preparation-site-specific-cytotoxic-protein-conjugates-via-maleimide

Preparation of Site-Specific Cytotoxic Protein Conjugates via Maleimide-thiol Chemistry and Sortase A-Mediated Ligation

Cancer is currently the second most common cause of death worldwide. The hallmark of cancer cells is the presence of specific marker proteins such as ...

University of Wroclaw

The blocking of specific protein-protein interactions using nanoparticles is an emerging alternative to small molecule-based therapeutic interventions. However, the nanoparticles designed as "artificial proteins" generally require modification of their surface with (bio)organic molecules and/or polymers to ensure their selectivity and specificity of action. Here, we show that nanosized diamond crystals (nanodiamonds, NDs) without any synthetically installed (bio)organic interface enable the specific and efficient targeting of the family of extracellular signalling molecules known as fibroblast growth factors (FGFs). We found that low nanomolar solutions of detonation NDs with positive ζ-potential strongly associate with multiple FGF ligands present at sub-nanomolar concentrations and effectively neutralize the effects of FGF signalling in cells without interfering with other growth factor systems and serum proteins unrelated to FGFs. We identified an evolutionarily conserved FGF recognition motif, ∼17 amino acids long, that contributes to the selectivity of the ND-FGF interaction. In addition, we inserted this motif into a de novo constructed chimeric protein, which significantly improved its interaction with NDs. We demonstrated that the interaction of NDs, as purely inorganic nanoparticles, with proteins can mitigate pathological FGF signalling and promote the restoration of cartilage growth in a mouse limb explant model. Based on our observations, we foresee that NDs may potentially be applied as nanotherapeutics to neutralize disease-related activities of FGFs in vivo.

Nanodiamonds as "artificial proteins": Regulation of a cell signalling system using low nanomolar solutions of inorganic nanocrystals.

Antibody-drug conjugates (ADCs) are a new class of anticancer therapeutics that combine the selectivity of targeted treatment, ensured by monoclonal antibodies, with the potency of the cytotoxic agent. Here, we applied an analogous approach, but instead of an antibody, we used fibroblast growth factor 2 (FGF2). FGF2 is a natural ligand of fibroblast growth factor receptor 1 (FGFR1), a cell-surface receptor reported to be overexpressed in several types of tumors. We developed and characterized FGF2 conjugates containing a defined number of molecules of highly cytotoxic drug monomethyl auristatin E (MMAE). These conjugates effectively targeted FGFR1-expressing cells, were internalized upon FGFR1-mediated endocytosis, and, in consequence, revealed high cytotoxicity, which was clearly related to the FGFR1 expression level. Among the conjugates tested, the most potent was that bearing three MMAE molecules, showing that the cytotoxicity of protein-drug conjugates in vitro is directly dependent on drug loading.

Cytotoxic Conjugates of Fibroblast Growth Factor 2 (FGF2) with Monomethyl Auristatin E for Effective Killing of Cells Expressing FGF Receptors.

In spite of significant progress in the field of targeted anticancer therapy, the FDA has approved only five ADC-based drugs. Hence the search for new targeted anticancer agents is an unfulfilled necessity. Here, we present novel types of protein-drug conjugates (PDCs) that exhibit superior anticancer activities. Instead of a monoclonal antibody, we used fibroblast growth factor 2 (FGF2) as a targeting molecule. FGF2 is a natural ligand of fibroblast growth factor receptor 1 (FGFR1), a transmembrane receptor overproduced in various types of cancers. We synthesized site-specific and stoichiometric-controlled conjugates of FGF2 with a highly potent, hydrophilic derivative of auristatin called auristatin Y. To increase the hydrophilicity and hydrodynamic radius of conjugates, we employed PEG4 and PEG27 molecules as a spacer between the targeting molecule and the cytotoxic payload. All conjugates were selective to FGFR1-positive cell lines, effectively internalized via the FGFR1-dependent pathway, and exhibited a highly cytotoxic effect only on FGFR1-positive cancer cell lines.

