JPS6012973 | SINGLE CLONE ANTIBODY OF HUMAN FROM LYMPH OF PATIENT HAVING MALIGNANT BLACK TUMOR |
WO/2021/133712 | HUMANIZED BCMA ANTIBODY AND BCMA-CAR-T CELLS |
WO/2017/053469 | CD3 BINDING POLYPEPTIDES |
MUTHUMANI KAR (US)
WO2010104749A2 | 2010-09-16 |
US20150004168A1 | 2015-01-01 |
CLAIMS: 1. A recombinant antibody or an epitope binding fragment thereof that specifically binds to a lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) epitope, said antibody or fragment comprising at least one of : (a) one or more of heavy chain complementarity-determining regions (CDRs) encoded by CDR-coding sequences of 6A10, 3D8, 9E12, 12D9, 12E4 or 4D6 identified in Table 1, or a nucleic acid sequence at least 85% identical thereto; or (b) one or more of heavy chain CDRs having CDR amino acid sequences of 6A10, 3D8, 9E12, 12D9, 12E4 or 4D6 identified in Table 2, or an amino acid sequence at least 85% identical thereto; or (c) one or more of light chain CDRs encoded by CDR-coding nucleic acid sequences of 6A10, 3D8, 9E12, 12D9, 12E4 or 4D6 identified in Table 3, or a nucleic acid sequence at least 85% identical thereto; or (d) one or more of light chain CDRs having CDR amino acid sequences of 6A10, 3D8, 9E12, 12D9, 12E4 or 4D6 clone 1 or 4D6 clone 2, identified in Table 4, or an amino acid sequence at least 85% identical thereto. 2. A recombinant antibody or an epitope binding fragment thereof that specifically binds to a lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) epitope, said antibody or fragment comprising at least one of : (a) a heavy chain variable region having an amino acid sequence of SEQ ID NOs: 3, 7, 11, 15, 19, or 25, or an amino acid sequence at least 85% identical thereto; or (b) a light chain variable region sequence having an amino acid sequence of SEQ ID NOs: 5, 9, 13, 17, 21, 23, or 27 or an amino acid sequence at least 85% identical thereto.; or (c) a heavy chain variable region encoded by a nucleic acid sequence of SEQ ID NOs: 2, 6, 10, 14, 18, or 24, or a nucleic acid sequence at least 85% identical thereto; or (d) a light chain variable region encoded by a nucleic acid sequence of SEQ ID NOs: 4, 8, 12, 16, 20, 22, or 26, or a nucleic acid sequence at least 85% identical thereto. 3. The antibody or epitope binding fragment thereof according to any one of claims 1 to 2, comprising a heavy chain variable region having an amino acid sequence of SEQ ID NO: 3 or an amino acid sequence at least 85% identical thereto and/or a light chain variable region having an amino acid sequence of SEQ ID NO: 5 or an amino acid sequence at least 85% identical thereto. 4. The antibody or epitope binding fragment thereof according to claim 1 to 3, comprising a heavy chain variable region encoded by a nucleic acid sequence of SEQ ID NO: 2 or a nucleic acid sequence at least 85% identical thereto and/or a light chain variable region encoded by a nucleic acid sequence of SEQ ID NO: 4 or a nucleic acid sequence at least 85% identical thereto. 5. The antibody or epitope binding fragment thereof according to claim 1 or 2, comprising a heavy chain variable region encoded by a nucleic acid sequence of SEQ ID NO: 6 or a nucleic acid sequence at least 85% identical thereto and/or a light chain variable region encoded by a nucleic acid sequence of SEQ ID NO: 8 or a nucleic acid sequence at least 85% identical thereto. 6. The antibody or epitope binding fragment thereof according to any one of claims 1, 2 and 5, comprising a heavy chain variable region having an amino acid sequence of SEQ ID NO: 7 or an amino acid sequence at least 85% identical thereto and/or a light chain variable region having an amino acid sequence of SEQ ID NO: 9 or an amino acid sequence at least 85% identical thereto. 7. The antibody or epitope binding fragment thereof according to claim 1 or 2, comprising a heavy chain variable region encoded by a nucleic acid sequence of SEQ ID NO: 10 or a nucleic acid sequence at least 85% identical thereto and/or a light chain variable region encoded by a nucleic acid sequence of SEQ ID NO: 12 or a nucleic acid sequence at least 85% identical thereto. 8. The antibody or epitope binding fragment thereof according to any one of claims 1, 2 and 7, comprising a heavy chain variable region having an amino acid sequence of SEQ ID NO: 11 or an amino acid sequence at least 85% identical thereto and/or a light chain variable region having an amino acid sequence of SEQ ID NO: 13 or an amino acid sequence at least 85% identical thereto. 9. The antibody or epitope binding fragment thereof according to claim 1 or 2, comprising a heavy chain variable region encoded by a nucleic acid sequence of SEQ ID NO: 14 or a nucleic acid sequence at least 85% identical thereto and/or a light chain variable region encoded by a nucleic acid sequence of SEQ ID NO: 16 or a nucleic acid sequence at least 85% identical thereto. 10. The antibody or epitope binding fragment thereof according to any one of claims 1, 2 and 9, comprising a heavy chain variable region having an amino acid sequence of SEQ ID NO: 15 or an amino acid sequence at least 85% identical thereto and/or a light chain variable region having an amino acid sequence of SEQ ID NO: 17 or an amino acid sequence at least 85% identical thereto. 11. The antibody or epitope binding fragment thereof according to claim 1 or 2, comprising a heavy chain variable region encoded by a nucleic acid sequence of SEQ ID NO: 18 or a nucleic acid sequence at least 85% identical thereto and/or a light chain variable region encoded by a nucleic acid sequence of SEQ ID NO: 20 or a nucleic acid sequence at least 85% identical thereto. 12. The antibody or epitope binding fragment thereof according to any one of claims 1, 2 and 11, comprising a heavy chain variable region having an amino acid sequence of SEQ ID NO: 19 or an amino acid sequence at least 85% identical thereto and/or a light chain variable region having an amino acid sequence of SEQ ID NO: 21 or an amino acid sequence at least 85% identical thereto. 13. The antibody or epitope binding fragment thereof according to claim 1 or 2, comprising a heavy chain variable region encoded by a nucleic acid sequence of SEQ ID NO: 18 or a nucleic acid sequence at least 85% identical thereto and/or a light chain variable region encoded by a nucleic acid sequence of SEQ ID NO: 22 or a nucleic acid sequence at least 85% identical thereto. 14. The antibody or epitope binding fragment thereof according to any one of claims 1, 2 and 13, comprising a heavy chain variable region having an amino acid sequence of SEQ ID NO: 19 or an amino acid sequence at least 85% identical thereto and/or a light chain variable region having an amino acid sequence of SEQ ID NO: 23 or an amino acid sequence at least 85% identical thereto. 15. The antibody or epitope binding fragment thereof according to claim 1 or 2, comprising a heavy chain variable region encoded by a nucleic acid sequence of SEQ ID NO: 24 or a nucleic acid sequence at least 85% identical thereto and/or a light chain variable region encoded by a nucleic acid sequence of SEQ ID NO: 26 or a nucleic acid sequence at least 85% identical thereto. 16. The antibody or epitope binding fragment thereof according to any one of claims 1, 2 and 15, comprising a heavy chain variable region having an amino acid sequence of SEQ ID NO: 25 or an amino acid sequence at least 85% identical thereto and/or a light chain variable region having an amino acid sequence of SEQ ID NO: 27 or an amino acid sequence at least 85% identical thereto. 17. The antibody or epitope binding fragment thereof according to any one of claims 1 to 16, which is an IgG or comprises an IgG backbone. 18. The antibody or epitope binding fragment thereof according to any one of claims 1 to 16, which is a humanized antibody, a mouse antibody, a rabbit antibody, a goat antibody, a donkey antibody, or a camelid antibody. 19. The antibody or epitope binding fragment thereof according to any one of claims 1 to 18, wherein the epitope is an epitope of LOX-1 protein in native conformation, optionally wherein the antibody or epitope binding fragment thereof does not specifically bind to an epitope of LOX-1 protein in denatured condition. 20. The antibody or epitope binding fragment thereof according to any one of claims 1 to 18, wherein the epitope is an epitope of LOX-1 protein in denatured condition. 21. The antibody or epitope binding fragment thereof according to any one of claims 1 to 20, wherein said antibody is a chimeric antibody, a humanized antibody, a human antibody, a CDR-grafted antibody, a multi-specific binding construct that can bind two or more targets, a dual specific antibody, a bi-specific antibody, an affinity matured antibody, a single-domain antibody (sdAb), a single antibody chain, an scFv fragment, a diabody, a single chain comprising complementary scFvs (tandem scFvs) or bispecific tandem scFvs, an Fv construct, a disulfide-linked Fv, a Fab construct, a Fab' construct, a F(ab')2 construct, a monovalent or bivalent construct from which domains non-essential to monoclonal antibody function have been removed, a single-chain molecule containing one light chain variable region (VL), one heavy chain variable region (VH) antigen-binding domain, and one or two constant “effector” domains optionally connected by linker domains, a univalent antibody lacking a hinge region, a single domain antibody, a dual variable domain immunoglobulin (DVD-Ig) binding protein or a nanobody, an aptamer, an affibody, an affilin, an affitin, an affimer, an alphabody, an anticalin, an avimer, a DARPin, a Fynomer, a Kunitz domain peptide, or a monobody. 22. The antibody or epitope binding fragment thereof according to any one of claims 1 to 21, which is coupled covalently or non-covalently to a detectable label, optionally the detectable label is selected from an enzyme, a fluorescent label, a radioisotope, or a chemiluminescent label. 23. The antibody or epitope biding fragment thereof according to any one of claims 1 to 22, which is coupled covalently or non-covalently to a chemotherapy drug or to a radioactive particle. 24. The antibody or epitope binding fragment thereof according to any one of claims 1 to 23, which is bi-specific and further comprises an epitope binding fragment which specifically binds to a cell surface biomarker of PMN-MDSC other than LOX-1. 25. A pharmaceutical composition comprising one, two, three, four, or more of the antibody or epitope binding fragment thereof according to any one of claims 1 to 24 and a pharmaceutically acceptable carrier or excipient. 26. A diagnostic composition comprising one, two, three, four, or more of the antibody or epitope binding fragment thereof according to any one of claims 1 to 24. 27. The composition according to claim 26, wherein the antibody or epitope binding fragment thereof is immobilized on a substrate, optionally the substrate is a plate, an enzyme linked immunosorbent assay (ELISA) plate, a slide, a pipette, a bead, a magnetic bead, a chip, or a microchip. 