Antibodies Containing Site-Specific Unnatural Amino Acid Residues, Methods of Making and Using Them
An unnatural amino acid, antibody technology, applied in chemical instruments and methods, medical preparations with non-active ingredients, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc. rate, inhomogeneity, etc.
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[0504] Antibody preparation
[0505] Antibodies described herein can be prepared by any technique apparent to those skilled in the art. without limitation. Useful techniques for preparation include in vivo synthesis (eg, using modified tRNAs and tRNA synthetases), cell-free synthesis (eg, using modified tRNAs and tRNA synthetases), solid-phase polypeptide synthesis, and solution-phase polypeptide synthesis. This section and the Examples below describe exemplary techniques.
[0506] In certain methods, antibodies are translated and / or transcribed from one or more polynucleotides encoding antibody polypeptide chains. Accordingly, provided herein are polynucleotides capable of encoding antibodies having one or more unnatural amino acids at site-specific positions in one or more polypeptide chains. In certain embodiments, the polynucleotide comprises a codon not normally associated with an amino acid at a nucleotide position corresponding to a site-specific polypeptide pos...
Embodiment 1
[0533] Synthesis and Interaction of Exemplary Antibodies Comprising Unnatural Amino Acids
[0534] Conjugation to Exemplary Cytotoxic Agents
[0535] The following examples describe exemplary antibodies containing unnatural amino acids at defined positions, and methods for their construction and expression. Antibodies were evaluated for expression, inhibition efficiency, conjugation efficiency to cell killing agents, cell binding, and cell killing as set forth below. In this example, an exemplary antibody is based on the parental antibody trastuzumab (US Pat. Nos. 6,165,464 and 2006 / 0018899A1; Carter et al., 1992, Proc. Natl. Acad. Sci. USA 10:4285-4289).
[0536] First, constructs were prepared by incorporating TAG amber codons at defined positions in the heavy chain of trastuzumab. Utilize the C-terminus (His) containing 6 Site-directed mutagenesis was performed using the pYD plasmid for the coding region of trastuzumab for the tag (SD02005) as a DNA template, an...
Embodiment 2
[0545] Characterization of Exemplary Antibody-Drug Conjugates
[0546] Hydrophobic interaction chromatography (HIC) was performed to quantify the samples and determine the drug-antibody ratios of the trastuzumab variant drug conjugates as follows. Samples and standards were diluted 1 :2 in 3M ammonium sulfate (EMD Chemical), in 50 mM sodium phosphate pH 7.0 (Mallinckrodt) prepared in MilliQ water. An Agilent 1100 binary pump HPLC system was equipped with Tosoh Bioscience LLC TSK-gel with a column oven temperature of 30°C (4.6mm x 3.5cm) column. Mobile phase A was 1.5M ammonium sulfate, 50 mM sodium phosphate pH 7.0. Mobile phase B was 50 mM sodium phosphate pH 7.0 in 80:20 water:isopropanol (Honeywell). The mobile phase was delivered at a flow rate of 1.0 mL / min. The separation was performed with a linear gradient from 15% mobile phase B to 100% mobile phase B in 10 minutes. UV data were acquired at both 210 nm and 280 nm. Peak areas were quantified using ChemStati...
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