Methods and compositions for treating cancer

Abstract

The present invention provides compositions and methods for treating cancer in a subject by administering to the subject an antibody or fragment thereof that binds to oxidized LDL. In some embodiments, the compositions and methods can reduce tumor size, reduce macrophage infiltration, and / or inhibit metastasis.

Description

[Technical Field]

[0001] The present invention relates generally to compositions and methods for treating cancer by inhibiting the biological activity of oxidized low density lipoprotein. [Background technology]

[0002] Obesity is thought to increase the risk of developing diabetes and some types of cancer. Obesity is associated with various adverse physiological changes, including adipose tissue dysfunction, low-grade and chronic inflammation, and alterations in lipid metabolism and circulating hormone levels. Oxidized low-density lipoprotein (oxLDL) is a proinflammatory mediator formed as a result of oxidative modification of LDL in the arterial wall. oxLDL is most commonly associated with the progression of atherosclerosis and may have adverse cardiovascular (CV) outcomes, including myocardial infarction, stroke, and death. oxLDL signals through various cellular scavenger receptors, including scavenger receptor A (SR-A), cluster of differentiation antigen 36 (CD36), CD68, mucin, and lectin-like oxLDL receptor-1 (LOX-1).

[0003] LOX-1 is expressed in endothelial cells, but is also found in macrophages, smooth muscle cells, fibroblasts, and platelets. In addition, LOX-1 is secreted in a soluble form. Several studies have shown that oxLDL signaling through LOX-1 plays a major role in the development and progression of atherosclerosis. The present invention provides methods and compositions for treating cancer using oxLDL-dependent mechanisms. Summary of the Invention

[0004] In one aspect, the present invention provides a method for inhibiting macrophage infiltration into a tumor in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody or fragment thereof that binds to oxidized low-density lipoprotein (oxLDL) (i.e., an anti-oxLDL antibody or fragment thereof).

[0005] In another aspect, the present invention provides a method for inhibiting tumor metastasis in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or fragment thereof that binds to oxidized low-density lipoprotein (oxLDL) (i.e., an anti-oxLDL antibody or fragment thereof).

[0006] In some embodiments, the subject is diagnosed with a tumor.

[0007] In some embodiments, the tumor is a LOX-1 positive tumor, hi other embodiments, the tumor is positive for one or more of LOX-1, SR-A, CD36, CD38, and mucin.

[0008] In some embodiments, the tumor is selected from the group consisting of ovarian cancer, bladder urothelial carcinoma, renal clear cell carcinoma, rectal adenocarcinoma, colon adenocarcinoma, prostate cancer, breast epithelial cell tumor, glioblastoma, pancreatic cancer, and esophageal cancer.

[0009] In some embodiments, the method reduces the growth rate of a tumor, macrophage infiltration of a tumor, and / or the metastatic potential of a tumor.

[0010] In another aspect, the present invention provides a method for treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody or fragment thereof that binds to oxidized low-density lipoprotein (oxLDL).

[0011] In another aspect, the present invention provides a method of treating cancer in a subject, comprising administering to the subject: (a) a therapeutically effective amount of a first-line anti-cancer therapy selected from the group consisting of chemotherapy, radiation therapy, and immunotherapy; and (b) administering a therapeutically effective amount of an antibody or fragment thereof that binds to oxidized low-density lipoprotein (oxLDL).

[0012] In some embodiments, the cancer is LOX-1 positive, hi other embodiments, the cancer is positive for one or more of LOX-1, SR-A, CD36, CD38, and mucin.

[0013] In some embodiments, the cancer is selected from the group consisting of ovarian cancer, bladder urothelial carcinoma, renal clear cell carcinoma, rectal adenocarcinoma, colon adenocarcinoma, prostate cancer, breast epithelial cell tumor, glioblastoma, pancreatic cancer, and esophageal cancer.

[0014] In some embodiments, the cancer is a hematological cancer, including, for example, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute monocytic leukemia, non-Hodgkin's lymphoma, and multiple myeloma.

[0015] In some embodiments, the antibody or fragment thereof is administered as an adjuvant to a primary anti-cancer therapy.

[0016] In some embodiments, the subject is diagnosed with serum hyperlipidemia, type 2 diabetes, or metabolic syndrome.

[0017] In some embodiments of any of the foregoing aspects, the anti-oxLDL antibody or fragment thereof inhibits binding of oxLDL to LOX-1. In other embodiments, the antibody or fragment thereof inhibits binding of oxLDL to one or more of SR-A, CD36, CD38, and mucin.

[0018] In some embodiments of any of the foregoing aspects, the antibody or fragment thereof binds with at least 10-fold, 50-fold, 100-fold, or 1000-fold greater affinity to oxLDL than to native (non-oxidized) LDL.

[0019] In some embodiments, the antibody is a human antibody, a humanized antibody, a murine antibody, or a rabbit antibody, or a fragment thereof.

[0020] In some embodiments, the antibody or fragment thereof comprises at least one light chain complementarity determining region (LCDR) substantially identical to an LCDR selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10.

[0021] In some embodiments, the antibody or fragment thereof comprises at least one heavy chain complementarity determining region (HCDR) substantially identical to an LCDR selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7.

[0022] In some embodiments, the antibody or fragment thereof comprises a variable heavy region (V) substantially identical to SEQ ID NO: 11. H ), a variable light region substantially identical to SEQ ID NO: 12 (V L ), or both.

[0023] In some embodiments, the antibody or fragment thereof EVQLLESGGGLVQPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVSSISVGGHRTYYADSVKGRSTISRDNSKNTLYLQMNSLRAEDTAVYYCARIRVGPSGGAFDYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP a heavy chain substantially identical to APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 3); a light chain substantially identical to QSVLTQPPSASGTPGQRVTISCSGSNTNIGKNYVSWYQQLPGTAPKLLIYANSNRPSGVPDRFSGSSKSGTSASLAISGLRSEDEADYYCASWDASLNGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 4), or both.

[0024] The heavy chain is

[0025] In some embodiments, the antibody is olticumab. In other embodiments, the antibody fragment is a fragment of olticumab.

[0026] In some embodiments, the antibody is administered intravenously at an initial dose of at least 5 mg / kg, followed by multiple subsequent doses of at least 2 mg / kg / week, at least 2.5 mg / kg / 2 weeks, or at least 6 mg / kg / month, respectively. In other embodiments, the antibody is administered subcutaneously at a dose of about 330 mg / month for at least 3 months.

[0027] As used herein, "adjuvant therapy" refers to a secondary therapy administered to treat a primary disease (i.e., the clinical indication for which the primary therapy is directed), to increase / maximize the effectiveness of the primary therapy, to alleviate side effects of the treatment or the primary disease, and / or to prevent disease recurrence, a secondary disease for which treatment is needed. In some embodiments, the antibody or antibody fragment is administered as an adjuvant to either treat the primary disease (e.g., cancer or tumor) or to reduce plasma concentrations of oxLDL.

[0028] As used herein, "administering" and / or "administering" refer to any route for delivering a pharmaceutical composition to a patient. Delivery routes can include non-invasive oral (through the mouth), topical (cutaneous), transmucosal (nasal, buccal / sublingual, vaginal, ocular, and rectal), and inhalation routes, as well as parenteral routes and other methods known in the art. Parenteral refers to delivery routes generally associated with injection, including intraorbital, infusion, intraarterial, intracarotid, intravesical, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, intrathecal, subcapsular, subcutaneous, transmucosal, or transtracheal. Via the parenteral route, the composition can be in the form of a solution or suspension for infusion or injection, or can be present as a lyophilized powder.

[0029] As used herein, the term "effective amount" refers to an amount of a pharmaceutical composition to reduce at least one or more symptoms of a disease or disorder and refers to a sufficient amount of a pharmacological composition to impart the desired effect. As used herein, the phrase "therapeutically effective amount" refers to an amount of a composition sufficient to treat a disease at a reasonable benefit-to-risk ratio applicable to any medical treatment.

[0030] A significant therapeutic or prophylactic reduction in symptoms is, for example, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150%, or more in a measured parameter compared to a control, or an untreated subject, or the subject's condition prior to administration of a peptide described herein. Measured or measurable parameters include increases or decreases in the level of clinically detectable markers of disease, e.g., biological markers, as well as parameters related to clinically accepted scales of atherosclerosis symptoms or markers. However, it will be understood that the total daily usage of the compositions and formulations disclosed herein will be determined by the attending physician within the bounds of good medical practice. The exact amount required will vary depending on factors such as the type of disease being treated and the subject's gender, age, and weight.

