Compositions and Methods for Binding Lysophosphatidic Acid

Abstract

Compositions and methods for making and using anti-LPA agents, for example, monoclonal antibodies, are described.

1 . A compound selected from the group consisting of:
 a. an isolated anti-LPA agent, which agent binds lysophosphatidic acid (LPA) under physiological conditions and comprises at least one CDR peptide having an amino acid sequence that has a sequence identity of at least 65 percent, optionally a sequence identity of at least 80 percent, at least 90 percent, at least 95 percent, and 100 percent identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 56, 57, 58, 59, 60, 61, 62, 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, 85, 86, 93, 94, 95, 96, 97, 98, 105, 106, 107, 108, 109, 110, and 111, wherein the anti-LPA agent is optionally:
 (i) selected from the group consisting of an antibody and a non-antibody-derived moiety; 
 (ii) selected from the group consisting of a chimeric antibody, a humanized antibody, a full-length antibody, an affinity matured antibody, an antibody derivative or an antibody fragment; 
 (iii) an antibody comprised of two heavy chains and two light chains, wherein each heavy chain independently comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 114, 118, 122, 126 and 130 and each light chain independently comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 115, 119, 123, 127, and 131; 
 (iv) combined with a second agent which is optionally selected from the group consisting of an antibody, an antibody fragment, an antibody derivative, and an antibody variant, wherein the second agent optionally comprises a binding moiety that binds a molecule other than LPA, wherein the anti-LPA agent and the second agent are optionally linked, optionally by a covalent linkage; and 
 (v) conjugated to a moiety selected from the group consisting of a polymer, a radionuclide, a chemotherapeutic agent, and a detection agent; 
   b. an isolated nucleic acid molecule comprising:
 (i) a sequence of nucleotide residues that encodes at least a first CDR peptide, and optionally a second CDR peptide or second and third CDR peptides, wherein the first, second, and third CDR peptides each independently have an amino acid sequence that has a sequence identity selected from the group consisting of at least 65 percent, at least 80 percent, at least 90 percent, at least 95 percent, and 100 percent identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 56, 57, 58, 62, 69, 70, 71, 81, 82, 83, 93, 94, 95, 105, 106, 107, and 111; and 
 (ii) a sequence of nucleotide residues that encodes at least a first CDR peptide, and optionally a second CDR peptide or second and third CDR peptides, wherein the first, second, and third CDR peptides each independently have an amino acid sequence that has a sequence identity selected from the group consisting of at least 65 percent, at least 80 percent, at least 90 percent, at least 95 percent, and 100 percent identity with an amino acid sequence selected from the group consisting of SEQ ID NO; 59, 60, 61, 72, 73, 74, 84, 85, 86, 96, 97, 98, 108, 109, and 110; 
   c. an isolated polypeptide comprising:
 (i) at least one framework region from a variable domain from an animal immunoglobulin heavy chain, which polypeptide binds LPA in a physiological context and comprises at least a first CDR peptide, and optionally a second CDR peptide or second and third CDR peptides, wherein the first, second, and third CDR peptides each independently have a sequence identity selected from the group consisting of at least 65 percent, at least 80 percent, at least 90 percent, at least 95 percent, and at least 100 percent identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 56, 57, 58, 62, 69, 70, 71, 81, 82, 83, 93, 94, 95, 105, 106, 107, and 111, wherein the polypeptide optionally is selected from the group consisting of a full length variable domain of an immunoglobulin heavy chain or fragment thereof and a full length immunoglobulin heavy chain or a fragment of an immunoglobulin heavy chain; and 
 (ii) one framework region from a variable domain from an animal immunoglobulin light chain, which polypeptide binds LPA in a physiological context and comprises at least a first CDR peptide, and optionally a second CDR peptide or second and third CDR peptides, wherein the first, second, and third CDR peptides each independently have a sequence identity selected from the group consisting of at least 65 percent, at least 80 percent, at least 90 percent, at least 95 percent, and at least 100 percent identity with an amino acid sequence selected from the group consisting of SEQ ID NO; 59, 60, 61, 72, 73, 74, 84, 85, 86, 96, 97, 98, 108, 109 and 110, wherein the polypeptide optionally is selected from the group consisting of a full length variable domain of an immunoglobulin light chain or fragment thereof and a full length immunoglobulin light chain or a fragment of an immunoglobulin light chain; and 
