`International Journal of
`
`Molecular Sciences
`
`Communication
`High Affinity Promotes Internalization of Engineered
`Antibodies Targeting FGFR1
`
`Łukasz Opali ´nski 1,*, Jakub Szymczyk 1, Martyna Szczepara 1, Marika Kuci ´nska 1,
`Daniel Krowarsch 2, Małgorzata Zakrzewska 1 and Jacek Otlewski 1,*
`
`1 Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Joliot-Curie 14a,
`50-383 Wroclaw, Poland; jakub.szymczyk@uwr.edu.pl (J.S.); martyna.szczepara@uwr.edu.pl (M.S.);
`kucinska.marika@gmail.com (M.K.); malgorzata.zakrzewska@uwr.edu.pl (M.Z.)
`2 Department of Protein Biotechnology, Faculty of Biotechnology, University of Wroclaw, Joliot-Curie 14a,
`50-383 Wroclaw, Poland; daniel.krowarsch@uwr.edu.pl
`* Correspondence: lukasz.opalinski@uwr.edu.pl (Ł.O.); jacek.otlewski@uwr.edu.pl (J.O.);
`Tel.: +48-71-375-2631 (Ł.O.); +48-71-375-2824 (J.O.)
`
`Received: 15 April 2018; Accepted: 8 May 2018; Published: 10 May 2018
`
`# & !*-
`0+/ .
`
`Abstract: Fibroblast growth factor receptor 1 (FGFR1) is a plasma membrane protein that transmits
`signals from the extracellular environment, regulating cell homeostasis and function. Dysregulation
`of FGFR1 leads to the development of human cancers and noncancerous diseases. Numerous tumors
`overproduce FGFR1, making this receptor a perspective target for cancer therapies. Antibody-drug
`conjugates (ADCs) are highly potent and selective anticancer agents. ADCs are composed of
`antibodies (targeting factors) fused to highly cytotoxic drugs (warheads). The efficiency of ADC
`strategy largely depends on the internalization of cytotoxic conjugate into cancer cells. Here, we have
`studied an interplay between affinity of anti-FGFR1 antibodies and efficiency of their cellular uptake.
`We have developed a unique set of engineered anti-FGFR1 antibodies that bind the same epitope
`in the extracellular part of FGFR1, but with different affinities. We have demonstrated that these
`antibodies are effectively taken up by cancer cells in the FGFR1-dependent manner. Interestingly,
`we have found that efficiency, defined as rate and level of antibody internalization, largely depends
`on the affinity of engineered antibodies towards FGFR1, as high affinity antibody displays fastest
`internalization kinetics. Our data may facilitate design of therapeutically relevant targeting molecules
`for selective treatment of FGFR1 overproducing cancers.
`
`Keywords: affinity; cancer therapy; engineered antibodies; FGFR1; internalization
`
`1. Introduction
`
`Cancer is one of the top causes of mortality worldwide. Currently, nearly one in six deaths is due
`to cancer and it is expected that the number of new cases will rise by 70% in the coming two decades [1].
`Traditional anti-cancer therapies usually aim for inhibition of high proliferative capacity of cancer cells.
`However, most of the targeted pathways are also critical for maintenance of normal cells, thus giving
`rise to numerous side effects of conventional anti-cancer drugs. In recent years, engineered monoclonal
`antibodies and antibody fragments have attracted attention as molecules that may ensure specificity
`of the cancer treatment [2]. Such antibodies inactivate the specific receptor on cancer cells, resulting
`in induction of apoptosis, or lead to cancer cell death by stimulation of the immune system of the
`patient [2]. Alternatively, engineered antibodies may be physically linked to highly potent cytotoxic
`drugs in the antibody-drug conjugates (ADCs). In the ADC approach, antigen-positive cancer cells
`are recognized by the antibody part of the ADCs. Next, ADCs bound to the cell surface antigen are
`internalized, utilizing one of cellular endocytic routes. Subsequently, ADCs traffic via cellular vesicular
`
`Int. J. Mol. Sci. 2018, 19, 1435; doi:10.3390/ijms19051435
`
`Lassen - Exhibit 1013, p. 1
`
`www.mdpi.com/journal/ijms
`
`

