Construction and Characterization of Single-Chain Variable Fragment Antibody Library Derived from Germline Rearranged Immunoglobulin Variable Genes

Construction and Characterization of Single-Chain Variable Fragment Antibody Library Derived from Germline Rearranged Immunoglobulin Variable Genes

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Abstract

Antibody repertoires for library construction are conventionally harvested from mRNAs of immune cells. To examine whether germline rearranged immunoglobulin (Ig) variable region genes could be used as source of antibody repertoire, an immunized phage-displayed scFv library was prepared using splenocytic genomic DNA as template. In addition, a novel frame-shifting PCR (fsPCR) step was introduced to rescue stop codon and to enhance diversity of the complementarity-determining region 3 (CDR3). The germline scFv library was initially characterized against the hapten antigen phenyloxazolone (phOx). Sequence analysis of the phOx-selective scFvs indicated that the CDRs consisted of novel as well as conserved motifs. In order to illustrate that the diversity of CDR3 was increased by the fsPCR step, a second scFv library was constructed using a single scFv clone L3G7C as a template. Despite showing similar binding characteristics towards phOx, the scFv clones that were obtained from the L3G7C-derived antibody library gave a lower non-specific binding than that of the parental L3G7C clone. To determine whether germline library represented the endogenous immune status, specific scFv clones for nucleocapsid (N) protein of SARS-associated coronavirus (SCoV) were obtained both from naïve and immunized germline scFv libraries. Both libraries yielded specific anti-N scFvs that exhibited similar binding characteristics towards recombinant N protein, except the immunized library gave a larger number of specific anti-N scFv, and clones with identical nucleotide sequences were found. In conclusion, highly diversified antibody library can be efficiently constructed using germline rearranged immunoglobulin variable genes as source of antibody repertoires and fsPCR to diversify the CDR3.

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Introduction

Phage-displayed antibody library has been widely used to derive high-affinity target-specific antibodies, such as antibodies that were specific for angiogenesis marker fibronectin [1], melanoma-specific B3 and B4 antigens [2], epidermal growth factor receptor [3], HIV Vpr protein [4], and spike protein of SCoV [5], [6]. Antibody repertoires of phage-displayed library are conventionally created by harvesting mRNAs from peripheral blood lymphocytes, spleen, bone marrow, tonsil or similar sources using RT-PCR and family-based oligonucleotides [7], [8], [9], [10]. The heavy and light chains are then randomly combined and cloned to construct a combinatorial scFv library, from which specific antibodies against not-yet-encountered antigen are selected [11], [12], [13], [14].

Although the use of mRNAs ensures functional antibody genes retrieval, there are some limitations. Potential Ig genes may not be recovered from antibody cDNA library due to reading frame shifted or the presence of stop codon(s) which are generated by imprecise somatic recombination and P- and N- additions [15], [16]; or the immunoglobulins are self-reactive and thus eliminated by the host immune system [17], [18]. Besides, like other somatic cells, B cells are diploid and therefore rearranged Ig genes can only be expressed from one of the sister chromosomes while the other is concealed [18], [19]. In addition, chance of getting Ig genes against poor immunogenic targets is hampered by poor humoral response of immunized host. On the other hand, activated B cells undergo clonal expansion and therefore the antibody repertoires of a cDNA-derived scFv library would be dominated by antigen-stimulated humoral response. Hence recombinant antibody repertoires of a cDNA-derived antibody library are limited.

Germline Ig variable region (V) genes, which are selected over millions of years for their compatibility with many different antigens, are poly-functional and capable of orchestrating an effective immune-response [20], [21]. Indeed, antibodies that encoded by a very limited VH and VL genes of inbred mice were found to react with different haptens, polysaccharides, and even protein antigens [22], [23], [24], [25], [26]. However, the potential use of rearranged germline Ig genes as source of antibody repertoires for construction of antibody library has never been explored.

