PEDS Advance Access published online on November 3, 2007
Protein Engineering Design and Selection, doi:10.1093/protein/gzm061
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A novel tri-functional antibody fusion protein with improved pharmacokinetic properties generated by fusing a bispecific single-chain diabody with an albumin-binding domain from streptococcal protein G
Institut für Zellbiologie und Immunologie, Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany
1 To whom correspondence should be addressed. E-mail: roland.kontermann{at}izi.uni-stuttgart.de
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Abstract |
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The therapeutic application of small recombinant antibody molecules is often limited by a short serum half-life. In order to improve the pharmacokinetic properties, we have investigated a strategy utilizing fusion with an albumin-binding domain (ABD) from streptococcal protein G. This strategy was applied to a bispecific single-chain diabody (scDb CEACD3) developed for the retargeting of cytotoxic T cells to CEA-expressing tumor cells. This novel tri-functional fusion protein (scDb–ABD) was expressed in mammalian cells and recognized both antigens as well as human and mouse serum albumin. scDb–ABD was capable to retarget T cells to CEA-expressing target cells in vitro and to activate the effector cells as measured by stimulation of IL-2 release. Although activity was reduced 3-fold compared with scDb and further reduced 4-fold in the presences of human serum albumin, this assay demonstrated that scDb–ABD is active when exposed to all three antigens. Compared with scDb, the circulation time of scDb–ABD in mice was prolonged 5- to 6-fold similar to a previously described scDb–HSA fusion protein. This strategy, which adds only a small protein domain (46 amino acids) and which utilizes high-affinity, non-covalent albumin interaction, should be broadly applicable to improve serum half-lives of small recombinant antibody molecules.
Keywords: albumin-binding domain/bispecific antibody/effector cells/pharmacokinetics/tri-functional antibody
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Introduction |
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Recombinant antibodies have found broad applications in therapy. While engineered whole antibodies, such as chimeric and humanized immunoglobulins, show pharmacokinetic properties similar to that of human antibodies, small recombinant antibody molecules, e.g. single-chain Fv (scFv) or bispecific molecules such as tandem scFv and single-chain diabodies (Müller and Kontermann, 2007
), are rapidly cleared from circulation (Weir et al., 2002; Müller et al., 2007). This is mainly due to their small size leading to rapid renal clearance and the lack of recycling processes mediated by the neonatal Fc receptor (FcRn) (Lencer and Blumberg, 2005). Thus, high doses and repeated injections or infusions are necessary to maintain therapeutically effective serum concentrations (Schlereth et al., 2005).
Several strategies have been developed in recent years to extend circulation time of small antibody molecules. One approach utilizes attachment of polyethylene glycol (PEG) chains. PEGylation has been shown to increase serum half-life of scFv and other molecules and to improve diagnostic or therapeutic efficacy (Chapman, 2002). However, this strategy involves a chemical coupling step and can lead to reduced binding and activity of the PEGylated protein (Kubetzko et al., 2005; Bailon et al., 2001). We have recently applied an albumin fusion strategy (Chuang et al., 2002) to improve the pharmacokinetic properties of small bispecific antibody molecules (single-chain diabody, tandem scFv) (Müller et al., 2007). By this approach, half-lives were increased leading to a 6- to 7-fold increase of the area-under-the-curve (AUC).
Similar strategies are based on the fusion of an albumin-binding moiety to the therapeutic protein (Makrides et al., 1996; Dennis et al., 2002). For example, albumin-binding antibody fragments have been employed to increase serum half-life of proteins (Smith et al., 2001; Roovers et al., 2007). Furthermore, albumin-binding peptides were isolated by phage or bacterial display and used to improve pharmacokinetics of proteins (Dennis et al., 2002; Sato et al., 2002; Bessette et al., 2004; Nguyen et al., 2006; Dennis et al., 2007).
Natural high-affinity albumin-binding domains are found in protein G of certain Streptococcus strains (Åkerström et al., 1987). For example, protein G of Streptococcus strain G148 contains three homologous albumin-binding domains. Albumin-binding domain 3 (ABD3) has been extensively studied. This domain consists of 46 amino acid residues forming a stable three-helix bundle (Kraulis et al., 1996). ABD3 has broad albumin species specificity and binds to human serum albumin (HSA) with a Kd of 
4 nM as determined by Biacore experiments (Johansson et al., 2002; Linhult et al., 2002). Residues involved in binding to HSA have been identified by mutational analysis in the second 
-helix of ABD3 (Linhult et al., 2002). Only a few reports have described the use of this domain or larger regions of protein G containing this domain to prolong the circulation time of proteins such as soluble complement receptor 1 (sCR1), CD4, Fab fragments and affibodies (Nygren et al., 1991; Makrides et al, 1996; Schlapschy et al., 2007; Tolmachev et al., 2007).
