P-cadherin antibodies
A cadherin and antibody technology, applied in the direction of antibodies, immunoglobulins, antibody medical components, etc., can solve the problems of tumor cell proliferation and survival reduction
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Embodiment 1
[0304] Example 1: Screening scFv phage display library
[0305]Recombinant human P-cadherin (R&D Systems 861-PC-100) was used as antigen to screen the scFv phage display library. Large scFv human antibody libraries cloned into phagemid vectors were used for selection (Vaughan, T. J. et al., Nat. Biotech. 14:309-314 (1996)). scFvs recognizing P-cadherin were isolated from the phage display library in a series of repeated selection cycles on confluent monolayers of recombinant human P-cadherin and P-cadherin-expressing HCT116 cells. Briefly, following incubation with the library, bound phage were recovered from P-cadherin and unbound phage were washed away. Bound phage are then recovered as described in Vaughan, T. J. et al., Nat. Biotech. 14:309-314 (1996), and the selection process repeated. A representative proportion of clones from the output of the selection cycle were subjected to phage enzyme-linked immunosorbent assay (ELISA) to detect binding to P-cadherin, essentia...
Embodiment 2
[0306] Example 2: Lead optimization
[0307] Oligonucleotide-directed mutagenesis of antibody variable heavy chain (V H ) and variable light chain (V L ) CD3 region to construct a phage display library from 129-1c4. Using eg Clackson and Lowman, Phage Display-A Practical Approach The library was constructed using standard molecular biology techniques as described in (Oxford University Press 2004). Affinity-based selection was thus performed; after incubation with the library, recombinant human P-cadherin (R&D Systems) was captured by protein G-coated paramagnetic beads (Dynal 100.03), and bound phages were recovered by magnetic separation, while not Bound complexes are washed away. The selection process was repeated with decreasing concentrations of recombinant human P-cadherin present (25 nM to 10 pM in 4 rounds). Additionally, from V H CDR3 and V L Selection output from the CDR3 library was recombined into another phage display library and selected in two addit...
Embodiment 3
[0308] Example 3: P-cadherin dependent adhesion assay
[0309] IC was determined using several optimized scFvs in a P-cadherin-dependent adhesion assay using the following protocol 50 value, the optimized scFv was converted to IgG in the antibody recovery optimization stage as described above in Example 2. The average measured IC of these antibodies 50 Values are shown in Table 4 below.
[0310] 24 hours before use, with 2mM CaCl 2 Recombinant human P-cadherin Fc (R&D Cat.861-PC) was reconstituted with MilliQ aqueous solution to a concentration of 1 mg / mL and stored at 4°C. A431 cells were cultured and prepared as follows. Routinely, A431 cells (ECACC No.85090402) were cultured in Nunc ternary flasks (3x175cm 2 area), the minimum essential medium contains 10% fetal bovine serum (Invitrogen Cat. 10100-147) and 1% non-essential amino acids (Invitrogen Cat. 11140-035). Cultured cells were about 80% confluent when harvested for assay. To prevent possible passage-related...
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