28. A method for monitoring population of polymorphonuclear myeloid derived suppressor cells (PMN-MDSCs) in a subject comprising: (a) obtaining a biological sample containing polymorphonuclear neutrophils (PMNs) and PMN-MDSCs; (b) contacting the biological sample with one or more of the antibody or epitope binding fragment thereof according to any one of claims 1 to 24, or the diagnostic composition according to claim 26 or 27; and (c) detecting and optionally distinguishing LOX-1 positive (LOX-1+ ) cells from LOX-1 negative (LOX-1-) cells in the sample, wherein the LOX-1+ cells are PMN- MDSCs substantially free of PMNs. 29. A method of diagnosing a cancer or monitoring progression or metastasis of a cancer in a subject comprising: (a) obtaining a biological sample from the subject; (b) contacting the sample with one or more of the antibody or epitope binding fragment thereof according to any one of claims 1 to 24, or the diagnostic composition according to claim 26 or 27; (c) detecting and optionally distinguishing LOX-1 positive cells from LOX-1 negative cells in the sample, and (d) diagnosing the subject with cancer or cancer metastasis if number of the LOX-1 positive cells is above that of a negative control, or determining cancer progression (for example, tumor size) in the subject by counting the LOX-1 positive cells and comparing it to a control. 30. The method according to claim 28 or 29, wherein the contacting step (b) further comprises contacting the sample with a ligand that specifically binds to or forms a complex with a neutrophil biomarker to identify neutrophil or polymorphonuclear neutrophils (PMNs), optionally wherein the neutrophil biomarker is CD15 or CD66b. 31. The method according to any one of claims 28 to 30, wherein step (c) comprises detecting and optionally distinguishing LOX-1 positive neutrophils from LOX-1 negative cells in the sample, and wherein the method comprises neutrophils. 32. The method according to any one of claim 28 to 31, wherein the contacting step (b) further comprises contacting the sample with a ligand that specifically binds to or forms a complex with a polymorphonuclear neutrophils (PMNs) biomarker to identify PMNs. 33. The method according to any one of claim 29 to 32, wherein step (c) comprises detecting and optionally distinguishing LOX-1 positive PMNs from LOX-1 negative cells in the sample, and wherein step (d) comprises diagnosing the subject with cancer or cancer metastasis if number of the LOX-1 positive PMNs is above that of a negative control, or determining cancer progression (for example, tumor size) in the subject by counting the LOX-1 positive PMNs and comparing it to a control. 34. The method according to any one of claims 28 to 33, further comprising counting or collecting the LOX-1 positive cells, or the LOX-1 positive neutrophils, or the LOX-1 positive PMNs. 35. The method according to any one of claims 28 to 34, wherein step (c) comprises washing to reduce or eliminate the LOX-1 negative cells and other debris in the sample. 36. The method according to any one of claims 28 to 35, comprising separating LOX-1 positive cells from LOX-1 negative cells in the sample and/or separating cells from other debris based on cell size. 37. The method according to any one of claims 28 to 36, further comprising a step of identifying cells with biomarkers shared by both PMN-MDSCs and PMNs, and isolating cells of both PMN-MDSCs and PMNs prior to the contacting step of (b). 38. The method according to any one of claims 28 to 37, wherein the biological sample is a fluid sample or a tumor tissue, optionally the biological sample is whole blood and wherein the method further comprises destroying or lysing any red blood cells in the sample. 39. The method according to any one of claims 28 to 38, further comprising collecting LOX-1 negative cells which contains PMNs and being substantially free from PMN- MDSCs. 40. The method according to any one of claims 28 to 39, wherein a population of LOX- 1 positive cells greater than 1% of the total neutrophil population in the sample of a subject indicates presence, progression or metastasis of a cancer. 41. A method for diagnosis of cancer in a subject comprising: (a) contacting a biological sample obtained from a patient with one or more of the antibody or epitope binding fragment thereof according to any one of claims 1 to 24, or the diagnostic composition according to claim 26 or 27; (b) detecting binding between a component of said biological sample and the antibody or epitope binding fragment thereof; and (c) diagnosing the subject with a cancer if a population of LOX-1+ cells in the total neutrophil population in the sample of a subject is greater than 1% or a control. 42. An assay method of evaluating differentiation of polymorphonuclear myeloid derived suppressor cells (PMN-MDSCs) from polymorphonuclear neutrophils (PMNs) in a biological sample containing both types of cells comprising: (a) contacting the sample with C-reactive protein (CRP), or oxidized-low density lipoprotein (ox-LDL), or other agent which induces differentiation of PMN-MDSC, or activators or regulators of ER stress response; and (b) detecting and optionally collecting PMN-MDSCs using one or more of the antibody or epitope binding fragment thereof according to any one of claims 1 to 24, or the diagnostic composition according to claim 26 or 27. 43. A method of identifying an antagonist or inhibitor of LOX-1 expression, comprising (a) contacting a biological sample or a cell population of PMN-MDSCs and PMNs with or without a test agent; (b) detecting expression of LOX-1 using one or more of the antibody or epitope binding fragment thereof according to any one of claims 1 to 24, or the diagnostic composition according to claim 26 or 27; and (c) the test agent is identified as a LOX-1 antagonist or inhibitor if LOX-1 expression on a single cell and/or number of LOX-1 positive cells decreases with the test agent compare to a control without the test agent. 44. A method of treating a cancer in a subject comprising administering an effective amount of one or more of the antibody or epitope binding fragment thereof according to any one of claims 1 to 24 to a patient in need thereof. 45. The method according to claim 44, further comprising co-administering a LOX-1 antagonist or inhibitor identified. 46. A method of treating a cancer in a subject comprising: (a) obtaining a biological sample from a subject; (b) contacting the sample with one or more of the antibody or epitope binding fragment thereof according to any one of claims 1 to 24, or the diagnostic composition according to claim 26 or 27; (c) detecting whether LOX-1 positive cells (for example, PMN-MDSCs) are present in the sample; and (d) when presence of LOX-1 is detected, administering an effective amount of a composition that reduces or inhibits ER stress response in mammalian neutrophils or reduces or inhibits LOX-1 expression on neutrophil populations, or administering an effective amount of the antibody or epitope binding fragment thereof according to any one of claims 1 to 24, or the pharmaceutical composition according to claim 25, or a LOX-1 antagonist or inhibitor identified using the method according to claim 43. 47. The method according to claim 44 or 46, wherein the composition that reduces or inhibits the ER stress response comprises one or more of the antibody or epitope binding fragment thereof according to any one of claims 1 to 24, or an antibody or functional antigen-binding fragment that binds to or inhibits the expression, activity or activation of at least one of IRE-1 RNase, sXBP1, DDIT3 (CHOP), ATF4, ATF3, SEC61A ARGI, MYCN, CSF3, ,/^^^7*)ȕ^, TNF, LDL, RAF1, APP, IL6 PDGFBB, EPO, CD40LG, Nek, IL13, AGT^^,/^ȕ^^(5%%^^^0$3^.^^^9(*)Į^^&6F1, FLI1, Fin, CD15, CD66b or CD33. 48. The method according to any one of claims 28 to 46, wherein the detecting step is performed in a form of an enzyme linked immunosorbent assay (ELISA), a competitive- binding assay, a capture assay, a western blot, a radioimmunoassay, or a fluorescence- activated cell sorting (FACS) assay. 49. A method of treating a cancer in a subject comprising (a) obtaining a biological sample from the subject; (b) removing LOX-1 positive PMN-MDSCs from the biological sample with an effective amount of one or more of the antibody or epitope binding fragment thereof according to any one of claims 1 to 24 or the diagnostic composition according to claim 26 or 27; (c) reintroducing the biological sample substantially free from PMN-MDSCs to the subject. 50. The method according to any one of claims 28 to 49, wherein the biological sample is selected from blood, plasma, serum, cerebrospinal fluid, a neuronal tissue sample, a tumor tissue, or cells derived therefrom. 51. An antibody of any one of claims 1-24 or a pharmaceutical composition of claim 25 for use in the treatment or inhibition of a LOX-1 related disease or cancer. |
Table 2. Amino Acid Sequences of Heavy Chain CDRs
Table 3. Coding Sequences for Light Chain CDRs
Table 4. Amino Acid Sequences of Light Chain CDRs
Table 5. Sequences of Clones 6A10, 9E12, 12D9, 12E4, 4D6 (clones 1 and 2) and 3D8 In certain embodiments, a CDR identified as an amino acid sequence in Tables 1-5 may be truncated with 1, 2, 3 or 4 amino acids in the N terminal and/or the C terminal. In certain embodiments, a CDR may be a CDR identified as an amino acid sequence in Tables 1-5 but shifted to the N terminal side or the C terminal side by 1, 2, 3 or 4 amino acids. In certain embodiments, a CDR may be a CDR identified as an amino acid sequence in Tables 1-5 but extended to the N terminal side or the C terminal side by 1, 2, 3 or 4 amino acids. In certain embodiments, a CDR is any combination of the CDRs as described in this paragraph. In certain embodiments, a CDR identified as a nucleic acid sequence in Tables 1-5 may be truncated with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides in the 5’ end and/or the 3’ end. In certain embodiments, a CDR may be a CDR identified as nucleic acid sequence in Tables 1-5 shifted to the 5’ and/or the 3’ side by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides. In certain embodiments, a CDR may be a CDR identified as nucleic acid sequence in Tables 1-5 but extended to the 5’ and/or the 3’ side by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides. In certain embodiments, a CDR is any combination of the CDRs as described in this paragraph. In certain embodiments, provided herein is an antibody, or a variant thereof, or an epitope binding fragment thereof comprising any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or all 33 CDR(s) as described. In certain embodiments, the antibody, or a variant thereof, or an epitope binding fragment thereof comprises one or more copies of the CDR(s). As used herein, the complementarity-determining region (CDR) refers to part of the variable chains in antibodies or T cell receptors, which binds to the corresponding epitope. Such CDR may be determined via experiments or via various predicating tools, such as www.imgt.org/IMGT_vquest/analysis. Also provided herein is a nucleic acid sequence encoding an antibody, or a variant thereof, or an epitope binding fragment thereof as described herein. As used herein, an epitope binding fragment refers to a fragment of an antibody which is determined to be bound to an epitope. Such determination may be performed experimentally using for example ELISA or other methods discussed herein or via various predicating tools such as IMGT.org. In certain embodiments, the antibody or fragment thereof comprising at least one of: a heavy chain variable region (V H ) encoded by a V H coding sequence of 3D8, 6A10, 