Novel Single-Chain Fv Formats for the Generation of

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Novel Single-Chain Fv′ Formats for the Generation of Immunoliposomes by Site-Directed Coupling Sylvia K. E. Messerschmidt,† Anke Kolbe,† Dafne Müller,† Michael Knoll,‡ Jürgen Pleiss,‡ and Roland E. Kontermann*,† Institut für Zellbiologie und Immunologie, Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany, and Institut für Technische Biochemie, Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany. Received September 10, 2007; Revised Manuscript Received October 2, 2007

Immunoliposomes generated by coupling of antibodies to the liposomal surface allow for an active targeting of entrapped compounds to diseased areas. Single-chain Fv fragments (scFv) represent the smallest part of an antibody containing the entire antigen-binding site. They can be coupled in a defined and site-directed manner through genetically engineered cysteine residues, for example, those added at the C-terminus. Here, we have performed a comparative analysis of various scFv′ variants with cysteine residues present at the end of a C-terminal extension of varying length and composition (HC variants) or introduced in the linker sequence connecting the variable heavy and light chain domain (LC variants). Using a scFv fragment directed against fibroblast activation protein (FAP) as a model antibody, we could show that all variants can be employed for the generation of active immunoliposomes, although the presence of three additional cysteine residues in one scFv′ molecule resulted in decreased binding of immunoliposomes compared to that of immunoliposomes generated with scFv′ molecules containing only one additional cysteine residue. In order to further improve the scFv′ format by reducing the number of additional amino acid residues, we also generated molecules with the hexahistidyl-tag incorporated into the linker sequence together with a cysteine residue either at position 1 or 3 of the linker sequence (LCH variants). These newly designed scFv′ molecules may be particularly suitable for the generation of immunoliposomes and other antibody conjugates, limiting the number of additional residues in these antibody molecules to a minimum.

INTRODUCTION Immunoliposomes have been developed for targeted drug delivery, for example, the delivery of chemotherapeutic drugs to tumor tissues (1). These immunoliposomes are generated by coupling antibodies or antibody fragments to the surface of liposomal drugs (2). Initially, whole antibodies were used for this purpose. However, several studies have shown that whole antibodies, for example, monoclonal antibodies of murine origin, induce immune responses, which can lead to neutralization (3, 4). Furthermore, immunoliposomes composed of whole antibodies are rapidly cleared from circulation because of Fc-receptor mediated uptake by cells of the reticulo-endothelial system (5). Hence, new approaches have emerged that employ antibody fragments, such as Fab′ fragments, which can be obtained from whole antibodies by proteolytic cleavage and subsequent reduction or by production in recombinant form, for example, in bacteria (6). Immunoliposomes composed of Fab′ fragments have shown reduced immunogenicity and improved therapeutic effects compared to IgG immunoliposomes (7). Progress in antibody engineering technologies has led to the establishment of various methods to obtain antibodies with desired binding properties from combinatorial antibody libraries, for example, using phage display technology (8). These approaches also allow for the isolation of human antibody molecules, which further reduce or even avoid immunogenicity in humans. Thus, a large variety of such antibodies has been already isolated and is available for the generation of targeted therapeutics. Routinely, these antibodies are present as single* Corresponding author. Tel: +49 711 685-66989. Fax: +49 711 685-67484. E-mail: [email protected]. † Institut für Zellbiologie und Immunologie. ‡ Institut für Technische Biochemie.

chain Fv (scFv) fragments. This format represents the smallest part of an antibody with full antigen-binding activity and is therefore sufficient for the generation of targeted therapeutics. Hence, scFv molecules are ideally suited for the generation of immunoliposomes, and various studies have already demonstrated their feasibility (6). To achieve site-directed and defined coupling, one or more additional cysteine residues have been introduced into the scFv molecule, mainly at the C-terminus, which allow conjugation to sulfhydryl-reactive reagents (9, 10). Such scFv′ molecules, that is, scFv possessing free sulfhydryl groups, have been successfully employed for the generation of immunoliposomes (9–11) but also for other applications, for example, site-directed PEGylation to improve pharmacokinetic properties and the generation of immunosensors and targeted gold nanoparticles (12–16). Different scFv′ formats have been described in the literature, differing in the position and number of additional cysteine residues. Thus, up to three cysteine residues have been added to the C-terminus of a scFv molecule (9, 10, 16–20). These scFv′ molecules also varied in the length and composition of the C-terminal extension. In addition, a few studies have described monovalent or bivalent scFv molecules with an additional cysteine residue introduced in the linker sequence connecting the variable domains (14, 21). Recent studies have shown that the position and number of the cysteine residues incorporated can influence the coupling efficiency and functionality of the generated immunoliposomes (ref 10; unpublished observations). Here, we performed a comparative analysis of scFv′ variants with one or three additional cysteine residues introduced either at a C-terminal extension of varying length or at different positions in the linker peptide using a scFv fragment directed against fibroblast activation protein (FAP) as model antibody (22). The novel