[0031] "Subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject for which diagnosis, prognosis, or treatment is desired, particularly a mammalian subject. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sport animals, pet animals (e.g., dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, dairy cows), primates (e.g., apes, monkeys, orangutans, and chimpanzees), canines (e.g., dogs and wolves), felines (e.g., cats, lions, and tigers), equines (e.g., horses, donkeys, and zebras), food plants (e.g., dairy cows, pigs, and sheep), ungulates (e.g., deer and giraffes), rodents (e.g., mice, rats, hamsters, and guinea pigs), and the like. In certain embodiments, the mammal is a human subject.

[0032] "Substantially identical" refers to a nucleic acid or amino acid sequence that shares at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a second nucleic acid or amino acid sequence when optimally aligned, for example, using the methods described below. "Substantially identical" can be used to refer to various types and lengths of sequences, including full-length sequences, epitopes or immunogenic peptides, functional domains, coding and / or regulatory sequences, exons, introns, promoters, and genomic sequences. Percent identity between two polypeptide or nucleic acid sequences can be determined using, for example, the Smith Waterman Alignment (Smith, T. F. and M. S. Waterman (1981) J. Mol. Biol. 147:195-7); GeneMatcher Plus™, incorporated in Schwarz and Dayhof (1979) Atlas of Protein Sequence and Structure, Dayhof, MO, Ed. pp 353-358; "BestFit" (Smith and Waterman, Advances in Applied Mathematics, 482-489 (1981)); the BLAST program (Basic Local Alignment Search Tool; (Altschul, S. F., W. Gish, et al. (1990) J. Mol. Biol. 215:403-10), BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, or Megalign (DNASTAR) software, and other commonly available computer software. In addition, those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the length of the sequences being compared.Generally, for proteins, the length of comparison sequences is at least 10 amino acids, preferably 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, or 400 amino acids or more. For nucleic acids, the length of comparison sequences is generally at least 25, 50, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1100, or 1200 or more. When comparing DNA sequences to RNA sequences, thymine nucleotides are understood to be equivalent to uracil nucleotides for purposes of determining sequence identity. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, lysine, arginine, and phenylalanine, tyrosine.

[0033] The present invention can be better understood with reference to the following drawings, in which components are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the present disclosure, and in which reference numbers refer to corresponding parts throughout the different views. [Brief explanation of the drawings]

[0034] [Figure 1] 1 is a line graph comparing the binding of olticumab to native (non-oxidized) LDL and MDA-LDL. [Figure 2A] 1 is a bar graph showing MCP-1 release from cells treated with olticumab and control antibody. [Figure 2B] 1 is a bar graph showing cumulative MCP-1 release over time. [Figure 3] 1 is a photomicrograph of a Western blot stained for IκBα. [Figure 4A]4A-4B are graphs showing macrophage infiltration (FIG. 4A) and total plaque burden (FIG. 4B) in a mouse model of atherosclerosis after treatment with olticumab or a control antibody. [Figure 4B] 4A-4B are graphs showing macrophage infiltration (FIG. 4A) and total plaque burden (FIG. 4B) in a mouse model of atherosclerosis after treatment with olticumab or a control antibody. DETAILED DESCRIPTION OF THE INVENTION

[0035] It is well accepted that a diet rich in fats and oils and lipid-related diseases increase the incidence of certain types of cancer, including breast, prostate, colon, and liver. Plasma lipids have been correlated with various types of cancer. In particular, total cholesterol (TC), triglycerides (TG), and LDL have been found to be significantly elevated in some breast cancer patients.

[0036] A high-fat diet may induce a shift to a favorable microenvironment that supports the growth and proliferation of cancer cells. This shift can be caused by changes in the hormonal environment, changes in cell membrane properties (the latter due to changes in lipid composition), or by regulating the immune response against tumor cells. As in other lipid-related metabolic diseases (e.g., obesity and NASH), oxidative stress leads to the oxidation of LDL to oxLDL, which causes a plethora of promutagenic and procarcinogenic effects. Various clinical studies have highlighted the role of oxLDL in carcinogenic processes, and a positive correlation between increased serum oxLDL concentrations and cancer risk has been reported for pancreatic, colon, breast, and esophageal cancers. Additionally, in clinical studies of colorectal cancer (CRC) patients, lipid oxidation products resulting from oxidative stress, including oxLDL, have been investigated as potential markers of CRC progression. Although this study did not observe a significant difference in oxLDL serum levels between CRC patients and healthy controls, it was shown that oxLDL levels were significantly higher in patients with early-stage primary tumors compared to patients whose primary tumors had progressed to advanced stages (Diakowska et al, Gastroenterol. Res. Pract. 2015:146819(2015)).

[0037] oxLDL-mediated signaling via LOX-1 leads to the upregulation of adhesion proteins, other proinflammatory mediators, and proangiogenic factors, all of which are pathogenic in cancer. Among these pathogenic signaling molecules are monocyte chemoattractant protein-1 (MCP-1) and nuclear factor-κB (NFκB). MCP-1 is a chemokine that drives macrophage recruitment to areas of inflammation, and its expression correlates with the degree of tumor-associated macrophage (TAM) infiltration. NFκB is a transcription factor that functions as a master regulator of proinflammatory gene expression. NFκB activity is upregulated in inflammatory conditions, including cancer, where it contributes to the transformation of normal cells into tumor cells, tumor cell survival, and the ongoing inflammatory cycle perpetuated in cancer. Importantly, oxLDL-Lox-1-mediated signaling is associated with both MCP-1 upregulation and NFκB activity.

[0038] The present invention is based, in part, on the characterization of the anti-cancer properties of anti-oxLDL antibodies. Direct interference with oxLDL can inhibit LOX-1-mediated tumor formation and / or progression. The present invention is also based on the discovery that olticumab specifically binds to the oxidized form of LDL (i.e., oxLDL) compared to native / non-oxidized LDL. Olticumab is a fully human recombinant monoclonal IgG1 antibody with an affinity for oxLDL of approximately 8±6 nM.

[0039] anti-oxLDL antibody As discussed herein, the present invention incorporates anti-oxLDL antibodies. One specific, useful anti-oxLDL antibody is olticumab. The synthesis and characterization of olticumab are described in WO2009 / 08205, where it is referred to as antibody 2D03. WO2009 / 08205 is incorporated by reference in its entirety.

[0040] Figure 2 of WO2009 / 08205 sets forth the amino acid sequences of the 2D03 heavy chain and the 2D03 light chain, with the complementarity determining regions (CDRs) underlined.

[0041] WO2007 / 025781, the entire contents of which are incorporated herein by reference, states that the invention also includes antibodies (i.e., olticumab) that selectively bind to the oxidized LDL epitope selectively bound by antibody 2D03, and also includes antibodies that contain at least one, two, three, four, five, or all six complementarity determining region(s) having the amino acid sequence of the corresponding CDR(s) of antibody 2D03. Furthermore, antibodies with three or four CDRs having sequences corresponding to the 2D03 antibody CDRs preferably have all three heavy chain or all three light chain CDRs with the sequences of the corresponding CDRs of antibody 2D03; i.e., this aspect of the invention includes antibodies comprising three light chain CDRs with the sequences of the corresponding three light chain CDRs of antibody 2D03, or three heavy chain CDRs with the sequences of the corresponding three heavy chain CDRs of antibody 2D03; i.e., even more preferably, the antibody comprises three light chain CDRs and three heavy chain CDRs with the sequences of the corresponding CDRs of antibody 2D03; i.e., if the antibody does not contain all six CDRs with the sequences of the corresponding CDRs of antibody 2D03, some or all of one, two, three, four, or five "non-identical" CDRs. preferably comprises a variant of the sequence of the corresponding CDR of antibody 2D03 (by "variant" WO2007 / 025781, the variant has at least 50%, more preferably at least 70%, even more preferably at least 80%, or at least 90%, or at least 95% sequence identity with the sequence of the corresponding CDR; most preferably, the variant has 96% or 97% or 98% or 99% sequence identity with the sequence of the corresponding CDR of antibody 2D03; typically, a "variant" CDR sequence includes the meaning of having 5 or 4 or 3 or 2, or even just 1 amino acid residue that differs from the sequence of the corresponding CDR of antibody 2D03); i.e., this aspect of the invention comprises antibody 2D03.

