Quantitative and qualitative differences in the activation of a fibroblast growth factor receptor by different FGF ligands.

The FGF system is the most complex of all receptor tyrosine kinase signaling networks with 18 FGF ligands and four FGFRs that deliver morphogenic signals to pattern most embryonic structures. Even when a single FGFR is expressed in the tissue, different FGFs can trigger dramatically different biological responses via this receptor. Here we show both quantitative and qualitative differences in the signaling of one of the FGF receptors, FGFR1c, in response to different FGFs. We provide an overview of the recent discovery that FGFs engage in biased signaling via FGFR1c. We discuss the concept of ligand bias, which represents qualitative differences in signaling as it is a measure of differential ligand preferences for different downstream responses. We show how FGF ligand bias manifests in functional data in cultured chondrocyte cells. We argue that FGF-ligand bias contributes substantially to FGF-driven developmental processes, along with known differences in FGF expression levels, FGF-FGFR binding coefficients and differences in FGF stability in vivo.

Investigations of membrane protein interactions in cells using fluorescence microscopy.

The interactions between proteins in membranes govern many cellular functions. Our ability to probe for such interactions has greatly evolved in recent years due to the introduction of new fluorescence techniques. As a result, we currently have a choice of methods that can be used to assess the spatial distribution of a membrane protein, its association state, and the thermodynamic stability of the oligomers in the native milieu. These biophysical measurements have revealed new insights into important biological processes in cellular membranes.

Targeting HER2 and FGFR-positive cancer cells with a bispecific cytotoxic conjugate combining anti-HER2 Affibody and FGF2.

Breast cancer remains a significant global health challenge, necessitating the development of effective targeted therapies. This study aimed to create bispecific targeting molecules against HER2 and FGFR1, two receptors known to play crucial roles in breast cancer progression. By combining the high-affinity Affibody ZHER2:2891 and a modified, stable form of fibroblast growth factor 2 (FGF2), we successfully generated bispecific proteins capable of simultaneously recognizing HER2 and FGFR1. Two variants were designed: AfHER2-sFGF2 with a shorter linker and AfHER2-lFGF2 with a longer linker between the HER2 and FGFR1-recognizing proteins. Both proteins exhibited selective binding to HER2 and FGFR1, with AfHER2-lFGF2 demonstrating simultaneous binding to both receptors. AfHER2-lFGF2 exhibited superior internalization compared to FGF2 in FGFR-positive cells and significantly increased internalization compared to AfHER2 in HER2-positive cells. To enhance their therapeutic potential, highly potent cytotoxic agent MMAE was conjugated to the targeting proteins, resulting in protein-drug conjugates. The bispecific AfHER2-lFGF2-vcMMAE conjugate demonstrated exceptional cytotoxic activity against HER2-positive, FGFR-positive, and dual-positive cancer cell lines that was significantly higher compared to monospecific conjugates. These data indicate the beneficial effect of simultaneous targeting of HER2 and FGFR1 in precise anticancer medicine and contribute valuable insights into the design and potential of bispecific targeting molecules for breast cancer treatment.

Dual-Warhead Conjugate Based on Fibroblast Growth Factor 2 Dimer Loaded with α-Amanitin and Monomethyl Auristatin E Exhibits Superior Cytotoxicity towards Cancer Cells Overproducing Fibroblast Growth Factor Receptor 1.

Targeting fibroblast growth factor receptor 1 (FGFR1) is a promising therapeutic strategy for various cancers associated with alterations in the FGFR1 gene. In this study, we developed a highly cytotoxic bioconjugate based on fibroblast growth factor 2 (FGF2), which is a natural ligand of this receptor, and two potent cytotoxic drugs-α-amanitin and monomethyl auristatin E-with completely independent mechanistic modes of action. Utilizing recombinant DNA technology, we produced an FGF2 N- to C-end dimer that exhibited superior internalization capacity in FGFR1-positive cells. The drugs were site-specifically attached to the targeting protein using SnoopLigase- and evolved sortase A-mediated ligations. The resulting dimeric dual-warhead conjugate selectively binds to the FGFR1 and utilizes receptor-mediated endocytosis to enter the cells. Moreover, our results demonstrate that the developed conjugate exhibits about 10-fold higher cytotoxic potency against FGFR1-positive cell lines than an equimolar mixture of single-warhead conjugates. The diversified mode of action of the dual-warhead conjugate may help to overcome the potential acquired resistance of FGFR1-overproducing cancer cells to single cytotoxic drugs.

