Field of the Invention

The field of the invention is cancer treatment and targeted therapies. More specifically, the present invention is directed to the new generation of therapeutics, being conjugates of a potent cytotoxic drug and a targeting delivery molecule, a human fibroblast growth factor 2 (FGF2), that are effective in killing cancer cells of FGFR-related cancers.

Technical Background

The fibroblast growth factors (FGFs) and their specific cell surface receptors - fibroblast growth factor receptors (FGFRs) are signalling molecules of high importance throughout all stages of development as well as adult life of metazoan organisms (Ornitz, 2001; Itoh 2004; Powers, 2000). FGFs are responsible for a wide spectrum of biological effects induced in numerous different cell types and a wide variety of tissues (Powers, 2000; Eswarakumar, 2005). FGFs through their receptors regulate many physiological processes such as cell proliferation, morphogenesis, differentiation, migration and cell survival (Dailey, 2005). They act in a paracrine or autocrine fashion and are responsible for angiogenesis, osteogenesis, neurogenesis and wound healing (Dailey, 2005). On the other hand, deregulations and abnormalities of FGFs or FGFRs such as mutations, protein overexpression or imbalanced signalling may lead to diverse pathological conditions including skeleton disorders, diabetic retinopathy, atherosclerosis and cancer (Wiedlocha, 2004; Wesche 2011).

All FGFs, except FHFs, signal by activating a family of tyrosine kinase cell surface receptors (FGFRs) encoded in distinct genes (Dailey, 2005; Mohammadi, 2005; Eswarakumar, 2005). Human fibroblast growth factor 2 (FGF2) is one of the best characterized members of the FGF superfamily. FGF2 is a powerful mitogen involved in diverse physiological processes such as cell migration, angiogenesis, osteogenesis and wound healing (Bikfalvi, 1997). Five FGF2 isoforms (18, 22, 22.5, 24 and 34 kDa) in human have been described.

Similar to other FGFs, the structure of FGF2 consists of 12 β-strands, arranged antiparallel in a β-trefoil motif with a pseudo-threefold internal symmetry (Zhu, 1991). Wild-type of FGF2 contains 4 cysteine residues: two of them (at positions 34 and 101) are buried within the protein core and inert, and two are solvent accessible (at position 78 and 96) and are highly reactive.

The biological action of FGF s, excluding FFIFs, is exerted through binding to and activation of four high-affinity cell-surface FGF receptors (FGFRl-4) that have intrinstic tyrosine kinase activity. FGF2 is more selective in binding and activation of FGFRs, compared with FGF1. FGF2 exhibits high affinity and ability to stimulate the following receptors: FGFRlb, FGFRlc, FGFR3c, FGFR2c and FGFR4, but does not activate FGFR2b and FGFR3b (Ornitz, 1996). The specificity of FGF2 to target tumor cells has been shown for several different cancer types, including neuroblastoma (Xu, 2010).

FGFRs overexpression has been found in different types of human cancer, including breast, bladder, prostate, endometrial, lung and hematologic cancers (Haugsten, 2010). This makes them promising candidates as potential targets for anticancer therapy.

The most common alteration of FGFRs in breast cancers involves amplification of FGFR1 (app. 10% of human breast cancers) and is associated with poor prognosis. Cell culture studies confirmed the oncogenic potential of FGFR1 and showed that activation of FGFR1 resulted in increased cell proliferation and invasion. It has been found that amplification of FGFR1 gene plays a role in resistance to endocrine therapy and it is a very promising therapeutic target in breast cancer. In some cases amplification of FGFR2 is also related to breast tumors, especially to triple-negative breast cancers, which are aggressive breast tumors negative for the oestrogen receptor, progesterone receptor and HER2, and not efficiently treated with current therapies (Schneider, 2008). Interestingly, proliferation potential of cell lines derived from these tumors were effectively decreased in the presence of FGFR inhibitor and siRNA (Wesche, 2011). Thus, anticancer therapy targeted to FGFR2 appears to be possible. Additionaly, overexpression of FGFR4 has been observed in human breast cancers and was reported to be associated with resistance to chemotherapy. When breast cancer cell line with upregulated expression of FGFR4 was treated with specific FGFR4 antibody, blocking signaling from the receptor, enhanced chemosensitivity of cancer cells was observed (Roidl, 2009). Therefore FGFR4 is also a very good potential therapeutic target.

FGFR1 is also often overexpressed in prostate cancer and is believed to disrupt the interplay between mesenchymal and epithelial cells of the prostate (Sahadevan, 2007; Giri, 1999). It was clearly showed that inhibition of FGFR1 led to reduced tumor cells proliferation and progression (Wesche, 2011). The same effects were observed in endometrial carcinomas cell lines, in which mutations enhancing receptor activation were identified in FGFR2 (Dutt, 2008). Similarly, the role of FGFRs has been indicated in both types of lung cancer: SCLC (small cell lung carcinoma) and NSCLC (non-small cell lung carcinoma). Recent study showed that amplification of FGFRl is frequent in human squamous cell lung cancer, the most common type of NSCLC (Weiss, 2010). The cell lines derived from this kind of tumor were sensitive to specific receptor kinase inhibitor and exhibited reduced growth upon treatment. Moreover, treatment with the inhibitor resulted in tumor reduction in xenografted mice (Turner, 2010). Overexpression of FGFRl and its impact on tumor progression was also indentified in SCLC (Voortman, 2010). These data suggested that targeting FGFRl can be a potential therapeutic solution in lung cancer. Overexpression of FGF receptors in bladder carcinoma was observed for FGFR3, as well as FGFRl and FGFR2. Results reported by Tomlinson, 2009 suggests that FGFRl displays carcinogenetic properties when overexpressed in urothelial cells and may be considered as a potential therapeutic target.

Description of State of the Art

There are several different approaches in the state of the art known to develop effective targeted anti-cancer therapy of FGFR-related tumors.

