High-Yield Site-Specific Conjugation of Fibroblast Growth Factor 1

Subscriber access provided by Binghamton University | Libraries

Article

High-yield site-specific conjugation of fibroblast growth factor 1 with monomethylauristatin E via cysteine flanked by basic residues Michal Lobocki, Malgorzata Zakrzewska, Anna Szlachcic, Mateusz Adam Krzyscik, Aleksandra Sokolowska-Wedzina, and Jacek Otlewski Bioconjugate Chem., Just Accepted Manuscript • Publication Date (Web): 09 Jun 2017 Downloaded from http://pubs.acs.org on June 10, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Bioconjugate Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Bioconjugate Chemistry

High-yield site-specific conjugation of fibroblast growth factor 1 with monomethylauristatin E via cysteine flanked by basic residues

Lobocki M.1, Zakrzewska M.1, Szlachcic A.1, Krzyscik M. A.1, Sokolowska-Wedzina A.1, Otlewski J.*,1 1Department

of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Joliot-Curie 14A, 50-

383 Wroclaw, Poland

*Corresponding author Prof. Jacek Otlewski University of Wroclaw Faculty of Biotechnology, Department of Protein Engineering Joliot-Curie 14A 50-383 Wroclaw, Poland

E-mail: [email protected]; Tel: +48 713752824

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Site-specific conjugation is a leading trend in the development of protein conjugates, including antibody-drug conjugates (ADCs), suitable for targeted cancer therapy. Here, we present a very efficient strategy for specific attachment of a cytotoxic drug to fibroblast growth factor 1 (FGF1), a natural ligand of FGF receptors (FGFRs), which are overexpressed in several types of lung, breast and gastric cancers and are therefore an attractive molecular target. Recently we showed that FGF1 fused to monomethylauristatin E (vcMMAE) was highly cytotoxic to cells presenting FGFRs on their surface and could be used as a targeting agent alternative to an antibody. Unfortunately, conjugation via maleimide chemistry to endogenous FGF1 cysteines or a cysteine introduced at the N-terminus proceeded with low yield led to non-homogeneous products. To improve the conjugation, we introduced a novel Lys-Cys-Lys motif at either FGF1 termini which increased cysteine reactivity and allowed us to obtain FGF1 conjugate with a defined site of conjugation and a yield exceeding 95%. Using FGFR-expressing cancer lines we confirmed specific cytotoxity of the obtained C-terminal FGF1-vcMMAE conjugate and its selective endocytososis, as compared with FGFR1-negative cells. This simple and powerful approach relying on the introduction of a short sequence containing cysteine and positively-charged amino acids could be used universally to improve the efficiency of site-specific chemical modification of other proteins.

Introduction Development of Antibody Drug Conjugates (ADCs) is among the most promising approaches in targeted treatment of cancer. An ADC molecule is composed of a strong cytotoxic drug and a monoclonal antibody that recognizes a specific target on malignant cells. The ADCs release the cytotoxic compound inside the tumor cell without affecting surrounding tissues and therefore causing less severe side effects compared to standard chemotherapies.1–3 The currently used conjugation methods based on lysine modification lead to non-homogeneous products, both in terms of the substituted sites and the number of drug molecules attached, defined as the drug-to-antibody ratio (DAR).4 Similarly, conjugation via reduced disulfide bonds results in conjugates with DAR values varying from 0 to 8.5 Since purification of a conjugate with a specific DAR value is economically inviable and does not solve the problem of different conjugation sites among individual molecules, the development of new conjugation strategies leading to well defined products is badly needed.


Page 2 of 30

Page 3 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Site-specific conjugation offers a solution to the heterogeneiety of ADCs. One intuitive approach involves engineering of a single reactive cysteine at a specific site. However, the location of the conjugation site may be crucial for the ADC function, including recognition, specificity and cytotoxicity. For example, Genentech researchers have applied phage display selection to identify optimal position for introducing an additional cysteine into an antibody molecule.6 Such engineered antibodies, named Thiomabs, allow for stoichiometric conjugation of a drug while preserving interchain disulfide bonds and proper antigen binding. We have previously tested the possibility of employing proteins alternative to antibodies as a vehicle to deliver a cytotoxic cargo into malignant cells. We used a conjugate of fibroblast growth factor 1 (FGF1) to deliver monomethyl auristatin E (MMAE) to cancer cell overexpressing FGF receptors.7Thereby,we demonstrated that natural ligands of cell surface receptors, other than antibody or antibody fragments, can be successfully combined with a potent drug to form an effective and selective weapon against cancer cells. However, in the previous report we experienced problems with conjugation yield and heterogeneiety of the conjugation product. Here, we present a simple yet very efficient solution to these problems. We show that introduction of basic residues flanking the cysteine provides excellent yield of maleimide conjugation and leads to a well-defined homogeneous conjugate. This specific sequence can be introduced at the N- or C-terminus of any protein, as well as within the protein sequence, being easily and universally applicable to cysteinemediated protein conjugation.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Results Design and preparation of FGF1 variants suitable for conjugation with MMAE Fibroblast growth factor 1 is a protein of relatively low stability, prone to proteolysis and with a high tendency to aggregate. Nevertheless, in a former study we showed that upon specific genetic modification FGF1 can be linked to a highly hydrophobic cytotoxic compound MMAE to provide a conjugate that selectively destroys cancer cells overproducing FGF receptor.7 The MMAE derivative used contained the Val-Cit linker (abbreviated as vc) cleavable with the lysosomal protease cathepsin B and a maleimide moiety for conjugation to the thiol group of cysteine. To obtain that conjugate we used an FGF1variant containing three stabilizing mutations (Q40P, S47I, H93G) that increased its Tden by 21 °C8and enabled conjugation in the harsh conditions of 20% acetonitrile. In addition, we mutated the exposed Cys117 to Ser, since conjugation of MMAE to Cys117 resulted in FGF1 unfolding, probably due to the hydrophobicity of MMAE. In parallel, to enable site-specific conjugation via maleimide chemistry we introduced a CGGG extension sequence at the N-terminus of FGF1. This variant, called FGF1V, could be conjugated with MMAE, however, depending on the reaction conditions we experienced problems with either low conjugation yield or unwanted modification of two buried cysteines, 16 and 83.7 In the case of the product with properly folded growth factor, the maximal yield reached barely 15%. The main goal of this study was increasing the modification yield and ensuring a defined conjugation stoichiometry. In order to provide high stability and resistance to degradation in all FGF1 variants described in this paper we preserved the three stabilizing mutations: Q40P, S47I, H93G. In the first step, we mutated not only the exposed Cys117 but also two remaining buried cysteines, Cys16 and Cys83, to serines to prevent their undesired substitution with MMAE. Next, we introduced a modified extension sequence containing a cysteine residue at the N- or C-terminus of FGF1 to increase the yield of the conjugation via maleimide chemistry. It is known that the pKa of cysteine residue can be lowered, and hence its reactivity increased, by the presence of positively charged groups in neighboring amino acids.9–11 Therefore, we flanked the terminal cysteine with two lysines to obtain KCKSGG or GGSKCK sequence for extending the N- or C-terminus of FGF1, respectively (Fig. 1). Moreover, it has been shown for MMAE-conjugated antibodies that flanking the conjugated cysteine with positively charged amino acids decreases the likelihood of hydrolysis of