10.1021/bc700349k CCC: $40.75  2008 American Chemical Society Published on Web 11/08/2007

Novel Single-Chain Fv′ Formats

finding that scFv′ molecules with a linker cysteine can be used for the generation of immunoliposomes prompted us to generate optimized scFv′ molecules with a hexahistidyl-tag sequence as well as the additional cysteine located in the linker sequence, thus reducing the number of additional residues in a scFv′ molecule to a minimum.

MATERIALS AND METHODS Materials. All lipids were purchased from Avanti Polar Lipids (Alabaster, USA). DiI was purchased from Sigma (Taufkirchen, Germany). HRP-conjugated anti-His-tag antibody was purchased from Santa Cruz Biotechnology (USA) and FITC-conjugated anti-His antibody from Dianova (Hamburg, Germany). Human plasma (stabilized with citrate/phosphate/ dextrose solution (CPD)) was kindly provided by the blood center of Katharinenhospital (Stuttgart, Germany). The stably transfected human FAP-expressing fibrosarcoma cell line HT108013.8 (HT1080-FAPmo) expressing mouse FAP as well as HT1080 wild-type cells was grown in RPMI, 5% FCS, and 2 mM glutamine. Cloning and Expression of scFv′ Variants. The scFv′ 36 HC constructs (HC2–4) were generated by PCR amplification of scFv′ 36-encoding DNA present in bacterial expression vector pABC4 (22) using primers LMB3 (5′-CAG GAA ACA GCT ATG ACC-3′) and HC2-Eco-For (5′-GCG CAT CAT CAC CAT CAC CAT GGC GGA TCG AGT GGC TCA GGA TGC TAA GAA TTC CAC TGG-3′), HC3-Eco-For (5′-GCG CAT CAT CAC CAT CAC CAT GGC GGA TCG AGT GGC TCA TGC GGA TGT AGT TGC TAA GAA TTC CAC TGG-3′), or HC4-Eco-For (5′-CAT CAT CAC CAT CAC CAC GGC GGA TCC AGC GGC GGA TCC AGC GGC TCC GGA TGC TAA GAA TTC CGG-3′), respectively. The resulting PCR products were digested with SfiI and EcoRI and cloned into vector pAB1 digested with the same enzymes. For generating the LC variants (LC1–3), scFv′ molecules were amplified from pAB1-scFv36 with primer LMB2 (5′-GTA AAA CGA CGG CCA GT-3′) and LC1-Xho-Back (5′-ACC GTC TCG AGT TGC GGA GGC GGT TCA GGC GGA GGT GGC TCT-3′), LC2-Xho-Back (5′-ACC GTC TCG AGT GGT TGC GGC GGT TCA GGC GGA GGT GGC TCT-3), and LC3-Xho-Back (5′-ACC GTC TCG AGT GGT GGA TGC GGT TCA GGC GGA GGT GGC TCT-3′), respectively. PCR products were digested with XhoI and NotI and cloned into pAB1-scFv36 digested with the same enzymes. LCH variants were generated in the same manner using primers LCH1-Xho-Back (5′-ACC GTC TCG AGT TGC GGA GGC GGT CAT CAT CAC CAT CAC CAT GGA GGC GGT AGT GCA CAA ATT CTG ATG3′) and LCH3-Xho-Back (5′-ACC GTC TCG AGT GGT GGA TGC GGT CAT CAT CAC CAT CAC CAT GGA GGC GGT AGT GCA CAA ATT CTG ATG-3′) as back primers. Antibody fragments were purified by immobilized metal ion affinity chromatography (IMAC) as described elsewhere (23). Protein concentration was determined by measuring the absorbance at 280 nm. scFv′ were analyzed by SDS-PAGE under reducing and nonreducing conditions and stained with Coomassie brilliant blue R250 or immunoblotted with a HRP-conjugated anti-Histag antibody. Generation of scFv Immunoliposomes by the Postinsertion Method. Immunoliposomes were generated by postinsertion of scFv′-conjugated micelles into preformed PEG-liposomes. All liposomes were prepared by the film hydration-extrusion method and were composed of EPC/Chol/mPEG-DSPE at a molar ratio of 6.5:3:0.5. In addition, all liposomes contained 0.3 mol % DiI as fluorescent dye. Lipids and DiI were dissolved in chloroform, and a thin lipid film was formed by removing the chloroform in a rotary evaporator at 42 °C and drying under vacuum for at least 1 h. The lipid film was hydrated with 1 mL

