events involving cell–cell and cell–matrix interactions (1). Integrins consist of an α and a β subunit and are important for cell adhesion and signal transduction. αvβ3 integrin is the most prominent receptor affecting tumor growth, tumor invasiveness, metastasis, tumor-induced angiogenesis, inflammation, osteoporosis, and rheumatoid arthritis (2-7). Expression of αvβ3 integrin is strong on tumor cells and activated endothelial cells, whereas expression is weak on resting endothelial cells and most normal tissues. αvβ3 antagonists are being studied as antitumor and antiangiogenic agents, and the agonists are being studied as angiogenic agents for coronary angiogenesis (6, 8, 9). A tripeptide sequence consisting of Arg-Gly-Asp (RGD) has been identified as a recognition motif used by extracellular matrix proteins (vitronectin, fibrinogen, laminin, and collagen) to bind to a variety of integrins, including αvβ3 and αvβ6. Various radiolabeled antagonists have been introduced for imaging of tumors and tumor angiogenesis (10).

Integrin αvβ6 plays an important role in the development of epithelial cells and is nearly undetectable on adult normal tissues. However, the levels of αvβ6 integrin can be upregulated during tissue remodeling and wound healing (11). On the other hand, αvβ6 integrin is strongly expressed on tumor cells of the oral cavity, pancreas, breast, ovary, colon, and stomach (12-14). αvβ6 integrin affects tumor growth, tumor invasiveness, and metastasis (13). αvβ6 binds to the RGD motif in fibronectin, tenascin, and the viral protein 1 (VP1) of foot-and-mouth disease virus (FMDV) (15). FMDV binds to cells through the RGD motif of the GH loop of the VP1. A consensus αvβ6-binding motif DLXXL was identified by using phage display screening with minimal binding to αvβ3, αIIbβ3, and αvβ5 (16). Engineered cysteine knot peptides (knots) comprise a rigid molecular scaffold of ~4 kDa with three disulfide bonds and a centrally located β sheet. Kimura et al. (17) grafted a αvβ6-binding peptide (RSLARTDLDHLRGR) into the loop 1 of an Arg-rich knot to produce the knot known as R02, which was radiolabeled with 64Cu via 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugation to form 64Cu-DOTA-R02 as a positron emission tomography (PET) probe for in vivo imaging of αvβ6 integrin in tumor-bearing nude mice.

Synthesis

[PubMed]

Kimura et al. (17) reported the synthesis of 64Cu-DOTA-R02. R02 was biosynthesized using Pichia pastoris. The N-terminus amine of R02 was conjugated with DOTA-N-hydroxysuccinimide ester for labeling with 64Cu. The radiochemical purity was >95%, with a specific activity of 18.5 MBq/nmol (0.5 mCi/nmol) at the end of purification. The labeling yield was >80%. Molecular masses of R02 (3.914 kDa) and DOTA-R02 (4.296 kDa) were confirmed with matrix-assisted laser desorption/ionization-mass spectrometry. 64Cu-DOTA-R02 remained >20% intact in mouse serum for 24 h at 37°C.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Competitive binding of soluble integrins αvβ6, αvβ3, αvβ5, and α5β1 to yeast cells expressing R02 was determined with flow cytometry (17). The binding affinity (Kd) value for αvβ6 was 3.2 ± 2.7 nM. Integrins αvβ3, αvβ5, and α5β1 (10–300 nM) exhibited little binding to R02. Cellular accumulation of 64Cu-DOTA-R02 was determined in A431 human epidermoid cancer and BxPC3 human pancreatic cancer cells (high αvβ6 expression), and HEK293 human embryonic kidney cells (low αvβ6 expression). A431 and BxPC3 cells exhibited one-fold higher radioactivity than that of HEK293 cells.

Animal Studies

Rodents

[PubMed]

Kimura et al. (17) performed biodistribution studies with 3.7 MBq (0.1 mCi, 0.15 nmol) 64Cu-DOTA-R02 in nude mice (n = 3/group) bearing A431 xenografts. Data were obtained at 1 h and 24 h after injection. 64Cu-DOTA-R02 had blood levels of 0.43% and 0.63% injected dose (ID)/g at 1 h and 24 h. The initial tracer accumulation in the αvβ6-integrin-expressing A431 tumor was about 4.43% ID/g at 1 h and increased to 5.18% ID/g at 24 h. At 60 min, the highest radioactivity concentration was found in the kidney (115% ID/g), followed by the stomach (3.58% ID/g), liver (3.18% ID/g), lung (2.52% ID/g), and intestine (2.48% ID/g). The spleen, bone, muscle, skin, pancreas, and heart showed low levels of radioactivity (<1% ID/g). The tumor/muscle ratios were 10.31 and 8.45 at 1 h and 24 h, respectively. No blocking studies were performed.