Page 4 of 30

Page 5 of 30

6

FGF1.A Q40P/S47I/H93G FGF1.B Q40P/S47I/H93G C16S/C83S CGGG FGF1.C Q40P/S47I/H93G C117S FGF1.D Q40P/S47I/H93G C16S/C83S/C117S CGGG FGF1.E Q40P/S47I/H93G C16S/C83S/C117S KCKSGG FGF1.F Q40P/S47I/H93G C16S/C83S/C117S

C117S

-

C83S H93G

FGF1 wt

Q40P S47I

the antibody-drug bond, giving the conjugate higher stability and therapeutic action.12 C16S

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


140

GGSKCK

Fig. 1. FGF1 variants used in this study Diagram shows introduced mutations in fibroblast growth factor 1 variants. Circles stand for stabilizing mutations (Q40P/S47I/H93G). Triangles represent cysteine substitution mutations (C16S/C83S/C117S). Two types of extensions were introduced: CGGG at the N-terminus and KCKSGG or GGSKCK either at the N- or Cterminus of protein respectively. In the case of the N-terminal KCKSGG extension we found out that the initiator methionine was not cleaved off, so the extension was in fact MKCKSGG.

Altogether, in this study we investigated six variants of FGF1named FGF1.A to FGF1.F that differed in the number of cysteine residues mutated, presence and type of the extension sequence, and its orientation, with FGF1.C corresponding to the previously described FGF1V7 (Fig. 1). The recombinant proteins were expressed in E. coli and purified on a heparin affinity column using FPLC. All variants were produced with a yield exceeding40 mg/L and over 95%purity, as judged by SDS-PAGE. The identity was confirmed by MALDI-MS. The analysis revealed that when the KCKSGG extension was introduced at the N-terminus (FGF1.E), the starting methionine was not cleaved off. In order to verify proper conformation of purified proteins, we employed tryptophan fluorescence measurements to monitor changes in FGF1 tertiary structure. In natively folded FGF1 Trp107 fluorescence is quenched by neighboring histidine and proline residues, whereas upon unfolding the quenching is no longer observed. The obtained fluorescence spectra showed that in general all the FGF1 variants are properly folded (Fig.2A), with small differences for variants FGF1.B, FGF1.D, FGF1.E and FGF1.Frevealinga slightly increased tryptophan fluorescence at 353nm. However, in the presence of 150mM ammonium sulfate, reported previously to partially mimic heparans naturally binding FGF1 and stabilizing it,13,14 the differences in fluorescence between the variants and wildtype FGF1 became minimal, with only FGF1.B exhibiting marginally higher tryptophan fluorescence (Fig.2B). Analysis of CD spectra of 5 µM protein in phosphate buffer pH 7.2, shows no significant difference in secondary structure between tested FGF1 mutants (Fig. S1). Moreover, we used size exclusion chromatography to confirm monomeric form of proteins, and the retention volume for all mutants was in the range of 12.9-13.1 mL, corresponding to monomeric FGF1 (Fig. S2). Finally, we tested if the introduced mutations affect protein stability. It was shown before that three mutations



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Q40P/S47I/H93G increase Tm by 20 degrees (from 40.9 for FGF1 wt to 61 °C for triple mutant). Substitution of all three cysteines (FGF1Q40P/S47I/H93G/C16S/C83S/C117S) leads to a decrease in stability (Tm = 47 °C), but the protein remains more stable than wild type; introduction of GGSKCK motif does not further affect protein thermostability (Tm = 46 °C , Fig. S3). Despite these differences in vitro test of FGF1 wt and FGF1.F stability in human serum shows no degradation during 144h observation and no significant difference between FGF1 wt and FGF1.F (Fig. S4).


