Construction of bacteriophage expressing mouse monoclonal Fab fragments directed against the human MN glycophorin blood group antigens

Czerwiński, Marcin; Siegel, D; Js, Moore; Spitalnik, PF; Spitalnik, SL

doi:10.1046/j.1537-2995.1995.35295125736.x

Cited by 12 publications

(26 citation statements)

References 50 publications

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“…A similar lack of H chain promiscuity has been demonstrated in shuffling experiments for several antibodies derived from combinatorial immunoglobulin gene libraries including, for example, murine antibodies to glycophorin A (63). Third, a three-way analysis (64) confirmed the epitopic relationships between our recombinant human Fab that identify the TPO immunodominant region (11), a panel of mouse TPO monoclonal antibodies (17), and a panel of TPO-specific Fab isolated independently in a separate laboratory (50,56).…”

Section: Discussionsupporting

confidence: 70%

Human Monoclonal Autoantibodies to B-Cell Epitopes Outside the Thyroid Peroxidase Autoantibody Immunodominant Region

Pichurin

Guo

Yan

et al. 2001

Thyroid

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Human autoantibodies to thyroid peroxidase (TPO) interact with a restricted or immunodominant region (IDR) on intact TPO. However, a smaller proportion of polyclonal serum TPO autoantibodies bind outside this region. To isolate monoclonal nonimmunodominant region (non-IDR) TPO autoantibodies, we screened a thyroid-derived immunoglobulin gene phage display library while "epitope masking" the TPO IDR with four human TPO monoclonal autoantibodies that define the IDR. Among 31 non-IDR autoantibodies obtained (expressed as Fab), 8 representatives were analyzed further based on their restriction digestion profiles. All are encoded by almost identical H chains (VH3 family), with extremely long D regions, paired with three different types of light chains. In contrast, IDR TPO Fab from the same patient utilize seven different heavy chains (VH1 and VH5 families) paired nonpromiscuously with different light chains. Use of VH5 genes has not been reported previously for TPO autoantibodies. Both non-IDR and IDR Fab bind specifically to TPO and not to other proteins. The non-IDR Fab affinities for TPO are moderately high (Kd 1-2 x 10(-9) M), somewhat lower than those for most IDR Fab (Kd 1-4 x 10(-10) M). The epitopes of the three types of non-IDR Fab overlap with each other, indicating a major role for their heavy chain in TPO binding. Most importantly, the epitopes of non-IDR Fab are recognized by patients' serum autoantibodies. In summary, we provide the first insight into the immunoglobulin genes, affinities and epitopes of human monoclonal autoantibodies that bind outside the TPO-immunodominant region.

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Section: Discussionsupporting

confidence: 70%

Human Monoclonal Autoantibodies to B-Cell Epitopes Outside the Thyroid Peroxidase Autoantibody Immunodominant Region

Pichurin

Guo

Yan

et al. 2001

Thyroid

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“…An IgG light chain leader sequence was introduced 5′ for protein secretion, followed by two identical gp350 1-470 sequences separated by a (Gly 4 Ser 1 ) 3 linker to allow protein folding [25]. P 2 and P 30 were introduced 3′ to the second gp350 1-470 followed by a leucine zipper sequence [26, 27] for homodimerization. A 3′ His 6 tag was included for purification.…”

Section: Resultsmentioning

confidence: 99%

“…The PCR product was cloned into the pOptiVEC-TOPO vector, and was verified by sequencing. Gp350F2R5 contained sequences encoding the (Gly 4 Ser) 3 linker, the universal tetanus toxoid (TT)-specific CD4+ T-cell epitopes P 2 and P 30 [31], a leucine zipper [26, 27] and a His6 tag, and was created by 3 rounds of PCR, adding the coding sequences sequentially. The PCR product was cloned into the PCRII-TOPO vector (Invitrogen), and was verified by sequencing.…”

Section: Methodsmentioning

confidence: 99%

“…However, approaches to multimerize proteins in a defined and cost-effective manner for vaccine use, with direct validation of a resultant increase in immunogenicity, have been limited. In this study, we constructed and expressed a plasmid DNA in which two identical gp350 sequences are separated by a (Gly 4 Ser 1 ) 3 linker to allow for proper protein folding [25], followed by a Saccharomyces cerevisiae GCN4 leucine zipper sequence [26, 27] to mediate self-association of the gp350 dimer, and thus formation of a tetrameric gp350 protein. The gp350 utilized was truncated, consisting of the first 470 amino acids (gp350 1-470 ) that contain the CD21 binding site recognized by the neutralizing mAb, 72A1 [28, 29].…”

