Institut für Molekularbiologie und Tumorforschung, Philipps-Universität Marburg, Emil-Mannkopff-Strasse 2, 35033 Marburg, Germany
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: antigen-binding site/bispecific/homodimer/linker/multivalent/phage display/single-chain diabodies
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We and others have recently modified the original dimeric diabody format by joining the two fragments by an additional middle linker (Brüsselbach et al., 1999; Kipriyanov et al., 1999
). The overall structure of these single-chain diabody (scDb) molecules is VHAlinkerAVLBlinkerMVHBlinkerBVLA, with linkers A and B consisting routinely of 56 residues and linker M of 1520 residues. The middle linker (linker M) resembles those connecting the two variable domains in scFv molecules. Monomeric single-chain diabodies (scDb) are readily assembled in bacterial and mammalian cells and show improved stability under physiological conditions (Brüsselbach et al., 1999
; Kipriyanov et al., 1999
). We could demonstrate that scDb molecules can be applied to recruit prodrug-converting enzymes and cytotoxic T lymphocytes to tumour cells (Brüsselbach et al., 1999
; T.Korn and R.E.Kontermann unpublished work). Furthermore, scDb can be expressed intracellularly in active form in the secretory pathway or displayed on the surface of mammalian cells opening additional therapeutic applications (Kontermann and Müller, 1999
). The functional affinity of scDb could be improved by fusion to the Fc or CH3 region of the immunoglobulin
1 chain generating IgG-like tetravalent and bispecific molecules (Alt et al., 1999
).
Recent findings indicate that the length and composition of the linkers used to connect the variable domains strongly influence the formation and stability of single-chain Fv fragments (scFv). Most scFv fragments are generated using a 15 amino acid residue linker of composition (Gly4Ser)3 (Huston et al., 1988). Several approaches have been described to obtain optimized linkers by phage display technology. These resulted in the selection of various new linkers, which improved secretion and antigen binding activity of the scFv fragments (Tang et al., 1996
; Turner et al., 1997
).
The length of the linker joining the two variable domains also dictates the formation of multimeric scFv molecules. Reduction of the length of the linker joining the two variable domains from 15 amino acid residues, generally used to produce single-chain Fv fragments (scFv), below 812 residues favours dimeric assembly of the VHVL fragments generating diabodies (Holliger et al., 1993; Kortt et al., 1994
; Aflthan et al., 1995
). Further reduction of the linker sequence to less than five amino acids has been shown to result in the generation of tri- or tetrameric molecules (triabodies, tetrabodies) (Iliades et al., 1997
; Kortt et al., 1997
; Pei et al., 1997
; Le Gall et al., 1999
; Dolezal et al., 2000
); a review is also available (Hudson and Kortt, 1999
).
Recently, Kipriyanov et al. showed that dimeric single-chain diabody molecules with four antigen-binding sites are formed by expressing polypeptides with a middle linker of 12 residues (Kipriyanov et al., 1999). It has been proposed that these so-called tandem diabodies (Tandabs) form by interchain pairing of the four variable domains of each chain. These molecules possess an increased functional affinity and improved pharmacokinetics compared with monomeric scDb (Cochlovius et al., 2000
).
In the present study, we applied phage display to isolate single-chain diabodies with optimized linker sequences. We first demonstrated that single-chain diabody molecules can be displayed on filamentous phage by fusion to gene 3, generating a single-gene encoded scDb-fusion protein. Libraries of scDb molecules containing randomized linker regions were then applied to isolate molecules with linkers optimized in length and sequence. Furthermore, we could show that functional dimeric scDb molecules are formed by reducing the lengths of either the middle linker M or the linkers A and B below a certain threshold.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ß-Galactosidase was purified as described (Kontermann et al., 1997a). CEA was kindly provided by Professor Sedlacek (HMR, Marburg, Germany) and anti-Myc-tag antibody 9E10 by Professor Eilers (IMT, Marburg, Germany). HRP- conjugated anti-M13 antibody was purchased from Amersham-Pharmacia (Freiburg, Germany) and HRP-conjugated anti-mouse IgG antibodies from Sigma (Deisenhofen, Germany).
