Broad Epitope Coverage of Therapeutic Multi-Antibody Combinations Targeting SARS-CoV-2 Boosts In Vivo Protection and Neutralization Potency to Corner an Immune-Evading Virus.

Therapeutic antibodies (Abs) which act on a broader range of epitopes may provide more durable protection against the genetic drift of a target, typical of viruses or tumors. When these Abs exist concurrently on the targeted antigen, several mechanisms of action (MoAs) can be engaged, boosting therapeutic potency. This study selected combinations of four and five Abs with non- or partially overlapping epitopes to the SARS-CoV-2 spike glycoprotein, on or outside the crucial receptor binding domain (RBD), to offer resilience to emerging variants and trigger multiple MoAs. The combinations were derived from a pool of unique-sequence scFv Ab fragments retrieved from two SARS-CoV-2-naïve human phage display libraries. Following recombinant expression to full-length human IgG1 candidates, a biolayer interferometric analysis mapped epitopes to bins and confirmed that up to four Abs from across the bins can exist simultaneously on the spike glycoprotein trimer. Not all the bins of Abs interfered with the spike protein binding to angiotensin converting enzyme 2 (ACE2) in competitive binding assays, nor neutralized the pseudovirus or authentic virus in vitro, but when combined in vivo, their inclusion resulted in a much stronger viral clearance in the lungs of intranasally challenged hamsters, compared to that of those treated with mono ACE2 blockers. In addition, the Ab mixtures activated in vitro reporter cells expressing Fc-gamma receptors (FcγRs) involved in antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). The best four-Ab combination neutralized seventeen variants of concern from Wuhan-Hu1 to Omicron BA.4/BA.5 in vitro.

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

Diffuse gliomas are primary brain cancers that are characterised by infiltrative growth. Whereas high-grade glioma characteristically presents with perinecrotic neovascularisation, large tumor areas thrive on pre-existent vasculature as well. Clinical studies have revealed that pharmacological inhibition of the angiogenic process does not improve survival of glioblastoma patients. Direct targeting of tumor vessels may however still be an interesting therapeutic approach as it allows pinching off the blood supply to tumor cells. Such tumor vessel targeting requires the identification of tumor-specific vascular targeting agents (TVTAs).Here we describe a novel TVTA, C-C7, which we identified via in vivo biopanning of a llama nanobody phage display library in an orthotopic mouse model of diffuse glioma. We show that C-C7 recognizes a subpopulation of tumor blood vessels in glioma xenografts and clinical glioma samples. Additionally, C-C7 recognizes macrophages and activated endothelial cells in atherosclerotic lesions. By using C-C7 as bait in yeast-2-hybrid (Y2H) screens we identified dynactin-1-p150Glued as its binding partner. The interaction was confirmed by co-immunostainings with C-C7 and a commercial anti-dynactin-1-p150Glued antibody, and via co-immunoprecipitation/western blot studies. Normal brain vessels do not express dynactin-1-p150Glued and its expression is reduced under anti-VEGF therapy, suggesting that dynactin-1-p150Glued is a marker for activated endothelial cells.In conclusion, we show that in vivo phage display combined with Y2H screenings provides a powerful approach to identify tumor-targeting nanobodies and their binding partners. Using this combination of methods we identify dynactin-1-p150Glued as a novel targetable protein on activated endothelial cells and macrophages.

Targeted therapies of cancer: angiogenesis inhibition seems not enough.

The therapeutic potential of targeting tumor endothelium to induce tumor regression is now widely recognized. Tumors obtain their blood supply by the formation of new vasculature and the incorporation of pre-existent vessels. Since anti-angiogenic therapy prevents formation of neovasculature, vessels in more matured stages are not affected by such therapies. Therefore, additional vascular targeting therapy, which aim at regression of existent tumor vasculature, seems an attractive approach to effectively deprive tumors from blood supply. In this review we present an overview of different strategies to target tumor endothelium. In addition, we discuss the pitfalls of anti-angiogenic therapies in clinical settings.

Isolation of targeting nanobodies against co-opted tumor vasculature.

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.

Circulating tumour tissue fragments in patients with pulmonary metastasis of clear cell renal cell carcinoma.

Tumour metastasis is the result of a complex sequence of events, including migration of tumour cells through stroma, proteolytic degradation of stromal and vessel wall elements, intravasation, transport through the circulation, extravasation and outgrowth at compatible sites in the body (the 'seed and soil' hypothesis). However, the high incidence of metastasis from various tumour types in liver and lung may be explained by a stochastic process as well, based on the anatomical relationship of the primary tumour with the circulation and mechanical entrapment of metastatic tumour cells in capillary beds. We previously reported that constitutive VEGF-A expression in tumour xenografts facilitates this type of metastatic seeding by promoting shedding of multicellular tumour tissue fragments, surrounded by vessel wall elements, into the circulation. After transport through the vena cava, such fragments may be trapped in pulmonary arteries, allowing them to expand to symptomatic lesions. Here we tested whether this process has clinical relevance for clear cell renal cell carcinoma (ccRCC), a prototype tumour in the sense of high constitutive VEGF-A expression. To this end we collected and analysed outflow samples from the renal vein, directly after tumour nephrectomy, in 42 patients diagnosed with ccRCC. Tumour fragments in venous outflow were observed in 33% of ccRCC patients and correlated with the synchronous presence or metachronous development of pulmonary metastases (p < 0.001, Fisher's exact test). In patients with tumours that, in retrospect, were not of the VEGF-A-expressing clear cell type, tumour fragments were never observed in the renal outflow. These data suggest that, in ccRCC, a VEGF-A-induced phenotype promotes a release of tumour cell clusters into the circulation that may contribute to pulmonary metastasis.