`

`Int. J. Mol. Sci. 2018, 19, 1435
`
`2 of 14
`
`compartments to their final lysosomal destination, where proteolytic degradation releases cytotoxic
`drugs from the ADCs. Drug moiety diffuses out from lysosomes and binds its intracellular target
`causing cell death [3,4]. Therefore, the effectiveness of ADC therapy depends on the selectivity and
`strength of antigen binding, tumor penetration and on the efficiency of ADCs internalization from the
`cell surface [5–9].
`The fibroblast growth factor receptors comprise a group of four conserved receptor tyrosine
`kinases (FGFR1-FGFR4) that, in conjunction with extracellular fibroblast growth factors (FGFs),
`transmit signals across the plasma membrane. Binding of FGFs to FGFRs (fibroblast growth factor
`receptors) leads to the activation of the receptor cytoplasmic tyrosine kinase domain that recruits
`numerous signaling molecules further propagating the signal [10,11]. The FGFR-dependent signaling
`cascades govern cell metabolism, proliferation, and apoptosis and are critical for angiogenesis,
`organogenesis, and wound healing [12]. The aberrations in the FGFRs such as gene amplification,
`rearrangements, and somatic mutations are often observed in cancer and can be found in over 7% of
`all tumors [13].
`FGFR1 is an attractive target for selective chemotherapy in ADC, as it is localized on the cell
`surface, thus being easily accessible to extracellular targeting molecules [10,11]. In numerous cancer
`cell types, the level of FGFR1 is elevated in comparison to the normal cells that may ensure selectivity of
`drug targeting [14]. Moreover, FGFR1 is rapidly internalized, mainly via clathrin-mediated endocytosis,
`providing the intracellular release of the drug after lysosomal degradation of ADC inside cancer
`cells [15]. The requirements for the design of highly internalizing antibodies against FGFR1 suitable as
`an ADC’s carrier are still largely undefined. We have recently developed novel antibody fragment
`scFvD2-Fc and have demonstrated that bivalency of scFvD2-Fc promotes internalization of this
`anti-FGFR1 engineered antibody by inducing receptor dimerization [16,17]. Here, we have assessed
`the importance of engineered antibodies affinities towards FGFR1 for their internalization. Using the
`phage display approach, we have selected two novel FGFR1-specific antibodies that bind to the
`same epitope within extracellular part of FGFR1 as scFvD2-Fc, but with different affinities. We have
`demonstrated that all these engineered antibodies are efficiently internalized via receptor-mediated
`endocytosis and are delivered through endosomes to lysosomes. Interestingly, our data show that an
`antibody with the highest affinity to FGFR1 displays the fastest internalization rate. Taken together,
`our data may facilitate the effective design of highly internalizing engineered antibodies suitable for
`ADC strategy of cancer treatment.
`
`2. Results
`
`2.1. Engineered Antibody Fragments Recognize the Same Epitope within D1 Domain of the FGFR1
`
`To select the panel of antibody fragments that specifically recognize FGFR1, we employed the
`phage display technique using Tomlinson I and Tomlinson J libraries and the extracellular part of
`FGFR1 (composed of D1, D2, and D3 domains) fused to the Fc fragment (FGFR1.D1-D2-D2-Fc) as an
`antigen [18]. For each library, we performed three consecutive rounds of selection. We obtained four
`novel scFv proteins that interact with the extracellular region of FGFR1: scFvK10, scFvL8, scFvL12
`and scFvP4.
`Our group has recently selected and characterized scFvD2 as a high affinity FGFR1 interactor [16].
`To obtain panel of scFv fragments that bind to the same region of the extracellular part of FGFR1
`as scFvD2 we employed epitope binning using surface plasmon resonance (SPR). We intended to
`select scFv proteins whose interaction with FGFR1-D1-D2-D3-Fc was blocked by the saturating
`concentrations of scFvD2. To this end, sensors with immobilized FGFR1-D1-D2-D3-Fc were incubated
`first with high concentration (1 µM) of scFvD2 and then with novel anti-FGFR1 scFv proteins at
`the same concentration. We observed that binding of scFvD2 to the extracellular part of FGFR1
`inhibited receptor interaction with scFvK10 and scFvL12 (Figure 1a). Moreover, the saturation of
`FGFR1-D1-D2-D3-Fc with scFvL12 blocked subsequent binding of scFvK10, suggesting that these three
`
`Lassen - Exhibit 1013, p. 2
`
`

