Patent Application
20070274985
The examples refer to the figures, wherein
In the examples, domains from the lysozyme binding antibody D 1.3 (Ward et al., 1989) were used as an example for any naturally occurring immunoglobulin, comprising the domains that are contained in the antibody according to the invention.
Using standard cloning procedures (Sambrook et al., Cold Spring Harbour Laboratory, 1989), expression cassettes for scFabs were cloned into a phagemid vector.
The coding sequence for the domains VH-CH1 was amplified by PCR from phagemid vector pHAL1-D 1.3 Fab (Kirsch et al., 2005), using sequence specific primers. In a separate PCR reaction, the sequence encoding the domains VL-CL was amplified from pHAL1-D 1.3 Fab, using specific primers. The PCR amplificates contained overlapping sections, allowing to generate their fusion amplificate in a third common PCR, comprising coding sequences for a VL-CL-linker-VH-CH1 fusion protein. This coding sequence was cloned into the NheI and NotII sites of pHAL1. The cloning procedure is schematically depicted in
ScFab encoding nucleic acid sequences additionally included at its 5′-end the signal peptide pIII encoding sequence that directs the translation product to the periplasm. Coding sequences for amino acid linkers are given below:
The nucleic acid sequences are given by way of example only and are not intended to limit the scope of the invention. Therefore, linker sequences can be changed, e.g. by exchanging amino acid residues, but it is preferred to maintain the length of linkers. The light and constant chain domains can also be exchanged for chains from different antibodies, having the same function as VH, VL, CH1 and CL, respectively. It is especially preferred to replace the VH and VL domains for VH and VL domains having an antigen specificity for a desired antigen. In detail, the variable domains VH and/or VL can be modified by replacing the CDR hypervariable regions and/or the frame work regions with respective regions from known natural or synthetic antibodies; the CDR and frame work regions are schematically indicated by horizontal stripes. Further, the light chain domains can be lambda or kappa.
XL1-Blue MRF′ E. coli (Stratagene, Amsterdam, Netherlands) were transformed with the phagemid constructs by electroporation. Cloning was verified by sequencing of phagemid vector.
The structure of expression vectors pHAL-D1.3Fab (comparative), pHAL1-D1.3scFv (comparative) and pHAL1-D1.3scFab are shown in
In
The dimerization of scFabs according to the invention is schematically depicted in
For production of antibody presenting phage, 50 mL 2× TY medium containing 100 μg/mL ampicillin and 100 μM glucose were inoculated with an overnight culture having an OD600 of about 0.025. Bacteria were incubated at 37° C. under agitation at 250 rpm to an OD600 of about 0.4 to 0.5. Of this culture, 2 mL were infected with 2×1010 helperphage VCSM13 (Stratagene), or Hyperphage (Rondot et al., 2001), incubated for an additional 30 minutes at 37° C. without shaking, followed by 30 minutes at 250 rpm. Infected cells were harvested by centrifugation for 10 minutes at 322× g and the cell pellet was resuspended in 13 mL 2× TY, 100 μg/mL ampicillin and 50 μg/mL kanamycin, containing various glucose concentrations. Phage were produced at 30° C. at 250 rpm for 16 hours. Cells were pelleted for 10 minutes at 10,000× g. The phage in the supernatant were precipitated with one fifth volume of a 20% by weight PEG/2.5 molar sodium chloride solution for one hour on ice with gentle shaking, followed by pelleting for one hour at 10000× g at 4° C. Precipitated phage were resuspended in 10 mL phage dilution buffer (10 mM Tris, 20 mM sodium chloride, 10 mM EDTA, pH adjusted to 7.5 using HCl), followed by a second precipitation with one fifth volume PEG solution as above for 20 minutes on ice and pelleted again at 10000× g for 30 minutes at 4° C. Precipitated phage were resuspended in 300 μL phage dilution buffer and cell debris was pelleted by an additional centrifugation for 5 minutes at 15400× g at 20° C. The supernatant containing the antibody presenting phage were stored at 4° C. Phage titration for the determination of plaque forming units (PFU) was done according to Koch et al. (2000), but packaging the infected bacteria directly onto LB-agar plates, omitting nitrocellulose sheets. When using an E. coli mutator strain for amplifying the phagemid containing the coding sequence for an scFab, a large variety of random mutants could be generated. Subsequent production of phage presenting the mutant scFabs could be used for expression of these mutant antibodies. The selection of an scFab having the desired antigen specificity could be done by standard procedures, e.g. by incubation of the phage presenting the mutant scFab population with the desired antigen that was linked to an immobilizing surface. Following interaction of the phage presented scFabs with the immobilized antigen, the coding sequence could be isolated from the isolated phage after removal of unbound phage species. Preferably, consecutive rounds of incubation of the scFab mutant phage population and the desired immobilized antigen were used to select the phagemid encoding the desired scFab.