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of 10 mM Hepes buffer at pH 6.7. The final lipid concentration was 10 µmol lipid per mL of buffer. The resulting multilamellar vesicle dispersion was extruded 21 times through 50 nm polycarbonate membranes using a LiposoFast extruder (Avestin, Ottawa, Canada). For the preparation of Mal-PEG-DSPE micelles, chloroform was removed by incubation at RT and lipid dissolved in ddH2O to a final concentration of 10 mg/mL. For the coupling of scFv′ to Mal-PEG-DSPE micelles, purified scFv were reduced in 2 mM tris(2-carboxyethyl)phosphine (TCEP) for 2 h at room temperature followed by the removal of TCEP by dialysis against O2 free 10 mM Na2HPO4/NaH2PO4 buffer, 0.2 mM EDTA, and 30 mM NaCl (pH 6.7) overnight at 4 °C. Micellar lipid and reduced scFv′ were mixed at a molar ratio of 4.67:1 (24). The coupling reaction was performed at room temperature for 30 min The reaction was quenched with 1 mM L-cysteine and 0.02 mM EDTA at pH 5.5. The scFv′-coupled micelles were inserted into preformed PEGylated liposomes by incubation at 55 °C for 30 min (0.6 mol % and 2 mol % micellar lipid with respect to liposomal lipid). Unbound scFv molecules were removed by gel filtration using a Sepharose CL4B column (Amersham, Braunschweig, Germany). Liposome size and ζ-potenial were measured using a ZetaSizer Nano ZS (Malvern, Herrenberg, Germany). Analysis of Coupling Efficiency. The scFv′-coupled micelles were analyzed by SDS-PAGE under reducing conditions and stained with Coomassie Brilliant Blue R250. The coupling efficiency was determined by quantifying the intensity of the protein bands before and after coupling to micelles using the software ImageQuant (GE Healthcare, Germany). Determination of Protein Melting Points. The melting point of the scFv′ 36 variants was determined with ZetaSizer Nano ZS (Malvern, Herrenberg, Germany). Approximately 150 µg of purified scFv′ protein was diluted in PBS to a total volume of 1 mL and sterile filtered into a quartz cuvette. Dynamic laser light scattering intensity was measured while the temperature was increased in 1 °C intervals from 30 to 70 °C with 2 min equilibration for each temperature step. The melting point was defined as the temperature at which the light scattering intensity dramatically increased. Flow Cytometry. Cells were detached from cell culture dishes using 0.05% trypsin and 0.02% EDTA (GIBCO). Approximately 250,000 cells were incubated with DiI-labeled immunoliposomes (10 nmol lipid) in PBS containing 2% FCS and 0.02% sodium azide (PBA) for 1 h at room temperature. After washing cells three times with PBA buffer (4 °C), cells were resuspended in 500 µL of PBA buffer and analyzed by flow cytometry (Epics XL-MCL, Beckmann). The binding of purified scFv′ molecules was determined by incubating detached cells with antibody molecules (10 µg/mL) in PBA buffer for 1 h at room temperature. Cells were then washed three times as described and incubated with FITC-conjugated anti-His-tag antibody diluted 1/200. Data were evaluated with WinMDI, version 2.8. In Vitro Plasma Stability. To analyze plasma stability, immunoliposomes (10 nmol lipid) were preincubated in the presence of 50% human plasma for 4 days at 37 °C in a total volume of 50 µL. Subsequently, the binding of immunoliposomes to cells was analyzed by flow cytometry as described above. Modeling of scFv 36 Variants. A model of scFv 36 without the linker was generated using the Web Antibody Modeling (WAM) server (25). A linker of 14 amino acids connecting the VH and VL subunits was modeled using MODELER 6v2 (26). The linker region did not contain secondary structure as expected from the distance between the C-terminus of VH and the N-terminus of VL along the surface of the protein. The quality of the scFv 36 model was checked by PROSA2003’s knowledge-

