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 Dbl homology nucleotide exchange factors (GEFs) activate Rho family cytosolic GTPases in a variety of physiological and pathophysiological events. These signaling molecules typically act downstream of tyrosine kinase receptors and often facilitate nucleotide exchange on more than one member of the Rho GTPase superfamily. Three unique GEFs, i.e. p115, PDZ-RhoGEF, and LARG, are activated by the G-protein coupled receptors via the Galpha(12/13), and exhibit very selective activation of RhoA, although the mechanism by which this is accomplished is not fully understood. Based on the recently solved crystal structure of the DH-PH tandem of PDZ-RhoGEF in complex with RhoA (Derewenda, U., Oleksy, A., Stevenson, A. S., Korczynska, J., Dauter, Z., Somlyo, A. P., Otlewski, J., Somlyo, A. V., and Derewenda, Z. S. (2004) Structure (Lond.) 12, 1955-1965), we conducted extensive mutational and functional studies of the molecular basis of the RhoA selectivity in PDZ-RhoGEF. We show that while Trp(58) of RhoA is intimately involved in the interaction with the DH domain, it is not a selectivity determinant, and its interaction with PDZ-RhoGEF is unfavorable. The key selectivity determinants are dominated by polar contacts involving residues unique to RhoA. We find that selectivity for RhoA versus Cdc42 is defined by a small number of interactions.

The molecular basis of RhoA specificity in the guanine nucleotide exchange factor PDZ-RhoGEF.

Fibroblast growth factors (FGFs) and their plasma membrane-localized receptors (FGFRs) transduce signals that regulate developmental processes and metabolism. In numerous cancer types genetic aberrations of FGFR1 lead to its uncontrolled activation. To circumvent the unrestrained signal transduction, several intramolecular inhibitory mechanisms within FGFR1 have evolved. In vitro experiments with receptor truncation have demonstrated that the N-terminal D1 domain of FGFR1 negatively regulates ligand binding to the receptor. Here, we show that D1-specific monovalent antibody fragments can activate FGFR1 and its downstream signaling cascades in the absence of ligand. These data suggest that the D1 domain of FGFR1 may play autoinhibitory role not only by controlling ligand binding, but also by regulating the overall conformation of FGFR1, keeping it in a state that disfavors autoactivation in the absence of its cognate growth factor.

The autoinhibitory function of D1 domain of FGFR1 goes beyond the inhibition of ligand binding.

Fibroblast growth factors (FGFs) and their plasma membrane-localized receptors (FGFRs) play a key role in the regulation of developmental processes and metabolism. Aberrant FGFR signaling is associated with the progression of serious metabolic diseases and human cancer. Binding of FGFs to FGFRs induces receptor dimerization and transphosphorylation of FGFR kinase domains that triggers activation of intracellular signaling pathways. Following activation, FGFRs undergo internalization and subsequent lysosomal degradation, which terminates transmission of signals. Although factors that regulate FGFR endocytosis are continuously discovered, little is known about the molecular mechanism that initiates the internalization of FGFRs. Here, we analyzed the internalization of antibody fragments in various formats that target FGFR1. We show that FGFR1-specific antibody fragments in the monovalent scFv format bind to FGFR1, but are not internalized into cells that overproduce FGFR1. In contrast, the same scFv proteins in the bivalent scFv-Fc format are efficiently internalized via FGFR1-mediated, clathrin and dynamin dependent endocytosis. Interestingly, the receptor tyrosine kinase activity is dispensable for endocytosis of scFv-Fc-FGFR1 complexes, suggesting that only dimerization of receptor is required to trigger endocytosis of FGFR1 complexes.

Antibody-induced dimerization of FGFR1 promotes receptor endocytosis independently of its kinase activity.

