METHODS AND KITS FOR DETECTING AND DIAGNOSING NEUROTRAUMA
20170254822 · 2017-09-07
Inventors
Cpc classification
G01N2800/2871
PHYSICS
International classification
Abstract
Methods and kits for detecting and diagnosing neurotrauma (e.g., traumatic brain injury, stroke, or spinal cord injury) are provided. These methods rely on the determination of lysophosphatidic acid (LPA) and/or LPA metabolite levels in patient samples following suspected injury.
Claims
1. A method of detecting and treating neurotrauma, if present, in a human subject suspected of having sustained neurotrauma, comprising: (a) providing a biological sample of tissue or bodily fluid from a human subject suspected of having sustained neurotrauma within about the previous 36 hours; (b) using an anti-LPA antibody or LPA-binding antibody fragment to determine a level of a first biomarker that is lysophosphatidic acid (LPA) or an LPA metabolite in a biological sample of tissue or bodily fluid from said human subject by contacting the biological sample with the anti-LPA antibody or LPA-binding antibody fragment and detecting a level of binding between LPA and/or an LPA metabolite and the anti-LPA antibody or LPA-binding antibody fragment to determine the level of the first biomarker in the biological sample; (c) determining a level of said first biomarker that is LPA or an LPA metabolite in a negative control, wherein said determining is performed using an antibody- or antibody fragment-based method; (d) comparing said level of said first biomarker to the level of LPA or LPA metabolite in said negative control, wherein an elevated level of said first biomarker from said human subject compared to the negative control is indicative of the subject having sustained neurotrauma; and, if neurotrauma is present, and (e) reducing the effective concentration of LPA in the subject by administering to the subject an anti-LPA antibody or LPA-binding antibody fragment that reduces that effective concentration of LPA in the subject, thereby treating the subject for neurotrauma.
2. A method according to claim 1 wherein the LPA is total LPA, or wherein the LPA is one or more of the group consisting of 16:0 acyl LPA, 18:0 acyl LPA, 18:1 acyl LPA, 18:2 acyl LPA, and 20:4 acyl LPA; and wherein the LPA metabolite is lysophosphatidylcholine (LPC) or lyso-platelet activating factor (lyso-PAF).
3. A method according to claim 1 wherein the antibody- or antibody fragment-based method is an enzyme-linked immunosorbent assay (ELISA) or lateral flow immunoassay.
4. A method according to claim 1 further comprising determining a level of at least one additional protein or lipid biomarker for neurotrauma in said biological sample or in another biological sample from said human subject, wherein the first biomarker and the at least one additional protein or lipid biomarker are not the same, and wherein the first biomarker and the at least one additional protein or lipid biomarker are detected in the same assay or a different assay.
5. A method according to claim 4 wherein the additional protein or lipid biomarker is selected from the group consisting of ubiquitin C-terminal hydrolase (UCH-L1), glial fibrillary acidic protein (GFAP), the phosphorylated form of the high-molecular-weight neurofilament subunit NF-H (pNF-H), LPA, an LPA metabolite and 12-hydroxyeicosatetraenoic acid (12-HETE).
6. A method according to claim 4 wherein if said first biomarker is LPA, said additional biomarker is an LPA metabolite or 12-HETE; wherein if said first biomarker is LPC, said additional biomarker is LPA, lyso-PAF, or 12-HETE; and wherein if said first biomarker is lyso-PAF, said additional biomarker is LPA, LPC, or 12-HETE.
7. A method of claim 1 wherein the first biomarker is LPA and wherein the determining of LPA levels is by an antibody-based method using an antibody which is specifically reactive with LPA, or an LPA-binding fragment thereof.
8. A method according to claim 7 wherein said method further comprises use of a derivatized LPA bound directly or indirectly to a solid support or a carrier moiety, wherein the derivatized LPA is optionally thiolated LPA and the carrier moiety is optionally selected from the group consisting of polyethylene glycol, colloidal gold, adjuvant, a silicone bead, and a protein, optionally wherein the carrier moiety is colored or carries a detectable label.
9. A kit for detecting neurotrauma in a human subject, wherein said kit comprises: an antibody- or antibody fragment-based means for determining a level of a first biomarker that is LPA or an LPA metabolite in a biological sample of tissue or bodily fluid from said human subject; instructions for using the kit, a negative control and an antibody- or antibody fragment-based means for determining a level of said first biomarker that is LPA or an LPA metabolite in said negative control, wherein an elevated level of LPA or an LPA metabolite compared to said negative control is indicative of neurotrauma.
10. A kit according to claim 9 wherein the first biomarker is LPA.
11. A kit according to claim 9 wherein the antibody- or antibody-fragment based means for determining LPA levels is optionally an enzyme-linked immunosorbent assay (ELISA) assay or a lateral flow immunoassay.
12. A kit according to claim 9 wherein the kit further comprises a derivatized LPA that is directly or indirectly bound to a solid support or a carrier moiety, wherein the carrier moiety is optionally selected from the group consisting of polyethylene glycol, colloidal gold, adjuvant, a silicone bead, a latex bead, a colored particle, and a protein.
13. A kit according to claim 9 further comprising a means for determining a level of at least one additional protein or lipid biomarker for neurotrauma in said biological sample or another biological sample from said human subject, wherein the first biomarker and the at least one additional protein or lipid biomarker are not the same and wherein the first biomarker and the at least one additional protein or lipid biomarker are detected in the same assay or a different assay.
14. A kit according to claim 13 wherein the additional protein or lipid biomarker is selected from the group consisting of ubiquitin C-terminal hydrolase (UCH-L1), glial fibrillary acidic protein (GFAP), the phosphorylated form of the high-molecular-weight neurofilament subunit NF-H (pNF-H), LPA, an LPA metabolite, and 12-hydroxyeicosatetraenoic acid (12-HETE).
15. A kit according to claim 9 wherein the LPA metabolite is LPC or lyso-PAF.
16. A kit according to claim 13 wherein if said first biomarker is LPA, said additional biomarker is an LPA metabolite or 12-HETE; wherein if said first biomarker is LPC, said additional biomarker is LPA, lyso-PAF, or 12-HETE; and wherein if said first biomarker is lyso-PAF, said additional biomarker is LPA, LPC, or 12-HETE.
17. A kit according to claim 9 wherein the neurotrauma is selected from the group consisting of traumatic brain injury, spinal cord injury and stroke.
18. A kit according to claim 9 wherein the biological sample of tissue or bodily fluid comprises central nervous system tissue, cerebrospinal fluid (CSF), blood, plasma, or urine.
19. A kit according to claim 9 further comprising a pharmaceutical composition for treating neurotrauma that comprises an agent that reduces the effective concentration of LPA.
20. A kit according to claim 19 wherein the agent that reduces the effective concentration of LPA is a humanized antibody, or fragment thereof, that binds LPA in a tissue or bodily fluid.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0134] A brief summary of each of the figures and tables described in this specification are provided below. This application contains at least one figure executed in color. Copies of this application with color drawings will be provided upon request and payment of the necessary fee.
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DETAILED DESCRIPTION OF THE INVENTION
[0154] The instant invention provides methods and kits for detection and diagnosis of neurotrauma, by measuring LPA and/or LPA metabolite levels in fluid or tissue samples from patients suspected of having sustained a CNS injury or damage. It has recently been shown (see examples below) that LPA levels and certain LPA metabolite levels rise significantly in cerebrospinal fluid (CSF) of human subjects following TBI, compared to normal control; in other words, LPA is a biomarker for neurotrauma. This observation will open doors to novel and highly useful diagnostic tests and procedures for rapid diagnosis of neurotrauma.
[0155] LPA and LPA metabolites can be measured using a variety of means, including enzymatic means, physical measurements (e.g., mass spectrometry, LC-MS), and methods which rely on specific LPA-binding agents such as antibodies to LPA, aptamers that bind LPA, LPA receptor fragments and the like. Derivatized LPA may also be useful in detection and measurement of LPA, such as in antibody-based methods such as ELISA or other immunochemical assays, and may be used in preparing labeled LPA (radiolabelled or otherwise).
[0156] Highly specific anti-LPA antibodies have been generated by Lpath, Inc. and have been demonstrated to have therapeutic utility against cancer, fibrosis and other conditions. More recently, anti-LPA antibodies have been shown to be neuroprotective in animal models of neurotrauma, e.g., TBI, stroke and SCI. Thus anti-LPA antibodies have both therapeutic and diagnostic uses related to neurotrauma, and may accordingly be used in companion diagnostics.
[0157] A. Derivatized and/or Conjugated LPA
[0158] 1. Compositions
[0159] The present invention may utilize LPA which has been derivatized in such a way as to facilitate the immunogenic response (i.e., antibody production) and/or to allow conjugation of the LPA molecule to a carrier molecule or other moiety such as a label or solid support. In one embodiment, a carbon atom within the hydrocarbon chain of the LPA is derivatized with a pendant reactive group [e.g., a sulfhydryl (thiol) group, a carboxylic acid group, a cyano group, an ester, a hydroxy group, an alkene, an alkyne, an acid chloride group or a halogen atom]. This derivatization serves to activate the bioactive lipid for reaction with a molecule, e.g., for conjugation to a carrier. In one embodiment, the derivatized LPA is thiolated LPA. In one embodiment, the derivatized LPA is derivatized C12 or C18 LPA. In one embodiment, the thiolated LPA is conjugated via a crosslinker, e.g., a bifunctional crosslinker such as IOA or SMCC, to a carrier, which may be a protein. It may be useful to conjugate the LPA in this way to a protein or other carrier that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin (KLH), serum albumin (including bovine serum albumin or BSA), bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl.sub.2, or R.sup.1N═C═NR, where R and R.sup.1 are different alkyl groups. Non-protein carriers (e.g., colloidal gold) are also known in the art for use in antibody production.
