Specificity grafting of human antibody frameworks selected from a phage display library: generation of a highly stable humanized anti-CD22 single-chain Fv fragment

Jürgen Krauss1, Michaela A.E. Arndt1, Andrew C.R. Martin2, Huaitian Liu1,3 and Susanna M. Rybak1,4

1National Cancer Institute at Frederick, Frederick, MD 21702, USA and 2School of Animal and Microbial Sciences, The University of Reading, Whiteknights, PO Box 228, Reading RG6 6AJ, UK 3Present address: National Cancer Institute, Biometric Research Branch, Bethesda, MD 20892-8315, USA

4 To whom correspondence should be addressed. e-mail: rybak{at}ncifcrf.gov


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A prerequisite for the enrichment of antibodies screened from phage display libraries is their stable expression on a phage during multiple selection rounds. Thus, if stringent panning procedures are employed, selection is simultaneously driven by antigen affinity, stability and solubility. To take advantage of robust pre-selected scaffolds of such molecules, we grafted single-chain Fv (scFv) antibodies, previously isolated from a human phage display library after multiple rounds of in vitro panning on tumor cells, with the specificity of the clinically established murine monoclonal anti-CD22 antibody RFB4. We show that a panel of grafted scFvs retained the specificity of the murine monoclonal antibody, bound to the target antigen with high affinity (6.4–9.6 nM), and exhibited exceptional biophysical stability with retention of 89–93% of the initial binding activity after 6 days of incubation in human serum at 37°C. Selection of stable human scaffolds with high sequence identity to both the human germline and the rodent frameworks required only a small number of murine residues to be retained within the human frameworks in order to maintain the structural integrity of the antigen binding site. We expect this approach may be applicable for the rapid generation of highly stable humanized antibodies with low immunogenic potential.

Keywords: antibody humanization/CD22/human phage display library/single-chain Fv/stability


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Recombinant antibodies have emerged as a novel class of highly efficient therapeutics within the last decade (reviewed in Glennie and Johnson, 2000Go). For many clinical applications, including radio-imaging studies and administration of recombinant cytotoxic fusion proteins, the employment of small antigen binding fragments such as single-chain Fv (scFv) antibodies is favorable over the use of antibodies in the IgG format. In contrast to whole immunoglobulins, scFv fragments are able to penetrate tumor tissue efficiently (Yokota et al., 1992Go) and are rapidly cleared from the circulation (Milenic et al., 1991Go). Furthermore, scFv antibodies can serve as building blocks for the generation of multivalent antigen binding fragments with improved affinities and tumor enrichment properties (reviewed in Todorovska et al., 2001Go).

Of major concern for the clinical application of scFv fragments generated from murine hybridoma cell lines is their remaining immunogenic potential as well as frequently exhibited low biophysical stability. Importantly, scFv fragments with insufficient stability were shown to fail enrichment at xenografted tumor sites in immunodeficient mice (Willuda et al., 1999Go). To improve biophysical properties of scFv antibodies they were stability engineered by the introduction of intermolecular disulfide bonds within the variable domain framework regions (Glockshuber et al., 1990Go; Reiter et al., 1994Go), rational mutagenesis of destabilizing residues (Chowdhury et al., 1998Go; Wörn and Plückthun, 1998Go) or grafting of the rodent antigen binding site into stable human antibody frameworks, resulting in a stable humanized scFv (Willuda et al., 1999Go). The application of the latter approach, however, is limited by the small number of stable acceptor frameworks currently available. As a result of this restriction, structural features between the murine antibody to be humanized and the human acceptor frameworks can substantially vary and thus may require the retention of a large number of potentially immunogenic murine residues within the human frameworks in order to preserve appropriate antigen binding of the humanized antibody (Willuda et al., 1999Go). Here we propose the use of stable antibody phage display library derived scaffolds for humanizing antibodies. Human antibody fragments directed against virtually any antigen of interest are now routinely obtained from antibody phage display libraries (reviewed in Hoogenboom, 2002Go). Stringent panning procedures enrich antibody fragments which are stably displayed on the phage during multiple rounds of selection on the antigen. As a consequence, not only antibodies with good binding properties, but also those with high biophysical stability are selected (Jung and Plückthun, 1997Go; Lee et al., 2002Go). To take advantage of this pre-selection for stability, we grafted the specificity of the clinically established anti-CD22 murine monoclonal antibody RFB4 (Campana et al., 1985Go) into antibody frameworks previously enriched from a small phage display library by four rounds of panning on Daudi lymphoma cells. A panel of humanized scFv antibodies is shown to recognize the same antigen epitope as the murine antibody, to bind to the target antigen with high affinity, and to exhibit exceptional stability when incubated in human serum at 37°C.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Generation of a patient-specific phage display library

An scFv antibody phage display library with a repertoire of 5x106 individual clones was generated from lymph node biopsies of two patients with B-cell non-Hodgkin lymphoma, as previously described (Mao et al., 1999Go). This library was subjected to four rounds of in vitro panning on Daudi tumor cells, as previously described (Schmidt et al., 1999Go). A pool of enriched scFv antibodies was obtained, sequenced and subsequently served as potential acceptor frameworks for specificity grafting.

