- Email: [email protected]
Display of somatostatin-related peptides in the complementarity determining regions of an antibody light chain
Archives of Biochemistry and Biophysics 440 (2005) 148–157 www.elsevier.com/locate/yabbi
Display of somatostatin-related peptides in the complementarity determining regions of an antibody light chain Peter J. Simon a,1, Kevin C. Brogle b, Baiyang Wang a,¤,2, Donald J. Kyle b, Daniel A. Soltis a a
b
Department of Immunotherapeutics, Purdue Pharma L.P., 6 Cedar Brook Drive, Cranbury, NJ 08512, USA Department of Computational, Combinatorial and Medicinal Chemistry, Purdue Pharma L.P., 6 Cedar Brook Drive, Cranbury, NJ 08512, USA Received 25 March 2005, and in revised form 14 June 2005 Available online 11 July 2005
Abstract Peptide display in antibody complementarity determining regions (CDRs) oVers several advantages over other peptide display systems including the potential to graft heterologous peptide sequences into multiple positions in the same backbone molecule. Despite the presence of six CDRs in an antibody variable domain, the majority of insertions reported have been made in heavy chain CDR3 (h-CDR3) which may be explained in part by the highly variable length and sequence diversity found in h-CDR3 in native antibodies. The ability to graft peptide sequences into CDRs is restricted by amino acids in these loops that make structural contacts to framework regions or are oriented towards the hydrophobic interior and are important for the proper folding of the antibody. To identify such positions in human -light chain CDR1 (-CDR1) and CDR2 (-CDR2), we performed alignments of 1330 -light chain variable region amino acid sequences and 19 variable region X-ray crystal structures. From analyses of these alignments, we predict insertion points where sequences can be grafted into -CDR1 and -CDR2 to prepare synthetic antibody molecules. We then tested these predictions by inserting somatostatin and somatostatin-related sequences into -CDR1 and -CDR2, and analyzing the expression and ability of the modiWed antibodies to bind to membranes containing somatostatin receptor 5. These results expand the repertoire of CDRs that can be used for the display of heterologous peptides in the CDRs of antibodies. 2005 Elsevier Inc. All rights reserved. Keywords: Synthetic antibody; Complementarity determining region; Somatostatin; Protein; Peptide; Display; ScaVold; Alignment; Receptor; Expression
The ability to insert biologically active peptide sequences and peptide libraries into the surface exposed loops of protein frameworks has been a signiWcant advance for engineering molecules with novel catalytic or binding properties [1,2]. Several scaVold
*
Corresponding author. Fax: +1 610 313 4039. E-mail address: [email protected] (B. Wang). 1 Present address: Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey, 195 Little Albany Street, New Brunswick, NJ 08903-2681, USA. 2 Present address: BioRexis Pharmaceutical Corp., 3400 Horizon Drive, King of Prussia, PA 19406, USA. 0003-9861/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2005.06.009
proteins have been evaluated including antibodies [3,4], lipocalins [5], CTLA-4 [6], and Wbronectin [7] that have enabled the construction of novel proteins that address some of the potential shortcomings of the native peptides including low binding aYnity, inadequate speciWcity or undesirable pharmacokinetic properties [4,8,9]. A key feature of these protein scaVolds is an ordered structure that stably displays surface exposed peptide loops. Antibodies are the prototypical scaVolds, presenting a wide array of amino acid sequences on a structured framework that is formed by the organized arrangement of -strands. Connecting these strands are six loops known as complementarity determining
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regions (CDRs)3 that show a high level of sequence diversity and form a binding surface for antigen interaction [10,11]. The insertion of peptide sequences into the CDR loops of full-length antibodies that maintain the basic antibody conformation has been performed previously. Sallazzo et al. [3] Wrst reported the expression of an antibody with a peptide epitope inserted in h-CDR3 and showed that the inserted peptide was recognized by a monoclonal antibody that bound to the same epitope in the native protein. In other experiments, synthetic antibodies containing adhesion molecule binding sequences (RGD) grafted in h-CDR3 have been shown to display the inserted peptide ligand in a functional conformation and bind to puriWed integrins and tumor cells whose adhesion to extracellular matrix is RGD dependent. These modiWed antibodies have also been shown to block cell adhesion to Wbronectin and inhibit natural killer cell mediated cytotoxicity [4,12–14]. In these and other experiments using the CDRs of antibodies for the functional display of peptide ligands, the majority of peptide grafts were made in h-CDR3 because of its diverse size range (up to 30 aa) and high sequence variability [15,16]. Even though the CDRs are surface exposed loops, and insertions and deletions of residues are a normal feature of the hypermutation process [17,18], not all CDRs exhibit the same length distribution. -CDR2 is 7 aa long in 99.9% of the sequences in the Kabat database whereas the lengths of the other Wve CDRs vary to a much greater extent ([19], unpublished results). Although the insertion of heterologous peptides has been previously reported for several of the CDRs [3,4,20–22], it is unclear whether peptide insertions that alter the length of the CDR can be made successfully in -CDR2. Expanding the repertoire of CDRs where peptide ligands can be grafted without signiWcantly diminishing the expression of the synthetic antibody would allow for peptide display in CDRs that may impose diVerent structural constraints. Although there are multiple deWnitions of CDRs based on the sequence diversity within the heavy and light chains [19,23], the structural loops between the -strands of the variable region framework [24,25], or the ability of each amino acid to contact antigen [26], none of these deWnitions were developed with the purpose of identifying positions within the CDRs where heterologous peptides could be inserted without disrupting the folding and stability of the modiWed antibody. As previous studies have shown, there are amino acid positions in the 3 Abbreviations used: aa, amino acids; CDR, complementarity determining region; CTLA-4, cytotoxic T-lymphocyte associated protein-4; ELISA, enzyme-linked immunosorbent assay; h-CDR, antibody heavy chain CDR; -CDR, antibody -light chain CDR; kDa, kiloDalton; SST-14, somatostatin; SSTR5, somatostatin receptor 5.
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CDRs where the sequence variability is very limited, that make contact with portions of the variable region framework or have side chains buried in the hydrophobic core [24,27–30]. It is likely that changing these residues would have a signiWcant impact on the folding and stability of the antibody as previously demonstrated [31–34]. In this report, we extend the structural analysis of the individual amino acid positions in the immunoglobulin variable domains focusing on the positions in the CDRs of the -light chain. Based on these analyses, we identiWed amino acid positions within -CDR1 and -CDR2 that do not appear to be involved in the folding and stability of the variable domain. We then prepared modiWed immunoglobulin molecules in which somatostatinrelated peptides were inserted into either -CDR1 or -CDR2. Insertions that did not disrupt residues involved with protein folding or stability were eYciently expressed and capable of binding membranes containing somatostatin receptor 5 (SSTR5).
Materials and methods Antibody sequence and structure alignment One thousand three hundred and thirty human -light chains were downloaded from the Kabat antibody database ([16], http://immuno.bme.nwu.edu) and aligned using the Wisconsin Package (Accelrys, San Diego). As PileUp is limited by the number of sequences it can process, an alignment was built from a randomly selected subset of 476 -light chain sequences. From this alignment, a Hidden Markov Model proWle was calculated using HmmerBuild. This proWle was used with HmmerAlign to align the remaining sequences. Finally, extraneous gaps created by HmmerAlign due to the sequence-variable CDRs were removed, and insertions in CDRs were Wlled from the left. Unique human antibody structures were downloaded from the Protein Data Bank [35]. Nineteen structures contained heavy chain and -light chain variable sequences. Using InsightII (Accelrys, San Diego), water and other molecules were removed and the variable regions from the antibodies were separated. The heavy chain variable and -variable regions were then separately aligned in three dimensions to compare side chain locations and interactions. Every position that Kabat and Chothia designated as occurring within a CDR [19,23–25] was evaluated for surface accessibility and participation in hydrogen bonds, salt-bridges, or hydrophobic interactions. The Protein Data Bank ID numbers for the structures used in this analysis are: 1AD0, 1AD9, 1BEY, 1BRE, 1DEE, 1DFB, 1DQL, 1FH5, 1GC1, 1IGM, 1LVE, 1OBE, 1QLR, 1QP1, 1REI, 1VGE, 1WTL, 2IMM, and 1HOU.
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Control antibody Control variable region sequences were derived from the frequency analysis of human light and heavy chains. The binding speciWcity of the control antibody is unknown. The amino acid sequence of the -light chain variable region is DIQMTQSPSSLSASVGDRVTIT CRASQSISNYLAWYQQKPGKAPKLLIYAASSLES GVPSRFSGSGSGTRFTLTISSLQPEDFATYYCQQ YNSLPWTFGQGTKVEI. The IgG heavy chain variable region amino acid sequence is QVQLVQSGAEVKKPGASVKVSCKAS GYTFTSYAISWNWVRQAPGQGLEWMGWINGN GDTNYAQKFQGRVTITADTSTSTAYMELSSLRS EDTAVYYCARAPGYGSDYWGQGTLVTVS. Antibody modiWcation DNA fragments encoding the peptide grafts in -CDR1 and -CDR2 were constructed by PCR using the method of gene splicing by overlap extension (reviewed in [36]). Primers were designed with two sections: the 3⬘ ends are complementary to the variable region sequence and the 5⬘ ends contain the ligand sequence to be inserted. A pUC19 vector containing the variable region of the -light chain was used as a template. For each construct, two PCRs were performed (one to amplify the 5⬘ end of the variable region to the CDR being modiWed, the other to amplify the CDR to the 3⬘ end). Products were gel puriWed and combined in a second PCR with M13 forward and reverse primers to amplify the full-length construct. After gel puriWcation, the fragments were digested and cloned into pUC19. The sequences of the constructs were then conWrmed by DNA sequencing and the variable regions cloned into the appropriate expression vectors containing either human heavy or light chain constant regions (1 and , obtained from S. Morrison, UCLA). The expression vectors used were the GS Gene Expression System plasmids pEE6.1 for the heavy chain and pEE14.1 for the light chain (Lonza Biologics, Slough, UK). Cell culture and transfections CHO-K1 cells selected to grow in suspension were cultured in serum-free ProCHO4 (Bio Whittaker) medium supplemented with L-glutamine (4 mM; Invitrogen). Vectors containing the cloned antibody genes were transfected into cells using CLONfectin (Clontech). For a 2 mL (Wnal volume) reaction, 250 ng of plasmid DNA was transfected at a 3:1 (weight/weight) light/heavy chain ratio with 4 g CLONfectin into 2.5 £ 106 cells. Transfected cells were incubated in ProCHO4 with growth supplements (JRH Biosciences) for 4 h before addition of an equal volume of ProCHO4 with 1£ growth supplements, 10% low IgG FBS (Invitrogen), penicillin (200 U/mL;
Invitrogen), and streptomycin (200 g/mL; Invitrogen). Transfections were scaled up to obtain suYcient material for binding assays. The control and modiWed antibodies were puriWed from Wltered cell culture supernatant on a protein G column (Amersham Biosciences), dialyzed, and concentrated using an Ultrafree-4 Biomax 10 kDa MWCO centrifugal Wlter unit (Millipore). Antibody quantitation Cell culture supernatants were collected at day 7, Wltered, and measured for assembled antibody concentration by sandwich ELISA using plate bound anti-human IgG antibody and anti-human -HRP conjugated antibody (Southern Biotechnology). Known concentrations of human IgG (Sigma) were used to generate a standard curve. Color generation from TMB peroxidase substrate (KPL) was measured on a Molecular Devices Vmax kinetic microplate reader. Data were collected and analyzed by SOFTmax PRO v3.0. The media from each transfection were assayed in triplicate at two dilutions in the linear range of the standard curve. Assembled antibody concentration was multiplied by the volume of recovered cell culture supernatant. All data are normalized to the expression level of the control antibody performed as a standard in each experiment. Receptor binding assays The aYnity of the peptide, SST-14 (Bachem), control antibody, and somatostatin-derived synthetic antibody constructs were tested using membranes derived from HEK-293 EBNA cells transfected with human SSTR5 (Perkin-Elmer) following the manufacturer’s protocol. BrieXy, membranes were incubated with ligand (SST-14, modiWed antibody or control antibody) and labeled compound [125I]Tyr11 SST-14 (Perkin-Elmer) for 1 h at room temperature. Reactions were Wltered and washed with ice-cold wash buVer (50 mM Tris, pH 7.5, 0.2% BSA). After drying at 50 °C, OptiPhase Supermix scintillation cocktail (Wallac) was added and plates were measured for 1 min/well in a Trilux 1450 MicroBeta liquid scintillation and luminescence counter (Wallac). Data were plotted and Ki values were calculated using the sigmoidal curve Wtting function in GraphPad Prism 3.0.