9E12, 12D9, 12E4 or 4D6 or a nucleic acid sequence at least 85% identical thereto; or a light chain variable region (V L ) encoded by a V H coding sequence of 3D8, 6A10, 9E12, 12D9, 12E4, 4D6 clone 1 or 4D6 clone 2 or a nucleic acid sequence at least 85% identical thereto; or a heavy chain variable region having a V H amino acid sequence of 3D8, 6A10, 9E12, 12D9, 12E4 or 4D6 or an amino acid sequence at least 85% identical thereto; or a light chain variable region having a V L amino acid sequence of 3D8, 6A10, 9E12, 12D9, 12E4, 4D6 clone 1 or 4D6 clone 2 or an amino acid sequence at least 85% identical thereto. In certain embodiments, V H or V L coding sequences or amino acid sequences are provided in Table 5 as well as in SEQ ID NOs: 2-27. In certain embodiments, a V H or V L identified as an amino acid sequence in Table 5 as well as in SEQ ID NOs: 2-27 may be truncated with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids in the N terminal and/or the C terminal. In certain embodiments, a V H or V L may be a V H or V L identified as an amino acid sequence in Table 5 as well as in SEQ ID NOs: 2-27 but shifted to the N terminal side or the C terminal side by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids. In certain embodiments, a V H or V L is any combination of the V H or V L s as described in this paragraph. In certain embodiments, a V H or V L identified as a nucleic acid sequence in in Table 5 as well as in SEQ ID NOs: 2-27 may be truncated with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39 or more nucleotides in the 5’ end and/or the 3’ end. In certain embodiments, a V H or V L may be a V H or V L identified as nucleic acid sequence in Table 5 as well as in SEQ ID NOs: 2-27 but shifted to the 5’ and/or the 3’ side by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39 or more nucleotides (nt). In certain embodiments, a V H or V L is any combination of the V H or V L as described in this paragraph. In certain embodiments, provided herein is an antibody, or a variant thereof, or an epitope binding fragment thereof comprising any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all 11 V H or V L as described. In certain embodiments, the antibody, or a variant thereof, or an epitope binding fragment thereof comprises one or more copies of the V H or V L . In certain embodiments, the antibody or fragment thereof comprising at least one of: a heavy chain variable region encoded by a nucleic acid sequence of SEQ ID NOs: 2, 6, 10, 14, 18, or 24 or a nucleic acid sequence at least 85% identical thereto; or a light chain variable region encoded by a nucleic acid sequence of SEQ ID NOs: 4, 8, 12, 16, 20, 22 or 26 or a nucleic acid sequence at least 85% identical thereto; or a heavy chain variable region having an amino acid sequence of SEQ ID NOs: 3, 7, 11, 15, 19 or 25 or an amino acid sequence at least 85% identical thereto; or a light chain variable region having an amino acid sequence of SEQ ID NOs: 5, 9, 13, 17, 21, 23 or 27 or an amino acid sequence at least 85% identical thereto. In certain embodiments, the antibody or epitope binding fragment thereof comprises a heavy chain variable region encoded by a nucleic acid sequence of SEQ ID NO: 2 or a nucleic acid sequence at least 85% identical thereto. Additionally, or alternatively, the antibody or epitope binding fragment thereof comprises a light chain variable region encoded by a nucleic acid sequence of SEQ ID NO: 4 or a nucleic acid sequence at least 85% identical thereto. In certain embodiments, the antibody or epitope binding fragment thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO: 3 or an amino acid sequence at least 85% identical thereto. Additionally, or alternatively, the antibody or epitope binding fragment thereof comprises a light chain variable region having an amino acid sequence of SEQ ID NO: 5 or an amino acid sequence at least 85% identical thereto. In certain embodiments, the antibody or epitope binding fragment thereof comprises a heavy chain variable region encoded by a nucleic acid sequence of SEQ ID NO: 6 or a nucleic acid sequence at least 85% identical thereto. Additionally, or alternatively, the antibody or epitope binding fragment thereof comprises a light chain variable region encoded by a nucleic acid sequence of SEQ ID NO: 8 or a nucleic acid sequence at least 85% identical thereto. In certain embodiments, the antibody or epitope binding fragment thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO: 7 or an amino acid sequence at least 85% identical thereto. Additionally, or alternatively, the antibody or epitope binding fragment thereof comprises a light chain variable region having an amino acid sequence of SEQ ID NO: 9 or an amino acid sequence at least 85% identical thereto. In certain embodiments, the antibody or epitope binding fragment thereof comprises a heavy chain variable region encoded by a nucleic acid sequence of SEQ ID NO: 10 or a nucleic acid sequence at least 85% identical thereto. Additionally, or alternatively, the antibody or epitope binding fragment thereof comprises a light chain variable region encoded by a nucleic acid sequence of SEQ ID NO: 12 or a nucleic acid sequence at least 85% identical thereto. In certain embodiments, the antibody or epitope binding fragment thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO: 11 or an amino acid sequence at least 85% identical thereto. Additionally, or alternatively, the antibody or epitope binding fragment thereof comprises a light chain variable region having an amino acid sequence of SEQ ID NO: 13 or an amino acid sequence at least 85% identical thereto. In certain embodiments, the antibody or epitope binding fragment thereof comprises a heavy chain variable region encoded by a nucleic acid sequence of SEQ ID NO: 14 or a nucleic acid sequence at least 85% identical thereto. Additionally, or alternatively, the antibody or epitope binding fragment thereof comprises a light chain variable region encoded by a nucleic acid sequence of SEQ ID NO: 16 or a nucleic acid sequence at least 85% identical thereto. In certain embodiments, the antibody or epitope binding fragment thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO: 15 or an amino acid sequence at least 85% identical thereto. Additionally, or alternatively, the antibody or epitope binding fragment thereof comprises a light chain variable region having an amino acid sequence of SEQ ID NO: 17 or an amino acid sequence at least 85% identical thereto. In certain embodiments, the antibody or epitope binding fragment thereof comprises a heavy chain variable region encoded by a nucleic acid sequence of SEQ ID NO: 18 or a nucleic acid sequence at least 85% identical thereto. Additionally, or alternatively, the antibody or epitope binding fragment thereof comprises a light chain variable region encoded by a nucleic acid sequence of SEQ ID NO: 20 or a nucleic acid sequence at least 85% identical thereto. In certain embodiments, the antibody or epitope binding fragment thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO: 19 or an amino acid sequence at least 85% identical thereto. Additionally, or alternatively, the antibody or epitope binding fragment thereof comprises a light chain variable region having an amino acid sequence of SEQ ID NO: 21 or an amino acid sequence at least 85% identical thereto. In certain embodiments, the antibody or epitope binding fragment thereof comprises a heavy chain variable region encoded by a nucleic acid sequence of SEQ ID NO: 18 or a nucleic acid sequence at least 85% identical thereto. Additionally, or alternatively, the antibody or epitope binding fragment thereof comprises a light chain variable region encoded by a nucleic acid sequence of SEQ ID NO: 22 or a nucleic acid sequence at least 85% identical thereto. In certain embodiments, the antibody or epitope binding fragment thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO: 19 or an amino acid sequence at least 85% identical thereto. Additionally, or alternatively, the antibody or epitope binding fragment thereof comprises a light chain variable region having an amino acid sequence of SEQ ID NO: 23 or an amino acid sequence at least 85% identical thereto. In certain embodiments, the antibody or epitope binding fragment thereof comprises a heavy chain variable region encoded by a nucleic acid sequence of SEQ ID NO: 24 or a nucleic acid sequence at least 85% identical thereto. Additionally, or alternatively, the antibody or epitope binding fragment thereof comprises a light chain variable region encoded by a nucleic acid sequence of SEQ ID NO: 26 or a nucleic acid sequence at least 85% identical thereto. In certain embodiments, the antibody or epitope binding fragment thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO: 25 or an amino acid sequence at least 85% identical thereto. Additionally, or alternatively, the antibody or epitope binding fragment thereof comprises a light chain variable region having an amino acid sequence of SEQ ID NO: 27 or an amino acid sequence at least 85% identical thereto. Utilizing any of the nucleotide sequences encoding the heavy chain variable region of 6A10 (SEQ ID NO: 2), 9E12 (SEQ ID NO: 6), 12D9 (SEQ ID NO: 10), 12E4 (SEQ ID NO: 14), 4D6 (SEQ ID NO: 18), and 3D8 (SEQ ID NO: 24); the nucleotide sequences encoding the light chain variable region of 6A10 (SEQ ID NO: 4), 9E12 (SEQ ID NO: 8), 12D9 (SEQ ID NO: 12), 12E4 (SEQ ID NO: 16), 4D6 clone 1(SEQ ID NO: 20), 4D6 clone 2(SEQ ID NO: 22), and 3D8 (SEQ ID NO: 26), their encoded amino acid sequences for the heavy chain variable region of 6A10 (SEQ ID NO: 3), 9E12 (SEQ ID NO: 7), 12D9 (SEQ ID NO: 11), 12E4 (SEQ ID NO: 15), 4D6 (SEQ ID NO: 19), and 3D8 (SEQ ID NO: 25); and their encoded amino acid sequences for the light chain variable region of 6A10 (SEQ ID NO: 5), 9E12 (SEQ ID NO: 9), 12D9 (SEQ ID NO: 13), 12E4 (SEQ ID NO: 17), 4D6 clone 1(SEQ ID NO: 21), 4D6 clone 2(SEQ ID NO: 23), and 3D8 (SEQ ID NO: 27), or nucleotide or amino acid sequences sharing at least about 80% (for example, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99%), at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.9% sequence identity therewith, other antibodies or fragments that specifically bind to a LOX-1 epitope, can be generated. In certain embodiments, the antibody or a epitope binding fragment thereof comprises an amino acid sequence sharing a certain percentage (which is less than 100%) identity to any one of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27 and is different from the sequence with the corresponding SEQ ID NO in the region other than CDR(s). In certain embodiments, the antibody or a epitope binding fragment thereof comprises an amino acid sequence sharing a certain percentage (which is less than 100%) identity to any one of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27 and is able to bind to LOX-1 epitope at an affinity of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold of the affinity of 6A10, or 9E12, or 12D9, or 12E4, or 4D6, or 3D8. As used herein, affinity of an antibody or peptide binding fragment thereof refers to the strength with which an epitope binds to the antibody or peptide binding fragment thereof. Such strength may be measured as described in the Example. The availability of these nucleic acid molecules encoding the heavy and light