Fibroblast growth factor receptor 1 (FGFR1) is a plasma membrane protein that transmits signals from the extracellular environment, regulating cell homeostasis and function. Dysregulation of FGFR1 leads to the development of human cancers and noncancerous diseases. Numerous tumors overproduce FGFR1, making this receptor a perspective target for cancer therapies. Antibody-drug conjugates (ADCs) are highly potent and selective anticancer agents. ADCs are composed of antibodies (targeting factors) fused to highly cytotoxic drugs (warheads). The efficiency of ADC strategy largely depends on the internalization of cytotoxic conjugate into cancer cells. Here, we have studied an interplay between affinity of anti-FGFR1 antibodies and efficiency of their cellular uptake. We have developed a unique set of engineered anti-FGFR1 antibodies that bind the same epitope in the extracellular part of FGFR1, but with different affinities. We have demonstrated that these antibodies are effectively taken up by cancer cells in the FGFR1-dependent manner. Interestingly, we have found that efficiency, defined as rate and level of antibody internalization, largely depends on the affinity of engineered antibodies towards FGFR1, as high affinity antibody displays fastest internalization kinetics. Our data may facilitate design of therapeutically relevant targeting molecules for selective treatment of FGFR1 overproducing cancers.

High Affinity Promotes Internalization of Engineered Antibodies Targeting FGFR1.

In the rapidly developing field of targeted cancer therapy there is growing interest towards therapeutics combining two or more compounds to achieve synergistic action and minimize the chance of cancer resistance to treatment. We developed a fibroblast growth factor 2 (FGF2)-conjugate bearing two cytotoxic drugs with independent mode of action: α-amanitin and monomethyl auristatin E. Drugs are covalently attached to the targeting protein in a site-specific manner via maleimide-thiol conjugation and Cu(I)-catalyzed alkyne-azide cycloaddition. The dual warhead conjugate binds to FGF receptor 1 (FGFR1) and utilizes receptor-mediated endocytosis for selective internalization into cancer cells with FGFR1. The developed conjugate displays high cytotoxicity towards all tested FGFR1-positive cell lines. Most importantly, the improved cytotoxic effect of both drugs is observed for lung cancer cell line NCI-H446. The single drug-FGF2 conjugates have no impact on the viability of NCI-H446 cells, whereas the dual warhead-FGF2 conjugate selectively and efficiently kills these FGFR1 positive cancer cells. Due to the diversified mode of action the dual warhead-FGF2 conjugate may overcome the potential acquired resistance of FGFR1-overproducing cancer cells towards single cytotoxic drugs.

FGF2 Dual Warhead Conjugate with Monomethyl Auristatin E and α-Amanitin Displays a Cytotoxic Effect towards Cancer Cells Overproducing FGF Receptor 1.

Fibroblast growth factor receptors (FGFRs) in response to fibroblast growth factors (FGFs) transmit signals across the cell membrane, regulating important cellular processes, like differentiation, division, motility, and death. The aberrant activity of FGFRs is often observed in various diseases, especially in cancer. The uncontrolled FGFRs' function may result from their overproduction, activating mutations, or generation of FGFRs' fusion proteins. Besides their typical subcellular localization on the cell surface, FGFRs are often found inside the cells, in the nucleus and mitochondria. The intracellular pool of FGFRs utilizes different mechanisms to facilitate cancer cell survival and expansion. In this review, we summarize the current stage of knowledge about the role of FGFRs in oncogenic processes. We focused on the mechanisms of FGFRs' cellular trafficking-internalization, nuclear translocation, and mitochondrial targeting, as well as their role in carcinogenesis. The subcellular sorting of FGFRs constitutes an attractive target for anti-cancer therapies. The blocking of FGFRs' nuclear and mitochondrial translocation can lead to the inhibition of cancer invasion. Moreover, the endocytosis of FGFRs can serve as a tool for the efficient and highly selective delivery of drugs into cancer cells overproducing these receptors. Here, we provide up to date examples how the cellular sorting of FGFRs can be hijacked for selective cancer treatment.

Targeting Cellular Trafficking of Fibroblast Growth Factor Receptors as a Strategy for Selective Cancer Treatment.

Mitogen-activated protein kinases (MAPK): Erk1 and Erk2 are key players in negative-feedback regulation of fibroblast growth factor (FGF) signaling. Upon activation, Erk1 and Erk2 directly phosphorylate FGF receptor 1 (FGFR1) at a specific serine residue in the C-terminal part of the receptor, substantially reducing the tyrosine phosphorylation in the receptor kinase domain and its signaling. Similarly, active Erks can also phosphorylate multiple threonine residues in the docking protein FGF receptor substrate 2 (FRS2), a major mediator of FGFR signaling. Here, we demonstrate that in NIH3T3 mouse fibroblasts and human osteosarcoma U2OS cells stably expressing FGFR1, in addition to Erk1 and Erk2, p38 kinase is able to phosphorylate FRS2. Simultaneous inhibition of Erk1/2 and p38 kinase led to a significant change in the phosphorylation pattern of FRS2 that in turn resulted in prolonged tyrosine phosphorylation of FGFR1 and FRS2 and in sustained signaling, as compared to the selective inhibition of Erks. Furthermore, excessive activation of p38 with anisomycin partially compensated the lack of Erks activity. These experiments reveal a novel crosstalk between p38 and Erk1/2 in downregulation of FGF-induced signaling.

