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.


■ INTRODUCTION
Conventional low molecular weight anticancer drugs do not effectively accumulate at the tumor site, leading to the high toxicity of the healthy tissues and, in consequence, exhibiting a narrow therapeutic window. 1,2 On the other hand, antibody− drug conjugates (ADCs), thanks to the monoclonal antibody as a targeting molecule, ensure the specific delivery of drugs to cancer cells. However, their large size limits the ability to penetrate solid tumors. 3 Therefore, small-format drug conjugates (SFDCs) 4 or small molecule−drug conjugates (SMDCs) 5 have been recently proposed as alternatives to ADCs. These conjugates utilize specific targeting molecules, usually much smaller than the full-length antibody, such as antibody fragments, 6−9 designed ankyrin repeat proteins (DARPins), 10,11 knottins, 12,13 affibodies, 14,15 bicyclic peptides, 16,17 or small organic ligands. 5,18 SFDCs or SMDCs retain the ability of ADCs to localize selectively in cancerous tissues, exhibiting at the same time favorable pharmacokinetics and accessibility. 4,5 Thus, many of them are currently entering clinical trials. 19 However, for effective delivery of the drug not only the size of the conjugate matters. 20−23 An import issue is the hydrophobicity of the cytotoxic payload and of the linker that connects the payload to the targeting molecule. Reduced hydrophobic properties of the toxic cargo increase a therapeutic window due to the better exposure and solubility, 20,24,25 but it has to be kept in mind that the potency of the conjugate increases with the higher drug loading (an increased drug-to-targeting-protein ratio, DPR (called DAR for ADCs)), leading to enhanced hydrophobicity and in consequence an unfavorable pharmacokinetic profile. Thus, there is a strong need in the ADC field for the reduction of offtarget cytotoxicity, and the optimization of linkers and payloads. 21,26,27 The most common approach used to improve the hydrophilicity of cargo is the attachment of polyethylene glycol moieties (PEGylation). 28−32 Other strategies utilize glucuronidation of the cytotoxic agent or preparation of its hydrophilic derivatives. 20,22,33,34 cell medium contained 50 μg/mL gentamicin sulfate from Thermo Fisher Scientific. All cell lines were cultured in a humidified incubator at 37°C in a 5% CO 2 atmosphere. The DMS114, NCI-H520, and U2OS cell lines were obtained from American Type Culture Collection (Manassas, VA). HCC15 cells were supplied by the Leibniz Institute DSMZ, German Collection of Microorganisms and Cell Cultures. The U2OS cells stably expressing FGFR1 (U2OS-R1) were a kind gift from Dr. Ellen M. Haugsten from The Norwegian Radium Hospital. 38 The E. coli expression strain Rosetta 2(DE3)pLysS was from Novagen-EMD Biosciences (Madison, WI).

Synthesis of PEGylated tvAY Moieties.
Step 1: Synthesis and Purification of L-Cys-tvAY. L-Cysteine (184 mg, 1.52 mmol, 20 equiv), maleimidocaproyl-threonine-valineauristatin Y (100 mg, 0.08 mmol), and DIPEA (26.5 μL, 0.16 mmol, 2 equiv) were added to 1 mL of DMAc. The reaction was conducted at 30°C for 12 h. Next, the solvent was removed under a vacuum. The crude product was dissolved in 500 μL of 30% acetonitrile/water with 0.1% TFA, then the NH 2 −Cys-tvAY was separated from an excess of Cys by RP-HPLC, and the solvent was removed by lyophilization. The identity of the product was confirmed by MALDI-MS.