chains of the antibody enables production of a recombinant antibody, fragment or modifications using in vitro expression methods and cell-free expression systems known in the art. In vitro transcription and translation systems are commercially available, e.g., from Promega Biotech (Madison, WI) or Gibco-BRL (Gaithersburg, MD). The antibodies, epitope-binding fragments or modifications thereof may also be produced by expression in a suitable prokaryotic or eukaryotic system. Similarly, modifications may be inserted into these sequences by use of a variety of CRISPR techniques and other related, e.g., zinc finger, methodologies for modifying amino acid and nucleotide sequences. These monoclonal antibodies 3D8, 6A10, 9E12, 12D9, 12E4, and 4D6 and their variable chain sequences identified herein can be further used to prepare other forms of antibodies, e.g., chimeric antibodies, humanized antibodies, human antibodies. Other antibody fragments or ligands can be produced by screening phage display libraries, antibody fragments and mixtures thereof. Techniques for generating these types of antibodies and ligands are well-known in the art and the ligands themselves may be generated using the disclosed amino acid sequences of the above-identified monoclonal antibodies. In certain embodiments, the antibody or epitope binding fragment thereof is an IgG or comprises an IgG backbone. In certain embodiments, the antibody or epitope binding fragment is an intact IgA, IgG, IgM, IgE, IgD, IgG1, IgG2, IgG3 or IgG4, or a fragment thereof, or comprises a backbone thereof. In certain embodiments, the antibody or epitope binding fragment thereof is a humanized antibody, a mouse antibody, a rabbit antibody, a goat antibody, a donkey antibody, a camelid antibody, or a fragment thereof. Chimeric antibodies may similarly be developed using known techniques. Chimeric antibodies are molecules in which different portions are derived from different animal species. Single chain antibodies may also be prepared by conventional methods, such as described in US Patent Nos.4,946,778 and 4,704,692 using the variable portions of the polyclonal or monoclonal antibodies produced according to this invention. Antibody fragments, such as the Fab, F(ab) 2 and scFv fragments and libraries thereof may also be employed in generation of the selective anti-LOX-1 antibodies as described herein. In certain embodiments, the antibody or epitope binding fragment thereof is a bi- specific antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, a CDR-grafted antibody, a multispecific binding construct that can bind two or more targets, a dual specific antibody, a bi-specific antibody or a multi-specific antibody, or an affinity matured antibody, a single-domain antibody (sdAb), a single antibody chain or an scFv fragment, a diabody, a single chain comprising complementary scFvs (tandem scFvs) or bispecific tandem scFvs, an Fv construct, a disulfide-linked Fv, a Fab construct, a Fab' construct, a F(ab') 2 construct, a monovalent or bivalent construct from which domains non-essential to monoclonal antibody function have been removed, a single-chain molecule containing one V L , one V H antigen-binding domain, and one or two constant “effector” domains optionally connected by linker domains, a univalent antibody lacking a hinge region, a single domain antibody, a dual variable domain immunoglobulin (DVD-Ig) binding protein or a nanobody, an aptamer, an affibody, an affilin, an affitin, an affimer, an alphabody, an anticalin, an avimer, a DARPin, a Fynomer, a Kunitz domain peptide, a monobody, or a fragment thereof. In certain embodiments, the antibodies and epitope binding fragment thereof may be further modified from those exemplified. For example, the antibodies may be humanized. In a particular embodiment, the selected sequences of the heavy or light chains of any of the antibodies disclosed herein (or a portion thereof) are inserted into the backbone of an antibody or antibody fragment construct. For example, the variable light domain and/or variable heavy domain of the antibodies described herein may be inserted into another antibody construct. In certain embodiments, the heavy and light chains/variable regions may be connected with a peptide linker. Still other antibody modifications employing the SEQ ID NOs disclosed herein, e.g., as taught by the techniques referenced in above-cited US Patent No.9,902,772, incorporated by reference herein. In certain embodiments, the antibody or epitope binding fragment thereof is bi- specific and comprises CDRs specifically recognizing and binding to more than one (for example, about 2, 3, 4, 5 or more) LOX-1 epitope. In certain embodiments, the antibody or epitope binding fragment thereof is bi-specific and comprises CDRs specifically recognizing and binding to a non-LOX-1 epitope, for example, an epitope of a biomarker for PMN-MDSC including CD15, CD66b, CD11b, CD33, or CD14. The production of bi- specific antibodies or ligands that specifically bind to two or more selected epitopes, can employ conventional techniques. See, e.g., Hornig N, Färber-Schwarz A., Production of bispecific antibodies: diabodies and tandem scFv., 2012, Methods Mol Biol., 907:713-27; Speiss, C. et al, Bispecific antibodies with natural architecture produced by co-culture of bacteria expressing two distinct half-antibodies, Jul 7, 2013, Nature Biotechnology, 31:753- 758; and Jonathan S Martin and Zhenping Zhu, Recombinant approaches to IgG-like bispecific antibodies, 2005 Acta Pharmacologica Sinica, 26: 649–658. In certain embodiments, the antibody or epitope binding fragment thereof is coupled covalently or non-covalently (or conjugated to) to a detectable label. In certain embodiments, the detectable label is an enzyme, a fluorescent label, a radioisotope, or a chemiluminescent label. In certain embodiments, the detectable label is a His tag. In certain embodiments, the antibody or epitope binding fragment thereof is an antibody-drug conjugate. In certain embodiments, the antibody or epitope binding fragment thereof is covalently or non-covalently attached to or associated with (or coupled to, or conjugated to) a cytotoxic agent, for example, a chemotherapy drug or a radioactive particle. In certain embodiments, the antibody or epitope binding fragment thereof recognizes and binds a LOX-1 epitope expressed on a cell surface of a target cell and the coupled cytotoxic agent induces cell death of the target cell. In certain embodiments, the target cell is a LOX-1 expressing neutrophil. In certain embodiments, the target cell is a LOX-1 expressing PMN. In certain embodiments, the target cell is a PMN- MDSC. In certain embodiments, the target cell is a LOX-1 positive PMN- MDSC. In certain embodiments, the antibody or epitope binding fragment thereof may be immobilized on a substrate, for example, a plate, a bead or a slide. COMPOSITIONS A variety of compositions are provided, such as the ones useful in producing the antibody or epitope binding fragment thereof. For example, a hybridoma cell or a host cell producing the described antibody or an epitope binding fragment thereof. In certain embodiments, the hybridoma cell or host cell comprises a hLOX-1 coding sequence as described herein.
Also provided are compositions useful in detecting expression of hLOX-1 protein, or in identifying, detecting and optionally separating cells (for example, PBMC, neutrophil or PMN) expressing hLOX-1 (for example on cell surface or in a cell) (i.e. LOX-1 positive cells) from cells not expressing hLOX-1 (i.e. LOX-1 negative cells) and other debris.
Additionally, provided are compositions useful in diagnosing presence, progression or metastasis of a cancer in a subject. In one embodiment, a diagnostic reagent composition or a kit is provided comprising one or more of the antibodies or epitope binding fragments thereof as described herein.
In a further embodiment, the diagnostic composition or the kit may further comprise a ligand that binds other biomarker/genetic signatures of the PMN-MDSCs, such as those listed in Table 1 in US Patent Application Publication No. US20180059115, and/or a biomarker or a regulator of pathways for ER stress response, such as IRE-1 RNase, sXBPl, DDIT3 (CHOP), ATF4, ATF3, SEC61A ARGI, NOS-2, MYCN, CSF3, IL3, TGFbl, TNF, LDL, RAFl, APP, IL6 PDGFBB, EPO, CD40LG, NFkB, IL13, AGT, IL1b, ERBB2, MAP2K1, VEGFa, CSF1, FL11, or IFNg. Still other likely biomarkers for pathways involved or activated in PMN-MDSC production may be included. In one embodiment, the ligand may be covalently or non-covalently joined with a detectable label or substrate. Selection and/or generation of suitable ligands with optional labels for use in this invention is within the skill of the art, provided with this specification, the documents incorporated herein, and the conventional teachings of the art In a further embodiment, the diagnostic reagent composition or the kit also contains miscellaneous reagents and apparatus for reading labels, e.g., certain substrates that interact with an enzymatic label to produce a color signal, etc., apparatus for taking blood samples, as well as appropriate vials and other diagnostic assay components.
Further provided are pharmaceutical compositions useful in reducing, inhibiting, retaining, or suppressing growth of the PMN-MDSC population, and/or useful in treating a cancer. For example, provided is a composition comprising the antibody or epitope binding fragment thereof as described herein and a pharmaceutically acceptable carrier and/or excipient. In certain embodiments, the pharmaceutical composition reduces or inhibits ER stress in mammalian neutrophils or reduces or inhibits LOX-1 expression on neutrophil populations. In one embodiment, this composition further comprises an antagonist or inhibitor of the expression, activity or activation of one or more of IRE- 1 RNase, sXBPl, DDIT3 (CHOP), ATF4, ATF3, SEC61A ARGI or NOS-2. In certain embodiments, this composition comprises an ER stress antagonist B-I09. In one embodiment, the composition further comprises an antagonist or inhibitor of LOX-1. In still further embodiments, the composition contains additional antagonist or inhibitor of the expression, activity or activation of one or more of IRE-1 RNase, MYCN, CSF3, 1L3, TGFbl, TNF, LDL, RAFl, APP, IL6 PDGFBB, EPO, CD40LG, NFkB, IL13, AGT, IL1b, ERBB2, MAP2K1, VEGFa, CSF1, FLI1, or IFNg, or of the pathways leading to the production of the immunosuppressive PMN-MDSC populations in vivo. In certain embodiments, the composition further comprises a chemotherapy drug for treating cancer. In certain embodiments, the composition is formulated with another effective compound or reagent for treatment of the cancers described herein, such as an antibiotic or bactericide, a surfactant, or other reagent commonly used in formulation of anti-cancer compositions.
The forms of the pharmaceutical compositions may be liquid, solid or a suspension or semi-solid and designed for use with a desired administrative route, such as those described herein. The doses and dosage regimens are adjusted for the particular cancer, and the stage of the cancer, physical status of the subject Such doses may range from about 1 to about 100 mg/kg subject body weight and include dosage regimens designed to administer the effective amount in smaller repeated doses.
