Functional humanization of an anti-CD30 Fab fragment for the immunotherapy of Hodgkin's lymphoma using an in vitro evolution approach

Martin Schlapschy1, Helga Gruber1, Oliver Gresch1, Claudia Schäfer1, Christoph Renner2, Michael Pfreundschuh2 and Arne Skerra1,3

1Lehrstuhl für Biologische Chemie, Technische Universität München, 85350 Freising-Weihenstephan and 2CR/MP Saarland University Medical School, 66421 Homburg/Saar, Germany

3 To whom correspondence should be addressed. E-mail: skerra{at}wzw.tum.de


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
CD30, the so-called Reed–Sternberg antigen, constitutes a promising cell-specific target for the treatment of Hodgkin's lymphoma. Starting from the previously characterized cognate HRS3 mouse monoclonal antibody, the bacterially produced functional Fab fragment was humanized by grafting the CDRs from the mouse antibody framework on to human immunoglobulin consensus sequences. This procedure led to a 10-fold decreased antigen affinity, which surprisingly was found to be mainly due to the VH domain. To improve the antigen-binding activity, an in vitro evolution strategy was employed, wherein random mutations were introduced into the humanized VH domain by means of error-prone PCR, followed by a filter sandwich Escherichia coli colony screening assay for functional Fab fragments using a recombinant extracellular domain of the CD30 antigen. After three cycles of in vitro affinity maturation, the optimized Fab fragment huHRS3-VH-EP3/1 was identified, which carried four exchanged residues within or close to the VH CDRs and had an affinity that was almost identical with that of the murine HRS3 Fab fragment. The resulting humanized Fab fragment was fully functional with respect to CD30 binding both in ELISA with the recombinant antigen and in FACS experiments with CD30-positive L540CY cells. In the light of the previously successful clinical application of an {alpha}CD30 x {alpha}CD16 bispecific mouse quadroma antibody derived from HRS3, the humanized Fab fragment comprises an important step towards the construction of a fully recombinant therapeutic agent. The combination of random mutagenesis and colony filter screening assay that was successfully applied here should be generally useful as a method for the rapid functional optimization of humanized antibody fragments.

Keywords: affinity maturation/antibody humanization/CD16/CD30/Hodgkin's lymphoma


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The CD30 receptor belongs to the tumor necrosis factor receptor superfamily and was originally identified on cultured Hodgkin–Reed–Sternberg (HRS) cells using the monoclonal antibody Ki-1 (Schwab et al., 1982Go). CD30 is a 120 kDa type I transmembrane glycoprotein of 577 amino acids (Swiss-Prot accession No.: P28908), which is expressed on different tumours, such as Hodgkin's and non-Hodgkin's lymphomas, embryonal carcinomas, malignant melanomas and mesenchymal tumours (Gruss and Kadin, 1996Go). Although its functional relevance in malignant diseases remains elusive, recent data indicate that the CD30 antigen, either as the native membrane protein on HRS cells and Chinese hamster ovary (CHO) cells or as a plate-bound chimeric protein, exerts inhibitory activity towards T-cell proliferation (Su et al., 2004Go).

Since overexpression of the CD30 antigen was first described for Hodgkin's lymphoma, CD30 antigen-specific monoclonal antibodies (MAbs) were originally raised against corresponding cell lines and most MAb-based studies have been performed in this entity (Chittal et al., 1988Go). In addition, MAbs against CD30 have been explored as vehicles for cytostatic drugs (Sahin et al., 1990Go) and for plant toxins (Terenzi et al., 1996Go) and a number of chemically linked immunotoxins have been developed and evaluated for clinical application (Engert et al., 1997Go; Schnell et al., 2000Go).

Clinical trials using anti-CD30 antibodies started in the early 1990s with biodistribution studies in patients suffering from Hodgkin's disease (da Costa et al., 1992Go). In these trials, specific tumour targeting with positive imaging was confirmed for the anti-CD30 antibody HRS3. Consequently, this antibody was used as the tumour-targeting component of a bispecific antibody in order to enhance its biological effector function (Hombach et al., 1993Go). Such bispecific antibodies are typically designed to bind simultaneously to the tumour cells and to a trigger receptor on immune effector cells (Carter, 2001Go), for example Fc{gamma}RIII (CD16) on Natural Killer (NK) cells. In the case of the {alpha}CD30 x {alpha}CD16 bispecific mouse quadroma antibody HRS3/A9, both in vitro and in vivo data and data from a clinical study impressively demonstrated the general applicability of this approach for the treatment of Hodgkin's disease (Hombach et al., 1993Go; Renner et al., 1994Go, 1997Go; Hartmann et al., 1997Go, 2001Go).

Unfortunately, a pronounced human anti-mouse antibody (HAMA) immune response occurred in the patients and prevented prolonged application of the mouse hybrid hybridoma protein. As an alternative, Arndt et al. (1999)Go decribed the construction of a bispecific antibody in the ‘diabody’ format (Holliger et al., 1993Go), which employs just the variable domains of the anti-CD30 and anti-CD16 murine antibodies. This diabody compared favourably with the parental {alpha}CD30 x {alpha}CD16 bispecific mouse quadroma antibody in its ability to activate and target NK cells both in cellular cytotoxicity experiments in vitro and in mice with xenotransplanted Hodgkin's lymphoma in vivo.

In general, immunogenicity of antibodies can be effectively reduced by grafting the hypervariable loops, known as complementarity-determining regions (CDRs), of the murine MAb on a human Ig framework in a process known as antibody ‘humanization’ (Jones et al., 1986Go; Winter and Harris, 1993Go). The generation of high-affinity humanized antibodies generally requires the transfer of one or more additional residues from the framework regions (FRs) of the mouse parent MAb (Carter et al., 1992Go; Foote and Winter, 1992Go). These back-mutations are thought to preserve (a) the canonical conformation of the CDRs, (b) the pairing between variable light and heavy chains and (c) the interaction with the antigen (Morea et al., 2000Go). Several humanized antibodies (for an overview, see Ross et al., 2003Go; Glennie and Winkel, 2003Go) have been successfully used in clinical trials or have already received approval from the FDA, such as the anti-Her2/neu antibody Trastuzumab/Herceptin or the anti-CD33 antibody Gemtuzumab/Mylotarg.

Here we describe the successful humanization and functional characterization of the mouse MAb HRS3, which previously served as the CD30-binding arm of the {alpha}CD30x{alpha}CD16 bispecific mouse quadroma antibody, as a bacterially secreted Fab fragment in conjunction with the extracellular domain of the CD30 target, which was also produced as a functional soluble antigen in the periplasm of Escherichia coli.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials

Escherichia coli K-12 strain JM83 (Yanisch-Perron et al., 1985Go) was used for cloning and production of recombinant Fab fragments and W3110 (Bachmann, 1972Go) was employed for the fermenter production of the CD30 antigen fragment. The vectors pASK102-D1.3 (A.Skerra, unpublished work), pASK111 (Vogt and Skerra, 2001Go), pASK88 (Schiweck and Skerra, 1995Go) and pASK106 (Fiedler et al., 2002Go) were all based on the generic expression plasmid pASK75 (Skerra, 1994bGo) carrying the chemically inducible tetracycline promoter/operator. The chromatography material with immobilized engineered streptavidin (Voss and Skerra, 1997Go) was kindly prepared by I.Theobald. Mass spectrometry was carried out by Dr J.Winkler using a Biflex III MALDI-TOF instrument (Bruker Daltonik, Bremen, Germany). The sequence for pASK88-huHRS3-VH-EP3/1 was submitted to the EMBL Nucleotide Sequence Database (accession number: AJ852004).