Protein Production and Purification. The plasmids encoding fibroblast growth factor 2 with the N-terminal KCKSGG linker and FGF2 with the N-terminal KCKSGG linker and the two-point mutations C78S and C96S were Molecular Pharmaceutics pubs.acs.org/molecularpharmaceutics Article used. 36 The plasmids were transformed into the E. coli Rosetta 2(DE3)pLysS expression strain. Protein production was carried out in the Biostat C fermentor system (B. Braun Biotech International, Germany). Bacteria were grown in a TB medium with 100 μg/mL ampicillin at 37°C, pO 2  Synthesis of the Conjugates. Maleimide-tvAY, maleimide-PEG4-tvAY, maleimide-PEG27-tvAY, and maleimide-PEG27-(tvAY) 3 were dissolved in DMAc at a concentration of 50 mg/mL. The attachment of the cytotoxic payload containing a maleimide moiety to the sulfhydryl group of protein was performed in the reaction buffer (25 mM monosodium phosphate, 10 mM Na 2 SO 4 , 10 mM methionine, 1 mM EDTA, pH 7.0) at a 0.5 mg/mL protein concentration and 5-fold molar excess of payload per sulfhydryl group. The reaction was incubated at 20°C for 1 h. Next, the reaction mixture was loaded onto the HiTrap CM Sepharose column, the unreacted payload was washed out using 25 mM monosodium phosphate pH 7.0 with 10 mM Na 2 SO 4 , and finally, the conjugate was eluted with the same buffer containing 0.5 M NaCl.
Fluorescence Labeling of Proteins and Conjugates. FGF2 and the conjugates were labeled with DyLight 488 or 550 coupled with NHS ester.
The 10 mM stock solutions of the fluorescence probes were prepared in N,N-dimethylacetamide (DMAc). Then, 100 μL of the protein samples at a concentration of 1 mg/mL in 25 mM HEPES pH 8.0 with 10 mM (NH 4 ) 2 SO 4 was mixed with 5 μL of fluorescence probes. The reactions were conducted at room temperature for 1 h in the dark. Labeled proteins or conjugates were purified using Zeba Spin Desalting columns using PBS supplemented with 10 mM (NH 4 ) 2 SO 4 as an elution buffer.
Spectrofluorimetry. To validate the folded state of protein and conjugates, spectrofluorimetry measurements were performed. 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 20°C using a 4 × 10 −6 M sample concentration in PBS.
Thermal Unfolding and Aggregation. The thermal denaturation and aggregation were analyzed using nanoDSF and light back-reflection, respectively. The measurements were conducted using the NanoTemper Technologies instrument Prometheus NT.48 fitted with back-reflection optics at the concentration of the proteins of 3 μM in PBS. The samples were loaded into Prometheus NT.48 Series nanoDSF grade high sensitivity capillaries and then heated at a defined, constant ramp, 1°C/min, over a defined temperature range from 20 to 95°C. Fluorescence was excited at 280 nm, and emission was recorded at 350 nm. The aggregation was measured in parallel, as light intensity loss due to scattering. Data recorded by back-reflection optics were expressed on the chart in milli-attenuation units.
Size-Exclusion Chromatography (SEC). Size-exclusion chromatography runs were performed to estimate the hydrodynamic-radius-based molecular mass of FGF2 and conjugates. The analysis was performed at 20°C using an ÄKTA explorer FPLC System with a Superdex 75 HR 10/30 column. Then, 2.5 mg/mL samples were loaded onto the column by a full 50 μL loop injection. The mobile phase (25 mM monosodium phosphate pH = 7.4, 10 mM Na 2 SO 4 ) was pumped at a flow rate of 1 mL/min, and absorption at 280 nm was recorded. Molecular weight standards containing BPTI, cytochrome C, carbonic anhydrases, human serum albumin, α-lactoglobulin, chymotrypsinogen A, ovalbumin, and albumin were used to generate a standard curve from which the effective size of the PEGylated conjugates was estimated.
Biolayer Interferometry (BLI). The kinetic rate constants were measured on ForteBio Octet K2 (Pall ForteBio, CA) and high-precision streptavidin biosensors (SAX) (Pall ForteBio, CA) were used. Biotinylated extracellular domains of FGFR1c fused to the Fc fragment were immobilized, and then binding of FGF2 or the conjugates was performed similarly as we reported previously. 36 Association of the samples at a concentration of 50 nM was conducted for 400 s, and then the dissociation was monitored for 400 s. Kinetic parameters were calculated using a 1:1 model with Octet Data Analysis software 11.0.
