Ch3 domain epitope tags
A technology of domains and structural loops, applied in peptides, immunoglobulins, hybrid immunoglobulins, etc.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0053] Example 1. Ig-fold protein derived epitope tags. Briefly, sequence modifications within the AB, CD, and EF loops are based on the substitution of AB, CD, or EF loop wild-type sequences with sequences derived from the corresponding domains of other structurally related Ig-fold proteins. This approach resulted in the identification of unique epitopes for incorporation into the human CH3 domain. Sequence modifications of the AB loop were generated in the context of the G1m1 and nG1m1 allotypes (DEL and (vs) EEM, respectively).
[0054] Identification of sequences corresponding to the AB, EF and CD loops by performing a multiple sequence alignment of the primary amino acid sequences of various Ig-fold proteins with the CH2 and CH3 domain sequences of the human IgG1 Fc region derived from the crystal structure associated with PDB 4WI2 Candidate Ig-fold CH3 loop sequences, summarized in Figures 2A-2C middle. up to April 11, 2018 , the alignment contains eight Ig-fold p...
Embodiment 2
[0065]Example 2. Sterically favorable amino acid substitutions in the AB and EF loops. Using the molecular visualization system software PyMOL, the positions of the loops were modeled using the human IgG1 Fc region derived from the PDB 4WI2 or 4NOU crystal structures and the solvent-exposed amino acid side chains within the AB and EF loops were identified. Modeling of amino acid substitutions using PyMOL's mutagenesis features allowed analysis of the steric effects of various amino acid substitutions for residues exposed on the inner surface of the AB and EF loops. Substitutions that did not cause steric hindrance were considered for further analysis. Tables 4 and 5 contain candidate AB and EF epitope amino acid sequences, respectively, formed by recognition of sterically favorable substitutions in the surface exposed loops of the AB and EF loops.
[0066] Table 4
[0067]
[0068] table 5
[0069]
[0070] * Amino acids with side chains pointing towards the interio...
Embodiment 3
[0071] Example 3. CD loop sequence modification. Using the molecular visualization system software PyMOL, the position of the loop was modeled using the human IgG1 Fc region derived from the crystal structure of PDB 4WI2 or 4NOU, and the solvent-exposed amino acid side chains within the CD loop sequence were identified. Using this method, the steric effects of sequence modifications in the context of the CD loop can be analyzed. Amino acid substitutions were selected based on similarity in charge, isoelectric point (pl), and polarity at each amino acid position. Another general consideration in designing CD epitopes is to avoid hydrophobic blocks on the outside of the antibody. Table 6 contains a list of candidate CD epitope tag sequences selected by using the aforementioned strategy.
[0072] Table 6
[0073]
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More