Here, we applied genetic fusion of the ABD3 domain of streptococcal protein G to a recombinant bispecific single-chain diabody (scDb) to generate a novel tri-functional antibody molecule with improved pharmacokinetic properties. We could show that the scDb–ABD fusion protein is expressed by mammalian cells and that it retains antigen-binding activity for both antigens and binds to human and mouse serum albumin. A comparative analysis with scDb demonstrated a 5- to 6-fold prolonged circulation time in vivo leading to a 14-fold increase of the AUC.
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Materials
HRP-conjugated anti-His-tag antibody was purchased from Santa Cruz Biotechnology (California, USA), unconjugated anti-His-tag antibody from Dianova (Hamburg, Germany) and anti-mouse IgG-FITC or PE-conjugated antibody from Sigma (Taufkirchen, Germany). Carcinoembryonic antigen (CEA) was obtained from Europa Bioproducts (Cambridge, UK). HSA and MSA were purchased from Sigma. The human colon adenocarcinoma cell line LS174T was purchased from ECACC (Wiltshire, UK) and cultured in EMEM (Invitrogen, Karlsruhe, Germany) supplemented with 2 mM glutamine, 1% non-essential amino acids and 10% FBS. LoVo cells were provided by Dr Bruno Robert (Montpellier, France) and cultured in RPMI, 10% FBS. Jurkat and HEK293 were cultured in RPMI, 10% and 5% FBS, respectively. Buffy coat (leukapheresis) from a healthy human donor was kindly provided by Prof G. Multhoff (Regensburg, Germany). IL-2 was purchased from Immunotools (Friesoythe, Germany) and phytohemagglutinin-L (PHA-L) from Boehringer-Mannheim (Germany). CD1 mice were purchased from Elevage Janvier (Le Genest St Isle, France).
Construction and expression of a scDb–ABD fusion protein
The construction of scDbCEACD3 has been described previously (Müller et al., 2007). DNA encoding the entire ABD3 domain of protein G of Streptococcus strain G148 was synthesized by GeneArt (Regensburg, Germany) adding a NotI and EcoRI restriction site at the 5' and 3' end, respectively, for cloning into plasmid pAB1 scDbCEACD3. The resulting DNA fragment encoding scDb–ABD was subcloned into mammalian expression vector pSecTagA (Invitrogen, Karlsruhe, Germany). HEK293 cells were stably transfected and scDb–ABD was purified from cell culture supernatant essentially as described previously (Müller et al., 2007).
CEA was immobilized in 96 well-plates (300 ng/well) overnight at 4°C. After 2 h blocking with 2% (w/v) dry milk/PBS, recombinant antibody fragments were titrated in duplicates and incubated for 1 h at RT. Detection was performed with mouse HRP-conjugated anti-His-tag antibody using TMB substrate (1 mg/ml TMB, sodium acetate buffer pH 6.0, 0.006% H2O2). The reaction was stopped with 50 µl of 1 M H2SO4. Absorbance was measured at 450 nm in an ELISA-reader. Binding of scDb–ABD to immobilized human and mouse serum albumin (10 µg/well) was analyzed accordingly. Competition-ELISA was performed according to the method described by Friguet et al. (Friguet et al., 1985). scDb–ABD at a concentration of 3 and 10 nM, respectively, was pre-incubated with increasing concentrations of HSA or MSA (0.1 nM–1 µM) for 3 h and subsequently added to microtiter plates coated with MSA. After a 15 min incubation step, plates were washed and bound scDb–ABD was detected as described above. PBS, 2.7% blocking reagent for ELISA (Roche Diagnostics) was used for blocking and incubation in these experiments.
5 x 105 cells/well were incubated with recombinant antibodies (10 µg/ml) for 2 h at 4°C. After washing, cells were incubated for 1 h at 4°C with mouse anti-His-tag antibody followed by washing and 30 min incubation with PE-labeled anti-mouse IgG. Wash cycles and incubation steps were performed in PBS, 2% FCS, 0.02% azide. Finally, cells were analyzed by flow cytometry using an EPICS XL-MCL (Beckman Coulter, Krefeld, Germany).
