FGFR3 binding molecules

Abstract

The present invention relates to a polypeptide binding to fibroblast growth factor receptor 3 isoforms 3b and 3c (FGFR3b and FGFR3c), wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: (a) GVTLFVALYDYEVYGPTPMLSFHKGEKFQIL(X.sup.1)(X.sup.2)(X.sup.3) (X.sup.4)GPYWEARSL(X.sup.5)TGETG(X.sup.6)IPSNYVAPVDSIQ (SEQ ID NO: 1), wherein amino acid positions (X1) to (X.sup.6) may be any amino acid sequence; (b) an amino acid sequence which is at least 95% identical to the amino acid sequence of (a), wherein the identity determination excludes amino acid positions (X.sup.1) to (X.sup.6) and provided that the amino acid sequence EVYGPTPM (SEQ ID NO: 2) in amino acid positions 12 to 19 of SEQ ID NO: 1 is conserved and the amino acids P and Y in amino acid positions 37 and 38 of SEQ ID NO: 1 are conserved; (c) GVTLFVALYDYEVMSTTALSFHKGEKF QILSQSPHGQYWEARSLTTGETG(X.sup.6)IPSNYVAPVDSIQ (SEQ ID NO: 19), wherein the amino acid position (X.sup.6) may be any amino acid; and (d) an amino acid sequence which is at least 95% identical to the amino acid sequence of (c), wherein the identity determination excludes amino acid position (X.sup.6) and provided that the amino acid sequences EVMSTTA (SEQ ID NO: 20) in amino acid positions 12 to 18 of SEQ ID NO: 19 and SQSPH (SEQ ID NO: 21) in amino acid positions 31 to 35 of SEQ ID NO: 19 are conserved and the amino acids Q and Yin amino acid positions 37 and 38 of SEQ ID NO: 19 are conserved.

Claims

1. A nucleic acid molecule encoding a polypeptide binding to fibroblast growth factor receptor 3 isoforms 3b and 3c (FGFR3b and FGFR3c), wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: TABLE-US-00010 (a) (SEQ ID NO: 1) GVTLFVALYDYEVYGPTPMLSFHKGEKFQIL(X.sup.1)(X.sup.2)(X.sup.3)(X.sup.4)GPYW EARSL(X.sup.5)TGETG(X.sup.6)IPSNYVAPVDSIQ; wherein amino acid positions (X.sup.1) to (X.sup.6) may be any amino acid sequence; and (b) an amino acid sequence which is at least 95% identical to the amino acid sequence of (a), wherein the identity determination excludes amino acid positions (X.sup.1) to (X.sup.6) and provided that the amino acid sequence EVYGPTPM (SEQ ID NO: 2) in amino acid positions 12 to 19 of SEQ ID NO: 1 is conserved and the amino acids P and Y in amino acid positions 37 and 38 of SEQ ID NO: 1 are conserved; TABLE-US-00011 (c) (SEQ ID NO: 19) GVTLFVALYDYEVMSTTALSFHKGEKFQILSQSPHGQYWEARSLTTGETG (X.sup.6)IPSNYVAPVDSIQ, wherein the amino acid position (X.sup.6) may be any amino acid; and (d) an amino acid sequence which is at least 95% identical to the amino acid sequence of (c), wherein the identity determination excludes amino acid position (X.sup.6) and provided that the amino acid sequences EVMSTTA (SEQ ID NO: 20) in amino acid positions 12 to 18 of SEQ ID NO: 19 and SQSPH (SEQ ID NO: 21) in amino acid positions 31 to 35 of SEQ ID NO: 19 are conserved and the amino acids Q and Y in amino acid positions 37 and 38 of SEQ ID NO: 19 are conserved.

2. The nucleic acid of claim 1, wherein (X.sup.1) is N, R, or K, and is preferably R or K; (X.sup.2) is S, G, K or R, and is preferably G, K or R; (X.sup.3) is S or G, and is preferably G; (X.sup.4) is E, Q, D, S or K, and is preferably Q, D, S or K; (X.sup.5) is T or A; and (X.sup.6) is Y, W or L, and is preferably L or W.