Small-molecule tyrosine kinase inhibitors (RTKs) inhibit the receptor activity by targeting the ATP-binding site of the tyrosine kinase domain, such inhibitors are currently in clinical trials and due to the unwanted broad specificity they need to be improved (Knights, 2010; Ahmad, 2012). Tasigna® (AMN107, Nilotinib) is a recently approved BCR-ABL kinase inhibitor for the treatment of drug-resistant chronic myelogenous leukemia (CML). Among others following molecules are under trials: AZD4547, FGFRl and 2 inhibitor against advanced solid malignancies (AstraZeneca), Dovitinib is an inhibitor of both VEGFR and FGFRs against renal cell carcinoma and advanced breast cancer, relapsed MM and bladder cancer, BGJ398 is a selective inhibitor of FGFRs (Novartis). As these inhibitors also inhibit VEGFR, due to the high structural similarity of the kinase domains many of these multiple targeting inhibitors may be relatively less potent as inhibitors against FGFRs. This also increases the side effect profile, limiting the deliverability of the drug at doses necessary for inhibition of FGF signaling (Ahmad, 2012). Off-target toxicities and induction of rapid resistance are major drawbacks that highly compromise successful long term administration.

FGF ligand traps are soluble fusion proteins consisting of the extracellular FGFRl domain fused to the Fc region of IgGl . Such protein fusion prevents FGFs from binding to their receptors and therefore decreases overall FGFR activation (Ellsworth, 2002). Five Prime drug FP-1039 is thought to bind to FGF ligands circulating in the extracellular space, thereby preventing these signaling proteins from reaching FGFR1 on the surface of tumor cells where they would otherwise stimulate cancer cell division and/or angiogenesis. They exhibit antiproliferative and anti-angiogenic effects (Ahmad, 2012).

WO2014179448 discloses a method of treating breast cancer in a subject comprising administering a therapeutically effective amount of a fibroblast growth factor receptor 1 (FGFR1) extracellular domain (ECD) or an FGFR1 ECD fusion molecule to the subject, wherein prior to administration at least a portion of the cells of the breast cancer were determined to have FGFR1 gene amplification, FGFR1 overexpression, FGFR3 overexpression, or FGF2 overexpression; and to be estrogen receptor (ER) positive, progesterone (PR) positive, or ER positive and PR positive.

Strategy using monoclonal antibodies against FGFs and FGFRs has been successfully tested to target different receptor tyrosine kinases and holds promise for the generation of highly specific antibodies against particular FGF or FGFR isoforms, however, up to now only several studies are undergoing and there are few antibodies developed against FGFRs. Moreover, since FGFR blocking antibodies are supposed to be specific to particular FGFRs, they limit pan- FGFR inhibition toxicity (Ahmad, 2012).

From Qing, 2009 a R3Mab antibody is known, which targets FGFR3, demonstrating antiproliferative effects on xenografts of bladder cancer and t(4; 14) myeloma cells (Qing, 2009). WO 2011143318 provides monoclonal antibodies that bind and inhibit biological activities of human FGFR2. The antibodies can be used to treat cell proliferative diseases and disorders, including certain forms of cancer, associated with activation or overexpression of FGFR2 in particular breast cancer, ovarian cancer, prostate cancer, cervical cancer, lung cancer, some forms of brain cancer, melanomas, and gastrointestinal cancers (e.g., colorectal, pancreatic, gastric, head and neck).

GP369, an FGFR2-IIIb-specific antibody has been used in vitro and in vivo to suppress the Illb isoform in FGFR2 amplified human breast and gastric cancer cell lines (Nord, 2009).

From WO2009148928 are known genetically engineered monoclonal antibodies (mAb) that bind and neutralize FGF2. The anti-FGF2 mAb completely inhibit binding of FGF2 to each of the FGF receptors FGFR1, FGFR2, FGFR3 and FGFR4. Exemplary antibodies are GAL-F2 and its chimeric or humanized forms, and those having the same epitope as GAL-F2 or competitive in binding with GAL-F2. The FGFly-coated Au Ps conjugates were used for photothermal therapy in cancer treatment (Szlachcic, 2012). FGFR-targeted gold nanoconjugates were designed for infrared-induced thermal ablation (localized heating lead to cancer cell death) based on gold nanoparticles (AuNPs). It was shown that a recombinant ligand of all FGFRs, human fibroblast growth factor 1 (FGF1), can be used as an agent targeting covalently bound AuNPs to cancer cells overexpressing FGFRs.

Recent studies and clinical trials have demonstrated that the most promising approach in cancer treatment is targeted therapy based on Antibody-Drug Conjugates (ADCs), consisting of monoclonal antibody as a targeting molecule and a highly cytotoxic agent.

Both, the site of conjugation and the amount of cytotoxic compound molecules per one antibody molecule (DAR parameter, drug to antibody ratio), are important for the properties of ADC. They affect, among others, the affinity of ADC to its target; ADC's level of aggregation and pharmacokinetic as well as pharmacodynamic parameters (Zaro, 2015). In the majority of ADCs that are currently in clinical trials, the reactive amino acids, naturally occurring in antibodies, are used for conjugation. These include amino groups of lysines and reduced thiol groups of cysteines.

Conjugation of cytotoxic compounds to lysine residues in antibody leads to formation of mixture, in which the cytotoxic molecules are randomly distributed within the antibody resulting in different DAR values (Kaur, 2013). It was calculated that conjugation of cytotoxic compounds via lysine residues occurring in forty different locations, may give 106 different variants of ADC (Wang, 2005). Despite the large number of lysines available for conjugation within single IgGl, the average DAR value obtained for Kadcyla, one of the two ADC drugs on the market, is about 3.5 of cytotoxic compound molecules per antibody and the actual number of substitutions ranges from 1 to 7 (Wakankar, 2010; Junutula, 2010).

Modification of cysteine residues requires the reduction of disulfide bonds within the hinge region of the antibody and leads to a conjugate with maximal 8 molecules of a cytotoxic compound per one antibody (for IgGl and IgG2). Conjugation via cysteine residues provides more homogeneous product, but still may generate over 100 different variants of the ADC (Hamblett, 2004). The internal disulfide bridges located within immunoglobulin domains are not exposed to solvent and exhibit high stability, thus they are not affected during conjugation reaction (Liu et al. 2010; Liu & May, 2012).

In Adcetris®, prepared using cysteines conjugation technology, there is about four molecules of the cytotoxic compound per single antibody (Sun, 2005; van de Donk & Dhimolea, 2012). Substitution of all eight cysteines is not desirable, because it leads to higher toxicity and increase removal of ADC from the body, without a corresponding improvement in the therapeutic efficiency. In addition, it was observed that a high number of cytotoxic drug has a negative impact on the structure and properties of antibodies, leading to the loss of their functionality (Singh, 2015). High DAR value increases the level of aggregation of the final product, which has a negative impact on overall pharmacokinetic properties and affinity for antigen. Conjugation process also alters the biological half-life of the antibody, usually reducing it from several weeks to several days (Chan, 2007; Sun, 2005; Hamblett, 2004).