`

`Int. J. Mol. Sci. 2018, 19, 1435
`
`3 of 14
`
`antibody fragments compete for the same binding site on FGFR1 (Figure 1a). To identify the epitope
`recognized by these three scFv proteins, we performed SPR binding studies with sensor-immobilized
`full length extracellular part of FGFR1 (FGFR1.D1-D2-D3-Fc) and truncated receptor variant lacking
`D1 domain (FGFR1.D2-D3-Fc). The lack of the D1 domain abolished the interaction of scFvD2,
`scFvK10, and scFvL12 with immobilized FGFR1 fragment, indicating that these proteins recognize
`epitope within the D1 domain of the receptor (Figure 1b). We confirmed this finding, showing
`that all three engineered antibodies bind to sensor-immobilized purified D1 domain fused to GST
`(GST-FGFR1.D125–124) (Figure 1b).
`To validate our experiments, we used FGF1, a natural FGFR1 ligand that recognizes binding site
`formed by D2 and D3 domains of the receptor. As expected, FGF1 interacted with FGFR1.D1-D2-D3-Fc
`and FGFR1.D2-D3-Fc, but did not bind to GST-FGFR1.D125–124 (Figure 1b). To identify the precise
`epitope within the D1 domain recognized by scFv proteins, we prepared a set of C-terminal truncations
`of D1 domain fused to glutathione S-transferase GST (Figure 1c). Next, we performed pull down
`experiments with purified GST-tagged D1 domain truncations and GST as a control. We found that
`scFvD2, scFvK10, and scFvL12 bound to the full length D1 domain (GST-FGFR1.D125–124) and to the D1
`truncation composed of residues 25–76 (GST-FGFR1.D125–76). The interaction was not observed when
`only N-terminal peptide, containing residues 25–40, was applied (GST-FGFR1.D125–40) (Figure 1c).
`These data demonstrate that engineered anti-FGFR1 antibodies recognize epitope that is located within
`residues 41–76 of the D1 domain of FGFR1.
`
`Figure 1. Cont.
`
`Lassen - Exhibit 1013, p. 3
`
`

`

`Int. J. Mol. Sci. 2018, 19, 1435
`
`4 of 14
`
`Figure 1. Engineered antibodies recognize the same epitope within the extracellular region of FGFR1.
`(a) SPR-based epitope binning. FGFR1.D1-D2-D3-Fc was immobilized on sensors and incubated with
`high concentrations of saturating antibodies. Next, competing antibodies were injected to assess
`if their binding to the extracellular region of FGFR1 is blocked by saturating antibodies. (b) SPR
`analysis of the interaction of scFv proteins with FGFR1 truncations. CM5 sensors were coated with
`FGFR1.D1-D2-D3-Fc, FGFR1.D2-D3-Fc, or GST-FGFR1-D125–124, and the association and dissociation
`were monitored for 240 s after injection of scFv proteins (1 µM), or FGF1 (1 µM). (c) Pull down
`experiments with scFv proteins and truncated variants of the D1 domain of FGFR1. GST and
`GST-tagged truncated versions of the D1 domain were bound to Glutathione Sepharose and incubated
`with scFv proteins. After extensive washing, bound proteins were eluted and analyzed by Western
`blotting. Membranes were first stained with CBB to visualize eluted proteins and then detected with
`anti-c-myc antibodies (for visualization of scFv proteins containing C-teminal c-myc).
`
`Next, we assessed specificity of scFvD2, scFvK10, and scFvL12 towards FGFR1 using SPR. Using
`extracellular regions of all four FGF receptors (FGFR1-4) immobilized on CM5 sensors, we observed
`binding of scFvD2, scFvK10, and scFvL12 only to FGFR1 (Figure 2). The high selectivity of scFv-Fc
`proteins towards FGFR1 can be attributed to the very low homology of FGF receptors within residues
`41–76 (13.9% identity, while sequence identity in the full extracellular region of FGF receptor is 36.7%)
`(Figure S1). To confirm that immobilized fragments of FGF receptors were functional, we employed
`developed in our group scFvF7 that recognizes FGFR2 [19] as well as commercial anti-FGFR3 and
`anti-FGFR4 antibodies as controls (Figure 2 and Figure S2).
`These results demonstrate that three independently selected scFv proteins display high specificity
`towards FGFR1 and recognize the same epitope (residues 40–76) within the D1 domain of FGFR1.
`
`Lassen - Exhibit 1013, p. 4
`
`

`

`Int. J. Mol. Sci. 2018, 19, 1435
`
`5 of 14
`
`Extracellular regions of FGFR1
`Specificity of scFv proteins towards FGFR1.
`Figure 2.
`(FGFR1.D1-D2-D3-Fc (a); FGFR2 (FGFR2.D1-D2-D3-Fc (b); FGFR3 (FGFR3.D1-D2-D3-Fc (c); and FGFR4
`(FGFR4.D1-D2-D3-Fc (d); were immobilized on CM5 sensors and incubated with scFvD2, scFvL10,
`scFvK12, scFvF7 (specific towards FGFR2), and commercial anti-FGFR3 and anti-FGFR4 antibodies.
`
`Lassen - Exhibit 1013, p. 5
`
`