[0160] The derivatized or derivatized and conjugated LPA may be used as an immunogen to generate anti-LPA antibodies (polyclonal and/or monoclonal). The derivatized or derivatized and conjugated LPA may also be used in the methods of the invention, particularly in diagnostic methods.
[0161] 2. Research and Diagnostic Uses for Derivatized LPA
[0162] The derivatized LPAs described above may be used to detect and/or purify anti-LPA antibodies and may be conjugated to a carrier as described above. The derivatives and conjugates may be conjugated to a solid support for use in diagnostic methods, including clinical diagnostic methods. For example, detection and/or quantitation of LPA antibodies, particularly autoantibodies, may be used in diagnosing various medical conditions in LPA plays a role. Quantitation of LPA antibodies is also useful in a clinical setting to detect and/or diagnose diseases and conditions characterized by aberrant levels of LPA, or to evaluate dosing, halflife and drug levels, or patient response, after treatment with, e.g., an anti-LPA antibody such as those described herein. Derivatized LPA made as described herein bears a reactive group that does not disable the epitope by which Lpath's anti-LPA antibodies recognize LPA. Thus, the derivatized LPA may be used as part of an assay method or kit that relies on anti-LPA antibodies for detection and/or quantitation of LPA. Derivatized LPA may also be used to allow attachment of a label (radiolabel or other label) to the LPA, for use in scintillation proximity assays (SPA) or other assays.
[0163] In one embodiment, the derivatized LPA conjugate (for example, thiolated LPA conjugated to BSA or KLH) is used as laydown material in ELISAs which are used to detect anti-LPA antibodies. In one embodiment the LPA is thiolated C12 LPA or thiolated C18 LPA conjugated to BSA. This embodiment is useful, for example, as laydown material (to coat the plate) in ELISA assays for detection of LPA. For example, in an LPA competitive ELISA, the plate is coated with derivatized and/or derivatized and conjugated LPA. A set of one or more LPA standards and one or more samples (e.g., serum or cell culture supernatant) is mixed with the mouse anti-LPA antibody and added to the derivitized-LPA-coated plate. The antibody competes for binding to either plate-bound LPA or LPA in the sample or standard. Following incubation and several ELISA steps, the absorbance at 450 nm is measured and the LPA concentration in the samples is determined by comparison to the standard curve.
[0164] The derivatized or derivatized and conjugated LPA may also be coupled to a solid support (e.g., resin or other column matrix, beads, membrane, plate) and used to isolate and/or purify anti-LPA antibodies, e.g., from blood or serum. Such anti-LPA antibodies may be newly generated antibodies (e.g., mammalian monoclonal or polyclonal antibodies to LPA) or may be native human anti-LPA antibodies.
[0165] Thus both derivatized LPA and derivatized and conjugated LPA are useful for research and in clinical diagnostics. In one embodiment, derivatized or derivatized and conjugated LPA is used in kits and methods for detection of neurotrauma by measurement of LPA in patient samples. In one embodiment, these kits and methods also employ an antibody which specifically binds LPA.
[0166] B. Anti-LPA Agents, Including Anti-LPA Antibodies
[0167] 1. Introduction
[0168] Effective inhibitors of LPA for therapeutic use have not been identified prior to Lpath's development of highly specific and potent antibodies to LPA. An alternative approach is the inhibition of autotaxin (ATX), a secreted nucleotide pyrophosphatase/phosphodiesterase that functions as a lysophospholipase D to produce LPA. The ATX-LPA signaling axis has been implicated in angiogenesis, chronic inflammation, fibrotic diseases and tumor progression, making this system an attractive target for therapy, but again, suitably potent and selective nonlipid inhibitors of ATX are currently not available. Inhibitors of LPA receptors such as the selective LPA1 receptor antagonist AM966 (Swaney et al. Br J Pharmacol. 2010 August; 160(7): 1699-1713) have also been tried as treatments for LPA-associated disease, particularly fibrosis. It is believed that direct neutralization of LPA itself is a more straightforward and favorable approach than inhibition of LPA synthesis or of one or more of the multiple LPA receptors. Thus compounds that bind LPA tightly and specifically are desired for use as therapeutics and in detection and diagnostics.
[0169] 2. Disease Associations of LPA and Therapeutic Uses for Anti-LPA Agents
[0170] LPA has been associated with a number of diseases and disorders. For review, see Gardell, et al. (2006), Trends Mol Med. 12(2):65-75, and Chun J. and Rosen, H., (2006) Curr. Pharma. Design 12:161-171. These include autoimmune disorders such as diabetes, multiple sclerosis and scleroderma; fibrotic diseases and conditions; hyperproliferative disorders including cancer; disorders associated with angiogenesis and neovascularization; obesity; neurodegenerative diseases including Alzheimer's disease; schizophrenia, immune-related disorders such as transplant rejection and graft-vs.-host disease, and others. Additional descriptions regarding LPA in disease and anti-LPA agents, particularly antibodies, in treatment and prevention of disease may be found, e.g., in U.S. patent application publication numbers: 20090136483, 20080145360, 20100034814 and 20110076269, all of which are commonly owned with the instant invention and are incorporated herein by reference in their entirety.
[0171] a. Hyperproliferative Disorders
[0172] LPA-associated hyperproliferative disorders include neoplasias, disorders associated with endothelial cell proliferation, and disorders associated with fibrogenesis. Most often, the neoplasia will be a cancer. Typical disorders associated with endothelial cell proliferation are angiogenesis-dependent disorders, for example, cancers caused by a solid tumors, hematological tumors, and age-related macular degeneration. Disorders associated with fibrogenesis include those than involve aberrant cardiac remodeling, such as cardiac failure.
[0173] Cancer is now primarily treated with one or a combination of three types of therapies, surgery, radiation, and chemotherapy. Surgery involves the bulk removal of diseased tissue. While surgery is sometimes effective in removing tumors located at certain sites, for example, in the breast, colon, and skin, it cannot be used in the treatment of tumors located in other areas, such as the backbone, nor in the treatment of disseminated neoplastic conditions such as leukemia. Radiation therapy involves the exposure of living tissue to ionizing radiation causing death or damage to the exposed cells. Side effects from radiation therapy may be acute and temporary, while others may be irreversible. Chemotherapy involves the disruption of cell replication or cell metabolism. Current therapeutic agents thus usually involve significant drawbacks for the patient in the form of toxicity and severe side effects. Therefore, many groups have recently begun to look for new approaches to fighting the war against cancer. These new so-called “innovative therapies” include gene therapy and therapeutic proteins such as monoclonal antibodies.
[0174] The first monoclonal antibody used in the clinic for the treatment of cancer was Rituxan (rituximab) which was launched in 1997, and has demonstrated the utility of monoclonal antibodies as therapeutic agents. Thus, not surprisingly, twenty monoclonal antibodies have since been approved for use in the clinic, including nine that are prescribed for cancer. The success of these products, as well as the reduced cost and time to develop monoclonal antibodies as compared with small molecules has made monoclonal antibody therapeutics the second largest category of drug candidates behind small molecules. Further, the exquisite specificity of antibodies as compared to small molecule therapeutics has proven to be a major advantage both in terms of efficacy and toxicity. Consequently, monoclonal antibodies are poised to become a major player in the treatment of cancer and they are estimated to capture an increasing share of the cancer therapeutic market. Generally therapeutic mAbs are targeted to proteins; only recently has it been feasible to raise mAbs to bioactive lipids (for example, antibodies to S1P, see Applicants' US Patent Application Publication No. 20070148168).
[0175] The identification of extracellular mediators that promote tumor growth and survival is a critical step in discovering therapeutic interventions that will reduce the morbidity and mortality of cancer. As described below, LPA is considered to be a pleiotropic, tumorigenic growth factor. LPA promotes tumor growth by stimulating cell proliferation, cell survival, and metastasis. LPA also promotes tumor angiogenesis by supporting the migration and survival of endothelial cells as they form new vessels within tumors. Taken together, LPA initiates a proliferative, pro-angiogenic, and anti-apoptotic sequence of events contributing to cancer progression. Thus, therapies that modulate, and, in particular, reduce LPA levels in vivo will be effective in the treatment of cancer.
Typically, methods for treating or preventing a hyperproliferative disorder such as cancer involve administering to a subject, such as a human subject or patient, an effective amount of each of an anti-LPA agent, such as an anti-LPA antibody, or a plurality of different agent species, and a cytotoxic agent. Cytotoxic agents include chemotherapeutic drugs.
[0176] A related method is intended to reduce toxicity of a therapeutic regimen for treatment or prevention of a hyperproliferative disorder. Such methods comprise administering to a subject, such as a human subject or patient, suffering from a hyperproliferative disorder an effective amount of an anti-LPA agent, such as an anti-LPA antibody, or a plurality of different agents, before, during, or after administration of a therapeutic regimen intended to treat or prevent the hyperproliferative disorder. It is believed that by using anti-LPA agents to sensitize cells, e.g., cancer cells, to chemotherapeutic drugs, efficacy can be achieved at lower doses and hence lower toxicity due to chemotherapeutic drugs.