A disadvantage of the strategies used for conjugation of cytotoxic compounds to antibodies is the heterogeneity of the final formulation of ADC. Currently several different companies make attempts to develop methods of specific conjugation that allow the ADSs' preparation of high homogeneity with defined DAR parameter. The main technology of site-specific conjugation includes the use of a modified cysteine residue (technology Thiomab, Genentech), the use of unnatural amino acids (Sutro Biopharma and Ambryx) and the application of the enzyme transglutaminase (Innate Pharma) (Junutula, 2008; Panowski, 2014; Zaro, 2015).

It was shown that only 1-2% of the dose of ADC accumulates in the tumor area (Teicher & Chan, 2011), therefore one of the main objectives of the pharmaceutical companies is to search for molecules with yet higher cytotoxicity. However, the development of new compounds is very difficult. The cytostatic suitable for use in the ADC should meet several conditions, including high cytotoxicity and stability in the bloodstream. Furthermore, it should be small, to reduce the risk of immunogenicity and provide good solubility in aqueous solutions, and also have a specific group for conjugation to the antibody.

Despite numerous approaches in targeted anticancer therapy there is still a critical need in prior art for cancer treatments that effectively target a specific cell population and deliver a powerful cytotoxin, while mitigating damage to healthy cells and tissue.

The aim of the invention is a provision of a new anticancer therapeutic suitable for targeted therapy of FGFR-related cancers that would be devoid of reported drawbacks.

Description of the Invention

To overcome above mentioned problems the subject invention provides strategy for destroying cancer cells using targeting molecules fused with highly cytotoxic agents. A natural ligand of FGF receptor, the fibroblast growth factor 2 (FGF2) or engineered variants thereof are used for bioconjugation with a potent cytotoxic compound such as monomethyl auristatin E (MMAE) or auristatin Y to establish a delivery technique for effective killing of cancer cells overexpressing FGFR. The FGF2 molecule and its engineered variants act as a delivery molecules specifically directing cytotoxic drug to cancer cell by binding to FGF receptors. Obtained conjugates showed a prominent cytotoxic effect toward FGFR - related cancers. The therapeutics of the invention prevent or inhibit the activation of (i.e. neutralize) human FGFRs. The conjugates of the invention can be used to inhibit the proliferation of tumor cells in vitro or in vivo. When administered to a human cancer patient (or an animal model), the conjugates have a potential to inhibit or reduce tumor growth in the human patient (or animal model).

The subject of the invention is a conjugate for targeted therapy of FGFR-related cancers comprising at least one molecule of a cytotoxic drug and a delivery molecule being a recombinant human fibroblast growth factor 2 (FGF2) protein or its variant, wherein the cytotoxic drug molecule binds at least one cysteine residue of the FGF2 or its variant.

Preferably, FGF2 protein variant is a polypeptide comprising amino acid sequence having at least 84% of homology or higher with the amino acid sequence of SEQ ID NO: 1.

Preferably, FGF2 protein variant comprises amino acid sequence wherein one or multiple amino acids are deleted, substituted, inserted, and/or added in the amino acid sequence of SEQ ID NO: 1.

Preferably, FGF2 variants are peptides selected from a group consisting of :

(i) FGF2 WT;

(ii) KCK-FGF2 protein variant having KCKSGG sequence on the N-terminus;

(iii) FGF2-KCK protein variant having GGSKCK sequence on the C-terminus;

(iv) KCK-FGF2[C78S/C96S] protein variant having KCKSGG sequence on the N- terminus, and substitutions of two surface cysteine residues to serine at positions 78 and 96;

(v) FGF2[C78S/C96S]-KCK protein variant having GGSKCK sequence on the C- terminus, and substitutions of two surface cysteine residues to serine at positions 78 and 96;

(vi) sFGF2 WT protein variant identified by Met-Ala-25-155 of FGF2 amino acid sequence;

(vii) sFGF2[C78S] protein variant identified by Met-Ala-25-155 of FGF2 amino acid sequence having a substitution of cysteine residues to serine at position 78; (viii) sFGF2[C96S] protein variant identified by Met-Ala-25-155 of FGF2 amino acid sequence having a substitution of cysteine residues to serine at position 96. "The human fibroblast growth factor 2 (FGF2)" term relates to all isoforms of FGF2 protein. In one embodiment the human fibroblast growth factor 2 (FGF2) protein is isoform 3 of FGF2. FGF2 is 155 aa lenght protein. FGF2 (FGF2 WT) is preferably identified by a SEQ ID: NO 1. During protein synthesis methionine in position 1 is cut off, which results in 2-155 aa polypeptide (Fig. la). KCK-FGF2 variant is identified by a SEQ ID NO: 2, FGF2-KCK variant is identified by a SEQ ID NO:3, KCK-FGF2[C78S/C96S] variant is identified by a SEQ ID NO:4, FGF2[C78S/C96S]-KCK variant is identified by a SEQ ID NO:5, sFGF2 WT variant is identified by a SEQ ID NO:6, sFGF2[C78S] variant is identified by a SEQ ID NO:7, sFGF2[C96S] is variant is identified by a SEQ ID NO:8.

In one embodiment, FGF2 proteins are identified by any of the amino acid sequences selected from SEQ ID NO: 1-8.

In one embodiment, the cytotoxic drugs are derivatives of dolastatin, A-amanitin, PBD dimers. Preferably, the drug is monom ethyl auri statin E or auri statin Y.

Preferably, the conjugate comprises PEG molecule.

The strongest drugs, currently used in ADC, are highly hydrophobic, thus lowering the solubility of ADC and leading to aggregation. One of the methods to overcome this problem is pegylation (addition of PEG molecules) of a conjugate. Polyethylene glycol (PEG), added to any molecule, greatly increases it water solubility. Moreover, pegylation is a well-known method for improving an action of therapeutic proteins. PEG molecules effectively protect protein from proteolysis, improve its pharmacokinetic parameters and increase its hydrodynamic radius, thus prolonging it circulation time in the blood (Veronese, 2008).