Construction of pASK111-CD30A

The coding sequence of CD30 corresponding to amino acids 185–335 of the mature protein (Swiss-Prot accession No.: P28908) was amplified from the plasmid pVL1392–CD30VL (C.Renner, unpublished work), containing the full-length cDNA for CD30 using a published procedure (Skerra, 1992Go). The primer sequences were 5'-GCACCCGGTCTCCGGCCGAAGCTGCTTCTAAACTGACG-3' and 5'-GCTCTGAGTGGGGCTGGT-3', introducing, respectively, a recognition sequence for BsaI and a blunt end compatible with Eco47III (both underlined). The unique amplification product was cut with BsaI and ligated with the vector fragment of pASK111, which had been cut with BsaI and Eco47III. The insert was checked by DNA sequencing using an ABI-Prism 310 Genetic Analyser (Perkin-Elmer Applied Biosystems, Weiterstadt, Germany) with the BigDye terminator kit.

Cloning of the variable region genes of HRS3

The genes for the variable (V) domains of the heavy (VH) and light (VL) chains of the murine (mu) monoclonal antibody HRS3 were cloned on a vector permitting the production of a corresponding chimaeric Fab fragment with human (hu) constant domains of subclass IgG1/{kappa}, carrying a His6-tag at the C-terminus of the heavy chain (Skerra, 1994aGo). Subcloning started from the vector pHRS3 (C.Renner, unpublished work) on which the VH and VL genes were encoded as an scFv fragment. The coding region for VH was already flanked by PstI and BstEII restriction sites compatible with the Fab expression vector. Thus, pHRS3 was first digested to completion with BstEII and then partially digested with PstI, since the VH gene contained an internal PstI restriction site. The 339 bp fragment was isolated and first inserted into pASK102-D1.3 (encoding the D1.3 Fab fragment equipped with a Strep-tag II containing an additional C-terminal Cys residue; for a description of the standardized cloning cassette, see Skerra, 1994aGo) resulting in pASK102-intHRS3.

Then, the coding sequence for the VL gene was amplified from pHRS3 via PCR using the PrimeZyme DNA Polymerase Kit 100 (Biometra, Göttingen, Germany) and primers 5'-CGGTCACCGTCTCCTCA-3' and 5'-TTTGATCTCGAGCTTGGTGCCCCCT-3'. The unique amplification product was cut with SstI (downstream of the first primer, within the amplified region) and XhoI (introduced by the second primer; underlined) and inserted into pASK102-intHRS3. Both inserts were confirmed by DNA sequencing and the resulting vector was designated pASK102-HRS3. The conserved CysL23 residue, which was missing in the HRS3 VL domain, was reconstituted by site-directed mutagenesis (Geisselsoder et al., 1987Go) using the oligodeoxynucleotide 5'-CCCACATTCTGAGAGGCCTTGCAGGTGACGTTGA-3'. The mutated VL gene sequence was confirmed by DNA sequencing and the resulting vector was designated pASK102–HRS3c. For bacterial production of a chimaeric HRS3 Fab fragment carrying the His6-affinity tag instead of the modified Strep-tag II, the VH and VL gene sequences were subcloned on pASK88 utilizing the conserved XbaI/BstEII and NcoI/XhoI restriction sites (Skerra, 1994aGo), yielding pASK88-muHRS3c.

Synthesis of the humanized VH and VL genes by PCR assembly

The humanized VH and VL domains were each assembled from six partially overlapping oligodeoxynucleotides (see Results). The PCR reaction mixture for the VH gene contained 0.5 µM VHx and VHy and 5 nM each of the VHa, VHb, VHc and VHd oligodeoxynucleotides in the presence of 0.2 mM of each dNTP, 1.5 mM MgCl2, 5 µl 10x Taq DNA polymerase buffer and 2.5 u Taq DNA polymerase (Promega, Mannheim, Germany) in a total volume of 50 µl. Twenty-five PCR cycles were performed at 94°C for 1 min, 55°C for 1 min and 72°C for 1.5 min in a thermocycler (UNO-Thermoblock, Biometra, Göttingen, Germany), followed by 5 min of incubation at 60°C. For synthesis of the VL gene, the PCR was performed with corresponding oligodeoxynucleotides under similar conditions.

In both cases, the unique amplification product was cut with PstI/BstEII (VH) and SacI/XhoI (VL), respectively. The DNA fragments were then separately inserted into pASK88. Cloned genes with a correct sequence from each chain were combined on the same plasmid utilizing the unique XbaI and NcoI restriction sites, yielding pASK88-huHRS3.

In order to prepare murine/human hybrid HRS3 Fab fragments, pASK88-muHRS3c and pASK88-huHRS3 were cut with NcoI and XhoI, thus excising the coding region for each VL domain together with the PhoA leader sequence. The huVL gene was ligated with the vector fragment of pASK88-muHRS3c, yielding pASK88-HRS3huVL/muVH. Similarly, the muVL gene was ligated with the vector fragment of pASK88-huHRS3, yielding pASK88-HRS3muVL/huVH.

Error-prone PCR

The VH gene was amplified from pASK88-huHRS3 under mutagenic conditions (Casson and Manser, 1995Go) with flanking primers VHx and VHyII (5'-GGAAACGGTGACCAGCGTGCCCTGACCCCAGTAAGCG-3'; see Results), which carried a compatible BstEII recognition site (underlined). The PCR mixture (100 µl) contained 10 ng pASK88-huHRS3 template DNA, 0.16 mM MnCl2, 0.2 mM dATP, 0.2 mM dGTP, 1 mM dCTP, 1 mM dTTP, 1 mM dITP, 0.5 µM of each primer, 5 u Taq DNA polymerase and Taq DNA polymerase buffer. PCR was performed with 30 cycles at 94°C for 1 min, 50°C for 1 min and 72°C for 1.5 min, followed by 5 min at 60°C. The PCR product of 366 bp was reamplified in a total volume of 50 µl with 1 µl from the first reaction mixture as template using the same conditions as in the PCR gene synthesis. The amplification product was digested with PstI and BstEII and ligated with the appropriately cut vector pASK106-huHRS3, which carries compatible restriction sites and codes for the humanized HRS3 Fab fragment as a fusion protein with an albumin-binding domain (ABD; König and Skerra, 1998Go).

Filter sandwich colony screening assay

The colony screening assay for antigen-binding activity of the bacterially produced Fab fragments was performed as described (Fiedler et al., 2002Go). In order to probe antigen-binding activity of the Fab variants that had been secreted by E.coli colonies and captured on a hydrophobic membrane purified recombinant CD30A was labeled with digoxigenin (DIG) at a molar ratio of 1:2 as described (Schlehuber et al., 2000Go). After washing the hydrophobic membrane three times with PBS/T, incubation was performed for 1 h with 10 ml of PBS/T containing either 1 µM or 500 nM of the CD30A–DIG conjugate. After washing, bound antigen was detected with anti-DIG Fab fragment conjugated with alkaline phosphatase (Roche Diagnostics, Penzberg, Germany) and the signals were developed in a chromogenic reaction. The colonies corresponding to the most intense signals were identified on the first membrane and propagated for subsequent analysis.