3. The nucleic acid of claim 1, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of any one of SEQ ID NOs 3 to 8 and 22.

4. A nucleic acid encoding a fusion construct comprising the polypeptide of claim 3 fused to a further compound, wherein the further compound comprises an antibody light chain, an antibody heavy chain, an Fc domain of an antibody, an antibody, or a combination thereof.

5. The nucleic acid of claim 1, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 22.

6. A nucleic acid encoding a fusion construct comprising the polypeptide of claim 5 fused to a further compound, wherein the further compound comprises an antibody light chain, an antibody heavy chain, an Fc domain of an antibody, an antibody, or a combination thereof.

7. A nucleic acid encoding a fusion construct comprising the polypeptide of claim 1 fused to a further compound, wherein the further compound comprises an antibody light chain, an antibody heavy chain, an Fc domain of an antibody, an antibody, or a combination thereof.

8. The nucleic acid of claim 7, wherein the antibody is directed against a target selected from the group consisting of GM-2, CD38, ICAM-1, SLAMF7, CD45, CD40, CD74, IGFR-1, CD20, BAFF, BCMA, CD66, GRP78, CXCR4, EGFR, EPCAM, TROP-2, B7H3 and CEACAM-1.

9. A vector comprising the nucleic acid molecule according to any one of claims 1 to 6.

10. An isolated cell comprising the vector of claim 9.

11. An isolated cell comprising the nucleic acid molecule according to any one of claims 1 to 6.

12. A method of producing the polypeptide encoded by the nucleic acid of any one of claims 1 to 6 comprising (a) culturing the isolated cell comprising the nucleic acid; and (b) isolating the produced polypeptide.

Description

(1) The above considerations apply mutatis mutandis to all appended claims. The figures show:

(2) FIG. 1: Alignment of the anti-FGFR3 Fynomers of SEQ ID NOs 3 to 8.

(3) FIG. 2: Internalization properties of anti-FGFR3 Fynomers.

(4) FIG. 3: Size exclusion profiles of Fyn SH3-derived FGFR3-binding polypeptides: A) FF2L4C3—SEQ ID NO. 3; B) FF44L65G12—SEQ ID NO. 4; C) FF44L65G7—SEQ ID NO. 5; D) FF48L66G7—SEQ ID NO. 6; E) FF43L65D5—SEQ ID NO. 7; F) FF44L65B7—SEQ ID NO. 8.

(5) FIG. 4: FACS binding of Fyn-SH3 derived polypeptides binding to FGFR3 to: A) FGFR3-positive KMS-11 cells and B) FGFR3-negative N87 cells.

(6) FIG. 5: Specificity ELISA of Fyn SH3-derived FGFR3-binding polypeptides: A) FF2L4C3—SEQ ID NO. 3; B) FF44L65G12—SEQ ID NO. 4; C) FF44L65G7—SEQ ID NO. 5; D) FF48L66G7—SEQ ID NO. 6; E) FF43L65D5—SEQ ID NO. 7; F) FF44L65B7—SEQ ID NO. 8. Antigens tested: huFGFR3b=human FGFR3 splice variant b; huFGFR3c=human FGFR3 splice variant c; cyFGFR3c=cynomolgus FGFR3 splice variant c; muFGFR3c=murine FGFR3 splice variant c; huFGFR3-D1=domain 1 of human FGFR3; huFGFR3-D2=domain 2 of human FGFR3; huFGFR3-D1D2=domain 1 and domain 2 of human FGFR3; IgG=polyclonal human IgG mix; PBS=phosphate buffered saline solution.

(7) FIG. 6: Internalization assay showing cytotoxic effect of Fyn SH3-derived FGFR3-binding polypeptides.

(8) The examples illustrate the invention.

Example 1: Fyn SH3-Derived Polypeptides Binding to FGFR3b and FGFR3c

(9) With the aim to be able to target FGFR3, we set out to obtain specific Fyn SH3-derived binding molecules thereto. Since the distribution and the expression of the two different splice variants (FGFR3b and FGFR3c) on different tumor cells is not fully understood, it is important that the Fyn SH3-derived polypeptides are able to bind both.

