Site-specific modification of ED-B-targeting antibody using intein-fusion technology
© Möhlmann et al; licensee BioMed Central Ltd. 2011
Received: 27 October 2010
Accepted: 21 July 2011
Published: 21 July 2011
A promising new approach in cancer therapy is the use of tumor specific antibodies coupled to cytotoxic agents. Currently these immunoconjugates are prepared by rather unspecific coupling chemistries, resulting in heterogeneous products. As the drug load is a key parameter for the antitumor activity, site-specific strategies are desired. Expressed protein ligation (EPL) and protein trans-splicing (PTS) are methods for the specific C-terminal modification of a target protein. Both include the expression as an intein fusion protein, followed by the exchange of the intein for a functionalized moiety.
A full-length IgG specific for fibronectin ED-B was expressed as fusion protein with an intein (Mxe GyrA or Npu DnaE) attached to each heavy chain. In vitro protocols were established to site-specifically modify the antibodies in high yields by EPL or PTS, respectively. Although reducing conditions had to be employed during the process, the integrity or affinity of the antibody was not affected. The protocols were used to prepare immunoconjugates containing two biotin molecules per antibody, attached to the C-termini of the heavy chains.
Full-length antibodies can be efficiently and site-specifically modified at the C-termini of their heavy chains by intein-fusion technologies. The described protocols can be used to prepare immunoconjugates of high homogeneity and with a defined drug load of two. The attachment to the C-termini is expected to retain the affinity and effector functions of the antibodies.
Monoclonal antibodies have been approved as therapeutic agents for indications including viral infections, immunological disorders, transplant rejection and cancer . They often act by blocking the function of their target molecule. More demanding is the therapy of cancer by antibodies requiring the specific recognition and subsequent elimination of tumor cells.
Several mechanisms have been described how therapeutic antibodies elicit cell death, including the triggering of apoptosis and the recruitment of the immune system. While therapeutic antibodies have been approved working by these mechanisms (e.g. Rituximab , Trastuzumab , Alemtuzumab ) their cytotoxic potential is generally not sufficient to completely eliminate the malignant cells. Higher efficacies have been observed if the antibody is coupled to toxic agents like radioisotopes (radioimmunoconjugates) or chemical drugs (antibody-drug-conjugates, ADC) . Several of these conjugates have been approved for cancer (Ibritomomab, Tositumomab) or are in clinical development (e.g. Trastuzumab-DM1).
Coupling of toxic agents to therapeutic antibodies also paves the way for new tumor associated antigens as these are not required to be present on the surface of the malignant cells. An example is the extra domain B (ED-B) of fibronectin, a protein of the extracellular matrix. ED-B-containing fibronectin is a splice variant associated with angiogenesis and tissue remodeling . High levels of ED-B expression have been detected in most solid tumors and in vivo studies with ED-B specific monoclonal antibody formats show the selective accumulation in tumors and metastases. Accordingly, ED-B is a promising target for antibody-based cancer treatment [7, 8] and the results of first clinical trials with ED-B specific antibody fragment conjugates are encouraging [9, 10].
Current methods for the preparation of immunoconjugates rely on the chemical coupling to lysine, cysteine or tyrosine side chains . These methods are rather unspecific and result in heterogeneous products. As the drug load - number of toxophore per antibody - is a key parameter for the antitumor activity of immunoconjugates [12–14] more site-specific coupling reactions are desired. Approaches employing the carbohydrate moieties [15, 16], the N- and the C-terminus [17, 18] of full-length IgG antibodies have been described. However, the carbohydrates are important for the effector functions of the Fc domain  and the N-terminus of antibodies is close to their antigen binding site which may result in decreased affinity after modification. This leads to the C-terminus as a preferred site for specific drug attachment.
Several enzymatic approaches have been described for the modification of protein C termini . They have in common that the target protein is expressed in fusion with a C-terminal tag containing the modification site. A common drawback of these methods is an incomplete conversion. Without the possibility for separation, this would result in heterogeneous preparations of low averaged drug loads. Interestingly, the intein tag is cleaved off from the target protein during modification, facilitating preparative separation of modified from non-modified protein. Inteins encompass catalytic domains which lead to the formation of a thioester bond at their junction to the target protein. This thioester bond can be employed to exchange the intein for a C-terminal probe. The probe is eventually connected via a native peptide bond .