[0042] Heavy chain complementarity determining regions (HCDRs) 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) are set forth in SEQ ID NOs: 5, 6, and 7, respectively, and light chain complementarity determining regions (LCDRs) 1 (LCDR1), 2 (LCDR2), and 3 (LCDR3) are set forth in SEQ ID NOs: 8, 9, and 10, respectively. Olticumab contains a variable heavy region (VH) amino acid sequence of SEQ ID NO: 11 and a variable light region (VL) amino acid sequence of SEQ ID NO: 12. Olticumab contains a heavy chain amino acid sequence of SEQ ID NO: 3 and a light chain amino acid sequence of SEQ ID NO: 4.

[0043] HCDR1 is FSNAWMSWVRQAPG (SEQ ID NO: 5).

[0044] HCDR2 is SSISVGGHRTYYADSVKGR (SEQ ID NO: 6).

[0045] HCDR3 is ARIRVGPSGGAFDY (SEQ ID NO: 7).

[0046] LCDR1 is CSGSNTNIGKNYVS (SEQ ID NO: 8).

[0047] LCDR2 is an ANSNRPS (SEQ ID NO: 9).

[0048] LCDR3 is CASWDASLNGWV (SEQ ID NO: 10).

[0049] Variable Heavy Region (V H ) is EVQLLESGGG LVQPGGSLRL SCAASGFTFS NAWMSWVRQA PGKGLEWVSS ISVGGHRTYY ADSVKGRSTI SRDNSKNTLY LQMNSLRAED TAVYYCARIR VGPSGGAFDY WGQGTLVTVS (SEQ ID NO: 11).

[0050] Variable light region (V L) is QSVLTQPPSA SGTPGQRVTI SCSGSNTNIG KNYVSWYQQL PGTAPKLLIY ANSNRPSGVP DRFSGSKSGT SASLAISGLR SEDEADYYCA SWDASLNGWV FGGGTKLTVL (SEQ ID NO: 12).

[0051] In some embodiments, the antibody or antibody fragment comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO:1 and / or SEQ ID NO:2.

[0052] In some embodiments, the present invention provides antibodies or antibody fragments that bind to oxLDL and one or more of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, as set forth in SEQ ID NOs: 5-10.

[0053] In some embodiments, the present invention provides antibodies comprising at least one CDR having the amino acid sequence of the corresponding CDR of olticumab. Preferably, the antibody has two, three, four, or five CDRs having the sequence of the corresponding CDR of olticumab. If the antibody has three or four CDRs having the sequence of the corresponding CDR of olticumab, it is preferred if the antibody has all three heavy chain or all three light chain CDRs having the sequence of the corresponding CDR of olticumab. Thus, this aspect of the method includes antibodies comprising three light chain CDRs having the sequence of the corresponding three light chain CDRs of olticumab, or three heavy chain CDRs having the sequence of the corresponding three heavy chain CDRs of olticumab. Even more preferably, the antibody comprises three light chain CDRs and three heavy chain CDRs having the sequence of the corresponding CDR of olticumab.

[0054] If the antibody does not contain all six CDRs with the sequences of the corresponding CDRs of olticumab, it is preferred that some or all of the 1, 2, 3, 4, or 5 "non-identical" CDRs contain variants of the sequences of the corresponding CDRs of olticumab. "Variant" includes the meaning that the variant has at least 50%, more preferably at least 70%, even more preferably at least 80%, or at least 90%, or at least 95% sequence identity with the sequence of the corresponding CDR. Most preferably, the variant has 96%, 97%, 98%, or 99% sequence identity with the sequence of the corresponding CDR of olticumab. Typically, the "variant" CDR sequence has 5, 4, 3, 2, or only 1 amino acid residue that differs from the sequence of the corresponding CDR of olticumab.