Short report galectins use N-glycans of FGFs to capture growth factors at the cell surface and fine-tune their signaling.

Fibroblast growth factors (FGFs) and their receptors (FGFRs) constitute complex signaling hubs that are crucial for the development and homeostasis of the human body. Most of FGFs are released by cells using the conventional secretory pathway and are N-glycosylated, yet the role of FGFs glycosylation is largely unknown. Here, we identify N-glycans of FGFs as binding sites for a specific set of extracellular lectins, galectins - 1, -3, -7 and - 8. We demonstrate that galectins attract N-glycosylated FGF4 to the cell surface, forming a reservoir of the growth factor in the extracellular matrix. Furthermore, we show that distinct galectins differentially modulate FGF4 signaling and FGF4-dependent cellular processes. Using engineered variants of galectins with altered valency we demonstrate that multivalency of galectins is critical for the adjustment of FGF4 activity. Summarizing, our data reveal a novel regulatory module within FGF signaling, in which the glyco-code in FGFs provides previously unanticipated information differentially deciphered by multivalent galectins, affecting signal transduction and cell physiology. Video Abstract.

Receptor clustering by a precise set of extracellular galectins initiates FGFR signaling.

FGF/FGFR signaling is critical for the development and homeostasis of the human body and imbalanced FGF/FGFR contributes to the progression of severe diseases, including cancers. FGFRs are N-glycosylated, but the role of these modifications is largely unknown. Galectins are extracellular carbohydrate-binding proteins implicated in a plethora of processes in heathy and malignant cells. Here, we identified a precise set of galectins (galectin-1, -3, -7, and -8) that directly interact with N-glycans of FGFRs. We demonstrated that galectins bind N-glycan chains of the membrane-proximal D3 domain of FGFR1 and trigger differential clustering of FGFR1, resulting in activation of the receptor and initiation of downstream signaling cascades. Using engineered galectins with controlled valency, we provide evidence that N-glycosylation-dependent clustering of FGFR1 constitutes a mechanism for FGFR1 stimulation by galectins. We revealed that the consequences of galectin/FGFR signaling for cell physiology are markedly different from the effects induced by canonical FGF/FGFR units, with galectin/FGFR signaling affecting cell viability and metabolic activity. Furthermore, we showed that galectins are capable of activating an FGFR pool inaccessible for FGF1, enhancing the amplitude of transduced signals. Summarizing, our data identify a novel mechanism of FGFR activation, in which the information stored in the N-glycans of FGFRs provides previously unanticipated information about FGFRs' spatial distribution, which is differentially deciphered by distinct multivalent galectins, affecting signal transmission and cell fate.

The Significance of Cell Surface N-Glycosylation for Internalization and Potency of Cytotoxic Conjugates Targeting Receptor Tyrosine Kinases.

Precise anticancer therapies employing cytotoxic conjugates constitute a side-effect-limited, highly attractive alternative to commonly used cancer treatment modalities, such as conventional chemotherapy, radiotherapy or surgical interventions. Receptor tyrosine kinases are a large family of N-glycoproteins intensively studied as molecular targets for cytotoxic conjugates in various cancers. At the cell surface, these receptors are embedded in a dense carbohydrate layer formed by numerous plasma membrane glycoproteins. The complexity of the cell surface architecture is further increased by galectins, secreted lectins capable of recognizing and clustering glycoconjugates, affecting their motility and activity. Cell surface N-glycosylation is intensively remodeled by cancer cells; however, the contribution of this phenomenon to the efficiency of treatment with cytotoxic conjugates is largely unknown. Here, we evaluated the significance of N-glycosylation for the internalization and toxicity of conjugates targeting two model receptor tyrosine kinases strongly implicated in cancer: HER2 and FGFR1. We employed three conjugates of distinct molecular architecture and specificity: AffibodyHER2-vcMMAE (targeting HER2), vcMMAE-KCK-FGF1.E and T-Fc-vcMMAE (recognizing different epitopes within FGFR1). We demonstrated that inhibition of N-glycosylation reduced the cellular uptake of all conjugates tested and provided evidence for a role of the galectin network in conjugate internalization. In vitro binding studies revealed that the reduced uptake of conjugates is not due to impaired HER2 and FGFR1 binding. Importantly, we demonstrated that alteration of N-glycosylation can affect the cytotoxic potential of conjugates. Our data implicate a key role for cell surface N-glycosylation in the delivery of cytotoxic conjugates into cancer cells.

