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.

Intracellular FGF1 protects cells from apoptosis through direct interaction with p53.

Fibroblast growth factor 1 (FGF1) acts by activating specific tyrosine kinase receptors on the cell surface. In addition to this classical mode of action, FGF1 also exhibits intracellular activity. Recently, we found that FGF1 translocated into the cell interior exhibits anti-apoptotic activity independent of receptor activation and downstream signaling. Here, we show that expression of FGF1 increases the survival of cells treated with various apoptosis inducers, but only when wild-type p53 is present. The p53-negative cells were not protected by either ectopically expressed or translocated FGF1. We also confirmed the requirement of p53 for the anti-apoptotic intracellular activity of FGF1 by silencing p53, resulting in loss of the protective effect of FGF1. In contrast, in p53-negative cells, intracellular FGF1 regained its anti-apoptotic properties after transfection with wild-type p53. We also found that FGF1 directly interacts with p53 in cells and that the binding region is located in the DBD domain of p53. We therefore postulate that intracellular FGF1 protects cells from apoptosis by directly interacting with p53.

Multivalent protein-drug conjugates - An emerging strategy for the upgraded precision and efficiency of drug delivery to cancer cells.

With almost 20 million new cases per year, cancer constitutes one of the most important challenges for public health systems. Unlike traditional chemotherapy, targeted anti-cancer strategies employ sophisticated therapeutics to precisely identify and attack cancer cells, limiting the impact of drugs on healthy cells and thereby minimizing the unwanted side effects of therapy. Protein drug conjugates (PDCs) are a rapidly growing group of targeted therapeutics, composed of a cancer-recognition factor covalently coupled to a cytotoxic drug. Several PDCs, mainly in the form of antibody-drug conjugates (ADCs) that employ monoclonal antibodies as cancer-recognition molecules, are used in the clinic and many PDCs are currently in clinical trials. Highly selective, strong and stable interaction of the PDC with the tumor marker, combined with efficient, rapid endocytosis of the receptor/PDC complex and its subsequent effective delivery to lysosomes, is critical for the efficacy of targeted cancer therapy with PDCs. However, the bivalent architecture of contemporary clinical PDCs is not optimal for tumor receptor recognition or PDCs internalization. In this review, we focus on multivalent PDCs, which represent a rapidly evolving and highly promising therapeutics that overcome most of the limitations of current bivalent PDCs, enhancing the precision and efficiency of drug delivery to cancer cells. We present an expanding set of protein scaffolds used to generate multivalent PDCs that, in addition to folding into well-defined multivalent molecular structures, enable site-specific conjugation of the cytotoxic drug to ensure PDC homogeneity. We provide an overview of the architectures of multivalent PDCs developed to date, emphasizing their efficacy in the targeted treatment of various cancers.

FGF1 Protects MCF-7 Cells against Taltobulin through Both the MEKs/ERKs and PI3K/AKT Signaling Pathway.

Breast cancer is a widespread and complex disease characterized by abnormal signaling pathways that promote tumor growth and progression. Despite significant medical advances and the development of increasingly effective therapies for breast cancer, drug resistance and reduced sensitivity to prior therapies remain persistent challenges. Dysregulation of growth factors such as FGFs and EGF and their receptors is a contributing factor to reduced response to treatment, promoting cell survival and proliferation, metastasis, EMT or increased expression of ABC transporters. Our study demonstrates a protective role for FGF1 in MCF-7 breast cancer cells against taltobulin-induced cytotoxicity, mediated by activation of its receptors and compares its activity to EGF, another growth factor involved in breast cancer development and progression. The mechanisms of action of these two proteins are different: FGF1 exerts its effects through the activation of both ERKs and AKT, whereas EGF acts only through ERKs. FGF1 action in the presence of the drug promotes cell viability, reduces apoptosis and increases cell migration. Although EGF and its receptors have received more attention in breast cancer research to date, our findings highlight the key role played by FGFs and their receptors in promoting drug resistance to tubulin polymerization inhibitors in FGFR-positive tumors.

N-glycosylation acts as a switch for FGFR1 trafficking between the plasma membrane and nuclear envelope.

Fibroblast growth factor receptor 1 (FGFR1) is a heavily N-glycosylated cell surface receptor tyrosine kinase that transmits signals across the plasma membrane, in response to fibroblast growth factors (FGFs). Balanced FGF/FGFR1 signaling is crucial for the development and homeostasis of the human body, and aberrant FGFR1 is frequently observed in various cancers. In addition to its predominant localization to the plasma membrane, FGFR1 has also been detected inside cells, mainly in the nuclear lumen, where it modulates gene expression. However, the exact mechanism of FGFR1 nuclear transport is still unknown. In this study, we generated a glycosylation-free mutant of FGFR1, FGFR1.GF, and demonstrated that it is localized primarily to the nuclear envelope. We show that reintroducing N-glycans into the D3 domain cannot redirect FGFR1 to the plasma membrane or exclude the receptor from the nuclear envelope. Reestablishment of D2 domain N-glycans largely inhibits FGFR1 accumulation in the nuclear envelope, but the receptor continues to accumulate inside the cell, mainly in the ER. Only the simultaneous presence of N-glycans of the D2 and D3 domains of FGFR1 promotes efficient transport of FGFR1 to the plasma membrane. We demonstrate that while disturbed FGFR1 folding results in partial FGFR1 accumulation in the ER, impaired FGFR1 secretion drives FGFR1 trafficking to the nuclear envelope. Intracellular FGFR1.GF displays a high level of autoactivation, suggesting the presence of nuclear FGFR1 signaling, which is independent of FGF. Using mass spectrometry and proximity ligation assay, we identified novel binding partners of the nuclear envelope-localized FGFR1, providing insights into its cellular functions. Collectively, our data define N-glycosylation of FGFR1 as an important regulator of FGFR1 kinase activity and, most importantly, as a switchable signal for FGFR1 trafficking between the nuclear envelope and plasma membrane, which, due to spatial restrictions, shapes FGFR1 interactome and cellular function. Video Abstract.

