Bi-modular fusion proteins, a versatile therapeutic tool for re-directing a pre-existing Epstein-Barr virus antibody response towards defined target cells


 Industrial production of therapeutic monoclonal antibodies is mostly performed in eukaryotic-based systems, allowing post-translational modifications mandatory for their functional activity. Nevertheless, the resulting elevated product cost limits therapy access to some patients, thus increasing medical inequality. To address this limitation, we conceptualized a novel immunotherapeutic approach aiming at redirecting a pre-existing polyclonal antibody response against Epstein-Barr virus (EBV) towards defined target cells. We engineered bi-modular fusion proteins (BMFPs), notably expressible in bacteria-based systems, comprising a Fc-deficient binding moiety (Nanobody, scFv) specifically targeting an antigen expressed at the surface of a target cell, fused to the P18 EBV antigen, which would recruit circulating endogenous anti-P18 IgG in individuals chronically infected by EBV. Opsonization of BMFP-coated target cells efficiently triggered antibody-mediated clearing effector mechanisms in vitro, such as the complement cascade, erythrophagocytosis by macrophages and FcγRIII-mediated activation of cellular pathways leading to antibody-dependent cell-mediated cytotoxicity (ADCC). When assessed in a mouse tumour model, therapy performed with an anti-huCD20 BMFP significantly led to increased mice survival and total cancer remission in some animals. These results indicate that BMFPs are versatile tools for redirecting an Epstein-Barr virus pre-existing immune antibody response towards pre-defined target cells and could represent potent and useful therapeutic molecules to treat a broad range of diseases.

macrophages promotes phagocytosis of IgG-opsonized target cells 30 . Thus, to assess if anti-P18F3 184 huIgG recruited by Nb-aDARC-P18F3 bound to DARC + RBCs were able to promote RBC 185 clearance by phagocytes, we performed an erythrophagocytosis assay using macrophage-like cells 186 derived from the monocytic leukemia THP1 cell line (CD64 + /CD32a + /CD16a -) (Supplementary 187 Fig. 6) 31 . 188 Carboxyfluorescein succimidyl ester (CFSE)-stained DARC + RBCs were incubated for 3h at 37°C 189 with macrophage-like cells (obtained by PMA treatment of THP1 cells) in presence of  aDARC-P18F3 and of human plasma pools exhibiting low, intermediate, or high antibody titers 191 against P18F3. Flow cytometry analysis of macrophage-like THP1 cells revealed that the 192 percentage of CFSE + THP1 was largely increased when the cells were incubated with  P18F3 and a human plasma pool exhibiting a high titer of anti-P18F3 antibodies (Fig. 4a). When 194 Nb-aDARC was used instead of Nb-aDARC-P18F3, no significant increase was observed (Fig. 195 4a). Four independent experiments confirmed that exposure of RBCs to  human plasma pools containing high or intermediate antibody titers against P18, provokes an 197 increased erythrophagocytosis by THP1-derived macrophage-like cells (mean fold change 6.3 and 198 2.6, respectively) as compared to the condition using untreated RBCs (Fig. 4b). No increase was 199 observed in absence of plasma in the assay where Nb-aDARC-P18F3 was used (Fig. 4b). Of note,200 erythrophagocytosis was more pronounced in presence of the plasma pool exhibiting the highest 201 antibody titer against P18F3 (as compared to the "no plasma" condition; p=0.0286). In contrast, 202 RBC treatment with Nb-aDARC did not modify the level of erythrophagocytosis directly exerted 203 by macrophage-like THP1 cells, regardless of the plasma pools tested (Fig. 4b). 204 Altogether, these results identified P18F3 as the most efficient P18-derived polypeptide able to 205 recruit specific IgG onto the cell surface of target cells. Fusion of P18F3 to a nanobody-based 206 binding moiety (Nb-aDARC) did not alter the intrinsic functionality of the 2 modules and 207 demonstrated a good capability to engage FcγRs on THP1-derived macrophages making it possible 208 to recruit anti-P18 antibodies, present in human plasma, that trigger RBC phagocytosis. 209 210 Engineering a BMFP against human CD20. 