Oligonucleotides
For LMB2, LMB3, fdSeq1 see Kontermann et al. (Kontermann et al., 1997a). CEAVHSfiBack, 5'-CTC GCG GCC CAG CCG GCC ATG GCC CAG GTG AAA-3'; CEAVLNotFor, 5'-TTC TGC GGC CGC CCG TTT CAG CTC CAG CTT GGT-3'; GalVLSeqBack, 5'-TCC TGG TAC CAA CAG CAC CCA GGC-3'; GalVHSeqBack, 5'-GCG AAA GAT CGC ATA GCA GGG-3'; CEAVHSeqBack, 5'-TGG ATT GAT CCT GAG AAT GGT-3'. Oligonucleotides for constructing the scDb linker M and scDb linker AB library are summarized in Tables I and II
.
|
|
For phage display of single-chain diabody scDb CEAGal, the coding region was isolated from plasmid pAB1 scDb CEAGal (Brüsselbach et al., 1999) digested with SfiI and NotI and cloned into phagemid vector pHEN2 (Krebs et al., 1998
). For display of diabody Db CEAGal, the coding region was isolated from plasmid pAB1 Db CEAGal (Kontermann et al., 1997a
) by digestion with NotI and partial digestion with SfiI and cloning into pHEN2. Positive clones were identified by polymerase chain reaction (PCR) screening with primers LMB2 and LMB3.
Construction of a bispecific scDb linker M phage display library
Plasmid pAB1 scDb CEAGal was digested with SfiI and AscI (fragment 1) or with AscI and NotI (fragment 2). Fragment 1 was PCR amplified with primers CEAVHSfiBack and ML1For, ML3For, ML5For or ML7For. The products were digested with SfiI and AscI. Fragment 2 was PCR amplified with primers CEAVLNotFor and ML1Back, ML3Back, ML5Back or ML7Back and digested with AscI and NotI. PCR products either were ligated by combining all eight fragments in one ligation reaction or the fragments were generated by combining ML1For and ML1Back, ML3For and ML3Back, ML5For and ML5Back or ML7For and ML7Back in four separate ligation reactions. The fragments were cloned into phagemid vector pHEN2 and six transformation reactions were performed using electrocompetent TG1 (Stratagene). Transformants were plated on two large (24x24 cm) LB plates containing 100 µg/ml ampicillin and 1% glucose. Phage were rescued as described (Marks et al., 1991). Diversity of linkers was confirmed by sequence analysis with primer GalVHSeqBack.
Construction of a bispecific scDb linker AB phage display library
Various positive clones isolated from the linker M library were digested with BstEII and SacI and the resulting GalVHlinkerMGalVL fragment was amplified with primers A0Back, A1Back, A2Back, A3Back, A4Back, A5Back or A6Back and B0For, B1For, B2For, B3For, B4For, B5For or B6For in all 49 possible combinations. PCR fragments were pooled and digested with BstEII and SacI and cloned into plasmid pHEN2 scDb CEAGal digested with BstEII and SacI. Diversity of linkers was confirmed by sequence analysis with primers GalVLSeqBack, GalVHSeqBack and CEAVHSeqBack.
Selections
Antigens were coated in 1 ml of PBS on Nunc Maxisorb immunotubes at a concentration of either 10 µg/ml for CEA or 25 µg/ml ß-galactosidase and selections were performed as described (Kontermann, 2001). One selection was performed using CEA in round 1 and ß-galactosidase in round 2 (selections 1a and 2a) and a second selection was performed using ß-galactosidase in round 1 and CEA in round 2 (selections 1b and 2b). Eluted phage were infected into suppressor strain TG1 for production of phage.