`

`Int. J. Mol. Sci. 2018, 19, 1435
`
`6 of 14
`
`2.2. Engineered Antibodies Differ in the Affinity towards FGFR1
`
`We have recently demonstrated that reformatting of the scFvD2 into bivalent scFvD2-Fc largely
`improved internalization of this engineered antibody by inducing FGFR1 dimerization [16,17]. To study
`the impact of antibody affinity to FGFR1 on the internalization of antibody-receptor complexes,
`we have also prepared Fc-fusions of scFvK10 and scFvL12 (scFvK10-Fc and scFvL14-Fc). Proteins were
`expressed in chinese hamster ovary (CHO) cells and purified using affinity chromatography. Next,
`the binding of scFvK10-Fc and scFvL12-Fc to the extracellular region of FGFR1 (FGFR1.D1-D2-D3-Fc)
`were analyzed by SPR technique. The calculated kinetic parameters (kon, koff and KD) clearly
`demonstrate that scFvD2-Fc displays the highest affinity towards FGFR1 (0.59 nM) [16], while
`scFvK10-Fc and scFvL12-Fc bind to the FGFR1 with over tenfold weaker affinities than scFvD2-Fc
`(9.41 nM and 13.4 nM, respectively) (Table 1, Figure S3).
`
`Table 1. Kinetic parameters of scFv-FGFR1 interaction measured with SPR. Sensor-immobilized
`FGFR1.D1-D2-D3-Fc was incubated with various concentrations of scFv-Fc proteins and kinetic
`parameters (ka, kd, and KD) were determined using BIAevaluation 4.1 software. * values from
`[16]. KD errors represent standard deviation.
`
`scFvD2-Fc *
`
`scFvK10-Fc
`
`scFvL12-Fc
`
`KD (nmol/L)
`kon (M−1s−1)
`koff (s−1)
`
`0.59
`6.47 × 105
`3.84 × 10−4
`
`9.41 ± 1.47
`6.82 × 105
`6.42 × 10−3
`
`11.6 ± 1.25
`4.08 × 105
`5.45 × 10−3
`
`2.3. Antibody Fragments Are Internalized via Receptor Mediated Endocytosis
`
`Next, we studied the internalization of engineered antibody fragments. For this purpose,
`we labeled scFvD2-Fc, scFvK10-Fc, and scFvL12-Fc with pH sensitive fluorophore pHrodo Red.
`The fluorescence intensity of pHrodo Red is very low at neutral or basic pH (outside the cells or on the
`cell surface), but largely increases in the acidic milieu of the endosomal and lysosomal lumen (when
`labeled antibody is internalized) [20]. Fluorescently labeled engineered antibodies were incubated
`with model U2OSR1 cells (overproducing FGFR1) and control U2OS cells (with very low level of FGF
`receptors) and analyzed by fluorescence microscopy. Internalization of scFv-Fc proteins was minimal
`for U2OS cells, while all three studied engineered antibodies were efficiently taken up by U2OSR1 cells
`(Figure 3a). Quantitative analysis of fluorescence intensities confirmed that all three scFv-Fc proteins
`required FGFR1 for their internalization (Figure 3b).
`Upon internalization, engineered antibodies can be directed to recycling endosomes, or to
`endosomes that fuse with lysosomes for degradation [15]. As epidermal growth factor (EGF) is
`a marker of endosomes following the degradation pathway, we simultaneously applied fluorescently
`labeled EGF (EGF-AlexaFluor-488) to visualize these compartments [21]. We observed a high
`degree of colocalization of pHrodo Red-labeled scFvD2-Fc, scFvK10-Fc, and scFvL12-Fc with
`EGF-AlexaFluor-488, which suggests that engineered antibodies are sequestered into endosomal
`compartments destined for fusion with lysosomes (Figure 4a). As expected, at longer time
`points (120 min) we observed colocalization of pHrodo Red-labeled antibody fragments with the
`lysosome-specific dye–Lysotracker Green DND-20 (Figure 4b). All these data demonstrate that
`engineered antibodies are internalized via FGF receptor-mediated endocytosis and are directed to
`lysosomes for degradation.
`
`Lassen - Exhibit 1013, p. 6
`
`