Page 6 of 30

Page 7 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig. 2. Characterization of FGF1 variants (A) Tryptophan fluorescence spectra of FGF1 variants in sodium phosphate buffer pH 7.4 and (B) in sodium phosphate buffer containing 150 mM ammonium sulfate. Spectra were normalized to the tyrosine signal at 304 nm. Spectrum of FGF1 fully denaturated by 6M guanidine hydrochloride is included as a reference. (C) Activation of FGFR-related signaling pathway (Erk1/2 kinases) by different variants of FGF1. Western blot analysis of Erk1/2 phosphorylation in starved NIH 3T3 cells after stimulation with FGF1 variants at 10 ng/mL.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

As expected, all the variants activated relevant signaling pathways in fibroblast cells to the same extent as wild-type FGF1 did, indicating that they are biologically functional and bind properly to FGF receptors on the cell surface (Fig. 2C). To confirm that introduced mutations did not interrupt ligand-receptor interaction we did in vitro binding analysis of FGF1 wt and FGF1-F variant to recombinant FGFR1. Using BLI technique (BioLayer Interferometry) we showed that introduced mutations virtually do not affect FGF1-to-FGFR1 biding affinity (11nM vs 29nM), while conjugation with vcMMAE decreased affinity approximately 2,5-fold compared to FGF1.F mutant (Fig. S5).

Optimization of vcMMAE conjugation To increase the efficiency of FGF1 conjugation with MMAE, first we decided to lower the organic solvent concentration to prevent FGF1 aggregation and/or unfolding. We found that MMAE can be dissolved in dimethylacetamide (DMAc) up to 50 mg/ mL, compared to only 1 mg/mL in acetonitrile. This allowed us to decrease the concentration of the organic solvent in the reaction mixture to 0.5%, compared with 20% acetonitrile used previously.7 However, the reaction yield analyzed by SDS-PAGE or HPLC for the FGF1.C variant studied earlier,7 remained relatively low: 18% in 0.5% DMAc versus 15% in 20% acetonitrile (Fig. 3). For variants with core cysteines mutated to serines we observed a significant improvement in conjugation yield. For the FGF1.D variant with all three cysteines mutated to serines (C16S/C83S/C117S) and extended at the N-terminus with CGGG, the yield was about 38%, i.e. 2-fold higher than that for FGF1.C. Only the single cysteine from the CGGG extension was modified with MMAE. The yield was further dramatically improved to over 90% when the introduced cysteine was flanked with two lysines, either at the N- or C-terminus of FGF1 (FGF1.E and FGF1.F). The KCK sequence was therefore highly reactive in conjugation with vcMMAE, whether at the N- or Cterminus.


Page 8 of 30

Page 9 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig.3. Yield of conjugation reaction of FGF1 variants with vcMMAE SDS-PAGE separation of conjugation reaction products for FGF1 variants. Conjugation yield was based on integration of chromatographic peaks after separation of reaction mixtures on a C18 RP-HPLC column. For FGF1 WT and FGF1.A yield was below detection, in case of FGF1.B precipitation made measurement impossible.

Purification and biophysical characterization of FGF1-vcMMAE conjugate We found that FGF1.F containing the KCK extension at the C-terminus showed no evidence of aggregation upon a freeze-thaw cycle, unlike the N-terminal KCK variant (FGF1.E) that precipitated after thawing at room temperature. Therefore, further studies were performed on FGF1.F and its vcMMAE conjugate. Following conjugation,hydrophobic interaction chromatography on phenyl-Sepharose was successfully used to remove traces of unconjugated protein (Fig. 4A), as confirmed by SDS-PAGE (Fig. 4B) and mass spectrometry. Similarly to unconjugated FGF1 variants, tryptophan fluorescence analysis showed thatafter conjugation with vcMMAEits the spatial structure remained native-like. Due to the presence of ammonium sulphate during purification on phenyl-Sepharose the FGF1.FvcMMAE conjugatehad a fluorescence spectrum very similar to that of unconjugated FGF1 WT (Fig. 4C), in agreement with findings of Middaugh and coworkers.13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 30

Fig. 4. Purification and characterization of FGF1.F-vcMMAE conjugate (A) HIC separation of conjugation reaction mixture. Separation was performed on TSKgel phenyl-5PW column in 100 mM phosphate buffer pH 7.2 in a gradient of ammonium sulfate from 1.2 M to 0 M. (B) SDS-PAGE evaluation of purification process. (C) Tryptophan fluorescence spectra of reaction mixture and FGF1.FvcMMAE conjugate after purification. Fluorescence intensity was normalized to tyrosine signal.

Effect of FGF1-vcMMAE conjugate on cell lines overexpressing FGFR When analyzing the expected cytotoxic effects of FGF1 conjugates one has to consider the proliferative action of unconjugated FGF1. Therefore, we first measured the proliferative effect of


Page 11 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


FGF1.F alone on NIH 3T3 fibroblasts. It exhibited a similar mitogenic activity as the wild-type FGF1 did (Fig. 5A). Next, we tested if the FGF1.F-vcMMAE conjugate binds FGFRs at the cell surface by analyzing the activation of FGFR downstream signaling in NIH3T3 cells. The main FGF1-induced signaling cascade, the MAPK pathway, was stimulated to similar extent by FGF1 WT, conjugated and unconjugated FGF1.F variant, as shown by western blot analysis (Fig. 5B). This indicates that the introduced mutations and the conjugation of the drug do not affect the interaction between FGF1 and its receptor. In the next step, we tested the efficacy and specificity of FGF1.F-vcMMAE internalization using confocal microscopy. FGFR1-negative cells, U2OS, stained with CellTrace Violet were co-cultured together with unstained U2OS stably transfected with FGFR1 gene (U2OS FGFR1). FGF1.F and its vcMMAE conjugate were stained with a red fluorescent dye, DyLight550. Early endosomes were visualized with a specific anti-EEA1 antibody stained with Alexa488. Internalization of FGF1.Fand of its conjugate was found only for the FGFR1-positive cells (Fig. 5C). Importantly, we observed high co-localization of the red and the green signal indicating the presence of the conjugate in early endosomes after a 15-minute treatment.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 30

Fig. 5. Biological activity of FGF1.F-vcMMAE (A) Mitogenic effect of FGF1 wt and FGF1.F expressed as viability of starved NIH 3T3 cells after 48 hours of exposure to FGF1. Results normalized to cells untreated with FGF1 that correspond to 0%. (B) Western blot analysis of Erk1/2 activation by FGF1 WT, FGF1.F and FGF1.F-vcMMAE conjugate at 10 ng/mL. (C) Internalization of FGF1.F and its vcMMAE conjugate in co-culture of FGFR-negative U2OS cells (blue) and U2OS cells stably transfected with FGFR1 (unstained). Cells were treated with 50 ng/mL of conjugate stained with DyLight550 (red). After 15 minutes, cells were fixed and stained with anti-EEA1 antibody (green).