Results Analysis of -light chain sequences and structures identiWes optimal locations for peptide insertion in -CDR1 and -CDR2 Although the CDRs of antibodies have been deWned by others as regions with high levels of sequence diversity [19,23], not all of the amino acids in these loops are
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highly variable and some make contacts with the antibody framework [27,29,30]. We therefore sought to identify residues in the -light chain CDRs that did not appear to be involved in maintaining structural integrity and thus may be amenable to modiWcation. To do this, 1330 human -light chain antibody sequences from the Kabat database [16] were aligned and every position in the variable region was analyzed for amino acid variability. CDR positions with a single residue or two stringently conserved residues (i.e., S/T, V/I, V/L, I/L or Y/F) occurring >80% were identiWed (Fig. 1). To ascertain if there was a structural role for these positions, an alignment of 19 human -light chain variable domain X-ray crystal structures was examined in detail. SpeciWcally, each side chain was analyzed for surface accessibility, residue orientation and involvement in hydrogen bonds, salt-bridges or hydrophobic interactions either between
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Table 1 Summary of structure analysis Position
Interactions
24
Side chain faces away from combining site. In 2 out of 19 structures, position 24 forms a salt-bridge with position 70 Side chain towards interior of protein H-bond with position 3 Hydrophobic interior Hydrophobic interior No direct salt-bridge seen between Arg at 54 and Asp/Ser at position 60. When Leu is present, it is part of the hydrophobic interior
25 26 29 33 54
the -sheets or chains. The amino acids in -CDR1 and -CDR2 identiWed by the structural analysis are shown in Table 1, and a representative crystal structure is shown in Fig. 2. All residue numbering is based on the deWnition of Chothia et al. [24,25]. Although some residues were identiWed by both methods (24, 26, 29, and 33 in -CDR1), other positions were identiWed in only one of the screens (positions 25 and 27 in -CDR1, and 52, 54, and 56 in -CDR2; Fig. 1). Similar observations have been made in other frequency and structural analyses [26,28–30,37,38]. For example, the CDR1 residues 25, 29, and 33, identiWed by Chothia and Lesk [24] and shown to be buried in the core, determine the canonical structure of the CDR. Our structural analysis of -CDR1 also identiWed the hydrogen bonding of residue 26 to the backbone –NH of position 3 [39], and the orientation of the side chain of amino acid 24 away from the combining site [26,40]. In -CDR2, a leucine residue present at position 54 appeared to be part of the hydrophobic core. Based on these analyses, we predict that the preferred positions for insertions in -CDR1 are after residue 29 and before residue 33, and in -CDR2, any insertion that maintains the amino acid at position 54. Selection of somatostatin as a ligand for insertion into light chain CDRs
Fig. 1. Variability of -light chain CDR1 and CDR2. One thousand three hundred and thirty -light chain sequences were aligned then adjusted by hand to left-justify insertion regions. The frequencies of amino acid(s) are displayed for each position (amino acids present at least 5% of the time are shown) of the Chothia and Kabat deWned CDR1 (A) and -CDR2 (B). Amino acids are presented in single letter code; (–), an amino acid not inserted at that location; 䊉, positions identiWed in the variability analysis; 䊐, residues that are part of the hydrophobic core or possessing contacts with other amino acids. Sequence grafting is best performed at the amino acids identiWed by the solid line. It may be possible to replace the entire -CDR2 sequence when grafting peptides (dashed line extension). Numbering is according to the Chothia convention [24,25].
To test our predictions of the positions in -CDR1 and -CDR2 where heterologous peptides may be successfully inserted, we selected the small peptide hormone somatostatin (amino acid sequence AGCKNFFWKTF TSC) as a model ligand. Somatostatin is a cyclic peptide which may be an advantage for these studies since many of the CDRs have a looped conformation. In fact, development of many somatostatin agonists demonstrates the importance of the FWKT motif in the context of a looped structure. The speciWc size of the loop does not seem to be critical, as agonists with loops as small as 6 aa (e.g., octreotide and lanreotide) have been identiWed (reviewed in [41–43]). In addition, since we are inserting the peptide sequence into the CDR of an antibody, it is also important that somatostatin contains no post-translational modiWcations such as phosphorylation,
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Fig. 2. Structural analysis of -light chain CDR1 and CDR2. The X-ray crystal structure of 2IMM, a representative variable region, is provided with relevant side chains displayed [55]. Blue ribbon, -CDR1; red ribbon, -CDR2; white ribbon, -CDR3. (A) Relevant -CDR1 residues are highlighted; yellow, side chains not facing combining site; gray, hydrophobic interior; red, hydrogen bonded residues. (B) Relevant -CDR2 residues shown. 2IMM has a -CDR1 sequence of KSSQSLLNSGNQKNFLA (length 17 aa), showing the insertion of 6 aa between positions 30 and 31. The sequence of 2IMM -CDR2 is GASTRES (length 7 aa).
adenylation or glycosylation and that the amino- and carboxyl-termini are not required for binding or function. -CDR1-modiWed antibodies with properly positioned insertions are eYciently expressed To test our hypothesis that disrupting residues involved in variable domain folding or stability would reduce antibody secretion, we inserted into -CDR1 a somatostatin-related peptide previously identiWed in a phage display experiment. This peptide, CRFWKTWC, was selected from a library of peptides consisting of six random amino acids Xanked by cysteine residues and shown to bind to somatostatin receptors [44]. We main-
tained -CDR1 length and replaced 8 aa from either the N- or C-terminal portion of the CDR with the amino acid sequence CRFWKTWC to create 24–31 and 27– 34, respectively (see Fig. 3 legend for nomenclature). The constructs were created by modiWcation of a control antibody that was designed to have no known antigen interaction. The sequence of the control antibody was derived from a frequency and linkage analysis of the presence of speciWc amino acid residues at every position of the antibody variable region sequence and attempts to include the more common amino acids at each position while maintaining apparent linkages to residues at other positions of the molecule (data not shown). Expression levels of the control antibody in our system (»4000 g/L)
Fig. 3. Sequence of -CDR1 and -CDR2 regions in control antibody and synthetic antibodies with somatostatin-related grafts. The sequence of CDR1 and -CDR2 in the control antibody is shown. Below are the names and sequences of the synthetic antibodies used in this study. The length of the CDR and change in size is provided in columns to the right of each sequence. Underlined residues are components of the hydrophobic interior or residues with internal contacts, as identiWed through frequency and structural analyses. The nomenclature is as follows: , phage display derived sequence (CRFWKTWC); S, somatostatin inner-loop sequence (KNFFWKTFTS); SC+, somatostatin amino acids 3–14 followed by an aspartic acid (CKNFFWKTFTSCD); SC¡, SC+ sequence with Wrst and last cysteine modiWed to G and A (GKNFFWKTFTSAD); the number in the construct name indicates the amino acid(s) removed when the ligand was grafted; ¤ indicates no sequence removed, with the insertion made after the position indicated. The boxed amino acids refer to the ligand sequence in the modiWed CDR.
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and 33 are important for expression. The threefold reduction in expression observed between 27–31 and 29* (Fig. 4) may be due to the absence of the isoleucine at position 29 that is normally buried in the hydrophobic core of the variable domain. It is also possible, however, that the poor expression of constructs 24–31 and 27–34 may be due to the formation of disulWde bonds within the inserted sequence that may alter antibody structure and prevent eYcient assembly and/or secretion. To test this hypothesis, we inserted KNFFWKTFTS, the sequence found between the two cysteines of the natural ligand SST-14, into CDR1. Two synthetic antibodies were made, S24–34 and S24–33 (S, somatostatin inner-loop sequence). The former replaces the entire -CDR1 with the inner-loop sequence (decreasing the CDR length by 1 aa) while the latter preserves the CDR length by maintaining the alanine at position 34 (Fig. 3). Similar to the results obtained above, modiWcation of residues identiWed as part of the hydrophobic interior or having framework contacts results in diminished expression levels (1 and 6% of control). However, when this sequence is used to replace aa 30–32 (the region predicted to tolerate peptide grafts), expression of the resulting construct (S30–32) is 50% of control, equivalent to 29*. These results suggest that the removal of internal contact residues, and not the presence of the cysteines, caused the reduction in expression found with 24–31 and 27–34. Thus, synthetic antibodies with peptide sequences grafted into -CDR1 in locations that do not disrupt amino acids involved in protein folding or stability can be eYciently expressed.
are similar to those of mouse monoclonal and chimeric antibodies expressed under similar conditions. Plasmids encoding the control or modiWed antibody were transfected into CHO-K1 cells selected to grow in suspension culture. Cell culture supernatants were assayed for assembled antibody in a sandwich ELISA and normalized to the expression levels measured from cells transfected with constructs expressing the control antibody. The data in Fig. 4 show synthetic antibodies 24–31 and 27–34 that are secreted at less than 2% of the control antibody. As these constructs modiWed amino acids that are part of the hydrophobic core (residues 29 and 33) or contact other portions of the protein (residue 26), the poor expression would indicate the importance of these residues in -CDR1 for proper antibody folding and secretion. To further test this hypothesis, CRFWKTWC was grafted after residue 29 (with no removal of sequence) to create an 8 aa insertion. Cells expressing this construct (29*) secreted signiWcantly more assembled antibody (»2000 g/L) than 24–31 and 27–34, indicating that the synthetic antibody containing the inserted sequence CRFWKTWC, when positioned in -CDR1 to avoid disrupting residues involved in maintaining proper antibody folding, can be eYciently expressed. The low levels of expression seen when the N- or Cterminal 3 aa of -CDR1 are removed (24–31 and 27– 34) suggests the importance of these residues (presumably 24–26 and 33, identiWed above) for proper folding and secretion. Therefore, 27–31, which maintains the 3 aa on the N- and C-termini of -CDR1 was created. Its expression is at least ninefold higher than 24–31 and 27–34 (17% compared to 1.0 and 1.8%, respectively; Fig. 4), suggesting that one or more of the residues 24–26
Insertions in -CDR2 do not signiWcantly reduce antibody expression
Fig. 4. Expression of synthetic antibodies containing somatostatinrelated sequence. Expression levels of synthetic antibodies are shown normalized to the control antibody. The concentration of assembled antibody secreted from transiently transfected CHO-K1 cells was determined by sandwich ELISA corrected for evaporation. Transfections were performed in triplicate in each of three independent experiments. Expression levels were measured in triplicate at two dilutions within the linear range of the assay. The average of all data points is shown; error bars indicate standard deviation. The expression level of the control antibody was determined to be 4000 g/L.
