Analysis of the V genes coding for a monospecific human antibody to myosin and functional expression of single chain Fv fragments

Laroche‐Traineau, Jeanny; Jacobin, Marie-Josée; Biard-Piechaczyk, Martine; Vuillemin, L; Chagnaud, Jean-Luc; Pau, Bernard; Nurden, Alan T.; Clofent‐Sanchez, Gisèle

doi:10.1016/s0014-5793(99)01308-3

Cited by 2 publications

(3 citation statements)

References 41 publications

(53 reference statements)

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“…Interestingly, both Ag-driven and germline clones specifically recognized the ␣ IIb ␤ 3 integrin, thus confirming previous results that germline chain genes can code highly specific Abs (59,60). Studies are in progress to determine whether the highly specific germline IgG clones really secrete pathogenic Abs that retain a germline sequence or whether they represent anergized autoreactive B cells also found in nonimmunized subjects that possess the genetic potential to produce anti-␣ IIb ␤ 3 Abs (61).…”

Section: Discussionsupporting

confidence: 83%

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Human IgG Monoclonal Anti-αIIbβ3-Binding Fragments Derived from Immunized Donors Using Phage Display

Jacobin

Laroche‐Traineau

Little

et al. 2002

The Journal of Immunology

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Previous studies of the immune response in polytransfused Glanzmann thrombasthenia (GT) patients and in autoimmune thrombocytopenic purpura (AITP) have relied on serum analysis and have shown the frequent development of Abs directed against the αIIbβ3 integrin. However, little is known about the molecular diversity of the humoral immune response to αIIbβ3 due to the paucity of mAbs issuing from these pathologies. We have isolated human IgG anti-αIIbβ3 binding fragments using combinatorial libraries of single-chain IgG created from the B cells of a GT and an AITP patient, both with serum Abs. Ab screening was performed using activated platelets or activated αIIbβ3-expressing Chinese hamster ovary cells. Sequencing of selected phage Abs showed that a broad selection of genes from virtually all V gene families had been used, indicating the diversity of the immune response. About one-half of the VH and VL segments of our IgG anti-αIIbβ3 fragments displayed extensive hypermutations in the complementarity-determining region, supporting the idea that an Ag-driven immune response was occurring in both patients. The H chain complementarity-determining region 3 analysis of phage Abs revealed motifs other than the well-known RGD and KQAGDV integrin-binding sequences. To our knowledge, our study is the first to illustrate multiple human IgG anti-αIIbβ3 reactivities and structural variations linked to the anti-platelet human immune response. Human αIIbβ3 Abs preferentially directed against the activated form of the integrin were further characterized because platelet αIIbβ3 inhibitors are potential therapeutic reagents for treating acute coronary syndromes. Currently available αIIbβ3 antagonists do not specifically recognize the activated form of the integrin.

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Section: Discussionsupporting

confidence: 83%

“…Each sample corresponded to the scFv in 1 ml of culture. As a control, the scFv fragment derived from an hybridoma secreting an antimyosine mAb (B7) was used, isolated from periplasmic extracts (60). The positions of the molecular mass markers are shown on the right.…”

Section: Figurementioning

confidence: 99%

Human IgG Monoclonal Anti-αIIbβ3-Binding Fragments Derived from Immunized Donors Using Phage Display

Jacobin

Laroche‐Traineau

Little

et al. 2002

The Journal of Immunology

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“…19 The first-strand cDNA was synthetized according to a standard methodology using a forward primer located on murine immunoglobulin heavy chain g CH1 gene (amino acid residues 115-118: Bi4: 59 -CCAGGGGCCAGTGGATAGA-CAAGCTTGGGTGTCGTTTT-39 ), Bi4 containing a HindIII restriction site indicated in bold. 2 0 The heavy variable domain was amplified by polymerase chain reaction (PCR) using primers as follows: backward primer located on the first amino acid residues in the FR1 region of chains (Bi3f : 59 -CAGCCGG-CCATGGCGCAGGT(GC)CAGCTGCAG(GC)AG-39 ) containing a NcoI restriction site indicated in bold and forward primer as described above.…”

Section: Cloning and Sequencing Of The Heavy Chain Xiif9 Hybridoma Genementioning

confidence: 99%

Characterisation, cloning and sequencing of a conformation-dependent monoclonal antibody to the α IIb β 3 integrin: interest for use in thrombus detection

et al. 2001

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The detection of newly formed thrombi is of primary importance in clinical medicine. The activated platelet is a potential target for the localization of thrombotic lesions in arteries. The integrin alpha(IIb)beta(3) membrane changes conformation upon activation. A novel anti-alpha(IIb)beta(3) monoclonal antibody (MAb), XIIF9, is described which recognizes an epitope whose expression was enhanced by activation. Radioiodinated XIIF9 bound to a single class of sites on the beta(3) subunit, with 13600 +/- 2000 molecules bound per unstimulated platelet and a K(d) of 34.5 nM. Platelets stimulated with 0.5 U/ml of thrombin bound 66000 +/- 4000 molecules/cell (K(d) = 51.6 nM). Moreover, XIIF9 binding to unstimulated platelets could be increased 4-fold by treatment of the alpha(IIb)beta(3) complex with 5 mM EDTA. Thus, XIIF9 recognized an epitope on the beta(3) subunit whose accessibility was increased upon thrombin activation or EDTA treatment. Sequence analysis of the gene segment encoding the XIIF9 heavy chain revealed interesting motifs shared with cyclic CX6-7C anti-alpha(IIb)beta(3) peptides or with AC7, a published MAb specific for activated alpha(IIb)beta(3). In vivo experiments in atherosclerotic rabbits followed by immunohistological analysis, revealed a specific binding of XIIF9 on platelets engaged in thrombus formation, demonstrating real clinical potential for such MAbs in imaging.

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Analysis of the V genes coding for a monospecific human antibody to myosin and functional expression of single chain Fv fragments

Cited by 2 publications

References 41 publications

Human IgG Monoclonal Anti-αIIbβ3-Binding Fragments Derived from Immunized Donors Using Phage Display

Human IgG Monoclonal Anti-αIIbβ3-Binding Fragments Derived from Immunized Donors Using Phage Display

Characterisation, cloning and sequencing of a conformation-dependent monoclonal antibody to the α IIb β 3 integrin: interest for use in thrombus detection

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