Structural analysis of antibody binding site suggests that only a few canonical conformations exist within the five CDRs except VH CDR3 loop which shows a wide range of variations in both length and space [27], [28]. Previous studies indicate that CDR3 diversity is the principle determinant of antigen-specificity and binding-affinity [29], [30], and the diversity of CDR3-FR4 junction determines how antibody undergoes affinity maturation [31]. Hence, modifications in CDR3 seem to be an efficient way of expanding antibody diversity beyond what are encoded by the germline Ig genes.

In the present study, we described the construction and characterization of germline scFv antibody fragment libraries using variable region of rearranged Ig genes as source of antibody repertoires. The germline Ig variable regions were further diversified using a novel frame-shifting PCR step to rescue stop codon(s) as well as to enhance diversity in the CDR3 loop. Indeed, in this proof-of-concept study, we showed that the germline scFv library was highly diverse, and specific antibodies against hapten and protein were obtained.

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Materials and Methods

Oligos and primers

All oligos and primers were custom synthesized by Invitrogen. The sequences and the relative position with respect to an scFv of various oligos and primers were detailed in Table 1 and Figure S1, respectively.

Table 1

Primer Pairs for Construction of Germline Phage-displayed scFv Library.

A. Mouse VH Forward Primers (FR1 Region)

FH1

5′- gAggTgMWgcTTRT-3′

FH2

5′-gAggTgMWgcTKVWgSAgTcTggA-3′

B. Mouse VH Reverse Primers (FR4 Region)

RH1

5′- gAcDgTgASHGWRGTYC -3′

RH2

5′-gAcDgTgASHRDRgTBccTKSRccccA-3′

C. Mouse V Forward Primers (FR1 Region)

FK11

5′-gAHRTYgTKMTSAc-3′

FK12

5′-gAHRTYgTKMTSAcMcARWcTMcA-3′

D. Mouse V Reverse Primers (FR4 Region)

RK11

5′-KATYTccARYYTKgT-3′

RK12

5′-KATYTccARYYTKgTSccHBcDccgAA-3′

E. Frame-shifting primers

RFSH

5′-gAcDgTgASHRDRgTBccTKSRccccANNNNNN-3′

RFSK

5′-YYTKgTSccHBcDccgAAYgTNNNNNN-3′

F. Linker primers for VH

SBS1

5′-cgAgcTcggATccggcccAgccggccSAggTgMWgcTKVWgSAg-3′

L1JP

5′-AgAAccgcTgccTgAAccgccTccAccAcTgAcDgTgASHRDRgTBccT-3′

G. Linker primers for V

L2JP

5′-cAggcAgcggTTcTAgcggcggTggcggAgAHRTYgTKMTSAcMcARWc-3′

KN1

5′-cggggTAccgcggccgcKATYTccARYYTKgTSccHBcDccgAA-3′

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For the primer nucleotide sequence, the non-standard bases are: N = A+G+C+T; R = A+G; Y = C+T; M = A+C; K = G+T; S = G+C; W = A+T; H = A+C+T; B = G+C+T; D = A+G+T; V = A+G+C.

Isolation of splenocytes

The protocol for animal work was approved by the Animal Experimentation Ethics Committee of the Chinese University of Hong Kong (Permit Number: 03/015/MIS). BALB/c mice (∼25 g) were sacrificed by cervical dislocation and the spleens were immediately removed. After punching holes with a G21 needle, splenocytes were squeezed out and resuspended in an ice-cold phosphate buffered saline (PBS) containing 4% BSA (Sigma). For isolation of CD19+ cells, splenocytes (2×107 cells/ml, 5 ml) were incubated with a fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse CD19+ monoclonal antibody (0.5 mg/ml, 100 µl, BD Bioscience) on ice for 30 min to label B-lymphocytes carrying rearranged immunoglobulin genes. After washing to remove unbound antibodies, labeled cells were resuspended in 5 ml of RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco). The CD19+ splenocytes were sorted and captured using a FACS VANTAGE SE cell sorter (Becton Dickinson).