Page 13 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Finally, we tested cytotoxicity of FGF1.F-vcMMAE on three cell lines expressing FGF1 receptors: BJ (human fibroblasts), U2OS FGFR1, and SNU-16 (human gastric carcinoma cell line showing FGFR2 overexpression (Kunii et al., 2008)). As a negative control the U2OS cell line was used. For the three FGFR-positive cell lines we observed high cytotoxicity with the IC50 values in the range from 2.3 x 10-8 M to 1.3 x 10-7M (Fig. 6). The cytotoxicity was the highest (the lowest IC50) for U2OS FGFR1 expressing more FGFR1 than BJ cells,7,15 with several-fold higher IC50. Notably, FGF1.F-vcMMAE proved to be cytotoxic also for the SNU-16 cell line expressing FGFR2. The IC50 value for a negative control line was higher than 1 x 10-6 M, i.e. at least one order of magnitude higher than for BJ cells.

Fig. 6. Cytotoxicity of FGF1.F-vcMMAE towards cells expressing FGFR Viability of BJ (A), U2OS FGFR1 (B) and SNU-16 cells (C) after treatment with different concentrations of FGF1.F-vcMMAE for 72 hours. U2OS cell line with very low FGFR level was used as a control. Cytotoxicity of the conjugate was determined by AlamarBlue assay.

Discussion During the last decade antibody-drug conjugates (ADCs) have reshaped the landscape of targeted cancer therapy. This efficacious group of therapeutics has provoked a large and rapidly growing clinical pipeline, with a dozen of them in late stages of clinical trials.16 However, despite their undeniable advantages, ADCs face certain challenges, such as insufficient internalization upon target binding17 and limited tumor tissue penetration, important especially in the treatment of solid tumors.18 Thus, there is a constant need for developing novel targeting molecules efficiently and selectively directing cytotoxic drugs to tumor cells. Here, we further explore the concept of targeting receptors overexpressed on cancer cells with their natural ligands conjugated with a cytotoxic drug using a growth factor as a protein scaffold alternative to antibodies. We have recently published a proof of concept study showing that FGF1, a natural ligand of FGFR, conjugated to a cytotoxic molecule is able to reach and effectively kill cells overexpressing the receptor.7 FGFR is a potential target for lung, gastric and breast cancer



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 30

therapy19,20 and its role in cancer development and therapy is increasingly being studied, with the main focus on small-molecule inhibitors and FGFR-targeting antibodies.21–23 The previously-developed FGF1 conjugate with vcMMAE attached via cysteine showed specific cytotoxicity towards several FGFR-expressing cell lines.7 However, preparation of the FGF1vcMMAE conjugate was compromised by the rather low yield of vcMMAE attachment and heterogeneity of conjugation products. These problems have been fully overcome in this report, by eliminating cysteines not intended for modification and introducing a short terminal peptide sequence containing KCK in which the lysines flanking the cysteine greatly enhance its reactivity. Various methods aiming at improving conjugation specificity and homogeneity of the resulting protein-drug product have been described, focusing primarily on antibodies.24,25 These methods involve either antibody engineering, such as mutating cysteines naturally occurring in the sequence26 or introducing reactive unnatural amino acid(s).27,28 Several enzyme-based approaches have been proposed relying on glycotransferases29,30, transglutaminases31,32 or sortases33 for drug attachment. Unlike those approaches, the method presented here is extremely simple as it involves introduction of two basic amino acids flanking the cysteine residue that is intended for conjugation. The approach is also versatile since both N- and C-terminal KCK extension prove to be equally efficient for conjugation. The increased reactivity of the cysteine flanked by basic residues in the KCK motif improves the conjugation product homogeneity and dramatically increases conjugation yield, in many cases allowing for lack of further steps of conjugate purification.9 Moreover, in the case of tested FGF1 variants, introduced point mutations did not significantly alter FGF-receptor binding affinity. The FGF1-vcMMAE conjugate obtained by the improved conjugation protocol shows clear difference in cytotoxicity versus cells expressing and lacking FGFR. Since FGF1 is a universal ligand of all FGFR isoforms, the conjugate is cytotoxic towards cells overexpressing FGFR1 (U2OS FGFR1) and those overexpressing FGFR2 (SNU-16). We expect that the level of this cytotoxicity is related to both the number of receptor molecules on the cell surface and the efficiency of the receptor-dependent internalization. Effective internalization of the conjugate, as was observed for U2OS FGFR1 cells, is a prerequisite for long-term cytotoxic response which should be the asset not only of FGF1, but also of other ligand-drug conjugates. It should be mentioned that a vast majority of natural ligands of cellsurface receptors induce efficient internalization upon ligand-receptor binding,34 whereas for antibodies the situation is not entirely clear, since in a number of cases they are internalized very poorly.17,35,36


Page 15 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Even though FGF1 is a protein naturally occurring in humans, every change in the protein sequence can potentially cause increased immune response upon administration. However, Blaber and coworkers have recently tested pharmacokinetic properties of multiple cysteine mutant of FGF1, and in their study based on rabbits they do not report significant increase in immunogenicity,37 which by similarity renders low immunogenicity of FGF1.F-based conjugate highly possible. It is also worth noting that due to its relatively small size (approximately 16 kDa), the obstacle in using FGF1.F-vcMMAE conjugate in in vivo models can be its fast kidney clearance, but this issue can be overcome by modifications leading to increase in apparent molecule size, eg. PEGylation. In summary, our FGF1-based conjugate with vcMMAE could be obtained with the expected stoichiometry and an excellent yield. Introduction of the short and universal KCK motif increases maleimide-based conjugation efficiency and may be applied to other proteins desired for productive and site-specific thiol-based chemical conjugation.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 30