(10) Using recombinant human FGFR3b-Fc (SEQ ID NO: 12) and FGFR3c-Fc (SEQ ID NO: 13) as targets, we successfully selected and isolated several families of Fyn SH3-derived binding proteins that are capable of binding to both splice variants of human FGFR3 (SEQ ID NOs 9 and 10). We continued with the most promising candidate family for further studies.

(11) Interestingly, a Fyn SH3-derived polypeptide referred to as FF2L4C3 (SEQ ID NO: 3), carrying the RT-loop sequence “EVYGPTP” (SEQ ID NO: 2), was enriched during the selection process and showed the most promising internalization properties among 29 tested anti-FGFR3 Fynomers (see FIG. 2). In more detail, 5 other sequence families were excluded from further analysis. The Fynomers belonging to the most promising sequence family showed the best affinities and internalization properties.

(12) In order to obtain Fyn SH3-derived FGFR3 binders with higher affinities and improved internalization properties, FF2L4C3 (SEQ ID NO: 3) was used as template for affinity maturation. The RT-loop sequence “EVYGPTP” (SEQ ID NO: 2) was kept constant and was combined with a randomized n-src-loop repertoire (where a stretch of 4 to 6 randomized amino acid residues were introduced at the positions (V) to (X.sup.4) in SEQ ID NO: 1). The process of affinity maturation library generation was essentially the same as described for cloning of the naïve library with a randomized n-src-loop (“library 0” as described in [25]).

(13) After naïve and affinity maturation selections, enriched Fyn SH3-derived polypeptides were screened for binding to FGFR3 by lysate ELISA. DNAs encoding the Fyn SH3-derived binding proteins were cloned into the bacterial expression vector pQE12 (Qiagen) so that the resulting constructs carried a C-terminal myc-hexahistidine tag as described in Grabulovski et al. [26]. The polypeptides were expressed in the cytosol of E. coli bacteria in a 96-well format and 200 μl of cleared lysate per well was prepared as described in Bertschinger et al. [27]. Briefly, transformed bacterial colonies were picked from the agar plate and grown in a round bottom 96-well plate (Nunc, cat. no. 163320) in 200 μl 2×YT medium containing 100 μg/ml ampicillin and 0.1% (w/v) glucose. Protein expression was induced after growth for 3 h at 37° C. and rotational shaking at 200 r.p.m. by adding 1 mM IPTG (Applichem, Germany). Proteins were expressed overnight in a rotary shaker (200 r.p.m., 30° C.). Subsequently, the 96-well plate was centrifuged at 1800 g for 10 min and the supernatant was discarded. Bacterial pellets were lysed using BugBuster® plus Benzonase® (Millipore 70750-3) and lysates were subsequently cleared by centrifugation for 10 min at 1800×g. 60 μl lysate were mixed with 170 μl PBS and filtered through a 0.45 μm Multiscreen filter plate (Millipore MSHVN4510), in order to eliminate any residual bacterial debris.

(14) Monoclonal bacterial lysates were used for ELISA. For the ELISA, Maxisorp plates were coated overnight with either 5 μg/ml huFGFR3b-Fc (SEQ ID NO: 12), 5 μg/ml huFGFR3c-Fc (SEQ ID NO: 13) or 5 μg/ml poly IgG and blocked for at least 1 h with 2% MPBS. Cleared lysates containing soluble Fynomer with a C-terminal myc- and hexahistidine peptide tag were added in 2% MPBS containing murine monoclonal anti-myc tag antibody, clone 9E10 (Roche Applied Science 11 667 203 001) to the maxisorp plates. Bound Fynomer was detected via 9E10 by an anti-mouse IgG-horse radish peroxidase conjugate (Sigma-Aldrich A2554). The detection of peroxidase activity was done by adding BM blue POD substrate (Roche) and the reaction was stopped by adding 1 M H2504.

(15) The DNA sequence of the specific binders was verified by DNA sequencing.