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Figure 1. Composition of scFv′ variants. (a) C-terminal sequences of constructs HC2, HC3, and HC4 as well as the linker sequences of constructs LC1, LC2, and LC3. Cysteine residues are marked with an asterisk. The Myc/His-tag has the sequence -EQKLISEEDLNGAAHHHHHH-. (b) Model structure of anti-FAP scFv′ 36 with the positions of the three linker modifications LC1–3 shown from two sides. Sulfur atoms of the three cysteine residues incorporated into the linker peptide are shown as spheres (1–3).

Messerschmidt et al.

Figure 2. SDS-PAGE and immunoblot analysis of purified scFv′ variants. Purified scFv′ variants HC2–4 and LC1–3 were analyzed by 15% SDS-PAGE under nonreducing (c and d) or reducing (a and b) conditions and either stained with Coomassie or immunoblotted with an anti-His-tag antibody (2 µg/lane were used for Coomassie staining and 1 µg/lane for immunoblotting).

based potentials (27), and stereochemical checks were carried out with the PROCHECK package (28, 29).

RESULTS Generation and Characterization of scFv′ Variants. Six different scFv′ variants of anti-FAP scFv 36 were designed (Figure 1a). The variants HC2 and HC4 contain one additional cysteine residue at the C-terminus and vary in the length of the C-terminal extension, which also contains a hexahistidyl-tag for purification. Variant HC3 contains three additional cysteine residues at the C-terminal extension. In addition, we designed three scFv′ variants (LC1–3) containing an additional cysteine residue in the peptide linker (position 1, 2, or 3, respectively). These variants are also provided with a vector-encoded Myctag and hexahistidy-tag at the C-terminus. In order to show that the cysteine side chains are accessible for coupling reactions, a model of scFv′ 36 including the linker connecting the variable domains was generated. This model structure of scFv′ 36 indicated that the linker modifications are located at the bottom of the scFv′ molecule opposite the antigen-binding site and that the side-chain sulfhydryl groups are accessible for coupling (Figure 1b). All six scFv′ variants could be expressed in bacteria and purified from periplasmic preparations by IMAC. Yields of 0.2–0.8 mg of purified protein per liter culture were obtained. These yields are similar to yields typically obtained for unmodified scFv 36 (0.3–0.7 mg/L culture). A single band with an apparent molecular weight of 32 kDa was detected in SDS-PAGE under reducing conditions (Figure 2a). Two bands of 30 and 60 kDa were seen under nonreducing conditions for the HC constructs, while the LC constructs showed only one prominent band of 30 kDa (Figure 2c). The identity of these protein bands was further confirmed by immunoblotting with an anti-His-tag antibody (Figure 2b and d). This experiment

Figure 3. Size exclusion chromatography. scFv′ molecules were analyzed by HPLC size exclusion chromatography using a BioSepSec-2000 column. Peak positions of standard proteins are indicated.

also revealed for the LC1–3 preparations a second protein band of approximately 20 kDa, probably due to proteolytic cleavage. Therefore, LC variants appeared to be more susceptible to proteolytic cleavage than the HC variants. The cleavage band was strongest for the LC2 variant. All scFv′ variants showed a single major peak in size exclusion chromatography (Figure 3). The three HC variants eluted with an apparent molecular mass of 22.5 kDa (calculated molecular mass, 28 kDa), while all LC variants eluted with an apparent molecular mass of 29 kDa (calculated molecular mass, 28 kDa). The thermal stability of the scFv′ variants was deduced from their melting points as measured by dynamic light scattering (Figure 4). For all HC constructs, the melting point was


Figure 4. Thermal stability. Protein melting curves of the scFv′ variants were determined by dynamic light scattering using 1 °C intervals.