Cyclic and dimeric fibroblast growth factor 2 variants with high biomedical potential.

Fibroblast growth factor 2 (FGF2) is a pleiotropic protein engaged in the regulation of key cellular processes in a wide spectrum of cells. FGF2 is an important object of basic research as well as a molecule used in regenerative medicine, in vitro cell culture maintenance, and as an anticancer drug carrier. However, the unsatisfactory stability and pleiotropic activities of the wild-type FGF2 largely limit its use as a medical product. To overcome these limitations, we have designed a set of FGF2-based macromolecules via sortase A-mediated cyclization and oligomerization. We obtained heparin-switchable FGF2 variants with enhanced stability and improved ability to stimulate cell proliferation and migration. We have shown that stimulation of glucose uptake by adipocytes is modulated by the architecture of FGF2 oligomers. Moreover, we used hyper-stable FGF2 variants for the construction of highly effective drug carriers for selective killing of FGFR1-overproducing cancer cells. The strategy for FGF2 engineering presented in this work provides novel insights into the design of growth factor variants for regenerative and anti-cancer precise medicine.

Drug Conjugation via Maleimide-Thiol Chemistry Does Not Affect Targeting Properties of Cysteine-Containing Anti-FGFR1 Peptibodies.

With a wide range of available cytotoxic therapeutics, the main focus of current cancer research is to deliver them specifically to the cancer cells, minimizing toxicity against healthy tissues. Targeted therapy utilizes different carriers for cytotoxic drugs, combining a targeting molecule, typically an antibody, and a highly toxic payload. For the effective delivery of such cytotoxic conjugates, a molecular target on the cancer cell is required. Various proteins are exclusively or abundantly expressed in cancer cells, making them a possible target for drug carriers. Fibroblast growth factor receptor 1 (FGFR1) overexpression has been reported in different types of cancer, but no FGFR1-targeting cytotoxic conjugate has been approved for therapy so far. In this study, the FGFR1-targeting peptide previously described in the literature was reformatted into a peptibody-peptide fusion with the fragment crystallizable (Fc) domain of IgG1. PeptibodyC19 can be effectively internalized into FGFR1-overexpressing cells and does not induce cells' proliferation. The main challenge for its use as a cytotoxic conjugate is a cysteine residue located within the targeting peptide. A standard drug-conjugation strategy based on the maleimide-thiol reaction involves modification of cysteines within the Fc domain hinge region. Applied here, however, may easily result in the modification of the targeting peptide with the drug, limiting its affinity to the target and therefore the potential for specific drug delivery. To investigate if this is the case, we have performed conjugation reactions with different auristatin derivatives (PEGylated and unmodified) under various conditions. By controlling the reduction conditions and the type of cytotoxic payload, different numbers of cysteines were substituted, allowing us to avoid conjugating the drug to the targeting peptide, which could affect its binding to FGFR1. The optimized protocol with PEGylated auristatin yielded doubly substituted peptibodyC19, showing specific cytotoxicity toward the FGFR1-expressing lung cancer cells, with no effect on cells with low FGFR1 levels. Indeed, additional cysteine poses a risk of unwanted modification, but changes in the type of cytotoxic payload and reaction conditions allow the use of standard thiol-maleimide-based conjugation to achieve standard Fc hinge region cysteine modification, analogously to antibody-drug conjugates.

Peptibody Based on FGFR1-Binding Peptides From the FGF4 Sequence as a Cancer-Targeting Agent.