[0055] Specifically, the present invention provides antibodies that contain "one or more of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3," including embodiments in which the antibody contains one, any two, any three, any four, any five, or all six CDRs (i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3). For example, one aspect of the embodiment provides that the antibody contains an HCDR1 set forth in SEQ ID NO:5. Another aspect provides that the antibody contains an HCDR2 set forth in SEQ ID NO:6. Another aspect provides that the antibody contains an HCDR3 set forth in SEQ ID NO:7. Yet another aspect provides that the antibody contains an LCDR1 set forth in SEQ ID NO:8. Another aspect provides that the antibody contains an LCDR2 set forth in SEQ ID NO:9. Another aspect provides that the antibody contains an LCDR3 set forth in SEQ ID NO:10. Yet another aspect provides that the antibody contains an HCDR1 set forth in SEQ ID NO:5 and an HCDR2 set forth in SEQ ID NO:6. Another aspect provides that the antibody contains an HCDR1 set forth in SEQ ID NO:5 and an HCDR3 set forth in SEQ ID NO:7. Another aspect provides that the antibody contains an HCDR1 set forth in SEQ ID NO:5 and an LCDR1 set forth in SEQ ID NO:8. Another aspect provides that the antibody contains an HCDR1 set forth in SEQ ID NO:5 and an LCDR2 set forth in SEQ ID NO:9. Another aspect provides that the antibody contains an HCDR1 set forth in SEQ ID NO:5 and an LCDR3 set forth in SEQ ID NO:10. Another aspect provides that the antibody contains an HCDR2 set forth in SEQ ID NO:6 and an HCDR3 set forth in SEQ ID NO:7. Another aspect provides that the antibody contains an HCDR2 set forth in SEQ ID NO:6 and an LCDR1 set forth in SEQ ID NO:8. Another aspect provides that the antibody contains an HCDR2 set forth in SEQ ID NO:6 and an LCDR2 set forth in SEQ ID NO:9. Another aspect provides that the antibody contains an HCDR2 set forth in SEQ ID NO:6 and an LCDR3 set forth in SEQ ID NO:10.Another aspect provides that the antibody contains an HCDR3 set forth in SEQ ID NO:7 and an LCDR1 set forth in SEQ ID NO:8. Another aspect provides that the antibody contains an HCDR3 set forth in SEQ ID NO:7 and an LCDR2 set forth in SEQ ID NO:9. Another aspect provides that the antibody contains an HCDR3 set forth in SEQ ID NO:7 and an LCDR3 set forth in SEQ ID NO:10. Another aspect provides that the antibody contains an LCDR1 set forth in SEQ ID NO:8 and an LCDR2 set forth in SEQ ID NO:9. Another aspect provides that the antibody contains an LCDR1 set forth in SEQ ID NO:8 and an LCDR3 set forth in SEQ ID NO:10. Another aspect provides that the antibody contains an LCDR2 set forth in SEQ ID NO:9 and an LCDR3 set forth in SEQ ID NO:10. Another aspect provides that the antibody contains an HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NOs:5-7, respectively. Another aspect provides that the antibody contains an HCDR1, HCDR2, and LCDR1 set forth in SEQ ID NOs:5, 6, and 8, respectively. Another aspect provides that the antibody contains an HCDR1, HCDR2, and LCDR2 set forth in SEQ ID NOs: 5, 6, and 9, respectively. Another aspect provides that the antibody contains an HCDR1, HCDR2, and LCDR3 set forth in SEQ ID NOs: 5, 6, and 10, respectively. Another aspect provides that the antibody contains an HCDR1, HCDR3, and LCDR1 set forth in SEQ ID NOs: 5, 7, and 8, respectively. Another aspect provides that the antibody contains an HCDR1, HCDR3, and LCDR2 set forth in SEQ ID NOs: 5, 7, and 9, respectively. Another aspect provides that the antibody contains an HCDR1, HCDR3, and LCDR3 set forth in SEQ ID NOs: 5, 7, and 10, respectively. Another aspect provides that the antibody contains an HCDR1, LCDR1, and LCDR2 set forth in SEQ ID NOs: 5, 9, and 10, respectively. Another aspect provides that the antibody contains an HCDR1, LCDR1, and LCDR3 set forth in SEQ ID NOs: 5, 8, and 10, respectively.Another aspect provides that the antibody contains an HCDR1, LCDR2, and LCDR3 set forth in SEQ ID NOs: 5, 9, and 10, respectively. Another aspect provides that the antibody contains an HCDR2, HCDR3, and LCDR1 set forth in SEQ ID NOs: 6, 7, and 8, respectively. Another aspect provides that the antibody contains an HCDR2, HCDR3, and LCDR2 set forth in SEQ ID NOs: 6, 7, and 9, respectively. Another aspect provides that the antibody contains an HCDR2, HCDR3, and LCDR3 set forth in SEQ ID NOs: 6, 7, and 10, respectively. Another aspect provides that the antibody contains an HCDR2, LCDR1, and LCDR2 set forth in SEQ ID NOs: 6, 8, and 10, respectively. Another aspect provides that the antibody contains an HCDR2, LCDR1, and LCDR3 set forth in SEQ ID NOs: 6, 8, and 10, respectively. Another aspect provides that the antibody contains an HCDR2, LCDR2, and LCDR3 set forth in SEQ ID NOs: 6, 9, and 10, respectively. Another aspect provides that the antibody contains an HCDR3, LCDR1, and LCDR2 set forth in SEQ ID NOs: 7, 8, and 9, respectively. Another aspect provides that the antibody contains an HCDR3, LCDR1, and LCDR3 set forth in SEQ ID NOs: 7, 8, and 10, respectively. Another aspect provides that the antibody contains an HCDR3, LCDR2, and LCDR3 set forth in SEQ ID NOs: 7, 9, and 10, respectively. Another aspect provides that the antibody contains an LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NOs: 8-10, respectively. Yet another aspect provides that the antibody contains an HCDR1, HCDR2, HCDR3, and LCDR1 set forth in SEQ ID NOs: 5-8, respectively. Another aspect provides that the antibody contains an HCDR1, HCDR2, HCDR3, and LCDR2 set forth in SEQ ID NOs: 5-7, and 10, respectively. Another embodiment provides that the antibody contains HCDR1, HCDR2, HCDR3, and LCDR3 set forth in SEQ ID NOs: 5-7, and 10, respectively.Another aspect provides that the antibody contains HCDR1, HCDR2, LCDR1, and LCDR2 set forth in SEQ ID NOs: 5, 6, 8, and 9, respectively. Another aspect provides that the antibody contains HCDR1, HCDR2, LCDR1, and LCDR3 set forth in SEQ ID NOs: 5, 6, 8, and 10, respectively. Another aspect provides that the antibody contains HCDR1, HCDR2, LCDR2, and LCDR3 set forth in SEQ ID NOs: 5, 6, 9, and 10, respectively. Another aspect provides that the antibody contains HCDR1, HCDR3, LCDR1, and LCDR2 set forth in SEQ ID NOs: 5, 7, 8, and 9, respectively. Another aspect provides that the antibody contains HCDR1, HCDR3, LCDR1, and LCDR3 set forth in SEQ ID NOs: 5, 7, 8, and 10, respectively. Another aspect provides that the antibody contains HCDR1, HCDR3, LCDR1, and LCDR3 set forth in SEQ ID NOs: 5, 7, 8, and 10, respectively. Another aspect provides that the antibody contains HCDR1, HCDR3, LCDR2, and LCDR3 set forth in SEQ ID NOs: 5, 7, 9, and 10, respectively. Another aspect provides that the antibody contains the HCDR1, LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NOs: 5, 8, 9, and 10, respectively. Another aspect provides that the antibody contains the HCDR2, HCDR3, LCDR1, and LCDR2 set forth in SEQ ID NOs: 6-9, respectively. Another aspect provides that the antibody contains the HCDR2, HCDR3, LCDR1, and LCDR3 set forth in SEQ ID NOs: 6-8, and 10, respectively. Another aspect provides that the antibody contains the HCDR2, HCDR3, LCDR2, and LCDR3 set forth in SEQ ID NOs: 6, 7, 9, and 10, respectively. Another aspect provides that the antibody contains the HCDR2, LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NOs: 6, 8, 9, and 10, respectively. Another aspect provides that the antibody contains the HCDR3, LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NOs: 7-10, respectively. Yet another embodiment provides that the antibody contains HCDR1, HCDR2, HCDR3, LCDR1, and LCDR2 set forth in SEQ ID NOs: 5-9, respectively.Another aspect provides that the antibody contains HCDR1, HCDR2, HCDR3, LCDR1, and LCDR3 set forth in SEQ ID NOs: 5-8, and 10, respectively. Yet another aspect provides that the antibody contains HCDR1, HCDR2, HCDR3, LCDR2, and LCDR3 set forth in SEQ ID NOs: 5-7, 9, and 10, respectively. Another aspect provides that the antibody contains HCDR1, HCDR2, LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NOs: 5, 6, 8-10, respectively. Another aspect provides that the antibody contains HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NOs: 5, 7-10, respectively. Another aspect provides that the antibody contains HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NOs: 6-10, respectively. Yet another embodiment provides that the antibody comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NOs: 5-10, respectively.

[0056] In making and using variants of any of the polypeptide sequences (e.g., CDRs) provided herein, it is understood that a given amino acid can be replaced with a residue having similar physiochemical characteristics, for example, replacing one aliphatic residue with another (e.g., Ile, Val, Leu, or Ala, for each other), or replacing one polar residue with another (e.g., Lys with Arg, Glu with Asp, or Gln with Asn). Other such conservative substitutions, such as the substitution of entire regions with similar hydrophobic characteristics, or the substitution of residues with similar side chain volumes, are well known. Isolated antibodies containing conservative amino acid substitutions can be tested in any one of the assays described herein to confirm the desired activity, as measured by the assays described elsewhere herein.

[0057] Amino acids can be grouped according to the similarity of their side chain properties (as in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile, Phe, Trp; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln, Ala, Tyr, His, Pro, Gly; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe, Pro, His, or hydroxyproline. Non-conservative substitutions would involve exchanging a member of one of these classes for another.

[0058] Particularly preferred conservative substitutions for use in the variants described herein are as follows: Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu or Asn; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln, or Glu; Met to Leu, Tyr, or Ile; Phe to Met, Leu, or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr or Phe; Tyr to Phe, or Trp; and / or Phe to Val, Tyr, Ile, or Leu. In general, conservative substitutions involve the exchange of a residue with a residue having similar physicochemical properties (i.e., the substitution of a hydrophobic residue with another hydrophobic amino acid).

[0059] Any cysteine residues not involved in maintaining the proper conformation of the isolated peptides described herein can also be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent ectopic cross-linking. Conversely, cysteine bond(s) can be added to the isolated peptides described herein to improve their stability or facilitate multimerization.

[0060] In some embodiments, the antibodies described herein can comprise naturally occurring amino acids commonly found in polypeptides and / or proteins produced by living organisms, such as Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M), Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q), Asp (D), Glu (E), Lys (K), Arg (R), and His (H). In some embodiments, the antibodies can comprise alternative amino acids. Non-limiting examples of alternative amino acids include D-amino acids; β-amino acids; homocysteine, phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, γ-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic acid, statins, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, penicillamine (3-mercapto-D-valine), ornithine, citrulline, α-methyl-alanine, para-benzoylphenylalanine, para-aminophenylalanine, p-fluorophenylalanine, phenylglycine, propargylglycine, sarcosine, and tert-butylglycine, diaminobutyric acid, 7-hydroxy-tetrahydroisoquinolinecarboxylic acid, naphthylalanine, biphenylsulfonyl methyl ... nylalanine, cyclohexylalanine, amino-isobutyric acid, norvaline, norleucine, tert-leucine, tetrahydroisoquinolinecarboxylic acid, pipecolic acid, phenylglycine, homophenylalanine, cyclohexylglycine, dehydroleucine, 2,2-diethylglycine, 1-amino-1-cyclopentanecarboxylic acid, 1-amino-1-cyclohexanecarboxylic acid, amino-benzoic acid, amino-naphthoic acid, γ-aminobutyric acid, difluorophenylalanine, nipecotic acid, α-aminobutyric acid, thienylalanine, t-butylglycine, trifluorovaline, hexafluoroleucine; fluorinated analogs; azide-modified amino acids; alkyne-modified amino acids; cyano-modified amino acids; and derivatives thereof.

