Plexin D1 Expression Is Induced on Tumor Vasculature and Tumor Cells: A Novel Target for Diagnosis and Therapy?

Abstract

We previously reported that during mouse embryogenesis, plexin D1 (plxnD1) is expressed on neuronal and endothelial cells. Endothelial cells gradually loose plxnD1 expression during development. Here we describe, using in situ hybridization, that endothelial plxnD1 expression is regained during tumor angiogenesis in a mouse model of brain metastasis. Importantly, we found PLXND1 expression also in a number of human brain tumors, both of primary and metastatic origin. Apart from the tumor vasculature, abundant expression was also found on tumor cells. Via panning of a phage display library, we isolated two phages that carry single-domain antibodies with specific affinity towards a PLXND1-specific peptide. Immunohistochemistry with these single-domain antibodies on the same tumors that were used for in situ hybridization confirmed PLXND1 expression on the protein level. Furthermore, both these phages and the derived antibodies specifically homed to vessels in brain lesions of angiogenic melanoma in mice after i.v. injection. These results show that PLXND1 is a clinically relevant marker of tumor vasculature that can be targeted via i.v. injections. (Cancer Res 2005; 65(18): 8317-23)

Semaphorin signaling in angiogenesis, lymphangiogenesis and cancer

“…Plexin-D1 is highly expressed in endothelium during development [48] and also in tumor-associated blood vessels [49]. Interestingly, the expression of plexin-D1 is dynamically regulated and increased in tip cells, which extend numerous filopodia and respond to attractive and repulsive guidance cues at the leading edge of the new branching blood vessels [50].…”

Isolation of targeting nanobodies against co-opted tumor vasculature

“…Nanobodies were collected by osmotic lysis as described. 16 Immunohistochemistry Soluble nanobodies, obtained via osmotic lysis of TG1 cells, were tested in immunostaining on 4 mm sections of paraffinembedded cerebral mouse xenografts (E98 or E434). After deparaffinization, endogenous peroxidase activity was blocked by incubation with 3% H 2 O 2 .…”

“…So far, identified targets with specificity for tumor vasculature are restricted to vessels in early stages of maturation. [14][15][16] Thus, there is a need for additional tumor vascular targeting agents, which recognize tumor vessels in more mature stages.…”

“…In that work, we used a nanobody phage display library, cloned from lymphocytes of an immune-naive Llama. 16 Nanobodies are composed of the variable domains of cameloid heavy-chain antibodies, which lack a light chain. As these consist of a single polypeptide chain, nanobodies can be cloned and displayed in a phage context without significant loss of affinity compared with the originating heavy-chain antibody, making them to an attractive class of compounds.…”

“…21 Nanobodies against EGFR have already been successfully used for in vivo tumor targeting purposes. [22][23][24] By in vitro biopannings, we previously isolated from our immune-naive library Plexin D1-specific nanobodies with affinities in the range of 10 -8 -10 -9 M, which suffice for immune stainings, 16 but are possibly of too low affinity for effective in vivo applications. Matured nanobodies, cloned from immunized animals, will obviously have the advantage of higher affinity.…”