64Cu-DOTA-R02 PET imaging studies were performed in nude mice (n = 3) bearing both BxPC3 and HEK293 xenografts at 1, 2, 4, and 24 h after injection 3.7 MBq (0.1 mCi, 0.15 nmol) 64Cu-DOTA-R02 (17). The BxPC3 tumors were clearly visualized as early as 1 h and as late as 24 h after injection. Major radioactivity was observed in the urinary bladder and kidneys. Region-of-interest analysis showed the tumor accumulation of 64Cu-DOTA-R02 was 4.4 ± 0.7% ID/g in BxPC3 tumors compared with 1.3 ± 0.1% ID/g in HEK293 tumors. Average tumor/background ratios were ~9 (BxPC3) and ~2 (HEK293). No blocking studies were performed.

Other Non-Primate Mammals

[PubMed]

No publication is currently available.

Non-Human Primates

[PubMed]

No publication is currently available.

Human Studies

[PubMed]

No publication is currently available.

References

1.
Hynes R.O. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992;69(1):11–25. [PubMed: 1555235]
2.
Jin H., Varner J. Integrins: roles in cancer development and as treatment targets. Br J Cancer. 2004;90(3):561–5. [PMC free article: PMC2410157] [PubMed: 14760364]
3.
Varner J.A., Cheresh D.A. Tumor angiogenesis and the role of vascular cell integrin alphavbeta3. Important Adv Oncol. 1996:69–87. [PubMed: 8791129]
4.
Wilder R.L. Integrin alpha V beta 3 as a target for treatment of rheumatoid arthritis and related rheumatic diseases. Ann Rheum Dis. 2002;61 Suppl 2:ii96–9. [PMC free article: PMC1766704] [PubMed: 12379637]
5.
Grzesik W.J. Integrins and bone--cell adhesion and beyond. Arch Immunol Ther Exp (Warsz) 1997;45(4):271–5. [PubMed: 9523000]
6.
Kumar C.C. Integrin alpha v beta 3 as a therapeutic target for blocking tumor-induced angiogenesis. Curr Drug Targets. 2003;4(2):123–31. [PubMed: 12558065]
7.
Ruegg C., Dormond O., Foletti A. Suppression of tumor angiogenesis through the inhibition of integrin function and signaling in endothelial cells: which side to target? Endothelium. 2002;9(3):151–60. [PubMed: 12380640]
8.
Kerr J.S., Mousa S.A., Slee A.M. Alpha(v)beta(3) integrin in angiogenesis and restenosis. Drug News Perspect. 2001;14(3):143–50. [PubMed: 12819820]
9.
Mousa S.A. alphav Vitronectin receptors in vascular-mediated disorders. Med Res Rev. 2003;23(2):190–9. [PubMed: 12500288]
10.
Haubner R., Wester H.J. Radiolabeled tracers for imaging of tumor angiogenesis and evaluation of anti-angiogenic therapies. Curr Pharm Des. 2004;10(13):1439–55. [PubMed: 15134568]
11.
Breuss J.M., Gallo J., DeLisser H.M., Klimanskaya I.V., Folkesson H.G., Pittet J.F., Nishimura S.L., Aldape K., Landers D.V., Carpenter W. et al. Expression of the beta 6 integrin subunit in development, neoplasia and tissue repair suggests a role in epithelial remodeling. J Cell Sci. 1995;108(Pt 6):2241–51. [PubMed: 7673344]
12.
Thomas G.J., Nystrom M.L., Marshall J.F. Alphavbeta6 integrin in wound healing and cancer of the oral cavity. J Oral Pathol Med. 2006;35(1):1–10. [PubMed: 16393247]
13.
Kawashima A., Tsugawa S., Boku A., Kobayashi M., Minamoto T., Nakanishi I., Oda Y. Expression of alphav integrin family in gastric carcinomas: increased alphavbeta6 is associated with lymph node metastasis. Pathol Res Pract. 2003;199(2):57–64. [PubMed: 12747466]
14.
Bates R.C. The alphaVbeta6 integrin as a novel molecular target for colorectal cancer. Future Oncol. 2005;1(6):821–8. [PubMed: 16556062]
15.
DiCara D., Rapisarda C., Sutcliffe J.L., Violette S.M., Weinreb P.H., Hart I.R., Howard M.J., Marshall J.F. Structure-function analysis of Arg-Gly-Asp helix motifs in alpha v beta 6 integrin ligands. J Biol Chem. 2007;282(13):9657–65. [PubMed: 17244604]
16.
Kraft S., Diefenbach B., Mehta R., Jonczyk A., Luckenbach G.A., Goodman S.L. Definition of an unexpected ligand recognition motif for alphav beta6 integrin. J Biol Chem. 1999;274(4):1979–85. [PubMed: 9890954]
17.
Kimura R.H., Teed R., Hackel B.J., Pysz M.A., Chuang C.Z., Sathirachinda A., Willmann J.K., Gambhir S.S. Pharmacokinetically stabilized cystine knot peptides that bind alpha-v-beta-6 integrin with single-digit nanomolar affinities for detection of pancreatic cancer. Clin Cancer Res. 2012;18(3):839–49. [PMC free article: PMC3271184] [PubMed: 22173551]