Dynamic human antibody light chain libraries
An antibody light chain, library technology, applied in libraries, chemical libraries, nucleotide libraries, etc., can solve problems such as increased possibility
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Embodiment 1
[0137] Example 1: Identification of a minimal set of dynamic motifs on hypervariable regions
[0138] To understand the variability of antibody variable domains at the structural level, algorithms were developed to map the geometric alignment of antibody variable domains and, moreover, to calculate structure and sequence entropy based on the geometric alignment. Using this approach reconciles the classical theory of antibody diversity determined by a well-defined V(D)J recombination process with conformational diversity from dynamic units (template-guided conformational selection proposed by Linus Pauling; see for example James, L . and Tawfik, D. "Conformational diversity and protein evolution–a 60-year-old hypothesis revisited", Trends Biochem Sci 2003 Jul;28(7):361-8) combinatorially to allow the sampling of the virtually unlimited epitope space by selection and adaptation of antibody binding sites. As an example, this algorithm was used to analyze the structural and sequ...
Embodiment 2
[0140] Example 2: Construction of common light chain library
[0141] Construction of light chain library DL280
[0142] To begin construction of the DL280 light chain library, 20 minigenes encoding 20 combinations of HVR-L1 and HVR-L2 sequences (Table 1) were synthesized. The synthetic minigene was then digested with Pvul and BamHI and ligated into the targeting vector Fad22 digested with the same two restriction enzymes. The ligation mix was transformed into DH10B cells, 20 constructs were purified, and sequence verified for next stage construction.
[0143] A total of 14 pairs of oligomers encoding 14 HVR-L3 sequences (Table 1) were designed and synthesized. Oligomer annealing was performed in the PCR machine with the following settings: 95°C for 3 minutes, followed by 35 cycles (each cycle lasting 15 seconds) starting at 95°C, each cycle reducing the temperature by 1°C . After annealing, each pair was individually digested with PstI and Acc65I and individually ligated ...
Embodiment 3
[0155] Example 3: Screening of Common Light Chain Libraries to Isolate Antibodies of Interest
[0156] Preparation of Common Light Chain Library Phagemid Particles
[0157] To prepare common light chain library phagemid particles for antigen panning, 1.6 liters of ER2738 cells with a common light chain library (described in Example 2 above) were incubated at a starting OD of 0.1 600 Inoculate in medium containing 2xYT, 2% glucose, 100 μg / mL ampicillin and 12.5 μg / mL tetracycline. Grow the culture at 37 °C with shaking at 250 rpm until it reaches an OD of 0.6-0.8 600 . Cells were then infected with M13KO7 helper phage at a multiplicity of infection (MOI) of 10 for 30 minutes at 37°C. Infected ER2738 cells were grown overnight at 22°C in 3.2 liters of medium containing 2xYT, 100 μg / mL ampicillin and 50 μg / mL kanamycin. The culture supernatant was then collected by centrifugation at 10,000 rpm for 15 minutes and filtered through a 0.45 μm low-binding membrane filter (Corning)...
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