Recombinant protein production and purification

The CD30A antigen fragment was produced in E.coli W3110 harbouring pASK111-CD30A using an 8 l bench-top fermenter with a synthetic glucose mineral medium supplemented with 30 mg/l chloramphenicol (Cam), in a similar manner as described for the production of Fab fragments (Schiweck and Skerra, 1995Go; Fiedler and Skerra, 2001Go). Recombinant gene expression was induced by the addition of 500 µg/l anhydrotetracycline (Skerra, 1994bGo) as soon as the culture reached OD550 = 20. After an induction period of 2.5 h, cells were harvested by centrifugation and a periplasmic extract was prepared. The recombinant protein was purified via the Strep-tag (Skerra and Schmidt, 2000Go), followed by gel filtration on a Superdex 75 HiLoad 16/60 prep grade column (Amersham Pharmacia Biotech, Uppsala, Sweden) with PBS as running buffer. For MALDI-TOF mass spectrometry, the purified protein was dialysed against water.

The chimaeric HRS3 Fab fragment or its humanized variants were produced in E.coli JM83 harbouring pASK88-muHRS3c or related plasmids using 2 l LB cultures (Sambrook et al., 1989Go) supplemented with 100 µg/ml ampicillin (Amp) at 22°C. Induction and periplasmic extraction were performed as described (Fiedler and Skerra, 1999Go). The recombinant protein was purified via immobilized metal affinity chromatography (IMAC) either on Zn(II)-charged IDA Sepharose (Skerra 1994aGo) or on a POROS MC column (PerSeptive Biosystems, Framingham, MA). Elution was achieved with an imidazole concentration gradient from 0 to 200 mM. Fractions were analysed for purity via SDS–PAGE (Fling and Gregerson, 1986Go), appropriately pooled and finally dialysed against PBS (4 mM KH2PO4, 16 mM Na2HPO4, 115 mM NaCl, pH 7.4).

Protein concentrations were determined according to the absorption at 280 nm using calculated extinction coefficients (Gill and von Hippel, 1989Go) of 15 640 M–1 cm–1 for the CD30A antigen fragment and of 72 130 and 73 410 M–1 cm–1 for the chimaeric and humanized HRS3 Fab fragments, respectively.

Enzyme-linked immunosorbent assay (ELISA)

Ninety-six-well microtitre plates (12 x 8-well ELISA strips with high binding capacity; Greiner, Frickenhausen, Germany) were coated overnight with 50 µl of purified CD30A at a concentration of 50 µg/ml in PBS. The wells were blocked with 3% (w/v) bovine serum albumin (BSA) in PBS containing 0.1% Tween 20 (PBS/T) for 1 h and washed three times with PBS/T. Anti-CD30 MAbs or Fab fragments were applied in a dilution series in PBS/T and incubated for 1 h. The wells were then washed three times with PBS/T and incubated for 1 h with 50 µl of a 1:1000 dilution in PBS/T of anti-mouse Fc-specific IgG/AP conjugate in the case of the mouse monoclonal antibodies or with anti-human C{kappa}-light chain IgG/AP conjugate in the case of the Fab fragments (both from DAKO, Glostrup, Denmark). After washing twice with PBS/T and twice with PBS, the enzymatic activity was detected in a chromogenic reaction and the data were fitted by non-linear least-squares regression according to the law of mass action with KaleidaGraph software (Voss and Skerra, 1997Go).

Real-time biomolecular interaction analysis

The affinities of the anti-CD30 Fab fragments for the recombinant CD30A antigen fragment were measured by surface plasmon resonance (SPR; Jonsson et al., 1991Go) on a BIAcore X system (BIAcore, Uppsala, Sweden). After buffer exchange to 10 mM sodium acetate pH 3.85 by gel filtration, purified CD30A was diluted to 50 µg/ml and immobilized on a ‘research grade’ CM5 sensor chip using the amine coupling kit (BIAcore), resulting in the immobilization of ~1700 response units (RU). The purified recombinant Fab fragments were applied in PBS/P (PBS containing 0.005% surfactant P-20; BIAcore) at a series of appropriate concentrations. Complex formation was observed under a continuous buffer flow of 5 µl/min. The sensorgrams were corrected by subtraction of the corresponding signals measured for the control blank channel No. 1, which was used in-line. The chip was regenerated by applying a 2 µl pulse of 1 mM NaOH at a flow-rate of 25 µl/min, followed by equilibration with PBS/P. For KD determination, both a kinetic and equilibrium state analysis was performed (Huber et al., 1999Go). Kinetic parameters were determined with BIAevalution software V 3.0 (BIAcore) using a 1:1 binding model with drifting baseline. For equilibrium state analysis, maximum resonance signals (RUmax) were plotted against the corresponding concentration of the Fab fragment. In this case the curve fitting was performed as described for the ELISA.

Flow cytometry

Flow cytometric analyses were performed with CD30-positive L540CY Hodgkin cells using a FACScan cytofluorimeter (Becton Dickinson, Mountain View, CA). 106 target cells were washed twice in ice-cold PBS and incubated with 50 µl of the purified Fab fragment (10 µg/ml) for 30 min at 4°C. Cells were pelleted at 1200 r.p.m. and 4°C for 5 min and washed twice with 1 ml of PBS. Then the cells were resuspended in 50 µl of PBS containing mouse anti-human light-chain IgG1 labeled with biotin (Dianova, Hamburg, Germany) at a dilution of 1:100 and incubated for 30 min at 4°C. After pelleting and washing as above the cells were resupended in 50 µl of PBS containing streptavidin–phycoerythrin conjugate (Dianova, Hamburg, Germany) at a dilution of 1:100 and again incubated for 30 min at 4°C. After another washing step the cells were resuspended in 1 ml of PBS/N (PBS containing 0.05% NaN3) and counted in the FACS instrument. The muA9 Fab fragment directed against the CD16 antigen (Hombach et al., 1993Go; M.Schlapschy and A.Skerra, to be published) was applied under the same conditions and served as negative control.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Preparation of the soluble recombinant CD30 antigen

CD30 has a large extracellular region of 361 amino acids (Dürkop et al., 1992Go), comprising two cysteine-rich domains, ecd1 with 18 cysteine residues and ecd2 with 14 cysteine residues. Prediction of the solvent accessibility from the primary sequence (ProtScal program ‘% accessible residues’; Janin, 1979Go) revealed that, at least in some regions, solvent accessibility of the first domain (residues 31–157 of the mature protein according to the sequence numbering in Dürkop et al., 1992Go) was lower than that of the second domain (residues 207–332), suggesting a greater likelihood of the presence of exposed epitopes in the second domain. A particularly high degree of solvent accessibility became apparent for the amino acid segment 185–206 between the two domains.