(16) Results

(17) The amino acid sequences of ELISA positive Fyn SH3-derived preferred polypeptides binding to FGFR3b and FGFR3c are presented in SEQ ID NOs: 3 to 8 as appended in the sequence listing. The Fyn-SH3 derived polypeptides SEQ ID NOs: 4 to 8 are a selection of binders from a large pool of molecules that were obtained after affinity maturation of FF2L4C3 (SEQ ID NO: 3), and are presented here because of their improved affinities and internalization properties (as shown in example 2 and 5).

(18) In more detail, more than 80 Fynomers derived from SEQ ID NO: 3 were characterized with respect to their biophysical properties, affinities and internalization properties. SEQ ID NOs: 4 to 8 are the binders with the best set of properties in terms of biophysical, affinities and internalization.

Example 2: Expression of Fyn SH3-Derived FGFR3 Binding Polypeptides of the Invention

(19) This example shows the expression yields of the preferred Fyn SH3-derived FGFR3-binding polypeptides and the characterization of these polypeptides by size exclusion chromatography.

(20) Methods

(21) a) Expression Yields of Fyn SH3-Derived FGFR3-Binding Polypeptides

(22) Fyn SH3-derived FGFR3-binding polypeptides were expressed in the cytosol of TG1 E. coli bacteria and were purified as described in Grabulovski et al. [2].

(23) b) Size Exclusion Chromatography (SEC)

(24) Samples were analyzed by size exclusion chromatography (TOSOH TSKgel PW G3000PWxL; Agilent 1260 Infinity HPLC; PBS pH 7.4 mobile phase). 10 μl of undiluted sample was injected onto the column, and the resulting profiles were analyzed.

(25) Results

(26) a) Expression Yields

(27) The expression yields for monomeric Fyn SH3-derived FGFR3-binding polypeptides of the invention ranged from 15 to 51 mg/liter of bacterial culture under non-optimized conditions in shake flasks (Table 1), and are in a typical range for a Fyn SH3-derived polypeptide.

(28) TABLE-US-00004 TABLE 1 Expression yields of Fyn SH3-derived FGFR3-binding polypeptides produced in TGI E. coli bacteria Fynomer SEQ ID NO Yield (mg/l) FF2L4C3 3 18 FF44L65G12 4 37 FF44L65G7 5 15 FF48L66G7 6 58 FF43L65D5 7 32 FF44L65B7 8 51
b) Size Exclusion Chromatography (SEC)

(29) Size exclusion chromatography (SEC) profiles demonstrated that all constructs eluted mainly as single, monomeric peaks (FIG. 3). This generally indicates good biophysical properties, which is advantageous from manufacturing perspective of Fynomers (Fyn SH3-derived binding molecules, including Fynomabs, which are fusion proteins with antibodies), and is in line with earlier observations of Fyn SH3-derived molecules.

Example 3: Fyn SH3-Derived Polypeptides of the Invention Bind to Human FGFR3b and FGFR3c with High Affinities

(30) This example shows the characterization of the preferred Fyn SH3-derived FGFR3-binding polypeptides by surface plasmon resonance and flow cytometry experiments.

(31) Methods

(32) a) Affinity Measurements by BIAcore

(33) Affinities were measured using a BIAcore T200 instrument. One flow cell on a CM5 series S chip (GE Healthcare BR-1005-30) was coated with the anti—myc antibody 9E10 (Roche 11 667 203 001; coating density ranging between 6000 and 8000 RU) using the amine coupling kit (GE Healthcare BR100633).