Screening and expression of soluble single-chain diabodies
Clones from round 1 and 2 were screened by ELISA for antigen binding of soluble scDb as described (Kontermann, 2001) using CEA and ß-galactosidase as antigens. Alternatively, phage-displayed scDb were produced by rescue with helper phage of single TG1 colonies grown on microtitre plates (Marks et al., 1991
). Clones isolated from the scDb linker M library were sequenced with primer GalVHSeqBack and clones isolated from the scDb linker AB library with primers CEAVHSeqBack and GalVHSeqBack. For soluble expression, scDb fragments were subcloned into pAB1 as SfiI/NotI fragment. Purification of scDb fragments was performed from 3 l of induced TG1 cultures by immobilized metal affinity chromatography (IMAC) as described (Kontermann et al., 1997a
). Purified fragments were dialysed against PBS and the concentrations were determined by spectrophotometry. Purity of fragments was analysed by 10% SDSPAGE under non-reducing conditions and by immunoblotting experiments using an anti-His-tag antibody. Gel filtration of scDb molecules was performed by FPLC on a Superose 12 column (Pharmacia) with a flow-rate of 0.33 ml/min in PBS and loading 200 µl of the purified proteins (corresponding to ~30 µg of protein).
ELISA and recruitment experiments
ELISA and recruitment experiments were performed as described (Kontermann et al., 1997a) using CEA coated in PBS on to Maxisorp microtitre plates (Nunc) at 10 µg/ml. For ELISA, bound proteins were detected with an HRP-conjugated anti-His-tag antibody (Santa Cruz). Recruitment experiments were performed with 50 µg/ml ß-galactosidase and bound enzyme was detected with o-nitrophenyl-ß-D-galactopyranoside (ONPG). The simultaneous binding of two molecules of the same antigen by dimeric single-chain diabodies was analysed in recruitment experiments using horseradish peroxidaseconjugated antigen (CEA, ß-galactosidase) labelled with EZ-Link Plus-activated HRP according to the manufacturer's protocol (Pierce). Antibody molecules at a concentration of 20 µg/ml were preincubated for 1 h with HRP-conjugated antigen at a molar ratio of antibody to antigen of 1:1. The solution was then added to immobilized CEA or ß-galactosidase coated at a concentration of 20 or 50 µg/ml, respectively. Bound antigen was directly detected with 3,3',5,5'-tetramethylbenzidineH2O2 as used for ELISA.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Both phage-displayed single-chain diabody scDb CEAGal and the dimeric diabody Db CEAGal bound with identical efficacy to the two antigens applying equal amounts of phage (Figure 1). Thus, single-chain diabody can be displayed on phage without interfering with antigen binding. However, in recruitment experiments no binding was observed with phage-displayed scDb CEAGal or Db CEAGal using 1012 t.u./ml, in contrast to soluble antibody fragments which efficiently recruited ß-galactosidase to immobilized CEA (not shown) (see also Brüsselbach et al., 1999
). This finding indicates that simultaneous binding of both antigens under the applied experimental conditions is abolished by phage display.
