Size and affinity kinetics of nanobodies influence targeting and penetration of solid tumours

Debie, Pieterjan; Lafont, Chrystel; Defrise, Michel; Hansen, I.; Willigen, Danny M. van; Leeuwen, Fijs W. B. van; Gijsbers, Rik; D’Huyvetter, Matthias; Devoogdt, Nick; Lahoutte, Tony; Mollard, Patrice; Hernot, Sophie

doi:10.1016/j.jconrel.2019.11.014

Cited by 133 publications

(113 citation statements)

References 39 publications

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“…11,12 Nanobodies also penetrate tumours more rapidly and have more favourable tumour distributions in comparison with monoclonal antibodies. 13 To translate nanobodies into therapies, however, there are some obstacles that need to be confronted. Nanobodies are eliminated rapidly from the human body because their molecular weight is below the renal cut-off of 60 kDa.…”

Section: Introductionmentioning

confidence: 99%

Anti-vimentin, anti-TUFM, anti-NAP1L1 and anti-DPYSL2 nanobodies display cytotoxic effect and reduce glioblastoma cell migration

et al. 2020

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Background: Glioblastoma is a particularly common and very aggressive primary brain tumour. One of the main causes of therapy failure is the presence of glioblastoma stem cells that are resistant to chemotherapy and radiotherapy, and that have the potential to form new tumours. This study focuses on validation of eight novel antigens, TRIM28, nucleolin, vimentin, nucleosome assembly protein 1-like 1 (NAP1L1), mitochondrial translation elongation factor (EF-TU) (TUFM), dihydropyrimidinase-related protein 2 (DPYSL2), collapsin response mediator protein 1 (CRMP1) and Aly/REF export factor (ALYREF), as putative glioblastoma targets, using nanobodies. Methods: Expression of these eight antigens was analysed at the cellular level by qPCR, ELISA and immunocytochemistry, and in tissues by immunohistochemistry. The cytotoxic effects of the nanobodies were determined using AlamarBlue and water-soluble tetrazolium tests. Annexin V/propidium iodide tests were used to determine apoptotsis/necrosis of the cells in the presence of the nanobodies. Cell migration assays were performed to determine the effects of the nanobodies on cell migration. Results: NAP1L1 and CRMP1 were significantly overexpressed in glioblastoma stem cells in comparison with astrocytes and glioblastoma cell lines at the mRNA and protein levels. Vimentin, DPYSL2 and ALYREF were overexpressed in glioblastoma cell lines only at the protein level. The functional part of the study examined the cytotoxic effects of the nanobodies on glioblastoma cell lines. Four of the nanobodies were selected in terms of their specificity towards glioblastoma cells and protein overexpression: anti-vimentin (Nb79), anti-NAP1L1 (Nb179), anti-TUFM (Nb225) and anti-DPYSL2 (Nb314). In further experiments to optimise the nanobody treatment schemes, to increase their effects, and to determine their impact on migration of glioblastoma cells, the anti-TUFM nanobody showed large cytotoxic effects on glioblastoma stem cells, while the anti-vimentin, anti-NAP1L1 and anti-DPYSL2 nanobodies were indicated as agents to target mature glioblastoma cells. The anti-vimentin nanobody also had significant effects on migration of mature glioblastoma cells. Conclusion: Nb79 (anti-vimentin), Nb179 (anti-NAP1L1), Nb225 (anti-TUFM) and Nb314 (anti-DPYSL2) nanobodies are indicated for further examination for cell targeting. The anti-TUFM nanobody, Nb225, is particularly potent for inhibition of cell growth after long-term exposure of glioblastoma stem cells, with minor effects seen for astrocytes. The anti-vimentin nanobody represents an agent for inhibition of cell migration.