[0177] Yet another aspect of the invention concerns methods of enhancing a survival probability of a subject treated for a hyperproliferative disorder by administering to a subject suffering from a hyperproliferative disorder an anti-LPA agent, such as an anti-LPA antibody, or a plurality of different agent species, before, during, or after administration of a therapeutic regimen intended to treat or prevent the hyperproliferative disorder to enhance the subject's survival probability.
[0178] 1. Fibrosis, Wound Healing and Scar Formation
[0179] Fibroblasts, particularly myofibroblasts, are key cellular elements in scar formation in response to cellular injury and inflammation (Tomasek et al. (2002), Nat Rev Mol Cell Biol, vol 3: 349-63, and Virag and Murry (2003), Am J Pathol, vol 163: 2433-40). Collagen gene expression by myofibroblasts is a hallmark of remodeling and necessary for scar formation (Sun and Weber (2000), Cardiovasc Res, vol 46: 250-6, and Sun and Weber (1996), J Mol Cell Cardiol, vol 28: 851-8).
[0180] Fibrosis can be described as the formation or development of excess or aberrant fibrous connective tissue in an organ or tissue as part of a pathological reparative or reactive process, in contrast to normal wound healing or development. The most common forms of fibrosis are: liver, lung, kidney, skin, uterine and ovarian fibroses. Some conditions, such as scleroderma, sarcoidosis and others, are characterized by fibrosis in multiple organs and tissues.
[0181] Recently, the bioactive lysophospholipid lysophosphatidic acid (LPA) has been recognized for its role in tissue repair and wound healing. Watterson et al., Wound Repair Regen. (2007) 15:607-16. As a biological mediator, LPA has been recognized for its role in tissue repair and wound healing (Watterson, 2007). In particular, LPA is linked to pulmonary and renal inflammation and fibrosis. LPA is detectable in human bronchioalveolar lavage (BAL) fluids at baseline and its expression increases during allergic inflammation Georas, S. N. et al. (2007) Clin Exp Allergy. (2007) 37: 311-22. Furthermore, LPA promotes inflammation in airway epithelial cells. Barekzi, E. et al (2006) Prostaglandins Leukot Essent Fatty Acids. 74:357-63. Recently, pulmonary and renal fibrosis have been linked to increased LPA release and signaling though the LPA type 1 receptor (LPA.sub.1). LPA levels were elevated in bronchialveolar lavage (BAL) samples from IPF patients and bleomycin-induced lung fibrosis in mice was dependent on activation of LPA.sub.1. Tager et al., (2008) Proc Am Thorac Soc. 5: 363. (2008) Following unilateral ureteral obstruction in mice, tubulointerstitial fibrosis was reduced in LPA.sub.1 knock-out mice and pro-fibrotic cytokine expression was attenuated in wild-type mice treated with an LPA.sub.1 antagonist. J. P. Pradere et al., (2007) J. Am. Soc. Nephrol. 18:3110-3118. LPA has been shown to have direct fibrogenic effects in cardiac fibroblasts by stimulating collagen gene expression and proliferation. Chen, et al. (2006) FEBS Lett. 580:4737-45. Combined, these studies demonstrate a role for LPA in tissue repair and fibrosis, and identify bioactive lipids as a previously unrecognized class of targets in the treatment of fibrotic disorders.
[0182] Examples of fibrotic disorders include scleroderma, pulmonary fibrosis, liver fibrosis, renal fibrosis, uterine fibrosis, fibrosis of the skin, and cardiac fibrosis. Agents that reduce the effective concentration of LPA, such as Lpath's anti-LPA mAb, are believed to be useful in methods for treating diseases and conditions characterized by aberrant fibrosis.
[0183] b. Cardiovascular and Cerebrovascular Disorders
[0184] Because LPA is involved in fibrogenesis and wound healing of liver tissue (Davaille et al., J. Biol. Chem. 275:34268-34633, 2000; Ikeda et al., Am J. Physiol. Gastrointest. Liver Physiol 279:G304-G310, 2000), healing of wounded vasculatures (Lee et al., Am. J. Physiol. Cell Physiol. 278:C612-C618, 2000), and other disease states, or events associated with such diseases, such as cancer, angiogenesis and inflammation (Pyne et al., Biochem. J. 349:385-402, 2000), the compositions and methods of the disclosure may be applied to treat not only these diseases but cardiac diseases as well, particularly those associated with tissue remodeling. LPA have some direct fibrogenic effects by stimulating collagen gene expression and proliferation of cardiac fibroblasts. Chen, et al. (2006) FEBS Lett. 580:4737-45.
[0185] c. Obesity and Diabetes
[0186] Autotaxin, a phospholipase D responsible for LPA synthesis, has been found to be secreted by adipocytes and its expression is up-regulated in adipocytes from obese-diabetic db/db mice as well as in massively obese women subjects and human patients with type 2 diabetes, independently of obesity (Ferry et al. (2003) JBC 278:18162-18169; Boucher et al. (2005) Diabetologia 48:569-577, cited in Pradere et al. (2007) BBA 1771:93-102. LPA itself has been shown to influence proliferation and differentiation of preadipocytes. Pradere et al., 2007. Together this suggests a role for anti-LPA agents in treatment of obesity and diabetes.
[0187] d. Pain
[0188] A significant role of LPA in the development of pain, including neuropathic pain, was established using various pharmacological and genetic approaches. LPA is responsible for long-lasting mechanical allodynia and thermal hyperalgesia as well as demyelination and upregulation of pain-related proteins through the LPA1 receptor. In addition, intrathecal injections of LPA induce behavioral, morphological and biochemical changes such as prolonged sensitivity to pain stimuli accompanied by demyelination of dorsal roots, similar to those observed after nerve ligation. Fujita, R., Kiguchi, N. & Ueda, H. (2007) Neurochem Int 50, 351-5. Wild-type animals with nerve injury develop behavioral allodynia and hyperalgesia paralleled by demyelination in the dorsal root and increased expression of both the protein kinase C isoform within the spinal cord dorsal horn and the 21 calcium channel subunit in dorsal root ganglia. It has been demonstrated that mice lacking the LPA1 receptor gene (Ipa1−/− mice) lose nerve injury-induced neuropathic pain behaviors and phenomena. Inoue, M. et al. (2004) Nat Med 10, 712-8. Heterozygous mutant mice for the autotaxin gene (atx+/−) showed approximately 50% recovery of nerve injury-induced neuropathic pain. The hyperalgesia was completely abolished in both Ipa1−/− and atx+/− mice. Furthermore, inhibitors of Rho and Rho kinase signaling pathways also prevented neuropathic pain. Mueller, B. K., Mack, H. & Teusch, N. (2005) Nat Rev Drug Discov 4, 387-98. Therefore, targeting LPA biosynthesis and/or LPA1 receptor may represent a novel approach to mitigating nerve-injury-induced neuropathic pain
[0189] At the cellular level, LPA is a potent inducer of morphological changes in neuronal and glial cells 66, 151-155. Kingsbury, M. A., et al. (2003) Nat Neurosci 6, 1292-9; Jalink, K. et al., (1993) Cell Growth Differ 4, 247-55; Tigyi, G. & Miledi, R. (1992) J Biol Chem 267, 21360-7 (1992); Fukushima, N. et al. (2000) Dev Biol 228, 6-18; Yuan, X. B. et al. (2003) Nat Cell Biol 5, 38-45; Fukushima, N., et al. (2007) Neurochem Int 50, 302-7.
[0190] In primary astrocytes, as well as in glioma-derived cell lines, LPA causes reversal of process outgrowth (‘stellation’), a process directed by active RhoA and accompanied by reassembly and activation of focal adhesion proteins. Ramakers, G. J. & Moolenaar, W. H. (1998). Exp Cell Res 245, 252-62. A role for LPA in myelination is also suggested by the finding that LPA promotes cell-cell adhesion and survival in Schwann cells. Weiner, J. A., et al. (2001) J Neurosci 21, 7069-78; Ramer, L. M. et al (2004) J Neurosci 24, 10796-805.
[0191] e. Neurotrauma and CNS Diseases/Conditions
[0192] Key components of the LPA pathway are modulated following CNS injury. In the adult mouse, LPA receptors are differentially expressed in the spinal cord and LPA receptors 1-3 (LPA1-3) are strongly upregulated in response to injury. Goldshmit, et al. (2010), Cell Tissue Res. 341:23-32. Examination of LPA receptors expression in the intact uninjured spinal cord showed that LPA1-3 are expressed at low but distinct levels in different areas of the spinal cord. LPA1 is expressed in the central canal by ependymal cells, while LPA2 is expressed in cells immediately surrounding the central canal and at low levels on some astrocytes in the grey matter. LPA3 is expressed at low levels on motor neurons of the ventral horn and throughout the grey matter neuropil. Following SCI, LPA1 is still expressed on a subpopulation of astrocytes near the injury site at 4 days following injury, although its level of expression is increased. LPA2 is expressed by astrocytes, with an upregulation on reactive astrocytes around the lesion site by 2 days, and further increased by 4 days. LPA3 expression remains confined to neurons but is upregulated in a small number of neurons by 2 days, and further increased by 4 days extending its expression to the neuronal processes. This upregulation is observed not only close to the lesion site, but also distal from both sides.