METHODS
In one aspect, methods for treatment of a cancer associated with LOX+1 or another disease or disorder associated with LOX+1 utilize the compositions and kits as described herein. In another aspect, these described compositions and kits are useful for monitoring differentiation and/or population of polymorphonuclear myeloid derived suppressor cells (PMN-MDSCs), for identifying a LOX-1 inhibitor and/or a PMN-MDSC inhibitor, and/or for cancer diagnosis, and diagnosis of other LOX-l+-related diseases or disorders.
A. Diagnostic Methods
In one aspect, a method is provided for monitoring the population of polymorphonuclear myeloid derived suppressor cells (PMN-MDSCs) in a subject comprising contacting a biological sample from the subject containing polymorphonuclear neutrophils (PMNs) and PMN-MDSC with one or more of the antibody or epitope binding fragment described herein or the diagnostic composition; and detecting and optionally- distinguishing LOX-1 positive (LOX-1* ) cells from LOX-1 negative (LOX-1 " ) cells in the sample, wherein the LOX-1 + cells are PMN-MDSCs substantially free of PMN. In one embodiment, the method may be used for diagnosing a cancer, a cancer progression or metastasis in a subject. In one embodiment, the subject is diagnosed with a cancer if percentage of LOX-1 + cells in the total neutrophils in the sample is greater than a control. In one aspect, provided is a method for monitoring population of polymorphonuclear myeloid derived suppressor cells (PMN-MDSCs) in a subject comprising obtaining a biological sample containing polymorphonuclear neutrophils (PMNs) and PMN-MDSC; contacting the biological sample with one or more of the antibody or epitope binding fragment thereof or the diagnostic composition as described herein; and detecting and optionally distinguishing LOX-1 positive (LOX-1 + ) cells from LOX-1 negative (LOX-1-) cells in the sample, wherein the LOX-1+ cells are PMN-MDSCs substantially free of PMN. In certain embodiments, LOX-1 positive cells refer to cells expressing LOX-1 protein, for example, on cell surface or in the cell. In certain embodiments, LOX-1 negative cells refer to cells which does not express LOX-1 protein or does not express LOX-1 protein on their cell surface. Also provided is a method of diagnosing a cancer or monitoring progression or metastasis of a cancer in a subject comprising obtaining a biological sample from the subject; contacting the sample with one or more of the antibody or epitope binding fragment thereof or the diagnostic composition as described herein; and detecting and optionally distinguishing LOX-1 positive cells from LOX-1 negative cells in the sample. In certain embodiments, the subject is diagnosed with cancer or cancer progression (for example, tumor size) in the subject by correlation with the concentration of LOX-1 + cells detected. In certain embodiments, the subject is diagnosed with cancer or cancer metastasis if number of the LOX-1 positive cells is above that of a negative control. In certain embodiments, cancer progression (for example, tumor size) of the subject is determined by counting the LOX-1 positive cells and comparing it to a control. In certain embodiments, cells of the methods are PBMC, neutrophil, or PMN. As used herein, in certain embodiments, PMN refers to cells showing biomarkers shared by PMN-MDSC and PMN which is not PMN-MDSC, i.e., PMN-MDSC is a subgroup of PMN. In certain embodiments, PMN only refers to the one that is not PMN-MDSC and/or is not LOX-1 positive. In certain embodiments of the methods, the subject is suspected of having a cancer, had a cancer, is having a cancer, is suspected of having a cancer progression, or is suspected of having a cancer metastasis. In some of the methods described herein, a control level is used as a reference point. The control level can be any of those described herein. In one embodiment, the control level is the level obtained from an individual, or a population of individuals, who are healthy (i.e., who do not have a cancer). In another embodiment, the control level is the level obtained from an individual, or a population of individuals, who have cancer that has not metastasized. In yet another embodiment, the control level is the level obtained from an individual, or a population of individuals, who have cancer at different progression stages (for example, having different tumor sizes). In certain embodiments, the method further comprises counting or collecting the LOX-1+ cells, or LOX-1+ neutrophils, or LOX-1+ PMNs. In a further embodiment, the method comprises counting the total number of the cells, or neutrophils, or PMNs and calculation rations of LOX-1+ cells/neutrophils/PMNs. In certain embodiments, the method comprises washing to reduce or eliminate LOX-1 negative cells and other debris in the sample. In certain embodiments, the method comprises separating LOX-1+ cells from LOX-1- cells or neutrophils from non-neutrophil cells in the sample based on cell size. In certain embodiments, the method further comprises destroying or lysing any red blood cells in the sample to permit their elimination from the sample and possible interference with the results of the assay. In certain embodiments, the method further comprises digesting a biological sample (such as a tissue) and releasing cells from the sample. Exemplary lytic reagents, stabilizing reagents and the method of use have been described, e.g., in U.S. Patent Nos.6,573,102 and 6,869,798. Alternatively, the reagent system can also be an isotonic lysing reagent as described in U.S. Pat. No.5,882,934. Other lytic reagents known in the art can also be used for the purpose of the present methods. In certain embodiments, the method further comprises collecting LOX-1 negative cells which contains PMNs and being substantially free from PMN-MDSCs. As used herein, A “being substantially free” from B refers to a mixture of A and B, wherein the ratio of number of A to that of B is at least about 5:1, or about 10:1, or about 20:1, or about 50:1, about 100:1, about 200:1, about 500:1, about 1000:1 or more. In one embodiment, the ratio of number of A to that of B is about 10:1. In certain embodiments, a population of LOX-1 positive cells greater than a control level, or about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, or about 20% of the total neutrophil population in the sample of a subject indicates the presence, progression or metastasis of a cancer. In certain embodiments, the population of LOX-1 positive cells greater than about 5% of the total neutrophil population in the sample of a subject indicates presence, progression, or metastasis of a cancer. In one embodiment, provided is an assay method for diagnosis of having a cancer in a subject comprising: contacting a biological sample obtained from a patient with one or more of the antibody or epitope binding fragment thereof as described herein; detecting the level of binding between a component of said biological sample and the antibody or epitope binding fragment thereof; and diagnosing the subject with a cancer if a population of LOX-1 + cells greater than a control level or 1% of the total neutrophil population in the sample of a subject . In another embodiment, these methods can diagnose the aggressiveness of a cancer. In another embodiment, these methods can diagnose the stage of a cancer. According to the inventors’ early studies, in most healthy individuals the proportion of LOX-1+ PMN is less than between 0.5% to 1% PMN. Patients with stage II diseases usually have between about 3 about 5% of LOX-1+ PMN and patients at stages III-IV have over 5% to about 12% PMN. In certain embodiments, the method described herein may further comprise contacting one or more of the antibody or epitope binding fragment thereof as described herein with a LOX-1 expressing cell, serving as a positive control. In certain embodiments, patients with a cancer/tumor, a cancer/tumor metastasis or at a specific progression stage are served as a positive control. In certain embodiments, the method may further comprise contacting a biological sample from a subject who does not have cancer, or a biological sample pooled from subjects who do not have cancer with one or more of the antibody or epitope binding fragment thereof as described herein, serving as a negative control. In certain embodiments, the control level is acquired from a pooled sample or is an average of more than one control levels. In certain embodiments, a patient is diagnosed as having a cancer, a cancer progression, or cancer metastasis, if amount/concentration of LOX-1+ cells/neutrophils/PMNs or LOX-1 expression level on the LOX-1+ cells/neutrophils/PMNs is greater than a negative control. Alternatively or additionally, the amount/concentration of LOX-1+ cells/neutrophils/PMNs or the LOX-1 expression level on the LOX-1+ cells/neutrophils/PMNs is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 1 fold, about 2 fold, about 5 fold, or about 10 fold of that of a positive control. In certain embodiments, detection of amount/concentration of LOX-1+ cells/neutrophils/PMNs in a biological sample or LOX-1 expression level on the LOX-1+ cells/neutrophils/PMNs may be performed by detecting level of the detectable label. In a further embodiment, the method further comprises diagnosing the patient as having a cancer, cancer progression or cancer metastasis when the detectable label is detected at a level greater than that in a negative control. Alternatively or additionally, the method further comprises diagnosing the patient as having a cancer, cancer progression or cancer metastasis when the detectable label is detected at a level which is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 1 fold, about 2 fold, about 5 fold, or about 10 fold of that of a positive control. A method for enhancing or inhibiting differentiating polymorphonuclear myeloid derived suppressor cells (PMN-MDSCs) from polymorphonuclear neutrophils (PMNs) or monocytic myeloid derived suppressor cells (M-MDSCs) in a biological sample containing these types of cells involves the following steps. The biological sample, e.g., whole blood or a cell suspension, or a tumor exudate, or tissue, e.g., biopsy material, is contacted with an antibody or epitope binding fragments thereof. Thus, one may detect antibody- conjugate complexes in the sample. Such detection can be based upon separation of the ligand-bound cells from unbound cells in the sample. The LOX-1-bound cells are PMN- MDSCs substantially free of PMN. The detection and separation of LOX-1 positive cells in the sample may be accomplished by a physical characteristic, such as the difference in size or weight of the LOX-1 positive cells vs. the LOX-1 negative cells. Such detection and/or separation techniques can thus employ appropriately sized filtration units, or the use of flow cytometry, or chromatographic or centrifugation techniques (size