Figure 6. Coupling of scFv′ variants to Mal-PEG-DSPE micelles. SDS-PAGE analysis of scFv′ fragments before (1) and after (2) coupling to Mal-PEG2000-DSPE micelles at a molar ratio of antibody to lipid of 1:4.67. Gels were stained with Coomassie. Coupling of the antibody molecules to the lipid is indicated by reduced mobility. Table 1. Coupling Efficiencies of scFv′ Variantsa construct scFv′ scFv′ scFv′ scFv′ scFv′ scFv′

coupling efficiency (%) 86.7 ( 4.5 97.8 ( 0.2 82.0 ( 7.5 79.7 ( 4.7 81.8 ( 3.9 90.2 ( 2.4

36-HC2 36-HC3 36-HC4 36-LC1 36-LC2 36-LC3

a Coupling efficiencies were determined by quantitative analysis of scFv′ fragments before and after coupling to micelles (see Figure 6).

Table 2. Size, Polydispersity Index (PDI) and ζ-Potential of scFv Immunoliposomesa construct

Figure 5. Binding of scFv′ variants to FAP-expressing cells. scFv′ molecules were analyzed by flow cytometry for binding to HT1080FAPmo cells. Gray areas, cells incubated without scFv′; solid line, cells incubated with scFv′ molecules.

determined to be around 56–57 °C. The LC constructs showed a slightly higher melting point with 58–59 °C. All scFv′ variants specifically recognized FAP-expressing cells (HT1080-FAPmo) in flow cytometry experiments, indicating that the incorporation of additional cysteine residues at the C-terminus or linker region does not interfere with antigen binding activity (Figure 5). Coupling of scFv′ Fragments to Mal-PEG-DSPE Micelles. Next, we analyzed the coupling of the scFv′ fragments to MalPEG-DSPE micelles using a molar scFv′ to lipid ratio of 1:4.67. The coupling reaction was performed at room temperature for 30 min. SDS-PAGE analysis demonstrated efficient coupling of all constructs (Figure 6). Coupling efficiency was in the range of 80–98% as determined by densitometric quantification (Table 1). Highest coupling efficiency was achieved with construct HC3. All constructs possessing one additional cysteine showed after coupling an increase in apparent molecular mass of approximately 3 kDa, while the mass of HC3 was increased by approximately 10 kDa. This finding indicates that one Mal-PEGDSPE chain is conjugated to HC2, HC4, and LC1–3, while three chains are conjugated to HC3. Generation of scFv Immunoliposomes by the Postinsertion Method. scFv′-coupled micelles were used to generate scFv immunoliposomes, applying the postinsertion method. In this method, scFv′-coupled micelles are inserted into preformed PEGylated liposomes by incubation at 55 °C for 30 min (30, 31). Two different micelle to liposome ratios (0.6 mol % and 2 mol

scFv′ scFv′ scFv′ scFv′ scFv′ scFv′

36-HC2 36-HC3 36-HC4 36-LC1 36-LC2 36-LC3

size (nm)

PDI

ζ-potential (mV)

108 ( 2.3 90 ( 1.5 96 ( 1.2 102 ( 2.8 106 ( 3.7 105 ( 7.6 105 ( 7.7

0.123 ( 0.038 0.159 ( 0.058 0.134 ( 0.032 0.156 ( 0.034 0.178 ( 0.021 0.149 ( 0.004 0.112 ( 0.054

5.2 ( 3.5 -12.6 ( 5.4 -12.5 ( 2.8 -15.0 ( 0.5 -8.8 ( 1.5 -11.2 ( 7.9 -2.8 ( 2.3

a Immunoliposomes were generated by postinsertion of scFv-conjugated micelles (2 mol % lipid) into preformed PEG-liposomes.

% micellar lipid with respect to liposomal lipid) were used for the preparation of immunoliposomes. In both cases, the generated immunoliposomes had a size of 90–106 nm, similar to the size of the acceptor liposomes used for the postinsertion of micelles (108 nm) (Table 2). All immunoliposomes had a slightly negative ζ-potential, while the ζ-potential of plain liposomes was slightly positive (Table 2). ScFv density was calculated to be in the range of 1.1–1.2 nmol scFv/µmol lipid at 0.6 mol % micellar lipid and 3.7–3.9 nmol scFv/µmol lipid at 2 mol % micellar lipid. All immunoliposomes specifically bound FAP-expressing HT1080-FAPmo cells (Figure 7). No binding to these cells was observed for plain (untargeted) liposomes. None of the liposomes showed binding to FAP-negative HT1080 cells. Binding was stronger when higher micellar concentrations were used for postinsertion. Binding intensity was similar for all immunoliposomes, except for scFv HC3-derived immunoliposomes, which showed a reduced binding activity (approximately 50% of the other immunoliposomes based on the mean fluorescence intensity) (Table 3). Furthermore, the binding of all immunoliposomes to HT1080-FAPmo cells could be completely blocked by soluble scFv36 (10 µg/mL), while no blocking was seen with an irrelevant scFv molecule directed against another antigen


Messerschmidt et al.