Another object of the invention is a recombinant human fibroblast growth factor 2 (FGF2) protein variant suitable for targeted anticancer therapy of FGFR- related cancers selected from:

(i) KCK-FGF2 protein variant having inserted KCKSGG sequence on the N- terminus;

(ii) FGF2-KCK protein variant having inserted GGSKCK sequence on the C- terminus;

(iii) KCK-FGF2[C78S/C96S] protein variant having inserted KCKSGG sequence on the N-terminus, and a substitution of two surface cysteine residues to serine at positions 78 and 96; (iv) FGF2[C78S/C96S]-KCK protein variant having inserted GGSKCK sequence on the C-terminus, and a substitution of two surface cysteine residues to serine at positions 78 and 96;

(v) sFGF2 WT protein variant identified by Met-Ala-25-155 of FGF2 amino acid sequence;

(vi) sFGF2[C78S] protein variant identified by Met-Ala-25-155 of FGF2 amino acid sequence having a substitution of cysteine residues to serine at position 78;

(vii) sFGF2[C96S] protein variant identified by Met-Ala-25-155 of FGF2 amino acid sequence having a substitution of cysteine residues to serine at position 96.

Preferably, KCK-FGF2 variant is identified by a SEQ ID NO: 2, FGF2-KCK variant is identified by a SEQ ID NO:3, KCK-FGF2[C78S/C96S] variant is identified by a SEQ ID NO:4, FGF2[C78S/C96S]-KCK variant is identified by a SEQ ID NO:5, sFGF2 WT variant is identified by a SEQ ID NO:6, sFGF2[C78S] variant is identified by a SEQ ID NO:7, sFGF2 [C96S] is variant is identified by a SEQ ID NO:8.

A variant sFGF2[C78S/C96S] is identified by Met-Ala-25-155 of FGF2 amino acid sequence having a substitution of cysteine residues to serine at position 78 and 96 (SEQ ID NO:9).

Within the scope of the invention there are proteins having identity to FGF2 or relevant FGF2 mutant of at least 84%, 85% 90%, 95%, 98%, 99% and that remain capable of binding to FGFRs. The % identity of two polypeptides can be measured by a similarity score determined by comparing the amino acid sequences of the two polypeptides using routine programs with the default settings for determining similarity (i.e. local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981)) to find the best segment of similarity between two sequences.

Preferably, the FGF2 protein variant is a peptide comprising amino acids having at least 84% of homology or higher with the amino acid sequence of SEQ ID NO: 1.

Preferably, the FGF2 protein variant comprises amino acids wherein one or multiple amino acids are deleted, substituted, inserted, and/or added in the amino acid sequence of SEQ ID NO: 1.

Another object of the invention is a method of obtaining the defined above conjugate of a cytotoxic drug and a human fibroblast growth factor 2 (FGF2) protein or its variant comprising performing conjugation reaction at the temperature range 4 - 40°C and pH 6.0 - 8.0. Another object of the invention is the conjugate as defined above for use in targeted treatment of cancers.

Preferably, the cancers are selected from cancers overexpressing FGFRs, particularly FGFR3 or FGFR1, more particularly III isoform of FGFR1.

Preferably, the cancer is selected from breast cancer, ovarian cancer, prostate cancer, cervical cancer, lung cancer, brain cancer, melanomas, colorectal, pancreatic, gastric, bladder, endometrial, hematologic cancer, head and neck cancer.

It has been demonstrated that FGF2 and FGF2 variants conjugates show high specificity and potency in killing FGFR1 expressed tumor cells.

The conjugates according to the invention show also many advantages over known therapies. For instance, the selectivity of FGF2 may cause less severe side-effects and lower the overall toxicity of cytotoxic FGF2-conjugates in comparison to FGF1 -conjugates. Moreover, FGF1 is moderately stable protein with denaturation temperature close to physiological, and contains three reactive cystienes localized within protein interior (Copeland, 1991; Blaber, 1999; Lee, 2009), so combination of low protein stability with reactivity of cysteine thiol group leads to irreversible denaturation, protein degradation or aggregation (Alsenaidy, 2011).

In contrary to FGF1, FGF2 shows higher denaturation temperature (Vemuri, 1994) and therefore is less prone to degradation (Chen, 2012). Increased resistance to degradation or to thermally induced unfolding leads to longer biological activity of FGF2 as compared to FGF1. FGF2 has a high specificity for selected isoforms of FGFRs. It exhibits high affinity to IIIc isoform of FGFR1 or FGFR3 and lower to FGFR2-IIIc, FGFRl-IIIb and FGFR4 (Zhang, 2006). Moreover, FGF2 is effectively internalized into a cell via receptor-depended pathway (Wiedlocha & Sorensen, 2004). In case of monoclonal antibodies as targeting molecules, there is no guarantee of their effective internalization. The selection of antibodies is based only on their affinity for the molecular target. Additional selection for their ability to be internalized is extremely complex and not always efficient (Rudnick, 2011). In addition, human FGF2 protein does not provoke an immune response, which may be the case with monoclonal antibodies (Harding, 2010).

FGF2 recombinant proteins for a conjugation with a cytotoxic drug

The following variants of FGF2 showed in Figure 1 a and b have been constructed:

1) FGF2 WT 2) KCK-FGF2 variant having KCKSGG sequence (abbreviated as KCK) on the N- terminus of FGF2;

3) FGF2-KCK variant having GGSKCK sequence (abbreviated as KCK) on the C- terminus of FGF2;

4) KCK-FGF2[C78S/C96S] variant having KCKSGG sequence (abbreviated as KCK) on the N-terminus of FGF2 and a substitution of two surface cysteine residues to serine at positions 78 and 96 in FGF2 sequence;

5) FGF2[C78S/C96S]-KCK variant having GGSKCK sequence (abbreviated as KCK) on the C-terminus a substitution of two surface cysteine residues to serine at positions 78 and 96 in FGF2 sequence;

6) sFGF2 WT identified by Met-Ala-25-155 of FGF2 amino acid sequence;

7) sFGF2[C78S] identified by Met-Ala-25-155 of FGF2 amino acid sequence having a substitution of cysteine residues to serine at position 78 in FGF2 sequence;

8) sFGF2[C96S] identified by Met-Ala-25-155 of FGF2 amino acid sequence having a substitution of cysteine residues to serine at position 96 in FGF2 sequence.

A "cell with FGFR overexpression" or a "cell that overexpresses FGFR" refers to a cell that has at least a 2-fold greater level of FGFR mRNA or protein than a reference cell. A "cancer with FGFR overexpression" or a "cancer that overexpresses FGFR" refers to a cancer in which at least a portion of the cells have at least a 2-fold greater level of FGFR mRNA or protein than a reference cell.

Any embodiment described herein or any combination thereof applies to any and all methods of the invention described herein.