An antigen binding ELISA could be employed for both antigen displaying phage and soluble antigen by using microtiter plates (Costar, Cambridge, USA), that were coated with 100 ng D 1.3 as the model antigen in 100 μL 0.1 M sodium carbonate solution at pH 9.6 per well overnight at 4° C. After coating, wells were washed three times with PBS and blocked with 2% by weight skim milk powder in PBS for 1.5 hours at room temperature, followed by three times washing with PBS. ScFab expressing phage or periplasmic fractions from E. coli cultures expressing the scFab were incubated on the coated microtiter plates after dilution in blocking solution, followed by five times washing with PBST (PBS containing 0.1% vol./vol. Tween-20).
Detection of bound antibody presenting phage was with monoclonal anti-m13 antibody, conjugated with HRP (Amersham Biosciences), diluted 1:5,000. In the case of periplasmic supernatants, e.g. soluble scFab antibody, detection was done with a mouse anti-Strep-tag antibody (Qiagen, Hilden, Germany), in a 1:10,000 dilution, followed by goat anti-mouse mAb, conjugated with HRP (1:50,000) or with protein L conjugated with HRP (Pearce, Bonn, Germany) in a 1:10,000 dilution, followed by visualisation with TMB substrate (Biorad, Munich, Germany). The staining reaction was stopped by addition of 100 μL 1N sulfuric acid. Absorbances at 450 and 620 nm were recorded on a Sunrise microtiter plate reader (Tecan, Germany). Absorbance of scattered light at 620 nm was substracted from absorbance at 450 nm.
Using the model scFab antibody, the antigen coated onto the microtiter plates for immobilization was lysozyme. When using VCSM13 was used in phage rescue, 5×106 phage per well were applied, whereas 107 phage per well were employed after packaging with Hyperphage to compensate for differences in antibody presentation efficiency.
For comparison, an scFv antibody was constructed, consisting of VH and VL domains only, as well as a phagemid encoding a Fab fragment, consisting of two expression cassettes, encoding the VH domain in connection with the CH1 domain, and the VL domain coupled to the CL domain, respectively.
Different results were obtained for Hyperphage packaging, wherein the scFv and the Fab gave best binding results, whereas the scFab variants of the invention achieved values of about a third of the activity. Among the scFabs, the scFabΔC achieved the best results. Phage titers are shown in
The results of the antigen presenting phage ELISA are shown in
Antibody presenting phage preparations were further analysed by SDS-PAGE under reducing conditions, followed by blotting onto PVDF membrane. The pIII leader peptide was visualized by immune staining using monoclonal mouse anti-pIII antibody. In SDS-PAGE, pIII runs at an apparent molecular mass of 65 kDa, although it has a calculated molecular mass of 42.5 kDa (Goldsmith and Konigsberg 1977). The Western blot is shown in
For expression of scFab antibodies according to the invention, bacteria can be used as host organisms to yield active conformation scFab.