211 We also developed a BMFP containing a scFv directed against huCD20 fused to the N-terminal 212 end of P18F3 to target Burkitt's lymphoma cells in vitro and huCD20-expressing tumor cells in an 213 in vivo mouse tumor model. 214 An anti-huCD20 scFv comprising the VH domain fused to the Vk domain with a (GGGGS)3 215 interdomain linker, derived from the mouse IgG2b,κ 2H7 monoclonal antibody 32 , was engineered 216 ( Fig. 5a) and expressed alone (scFv2H7) or in fusion with P18F3 (scFv2H7-P18F3) ( Fig. 5b and 217 Supplementary Fig. 7). Both constructs included a 6xHistidine tag (6xHis tag) at the C-terminus. 218 The binding of the scFv2H7 and scFv2H7-P18F3 to native huCD20 expressed at the surface of cells 219 from the Burkitt's lymphoma cell line RAJI (Supplementary Fig. 8a) was assessed by an indirect 220 anti-His-tag fluorescence assay and flow cytometry (Supplementary Fig. 8b). Both scFv2H7 and 221 scFv2H7-P18F3 bound to huCD20-expressing RAJI cells and a similar binding was observed when 222 used at an equimolar concentration of 0.48 μM. As already observed with Nb-aDARC and Nb-223 aDARC-P18F3 (Supplementary Fig. 4), a difference in the binding curves of the two molecules 224 was seen (Supplementary Fig. 8b). This may reflect a steric hindrance for the binding of the anti-225 6xHis antibody to the recombinant molecules when P18F3 is fused at their C-terminus rather than 226 a change in the ability of BMFPs to bind the target molecules (DARC and huCD20). 227

ScFv2H7-P18F3-mediated anti-P18 IgG opsonization of Burkitt's Lymphoma cells activates 229
antibody-dependent complement cascade and triggers FcgRIIIa-mediated activation of 230 intracellular signaling pathways. 231 We then evaluated if the binding of scFv2H7-P18F3 to Burkitt's lymphoma cells induced the 232 activation of the complement cascade in presence of a pool of heat-inactivated human plasma 233 exhibiting a high titer of anti-P18F3 antibodies. This activation leads to the formation of the C5b-234 8 complex that binds to C9 to form the membrane attack complex (MAC), C5b-9. Incubation of 235 RAJI cells with scFv2H7-P18F3 in the presence of plasma led to more C5b-8/9 deposition than 236 when untreated cells or cells coated with scFv2H7 were tested as shown by an increased detection 237 of C5b-8/9 using either rabbit anti-C5b-9 antibodies (Mann-Whitney test, p=0.0286) (Fig. 5c) or 238 a mouse anti-C5b-8/9 mAb (Supplementary Fig. 9a). 239 The Fcγ region of immuno-complexed IgG can bind to FcγRIIIa/CD16a expressed at the surface 240 of Natural Killer (NK) cells and trigger an intracellular signaling cascade leading to the release of 241 IFN-γ, TNF-a and of perforin and granzymes from cytotoxic granules. Thus, we examined if 242 scFv2H7-P18F3-mediated anti-P18 IgG opsonization of Burkitt's Lymphoma cells could provoke 243 the crosslinking of FcγRIIIa, hence triggering a signalling cascade that ultimately leads to ADCC. 244 Jurkat cells stably expressing human FcγRIIIa-V158, that binds IgG1 more efficiently than 245 FcγRIIIa-F158, were used as effector cells to monitor cell activation. In this reporter assay, 246 FcγRIIIa engagement by immune-complexes transduces intracellular signals resulting in NFAT-247 mediated luciferase activity, which represents a robust and valid downstream readout for ADCC 248 induction by IgG1 antibodies 33 . Thus, huCD20 + RAJI cells were first opsonized with scFv2H7-249 P18F3 or scFv2H7 and then co-cultured with FcgRIIIa + Jurkat cells in presence of a pool of plasma 12 exhibiting a high titer of anti-P18F3 antibodies for 6 h. Treatment of RAJI cells with scFv2H7-251 P18F3 drastically increased NFAT-mediated luciferase activity in Jurkat cells in the presence of 252 plasma as compared to untreated cells or scFv2H7-coated cells ( Fig. 5d and Supplementary Fig.  253 9b), demonstrating that P18F3-mediated anti-P18 IgG opsonization of Burkitt' Lymphoma cells 254 triggers FcγRIIIa-mediated activation of intracellular signaling pathways that leads to ADCC. 255 256 Treatment with scFv2H7-P18F3 reduces cancer progression in mice bearing huCD20 + tumor 257

cells. 258