Brief description of the Figures

Fig. la and lb FGF2 constructs with marked conjugation sites.

Fig. 2 Conjugation reaction of FGF2 variants with vcMMAE. (A) Electrophoretic separation and (B) mass spectra of FGF2 variants before and after the reaction performed for 1 h at 25°C in 50 mM monosodium phosphate buffer, pH 7.0, in the presence of 10 mM Na ₂ S0 ₄ , 10 mM methionine, 1 mM EDTA.

Fig. 3 Conjugation reaction of sFGF2 (truncated form of FGF2) variants with vcMMAE. Electrophoretic separation and mass spectra of sFGF2 variants and their conjugates. Fig. 4 Functional competence of FGF2 conjugates. (A) Fluorescence emission spectra of FGF2 variants and their conjugates. Measurements were performed at a protein concentration 4xl0 ^"6 M upon excitation at 280 nm. Curves were normalized to tyrosine emission at 303 nm. (B) Activation of ERK 1/2 (phosho-ERK 1/2) in NIH 3T3 cells after stimulation with 20 ng/ml of FGF2 or their conjugates in the presence of 10 U/ml heparin detected with Western blot analysis. Total amount of ERK 1/2 and γ-tubulin served as a loading control.

Fig. 5 Specific internalization of FGF2 WT, KCK-FGF2[C78S/C96S]-(vcMMAE)i and KCK- FGF2-(vcMMAE) ₃ conjugates into cells expressing FGFR1. Representative images of specific internalization of FGF2 WT, KCK-FGF2[C78S/C96S]-(vcMMAE)i or KCK-FGF2- (vcMMAE) ₃ into U20S-R1 cells versus U20S cells. Equal number of U20S stably stained with CellTrace™Violet (blue) and U20S-R1 (non-stained) were grown together and then incubated with 50 μg of FGF2 WT, KCK-FGF2[C78S/C96S]-(vcMMAE)i or KCK-FGF2- (vcMMAE) ₃ labeled with DyLight550 (red) at 37 °C for 15 min. The cells were fixed, stained with anti-EEAl antibody (green) and examined by confocal microscopy. U20S-R1 cells are marked with dashed line. Bar corresponds to 10 μπι.

Fig. 6 Viability of BJ, U20S and U20S-R1 cells treated with FGF2 WT and FGF2-vcMMAE conjugates for 96 h, assessed with AlamarBlue assay. Results shown are mean values from three experiments ± SD.

Fig. 7 Conjugation reaction of FGF2 variants with vcMMAE and YCP. Electrophoretic separation of FGF2 variants before and after the reaction performed for 1 h at 25°C in 50 mM monosodium phosphate buffer, pH 7.0, in the presence of 10 mM Na ₂ S0 ₄ , 10 mM methionine, 1 mM EDTA.

Fig. 8 Dependence of number of cytotoxic molecules per FGF2 molecule on the cytotoxicity of conjugate towards cells overexpressing FGFR1.

Fig. 9 Dependence of number of cytotoxic molecules per FGF2 molecule on the cytotoxicity of conjugate towards cells non-producing the FGFRl .Fig. 10 Fluorescence emission spectra of FGF2 variants and their conjugates. Measurements were performed at a protein concentration 4xl0 ^"6 M upon excitation at 280 nm. Curves were normalized to tyrosine emission at 303 nm. Fig. 11 Viability of U20S and U20S-R1 cells treated with KCK-FGF2-YCP ₃ and FGF2- vcMMAE ₃ for 96 h, assessed with AlamarBlue assay. Results shown are mean values from three experiments ± SD

Example 1 Cell lines and bacterial strains

BJ cells (CRL-2522) were grown in Eagle's Minimum Essential Medium. U20S (HTB-96™) and U20S stably transfected with FGFR1 (U20S-R1) were grown in McCoy's 5A Modified Medium. All media were supplemented with 10% fetal bovine and 1% penicillin/streptomycin mix. Additionally, U20S-R1 cell culture contained 50 μg/mL gentamicin sulfate. All cell lines were cultured in a humidified incubator at 37 °C in 5% C02 atmosphere. BJ and U20S cell lines were obtained from American Type Culture Collection ATCC (Manassas, VA, USA). The U20S cells stably expressing FGFR1 (U20S-R1) was a kind gift from Dr. Ellen M. Haugsten from The Norwegian Radium Hospital (Haugsten, 2008).

E. coli strains Rosetta 2(DE3)pLysS was from Novagen-EMD Biosciences (Madison, WI, USA).

Plasmids

The sequence encoding human fibroblast growth factor 2 (FGF2) (residues 1-155) or its truncated form (Met-Ala-residues 25-155) was cloned into the pET-3c expression vector from Stratagene (La Jolla, CA, USA). Insertion of KCKSGG N- and GGSKCK C-terminal linker and the point mutations C78S and C96S were introduced using QuikChange Site-Directed Mutagenesis Kit from Agilent Technologies (Santa Clara, CA, USA) according to manufacturer's protocol.

Protein expression and purification

The proteins FGF2 WT, KCK-FGF2, FGF2-KCK, KCK-FGF2[C78S/C96S], FGF2[C78S/C96S]-KCK, sFGF2 WT, sFGF2 FGF2[C78S], sFGF2 FGF2[C96S], sFGF2[C78S/C96S] were expressed in E. coli Rosetta™ 2(DE3)pLysS expression strain. Bacterial cells were grown in TB medium in the presence of 100 μg/mL ampicillin in 37 °C until OD ₆ oo=0.6 was reached. Then protein expression was induced by the addition of IPTG to the final concentrations of 0.3 mM and the culture was incubated at 25 °C for 12 h. Next, bacterial cells were harvested by centrifugation at 8000xg, resuspended in the lysis buffer (50 mM monosodium phosphate, 0.15 M NaCl, 1 mM DTT, 1 mM EDTA, 0.1% Triton X-100, 1 mM PMSF, pH=7.2), and homogenized using the French's press. Total cell extract was centrifuged at 50 000xg at 4 °C for 1 h. The supernatant was diluted in the binding buffer (monosodium phosphate 50 mM, 1 mM DTT, 1 mM EDTA, 0.7 M NaCl, pH=7.2) and loaded on the HiTrap™ Heparin HP Column. The column was washed with washing buffer (monosodium phosphate 50 mM, 1 mM DTT, 1 mM EDTA, 1.0 M NaCl, pH=7.2) and then the proteins were eluted with a linear gradient of NaCl (monosodium phosphate 50 mM, 1 mM DTT, 1 mM EDTA, 1.0 - 2.0 M NaCl, pH=7.2).