(34) The parental Fynomer FF2L4C3 (SEQ ID NO:3), at a concentration of 500 nM, and the Fynomers with SEQ ID NOs: 4-8, at a concentration of 100 nM, were captured on the 9E10 surface followed by injections of different concentrations of huFGFR3b-Fc (SEQ ID NO: 12), huFGFR3c-Fc (SEQ ID NO: 13) or cynoFGFR3c-Fc (SEQ ID NO: 14) (0 nM, 3.9 nM, 7.8 nM, 15.6 nM, 31.25 nM, 62.5 nM, 125 nM, 250 nM and 500 nM for the measurements of the parental Fynomer FF2L4C3, 0 nM, 0.046 nM, 0.14 nM, 0.41 nM, 1.2 nM, 3.7 nM, 11.1 nM, 33.3 nM and 100 nM for Fynomers with SEQ ID NO: 4-8). Sensograms were recorded and apparent kinetics constants were determined by curve fitting using the 1.1 Langmuir interaction model in the BIAevaluation 2.1 software.

(35) b) Affinity Measurements by Flow Cytometry

(36) Binding of Fynomers to huFGFR3 on cells was analyzed by flow cytometry using KMS-11 cells (JCRB1179) as FGFR3-positive cells and N87 (ATCC, CRL-5822) as a FGFR3-negative control cell line. Both KMS-11 and N87 cells were maintained in RPMI1640 medium (Invitrogen 52400-25). All media were supplemented with 25 U/ml penicillin, 25 μg/ml streptomycin and 10% FCS. To harvest the semi-adherent KMS-11 cells from a T150 flask, the supernatant was removed into a 50 ml falcon tube, and the cells were washed with 10 ml PBS, which was also added to the falcon tube. 2 ml of Accutase (Sigma A6964) was added to the flask, and incubated for 10 min at 37° C. The Accutase was inactivated with the addition of 10 ml medium and added to the falcon tube, which was then centrifuged (250×g, 5 min) to pellet the cells. The cells were resuspended in FACS buffer (PBS+1% FCS+0.2% sodium azide) to a cell concentration of 1×10.sup.6 cells/ml and 100 μl was used per well (1×10.sup.5 cells/well) for the flow cytometry staining in a 96-well round bottomed plate (Nunc 163320). For adherent N87 cells the supernatant and wash were discarded, and only the Accutase-detached cells were collected and prepared.

(37) Fynomers were co-incubated with the mouse anti-myc antibody (clone 9E10; Roche 11667149001) to allow cross linking of myc-tagged Fynomers prior to cell binding. Fynomers were diluted to 1 μM and co-incubated with 667 nM 9E10 anti-myc antibody (3:2 molar ratio) in FACS buffer, for approximately 10 minutes on ice.

(38) This mixture was serially diluted 1 in 4 down to a Fynomer concentration of 0.06 nM (8 concentrations in total). Controls included the secondary antibody 9E10 only (no Fynomer; 667 nM 9E10), cells only (FACS buffer only) and an anti-FGFR3 antibody (R&D systems; cat. No. MAB766) at a concentration of 10 nM. Cells were centrifuged in the 96-well plate (250×g, 5 min) and were resuspended with the samples indicated above, before incubation on ice for 1 hr. The plate was centrifuged and washed (PBS+0.2% sodium azide), before centrifuging again. Then 50 μl of the secondary antibody anti-mIgG Alexa488 (Life Technologies A21202) was added to the cells at a concentration of 4 μg/ml, before incubating in the dark, on ice, for 45 min. The plate was centrifuged and washed twice with PBS+0.2% sodium azide, before resuspending in FACS buffer and FACS analysis (Millipore Guava easyCyte 8HT).

(39) FACS data analysis was performed using Prism 6. The data was transformed (X=log X), and analysed using a non-linear fit, log(agonist) vs. response—Variable slope (4 parameters).

(40) Results

(41) a) Affinity Measurements by BIAcore

(42) The binding properties were analyzed by real-time interaction analysis on a BIAcore chip revealing the following dissociation constants (1(D) for selected FGFR3-binding polypeptides:

(43) TABLE-US-00005 TABLE 2 Apparent kinetics constants of the binding of Fyn SH3- derived FGFR3-binding polypeptides to recombinant human FGFR3b, human FGFR3c and cynomolgus FGFR3c. SEQ ID huFGFR3b KD.sub.app huFGFR3c KD.sub.app cyFGFR3c KD.sub.app Fynomer NO. (pM) (pM) (pM) FF2L4C3 3 4700 4600 5900 FF44L65G12 4 690 685 110 FF44L65G7 5 470 335 280 FF48L66G7 6 260 190 210 FF43L65D5 7 335 230 160 FF44L65B7 8 100 250 260