Firstly, in order to raise mouse antibodies directed against P18F3, 12 BALB/cByJ were immunized 259 with the P18FL protein fused to MBP (MBP-P18FL). Analysis of IgG subclasses 52 days after the 260 first injection revealed that around 46.8% of anti-P18F3 IgG are IgG1 and 34.9% IgG2b (mean 261 values) (Supplementary Fig. 10). A smaller proportion of anti-P18 IgG belonged to the IgG3 and 262 IgG2a subclasses (10.4% and 7.9%, respectively) (mean values). Individual pre-immune and 263 immune sera from the 12 mice were then used to perform an opsonization assay with EL4-WT 264 cells and transduced EL4-huCD20 cells that stably express human CD20 34 (Supplementary Fig.  265 11a). In the presence of immune mouse sera, binding of scFv2H7-P18F3 to EL4-huCD20 cells led 266 to the recruitment of anti-P18 mouse IgG as detected by an immunofluorescence assay using 267 specific goat anti-mouse IgG (Fc-specific) antibodies conjugated to allophycocyanine (APC) and 268 hence, to opsonization of target cells (Fig. 6a, right panel). No noticeable recruitment of IgG was 269 observed without scFv (Fig. 6a, left panel), when the scFv2H7 was tested (Fig. 6a,

middle panel) 270
and when EL4-WT were treated with scFv2H7 or scFv2H7-P18F3 and incubated with pre-immune 271 sera (Supplementary Fig. 11b, middle and right panels). 272 13 Second, to determine whether or not scFv2H7-P18F3 therapy can protect mice from tumor 273 challenge, C57Bl/6 immunocompetent mice were immunized with MBP-P18FL to generate 274 endogenous anti-P18 antibodies (Fig. 6b). Mice were then injected intravenously with 2.5 x 10 5 275 EL4-huCD20 cells (Day 0) and received scFv2H7-P18F3 therapy (group G1.1), consisting in four 276 intraperitoneal injections at days 1, 4, 7 and 10. In a first set of experiments, 3 additional control 277 groups (n=5) were injected with tumor cells. Mice from group G1.2 were left untreated, whereas 278 mice from group G1.3 received 4 injections of scFv2H7. Mice from group G1.4 received scFv2H7-279 P18F3 therapy but were not pre-immunized with MBP-P18FL (Fig. 6c). All mice from G1.2, G1.3 280 and G1.4 died within 35-50 days post tumor cell injection. In contrast, the overall long-term 281 survival in the G1.1 group (scFv2H7-P18F3 therapy) was 40% (Fig. 6c). In a second set of 282 experiments involving a larger number of animals, 2 groups were designed. The G2.1 group (n=11) 283 did not receive any treatment after injection of tumor cells whereas the animals from the G2.2 284 group (n=12) received scFv2H7-P18F3 therapy (Fig. 6d). All mice from the untreated group died 285 before Day 60 (median survival 35 days). ScFv2H7-P18F3 therapy significantly increased mice 286 survival (median survival 51.5 days; Log-rank Mantel-Cox test, p=0.0387) and led to an overall 287 17% long-term survival. Two days before EL4-huCD20 cell injection, most of circulating anti-288 P18F3 IgG in mice from G1.1 and G2.1 belonged to the IgG1 and IgG2b subclasses (Fig. 6c, 6d  289 -right panels) as already observed in sera from MBP-P18FL-immunized mice used to test IgG 290 opsonization (Supplementary Fig. 10). In addition, scFv2H7-P18F3 treatment led to increased 291 levels of circulating antibodies directed against P18F3 at Day 15 post-treatment (Supplementary 292 Here, we describe a strategy for re-directing a pre-existing EBV antibody response towards defined 296 pathogenic cells using bi-modular fusion proteins comprising a specific binding moiety and an 297 EBV-derived Ig recruiting antigen, P18. As a proof of concept, we first generated a set of BMFPs 298 targeting RBCs via an anti-DARC nanobody. Treatment of RBCs with BMFPs in presence of 299 human plasma from EBV + donors mediated target opsonization by circulating anti-P18 IgG and 300 the subsequent induction of erythrophagocytosis by macrophage-like cells. We then developed a 301 scFv-based BMFP directed towards huCD20 (scFv2H7-P18F3) and analysed its efficacy in vitro 302 and in vivo in a mouse tumour model. ScFv2H7-P18F3-mediated opsonization of Burkitt's 303 lymphoma cells, activated the antibody-dependent cascade of the complement system and engaged 304 FcγRIII in a cell assay recapitulating the first steps of ADCC triggering. When assessed in a tumour 305 model, scFv2H7-P18F3 therapy significantly increased mice survival, leading to total cancer 306 remission in some animals. 