exclusion or weight exclusion), among others known to the art. The method of identifying and separating PMN-MDSCs from a sample can also include contacting the biological sample with other biomarkers that identify as a single population both PMN-MDSCs and PMNs and/or M-MDSCs and isolating a cell suspension containing PMN-MDSCs and PMNs (and/or M-MDSCs) prior to, or simultaneously with, contacting the cell suspension with the LOX-1 antibody or epitope binding fragment thereof. In still other embodiments of the methods, the sample may be contacted (with or without RBC lysis) with a LOX-1 antibody or epitope binding fragment thereof and a ligand that identifies neutrophils, i.e., other PMN that are not LOX-1 + . In one embodiment, the sample is contacted with a LOX-1 ligand and a CD15 ligand. In still other embodiments of the methods, the sample may be contacted with a LOX-1 antibody or epitope binding fragment thereof and a CD66b ligand. Still other ligands that identify neutrophils generally may be useful in this context. In certain embodiments, the contacting step further comprises contacting the sample with a ligand that specifically binds to or forms a complex with a neutrophil biomarker to identify neutrophil or polymorphonuclear neutrophils (PMNs). In one embodiment, the neutrophil biomarker is CD33, CD11b, CD 14, CD15 or CD66b. In one embodiment, the detecting step comprises detecting and optionally distinguishing LOX-1 positive neutrophils from LOX-1 negative cells in the sample. In a further embodiment, the diagnosing step comprises diagnosing the subject with cancer or determining cancer progression (for example, tumor size) in the subject by correlation with the concentration of LOX-1+ neutrophils detected. In certain embodiments, the contacting step further comprises contacting the sample with a ligand that specifically binds to or forms a complex with a polymorphonuclear neutrophils (PMNs) biomarker to identify PMNs. In one embodiment, the biomarker may be any one or more of CD33, CD11b, CD 14, CD15 or CD66b. In one embodiment, the method further comprises a step of identifying cells with biomarkers shared by both PMN-MDSCs and PMNs, and isolating cells of both PMN-MDSCs and PMNs prior to the contacting step. In one embodiment, the detecting step comprises detecting and optionally distinguishing LOX-1 positive PMNs from LOX-1 negative cells in the sample. In a further embodiment, the diagnosing step comprises diagnosing the subject with cancer or determining cancer progression (for example, tumor size) in the subject by correlation with the concentration of LOX-1+ PMNs detected. The method of identifying and separating PMN-MDSCs from a sample can also include contacting the biological sample with the other biomarkers forming the distinguishing signature of PMN-MDSC or other biomarkers that identify as a single population both PMN-MDSCs and PMNs and/or M-MDSCs and isolating a cell suspension containing PMN-MDSCs and PMNs (and/or M-MDSCs) prior to, or simultaneously with, contacting the cell suspension with the LOX-1 antibody or epitope binding fragment thereof. In still other embodiments of the methods, the sample may be contacted (with or without RBC lysis) with a LOX-1 antibody or epitope binding fragment thereof and a ligand that identifies neutrophils, i.e., other PMN that are not LOX-1 + . In one embodiment, the sample is contacted with a LOX-1 ligand and a CD15 ligand. In still other embodiments of the methods, the sample may be contacted with a LOX-1 antibody or epitope binding fragment thereof and a CD66b ligand. Still other ligands that identify neutrophils generally may be useful in this context. In one embodiment, therefore, the method involves contacting the biological sample with the ligand for CD15 prior to, or simultaneously with, the use of the LOX-1 antibody or epitope binding fragment thereof. In one embodiment, therefore, the method involves contacting the biological sample with a ligand for CD66b prior to, or simultaneously with, the use of the LOX-1 antibody or epitope binding fragment thereof. In one embodiment, therefore, the method involves contacting the biological sample with a ligand for CD14 prior to, or simultaneously with, the use of the LOX-1 antibody or epitope binding fragment thereof. In one embodiment, therefore, the method involves contacting the biological sample with a ligand for CD11b prior to, or simultaneously with, the use of the LOX-1 antibody or epitope binding fragment thereof. In one embodiment, therefore, the method involves contacting the biological sample with the ligand for CD33, prior to, or simultaneously with, the use of the LOX-1 antibody or epitope binding fragment thereof. In one embodiment, therefore, the method involves contacting the biological sample with a ligand for CD14 and a ligand for CD15 prior to, or simultaneously with, the use of the LOX-1 antibody or epitope binding fragment thereof. In another embodiment, therefore, the method involves contacting the biological sample with a ligand for CD14, and a ligand for CD11b prior to, or simultaneously with, the use of the LOX-1 antibody or epitope binding fragment thereof. In another embodiment, therefore, the method involves contacting the biological sample with a ligand for CD14 and a ligand for CD33 prior to, or simultaneously with, the use of the LOX-1 antibody or epitope binding fragment thereof. In another embodiment, therefore, the method involves contacting the biological sample a ligand for CD15 and a ligand for CD11b prior to, or simultaneously with, the use of the LOX-1 antibody or epitope binding fragment thereof. In another embodiment, therefore, the method involves contacting the biological sample with a ligand for CD15 and a ligand for CD33 prior to, or simultaneously with, the use of the LOX-1 antibody or epitope binding fragment thereof. In another embodiment, therefore, the method involves contacting the biological sample with a ligand for CD15, a ligand for CD11b and a ligand for CD33 prior to, or simultaneously with, the use of the LOX-1 antibody or epitope binding fragment thereof. In another embodiment, therefore, the method involves contacting the biological sample with a ligand for CD14, a ligand for CD11b and a ligand for CD33 prior to, or simultaneously with, the use of the LOX-1 antibody or epitope binding fragment thereof. In one embodiment of the method, any of these biomarkers may be detected prior to, or simultaneously with, the detection of the LOX-1 biomarker. The use of these other ligands assists in identifying all PMNs from other cells in the sample. Subsequent exposure of this population of cells from the sample with the LOX-1 ligands enables further separation of the PMN-MDSCs from the PMN population. In one embodiment, following contact with the LOX-1 antibody or epitope binding fragment thereof and a second neutrophil specific biomarker ligand, such as a CD15 ligand or CD66b ligand, one may calculate the number of LOX-1+ vs. CD15+ or the number of LOX-1+ vs. CD66b+ cells are present in the sample. Such calculation can involve cell counting systems known to those of skill in the art. In another embodiment, the method involves collecting as a second population, the cells which did not form complexes with the ligands, e.g., are not providing a detectable signal or are not immobilized on the substrate. This second population contains PMNs and other cells substantially free from PMN-MDSCs. In still another embodiment, the methods described herein permit the obtaining of a population of cells enriched in human polymorphonuclear myeloid derived suppressor cells (PMN-MDSCs) by isolating from a cell suspension those cells which express LOX-1 to provide a population of cells enriched with PMN-MDSCs. In still another embodiment, the methods involve measuring the amount/concentration of LOX-1 (for example, soluble LOX-1) in the serum and correlating that amount/concentration with the number of LOX-1+ PMN-MDSC. These methods also permit the removal of human PMN-MDSCs from a cell population, comprising isolating from the cell population those cells which express LOX-1. These methods are useful in one embodiment for monitoring of the progression or metastasis of a cancer or the monitoring of therapy in a cancer patient by permitting the evaluation of an increase in the LOX-1 cell surface receptor in a biological fluid of a patient having a cancer or under treatment for cancer. The increase of LOX-1+ cell number is indicative of metastasizing cancer or a progression of cancer. In other embodiments, this method may be useful diagnostically to initially detect the presence of cancer. These methods depend initially upon obtaining an accurate enumeration or concentration of a PMN-MCSC cell population, substantially free of any PMNs, from a suitable biological sample of a subject. In one embodiment, these methods of determining an accurate cell count/concentration of cells expressing LOX-1 in a subject having a cancer or being treated for a cancer can be used to monitor the progression of the cancer (with or without treatment). In still another embodiment, the use of these methods to determine an accurate measurement of LOX-1+ cells enable the monitoring of metastasis in a cancer, e.g., an increase in the LOX-1+ cell number indicates metastatic cancer. In another embodiment, these methods are useful to monitor and/or influence cancer treatment. For example, where the LOX-1+ cell number is increasing prior to cancer therapy, and subsequent performance of the method on a similar sample in the subject does not show a decrease in LOX-1+ cell number, the method can indicate that a change in therapeutic method or dosage is necessary. In still another aspect, the method of measuring the LOX-1+ population in a fluid sample, such as whole blood, can be employed as a research method to determine the cause of the increase in such cells during the progression of a cancer. In still other aspects of the diagnostic methods identified above, additional diagnostic steps include contacting the sample with a reagent that identifies activators or regulators of ER stress response in said cells. In one embodiment, the activators or regulators so identified are one or more of sXBP1, DDIT3 (CHOP), ATF4, ATF3, SEC61A ARGI or NOS-2. In another embodiment, the regulators are one or more of one or more of MYCN, CSF3, IL3, T*)ȕ^^^71), LDL, RAF1, APP, IL6 PDGFBB, EPO, CD40LG, NFkB, IL13, A*7^^,/^ȕ^^(5%%^^^MAP2K1, VE*)Į^^&6)^^^)/,^^^RU^,)1Ȗ^ Yet another embodiment of a diagnostic method for a mammalian subject with a cancer comprises the additional step of determining the size of a tumor in the subject by correlation with the number of LOX-1+ PMN or PMN-MDSC detected. This method step includes obtaining a biological sample from the subject; detecting whether LOX-1 is present in the sample by contacting the sample with an antibody or epitope binding fragment