Selection of acceptor frameworks and sequence alignment strategies

Framework amino acid sequences of the variable light chains and the variable heavy chains of the pre-selected scFvs were aligned to corresponding sequences of the monoclonal antibody RFB4 (Mansfield et al., 1997Go). Human framework sequences showing at least 70% sequence identity to the murine counterparts were considered for specificity grafting. These human frameworks were screened against V-Base (http://www.mrc-cpe.cam.ac.uk/DNAPLOT.php?menu=901) to reveal their closest corresponding human germline sequences. J-genes were analyzed separately.

With the exception of CDR-H1, the antigen binding loops to be grafted into the human framework regions were defined according to Kabat et al. (Kabat et al., 1991Go). Since residues H26–H32 comprise the structural loop of CDR-H1 (Chothia et al., 1989Go), we applied residues H26–H35 as CDR-H1 according to the combined Kabat–Chothia definition of CDR-H1.

To identify residues within the human frameworks likely to influence the integrity of the antigen binding site, and therefore antigen binding, both murine donor and selected human acceptor sequences were aligned to several sequence templates derived from antibody repertoires. ‘Invariant residues’ (Kabat et al., 1991Go) and ‘key residues’ (Chothia et al., 1989Go) were identified, and canonical-class assignments of the donor antigen binding loops L1–L3, H1 and H2, respectively, were determined by screening the sequence against sequence templates (Martin and Thornton, 1996Go) at http://www.bioinf. org.uk/. Furthermore, residues at the VH/VL interface (Chothia et al., 1985Go) and residues known to be structurally conserved at core sites (Chothia et al., 1998Go), were compared with corresponding donor and acceptor residues. Non-matching donor and acceptor framework residues at these sites were analyzed on the basis of information from other antibodies of known structure from the Protein Data Bank (Berman et al., 2000Go).

Construction of grafted scFv variants

CDR-grafted variant scFv SG0 was generated by sequential PCRs using eight overlapping oligonucleotides each, for construction of VH and VL, respectively, and overlap extension techniques as described elsewhere (Ye et al., 1992Go). A standard (Gly4Ser)3 linker connecting the VH and VL domains was likewise introduced by PCR. Silent mutations were introduced into primers such that the codon usage was adapted for optimized expression of the constructs in Escherichia coli by eliminating the most unusual codons for prokaryotic protein expression: Leu-CTA, Pro-CCC, Ile-ATA, Arg-AGA and Arg-AGG. PCR products encoding scFv SG0 were cleaved with appropriate restriction enzymes and ligated into pHOG 21 (Kipriyanov et al., 1996Go) for soluble expression. Variants SGI–SGV were constructed by site-directed mutagenesis and overlap extension PCR, as described (Ho et al., 1989Go), using scFv SG0-DNA as a template.

Periplasmic expression and purification of scFv fragments

The E.coli strain TG1 (Stratagene, La Jolla, CA), transformed with the scFv expression plasmid, was grown at 37°C, shaken at 230 r.p.m. in 1000 ml of 2YT medium containing 100 µg/ml ampicillin and 100 mM glucose (2YTGA). Cells were pelleted by centrifugation after reaching an OD600 of 0.8–1.0 at 1500 g for 20 min at 20°C and resuspended in the same volume of fresh 2YT medium containing 100 µg/ml ampicillin, 0.4 M sucrose and 1 mM IPTG. Induction was performed at 19°C for 18–20 h. Bacteria were pelleted by centrifugation at 7000 g for 30 min at 4°C, resuspended in 5% of the initial volume in periplasmic extraction buffer (50 mM Tris, 1 mM EDTA, 20% sucrose, pH 8.0) and incubated for 1 h on ice. The suspension was centrifuged at 30 000 g at 4°C for 1 h and the soluble scFv-containing supernatant was thoroughly dialyzed against SP10 buffer (300 mM NaCl, 50 mM NaH2PO4, 10 mM imidazole, pH 8.0). The dialyzed crude periplasmic extract was purified by immobilized metal affinity chromatography using Ni-NTA columns according to the protocol of the manufacturer (Qiagen, Valencia, CA). Eluted, purified scFv antibodies were extensively dialyzed against PBS, 50 mM imidazole. Monomeric scFv fragments were separated from higher molecular forms by size-exclusion chromatography in the same buffer using a calibrated Superdex 75 HR 10/30 column (Amersham Pharmacia, Piscataway, NJ). Monomeric scFv fractions were analyzed on 4–20% SDS–PAGE under reducing conditions and stained with Simply BlueTM Safe Stain (Invitrogen, Carlsbad, CA) or by western blot, using anti-c-myc mAb 9E10 (Roche, Indianapolis, IN) as primary, and alkaline phosphatase conjugated anti-mouse IgG (Sigma, St Louis, MO) as secondary, antibody. Concentrations of monomeric scFv proteins were determined after measuring the absorbance at A280nm with a spectrophotometer and calculating the specific extinction coefficient {epsilon} with the program Gene InspectorTM (Textco BioSoftware Inc., West Lebanon, NH).