   d. an isolated anti-LPA antibody heavy chain, which anti-LPA antibody heavy chain comprises a variable domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 114, 118, 122, 126, and 130;   e. an isolated anti-LPA antibody light chain, which anti-LPA antibody light chain comprises a variable domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 115, 119, 123, 127, and 131;   f. an isolated anti-LPA antibody having two heavy chains and two light chains, wherein each immunoglobulin heavy chain comprises a variable domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 114, 118, 122, 126, and 130, and each immunoglobulin light chain comprises a variable domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 115, 119, 123, 127, and 131, and wherein said heavy chains and said light chains are independently derived from two or more different hybridoma cells;   g. a multivalent binding molecule that comprises:
 (i) at least first and second ligand binding elements, wherein the first ligand binding element, and optionally the second ligand binding element, binds LPA in a physiological context and comprises at least a first CDR peptide, and optionally a second CDR peptide or second and third CDR peptides, wherein the first, second, and third CDR peptides each independently have a sequence identity selected from the group consisting of at least 65 percent, at least 80 percent, at least 90 percent, at least 95 percent, and 100 percent identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 56, 57, 58, 62, 69, 70, 71, 81, 82, 83, 93, 94, 95, 105, 106, 107, and 111, wherein the multivalent binding molecule optionally is selected from the group consisting of a full length variable domain of an immunoglobulin heavy chain or fragment thereof and a full length immunoglobulin heavy chain or a fragment of an immunoglobulin heavy chain; and/or 
 (ii) at least first and second ligand binding elements, wherein the first ligand binding element, and optionally the second ligand binding element, binds LPA in a physiological context and comprises at least a first CDR peptide, and optionally a second CDR peptide or second and third CDR peptides, wherein the first, second, and third CDR peptides each independently have a sequence identity selected from the group consisting of at least 65 percent, at least 80 percent, at least 90 percent, at least 95 percent, and 100 percent identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 59, 60, 61, 72, 73, 74, 84, 85, 86, 96, 97, 98, 108, 109, and 110, wherein the multivalent binding molecule optionally is selected from the group consisting of a full length variable domain of an immunoglobulin light chain or fragment thereof and a full length immunoglobulin light chain or a fragment of an immunoglobulin light chain; and/or 
 (iii) a scaffold to which is linked at least first and second ligand binding elements that each binds LPA in a physiological context, wherein the first ligand binding element comprises functionally associated first and second polypeptides, wherein the first polypeptide comprises at least one CDR peptide having an amino acid sequence that has a sequence identity of at least 50 percent with an amino acid sequence selected from the group consisting of SEQ ID NOs: 56, 57, 58, 62, 69, 70, 71, 81, 82, 83, 93, 94, 95, 105, 106, 107, and 111, and the second polypeptide comprises at least one CDR peptide having an amino acid sequence that has a sequence identity of at least 50 percent with an amino acid sequence selected from the group consisting of SEQ ID NO; 59, 60, 61, 72, 73, 74, 84, 85, 86, 96, 97, 98, 108, 109, and 110; and 
   h. diagnostic reagent comprising a derivatized lysophosphatidic acid which comprises a polar head group and at least one hydrocarbon chain, wherein a carbon atom within at least one hydrocarbon chain is derivatized, optionally with a protected pendant reactive group optionally selected from the group consisting of a sulfhydryl (thiol) group, a carboxylic acid group, a cyano group, an ester, a hydroxy group, an alkene, an alkyne, an acid chloride group, and a halogen atom, and wherein the derivatized lysophosphatidic acid is optionally associated with a solid support, optionally covalently, or is conjugated to a carrier moiety optionally selected from the group consisting of polyethylene glycol, colloidal gold, adjuvant, a silicone bead, and a protein, wherein the protein is optionally selected from the group consisting of keyhole limpet hemocyanin, albumin, bovine thyroglobulin, and soybean trypsin inhibitor, wherein the carrier moiety is optionally attached to a solid support.   
     