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Section: Introductionmentioning

confidence: 99%

Anti-vimentin, anti-TUFM, anti-NAP1L1 and anti-DPYSL2 nanobodies display cytotoxic effect and reduce glioblastoma cell migration

et al. 2020

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“…For IgG, however, the long plasma half-life requires an extended time between injection and illumination to yield sufficient systemic clearance, which is impractical and raises the risk for off-target effects [ 34 ]. In parallel, IgGs have poor tumor penetration due to their large size [ 35 , 36 ]. Low molecular weight non-antibody formats, such as designed ankyrin repeat proteins (DARPins), represent attractive alternatives that overcome these disadvantages [ 37 , 38 , 39 , 40 , 41 ].…”

Section: Introductionmentioning

confidence: 99%

EpCAM-Binding DARPins for Targeted Photodynamic Therapy of Ovarian Cancer

Brand

Lith

Jong

et al. 2020

Cancers

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Ovarian cancer is the most lethal gynecological malignancy due to late detection associated with dissemination throughout the abdominal cavity. Targeted photodynamic therapy (tPDT) aimed at epithelial cell adhesion molecule (EpCAM), overexpressed in over 90% of ovarian cancer metastatic lesions, is a promising novel therapeutic modality. Here, we tested the specificity and activity of conjugates of EpCAM-directed designed ankyrin repeat proteins (DARPins) with the photosensitizer IRDye 700DX in in vitro and in vivo ovarian cancer models. EpCAM-binding DARPins (Ec1: Kd = 68 pM; Ac2: Kd = 130 nM) and a control DARPin were site-specifically functionalized with fluorophores or IRDye 700DX. Conjugation of anti-EpCAM DARPins with fluorophores maintained EpCAM-specific binding in cell lines and patient-derived ovarian cancer explants. Penetration of DARPin Ec1 into tumor spheroids was slower than that of Ac2, indicative of a binding site barrier effect for Ec1. DARPin-IRDye 700DX conjugates killed EpCAM-expressing cells in a highly specific and illumination-dependent fashion in 2D and 3D cultures. Furthermore, they effectively homed to EpCAM-expressing subcutaneous OV90 xenografts in mice. In conclusion, the high activity and specificity observed in preclinical ovarian cancer models, combined with a high specificity in patient material, warrant a further investigation of EpCAM-targeted PDT for ovarian cancer.

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“…This can contribute to the heterogeneous tissue distribution of ADC and nano-formulations and can impede a successful anti-tumour response [ 29 ]. The combination of rapidly dividing cancer cells with the production and deposition of ECM constituents such as collagen and hyaluronic acid leads to a high solid stress in tumour ECM [ 30 ]. Solid stress arises from the mechanical forces associated with the solid phase of the tumour, and is distinct from interstitial fluid pressure, which is derived from the leaky vasculature and poor lymphatic drainage.…”

Section: Vascular Endothelial Transport and Target Tissue Distribumentioning

confidence: 99%

Antibody-Drug Conjugates and Targeted Treatment Strategies for Hepatocellular Carcinoma: A Drug-Delivery Perspective

Dahlgren

Lennernäs

2020

Molecules

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Increased understanding of cancer biology, pharmacology and drug delivery has provided a new framework for drug discovery and product development that relies on the unique expression of specific macromolecules (i.e., antigens) on the surface of tumour cells. This has enabled the development of anti-cancer treatments that combine the selectivity of antibodies with the efficacy of highly potent chemotherapeutic small molecules, called antibody-drug conjugates (ADCs). ADCs are composed of a cytotoxic drug covalently linked to an antibody which then selectively binds to a highly expressed antigen on a cancer cell; the conjugate is then internalized by the cell where it releases the potent cytotoxic drug and efficiently kills the tumour cell. There are, however, many challenges in the development of ADCs, mainly around optimizing the therapeutic/safety benefits. These challenges are discussed in this review; they include issues with the plasma stability and half-life of the ADC, its transport from blood into and distribution throughout the tumour compartment, cancer cell antigen expression and the ADC binding affinity to the target antigen, the cell internalization process, cleaving of the cytotoxic drug from the ADC, and the cytotoxic effect of the drug on the target cells. Finally, we present a summary of some of the experimental ADC strategies used in the treatment of hepatocellular carcinoma, from the recent literature.

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Size and affinity kinetics of nanobodies influence targeting and penetration of solid tumours

Cited by 133 publications

References 39 publications

Anti-vimentin, anti-TUFM, anti-NAP1L1 and anti-DPYSL2 nanobodies display cytotoxic effect and reduce glioblastoma cell migration

Anti-vimentin, anti-TUFM, anti-NAP1L1 and anti-DPYSL2 nanobodies display cytotoxic effect and reduce glioblastoma cell migration

EpCAM-Binding DARPins for Targeted Photodynamic Therapy of Ovarian Cancer

Antibody-Drug Conjugates and Targeted Treatment Strategies for Hepatocellular Carcinoma: A Drug-Delivery Perspective

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