`

`Int. J. Mol. Sci. 2018, 19, 1435
`
`7 of 14
`
`Figure 3. FGFR1-dependence of scFv proteins internalization. (a) U2OS (control) and U2OSR1 cells
`(overproducing FGFR1) were incubated for 2 h with pHrodo Red labeled scFv proteins (red). Nuclei
`were stained with NucBlue Live (blue) and cells were analyzed by fluorescence microscopy. Scale
`bars represent 20 µm. (b) Quantification of internalization of pHrodo Red-labeled scFvs into U2OS
`and U2OSR1 cells. pHrodo Red fluorescence intensity inside single cells was measured using Zen
`2.3 software (Zeiss, Oberkochen, Germany). At least 30 cells were measured from five fields of view
`for U2OS and U2OSR1 cells. Each square on the graph represents fluorescence intensity of single cell.
`Black squares represent average fluorescence intensity for particular cell line.
`
`Lassen - Exhibit 1013, p. 7
`
`

`

`Int. J. Mol. Sci. 2018, 19, 1435
`
`8 of 14
`
`Figure 4. Intracellular trafficking of engineered antibodies. (a) U2OSR1 cells were incubated for 1 h
`with pHrodo Red labeled scFv proteins (red) and EGF-AlexaFluor-488 (green) (marker of internalization
`pathway directing cargo for lysosomal degradation). Nuclei were stained with NucBlue Live (blue)
`and cells were analyzed by fluorescence microscopy. Scale bars represent 20 µm. (b) U2OSR1 cells
`were incubated for 2 h with pHrodo Red labeled scFv proteins (red). Lysosomes were subsequently
`stained with Lysotracker Green DND-20 (green) and nuclei were labeled with NucBlue Live (blue).
`Cells were analyzed by fluorescence microscopy. Arrowheads mark selected regions of high degree of
`signal colocalization. Scale bars represent 20 µm.
`
`2.4. High Affinity Promotes Internalization of Engineered Antibodies
`
`As differences in the efficiency of chemical labeling made the quantitative comparison of the
`internalization rates of fluorescently labeled antibodies impossible, we developed a fluorescence
`
`Lassen - Exhibit 1013, p. 8
`
`

`

`Int. J. Mol. Sci. 2018, 19, 1435
`
`9 of 14
`
`microscopy assay where we utilized non-labeled scFv-Fc proteins. Engineered antibodies were
`incubated with the U2OSR1 cells and the internalization was blocked at various time points by placing
`the cells on ice. Surface-bound antibodies were subsequently removed by washing the cells with
`buffer containing high salt concentration and low pH (HSLP). Cells were then fixed, permeabilized,
`and incubated with Zenon-AF-488, fluorescently labeled Fab fragment specifically recognizing the Fc
`fragment of the human origin, to visualize internalized scFv-Fc proteins (Figure 5a).
`
`(a) Scheme of the experimental
`Figure 5. Kinetics of engineered antibodies internalization.
`setup. Non-labeled scFv-Fc proteins were incubated with U2OSR1 cells to allow for binding and
`internalization. Next, cell surface-bound engineered antibodies were removed by washing, cells
`were permeabilized and incubated with Zenon-AlexaFluor-488, a fluorescently labeled Fab fragment
`specifically recognizing Fc fragment of human origin that detects non-labeled, internalized scFv-Fc
`proteins.
`(b) U2OSR1 cells were incubated with scFv-Fc proteins for various time points and
`internalized engineered antibodies were detected with Zenon-AlexaFluor-488 (green), as described
`above. Nuclei were labeled with NucBlue Live (blue). Scale bars represent 20 µm. (c) Kinetics of scFv-Fc
`proteins internalization. Fluorescence intensity of Zenon-AlexaFluor-488 (visualizing internalized
`scFv-Fc proteins) was measured within single U2OSR1 cells at various time points. At least 20 cells
`were measured from three fields of view for each time point. The values represent average fluorescence
`intensity from three experiments per cell. Error bars represent +/− SEM.
`
`Lassen - Exhibit 1013, p. 9
`
`