(44) The measured apparent affinities (Table 2) of the Fyn SH3-derived polypeptides (SEQ ID NOs: 3 to 8) binding to FGFR3b and FGFR3c (SEQ ID NOs 9 and 10) are surprisingly high considering the fact that sub-nanomolar values were obtained after only one round of affinity maturation. Moreover, these measurements confirmed the comparable binding properties of the Fyn SH3 derived polypeptides (SEQ ID NOs: 3 to 8) to both human isoforms of FGFR3 (FGFR3b and FGFR3c; SEQ ID NOs 9 and 10), and to cynomolgus FGFR3c (binding to cynomolgus FGFR3b was not tested).

(45) b) Affinity Measurements by Flow Cytometry

(46) The binding properties were analyzed by flow cytometry using FGFR3-positive KMS-11 cells and FGFR3-negative N87 cells as negative control. The following EC50 values for selected FGFR3-binding polypeptides were measured as shown in Table 3 and FIG. 4.

(47) TABLE-US-00006 TABLE 3 EC50 values determined on FGFR3-positive KMS-11 cells for Fyn SH3-derived FGFR3-binding polypeptides. Fynomer SEQ ID NO EC50 (nM) FF2L4C3 3 5.9 FF44L65G12 4 2.2 FF44L65G7 5 4.2 FF48L66G7 6 2.3 FF43L65D5 7 1.2 FF44L65B7 8 0.6

(48) EC50 values, in the low nanomolar range (Table 3), measured on a cell line expressing FGFR3 (FIG. 4A, KMS-11) confirmed the high apparent affinities measured by surface plasmon resonance (Table 2), and demonstrate binding to FGFR3 in the natural context of a cell surface. All the Fyn SH3-derived polypeptides (SEQ ID NOs: 3 to 8) binding to FGFR3 did not show unspecific binding on a cell line not expressing FGFR3 (FIG. 4B).

Example 4: Fyn SH3-Derived Polypeptides of the Invention Specific to FGFR3 do not Interfere with Ligand Binding

(49) It would be preferred if the Fyn SH3-derived polypeptides for binding both isoforms FGFR3b and FGFR3c to not interfere with ligand (e.g. FGF1) binding, as the ligand binding site is located in proximity to the splice site give rise to either FGFR3b or FGFR3c.

(50) For the purpose of verifying the ability of the Fyn SH3-derived polypeptides to bind to FGFR3 in presence of one of its ligands, a BIAcore experiment was set up to measure the affinity of the Fynomers to FGFR3 in presence or absence of FGF1 (Fibroblast Growth Factor 1 is one of the major ligands of FGFR3).

(51) In analogy to the method used for measuring the affinities (as described in Example 3) Fynomers at concentration of 100 nM (with the exception of FF2L4C3—SEQ ID NO. 3 used at a concentration of 500 nM) were captured on the 9E10 surface followed by injections of different concentrations of huFGFR3c-Fc (SEQ ID NO: 13) (0 nM, 11 nM, 33 nM, 100 nM) in presence or absence of 200 nM FGF1 (R&D systems 232-FA-025/CF). Sensograms were recorded and apparent kinetics constants were calculated using the BIAevaluation 2.1 software.

(52) Results

(53) Independently of the presence or absence of 200 nM FGF1 in solution binding of the Fyn SH3-derived polypeptides to huFGFR3c was unchanged, showing that binding of the Fynomers to FGFR3 did not interfere with ligand binding.