|
A library of scDb fragments containing a randomized middle linker (linker M) of varying length was generated by a two fragment ligation procedure (see Figure 2a for details). For randomization we used triplets with the sequence RVT (R = A or G, V = A, C or G), with the six possible triplets coding for only six amino acids (T, N, S, D, G, A), thus avoiding all hydrophobic and the positively charged amino acids and reducing the total number of possible permutations. The randomized linker M was inserted between glycine 108 of the anti-ß-galactosidase VL
domain and glutamine 1 of the anti-ß-galactosidase VH domain. Based on homology to VL
domains, where arginine or lysine 107 was defined as a C-terminal residue (Malby et al., 1998
; Dolezal et al., 2000
), leucine 107 can be defined as the C-terminus of the VL
domain (P.Hudson, personal communication). We therefore considered glycine 108 as part of the linker sequence. Thus, the combination of the possible linker fragments resulted in middle linkers of 7, 9, 11, 13, 15, 17 and 19 amino acid residues in length. The scDb linker M library with a total diversity of 1.7x106 clones was subjected to two rounds of selections on CEA and ß-galactosidase (see Table III
). Between 95 and 100% of the clones of round 2 recognized both antigens in ELISA. Over 80% of the sequenced clones contained a linker M with a total length of 15 or more residues. The majority (63%) had a linker M of 15 residues (Table III
). Of 11 clones analysed only two had linkers with a length <15 residues (13 and 9 residues, respectively). In contrast, sequencing of randomly picked clones of the unselected library showed that the majority of clones (6/9) had a linker M with a length <15 residues. One clone (M14) possessed an unexpected linker length of 18 residues, which was most likely generated by mispriming at the 5' end of the linker-encoded sequence during PCR. Except for a slight under-representation of serine residues, there was no significant deviation from the statistical usage of the six amino acids (16.7%) in the selected clones.
|
|
The linkers A and B of several scDb molecules (clones M1, M3, M4, M9, M11 and M19; see Table III) isolated from the linker M library were randomized in length and sequence introducing between 0 and 6 amino acids into the two linker regions in all possible combinations and the same degenerated triplets as for the linker M library (Figure 2b
). Serine 112 was defined as the C-terminus of the VH domains and residue 1 as the N-terminus of the VL domains, as suggested earlier (Hudson and Kortt, 1999
) (see Figure 2b
). This linker AB library with a diversity of 2.7x107 was subjected to two rounds of alternating selection on CEA and ß-galactosidase. All clones analysed from round 1 and 2 recognized both antigens in ELISA and 100% of the clones of round 2 were positive. Some differences in length were observed for linkers A and B (Table IV
). The length of linker A was diverse, varying from one to seven amino acid residues. However, except for one clone, all linkers had a length of three or more amino acids. The seven amino acid residue linker of clone AB13 was, as for clone M14, most likely generated by mispriming during PCR, since this length was not originally present in the primers used for cloning. For linker B, all clones had a length of four, five or six amino acid residues. Differences were also observed for the amino acid usage. In linker A, only 3% threonine residues were found, whereas all other amino acids were within the expected range. The three clones with a linker A of one or three amino acids (clones AB16, AB6, AB8) all possessed a linker B of the maximum possible length of six residues. These data led us to suggest that a short linker at one site forces a longer linker at the other site in order to retain correct folding and full antigen binding activity. In order to investigate the assumption further, we generated a library of clones having linker M3 as middle linker and either a linker A of one residue and a linker B of 06 residues (linker A1Bx library) or a linker B of one residue and a linker A of 06 residues (linker AxB1 library). These libraries with diversities of 6x104 and 4x104, respectively, were subjected to one round of selection on CEA or ß-galactosidase. Interestingly, 68% of the clones selected from the A1Bx library with CEA and 13% of the clones selected on ß-galactosidase reacted with both antigens, whereas fewer positive clones were isolated from the AxB1 library (26% from the CEA selection and 0% from the ß-galactosidase selection). All clones isolated from the A1Bx library had a linker B of five or six residues (Table V
). These clones had preferentially (four out of six) an asparatic acid residue in the linker A1. Different results were obtained from the selection of the AxB1 library. Here, the selected linkers A had a length between one and five residues with no observed preference for a certain length. A preference for glycine residues was found for linker A of 13 amino acid residues. Asparagine or aspartic acid residues were mainly found at the B1 linker.
|
|
Similar yields of purified protein were obtained for various clones (wt, M3, M9, M2, M13, AB16, AB3, B129 and B130) containing linkers of different length and composition (Table VI). Thus, the selected linker sequences did not significantly improve the yields of soluble protein. SDSPAGE and immunoblotting experiments confirmed the correct size of the purified scDb polypeptides (Figure 3a and b
). All the purified scDb molecules were active in ELISA with plastic-bound CEA (not shown). Simultaneous binding of both antigens was demonstrated in experiments recruiting ß-galactosidase to CEA by the scDbs and the detection of bound enzyme by substrate conversion (Figure 3c
).