Binding and competition assays

Specific binding of the constructs was determined by flow cytometry using the human CD22+ B-cell lines Raji, Ramos, Daudi and CA46 (ATTC, Manassas, VA, USA). Human T-cell lines Jurkat and HUT102 (ATTC) were used as negative controls. A total of 5x105 cells were incubated with 100 µl of a sample containing either the scFv fragments or control antibodies, in FACS buffer (PBS, 0.1% NaN3, 2% FBS) for 45 min at 4°C in round-bottom 96-well microtiter plates. Cells were pelleted at 200 g at 4°C for 5 min and washed twice with 200 µl of FACS buffer. For detection of bound antibodies, cells were first incubated for 30 min at 4°C with saturating concentrations of the anti-c-myc mAb 9E10 (10 µg/ml; Roche), followed by two washes and incubation with saturating amounts of FITC-labeled anti-mouse IgG (13 µg/ml; Jackson Immuno Research, West Grove, PA) for 30 min at 4°C. To exclude dead cells from the analysis, cells were washed as above and resuspended in FACS buffer containing 10 µg/ml propidium iodide (Sigma). Background fluorescence was determined by using cells incubated with 9E10 antibody and FITC-labeled anti-mouse antibody under the same conditions. Stained cells were analyzed on a FACScan Flow Cytometer (BD Bioscience, San Jose, CA), and median fluorescence intensity (MFI) was calculated using the CellQuestTM software (BD Bioscience). For competition experiments, Raji cells were pre-incubated with a 200-fold excess of scFv (1 µM) in FACS buffer for 1 h at 4°C. The mAb RFB4 (SouthernBiotech, Birmingham, AL) was added at a concentration of 5 nM and cells were incubated for an additional hour at 4°C. After two washes with FACS buffer, bound RFB4 was detected using FITC-labeled anti-mouse IgG. Samples were analyzed as described above. Inhibition of RFB4 for binding to Raji cells in the presence of competing scFv was determined as a percentage of maximal MFI of RFB4 in the absence of competing antibodies or the presence of an irrelevant scFv.

Determination of affinity constants (Kd)

Affinity measurements were performed as previously described (Benedict et al., 1997Go) with the following modifications. Varying concentrations of antibodies were incubated in triplicate with 5x105 Raji cells at room temperature in FACS buffer for 2 h. Bound antibodies were detected under the same conditions, as described above. After two final washing steps, cells were fixed in PBS containing 2% paraformaldehyde for 15 min at room temperature and analyzed by flow cytometry. The MFI was determined as described above and background fluorescence was subtracted. Equilibrium constants were determined by using the Marquardt–Levenberg algorithm for non-linear regression with GraphPad Prism version 3.0a for Macintosh (GraphPad Software, San Diego, CA).

Biophysical stability

ScFv fragments were incubated at 37°C in 90% human serum at a concentration of 12 µg/ml for up to 144 h. Samples were taken at different time points and stored at –20°C. Binding activity of the samples to CD22+ Raji cells was determined by flow cytometry. The MFI was determined as described above. Temperature-dependent degradation of monomeric scFv variants was determined by incubation of samples at 4 or 37°C in 1x PBS, at a concentration of 12 µg/ml for 120 h, followed by analytical gel filtration.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
To test our strategy, a small human library was used and, because of this small size, the number of available pre-selected human frameworks was limited. In future applications of the methodology, a large human library would be employed, allowing the selection of suitable scaffolds from a larger repertoire for grafting the desired specificity.

Selection of antibody acceptor frameworks for specificity grafting

A pool of scFv antibodies previously isolated from a lymphoma-patient-specific phage display library after four rounds of panning on Daudi tumor cells was sequenced. Variable domain framework sequences of these clones were aligned to corresponding sequences of the murine monoclonal antibody RFB4. A subset of four VH (all human VHIII subgroup) and four VL (all human V{kappa}1 subgroup) human scaffolds each showed >=70% sequence identity to corresponding sequences of the murine mAb RFB4 (Mansfield et al., 1997Go). Aligning the best matching human FR-VL clone #19 (73.8% sequence identity to murine FR-VL-RFB4) to the closest human variable domain germline sequence (HSIGKLA30, V{kappa}1–17) also revealed the lowest number of somatic framework mutations when compared with the other pre-selected light chains. Separate alignment of the human J-gene segment showed a complete match to human J{kappa}2 (Figure 1). Likewise, human clone VH#8 with the highest sequence identity to murine FR-VH-RFB4 (81.7%) also showed, in comparison with the other VH candidates, the best match to the closest human V gene germline segment (HSIGVH81B, VH3-3-66). Three non-identical residues within CDR-H3 of the rearranged human JH4 gene segment were identified (Figure 1). Thus, frameworks of VL#19 and VH#8 showed the best match to both the murine mAb to be humanized and corresponding human germline sequences and were therefore selected for specificity grafting.