Crosstalk between p38 and Erk 1/2 in Downregulation of FGF1-Induced Signaling.

Fibroblast growth factors (FGFs) and their receptors (FGFRs) constitute signaling circuits that transmit signals across the plasma membrane, regulating pivotal cellular processes like differentiation, migration, proliferation, and apoptosis. The malfunction of FGFs/FGFRs signaling axis is observed in numerous developmental and metabolic disorders, and in various tumors. The large diversity of FGFs/FGFRs functions is attributed to a great complexity in the regulation of FGFs/FGFRs-dependent signaling cascades. The function of FGFRs is modulated at several levels, including gene expression, alternative splicing, posttranslational modifications, and protein trafficking. One of the emerging ways to adjust FGFRs activity is through formation of complexes with other integral proteins of the cell membrane. These proteins may act as coreceptors, modulating binding of FGFs to FGFRs and defining specificity of elicited cellular response. FGFRs may interact with other cell surface receptors, like G-protein-coupled receptors (GPCRs) or receptor tyrosine kinases (RTKs). The cross-talk between various receptors modulates the strength and specificity of intracellular signaling and cell fate. At the cell surface FGFRs can assemble into large complexes involving various cell adhesion molecules (CAMs). The interplay between FGFRs and CAMs affects cell-cell interaction and motility and is especially important for development of the central nervous system. This review summarizes current stage of knowledge about the regulation of FGFRs by the plasma membrane-embedded partner proteins and highlights the importance of FGFRs-containing membrane complexes in pathological conditions, including cancer.

Cross-Talk between Fibroblast Growth Factor Receptors and Other Cell Surface Proteins.

Fibroblast growth factor receptors (FGFRs) are integral membrane proteins that transmit signals through the plasma membrane. FGFRs signaling needs to be precisely adjusted as aberrant FGFRs function is associated with development of human cancers or severe metabolic diseases. The subcellular localization, trafficking and function of FGFRs rely on the formation of multiprotein complexes. In this study we revealed galectins, lectin family members implicated in cancer development and progression, as novel FGFR1 binding proteins. We demonstrated that galectin-1 and galectin-3 directly bind to the sugar chains of the glycosylated extracellular part of FGFR1. Although both galectins compete for the same binding sites on FGFR1, these proteins elicit different impact on FGFR1 function and cellular trafficking. Galectin-1 mimics fibroblast growth factor as it efficiently activates FGFR1 and receptor-downstream signaling pathways that result in cell proliferation and apoptotic evasion. In contrast, galectin-3 induces extensive clustering of FGFR1 on the cell surface that inhibits constitutive internalization of FGFR1. Our data point on the interplay between extracellular galectins and FGFRs in the regulation of cell fate.

Differential regulation of fibroblast growth factor receptor 1 trafficking and function by extracellular galectins.

Fibroblast growth factor 1 (FGF1) has been shown to interact with integrin αβ through a specific binding site, involving Arg35 residue. The FGF1 mutant (R35E) with impaired integrin binding was found to be defective in its proliferative response, although it was still able to interact with FGF receptors (FGFR) and heparin and induce the activation of downstream signaling pathways. Here, we demonstrate that the lack of mitogenic potential of R35E mutant is directly caused by its decreased thermodynamic stability and susceptibility to proteolytic degradation. Introduction of three stabilizing mutations into R35E variant compensated the effect of destabilizing R35E mutation and restored the proliferation potential of FGF1. Moreover, the stabilized R35E variant regained both anti-apoptotic and wound healing activities, while remaining defective in binding to integrin αβ. Our results suggest that the thermodynamic stability and resistance to degradation, rather than the interaction with integrin are required for mitogenic response of FGF1.

Low Stability of Integrin-Binding Deficient Mutant of FGF1 Restricts Its Biological Activity.