Soluble antibody was expressed in shake flasks using 2× TY medium (Sambrook et al., 1989), supplemented with 100 μg/mL ampicillin, 100 mM glucose with an inoculation of 1:20 vol./vol. with an overnight culture of the transformed XL1-Blue. Cultivation was at 37° C. at 350 rpm for 2 hours. Bacteria were harvested by centrifugation at 3,900× g for 20 minutes. The pellet was resuspended in 100 mL 2× TY medium with 100 μg/mL ampicillin and 20 μM IPTG and incubated at 30° C. at 350 rpm overnight. Following harvesting by centrifugation, the pellet was resuspended in 13 mL PBS (phosphate buffered saline, Sambrook et al. 1989), supplemented 1% Tween-20, and incubated at 30° C. at 350 rpm for a further 2.5 hours. Centrifugation for 10 minutes at 7,000× g separated cells from supernatant, which contained the antibody.
Alternatively, expression was done in microtiter plates using 200 μL 2× TY medium with 100 μg/mL ampicillin and 100 μM glucose, inoculated with 10 μL overnight culture, and incubation at 37° C. with agitation at 1,400 rpm for 2 hours. Bacteria were harvested by centrifugation for 10 minutes at 2,500× g. After resuspension of the pellet in 200 μL 2× TY with 100 μg/mL ampicillin and 20 μM IPTG at 30° C. at 1,400 rpm overnight, 50 μL PBS including 1% Tween-20 was added, followed by incubation at 30° C. and 1,400 rpm for an additional 3.5 hours. Again, cells could be separated from the antibody containing supernatant by centrifugation for 10 minutes at 3,200× g.
For production, E. coli strain XL1-Blue was transformed with pHAL1-D1.3 constructs, encoding comparative antibodies scFv and Fab, as well scFab, scFab −2, scFab +2 and scFabΔC according to the invention, respectively. After centrifugation, periplasmic fractions were analysed by antigen ELISA, using protein L for detection.
The result is shown in
This is a demonstration of the feasibility to produce antibody according to the invention in its active conformation within a bacterial host organism. Accordingly, mammalian cell culture is no prerequisite for efficiently expressing scFab according to the invention in its natural conformation, and bacterial expression can be used without subsequent denaturing and refolding of originally inactive protein that has been obtained for state of the art antibody constructs as inclusion bodies.
Similar results were obtained when expressing the scFab antibodies of the invention in Pichia, using a yeast expression cassette.
The preferred embodiment of the present invention, namely the scFab having a 34 amino acid linker with at least one cystein within the VH domain and/or the CL domain deleted, which cysteines are capable of forming a disulfide bond, is currently termed scFabΔC. When testing serial dilutions of periplasmic E. coli supernatant containing comparative scFv antibody and scFabΔC according to the invention, an ELISA using lysozyme as the antigen could demonstrate the production of active conformation antibody. The results are shown in
Analysis of the association of antibodies was done for comparative constructs scFv, Fab, and for antibodies of the invention, namely scFabΔC and scFab.
The antibody constructs of the invention were expressed in E. coli and isolated from the periplasmic fraction of the culture by SEC, whereas the comparative antibodies were expressed in mammalian cell culture and isolated from culture supernatant by SEC:
scFv (comparative): VH-linker-VL
Fab (comparative): VH-CH I VL-CL, connected by a disulfide bridge
scFabΔC: VL-CL-linker-VH-CH1, wherein both C-terminal cysteins of the CL and CH1 domains were deleted,
scFab: VL-CL-linker-VH-CH1,
wherein C1, C2, C5, C6, and C8 indicate SEC fractions. In accordance with SEC fractions, antibodies are designated as dimers or multimers in
The results of an ELISA as described in Example 2, using increasing concentrations of the respective antibody, are depicted in
The SEC fractions were further analyzed by an ELISA using the model antigen lysozyme adsorbed onto plates as described in Example 2. Results are shown in