View larger version (40K):
[in this window]
[in a new window]
 
Fig. 1. Sequence alignment of the VH and VL domains. The antigen binding site is defined according to Chothia et al. (Chothia et al., 1989Go) (dotted line) and Kabat et al. (Kabat et al., 1991Go) (dashed line); germ, human germline sequences; #8/#19, human VH/VL acceptor sequence; RFB4, murine VH/VL donor sequence; J-gene segments are underlined. (A) ‘Invariant residues’ (Kabat et al., 1991Go), (B) ‘key residues’ (Chothia et al., 1989Go) and (C) residues at the VH/VL interface (Chothia et al., 1985Go) are marked with (+) for matching or (–) for non-matching residues between murine and human sequence, respectively. (D) Residues at core sites as defined by Chothia et al. (Chothia et al., 1998Go) as: invariant (i) residue sites; similar (r) residue sites; surface (s) residues R, K, E, D, Q, N; neutral (n) residues P, H, Y, G, A, S, T; and buried (b) residues C, V, L, I, M, F, W, respectively; x, buried (b) or neutral (n) residues; y, surface (s) or neutral (n) residues; non-matching residue sites between the murine and human sequence are marked in bold letters; hum, specificity grafted sequences with murine back-mutated framework and CDR residues shown in bold letters. All residues are shown in the single letter code and numbered according to Kabat et al. (Kabat et al., 1991Go).

 
Identification of critical framework residues

Our general strategy was to identify unusual framework residues in the human sequence which may influence the structural integrity of the antigen binding site and to back-mutate these to murine residues, especially if this restored the human germline residue at the same time. By multiple alignment of both the murine donor and selected human acceptor sequences to several sequence reference templates, uncommon residues within both the VH and VL framework regions were identified (Figure 1). The potential significance of these residues was analyzed on the basis of structural information of antibody repertoires. Nine residues were predicted to affect antigen binding and thus considered back-mutation candidates. Each selected residue is now discussed in turn.

VL-3E->Q. In all human sequences in Kabat, valine, glutamate, glutamine and alanine are all common at VL-3. However, glutamine (Q) is the most common residue of human subgroup V{kappa}1 at this position and is present in the germline sequence corresponding to VL#19. Thus, glutamate at VL-3 in the acceptor sequence was most likely to have been introduced by the use of a degenerate 5' primer and this location was thus considered a prime candidate for back-mutation to the murine donor residue which is glutamine.

VL-40V->P. The valine (V) at VL-40 in the acceptor sequence was particularly unusual for human or mouse sequences and did not match the closest corresponding germline sequence. The Kabat database contains only three sequences with valine at this location, whereas proline is extremely common. This proline is involved in a hairpin structure at the rear end of FRII (away from the combining site) which, in turn, may have an effect on the conformation of the framework supporting CDR-L1 and CDR-L2. Hence, this residue was predicted to be critical.

VL-46R->L. Residue VL-46 is involved in an interface contact with VH-47 and also has a minor packing role in canonical class 2 CDR-L1 (Martin and Thornton, 1996Go). These two factors suggest that it will be critical in defining the conformation of CDR-L1 and will have an influence on VH/VL packing. A back-mutation from arginine to leucine at VL-46 was therefore predicted as potentially important.

VL-49S->Y. The VL#19 sequence was predicted to adopt a CDR-L1 conformation similar to canonical class 2/11A as defined by Martin and Thornton (Martin and Thornton, 1996Go). However, their analysis of conformational classes requires tyrosine, histidine, phenyl-alanine or lysine at VL-49, a residue against which CDR-L1 packs. The human VL#19 sequence has a serine at this location, whereas the mouse donor sequence has tyrosine. Back-mutation of this residue was therefore considered important.

VL-71F->Y. Residue VL-71 is also involved in packing with CDR-L1. However, both VL-71Y in the murine donor sequence and VL-71F in the VL#19 acceptor framework sequence allow CDR-L1 to adopt canonical class 2. This residue was predicted to have only a minor effect on the conformation of CDR-L1.

VH-6Q->E. Residue VH-6, a major determinant of the framework H1 conformation, was previously shown to be highly critical for antigen binding (de Haard et al., 1998Go; Honegger and Plückthun, 2001Go). Mutation from glutamine to glutamate was therefore predicted to be of crucial importance.

VH-40V->T. Hydrophobic residues at VH-40 such as the valine in the acceptor sequence are only seen in 8% of rearranged and 6% of germline genes, respectively. However, since VH-40 is located at the back of the Fv away from the combining site, a back-mutation to the donor sequence was predicted to have only a minor effect.

VH-79D->Y. The aspartate present in the human acceptor is unusual with the most common residue at VH-79 in human sequences being tyrosine, as seen in the mouse donor. A neutral residue is present in 89% of sequences (95% of germline). While not interacting directly with the CDRs, VH-79 packs against the H0 loop against which the CDRs pack and may thus have an indirect effect on CDR conformation. (The H0 and L0 loops occur between the fifth and sixth strands of the immunoglobulin fold and thus lie on the same side of the antibody as the CDRs, but are not hypervariable and do not form part of the combining site.) This residue is in a location similar to VH-23 which has been shown previously to have a small effect on binding (Adair et al., 1994Go).