     
         2 . A composition comprising a carrier, optionally a pharmaceutically acceptable carrier, and a compound according to  claim 1  that is an anti-LPA agent, optionally an anti-LPA antibody. 
     
     
         3 . A compound according to  claim 1  that is an isolated nucleic acid molecule encoding a fragment of an immunoglobulin heavy chain or a full length immunoglobulin heavy chain, wherein the immunoglobulin heavy chain is optionally derived from an animal selected from the group consisting of a fish, a bird, and mammal, wherein the mammal optionally is a primate, optionally a human. 
     
     
         4 . A vector comprising a first or a first and a second isolated nucleic acid molecule species each according to  claim 1 . 
     
     
         5 . A host cell transfected with a first or a first and a second nucleic acid molecule species each according to  claim 1 , wherein the host cell is optionally transfected with a first vector or first and second vectors, wherein when the host cell is transfected with a first vector or a second vector but not first and second vectors, the first or second vector comprises the first or second nucleic acid molecule species, and wherein when the host cell is transfected with both first and second vectors, the first vector comprises the first nucleic acid molecule species and the second vector comprises the second nucleic acid molecule species. 
     
     
         6 . A compound according to  claim 1  that is an isolated anti-LPA agent that is an antibody molecule capable of binding at least one LPA molecule in a physiological context, comprising:
 a. two immunoglobulin heavy chains each of which comprises at least one framework region from a variable domain of an animal immunoglobulin heavy chain and comprises at least a first CDR peptide, and optionally a second CDR peptide or second and third CDR peptides, wherein the first, second, and third CDR peptides each independently have a sequence identity selected from the group consisting of at least 50 percent, at least 65 percent, at least 80 percent, at least 90 percent, at least 95 percent, and at least 100 percent identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 56, 57, 58, 62, 69, 70, 71, 81, 82, 83, 93, 94, 95, 105, 106, 107, and 111; and, 
 b. functionally associated with the two immunoglobulin heavy chains, two immunoglobulin light chains each of which comprises at least one framework region from a variable domain of an animal immunoglobulin light chain and comprises at least a first CDR peptide, and optionally a second CDR peptide or second and third CDR peptides, wherein the first, second, and third CDR peptides each independently have a sequence identity selected from the group consisting of at least 50 percent, at least 65 percent, at least 80 percent, at least 90 percent, at least 95 percent, and at least 100 percent identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 59, 60, 61, 72, 73, 74, 84, 85, 86, 96, 97, 98, 108, 109 and 110. 
 
     
     
         7 . A composition comprising a carrier, optionally a pharmaceutically acceptable carrier, and an antibody according to  claim 6 . 
     
     
         8 . A compound according to  claim 1  that is an isolated anti-LPA agent that is a humanized antibody molecule capable of binding at least one LPA molecule in a physiological context, comprising
 a. two immunoglobulin heavy chains each of which comprises at least one framework region from a variable domain from a human immunoglobulin heavy chain and comprises at least a first CDR peptide, and optionally a second CDR peptide or second and third CDR peptides, wherein the first, second, and third CDR peptides each independently have a sequence identity selected from the group consisting of at least 50 percent, at least 65 percent, at least 80 percent, at least 90 percent, at least 95 percent, and at least 100 percent identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 56, 57, 58, 62, 69, 70, 71, 81, 82, 83, 93, 94, 95, 105, 106, 107, and 111; and, 
 b. functionally associated with the two immunoglobulin heavy chains, two immunoglobulin light chains each of which comprises at least one framework region from a variable domain from a human immunoglobulin light chain and comprises at least a first CDR peptide, and optionally a second CDR peptide or second and third CDR peptides, wherein the first, second, and third CDR peptides each independently have a sequence identity selected from the group consisting of at least 50 percent, at least 65 percent, at least 80 percent, at least 90 percent, at least 95 percent, and at least 100 percent identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 59, 60, 61, 72, 73, 74, 84, 85, 86, 96, 97, 98, 108, 109 and 110. 
 
     
     
         9 . A composition comprising a carrier, optionally a pharmaceutically acceptable carrier, and a humanized antibody according to  claim 8 . 
     
     
         10 . An ELISA kit for use in a method for detecting lysophosphatidic acid (LPA) comprising a diagnostic reagent according to  claim 1  and an anti-LPA agent that binds LPA under physiological conditions and comprises at least one CDR peptide having an amino acid sequence that has a sequence identity of at least 65 percent, optionally a sequence identity of at least 80 percent, at least 90 percent, at least 95 percent, and 100 percent identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 56, 57, 58, 59, 60, 61, 62, 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, 85, 86, 93, 94, 95, 96, 97, 98, 105, 106, 107, 108, 109, 110, and 111, wherein the diagnostic reagent is optionally a thiolated LPA conjugated to bovine serum albumin or keyhole limpet hemocyanin and the anti-LPA agent is optionally an anti-LPA monoclonal antibody. 
     