Conjugation of FGF2 variants with a cytotoxic drug

Maleimidocaproyl-Val-Cit-PABC-monomethylauristatin E (abbreviated vcMMAE) was used as a cytotoxic compound delivered by FGF2. MMAE is a highly cytotoxic derivative of dolastatin containing a maleimide moiety suitable for conjugation to cysteines residue and a protease-sensitive valine-citrulline dipeptide sequence designed for optimal stability in human plasma and effective cleavage by human cathepsin B.

The conjugation reaction was optimized to provide high yield and optimal conditions, avoiding protein unfolding and a loss of receptor binding activity. For optimal reaction conditions few temperature points between 4 and 40°C were tested in several time points. The conjugation reaction occurs in every temperature in this range with different speeds and efficiencies, but the reaction conducted by 1 h at 20 °C leads to the best results. Moreover, wide range of buffers pH can be used in reaction (from 6 to 8). Purified proteins were desalted using Zeba Spin Desalting column into reaction buffer (50 mM monosodium phosphate, 10 mM Na ₂ S0 ₄ , 10 mM methionine, 1 mM EDTA, pH 7.0). Maleimide derivative of vcMMAE dissolved in DMAc (Ν,Ν-Dimethylacetamide) at concentration of 50 mg/mL was added to 1.5 mg/mL protein solutions. There was a two-fold molar excess of the drug over protein SH-groups. The conjugation reaction mixture was incubated for 1 h at 20 °C.

Reaction progress was monitored by SDS-PAGE and MALDI-TOF MS. Molecular masses of proteins and their conjugates were verified by MALDI-TOF MS (Applied Biosystems AB 4800+) using a-cyano-4-hydroxycinnamic acid (a-CHCA) as a matrix.

As a result of conjugation reaction highly homogenous preparations were obtained that contained negligible amount of unconjugated species, as confirmed by SDS PAGE (Figure 2a, 3). The identities of conjugates were verified by MS (Figure 2b, 3).

Different variants of FGF2 allowed controlling the number of drug molecules attached to FGF2. When vcMMAE was added to FGF2 wild type (FGF2 WT) or truncated form of FGF2 (sFGF2 WT), two exposed cysteines of growth factor were substituted and two other remained intact (FGF2 WT-(vcMMAE) _2, sFGF2 WT-(vcMMAE) ₂ ) (Figure 2, 3). Also, in the case of all other FGF2 constructs, two buried cysteines were not reactive and expected drug molecule loading was achieved (drug to protein ratio (DPR) value from 1 to 3) (Figure 2, 3).

Substitution of two surface cysteine residues to serines (Cys78Ser and Cys96Ser) together with the introduction of KCKSGG sequence on the N-terminus or GGSKCK on the C-terminus (in both cases abbreviated as KCK) allowed obtaining mono-substituted FGF2 (KCK- FGF2[C78S/C96S]-(vcMMAE)i and FGF2[C78S/C96S]-KCK-(vcMMAE)i). Similarly, for sFGF2 variants (sFGF2[C78S] and sFGF2[C96S]), two monosubstituted conjugates were obtained, sFGF2[C78S]-(vcMMAE)i and sFGF2[C96S]-(vcMMAE)i (Figure 3). In addition, to obtain triply substituted conjugates, the WT sequences of FGF2 extended with KCK sequence at each terminus were used (KCK-FGF2-(vcMMAE) ₃ ). The cysteine residue flanked with lysines is highly reactive and ensures excellent yield of conjugation reaction.

The yield of protein expression was between 8 and 40 mg per liter of culture. The variants exhibited a very similar elution profile during purification on a heparin-Sepharose column. Finally, the excess of unconjugated vcMMAE was removed from reaction mixture by buffer exchange to Dulbecco's PBS using Zeba Spin Desalting column.

Spectrofluorimetry

To assess the native conformation of all FGF2 variants before and after conjugation fluorescence analysis was performed that is a useful indicator of a proper folding of FGF2 (Figure 4a). The folded state of proteins and their conjugates were verified by spectrofluorimetry. The fluorescence spectra were acquired using an FP-8500 spectrofluorimeter (Jasco, Japan) with excitation at 280 nm and emission in the 300-450 nm range, at a protein concentration ~4 ^χ 10 ^"6 M in Dulbecco's PBS.

The fluorescence spectrum of properly folded wild-type FGF2 showed very low emission at around at 353 nm, since the signal from the single tryptophan residue is completely quenched and the spectrum is dominated by emission of tyrosine residues (maximum at around 303 nm). Upon unfolding, the quenching effect is abolished, resulting in a significant increase in the fluorescence at 353 nm. All fluorescence emission spectra were similar to the spectrum of native FGF2 WT, showing no changes in the tertiary structure of proteins conjugated to vcMMAE.

Biological competence of FGF2 variants and their conjugates In order to verify if introduced mutations (Cys to Ser substitution(s), N- or C-terminus extension) or the drug fusion (introduction of vcMMAE molecule(s)) did not affect the binding of FGF2 proteins to FGF receptors, activation of signaling pathways in cells was analyzed upon treatment with modified growth factors. Serum-starved NIH 3T3 cells were stimulated for 15 min with 100 ng/ml FGF2 proteins or their conjugates in the presence of heparin (10 U/ml). The cells were then washed with PBS, lysed by Laemmli Sample Buffer and sonicated. Total cell lysates was separated by SDS-PAGE (12%) and analyzed by Western blotting using following antibodies: anti-phospho-Erkl/2 , anti-Erkl/2 and anti-y-tubulin. Specific protein bands were visualized using HRP-conjugated secondary antibodies and an enhanced chemoluminescence substrate in a ChemiDoc station (BioRad, Hercules, CA, USA ).

All conjugates stimulated the downstream signaling at the same level as did FGF2 WT, as detected by Western blot analysis using anti-phospho-ERK 1/2 antibodies (Figure 4b). Those results indicated that all applied modifications of FGF2 molecule not only did not affect its conformation but also did not impair its binding to FGF receptor. To assure equal loading of the gel, the membranes were reprobed with antibodies recognizing the total amount of ERK 1/2 and γ-tubulin.

