ANTIBODY DESIGN USING ANTI-LIPID ANTIBODY CRYSTAL STRUCTURES
First Claim
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1. A method of designing an optimized 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, optionally a humanized antibody, wherein the anti-lipid antibody is specific for a first lipid, optionally a sphingolipid or a lysolipid, and wherein optionally at least one complementarity-determining region within the variable region is identified;
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;
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 antibody, optionally by molecular modeling, ELISA, or surface plasmon resonance, wherein at least one of the activity criteria is optionally binding affinity for the first lipid, binding affinity for a second lipid, or specificity for the first lipid or specificity for a second lipid, wherein the first and second lipids are different lipid species; and
e. selecting an optimized antibody based on one or more of the activity criteria, wherein the optimized antibody is the second antibody,wherein the method is optionally performed in silico.
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Abstract
Methods for designing optimized antibodies, including optimized humanized 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.
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Citations
12 Claims
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1. A method of designing an optimized antibody to a lipid, comprising:
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a. providing an amino acid sequence of at least one variable region of a heavy or light chain of a first anti-lipid antibody, optionally a humanized antibody, wherein the anti-lipid antibody is specific for a first lipid, optionally a sphingolipid or a lysolipid, and wherein optionally at least one complementarity-determining region within the variable region is identified; 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; 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 antibody, optionally by molecular modeling, ELISA, or surface plasmon resonance, wherein at least one of the activity criteria is optionally binding affinity for the first lipid, binding affinity for a second lipid, or specificity for the first lipid or specificity for a second lipid, wherein the first and second lipids are different lipid species; and e. selecting an optimized antibody based on one or more of the activity criteria, wherein the optimized antibody is the second antibody, wherein the method is optionally performed in silico. - View Dependent Claims (2, 3, 4, 5, 12)
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6. A method selected from the group consisting of:
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a. a method of designing a consensus anti-lipid antibody specifically reactive with a target bioactive lipid, comprising; (i) identifying at least a first CDR amino acid sequence from a first parent antibody species specifically reactive with a target bioactive lipid and at least a second CDR amino acid sequence from a second parent antibody species specifically reactive with the target bioactive lipid, wherein the first and second CDR amino acid sequences are both CDRH1, both CDRH2, both CDRH3, both CDRL1, both CDRL2 or both CDRL3 CDR amino acid sequences; (ii) aligning the first CDR amino acid sequence and second CDR amino acid sequence to determine a consensus CDR amino acid sequence; and (iii) engineering a nucleic acid sequence that encodes the consensus CDR amino acid sequence into a gene comprising a variable region of an antibody heavy or light chain, thereby designing a consensus anti-lipid antibody specifically reactive with a target bioactive lipid, and, optionally, (iv) producing an antibody or antibody fragment that binds the target bioactive lipid; and b. a method of designing an antibody variant or antibody fragment variant specifically reactive with a target bioactive lipid, comprising; (i) providing a first structural representation comprising an initial representation of a target bioactive lipid in binding association with an antibody or antibody fragment specifically reactive with a source bioactive lipid, wherein the target bioactive lipid and the source bioactive lipid are the same or a different bioactive lipid species, wherein the initial representation comprises three-dimensional structural information for the antibody or antibody fragment optionally derived from molecular modeling data or x-ray crystallography data; and (ii) substituting at least one amino acid residue represented in the first structural representation with a different amino acid residue in order to identify a second structural representation comprising a subsequent representation of the target bioactive lipid in modified binding association with the modified antibody or antibody fragment, thereby designing an antibody variant or antibody fragment variant that is specifically reactive with the target bioactive lipid, wherein the antibody variant or antibody fragment optionally has a modified binding association, optionally an improved binding association, for the bioactive lipid. - View Dependent Claims (7, 8, 9, 10, 11)
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Specification