Pac1 antibodies and uses thereof
An antibody and antigen technology, applied in the direction of antibodies, antibody medical components, hormone receptors, etc., can solve problems such as intolerance, unmet medical needs of migraine, and ineffectiveness
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example 1
[0064] The crystal structure of the human PAC1 ECD-Fab complex described in Example 1 also reveals important residues in the CDRs of the heavy and light chains of the Fab that interact with amino acids in the human PAC1 ECD, thereby identifying the paratope of the antibody. key amino acids. A "paratope" is the region of an antibody that recognizes and binds a target antigen. Paratope residues include Gln27, Gly30, Arg31 and Ser32 in the light chain variable region (SEQ ID NO:3) and Arg31, Phe32, Tyr53, Asp54, Gly56 in the heavy chain variable region (SEQ ID NO:2) . Specific mutations of several of these residues in the paratope were designed to improve the interaction with the core interface residues (i.e., those in the epitope) in the human PAC1 ECD, resulting in enhanced binding affinity and inhibitory potency compared to the parental antibody ( See Examples 1-3). Gln27, Gly30, Arg31 and Ser32 in the light chain variable region of SEQ ID NO:3 or SEQ ID NO:52 correspond to...
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[0348] Example 1. Crystal structure of human PAC1 antibody-guided design
[0349] The crystal structure of the complex between the extracellular domain (ECD) of human PAC1 and the Fab fragment of a human anti-PAC1 neutralizing antibody (29G4v9) was determined. Human PAC1 ECD and anti-PAC1 Fab were purified separately and then complexed together in a 1:1 molar ratio. The sample was then run through a gel filtration column, equilibrated in 20 mM TRIS (pH 7.5), 50 mM NaCl, 5 mM EDTA, concentrated to 35 mg / ml and filtered.
[0350] The heavy chain (comprising variable region (VH), CH1 constant region, upper hinge and caspase III cleavage site) and light chain (comprising variable region (VL) and CL constant region) of the Fab fragment are listed below )the sequence of. The sequence of the human PAC1 ECD construct is provided below and comprises the region between amino acids 26 to 143 minus amino acids 89-109 of human PAC1 (SEQ ID NO: 1).
[0351] Amino acid sequence of the hea...
example 2
[0374] Example 2. Yeast Display Affinity Maturation of 29G4v10 Human PAC1 Antibody
[0375] An efficient approach to affinity maturation involves constructing a yeast-displayed library of antibody Fab mutants and selecting for improved binders by fluorescence-assisted cell sorting (FACS) ( Figure 4 ). To generate anti-PACl antibodies with improved inhibitory potency, the 29G4v10 antibody (VH region of SEQ ID NO: 191; VL region of SEQ ID NO: 52) was affinity matured by FACS of a yeast display Fab library. The library design was guided by the resolved co-crystal structure described in Example 1 of a complex between human PAC1 ECD and 29G4v9 Fab whose CDRs are nearly identical to those of 29G4v10. Structural analysis initially identified six and seven CDR positions for mutagenesis within the light chain (LC) and heavy chain (HC), respectively, and suggested a strategy for affinity enhancement. Mutants were designed to improve hydrophobic interactions and charge complementarity...
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