|
|
Wild-type scDb and also clones M3, M9, M2, AB3, AB16 and B130 showed a predominant peak after separation by size exclusion chromatography on a Superose 12 column corresponding in size to monomeric molecules (~55 kDa) (Table VI). In contrast, scDb M13, possessing a nine-residue middle linker and scDb B129, with one-residue linkers A and B, showed two peaks corresponding in size to monomeric and dimeric molecules (Figure 4
). Fractions of monomeric and dimeric scDb contained functional molecules as demonstrated by ELISA and recruitment experiments (not shown). The purified scDb also contained to various extents some low molecular weight contaminants (>20 kDa) with no antigen-binding activity.
|
In order to analyse further the influence of the length of linkers A and B on the transition from monomeric to dimeric molecules, we analysed clones B117, B126 and B119 containing a linker A of two, three or four residues, respectively (see Table V). In addition, we generated two constructs (A0B1D, A0B0) containing a zero-linker A and either a one-residue (Asp) or no-residue linker B. Thus, in construct A0B0 the VH domains are directly linked to the VL domains without the presence of any additional residues. Size exclusion chromatography of purified proteins showed monomeric molecules for B117, B126 and B119 (Figure 4b
; Table VI
). In contrast, A0B1D contained mainly dimeric molecules. Only a small shoulder at the right side of the main peak indicated the presence of some monomeric molecules. The A0B0 preparation did not lead to significant amounts of soluble protein and was therefore not further analysed by size exclusion chromatography.
Recruitment experiments of the fractionated samples of M132 and A0B1D confirmed the presence of active bispecific molecules in the fractions containing dimeric molecules (Figure 4c). These could be clearly separated from the active fractions of M9, which were included as a representative sample of monomeric scDb.
In order to analyse further whether the dimeric scDb molecules exhibit an increased binding to their antigens compared with monomeric scDb, we performed additional recruitment experiments. A titration of antibody molecules showed an identical recruitment efficiency for M9 and M132, whereas that of A0B1D was slightly reduced compared with M9 (Figure 5a). This finding indicates that simultaneous binding of the two identical antigen-binding sites of dimeric scDbs is sterically hindered under the applied assay conditions. We therefore analysed the possibility of the antibody molecules recruiting soluble horseradish peroxidase (HRP)-conjugated antigen to immobilized antigen. This experiment showed that the two dimeric scDbs M132 and A0B1D were able to recruit HRP-conjugated ß-galactosidase to plastic-bound ß-galactosidase, whereas the monomeric scDb M9 did not mediate any recruitment (Figure 5b
). Hence this experiment confirmed the presence of two active ß-galactosidase binding sites in scDb M132 and A0B1D. As expected, recruitment of ß-galactosidase to plastic-bound CEA, i.e. bispecific interaction, was observed for all three constructs. Similar results were found for HRP-conjugated CEA, although some weak recruitment of CEA-HRP to immobilized CEA was also seen with M9 (Figure 5c
).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
One of the selected clones (M13) possessed a middle linker of nine residues. These nine residues can span a maximum distance of ~32 Å, which is probably too short to form an Fv domain from the adjacent variable domains. In accordance with this finding, M13 contained a significant amount of dimeric molecules. Further reduction of linker M to seven residues increased the amount of dimeric molecules. The observed transition from monomeric to dimeric scDb by reduction of the length of linker M from 13 to seven residues resembles the transition from monomeric scFv to bivalent diabodies. This indicates that the core region of dimeric scDb, with a short linker M connecting a VL and a VH domain, may resemble the structure of a bivalent diabody (see Figure 6). Hence the overall structure of these dimeric molecules would be different from that postulated by Kipriyanov et al. (Kipriyanov et al., 1999
). They generated dimeric bispecific single-chain diabodies possessing a middle linker of 12 residues and linkers A and B of nine residues. They presented a model of homodimers (assigned tandem diabodies or Tandabs) with all four variable domains of one chain interacting with the variable domains of the second chain. In this model, two diabody-like structures are connected in a flexible manner by the middle linkers (see Figure 6
). Assuming a diabody-like structure for the core region, it is not possible, however, for the adjacent variable domains to fold with the core domains into a diabody-like structure owing to the occupation of the VHVH interface by this core structure (Peresic et al., 1994
). Thus, in a model with a central diabody-like core structure, the flanking variable domains must fold into a molecule resulting in a yet unknown overall structure. We assigned these molecules linear dimeric single-chain diabodies (LD-scDb).