`

`Int. J. Mol. Sci. 2018, 19, 1435
`
`10 of 14
`
`We observed that scFvD2-Fc displayed the fastest kinetics of internalization and the highest
`accumulation within U2OSR1 cells (Figure 5b,c). The internalization of the other two engineered
`antibodies (scFvK10-Fc and scFvL12-Fc) that bind to the same antigen within FGFR1, but with about
`15 times lower affinity, was much slower and lower amounts of antibodies were taken up by the
`cells (Figure 5b,c). These data demonstrate that efficiency of the receptor-mediated endocytosis of
`anti-FGFR1 antibodies largely depends on the affinity of antibodies towards the receptor.
`
`3. Discussion
`
`Success in selective targeting of tumors with engineered antibodies and their conjugates largely
`depends on the properties of the cancer antigen and applied antibody. The characteristics of an “ideal”
`antigen for ADC strategy are still under the debate. Nevertheless, the “ideal” antigen is believed to
`be present at an elevated level on the surface of cancer cells, efficiently endocytosed and targeted to
`lysosomes [3–5]. On the other hand, the perfect targeting molecule (antibody), especially in the ADC
`approach, should display high affinity towards specific cancer marker (ensuring selectivity) and high
`internalization rate, facilitating the efficient delivery of its cargo (cytotoxic drug) [3–5]. The relationship
`between affinity of antibodies to cell surface antigens and their internalization kinetics is still not
`established. It was demonstrated that high affinity to antigen promotes internalization of antibodies
`directed against CD44 and HER2 receptors [22,23]. In contrast, differences in binding to target had
`no influence of the internalization rates of anti-CEA scFv proteins [24]. Moreover, the significance
`of antibodies affinities for tumor targeting in vivo is controversial. Some high affinity antibodies
`display poor tumor penetration, while in the other cases high affinity supports tumor targeting by
`antibodies [6–9]. Therefore, in case of each antigen–antibody pair it is important to assess the antigen
`properties and the significance of interaction strength for the efficiency of antibody internalization and
`for tumor targeting in vivo.
`Here, we have analyzed the interplay between the affinity of anti-FGFR1 antibody fragments to
`the receptor and their cellular uptake. We have developed a unique set of engineered antibodies that
`bind to the same epitope of extracellular part of FGFR1, but with different strength. All these antibodies
`are internalized by FGFR1-dependent endocytosis, however high affinity antibody is taken up by
`the cells more rapidly than antibodies displaying lower affinities towards FGFR1. We have recently
`demonstrated that bivalent anti-FGFR1 antibodies are rapidly internalized by promoting receptor
`dimerization that constitutes the trigger for receptor endocytosis [17]. In our experiments, we used
`excessive concentrations of scFv-Fc proteins (over 20 times higher than equilibrium dissociation
`constants), thus a vast majority of the cell surface FGFR1 was occupied by antibody fragments.
`Therefore, we hypothesize that rapid internalization of the high affinity bivalent antibody-FGFR1
`complexes is caused by enhanced FGFR1 dimerization on the cell surface.
`Taken together, our data imply that bivalency and high affinity ensure efficient internalization of
`engineered anti-FGFR1 antibodies. These results may facilitate design of targeting molecules suitable
`for ADCs directed against FGFR1-overproducing cancers.
`
`4. Materials and Methods
`
`4.1. Antibodies and Recombinant Proteins
`
`The primary anti-c-myc antibodies (sc-40), anti-FGFR3 (sc-73994), and anti-FGFR4 (sc-73995)
`were from Santa Cruz Biotechnology (Dallas, TX, USA). The primary antibody against FGFR1
`was generated by Davids Biotechnologie GmbH (Regensburg, Germany) [17]. Recombinant FGF1
`(Met-Ala-FGF122-155) was produced in E. coli, as described previously [25]. The Fc fragment and
`the full length extracellular region of FGFR1 and the extracellular part of FGFR1 lacking N-terminal
`D1 domain fused to the Fc were produced in CHO cells and purified using Protein A Sepharose
`(GE Healthcare, Piscataway, NJ, USA) [18].
`
`Lassen - Exhibit 1013, p. 10
`
`