[0061] In some embodiments, antibodies can be modified, e.g., moieties can be added to one or more amino acids. In some embodiments, antibodies can include one or more moieties, e.g., one or more moieties per peptide, two or more moieties per peptide, five or more moieties per peptide, ten or more moieties per antibody, or more than one moiety per antibody. In some embodiments, antibodies described herein can include one or more modifications and / or moieties, e.g., one modification, two modifications, three modifications, or more modifications. Non-limiting examples of modifications and / or moieties include PEGylation, glycosylation, hydroxylase (HES)-linked phospholipidation (ELP)-linked phospholipidation, lipidation, acetylation, amidation, end-capping modification, cyano group, phosphorylation, and cyclization. In some embodiments, end-capping modification can include acetylation at the N-terminus, N-terminal acylation, and N-terminal formylation. In some embodiments, end-capping modification can include amidation at the C-terminus, introduction of a C-terminal alcohol, aldehyde, ester, or thioester moiety.

[0062] Treatment method The present invention also provides methods for treating or preventing cancer by administering to a subject a therapeutically effective amount of an antibody or antibody fragment that specifically binds to oxLDL. The present invention also provides methods for preventing metastasis in a subject diagnosed with cancer by administering to the subject a therapeutically effective amount of an antibody or antibody fragment that specifically binds to oxLDL. In some embodiments, binding of the antibody or antibody fragment inhibits or blocks at least one biological function of oxLDL. In some embodiments, the cancer is pancreatic cancer, breast cancer, colorectal cancer, including rectal adenocarcinoma and colon adenocarcinoma, ovarian cancer, bladder urothelial carcinoma, renal clear cell carcinoma, or prostate cancer (prostate adenocarcinoma). In some embodiments, the cancer cells express LOX-1 and / or are identified as expressing LOX-1. In some embodiments, the antibody or antibody fragment inhibits, reduces, or blocks binding of oxLDL to LOX-1. In some embodiments, the antibody or antibody fragment inhibits, reduces, or blocks binding of oxLDL to SR-A, CD36, CD38, and / or mucin. In some embodiments, the antibody or antibody fragment is olticumab, a fragment of olticumab, a derivative of olticumab, or any anti-oxLDL antibody or antibody fragment described herein.

[0063] In various embodiments, the compositions administered in the disclosed methods are formulated for delivery via any route of administration. For example, methods include administration via aerosol, nasal, oral, transmucosal, transdermal, parenteral, or enteral routes. "Parenteral" refers to administration routes generally associated with injection, including intraorbital, infusion, intraarterial, intravesical, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, intrathecal, subcapsular, subcutaneous, transmucosal, or transtracheal. Via the parenteral route, the composition can be in the form of a solution or suspension for injection or infusion, or can exist as a lyophilized powder. Via the parenteral route, the composition can be in the form of a solution or suspension for injection or infusion. Via the enteral route, the pharmaceutical composition can be in the form of a tablet, gel capsule, dragee, syrup, suspension, solution, powder, granule, emulsion, microparticles or nanospheres or lipid or polymer vesicles that allow controlled release. Typically, the composition is administered by injection.

[0064] Typically, an effective amount of anti-oxLDL in the methods disclosed herein provides a plasma concentration in a subject of at least 4 μg / mL, preferably at least 12 μg / mL.

[0065] The methods of the present invention can include subcutaneously administering to a subject, the subject being an adult, the above-disclosed antibody or antibody fragment at about 330 mg / month for about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer. Other embodiments provide weekly administration of the antibody or antibody fragment at 2 mg / kg / week or more (166 mg for an average human patient weighing 83 kg); preferably, about 4 mg / kg / week (332 mg for an average human patient weighing 83 kg). In another aspect, the anti-oxLDL antibody composition is administered biweekly at >2.5 mg / kg / 2 weeks (e.g., 208 mg for an average human patient weighing 83 kg). In yet another aspect, the anti-oxLDL antibody composition is administered monthly at about 6 mg / kg / month (e.g., about 498 mg for an average human patient weighing 83 kg). For example, monthly administration can be performed for 12 months or 3 months. Still other embodiments provide for administering to a subject an initial dose of 800-900 mg, 900-1000 mg, 1000-1100 mg, 1100-1200 mg, 1200-1300 mg, 1300-1400 mg, 1400-1500 mg, or 1500-1600 mg of the antibody or antibody fragment. In some aspects, the effective amount in the methods described herein comprises an initial dose of about 1000-1500 mg of olticumab administered weekly for 2, 3, 4, or 5 weeks, and / or further administered monthly for 1, 2, or 3 months, followed by subsequent doses of the antibody of 700-900 mg.

[0066] Another embodiment provides for administering an anti-oxLDL antibody or fragment in escalating doses. In this embodiment, an exemplary (starting) dose of an antibody (e.g., olticumab) against oxLDL is 0.005 to 0.01 mg / kg (e.g., intravenously) for a single administration; other exemplary dose levels administered in a single administration are 0.01 to 0.15, 0.15 to 0.75, 0.75 to 2.5, 2.5 to 7.5, and 7.5 to 30 mg / kg (e.g., intravenously). For example, a starting dose of olticumab for a single intravenous administration is 0.007 mg / kg; other exemplary doses may be 0.05, 0.25, 1.25, 5.0, or 15.0 mg / kg for subsequent single intravenous administrations. In another embodiment, a single subcutaneous administration of the antibody is 0.5-5 mg / kg, and multiple subcutaneous administrations are also 0.5-5 mg / kg. For example, 1.25 mg / kg of antibody is administered subcutaneously. In various embodiments, doses are administered within a specific time range within the day for each administration, and each dose in a multiple treatment (e.g., 4 doses, 3 doses, 5 doses, or 6 doses) is administered at weekly intervals that are ±1 day apart. In another example, an antibody (e.g., olticumab) is administered to a human subject at 300 mg-450 mg (e.g., 360 mg), and optionally subsequently, another dose of 300 mg-450 mg (e.g., 360 mg) is administered to the human subject, the second dose being separated from the first by at least 70 days (up to 91 days). The antibody can be formulated for use in subcutaneous administration at a concentration of 100-170 mg / mL (e.g., 150 mg / mL) and without further dilution, or can be diluted to a large volume for intravenous injection.

[0067] Further embodiments provide a subject with approximately 10-50 μg / period, 50-100 μg / period, 100-150 μg / period, 150-200 μg / period, 100-200 μg / period, 200-300 μg / period, 300-400 μg / period, 400-500 μg / period, 500-600 μg / period, 600-700 μg / period, 700-800 μg / period, 800-900 μg / period, 900-1000 μg / period, 1000-1100 μg / period, 1100-1200 μg / period, 1200-1300 μg / period, 1300-1400 μg / period, 1400-1500 μg / period This includes administering an effective amount of antibody in the range of 1500-1600 μg / period, 1600-1700 μg / period, 1700-1800 μg / period, 1800-1900 μg / period, 1900-2000 μg / period, 2000-2100 μg / period, 2100-2200 μg / period, 2200-2300 μg / period, 2300-2400 μg / period, 2400-2500 μg / period, 2500-2600 μg / period, 2600-2700 μg / period, 2700-2800 μg / period, 2800-2900 μg / period, or 2900-3000 μg / period, where the period may be days, weeks, months, or another length of time. In one embodiment, the antibody (e.g., olticumab) is administered weekly, biweekly, or monthly at any of the doses per period described above.

[0068] In some embodiments, the method includes administering an anti-oxLDL antibody (e.g., olticumab) to a subject for 1 to 5 days, 1 to 5 weeks, 1 to 5 months, or 1 to 5 years. For example, the antibody is administered to the subject in 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 doses, each dose separated by at least 3 days, 5 days, 1 week, 2 weeks, 1 month, 2 months, or a combination thereof. In other embodiments, a second dose is administered about 2 to 3 weeks or about 3 weeks after the first dose, a third dose is administered about 5 to 6 weeks or about 6 weeks after the first dose, etc. In another embodiment, the second dose is administered about 2-3 months, about 2 months, about 3 months, or about 4 months after the first dose, and the third dose is administered about 4-6 months, about 5-6 months, about 5 months, or about 6 months after the first dose.