[0193] Considering the pleiotropic effects of LPA on most neural cell types, especially on cell morphology, proliferation and survival, together with demonstration of a localized upregulation of LPA1-3 following injury, it is likely that LPA regulates essential aspects of the cellular reorganization following neurotrauma by being a key player in reactive astrogliosis, neural regeneration and axonal re-growth.
[0194] Data strongly suggest that neural responses to LPA stimuli are likely to significantly influence the amount of ensuing damage or repair following brain and/or spinal cord injury. Elevated levels of LPA are observed in certain pathological states including brain and spinal cord injury. LPA injections into mouse brain induce astrocyte reactivity at the site of the injury, while in the spinal cord, LPA induces neuropathic pain and demyelination. LPA can stimulate astrocytic proliferation and can promote death of hippocampal neurons. Moreover, LPA mediates microglial activation and is cytotoxic to the neuromicrovascular endothelium.
[0195] Following injury, LPA is synthesized in the mouse spinal cord in a model of sciatic nerve ligation (Ma, Uchida et al. 2010) and LPA-like activity is increased in the cerebrospinal fluid following intrathecal injection of autologous blood to mimic cerebral hemorrhage in newborn pigs (Tigyi, et al. (1995), Am J. Physiol. 268:H2048-2055; Yakubu, et al. (1997), Am J. Physiol. 273:R703-709). Normally undetectable, levels of ATX increase in astrocytes neighboring a lesion of the adult rat brain (Savaskan, et al. (2007), Cell Mol. Life Sci. (2007) 64:230-43). In humans, the presence of ATX in cerebrospinal fluid has been demonstrated in multiple sclerosis patients (Hammack, et al. (2004), Mult Scler. 10:245-60 and higher levels of LPA in human plasma might predict silent brain infarction (Li, et al. (2010), Int J Mol Sci. 11:3988-98). Further, in human cerebrospinal fluid from traumatic brain injury (TBI) patients (Farias, et al. (2011), J Trauma. 71:1211-8) increased levels of arachidonic acid, a lipid generated from the hydrolysis of phosphatidic acid into LPA and arachidonic acid, have been reported.
[0196] Following injury, hemorrhage, or trauma to the nervous system, levels of LPA within the nervous system are believed to increase to 10 μM. Dottori, et al. [(2008) Stem Cells 26:1146-54] have shown that 10 μM LPA can inhibit neuronal differentiation of human NSC, while lower concentrations do not, suggesting that high levels of LPA within the CNS following injury might inhibit differentiation of NSC toward neurons, thus inhibiting endogenous neuronal regeneration. Modulating LPA signaling may thus have a significant impact in nervous system injury, allowing new potential therapeutic approaches. Antibodies to LPA have now been shown (see examples below) to decrease infarct size, neuroinflammation (including gliogenesis) and neurodegeneration.
[0197] LPA and LPA metabolites have now been shown to be a biomarker for neurotrauma, such as TBI, as is shown in the examples below. LPA is elevated in CSF following TBI, and thus can be used diagnostically to indicate the presence of neurotrauma. This allows the development of rapid methods and kits as are described herein, to aid in detecting neurotrauma independently of neurological symptoms. Such methods and kits can be used, for example, by emergency medical personnel, emergency room physicians, and in combat situations, to aid in patient triage. The rapidity of the method allows treatment to begin soon after injury, which is believed to minimize (to the extent possible) the CNS damage that occurs subsequent to the initial injury. Treatment may be with anti-LPA agents such as the antibodies described herein, which have shown efficacy in models of neurotrauma (see examples herein).
[0198] In addition to LPA, other markers for neurotrauma are known, and these may be used in combination with LPA in methods and kits for detecting and diagnosing neurotrauma. For example, comparison of albumin levels in CSF and serum can be used to assess BBB disruption, and the astrocytic protein S100B and monomeric transthyretin have been reported as serum markers for BBB disruption. Blyth et al. (2009) J. Neurotrauma 26:1497-1507. A proteomics approach has been used to identify 30 putative prognostic biomarkers for TBI, including cerebellin, FGF-13, glutathione peroxidase 3, serpinA3, murinoglobin, ApoA4, Clusterin/ApoJ, complement proteins C1 QB, C8B and CBG, fibrinogen alpha and beta chains, prothrombin, hemoglobin subunits alpha, beta and delta, hemopexin, and ten immunoglobin (or related) proteins: IGHG, IGK5, EP3-6, LOC100047628, IGHM, IGL3C, IGH2, IGK8, IGG3C, and EALC. Crawford et al. (2012) J. Neurotrauma 29:246-60. The phosphorylated form of the high-molecular-weight neurofilament subunit NF-H (pNF-H), has been reported to be elevated in blood after SCI, with levels reflecting the degree of axonal damage. Hayakawa et al. (2012) 1-4. Serum levels of ubiquitin C-terminal hydrolase (UCH-L1) have been shown to distinguish mild TBI from controls. UCH-L1 is detectable within an hour of injury and levels correlate with Glasgow score, existence of intracranial lesions detectable by CT, and need for neurosurgical intervention. Papa et al. (2012) J. Trauma 72:1335-1344. Glial fibrillary acidic protein (GFAP) is a brain-specific biomarker that is released into the blood following TBI and stroke and is a putative biomarker for these conditions. Schiff, L. et al., Mol Diagn Ther. 2012 Apr. 1; 16(2):79-92 (abstract). Plasma GFAP analysis performed within 4.5 h of symptom onset can differentiate intracranial hemorrhage from ischemic stroke. Foerch et al. (2012) Clinical Chemistry 58: 237-245 and US patent application publication 20060240480. Additional putative protein biomarkers for TBI include SBDP150, SBDP120, MBPlfrag, MAP2, BA0293, S100B, NSA, MMP9, VCAM and IL-12.
[0199] In addition to LPA, other lipid biomarkers for neurotrauma also exist, such as LPA metabolites as described herein (e.g., lyso-PAF and LPC), as well as other lipid biomarkers such as 12-hydroxyeicosatetraenoic acid (12-HETE), which has been shown to be elevated in CSF of patients after TBI [Farias et al., (2011) J. Trauma 71:1211-1218]. These lipid biomarkers may also be used alone or in combination for detection and diagnosis of neurotrauma. In one embodiment, levels of LPA and of one or more LPA metabolites are determined in one or more biological samples from a subject to detect and/or diagnose neurotrauma. In another embodiment, levels of LPA and/or levels of an LPA metabolite in addition to levels of 12-HETE are determined in one or more biological samples from a subject to detect and/or diagnose neurotrauma.
[0200] Other examples of protein and lipid biomarkers for neurotrauma exist. The methods and kits for detecting and diagnosing neurotrauma as disclosed herein may rely on determination of LPA alone or in combination with detection and/or measurement of one or more additional markers of neurotrauma. In some embodiments, determination of multiple biomarkers is desired.
[0201] 3. Antibodies to LPA
[0202] Polyclonal antiserum against naturally-occurring LPA has been reported in the literature (Chen J H, et al., Bioorg Med Chem Lett. 2000 Aug. 7; 10(15):1691-3). The examples hereinbelow describe the production of monoclonal anti-LPA antibodies with desirable properties from a therapeutic perspective including: (a) binding affinity for LPA and/or its variants, including 18:2, 18:1, 18:0, 16:0, 14:0, 12:0 and 20:4 LPA. Antibody affinities may be determined as described in the examples herein below. Preferably antibodies bind LPA with a high affinity, e.g., a K.sub.d value of no more than about 1×10.sup.−7 M; possibly no more than about 1×10.sup.−8 M; and possibly no more than about 5×10.sup.−9 M. In a physiological context, it is preferable for an antibody to bind LPA with an affinity that is higher than the LPA's affinity for an LPA receptor. It will be understood that this need not necessarily be the case in a nonphysiological context such as a diagnostic assay.
[0203] Aside from antibodies with strong binding affinity for LPA, it may also be desirable to select chimeric, humanized or variant antibodies which have other beneficial properties from a therapeutic perspective. For example, the antibody may be one that reduces scar formation or alters tumor progression. One assay for determining the activity of the anti-LPA antibodies is ELISA. Preferably the humanized or variant antibody fails to elicit an immunogenic response upon administration of a therapeutically effective amount of the antibody to a human patient. If an immunogenic response is elicited, preferably the response will be such that the antibody still provides a therapeutic benefit to the patient treated therewith.
[0204] More information about antibodies to LPA, including antigen-binding antibody fragments and variants, can be found in applicant's patent applications, e.g., US Patent Application Publication Nos: 20090136483, 20080145360, 20100034814 and 20110076269, all of which are commonly owned with the instant invention and are incorporated herein by reference in their entirety, and in the examples below. Antibodies to LPA may be polyclonal or monoclonal, and may be humanized. Isolated nucleic acid encoding the anti-LPA antibody, vectors and host cells comprising the nucleic acid, and recombinant techniques for the production of the antibody are also described in the above patent applications.
[0205] a. Pharmaceutical Formulations, Dosing and Routes of Administration
[0206] One way to control the amount of undesirable LPA in a patient is by providing a composition that comprises one or more anti-LPA antibodies to bind one or more LPAs, thereby acting as therapeutic “sponges” that reduce the level of free LPA. When a compound is stated to be “free,” the compound is not in any way restricted from reaching the site or sites where it exerts its undesirable effects. Typically, a free compound is present in blood and tissue, which either is or contains the site(s) of action of the free compound, or from which a compound can freely migrate to its site(s) of action. A free compound may also be available to be acted upon by any enzyme that converts the compound into an undesirable compound.