Methods for intein-mediated C-terminal protein modification encompass expressed protein ligation (EPL) and protein trans-splicing (PTS). In EPL, the target protein is fused to a modified full-length intein. The intein is cleaved off by the addition of a thiol reagent, leaving a thioester bond (first step), and the target protein is ligated to a probe functionalized with an N-terminal cysteine residue (second step) [22, 23]. In PTS, inteins are used which are split into two parts with high affinity to each other. The large N-terminal part is fused to the target protein. The probe is functionalized with the small C-terminal part of the intein. Their combination results in a functional intein, which splices itself out and concomitantly fuses the target protein to the probe [24–26].
Results and Discussion
Production of L19 IgG antibodies with C-terminal intein domains
Antibodies require an oxidizing environment for their expression as they contain several disulfide bonds important for proper folding. In addition, glycosylation has been shown to be important for the effector functions of antibodies . They are therefore usually produced by mammalian expression systems. We chose transiently transfected HEK293-6E cells for the expression of L19 IgG antibody which is specific for the ED-B domain of fibronectin. Endogenous signal sequences ensure the secretion into the extracellular medium and therefore the proper folding of the immunoglobulin domains. Connected via an 11 residue linker, the heavy chain was fused to an intein domain. We tested two distinct inteins, GyrA from Mycobacterium xenopi  and the split intein DnaE from Nostoc punctiforme . In case of DnaE, L19 IgG was fused to the N-terminal domain DnaEN (residues 1-102). Both inteins were extended C-terminally by a decahistidine tag for purification purposes.
Preparation of immunoconjugates by MxeGyrA intein mediated EPL
In vitro cleavage of L19 IgG-GyrA fusion protein was induced by the addition of 50 mM Mesna which has been reported to form a long-living thioester at the C-terminus. This thioester can be employed to link to the IgG a probe that is functionalized with an N-terminal cysteine residue. It is added concomitantly with Mesna but in lower concentration (5 mM). The cysteine replaces the Mesna-thioester, resulting in a stable peptide bond between the IgG and the probe (Figure 1).
Preparation of immunoconjugates by NpuDnaE intein catalyzed PTS
L19 IgG-DnaEN contains an incomplete intein which was activated in vitro by the addition of a peptide comprising the missing C-terminal 36 residues, followed by the naturally occurring post-intein sequence CFN. This DnaEC peptide can be employed to transfer a C-terminally attached probe to L19 IgG by PTS (Figure 1). Importantly, trans-splicing was only observed under reducing conditions i.e. after the addition of 5 mM DTT. We propose that this is due to the requirement of a free cysteine in the reactive center of DnaE intein.
L19 IgG heavy chain conjugated to GST (80 kDa) was the main product after PTS with DnaEC-GST as probe. However, also a side product of 50 kDa was observed. It corresponded to the heavy chain of L19 IgG produced by hydrolysis of the thioester intermediate formed during intein catalysis and allowed evaluation of its magnitude compared to probe conjugation. We estimate from the gel that about 25% of processed heavy chain was hydrolyzed while about 75% encompassed the desired conjugation product. Due to this hydrolysis, the drug load of immunoconjugates prepared by our DnaE PTS protocol reached only about 1.5. It is likely that hydrolysis was promoted by attack of the thiol reagent DTT on the intein-derived thioester . DTT had to be added to ensure reducing conditions for optimal intein activity. Alternative reducing agents like tris(2-carboxyethyl)phosphine (TCEP) could decrease hydrolysis and further optimize the PTS protocol.