[0069] Pharmaceutical compositions and drugs In various embodiments, the present invention provides pharmaceutical compositions for use in the methods. The pharmaceutical composition comprises an anti-oxLDL antibody or fragment thereof and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle involved in the transport or delivery of a compound of interest from one tissue, organ, or part of the body to another tissue, organ, or part of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Examples of excipients include, but are not limited to, starch, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrants, wetting agents, emulsifiers, colorants, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives, antioxidants, plasticizers, gelling agents, thickening agents, hardening agents, setting agents, suspending agents, surfactants, wetting agents, carriers, stabilizers, and combinations thereof. Generally, each component of a carrier must be "pharmaceutically acceptable" in that it must be compatible with the other components of the formulation. Each component of a carrier must also be suitable for use in contact with any tissue or organ with which it may come into contact, meaning that it must not pose a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that unduly outweighs its therapeutic benefit.

[0070] The pharmaceutical compositions of the present invention can be delivered in a therapeutically effective amount. The precise therapeutically effective amount is the amount of the composition that will produce the most effective results in terms of therapeutic efficacy in a given subject. This amount will vary depending on various factors, including, but not limited to, the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (age, sex, type and stage of disease, overall health, response to a given dose, and type of administration), the nature of the pharmaceutically acceptable carrier(s) in the formulation, and the route of administration. Those skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, for example, by monitoring the subject's response to administration of the compound and adjusting the dose accordingly. For further guidance, see Remington: The Science and Practice of Pharmacy (Gennaro ed. 20th edition, Williams & Wilkins PA, USA) (2000).

[0071] antibody preparation The present invention is based, in part, on the use of anti-oxLDL antibodies, fragments, and binding proteins. Modern recombinant library technology is used to prepare therapeutic antibodies against oxLDL. Although mouse hybridoma cells produce large amounts of identical antibodies, these non-human antibodies are recognized by the human body as foreign, and as a result, their efficacy and plasma half-life are reduced, in addition to inducing allergic reactions. To solve this problem, one approach is to create chimeric antibodies, in which the mouse variable domains of an antibody are transferred to human constant regions, resulting in an antibody that is primarily human. A further refinement of this approach is to develop humanized antibodies, in which the regions of the mouse antibody that contact the antigen, the complementarity-determining region (CDR), are transferred to a human antibody framework, resulting in a humanized antibody. Another approach is to use recombinant technology to produce fully human antibodies, which does not rely on immunizing animals to generate specific antibodies. Instead, recombinant libraries contain a large number of pre-created antibody variants, and it is highly likely that the library will contain at least one antibody specific to any given antigen. Phage display systems can be used when antibody fragments are expressed and displayed as fusions with phage coat proteins on the surface of filamentous phage particles, while simultaneously carrying the genetic information encoding the displayed molecules. Phage-displayed antibody fragments specific for a particular antigen can be selected through binding to the antigen of interest. Isolated phage are then amplified, and the genes encoding selected antibody variable domains can optionally be transferred to other antibody formats, such as full-length immunoglobulins, and expressed in large quantities using appropriate vectors and host cells well known in the art. The format of the antibody specificity displayed on the phage particle can vary. The most commonly used formats are Fab and single-chain (scFv), both of which contain the variable antigen-binding domains of an antibody. The single-chain format consists of a variable heavy domain (VH) connected to a variable light domain (VL) via a flexible linker.Prior to use as analytical reagents or therapeutic agents, the displayed antibody specificities are transferred to a soluble format, e.g., Fab or scFv, and analyzed in this manner. At a later stage, antibody fragments identified as having desirable characteristics can be transferred to yet another format, such as a full-length antibody.

[0072] Antibody production from hybridomas Cell fusion is accomplished by standard procedures well known to those skilled in the art of immunology. Fusion partner cell lines, as well as methods for fusing and selecting hybridomas and screening for mAbs, are well known in the art. See, e.g., Ausubel, Harlow, and Colligan, the contents of which are incorporated herein by reference in their entirety.

[0073] Anti-oxLDL antibodies can be produced in large quantities by injecting antibody-secreting hybridoma or transfectoma cells into the peritoneal cavity of mice, collecting ascites fluid containing high-titer mAbs after a suitable time, and isolating the mAbs from the fluid. For such in vivo production of mAbs containing non-murine hybridomas (e.g., rat or human), hybridoma cells are preferably grown in irradiated nude mice or athymic nude mice. Alternatively, antibodies can be produced by culturing hybridoma or transfectoma cells in vitro and isolating the secreted mAbs from the cell culture medium or recombinantly from eukaryotic or prokaryotic cells.

[0074] Recombinant expression of antibodies Recombinant mouse or chimeric mouse-human or human-human antibodies that inhibit oxidized LDL can be provided according to the present invention using known techniques based on the teachings provided herein (see, e.g., Ausubel et al., eds. Current Protocols in Molecular Biology, Wiley Interscience, NY (1987, 1992, 1993); and Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)).

[0075] The DNA encoding the anti-oxLDL antibody can be genomic DNA or cDNA encoding at least one of the heavy chain constant region (Hc), heavy chain variable region (Hc), light chain variable region (Lv), and light chain constant region (Lc). A convenient alternative to using chromosomal gene fragments as a source of DNA encoding mouse V-region antigen-binding segments is the use of cDNA to construct chimeric immunoglobulin genes, as reported, for example, by Liu et al. (Proc. Natl. Acad. Sci., USA 84:3439 (1987) and J. Immunology 139:3521 (1987)). The use of cDNA requires that gene expression elements appropriate for the host cell be combined with the gene to achieve synthesis of the desired protein. The use of cDNA sequences has an advantage over genomic sequences (containing introns) in that cDNA sequences can be expressed in bacteria or other hosts lacking a suitable RNA splicing system. [Example]

[0076] The following examples are not intended to limit the scope of the claimed invention, but rather to illustrate particular embodiments. Any variations of the exemplified methods that occur to those skilled in the art are intended to fall within the scope of the invention.

[0077] Example 1: Olticumab specifically binds to oxLDL The binding specificity of olticumab to various forms of LDL was investigated. In this experiment, the binding of olticumab to native (non-oxidized) LDL and malondialdehyde-modified LDL (MDA-LDL) was measured across various concentrations of olticumab, and the dissociation constant (K d ) was calculated. For this experiment, MDA-LDL was chosen because it is an endogenous LDL species that is thought to reflect naturally occurring levels of oxLDL.

[0078] Olticumab was tested in an ELISA assay for binding to malondialdehyde (MDA)-modified and native human LDL prepared from blood donor serum. LDL samples were immobilized on plates, and antibody concentrations were titrated. Multiwell plates were coated with 50 μL of MDA-LDL or native LDL per well, diluted to 2 μg / mL in PBS + 1 mM EDTA. Plates were incubated overnight at 4°C. Purified antibodies were diluted and titrated in ELISA blocking buffer (0.2% non-fat dry milk) and applied. Plates were incubated for 1 hour at room temperature. Bound antibodies were detected with horseradish peroxidase (HRP)-conjugated rabbit anti-human IgG antibody (P0214 DAKO).

[0079] Figure 1 shows exemplary binding curves from olticumab (0.01-50 μg / mL) to native LDL and MDA-LDL. Olticumab lacked any significant affinity for native LDL, but exhibited a K of approximately 8±6 nM. d showed robust and specific binding of MDA-LDL at 1000 kJ / mL.

[0080] Example 2: Olticumab blocks oxLDL-induced release of MCP-1 by macrophages. To evaluate potential anti-inflammatory effects, we measured the effect of olticumab on oxLDL-induced release of MCP-1 by macrophages. MCP-1 is a macrophage chemoattractant secreted by activated macrophages in inflammatory areas and plays a role in enhancing the inflammatory response. MCP-1 expression correlates with tumor-associated macrophage infiltration.

[0081] Freshly isolated CD14+ macrophages were preactivated with 0.1 ng / mL lipopolysaccharide (LPS) to generate proinflammatory M1 macrophages. After 20 h of preactivation, olticumab, FITC-8, or an equal volume of medium was added to a final concentration of 40 nM, and cells were cultured for an additional 24 h. Culture supernatants were collected and MCP-1 levels were analyzed using a cytometric bead array (CBA; BD Bioscience, Franklin Lakes, NJ, USA). Figure 2A shows mean (±SD) or pooled normalized data from two different donors in two separate experiments treated in triplicate. Values were normalized to the mean MCP-1 value of samples without antibody treatment. Statistical analysis was performed using ANOVA followed by a Tukey-Kramer multiple comparison test (***p<0.001) using GraphPad Instat 3 software. (Data on file, Report BI 209-68) The results show that olticumab treatment significantly reduced MCP-1 secretion in ox-LDL-stimulated macrophages compared to treatment with a control antibody (FITC-8) and a vehicle control ("no antibody").