[0207] Anti-LPA antibodies may be formulated in a pharmaceutical composition that is useful for a variety of purposes, including the treatment of diseases, disorders or physical trauma. Pharmaceutical compositions comprising one or more anti-LPA antibodies may be incorporated into kits and medical devices for such treatment. Medical devices may be used to administer the pharmaceutical compositions to a patient in need thereof, and according to one embodiment, kits are provided that include such devices. Such devices and kits may be designed for routine administration, including self-administration, of the pharmaceutical compositions. Such devices and kits may also be designed for emergency use, for example, in ambulances or emergency rooms, or during surgery, or in activities where injury is possible but where full medical attention may not be immediately forthcoming (for example, hiking and camping, or combat situations).
[0208] Therapeutic formulations of the antibody are prepared for storage by mixing the antibody having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).
[0209] The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
[0210] The active ingredients may also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
[0211] The formulations to be used for in vivo administration must be sterile. This is readily accomplished for instance by filtration through sterile filtration membranes.
[0212] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the Lupron Depot™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37° C., resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S—S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
[0213] For therapeutic applications, the anti-LPA agents, e.g., antibodies, are administered to a mammal, preferably a human, in a pharmaceutically acceptable dosage form such as those discussed above, including those that may be administered to a human intravenously as a bolus or by continuous infusion over a period of time, or by intramuscular, intraperitoneal, intra-cerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, intranasal or inhalation routes. For CNS applications, intracerebrosponal, intrathecal and intranasal administration may be particularly useful. Although the blood-brain barrier (BBB) is impermeable to most drugs, intranasal delivery allows efficient drug delivery into the CNS; this is believed to occur via the rostral migratory stream, trigeminal nerve and/or olfactory nerve. Scranton et al. (2011) PLoS ONE 6:e18711. For a review of intranasal administration, including routes and devices, see Dhuria et al. (2010) J Pharm Sci. 99(4):1654-73. It may be preferable to administer the drug intranasally into the upper third of the nasal cavity (U.S. Pat. No. 6,313,093, Frey, W H). In addition to these targeted CNS routes, the possibility of CNS administration through systemic or other routes also exists in patients with neurotrauma because the BBB is often compromised for a window of time following neurotrauma.
[0214] For the prevention or treatment of disease, the appropriate dosage of antibody will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments.
[0215] Depending on the type and severity of the disease, about 1 .mu.g/kg to about 50 mg/kg (e.g., 0.1-20 mg/kg) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily or weekly dosage might range from about 1 μg/kg to about 20 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays, including, for example, radiographic imaging. Detection methods using the antibody to determine LPA levels in bodily fluids or tissues may be used in order to optimize patient exposure to the therapeutic antibody.
[0216] According to another embodiment, the composition comprising an agent, e.g, a mAb, that interferes with LPA activity is administered as a monotherapy, while in other preferred embodiments, the composition comprising the agent that interferes with LPA activity is administered as part of a combination therapy. In some cases the effectiveness of the antibody in preventing or treating disease may be improved by administering the antibody serially or in combination with another agent that is effective for those purposes, such as a chemotherapeutic drug for treatment of cancer. In other cases, the anti-LPA agent may serve to enhance or sensitize cells to chemotherapeutic treatment, thus permitting efficacy at lower doses and with lower toxicity. Preferred combination therapies include, in addition to administration of the composition comprising an agent that interferes with LPA activity, delivering a second therapeutic regimen selected from the group consisting of administration of a chemotherapeutic agent, radiation therapy, surgery, and a combination of any of the foregoing.
[0217] Such other agents may be present in the composition being administered or may be administered separately. Also, the antibody is suitably administered serially or in combination with the other agent or modality, e.g., chemotherapeutic drug or radiation for treatment of cancer.
[0218] b. Research and Diagnostic, Including Clinical Diagnostic, Uses for Anti-LPA Agents
[0219] Anti-LPA agents, e.g., aptamers, receptor fragments, small molecules and antibodies, are molecules which specifically bind LPA. As such they may be used to detect and/or purify LPA, e.g., from bodily fluid(s). For use of anti-LPA antibodies as affinity purification agents, the antibodies are immobilized on a solid support such as beads, a Sephadex resin or filter paper, using methods well known in the art. The immobilized antibody (or other anti-LPA detection reagent) is contacted with a sample containing the LPA to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the LPA, which is bound to the immobilized antibody. Finally, the support is washed with another suitable solvent, such as glycine buffer, for instance between pH 3 to pH 5.0, that will release the LPA from the antibody.
[0220] Anti-LPA antibodies are useful in diagnostic assays for LPA, e.g., detecting its presence in specific cells, tissues, or bodily fluids. Such diagnostic methods may be useful in diagnosis, e.g., of a hyperproliferative disease or disorder. Thus, clinical diagnostic uses as well as research uses are comprehended by the invention. In these methods, the anti-LPA antibody is preferably attached to a solid support, e.g., bead, column, plate, gel, filter, membrane, etc.
[0221] For diagnostic applications, the antibody may be labeled with a detectable moiety. Numerous labels are available which can be generally grouped into the following categories:
[0222] (a) Radioisotopes, such as .sup.35S, .sup.14C, .sup.125I, .sup.3H, and .sup.131I. The antibody can be labeled with the radioisotope using the techniques described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al., Ed. Wiley-Interscience, New York, N.Y., Pubs. (1991), for example, and radioactivity can be measured using scintillation counting.
[0223] (b) Fluorescent labels such as rare earth chelates (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, Lissamine, phycoerythrin and Texas Red are available. The fluorescent labels can be conjugated to the antibody using the techniques disclosed in Current Protocols in Immunology, supra, for example. Fluorescence can be quantified using a fluorimeter.
[0224] (c) Various enzyme-substrate labels are available and U.S. Pat. No. 4,275,149 provides a review of some of these. The enzyme generally catalyzes a chemical alteration of the chromogenic substrate that can be measured using various techniques. For example, the enzyme may catalyze a color change in a substrate, which can be measured spectrophotometrically. Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying a change in fluorescence are described above. The chemiluminescent substrate becomes electronically excited by a chemical reaction and may then emit light that can be measured (using a chemiluminometer, for example) or donates energy to a fluorescent acceptor. Examples of enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclicoxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Techniques for conjugating enzymes to antibodies are described in O'Sullivan et al., Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym. (ed J. Langone & H. Van Vunakis), Academic press, New York, 73:147-166 (1981).
[0225] Examples of enzyme-substrate combinations include, for example:
[0226] (i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate, wherein the hydrogen peroxidase oxidizes a dye precursor (e.g., orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethyl benzidine hydrochloride (TMB));
[0227] (ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate as chromogenic substrate; and (iii) .beta.-D-galactosidase (.beta.-D-Gal) with a chromogenic substrate (e.g., p-nitrophenyl-β-D-galactosidase) or fluorogenic substrate 4-methylumbelliferyl-.beta.-D-galactosidase.
[0228] Numerous other enzyme-substrate combinations are available to those skilled in the art. For a general review of these, see U.S. Pat. Nos. 4,275,149 and 4,318,980.
[0229] Sometimes, the label is indirectly conjugated with the antibody. The skilled artisan will be aware of various techniques for achieving this. For example, the antibody can be conjugated with biotin and any of the three broad categories of labels mentioned above can be conjugated with avidin, or vice versa. Biotin binds selectively to avidin and thus, the label can be conjugated with the antibody in this indirect manner. Alternatively, to achieve indirect conjugation of the label with the antibody, the antibody is conjugated with a small hapten (e.g., digoxin) and one of the different types of labels mentioned above is conjugated with an anti-hapten antibody (e.g., anti-digoxin antibody). Thus, indirect conjugation of the label with the antibody can be achieved.
[0230] In another embodiment, the anti-LPA antibody need not be labeled, and the presence thereof can be detected, e.g., using a labeled antibody which binds to the anti-LPA antibody.
[0231] The antibodies may be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987). ELISA assays (competitive or direct) using the anti-LPA antibodies are useful for detecting LPA and assessing its binding and antigen specificity. An LPA ELISA kit incorporating applicant's anti-LPA antibody is commercially available from Echelon Biosciences, Salt Lake City Utah (cat no. K-2800).
[0232] Competitive binding assays rely on the ability of a labeled standard to compete with the test sample analyte for binding with a limited amount of antibody. The amount of LPA in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies. To facilitate determining the amount of standard that becomes bound, the antibodies generally are insoluble before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently be separated from the standard and analyte that remain unbound.
[0233] Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected. In a sandwich assay, the test sample analyte is bound by a first antibody that is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three-part complex. See, e.g., U.S. Pat. No. 4,376,110. The second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme.
[0234] For immunohistochemistry, the blood or tissue sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin, for example.
[0235] The antibodies may also be used for in vivo diagnostic assays. The antibody may be radiolabeled (such as with .sup.111In, .sup.99Tc, .sup.14C, .sup.131I, .sup.125I, .sup.3H, .sup.32P, or .sup.35S) so that the bound target molecule can be localized using immunoscintillography.