Figure 7. Binding of scFv-immunoliposomes to FAP-expressing cells. Micelle-coupled scFv′s were inserted at two concentrations (0.6 mol % and 2 mol % micellar lipids) into preformed PEGylated liposomes and analyzed for binding to FAP-expressing HT1080 cells (HT1080-FAPmo) or to wild-type HT1080 cells. Plain liposomes (-), i.e., untargeted liposomes, were included as the negative control. Gray areas, cells alone; thick line, cells incubated with liposomes at 10 nmol lipid per 250.000 cells.

expressed by these cells (not shown). All immunoliposomes were highly stable in human plasma, showing no or only marginal reduction in binding after incubation in human plasma at 37 °C for 4 days (not shown). scFv′ Formats Combining a Cysteine Residue and Hexahistidyl-Tag in the Linker. In order to reduce the number of additional amino acids in the scFv′ molecule, we generated new scFv′ variants with the hexahistidyl-tag incorporated into the linker sequence together with an additional cysteine residue either at position 1 or 3 of the linker sequence (Figure 8a). These LCH variants (LCH1 and LCH3) terminate at the C-terminus with residue Arg107 of the VL domain, thus containing only 14 amino acid residues

of the linker sequence as additional residues. Both variants could be expressed in bacteria and purified by IMAC with yields of 0.3 mg/L culture, similar to the HC and LC variants (Figure 8b and c). For both variants, SDS-PAGE analysis showed a predominant band of 26 kDa under reducing and nonreducing conditions. Both variants also formed dimers. In addition, some proteolytic cleavage products were observed (corresponding to 15–25% of the intact molecules). Although detection with anti-His-tag antibodies of cell-bound scFv′ LCH variants was strongly reduced (not shown), immunoliposomes generated with both LCH variants (size, 103.0 ( 2.1 nm for LCH1 and 105.6 ( 1.7 nm for LCH3) showed strong and specific binding to FAP-expressing cells (Figure



Table 3. Binding of scFv Immunoliposomes to FAP-Expressing Cellsa construct scFv′ scFv′ scFv′ scFv′ scFv′ scFv′

36-HC2 36-HC3 36-HC4 36-LC1 36-LC2 36-LC3

MFI 9.0 ( 3.1 104.8 ( 20.5 48.5 ( 10.4 108.2 ( 35.4 97.7 ( 35.5 109.8 ( 52.2 96.8 ( 41.8

a Binding is shown as mean fluorescence intensity (MFI) derived from four preparations of each immunoliposome.

8d). Binding was similar to that observed for LC variants (Figure 8d) and could be blocked with soluble anti-FAP scFv (not shown).

DISCUSSION Here, we investigated two strategies for the generation of scFv′ molecules suitable for site-directed coupling via reactive sulfhydryl groups. Strategy 1 is based on scFv′ molecules having one or more additional cysteine residues at the end of a C-terminal extension of varying length. These C-terminal extensions introduce some additional spacing as well as flexibility between the antibody molecule and the reactive partner. Strategy 2 is based on molecules having one additional cysteine residue incorporated into the peptide linker connecting the VH and VL domains, thus not requiring any additional C-terminal extension. Using an anti-FAP scFv as the model antibody, we could show that all these scFv′ derivatives could be expressed in E. coli and purified in soluble form with the majority of the molecules present in monomeric form. Yields of the scFv′ molecules were similar to those obtained for the unmodified scFv 36. Thus, the addition of one or three cysteine residues either to the C-terminus or to the linker sequences does not lead to reduced expression levels of this scFv molecule, as described for other scFv′ molecules that showed markedly reduced expression levels (32). Some scFv′ dimers could be observed in SDS-PAGE and immunoblotting analysis, especially for the three HC constructs but also for the LCH constructs, indicating that the cysteine residues are accessible for the formation of disulfide-linked homodimers. Furthermore, all molecules were functional, active, and possessed similar melting points. These findings demonstrate that the addition of one or three cysteine residues as well as C-terminal extensions does not influence expression, conformation, stability, and binding activity. All scFv′ constructs could be coupled to maleimide-containing lipids. Coupling efficiencies and binding activity of the resulting immunoliposomes were similar for all constructs containing one additional cysteine residue, irrespective of whether it is present at the C-terminus or in the linker sequence. In contrast, the presence of three cysteine residues at the C-terminus resulted in immunoliposomes with reduced binding activity. SDS-PAGE analysis indicated that this variant (HC3) reacts with three MalPEG-DSPE molecules. After insertion into liposomes, this might lead to unfavorable orientation on the lipid bilayer, resulting in reduced accessibility and cell binding. All scFv immunoliposomes described in the literature so far have been prepared from scFv′ molecules with the additional cysteine(s) located at the C-terminus (9, 10, 18, 20, 22, 33, 34) or have been conjugated through amino-reactive coupling reagents (35, 36). The use of cysteine residues engineered into the recombinant antibody molecule allows for a site-directed and oriented coupling compared to coupling to amino groups, which is undirected and might thus influence antigen-binding activity. Because the C-terminus of a scFv molecule is located