Endocytosis of FGF2 conjugate

Since the main aim of the study was the specific delivery of cytotoxic cargo into FGFR- positive cells, confocal microscopy study was performed to check whether FGF2 conjugates are entering the cell. Wild-type of FGF2 and two FGF2 conjugates, KCK-FGF2[C78S/C96S]- (vcMMAE)i (DPR=1) and KCK-FGF2-(vcMMAE) ₃ (DPR=3) were labeled with fluorescent dye (DyLight550). Endocytic uptake was analyzed in U20S cells stably transfected with FGF receptor 1 (U20S-R1) versus untransfected cells (U20S).

U20S cells were pre-stained with CellTrace™Violet fluorescent dye and co-cultured with equal number of non-stained U20S-R1 cells that allowed us to discriminate between two cell lines on the same coverslip.

In more detail, U20S cells stained with CellTrace™ Violet according the manufacturer's protocol were seeded on coverslips with equal number of non-stained U20S-R1 cells and grown together up to 70% of confluence. The cells were incubated with 100 ng/mL of labeled samples in the presence of 10 U/ml heparin at 37 °C for 15 min. Then, the cells were washed with PBS, fixed in 4% formaldehyde for 15 min at RT, permeabilized in 0.5% Triton X-100 for 10 min at 4 °C and blocked with blocking buffer (1% BSA, 10% Normal Goat Serum, 0.2% Tween-20 and 0.3 M glycine in PBS) for 1 h at RT. Next, the cells were incubated with primary rabbit anti-EEAl antibody overnight at 4°C, and then incubated with an AlexaFluor488 goat anti-rabbit secondary antibody at RT for 1 h. Nuclei were stained with DAPI and cover slips were mounted with ProLongGold Antifade Mountant. The cell staining was analyzed using Cell Observer SD confocal system (Zeiss, Germany) equipped with EMCCD Qlmaging Rolera EM-C2 camera with a 63 x oil immersion objective. All images data were processed in Fiji software (Schindelin et al. 2012).

Using this approach, it was demonstrated that in case of all constructs applied fluorescence of DyLight550 was observed only in U20S-R1 cells thus indicating that internalization of FGF2 and FGFvariants conjugates occurs effectively in FGF receptor-specific manner, similarly to FGF2 WT.

Cytotoxic effect of FGF2 conjugates

To assess the toxicity of FGF2 conjugates, three cell lines that differ in FGFRl level: BJ (non- malignant cells naturally expressing relatively high level of FGFRl), U20S (cells that show hardly detectable level of FGFRl and serve as a negative control) and U20S-R1 (U20S cells stably expressing very high level of FGFRl) were used. Cells viability was assessed with the Alamar Blue assay.

Cells cultured on the 96-well plates (5000 cells/well in the required media supplemented with 10 U/mL heparin sulphate were treated with FGF2 variants and their cytotoxic conjugates. Cells were treated with FGF2 WT and FGF2 conjugates KCK-FGF2-(vcMMAE) _3, KCK- FGF2[C78S/C96S]-(vcMMAE)i, FGF2[C78S/C96S]-KCK-(vcMMAE)i, FGF2 -(vcMMAE) ₂ in 0.04 to 4000 nM concentration range for 96 h. After 96 hours of continuous exposure to the drug, the medium was removed and replaced with a fresh medium containing 10% of Alamar Blue. Fluorescence emission at 590 nm (excitation at 560 nm), reflecting the viability of the cells, was measured 4 h later using En Vision Multilabel Reader fluorescence plate reader (PerkinElmer, Waltham MA, USA).

The sensitivity of the cells towards FGF2 conjugates differed considerably between the cell lines and correlated with the level of FGFR on cellular surface (Figure 4). The strongest cytotoxic effect of FGF2-vcMMAE conjugates in the case of U20S-R1 cells was found, whereas in U20S cells the toxicity was more than two orders of magnitude lower (Table 1). In U20S-R1 cells EC50 value was equal to 2.2 nM and 4.1 nM for KCK-FGF2-(vcMMAE) ₃ and FGF2-(vcMMAE) ₂ , respectively (Table 1). As a positive control we used free MMAE at concentration of 11 μΜ that exhibited very similar toxicity in all cell lines tested (Figure 6). Moreover, a strong correlation between the drug to protein ratio and toxic effect of the conjugates was observed. Among all tested conjugates KCK-FGF2-(vcMMAE) ₃ construct revealed the highest cytotoxity, whereas singly substituted conjugates (KCK- FGF2[C78S/C96S]-(vcMMAE)i and FGF2[C78S/C96S]-KCK-(vcMMAE)i) the lowest. Table 1 shows the half maximal effective concentration (EC ₅₀ ) of FGF2-vcMMAE conjugates in BJ, U20S and U20S-R1 cells.

Table 1

In summary, our results demonstrate high specificity and potency of FGF2 and FGF2 variants conjugates in killing FGFRl expressed tumor cells.

Example 2

Conjugation FGF2 with PEGylated auristatin Y. Cell lines and bacterial strains

U20S (HTB-96™) and U20S stably transfected with FGFRl (U20S-R1) were grown in McCoy's 5A Modified Medium. All media were supplemented with 10% fetal bovine and 1% penicillin/streptomycin mix. Additionally, U20S-R1 cell culture contained 50 μg/mL gentamicin sulfate. All cell lines were cultured in a humidified incubator at 37 °C in 5% C02 atmosphere. U20S cell lines was obtained from American Type Culture Collection ATCC (Manassas, VA, USA). The U20S cells stably expressing FGFRl (U20S-R1) was a kind gift from Dr. Ellen M. Haugsten from The Norwegian Radium Hospital (Haugsten, 2008). E. coli strains Rosetta 2(DE3)pLysS was from Novagen-EMD Biosciences (Madison, WI, USA).

Plasmids

The sequence encoding human fibroblast growth factor 2 (FGF2) (residues 1-155) was cloned into the pET-3c expression vector from Stratagene (La Jolla, CA, USA). Insertion of KCKSGG N- and GGSKCK C-terminal linker and the point mutations C78S and C96S were introduced using QuikChange Site-Directed Mutagenesis Kit from Agilent Technologies (Santa Clara, CA, USA) according to manufacturer's protocol.