VH-84V->S. Position VH-84 is a valine in the human acceptor. Hydrophobic residues are seen in only 9% of human sequences (3% of germline) at this location. However, like VH-40, residue VH-84 is located at the rear of the Fv and was therefore expected to have only a minor effect on binding.

Based on this analysis, a hierarchy of the expected importance of mutations was generated (Table I) and six specificity grafted versions with successive donor residue back-mutations were designed accordingly (Table II).


View this table:
[in this window]
[in a new window]
 
Table I. Hierarchy of critical framework residues
 

View this table:
[in this window]
[in a new window]
 
Table II. Specificity grafted variants with back-mutations to the murine donor sequence
 
Generation, expression and purification of specificity grafted scFv variants

We first constructed an scFv comprising the human VH#8 heavy chain and the VL#19 light chain, respectively (Figure 1). This construct was produced as soluble protein but binding was not specific to the CD22 antigen (data not shown). Six specificity grafted scFv mutants were generated subsequently (Table II).

Monomeric protein fractions with apparent molecular masses of ~29 kDa could be well separated from a small fraction of dimers (<8%, except variant SGII producing 28% dimeric protein) by size-exclusion chromatography.

Immunoreactivity and antigen affinity

Flow cytometry analysis revealed a specific binding of all specificity grafted versions to several CD22+ lines and no binding to CD22 cell lines (data not shown). Grafting the antigen binding site of mAb RFB4 onto the selected frameworks VH#8/VL#19 (variant SG0) was not sufficient to generate a molecule with appropriate antigen binding properties. Variant SGI contained two back-mutations—the residues predicted to be of the highest importance. SGI showed an antibody concentration-dependent increase in fluorescence intensity, but saturation on CD22+ tumor cells was not reached at concentrations up to 3.3 µM. Two further light chain back-mutations in variant SGII resulted in a moderate binding affinity (Kd 463 nM; Figure 2 and Table II) to CD22. Variant SGIII contained one VH and four VL framework back-mutations and had an apparent Kd of 9.8 nM (Figure 2 and Table II). The 47-fold increase in affinity of variant SGIII in comparison with SGII is caused by a single back-mutation of interface framework residue VL-46R->L. Importantly, four out of five back-mutated residues of variant SGIII also restored respective human germline sites (Table I). Hence, only one of the back-mutations (VL-46R->L) generated a potentially immunogenic site in the humanized antibody SGIII.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 2. Equilibrium binding curves of specificity grafted scFv monomers. Raji cells were incubated with various concentrations of SGII (diamonds), SGIII (boxes), SGIV (triangles), SGV (closed circles) or mAb RFB4 (open circles). Specific binding of antibodies was determined by flow cytometry. Binding activity at indicated concentrations is given as percent of the MFI. Measurements were performed in triplicate; standard deviations are shown as bars. Binding affinity constants (Kd) were determined by fitting the cell binding data to the non-linear regression model according to the Levenberg–Marquard method.

 
In variant SGIV, additional back-mutations were made at VH-79D->Y (which also restored the human germline residue) and VL-71F->Y. This variant had the highest affinity for the target antigen (Kd 6.5 nM; Figure 2 and Table II). The back-mutations VH-40V->T and VH-84V->S were expected to have only a minor effect on binding. Surprisingly, when mutated together (variant SGV), this lead to a 1.5-fold decrease in affinity when compared with SGIV (Figure 2 and Table II). The murine mAb RFB4 revealed an apparent Kd of 2 nM (Figure 2).

Epitope specificity

The epitope specificity of variants SGIII, SGIV and SGV, which bound to the target antigen with high affinity, was tested by binding competition with mAb RFB4 on living tumor cells by flow cytometry. Incubation of CD22+ Raji cells with a 200-fold molar excess of the specificity grafted variants SGIII, SGIV or SGV, respectively, almost completely prevented mAb RFB4 from binding to the target cells (>95% inhibition; Figure 3), indicating that the variants recognize the same CD22 epitope as the murine antibody.



View larger version (10K):
[in this window]
[in a new window]
 
Fig. 3. Epitope specificity of variants SGIII, SGIV and SGV. Competition of scFv variants with mAb RFB4 for binding to CD22+ Raji cells was determined by flow cytometry. Results are shown as percent binding inhibition of mAb (5 nM) when incubating tumor cells with a 200-fold molar excess of scFv variants.

 
Biophysical stability

Since scFv antibodies of clinical relevance must be stable at body temperature and resistant towards human serum proteases, we assessed the stability of the specificity grafted variants by determination of their actual binding activity to living tumor cells after incubation in human serum at 37°C for various time periods. The variants with appropriate antigen binding affinities, SGII, SGIII, SGIV and SGV, respectively, showed exceptional stability with 89–93% of their initial binding activity to tumor cells after a 6 day incubation period (Figure 4). Analytical size-exclusion chromatography after 5 days incubation of samples from each variant at 37°C in PBS revealed a decrease in monomeric protein fractions of between only 4 and 11% (Figure 5).