`

`Int. J. Mol. Sci. 2018, 19, 1435
`
`11 of 14
`
`Antibody fragments in the scFv format (scFvD2, scFvI4, scFvK10, scFvL8, scFvL12, scFvP4,
`scFvF7 containing c-myc) were expressed in E. coli HB2151 and purified using Protein A Sepharose.
`scFv proteins in the bivalent Fc format (scFvD2-Fc, scFvK10-Fc and scFvL12-Fc) were produced in
`CHO cells and purified with Protein A Sepharose [16].
`The DNA sequence encoding the D1 domain of FGFR1 (residues 25–124) was cloned into
`pDEST15 using Gateway Cloning (Thermo Fisher Scientific; Waltham, MA, USA), resulting in
`plasmid pEXP15-D125–124 (allowing for production of GST-FGFR1-D125–124). Plasmids for production
`of truncated variants of D1 domain: pEXP-D125–76 (for production of GST-FGFR1-D125–76) and
`pEXP-D125–40 (for production of GST-FGFR1-D125–40) were generated by introducing stop codon
`in the vector pEXP15-D125–124 by site directed mutagenesis. GST-tagged truncated variants of the D1
`domain were produced in E. coli BL21 CodonPlus(DE3)-RIL (Agilent Technologies; Santa Clara, CA,
`USA) and purified with Glutathione Sepharose (GE Healthcare, Piscataway, NJ, USA), according to the
`standard procedure.
`
`4.2. Cells
`
`U2OS (human osteosarcoma, ATCC #HTB-96) and U2OSR1 cells (U2OS cells stably expressing
`FGFR1) were a kind gift of Dr. E.M. Haugsten from the Department of Molecular Cell Biology, Institute
`for Cancer Research, Oslo University Hospital, Norway. Cells were cultured in 5% CO2 atmosphere at
`37 ◦C in Dulbecco’s Modified Eagle’s Medium (Biowest, Nuaille, France) supplemented with 10% fetal
`bovine serum (Thermo Fisher Scientific, Waltham, MA, USA), antibiotics mix (100 U/mL penicillin,
`100 µg/mL streptomycin (Thermo Fisher Scientific; Waltham, MA, USA), and 1 mg/mL geneticin
`(in the case of U2OSR1 cells) (Thermo Fisher Scientific; Waltham, MA, USA). Cells were seeded into
`tissue culture plates one day prior start of the experiments.
`
`4.3. Selection of Antibody Fragments against FGFR1 by Phage Display Technology
`
`The selection of scFv proteins recognizing an extracellular region of FGFR1 was carried out using
`two commercial phage libraries of human scFv (Tomlinson I and J) as described previously [16].
`Three cycles of panning were performed on MaxiSorp 96-well plates (Thermo Fisher Scientific;
`Waltham, MA, USA) coated with FGFR1.D1-D2-D3-Fc with counter selection using purified Fc
`protein. To confirm the interaction with FGFR1 bacterial supernatants containing selected scFv proteins
`were subjected to the monoclonal ELISA and the SPR assay with immobilized FGFR1.D1-D2-D3-Fc,
`as described in [16].
`
`4.4. SPR-Based Interaction Studies and Affinity Measurements
`
`SPR experiments were performed on the Biacore 3000 instrument (GE Healthcare, Piscataway,
`NJ, USA) at 25 ◦C. For epitope binning FGFR1.D1-D2-D3-Fc was immobilized on a CM5 sensor (GE
`Healthcre) at 825 RU and scFv proteins (1 µM) were sequentially injected at flow rate of 30 µL/min.
`The association and dissociation were monitored for 240 s.
`For analysis of the binding site of scFv proteins within FGFR1, CM5 sensors were coated with
`FGFR1.D1-D2-D3-Fc (at 825 RU), FGFR1.D2-D3-Fc (at 900 RU), or GST-FGFR1-D125–124 (at 300 RU).
`Next, scFv proteins (1 µM) and FGF1 (1 µM) were injected and the association and dissociation were
`monitored for 240 s.
`For specificity analysis of generated scFv proteins, FGFR1.D1-D2-D3-Fc (at 825 RU),
`FGFR2.D1-D2-D3-Fc (at 1000 RU), FGFR3.D1-D2-D3-Fc (at 1000 RU), and FGFR4.D1-D2-D3-Fc (at
`1000 RU) were immobilized on CM5 sensors. Next, scFv proteins (1 µM), anti-FGFR1 antibody (as a
`positive control for interaction with FGFR1.D1-D2-D3-Fc) [17], scFvF7 (1 µM) (as a positive control
`for interaction with FGFR2.D1-D2-D3-Fc) [15] and two commercial antibodies against FGFR3 and
`FGFR4 (0.1 µM; as positive controls for interaction with FGFR3.D1-D2-D3-Fc and FGFR4.D1-D2-D3-Fc,
`respectively) were injected independently over all sensors at 30 µL/min flow rate. The association
`
`Lassen - Exhibit 1013, p. 11
`
`