Confocal Microscopy. Specific Internalization of FGF2 and Conjugates into Cells Expressing FGFR1. U2OS cells were stained with CellTracker Red CMTPX, according to the manufacturer's protocol, then seeded on coverslips with an equal number of nonstained U2OS-R1 cells, and grown together to 70% confluence. Next, the cells were starved in a serum-free medium for 2 h. Subsequently, the cells were incubated with 500 ng/mL of FGF2 or conjugates labeled with DyLight 488 in serum-free medium supplemented with 1% BSA and 10 U/mL heparin at 4°C for 40 min. Then, the cells were incubated at 37°C for 35 min. Next, the cells were washed with PBS, fixed with 4% formaldehyde in PBS, and blocked with 1% BSA, and 0.1 M glycine in PBS, and DNA was stained with DAPI. The coverslips were mounted with the ProLong Gold antifade mountant and viewed under a ZEISS LSM 880 microscope using a Plan-Apochromat 63×/1.4 Oil DIC M27 objective and GaAsP PMT combined with 2 multialkali PMT spectral detectors (Zeiss, Germany). Images were processed with Zeiss ZEN 2.6 software (Zeiss, Germany).
Endocytosis of FGF2 and Conjugates. U2OS-R1 cells were seeded on coverslips and grown to 70% confluence. ProLong Gold antifade mountant, and images were collected as described above. Raw data images were analyzed using Zeiss ZEN 2.6 software (Zeiss, Germany). Flow Cytometry Analysis of Internalization. U2OS and U2OS-R1 cells were seeded onto 12-well plates (0.15 × 10 6 cells per well) in full medium and left to attach overnight. Then the medium was removed, and the cells were washed with PBS and starved with serum-free medium for 2 h. Next, plates were cooled on ice, and FGF2 or conjugates (500 ng/ mL) labeled with DyLight 488 were added to the cells. After 40 min of incubation on ice, the cells were moved to 37°C for 35 min. Then the medium was removed; the cells were washed with PBS (three times, 5 min) and subsequently detached by 10 mM EDTA in PBS, pH = 8.0. The cells were harvested by Cell Viability Assay. The cells in the appropriate full medium were seeded on 96-well plates and left overnight to attach. Then, the medium was removed, replaced with a fresh one, supplemented with 10 U/mL heparin, and cells were treated with six different concentrations of FGF2, conjugates, or free AY for 96 h. Next, the medium was removed, and the cells were incubated for 4 h in fresh, full medium with 10% alamarBlue. The fluorescence signal (excitation at 560 nm and emission at 590 nm) was measured by a TECAN Infinite M1000 Pro microplate reader (TECAN Group Ltd., Switzer-land). The data were fitted to the Hill equation to calculate EC 50 values using OriginPro 8 software (Northampton, MA). Statistical analyses were performed using SigmaPlot software for three independent experiments using the t test. The statistical significance of differences in cytotoxicity between the FGF2 1xC -PEG27-(tvAY) 3 conjugate and other conjugates was assessed.
■ RESULTS Design Schemes of Novel FGF2 Conjugates. We applied a hydrophilic derivative of auristatin, auristatin Y (AY), as a cytotoxic agent. This compound contains a dimethylamine group at the N-terminus and L-norleucine at

Molecular Pharmaceutics
pubs.acs.org/molecularpharmaceutics Article the C-terminus of the auristatin backbone ( Figure 1A). The linker, which allows the conjugation of FGF2 to the The C-terminus of auristatin Y contains hydrolyzable L-Thr-D-Val dipeptide (tv) and a maleimide moiety ( Figure 1A). 20,39 In order to further increase the hydrophilicity of the cytotoxic payload and to elevate the hydrodynamic radius of conjugates, we used a PEG molecule (either 4 or 27 mer) as a spacer between tvAY and FGF2. We applied two different schemes of FGF2-based conjugate synthesis. In both schemes, always three tvAY molecules were attached to a single FGF2 molecule to enable direct comparisons.
We used two previously described FGF2 variants with one or three cysteines exposed to a solvent. 36 The first one, FGF2 3xC , contained three exposed Cys residues, two naturally occurring (C78, C96) and one introduced at the N-terminus (in the KCK extension) to facilitate the thiol-maleimide reaction. Previously we reported that cysteine surrounded by basic residues Lys or Arg, as in the KCK sequence, shows significantly higher reactivity. 40 The second FGF2 variant (FGF2 1xC ) contained a single solvent-exposed Cys residue, located in the N-terminal KCK extension, and C78 and C96 were mutated to serines (C78S/C96S) ( Figure 1C).