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 4. Serum stability of humanized scFvs. Constructs SGII (diamonds), SGIII (boxes), SGIV (triangles) and SGV (circles) were incubated at 37°C for various time periods, followed by determination of immunoreactivity with CD22+ Raji cells by flow cytometry.

 


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 5. Biophysical stability of specificity grafted scFv monomers. Analytical size-exclusion FPLC (fast protein liquid chromatography) on a calibrated Superdex 75 column was performed before (dashed line) and after (solid line) incubation of 12 µg/ml scFv in PBS at 37°C for 5 days.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study, we generated a humanized scFv antibody by grafting the specificity of the murine anti-CD22 monoclonal antibody RFB4 into human frameworks pre-selected for stability from a phage display library. Effects on antigen binding and stability properties of specificity grafted scFv antibodies were analyzed.

Grafting the antigen binding loops of the murine monoclonal antibody RFB4 into the pre-selected human frameworks did not result in a humanized scFv fragment which retained sufficient antigen binding. Loss of avidity of initial CDR grafted antibodies is observed very commonly and the introduction of additional murine donor residues into the human acceptor antibody frameworks is often required to maintain the structural integrity of the antigen binding site and thus appropriate antigen binding (Riechmann et al., 1988Go; Queen et al., 1989Go; Kettleborough et al., 1991Go; Carter et al., 1992Go; Foote and Winter, 1992Go). In most cases, the potential of human acceptor framework residues to compromise antigen binding properties is assessed by a computer homology model (Riechmann et al., 1988Go; Queen et al., 1989Go; Kettleborough et al., 1991Go; Carter et al., 1992Go). In the present study, all antigen binding loops adopted known canonical structures (except CDR-H3 for which canonical structures cannot yet be defined) of the antigen binding loops (Chothia et al., 1989Go). Therefore, we applied a sequence alignment strategy to identify residues with possible detrimental effects on antigen binding. After identifying uncommon amino acids within the human acceptor frameworks by alignment to several sequence reference templates, the structural role of each of these residues was examined on the basis of information on antibodies with known crystal structures. Our data show that this procedure allowed a very accurate prediction of the critical potential of each identified unusual framework residue to affect antigen binding of the molecule. As a result, grafted scFvs SGIII, SGIV and SGV, respectively, retained antigen binding properties comparable with the donor mAb RFB4. The ~3–5-fold lower affinity constants of these scFv variants when compared with the murine parental mAb RFB4 (Figure 2) most likely reflect avidity loss owing to the monovalency of the constructs. In comparison, an scFv antibody generated from the murine monoclonal antibody CC49 with specificity for the pancarcinoma antigen TAG-72 exhibited an 8-fold lower relative binding affinity than the corresponding murine IgG and a dimeric F(ab')2 derivative (Milenic et al., 1991Go).

Non-covalently associated variable domains of scFv formatted antibodies frequently show a high tendency for aggregation (Essig et al., 1993Go). Temperature-dependent aggregation and failure to enrich at tumor xenografts of a high-affinity scFv fragment with specificity for the epithelial glycoprotein-2 was shown to be caused by its low biophysical stability (Willuda et al., 1999Go). Grafting the antigen binding loops and several structurally important framework residues of this murine scFv into stable human acceptor frameworks resulted in a humanized scFv fragment with markedly improved biophysical properties (Willuda et al., 1999Go). This construct was able to enrich at the tumor site efficiently with a tumor to blood ratio of 5.25 after 24 h, while retaining the specificity and affinity of the murine scFv. The engineered humanized scFv antibody retained 48.3% of its initial binding activity after 20 h incubation in human serum at 37°C and revealed no temperature-induced degradation at this time point, as demonstrated by analytical gel filtration. In the present study, we wished to make use of similar stable scaffolds for humanizing an anti-CD22 antibody. Our approach was based on the assumption that stringent panning procedures of antibody phage display libraries not only enrich for molecules with good antigen binding properties, but also favor the selection of biophysically stable molecules displayed on the phage during several selection rounds. As reported here, the selection of such stable scaffolds which derived from a small patient-specific phage display library, and thus very limited diversity, could not necessarily be expected. Hence, these results indicate that panning procedures indeed enriched molecules with extraordinary stability and this stability could be maintained in the grafts. Biophysical properties of human germline family consensus variable domains were recently systematically examined (Ewert et al., 2003Go). It was shown that VH3 subgroup domains alone and in combination with VL domains in scFv formatted antibodies exhibited very high stability. The most stable scFv fragments were VH3/VL{kappa}3, VH1b/VL{kappa}3, VH5/VL{kappa}3 and VH3/VL{kappa}1, respectively. As reported earlier, grafting the specificities of murine monoclonal antibodies into human VH3{kappa}1 consensus frameworks derived from the humanized anti-p185Her2 antibody Trastuzumab (HerceptinTM) (Carter et al., 1992Go) resulted in scFv molecules with high stability (Jung and Plückthun, 1997Go; Willuda et al., 1999Go). The most abundant human variable domains in the B-cell repertoire belong to the VH3 and, to a lesser extent, VL{kappa}1 and VL{lambda}3 subgroup families (Kabat et al., 1991Go). As a consequence, these V-gene families are over-represented in phage display libraries generated from B lymphocytes of human donors. This bias was even shown to be increased after enriching binders by panning, and VH3 frameworks in particular were more often selected than theoretically expected (Hoogenboom and Winter, 1992Go; Sheets et al., 1998Go; Ewert et al., 2003Go). Notably, VH3 family gene segments were recently shown to be exclusively used in phage enriched intrabodies which are required to possess high intrinsic stability in order to compensate for the missing disulfide bonds which cannot form in the reducing environment of the cytosol (Visintin et al., 2002Go). Hence, the preferred selection of VH3 family consensus heavy chains from phage display libraries strongly indicates a co-selection for molecules with high intrinsic stability. Since the murine donor antibody in the present study exhibited a high structural similarity to the human pre-selected VH3/VL{kappa}1 consensus frameworks, some caution should be exercised, as to conclude our approach may invariantly result in humanized scFv fragments with similar exceptional biophysical stability. Therefore, it remains to be investigated in the future as to whether stringent panning procedures would also select for similar stable molecules of human subclass families known to exhibit less favorable biophysical properties than VH3/VL{kappa}3, VH1b/VL{kappa}3, VH5/VL{kappa}3 and VH3/VL{kappa}1, and thus would allow to graft structurally related antigen binding sites of any murine monoclonal antibody into such robust pre-selected scaffolds.