thereof as described herein; and detecting and distinguishing the complexes of antibody-bound LOX-1-cells from other cells not bound to the antibody in the sample. The size of the tumor is then determined based upon the increase of LOX-1+ PMNs or PMN- MDSCs over a baseline level. The baseline level is readily determined based upon enumeration of patient samples to create a standard. The presence of LOX-1 (and any of the PMN-MDSC signature biomarkers) in the sample may be detected using any assay format known in the art or described herein. There are a variety of assay formats known to the skilled artisan for using the antibody or epitope binding fragment thereof and optionally a ligand to detect a signature biomarker in a sample. For example, see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, the presence or absence of LOX-1 in a sample may be determined by (a) contacting the sample with the antibody or epitope binding fragment thereof; and (b) determining the presence or level of LOX-1 in the sample, wherein the presence of LOX-1 in the sample is indicative of cancer or where an increase in the level of LOX-1 in the sample as compared to a control, is indicative of cancer. Methods of detection, diagnosis, monitoring, and prognosis of cancer, or the status of cancer, and for the identification of subjects with an increased risk of cancer metastasis by detecting the presence of, or measuring the level of, LOX-1 protein or another biomarker described herein. Such methods may employ the antibodies or epitope binding fragments thereof as described herein. The particular assay format used to measure the LOX-1 in a biological sample may be selected from among a wide range of immunoassays, such as enzyme-linked immunoassays, sandwich immunoassays, homogeneous assays, immunohistochemistry formats, an enzyme linked immunosorbent assay (ELISA), a lateral flow assay, a radioimmunoassay (RIA), Fluorescence-activating cell sorting (FACS), a western blot, an immunoprecipitation, or other conventional assay formats. One of skill in the art may readily select from any number of conventional immunoassay formats to perform this invention. Other reagents for the detection of protein in biological samples, such as peptide mimetics, synthetic chemical compounds capable of detecting LOX-1 may be used in other assay formats for the quantitative detection of LOX-1 protein in biological samples, such as high-pressure liquid chromatography (HPLC), immunohistochemistry, etc. The diagnostic methods described herein can employ contacting a patient’s sample with a diagnostic reagent, as described above, which forms a complex or association with LOX-1 in the patients’ sample. Detection or measurement of the sample LOX-1 may be obtained by use of a variety of apparatus or machines, such as computer-programmed instruments that can transform the detectable signals generated from the diagnostic reagents complexed with the LOX-1 or other biomarker in the biological sample into numerical or graphical data useful in performing the diagnosis. Such instruments may be suitably programmed to permit the comparison of the measured LOX-1 in the sample with the appropriate reference standard and generate a diagnostic report or graph. In another aspect, provided is a method of evaluating differentiation of polymorphonuclear myeloid derived suppressor cells (PMN-MDSCs) from polymorphonuclear neutrophils (PMNs) in a biological sample containing both types of cells comprising: contacting the sample with C-reactive protein (CRP), or Oxidized-low density lipoprotein (ox-LDL), or other agent which induces differentiation of PMN-MDSC, or activators or regulators of ER stress response; and detecting and optionally collecting PMN-MDSCs using one or more of the antibody or epitope binding fragment thereof as described herein. In certain embodiments, the agent is identified as an antagonist or inhibitor of PMN-MDSC differentiation if treatment with the agent decreases expression of LOX-1 or numbers/concentration of LOX-1 positive cells/neutrophils/PMNs compared to a no-treatment control. In certain embodiments, the agent is identified as an activator of PMN-MDSC differentiation if treatment with the agent increases expression of LOX-1 or numbers/concentration of LOX-1 positive cells/neutrophils/PMNs compared to a no- treatment control. Also provided is an assay method of identifying an antagonist or inhibitor of LOX- 1 expression, comprising contacting a biological sample or a cell population of PMN- MDSCs and PMNs with a test agent; and detecting expression of LOX-1 using one or more of the antibody or epitope binding fragment thereof as described herein. In certain embodiments, the test agent is identified as an antagonist or inhibitor of LOX-1 expression if treatment with the test agent decreases expression of LOX-1 or LOX-1 positive cells/neutrophils/PMNs compared to a no-treatment control. B. Therapeutic Methods In one aspect, a therapeutic method involves administering to a patient in need thereof an effective amount of a composition that reduces or inhibits ER stress response in mammalian neutrophils or reduces or inhibits LOX-1 expression on neutrophil populations In another embodiment, the antibodies and fragments described herein are useful in an amount that reduces or inhibits the growth or metastasis of cancer cells that express LOX- 1. In another embodiment, the amounts of the compositions containing the antibodies, fragments, or inhibits is effective to reduce the growth, spread or amount of LOX-1+ cells in any disorder characterized by cells expressing LOX-1. In one embodiment, the patient has a LOX-1+ associated cancer. In another embodiment, the patient has a LOX-1+ associated disease. In another aspect, a method of treating a cancer in a subject is provided comprising administering an effective amount of one or more of the antibody or epitope binding fragments thereof as described herein, with an additional inhibitor of LOX-1 expression or inhibitor of the ER stress response. In a further embodiment, B-I09 is administered to the subject. In other embodiments, the composition that reduces or inhibits the ER stress response comprises one or more of the antibody or epitope binding fragment thereof as described herein, and/or an antibody or functional antigen-binding fragment that binds to or inhibits the expression, activity or activation of at least one of sXBPl, DDIT3 (CHOP), ATF4, ATF3, SEC61A ARGI, MYCN, CSF3, IL3, TGFbI, TNF, LDL, RAF1, APP, IL6 PDGFBB, EPO, CD40LG, Nek, IL13, AGT, IL1b, ERBB2, MAP2K1, VEGFa, CSF1, FLI1, Fin, CD15, CD66b or CD33.
In still other embodiments, the treatment step may be combined with the diagnostic steps in a combined method. Such a method combines diagnosing and treating a cancer and combines the steps, such as obtaining a biological sample from a subject; detecting whether PMN-MDSC are present in the sample via detecting LOX-1 expressing cells/neutrophils/PMNs; diagnosing the subject with cancer when the presence of LOX-1+ (optionally with any other of the PMN-MDSC signature biomarkers identified herein or in the references cited) is detected at a level that indicates PMN-MDSC are present; and administering an effective amount of a composition that reduces or inhibits ER stress response in mammalian neutrophils or reduces or inhibits LOX-1 expression on neutrophil populations.
In another aspect, provided is a method of treating a cancer in a subject comprising: obtaining a biological sample from a subject; contacting the sample with one or more of the antibody or epitope binding fragment thereof or the composition as described herein; detecting whether LOX-1 positive cells are present in the sample; and the presence of LOX-1+ cell is detected, administering an effective amount of a composition that reduces or inhibits ER stress response in mammalian neutrophils or reduces or inhibits LOX-1 expression on neutrophil populations (for example, B-I09), and/or an identified LOX-1 inhibitor, and/or an identified PMN-MDSC differentiation inhibitor, and/or the antibody or epitope binding fragment thereof. In certain embodiments, the LOX-1 positive cells are LOX-1 positive neutrophils. In certain embodiments, the LOX-1 positive cells are LOX-1 positive MDSC. In certain embodiments, the LOX-1 positive cells are PMN-MDSC.
In yet another aspect, provided is a method of treating a cancer in a subject comprising: obtaining a biological sample from a subject; contacting the sample with one or more of the antibody or epitope binding fragment thereof or the composition as described herein; detecting whether PMN-MDSC are present in the sample; and the presence of LOX-1+ is detected at a level that indicates PMN-MDSC are present, administering an effective amount of a composition that reduces or inhibits ER stress response in mammalian neutrophils or reduces or inhibits LOX-1 expression on neutrophil populations, and/or an identified LOX-1 inhibitor, and/or an identified PMN-MDSC differentiation inhibitor, and/or the antibody or epitope binding fragment thereof. In certain embodiments, the composition that reduces or inhibits the ER stress response comprises one or more of the antibody or epitope binding fragment thereof as described herein, and/or an antibody or functional antigen-binding fragment that binds to or inhibits the expression, activity or activation of at least one of sXBPl, DDIT3 (CHOP), ATF4, ATF3, SEC61A ARG1, MYCN, CSF3, IL3, Tgf1I, TNF, LDL, RAF1, APP, IL6 PDGFBB, EPO, CD40LG, Nek, IL13, AGT, IL1b, ERBB2, MAP2K1, VEGFa, CSF1, FLI1, Fin, CD15, CD66b or CD33.
In certain embodiments, provided is a method of treating a cancer in a subject comprising obtaining a biological sample from the subject; removing and deleting LOX-1+ PMN-MDSC with an effective amount of one or more of the antibody or epitope binding fragment thereof or a composition as described herein; and administering the biological sample substantially free from PMN-MDSCs to the subject.
Whether the LOX-1+ antibodies and/or epitope binding fragments are administered alone to treat the LOX-1+ associated cancer or disease, or are coupled with diagnostic and therapeutic steps as described above, the LOX-1 + antibodies and/or fragments may be administered by any suitable method or route. Similarly, the same or other routes can be used to co-administer other active drugs or therapies in conjunction with the composition described herein. Routes of administration include, for example, systemic, oral, intravenous, intraperitoneal, subcutaneous, intramuscular, or intratumor administration. In certain embodiments, administration involves directly introducing the compositions as described into a tumor microenvironment. In still other embodiments the LOX-1 antibodies or fragments may be coupled with other therapeutic moieties to target the other therapeutic moieties to the environment of LOX-1+ expressing cells or tissues.