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.

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.

FGF1 protects FGFR1-overexpressing cancer cells against drugs targeting tubulin polymerization by activating AKT via two independent mechanisms.

Cancer drug resistance is a common, unpredictable phenomenon that develops in many types of tumors, resulting in the poor efficacy of current anticancer therapies. One of the most common, and yet the most complex causes of drug resistance is a mechanism related to dysregulation of tumor cell signaling. Abnormal signal transduction in a cancer cell is often stimulated by growth factors and their receptors, including fibroblast growth factors (FGFs) and FGF receptors (FGFRs). Here, we investigated the effect of FGF1 and FGFR1 activity on the action of drugs that disrupt tubulin polymerization (taltobulin, paclitaxel, vincristine) in FGFR1-positive cell lines, U2OS stably transfected with FGFR1 (U2OSR1) and DMS114 cells. We observed that U2OSR1 cells exhibited reduced sensitivity to the tubulin-targeting drugs, compared to U2OS cells expressing a negligible level of FGFRs. This effect was dependent on receptor activation, as inhibition of FGFR1 by a specific small-molecule inhibitor (PD173074) increased the cells' sensitivity to these drugs. Expression of functional FGFR1 in U2OS cells resulted in increased AKT phosphorylation, with no change in total AKT level. U2OSR1 cells also exhibited an elevated MDR1 and blocking MDR1 activity with cyclosporin A increased the toxicity of paclitaxel and vincristine, but not taltobulin. Analysis of tubulin polymerization pattern using fluorescence microscopy revealed that FGF1 in U2OSR1 cells partially reverses the drug-altered phenotype in paclitaxel- and vincristine-treated cells, but not in taltobulin-treated cells. Furthermore, we showed that FGF1, through activation of FGFR1, reduces caspase 3/7 activity and PARP cleavage, preventing apoptosis induced by tubulin-targeting drugs. Next, using specific kinase inhibitors, we investigated which signaling pathways are responsible for the FGF1-mediated reduction of taltobulin cytotoxicity. We found that AKT kinase is a key factor in FGF1-induced cell protection against taltobulin in U2OSR1 and DMS114 cells. Interestingly, only direct inhibition of AKT or dual-inhibition of PI3K and mTOR abolished this effect for cells treated with taltobulin. This suggests that both canonical (PI3K-dependent) and alternative (PI3K-independent) AKT-activating pathways may regulate FGF1/FGFR1-driven cancer cell survival. Our findings may contribute to the development of more effective therapies and may facilitate the prevention of drug resistance in FGFR1-positive cancer cells.

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.

Nuclear Localization Sequence of FGF1 Is Not Required for Its Intracellular Anti-Apoptotic Activity in Differentiated Cells.

Fibroblast growth factor 1 (FGF1) is considered primarily as a ligand for FGF surface receptors (FGFRs) through which it activates a number of cellular responses. In addition to its canonical mode of action, FGF1 can act intracellularly, before secretion or after internalization and translocation from the cell exterior. The role of FGF1 inside the cell is to provide additional protection against apoptosis and promote cell survival. The FGF1 protein contains a specific N-terminal nuclear localization sequence (NLS) that is essential for its efficient transport to the nucleus. Here, we investigated the role of this sequence in the anti-apoptotic response of FGF1. To this end, we produced recombinant FGF1 variants with mutated or deleted NLS and added them to apoptosis-induced cells in which FGFR1 was inactive, either as a result of chemical inhibition or kinase-dead mutation. After internalization, all FGF1 variants were able to protect the differentiated cells from serum starvation-induced apoptosis. To verify the results obtained for NLS mutants, we knocked down LRRC59, a protein that mediates the nuclear transport of FGF1. Upon LRRC59 silencing, we still observed a decrease in caspase 3/7 activity in cells treated exogenously with wild-type FGF1. In the next step, FGF1 variants with mutated or deleted NLS were expressed in U2OS cells, in which apoptosis was then induced by various factors (e.g., starvation, etoposide, staurosporine, anisomycin and actinomycin D). Experiments were performed in the presence of specific FGFR inhibitors to eliminate FGFR-induced signaling, potentially activated by FGF1 proteins released from damaged cells. Again, we found that the presence of NLS in FGF1 is not required for its anti-apoptotic activity. All NLS variants tested were able to act as wild type FGF1, increasing the cell viability and mitochondrial membrane potential and reducing the caspase 3/7 activity and PARP cleavage in cells undergoing apoptosis, both transiently and stably transfected. Our results indicate that the nuclear localization of FGF1 is not required for its intracellular anti-apoptotic activity in differentiated cells and suggest that the mechanism of the stress response differs according to the level of cell differentiation.