`

`Int. J. Mol. Sci. 2018, 19, 1435
`
`12 of 14
`
`and dissociation were monitored for 240 s. All sensograms were analyzed using BIAevaluation 4.1
`software (GE Healthcare, Piscataway, NJ, USA).
`interaction,
`antibodies–FGFR1
`engineered
`of
`To
`determine
`kinetic
`parameters
`FGFR1.D1-D2-D3-Fc was immobilized at 825 RU on a CM5 sensor. Various concentrations of
`scFv-Fc proteins (2.5–320 nM) were measured for 300 s at 30 µL/min flow rate. Kinetic constants (ka,
`kd, and KD) were calculated using BIAevaluation 4.1 software using 1:1 Langmuir binding model with
`drifting baseline.
`
`4.5. Pull-Down
`
`To study the interaction of scFv proteins with purified GST-tagged full length D1 domain of
`FGFR1 and the D1 domain truncations, purified GST (control, 10 µg) and GST-tagged D1 domain
`variants (10 µg) were diluted in washing buffer (WB): 50 mM Tris, 150 mM NaCl, pH 7.5, and bound
`to Glutathione Sepharose. Next, scFv proteins (10 µg) in WB were incubated with resin-bound scFv
`proteins. Resins were extensively washed with WB and bound proteins were eluted with elution
`buffer: 50 mM Tris, 150 mM NaCl, 20 mM glutathione, pH 7.5. Eluates were analyzed by Western
`blotting. Before detection membranes were stained with CBB to visualize the amount of eluted
`GST-tagged truncated variants of the D1 domain and GST. After destaining, membranes were probed
`with anti-c-myc antibodies.
`
`4.6. Fluorescence Microscopy
`
`Purified scFvD2-Fc, scFvK10-Fc, and scFvD12-Fc were chemically labeled with pHrodo Red
`(Thermo Fisher Scientific; Waltham, MA, USA) according to manufacturer’s protocol. For analysis of
`the FGFR1-dependence engineered antibodies internalization, U2OS and U2OSR1 cells were incubated
`with pHrodo Red-labeled engineered antibodies (10 µg/mL) for 2 h at 37 ◦C. Cells were extensively
`washed with PBS, fixed with 4% PFA, nuclei were stained with NucBlue Live (Thermo Fisher Scientific;
`Waltham, MA, USA) and cells were analyzed by fluorescence microscopy. For quantitation of
`engineered antibodies internalization, pHrodo Red fluorescence intensity was measured within single
`cells using ZEN 2.3 software (Zeiss, Oberkochen, Germany). At least 30 cells were measured from five
`fields of view for U2OS and U2OSR1 cells.
`For analysis of the intracellular trafficking of engineered antibodies, serum-starved U2OSR1
`cells were incubated for 1 h at 37 ◦C with pHrodo Red-labeled scFv-Fc proteins (10 µg/mL) and
`EGF-AlexaFluor-488 (15 µg/mL) (Thermo Fisher Scientific; Waltham, MA, USA). Alternatively,
`U2OSR1 cells were incubated for 2 h with pHrodo Red labeled scFv-Fc proteins (10 µg/mL) and
`stained with lysosome-specific dye Lysotracker Green DND-20 (Thermo Fisher Scientific; Waltham,
`MA, USA) according to manufacturer’s protocol. Nuclei were stained with NucBlue Live and cells
`were analyzed by fluorescence microscopy.
`For analysis of the kinetics of engineered antibodies internalization, non-labeled scFv-Fc proteins
`and U2OSR1 cells were used. Cells were incubated with scFv-Fc proteins (15 µg/mL) at 37 ◦C and
`internalization was stopped at various time points by placing the cells on ice. Cells were then washed
`extensively with PBS and with HSLP buffer (high salt low pH) (2 M NaCl, 40 mM NaAc, pH 4.0) to
`remove cell surface-bound antibodies. Next, cells were fixed with 4% PFA and permeabilized with 0.1%
`Triton X-100 in PBS. Cells were subsequently blocked with 2% BSA in PBS and internalized scFv-Fc
`proteins were visualized by addition of Zenon-AlexaFluor-488 (Thermo Fisher Scientific; Waltham,
`MA, USA) that recognizes the Fc fragment of human origin, accordingly to supplier’s protocol.
`The excessive Zenon-AlexaFluor-488 was blocked with Blocking Reagent and after extensive washing
`cells were fixed again to preserve scFv-Fc- Zenon-AlexaFluor-488 complexes. Next, nuclei were stained
`with NucBlue Live and cells were analyzed with fluorescence microscopy. Zenon-AlexaFluor-488
`fluorescence intensity within single U2OSR1 cells was measured using ZEN 2.3 software. At least
`20 cells were measured from three fields of view for three experiments. Average intensity +/− SEM
`were plotted.
`
`Lassen - Exhibit 1013, p. 12
`
`

`

`Int. J. Mol. Sci. 2018, 19, 1435
`
`13 of 14
`
`Wide-field fluorescence microscopy was carried out using a Zeiss Axio Observer Z1 fluorescence
`microscope (Zeiss, Oberkochen, Germany). Images were taken using LD-Plan-Neofluar 40×/0.6
`Korr M27 objective and Axiocam 503 camera. pHrodo Red signal was visualized with a 540/552 nm
`bandpass excitation filter and a 575/640 nm bandpass emission filter, EGF-AlexaFluor-488, Lysotracker
`Green DND-20 and Zenon-AlexaFluor-488 signal was visualized with a 450/490 nm bandpass
`excitation filter and a 550/590 nm bandpass emission filter. NucBlue Live signal was visualized
`with a 335/383-nm bandpass excitation filter and