307 The initial part of this work consisted in establishing that BMFPs, expressed in a bacteria-based 308 system, could be designed to efficiently target defined cellular elements. The conservation of the 309 intrinsic biochemical and functional properties of both modules (P18 and binding moiety), is 310 crucial when designing BMFPs aiming at re-directing a pre-existing EBV antibody response 311 towards specific cells. In order to create a versatile therapeutic tool, in which the binding moiety 312 could be easily substituted, we first optimized the nature of the P18 antigen using a model targeting 313 DARC expressed at the surface of RBCs with a nanobody-based BMFP (Nb-aDARC). The EBV 314 antigen P18 has been selected primarily for its capability to be strongly bound by circulating IgG 315 from EBV-infected individuals 18 . Another important criterion for selecting P18 was the absence 316 of any post-translational addition of sugar moieties. P18 is a non-glycosylated protein of small size 317 (18 kDa) that could therefore be produced in a heterologous bacterial system. However, protein 318 15 sequence analysis of P18FL revealed that its N-terminus part displays several stretches of 319 hydrophobic residues and numerous prolines that could potentially interfere with the expression 320 of stable proteins in physiological buffers. Furthermore, P18FL harbors a cysteine in position 56 321 that could unwillingly engage in disulfide bond formation with other cysteines present within the 322 binding moiety sequence during folding of the fusion constructs as antibody fragments possess 323 structuring disulfide-bonds mandatory for their functionality. To overcome these potential issues, 324 we designed P18 fragments lacking the N-terminus region that contains Cys56. In addition, we 325 used a mutant E. coli strain (SHuffle) that promotes disulfide bond formation in the cytoplasm, 326 leading to more efficient folding of recombinant proteins, improved activity and increased 327 production yields 29 . We also optimized the P18 antigen to efficiently recruit circulating anti-P18 328 IgG, without affecting the functionality of the binding moiety. P18F3 was down-selected as the 329 prime P18-derived antigen, presenting binding titers by circulating anti-P18 IgG comparable to 330 that of P18FL, thus confirming the presence of immunodominant epitopes within the C-terminus 331 part of the protein 35 . Notably, fusion of P18F3 to Nb-aDARC did not significantly affect the 332 affinity of the Nb for its cognate target, DARC. Furthermore, RBC treatment with Nb-aDARC-333 P18F3 triggered erythrophagocytosis by THP1-derived macrophages. The amplitude of 334 erythrophagocytosis correlated with the P18F3-binding titers of the circulating IgG, strongly 335 suggesting that erythrophagocytosis was driven by IgG opsonization rather than by pattern 336 recognition receptors or mannose receptor engagement. 337 Based on these results, we then generated a P18F3-derived BMFP comprising a scFv binding 338 moiety targeting huCD20 that is expressed at the surface of B lymphocytes from early 339 developmental stages (late pro-B cell) to late stages (memory B cell) as well as in most non-340 Hodgkin's B lymphomas. MAb therapy targeting huCD20 has revolutionized the treatment of B-341 16 cell malignancies for more than 30 years, becoming the leading therapeutic agents for the care of 342 numerous B cell-related cancers such as follicular lymphoma, diffuse large B-cell lymphoma, 343 mantle cell lymphoma as well as Burkitt's lymphoma 36 . The most striking example of such success 344 is rituximab 37 . Several other therapeutic anti-huCD20 mAbs such as ofatumumab, obinutuzumab, 345 and ublituximab (derived from the EMAB-6 antibody 38 ) have been developed since the advent of 346 rituximab (with alternate binding epitopes, additional humanization and modified 347 glycosylations) 39,40 . We have developed herein a binding moiety targeting the large extracellular 348 loop of huCD20 based on the C2H7 (2H7) chimeric antibody, which presents a different binding 349 epitope profile than rituximab 41 . ScFv2H7-P18F3 expression in SHuffle resulted in the production 350 of a soluble protein, with a production yield reaching 0.7-1 mg ml -1 of bacteria culture, in line with 351 previous work highlighting the difficulty to express stable and soluble scFv molecules 42,43 . We The DNA sequence ORF BFRF3 encoding for P18 was recoded and optimized for E. coli codon 564 usage (Integrated DNA Technology) to allow maximal expression in E. coli-based systems. The 565 full-length P18 (P18FL) recoded sequence as well as the truncated fragment (P18F2, P18F3, 566 P18F4) sequences were cloned into a pet28a-derived plasmid (Novagen) in order to express C-567 terminus His-tagged proteins. 