Experimental procedures Recombinant FGF1 expression and purification cDNA encoding human FGF1 truncated form (residues 21–154) in the pET-3cvector (Invitrogen) was used. All FGF1 variants were prepared using QuikChange system (Stratagene) and their residue numbering is according to Gimenez-Gallego.38Expression was performed in E. coli BL21(DE3) pLysS as previously described.39FGF1 and its variants were purified by heparin affinity chromatography with FPLC on a HiTrap Heparin HP 5-mL column (GE) eluted with PBS containing 2M NaCl, 1mMDTT, 1mM EDTA. Protein purity was determined by SDS-PAGE analysis. Introduced mutations were

confirmed

by

MALDI-TOF

MS

(Applied

Biosystems

4800+)

with

α-cyano-4-

hydroxycinnamicacid(α-CHCA) as a matrix.

FGF1-vcMMAE conjugate preparation Protein buffer was exchanged to 50 mM sodium phosphate, pH 6.9 with 0.5 M NaCl and 1mM EDTA on a Zeba spin column (Thermo Scientific). Maleimide-functionalized vcMMAE dissolved in dimethylacetamide (DMAc) at50 mg/mL was added to FGF1 in a 1.5-fold molar excess over protein thiol groups. The reaction was incubated at 4°C or room temperature (23°C) overnight. During optimization, reaction progress and yield were determined by HPLC and SDS-PAGE.

FGF1-vcMMAE yield determination To determine the yield of conjugation, HPLC analysis on a DIONEX 3000 UltiMate system with an Aeris Peptide 3.6 µm XB-C18 column was performed, at 1 mL/min flow, with 0.1% TFA in water (A) and 0.1% TFA in acetonitrile (B) as solvents. Solvent gradient consisted of 3.5 minutes of isocratic flow of 1% B, then increase to 30% B and 6.5 minutes of gradient from 30% to 55%B. Absorbance was monitored at257 nm and 280 nm for vcMMAE and protein detection, respectively. Integrated 280 nm chromatograms were used for conjugation yield determination.

FGF1-vcMMAE conjugate purification To purify the desired product preparative hydrophobic interaction chromatography (HIC) was performed on an Äkta Purifier with a TSK gel Phenyl-5PW 10µm 7.5mm x 75mm column. To the reaction mixture, 4 M ammonium sulfate was gradually added to a final 1.2 M concentration. A 50-mL superloop was used for injection. MQ water (A) and 100 mM phosphate buffer with 1.2 M ammonium sulfate (B) were used as solvents, at 0.8 mL/min flow and a gradient from 100% to 0% B in 20 minutes. Absorbance was monitored as above.


Page 17 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Biophysical characterization of FGF1, mutants and conjugate The folding state of FGF1 variants and their conjugates was determined by tryptophan fluorescence spectra using a Jasco FP-8500 spectrofluorimeter. Five-micromolar protein or conjugate in 25 mM phosphate buffer, pH 7.3 was assayed. Emission in the range from 300 to 450 nm was acquired after excitation at 280 nm.39 CD spectra were acquired for 4 µM protein in 20 mM phosphate buffer pH 7.2 at 20 °C in 202 – 270 nm range by JASCO J-815 CD spectropolarimeter. Thermal stability was determined by tracking changes in ellipticity at 227 nm, during the 0.5 °C/min temperature gradient from 15 to 90°C in 20 mM phosphate buffer pH 7.2 with 0.7M guanidine hydrochloride. Size exclusion chromatography was performed on an Äkta Purifier with a Superdex® 75 10/300 GL column, at 20 °C in PBS, at 1 mL/min flow. A 100 µL of protein at ≈1 mg/mL concentration was injected and absorbance at 280 nm was monitored. Binding affinity of FGF1 wt, FGF1.F and FGF1.F-vcMMAE to FGFR1c was analyzed by Bio-Layer Interferometry (BLI). Analysis was performed at ForteBio Octet K2 and using Streptavidin Biosensors (SA) with immobilized biotinylated extracellular domain of FGFR1c fused to Fc fragment. Analysis was performed at 25 °C in PBS supplemented with 0.2% (w/v) BSA, 0.1% (w/v) PEG 3.5 kDa, 0.05% (v/v) Triton X-100 and 10 mM (NH4)2SO4. Standard black 96-well plates were used kept at 25 °C for 10 minutes for equilibration. Sensor was loaded with FGFR1c-Fc for 300 s, then blocked for next 300 s with biocytin at 0.04 mg/mL concentration. Sensor not loaded with FGFR1c-Fc was used as a reference. After 60 s of washing in a buffer, 200 s association started followed by 200 s of dissociation. For each protein/conjugate 20 nM, 40 nM and 80 nM concentrations were tested.

FGF1 stability in human serum FGF1 wt and FGF1.F were incubated at 5 ug/mL in human serum (H4522, Sigma-Aldrich) at 37 °C. Samples were taken after 0, 24, 48, 96 and 144 hours of incubation, boiled in Laemmli buffer, separated on SDS-PAGE and transferred to PVDF membrane. Membrane was probed with anti-FGF1 antibody (sc-1884, Santa Cruz Biotechnology) and goat secondary antibodies conjugated to HRP (Jackson ImmunoResearch) and a chemiluminescent substrate (Pierce) were used for visualization in a ChemiDoc station (BioRad).