We next examined whether a CDR that is less often involved in antigen contact [26,45,46] and has a constant size could also be modiWed. -CDR2 was chosen, as its length is 7 aa in over 99.9% of antibody sequences in the Kabat database ([19], unpublished data). Since leucine is present at position 54 in the control antibody sequence and the structural alignment suggests this may be part of the hydrophobic interior, residues 54–56 were maintained and 50–53 were replaced with the SST-14 innerloop sequence KNFFWKTFTS (note, the serine at position 53 matches the Wnal residue of the ligand). S50– 53 expressed at levels t40% of the control antibody (Fig. 4). Construct S50–56, that replaces the entire CDR2 sequence, was expressed at a similar level (50% of control) as S50–53. This suggests that any structural interactions present at position 54 may be modiWed with minimal impact on protein stability. Alternatively, the phenylalanine in position 8 of the inserted peptide may serve as a mimic of the hydrophobic leucine found at position 54 of the control antibody sequence. In either case, these data show that modiWed antibodies, and
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Fig. 5. Binding of synthetic antibodies to membranes containing SSTR5. The ability of synthetic antibodies containing somatostatinrelated sequence to compete with a radioligand for somatostatin receptor 5 membranes is shown. Increasing concentrations of competitor (SST-14, control or synthetic antibody) were mixed with SSTR5 membranes. [125I]Tyr11 SST-14 was added, and after 1 h the membranes were washed to remove unbound radioligand. The amount of radioligand bound to membranes was measured in a scintillation counter. Plotted values represent means § SD of triplicate reactions. Data from a single representative experiment are shown. The log molar concentration is shown on the x-axis, decays per minute on the y-axis.
particularly those where the length of the normally highly size restricted -CDR2 has been increased, can be eYciently expressed in CHO-K1 cells. These results are not unique to somatostatin, as synthetic antibodies with other peptide ligands grafted into -CDR2 (with lengths up to 47 aa)4 are capable of being expressed at levels >1000 g/L. Synthetic antibody molecules containing somatostatinrelated peptides bind to membranes containing somatostatin receptor 5 For applications where the inserted peptide is a ligand that normally binds to a receptor, the modiWed antibody must present the ligand in a biologically active conformation to interact eVectively with its relevant target. Therefore, we measured the ability of selected constructs to bind membranes containing SSTR5 in a competition assay using constant levels of the natural ligand labeled with 125I ([125I]Tyr11 SST-14; Fig. 5). In these experiments, the control peptide SST-14 shows binding to the membranes with an average Ki »6 nM, similar to the reported Ki of 1.4 nM [42]. As expected, the control antibody, devoid of somatostatin sequence, shows no detectable binding to the membranes. All of the synthetic antibodies containing somatostatin-related sequence tested can compete with the radiolabeled ligand and show binding to the membranes containing SSTR5, albeit to diVerent degrees. S30–32 has weak aYnity and is unable to fully compete away the radioligand even at high concentrations. 29*, another 4
Earl Albone, personal communication.
Fig. 6. Synthetic antibodies containing cysteine residues have improved binding proWles for membranes containing SSTR5. The aYnity assays of SC+30 and SC¡30 and natural ligand SST-14 are shown. See legend to Fig. 5.
-CDR1-modiWed antibody, has similar (but reproducibly slightly higher) aYnity for membranes containing SSTR5 as S30–32. In this experiment, S50–56 shows the strongest binding to SSTR5-containing membranes of the three synthetic antibodies tested. It should be noted, however, that the binding of these constructs to the SSTR5-containing membranes may be inXuenced by the presence of two light chain-displayed ligands that may enhance the binding aYnity by introducing an avidity component. Consequently, the actual binding aYnity of an individual, modiWed CDR may be less than what is observed in these experiments. In an attempt to enhance the binding of 29* to the membranes containing SSTR5, we restored the cysteines from SST-14 with the expectation that this may further constrain the somatostatin peptide structure and more accurately mimic the structure of the natural ligand. SC+30 (SC+, somatostatin sequence including cysteines) was made by replacing position 30 of -CDR1 with amino acids 3–14 of SST-14 (CKNFFWKTFTSC) and inserting an aspartic acid after the second cysteine to mimic an exposed carboxyl terminus (Fig. 3). As a control, SC¡30 maintains the aspartic acid, but modiWes both cysteines to prevent the somatostatin ligand from forming an internal disulWde bond or a disulWde bond to other portions of the antibody structure. Constructs SC+30 and SC¡30 were eYciently expressed (60 and 65% of control, respectively; Fig. 4) and tested for their ability to bind membranes containing SSTR5 (Fig. 6). In these experiments, SST-14 exhibited a Ki of 1.2 nM for the membranes. The results show that SC+30 has a slightly improved binding proWle compared to SC¡30. As these constructs diVer in their ability to form a disulWde bond within the grafted sequence, the results may reXect the importance of mimicking the structure of the natural ligand. In the case of SC+30, the cysteine residues in the inserted somatostatin sequence may be capable of forming a disulWde bond, constrain-
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ing the peptide in a cyclic structure similar to the natural SST-14 structure.
Discussion We sought to determine whether we could expand the repertoire of CDR usage for the display of bioactive peptide sequences. Studies from other investigators have shown that heterologous peptides can be inserted into several diVerent protein scaVolds and that the inserted peptides can retain their biological function. However, where antibodies have been used as the scaVold, most of these studies describe constructs where the heterologous peptides were inserted into h-CDR3. Since some amino acids in the framework contribute to a limited set of canonical structures for each CDR [47], identifying positions in each CDR where insertions are tolerated should enhance the likelihood that a synthetic antibody could be prepared where the inserted peptide adopts a bioactive conformation. By identifying and maintaining residues involved in folding and structural stability when grafting somatostatin-related sequences into -CDR1 and -CDR2, we have obtained eYciently expressed synthetic antibodies capable of binding to membranes containing SSTR5. We initially evaluated the eVects of grafting amino acid sequences into antibody CDRs by monitoring their secretion as assembled proteins from transiently transfected CHO-K1 cells. This analysis allowed us to identify sites for grafting peptide ligands into CDRs while still maintaining reasonable (>500 g/L) expression levels. In these studies, grafting of somatostatin-related peptides into the insertion points predicted by our sequence and structure alignment analyses resulted in expression from transiently transfected CHO-K1 cells at levels >1600 g/ L. In -CDR1, the peptide insertions tested were tolerated when localized after residue 29 and before residue 33. Notably, when -CDR1 size variation naturally occurs, this is the region that accommodates the additional sequence by bulging away from the surface. For CDR2, synthetic antibodies containing peptide grafts that were inserted after residue 49 and before residue 54 or those that replaced the entire CDR were also eYciently expressed. Our prediction that the insertion of peptide sequences in -CDR1 would be successful when grafted after position 29 and before position 33 is supported by the eYcient expression of 29*, S30–32, SC+30, and SC¡30. When Chothia/Kabat deWnitions of -CDR1 were used to determine sequence graft sites (24–31, 27–34, 27– 31, S24–34, and S24–33), assembled antibody, as assayed by sandwich ELISA (Fig. 4), was secreted 16- to 100-fold lower than control antibody. The reason for this decreased production is not known. A Western blot analysis of these cell culture supernatants shows light
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chain monomer and dimer, and fully assembled synthetic antibody are secreted in similar proportions to the eYciently expressed -CDR1-modiWed antibodies with grafts made after position 29 and before position 33 (data not shown). Light chain assembly with heavy chain is a prerequisite to antibody secretion, and although some residues may be positioned near the VH/VL interface [48], the -CDR1 residues that were modiWed in the constructs presented here (or the amino acids they contact) are not known to be involved in binding heavy chain [33,49]. Alternatively, the reduced expression of constructs 24–31, 27–34, 27–31, S24–34, and S24–33 could reXect a defect in light chain folding. The hsp70 chaperone BiP binds unfolded and unassembled light and heavy chains prior to their assembly [50,51]. Blond-Elguindi et al. [52] devised an algorithm for predicting peptide binding to the 7 aa long BiP binding cleft [53]. The program calculates a BiP score, with higher scores correlated to increases in the probability of BiP binding. Davis et al. [54] found the 7 aa peptide corresponding to positions 31–37 of the -light chain was able to bind BiP, suggesting this stretch of sequence may be a binding site in the light chain. Although it can be proposed that an altered BiP binding site may account for the decreased expression of some constructs, we have found no correlation between BiP score and expression of our modiWed antibodies (data not shown). Therefore, although light chain folding may be aVected in these -CDR1-modiWed antibodies, their diminished expression does not appear to correlate with reductions in BiP score. Peptide grafts in -CDR2 that replaced the entire CDR did not signiWcantly decrease expression levels even though a leucine at position 54 is part of the hydrophobic interior (Table 1, Fig. 2). Its modiWcation in S50– 56 did not signiWcantly decrease expression levels, suggesting its contribution to antibody stability may be minimal. Alternatively, an argument can be made whereby the last phenylalanine of the inserted sequence (KNFFWKTFTS) in S50–56 replaces the function of the leucine at position 54, maintaining the presence of a hydrophobic residue in the core. This model raises an intriguing possibility where some ligands may be able to mimic or overlap the sequence already present in the CDR. Further work is needed to distinguish these models. In our experiments, insertions of peptide sequence that increased -CDR2 length from 7 to 10 or 13 aa did not have a signiWcant aVect on secretion of full-length assembled antibody. Although the -CDR2 length is almost always 7 aa in germline V genes and functionally rearranged cDNA molecules that have been sequenced and submitted to the Kabat database, this restriction does not appear to prevent the successful insertion of peptide grafts. The lack of -CDR2 length variability observed in naturally occurring antibodies may reXect a
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decreased selection for somatic hypermutations in this region due to the limited contribution of residues in -CDR2 to antigen binding [26,45,46]. The presentation of a ligand in its biologically active conformation is important for binding to its receptor. The data presented in Fig. 6 show SC+30, displaying a somatostatin ligand with two cysteines, binds slightly stronger to SSTR5 membranes than SC¡30, which is the same size, but can no longer form a disulWde bond within the inserted somatostatin sequence. This slight increase in binding aYnity may result from the formation of a disulWde bond between the two cysteines in SC+30 that are not present in SC¡30 and constrain the somatostatin sequence in a cyclic conformation that is more similar to the natural ligand. One should note that the binding of synthetic antibodies to membranes containing SSTR5 presented here may not be universal across all SSTRs. In experiments performed by Nuttall et al., somatostatin was grafted into the loop of human cytotoxic T-lymphocyte associated protein-4 (CTLA-4) that is equivalent to CDR3 of an antibody variable domain. Phage displaying the modiWed CTLA-4 fused to the pIII protein was able to speciWcally bind SSTR4 containing membranes. Partial binding to membranes containing SSTR5 was also observed, but no binding was detected with membranes containing SSTR1, SSTR2 or SSTR3 [6]. It is likely that ligand-independent sequences interact with the extracellular surface of the receptor and aVect binding, a feature that could be useful in designing or selecting receptor speciWc synthetic antibodies. We attempted to examine the binding proWle of our constructs with other SSTRs (including membranes containing SSTR4 and those from the AtT-20 cell line which express multiple SSTRs), however, these studies were hampered by the poor binding of the natural ligand SST-14 (data not shown). Our work provides evidence for the eYcient expression of full-length antibody sequences with peptide insertions in CDRs that have not been extensively exploited previously for this use. When the peptide grafts do not interfere with amino acids involved in maintaining the proper folding and stability of the antibody variable domain, the resulting constructs are eYciently expressed from transiently transfected CHO-K1 cells and bind in a ligand-speciWc manner to the cognate receptor. The insertion of peptide ligands in multiple CDRs could also increase the binding aYnity of the modiWed antibodies displaying a single ligand species, or, when two or more distinct ligands are used, may target several molecules on one cell and could juxtapose proteins for tailored receptor co-activation. The preparation of these synthetic antibodies also may provide an approach for improving the pharmacokinetic properties of the inserted peptides which often have short half-lives in circulation that limit their potential utility as therapeutic agents [8,9]. In addition, by using the antibody
molecule as the scaVold for peptide display, one also may be able to direct immune eVector functions against targeted cells for the treatment of diVerent diseases including cancer.