In summary, we have generated a panel of humanized scFv antibodies by grafting the specificity of the murine monoclonal anti-CD22 antibody RFB4 into frameworks pre-selected for stability from a phage display library. The constructs exhibit excellent antigen binding and stability properties and can be expected to possess a low immunogenic potential. These features predict them as promising building blocks for the generation of novel reagents for the diagnosis and treatment of CD22+ B-cell malignancies.


    Acknowledgements
 
We thank Drs L.Kwak and A.Biragyn for providing us with tumor biopsies from lymphoma patients in order to construct the human phage display library. The help and encouragement of Dr D.Newton, D.Ruby and M.Hursey is greatly appreciated. We thank Dr E.Sausville for his continued interest and support. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the US Government. The publisher or recipient acknowledges the right of the US government to retain a non-exclusive, royalty-free license in and to any copyright covering the article. This project has been funded in whole or in part with Federal Funds from the National Cancer Institute, National Institutes of Health, under contract number N01-CO-12400, and grants from the Deutsche Krebshilfe/Dr Mildred-Scheel-Stiftung Grant D/00/39301 (to J.K.), and Deutsche Akademie der Naturforscher Leopoldina Grant BMBF-LPD 9901/8-33 (to M.A.E.A.).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Adair,J.R. et al. (1994) Hum. Antibodies Hybridomas, 5, 41–47.[Medline]

Benedict,C.A., MacKrell,A.J. and Anderson,W.F. (1997) J. Immunol. Methods, 201, 223–231.[CrossRef][ISI][Medline]

Berman,H.M., Westbrook,J., Feng,Z., Gilliland,G., Bhat,T.N., Weissig,H., Shindyalov,I.N. and Bourne,P.E. (2000) Nucleic Acids Res., 28, 235–242.[Abstract/Free Full Text]

Campana,D., Janossy,G., Bofill,M., Trejdosiewicz,L.K., Ma,D., Hoffbrand,A.V., Mason,D.Y., Lebacq,A.M. and Forster,H.K. (1985) J. Immunol., 134, 1524–1530.[Abstract/Free Full Text]

Carter,P. et al. (1992) Proc. Natl Acad. Sci. USA, 89, 4285–4289.[Abstract]

Chothia,C., Novotny,J., Bruccoleri,R. and Karplus,M. (1985) J. Mol. Biol., 186, 651–663.[ISI][Medline]

Chothia,C. et al. (1989) Nature, 342, 877–883.[CrossRef][ISI][Medline]

Chothia,C., Gelfand,I. and Kister,A. (1998) J. Mol. Biol., 278, 457–479.[CrossRef][ISI][Medline]

Chowdhury,P.S., Vasmatzis,G., Beers,R., Lee,B. and Pastan,I. (1998) J. Mol. Biol., 281, 917–928.[CrossRef][ISI][Medline]

de Haard,H.J., Kazemier,B., van der Bent,A., Oudshoorn,P., Boender,P., van Gemen,B., Arends,J.W. and Hoogenboom,H.R. (1998) Protein Eng., 11, 1267–1276.[Abstract]

Essig,N.Z., Wood,J.F., Howard,A.J., Raag,R. and Whitlow,M. (1993) J. Mol. Biol., 234, 897–901.[CrossRef][ISI][Medline]

