Anti-HIV vaccine antibodies with reduced multi-reactivity
A technology of antibodies and antigens, applied in the direction of antibodies, antiviral immunoglobulins, medical preparations containing active ingredients, etc., can solve problems such as difficulties in improvement
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Embodiment 1
[0420] This example describes the materials and methods used in the subsequent examples below.
[0421] Polyreactivity assay: as described ( et al. 2012, mAbs, 4:6, 753-760), polyreactivity assay using ELISA to detect non-specific binding to baculovirus extract. Briefly, a solution of 1% baculovirus particles in 100 mM sodium bicarbonate buffer pH 9.6 was absorbed into the wells of a 384-well ELISA plate (Nunc Maxisorp) using a Tecan Freedom Evo liquid handling robot and the plate was incubated at 4°C. Incubate overnight. Plates were blocked with 0.5% BSA in PBS for 1 hour at room temperature. Purified IgG (diluted to 1 μg / mL in PBS with 0.5% BSA) was added to blocked assay plates and incubated for 3 hours at room temperature. Bound IgG was detected with a luminescent signal at 425 nm using HRP-conjugated anti-human IgG (H&L) secondary antibody (Genscript) and SuperSignal ELISA Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific).
[0422] Neutralization assay: ...
Embodiment 2
[0426] The parental antibody NIH45-46 G54W consists of NIH45-46 HC paired with NIH45-46 LC with the G54W mutation. New antibody variants have mutations in one or both of the chains.
[0427] The single chain used to construct the new antibody variant is:
[0428] Chain name description (mutations listed using standard Kabat numbering)
[0429] HC1 HC NIH45-46 G54W HC with T68D SAP10 LC NIH45-46LC with Q27E, S28H and S30D SAP10T LC NIH45-46LC with Q27E, S28H, S30D and S74T SAP10T-LS LC NIH45-46LC with Q27E, S28H, S30D, S74T, M428L and N434S SAP3 LC NIH45-46LC with Q27E, S28Y and S30D SAP8 LC NIH45-46LC with Q27D, S28H m2 LC NIH45-46LC with S28Y
[0430] The new antibody reagent consists of the following HC / LC pairings (LS denotes a mutation in the Fc region previously described, eg M428L / N434S):
[0431] HC1 / SAP10
[0432] HC1 / SAP10-LS
[0433] HC1 / SAP10T
[0434] HC1 / SAP10T-LS
[0435] HC1 / SAP8
[0436] HC1 / SAP...
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