Section: Introductionmentioning

confidence: 99%

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A novel tetrameric gp3501–470 as a potential Epstein–Barr virus vaccine

Cui

Cao

Sen

et al. 2013

Vaccine

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Infectious mononucleosis and B-cell transformation in response to infection with Epstein-Barr virus (EBV) is dependent upon binding of the EBV envelope glycoprotein gp350 to CD21 on B-cells. Gp350-specific antibody comprises most of the EBV neutralizing activity in the serum of infected patients, making this protein a promising target antigen for a prophylactic EBV vaccine. We describe a novel, tetrameric gp350-based vaccine that exhibits markedly enhanced immunogenicity relative to its monomeric counterpart. Plasmid DNA was constructed for synthesis, within transfected CHO cells, of a tetrameric, truncated (a.a. 1-470) gp350 protein (gp3501-470). Tetrameric gp3501-470 induced ~20-fold higher serum titers of gp3501-470-specific IgG and >19-fold enhancements in neutralizing titers at the highest dose, and was >25-fold more immunogenic on a per-weight basis than monomeric gp3501-470. Further, epidermal immunization with plasmid DNA encoding gp3501-470 tetramer induced 8-fold higher serum titers of gp3501-470-specific IgG relative to monomer. Tetrameric gp3501-470 binding to human CD21 was >24-fold more efficient on a per-weight basis than monomer, but neither tetramer nor monomer mediated polyclonal human B-cell activation. Finally, the introduction of strong, universal tetanus toxoid (TT)-specific CD4+ T-cell epitopes into the tetrameric gp3501-470 had no effect on the gp3501-470-specific IgG response in naïve mice, and resulted in suppressed gp3501-470-specific IgG responses in TT-primed mice. Collectively, these data suggest that tetrameric gp3501-470 is a potentially promising candidate for testing as a prophylactic EBV vaccine, and that protein multimerization, using the approach described herein, is likely to be clinically relevant for enhancing the immunogenicity of other proteins of vaccine interest.

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“…The screening involved up to 3 rounds of enrichment on an immobilized target protein. After each cycle of enrichment about 15 individual clones were sequenced to In order to produce the phage particles presenting variants of BPTI, male cells of E. coli SURE transformed with the phagemid were infected with VCS-M13 helper phage at the rate of approximately 100 phages per bacterial cell (Czerwinski et al, 1995). The phages were cultured in SB medium containing 10 g/ml tetracycline and 100 g/ml ampicillin at 30°C.…”

mentioning

confidence: 99%

Phage Display Selection of P1 Mutants of BPTI Directed against Five Different Serine Proteinases

Kiczak¹,

Koscielska²,

Otlewski³

et al. 1999

Biological Chemistry

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The P1 position of protein inhibitors and oligopeptide substrates determines, to a large extent, association energy with many serine proteinases. To test the agreement of phage display selection with the existing thermodynamic data, a small library of all 20 P1 mutants of basic pancreatic trypsin inhibitor (BPTI) was created, fused to protein III, and displayed on the surface of M13 phage. The wild type of displayed inhibitor monovalently and strongly inhibited trypsin with an association constant of K a = 3 • 10 11 M -1 . The library was applied to select BPTI variants active against five serine proteinases of different specificity (bovine trypsin and chymotrypsin, human leukocyte and porcine pancreatic elastases, human azurocidin). The results of enrichment with four proteinases agreed well with the available thermodynamic data. In the case of azurocidin, the phage display selection allowed determination of the P1 specificity of this protein with the following frequencies for selected P1 variants: 43% Lys, 36% Leu, 7% Met, 7% Thr, 7% Gln.

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Construction of bacteriophage expressing mouse monoclonal Fab fragments directed against the human MN glycophorin blood group antigens

Abstract: These results suggest that Fab-phage may provide novel reagents with applications in immunohematology and may be useful in the study of the immune response to human blood group antigens.

Cited by 12 publications

References 50 publications

Human Monoclonal Autoantibodies to B-Cell Epitopes Outside the Thyroid Peroxidase Autoantibody Immunodominant Region

Human Monoclonal Autoantibodies to B-Cell Epitopes Outside the Thyroid Peroxidase Autoantibody Immunodominant Region

A novel tetrameric gp3501–470 as a potential Epstein–Barr virus vaccine

Phage Display Selection of P1 Mutants of BPTI Directed against Five Different Serine Proteinases

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