Cell lines



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 30

Human fibroblasts BJ (ATCC) were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% bovine serum. Murine NIH 3T3fibroblasts (ATCC)were cultured in DMEM with 10% fetal bovine serum (FBS). Human gastric carcinoma SNU-16 (ATCC) cells were grown in RPMI-1640 medium with 10% FBS. Human osteosarcoma U2OS (ATCC) cells were cultured in McCoy’s 5a medium with 10% FBS. U2OS cells stably transfected with FGFR1 (U2OS-FGFR1) were a kind gift from Dr. Ellen M. Haugsten from Oslo University Hospital. They were cultured as untransfected U2OS apart from the addition of geneticin to 0.2 mg/mL.

Activation of FGF1 signaling pathways NIH 3T3 cells were seeded in a 6-well plate (250 000 cells/well). After 24 hours medium was changed for DMEM without serum. After 24 hours of serum starvation FGF1 variants or their conjugates were added to a final concentration of 100 ng/mL in the presence of 10 U/mL heparin. After 15 minutes of stimulation, cells were lysed with Laemmli buffer. The lysates were used for SDS-PAGE separation and Western blot analysis with anti-phospho-Erk1/2 (#9106, Cell Signaling Technology), anti-Erk1/2 kinase (#9102, Cell Signaling Technology) and anti-γ-tubulin antibodies (T6557, Sigma-Aldrich). Appropriate secondary antibodies conjugated to HRP (Jackson ImmunoResearch) and a chemiluminescent substrate (Pierce) were used for visualization in a ChemiDoc station (BioRad).

Cell viability assays NIH 3T3 cells were seeded in a 96-well plate (10 000 cells/well), after 24 hours medium was changed for DMEM without serum and cells were starved for the next 24 hours. The cells were then incubated for 48 hours with dilutions of FGF1 variants (6 pM – 60 nM) in the presence of 100 U/mL heparin. Three hours before the end of incubation AlamarBlue (Invitrogen) was added and the amount of reduced fluorescent product forming in live cells only was measured in an EnVision Multilabel Reader. U2OS, U2OS FGFR1, BJ and SNU-16 cells were seeded in 96-well plates (5000 cells/well) and after 24 hours dilutions ofFGF1.F-vcMMAE with 100 U/mL heparin and 100 µg/mL BSA were added. Cells were incubated for 72 hours, the last 3 hours in the presence of AlamarBlue and fluorescence was measured as above.

Confocal microscopy FGF1.F and its vcMMAE conjugate were labeled with DyLight 550 NHS ester according to manufacturer’s protocol. U2OS cells were stained with CellTrace Violet (Thermo Fisher Scientific),


Page 19 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


mixed with unlabeled U2OS-FGFR1 cells in a 1:1 ratio, seeded on coverslips and grown to 70% confluence. Then, DyLight™ 550 labeled FGF1.F or its conjugate were added to 5 µg/mL and heparin to 50 U/mL and the cells were incubated at 37°C for 15 minutes. Cells were washed with PBS, fixed with 4% paraformaldehyde, then permeabilized at 4°C with 0.5% Triton 100-X and blocked in PBS containing 0.01% Triton X100 and 1% BSA, 10% normal goat serum and 0.3M glycine. Next, the cells were treated with rabbit anti-EEA1 antibodies (2411S, BD Biosciences Transduction Laboratories) followed by secondary antibodies labeled with AlexaFluor488(ab15150077, Abcam). Slides were mounted in ProLongGold Antifade Mountant and viewed under Zeiss Cell Observer SD confocal microscope with an EMCCD QImaging Rolera EM-C2 camera using 1x1 binning (0.106 µm image pixel size) and a 63× oil immersion objective. Images were processed with a Fiji software. Pictures shown are merged z-stacks.

Acknowledgements This

work

was

supported

by

the

National

Science

Centre,

Poland,

grant

number

2011/02/A/NZ1/00066 (NCN Maestro). We would like to thank Dr. Grzegorz Chodaczek for technical expertise with fluorescence microscopy, Ms. Marta Minkiewicz for her skillful help with cell culture, and Dr. Ellen M. Haugsten for the stably transfected U2OS FGFR1 cell line.

Abbreviations ADC, Antibody-Drug Conjugates; FGF1, fibroblast growth factor 1; FGFR, fibroblast growth factor receptor; MMAE, monomethylauristatin E

Notes The authors declare no competing financial interest.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 30

Literature (1) Chari, R. V. J. (2008) Targeted cancer therapy: Conferring specificity to cytotoxic drugs. Acc. Chem. Res. 41, 98–107. (2) Sievers, E. L., and Senter, P. D. (2013) Antibody-drug conjugates in cancer therapy. Annu. Rev. Med. 64, 15–29. (3) Tolcher, A. W. (2016) Antibody drug conjugates: lessons from 20 years of clinical experience. Ann. Oncol. 27, 2168–2172. (4) Lazar, A. C., Wang, L., Blattler, W. A., Amphlett, G., Lambert, J. M., and Zhang, W. (2005) Analysis of the composition of immunoconjugates using size-exclusion chromatography coupled to mass spectrometry. Rapid Commun. Mass Spectrom. 19, 1806–1814. (5) Hamblett, K. J., Senter, P. D., Chace, D. F., Sun, M. M., Lenox, J., Cerveny, C. G., Kissler, K. M., Bernhardt, S. X., Kopcha, A. K., Zabinski, R. F., et al. (2004) Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate. Clin Cancer Res 10, 7063–7070. (6) Junutula, J. R. J. R., Raab, H., Clark, S., Bhakta, S., Leipold, D. D. D., Weir, S., Chen, Y., Simpson, M., Tsai, S. P. S. P., Dennis, M. S. M. S., et al. (2008) Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat. Biotechnol. 26, 925–32. (7) Szlachcic, A., Zakrzewska, M., Lobocki, M., Jakimowicz, P., and Otlewski, J. (2016) Design and characteristics of cytotoxic fibroblast growth factor 1 conjugate for fibroblast growth factor receptor-targeted cancer therapy. Drug Des. Devel. Ther. 10, 2547–2560. (8) Zakrzewska, M., Krowarsch, D., Wiedlocha, A., Olsnes, S., and Otlewski, J. (2005) Highly stable mutants of human fibroblast growth factor-1 exhibit prolonged biological action. J. Mol. Biol. 352, 860–875. (9) Heitmann, P. (1968) Reactivity of sulfhydryl groups in micelles. A model for protein. Eur. J. Biochem. 5, 305–15. (10) Parente, A., Merrifield, B., Geraci, G., and D’Alessio, G. (1985) Molecular basis of superreactivity of cysteine residues 31 and 32 of seminal ribonuclease. Biochemistry 24, 1098–1104. (11) Jensen, K. S., Pedersen, J. T., Winther, J. R., and Teilum, K. (2014) The pKa value and accessibility of cysteine residues are key determinants for protein substrate discrimination by glutaredoxin. Biochemistry 53, 2533–2540.


