Cy5.5-Anti-ephrin receptor B4 (EphB4) humanized monoclonal antibody hAb47

Cy5.5-hAb47

Leung K.

Image

Table

In vitro Rodents

Background

[PubMed]

The ephrin (Eph) receptors constitute the largest group in the receptor tyrosine kinase family (1, 2). The Eph receptors and their ligands (ephrins) mediate numerous biological processes in normal development, particularly in the nervous and cardiovascular systems (3-5). On the basis of their structures and sequence relationships, ephrins are divided into two classes: the ephrin-A class, Ephs that are anchored to the cell membrane by a glycosylphosphatidylinositol linkage, and the ephrin-B class, Ephs that are transmembrane proteins. The Eph family of receptors is divided into two groups, EphA and EphB, on the basis of the similarity of their extracellular domain sequences and their affinities for binding ephrin-A and ephrin-B ligands. The Eph receptors transmit forward signals via their kinase domains and reverse signals via their transmembrane ephrin ligands (6). EphB–ephrin-B interactions are capable of mediating bidirectional signaling events upon cell–cell contact, either into the receptor-expressing cell as "forward signaling" or into the ligand-expressing cell as "reverse signaling" (7).

Eph-2 is expressed on arterial and activated endothelial cells, whereas EphB4 is normally expressed on venous endothelial cells and various blood cells (8). EphB4 selectively binds to ephrin-2 to promote cell signaling and angiogenesis. EphB4 has been implicated in cancer progression and in pathological forms of angiogenesis. Overexpression of EphB4 has been observed in cancer cells and is associated with tumorigenesis via forward signaling and with angiogenesis via reverse signaling through Eph-2 interaction (9). EphB4 forward signaling stimulates cellular proliferation. Koolpe et al. (10) identified a 15-mer peptide, Tyr-Asn-Tyr-Leu-Phe-Ser-Pro-Asn-Gly-Pro-Ile-Ala-Arg-Ala-Trp (TNYL-RAW), to be a selective antagonist of EphB4 with the use of phage display screening. Xiong et al. (11) reported the development of 64Cu-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid-TNYL-RAW (64Cu-DOTA-TNYL-RAW) for use with positron emission tomography (PET) imaging of EphB4 in nude mice bearing tumor xenografts. A monoclonal antibody mAb47 was found to have high affinity (0.8 nM) for the fibronectin-like extracellular domain of both human and mouse EphB4. Li et al. (12) conjugated Cy5.5 to the humanized Ab47 (hAb47) antibody to produce Cy5.5-hAb47 for use with near-infrared (NIR) fluorescence imaging of EphB4 expression in tumors.

Synthesis

[PubMed]

A solution of hAb47 (3.3. nmol) and Cy5.5-NHS (3.3 nmol) in borate buffer (pH 8.5) was incubated for 1.5 h at room temperature (12). Cy5.5-hAb47 was isolated with PD-10 column chromatography. Cy5.5-hIgG was prepared similarly as a control. Gel electrophoresis showed that both the heavy and the light chains of the antibodies were labeled with Cy5.5. The Cy5.5 moiety per antibody was not reported.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Li et al. (12) performed binding experiments with Cy5.5-hAb47 immobilized with EphB4-HSA. Fluorescence signal increased with increasing amounts of Cy5.5-hAb47 (0.066–3.3 nmol). The immunoreactivity values for Cy5.5-hAB47 and Cy5.5-hIgG were 81.62 ± 2.08% and 0.024 ± 0.07%, respectively.