568 The anti-human CD20 (huCD20) VH and Vk sequences of scFv2H7 were obtained from the 569 sequences of an anti-human-CD28 x anti-huCD20 bispecific scFv antibody (clone r2820) 570 (Genebank AJ937362) and synthesized according to the following orientation: VH-(GGGGS)3-Vk. 571 The DNA sequences of Nb-aDARC and scFv2H7 were inserted between the NheI and NcoI 572 restriction sites of Pet28a-NC (Supplementary Fig. 2). For each BMFP set, a construct 573 comprising the binding moiety but lacking P18 was also generated. 574 For protein expression, E. coli SHuffle (New England Biolabs) were transformed with the different 575 constructs. Bacteria cultures were induced with 0.2 mM IPTG at OD600nm 0.8 and protein 576 expression was carried out at 20°C for 16 h. The bacteria pellets were resuspended in 50 mM Tris, 577 500 mM NaCl at pH 7.2 and the samples were then frozen at -80°C until further use. For protein 578 purification, bacteria suspensions were thawed on ice and supplemented with EDTA-free 579 cOmplete Protease Inhibitors (Roche) and with 1 mg ml -1 lysozyme from chicken egg white 580 (Sigma). Bacteria lysis was achieved by passing the cell suspensions through an EmulsiFlex-C5 581 high-pressure homogenizer (Avestin), three times at 4°C. Following centrifugation at 12,000 x g, 582  Left panel. Following expression and puri cation, the purity of the MBP-P18FL fusion protein and of MBP alone was assessed by SDS-PAGE followed by Coomassie blue staining. Right panel. ELISA results of the binding of IgG present in plasma samples from 22 EBV+ individuals (1:100 dilution) to αDARC-P18 BMFPs. The full-length P18 polypeptide was included in the assay (MBP-P18FL). OD: Optical Density. MBP: Maltose-Binding Protein. Seven plasma samples displaying low, mild or high binding levels to Nb-αDARC-P18F3 (red, dark blue and light blue dots, respectively) were mixed together to obtained 3 different plasma pools (low, mild, high) to be used in further experiments (Fig. 3). The plasma sample recognizing both MBP (white dot) and Nb-αDARC alone (white dot) was excluded from pooling.

Figure 3
Nb-αDARC-P18F3 binds to native DARC and recruits anti EBV-human IgG (huIgG) to the RBC surface promoting target opsonisation. (a) The ability of Nb-αDARC-P18F3 to promote RBC opsonization by IgG present in the human plasma pools exhibiting different antibody titers against P18F3 was assessed by an indirect immuno uorescence assay and ow cytometry. Nb-αDARC and Nb-α DARC-P18F3 were used at a concentration that results in a similar binding to RBCs, 9.6 nM ( Supplementary Fig 4). Membrane bound huIgG were detected using anti-huIgG antibodies conjugated to phycoerythrin (PE). Plotted data represent the mean values of duplicates. (b) A qualitative analysis was performed by confocal microscopy to con rm that the signal resulting from huIgG detection was localized at the RBC surface. Upper panels. Confocal laser scanning microscopy images of huIgG distribution on the RBC membrane after incubation of RBCs with Nb-αDARC or Nb-α DARC-P18F3 in presence of plasma exhibiting a high antibody titer to P18F3. The binding of huIgG was revealed with goat Alexa Fluor-488-anti-huIgG antibodies (indicated as A-488). (c) 2.5D representation of the A-488 signal obtained when RBCs were incubated with Nb-α DARC-P18F3 in presence of plasma exhibiting a high antibody titer to P18F3. The right inset shows the enlarged 2.5D representation. Fluorescence is located at the RBC surface. Due to the biconcave shape of RBCs, membrane uorescence located within the cell structure on the scanning microscopy images must not be mistakenly interpretated as cytosolic uorescence. Confocal microscopy was performed with a Zeiss LSM700 microscope and images were analyzed with ZEN 2.0 software. AF-488: Alexa 488.