Page 21 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(12) Shen, B.-Q., Xu, K., Liu, L., Raab, H., Bhakta, S., Kenrick, M., Parsons-Reponte, K. L., Tien, J., Yu, S.F., Mai, E., et al. (2012) Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates. Nat. Biotechnol. 30, 184–189. (13) Dabora, J. M., Sanyal, G., and Middaugh, C. R. (1991) Effect of polyanions on the refolding of human acidic fibroblast growth factor. J. Biol. Chem. 266, 23637–23640. (14) Wiedlocha, A., Madshus, I. H., Mach1, H., Middaugh ’, R., and Oisnes, S. (1992) Tight folding of acidic fibroblast growth factor prevents its translocation to the cytosol with diphtheria toxin as vector. EMBO J. 1, 4835–4842. (15) Shin, E. Y., Lee, B. H., Yang, J. H., Shin, K. S., Lee, G. K., Yun, H. Y., Song, Y. J., Park, S. C., and Kim, E. G. (2000) Up-regulation and co-expression of fibroblast growth factor receptors in human gastric cancer. J. Cancer Res. Clin. Oncol. 126, 519–528. (16) Polakis, P. (2016) Antibody Drug Conjugates for Cancer Therapy. Pharmacol. Rev. 68, 3–19. (17) Xu, S. (2015) Internalization, Trafficking, Intracellular Processing and Actions of Antibody-Drug Conjugates. Pharm. Res. 32, 3577–3583. (18) Firer, M. A., and Gellerman, G. (2012) Targeted drug delivery for cancer therapy: the other side of antibodies. J. Hematol. Oncol. 5, 70. (19) Katoh, M., and Nakagama, H. (2014) FGF Receptors: Cancer Biology and Therapeutics. Med. Res. Rev. 34, 280–300. (20) Inokuchi, M., Fujimori, Y., Otsuki, S., Sato, Y., Nakagawa, M., and Kojima, K. (2015) Therapeutic Targeting of Fibroblast Growth Factor Receptors in Gastric Cancer. Gastroenterol. Res. Pract. 2015, 1–8. (21) Hynes, N. E., and Dey, J. H. (2010) Potential for targeting the fibroblast growth factor receptors in breast cancer. Cancer Res 70, 5199–5202. (22) Dieci, M. V, Arnedos, M., Andre, F., and Soria, J. C. (2013) Fibroblast Growth Factor Receptor Inhibitors as a Cancer Treatment: From a Biologic Rationale to Medical Perspectives. Cancer Discov. 3, 264–279. (23) Hallinan, N., Finn, S., Cuffe, S., Rafee, S., O’Byrne, K., and Gately, K. (2016) Targeting the fibroblast growth factor receptor family in cancer. Cancer Treat. Rev. 46, 51–62. (24) Kim, E. G., and Kim, K. M. (2015) Strategies and Advancement in Antibody-Drug Conjugate Optimization for Targeted Cancer Therapeutics. Biomol. Ther. (Seoul). 23, 493–509.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 30

(25) Sochaj, A. M., Świderska, K. W., and Otlewski, J. (2015) Current methods for the synthesis of homogeneous antibody-drug conjugates. Biotechnol. Adv. 33, 775–784. (26) McDonagh, C. F., Turcott, E., Westendorf, L., Webster, J. B., Alley, S. C., Kim, K., Andreyka, J., Stone, I., Hamblett, K. J., Francisco, J. A., and Carter, P. (2006) Engineered antibody-drug conjugates with defined sites and stoichiometries of drug attachment. Protein Eng. Des. Sel. 19, 299–307. (27) Axup, J. Y., Bajjuri, K. M., Ritland, M., Hutchins, B. M., Kim, C. H., Kazane, S. A., Halder, R., Forsyth, J. S., Santidrian, A. F., Stafin, K., et al. (2012) Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proc. Natl. Acad. Sci. 109, 16101–16106. (28) Hallam, T. J., Wold, E., Wahl, A., and Smider, V. V. (2015) Antibody conjugates with unnatural amino acids. Mol. Pharm. 12, 1848–1862. (29) Boeggeman, E., Ramakrishnan, B., Pasek, M., Manzoni, M., Puri, A., Loomis, K. H., Waybright, T. J., and Qasba, P. K. (2009) Site specific conjugation of fluoroprobes to the remodeled Fc N-glycans of monoclonal antibodies using mutant glycosyltransferases: Application for cell surface antigen detection. Bioconjug. Chem. 20, 1228–1236. (30) Zhu, Z., Ramakrishnan, B., Li, J., Wang, Y., Feng, Y., Prabakaran, P., Colantonio, S., Dyba, M. A., Qasba, P. K., and Dimitrov, D. S. (2014) Site-specific antibody-drug conjugation through an engineered glycotransferase and a chemically reactive sugar. MAbs 6, 1190–1200. (31) Strop, P., Liu, S. H., Dorywalska, M., Delaria, K., Dushin, R. G., Tran, T. T., Ho, W. H., Farias, S., Casas, M. G., Abdiche, Y., et al. (2013) Location matters: Site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates. Chem. Biol. 20, 161–167. (32) Dennler, P., Chiotellis, A., Fischer, E., Brégeon, D., Belmant, C., Gauthier, L., Lhospice, F., Romagne, F., and Schibli, R. (2014) Transglutaminase-based chemo-enzymatic conjugation approach yields homogeneous antibody-drug conjugates. Bioconjug. Chem. 25, 569–578. (33) Beerli, R. R., Hell, T., Merkel, A. S., and Grawunder, U. (2015) Sortase Enzyme-Mediated Generation of Site-Specifically Conjugated Antibody Drug Conjugates with High In Vitro and In Vivo Potency. PLoS One (Hagemeyer, C. E., Ed.) 10, e0131177. (34) Goh, L. K., and Sorkin, A. (2013) Endocytosis of Receptor Tyrosine Kinases. Cold Spring Harb. Perspect. Biol. 5, a017459–a017459. (35) Harper, J., Mao, S., Strout, P., and Kamal, A. (2013) Selecting an optimal antibody for antibodydrug conjugate therapy: Internalization and intracellular localization. Methods Mol. Biol. 1045, 41–