[0082] In another experiment, human monocyte-derived macrophages were grown for 14 days in the presence of oxLDL-containing human serum. These cells were then treated with control IgG (FITC-8) or olticumab for the indicated time periods. Supernatants were removed daily and analyzed for MCP-1 levels. Figure 2B shows that in the presence of olticumab, MCP-1 levels did not increase over time, as observed in cultures treated with control antibody, indicating that olticumab effectively blocked MCP-1 secretion. For example, after 4 days (96 hours), olticumab blocked the increase in MCP-1 release by macrophages, which were reduced in MCP-1, by up to 60% compared to cells treated with control IgG.

[0083] Example 3: Olticumab inhibits NFκB signaling by increasing IκBα expression. NFκB is a well-known transcription factor that responds to stressful stimuli in most cell types, generally upregulating immune responses and genes involved in inflammation. In unstimulated cells, NFκB is primarily cytoplasmic and is sequestered by inhibitors, including IκB, masking its nuclear localization signal. Upon cell stimulation, IκB is rapidly phosphorylated by IκB kinase ("IKK") and subsequently ubiquitinated and degraded. Upon release from IκB inhibition, NFκB rapidly translocates to the nucleus.

[0084] The effect of olticumab on oxLDL signaling was investigated in the NFκB pathway. Macrophages were stimulated with lipopolysaccharide (LPS) in the presence of oxLDL and treated with either olticumab or a mutant olticumab-like antibody that significantly lacks affinity for oxLDL and / or native LDL. Monocytes were isolated from healthy subjects and incubated in the absence or presence of 0.3 ng / mL lipopolysaccharide (LPS). Cells were simultaneously treated with a control antibody, olticumab, or mutant olticumab. Cells were then harvested and immunoblotted for total IκBα or a control protein (actin). Whole cell lysates from primary human monocytes were extracted with RIPA buffer (150 mM NaCl, 1.0% IGEPAL CA-630, 0.5% v / v sodium deoxycholate, 0.1% w / v SDS, and 50 mM Tris-chloride, pH 8.0). After centrifugation to remove cell debris, aliquots of the supernatants at 20,000 g were subjected to 10% SDS / PAGE, after which proteins were transferred to Hybond-C Extra nitrocellulose filters (Amersham Biosciences, Piscataway, NJ). Filters were incubated with primary antibodies at room temperature. Bound antibodies were visualized by chemiluminescence (Super Signal Substrate; Thermo Fisher Scientific, Waltham, MA) using a 1:5000 dilution of donkey anti-rabbit IgG or donkey anti-mouse IgG conjugated to horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA). Filters were exposed to Kodak X-Omat BlueXB-1 film for 1–60 s at room temperature.

[0085] As shown in Figure 3, olticumab treatment was found to induce the expression of IκBα in both LPS-stimulated and control macrophages. These results suggest that olticumab has anti-inflammatory activity in oxLDL-stimulated macrophages by inhibiting NFκB through increasing IκBα expression.

[0086] Example 4: Olticumab inhibits macrophage infiltration Based on the biochemical evidence, we investigated the net effect of olticumab on oxLDL-induced macrophage infiltration. A well-characterized mouse model of atherosclerosis was used. Apobec-1- / - / LDLR- / - mice on a C57BL / 6 background (Jackson Laboratories, Bar Harbor, Maine) express full-length apoB-100 in LDL particles and have plasma levels of apoB-100 threefold higher than those of LDLR- / - mice. Starting at 4 weeks of age, Apobec-1- / - / LDLR- / - mice were fed a high-cholesterol diet (15% cholesterol, 21% fat, Lactamin AB, Kimstad, Sweden) offered ad libitum. One week before the first treatment (at 24 weeks of age), the diet was changed to normal chow. One week later (at 25 weeks of age), a group of mice was sacrificed as baseline controls (baseline 25w). The remaining animals were either left untreated (control 29w) or received 1 mg (0.5 mL) of a control IgG antibody (fluorescein isothiocyanate-8 (FITC-8)) or olticumab via intraperitoneal (IP) injection. Injections were repeated at weekly intervals for a total of three doses, and mice were sacrificed 2 weeks after the last injection (at 29 weeks of age).

[0087] The efficacy of olticumab in limiting macrophage infiltration was evaluated using two endpoints. Figure 4A shows that 4 weeks of olticumab treatment after plaque formation significantly reduced the observed number of infiltrating macrophages compared with either untreated or control IgG-treated controls. This inhibition resulted in beneficial physiological changes in the subjects studied. Macrophage infiltration was assessed in the innominate artery by MOMA2 monoclonal antibody staining (*p<0.05 vs. FITC-8). Figure 4B shows that olticumab treatment significantly reduced plaque burden compared with pretreatment conditions and various controls. Plaque burden was assessed in the descending aorta by Oil Red O staining and calculation of the ratio of total plaque area to the total area of the descending aorta. P values were calculated relative to fluorescein isothiocyanate-8 (FITC-8) (***p<0.001 vs. FITC-8).

[0088] All references cited herein are incorporated by reference in their entirety as if fully set forth. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Allen et al.,Remington: The Science and Practice of Pharmacy 22nd ed.,Pharmaceutical Press(Sep.15,2012);Hornyak et al.,Introduction to Nanoscience and Nanotechnology,CRC Press(2008);Singleton and Sainsbury,Dictionary of Microbiology and Molecular Biology 3rd ed.,revised ed.,J.Wiley & Sons(New York, NY2006); Smith, March's Advanced Organic Chemistry Reactions, Mechanisms and Structure 7th ed., J. Wiley & Sons (New York, NY2013); Singleton, Dictionary of DNA and Genome Technology 3rd ed., Wiley-Blackwell (Nov. 28, 2012); and Green and Sambrook, Molecular Cloning: A Laboratory Manual 4th ed.,Cold Spring Harbor Laboratory Press(Cold Spring Harbor, NY 2012) provides one of ordinary skill in the art with a general guide to many of the terms used in this application.For references on methods for preparing antibodies, see Greenfield, Antibodies A Laboratory Manual 2nd ed., Cold Spring Harbor Press (Cold Spring Harbor, NY, 2013); Kohler and Milstein, Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, Eur. J. Immunol. 1976 July 6(7):511-9; Queen and Selick, Humanized immunoglobulins, US Pat. No. 5,585,089 (1996 December); and Riechmann et al., Reshaping human antibodies for therapy, Nature 1988 March 24,332(6162):21-7.

[0089] The foregoing specification describes the present invention and its methods of use in several embodiments. Those skilled in the art may be able to make changes and modifications to what is described herein without departing from its spirit and scope. While the present invention is susceptible to different embodiments in different forms, preferred embodiments of the present invention are shown in the drawings and will be described in detail herein, with the understanding that the present disclosure can be considered as an exemplification of the principles of the invention and is not intended to limit the broader aspects of the invention to the embodiments shown. It is intended that all features, elements, components, functions, and steps described with respect to any embodiment provided herein can be freely combined with and substituted for any other embodiment, unless otherwise specified. Therefore, it should be understood that what is shown in the drawings is set forth for illustrative purposes only and should not be construed as limiting the scope of the invention.

[0090] The invention has been described broadly and generically herein. Each narrower species and subgeneric grouping falling within a generic disclosure also forms part of the method. This includes any general description of the method that is subject to a qualification or negative limitation excluding any subject matter from that genus, regardless of whether the excluded material is specifically recited herein.