Targeted therapies are a promising alternative to conventional chemotherapy, with an increasing number of therapeutics targeting specific molecular aberrancies in cancer cells. One of the emerging targets for directed cancer treatments is fibroblast growth factor receptors (FGFRs), which are known to be involved in the pathogenesis and progression of multiple cancer types, specially in lung, bladder, and breast cancers. Here, we are demonstrating the development of the FGFR1-targeting agent based on the interactome screening approach, based on the isolation of binding regions from ligands interacting with the receptor. The parallel analysis by FGFR1 pull-down of chymotryptic peptides coupled with MS analysis, and PepSpot analysis yielded equivalent peptide sequences from FGF4, one of the FGFR1 ligands. Three sequences served as a basis for peptibody (Fc-fusion) generation, to overcome clinical limitations of peptidic agents, and two of them showed favorable FGFR1-binding in vitro and FGFR1-dependent internalization into cells. To validate if developed FGFR1-targeting peptibodies can be used for drug delivery, similar to the well-established concept of antibody-drug conjugates (ADCs), peptibodyF4_1 was successfully conjugated with monomethylauristatin E (MMAE), and has shown significant and specific toxicity toward FGFR1-expressing lung cancer cell lines, with nanomolar EC50 values. Essentially, the development of new effective FGFR1 binders that comprise the naturally occurring FGFR-recognition peptides and Fc region ensuring high plasma stability, and long bloodstream circulation is an interesting strategy expanding targeted anticancer agents' portfolio. Furthermore, identifying peptides effectively binding the receptor from sequences of its ligands is not limited to FGFRs and is an approach versatile enough to be a basis for a new peptide/peptibodies development strategy.

Intrinsically Fluorescent Oligomeric Cytotoxic Conjugates Toxic for FGFR1-Overproducing Cancers.

Fibroblast growth factor receptor 1 (FGFR1) is an integral membrane protein that transmits prolife signals through the plasma membrane. Overexpression of FGFR1 has been reported in various tumor types, and therefore, this receptor constitutes an attractive molecular target for selective anticancer therapies. Here, we present a novel system for generation of intrinsically fluorescent, self-assembling, oligomeric cytotoxic conjugates with high affinity and efficient internalization targeting FGFR1. In our approach, we employed FGF1 as an FGFR1 recognizing molecule and genetically fused it to green fluorescent protein polygons (GFPp), a fluorescent oligomerization scaffold, resulting in a set of GFPp_FGF1 oligomers with largely improved receptor binding. To validate the applicability of using GFPp_FGF1 oligomers as cancer probes and drug carriers in targeted therapy of cancers with aberrant FGFR1, we selected a trimeric variant from generated GFPp_FGF1 oligomers and further engineered it by introducing FGF1-stabilizing mutations and by incorporating the cytotoxic drug monomethyl auristatin E (MMAE) in a site-specific manner. The resulting intrinsically fluorescent, trimeric cytotoxic conjugate 3xGFPp_FGF1E_LPET_MMAE exhibits nanomolar affinity for the receptor and very high stability. Notably, the intrinsic fluorescence of 3xGFPp_FGF1E_LPET_MMAE allows for tracking the cellular transport of the conjugate, demonstrating that 3xGFPp_FGF1E_LPET_MMAE is efficiently and selectively internalized into cells expressing FGFR1. Importantly, we show that 3xGFPp_FGF1E_LPET_MMAE displays very high cytotoxicity against a panel of different cancer cells overproducing FGFR1 while remaining neutral toward cells devoid of FGFR1 expression. Our data implicate that the engineered fluorescent conjugates can be used for imaging and targeted therapy of FGFR1-overproducing cancers.

Modular self-assembly system for development of oligomeric, highly internalizing and potent cytotoxic conjugates targeting fibroblast growth factor receptors.

