Fully human influenza m2 specific antibodies
a technology of m2 and specific antibodies, applied in the field of human antibodies, can solve the problems of the same disadvantages, and achieve the effects of less severe side effects, high affinity, and high affinity
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example 1
Identification of M2-Specific Single-Chain Antibodies by Mammalian Cell Display
[0168]Peripheral blood mononuclear cells (PBMC) were isolated from 10 ml of heparinized blood of an individual with high M2-titers using the BD Vacutainer™ CPT™ Tube method (BD Biosciences, Franklin Lakes, N.J.). PBMC were pre-incubated with Alexa 647 nm-labeled Qβ (5.5 μg / ml) and human gamma globulin (11 μg / ml; Jackson ImmunoResearch) and then stained with: (1) an M2 extracellular domain consensus peptide (M2e; SEQ ID NO:48, cf. Table 7) coupled to Qβ (2.4 μg / ml) in combination with a Alexa 488 nm-labeled Qβ-specific mouse mAb (2 μg / ml), as well as a M2-specific mouse mAb (0.5 μg / ml) in combination with FITC-labeled donkey anti-mouse IgG antibody (1 μg / ml; Jackson ImmunoResearch); (2) PE-labeled mouse anti-human IgM (diluted 1:50; BD Biosciences / Pharmingen), mouse anti-human IgD (diluted 1:100; BD Biosciences / Pharmingen), mouse anti-human CD14 (diluted 1:50; BD Biosciences / Pharmingen), and mouse anti-hum...
example 2
Gene Rescue, ELISA Screening and Sequencing of M2-Specific Antibodies
[0170]The supernatants of BHK cells encoding putative M2e-specific antibodies, each containing a monoclonal recombinant Sindbis virus, were subjected to RT-PCR as described (see WO 2008 / 055795 A1). The resulting PCR products, each comprising a scFv coding region, were digested with the restriction endonuclease Sfi1 and cloned into the expression vector pCEP-SP-Sfi-Fc (disclosed as SEQ ID NO:37 in WO 2008 / 055795 A1), allowing for expression of scFv proteins fused to a C-terminal human Fc-γ1 domain under the control of a CMV promoter.
[0171]For ELISA analysis, each of the clones was transfected into HEK-293T cells in a 24-well plate format, using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. 2-3 days post transfection, supernatants containing transiently expressed scFv-Fc fusion proteins were harvested. To check for M2e-specific binding, ELISA plates were coated with M2e-conjugated R...
example 3
Expression and Purification of M2-Specific scFv-msFcγ2c Fusion Proteins
[0174]To investigate the effect of M2e-specific human antibodies on Influenza A infection in a mouse model, clones D005, E040 and F052 were expressed and purified as scFv-mouse Fc-γ2c (msFcγ2c) fusion proteins. The nucleotide sequences encoding the D005, E040 and F052 scFv-mouse Fc-γ2c (msFcγ2c) fusion proteins correspond to SEQ ID NOs:42, 44 and 46, respectively. The amino acid sequences of the D005, E040 and F052 scFv-mouse Fc-γ2c (msFcγ2c) fusion proteins correspond to SEQ ID NOs: 43, 45 and 47, respectively. The corresponding scFv coding regions were excised from the pCEP-SP-Sfi-Fc expression vectors with the restriction endonuclease Sfi1 and the resulting fragments were cloned into the expression vector pCEP-SP-Sfi-msFcγ2c (SEQ ID NO:41). The Sfi-digested fragments of monoclonal antibodies D005, E042, and F052 correspond to SEQ ID NOs 38, 39 and 40, respectively. These Sfi-fragments encode the entire scFv fr...
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