(54) TABLE-US-00007 TABLE 4 Shows the kinetics constants obtained in presence or absence of 200 nM FGF1. KD.sub.app (pM) to huFGFR3c-Fc KDapp (pM) to huFGFR3c- (SEQ ID NO: 13) in presence Fynomer SEQ ID NO. Fc (SEQ ID NO: 13) of 200 nM GF1 FF2L4C3 3 4000 3800 FF44L65G12 4 140 120 FF44L65G7 5 320 230 FF48L66G7 6 170 130 FF43L65D5 7 60 70 FF44L65B7 8 170 130

(55) Even though, due to assay variability the values for KD.sub.app in absence of FGF1 are slightly different than the values obtained in the experiment shown in Example 2 (Table 2), this experiment shows that the Fyn SH3-derived polypeptides are able bind to FGFR3 even if the ligand (in this case FGF1) is bound to the ligand binding site.

(56) From this we conclude that the epitope bound by the Fyn SH3-derived polypeptides described here is located in a constant region of FGFR3.

Example 5: Fyn SH3-Derived Polypeptides of the Invention Bind to the Domains D1-D2 of FGFR3

(57) Specificity of Fyn-SH3 derived polypeptides binding to FGFR3 was tested by ELISA.

(58) Different antigens were coated on the plate (Maxisorp plate; Nunc 439454): huFGFR3b-Fc (SEQ ID NO: 12), huFGFR3c-Fc (SEQ ID NO: 13), cyFGFR3c-Fc (SEQ ID NO: 14), muFGFR3c-His (SEQ ID NO: 15), huD1-Fc (SEQ ID NO: 16), huD2-Fc (SEQ ID NO: 17), huD1-D2-Fc (SEQ ID NO: 18).

(59) The plate was coated with 100 ul antigen at 5 μg/ml (0.5 μg/well), and incubated at 4° C. overnight. The wells were washed 3× with PBS before being blocked with 200 μl 4% MPBS for 1 hr at RT. The wells were washed again, and 20 μl 10% MPBS containing 15 μg/ml 9E10 was added, before the addition of 80 μl Fynomer at 250 nM (200 nM final Fynomer concentration). The wells were incubated for 45 min at RT, before washing and the addition of 100 μl anti-mouse IgG-HRP (Sigma A2554) diluted 1:1000 in 2% MPBS. The wells were incubated for 30 min at RT, before washing 3× with 0.1% Tween-20 in PBS, and then 3×PBS. 100 μl BM POD Blue substrate (Roche 11 484 281 001) was added to each well followed by 50 μl 1M H2504 to stop the reaction. The absorbance 450 nm-650 nm was recorded using a Tecan M1000 instrument.

(60) Results

(61) As shown in FIG. 5 A-F, the Fyn SH3-derived polypeptides all are cross-reactive to cynomolgus and murine FGFR3c. Interestingly all binders are specific for an epitope present only when the domains D1 and D2 are physically linked (see FIG. 5 A-F bar huFGFR3-D1D2), in fact no binding is observed if the single domains D1 or D2 (hFGFR3-D1 or huFGFR3-D2) are immobilized on the ELISA plate.

Example 6: Fyn SH3-Derived Polypeptides of the Invention Cause Efficient Internalization of FGFR3

(62) Internalization is a central feature of the Fyn SH3-derived polypeptides described here, and provides the opportunity to use these binders to deliver toxic payloads and/or fused proteins such as antibodies intracellularly.

(63) In order to assess the ability of the Fyn SH3-derived polypeptides binding to FGFR3 to internalize upon binding to the target, an internalization assay based on the intracellular delivery of a cytotoxic agent was established.