Acknowledgments The authors thank Drs. Namit Ghildyal, Andrew Gordon, and Jamshid Arjomand for their critical review of the manuscript and Dr. Rajiv Shukla for cloning the 29* expression plasmid.
References [1] [2] [3] [4] [5] [6] [7]
[8] [9] [10] [11] [12]
[13] [14] [15] [16] [17] [18] [19]
[20] [21] [22] [23] [24] [25] [26] [27] [28] [29]
A. Skerra, P.A. Nygren, J. Immunol. Methods 290 (2004) 3–28. A. Skerra, J. Mol. Recognit. 13 (2000) 167–187. M. Sollazzo, R. Billetta, M. Zanetti, Protein Eng. 4 (1990) 215–220. C.F. Barbas 3rd, L.R. Languino, J.W. Smith, Proc. Natl. Acad. Sci. USA 90 (1993) 10003–10007. A. Skerra, Biochim. Biophys. Acta 1482 (2000) 337–350. S.D. Nuttall, M.J. Rousch, R.A. Irving, S.E. Hufton, H.R. Hoogenboom, P.J. Hudson, Proteins 36 (1999) 217–227. L. Xu, P. Aha, K. Gu, R.G. Kuimelis, M. Kurz, T. Lam, A.C. Lim, H. Liu, P.A. Lohse, L. Sun, S. Weng, R.W. Wagner, D. Lipovsek, Chem. Biol. 9 (2002) 933–942. W. Wang, Y. Ou, Y Shi, Pharm. Res. 21 (2004) 2105–2111. L.L. Baggio, Q. Huang, T.J. Brown, D.J. Drucker, Diabetes 53 (2004) 2492–2500. L.M. Amzel, R.J. Poljak, Annu. Rev. Biochem. 48 (1979) 961–997. E.A. Padlan, Mol. Immunol. 31 (1994) 169–217. M. Zanetti, G. Filaci, R.H. Lee, P. del Guercio, F. Rossi, R. Bacchetta, F. Stevenson, V. Barnaba, R. Billetta, EMBO J. 12 (1993) 4375–4384. J. Chung, C. Rader, M. Popkov, Y.M. Hur, H.K. Kim, Y.J. Lee, C.F. Barbas III, FASEB J. 18 (2004) 361–363. P. Lanza, B. Felding-Habermann, Z.M. Ruggeri, M. Zanetti, R. Billetta, Blood Cells Mol. Dis. 23 (1997) 230–241. T.T. Wu, G. Johnson, E.A. Kabat, Proteins 16 (1993) 1–7. G. Johnson, T.T. Wu, Nucleic Acids Res. 28 (2000) 214–218. M. Ohlin, C.A. Borrebaeck, Mol. Immunol. 35 (1998) 233–238. P.C. Wilson, O. de Bouteiller, Y.J. Liu, K. Potter, J. Banchereau, J.D. Capra, V. Pascual, J. Exp. Med. 187 (1998) 59–70. E.A. Kabat, National Institutes of Health (U.S.), Sequences of proteins of immunological interest, U.S. Department of Health and Human Services Public Health Service National Institutes of Health, Bethesda, MD, 1991. S. Xiong, M. Gerloni, M. Zanetti, Nat. Biotechnol. 15 (1997) 882–886. T.D. Brumeanu, A. Bot, C.A. Bona, P. Dehazya, I. Wolf, H. Zaghouani, Immunotechnology 2 (1996) 85–95. T. van den Beucken, N. van Neer, E. Sablon, J. Desmet, L. Celis, H.R. Hoogenboom, S.E. Hufton, J. Mol. Biol. 310 (2001) 591–601. T.T. Wu, E.A. Kabat, J. Exp. Med. 132 (1970) 211–250. C. Chothia, A.M. Lesk, J. Mol. Biol. 196 (1987) 901–917. C. Chothia, A.M. Lesk, M. Levitt, A.G. Amit, R.A. Mariuzza, S.E. Phillips, R.J. Poljak, Science 233 (1986) 755–758. R.M. MacCallum, A.C. Martin, J.M. Thornton, J. Mol. Biol. 262 (1996) 732–745. E.A. Padlan, Proc. Natl. Acad. Sci. USA 74 (1977) 2551–2555. E.A. Padlan, C. Abergel, J.P. Tipper, FASEB J. 9 (1995) 133–139. E.A. Kabat, T.T. Wu, H. Bilofsky, J. Biol. Chem. 252 (1977) 6609– 6616.
P.J. Simon et al. / Archives of Biochemistry and Biophysics 440 (2005) 148–157 [30] G.M. Studnicka, S. Soares, M. Better, R.E. Williams, R. Nadell, A.H. Horwitz, Protein Eng. 7 (1994) 805–814. [31] C. Chen, T.M. Martin, S. Stevens, M.B. Rittenberg, J. Exp. Med. 180 (1994) 577–586. [32] K. Leitzgen, M.R. Knittler, I.G. Haas, J. Biol. Chem. 272 (1997) 3117–3123. [33] J. Chatellier, M.H. Van Regenmortel, T. Vernet, D. Altschuh, J. Mol. Biol. 264 (1996) 1–6. [34] A.H. Horwitz, R. Nadell, F. Preugschat, M. Better, Mol. Immunol. 31 (1994) 683–692. [35] F.C. Bernstein, T.F. Koetzle, G.J. Williams, E.F. Meyer Jr., M.D. Brice, J.R. Rodgers, O. Kennard, T. Shimanouchi, M. Tasumi, J. Mol. Biol. 112 (1977) 535–542. [36] R.M. Horton, Methods Mol. Biol. 67 (1997) 141–149. [37] I.M. Tomlinson, J.P. Cox, E. Gherardi, A.M. Lesk, C. Chothia, EMBO J. 14 (1995) 4628–4638. [38] A. Honegger, A. Pluckthun, J. Mol. Biol. 309 (2001) 657–670. [39] A. Tramontano, C. Chothia, A.M. Lesk, Proteins 6 (1989) 382–394. [40] C. Chothia, I. Gelfand, A. Kister, J. Mol. Biol. 278 (1998) 457–479. [41] J.P. Hannon, C. Nunn, B. Stolz, C. Bruns, G. Weckbecker, I. Lewis, T. Troxler, K. Hurth, D. Hoyer, J. Mol. Neurosci. 18 (2002) 15–27. [42] W. Meyerhof, Rev. Physiol. Biochem. Pharmacol. 133 (1998) 55– 108. [43] Y.C. Patel, M.T. Greenwood, R. Panetta, L. Demchyshyn, H. Niznik, C.B. Srikant, Life Sci. 57 (1995) 1249–1265.
157
[44] R.M. Wright, H. Gram, A. Vattay, S. Byme, P. Lake, D. Dottavio, Biotechnology (N Y) 13 (1995) 165–169. [45] I.A. Wilson, R.L. StanWeld, Curr. Opin. Struct. Biol. 3 (1993) 113– 118. [46] I.A. Wilson, R.L. StanWeld, Curr. Opin. Struct. Biol. 4 (1994) 857– 867. [47] C. Chothia, A.M. Lesk, A. Tramontano, M. Levitt, S.J. Smith-Gill, G. Air, S. SheriV, E.A. Padlan, D. Davies, W.R. Tulip, et al., Nature 342 (1989) 877–883. [48] C. Chothia, J. Novotny, R. Bruccoleri, M. Karplus, J. Mol. Biol. 186 (1985) 651–663. [49] J. Novotny, E. Haber, Proc. Natl. Acad. Sci. USA 82 (1985) 4592– 4596. [50] D.G. Bole, L.M. Hendershot, J.F. Kearney, J. Cell Biol. 102 (1986) 1558–1566. [51] J. Ma, J.F. Kearney, L.M. Hendershot, Mol. Immunol. 27 (1990) 623–630. [52] S. Blond-Elguindi, S.E. Cwirla, W.J. Dower, R.J. Lipshutz, S.R. Sprang, J.F. Sambrook, M.J. Gething, Cell 75 (1993) 717–728. [53] G.C. Flynn, J. Pohl, M.T. Flocco, J.E. Rothman, Nature 353 (1991) 726–730. [54] D.P. Davis, R. Khurana, S. Meredith, F.J. Stevens, Y. Argon, J. Immunol. 163 (1999) 3842–3850. [55] B. Steipe, A. Pluckthun, R. Huber, J. Mol. Biol. 225 (1992) 739– 753.