We have shown here for the first time that full-length IgGs can be specifically functionalized at the C-terminus of their heavy chains by EPL and PTS. Antibodies processed on both chains were obtained in good yields and purified from incompletely processed proteins. The attachment of two probes per antibody and in vicinity to each other yielded an additional disulfide bond, circumventing problems with free thiol groups introduced by intein-fusion technologies. These methods could be employed to generate immunoconjugates of high homogeneity and with a drug load of two. The modification at the C-termini of the heavy chains does not interfere with antigen binding and, judged by binding to Fcγ RI and Fcγ RIIIA, it does not compromise the effector functions of the Fc domain. Provided with an effective toxophore, this would result in therapeutic antibodies fully equipped for the elimination of malignant cells.
Chemicals were purchased from Sigma Aldrich if not stated otherwise. Chromatography material (Glutathione Sepharose 4 Fast Flow, HiTrap Chelating HP, Superdex 200) was obtained from GE Healthcare (Munich).
L19 IgG was derived by transferring the variable domains of L19 scFv  into a human IgG1/к format.
Mxe GyrA (from vector pTXB1, New England Biolabs) or Npu DnaEN (from vector pSKDuet, provided by Hideo Iwai, University of Helsinki) were cloned behind the heavy chain of L19 IgG, separated by a linker coding for GLEGGSGGSEY. GyrA and DnaEN were extended C-terminally to include a His tag (GSA-H10 and AS-H10, respectively). Constructs encoding L19 IgG (control, without linker) and L19 IgG fused to GyrA or DnaEN were transferred into pTT5 vector (National Research Council Canada). In the resulting expression vectors, both, heavy and light chain, were under the control of an hCMV promoter.
DNA encoding DnaEC from Nostoc punctiforme (MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN ), followed by a linker (CFNPAGSSGVIM) and Glutathione-S-Transferase (GST, Schistosoma japonicum) was purchased from Geneart (Regensburg, Germany), amplified by PCR and ligated into vector pET29a (Novagen) by NdeI/NotI.
Expression and Purification of L19 IgG constructs
0.5 L of HEK293-6E cells were grown in F17 medium with 4 mM GlutaMAX-I and 0.1% Pluronic F-68 (Invitrogen) to a density of 1.5 - 2.0 × 106 cells/mL and transfected with 500 μg plasmid DNA (L19 IgG constructs) as described , with minor modifications. After five days cells were harvested by centrifugation (8000 × g, 5'). The supernatant was concentrated to 50 mL by crossflow filtration (Sartorius) and dialyzed against column buffer (500 mM NaCl, 50 mM Hepes-NaOH, pH 7.5). For purification, Ni-loaded IMAC (elution with imidazole gradient) and subsequent gel filtration was applied.
Expression and Purification of DnaEC-GST
E. coli BL21-Star (DE3) (Invitrogen) was transformed with vector DnaEC-GST-pET29a. Medium 2xYT (Difco) containing 50 μg/mL kanamycin was inoculated with an overnight culture and grown at 37°C, 220 rpm. At an OD600 of 0.4 temperature was decreased to 16°C. Protein expression was induced with 0.3 mM IPTG at an OD600 of 0.8. After 48 h cells were harvested (3,000 × g, 20') and suspended in buffer (500 mM NaCl, 50 mM Tris-HCl pH 7.5, 2 mM β-mercaptoethanol). Cell lysate was obtained by high pressure homogenization (Constant cell disruption systems, Constant Systems Ltd, UK) and subsequent centrifugation (20,000 × g, 30'). Purification of DnaEC-GST protein was achieved by applying Glutathione Sepharose (500 mM NaCl, 50 mM Tris-HCl pH 7.5, 2 mM β-mercaptoethanol, elution with 10 mM reduced glutathione), followed by gel filtration (500 mM NaCl, 50 mM Hepes-NaOH, pH 7.5).
