Antibody design using anti-lipid antibody crystal structures
View Patent ↗Methods for designing optimized antibodies, including optimized humainized or human antibodies, to target bioactive lipids are provided. These methods may be performed in silico and may be intended to enhance binding affinity of an antibody to its original target lipid, and/or to alter binding specificity. Antibodies produced by these methods are also provided, as are methods for using them.
1. A method of generating an optimized humanized antibody to a lipid, comprising:
a. providing an amino acid sequence of at least one variable region of a heavy or light chain of a first anti-lipid antibody, wherein the first anti-lipid antibody is specific for a first lipid, and wherein the first anti-lipid antibody is LT1009;
b. replacing one or more amino acids within the at least one variable region with a different amino acid to yield a variant amino acid sequence, wherein the amino acid replacement(s) is(are) within a complementarity-determining region, and wherein the one or more amino acids replaced is/are selected from the group consisting of: aspartic acid at positions 30, 31, and 32, glutamic acid at position 50, aspartic acid at position 92, leucine at position 94 and phenylalanine at position 96, all of the light chain of LT1009; and threonine at position 33, histidine at position 35, alanine at position 50, serine at position 52, histidine at position 54, isoleucine at position 56, lysine at position 58, phenylalanine at position 97, tyrosine at position 98, threonine at position 100A or tryptophan at position 100C, all of the heavy chain of LT1009;
c. preparing a second anti-lipid antibody containing the variant amino acid sequence, wherein the amino acid sequences of the first and second anti-lipid antibodies differ only in the variant amino acid sequence;
d. determining one or more activity criteria of the second anti-lipid antibody;
e. selecting an optimized anti-lipid antibody based on one or more of the activity criteria, wherein the optimized anti-lipid antibody is the second anti-lipid antibody; and
f. synthesizing the optimized anti-lipid antibody.
2. A method according to claim 1 wherein steps (a)-(e) are performed in silico.
3. A method according to claim 1 wherein the first lipid is a sphingolipid or a lysolipid.
4. A method according to claim 1 wherein determining the one or more activity criteria of the second antibody is selected from the group consisting of molecular modeling, ELISA, or surface plasmon resonance.
5. A method according to claim 1 wherein the one or more activity criteria of the second antibody is selected from the group consisting of binding affinity for the first lipid, binding affinity for a second lipid, and specificity for the first lipid or specificity for a second lipid, wherein the first and second lipids are different lipid species.
6. A method according to claim 1 further comprising use of three-dimensional structural information about the binding of the first anti-lipid antibody and the first lipid to select a location and/or identity of the amino acid replacement(s).
7. A method according to claim 6 wherein the three-dimensional structural information is molecular modeling data or x-ray crystallography data.