     
         11 . A method selected from the group consisting of:
 a. a method of treating or preventing a disease or disorder associated with aberrant levels of LPA, comprising administering to a subject in need of such treatment a compound according to  claim 1 , other than a nucleic acid molecule or a diagnostic reagent, in an amount effective to reduce in vivo the effective concentration of LPA, thereby effecting treatment or prevention of the disease or disorder, wherein the disease or disorder is optionally selected from the group consisting of a hyperproliferative disease, including cancer; an immune-related disease, including an autoimmune disease, allograft rejection and graft-vs-host disease; a neurodegenerative disease; obesity; type 2 diabetes; an ocular disease, including macular degeneration; pain; a disease associated with aberrant angiogenesis or neovascularization; apoptosis; fibrogenesis or fibrosis, including scleroderma, pulmonary fibrosis, renal fibrosis, skin fibrosis, cardiac fibrosis, and hepatic fibrosis; wound repair and healing; and a spider bite;   b. a method of treating or preventing a disease or disorder associated with aberrant levels of LPA, comprising administering to a subject in need of such treatment an anti-LPA antibody molecule capable of binding at least one LPA molecule in a physiological context, wherein the antibody comprises:
 (i) two immunoglobulin heavy chains each of which comprises at least one framework region from a variable domain of an animal immunoglobulin heavy chain and comprises at least a first CDR peptide, and optionally a second CDR peptide or second and third CDR peptides, wherein the first, second, and third CDR peptides each independently have a sequence identity selected from the group consisting of at least 50 percent, at least 65 percent, at least 80 percent, at least 90 percent, at least 95 percent, and at least 100 percent identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 56, 57, 58, 62, 69, 70, 71, 81, 82, 83, 93, 94, 95, 105, 106, 107, and 111; and, 
 (ii) functionally associated with the two immunoglobulin heavy chains, two immunoglobulin light chains each of which comprises at least one framework region from a variable domain of an animal immunoglobulin light chain and comprises at least a first CDR peptide, and optionally a second CDR peptide or second and third CDR peptides, wherein the first, second, and third CDR peptides each independently have a sequence identity selected from the group consisting of at least 50 percent, at least 65 percent, at least 80 percent, at least 90 percent, at least 95 percent, and at least 100 percent identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 59, 60, 61, 72, 73, 74, 84, 85, 86, 96, 97, 98, 108, 109 and 110, 
    wherein the antibody is administered in an amount effective to reduce in vivo the effective concentration of LPA, thereby effecting treatment or prevention of the disease or disorder, and wherein the disease or disorder is optionally selected from the group consisting of a hyperproliferative disease, including cancer; an immune-related disease, including an autoimmune disease, allograft rejection and graft-vs-host disease; a neurodegenerative disease; obesity; type 2 diabetes; an ocular disease, including macular degeneration; pain; a disease associated with aberrant angiogenesis or neovascularization; apoptosis; fibrogenesis or fibrosis, including scleroderma, pulmonary fibrosis, renal fibrosis, skin fibrosis, cardiac fibrosis, and hepatic fibrosis; wound repair and healing; and a spider bite;   c. a method of decreasing an aberrant condition selected from the group consisting of aberrant hyperproliferation, immune response, neurodegeneration, angiogenesis, neovascularization, apoptosis, fibrogenesis, and fibrosis in an animal, optionally a mammal, optionally a human, the method comprising administering to the animal a compound according to  claim 1 , other than a nucleic acid molecule or a diagnostic reagent, in an amount effective to reduce in vivo the effective concentration of LPA, thereby decreasing the aberrant condition;   d. a method of decreasing an aberrant condition selected from the group consisting of aberrant hyperproliferation, immune response, neurodegeneration, angiogenesis, neovascularization, apoptosis, fibrogenesis, and fibrosis in an animal, optionally a mammal, optionally a human, the method comprising administering to the animal an anti-LPA antibody molecule capable of binding at least one LPA molecule in a physiological context, wherein the antibody comprises:
 (i) two immunoglobulin heavy chains each of which comprises at least one framework region from a variable domain of an animal immunoglobulin heavy chain and comprises at least a first CDR peptide, and optionally a second CDR peptide or second and third CDR peptides, wherein the first, second, and third CDR peptides each independently have a sequence identity selected from the group consisting of at least 50 percent, at least 65 percent, at least 80 percent, at least 90 percent, at least 95 percent, and at least 100 percent identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 56, 57, 58, 62, 69, 70, 71, 81, 82, 83, 93, 94, 95, 105, 106, 107, and 111; and, 