Display of somatostatin-related peptides in the complementarity determining regions of an antibody light chain Peter J. Simon a,1, Kevin C. Brogle b, Baiyang Wang a,¤,2, Donald J. Kyle b, Daniel A. Soltis a a
b
Department of Immunotherapeutics, Purdue Pharma L.P., 6 Cedar Brook Drive, Cranbury, NJ 08512, USA Department of Computational, Combinatorial and Medicinal Chemistry, Purdue Pharma L.P., 6 Cedar Brook Drive, Cranbury, NJ 08512, USA Received 25 March 2005, and in revised form 14 June 2005 Available online 11 July 2005
Abstract Peptide display in antibody complementarity determining regions (CDRs) oVers several advantages over other peptide display systems including the potential to graft heterologous peptide sequences into multiple positions in the same backbone molecule. Despite the presence of six CDRs in an antibody variable domain, the majority of insertions reported have been made in heavy chain CDR3 (h-CDR3) which may be explained in part by the highly variable length and sequence diversity found in h-CDR3 in native antibodies. The ability to graft peptide sequences into CDRs is restricted by amino acids in these loops that make structural contacts to framework regions or are oriented towards the hydrophobic interior and are important for the proper folding of the antibody. To identify such positions in human -light chain CDR1 (-CDR1) and CDR2 (-CDR2), we performed alignments of 1330 -light chain variable region amino acid sequences and 19 variable region X-ray crystal structures. From analyses of these alignments, we predict insertion points where sequences can be grafted into -CDR1 and -CDR2 to prepare synthetic antibody molecules. We then tested these predictions by inserting somatostatin and somatostatin-related sequences into -CDR1 and -CDR2, and analyzing the expression and ability of the modiWed antibodies to bind to membranes containing somatostatin receptor 5. These results expand the repertoire of CDRs that can be used for the display of heterologous peptides in the CDRs of antibodies. 2005 Elsevier Inc. All rights reserved. Keywords: Synthetic antibody; Complementarity determining region; Somatostatin; Protein; Peptide; Display; ScaVold; Alignment; Receptor; Expression
The ability to insert biologically active peptide sequences and peptide libraries into the surface exposed loops of protein frameworks has been a signiWcant advance for engineering molecules with novel catalytic or binding properties [1,2]. Several scaVold
*
Corresponding author. Fax: +1 610 313 4039. E-mail address: [email protected] (B. Wang). 1 Present address: Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey, 195 Little Albany Street, New Brunswick, NJ 08903-2681, USA. 2 Present address: BioRexis Pharmaceutical Corp., 3400 Horizon Drive, King of Prussia, PA 19406, USA. 0003-9861/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2005.06.009
proteins have been evaluated including antibodies [3,4], lipocalins [5], CTLA-4 [6], and Wbronectin [7] that have enabled the construction of novel proteins that address some of the potential shortcomings of the native peptides including low binding aYnity, inadequate speciWcity or undesirable pharmacokinetic properties [4,8,9]. A key feature of these protein scaVolds is an ordered structure that stably displays surface exposed peptide loops. Antibodies are the prototypical scaVolds, presenting a wide array of amino acid sequences on a structured framework that is formed by the organized arrangement of -strands. Connecting these strands are six loops known as complementarity determining
P.J. Simon et al. / Archives of Biochemistry and Biophysics 440 (2005) 148–157
regions (CDRs)3 that show a high level of sequence diversity and form a binding surface for antigen interaction [10,11]. The insertion of peptide sequences into the CDR loops of full-length antibodies that maintain the basic antibody conformation has been performed previously. Sallazzo et al. [3] Wrst reported the expression of an antibody with a peptide epitope inserted in h-CDR3 and showed that the inserted peptide was recognized by a monoclonal antibody that bound to the same epitope in the native protein. In other experiments, synthetic antibodies containing adhesion molecule binding sequences (RGD) grafted in h-CDR3 have been shown to display the inserted peptide ligand in a functional conformation and bind to puriWed integrins and tumor cells whose adhesion to extracellular matrix is RGD dependent. These modiWed antibodies have also been shown to block cell adhesion to Wbronectin and inhibit natural killer cell mediated cytotoxicity [4,12–14]. In these and other experiments using the CDRs of antibodies for the functional display of peptide ligands, the majority of peptide grafts were made in h-CDR3 because of its diverse size range (up to 30 aa) and high sequence variability [15,16]. Even though the CDRs are surface exposed loops, and insertions and deletions of residues are a normal feature of the hypermutation process [17,18], not all CDRs exhibit the same length distribution. -CDR2 is 7 aa long in 99.9% of the sequences in the Kabat database whereas the lengths of the other Wve CDRs vary to a much greater extent ([19], unpublished results). Although the insertion of heterologous peptides has been previously reported for several of the CDRs [3,4,20–22], it is unclear whether peptide insertions that alter the length of the CDR can be made successfully in -CDR2. Expanding the repertoire of CDRs where peptide ligands can be grafted without signiWcantly diminishing the expression of the synthetic antibody would allow for peptide display in CDRs that may impose diVerent structural constraints. Although there are multiple deWnitions of CDRs based on the sequence diversity within the heavy and light chains [19,23], the structural loops between the -strands of the variable region framework [24,25], or the ability of each amino acid to contact antigen [26], none of these deWnitions were developed with the purpose of identifying positions within the CDRs where heterologous peptides could be inserted without disrupting the folding and stability of the modiWed antibody. As previous studies have shown, there are amino acid positions in the 3 Abbreviations used: aa, amino acids; CDR, complementarity determining region; CTLA-4, cytotoxic T-lymphocyte associated protein-4; ELISA, enzyme-linked immunosorbent assay; h-CDR, antibody heavy chain CDR; -CDR, antibody -light chain CDR; kDa, kiloDalton; SST-14, somatostatin; SSTR5, somatostatin receptor 5.
149
CDRs where the sequence variability is very limited, that make contact with portions of the variable region framework or have side chains buried in the hydrophobic core [24,27–30]. It is likely that changing these residues would have a signiWcant impact on the folding and stability of the antibody as previously demonstrated [31–34]. In this report, we extend the structural analysis of the individual amino acid positions in the immunoglobulin variable domains focusing on the positions in the CDRs of the -light chain. Based on these analyses, we identiWed amino acid positions within -CDR1 and -CDR2 that do not appear to be involved in the folding and stability of the variable domain. We then prepared modiWed immunoglobulin molecules in which somatostatinrelated peptides were inserted into either -CDR1 or -CDR2. Insertions that did not disrupt residues involved with protein folding or stability were eYciently expressed and capable of binding membranes containing somatostatin receptor 5 (SSTR5).
Materials and methods Antibody sequence and structure alignment One thousand three hundred and thirty human -light chains were downloaded from the Kabat antibody database ([16], http://immuno.bme.nwu.edu) and aligned using the Wisconsin Package (Accelrys, San Diego). As PileUp is limited by the number of sequences it can process, an alignment was built from a randomly selected subset of 476 -light chain sequences. From this alignment, a Hidden Markov Model proWle was calculated using HmmerBuild. This proWle was used with HmmerAlign to align the remaining sequences. Finally, extraneous gaps created by HmmerAlign due to the sequence-variable CDRs were removed, and insertions in CDRs were Wlled from the left. Unique human antibody structures were downloaded from the Protein Data Bank [35]. Nineteen structures contained heavy chain and -light chain variable sequences. Using InsightII (Accelrys, San Diego), water and other molecules were removed and the variable regions from the antibodies were separated. The heavy chain variable and -variable regions were then separately aligned in three dimensions to compare side chain locations and interactions. Every position that Kabat and Chothia designated as occurring within a CDR [19,23–25] was evaluated for surface accessibility and participation in hydrogen bonds, salt-bridges, or hydrophobic interactions. The Protein Data Bank ID numbers for the structures used in this analysis are: 1AD0, 1AD9, 1BEY, 1BRE, 1DEE, 1DFB, 1DQL, 1FH5, 1GC1, 1IGM, 1LVE, 1OBE, 1QLR, 1QP1, 1REI, 1VGE, 1WTL, 2IMM, and 1HOU.
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Control antibody Control variable region sequences were derived from the frequency analysis of human light and heavy chains. The binding speciWcity of the control antibody is unknown. The amino acid sequence of the -light chain variable region is DIQMTQSPSSLSASVGDRVTIT CRASQSISNYLAWYQQKPGKAPKLLIYAASSLES GVPSRFSGSGSGTRFTLTISSLQPEDFATYYCQQ YNSLPWTFGQGTKVEI. The IgG heavy chain variable region amino acid sequence is QVQLVQSGAEVKKPGASVKVSCKAS GYTFTSYAISWNWVRQAPGQGLEWMGWINGN GDTNYAQKFQGRVTITADTSTSTAYMELSSLRS EDTAVYYCARAPGYGSDYWGQGTLVTVS. Antibody modiWcation DNA fragments encoding the peptide grafts in -CDR1 and -CDR2 were constructed by PCR using the method of gene splicing by overlap extension (reviewed in [36]). Primers were designed with two sections: the 3⬘ ends are complementary to the variable region sequence and the 5⬘ ends contain the ligand sequence to be inserted. A pUC19 vector containing the variable region of the -light chain was used as a template. For each construct, two PCRs were performed (one to amplify the 5⬘ end of the variable region to the CDR being modiWed, the other to amplify the CDR to the 3⬘ end). Products were gel puriWed and combined in a second PCR with M13 forward and reverse primers to amplify the full-length construct. After gel puriWcation, the fragments were digested and cloned into pUC19. The sequences of the constructs were then conWrmed by DNA sequencing and the variable regions cloned into the appropriate expression vectors containing either human heavy or light chain constant regions (1 and , obtained from S. Morrison, UCLA). The expression vectors used were the GS Gene Expression System plasmids pEE6.1 for the heavy chain and pEE14.1 for the light chain (Lonza Biologics, Slough, UK). Cell culture and transfections CHO-K1 cells selected to grow in suspension were cultured in serum-free ProCHO4 (Bio Whittaker) medium supplemented with L-glutamine (4 mM; Invitrogen). Vectors containing the cloned antibody genes were transfected into cells using CLONfectin (Clontech). For a 2 mL (Wnal volume) reaction, 250 ng of plasmid DNA was transfected at a 3:1 (weight/weight) light/heavy chain ratio with 4 g CLONfectin into 2.5 £ 106 cells. Transfected cells were incubated in ProCHO4 with growth supplements (JRH Biosciences) for 4 h before addition of an equal volume of ProCHO4 with 1£ growth supplements, 10% low IgG FBS (Invitrogen), penicillin (200 U/mL;
Invitrogen), and streptomycin (200 g/mL; Invitrogen). Transfections were scaled up to obtain suYcient material for binding assays. The control and modiWed antibodies were puriWed from Wltered cell culture supernatant on a protein G column (Amersham Biosciences), dialyzed, and concentrated using an Ultrafree-4 Biomax 10 kDa MWCO centrifugal Wlter unit (Millipore). Antibody quantitation Cell culture supernatants were collected at day 7, Wltered, and measured for assembled antibody concentration by sandwich ELISA using plate bound anti-human IgG antibody and anti-human -HRP conjugated antibody (Southern Biotechnology). Known concentrations of human IgG (Sigma) were used to generate a standard curve. Color generation from TMB peroxidase substrate (KPL) was measured on a Molecular Devices Vmax kinetic microplate reader. Data were collected and analyzed by SOFTmax PRO v3.0. The media from each transfection were assayed in triplicate at two dilutions in the linear range of the standard curve. Assembled antibody concentration was multiplied by the volume of recovered cell culture supernatant. All data are normalized to the expression level of the control antibody performed as a standard in each experiment. Receptor binding assays The aYnity of the peptide, SST-14 (Bachem), control antibody, and somatostatin-derived synthetic antibody constructs were tested using membranes derived from HEK-293 EBNA cells transfected with human SSTR5 (Perkin-Elmer) following the manufacturer’s protocol. BrieXy, membranes were incubated with ligand (SST-14, modiWed antibody or control antibody) and labeled compound [125I]Tyr11 SST-14 (Perkin-Elmer) for 1 h at room temperature. Reactions were Wltered and washed with ice-cold wash buVer (50 mM Tris, pH 7.5, 0.2% BSA). After drying at 50 °C, OptiPhase Supermix scintillation cocktail (Wallac) was added and plates were measured for 1 min/well in a Trilux 1450 MicroBeta liquid scintillation and luminescence counter (Wallac). Data were plotted and Ki values were calculated using the sigmoidal curve Wtting function in GraphPad Prism 3.0.