In the second scheme, three tvAY molecules were first attached to a short synthetic peptide scaffold (SRCRCRC),  Table 1. (E) Stability of FGF2 and FGF2 conjugates in human serum. The FGF2 and the conjugates were incubated at a concentration of 1 μg/mL without heparin in the human serum at 37°C for indicated times. The samples were analyzed by SDS-PAGE and immunoblotting using the anti-FGF2 antibody. Experiments were performed in triplicate, and representative results are shown.

Molecular Pharmaceutics
pubs.acs.org/molecularpharmaceutics Article again containing highly reactive cysteines, and the peptide conjugate was attached to the N-terminal KCK extension of FGF2 1xC via PEG27. First, we synthesized a peptide scaffold comprising three Cys residues (H 2 N-SRCRCRC−CONH 2 ) and attached three tvAY molecules to peptide sulfhydryl groups. Next, we coupled the NHS-PEG27-mal moiety to the α-amine group of the peptide scaffold via an NHS-primary amine reaction. We obtained a PEGylated, triply substituted with tvAY peptide scaffold (maleimide-PEG27-(tvAY) 3 ) ready for conjugation with cysteine residues of proteins. ( Figure 1B). Finally, we coupled one molecule of the peptide scaffold (maleimide-PEG27-(tvAY) 3 ) with one cysteine residue of FGF2 1xC ( Figure 1C). As a consequence, in both schemes, the drug-to-protein ratio (DPR) was three, but the payloads were located differently on the FGF2 molecule. Unless otherwise stated, in all further experiments, we compared obtained conjugates with the wildtype FGF2 denoted as FGF2. Basic properties of FGF2 1xC and FGF2 3xC were examined previously 36 and were similar to FGF2.
Biophysical Properties of Proteins and Conjugates. The Native State of the FGF2 Proteins and Their Conjugates. The conformation of FGF2 3xC and FGF2 1xC before and after conjugation was evaluated by the fluorescence measurement of Trp123 emission. In the native state, the fluorescence signal of single Trp (at 353 nm) is quenched, and the spectrum is dominated by the emission of several Tyr residues (at 303 nm). Upon unfolding, the Trp residue recovers strong emission at 353 nm. The fluorescence spectra of all tested samples showed the highest emission at 303 nm and a very low signal at 353 nm ( Figure 3A), in clear contrast to the spectrum of FGF2 chemically unfolded in Six M GdmCl ( Figure 3A dashes line). These results confirmed the native conformation of FGF2 variants before and after conjugation.
Stability and Susceptibility to the Aggregation of the Conjugates. The thermal stability of FGF2 and all tvAY conjugates was studied by the nanoDSF technique. In all cases, protein samples showed cooperative unfolding determined by the change of the intrinsic fluorescence emission at 350 nm in the temperature range from 20 to 95°C ( Figure 3B). Denaturation temperature of all conjugates differed only slightly from the T den of FGF2 wild type, indicating that cytotoxic payloads do not substantially influence the stability of FGF2 ( Figure 3B, Table 1). The most stable conjugate was FGF2 3xC -(PEG4-tvAY) 3 − T den was 3°C higher than that observed for the wild type (T denFGF2 = 54.5°C). FGF2 1xC with three tvAY molecules located at the peptide scaffold linked by the PEG27 molecule (FGF2 1xC -PEG27-(tvAY) 3 ) exhibited the lowest T den value, equal to 50.5°C.
To verify how cytotoxic payloads affect FGF2 aggregation, we analyzed the change of light back-reflection during the thermal unfolding of conjugates. None of the tested samples aggregated under the applied experimental conditions ( Figure  3C).
Since it is well-known that PEGylation increases the hydrodynamic radius of a protein in a nonlinear manner, 41 we performed a size-exclusion chromatography (SEC) analysis to determine the size of FGF2 conjugates ( Figure 3D) and to estimate their equivalent molecular weights (Table 1). In contrast to the hydrophobic auristatin derivative (MMAE), 20 triple substitutions of FGF2 with hydrophilic tvAY increased the hydrodynamic size of FGF2 protein ( Figure 3D, Table 1). The addition of PEG molecules further enlarged the hydrodynamic radius of conjugates up to the equivalent protein molecular weight of 58.6 kDa, in the case of FGF2 3xC -(PEG27-tvAY) 3 . Additionally, we analyzed the stability of FGF2 and FGF2 conjugates over time in human serum in the absence of heparin ( Figure 2E). We observed that all conjugates were intact after 120-h incubation in human serum.