 (ii) functionally associated with the two immunoglobulin heavy chains, two immunoglobulin light chains each of which comprises at least one framework region from a variable domain of an animal immunoglobulin light chain and comprises at least a first CDR peptide, and optionally a second CDR peptide or second and third CDR peptides, wherein the first, second, and third CDR peptides each independently have a sequence identity selected from the group consisting of at least 50 percent, at least 65 percent, at least 80 percent, at least 90 percent, at least 95 percent, and at least 100 percent identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 59, 60, 61, 72, 73, 74, 84, 85, 86, 96, 97, 98, 108, 109 and 110, 
    wherein the antibody is administered in an amount effective to reduce in vivo the effective concentration of LPA and thereby decrease the aberrant condition;   e. a method of decreasing fibrosis in a human subject, comprising administering to the human subject an anti-LPA antibody in an amount sufficient to reduce in the human subject the effective concentration of LPA, so that fibrosis is decreased, wherein the fibrosis to be decreased is optionally selected from the group consisting of hepatic, renal, pulmonary, cardiac, uterine, and skin fibrosis, and wherein the method optionally further comprises detecting at least one fibrosis marker and detecing an amount of LPA in a fluid or tissue sample from the human subject;   f. a method of detecting an anti-LPA agent in a biological sample comprising detecting binding of an anti-LPA agent in a biological sample to a diagnostic reagent under conditions that allow the diagnostic reagent to bind the anti-LPA agent, if present in the sample, wherein the diagnostic reagent comprises a derivatized lysophosphatidic acid which comprises a polar head group and at least one hydrocarbon chain, wherein a carbon atom within at least one hydrocarbon chain is derivatized, optionally with a protected pendant reactive group optionally selected from the group consisting of a sulfhydryl (thiol) group, a carboxylic acid group, a cyano group, an ester, a hydroxy group, an alkene, an alkyne, an acid chloride group, and a halogen atom, and wherein the derivatized lysophosphatidic acid is optionally associated with a solid support, optionally covalently, or is conjugated to a carrier moiety optionally selected from the group consisting of polyethylene glycol, colloidal gold, adjuvant, a silicone bead, and a protein, wherein the protein is optionally selected from the group consisting of keyhole limpet hemocyanin, albumin, bovine thyroglobulin, and soybean trypsin inhibitor, wherein the carrier moiety is optionally attached to a solid support, wherein
 (i) the biological sample is optionally selected from the group consisting of a tissue sample, optionally a biopsy sample, and a fluid sample, optionally selected from the group consisting of whole blood, plasma, serum, urine, semen, bile, aqueous humor, vitreous humor, bronchioalveolar lavage fluid, mucous, and sputum; and 
 (ii) the anti-LPA agent isoptionally selected from the group consisting of an antibody, optionally a human anti-LPA antibody; an antibody fragment; an antibody derivative; and a non-antibody-derived moiety; and 
   g. a method of detecting lysophosphatidic acid or a metabolite thereof in a sample, comprising detecting binding of lysophosphatidic acid or a metabolite thereof in a sample to an anti-LPA agent under conditions that allow the anti-LPA agent to bind to the LPA, if present in the sample, wherein the anti-LPA agent binds LPA under physiological conditions and comprises at least one CDR peptide having an amino acid sequence that has a sequence identity of at least 65 percent, optionally a sequence identity of at least 80 percent, at least 90 percent, at least 95 percent, and 100 percent identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 56, 57, 58, 59, 60, 61, 62, 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, 85, 86, 93, 94, 95, 96, 97, 98, 105, 106, 107, 108, 109, 110, and 111, wherein
 (i) the sample is optionally an animal-derived sample selected from the group consisting of a tissue sample, optionally a biopsy sample, and a bodily fluid sample, optionally selected from the group consisting of whole blood, plasma, serum, urine, semen, bile, aqueous humor, vitreous humor, mucus, bronchioalveolar lavage fluid, and sputum; 
 (ii) the anti-LPA agent is optionally selected from the group consisting of a polyclonal antibody; a monoclonal antibody; a chimeric antibody; a fragment of a polyclonal, monoclonal, or chimeric antibody; a variant of a polyclonal, monoclonal, or chimeric antibody; and a derivative of a polyclonal, monoclonal, or chimeric antibody 
 (iii) the method optionally further comprises detecting at least one fibrosis marker; 
 (iv) the method is performed on an animal-derived sample wherein the method optionally further comprises:
 A. comparing a level of LPA in the sample to a reference level of LPA obtained from a normal animal of the same species, wherein the presence of an increased level of LPA relative to the reference level correlates with the presence of disease; and/or 
 B. comparing a level of LPA in the sample to a desired level of LPA, and, if necessary, altering a therapeutic dosage of an anti-LPA agent administered to the animal, wherein the anti-LPA agent modulates the effective concentration of LPA, in order to regulate the effective concentration of LPA in the animal; and 
 
   h. a method of detecting in a sample an anti-LPA agent, comprising contacting a sample with a diagnostic device bearing a diagnostic reagent capable of detecting LPA under conditions that allow the anti-LPA agent, if present, to be detected, wherein the diagnostic reagent comprises a derivatized lysophosphatidic acid.