Isolation of targeting nanobodies against co-opted tumor vasculature

Abstract

Tumor vasculature is in general highly heterogeneous. This characteristic is most prominent in high-grade gliomas, which present with areas of angiogenic growth, next to large areas of diffuse infiltrative growth in which tumor cells thrive on pre-existent brain vasculature. This limits the effectiveness of anti-angiogenic compounds as these will not affect more matured and co-opted vessels. Therefore, additional destruction of existing tumor vasculature may be a promising alternative avenue to effectively deprive tumors from blood. This approach requires the identification of novel tumor vascular targeting agents, which have broad tumor vessel specificities, ie are not restricted to newly formed vessels. Here, we describe the generation of a phage library displaying nanobodies that were cloned from lymphocytes of a Llama which had been immunized with clinical glioma tissue. In vivo biopanning with this library in the orthotopic glioma xenograft models E98 and E434 resulted in the selection of various nanobodies which specifically recognized glioma vessels in corresponding glioma xenografts. Importantly, also nanobodies were isolated which discriminated incorporated pre-existent vessels in highly infiltrative cerebral E434 xenografts from normal brain vessels. Our results suggest that the generation of nanobody-displaying immune phage libraries and subsequent in vivo biopanning in appropriate animal models is a promising approach for the identification of novel vascular targeting agents.

In vivo phage display screening for tumor vascular targets in glioblastoma identifies a llama nanobody against dynactin-1-p150Glued

“…We chose to use mice carrying orthotopic E98 xenografts because these tumors display both areas of angiogenesis and diffuse infiltrative growth [39]. Similarly to our previous work using different tumor xenograft models and other phage libraries [29], anti-M13 immunostainings demonstrated already a tumor-specific vessel localization of phages after the first round of biopanning (Figure 1, compare the anti-M13 immunostaining in panel A to the endothelial cell CD34 staining in panel B). After collection of tumor areas from brain sections by laser capture dissection microscopy and subsequent trypsin treatment, a total of 453 colony-forming phages was rescued of which 192 clones were randomly picked and analysed for full length nanobody expression and diversity.…”

“…Biopanning of peptide or single chain-antibody phage display libraries is a powerful technique that allows identification and isolation of tumor endothelium binding partners [29–32]. Nanobodies ® ( V ariable Heavy chain domains of Heavy chain antibodies or VHHs) are recombinant antibodies, cloned from cameloid IgG2 and IgG3 heavy chain-only antibodies (V-H) and consist of a single polypeptide chain, making this class of antibodies suitable for display on phages without significant loss of affinity [33, 34].…”

“…In a search for novel relevant nanobody-based TVTAs we performed in vivo biopannings with a nanobody phage display library [29]. As a tumor model we utilized mice carrying orthotopic E98 human glioma xenografts that characteristically display both angiogenesis-dependent growth and diffuse infiltrative growth [18, 39].…”

Combinatorial Approaches for the Identification of Brain Drug Delivery Targets

“…These two studies sought to partially address the second problem by using an experimentally optimized blood circulation time that maximized the brain to blood ratio of phage, increasing the likelihood of identifying brain targeting phage [56]. Interestingly, this time point was found to be at 24 hrs when a large component of the initial library was likely already cleared by liver and spleen, and hence, most other in vivo binding screens have been performed on the 3-15 minute time scale [28, 58, 59]. The first study, used the Ph.D. linear 12mer phage display peptide library, and after four rounds of in vivo screening, a 250-fold increase in the amount of phage recovered from the brain was observed.…”

“…One such study was performed by Hong et al in 2008 [65], in which a cyclic peptide that homes to ischemic stroke-compromised brain tissue was isolated from a T7 phage displayed library. Another study performed by Roodnik et al in 2010 [59] was particularly interesting in that unlike the other studies listed thus far, it employed an immunized sdAb library rather than a nonimmune antibody pool. An sdAb library recognizing glioma tissue was screened in vivo after intravenous injection to identify an sdAb that preferentially binds brain tumor vasculature over normal brain vasculature.…”