Protein melting points were determined by dynamic light scattering using a Zetasizer Nano ZS (Malvern, UK). Proteins were diluted in PBS to a concentration of 150 µg/ml and analyzed at increasing temperatures (30–70°C) using 1°C intervals and an equilibration time of 2 min. Furthermore, stability was determined by incubating scDb–ABD in mouse serum or human plasma at 37°C for up to 18 days. Aliquots were taken at different time points and concentration of active antibody molecules was determined by ELISA as described above.
Apparent molecular masses and formation of scDb–ABD albumin complexes were analyzed by HPLC size exclusion chromatography (SEC) using a BioSep-Sec-3000 column (Phenomenex, Torrance, USA) and a flow rate of 0.5 ml/min. The following standard proteins were used: thyroglobulin, apoferritin, ß-amylase, bovine serum albumin, carbonic anhydrase, cytochrome c. Complex formation of scDb–ABD with HSA or MSA was analyzed by incubating equimolar amounts of scDb–ABD and albumin (8.5 µM) in PBS at room temperature and subsequent analysis by SEC.
Peripheral blood mononuclear cells (PBMCs) from a healthy donor were isolated from buffy coat (leukapheresis) as described before (Müller et al., 2007). 1 x 105 LS174T or HT1080#13.8 cells/100 µl/well were seeded in 96-well plates. The next day, supernatant was removed and 100 µl of recombinant antibody added. After 1 h pre-incubation at RT, 2 x 105 PBMC/100 µl/well were added. PBMCs had been thawed the day before and seeded on a culture dish to remove monocytes by the attachment to the plastic surface. Only cells that remained in suspension were used for the assay. After addition of PBMCs, the 96-well plate was incubated for 22–24 h at 37°C, 5% CO2. Plates were centrifuged and cell-free supernatant collected. Concentration of human IL-2 in the supernatant after T-cell retargeting was determined by an IL-2 sandwich-ELISA. Anti-human IL-2 antibodies as well as the standard of recombinant human IL-2 was provided by DuoSet IL-2 ELISA Development System kit (R&D Systems, Nordenstadt, Germany) and the assay was performed following the manufacturer’s protocol.
Animal care and all experiments performed were in accordance with federal guidelines and had been approved by university and state authorities. CD1 mice (female, 15–36 weeks, weight between 39 and 54 g) received i.v. injections of 25 µg scDb, scDb–ABD or scDb–HSA in a total volume of 200 µl. In time intervals of 3, 30, 60, 120, 360 min, 24 h and 72 h blood samples (100 µl) were taken from the tail and incubated on ice. Clotted blood was centrifuged at 10.000 g for 10 min, 4°C and serum samples stored at –20°C. Serum concentrations of CEA-binding recombinant antibodies were determined by ELISA (as described above), interpolating the corresponding calibration curves. For comparison, the first value (3 min) was set to 100%. Pharmacokinetic parameters AUC, t1/2 and t1/2ß were calculated with Excel using the first three times points to calculate t1/2 and the last three time points to calculate t1/2ß. Six animals were analyzed for scDb–ABD (72 h value measured only for 3 animals). Pharmacokinetics of scDb and scDb–HSA were measured for three animals, including data for 72 h, and combined with previously obtained data (Müller et al., 2007). For statistics, Student’s t test was applied.
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Results |
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Production of a scDb–ABD fusion protein
ScDbCEACD3–ABD directed against the CEA and the T cell receptor complex protein CD3 was generated by fusing the albumin-binding domain ABD3 of streptococcal protein G (composed of 46 aa) to scDbCEACD3 including a C-terminal hexahistidyl tag (Fig. 1A–C). The resulting scDbCEACD3–ABD fusion protein is composed of 550 amino acid residues with a calculated mass of 59.3 kDa. Thus, scDbCEACD3–ABD is 5 kDa larger than scDbCEACD3, which contains also a Myc-tag (505 aa, 54.5 kDa). Both scDbCEACD3 and scDbCEACD3–ABD were purified from the supernatant of stably transfected HEK293 cells with yields of 10 and 15 mg/l culture medium, respectively. SDS–PAGE and immunoblot analysis showed a single protein band for scDbCEACD3 and scDbCEACD3–ABD under non-reducing and reducing conditions (Fig. 1D and E). The apparent molecular masses were 59 kDa for scDbCEACD3 and 62 kDa for scDbCEACD3–ABD.