Plexin D1 is ubiquitously expressed on tumor vessels and tumor cells in solid malignancies.

BACKGROUND: Plexin D1 is expressed on both tumor-associated endothelium and malignant cells in a number of clinical brain tumors. Recently we demonstrated that Plexin D1 expression is correlated with tumor invasion level and metastasis in a human melanoma progression series. The objective of this study was to examine whether Plexin D1 might be clinically useful as a pan-tumor vessel and pan-tumor cell target in solid tumors. METHODS: We examined Plexin D1 expression in clinical solid tumors (n = 77) of different origin, a selection of pre-malignant lesions (n = 29) and a variety of non-tumor related tissues (n = 52) by immunohistochemistry. Signals were verified in a selection of tissues via mRNA in situ hybridization. RESULTS: Plexin D1 is abundantly expressed on both activated established tumor vasculature and malignant cells in the majority of primary and metastatic clinical tumors, as well as on macrophages and fibroblasts. Importantly, in non-tumor related tissues Plexin D1 expression is restricted to a subset of, presumably activated, fibroblasts and macrophages. CONCLUSION: We demonstrate that Plexin D1 is in general ubiquitously expressed in tumor but not normal vasculature, as well as in malignant cells in a wide range of human tissues. This expression profile highlights Plexin D1 as a potentially valuable therapeutic target in clinical solid tumors, enabling simultaneous targeting of different tumor compartments.

Semaphorin 3E expression correlates inversely with Plexin D1 during tumor progression.

Plexin D1 (PLXND1) is broadly expressed on tumor vessels and tumor cells in a number of different human tumor types. Little is known, however, about the potential functional contribution of PLXND1 expression to tumor development. Expression of semaphorin 3E (Sema3E), one of the ligands for PLXND1, has previously been correlated with invasive behavior and metastasis, suggesting that the PLXND1-Sema3E interaction may play a role in tumor progression. Here we investigated PLXND1 and Sema3E expression during tumor progression in cases of melanoma. PLXND1 was not expressed by melanocytic cells in either naevi or melanomas in situ, whereas expression increased with invasion level, according to Clark's criteria. Furthermore, 89% of the metastatic melanomas examined showed membranous PLXND1-staining of tumor cells. Surprisingly, expression of Sema3E was inversely correlated with tumor progression, with no detectable staining in melanoma metastasis. To functionally assess the effects of Sema3E expression on tumor development, we overexpressed Sema3E in a xenograft model of metastatic melanoma. Sema3E expression dramatically decreased metastatic potential. These results show that PLXND1 expression during tumor development is strongly correlated with both invasive behavior and metastasis, but exclude Sema3E as an activating ligand.

Development of the tumor vascular bed in response to hypoxia-induced VEGF-A differs from that in tumors with constitutive VEGF-A expression.

Tumors arise initially as avascular masses in which central hypoxia induces expression of vascular endothelial growth factor-A (VEGF-A) and subsequently tumor vascularization. However, VEGF-A can also be constitutively expressed as a result of genetic events. VEGF-A is alternatively spliced to yield at least 6 different isoforms. Of these, VEGF-A(121) is freely diffusible whereas basically charged domains in the larger isoforms confer affinity for cell surface or extracellular matrix components. We previously reported that in a mouse brain metastasis model of human melanoma, VEGF-A(121) induced a qualitatively different tumor vascular phenotype than VEGF-A(165) and VEGF-A(189): in contrast to the latter ones, and VEGF-A(121) did not induce a neovascular bed but rather led to leakage and dilatation of preexistent brain vessels. Here, we correlate vascular phenotypes with spatial VEGF-A expression profiles in clinical brain tumors (low grade gliomas; n = 6, melanoma metastases; n = 4, adenocarcinoma metastases; n = 4, glioblastoma multiforme; n = 3, sarcoma metastasis; n = 1, renal cell carcinoma metastasis; n = 1). We show that tumors that constitutively express VEGF-A present with different vascular beds than tumors in which VEGF-A is expressed as a response to central hypoxia. This phenotypic difference is consistent with a model where in tumors with constitutive VEGF-A expression, all isoforms exert their effects on vasculature, resulting in a classical angiogenic phenotype. In tumors where only central parts express hypoxia-induced VEGF-A, the larger angiogenic isoforms are retained by extracellular matrix, leaving only freely diffusible VEGF-A(121) to exert its dilatation effects on distant vessels.

Plexin D1 expression is induced on tumor vasculature and tumor cells: a novel target for diagnosis and therapy?

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.