|
For linkers A and B, which are usually chosen to be 56 amino acid residues for expression of bivalent or bispecific diabodies or single-chain diabodies, we found a preferred length of 36 residues. This is in agreement with various findings obtained with bivalent diabodies which demonstrated that a reduction of these linkers below 23 residues results in the formation of tri- or tetrameric molecules (Atwell et al., 1999; Hudson and Kortt, 1999
; Le Gall et al., 1999
). Again, this was dependent on the antibody fragment used and the orientation of the VH and VL domains (Dolezal et al., 2000
).
Reduction of the length of linkers A and B below two residues results in the formation of dimeric molecules. Since linker M is of sufficient length, the two variable domains of one chain connected by linker M can fold into an antigen-binding site. However, owing to short linkers A and B, the flanking variable domains within one chain cannot assemble into antigen-binding sites leading to interchain pairing of these domains (Figure 6). We assume that the structure of these dimeric single-chain diabodies closely resembles that of tetrameric scFv molecules (tetrabodies) (Hudson and Kortt, 1999
). Hence these molecules are structurally different from the above-described linear dimeric single-chain diabody molecules. We assigned these molecules as circular dimeric single-chain diabodies (CD-scDb). Compared with tetrabodies, which are monospecific homotetramers, CD-scDb are bispecific homodimers. Owing to random association of the four polypeptide chains forming a tetrabody, the generation of bispecific tetrabodies is limited and has not yet been described. Hence the CD-scDb format allows the generation of small bispecific and tetravalent molecules with well-defined antigen-binding sites. It will, of course, also be possible to use this format to generate monospecific tetravalent molecules. As has been shown for various multimeric scFv molecules and also tandem diabodies, such molecules possess an increased functional affinity due to di- or multivalent antigen binding and improved pharmacokinetic properties (Hudson and Kortt, 1999
; Kipriyanov et al., 1999
). No improved functional affinity was observed in our experiments analysing the binding of the scDb molecules to plastic-bound antigen. This indicates that under these conditions only one binding site of the molecules can interact at the same time. However, our experiments analysing the recruitment of soluble HRP-conjugated antigen to immobilized antigen demonstrated that the dimeric scDb molecules possessed two functional and accessible binding sites for each antigen, thus confirming the tetravalent and bispecific nature of these molecules.
Selection of the A1Bx library with a one-residue linker A resulted in the isolation of clones possessing a long linker B. Thus, a short linker A in our scDb molecule promotes the selection of a long linker B. This indicates that short linkers A and B in a monomeric molecule are disfavoured, presumably for steric reasons. Although we could show that polypeptides with short linkers A and B assemble into functional dimeric molecules, scDb which form exclusively dimeric molecules (e.g. M132) were not selected from phage display libraries. This indicates that phage display forces the selection of scDb with linkers which allow monomeric assembly.
The selection of the AxB1 library, with linker B restricted to one residue, did result in the isolation of clones with linkers A having a length of 15 residues. Thus, in contrast to the observation with a one-residue linker A, one-residue linker B does not necessitate a long linker A. This might be explained by the introduction of some structural constrains by a short linker B. For example, it might be possible that a short linker B leads to a tight back-to-back packing of the VH domains, which also brings the two domains connected by linker A into close proximity. Thus linker A can be very short without interfering with the assembly of functional molecules.