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Antigen binding of scDb–ABD
Both scDbCEACD3 and scDbCEACD3–ABD recognized CEA in ELISA in the absence or presence of HSA (1 mg/ml) (Fig. 2). Binding of scDbCEACD3 to CEA was not reduced by HSA (half maximal binding at 2.2 nM without HSA and 1.5 nM with HSA), whereas binding of scDbCEACD3–ABD was slightly reduced (half maximal binding at 4.1 nM without HSA and 6.6 nM with HSA). Both proteins bound with similar efficiency to CEA-expressing tumor cell lines (LS174T, LoVo) and to activated CD3-positive PBMCs. No reduction of binding was observed in the presence of HSA (1 mg/ml) or 50% mouse serum (Fig. 3). No binding was seen with antigen-negative control cells (HEK293). Thus, both antigen-binding sites in scDbCEACD3–ABD are accessible for antigen binding.
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Stability of scDb–ABD
Thermal stability of scDbCEACD3–ABD was found to be similar to that of scDbCEACD3. Melting points were determined to be 48°C for scDbCEACD3–ABD and 49°C for scDbCEACD3. Similar values were measured for scFv CEA (48°C) and scFv CD3 (47°C) (data not shown). ScDbCEACD3–ABD was highly stable in human plasma or mouse serum at 37°C with a half-life >3 days (data not shown).
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Binding of scDb–ABD to albumin
SEC was applied to demonstrate that scDbCEACD3–ABD interacts with human and mouse serum albumin in solution (Fig. 4). For this study, scDbCEACD3–ABD was incubated with HSA or MSA at equimolar concentrations (8.5 µM) and then separated by SEC. ScDbCEACD3–ABD alone eluted with a major peak (88%) corresponding to an apparent molecular mass of 40 kDa. HSA showed a major peak (92.5%) of 65 kDa and a minor peak of 220 kDa. After incubation with scDbCEACD3–ABD both peaks were shifted to an apparent molecular mass of 150 and 450 kDa, whereas the peak of the unbound scDbCEACD3–ABD was almost completely diminished (approximately 5% left). Similar results were obtained with MSA. MSA alone showed a major peak (50.6%) of 60 kDa and several peaks with higher molecular masses. These peaks were shifted towards the left after incubation with scDbCEACD3–ABD. Only a small fraction (11%) corresponded in size to free scDbCEACD3–ABD. A control experiment with scDbCEACD3 incubated with HSA showed no apparent interaction of these molecules (data not shown). In a further experiment, we analyzed inhibition of binding of scDbCEACD3–ABD to immobilized MSA by soluble HSA or MSA. In this competition-ELISA, we determined IC50 values of 4.4 nM for MSA and 3.4 nM for HSA, respectively (Fig. 5A). Next, we compared binding of scDbCEACD3–ABD to MSA at pH 7.4 and pH 6.0 in ELISA in order to investigate if scDbCEACD3–ABD albumin complexes are stable in an acidic environment. Binding of scDbCEACD3–ABD to MSA at pH 6.0 was not affected (using 10 µg/ml scDb–ABD) or slightly increased (1.2- to 1.9-fold at 3 and 1 µg/ml, respectively) compared to binding at pH 7.4 (Fig. 5B).
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T cell stimulatory activity of scDb–ABD
An IL-2 release assay was performed to demonstrate simultaneous binding of scDbCEACD3–ABD to CEA-expressing target cells and CD3-positive effector cells leading to activation of the effector cells. Both molecules, scDbCEACD3 and scDbCEACD3–ABD, activated human effector cells in a concentration-dependent manner in the presence or absence of HSA (Fig. 6A). For scDbCEACD3, maximal IL-2 release was induced between 2 and 10 nM (EC50 = 0.5 nM). In the absence of HSA, activation induced by scDbCEACD3–ABD was approximately 3-fold reduced compared with scDbCEACD3 (EC50 = 1.5 nM). The presence of HSA in the cell culture medium (1 mg/ml) had no or only a marginal effect on activation by scDbCEACD3, while activation by scDbCEACD3–ABD was further reduced 4-fold (EC50 = 6 nM). Selectivity of activation in this target cell-based assay was demonstrated with a control scDb directed against fibroblast activation protein and CD3 (scDb33CD3), which when compared with scDbCEACD3 and scDbCEACD3–ABD induced only low IL-2 release at a concentration of 25 nM (Fig. 6B).