Consequently, the coding sequence for residues 185–335 was cloned on the bacterial expression vector pASK111 (Vogt and Skerra, 2001Go). On the resulting plasmid, pASK111-CD30A, the structural gene was precisely fused at its 5'-end to the coding sequence for the bacterial OmpA signal peptide in order to effect secretion of the extracellular receptor domain into the periplasm of E.coli. At its 3'-end it was fused with the Strep-tag for subsequent purification by streptavidin affinity chromatography (Skerra and Schmidt, 2000Go). The whole structural gene was placed under transcriptional control of the tightly regulated tetp/o (Skerra, 1994bGo). Escherichia coli W3110 harbouring pASK111-CD30A was employed for the 8 l fermenter production of the antigen fragment. The recombinant protein was isolated from the periplasmic cell fraction as a soluble protein via the Strep-tag and purified to homogeneity by gel filtration with a total yield of 5.6 mg.

SDS–PAGE analysis revealed (Figure 1) that, in the reduced state, the recombinant CD30A appeared as a single homogeneous band with an apparent molecular size of 28 kDa. This is significantly larger than the calculated mass of 16 692 Da, which was however confirmed by MALDI-TOF mass spectrometry, yielding 16 683 ± 11 Da. When not reduced prior to gel electrophoresis, the mobility was significantly increased, also giving rise to some heterogeneity with respect to the disulfide bond pattern. Nevertheless, there was a predominant form corresponding to ~90% of the protein which exhibited maximum electrophoretic mobility (~21 kDa), thus indicating complete formation of the disulfide bonds. A second distinct form (~24 kDa) representing ~10% of the protein appeared to be less compact, indicating that some of the intramolecular disulfide bonds were not properly formed. Attempts to separate these two forms by size-exclusion chromatography failed, even though several high molecular weight oligomers, which were also seen in SDS–PAGE, could be quantitatively removed (data not shown). Further attempts to coexpress the bacterial disulfide isomerases DsbC or DsbA (Schmidt et al., 1998Go) had no effect on the composition of the recombinant protein.



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Fig. 1. Characterization of the bacterially secreted and purified CD30A antigen fragment by SDS–PAGE, followed by staining with Coomassie Brilliant Blue R-250. Lanes 1 and 3, CD30A purified from the periplasmic cell fraction of E.coli W3110 harbouring pASK111-CD30A via streptavidin-affinity chromatography; lanes 2 and 4, CD30A after subsequent gel filtration. Lanes 1 and 2 were reduced with 2-mercaptoethanol before application to the gel whereas in lanes 3 and 4 the disulfide bonds were not reduced. Two distinct disulfide isomers that are visible in lane 4 are labeled.

 
The functionality of CD30A was first investigated in an ELISA (Figure 2). Both the mouse anti-CD30 MAb and the {alpha}CD30 x {alpha}CD16 bispecific mouse quadroma antibody HRS3/A9 (Hombach et al., 1993Go) gave rise to a pronounced binding signal with typical saturation curves, whereas no signal was detected with the secondary antibody conjugate alone, indicating that the epitope recognized in vivo is present on the second extracellular domain of the CD30 cell surface marker. The affinity of the bispecific—and hence monovalent—{alpha}CD30 x {alpha}CD16 bispecific mouse quadroma antibody HRS3/A9 for the recombinant CD30A antigen was determined by SPR, revealing a dissociation constant (KD) of 82 ± 9 nM (cf. Table I).



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Fig. 2. Detection of the epitope of the anti-CD30 MAb (circles) and the {alpha}CD30 x {alpha}CD16 bispecific MAb (triangles) in the recombinant CD30A antigen fragment by ELISA. The wells of a microtitre plate were coated with CD30A and blocked with BSA and the muMAbs were applied in a dilution series. Bound antibody was detected by a second antibody, specific for the Fc part of mouse IgG, conjugated with alkaline phosphatase. The chromogenic reaction was measured in the presence of p-nitrophenyl phosphate ({Delta}A405/{Delta}t) and plotted against antibody concentration. No interaction of the second antibody with the BSA-coated matrix alone (squares) could be detected.

 

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Table I. Affinities of the engineered Fab fragments and the {alpha}CD30 x {alpha}CD16 bispecific antibody for the recombinant CD30A antigen as determined by SPR analysis

 
Cloning of the VH and VL domains of HRS3 and production as a recombinant Fab fragment

The genes for the VH and VL domains of HRS3 were first cloned on pASK102, encoding human constant genes of subclass IgG1/{kappa}. Surprisingly, upon DNA sequencing (Figure 3), it was found that the base triplet 67–69 of VL encoded a tyrosine residue instead of the conserved cysteine at amino acid position L23, which normally gives rise to the intrachain disulfide bond with CysL88. Because it was assumed that the CysL23Tyr mutation had a negative effect on the folding of the recombinant Ig fragment (Proba et al., 1997Go), CysL23 was reconstituted by site-directed mutagenesis and the mutant protein was dubbed HRS3c. After subcloning on pASK88, which encoded the His6-tag at the C-terminus of the human heavy chain constant region (Schiweck and Skerra, 1995Go), the chimaeric Fab fragment was produced in E.coli JM83 and purified from the periplasmic cell fraction by IMAC (Skerra, 1994aGo,bGo). The yield was ~0.4 mg/l shake-flask culture.



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Fig. 3. Nucleotide and amino acid sequences of the muHRS3c VH and VL domains before and after humanization. Top: amino acid and nucleotide sequence derived from the hybridoma cell line; Kabat numbering of amino acid sequence. Bottom: synthetic nucleotide and amino acid sequence designed for humanization. The CDR residues according to Kabat et al. (1991)Go are underlined both in the murine and humanized amino acid sequence. During gene synthesis for the humanized variable domains, both coding regions were assembled via PCR from four long oligodeoxynucleotides (boxed and labeled at each 5'-end with VH/La, VH/Lb, VH/Lc and VH/Ld, respectively) in the presence of an excess of two flanking PCR primers VH/Lx and VH/Ly (bold letters). The sequences of the altogether six oligodeoxynucleotides correspond to the coding and the non-coding strand of the V gene in an alternating manner, with overlaps between 18 and 20 bases (containing at least 10 G/C pairs), thus covering the entire sequence. Because of the terminal transferase activity of the Taq DNA polymerase (Hu, 1993Go), oligodeoxynucleotides were designed in a manner that the base following the 3'-end of intermediate PCR products was always an adenine. The restriction sites PstI, BstEII and SacI, XhoI that served for cloning of both synthetic genes are shown at the beginning and the end of each sequence. Additional restriction sites that were introduced into the framework coding regions for subcloning purposes are also indicated.

 
The antigen-binding activity of the HRS3c Fab fragment was investigated in an ELISA. A pronounced binding signal with a typical saturation was observed (see below), demonstrating that the Fab fragment recognized the epitope on the recombinant CD30A antigen, similarly to the intact murine antibodies before. Measurement of its antigen affinity by SPR revealed a KD value of 25.8 ± 2.8 nM, indicating slightly better affinity than for the bispecific, monovalent HRS3/A9 MAb (Table I).