Site-Specific, Stoichiometric-Controlled, PEGylated Conjugates of Fibroblast Growth Factor 2 (FGF2) with Hydrophilic Auristatin Y for Highly Selective Killing of Cancer Cells Overproducing Fibroblast Growth Factor Receptor 1 (FGFR1).

Fibroblast growth factor receptor 1 (FGFR1) transmits signals through the plasma membrane regulating essential cellular processes like division, motility, metabolism, and death. Overexpression of FGFR1 is observed in numerous tumors and thus constitutes an attractive molecular target for selective cancer treatment. Targeted anti-cancer therapies aim for the precise delivery of drugs into cancer cells, sparing the healthy ones and thus limiting unwanted side effects. One of the key steps in targeted drug delivery is receptor-mediated endocytosis. Here, we show that the efficiency and the mechanism of FGFR1 internalization are governed by the spatial distribution of the receptor in the plasma membrane. Using engineered antibodies of different valency, we demonstrate that dimerization of FGFR1 with bivalent antibody triggers clathrin-mediated endocytosis (CME) of the receptor. Clustering of FGFR1 into larger oligomers with tetravalent antibody stimulates fast and highly efficient uptake of the receptor that occurs via two distinct mechanisms: CME and dynamin-dependent clathrin-independent endocytic routes. Furthermore, we show that all endocytic pathways engaged in FGFR1 internalization do not require receptor activation. Our data provide novel insights into the mechanisms of intracellular trafficking of FGFR1 and constitute guidelines for development of highly internalizing antibody-based drug carriers for targeted therapy of FGFR1-overproducing cancers.

FGFR1 clustering with engineered tetravalent antibody improves the efficiency and modifies the mechanism of receptor internalization.

Fibroblast growth factor 2 (FGF2) is a heparin-binding growth factor with broad mitogenic and cell survival activities. Its effector functions are induced upon the formation of 2:2 FGF2:FGFR1 tetrameric complex. To facilitate receptor activation, and therefore, to improve the FGF2 biological properties, we preorganized dimeric ligand by a covalent linkage of two FGF2 molecules. Mutations of the FGF2 WT protein were designed to obtain variants with a single surface-exposed reactive cysteine for the chemical conjugation via maleimide-thiol reaction with bis-functionalized linear PEG linkers. We developed eight FGF2 dimers of defined topology, differing in mutual orientation of individual FGF2 molecules. The engineered proteins remained functional in terms of FGFR downstream signaling activation and were characterized by the increased stability, mitogenic potential and anti-apoptotic activity, as well as induced greater migration responses in normal fibroblasts, as compared to FGF2 monomer. Importantly, biological activity of the dimers was much less dependent on the external heparin administration. Moreover, some dimeric FGF2 variants internalized more efficiently into FGFR overexpressing cancer cells. In summary, in the current work, we showed that preorganization of dimeric FGF2 ligand increased the stability of the growth factor, and therefore, enhanced its biological activity.

Article	Year
Nanodiamonds as "artificial proteins": Regulation of a cell signalling system using low nanomolar solutions of inorganic nanocrystals. Biomaterials\| PubMed ID: 29879652	2018
Cytotoxic Conjugates of Fibroblast Growth Factor 2 (FGF2) with Monomethyl Auristatin E for Effective Killing of Cells Expressing FGF Receptors. ACS omega\| PubMed ID: 30023704	2017
Site-Specific, Stoichiometric-Controlled, PEGylated Conjugates of Fibroblast Growth Factor 2 (FGF2) with Hydrophilic Auristatin Y for Highly Selective Killing of Cancer Cells Overproducing F... Molecular pharmaceutics\| PubMed ID: 32501706	2020
FGFR1 clustering with engineered tetravalent antibody improves the efficiency and modifies the mechanism of receptor internalization. Molecular oncology\| PubMed ID: 32511887	2020
Stable Fibroblast Growth Factor 2 Dimers with High Pro-Survival and Mitogenic Potential. International journal of molecular sciences\| PubMed ID: 32526859	2020

Mateusz Adam Krzyscik

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