It can be envisaged that the use of linkers of different length, with one linker being very short, results in scDb with a bend structure. Indeed, electron microscopy studies by Hudson's group in Australia revealed boomerang-shaped projections of bivalent diabodies. The angle between the two arms ranged from 60 to 180°, with a mean angle of 122° (Lawrence et al., 1998). This variation in the angle between the arms points to a considerable flexibility in the linker region.
Our approach can be applied to identify a set of minimal linker sequences. This is particularly interesting for therapeutic applications to reduce the number of additional amino acids, which might be immunogenic. However, immunogenicity may also be influenced by the amino acid composition of the linkers. We isolated a large variety of different linkers using a minimal set of codons encoding only six different amino acids. Since the linker regions are exposed to the solvent, we focused in this study on small aliphatic, hydrophilic and negatively charged amino acids. In a similar approach, Hennecke et al. generated a library of an anti-fluorescein scFv with sequences at the beginning and end of the linker encoding ß-turns and a randomized middle part of the linker where glycines, prolines and polar and charged amino acids were allowed to occur at certain positions (Hennecke et al., 1998). Selection of this library applying the selectively infective phage (SIP) technology resulted in various non-repetitive sequences with no obvious bias towards certain amino acid residues. These new linkers performed in a manner identical with that of the original (Gly4Ser)3 linker. Similarly, no obvious bias towards a specific linker sequence was observed in our study and the linkers that we selected did not significantly improve the yields of soluble protein. This indicates that the variable domains of scDb CEAGal are responsible for the low expression levels oberserved in our study. Other groups used triplets coding for all 20 amino acids and phage display to isolate new linkers for the generation of scFv fragments. Although one study described that proline and positively charged amino acids were more abundant and negatively charged amino acids disfavoured (Tang et al., 1996
), another study did not result in the isolation of linkers with any amino acid preference or a significant sequence similarity (Turner et al., 1997
). Starting from poorly expressed scFv, in these two studies an improved secretion and solubility of scFv fragments were described compared with the parental scFv fragments containing a (Gly4Ser)3 linker.
Further applications of phage displayed scDb include the generation of naive libraries for the direct selection of bispecific scDb. Such libraries can be either generated by random assembly of all four variable domains or by keeping one antigen-binding site fixed and varying only the variable domains of the second antigen-binding site. For example, bispecific scDb used for the retargeting of cytotoxic T lymphocytes (T.Korn and R.E.Kontermann, unpublished work) could be isolated from a library containing the variable domains directed against CD3. These libraries could be directly used to select correctly folded scDb molecules against a variety of different target cells or molecules.
![]() |
Notes |
---|
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alt, M, Müller,R. and Kontermann,R.E. (1999) FEBS Lett., 454, 9094.[ISI][Medline]
Atwell,J.L., Breheney,K.A., Lawrence,L.J., McCoy,A.J., Kortt,A.A., Hudson,P.J. (1999) Protein Eng., 12, 597604.
Brüsselbach,S., Korn,T., Völkel,T., Müller,R. and Kontermann,R.E. (1999) Tumor Targeting, 4, 115123.[ISI]
Cochlovius,B., Kipriyanov,S.M., Stassar,M.J., Schuhmacher,J., Benner,A., Moldenhauer,G. and Little,M. (2000) Cancer Res., 60, 43364341.
Dolezal,O., Pearce,L.A., Lawrence,L.J., McCoy,A.J., Hudson,P.J. and Kortt,A.A. (2000) Protein Eng., 13, 565574.