Animal Studies

Rodents

[PubMed]

Li et al. (12) performed in vivo optical imaging studies of Cy5.5-hAb47 or Cy5.5-hIgG (0.2 nmol/mouse) in nude mice (n = 6) bearing HT29 human colorectal tumors. Whole-body optical imaging scans were performed at 6, 24, 48, 72, 96, and 120 h after injection. Cy5.5-hAb47 exhibited a relatively low tumor fluorescence signal of ~1 × 109 photons/s/cm2/sr at 6 h, and the signal increased to a plateau of ~2 × 109 photons/s/cm2/sr at 48 h. Tumor accumulation of the control Cy5.5-IgG was low at 6 h (~0.6 × 109 photons/s/cm2/sr) and reached a peak at 48 h (~0.8 × 109 photons/s/cm2/sr). The accumulation of Cy5.5-hAb47 was significantly higher than that of Cy5.5-hIgG at all the time points studied (P < 0.05). Ex vivo imaging studies showed that the tumor fluorescence intensity of mice injected with Cy5.5-hAb47 was 0.8-fold higher than that of Cy5.5-hIgG at 120 h after injection. Of the major normal tissues, only the liver showed a moderate and similar accumulation of both tracers (~0.4 × 109 photons/s/cm2/sr). 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.

NIH Support

R21 EB012294-01a1

References

1.
Dodelet V.C., Pasquale E.B. Eph receptors and ephrin ligands: embryogenesis to tumorigenesis. Oncogene. 2000;19(49):5614–9. [PubMed: 11114742]
2.
Pasquale E.B. The Eph family of receptors. Curr Opin Cell Biol. 1997;9(5):608–15. [PubMed: 9330863]
3.
Adams R.H., Klein R. Eph receptors and ephrin ligands. essential mediators of vascular development. Trends Cardiovasc Med. 2000;10(5):183–8. [PubMed: 11282292]
4.
Cheng N., Brantley D.M., Chen J. The ephrins and Eph receptors in angiogenesis. Cytokine Growth Factor Rev. 2002;13(1):75–85. [PubMed: 11750881]
5.
Murai K.K., Pasquale E.B. Eph receptors and ephrins in neuron-astrocyte communication at synapses. Glia. 2011;59(11):1567–78. [PubMed: 21850709]
6.
Noren N.K., Lu M., Freeman A.L., Koolpe M., Pasquale E.B. Interplay between EphB4 on tumor cells and vascular ephrin-B2 regulates tumor growth. Proc Natl Acad Sci U S A. 2004;101(15):5583–8. [PMC free article: PMC397426] [PubMed: 15067119]
7.
Pasquale E.B. Eph receptors and ephrins in cancer: bidirectional signalling and beyond. Nat Rev Cancer. 2010;10(3):165–80. [PMC free article: PMC2921274] [PubMed: 20179713]
8.
Pfaff D., Heroult M., Riedel M., Reiss Y., Kirmse R., Ludwig T., Korff T., Hecker M., Augustin H.G. Involvement of endothelial ephrin-B2 in adhesion and transmigration of EphB-receptor-expressing monocytes. J Cell Sci. 2008;121(Pt 22):3842–50. [PubMed: 18957513]
9.
Noren N.K., Pasquale E.B. Paradoxes of the EphB4 receptor in cancer. Cancer Res. 2007;67(9):3994–7. [PubMed: 17483308]
10.
Koolpe M., Burgess R., Dail M., Pasquale E.B. EphB receptor-binding peptides identified by phage display enable design of an antagonist with ephrin-like affinity. J Biol Chem. 2005;280(17):17301–11. [PubMed: 15722342]
11.
Xiong C., Huang M., Zhang R., Song S., Lu W., Flores L. 2nd, Gelovani J., Li C. In vivo small-animal PET/CT of EphB4 receptors using 64Cu-labeled peptide. J Nucl Med. 2011;52(2):241–8. [PubMed: 21233177]
12.
Li D., Liu S., Liu R., Park R., Hughes L., Krasnoperov V., Gill P.S., Li Z., Shan H., Conti P.S. Targeting the EphB4 Receptor for Cancer Diagnosis and Therapy Monitoring. Mol Pharm. 2013;10(1):329–36. [PMC free article: PMC3540204] [PubMed: 23211050]