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Pharmacokinetic properties of scDb–ABD
Serum concentrations of scDbCEACD3 and scDbCEACD3–ABD were determined at different time points after a single dose i.v. injection into CD1 mice. Twenty-five micrograms were used per animal in accordance with doses applied in studies of other recombinant bispecific antibodies in mice (Cochlovius et al., 2000; Kipriyanov et al., 2002). Initial serum concentrations were found to be between 11.8 and 65.7 µg/ml for scDb and between 4.1 and 27.0 µg/ml for scDb–ABD. Compared with scDbCEACD3, scDbCEACD3–ABD showed a prolonged circulation time, with an increase in initial and terminal half-lives by a factor of 5–6 and the AUC(0–24) by a factor of 14 (Fig. 7, Table I). Half-lives determined for scDbCEACD3–ABD were similar to those of an scDbCEACD3-HSA fusion protein (Müller et al., 2007). The AUC(0–24) of scDbCEACD3–ABD was significantly increased by a factor of 1.4 (P < 0.05) compared with scDbCEACD3-HSA.
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Discussion |
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Fusion of the albumin-binding domain 3 (ABD3) of streptococcal protein G to scDb CEACD3 prolonged circulation half-lives in mice by a factor of 5–6 and the AUC increased by a factor of 14 compared with scDb. This finding indicates that scDb–ABD utilizes binding to mouse albumin to achieve prolongation of circulation time most likely caused by reduced renal clearance and FcRn-mediated recycling effects of scDb–ABD albumin complexes formed in vivo. This assumption is supported by the observation that scDb–ABD formed complexes with MSA and HSA in vitro when incubated at equimolar concentrations. Furthermore, binding of scDbCEACD3–ABD to MSA in ELISA was not reduced at pH 6.0 indicating that scDb–ABD albumin complexes are stable at low pH, e.g. found in endosomes. Using proteolytically and chemically produced fragments of HSA, the binding site(s) of streptococccal protein G was found to reside in a segment spanning residues 330–446, indicating that protein G binds to domains II and III of HSA (Falkenberg et al., 1992). Crystallographic studies of a complex of HSA and the ABD of protein PAB of Finegoldia magna, which is homologous to the ABD3 of streptococcal strain G148 (60% identity), revealed that the ABD binding site is located in domain II of albumin involving albumin residues 212, 309, 318, 321, 326 and 329 (Lejon et al., 2004
). The exact FcRn binding site on albumin is not known but has been suggested to be contained within domain III of albumin (Anderson et al., 2006; Chaudhury et al., 2006). Thus, most likely the ABD and FcRn binding sites on albumin are located on separate regions of albumin. Therefore, FcRn-mediated recycling should not be impaired by binding of ABD to albumin. However, currently we do not have direct evidence that FcRn-mediated recycling is involved in prolongation of circulation time in mice.
Prolonged circulation time was also observed for a fusion protein consisting of two fragments of streptococcal protein G fused N- and C-terminally of CD4. This bacterially expressed fusion protein showed a half-life of 15–24 h after single-dose injection into mice similar to a CD4-Fc fusion protein (Nygren et al., 1991). A CD4-HSA fusion protein showed after a single-dose injection into rabbits a terminal half-life of 34 ± 4 h compared with 47 ± 6 h for HSA and 0.25 ± 0.1 h for soluble CD4 (Yeh et al., 1992). In another study, fusion of sCR1 to an albumin-binding domain of protein G increased terminal half-life by a factor of 2.9–5 h (Makrides et al., 1996). Recently, this albumin-binding domain was fused to a monovalent or bivalent affibody directed against HER2 and used to generate radiolabeled conjugates (Tolmachev et al., 2007). Compared with the non-fused affibody dimer half-life was prolonged from 0.64 to 35.8 h. Furthermore, a 177Lu-labeled ABD-affibody dimer showed high and specific tumor uptake and completely prevented growth of SKOV-3 microxenografts in mice. These studies together with the present work clearly demonstrate that fusion of one or more albumin-binding domains of protein G is able to increase circulation time in animals.