Humanization of the muHRS3c Fab fragment

The variable domains of HRS3 were humanized via CDR grafting (Riechmann et al., 1988Go). First, the Kabat database (Johnson and Wu, 2001Go) and the protein data bank (Berman et al., 2000Go) were searched for human antibodies with closest sequence similarity to the murine VL and VH domains, especially concerning their framework regions (FR). Significant sequence similarity was found for the framework positions of the hu4D5ver8 antibody (Carter et al., 1992Go), i.e. 61% (for 87 aligned residues) in the case of VH and 71% (for 80 aligned residues) in the case of VL (Figure 3). This antibody, nowadays known as Trastuzumab/Herceptin (Carter, 2001Go), is a humanized version of the murine anti-p185HER2 monoclonal antibody 4D5 (Fendly et al., 1990Go) based on human framework consensus sequences of VH subgroup III and V{kappa} subgroup I (Kabat et al., 1991Go). Except for CDR-H3, which had three additional residues in HRS3 at positions 100D, 100E and 100F (according to Kabat numbering; Kabat et al., 1991Go; residues 108–110 in sequential numbering) all framework and CDR segments were mutually of equal lengths for both domains. Since the same canonical CDR conformations were likely (Al-Lazikani et al., 1997Go), the crystal structure of the Fv fragment hu4D5ver8 (Eigenbrot et al., 1993Go; pdb code 1FVC) was employed as guidance for the design of the humanized VH and VL domains of HRS3.

The three CDR sequences of each HRS3 variable domain were grafted on the corresponding human consensus sequences (Figure 3). The framework of the muHRS3c VL domain, comprising residues 1–23, 35–49, 57–88, 98–107, had 22 mismatches with the V{kappa} subgroup I whereas the muHRS3c VH framework, comprising residues 1–30, 36–49, 66–94 (residues 67–98 in sequential numbering), 103–120 (residues 113–123 in sequential numbering), showed 33 mismatches compared with the human consensus sequence of VH subgroup III. Framework residues that were likely to interact with the CDRs were identified by assessing their structural influence using the hu4D5ver8 crystal structure and, thus, several residues of the human sequence were restored by the original murine amino acids.

In the case of VL, ValL46 might interact with CDR-H3 of VH and its hydrophobic side chain seems also to be important for the contact with the VH framework. Therefore, Val was kept at this position of the ‘vernier’ region (Foote and Winter, 1992Go) instead of Leu, which is present in the human consensus sequence. Furthermore, owing to practical considerations with respect to the cloning of the VL gene on the expression vector pASK88, Gln and Met from the human consensus sequence at positions L3 and L4 close to the N-terminus were replaced by Glu and Leu, respectively, whereas Val at position L104 close to the C-terminus was replaced by Leu, thus allowing the introduction of SacI and XhoI restriction sites at the DNA level.

In the case of the VH domain, ThrH28, PheH29 and ThrH30 of the murine HRS3 sequence, again forming part of the ‘vernier’ region, are likely to be in contact with CDR-H1 and to influence its proper orientation. Whereas ThrH28 and PheH29 are present in the human consensus sequence, there is a Ser at position H30 so that the murine Thr was kept. VH residue 71 (residue 72 in sequential numbering) was previously proposed to be critical for the conformation of CDR-H2 (Tramontano et al., 1990Go; Carter et al., 1992Go). Therefore, we kept the murine Ala at this position instead of Arg from the human consensus sequence. At positions H73 and H78 (residues 74 and 79, respectively, in sequential numbering) of the ‘vernier’ region, the murine residues Lys and Ala were retained (instead of Asn and Leu), because they are part of the same loop as AlaH71 (residue 72 in sequential numbering) and may stabilize the structure of the antigen-binding site. Again, for cloning of the VH gene on pASK88, a PstI restriction site was introduced close to the 5'-end, generating a codon for Gln at position H5 instead of Val from the human consensus sequence. The three amino acids at the N-terminus (GluValLys) of the final construct were plasmid encoded, leading to a Lys at position H3 instead of Gln from the human consensus sequence. The BstEII restriction site could be introduced close to the 3'-end of the VH gene without changing the amino acid sequence.

Amino acids at positions corresponding to the VL/VH interface as defined by Chothia et al. (1985)Go were identical between HRS3 and the murine and human consensus sequences, except for positions 36 and 87 in huVL, where in both cases Tyr occurred instead of the murine Phe.

In summary, humanization of the muHRS3c VL domain required 18 amino acid exchanges while one amino acid from the murine framework sequence was retained, whereas in the muHRS3c VH domain 27 amino acids were exchanged and four murine residues were kept by purpose.

The amino acid sequences of both humanized variable domains were back-translated into a nucleotide sequence (Figure 3) using preferred codon frequencies of E.coli (Bennetzen and Hall, 1982Go) and potential mRNA secondary structure was minimized. For each domain a set of six overlapping oligodeoxynucleotides with lengths between 52 and 91 bp was designed following the strategy of Essen and Skerra (1994)Go and gene synthesis was performed in a PCR assembly reaction for each variable domain. In both cases a single amplification product was obtained, which was cut with appropriate restriction enzymes (Figure 3) and separately ligated with the likewise cut expression vector pASK88. After sequencing the synthetic variable genes of several clones, inserts with the correct nucleotide composition from one VL clone and from one VH clone were combined on a single plasmid, yielding pASK88-huHRS3.

The humanized HRS3 Fab fragment was produced from this vector in E.coli JM83 under the same conditions as the chimaeric Fab fragment, carrying the murine variable domains, before. The yield was ~0.2 mg/l shake-flask culture (OD550 = 1). SDS–PAGE under reducing conditions revealed two bands at 25 kDa with high purity (>98%), consistent with the expected masses of the light and heavy chains (see below). A single band was observed under non-reducing conditions with the expected mass of ~50 kDa. The affinity of the humanized Fab fragment towards the recombinant CD30A antigen was analysed by SPR, revealing a KD of 278 ± 61 nM under equilibrium conditions. Hence, antigen-binding activity was in principle retained upon humanization, but there was a 10-fold decrease in affinity compared with the chimaeric muHRS3c Fab fragment (Table I).

Affinity maturation of the humanized HRS3 Fab fragment by random mutagenesis and colony screening

In order to elucidate which of the two variable domains has the larger contribution to the observed loss in affinity, we produced two Fab fragments with murine/human hybrid variable domain pairing, both carrying the human constant domains as before, and analysed their binding activity for CD30A. Both ‘partially’ humanized Fab fragments could be successfully produced in E.coli and purified by IMAC, with yields of 1 mg/l shake-flask culture for the combination huVL/muVH and of 100 µg/l for the combination muVL/huVH. During functional comparison in a qualitative SPR experiment of both versions in conjunction with Fab fragments carrying the fully murine or the humanized pair of variable domains, the combination huVL/muVH showed an identical binding activity towards the immobilized CD30A antigen as the wild-type muHRS3c Fab fragment (data not shown). In contrast, the combination muVL/huVH showed precisely the same lowered association rate as the fully humanized HRS3 Fab fragment. These findings indicated that the loss of antigen affinity was to be attributed to the huVH domain, which therefore provided the target for functional improvement by protein engineering.