[0236] c. Kits for Detection and Diagnosis of LPA-Associated Diseases or Conditions
[0237] As a matter of convenience for methods of detecting and diagnosing neurotrauma, particularly for emergency medical use, antibodies to LPA can be provided in a kit, e.g., a packaged combination of reagents in predetermined amounts with instructions for performing the method. In some embodiments the kit also includes patient sample collection equipment, e.g., syringes, for collecting, e.g., blood or CSF, vials for collection of bodily fluids such as urine, tears, saliva, etc. In one embodiment the kit comprises materials for an antibody-based assay for detection and quantitation of LPA, and preferably contains means and materials (such as standards) for quantitation of the LPA in the patient sample to determine whether LPA levels are elevated to levels indicative of neurotrauma.
[0238] In one embodiment, the LPA assay is performed on a solid support that is compact and portable in a kit, such as a microtiter plate. In one embodiment, the assay for detecting and quantitating LPA for diagnosis of neurotrauma is an ELISA assay. In one embodiment, this assay uses both anti-LPA antibody and derivatized LPA as described herein. The derivatized LPA conjugate (e.g., thiolated LPA conjugated to BSA or KLH) may be used as laydown material (to coat the plate) in ELISA kits that are used to detect anti-LPA antibodies. As one example, in an LPA competitive ELISA kit, the plate (provided) is coated with derivatized and/or derivatized and conjugated LPA. A set of one or more LPA standards (generally provided in the kit) and one or more samples (e.g., urine, blood, serum, cells or tissue) is mixed with the mouse anti-LPA antibody and added to the derivitized-LPA-coated plate. The antibody competes for binding to either plate-bound LPA or LPA in the sample or standard. Following incubation and several ELISA steps (instructions and reagents for which are provided in the kit), the LPA concentration in the samples is determined by comparison to the standard curve, for example, using a colorimetric assay. In one nonlimiting embodiment the LPA used for laydown material in the ELISA kit is thiolated C12 LPA or thiolated C18 LPA conjugated to BSA. The antibody used in the kit may be a polyclonal or monoclonal antibody, preferably a monoclonal antibody.
[0239] A kit incorporating a derivatized and conjugated LPA and an anti-LPA antibody, both of which were developed by Lpath Inc., is commercially available from Echelon Biosciences, Inc., Salt Lake City, Utah (Lysophosphatidic Assay Kit, Cat. No. K-2800).
[0240] As a matter of convenience, anti-LPA antibodies (or antigen-binding fragments thereof) can be provided in a kit, for example, a packaged combination of reagents in predetermined amounts with instructions for performing the diagnostic assay. Where the antibody is labeled with an enzyme, the kit will include substrates and cofactors required by the enzyme (e.g., a substrate precursor which provides the detectable chromophore or fluorophore). In addition, other additives may be included such as stabilizers, buffers (e.g., a block buffer or lysis buffer) and the like. The relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents that substantially optimize the sensitivity of the assay. Particularly, the reagents may be provided as dry powders, usually lyophilized, including excipients that on dissolution will provide a reagent solution having the appropriate concentration. The kit may also contain materials for convenient and safe patient sample collection—sterile gloves, sterilizing wipes, washes, rinses or swabs for ensuring patient safety and/or sterile sample collection, etc.
[0241] In one embodiment, the kit uses a lateral flow test [immunochromatographic strip (ICS) or lateral flow immunoassay (LFIA)] format which is widely used for rapid diagnostics and has the advantage of being easiest to use, particularly in the field, and can test for several analytes (e.g., LPA and one or more additional biomarkers) at once. Such lateral flow kits and methods are particularly suited to point-of-care testing. In one embodiment, a sample of blood or urine is tested using a lateral flow test to detect LPA and/or an LPA metabolite(s) to detect and diagnose neurotrauma.
[0242] In some embodiments, the diagnostic kit also contains therapeutic materials, including an anti-LPA antibody or antigen-binding fragment thereof, for treatment of neurotrauma. This kit is intended to provide rapid neuroprotective treatment of a patient who has been determined, using the diagnostic portion of the kit as described above, to have sustained neurotrauma. The therapeutic portion of the kit preferably contains materials for patient dosing, such as sterile syringes, sterile gloves, etc. along with dosing information and necessary materials for dissolving and/or diluting the antibody, if needed. In some embodiments the kit contains solutions and devices for intranasal administration of the antibody.
[0243] The great advantage of the kits as described herein is the rapid diagnosis of neurotrauma, which allows treatment during the critical window of time during which neuroprotection is possible, before the second phase of brain injury causes maximal damage, and possibly while the BBB is still compromised. In some cases the kit also provides necessary materials for treatment, allowing treatment to begin immediately upon diagnosis, even before reaching an emergency room setting. Because there is no single TBI symptom or pattern of symptoms that characterize mild TBI, for example, a rapid screening test, ideally one (such as a kit described herein) that can be used in the field or in a rescue vehicle. Undiagnosed and untreated TBI presents a risk because some signs and symptoms may be delayed from days to months after injury, and may have significant impact on the patient's physical, emotional, behavioral, social, or family status if untreated, and may result in a functional impairment.
[0244] d. Other Articles of Manufacture
[0245] In another aspect, an article of manufacture containing materials useful for the treatment of the disorders described above is provided. The article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agent in the composition is the anti-sphingolipid antibody. The label on, or associated with, the container indicates that the composition is used for treating the condition of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
[0246] The invention will be better understood by reference to the following Examples, which are intended to merely illustrate the best mode now known for practicing the invention. The scope of the invention is not to be considered limited thereto.
Examples
[0247] The invention will be further described by reference to the following detailed examples. These Examples are in no way to be considered to limit the scope of the invention in any manner.
Example 1: Synthetic Scheme for Making a Representative Thiolated Analog of S1P
[0248] The synthetic approach described in this example results in the preparation of an antigen by serial addition of structural elements using primarily conventional organic chemistry. A scheme for the approach described in this example is provided in
[0249] This synthetic approach began with the commercially available 15-hydroxyl pentadecyne, 1, and activation by methyl sulphonyl chloride of the 15-hydroxy group to facilitate hydroxyl substitution to produce the sulphonate, 2. Substitution of the sulphonate with t-butyl thiol yielded the protected thioether, 3, which was condensed with Garner's aldehyde to produce 4. Gentle reduction of the alkyne moiety to an alkene (5), followed by acid catalyzed opening of the oxazolidene ring yielded S-protected and N-protected thiol substituted sphingosine, 6. During this last step, re-derivatization with di-t-butyl dicarbonate was employed to mitigate loss of the N—BOC group during the acid-catalyzed ring opening.
[0250] As will be appreciated, compound 6 can itself be used as an antigen for preparing haptens to raise antibodies to sphingosine, or, alternatively, as starting material for two different synthetic approaches to prepare a thiolated S1P analog. In one approach, compound 6 phosphorylation with trimethyl phosphate produced compound 7. Treatment of compound 7 with trimethylsilyl bromide removed both methyl groups from the phosphate and the t-butyloxycarbonyl group from the primary amine, leaving compound 8 with the t-butyl group on the sulfur as the only protecting group. To remove this group, the t-butyl group was displaced by NBS to form the disulfide, 9, which was then reduced to form the thiolated S1P analog, 10.
[0251] Another approach involved treating compound 6 directly with NBSCl to form the disulfide, 11, which was then reduced to form the N-protected thiolated S1P analog, 12. Treatment of this compound with mild acid yielded the thiolated sphingosine analog, 13, which can be phosphorylated enzymatically with, e.g., sphingosine kinase, to yield the thiolated S1P analog, 10.
[0252] Modifications of the presented synthetic approach are possible, particularly with regard to the selection of protecting and de-protecting reagents, e.g., the use of trimethyl disulfide triflate described in Example 3 to de-protect the thiol.
[0253] Compound 2.
[0254] DCM (400 mL) was added to a 500 mL RB flask charged with 1 (10.3 g, 45.89 mmol), and the resulting solution cooled to 0° C. Next, TEA (8.34 g, 82.60 mmol, 9.5 mL) was added all at once followed by MsCl (7.88 g, 68.84 mmol, 5.3 mL) added drop wise over 10 min. The reaction was allowed to stir at RT for 0.5 h or until the disappearance of starting material (R.sub.f=0.65, 5:1 hexanes:EtOAc). The reaction was quenched with NH.sub.4Cl (300 mL) and extracted (2×200 mL) DCM. The organic layers were dried over MgSO.sub.4, filtered and the filtrate evaporated to a solid (13.86 g, 99.8% yield). .sup.1H NMR (CDCl.sub.3) δ 4.20 (t, J=6.5 Hz, 2H), 2.98 (s, 3H), 2.59 (td, J=7 Hz, 3 Hz, 2H), 1.917 (t, J=3 Hz, 1H), 1.72 (quintet, J=7.5 Hz, 2H), 1.505 (quintet, J=7.5 Hz, 2H), 1.37 (br s, 4H), 1.27 (br s, 14H). .sup.13C{.sup.1H} NMR (CDCl.sub.3) δ 85.45, 70.90, 68.72, 46.69, 38.04, 30.22, 30.15, 30.14, 30.07, 29.81, 29.76, 29.69, 29.42, 29.17, 26.09, 19.06, 9.31. The principal ion observed in a HRMS analysis (ES-TOF) of compound 2 was m/z=325.1804 (calculated for C.sub.16H.sub.30O.sub.3S: M+Na.sup.+ 325.1808).