Protein expression and purification

The proteins FGF2, KCK-FGF2, FGF2-KCK , KCK-FGF2[C78S/C96S], FGF2[C78S/C96S]- KCK were expressed in E. coli Rosetta™ 2(DE3)pLysS expression strain. Bacterial cells were grown in TB medium in the presence of 100 μg/mL ampicillin in 37 °C until OD600=0.6 was reached. Then protein expression was induced by the addition of IPTG to the final concentrations of 0.3 mM and the culture was incubated at 25 °C for 12 h. Next, bacterial cells were harvested by centrifugation at 8000xg, resuspended in the lysis buffer (50 mM monosodium phosphate, 0.15 M NaCl, 1 mM DTT, 1 mM EDTA, 0.1% Triton X-100, 1 mM PMSF, pH=7.2), and homogenized using the French's press. Total cell extract was centrifuged at 50 000xg at 4 °C for 1 h. The supernatant was diluted in the binding buffer (monosodium phosphate 50 mM, 1 mM DTT, 1 mM EDTA, 0.7 M NaCl, pH=7.2) and loaded on the HiTrap™ Heparin HP Column. The column was washed with washing buffer (monosodium phosphate 50 mM, 1 mM DTT, 1 mM EDTA, 1.0 M NaCl, pH=7.2) and then the proteins were eluted with a linear gradient of NaCl (monosodium phosphate 50 mM, 1 mM DTT, 1 mM EDTA, 1.0 - 2.0 M NaCl, pH=7.2).

Conjugation of FGF2 variants with a cytotoxic drug

Maleimid-PEG-Val-Thr-auristatin Y (abbreviated YCP) was used as a cytotoxic compound delivered by FGF2. Auristatin Y is a highly cytotoxic derivative of dolastatin, more hydrophilic than MMAE (Doronina, 2012). It contains a maleimide moiety suitable for conjugation to cysteines residue. To enhance further the hydrophilicity of toxic agent, PEG molecule was added to auristatin Y. Any of PEG 513, PEG 1570 or PEG 3000 was suitable for this use. The conjugation reaction was optimized, as in example 1, to provide high yield and optimal conditions, protecting against protein unfolding and a loss of biological activity. Purified proteins were desalted using Zeba Spin Desalting column into reaction buffer (50 mM monosodium phosphate, 10 mM Na2S04, 10 mM methionine, 1 mM EDTA, pH 7.0). Maleimide derivative of PEGylated auristatin Y (YCP) dissolved in DMAc (N,N- Dimethylacetamide) at concentration of 50 mg/mL was added to 1.5 mg/mL protein solutions. There was a two-fold molar excess of the drug over protein SH-groups. The conjugation reaction mixture was incubated for 1 h at 20 °C.

As a result of conjugation reaction highly homogenous preparations were obtained that contained negligible amount of unconjugated species, as confirmed by SDS PAGE (Figure 7). Different variants of FGF2 allowed us to control the number of drug molecules attached to FGF2. When YCP was added to KCK-FGF2, three exposed cysteines of growth factor were substituted and two other remained intact (Figure 7). Similarly, in the case of all other FGF2 constructs, two buried cysteines were not reactive and expected drug molecule loading was achieved (DPR = 1 in the case of KCK-FGF2[C78S/C96S] and FGF2[C78S/C96S]-KCK) (Figure 7).

Substitution of two surface cysteine residues to serines (C78S and C96S) together with the introduction of KCKSGG sequence on the N-terminus or GGSKCK on the C-terminus (in both cases abbreviated as KCK) allowed to obtain two mono-substituted FGF2 (KCK- FGF2[C78S/C96S]-YCPi and FGF2[C78S/C96S]-KCK-YCPi). In addition, to generate triply substituted conjugates, the WT sequence of FGF2 extended with either KCKSGG sequence on the N-terminus or GGSKCK on the C-terminus were used (KCK-FGF2-YCP ₃ and FGF2-KCK- YCP ). The cysteine residue flanked with lysines is highly reactive and ensures excellent yield of conjugation reaction (Table 2).

Table 2. The yields of the reaction based on HPLC analysis.

KCK-FGF2 [C78S/C96S]- FGF2[C78S/C96S]-KCK -

Conjugate KCK-FGF2-YCP

YCP YCP

Yield [%] 100 97 98 Spectrofluorimetry

To assess the native conformation of all FGF2 variants before and after conjugation fluorescence analysis was performed that is a useful indicator of a proper folding of FGF2 (Figure 10). The folded state of proteins and their conjugates were verified by spectrofluorimetry. The fluorescence spectra were acquired using an FP-8500 spectrofluorimeter (Jasco, Japan) with excitation at 280 nm and emission in the 300-450 nm range, at a protein concentration ~4x 10 ^"6 M in Dulbecco's PBS.

The fluorescence spectrum of properly folded wild-type FGF2 showed very low emission at around at 353 nm, since the signal from the single tryptophan residue is completely quenched and the spectrum is dominated by emission of tyrosine residues (maximum at around 303 nm). Upon unfolding, the quenching effect is abolished, resulting in a significant increase in the fluorescence at 353 nm. All fluorescence emission spectra were similar to the spectrum of native FGF2 WT, showing no changes in the tertiary structure of proteins conjugated to YCP.

Cytotoxic effect of FGF2 conjugates

To assess the toxicity of FGF2 conjugates, two cell lines that differ in FGFR1 level: U20S (cells that show hardly detectable level of FGFR1 and serve as a negative control) and U20S- Rl (U20S cells stably expressing very high level of FGFR1) were used. Cells viability was assessed with the Alamar Blue assay.

Cells cultured on the 96-well plates (5000 cells/well in the required media supplemented with 10 U/mL heparin sulphate were treated with tested samples.

Cells were treated with two FGF2 conjugates KCK-FGF2-(vcMMAE) ₃ and KCK-FGF2-YCP ₃ in 0.04 to 4000 nM concentration range for 96 h. After 92 h of continuous exposure to the drug, the medium was removed and replaced with a fresh medium containing 10% of Alamar Blue. Fluorescence emission at 590 nm (excitation at 560 nm), reflecting the viability of the cells, was measured 4 h later using En Vision Multilabel Reader fluorescence plate reader (PerkinElmer, Waltham MA, USA).

The sensitivity of the cells towards FGF2 conjugates differed considerably between the cell lines and correlated with the level of FGFR expression (Figure 11). Significantly stronger cytotoxic effect U20S-R1 cells was found for KCK-FGF2-YCP ₃ than KCK-FGF2- (vcMMAE) ₃ , whereas in U20S cells the toxicity was more than one order of magnitude lower for KCK-FGF2-YCP ₃ than KCK-FGF2-(vcMMAE) ₃ (Figure 11). These results show that increased hydrophilicity of cytotoxic drug provides higher efficacy and specificity of conjugates by widening the therapeutic window and reducing side effects for non-malignant cells.

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