To this end, we chose a strategy wherein error-prone PCR was used to introduce random amino acid substitutions into the huVH domain, followed by a filter sandwich colony screening assay (Skerra et al., 1991Go; Schlehuber et al., 2000Go) for mutated Fab fragments with enhanced binding activity for the recombinant CD30A antigen. The mutagenic PCR was performed with pASK88-huHRS3 as template DNA and the oligodeoxynucleotides VHx and VHyII as primers (cf. Figure 4). The PCR product was ligated with the vector pASK106-huHRS3 (see Materials and methods), which allowed secretion of the huHRS3 Fab fragment as a fusion with the bacterial albumin-binding domain (ABD) into the periplasm of E.coli (König and Skerra, 1998Go). Following transformation, a filter sandwich colony screen was performed such that the secreted Fab fragments were released from the colonies on a first filter membrane and allowed to diffuse to a second capture membrane that had been coated with HSA. There, the Fab fragments became locally immobilized by ABD/HSA complex formation. Using CD30A labeled with digoxigenin, the functionally immobilized Fab fragments were subsequently probed for binding of the antigen, which was detected as coloured spots with an anti-digoxigenin Fab–alkaline phosphatase conjugate.



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Fig. 4. DNA sequences and corresponding amino acid translation for the mutant Fab fragments that were selected from the affinity maturation in comparison with the original huHRS3 VH gene sequence (Kabat numbering of amino acid sequence). CDR residues are underlined. In the mutants derived from error-prone PCR cycles 1–3 (huHRS3-VH-EP1/1 to huHRS3-VH-EP3/7), only the mutated nucleotides and correspondingly changed amino acids with respect to huHRS3 are given, while unchanged bases are marked by dots. Primer regions (VHx and VHyII) at the 5'- and 3'-ends are shown in bold. The best mutant of each cycle is underlined.

 
More than 500 transformands were plated on a filter on one Petri dish and of those the majority (>90%) gave rise to coloured signals on the second membrane, with slight variations in signal strength and morphology. Eight colonies corresponding to the most intense spots were recovered from the upper membrane and propagated and their plasmid DNA was isolated. Sequencing of the mutagenized huVH inserts revealed that five of the clones carried one or two amino acid substitutions (Figure 4), disregarding several silent mutations, while the other variants had the original huVH gene sequence retained.

All five of the mutated VH gene cassettes were subcloned on pASK88-huHRS3 for the production of the corresponding Fab fragments, without ABD, as before. After purification by IMAC, their antigen-binding properties were investigated in an ELISA and compared with the huHRS3 Fab fragment. The mutant Fab fragment huHRS3-VH-EP1/2 exhibited signals that were ~3-fold higher than those of huHRS3 (Figure 5). huHRS3-VH-EP1/2 and huHRS3-VH-EP1/7 showed higher ELISA signals by a factor of two (not shown), whereas the other mutants, huHRS3-VH-EP1/1, huHRS3-VH-EP1/4 and huHRS3-VH-EP1/6, did not exhibit any improved behaviour. Qualitative SPR measurements with huHRS3-VH-EP1/2 and huHRS3-VH-EP1/7 confirmed this affinity ranking. However, comparison with the original muHRS3c Fab fragment in an ELISA still revealed much better antigen affinity for the latter (Figure 6).



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Fig. 5. Comparative analysis of mutant Fab fragments with the huHRS3 and muHRS3c Fab fragments. (A) ELISA of Fab fragments huHRS3-VH-EP1/2 (triangles) and huHRS3 (circles) with the CD30A antigen fragment. The wells of a microtitre plate were coated with the recombinant CD30A protein and the purified Fab fragment was applied in a dilution series. Bound antibody fragment was detected with anti-human C{kappa} IgG/AP conjugate as secondary antibody. (B) ELISA of Fab fragments muHRS3c (squares), huHRS3-VH-EP1/2 (triangles) and huHRS3-VH-EP2/1 (diamonds) with the CD30A antigen fragment (note the differing concentration range).

 


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Fig. 6. Comparative analysis of muHRS3c, huHRS3 and mutant humanized Fab fragments. (A) 12% SDS–PAGE of the bacterially produced Fab fragments after purification via IMAC: lane 1, muHRS3c; lane 2, huHRS3; lane 3, huHRS3-VH-EP1/2; lane 4, huHRS3-VH-EP2/1; lane 5, huHRS3-VH-EP3/1. All samples were reduced with 2-mercaptoethanol. Heavy and light chains of the muHRS3c Fab fragment co-migrate, but can be separated in a 12% urea–SDS/polyacrylamide gel (Skerra, 1994aGo; not shown). (B) ELISA of Fab fragments huHRS3 (crosses), huHRS3-VH-EP1/2 (triangles), huHRS3-VH-EP2/1 (diamonds), huHRS3-VH-EP3/1 (circles) and muHRS3c (squares) with the CD30A antigen fragment (note that signals for huHRS3 and huHRS3-VH-EP1/2 coincide). For experimental details, see Figure 5 and Materials and methods.

 
Consequently, a second cycle of error-prone PCR was performed, this time starting from pASK88-huHRS3-VH-EP1/2 as template and followed by the filter-sandwich colony screening assay as before. When analysing eight colonies with intense signals, amino acid substitutions appeared predominantly in the following regions of the huVH domain: within and around CDR-H1, in FR-H3 and in CDR-H3 (cf. Figure 4). Interestingly, apart from other amino acid replacements, huHRS3-VH-EP2/1 exhibited the same substitution GluH100BLys (residue 106 in sequential numbering) that had been observed for huHRS3-VH-EP1/7 from the first affinity maturation cycle (see above).

Six of the mutated VH genes (EP2/1–6) were chosen for subcloning on pASK88-huHRS3 and the corresponding Fab fragments were produced in E.coli, purified by IMAC and analysed by qualitative SPR in comparison with huHRS3-VH-EP1/2. The Fab fragment huHRS3-VH-EP2/1 gave rise to the highest binding signal among all mutants (data not shown) and was further analysed in an ELISA together with muHRS3c and huHRS3-VH-EP1/2 (Figure 5B). Here, huHRS3-VH-EP2/1 revealed significantly improved binding activity compared with huHRS3-VH-EP1/2; however, its affinity still appeared somewhat lower than that of the original muHRS3c Fab fragment. The precise affinity of huHRS3-VH-EP2/1 for CD30A was determined by SPR, revealing a KD of 101 ± 30 nM (Table I).

To investigate the role of the amino acid substitution AspH72Asn (residue 73 in sequential numbering), we constructed a mutant carrying just the replacements IleH69Thr from huHRS3-VH-EP1/2 (residue 70 in sequential numbering) and GluH100BLys from huHRS3-VH-EP1/7 (residue 106 in sequential numbering) using the unique EcoRI and XhoI restriction sites that had been introduced into the synthetic gene. Compared with huHRS3-VH-EP2/1, the resulting mutant showed a significantly decreased binding signal in an ELISA (data not shown), indicating the functional importance of the modest side chain substitution AspH72Asn within FR-H3.

