Method for preparing antibodies having improved properties
A characteristic and antibody technology, applied in the direction of antibodies, chemical instruments and methods, anti-inflammatory agents, etc., can solve problems such as reduced affinity
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Embodiment 13
[0168] Example 13 provides an in vitro assay for measuring ADCC of B cell depletion and released fluorescence.
[0169] Bioavailability of Fc muteins
[0170] Bioavailability refers to the extent and rate at which the active moiety, whether it is a drug or a metabolite, enters the human circulatory system and thus accesses the site of action. In contrast to the physicochemical properties of the drug, the properties of the dosage form mainly affect the bioavailability of the drug, which in turn determines its absorption potential. Chemical equivalence indicates that the drug product contains the same active ingredient in the same amount, although other inactive ingredients may differ. Likewise, bioequivalence indicates that two drug products, when administered to the same patients on the same dosing regimen, will produce equivalent drug concentrations in the circulation and tissues. Conversely, two drug products that are not identical may be able to produce the same therapeut...
Embodiment 1
[0233] Strains and Reagents
[0234] Escherichia coli strain TOP 10 or DH5α (Invitrogen, CA) were used for recombinant DNA experiments. Restriction endonucleases, DNA modifying enzymes and PNGase F were obtained from New England Biolabs, Ipswich, MA. Oligonucleotides were ordered from Integrated DNA Technologies, Coralville, LA.
Embodiment 2
[0236] Construction of Anti-Her 2 IgG1 Fc Mutant Protein and Recombinant Expression Vector of Pichia pastoris
[0237] Production of single and double Fc muteins of the Her2 IgG1 monoclonal antibody in Pichia pastoris was performed using the sequences and protocols listed below.
[0238] A. Heavy and light chains
[0239] The sequences of the heavy and light chains, SEQ ID NO: 1 and 2, used to prepare the Her2 monoclonal IgG1 antibody, respectively, are described below. The amino acid sequence of the heavy chain anti-Her2 double mutein antibody is shown in SEQ ID NO:9. The heavy and light chains were codon optimized according to Pichia pastoris codon usage, synthesized by GeneArt AG (Josef-Engert-Str. 11, D-93053 Regensburg, Germany) and cloned into pUC19.
[0240] Using forward primer, reverse primer, FcF243A-F (SEQ ID NO: 3) and FcF243A-R (SEQ ID NO: 4) to carry out F243 aminoacid profiling with QuikChange? Site-Directed Mutagenesis Kit (Strategene, CA) amino acid Fc muta...
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