Results Analysis of -light chain sequences and structures identiWes optimal locations for peptide insertion in -CDR1 and -CDR2 Although the CDRs of antibodies have been deWned by others as regions with high levels of sequence diversity [19,23], not all of the amino acids in these loops are
P.J. Simon et al. / Archives of Biochemistry and Biophysics 440 (2005) 148–157
highly variable and some make contacts with the antibody framework [27,29,30]. We therefore sought to identify residues in the -light chain CDRs that did not appear to be involved in maintaining structural integrity and thus may be amenable to modiWcation. To do this, 1330 human -light chain antibody sequences from the Kabat database [16] were aligned and every position in the variable region was analyzed for amino acid variability. CDR positions with a single residue or two stringently conserved residues (i.e., S/T, V/I, V/L, I/L or Y/F) occurring >80% were identiWed (Fig. 1). To ascertain if there was a structural role for these positions, an alignment of 19 human -light chain variable domain X-ray crystal structures was examined in detail. SpeciWcally, each side chain was analyzed for surface accessibility, residue orientation and involvement in hydrogen bonds, salt-bridges or hydrophobic interactions either between
151
Table 1 Summary of structure analysis Position
Interactions
24
Side chain faces away from combining site. In 2 out of 19 structures, position 24 forms a salt-bridge with position 70 Side chain towards interior of protein H-bond with position 3 Hydrophobic interior Hydrophobic interior No direct salt-bridge seen between Arg at 54 and Asp/Ser at position 60. When Leu is present, it is part of the hydrophobic interior
25 26 29 33 54
the -sheets or chains. The amino acids in -CDR1 and -CDR2 identiWed by the structural analysis are shown in Table 1, and a representative crystal structure is shown in Fig. 2. All residue numbering is based on the deWnition of Chothia et al. [24,25]. Although some residues were identiWed by both methods (24, 26, 29, and 33 in -CDR1), other positions were identiWed in only one of the screens (positions 25 and 27 in -CDR1, and 52, 54, and 56 in -CDR2; Fig. 1). Similar observations have been made in other frequency and structural analyses [26,28–30,37,38]. For example, the CDR1 residues 25, 29, and 33, identiWed by Chothia and Lesk [24] and shown to be buried in the core, determine the canonical structure of the CDR. Our structural analysis of -CDR1 also identiWed the hydrogen bonding of residue 26 to the backbone –NH of position 3 [39], and the orientation of the side chain of amino acid 24 away from the combining site [26,40]. In -CDR2, a leucine residue present at position 54 appeared to be part of the hydrophobic core. Based on these analyses, we predict that the preferred positions for insertions in -CDR1 are after residue 29 and before residue 33, and in -CDR2, any insertion that maintains the amino acid at position 54. Selection of somatostatin as a ligand for insertion into light chain CDRs
Fig. 1. Variability of -light chain CDR1 and CDR2. One thousand three hundred and thirty -light chain sequences were aligned then adjusted by hand to left-justify insertion regions. The frequencies of amino acid(s) are displayed for each position (amino acids present at least 5% of the time are shown) of the Chothia and Kabat deWned CDR1 (A) and -CDR2 (B). Amino acids are presented in single letter code; (–), an amino acid not inserted at that location; 䊉, positions identiWed in the variability analysis; 䊐, residues that are part of the hydrophobic core or possessing contacts with other amino acids. Sequence grafting is best performed at the amino acids identiWed by the solid line. It may be possible to replace the entire -CDR2 sequence when grafting peptides (dashed line extension). Numbering is according to the Chothia convention [24,25].
To test our predictions of the positions in -CDR1 and -CDR2 where heterologous peptides may be successfully inserted, we selected the small peptide hormone somatostatin (amino acid sequence AGCKNFFWKTF TSC) as a model ligand. Somatostatin is a cyclic peptide which may be an advantage for these studies since many of the CDRs have a looped conformation. In fact, development of many somatostatin agonists demonstrates the importance of the FWKT motif in the context of a looped structure. The speciWc size of the loop does not seem to be critical, as agonists with loops as small as 6 aa (e.g., octreotide and lanreotide) have been identiWed (reviewed in [41–43]). In addition, since we are inserting the peptide sequence into the CDR of an antibody, it is also important that somatostatin contains no post-translational modiWcations such as phosphorylation,
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Fig. 2. Structural analysis of -light chain CDR1 and CDR2. The X-ray crystal structure of 2IMM, a representative variable region, is provided with relevant side chains displayed [55]. Blue ribbon, -CDR1; red ribbon, -CDR2; white ribbon, -CDR3. (A) Relevant -CDR1 residues are highlighted; yellow, side chains not facing combining site; gray, hydrophobic interior; red, hydrogen bonded residues. (B) Relevant -CDR2 residues shown. 2IMM has a -CDR1 sequence of KSSQSLLNSGNQKNFLA (length 17 aa), showing the insertion of 6 aa between positions 30 and 31. The sequence of 2IMM -CDR2 is GASTRES (length 7 aa).
adenylation or glycosylation and that the amino- and carboxyl-termini are not required for binding or function. -CDR1-modiWed antibodies with properly positioned insertions are eYciently expressed To test our hypothesis that disrupting residues involved in variable domain folding or stability would reduce antibody secretion, we inserted into -CDR1 a somatostatin-related peptide previously identiWed in a phage display experiment. This peptide, CRFWKTWC, was selected from a library of peptides consisting of six random amino acids Xanked by cysteine residues and shown to bind to somatostatin receptors [44]. We main-
tained -CDR1 length and replaced 8 aa from either the N- or C-terminal portion of the CDR with the amino acid sequence CRFWKTWC to create 24–31 and 27– 34, respectively (see Fig. 3 legend for nomenclature). The constructs were created by modiWcation of a control antibody that was designed to have no known antigen interaction. The sequence of the control antibody was derived from a frequency and linkage analysis of the presence of speciWc amino acid residues at every position of the antibody variable region sequence and attempts to include the more common amino acids at each position while maintaining apparent linkages to residues at other positions of the molecule (data not shown). Expression levels of the control antibody in our system (»4000 g/L)
Fig. 3. Sequence of -CDR1 and -CDR2 regions in control antibody and synthetic antibodies with somatostatin-related grafts. The sequence of CDR1 and -CDR2 in the control antibody is shown. Below are the names and sequences of the synthetic antibodies used in this study. The length of the CDR and change in size is provided in columns to the right of each sequence. Underlined residues are components of the hydrophobic interior or residues with internal contacts, as identiWed through frequency and structural analyses. The nomenclature is as follows: , phage display derived sequence (CRFWKTWC); S, somatostatin inner-loop sequence (KNFFWKTFTS); SC+, somatostatin amino acids 3–14 followed by an aspartic acid (CKNFFWKTFTSCD); SC¡, SC+ sequence with Wrst and last cysteine modiWed to G and A (GKNFFWKTFTSAD); the number in the construct name indicates the amino acid(s) removed when the ligand was grafted; ¤ indicates no sequence removed, with the insertion made after the position indicated. The boxed amino acids refer to the ligand sequence in the modiWed CDR.
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and 33 are important for expression. The threefold reduction in expression observed between 27–31 and 29* (Fig. 4) may be due to the absence of the isoleucine at position 29 that is normally buried in the hydrophobic core of the variable domain. It is also possible, however, that the poor expression of constructs 24–31 and 27–34 may be due to the formation of disulWde bonds within the inserted sequence that may alter antibody structure and prevent eYcient assembly and/or secretion. To test this hypothesis, we inserted KNFFWKTFTS, the sequence found between the two cysteines of the natural ligand SST-14, into CDR1. Two synthetic antibodies were made, S24–34 and S24–33 (S, somatostatin inner-loop sequence). The former replaces the entire -CDR1 with the inner-loop sequence (decreasing the CDR length by 1 aa) while the latter preserves the CDR length by maintaining the alanine at position 34 (Fig. 3). Similar to the results obtained above, modiWcation of residues identiWed as part of the hydrophobic interior or having framework contacts results in diminished expression levels (1 and 6% of control). However, when this sequence is used to replace aa 30–32 (the region predicted to tolerate peptide grafts), expression of the resulting construct (S30–32) is 50% of control, equivalent to 29*. These results suggest that the removal of internal contact residues, and not the presence of the cysteines, caused the reduction in expression found with 24–31 and 27–34. Thus, synthetic antibodies with peptide sequences grafted into -CDR1 in locations that do not disrupt amino acids involved in protein folding or stability can be eYciently expressed.
are similar to those of mouse monoclonal and chimeric antibodies expressed under similar conditions. Plasmids encoding the control or modiWed antibody were transfected into CHO-K1 cells selected to grow in suspension culture. Cell culture supernatants were assayed for assembled antibody in a sandwich ELISA and normalized to the expression levels measured from cells transfected with constructs expressing the control antibody. The data in Fig. 4 show synthetic antibodies 24–31 and 27–34 that are secreted at less than 2% of the control antibody. As these constructs modiWed amino acids that are part of the hydrophobic core (residues 29 and 33) or contact other portions of the protein (residue 26), the poor expression would indicate the importance of these residues in -CDR1 for proper antibody folding and secretion. To further test this hypothesis, CRFWKTWC was grafted after residue 29 (with no removal of sequence) to create an 8 aa insertion. Cells expressing this construct (29*) secreted signiWcantly more assembled antibody (»2000 g/L) than 24–31 and 27–34, indicating that the synthetic antibody containing the inserted sequence CRFWKTWC, when positioned in -CDR1 to avoid disrupting residues involved in maintaining proper antibody folding, can be eYciently expressed. The low levels of expression seen when the N- or Cterminal 3 aa of -CDR1 are removed (24–31 and 27– 34) suggests the importance of these residues (presumably 24–26 and 33, identiWed above) for proper folding and secretion. Therefore, 27–31, which maintains the 3 aa on the N- and C-termini of -CDR1 was created. Its expression is at least ninefold higher than 24–31 and 27–34 (17% compared to 1.0 and 1.8%, respectively; Fig. 4), suggesting that one or more of the residues 24–26
Insertions in -CDR2 do not signiWcantly reduce antibody expression
Fig. 4. Expression of synthetic antibodies containing somatostatinrelated sequence. Expression levels of synthetic antibodies are shown normalized to the control antibody. The concentration of assembled antibody secreted from transiently transfected CHO-K1 cells was determined by sandwich ELISA corrected for evaporation. Transfections were performed in triplicate in each of three independent experiments. Expression levels were measured in triplicate at two dilutions within the linear range of the assay. The average of all data points is shown; error bars indicate standard deviation. The expression level of the control antibody was determined to be 4000 g/L.