Hennecke,F., Krebber,C. and Plückthun,A. (1998) Protein Eng., 11, 405410.[Abstract]
Holliger,P. and Winter,G. (1997) Cancer Immunol. Immunother., 45, 128130.[ISI][Medline]
Holliger,P., Prospero,T.D. and Winter,G. (1993) Proc. Natl Acad. Sci. USA, 90. 64446448.[Abstract]
Holliger,P., Brissinck,J., Williams,R.L., Thielemans,K. and Winter,G. (1996) Protein Eng., 9, 299305.[Abstract]
Holliger,P., Wing,M., Pound,J.D., Bohlen,H. and Winter,G. (1997) Nat. Biotechnol. 15, 632636.[ISI][Medline]
Hudson,P.J. and Kortt,A.A. (1999) J. Immunol. Methods, 231, 177189.[ISI][Medline]
Huston,J.S. et al. (1988) Proc. Natl Acad. Sci. USA, 85, 58795883.[Abstract]
Iliades,P. Kortt,A.A. and Hudson,P.J. (1997) FEBS Lett., 409, 437441.[ISI][Medline]
Kipriyanov,S., Moldenhauer,G., Schumacher,J., Cochlovius,B., von der Lieth,C.-W., Matys,E.R. and Little,M. (1999) J. Mol. Biol., 293, 4156.[ISI][Medline]
Kontermann,R.E. (2001) In Kontermann,R.E. and Dübel,S. (eds), Antibody Engineering, a Laboratory Manual. Springer, Heidelberg, pp. 137148.
Kontermann,R.E. and Müller,R. (1999) J. Immunol. Methods, 226, 179188.[ISI][Medline]
Kontermann,R.E., Martineau,P., Cummings,C.E., Karpas,A., Allen,D., Derbyshire,E. and Winter,G. (1997a) Immunotechnology, 3, 137144.[ISI][Medline]
Kontermann,R.E., Wing,M.G. and Winter,G. (1997b) Nature Biotechnol., 15, 629632.[ISI][Medline]
Kortt,A.A. et al. (1994) Eur. J. Biochem., 221, 151157.[Abstract]
Kortt,A.A. et al. (1997) Protein Eng., 10, 423433.[Abstract]
Krebber,A., Bornhauser,S., Burmester,J., Honegger,A., Willuda,J., Bosshard,H.R. and Plückthun,A. (1997) J. Immunol. Methods, 201, 3555.[ISI][Medline]
Krebs,B., Ackermann,B. and Rose-John,S. (1998) J. Interferon Cytokine Res., 18, 783791.[ISI][Medline]
Lawrence,L.J., Kortt,A.A., Iliades,P., Tulloch,P.A. and Hudson,P.J. (1998) FEBS Lett., 425, 479484.[ISI][Medline]
Le Gall,F., Kipriyanov,S.M., Moldenhauer,G. and Little,M. (1999) FEBS Lett., 453, 164168.[ISI][Medline]
Malby,R.L., McCoy,A.J., Kortt,A.A., Hudson,P.J. and Colman,P.M. (1998) J. Mol. Biol., 279, 901910.[ISI][Medline]
Marks,J.D., Hoogenboom,H.R., Bonnert,T.P., McCafferty J., Griffiths,A.D. and Winter,G. (1991) J. Mol. Biol., 222, 581597.[ISI][Medline]
Pei,X.Y., Holliger,P., Murzin,A.G. and Williams,R.L. (1997) Proc. Natl Acad. Sci. USA, 94, 96379642.
Peresic,O., Webb,P.A., Holliger,P., Winter,G. and Williams,R.L. (1994) Structure, 2, 12171226.[ISI][Medline]
Plückthun,A. and Pack,P. (1997) Immunotechnology, 3, 83105.[ISI][Medline]
Tang,Y., Jiang,N., Parakh,C. and Hilvert,D. (1996) J. Biol. Chem., 271, 1568215686.
Turner,D.J., Ritter,M.A. and George,A.J.T. (1997) J. Immunol. Methods, 205, 4354.[ISI][Medline]
Received February 9, 2001; revised June 26, 2001; accepted July 10, 2001.