Affinity of FGF2 Conjugates to FGFR1. PEGylation is a useful method to reduce nonspecific interactions of biomolecules. 41−43 However, in some cases, PEGylated proteins exhibit lower affinity to their molecular targets.
To examine the impact of introduced payloads on the interaction of FGF2-based conjugates with the extracellular domain of FGFR1, we performed biolayer interferometry (BLI) measurements.

Molecular Pharmaceutics
pubs.acs.org/molecularpharmaceutics Article the uptake was at least 10-fold lower. Interestingly, in U2OS-R1 cells, we observed a slightly higher uptake of FGF2 3xC -(PEG27-tvAY) 3 as compared to FGF2 3xC -(PEG4-tvAY) 3 . Differences between other preparations were statistically insignificant ( Figure 5C). Intracellular Trafficking of FGF2 and Conjugates. After binding of FGF2 to FGFR1, dimerized receptors together with the ligands undergo internalization from the cell membrane, and then the complexes are sorted into endosomes and directed to lysosomes. 45 Endosomal and lysosomal proteases hydrolyze peptide bonds of internalized cargo, facilitating, in the case of protein−drug conjugates, the release of the active form of the cytotoxic agent. 46,47 To verify whether FGF2 conjugates reach the acidic compartment within the cell, we studied their intracellular localization in U2OS-R1 cells using confocal microscopy. We labeled early endosomes using a green fluorescent protein (GFP) fused to Rab5 (Rab5-GFP) and lysosomes with GFP fusions of lysosomal membrane-associated protein 1 (LAMP1-GFP). Next, the cells were incubated at 37°C for 35 or 50 min with FGF2 or conjugates labeled with DyLight550. In the case of FGF2 wild type as well as FGF2 conjugates, the red fluorescent signal colocalized with green fluorescence of Rab5-GFP or LAMP1-GFP after 35 or 50 min of incubation, respectively ( Figure 6). These experiments show that FGF2 and its conjugates are efficiently internalized into U2OS-R1 cells and traffic via endosomes to lysosomes.
Cytotoxic Effect of the Conjugates. To evaluate in vitro selectivity and cytotoxicity of the conjugates, we used a panel of five human cancer cell lines differing in expression level 8,48 and splicing isoform of FGFR1. We used two FGFR1-negative cell lines (U2OS and HCC15) and three FGFR1-positive cell lines (U2OS-R1 expressing FGFR1-α-IIIc, DMS114 expressing FGFR1-β-IIIc, and NCI-H520 expressing FGFR1-α-IIIb). 49 The cells were treated with FGF2, the conjugates and free AY in the concentration range from 0.003 nM to 273 nM for 96 h. Then, cell viability was assessed with the alamarBlue assay. The EC 50 values were calculated from the Hill equation.  Table 2). In the case of the U2OS-R1 cell line, the conjugates exhibited EC 50 in the subnanomolar range. FGF2 1xC -PEG27-(tvAY) 3 demonstrated an 8.6 and 5.3 times higher EC 50 value, as compared with FGF2 3xC -(tvAY) 3 and FGF2 3xC -(PEG4-tvAY) 3 , respectively. FGF2 conjugates showed the largest cytotoxic potency in DMS114 cells, presenting the highest natural level of FGFR1 among tested cell lines, with EC 50 in the low subnanomolar range. The NCI-H520 cell line, expressing relatively low amounts of FGFR1, was less sensitive to conjugates with EC 50 values more than 100 times higher than that observed in U2OS-R1 cells. Notably, in this case, the FGF2 1xC -PEG27-(tvAY) 3 conjugate exhibited 3.1 and 5.7 times higher EC 50 values than FGF2 3xC -(PEG4-tvAY) 3 and FGF2 3xC -(PEG27-tvAY) 3 conjugates, respectively.
Interestingly, free auristatin Y (AY) was nontoxic to all tested cell lines. We found that the cytotoxicity of FGF2 conjugates is determined by the FGFR1 expression level on the cell surface, 8,48 and confirmed that auristatin Y needs to be specifically delivered into the cell interior in order to evoke its toxic effect.