We next examined whether a CDR that is less often involved in antigen contact [26,45,46] and has a constant size could also be modiWed. -CDR2 was chosen, as its length is 7 aa in over 99.9% of antibody sequences in the Kabat database ([19], unpublished data). Since leucine is present at position 54 in the control antibody sequence and the structural alignment suggests this may be part of the hydrophobic interior, residues 54–56 were maintained and 50–53 were replaced with the SST-14 innerloop sequence KNFFWKTFTS (note, the serine at position 53 matches the Wnal residue of the ligand). S50– 53 expressed at levels t40% of the control antibody (Fig. 4). Construct S50–56, that replaces the entire CDR2 sequence, was expressed at a similar level (50% of control) as S50–53. This suggests that any structural interactions present at position 54 may be modiWed with minimal impact on protein stability. Alternatively, the phenylalanine in position 8 of the inserted peptide may serve as a mimic of the hydrophobic leucine found at position 54 of the control antibody sequence. In either case, these data show that modiWed antibodies, and
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Fig. 5. Binding of synthetic antibodies to membranes containing SSTR5. The ability of synthetic antibodies containing somatostatinrelated sequence to compete with a radioligand for somatostatin receptor 5 membranes is shown. Increasing concentrations of competitor (SST-14, control or synthetic antibody) were mixed with SSTR5 membranes. [125I]Tyr11 SST-14 was added, and after 1 h the membranes were washed to remove unbound radioligand. The amount of radioligand bound to membranes was measured in a scintillation counter. Plotted values represent means § SD of triplicate reactions. Data from a single representative experiment are shown. The log molar concentration is shown on the x-axis, decays per minute on the y-axis.
particularly those where the length of the normally highly size restricted -CDR2 has been increased, can be eYciently expressed in CHO-K1 cells. These results are not unique to somatostatin, as synthetic antibodies with other peptide ligands grafted into -CDR2 (with lengths up to 47 aa)4 are capable of being expressed at levels >1000 g/L. Synthetic antibody molecules containing somatostatinrelated peptides bind to membranes containing somatostatin receptor 5 For applications where the inserted peptide is a ligand that normally binds to a receptor, the modiWed antibody must present the ligand in a biologically active conformation to interact eVectively with its relevant target. Therefore, we measured the ability of selected constructs to bind membranes containing SSTR5 in a competition assay using constant levels of the natural ligand labeled with 125I ([125I]Tyr11 SST-14; Fig. 5). In these experiments, the control peptide SST-14 shows binding to the membranes with an average Ki »6 nM, similar to the reported Ki of 1.4 nM [42]. As expected, the control antibody, devoid of somatostatin sequence, shows no detectable binding to the membranes. All of the synthetic antibodies containing somatostatin-related sequence tested can compete with the radiolabeled ligand and show binding to the membranes containing SSTR5, albeit to diVerent degrees. S30–32 has weak aYnity and is unable to fully compete away the radioligand even at high concentrations. 29*, another 4
Earl Albone, personal communication.
Fig. 6. Synthetic antibodies containing cysteine residues have improved binding proWles for membranes containing SSTR5. The aYnity assays of SC+30 and SC¡30 and natural ligand SST-14 are shown. See legend to Fig. 5.
-CDR1-modiWed antibody, has similar (but reproducibly slightly higher) aYnity for membranes containing SSTR5 as S30–32. In this experiment, S50–56 shows the strongest binding to SSTR5-containing membranes of the three synthetic antibodies tested. It should be noted, however, that the binding of these constructs to the SSTR5-containing membranes may be inXuenced by the presence of two light chain-displayed ligands that may enhance the binding aYnity by introducing an avidity component. Consequently, the actual binding aYnity of an individual, modiWed CDR may be less than what is observed in these experiments. In an attempt to enhance the binding of 29* to the membranes containing SSTR5, we restored the cysteines from SST-14 with the expectation that this may further constrain the somatostatin peptide structure and more accurately mimic the structure of the natural ligand. SC+30 (SC+, somatostatin sequence including cysteines) was made by replacing position 30 of -CDR1 with amino acids 3–14 of SST-14 (CKNFFWKTFTSC) and inserting an aspartic acid after the second cysteine to mimic an exposed carboxyl terminus (Fig. 3). As a control, SC¡30 maintains the aspartic acid, but modiWes both cysteines to prevent the somatostatin ligand from forming an internal disulWde bond or a disulWde bond to other portions of the antibody structure. Constructs SC+30 and SC¡30 were eYciently expressed (60 and 65% of control, respectively; Fig. 4) and tested for their ability to bind membranes containing SSTR5 (Fig. 6). In these experiments, SST-14 exhibited a Ki of 1.2 nM for the membranes. The results show that SC+30 has a slightly improved binding proWle compared to SC¡30. As these constructs diVer in their ability to form a disulWde bond within the grafted sequence, the results may reXect the importance of mimicking the structure of the natural ligand. In the case of SC+30, the cysteine residues in the inserted somatostatin sequence may be capable of forming a disulWde bond, constrain-
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ing the peptide in a cyclic structure similar to the natural SST-14 structure.
Discussion We sought to determine whether we could expand the repertoire of CDR usage for the display of bioactive peptide sequences. Studies from other investigators have shown that heterologous peptides can be inserted into several diVerent protein scaVolds and that the inserted peptides can retain their biological function. However, where antibodies have been used as the scaVold, most of these studies describe constructs where the heterologous peptides were inserted into h-CDR3. Since some amino acids in the framework contribute to a limited set of canonical structures for each CDR [47], identifying positions in each CDR where insertions are tolerated should enhance the likelihood that a synthetic antibody could be prepared where the inserted peptide adopts a bioactive conformation. By identifying and maintaining residues involved in folding and structural stability when grafting somatostatin-related sequences into -CDR1 and -CDR2, we have obtained eYciently expressed synthetic antibodies capable of binding to membranes containing SSTR5. We initially evaluated the eVects of grafting amino acid sequences into antibody CDRs by monitoring their secretion as assembled proteins from transiently transfected CHO-K1 cells. This analysis allowed us to identify sites for grafting peptide ligands into CDRs while still maintaining reasonable (>500 g/L) expression levels. In these studies, grafting of somatostatin-related peptides into the insertion points predicted by our sequence and structure alignment analyses resulted in expression from transiently transfected CHO-K1 cells at levels >1600 g/ L. In -CDR1, the peptide insertions tested were tolerated when localized after residue 29 and before residue 33. Notably, when -CDR1 size variation naturally occurs, this is the region that accommodates the additional sequence by bulging away from the surface. For CDR2, synthetic antibodies containing peptide grafts that were inserted after residue 49 and before residue 54 or those that replaced the entire CDR were also eYciently expressed. Our prediction that the insertion of peptide sequences in -CDR1 would be successful when grafted after position 29 and before position 33 is supported by the eYcient expression of 29*, S30–32, SC+30, and SC¡30. When Chothia/Kabat deWnitions of -CDR1 were used to determine sequence graft sites (24–31, 27–34, 27– 31, S24–34, and S24–33), assembled antibody, as assayed by sandwich ELISA (Fig. 4), was secreted 16- to 100-fold lower than control antibody. The reason for this decreased production is not known. A Western blot analysis of these cell culture supernatants shows light
155
chain monomer and dimer, and fully assembled synthetic antibody are secreted in similar proportions to the eYciently expressed -CDR1-modiWed antibodies with grafts made after position 29 and before position 33 (data not shown). Light chain assembly with heavy chain is a prerequisite to antibody secretion, and although some residues may be positioned near the VH/VL interface [48], the -CDR1 residues that were modiWed in the constructs presented here (or the amino acids they contact) are not known to be involved in binding heavy chain [33,49]. Alternatively, the reduced expression of constructs 24–31, 27–34, 27–31, S24–34, and S24–33 could reXect a defect in light chain folding. The hsp70 chaperone BiP binds unfolded and unassembled light and heavy chains prior to their assembly [50,51]. Blond-Elguindi et al. [52] devised an algorithm for predicting peptide binding to the 7 aa long BiP binding cleft [53]. The program calculates a BiP score, with higher scores correlated to increases in the probability of BiP binding. Davis et al. [54] found the 7 aa peptide corresponding to positions 31–37 of the -light chain was able to bind BiP, suggesting this stretch of sequence may be a binding site in the light chain. Although it can be proposed that an altered BiP binding site may account for the decreased expression of some constructs, we have found no correlation between BiP score and expression of our modiWed antibodies (data not shown). Therefore, although light chain folding may be aVected in these -CDR1-modiWed antibodies, their diminished expression does not appear to correlate with reductions in BiP score. Peptide grafts in -CDR2 that replaced the entire CDR did not signiWcantly decrease expression levels even though a leucine at position 54 is part of the hydrophobic interior (Table 1, Fig. 2). Its modiWcation in S50– 56 did not signiWcantly decrease expression levels, suggesting its contribution to antibody stability may be minimal. Alternatively, an argument can be made whereby the last phenylalanine of the inserted sequence (KNFFWKTFTS) in S50–56 replaces the function of the leucine at position 54, maintaining the presence of a hydrophobic residue in the core. This model raises an intriguing possibility where some ligands may be able to mimic or overlap the sequence already present in the CDR. Further work is needed to distinguish these models. In our experiments, insertions of peptide sequence that increased -CDR2 length from 7 to 10 or 13 aa did not have a signiWcant aVect on secretion of full-length assembled antibody. Although the -CDR2 length is almost always 7 aa in germline V genes and functionally rearranged cDNA molecules that have been sequenced and submitted to the Kabat database, this restriction does not appear to prevent the successful insertion of peptide grafts. The lack of -CDR2 length variability observed in naturally occurring antibodies may reXect a
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decreased selection for somatic hypermutations in this region due to the limited contribution of residues in -CDR2 to antigen binding [26,45,46]. The presentation of a ligand in its biologically active conformation is important for binding to its receptor. The data presented in Fig. 6 show SC+30, displaying a somatostatin ligand with two cysteines, binds slightly stronger to SSTR5 membranes than SC¡30, which is the same size, but can no longer form a disulWde bond within the inserted somatostatin sequence. This slight increase in binding aYnity may result from the formation of a disulWde bond between the two cysteines in SC+30 that are not present in SC¡30 and constrain the somatostatin sequence in a cyclic conformation that is more similar to the natural ligand. One should note that the binding of synthetic antibodies to membranes containing SSTR5 presented here may not be universal across all SSTRs. In experiments performed by Nuttall et al., somatostatin was grafted into the loop of human cytotoxic T-lymphocyte associated protein-4 (CTLA-4) that is equivalent to CDR3 of an antibody variable domain. Phage displaying the modiWed CTLA-4 fused to the pIII protein was able to speciWcally bind SSTR4 containing membranes. Partial binding to membranes containing SSTR5 was also observed, but no binding was detected with membranes containing SSTR1, SSTR2 or SSTR3 [6]. It is likely that ligand-independent sequences interact with the extracellular surface of the receptor and aVect binding, a feature that could be useful in designing or selecting receptor speciWc synthetic antibodies. We attempted to examine the binding proWle of our constructs with other SSTRs (including membranes containing SSTR4 and those from the AtT-20 cell line which express multiple SSTRs), however, these studies were hampered by the poor binding of the natural ligand SST-14 (data not shown). Our work provides evidence for the eYcient expression of full-length antibody sequences with peptide insertions in CDRs that have not been extensively exploited previously for this use. When the peptide grafts do not interfere with amino acids involved in maintaining the proper folding and stability of the antibody variable domain, the resulting constructs are eYciently expressed from transiently transfected CHO-K1 cells and bind in a ligand-speciWc manner to the cognate receptor. The insertion of peptide ligands in multiple CDRs could also increase the binding aYnity of the modiWed antibodies displaying a single ligand species, or, when two or more distinct ligands are used, may target several molecules on one cell and could juxtapose proteins for tailored receptor co-activation. The preparation of these synthetic antibodies also may provide an approach for improving the pharmacokinetic properties of the inserted peptides which often have short half-lives in circulation that limit their potential utility as therapeutic agents [8,9]. In addition, by using the antibody
molecule as the scaVold for peptide display, one also may be able to direct immune eVector functions against targeted cells for the treatment of diVerent diseases including cancer.