Intein catalyzed protein modification
For GyrA mediated EPL, 5 μM L19 IgG-GyrA was incubated in 500 mM NaCl, 50 mM Hepes-NaOH, pH 8.0, 50 mM Mesna (2-mercaptoethane sulfonate-sodium), 5 mM cysteine or Bio-P1 Peptide (NEB). For DnaE mediated protein trans-splicing, 5 μM L19 IgG-DnaEN was mixed with 50 μM DnaEC Peptide (MVKVIGRRSLGVQRIFDIGLPQDHNFLLANGAIAANCFN, kind gift from Prof. Christian Becker, TU Munich, Germany. The sequence corresponds to DnaEC from Synechocystis sp. strain PCC6803 (Ssp) which is interchangeable with Npu DnaEC ), DnaEC-Biotin (MVKVIGRRSLGVQRIFDIGLPQDHNFLLANGAIAANCFNK-Biotin, synthesis by JPT Peptide Technologies GmbH, Berlin, Germany) or DnaEC-GST in 500 mM NaCl, 50 mM Hepes-NaOH, pH 7.5, 5 mM DTT. After incubation for 22-24h at room temperature (RT), intein activity was stopped by removing reducing agents (Mesna, DTT, cysteine) by dialysis over night at RT. For purification, the sample was loaded onto a Ni-IMAC column (500 mM NaCl, 50 mM Hepes-NaOH, pH 7.5). Completely processed L19 IgG was not in the flow through but was eluted at 30 mM imidazole. Partly- or non-processed L19 IgG-GyrA or -DnaEN was eluted with 300 mM imidazole. For subsequent analysis, protein was dialyzed against 500 mM NaCl, 50 mM Tris-HCl, pH 7.5 and concentrated to 1-2 mg/mL.
A sample (~50 μg) was loaded onto a gel filtration column connected to an HPLC instrument (Agilent Technologies 1200 Series) equipped with detectors for refractive indices (RI) and multi angle light scattering (MALS) (Optilab rEX and miniDAWN TREOS, Wyatt Technology Corp., Santa Barbara, CA). To calculate the yield of intein processing, a sample after EPL/PTS but before purification was analyzed. The mass of the excised intein was determined by its RI signal and set into relation to the total loaded mass.
Pepsin digestion and mass spectrometry
L19 IgG was dialyzed against 20 mM NaAcetat pH 4.5. Pepsin (Protea Biosciences) was added to give an enzyme-to-antibody ratio of 1:25 (w:w) and incubated for 24h at 37°C. A sample was directly analyzed by ESI-TOF to assess masses of peptides derived by the Pepsin digest. Further samples were separated by reducing and non-reducing SDS-PAGE and gels were Coomassie stained. Protein bands were excised, de-stained, reduced with DTT, alkylated with iodacetamide and digested with trypsine. Protein sequences were determined by ESI-Q-TOF (MS and MS/MS) and search in Sequest (Finnigan) and Mascot Databases.
After SDS-PAGE, gels were blotted onto a nitrocellulose membrane (iBlot, Invitrogen) and incubated for 1h in blocking buffer (TBS with 0.05% (v/v) Tween-20 and 5% (w/v) milk powder). Biotin was detected by incubating with 1 μg/ml Streptavidin-Alkaline Phosphatase (Sigma) in blocking buffer over night. GST was detected by over night incubation with anti-GST antibody (GE Healthcare, 1:5000 in blocking buffer) followed by 1h incubation with anti-Goat IgG-Alkaline Phosphatase (Sigma, 1:1000 in blocking buffer) as secondary antibody. BCIP/NBT (SigmaFAST tablet) was used for membrane staining.
Surface Plasmon resonance (SPR)
A Biacore T100 instrument (GE Healthcare, Munich) was employed to study the interaction between L19 IgG or modified versions thereof and ED-B, Fcγ RI/CD64 and Fcγ RIIIA/CD16a. L19 IgG or modified versions thereof were immobilized on a Biacore Sensor Chip (Series S Sensor Chip CM5 treated with antibody capture kit, GE Healthcare). Soluble ED-B domain in several concentrations (1.56 - 200 nM) or soluble Fc receptor fragments Fcγ RI/CD64 and Fcγ RIIIA/CD16a (R&D Systems) in several concentrations (1.56 - 50 nM) were used as analytes. Association and dissociation kinetics were detected to calculate the dissociation constant.
The authors thank Prof. Dario Neri (Philogen) for providing the L19 single chain sequence and Prof. Christian Becker (TU Munich) for his initial support with the DnaE intein system and for synthesis of the DnaEC peptide.
This study was funded by Bayer Healthcare, Berlin, Germany.
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