BACKGROUND: Overexpression of FGFR1 is observed in numerous tumors and therefore this receptor constitutes an attractive molecular target for selective cancer treatment with cytotoxic conjugates. The success of cancer therapy with cytotoxic conjugates largely relies on the precise recognition of a cancer-specific marker by a targeting molecule within the conjugate and its subsequent cellular internalization by receptor mediated endocytosis. We have recently demonstrated that efficiency and mechanism of FGFR1 internalization are governed by spatial distribution of the receptor in the plasma membrane, where clustering of FGFR1 into larger oligomers stimulated fast and highly efficient uptake of the receptor by simultaneous engagement of multiple endocytic routes. Based on these findings we aimed to develop a modular, self-assembly system for generation of oligomeric cytotoxic conjugates, capable of FGFR1 clustering, for targeting FGFR1-overproducing cancer cells. METHODS: Engineered FGF1 was used as FGFR1-recognition molecule and tailored for enhanced stability and site-specific attachment of the cytotoxic drug. Modified streptavidin, allowing for controlled oligomerization of FGF1 variant was used for self-assembly of well-defined FGF1 oligomers of different valency and oligomeric cytotoxic conjugate. Protein biochemistry methods were applied to obtain highly pure FGF1 oligomers and the oligomeric cytotoxic conjugate. Diverse biophysical, biochemical and cell biology tests were used to evaluate FGFR1 binding, internalization and the cytotoxicity of obtained oligomers. RESULTS: Developed multivalent FGF1 complexes are characterized by well-defined architecture, enhanced FGFR1 binding and improved cellular uptake. This successful strategy was applied to construct tetrameric cytotoxic conjugate targeting FGFR1-producing cancer cells. We have shown that enhanced affinity for the receptor and improved internalization result in a superior cytotoxicity of the tetrameric conjugate compared to the monomeric one. CONCLUSIONS: Our data implicate that oligomerization of the targeting molecules constitutes an attractive strategy for improvement of the cytotoxicity of conjugates recognizing cancer-specific biomarkers. Importantly, the presented approach can be easily adapted for other tumor markers.

Fibroblast Growth Factor 2 Conjugated with Monomethyl Auristatin E Inhibits Tumor Growth in a Mouse Model.

Worldwide, cancer is the second leading cause of death. Regardless of the continuous progress in medicine, we still do not have a fully effective anti-cancer therapy. Therefore, the search for new targeted anti-cancer drugs is still an unmet need. Here, we present novel protein-drug conjugates that inhibit tumor growth in a mouse model of human breast cancer. We developed conjugates based on fibroblast growth factor (FGF2) with improved biophysical and biological properties for the efficient killing of cancer cells overproducing fibroblast growth factor receptor 1 (FGFR1). We used hydrophilic and biocompatible PEG4 or PEG27 molecules as a spacer between FGF2 and the toxic agent monomethyl auristatin E. All conjugates exhibited a cytotoxic effect on FGFR1-positive cancer cell lines. The conjugate with the highest hydrodynamic size (42 kDa) and cytotoxicity was found to efficiently inhibit tumor growth in a mouse model of human breast cancer.

The cytotoxic conjugate of highly internalizing tetravalent antibody for targeting FGFR1-overproducing cancer cells.

BACKGROUND: Antibody drug conjugates (ADCs) represent one of the most promising approaches in the current immuno-oncology research. The precise delivery of cytotoxic drugs to the cancer cells using ADCs specific for tumor-associated antigens enables sparing the healthy cells and thereby reduces unwanted side effects. Overexpression of fibroblast growth factor receptor 1 (FGFR1) has been demonstrated in numerous tumors and thereby constitutes a convenient molecular target for selective cancer treatment. We have recently engineered tetravalent anti-FGFR1 antibody, T-Fc, and have demonstrated that it displays extremely efficient internalization into FGFR1 producing cells, a feature highly desirable in the ADC approach. We have revealed that T-Fc mediates clustering of FGFR1, largely enhancing the uptake of FGFR1-T-Fc complexes by induction of clathrin-independent endocytic routes. The aim of this study was to obtain highly internalizing cytotoxic conjugate of the T-Fc for specific delivery of drugs into FGFR1-positive cancer cells. METHODS: Conjugation of the T-Fc to a cytotoxic payload, vcMMAE, was carried out via maleimide chemistry, yielding the T-Fc-vcMMAE. The specific binding of the T-Fc-vcMMAE conjugate to FGFR1 was confirmed in vitro with BLI technique. Confocal microscopy and flow cytometry were applied to determine FGFR1-dependence of the T-Fc-vcMMAE internalization. Western blot analyses of FGFR1-dependent signaling were conducted to assess the impact of the T-Fc-vcMMAE on FGFR1 activation and initiation of downstream signaling cascades. Finally, using FGFR1-negative and FGFR1-possitive cell lines, the cytotoxic potential of the T-Fc-vcMMAE was evaluated. RESULTS: We have performed the efficient conjugation of the tetravalent engineered antibody with a cytotoxic drug and generated FGFR1-specific ADC molecule, T-Fc-vcMMAE. We have demonstrated that T-Fc-vcMMAE conjugate exhibits high selectivity and affinity for FGFR1, similarly to T-Fc. Furthermore, we have shown that T-Fc constitutes an effective drug delivery vehicle as T-Fc-vcMMAE was efficiently and selectively internalized by FGFR1-producing cells leading to their death. Interestingly, we show that the efficiency of the uptake of T-Fc-vcMMAE corresponds well with the cytotoxicity of the conjugate, but doesn't correlate with the FGFR1expression level. CONCLUSION: Our results show that T-Fc-vcMMAE fulfills the key criteria for the successful cytotoxic drug carrier in a targeted approach against FGFR1-positive cancer cells. Furthermore, our data implicate that not solely expression level of the receptor, but rather its cellular trafficking should be taken into account for selection of suitable molecular targets and cancer models for successful ADC approach.