Figure 4
Nb-αDARC-P18F3-mediated huIgG RBC opsonization triggers erythrophagocytosis by macrophage-like THP1 cells. The ability of Nb-αDARC-P18F3 to promote RBC phagocytosis by THP1-derived macrophages was assessed in an erythrophagocytosis assay. CFSE-stained DARC+ RBCs were rst incubated with Nb-αDARC or Nb-αDARC-P18F3 and with plasma pools exhibiting different antibody titers against P18F3. Following a 3h culture with THP1-derived macrophages, non-phagocytized RBCs were lysed and THP1 cells were then subjected to immuno uorescence analysis. (a) Representative data obtained from one experiment. A CFSE+ THP1 cell was regarded as a cell having phagocytized at least one RBC. Untreated cells (left panel) served as a reference for basal erythrophagocytosis by macrophage-like THP1-derived cells. Nb-αDARC-P18F3 treatment of RBCs in presence of plasma exhibiting a high antibody titer against P18 (indicated plasma high) led to a marked increase in the percentage of CFSE+ THP1 cells (right panel) as compared to Nb-αDARC-treated RBCs (middle panel). CFSE+ THP1 represents CFSE+ macrophage-like THP1-derived cells. SCC, Side SCatter. (b) Results obtained from 4 independent experiments are expressed as a fold increase (ordinate) in the percentage of CFSE+ macrophage-like THP1-derived cells incubated with RBCs coated with Nb-αDARC or Nb-αDARC-P18F3 and plasma pools exhibiting different antibody titers against P18F3 (high, intermediate, low) compared to the percentage of CFSE+ cells incubated with untreated RBCs (i.e., no incubation with Nb-αDARC or Nb-αDARC-P18F3 and no plasma).
Box plots include the mean horizontal line and interquartile range (box), whereas the whiskers represent the minimal and maximal values. Group comparison was performed using the non-parametric Mann-Whitney test. P-values < 0.05 were regarded as statistically signi cant (*). ns: non signi cant. Inter : intermediate.

Figure 5
ScFv2H7-P18F3-mediated opsonization of Burkitt's Lymphoma cells induces activation of the antibodydependent complement cascade and triggers FcγRIIIa-mediated activation of intracellular signaling pathways that leads to ADCC. (a) Architecture of BMFP (scFv2H7-P18F3) and of scFv2H7 targeting huCD20. MW: Molecular weight. (b) Gel ltration pro les of scFv2H7 and scFv2H7-P18F3. For each elution, red bars delimit the protein of interest (POI) pick. AU: Arbitrary Units. (c) The capability of scFv2H7-P18F3 to promote complement activation through the classical pathway (antibody-dependent) was assessed by monitoring the deposition of the membrane attack complex (MAC) C5b-9 at the surface of RAJI cells. RAJI cells were incubated with saturating concentration of scFv2H7 or scFv2H7-P18F3 and with a pool of human plasma exhibiting a high titer of anti-P18 antibodies. Ø: no scFv protein. Results obtained from 4 independent experiments are depicted. Box plots include the mean horizontal line and interquartile range (box), whereas the whiskers represent the minimal and maximal values. Group comparison was performed using the non-parametric Mann-Whitney test. P-values < 0.05 were regarded as statistically signi cant (*). ns: non-statistically signi cant. (d) The capability of scFv2H7-P18F3 to promote the early events that lead to ADCC was assessed using a reporter assay. Activation of gene transcription through the NFAT pathway in competent effector Jurkat cells was quanti ed using a luciferase assay. RAJI cells were incubated with saturating concentration of scFv2H7 or scFv2H7-P18F3 and with a pool of human plasma exhibiting a high titer of anti-P18 antibodies and put in presence of Jurkat cells expressing human FcγRIIIa-V158 (ratio: 1:6) for 6 h. NFAT pathway activation was monitored by reading the luminescence of each plate well. CPS: counts per second. Ø: no scFv protein. Results obtained from 2 independent experiments performed in triplicates are depicted (exp.1 , exp.2 ). Boxes represent the mean values of the six measurements and the error bars depict the associated standard deviations.