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.

FGF/FGFR-Dependent Molecular Mechanisms Underlying Anti-Cancer Drug Resistance.

Increased expression of both FGF proteins and their receptors observed in many cancers is often associated with the development of chemoresistance, limiting the effectiveness of currently used anti-cancer therapies. Malfunctioning of the FGF/FGFR axis in cancer cells generates a number of molecular mechanisms that may affect the sensitivity of tumors to the applied drugs. Of key importance is the deregulation of cell signaling, which can lead to increased cell proliferation, survival, and motility, and ultimately to malignancy. Signaling pathways activated by FGFRs inhibit apoptosis, reducing the cytotoxic effect of some anti-cancer drugs. FGFRs-dependent signaling may also initiate angiogenesis and EMT, which facilitates metastasis and also correlates with drug resistance. Therefore, treatment strategies based on FGF/FGFR inhibition (using receptor inhibitors, ligand traps, monoclonal antibodies, or microRNAs) appear to be extremely promising. However, this approach may lead to further development of resistance through acquisition of specific mutations, metabolism switching, and molecular cross-talks. This review brings together information on the mechanisms underlying the involvement of the FGF/FGFR axis in the generation of drug resistance in cancer and highlights the need for further research to overcome this serious problem with novel therapeutic strategies.

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.

FGF1 Fusions with the Fc Fragment of IgG1 for the Assembly of GFPpolygons-Mediated Multivalent Complexes Recognizing FGFRs.

FGFRs are cell surface receptors that, when activated by specific FGFs ligands, transmit signals through the plasma membrane, regulating key cellular processes such as differentiation, division, motility, metabolism and death. We have recently shown that the modulation of the spatial distribution of FGFR1 at the cell surface constitutes an additional mechanism for fine-tuning cellular signaling. Depending on the multivalent, engineered ligand used, the clustering of FGFR1 into diverse supramolecular complexes enhances the efficiency and modifies the mechanism of receptor endocytosis, alters FGFR1 lifetime and modifies receptor signaling, ultimately determining cell fate. Here, we present a novel approach to generate multivalent FGFR1 ligands. We functionalized FGF1 for controlled oligomerization by developing N- and C-terminal fusions of FGF1 with the Fc fragment of human IgG1 (FGF1-Fc and Fc-FGF1). As oligomerization scaffolds, we employed GFPpolygons, engineered GFP variants capable of well-ordered multivalent display, fused to protein G to ensure binding of Fc fragment. The presented strategy allows efficient assembly of oligomeric FGFR1 ligands with up to twelve receptor binding sites. We show that multivalent FGFR1 ligands are biologically active and trigger receptor clustering on the cell surface. Importantly, the approach described in this study can be easily adapted to oligomerize alternative growth factors to control the activity of other cell surface receptors.

Purification and In Vitro Evaluation of an Anti-HER2 Affibody-Monomethyl Auristatin E Conjugate in HER2-Positive Cancer Cells.

A promising approach for the development of high-affinity tumor targeting ADCs is the use of engineered protein drugs, such as affibody molecules, which represent a valuable alternative to monoclonal antibodies (mAbs) in cancer-targeted therapy. We developed a method for a more efficient purification of the ZHER2:2891DCS affibody conjugated with the cytotoxic antimitotic agent auristatin E (MMAE), and its efficacy was tested in vitro on cell viability, proliferation, migration, and apoptosis. The effects of ZHER2:2891DCS-MMAE were compared with the clinically approved monoclonal antibody trastuzumab (Herceptin®). To demonstrate that ZHER2:2891DCS-MMAE can selectively target HER2 overexpressing tumor cells, we used three different cell lines: the human adenocarcinoma cell lines SK-BR-3 and ZR-75-1, both overexpressing HER2, and the triple-negative breast cancer cell line MDA-MB-231. MTT assay showed that ZHER2:2891DCS-MMAE induces a significant time-dependent toxic effect in SK-BR-3 cells. A 30% reduction of cell viability was already found after 10 min exposure at a concentration of 7 nM (IC50 of 80.2 nM). On the contrary, MDA-MB-231 cells, which express basal levels of HER2, were not affected by the conjugate. The cytotoxic effect of the ZHER2:2891DCS-MMAE was confirmed by measuring apoptosis by flow cytometry. In SK-BR-3 cells, increasing concentrations of conjugated affibody induced cell death starting from 10 min of treatment, with the strongest effect observed after 48 h. Overall, these results demonstrate that the ADC, formed by the anti-HER2 affibody conjugated to monomethyl auristatin E, efficiently interacts with high affinity with HER2 positive cancer cells in vitro, allowing the selective and specific delivery of the cytotoxic payload.

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.