Retrieval of variable regions from germline rearranged Ig genes

Splenocytic genome DNA was extracted using DNAzol® reagent (Invitrogen) as described by the manufacture. The variable regions of germline rearranged Ig genes were then retrieved using semi-nested PCR (snPCR). The 1st round snPCR aimed at amplifying the rearranged Ig variable regions using genome DNA as template, and reaction was carried out in a final volume of 50 µl containing 1X PCR buffer (Perkin-Elmer), 1.5 mM MgCl2 (Perkin-Elmer), 0.2 mM dNTP (Invitrogen), 5% DMSO (MB grade from Sigma), 2.5 U non-proof reading Taq polymerase, 200 ng of extracted splenocytic genome DNA and VH degenerate primer pair of 0.3 µM FH1 and 1.5 µM RH1, or V degenerate primer pair of 0.45 µM FK11 and 0.3 µM RK11. Following pre-denatured at 94°C for 60 s, the reaction mixture was subjected to PCR amplification that consisted of two stages. The first stage was composed of 10 amplification cycles each consisting of 4 amplification steps, including denaturation at 94°C for 30 s, annealing at 40°C for 60 s, a ramping up extension step from 40°C to 72°C at a rate of 0.2°C/s, and then followed by an extended incubation step at 72°C for 1 min. Then the samples were subjected to another stage of 20 amplification cycles each consisting of denaturation at 94°C for 30 s, a touch up annealing step from 40°C to 50°C at a rate of 0.5°C/cycle for 1 min, and an extension step at 72°C for 60 s. Following the amplification cycles, the reaction mixture was further subjected to a post-extension step at 72°C for 2 min. The PCR reactions were carried out in a GeneAmp® 9700 PCR thermocycler with 96-well aluminum plate (Applied Biosystems). The PCR products were stored at 4°C until use.

To enrich Ig variable region fragments, PCR products of 1st round snPCR were used as templates for 2nd round of snPCR with primer pair carrying additional oligonucleotides at the 3′-end that bracketed only Ig-like templates being further amplified (Figure S1). The 2nd round snPCR was carried out in a final volume of 50 µl containing 1X PCR buffer; 1.5 mM MgCl2, 0.2 mM dNTP, 5% DMSO, 2.5 U non-proof reading Taq polymerase, and 4 µl of the 1st round PCR products. For retrieving VH segments, 0.3 µM FH2 and 1.5 µM RH2 were used. For V semi-nested PCR, 0.5 µM FK12 and 1.125 µM RK12 were used. Reaction mixture was pre-denatured at 94°C for 2 min, and then subjected to PCR amplification that consisted of three stages. The first stage is composed of 5 amplification cycles each consisting of denaturation at 94°C for 30 s, annealing at 65°C for 30 s and extension at 72°C for 30s. The second stage was composed of 25 amplification cycles each consisting of a denaturation step at 94°C for 30s, a touch-down annealing step from 65°C to 55°C at a rate of 0.4°C/cycle for 30 s and an extension step at 72°C for 30 s. The third stage was composed of 5 amplification cycles each consisting of a denaturation step at 94°C for 30s, an annealing step at 55°C for 30 s and an extension step at 72°C for 30 s. Following the amplification cycles, the reaction mixture was further subjected to a post-extension step at 72°C for 2 min. The PCR products were stored at 4°C until use.

To purify the Ig variable region gene fragments, PCR products were pooled and subjected to agarose gel electrophoresis. Agarose gel stripes containing the Ig gene fragments were placed into a dialysis tubing (MWCO 3,500 from Spectrum) together with 1 ml of TAE buffer, DNA fragments were electro-eluted, purified by phenol chloroform extraction and then precipitated by ethanol. The purified PCR products were either cloned into TOPO TA (Invitrogen) or pGEM-T Easy (Promega) cloning vectors for nucleotide sequence determination, or used for subsequent CDR3 diversity enhancement.