[0091] Other embodiments are found in the following claims. Additionally, when features or aspects of the present methods are described in terms of a Markush group, those skilled in the art will recognize that the invention may be described in terms of any individual member or subgroup of members of that Markush group. The present application provides the following: 1. A method for inhibiting macrophage infiltration into a tumor in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody or fragment thereof that binds to oxidized low-density lipoprotein (oxLDL). 2. A method for inhibiting tumor metastasis in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or fragment thereof that binds to oxidized low-density lipoprotein (oxLDL). 3. The method according to any one of 1 to 2 above, wherein the tumor is LOX-1 positive. 4. The method according to any one of 1 to 3 above, wherein the tumor is a CD36-positive tumor. 5. The method according to any one of 1 to 4 above, wherein the tumor is selected from the group consisting of ovarian cancer, bladder urothelial carcinoma, renal clear cell carcinoma, rectal adenocarcinoma, colon adenocarcinoma, prostate cancer, breast epithelial cell tumor, glioblastoma, pancreatic cancer, and esophageal cancer. 6. A method according to any one of 1 to 5 above, wherein the method reduces the growth rate of the tumor. 7. A method for treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody or fragment thereof that binds to oxidized low-density lipoprotein (oxLDL). 8. A method of treating cancer in a subject, comprising administering to the subject: (a) a therapeutically effective amount of a first-line anti-cancer therapy selected from the group consisting of chemotherapy, radiation therapy, and immunotherapy; and (b) administering a therapeutically effective amount of an antibody or fragment thereof that binds to oxidized low-density lipoprotein (oxLDL). 9. The method according to any one of 7 to 8 above, wherein the cancer is LOX-1 positive. 10. The method according to any one of 7 to 8 above, wherein the cancer is a CD36-positive tumor. 11. The method according to any one of 7 to 10 above, wherein the cancer is selected from the group consisting of ovarian cancer, bladder urothelial carcinoma, renal clear cell carcinoma, rectal adenocarcinoma, colon adenocarcinoma, prostate cancer, breast epithelial cell tumor, glioblastoma, pancreatic cancer, and esophageal cancer. 12. The method according to any one of 7 to 10 above, wherein the cancer is a hematological cancer. 13. The method of claim 12, wherein the hematological cancer is selected from the group consisting of acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute monocytic leukemia, non-Hodgkin's lymphoma, and multiple myeloma. 14. The method according to any one of claims 7 to 13, wherein the antibody or fragment thereof is an adjuvant to the first-line anti-cancer therapy. 15. A method according to any one of 7 to 14 above, wherein the subject is diagnosed with serum hyperlipidemia, type 2 diabetes, or metabolic syndrome. 16. The method according to any one of 1 to 15 above, wherein the antibody or fragment thereof inhibits the binding of oxLDL to LOX-1. 17. The method according to any one of 1 to 16 above, wherein the antibody or fragment thereof binds with at least 100 times greater affinity to oxLDL than to native LDL. 18. The method according to any one of 1 to 17 above, wherein the antibody is a human antibody. 19. The method described in any one of 1 to 18 above, wherein the antibody or fragment thereof comprises at least one light chain complementarity-determining region (LCDR) that is at least 90% identical to an LCDR selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. 20. The method of claim 19, wherein the antibody or fragment thereof comprises at least one LCDR selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10. 21. The method described in any one of 1 to 20 above, wherein the antibody or fragment thereof comprises at least one heavy chain complementarity-determining region (HCDR) that is at least 90% identical to an LCDR selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7. 22. The method of claim 21, wherein the antibody or fragment thereof comprises at least one LCDR selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7. 23. The antibody or fragment thereof comprises a variable heavy region (V) of SEQ ID NO: 11. H ), the variable light region of SEQ ID NO: 12 (V L 23. The method according to any one of 1 to 22 above, comprising: 24. The method described in any one of 1 to 23 above, wherein the antibody or fragment thereof comprises a heavy chain of SEQ ID NO: 3, a light chain of SEQ ID NO: 4, or both. 25. The method according to any one of 1 to 24 above, wherein the antibody is olticumab. 26. The method of any one of 1 to 25 above, wherein the antibody or fragment thereof is administered intravenously at an initial dose of at least 5 mg / kg, followed by multiple subsequent doses of at least 2 mg / kg / week, at least 2.5 mg / kg / 2 weeks, or at least 6 mg / kg / month, respectively. 27. The method according to any one of 1 to 26 above, wherein the antibody or fragment thereof is administered subcutaneously at a dose of about 330 mg / month for at least 3 months.

Claims

1. A pharmaceutical composition comprising an antibody or fragment thereof that binds to oxidized low-density lipoprotein (oxLDL), for use in a method for inhibiting macrophage infiltration into a tumor in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody or fragment thereof that binds to oxLDL, the antibody or fragment thereof comprising heavy chain complementarity-determining regions (HCDRs) of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, and light chain complementarity-determining regions (LCDRs) of SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:

10.

2. A pharmaceutical composition comprising an antibody or fragment thereof that binds to oxLDL for use in a method for inhibiting tumor metastasis in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody or fragment thereof that binds to oxLDL, the antibody or fragment thereof comprising the HCDRs of SEQ ID NOs: 5, 6, and 7, and the LCDRs of SEQ ID NOs: 8, 9, and 10.

3. The composition for use according to claim 1 or 2, wherein the tumor is LOX-1 positive.

4. The composition for use according to any one of claims 1 to 3, wherein the tumor is a CD36-positive tumor.

5. The composition for use according to any one of claims 1 to 4, wherein the tumor is selected from the group consisting of ovarian cancer, bladder urothelial carcinoma, renal clear cell carcinoma, rectal adenocarcinoma, colon adenocarcinoma, prostate cancer, breast epithelial cell tumor, glioblastoma, pancreatic cancer, and esophageal cancer.

6. The composition for use according to any one of claims 1 to 5, wherein said method reduces the growth rate of said tumor.

7. A pharmaceutical composition comprising an antibody or fragment thereof that binds to oxLDL for use in a method for treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody or fragment thereof that binds to oxLDL, wherein the antibody or fragment thereof comprises the HCDRs of SEQ ID NOs: 5, 6, and 7, and the LCDRs of SEQ ID NOs: 8, 9, and 10.

8. 1. A pharmaceutical composition comprising an antibody or fragment thereof that binds to oxLDL for use in a method of treating cancer in a subject, said method comprising administering to said subject: (a) a therapeutically effective amount of a first-line anti-cancer therapy selected from the group consisting of chemotherapy, radiation therapy, and immunotherapy; and (b) a therapeutically effective amount of an antibody or fragment thereof that binds to oxLDL. wherein the antibody or fragment thereof comprises an HCDR of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, and an LCDR of SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:

10.

9. The composition for use according to claim 7 or 8, wherein the cancer is LOX-1 positive.

10. The composition for use according to any one of claims 7 to 9, wherein the cancer is a CD36-positive tumor.

11. The composition for use according to any one of claims 7 to 10, wherein the cancer is selected from the group consisting of ovarian cancer, bladder urothelial carcinoma, renal clear cell carcinoma, rectal adenocarcinoma, colon adenocarcinoma, prostate cancer, breast epithelial cell tumor, glioblastoma, pancreatic cancer, and esophageal cancer.

12. The composition for use according to any one of claims 7 to 10, wherein the cancer is a hematological cancer.

13. 13. The composition for use of claim 12, wherein the hematological cancer is selected from the group consisting of acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute monocytic leukemia, non-Hodgkin's lymphoma, and multiple myeloma.

14. The composition for use according to any one of claims 7 to 13, wherein said antibody or fragment thereof is an adjuvant to said first line anti-cancer therapy.

15. The composition for use according to any one of claims 7 to 14, wherein the subject is diagnosed with serum hyperlipidemia, type 2 diabetes, or metabolic syndrome.

16. The composition for use according to any one of claims 1 to 15, wherein the antibody or fragment thereof inhibits the binding of oxLDL to LOX-1.

17. The composition for use according to any one of claims 1 to 16, wherein the antibody or fragment thereof binds with at least 100 times greater affinity to oxLDL than to native LDL.

18. The composition for use according to any one of claims 1 to 17, wherein the antibody is a human antibody.

19. The antibody or fragment thereof comprises a variable heavy region (V Ｈ ), the variable light region of SEQ ID NO: 12 (V Ｌ 19. The composition for use according to any one of claims 1 to 18, comprising:

20. The composition for use according to any one of claims 1 to 19, wherein the antibody or fragment thereof comprises a heavy chain of SEQ ID NO: 3, a light chain of SEQ ID NO: 4, or both.

21. The composition for use according to any one of claims 1 to 20, wherein the antibody is olticumab.

22. 22. The composition for use according to any one of claims 1 to 21, wherein the antibody or fragment thereof is administered intravenously at an initial dose of at least 5 mg / kg, followed by multiple subsequent doses of at least 2 mg / kg / week, at least 2.5 mg / kg / 2 weeks, or at least 6 mg / kg / month, respectively.

23. The composition for use according to any one of claims 1 to 21, wherein the antibody or fragment thereof is administered subcutaneously at a dose of about 330 mg / month for at least 3 months.

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