Figure 8. LCH variants. (a) Sequence of the linker of constructs LCH1 and LCH3 with the additional cysteine and the hexahistidyl-tag present in the linker peptide. (b) Coomassie-stained SDS-PAGE (2 µg/lane) and (c) immunoblot experiments (1 µg/lane) with the anti-His-tag antibody of purified LCH1 and LCH3 analyzed under reducing (left side of marker) or nonreducing (right side of marker) conditions. (d) Flow cytometry analysis of binding of LCH1 and LCH3 immunoliposomes to FAP-expressing HT1080 cells or FAP-negative HT1080 wildtype cells. The binding of LC1 and LC3 immunoliposomes was included for direct comparison. Gray areas, cells alone; thick line, cells incubated with liposomes at 10 nmol lipid per 250.000 cells.

opposite the antigen-binding site, coupling to reactive lipids or PEG chains should not interfere with the recognition of target cells. This was further confirmed in our study applying three constructs with C-terminal cysteines. Interestingly, functional immunoliposomes could also be prepared with scFv′ molecules with an additional cysteine residue present in the linker sequence. For this approach, we compared three positions (1–3) of the linker peptide. No


differences in coupling efficiency and binding to target cells were observed between these three constructs or in comparison to the HC2 and HC4 constructs. The structural models of the anti-FAP scFv′ molecules indicate that these three positions are located at the bottom of the scFv′ molecule opposite the antigenbinding site. Recently, a similar scFv′ fragment possessing an additional cysteine residue at position 1 of a 15 residue linker was applied for the generation of a piezoimmunosensor for the detection of low concentrations of antigen in complex samples (14). Our study confirmed the suitability of site-directed coupling and has extended the application of scFv′ linker variants for the generation of targeted nanoparticulate carrier systems. This approach was further developed by applying scFv′ molecules with the additional cysteine residue and the hexahistidyl-tag present in the linker sequence. These novel molecules have the advantage that except for the linker sequence no additional amino acid residues are present. Thus, these scFv′ molecules are reduced to the minimal length and composition required for easy one-step purification via IMAC and sitedirected coupling to sulfhydryl-reactive molecules. Indeed, we could show that these LCH variants are produced in soluble and active forms in bacteria and that these molecules could be efficiently purified from periplasmic extracts by IMAC. Although recognition by anti-His-tag antibodies is impaired, they could be used to generate immunoliposomes that bind strongly to target cells. These newly designed scFv′ molecules may be particularly suitable for the generation of immunoliposomes and other antibody conjugates, limiting the number of additional residues in these antibody molecules. Instead of the hexahistidyl sequence, one can envisage that other short purification and/or detection tags, such as Strep-tag, poly Arg-tag, or FLAG-tag, may be integrated into the linker sequence, thus implementing further approaches for detection and purification (37). In summary, we performed a comparative study of various scFv′ molecules designed for defined and oriented conjugation to sulfhydryl-reactive molecules. We could show that both genetically introduced single C-terminal cysteine residues as well as single cysteine residues in the linker sequence are equally suitable for the generation of scFv immunoliposomes for targetcell-specific binding and delivery.

ACKNOWLEDGMENT We would like to thank Ronny Rüger (University of Jena) for helpful suggestions and discussions. This work was supported by a grant from Landesstiftung Baden-Württemberg (“Kompetenznetz funktionelle Nanostrukturen”, project C11).

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