Frame-shifting PCR for CDR3 diversity enhancement

The frame-shifting PCR (fsPCR) was performed as described in our US patent [32]. Briefly, the frame-shifting reaction was carried out in a final volume of 50 µl containing 1X PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTP, 2.5 U non-proof reading Taq polymerase, 60-100 ng gel-extracted VH or V DNA fragments, and VH primer pair of 0.5 µM FH1 and 4 µM VH frame-shifting reverse primer RFSH; or V primer pair of 0.5 µM FK12 and 4 µM V frame-shifting reverse primer RFSK. For both VH and V fsPCR, the reaction mixture was pre-denatured at 94°C for 2 min. The 25 fsPCR cycles were carried out in a GeneAmp® 9700 PCR thermocycler with 96-well aluminum plate (Applied Biosystems). Each fsPCR cycle consisted of a denaturation step at 94°C for 30 s, an annealing step at 20°C for 2 min and an extension step with temperature ramping up from 20 to 94°C at a rate of +0.1°C per second (or 5% ramping-speed of GeneAmp® 9700 PCR thermocycler with 96-wells aluminum plate). The fsPCR products were stored at 4°C until use. After separation on a 1.5% agarose gel, DNA fragments with a size range of 300–400 bp were electro-eluted, phenol-chloroform purified, and ethanol precipitated. The purified PCR products were either cloned into TOPO TA (Invitrogen) or pGEM-T Easy (Promega) cloning vectors for nucleotide sequence determination, or used for subsequent scFv construction.

ScFv construction

The CDR3-diversified germline Ig VH and V DNA fragments were randomly linked together using a two-stage overlap extension PCR (oePCR). The 1st-stage oePCR was carried out in a reaction volume of 50 µl containing 1X PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTP, 2.5 U non-proof reading Taq polymerase, 120 ng of frame-shifted Ig VH or V DNA fragments, and 0.2 µM each SBS1 and L1JP (VH primer pair); or L2JP and KN1 (V primer pair). The 1st-stage oePCR was used to add an adaptor and a linker to the retrieved Ig variable region of heavy or light chains. After a pre-denaturing step at 94°C for 2 min, 15 amplification cycles were carried out. Each cycle consisted of a denaturation, an annealing, and an extension steps at 94°C, 54°C, and 72°C for 30 s, respectively. After an extended incubation at 72°C for 5 min, the PCR products were stored at 4°C until use. The PCR products (5 µl) were used as templates for the subsequent 2nd-stage oePCR for scFv construction.

The 2nd-stage of oePCR was used to join the heavy and light chains randomly into an scFv. The reaction was carried out in a reaction volume of 50 µl containing 1X PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTP, 2.5 U non-proof reading Taq polymerase, 5 µl of PCR products of the 1st-stage oePCR (both VH and V) and 0.2 µM each SBS1 and KN1 primers. The mixture was pre-denatured at 94°C for 2 min, then subjected to 20 amplification cycles. Each cycle consisted of denaturation at 94°C for 30 s, annealing at 62°C for 30s, and extension at 72°C for 90s. After an extended incubation at 72°C for 5 min, the PCR products were stored at 4°C until use.

ScFv Library construction and specificity assessment

The Not 1- and Sfi 1- restricted scFv repertoires were cloned into pCANTAB 5E phagemid vector (GE Healthcare). Library was constructed by electroporation of the ligated products into competent TG1 E. coli as described in our previous publication [33]. Subsequently, log-phase TG1 transformants were super-infected with M13KO7 helper phage (GE Healthcare) in a multiplicity of infection (moi) ratio of 3∶1, and phages were rescued at 30°C overnight with gentle shaking. The overnight culture of scFv-phages was purified by polyethylene glycol precipitation (20% PEG8000 and 2.5 M NaCl). Purified phages were resuspended in 4 ml of a pre-blocking buffer (1X PBS, 0.2% Triton X-100, 0.01% NaN3, 0.1% BSA and 10% non-fat milk) and incubated at room temperature for 30 min prior panning. Phages (0.5 ml/well) were panned against immobilized antigen as described previously [34], [35]. Briefly, antigen (5 – 200 µg) was immobilized in a 24-well plate and incubated with the scFv library. After incubation at room temperature for 2 hr with gentle shaking, bound scFv-phages were eluted with 100 µl of 0.1 M glycine-HCl, pH 2.2. After 10 min acid-incubation at room temperature, the eluant was neutralized with 10 µl of 1 M Tris-HCl, pH 8.0. Specificity of eluted phages was confirmed by phageELISA and competitive phageELISA with free ligand in antigen-competition manner, bound phages were detected with an anti-M13 peroxidase-conjugated antibody (GE Healthcare).