■ DISCUSSION
Despite continuous progress in the field of targeted cancer therapy, only five ADC-based drugs have been approved by the FDA. 21,50 Immunogenicity of mAb, drug resistance (especially P-glycoprotein-mediated efflux of hydrophobic drugs), nonoptimal bioavailability, and off-target toxicity are among major limitations of ADCs. 51−57 Thus, the development of novel targeted approaches suitable for cancer treatment remains a vital need.
There are numerous protein scaffolds that could potentially replace monoclonal antibodies as the targeting molecules, including antibody fragments, 6,7 DARPins, 10,11 knottins, 12,13 and affibodies. 14,15 Previously, we demonstrated that affibody, 58,59 diaffibody, 60 scFv-Fc, 8,9 FGF1,40,61,62 and FGF2 36,48,63,64 are effective proteins in terms of specific delivery of the cytotoxic drug vcMMAE to tumor cells.  One of the problems in applying vcMMAE, especially in conjugates with high drug loading, is its hydrophobicity, a factor greatly influencing tissue penetration and specificity of cellular internalization. 65− 68 We previously found that an FGF2 conjugate containing three vcMMAE molecules (FGF2 3xC -(vcMMAE) 3 ) at a concentration higher than 125 nM exhibits nonspecific cytotoxicity toward the FGFR1negative U2OS cell line. 36 However, there are numerous chemical derivatives of auristatin, i.e., MMAE, MMAF, MMAU, and AY, which differ in hydrophobicity, charge, and cytotoxicity. 20,22,39,69 Taking into account the plan to load FGF2 with three auristatin molecules, we decided to use more hydrophilic AY. Unlike MMAE, 39 AY did not show toxicity as a free drug (Figure 7, Table 2). In general, increased hydrophilicity of cytotoxic payloads affects their efflux from the cell interior, which reduces multiple drug resistance (MDR). 70 Moreover, the cytotoxic agents coupled with polar amino acids (such as tvAY, Figure 1A) cannot be a substrate of the ABCB1 transporter, which further overcomes MDR. 71−74 Concomitantly, cytotoxic payloads with lower hydrophobicity exhibit lower bystander killing, 75,76 which can be considered as a possible drawback of AY manifesting in reduced potency against FGFR-negative cells in the tumor environment.
Replacement of hydrophobic vcMMAE with hydrophilic vtAY increased polarity of FGF2 conjugates, which manifested in shorter retention time observed in SEC experiments ( Figure  3D, Table 1. Importantly, the FGF2 3xC -(tvAY) 3 conjugate was nontoxic to FGFR1-negative cell lines (Figure 7, Table 2). Finally, in agreement with ADC data, 20,22,65 the therapeutic index was increased due to the hydrophilic nature of the (tvAY) 3 payload.
To further increase the hydrophilicity of cytotoxic cargo and to increase the hydrodynamic radius of conjugates, we attached PEG linkers in a series of FGF2 conjugates. Both properties play a key role in pharmacokinetics and in vivo potency of conjugates. 20,25,65,77,78 The short PEG4 linker introduced between FGF2 and vtAY (FGF2 3xC -(PEG4-tvAY) 3 ) increased hydrodynamic molecular weight, in agreement with the PEGhydration model. 41 Further, a large increase in the hydrodynamic radius corresponding to a globular protein of 58 kDa was observed when PEG27 was applied (FGF2 3xC -(PEG27-tvAY) 3 ). Using PEG4 and PEG27 Burke et al. and Simmons et al. observed comparable hydrodynamic radius of classical ADCs; 33,79 thus, it is likely that the size of FGF2 3xC -(PEG27-tvAY) 3 exceeds the limit of renal filtration. 80,81 However, a further pharmacokinetic study should be performed to confirm this.
FGF2 3xC -(PEG27-tvAY) 3 showed cytotoxic effects comparable to non-PEGylated FGF2 3xC -(tvAY) 3 or a conjugate containing a short PEG4 linker (FGF2 3xC -(PEG4-tvAY) 3 ). We decided to study whether conjugation via a single cysteine present in the KCK extension at the N-terminus with tvAY 3 attached to the PEG27-SRCRCRC scaffold peptide would show comparable cytotoxicity as observed in FGF2 3xC -(PEG27-tvAY) 3 . This might be beneficial for loading proteins that contain a single exposed cysteine in their sequence with three (or even more) payloads.