Ewert,S., Huber,T., Honegger,A. and Plückthun,A. (2003) J. Mol. Biol., 325, 531–553.[CrossRef][ISI][Medline]

Foote,J. and Winter,G. (1992) J. Mol. Biol., 224, 487–499.[ISI][Medline]

Glennie,M.J. and Johnson,P.W. (2000) Immunol. Today, 21, 403–410.[CrossRef][ISI][Medline]

Glockshuber,R., Malia,M., Pfitzinger,I. and Plückthun,A. (1990) Biochemistry, 29, 1362–1367.[ISI][Medline]

Ho,S.N., Hunt,H.D., Horton,R.M., Pullen,J.K. and Pease,L.R. (1989) Gene, 77, 51–59.[CrossRef][ISI][Medline]

Honegger,A. and Plückthun,A. (2001) J. Mol. Biol., 309, 687–699.[CrossRef][ISI][Medline]

Hoogenboom,H.R. (2002) Methods Mol. Biol., 178, 1–37.[Medline]

Hoogenboom,H.R. and Winter,G. (1992) J. Mol. Biol., 227, 381–388.[ISI][Medline]

Jung,S. and Plückthun,A. (1997) Protein Eng., 10, 959–966.[Abstract]

Kabat,E.A., Wu,T.T., Perry,H., Gottesman,K. and Foeller,C. (1991) Sequences of Proteins of Immunological Interest. NIH Publication No. 91-3242, US Department of Health and Human Services, Bethesda, MD.

Kettleborough,C.A., Saldanha,J., Heath,V.J., Morrison,C.J. and Bendig,M.M. (1991) Protein Eng., 4, 773–783.[Abstract]

Kipriyanov,S.M., Kupriyanova,O.A., Little,M. and Moldenhauer,G. (1996) J. Immunol. Methods, 196, 51–62.[CrossRef][ISI][Medline]

Lee,Y.C., Boehm,M.K., Chester,K.A., Begent,R.H. and Perkins,S.J. (2002) J. Mol. Biol., 320, 107–127.[CrossRef][ISI][Medline]

Mansfield,E., Amlot,P., Pastan,I. and FitzGerald,D.J. (1997) Blood, 90, 2020–2026.[Abstract/Free Full Text]

Mao,S., Gao,C., Lo,C.H., Wirsching,P., Wong,C.H. and Janda,K.D. (1999) Proc. Natl Acad. Sci. USA, 96, 6953–6958.[Abstract/Free Full Text]

Martin,A.C. and Thornton,J.M. (1996) J. Mol. Biol., 263, 800–815.[CrossRef][ISI][Medline]

Milenic,D.E., Yokota,T., Filpula,D.R., Finkelman,M.A., Dodd,S.W., Wood,J.F., Whitlow,M., Snoy,P. and Schlom,J. (1991) Cancer Res., 51, 6363–6371.[Abstract]

Queen,C. et al. (1989) Proc. Natl Acad. Sci. USA, 86, 10029–10033.[Abstract]

Reiter,Y., Brinkmann,U., Jung,S.H., Lee,B., Kasprzyk,P.G., King,C.R. and Pastan,I. (1994) J. Biol. Chem., 269, 18327–18331.[Abstract/Free Full Text]

Riechmann,L., Clark,M., Waldmann,H. and Winter,G. (1988) Nature, 332, 323–327.[CrossRef][ISI][Medline]

Schmidt,S., Braunagel,M., Kurschner,T. and Little,M. (1999) Biotechniques, 26, 697–702.[ISI][Medline]

Sheets,M.D. et al. (1998) Proc. Natl Acad. Sci. USA, 95, 6157–6162.[Abstract/Free Full Text]

Todorovska,A., Roovers,R.C., Dolezal,O., Kortt,A.A., Hoogenboom,H.R. and Hudson,P.J. (2001) J. Immunol. Methods, 248, 47–66.[CrossRef][ISI][Medline]

Visintin,M., Settanni,G., Maritan,A., Graziosi,S., Marks,J.D. and Cattaneo,A. (2002) J. Mol. Biol., 317, 73–83.[CrossRef][ISI][Medline]

Willuda,J., Honegger,A., Waibel,R., Schubiger,P.A., Stahel,R., Zangemeister-Wittke,U. and Plückthun,A. (1999) Cancer Res., 59, 5758–5767.[Abstract/Free Full Text]

Wörn,A. and Plückthun,A. (1998) Biochemistry, 37, 13120–13127.[CrossRef][ISI][Medline]

Ye,Q.Z., Johnson,L.L. and Baragi,V. (1992) Biochem. Biophys. Res. Commun., 186, 143–149.[ISI][Medline]

Yokota,T., Milenic,D.E., Whitlow,M. and Schlom,J. (1992) Cancer Res., 52, 3402–3408.[Abstract]

Received June 10, 2003; accepted August 27, 2003.





This Article
Abstract
FREE Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (1)
Request Permissions
Google Scholar
Articles by Krauss, J.
Articles by Rybak, S. M.
PubMed
PubMed Citation
Articles by Krauss, J.
Articles by Rybak, S. M.