Finally, a third cycle of error-prone PCR mutagenesis was performed starting from huHRS3-VH-EP2/1. This time, the stringency of selection was raised by using a lowered concentration of the CD30A-DIG conjugate in the filter sandwich colony screening assay. Ten colonies that gave rise to intense spots were recovered and their plasmid DNA was sequenced (cf. Figure 4). Interestingly, of those huHRS3-VH-EP3/1 and huHRS3-VH-EP3/6 revealed amino acid substitutions that were already observed in previous cycles, such as IleH34Thr and AlaH101Pro (residue 111 in sequential numbering). The other six mutants corresponded to the previous version huHRS3-VH-EP2/1, in some cases carrying silent mutations.

The VH genes of the four novel mutants were again subcloned on pASK88-huHRS3 and the corresponding Fab fragments were produced in E.coli JM83, purified by IMAC and analysed by ELISA. Therein, the mutant huHRS3-VH-EP3/1, which carried the mutation AlaH101Pro (residue 111 in sequential numbering), revealed particularly strong binding activity, with a steep saturation curve almost identical with that of muHRS3c. Also, HRS3-VH-EP3/5 showed improved binding compared with huHRS3-VH-EP2/1, but clearly not approaching that of the original muHRS3c Fab fragment.

The best mutants from each mutagenesis cycle were compared with each other and with the huHRS3 and muHRS3c Fab fragments in another ELISA and also analysed on a 12% SDS gel (Figure 6). The affinity of huHRS3-VH-EP3/1 for the recombinant CD30A antigen was also determined by SPR (Figure 7), revealing a KD of 58 ± 10 nM (Table I). Analysis of the kinetic parameters (Table I) revealed a slightly slower kon but identical koff value with that of the muHRS3c Fab fragment. No significant difference in the strength of antigen binding was observed in the ELISA (Figure 6B).



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Fig. 7. Quantitative affinity analysis of the anti-CD30 Fab fragment muHRS3c and its humanized variant huHRS3-VH-EP3/1 with respect to the recombinant CD30A antigen fragment by surface plasmon resonance (SPR) spectroscopy. (A and C) Overlay of sensorgrams for a concentration series of the muHRS3c Fab fragment (A) and huHRS3-VH-EP3/1 (C). The dotted line at the end of the injection phase—which was followed by buffer flow—indicates the maximum resonance signals that were used for equilibrium analysis. (B and D) Determination of the dissociation constant (KD) by equilibrium analysis. Maximum resonance signals (RUmax) from the equilibrium phase of (A) and (C), respectively, were plotted against the corresponding concentration of each Fab fragment. KD values derived from curve fitting are given in Table I.

 
The same was the case in a final FACScan analysis with CD30+ L540CY cells (Figure 8). In this experiment, the binding signal of the mutant Fab fragment huHRS3-VH-EP3/1 appeared to be even slightly better than that of the original muHRS3c version. Hence, based on the pair of synthetic variable domains that carry four additional amino acid substitutions introduced during the affinity maturation, a humanized version of the anti-CD30 antibody was obtained, which is fully functional in terms of recognizing its native receptor antigen.



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Fig. 8. FACS analysis of the binding of the engineered Fab fragments toward CD30+ L540CY Hodgkin cells. (A) muA9 (Hombach et al., 1993Go; negative control); (B) muHRS3c; (C) huHRS3; (D) huHRS3-VH-EP3/1. Bound Fab fragment was detected with a mouse anti-human light chain IgG1 labeled with biotin followed by a streptavidin–phycoerythrin conjugate in a FACScan cytofluorimeter.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Humanization of the Fv region of the murine antibody HRS3, which is directed against the CD30 receptor on Hodgkin's lymphoma cells, has been accomplished using the strategy of CDR grafting. The success of CDR grafting depends largely on the ability of the chosen human framework to support CDRs from the original MAb in a conformation that is compatible with antigen binding. In this study, the framework was chosen on the basis of sequence homology.

Consensus sequences of the most prevalent human subclasses VHIII and V{kappa}I were previously employed as framework for the humanization of several antibodies, including anti-p185HER2 (4D5ver8, Herceptin; Carter et al., 1992Go), anti-CD3 (Shalaby et al., 1992Go) and anti-CD18 (Eigenbrot et al., 1994Go). In the case of the anti-p185HER2 and anti-CD18 humanized antibodies, the crystal structures of the Fab or Fv fragments were solved (Eigenbrot et al., 1993Go, 1994Go). Mutual superposition of the Fv regions revealed high similarity of the framework regions, indicating that their conformation is fairly independent of the CDRs supported (Eigenbrot et al., 1994Go). Furthermore, antibodies humanized on the basis of these human consensus sequences could be produced with exceptionally high yields both in E.coli and in eukaryotic expression systems, which is beneficial with respect to the required amounts for therapeutic application (Baca et al., 1997Go).

Simple CDR substitution alone generally does not lead to complete reconstitution of the binding activity of the original antibody (Carter et al., 1992Go). Additional murine residues belonging to the framework region must often be reintroduced in order to restore the initial affinity. Critical positions are the residues of the ‘vernier’ region (Foote and Winter, 1992Go), which influence the structure and mutual arrangement of the CDRs, residues at the VL/VH interface and those residues within the framework that are determinants of the canonical CDR conformations (Chothia et al., 1989Go).

Compared with the murine HRS3 sequence, the human V{kappa}I consensus sequence showed only two differences within the ‘vernier’ region (L36, huTyr/muPhe; L46, huLeu/muVal). No experience on the role of residue L36 as part of the ‘vernier’ region has been described in the literature, hence the human Tyr was kept in the final version of the huVL amino acid sequence. However, analysis of position L46 in the crystal structure of hu4D5ver8 (Eigenbrot et al., 1993Go) revealed that Leu is positioned at the VL/VH contact region and may influence the conformation of CDR-H3. Baca et al. (1997)Go described an affinity improvement by a factor of six in their humanized anti-EGF antibody upon substitution of the human Leu with Val, as it was present in the original murine antibody. Based on these observations, position L46 was assigned to Val in the humanized HRS3 VL domain.

The human VHIII consensus sequence showed nine differences from HRS3 at positions of the ‘vernier’ region, which were clustered especially at the N-terminal side of CDR-H1 and the C-terminal side of CDR-H2. According to Foote and Winter (1992)Go, positions 27–30 are part of a larger loop comprising CDR-H1 and thus influencing its correct positioning. The structural definition of Chothia and Lesk (1987)Go includes all positions from 26 to 32 for this hypervariable loop. Consequently, all amino acids of the murine sequence were kept in this region, except for H27 (see below), similarly as in the strategy followed by Carter et al. (1992)Go and Baca et al. (1997)Go.

The second cluster of differing amino acids in the ‘vernier’ region of the VH domain occurred between positions 66 and 80 in a loop flanking CDR-H2. Hence a stabilizing effect on this hypervariable loop or even a direct contact with the antigen was possible (Baca et al., 1997Go). In the human VHIII consensus sequence, the positions H71, H73 and H78 (residues 72, 74 and 79 in sequential numbering) are occupied by Arg, Asn and Leu, respectively, whereas in the huHRS3 VH domain the corresponding residues Ala, Lys and Ala of the murine sequence were kept at these positions. Tramontano et al. (1990)Go observed that the residue at position H71 is crucial for the conformation of both CDR-H2 and CDR-H3. CDR-H2 can contact CDR-H3 in different ways depending on the side chain at this position. A large side chain, such as Arg, separates the two CDRs, whereas a small one, such as Ala, permits an approximation of both hypervariable loops.