All studied FGF2-based conjugates showed several desirable characteristics, such as homogeneity and defined DPR ( Figure  2A), stability in human serum ( Figure 3E), resistance to aggregation ( Figure 3C), selective affinity to FGFR1-positive cells ( Figure 5), specific internalization via FGFR-mediated endocytosis, and trafficking from endosomes to lysosomes ( Figure 6). All conjugates were highly efficient in killing FGFR-expressing cancer cells and nontoxic for FGFR-negative cell lines (Figure 7, Table 2). Since free AY cannot easily diffuse across cell membranes, it has to be actively delivered into the cell (Figure 7, Table 2).
So far, the FDA has approved 15 PEGylated protein drugs. Most of them contain heterogenic PEG moieties bigger than 5 kDa, and only four are generated by site-specific PEGylation. The latter show a significant advantage since they are easier to purify and are homogenic. 42,82,83 In the ADC field, PEG molecules are used mainly for increasing the hydrophilicity of the payload and the bioavailability of the conjugate. Unfortunately, PEG molecules bring several disadvantages, including the possible loss of biological activity and the decrease in affinity to the molecular target. 41,82 FGFRs are abundant in normal adult tissues (e.g., skin, cornea, lung, heart, placenta, kidney, ureter, and retina). 84,85 Nonetheless, the level of FGFRs in many cancer cells is significantly elevated. 86−88 Therefore, the development of drugs targeting FGFRs is a well-justified anticancer strategy. However, so far, only one ADC targeting the FGF receptor (Aprutumab Ixadotin, BAY 1187982) has been evaluated in a phase 1 clinical study. 89 BAY 1187982, targeted at FGFR2-IIIb and FGFR2-IIIc, contained a derivative of auristatin W as a cytotoxic drug, not specifically conjugated to lysine residues via a noncleavable linker. 90 The trial was terminated because BAY 1187982 was poorly tolerated. The study suggests that observed side effects were associated with the toxicity of the payload. 89 Thus, novel approaches to modify the cytotoxic drugs in therapeutic conjugates are of high importance.
Another important issue is the systemic elimination half-life of conjugates. A significant impact on prolongation of ADCs' half-life is exerted by the Fc region that binds to the neonatal Fc receptor 91 expressed in various tissues, including the endothelial cells, interstitial macrophages, Kupffer cells, alveolar macrophages, enterocytes, and choroid plexus epithelium. 92 FGF2-conjugates lack the Fc fragment, but FGF2 protein has a high affinity to heparans occurring on the surface of many types of cells and in body fluids. Interaction with sugar moiety highly stabilizes FGF2 and protects the FGF2-conjugates from degradation. We have shown that FGF2-tvAY conjugates are stable in human plasma for at least 5 days ( Figure 3E); thus, coadministration of heparin along with FGF2 conjugates should result in an increased half-life in comparison to other targeting scaffolds that do not have systemic stabilizing/recyclizing partners. Nevertheless, this issue needs to be clarified in a further detailed pharmacokinetic study.
Here, we used small-molecular-weight, homogeneous PEG molecules with a defined chain length to couple auristatin Y to the FGF2 molecule in a site-specific manner. The analysis of the tertiary structure of FGF2 conjugates ( Figure 3A), the receptor affinity measurements (Figure 4), cell internalization experiments ( Figure 5) and the analysis of the intracellular trafficking ( Figure 6) clearly showed that payloads introduced via PEG4 or PEG27 linkers do not alter the biophysical and biological properties of FGF2.

Molecular Pharmaceutics
pubs.acs.org/molecularpharmaceutics Article All conjugates were characterized by the defined drug-toprotein ratio (DAR = 3), due to a site-specific attachment of the defined PEGylated derivative of vtAY. They exhibited a highly toxic effect toward FGFR1-positive cell lines, especially those expressing isoform IIIc of FGFR1 specific for FGF2. 93,94 The highest cytotoxicity was observed in the case of DMS114 cells, which expressed isoform β of FGFR1, which is correlated with reduced survival of a patient. 93,95 The most potent conjugate, FGF2 3xC -(PEG27-tvAY) 3 , with highly desirable hydrodynamic properties, possesses very high potential for a further in vivo study in order to validate its feasibility for anticancer targeted therapy.