Page 23 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


49. (36) Liao-Chan, S., Daine-Matsuoka, B., Heald, N., Wong, T., Lin, T., Cai, A. G., Lai, M., D’Alessio, J. A., and Theunissen, J.-W. (2015) Quantitative Assessment of Antibody Internalization with Novel Monoclonal Antibodies against Alexa Fluorophores. PLoS One 10, e0124708. (37) Xia, X., Babcock, J. P., Blaber, S. I., Harper, K. M., and Blaber, M. (2012) Pharmacokinetic Properties of 2nd-Generation Fibroblast Growth Factor-1 Mutants for Therapeutic Application. PLoS One (Houtman, J. C., Ed.) 7, e48210. (38) Thomas, K. A., and Gimenez-Gallego, G. (1986) Fibroblast growth factors: broad spectrum mitogens with potent angiogenic activity. Trends Biochem. Sci. 11, 81–84. (39) Zakrzewska, M., Krowarsch, D., Wiedlocha, A., and Otlewski, J. (2004) Design of fully active FGF1 variants with increased stability. Protein Eng. Des. Sel. 17, 603–611.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table of Contents Graphic


Page 24 of 30

Page 25 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Diagram shows introduced mutations in fibroblast growth factor 1 variants. Circles stand for stabilizing mutations (Q40P/S47I/H93G). Triangles represent cysteine substitution mutations (C16S/C83S/C117S). Two types of extensions were introduced: CGGG at the N-terminus and KCKSGG or GGSKCK either at the Nor C- terminus of protein respectively. In the case of the N-terminal KCKSGG extension we found out that the initiator methionine was not cleaved off, so the extension was in fact MKCKSGG. 82x38mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(A) Tryptophan fluorescence spectra of FGF1 variants in sodium phosphate buffer pH 7.4 and (B) in sodium phosphate buffer containing 150 mM ammonium sulfate. Spectra were normalized to the tyrosine signal at 304 nm. Spectrum of FGF1 fully denaturated by 6M guanidine hydrochloride is included as a reference. (C) Activation of FGFR-related signaling pathway (Erk1/2 kinases) by different variants of FGF1. Western blot analysis of Erk1/2 phosphorylation in starved NIH 3T3 cells after stimulation with FGF1 variants at 10 ng/mL. 81x188mm (300 x 300 DPI)


Page 26 of 30

Page 27 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


SDS-PAGE separation of conjugation reaction products for FGF1 variants. Conjugation yield was based on integration of chromatographic peaks after separation of reaction mixtures on a C18 RP-HPLC column. For FGF1 WT and FGF1.A yield was below detection, in case of FGF1.B precipitation made measurement impossible. 149x97mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(A) HIC separation of conjugation reaction mixture. Separation was performed on TSKgel phenyl 5PW column in 100 mM phosphate buffer pH 7.2 in a gradient of ammonium sulfate from 1.2 M to 0 M. (B) SDSPAGE evaluation of purification process. (C) Tryptophan fluorescence spectra of reaction mixture and FGF1.F-vcMMAE conjugate after purification. Fluorescence intensity was normalized to tyrosine signal. 133x170mm (300 x 300 DPI)


Page 28 of 30

Page 29 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(A) Mitogenic effect of FGF1 wt and FGF1.F expressed as viability of starved NIH 3T3 cells after 48 hours of exposure to FGF1. (B) Western blot analysis of Erk1/2 activation by FGF1 WT, FGF1.F and FGF1.F-vcMMAE conjugate at 10 ng/mL. (C) Internalization of FGF1.F and its vcMMAE conjugate in co-culture of FGFRnegative U2OS cells (blue) and U2OS cells stably transfected with FGFR1 (unstained). Cells were treated with 50 ng/mL of conjugate stained with DyLight550 (red). After 15 minutes, cells were fixed and stained with anti-EEA1 antibody (green). 313x234mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Viability of BJ (A), U2OS FGFR1 (B) and SNU-16 cells (C) after treatment with different concentrations of FGF1.F-vcMMAE for 72 hours. U2OS cell line with very low FGFR level was used as a control. Cytotoxicity of the conjugate was determined by AlamarBlue assay. 177x46mm (300 x 300 DPI)


Page 30 of 30

High-Yield Site-Specific Conjugation of Fibroblast Growth Factor 1

Recommend Documents