Constructs and methods to identify antibodies that target glycans
View Patent ↗Nucleic acid constructs for expressing an antibody on the surface of bacteriophage are disclosed, as are methods for using the constructs to identify antibodies that target glycans.
1. A nucleic acid construct comprising HindIII (AAGCTT), 2G12 VL (SEQ ID NO:1), C L (SEQ ID NO:2), Intragenic region (SEQ ID NO:3), pel B (SEQ ID NO:4), Region X (SEQ ID 5), spe I (ACTAGT), VH (SEQ ID NO:6), C H (SEQ ID NO:7), Hinge (SEQ ID NO:8), GCN4 (SEQ ID NO:9), and SalI (GTCGAC), or a modified version thereof wherein the modification consists of one or more modification in the sequence of the 2G12 VL region and/or one or more modification in the sequence of the VH region, and wherein the modification consists of a substitution of one codon for another codon, up to nine such substitutions, and/or insertion of up to nine additional codons.
2. The nucleic acid construct of claim 1 , comprising one or more modification in the sequence of the 2G12 VL region.
3. The nucleic acid construct of claim 2 , comprising a modification in the “TAC” nucleotides at nucleotide positions 277-279 of SEQ ID NO:1.
4. The nucleic acid construct of claim 1 , comprising one or more modification in the sequence of the VH region.
5. The nucleic acid construct of claim 4 , comprising a modification in one or more of the “CGT” nucleotides at nucleotide positions 81-83, the “CTGGAAGAC” nucleotides at nucleotide positions 223-231, and/or the GAC” nucleotides at nucleotide positions 316-318 of SEQ ID NO:6.
6. The nucleic acid construct of claim 1 , comprising one or more modification in the sequence of the 2G12 VL region and one or more modification in the sequence of the VH region.
7. The nucleic acid construct of claim 1 , wherein the codon that is added to, or substituted in, the sequence of the VL and/or VH region is selected from the group consisting of one or more of NNS, DVK, WNY, KVK, KMT and TMT.
8. A method for identifying an antibody that targets a glycan of interest, comprising:
a) expressing an antibody encoded by the nucleic acid construct of claim 1 on the surface of bacteriophage; and
b) contacting the bacteriophage with the glycan of interest;
wherein binding of the glycan to the antibody expressed on the bacteriophage identifies the antibody as an antibody that targets the glycan of interest, and wherein lack of binding of the glycan to the antibody expressed on the bacteriophage indicates that the antibody is not an antibody that targets the glycan of interest.
9. The method of claim 8 , wherein the bacteriophage is a M13, fd filamentous phage, T4, T7 or λ bacteriophage.
10. The method of claim 8 , wherein the glycan is immobilized to a fixed surface.
11. The method of claim 8 , wherein the glycan of interest is oligomannose on HIV-1 gp120 or Le Y antigen.
12. The method of claim 8 , wherein the antibody binds the glycan with a K D of 50 nM or lower.