Nucleotide sequence analysis

Nucleotide sequence determinations were performed by dye-terminator cycle sequencing using Beckman CEQ DTCS Kit as recommended by manufacturer, or by custom sequencing service. Sequences obtained were compared with NCBI IgBLAST® and analyzed against VBASE2 Ig database [36]. Multiple sequence alignment was performed by ClustalW® from EMBL-EBI server with following default conditions: matrix, BLOSUM; gap opening penalty, 10.0; gap extension penalty, 0.05; gap separation penalty, 8; maxdiv, default; no end gap separation penalty. Alignment in the CDR3 was further adjusted manually in accordance with physical property of amino acid residues.

PhageELISA

PhageELISA was performed as described previously [35]. Briefly, the phageELISA assay was carried out in a 96-well ELISA plate which was pre-coated with an antigen (0.3–50 µg). After incubation with 100 µl of scFv-phages at 37°C for 1 hr, in the absence or presence of free ligand, bound phages were detected by incubation with 100 µl of a horseradish peroxidase-conjugated anti-M13 mouse antibody (GE Healthcare) at 37°C for 1 hr. Activity of horseradish peroxidase was measured by a colorimetric method with o-phenylenediamine/H2O2 as substrates. Color was allowed to develop for 1 hr at room temperature, and absorbance at 450 nm was measured with a µQuant™ micro-plate reader (Bio-Tek).

His-tagged scFv expression

For expression analysis, scFv clones were subcloned into pCantab His vector, transformed into E Coli HB2151, and periplasmic His-tagged scFvs were purified using Ni-NTA agarose beads as described in our previous publication [35].

Recombinant N protein

Recombinant SCoV-N protein was cloned, bacterially expressed and purified as described in our previous publication [34].

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Results

Retrieval of variable regions of germline rearranged Ig genes

To retrieve the variable regions of rearranged Ig genes from genomic DNA, degenerate primer pair that is complementary to the antibody framework segment 1 (FR1) and FR4 of immunoglobulin chain was used for semi-nested PCR. As shown in Figure 1A, semi-nested PCR generated DNA fragments that displayed a size corresponding to VH (lanes 2 and 5) and V (lanes 4 and 6) variable segments. To confirm the DNA fragments being Ig genes, the PCR products deriving from splenocytic genomic DNA of naïve non-immunized mice were gel-purified and then cloned into vectors. The VH or V transformants (∼100 each) were randomly picked for sequencing. Based on successfully sequenced clones, 92.0% (96/104) and 100% (84/84) of clones were found to be murine Ig VH, and murine Ig V, respectively (Figure 1B).

Figure 1

Retrieval of the variable region of rearranged germline immunoglobulin genes by semi-nested PCR.

(A) Genomic DNA (200 ng) from CD19+ splenocytes was used as templates for semi-nested PCR to retrieve the variable region of rearranged Ig genes. The 1st round PCR products of heavy- (lane 1) and κ light- (lane 3) chains were used as templates for the 2nd round semi-nest PCR (lane 2 and 4). Sizes of PCR products were estimated against the 1 Kbp DNA ladders (Marker). Primers used and PCR protocols are described in Methods. After gel purification, the 2nd round semi-nested PCR products of heavy- (lane 5) and κ light- (lane 6) chains were cloned into Topo TA or pGEM-T vectors for nucleotide sequence determination. (B) Sequence analysis indicates that 92% and 100% of the randomly picked VH or V clones are mouse immunoglobulin genes, respectively. The sequence VH (C) or V (D) clones were further analyzed against the VBASE2 Ig database, indicating all the sequenced clones are germline-derived. Class 1 genes are with genomic and rearranged evidence; Class 2 genes are with genomic evidence only; Class 3 genes are with rearranged evidence only as defined by VBASE2.