[0255] Compound 3.
[0256] A three-neck 1 L RB flask was charged with t-butylthiol (4.54 g, 50.40 mmol) and THF (200 mL) and then placed into an ice bath. n-BuLi (31.5 mL of 1.6 M in hexanes) was added over 30 min. Next, compound 2 (13.86 g, 45.82 mmol), dissolved in THF (100 mL), was added over 2 min. The reaction is allowed to stir for 1 hour or until starting material disappeared (R.sub.f=0.7, 1:1 hexanes/EtOAc). The reaction was quenched with saturated NH.sub.4Cl (500 mL) and extracted with EtO.sub.2 (2×250 mL), dried over MgSO.sub.4, filtered, and the filtrate evaporated to yield a yellow oil (11.67 g, 86% yield). .sup.1H NMR (CDCl.sub.3) δ 2.52 (t, J=7.5 Hz, 2H), 2.18 (td, J=7 Hz, 2.5 Hz, 2H), 1.93 (t, J=2.5 Hz, 1H), 1.55 (quintet, J=7.5 Hz, 2H), 1.51 (quintet, J=7 Hz, 2H), 1.38 (br s, 4H), 1.33 (s, 9H), 1.26 (s, 14H). .sup.13C{.sup.1H} NMR (CDCl.sub.3) δ 85.42, 68.71, 68.67, 54.07, 42.37, 31.68, 30.58, 30.28, 30.26, 30.19, 30.17, 29.98, 29.78, 29.44, 29.19, 29.02, 19.08.
[0257] Compound 4.
[0258] A 250 mL Schlenk flask charged with compound 3 (5.0 g, 16.85 mmol) was evacuated and filled with nitrogen three times before dry THF (150 mL) was added. The resulting solution cooled to −78° C. Next, n-BuLi (10.5 mL of 1.6M in hexanes) was added over 2 min. and the reaction mixture was stirred for 18 min. at −78° C. before the cooling bath was removed for 20 min. The dry ice bath was returned. After 15 min., Garner's aldeyde (3.36 g, 14.65 mmol) in dry THF (10 mL) was then added over 5 min. After 20 min., the cooling bath was removed. Thin layer chromatography (TLC) after 2.7 hr. showed that the Garner's aldehyde was gone. The reaction was quenched with saturated aqueous NH.sub.4Cl (300 mL) and extracted with Et.sub.2O (2×250 mL). The combined Et.sub.2O phases were dried over Na.sub.2SO.sub.4, filtered, and the filtrate evaporated to give crude compound 4 and its syn diastereomer (not shown in
[0259] Compound 5.
[0260] To reduce the triple bond in compound 4, the oil was dissolved in dry Et.sub.2O (100 mL) under nitrogen. RED-Al (20 mL, 65% in toluene) was slowly added to the resulting solution at RT to control the evolution of hydrogen gas (H.sub.2). The reaction was allowed to stir at RT overnight or when TLC showed the disappearance of the starting material (R.sub.f=0.6 in 1:1 EtOAc:hexanes) and quenched slowly with cold MeOH or aqueous NH.sub.4Cl to control the evolution of H.sub.2. The resulting white suspension was filtered through a Celite pad and the filtrate was extracted with EtOAc (2×400 mL). Combined EtOAc extracts were dried over MgSO.sub.4, filtered, and the filtrate evaporated to leave crude compound 5 and its syn diastereomer (not shown in
[0261] Compound 6.
[0262] The oil containing compound 5 was dissolved in MeOH (200 mL), PTSA hydrate (0.63 g) was added, and the solution stirred at RT for 1 day and then at 50° C. for 2 days, at which point TLC suggested that all starting material (5) was gone. However, some polar material was present, suggesting that the acid had partially cleaved the BOC group. The reaction was worked up by adding saturated aqueous NH.sub.4Cl (400 mL), and extracted with ether (3×300 mL). The combined ether phases were dried over Na.sub.2SO.sub.4, filtered, and the filtrate evaporated to dryness, leaving 5.14 g of oil. In order to re-protect whatever amine had formed, the crude product was dissolved in CH.sub.2Cl.sub.2 (150 mL), to which was added BOC.sub.2O (2.44 g) and TEA (1.7 g). When TLC (1:1 hexanes/EtOAc) showed no more material remaining on the baseline, saturated aqueous NH.sub.4Cl (200 mL) was added, and, after separating the organic phase, the mixture was extracted with CH.sub.2Cl.sub.2 (3×200 mL). Combined extracts were dried over Na.sub.2SO.sub.4, filtered, and the filtrated concentrated to dryness to yield a yellow oil (7.7 g) which was chromatographed on a silica column using a gradient of hexanes/EtOAc (up to 1:1) to separate the diastereomers. By TLC using 1:1 PE/EtOAc, the R.sub.f for the anti isomer, compound 6, was 0.45. For the syn isomer (not shown in
[0263] Anal. Calculated for C.sub.27H.sub.53NO.sub.4S: C, 66.48; H, 10.95; N, 2.87. Found: C, 65.98; H, 10.46; N, 2.48.
[0264] Compound 7.
[0265] To a solution of the alcohol compound 6 (609.5 mg, 1.25 mmol) dissolved in dry pyridine (2 mL) was added CBr.sub.4 (647.2 mg, 1.95 mmol, 1.56 equiv). The flask was cooled in an ice bath and P(OMe).sub.3 (284.7 mg, 2.29 mmol, 1.84 equiv) was added drop wise over 2 min. After 4 min. the ice bath was removed and after 12 hr. the mixture was diluted with ether (20 mL). The resulting mixture washed with aqueous HCl (10 mL, 2 N) to form an emulsion which separated on dilution with water (20 mL). The aqueous phase was extracted with ether (2×10 mL), then EtOAc (2×10 mL). The ether extracts and first EtOAc extract were combined and washed with aqueous HCl (10 mL, 2 N), water (10 mL), and saturated aqueous NaHCO.sub.3 (10 mL). The last EtOAc extract was used to back-extract the aqueous washes. Combined organic phases were dried over MgSO.sub.4, filtered, and the filtrate concentrated to leave crude product (1.16 g), which was purified by flash chromatography over silica (3×22 cm column) using CH.sub.2Cl.sub.2, then CH.sub.2Cl.sub.2-EtOAc (1:20, 1:6, 1:3, and 1:1-product started to elute, 6:4, 7:3). Early fractions contained 56.9 mg of oil. Later fractions provided product (compound 7, 476.6 mg, 64%) as clear, colorless oil.
[0266] Anal. Calculated for C.sub.29H.sub.58NO.sub.7PS (595.82): C, 58.46; H, 9.81; N, 2.35. Found: C, 58.09; H, 9.69; N, 2.41.
[0267] Compound 8.
[0268] A flask containing compound 7 (333.0 mg, 0.559 mmol) and a stir bar was evacuated and filled with nitrogen. Acetonitrile (4 mL, distilled from CaH.sub.2) was injected by syringe and the flask now containing a solution was cooled in an ice bath. Using a syringe, (CH.sub.3).sub.3SiBr (438.7 mg, 2.87 mmol, 5.13 equiv.) was added over the course of 1 min. After 35 min., the upper part of the flask was rinsed with an additional portion of acetonitrile (1 mL) and the ice bath was removed. After another 80 min., an aliquot was removed, the solution dried by blowing nitrogen gas over it, and the residue analyzed by .sup.1H NMR in CDCl.sub.3, which showed only traces of peaks ascribed to P—OCH.sub.3 moieties. After 20 min., water (0.2 mL) was added to the reaction mixture, followed by the CDCl.sub.3 solution used to analyze the aliquot, and the mixture was concentrated to ca. 0.5 mL volume on a rotary evaporator. Using acetone (3 mL) in portions the residue was transferred to a tared test tube, forming a pale brown solution. Water (3 mL) was added in portions. After addition of 0.3 mL, cloudiness was seen. After a total of 1 mL, a gummy precipitate had formed. As an additional 0.6 mL of water was added, more cloudiness and gum separated, but the final portion of water seemed not to change the appearance of the mixture. Overall, this process was accomplished over a period of several hours. The tube was centrifuged and the supernatant removed by pipet. The solid, no longer gummy, was dried over P.sub.4O.sub.10 in vacuo, leaving compound 8 (258.2 mg, 95%) as a monohydrate.
[0269] Anal. Calculated. for C.sub.22H.sub.46NO.sub.5PS+H.sub.2O (485.66): C, 54.40; H, 9.96; N, 2.88. Found: C, 54.59; H, 9.84; N, 2.95.
[0270] Compound 9.
[0271] Compound 8 (202.6 mg, 0.417 mmol) was added in a glove box to a test tube containing a stir bar, dry THF (3 mL) and glacial HOAc (3 mL). NBSCl (90 mg, 0.475 mmol, 1.14 equiv) were added, and after 0.5 hr., a clear solution was obtained. After a total of 9 hr., an aliquot was evaporated to dryness and the residue analyzed by .sup.1H NMR in CDCl.sub.3. The peaks corresponding to CH.sub.2StBu and CH.sub.2SSAr suggested that reaction was about 75% complete, and comparison of the spectrum with that of pure NBSCl in CDCl.sub.3 suggested that none of the reagent remained in the reaction. Therefore, an additional portion (24.7 mg, 0.130 mmol, 0.31 equiv) was added, followed 3 hr. later by an additional portion (19.5 mg, 0.103 mmol, 0.25 equiv). After another 1 hr., the mixture was transferred to a new test tube using THF (2 mL) to rinse and water (1 mL) was added.