Fibroblast growth factors (FGFs) via their receptors (FGFRs) transduce signals from the extracellular space to the cell interior, modulating pivotal cellular processes such as cell proliferation, motility, metabolism and death. FGF superfamily includes a group of fibroblast growth factor homologous factors (FHFs), proteins whose function is still largely unknown. Since FHFs lack the signal sequence for secretion and are unable to induce FGFR-dependent cell proliferation, these proteins were considered as intracellular proteins that are not involved in signal transduction via FGFRs. Here we demonstrate for the first time that FHF1 directly interacts with all four major FGFRs. FHF1 binding causes efficient FGFR activation and initiation of receptor-dependent signaling cascades. However, the biological effect of FHF1 differs from the one elicited by canonical FGFs, as extracellular FHF1 protects cells from apoptosis, but is unable to stimulate cell division. Our data define FHF1 as a FGFR ligand, emphasizing much greater similarity between FHFs and canonical FGFs than previously indicated. Video Abstract. (MP4 38460 kb).

FHF1 is a bona fide fibroblast growth factor that activates cellular signaling in FGFR-dependent manner.

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.

Stable Fibroblast Growth Factor 2 Dimers with High Pro-Survival and Mitogenic Potential.

Fibroblast growth factor receptors (FGFRs) are emerging targets for directed cancer therapy. Presented here is a new FGFR1-targeting conjugate, the peptibodyF2, which employs peptibody, a fusion of peptide and the Fc fragment of human IgG as a selective targeting agent and drug carrier. Short peptide based on FGF2 sequence was used to construct a FGFR1-targeting peptibody. We have shown that this peptide ensures specific delivery of peptibodyF2 into FGFR1-expressing cells. In order to use peptibodyF2 as a delivery vehicle for cytotoxic drugs, we have conjugated it with MMAE, a drug widely used in antibody-drug conjugates for targeted therapy. Resulting conjugate shows high and specific cytotoxicity towards FGFR1-positive cells, i.e., squamous cell lung carcinoma NCI-H520, while remaining non-toxic for FGFR1-negative cells. Such peptibody-drug conjugate can serve as a basis for development of therapy for tumors with overexpressed or malfunctioning FGFRs.

Article	Year
The molecular basis of RhoA specificity in the guanine nucleotide exchange factor PDZ-RhoGEF. The Journal of biological chemistry\| PubMed ID: 16954208	2006
The autoinhibitory function of D1 domain of FGFR1 goes beyond the inhibition of ligand binding. The international journal of biochemistry & cell biology\| PubMed ID: 28652212	2017
Antibody-induced dimerization of FGFR1 promotes receptor endocytosis independently of its kinase activity. Scientific reports\| PubMed ID: 28769084	2017
High Affinity Promotes Internalization of Engineered Antibodies Targeting FGFR1. International journal of molecular sciences\| PubMed ID: 29748524	2018
FGF2 Dual Warhead Conjugate with Monomethyl Auristatin E and α-Amanitin Displays a Cytotoxic Effect towards Cancer Cells Overproducing FGF Receptor 1. International journal of molecular sciences\| PubMed ID: 30029518	2018
Targeting Cellular Trafficking of Fibroblast Growth Factor Receptors as a Strategy for Selective Cancer Treatment. Journal of clinical medicine\| PubMed ID: 30577533	2018
Crosstalk between p38 and Erk 1/2 in Downregulation of FGF1-Induced Signaling. International journal of molecular sciences\| PubMed ID: 31013829	2019
Cross-Talk between Fibroblast Growth Factor Receptors and Other Cell Surface Proteins. Cells\| PubMed ID: 31091809	2019
Differential regulation of fibroblast growth factor receptor 1 trafficking and function by extracellular galectins. Cell communication and signaling : CCS\| PubMed ID: 31208421	2019
Low Stability of Integrin-Binding Deficient Mutant of FGF1 Restricts Its Biological Activity. Cells\| PubMed ID: 31443196	2019
FHF1 is a bona fide fibroblast growth factor that activates cellular signaling in FGFR-dependent manner. Cell communication and signaling : CCS\| PubMed ID: 32357892	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
FGF2-Derived PeptibodyF2-MMAE Conjugate for Targeted Delivery of Cytotoxic Drugs into Cancer Cells Overexpressing FGFR1. Cancers\| PubMed ID: 33076489	2020

Lukasz Opalinski

Biography

JoVE Journal Publications

Other Publications