To analyze the diversity of retrieved Ig genes and to confirm their germline origin, VH and V clones were searched for homology with germline Ig variable region genes against the VBASE2 database (Table S1). Sequence analysis indicated that the retrieved VH and V genes were mainly derived from 6 out of the 15 VH (Figure 1C) and 4 out of the 19 V (Figure 1D) subfamilies, respectively. Intriguingly, in some case the number of Ig clone with unique sequence exceeded the number of germline variable region genes. For instance, a total of 59 unique V clones were identified as members of the V 21 subfamily while the estimated germline Ig gene number of the V 21 subfamily is only 6-13, suggesting retrieved Ig genes might have derived from both rearranged germline and hypermutated Ig genes.

Diversification of CDR3 by frame-shifting PCR

To rescue the non-functional rearranged Ig genes and to enhance the diversity of CDR3, retrieved variable regions of Ig genes were subjected to frame-shifting PCR (fsPCR) that aimed at introducing sequences variation in CDR3. Essentially, the frame-shifting reverse primers (RFSH for VH and RFSK for V) are composed of two portions. In the 5′-portion of the frame-shifting reverse primer is a 5′-degenerate sequence of J gene-segments while the 3′-portion is a random hexamer. We reasoned that the 5′-degenerate J gene sequence would bring the frame-shifting reverse primer close to the CDR3-J region of the Ig templates while the 3′-random hexamer would imperfectly anneal to the CDR3 (Figure 2A). As for the low sequence similarity and the sequence heterogeneity of 3′-random hexamer, annealing between templates and the frame-shifting reverses primer would not be precise and resulted in generating a library of immunoglobulin chains with different CDR3 nucleotide sequences. Indeed, as illustrated in Figure 2B, fsPCR generated DNA fragments that spread around 220-396 bp for VH and 250-344 bp for V Ig genes.

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Figure 5

Panning performance and nucleotide sequence analysis of single temple-derived scFv library.

Phage clone L3G7C with moderate affinity towards phOx was subjected to fsPCR and the products were used to construct a phage-displayed scFv library. (A) Panning performance of L3G7C-derived scFv library against immobilized phOx-CSA conjugate. Phage clones of the L3G7C-derived scFv library were randomly picked for sequence determination. Nucleotide and putative amino acid sequences of the (B) heavy- and (C) κ light-chain CDR3 (H3 and L3, respectively) were aligned. Nucleotide sequence variation was noted in the CDR3, and lack of consensus antibody FR4 was noted in some of the phage clones.

Reactivity of the selected clones was further evaluated by phageELISA, and 7 distinct clones that exhibited high phageELISA signal were identified (Table S4). PhageELISA indicated the selected clones bound phOx avidly with minimum binding to other control antigens, except LPS (Figure 6A). There was no apparent changes in specific binding (Figure 6B), but clones L10D5A and L10D10A gave a significant lower binding than the parental L3G7C to the unrelated antigens insulin, angiotensin and ssDNA (Figure 6A). Despite clones L10D5A and L10D10A still gave noticeable non-specific binding towards LPS, the binding was substantially lower than the parental L3G7C. These results suggested that fsPCR diversified the CDR3 sequences, which resulted in decreasing the non-specific binding and therefore enhancing the signal-to-noise ratio of antigen binding. Hence, fsPCR could be used for optimizing the binding selectivity of antibody.

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Figure 7

Characterization of anti-N scFv-phages.

(A) Western protein analysis of scFv-phage binding to recombinant SCoV-N protein. Purified His6-SCoV-N recombinant protein was separated on gradient sodium dodecyl sulfate (5% - 20%) polyacrylamide gel and transferred onto a 0.2 µm nitrocellulose membrane. The stripes were probed with different anti-N scFv-phages, including L9A6B, L9A11B, L4A3A, L4A3B, L4A8B, and M13KO7 helper phage (negative control) as indicated. Purified SCoV-N protein was stained with Coomassie Blue for reference. (B) Cross-reactivity of selected anti-N scFv clones was evaluated using phageELISA against different antigens, including BSA, SCoV-N, insulin, thyroglobulin, Ang II-BSA conjugate, LPS, ssDNA as indicated. Error bars represent SEM of three separate experiments each performed in duplicate. Difference between means of binding to SCoV-N protein versus to other unrelated antigens is evaluated by One Way ANOVA using GraphPad Prism 4. Statistical significance is indicated as *** (P<0.001).

Jawahar Raina

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