A reduced tumor cell binding was described for a monomeric affibody ABD fusion protein indicating that binding to albumin can influence additional binding to target cells probably due to a sterical hindrance of binding (Tolmachev et al., 2007). We found that HSA or mouse serum had no effect on binding of scDbCEACD3–ABD to CEA-expressing tumor cells or IL-2 stimulated CD3-positive PBMCs, although binding of scDbCEACD3–ABD to CEA in ELISA was reduced in the presence of HSA. Further experiments showed that activation of effector cells by target cell-bound scDb–ABD is reduced in the presence of albumin by a factor of 4. This finding demonstrated that scDbCEACD3–ABD is still active when exposed to effector and target cells in the presence of albumin but also showed that overall activity of scDbCEACD3–ABD is influenced by albumin.
The approach of using an albumin-binding domain derived from protein G is similar to approaches based on albumin-binding peptides and other small binding domains such as single-domain antibodies, i.e. are utilizing complexation with naturally occurring albumin. A 20mer albumin-binding peptide with the sequence QRLMEDICLPRWGCLWEDDF was isolated from a phage display library (Dennis et al., 2002). This peptide was applied to prolong circulation time of an anti-HER2 Fab fragment derived from antibody 4D5 leading to rapid accumulation and retention in tumors and favorable tumor to normal tissue ratios (Dennis et al., 2007). This peptide has an affinity for MSA of 44 nM and for HSA of 556 nM. In mice, AUC of the fusion protein (AB.Fab4D5-H) was increased by a factor of 114 compared with Fab4D5 and terminal half-life increased from 1.28 to 19.7 h (Nguyen et al., 2006). Furthermore, using shorter sequences of this albumin-binding peptide with lower affinity for albumin, it was found that reduced affinity correlates with reduced half-life. We have measured the affinity of scDbCEACD3–ABD for HSA and MSA by competition ELISA (Friguet et al., 1985). The IC50 values of 3.4 nM for HSA and 4.4 nM for MSA are close to previously determined IC50 values for the recombinant ABD3 domain (1.6 nM for HSA and 10.2 nM for MSA) (Johansson et al., 2002) and a Kd of 3.86 nM for HSA as measured by surface plasmon resonance (Linhult et al., 2002). This finding indicates that fusion of ABD to scDb does not reduce affinity for albumin. Thus, ABD fusion proteins exhibit a high affinity for HSA and should be highly suitable to improve pharmacokinetics of proteins in human.
Concerns of using a streptococcal ABD domain for improvement of pharmacokinetics may arise from the risk of being immunogenic in humans. Indeed immunogenicity of the minimal albumin-binding domain (aa 254–299) of protein G was found in various mice strains (Sjölander et al., 1997). Furthermore, an increased antibody response was observed for a fusion protein of the albumin-binding region (BB; aa 113–326) of protein G and part of an RSV-A antigen (Libon et al., 1999). For therapeutic applications, especially involving repeated injections, it is essential that immunogenicity of the ABD is reduced or ideally eliminated. It was speculated that the BB domain is a T cell-dependent protein acting as a carrier and supplying additional T cell epitopes. Hence, deimmunization strategies should be applicable to reduce immunogenicity of the ABD (De Groot et al., 2005; Baker and Jones, 2007).
In summary, we found that scDbCEACD3–ABD recruited effector cells to target cells in vitro even in the presence of excess amounts of HSA, leading to activation of the effector cells as measured by IL-2 release. These experiments also showed that activation of effector cells is influenced by the ABD moiety and interaction with albumin, resulting in a 3- to 12-fold reduced activity compared with scDb. Nevertheless, this data clearly demonstrate that scDb–ABD is active when exposed to effector and target cells in the presence of albumin. Further in vivo studies have to demonstrate potency of this novel tri-functional scDb–ABD fusion proteins in respect to effector cell retargeting and the effects of prolonged circulation time on induction of anti-tumor responses.
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This work was supported in part by a grant from the Deutsche Forschungsgemeinschaft (Ko1461/2).
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Footnotes |
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Edited by Paul Carter
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Acknowledgement |
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We would like to thank Katja Stolpa for technical assistance. Furthermore, we would like to thank Prof. Gabriele Multhoff (Regensburg) for supplying buffy coat from a healthy human donor, Dr Bruno Robert (CRLC, Montpellier) for providing LoVo cells and Sabine Münkel (IZI, Stuttgart) for the HPLC analysis.
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Received July 12, 2007; revised September 28, 2007; accepted October 2, 2007.
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