(64) The assay measures the cytotoxic effect of anti-FGFR3 Fynomers cross-linked with MMAF (Monomethyl auristatin F)-conjugated 9E10, on KMS-11 cells. MMAF is an antimitotic agent (blocks tubulin polymerization) and is active only upon internalization into the cells. Therefore, this assay indicates how well the Fynomer facilitates internalization of MMAF. 50 μl of KMS-11 cells at 2×10.sup.5 cells/ml were seeded into a 96-well flat bottomed plate (Corning Costar 3610), to give 10,000 cell per well. The cells were incubated for 4 hours to allow the cells to adhere (37° C., 5% CO2). Fynomers and 9E10-MMAF were mixed at a 3:1 ratio. A 4× stock of Fynomer (4 μM) and a 4× stock of 9E10-MMAF (1.33.sub.NM) were prepared in RPMI media (see section 5.4.1) and mixed 1:1 (40 μl+40 μl). This mixture was then serially diluted 1 in 3, to give a concentration range 1000 nM-50 pM. 50 μl of the sample was added to the 50 μl of cells (as seeded above), and incubated for 5 days (37° C., 5% CO2). Appropriate controls, the wild-type Fynomer FynSH3, MMAF-9E10 without Fynomer and also cells without addition of any reagents, were included. All samples were prepared in duplicate. After 5 days, 100 μl Cell titer glo (Promega G7573) was added to each well and incubated with gentle shaking for 10 min in the dark. As a read-out for cell viability, luminescence was measured using a Tecan M1000 instrument. Analysis was performed using Prism 6. The data was transformed (X=log X), and analysed using a non-linear fit, log(inhibitor) vs. response—Variable slope (4 parameters).

(65) Results

(66) All Fyn SH3-derived FGFR3-binding polypeptides described here show increased cytotoxicity (e.g. internalization) compared to the cells treated with the MMAF-labeled secondary antibody only (9E10 in FIG. 6A) or the wild-type Fynomer FynSH3 in combination with MMAF-labeled 9E10 shown in all 3 experiments (FIG. 6 A-C indicated as FynSH3), that show cytotoxicity only at the highest concentration tested, probably due to the toxicity of MMAF itself. FIG. 6 shows the cytotoxicity profiles obtained in different experiments, and Table 5 shows the EC50 obtained for the different Fyn SH3-derived FGFR3-binding polypeptides.

(67) TABLE-US-00008 TABLE 5 EC50 values determined in internalization assays using FGFR3 + KMS-11 cells for Fyn SH3-derived FGFR3-binding polypeptides. Fynomer SEQ ID NO. EC50 (nM) FF2L4C3 3 28.5/21/26.4 FF44L65G12 4 2.5 FF44L65G7 5 2.6 FF48L66G7 6 2.4 FF43L65D5 7 0.8 FF44L65B7 8 1.8 Note: for FF2L4C3 the 3 values obtained in the 3 experiments shown in FIG. 6 are indicated.

(68) The data shown in FIG. 6 and Table 5 show that increased affinity also leads to more efficient internalization.

Example 7: Alternative Fyn-SH3 Derived Polypeptide that Shows Excellent Binding and Internalization Properties and which is Derived of a Different Family

(69) In addition to the preferred family of sequences (SEQ ID NO: 1), we identified one alternative Fynomer, FF40L54A5 (SEQ ID NO: 22), that surprisingly also shows excellent binding and internalization properties and shares manufacturability and cross-reactivity properties with the Fynomers derived from SEQ ID NO: 1 (see Table 6). Fynomer FF40L54A5 was not expected to have excellent internalization properties as its sequence derived from a Fynomer that showed only very poor internalization properties. Table 6 summarizes the properties of Fynomer FF40L54A5.

(70) TABLE-US-00009 TABLE 6 part 1 Affinities measured by BIAcore SEQ ID huFGFR3b huFGFR3c cyFGFR3c Fynomer NO. Yield (mg/L) KD.sub.app (pM) KD.sub.app (pM) KD.sub.app (pM) FF40L54A5 22 5.4 170 170* 160 part2 Affinities measured by BIAcore (competition with FGF1) EC50 (nM) KD.sub.app (pM) KD.sub.app (pM) to for binding to to huFGFR3c-Fc in EC50 (nM) in SEQ ID FGFR3.sup.+ huFGFR3c- presence of 200 internalization Fynomer NO. KMS-11 cells Fc nM FGF1 assay FF40L54A5 22 6.6 210* 230 4.7 part 3 Specificity ELISA Binding to Binding to Binding to Cross-reactivity domain D1 domain D2 domains D1- SEQ Cross-reactivity to to of of D2 of Fynomer ID NO. cyFGFR3c muFGFR3c huFGFR3 huFGFR3 huFGFR3 FF40L54A5 22 +++ +++ − − +++ *differences due to experimental variability

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