According to Chothia et al. (1989)Go, the hypervariable loops of an antibody—with the exception of CDR-H3—possess a limited number of defined main chain conformations. Crucial for these ‘canonical’ structures are the loop lengths and also side chains at characteristic positions both in the CDRs and in adjacent FR segments. In the case of the huHRS3 VL domain, all amino acids of the murine HRS3 sequence and the human consensus sequence that were considered important for the canonical structures were identical both in the framework regions and within the CDRs. However, in the case of the HRS3 VH domain, key residues were different between the murine and the human consensus sequences at positions H27 (huPhe/muTyr) and H72 (huArg/muAla). Position H27 may be occupied either with Tyr or with Phe without changing the canonical structure of CDR-H1 (Bendig and Jones, 1996Go); hence the human amino acid was used at this position. However, position H72 is considered essential for the proper canonical structure of CDR-H2 (Chothia and Lesk, 1987Go). Use of the murine Ala was therefore preferred instead of Arg from the human consensus sequence.

Finally, the correct association of the humanized VH und VL domains is important for the formation of a functional antigen-binding site. Chothia et al. (1985)Go defined positions in the VL/VH interface that are highly conserved both among individual antibodies and also across species. In this context, the amino acid sequences for the humanized and the murine VL and VH domains differ only at positions L36 and L87. In both cases the murine Phe was substituted with Tyr from the human consensus sequence as Tyr at these positions is predominantly conserved (Bendig and Jones, 1996Go). A 90% sequence homology at altogether 20 positions of the VL/VH contact region and 100% identity at the six positions that form the core region of the VL/VH interface (Chothia et al., 1985Go) should ensure proper association of the two humanized variable domains.

The observed sub-micromolar antigen affinity of the initially humanized HRS3 Fab fragment demonstrates that the antigen-binding site was in principle correctly formed. The moderate decrease in affinity by a factor of 10 compared with the original murine antibody was probably due to individual amino acid positions in the framework whose influence on antigen binding cannot be theoretically predicted (Baca et al., 1997Go). Interestingly, just the VH domain needed optimization, since an huVL/muVH hybrid Fab fragment showed essentially the same binding activity as the original muHRS3c Fab fragment. This finding furthermore indicated that the association between the VH and VL domains was actually retained between the murine and humanized versions because just the huVL domain carried side chain replacements (PheL36Tyr and PheL87Tyr) in the interface region. The use of mu/hu hybrid antibodies for the analysis of an affinity loss after antibody humanization has already been successfully used by Mateo et al. (1997)Go, leading to a similar result in so far as only their humanized VH domain had to be optimized.

In order to improve the huVH domain functionally, directed in vitro evolution was performed, thus imitating the natural mutagenesis and selection process during antibody affinity maturation in B lymphocytes (Skerra, 2003Go). Experimental conditions were chosen such as to introduce approximately one base substitution per 100 bp, which corresponds to the frequency of mutations in B-cells (Berek and Milstein, 1987Go). In order to select for improved antigen-binding activity, a filter sandwich colony screening assay (Skerra et al., 1991Go) in conjunction with the recombinant extracellular domain of CD30 was employed as a novel strategy. The humanized HRS3 Fab fragment with an initial KD value of 278 nM provided an appropriate submicromolar baseline affinity to generate already weak colour signals within the dynamic range of this assay, thus permitting the visual detection of mutants with increased antigen-binding activity. In the course of three mutagenesis and selection cycles, 17 mutants were isolated, each time using the best mutant from one cycle as the starting point for the following cycle and hence accumulating favourable mutations in an evolutionary manner.

In the first cycle, the mutated Fab fragment huHRS3-VH-EP1/2 carrying the substitution IleH69Thr (residue 70 in sequential numbering) was identified. When applied as a purified protein, it clearly showed better binding signals in an ELISA compared with the original huHRS3 Fab fragment. The side chain replacement at this position of the ‘vernier’ region indicates the importance of the loop formed by amino acids 66–80, which flanks the antigen-binding site as can be seen in the crystal structure of the hu4D5ver8 antibody (Eigenbrot et al., 1993Go). Substitution of the hydrophobic and more voluminous Ile by the hydrophilic Thr residue may favourably influence the conformation of this segment together with the neighbouring CDR-H2.

The substitution AspH72Asn (residue 73 in sequential numbering), which was identified during the second affinity maturation cycle in huHRS3-VH-EP2/1, is similarly flanked by two positions of the ‘vernier’ region. Although being modest in its size effect, this side chain substitution has a critical influence on antigen affinity. In additon to this substitution in FR-H3, the mutated Fab fragment huHRS3-VH-EP2/1 carries a side chain replacement GluH100BLys (residue 106 in sequential numbering) directly in CDR-H3. Interestingly, the same exchange of charged side chains was observed in the variant huHRS3-VH-EP1/7 from the first affinity maturation cycle.

Finally, in the third cycle the mutated Fab fragment huHRS3-VH-EP3/1 was identified, which carries the additional substitution AlaH101Pro (residue 111 in sequential numbering) in CDR-H3 and exhibits further improvement in antigen affinity by a factor of two. Again, the substitution AlaH101Pro had been identified before in the variant huHRS3-VH-EP1/6; however, then this substitution alone had no clear positive effect on the affinity of the purified Fab fragment. The rigid Pro imino acid is likely to affect the backbone conformation of CDR-H3. Interestingly, Wu et al. (1999)Go noted a critical role for position 101 in CDR-H3 before and they could show that there is a cooperative effect with position 49 in the VL domain when humanizing an anti-CD40 antibody.

In conclusion, in vitro evolution using error-prone gene amplification in conjunction with a functional colony screening assay permitted the quick improvement of the humanized anti-CD30 Fab fragment. In this manner, beneficial amino acid substitutions were found that can be well explained based on previous experience with antibody humanization but would have been difficult to predict on a rational basis. By introducing just four amino acid substitutions—two in FR-H3 and two in CDR-H3—the affinity could be increased by almost a factor of 10 with respect to the original humanized HRS3 Fab fragment. Comparison of the dissociation constant determined by SPR via analysis of the kinetic parameters revealed that it was almost identical between the clinically validated bispecific HRS3/A9 MAb and the optimized huHRS3-VH-EP3/1 Fab fragment. Most importantly, the antigen-binding behaviour of the humanized Fab fragment and of the recombinant muHRS3c Fab fragment as revealed by FACS analysis of live cells expressing the CD30 receptor was indistinguishable. Hence our strategy of combining error-prone PCR and a colony-filter screening assay proved to be an efficient procedure for the affinity maturation of a rationally humanized Fab fragment and, therefore, this technique should be of interest as a general tool for the functional engineering of recombinant antibodies (Pini et al., 2002Go).


    Acknowledgments
 
The authors thank Biotest Pharma GmbH, especially Dr Matthias Germer and Dr Michael Kloft, for a financial contribution and general advice. This work was supported by the Bundesministerium für Bildung und Forschung (BMBF project No. 0311759).


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 Introduction
 Materials and methods
 Results
 Discussion
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Received December 30, 2004; accepted January 5, 2005.

Edited by Dario Neri





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