EXAMPLES
The invention is now described with reference to the following examples. These examples are provided for the purpose of illustration only. The compositions, experimental protocols and methods disclosed and/or claimed herein can be made and executed without undue experimentation in light of the present disclosure. The protocols and methods described in the examples are not considered to be limitations on the scope of the claimed invention. Rather this specification should be construed to encompass any and all variations that become evident as a result of the teaching provided herein. One of skill in the art will understand that certain changes or variations can be made in the disclosed embodiments of the examples and expected similar results can be obtained. For example, the substitutions of reagents that are chemically or physiologically related for the reagents described herein are anticipated to produce the same or similar results. All such similar substitutes and modifications are apparent to those skilled in the art and fall within the scope of the invention. EXAMPLE 1: LOX1 MAB DEVELOPMENT. Monoclonal antibodies against LOX-1 is developed as summarized as follows and further detailed in the Examples: (1) Lox-1 Immunogen was generated including construction of DNA and production of recombinant Lox1 protein; (2) animal was immunized and seroconversion was evaluated; (3) spleen cells were isolated and antibody secreting cells were enriched; (4) antibody secreting cells were fused with myeloma cells followed by hybridoma positive selection and expansion screen for specificity; (5) variable regions of both heavy and light chains were amplified via PCR and sequenced; and (6) characterization and modification of heavy and light chains were performed for large scale amplification. Construction of human Lox1 DNA The human LOX1 plasmid DNA construct was synthesized. The DNA immunogen was computationally aligned and optimized via codon and RNA optimization for enhanced expression. Additionally, an IgE leader sequence (aa 1 - 18 of SEQ ID NO: 1) was inserted to enhance immunogen expression. This optimized hLOX1 immunogen (with amino acid sequence of SEQ ID NO: 1) was constructed by subcloning into pMV101 expression vector with the cytomegalovirus immediate-early promoter. The large-scale DNA production was carried out and DNA gel electrophoresis technique used to separate DNA fragments to confirm the insert. Generation and characterization of hLOX1 recombinant protein. A recombinant protein spanning the entire human LOX1 sequence was synthesized in-house since commercial reagents only encode portions of the full-length protein. The hLOX1 immunogen was cloned into pET30a Escherichia coli expression vector and produced. Nickel column chromatography method was used to purify the recombinant hLOX1 protein. SDS-PAGE was used to analyze the predicted size of overexpressed proteins in lysates using anti-His tag antibodies. DNA immunization and Electroporation delivery in mice Six- to eight-week old female BALB/c mice (The Jackson Laboratory, ME, USA) were housed in the Wistar Institute Animal Facility in a light-cycled, temperature- and humidity-controlled condition. All animal studies were performed in accordance with the recommendations and guidelines from the National Institute of Health (NIH) and the Wistar Institute Institutional Animal Care and Use Committee. Mice were injected by a 30cc syringe with 50ug of DNA diluted in 30ul of sterile water into the anterior tibialis (TA) muscle. The injection site is immediately electroporated using the CELLECTRA electroporation (EP) delivery device (Inovio Pharmaceuticals, PA, USA). This 26-gauge, stainless steel three-pronged probe is inserted 2mm into the injection site into the TA muscle and delivered 0.1Amps of triangulated square-wave pulses for 52 msec twice. Blood via the submandibular method is collected prior to the DNA injection and EP delivery to assess antibody levels in circulation. Mice under all experimental manipulations were anesthetized with 2-5% isoflurane (Pheonix, MO, USA). In addition to the DNA immunization as described above, mice were subcutaneously injected 5ug of hLOX1 recombinant protein mixed with incomplete Freund’s adjuvant (Sigma, USA) in a total volume of 100ul per injection. All animal studies were performed in accordance with the recommendations and guidelines from the National Institute of Health (NIH) and the Wistar Institute Institutional Animal Care and Use Committee. ELISA for the detection of antibodies and seroconversion Flat-bottomed MaxiSorp 96-well ELIS plates (ThermoFisher, USA) were coated with 1mg/ml of hLOX1 recombinant protein diluted in PBS and incubated at 4ºC overnight. Plates were washed 4 times with PBS buffer solution (PBS+ 0.01% Tween-20 (ThermoFisher, USA)) and blocked with 10% FBS diluted in PBS for 1 hour at room temperature. On a separate, non-absorbent U-bottom 96-well plate, serum samples are diluted on a half-log scale starting with the initial dilution of 1:50. One hundred microliter (ml) of the diluted samples in each well are transferred to the Maxisorp plate after 5 washes. The plate is incubated for 2-hours room temperature, then washed 5 times with PBST. The plate is then incubated with HRP-labeled goat anti-mouse IgG (Sigma-Aldrich, USA) for one hour. The plate is washed 5 times and 100ml of 3,3’5,5’-Tetramethylbenzidine (TMB) Substrate (Sigma-Aldrich, USA) is added to each well for 10 minutes before the reaction being stopped with 2M H 2 SO 4 solution. The plate is read at 450nm by Biotek ELISA plate reader. The antibody endpoint titer is defined as the highest dilution of a sample with OD values >(mean+3SD) of vehicle sample. Samples with a titer less than 50 were given the endpoint titer of 1. Lox-1 hybridoma fusion & Characterization of specificity of hLOX-1 mAbs Based on the ELISA result for the highest-binding sera, immunogen-boosted mice were preliminarily screened for the highest OD value as well as the highest endpoint titer then sacrificed to harvest the spleen. Single-cell suspensions of splenocytes in R10 were prepared using a Stomacher 80 paddle blender (AJ. Seward and Co. Ltd.) for 30 seconds on high speed. The samples were filtered through 45mm nylon filters and the cell pellets were collected at 1,500g centrifuged 10min at 4ºC. The collected cell pellets were lysed using ammonium-chloride-potassium lysis buffer (ACK) (Life Technologies, USA). The splenocytes in single-cell suspension was sent to Fox Chase Cell Culture Center (Philadelphia, PA) for a fusion process with P3X63.Ag.6.5.3 myeloma cells. The initial fusion produces approximately 1400 hybridoma candidates. After antibody screening through ELISA as previously mentioned, approximately 50-60 antibody-producing mouse hybridoma clones were identified as having antibody binding at least four-fold greater than the background level reactivity. The cell lines of top 5 hybridoma candidates are amplified, and the extracted RNA of each cell line is sequenced for HL and VL variable regions using PCR. Total RNA was isolated from the hybridoma cells following the technical manual of TRIzol® Reagent (TRIzol® Reagent (Ambion, Cat. No.15596-026). Total RNA was then reverse-transcribed into cDNA using either isotype-specific anti-sense primers or universal primers with 1st Strand cDNA Synthesis Kit. (PrimeScriptTM 1st Strand cDNA Synthesis Kit, Takara, Cat. No.: 6110A). Antibody fragments of heavy chain and light chain were amplified by rapid amplification of cDNA ends (RACE). Amplified antibody fragments were cloned into a standard cloning vector separately. Colony PCR was performed to screen for clones with inserts of correct sizes. Individual positive clones with correct VH and VL insert sizes were sequenced. Comparison of the hLox-1 IgG avidity test results for hybridoma. Microtiter plates previously coated with recombinant Lox-1 antigens were washed 3 times with PBS plus 0.05% tween 20 (PBST). Hybridomas 1B6, 2A10, 3D8, 3E6, 4D6, 5C2, 5F5, 6A10, 8E7, 9E12, 10H11, 11A6, 12D9, 11C5, 12A10, 12E4, 14A12, and 12G6 samples were diluted as indicated and added (100ml/well). The avidity of antibodies against hLOX-1 protein was determined by a particle disruption ELISA using 4M Urea. The binding of hLOX1 protein and incubation with mAbs were performed followed by a 5- minute incubation with urea after washing five times with 0.05% PBST. Control (urea- untreated) wells were treated with PBS during the 5-minute incubation. The anti-human IgG conjugated with horseradish peroxidase (HRP) was added with the dilution of 1/5000 in PBS for 1 hour. Afterwards, the plate is washed 5 times, and 100ml of 3,3’5,5’- Tetramethylbenzidine (TMB) Substrate (Sigma-Aldrich, USA) is added to each well for 10 minutes before the reaction being stopped with 2M H 2 SO 4 solution. The plate is read at 450nm by Biotek ELISA plate reader. Binding properties of hLOX-1 mAbs-Western blot analysis Immunological reactivity of hLox-1 antibodies was characterized by Western blot analysis against native and denatured full-length recombinant proteins. Protein samples were electrophoretically separated under native or denaturing conditions on SDS-PAGE, performed using Novex gels with reagents as followed: 4-10% mini-gel, 10× Native-PAGE running buffer and 2× Native-PAGE sample buffer. Denatured protein was heated in a heat block for 10 minutes at 57°C. Two micrograms (2mg) of recombinant full length hLOX1 protein or non-specific POWV-Env viral protein was loaded to each well along with a protein size marker as a ladder. Gels were run at 150 V for 1 hour in a running buffer. Protein was transferred to a nitrocellulose membrane using the iBlot 2 Gel Transfer Device (Life Technologies). Membranes were blocked in Odyssey blocking buffer (Licor) for 1 hour at room temperature. Indicated hybridomas were diluted 1:250 in 0.5X Odyssey blocking buffer with 0.1% Tween-20 (BioRad) and incubated with the membranes overnight at 4°C. Membranes were washed and then incubated with the appropriate secondary antibody (goat anti-mouse IRDye680RD) for hybridoma samples for 1 hour at room temperature. After washing with PBST 5 times, membranes were imaged on the Odyssey infrared imager. In summary, an immunogen strategy with a DNA immunization followed by a recombinant protein boost approach was developed to generate monoclonal antibodies (mAbs) against human LOX-1. Over 1000 clones were screened and 20 monoclones of binders were identified by ELISA. The antibodies were then characterized for Avidity and conformational binding by ELISA and Western Blotting (WB). Clones 6A10 and 12D9 were finalized as having strong avidity. Also, the preserved binding epitopes are highly conserved by both native as well as denatured conditions. All the group of clones are studied in Fluorescence-activated cell sorting (FACS) against human cell targets. Without wishing to be bound by the theory, native conditions are likely most important for Fluorescence-activated cell sorting (FACS), cell staining and functional assays, while WB denatured antibodies likely more important in diagnostic assays. Functionality of antibodies and their ability to function in immune assays are investigated in cancer models described below. EXAMPLE 2: USES OF ANTI-LOX1 ANTIBODIES Expression of LOX1 on PMN-MDSC by flow cytometry using the antibodies described herein or another available anti-LOX-1 monoclonal antibody (for example, clone 15C4; Biolegend Inc., San Diego, CA) is analyzed in blood samples from patients different types of cancer, for example, head and neck, breast, non-small lung, or colon cancer. Blood samples form healthy donors are served as negative control. PMN-MDSC are identified using classical definition, i.e., cells with CD11b+ CD14- CD15+ and CD33+ from the low- density mononuclear cells fraction. Additionally, blood sample from heathy donor and cancer patients are incubated with C-reactive protein (CRP), or Oxidized-low density lipoprotein (ox-LDL) or other factors which induces expression of LOX1 (for example, as identified in US Patent Application Publication with Publication No.20180059115). The antibodies as described herein are then used to evaluate up-regulation of LOX1 expression. Further, the antibodies as described herein are used to treat LOX1+ PMN together with a factor which induces expression of LOX1. LOX1 expression and ROS production are then evaluated. In certain embodiments, neutrophils from healthy donors are treated with 100 ug/ml immune complexes (for example, one or more of the described LOX-1 antibodies) for 18 hours to induce upregulation of LOX-1. In certain embodiments, one or more of the LOX-1 antibodies or epitope binding fragments is added at different concentrations (from 5 mg to 50 mg/ml) together with a reagent to measure reactive oxygen species (ROS) response (for example, 2’,7’ – dichlorofluorescin diacetate, i.e., DCFDA).30 minutes later, LOX^1 ligand oxidized LDL is added, and ROS production is measured. Reduction in ROS production indicates anti- ROS effects of the antibody or epitope binding fragment. Suppressive activity of PMN-MDSC is assessed in mixed leukocyte reaction where T cells from one donor are incubated at 10:1 ratio with dendritic cells from another donor. PMN-MDSC after treatment described above are added at different ratios to T cells (1:1 – 1:4) and T cell proliferation is measured 5 days later using 3 H-thymidine uptake. An immunohistochemical analysis of LOX- expression in tumor tissues was performed.5 mm sized paraffin embedded tissue sections were deparaffinized. Standard IHC protocol was followed to stain the tumor tissue samples using the monoclonal antibody against LOX-1(IgG1) for the detection of LOX-1 followed by staining with hematoxylin-eosin. LOX-1 expression was observed in tumor tissues from the liver, breast, ovarian, pancreatic, kidney and lung tissues at 20X, but not in colon or bladder tumor tissues. (see Fig.9). All documents, including websites, cited in this specification are incorporated herein by reference. The Sequence Listing filed herewith, labelled “WST183PCT _ST25.txt”, and the sequences and text therein are incorporated by reference. While the invention has been described with reference to particular embodiments, it will be appreciated that modifications can be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.
TABLE 6 (Sequence Listing Free Text) The following information is provided for sequences containing free text under numeric identifier <223>.