Dissecting biological activities of fibroblast growth factor receptors by the coiled-coil-mediated oligomerization of FGF1.

Fibroblast growth factor receptors (FGFRs) are integral membrane proteins involved in various biological processes including proliferation, migration and apoptosis. There are a number of regulatory mechanisms of FGFR signaling, which tightly control the specificity and duration of transmitted signals. The effect of the FGFRs spatial distribution in the plasma membrane on receptor-dependent functions is still largely unknown. We have demonstrated that oligomerization of FGF1 with coiled-coil motifs largely improves FGF1 affinity for FGFRs and heparin. Set of developed FGF1 oligomers evoked prolonged activation of FGFR1 and receptor-downstream signaling pathways, as compared to the wild type FGF1. The majority of obtained oligomeric FGF1 variants showed increased stability, enhanced mitogenic activity and largely improved internalization via FGFR1-dependent endocytosis. Importantly, FGF1 oligomers with the highest oligomeric state exhibited reduced ability to stimulate FGFR-dependent glucose uptake, while at the same time remained hyperactive in the induction of cell proliferation. Our data implicate that oligomerization of FGF1 alters the biological activity of the FGF/GFR1 signaling system. Furthermore, developed FGF1 oligomers, due to improved stability and proliferative potential, can be applied in the regenerative medicine or as drug delivery vehicles in the ADC approach against FGFR1-overproducing cancers.

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 growth factor receptors on their surface. This feature enables development of highly selective therapeutics, the protein bioconjugates, composed of targeting proteins (antibodies or receptor ligands) connected to highly cytotoxic drugs by a specific linker. Due to very high affinity and selectivity of targeting proteins the bioconjugates recognize marker proteins on the cancer cells surface and utilize receptor-mediated endocytosis to reach the cell interior. Intracellular vesicular transport system ultimately delivers the bioconjugates to the lysosomes, where proteolysis separates free cytotoxic drugs from the proteinaceous core of the bioconjugates, triggering drug-dependent cancer cell death. Currently, there are several protein bioconjugates approved for cancer treatment and large number is under development or clinical trials. One of the main challenges in the generation of the bioconjugates is a site-specific attachment of the cytotoxic drug to the targeting protein. Recent years have brought a tremendous progress in the development of chemical and enzymatic strategies for protein modification with cytotoxic drugs. Here we present the detailed protocols for the site-specific incorporation of cytotoxic warheads into targeting proteins using a chemical method employing maleimide-thiol chemistry and an enzymatic approach that relies on sortase A-mediated ligation. We use engineered variant of fibroblast growth factor 2 and fragment crystallizable region of human immunoglobulin G as an exemplary targeting proteins and monomethyl auristatin E and methotrexate as model cytotoxic drugs. All the described strategies allow for highly efficient generation of biologically active cytotoxic conjugates of defined molecular architecture with potential for selective treatment of diverse cancers.

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

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.

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).

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.

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

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.