Acknowledgments The authors thank Drs. Namit Ghildyal, Andrew Gordon, and Jamshid Arjomand for their critical review of the manuscript and Dr. Rajiv Shukla for cloning the 29* expression plasmid.
References [1] [2] [3] [4] [5] [6] [7]
[8] [9] [10] [11] [12]
[13] [14] [15] [16] [17] [18] [19]
[20] [21] [22] [23] [24] [25] [26] [27] [28] [29]
A. Skerra, P.A. Nygren, J. Immunol. Methods 290 (2004) 3–28. A. Skerra, J. Mol. Recognit. 13 (2000) 167–187. M. Sollazzo, R. Billetta, M. Zanetti, Protein Eng. 4 (1990) 215–220. C.F. Barbas 3rd, L.R. Languino, J.W. Smith, Proc. Natl. Acad. Sci. USA 90 (1993) 10003–10007. A. Skerra, Biochim. Biophys. Acta 1482 (2000) 337–350. S.D. Nuttall, M.J. Rousch, R.A. Irving, S.E. Hufton, H.R. Hoogenboom, P.J. Hudson, Proteins 36 (1999) 217–227. L. Xu, P. Aha, K. Gu, R.G. Kuimelis, M. Kurz, T. Lam, A.C. Lim, H. Liu, P.A. Lohse, L. Sun, S. Weng, R.W. Wagner, D. Lipovsek, Chem. Biol. 9 (2002) 933–942. W. Wang, Y. Ou, Y Shi, Pharm. Res. 21 (2004) 2105–2111. L.L. Baggio, Q. Huang, T.J. Brown, D.J. Drucker, Diabetes 53 (2004) 2492–2500. L.M. Amzel, R.J. Poljak, Annu. Rev. Biochem. 48 (1979) 961–997. E.A. Padlan, Mol. Immunol. 31 (1994) 169–217. M. Zanetti, G. Filaci, R.H. Lee, P. del Guercio, F. Rossi, R. Bacchetta, F. Stevenson, V. Barnaba, R. Billetta, EMBO J. 12 (1993) 4375–4384. J. Chung, C. Rader, M. Popkov, Y.M. Hur, H.K. Kim, Y.J. Lee, C.F. Barbas III, FASEB J. 18 (2004) 361–363. P. Lanza, B. Felding-Habermann, Z.M. Ruggeri, M. Zanetti, R. Billetta, Blood Cells Mol. Dis. 23 (1997) 230–241. T.T. Wu, G. Johnson, E.A. Kabat, Proteins 16 (1993) 1–7. G. Johnson, T.T. Wu, Nucleic Acids Res. 28 (2000) 214–218. M. Ohlin, C.A. Borrebaeck, Mol. Immunol. 35 (1998) 233–238. P.C. Wilson, O. de Bouteiller, Y.J. Liu, K. Potter, J. Banchereau, J.D. Capra, V. Pascual, J. Exp. Med. 187 (1998) 59–70. E.A. Kabat, National Institutes of Health (U.S.), Sequences of proteins of immunological interest, U.S. Department of Health and Human Services Public Health Service National Institutes of Health, Bethesda, MD, 1991. S. Xiong, M. Gerloni, M. Zanetti, Nat. Biotechnol. 15 (1997) 882–886. T.D. Brumeanu, A. Bot, C.A. Bona, P. Dehazya, I. Wolf, H. Zaghouani, Immunotechnology 2 (1996) 85–95. T. van den Beucken, N. van Neer, E. Sablon, J. Desmet, L. Celis, H.R. Hoogenboom, S.E. Hufton, J. Mol. Biol. 310 (2001) 591–601. T.T. Wu, E.A. Kabat, J. Exp. Med. 132 (1970) 211–250. C. Chothia, A.M. Lesk, J. Mol. Biol. 196 (1987) 901–917. C. Chothia, A.M. Lesk, M. Levitt, A.G. Amit, R.A. Mariuzza, S.E. Phillips, R.J. Poljak, Science 233 (1986) 755–758. R.M. MacCallum, A.C. Martin, J.M. Thornton, J. Mol. Biol. 262 (1996) 732–745. E.A. Padlan, Proc. Natl. Acad. Sci. USA 74 (1977) 2551–2555. E.A. Padlan, C. Abergel, J.P. Tipper, FASEB J. 9 (1995) 133–139. E.A. Kabat, T.T. Wu, H. Bilofsky, J. Biol. Chem. 252 (1977) 6609– 6616.
P.J. Simon et al. / Archives of Biochemistry and Biophysics 440 (2005) 148–157 [30] G.M. Studnicka, S. Soares, M. Better, R.E. Williams, R. Nadell, A.H. Horwitz, Protein Eng. 7 (1994) 805–814. [31] C. Chen, T.M. Martin, S. Stevens, M.B. Rittenberg, J. Exp. Med. 180 (1994) 577–586. [32] K. Leitzgen, M.R. Knittler, I.G. Haas, J. Biol. Chem. 272 (1997) 3117–3123. [33] J. Chatellier, M.H. Van Regenmortel, T. Vernet, D. Altschuh, J. Mol. Biol. 264 (1996) 1–6. [34] A.H. Horwitz, R. Nadell, F. Preugschat, M. Better, Mol. Immunol. 31 (1994) 683–692. [35] F.C. Bernstein, T.F. Koetzle, G.J. Williams, E.F. Meyer Jr., M.D. Brice, J.R. Rodgers, O. Kennard, T. Shimanouchi, M. Tasumi, J. Mol. Biol. 112 (1977) 535–542. [36] R.M. Horton, Methods Mol. Biol. 67 (1997) 141–149. [37] I.M. Tomlinson, J.P. Cox, E. Gherardi, A.M. Lesk, C. Chothia, EMBO J. 14 (1995) 4628–4638. [38] A. Honegger, A. Pluckthun, J. Mol. Biol. 309 (2001) 657–670. [39] A. Tramontano, C. Chothia, A.M. Lesk, Proteins 6 (1989) 382–394. [40] C. Chothia, I. Gelfand, A. Kister, J. Mol. Biol. 278 (1998) 457–479. [41] J.P. Hannon, C. Nunn, B. Stolz, C. Bruns, G. Weckbecker, I. Lewis, T. Troxler, K. Hurth, D. Hoyer, J. Mol. Neurosci. 18 (2002) 15–27. [42] W. Meyerhof, Rev. Physiol. Biochem. Pharmacol. 133 (1998) 55– 108. [43] Y.C. Patel, M.T. Greenwood, R. Panetta, L. Demchyshyn, H. Niznik, C.B. Srikant, Life Sci. 57 (1995) 1249–1265.
157
[44] R.M. Wright, H. Gram, A. Vattay, S. Byme, P. Lake, D. Dottavio, Biotechnology (N Y) 13 (1995) 165–169. [45] I.A. Wilson, R.L. StanWeld, Curr. Opin. Struct. Biol. 3 (1993) 113– 118. [46] I.A. Wilson, R.L. StanWeld, Curr. Opin. Struct. Biol. 4 (1994) 857– 867. [47] C. Chothia, A.M. Lesk, A. Tramontano, M. Levitt, S.J. Smith-Gill, G. Air, S. SheriV, E.A. Padlan, D. Davies, W.R. Tulip, et al., Nature 342 (1989) 877–883. [48] C. Chothia, J. Novotny, R. Bruccoleri, M. Karplus, J. Mol. Biol. 186 (1985) 651–663. [49] J. Novotny, E. Haber, Proc. Natl. Acad. Sci. USA 82 (1985) 4592– 4596. [50] D.G. Bole, L.M. Hendershot, J.F. Kearney, J. Cell Biol. 102 (1986) 1558–1566. [51] J. Ma, J.F. Kearney, L.M. Hendershot, Mol. Immunol. 27 (1990) 623–630. [52] S. Blond-Elguindi, S.E. Cwirla, W.J. Dower, R.J. Lipshutz, S.R. Sprang, J.F. Sambrook, M.J. Gething, Cell 75 (1993) 717–728. [53] G.C. Flynn, J. Pohl, M.T. Flocco, J.E. Rothman, Nature 353 (1991) 726–730. [54] D.P. Davis, R. Khurana, S. Meredith, F.J. Stevens, Y. Argon, J. Immunol. 163 (1999) 3842–3850. [55] B. Steipe, A. Pluckthun, R. Huber, J. Mol. Biol. 225 (1992) 739– 753.