[0272] Compound 10.
[0273] PMe.sub.3 (82.4 mg, 1.08 mmol, 1.52 times the total amount of 2-nitrobenzenesulfenyl chloride added) was added to the clear solution compound 9 described above. The mixture grew warm and cloudy, with precipitate forming over time. After 4.5 hr., methanol was added, and the tube centrifuged. The precipitate settled with difficulty, occupying the bottom 1 cm of the tube. The clear yellow supernatant was removed using a pipet. Methanol (5 mL, deoxygenated with nitrogen) was added, the tube was centrifuged, and the supernatant removed by pipet. This cycle was repeated three times. When concentrated, the final methanol wash left only 4.4 mg of residue. The bulk solid residue was dried over P.sub.4O.sub.10 in vacuo, leaving compound 10 (118.2 mg, 68%) as a monohydrochloride.
[0274] Anal. Calculated for C.sub.18H.sub.38NO.sub.5S+HCl (417.03): C, 51.84; H, 9.43; N, 3.36. Found: C, 52.11; H, 9.12; N, 3.30.
[0275] Compound 11.
[0276] Compound 6 (1.45 g, 2.97 mmol) was dissolved in AcOH (20 mL), and NBSCl (0.56 g, 2.97 mmol) was added all at once. The reaction was allowed to stir for 3 hr. or until the disappearance of the starting material and appearance of the product was observed by TLC [product R.sub.f=0.65, starting material R.sub.f=0.45, 1:1 EtOAc/hexanes]. The reaction was concentrated to dryness on a high vacuum line and the residue dissolved in THF/H.sub.2O (100 mL of 10:1).
[0277] Compound 12.
[0278] Ph.sub.3P (0.2.33 g, 8.91 mmol) was added all at once to the solution above that contained compound 11 and the reaction was allowed to stir for 3 hr. or until the starting material disappeared. The crude reaction mixture was concentrated to dryness on a high vacuum line, leaving a residue that contained compound 12.
[0279] Compound 13.
[0280] The residue above containing compound 12 was dissolved in DCM (50 mL) and TFA (10 mL). The mixture was stirred at RT for 5 hr. and concentrated to dryness. The residue was the loaded onto a column with silica gel and chromatographed with pure DCM, followed by DCM containing 5% MeOH, then 10% MeOH, to yield the final product, compound 13, as a sticky white solid (0.45 g, 46% yield from 5). .sup.1H NMR (CDCl.sub.3) δ 1.27 (s), 1.33 (br m), 1.61 (p, 2H, J=7.5 Hz), 2.03 (br d, 2H, J=7 Hz), 2.53 (q, 2H, J=7.5 Hz), 3.34 (br s, 1H), 3.87 (br d, 2H, J=12 Hz), 4.48 (br s, 2H), 4.58 (br s, 2H), 5.42 (dd, 1H, J=15 Hz, 5.5 Hz), 5.82 (dt, 1H, J=15 Hz, 5.5 Hz), 7.91 (br s, 4H). .sup.13C{.sup.1H} NMR (CDCl.sub.3) δ 136.85, 126.26, 57.08, 34.76, 32.95, 30.40, 30.36, 30.34, 30.25, 30.19, 30.05, 29.80, 29.62, 29.09, 25.34.
Example 2: Synthetic Schemes for Making Thiolated Fatty Acids
[0281] The synthetic approach described in this example details the preparation of a thiolated fatty acid to be incorporated into a more complex lipid structure that could be further complexed to a protein or other carrier and administered to an animal to elicit an immune response. The approach uses using conventional organic chemistry. A scheme showing the approach taken in this example is provided in
[0282] Two syntheses are described. The first synthesis, for a C-12 thiolated fatty acid, starts with the commercially available 12-dodecanoic acid, compound 14. The bromine is then displaced with t-butyl thiol to yield the protected C-12 thiolated fatty acid, compound 15. The second synthesis, for a C-18 thiolated fatty acid, starts with the commercially available 9-bromo-nonanol (compound 16). The hydroxyl group in compound 16 is protected by addition of a dihydroyran group and the resulting compound, 17, is dimerized through activation of half of the brominated material via a Grignard reaction, followed by addition of the other half. The 18-hydroxy octadecanol (compound 18) produced following acid-catalyzed removal of the dihydropyran protecting group is selectively mono-brominated to form compound 19. During this reaction approximately half of the alcohol groups are activated for nucleophilic substitution by formation of a methane sulfonic acid ester. The alcohol is then oxidized to form the 18-bromocarboxylic acid, compound 20, which is then treated with t-butyl thiol to displace the bromine and form the protected, thiolated C-18 fatty acid, compound 21.
[0283] The protected thiolated fatty acids, each a t-butyl thioether, can be incorporated into a complex lipid and the protecting group removed using, e.g., one of the de-protecting approaches described in Examples 1 and 3. The resulting free thiol then can be used to complex with a protein or other carrier prior to inoculating animal with the hapten.
[0284] A. Synthesis of a C-12 Thiolated Fatty Acid
[0285] Compound 15.
[0286] t-Butyl thiol (12.93 g, 143 mmol) was added to a dry Schlenk flask, and Schlenk methods were used to put the system under nitrogen. Dry, degassed THF (250 mL) was added and the flask cooled in an ice bath. n-BuLi (55 mL of 2.5 M in hexanes, 137.5 mmol) was slowly added over 10 min by syringe. The mixture was allowed to stir at 0° C. for an hour. The bromoacid, compound 14 (10 g, 36 mmol), was added as a solid and the reaction heated and stirred at 60° C. for 24 hr. The reaction was quenched with 2 M HCl (250 mL), and extracted with ether (2×300 mL). The combined ethereal layers were dried with magnesium sulfate, filtered, and the filtrate concentrated by rotary evaporation to yield the thioether acid, compound 15 (10 g, 99% yield) as a beige powder. .sup.1H NMR (CDCl.sub.3, 500 MHz) δ 1.25-1.35 (br s, 12H), 1.32 (s, 9H), 1.35-1.40 (m, 2H), 1.50-1.60 (m, 2H), 1.60-1.65 (m, 2H), 2.35 (t, 2H, J=7.5 Hz), 2.52 (t, 2H, J=7.5 Hz). Principal ion in HRMS (ES-TOF) was observed at m/z 311.2020, calculated for M+Na.sup.+ 311.2015.
[0287] B. Synthesis of a C-12 Thiolated Fatty Acid
[0288] Compound 17.
[0289] A dry Schlenk flask was charged with compound 16 (50 g, 224.2 mmol) and dissolved in dry degassed THF (250 mL) distilled from sodium/benzophenone. The flask was cooled in an ice bath and then PTSA (0.5 g, 2.6 mmol) was added. Dry, degassed DHP (36 g, 42.8 mmol) was then added slowly over 5 min. The mixture was allowed to warm up to RT and left to stir overnight and monitored by TLC (10:1 PE:EtOAc) until the reaction was deemed done by the complete disappearance of the spot for the bromoalcohol. TEA (1 g, 10 mmol) was then added to quench the PTSA. The mixture was then washed with cold sodium bicarbonate solution and extracted with EtOAc (3×250 mL). The organic layers were then dried with magnesium sulfate and concentrated to yield 68.2 g of crude product which was purified by column chromatography (10:1 PE:EtOAc) to yield 60 g (99% yield) of a colorless oil. .sup.1H NMR (CDCl.sub.3, 500 MHz) δ 1.31 (br s, 6H), 1.41-1.44 (m, 2H), 1.51-1.62 (obscured multiplets, 6H), 1.69-1.74 (m, 1H), 1.855 (quintet, J=7.6 Hz, 2H), 3.41 (t, J=7 Hz, 2H), 3.48-3.52 (m, 2H), 3.73 (dt, 2H, J=6.5 Hz), 3.85-3.90 (m, 2H), 4.57 (t, 2H, J=3 Hz).
[0290] Compound 18.
[0291] Magnesium shavings (2.98 g, 125 mmol) were added to a flame-dried Schlenk flask along with a crystal of iodine. Dry THF (200 mL) distilled from sodium was then added and the system was degassed using Schlenk techniques. Compound 17 (30 g, 97 mmol) was then slowly added to the magnesium over 10 min. and the solution was placed in an oil bath at 65° C. and allowed to stir overnight. The reaction was deemed complete by TLC by quenching an aliquot with acetone and observing the change in RF in a 10:1 PE:EtOAc mixture. The Grignard solution was then transferred by cannula to a three-necked flask under nitrogen containing additional compound 17 (30 g, 97 mmol). The flask containing the resulting mixture was then cooled to 0° C. in an ice bath and a solution of Li.sub.2CuCl.sub.4 (3 mL of 1 M) was then added via syringe. The reaction mixture turned a very dark blue within a few minutes. This mixture was left to stir overnight. The next morning the reaction was deemed complete by TLC (10:1 PE:EtOAc), quenched with a saturated NH.sub.4Cl solution, and then extracted into ether (3×250 mL). The ether layers were dried with magnesium sulfate and concentrated to yield crude product (40 g), which was dissolved in MeOH. Concentrated HCl (0.5 mL) was then added, which resulted in the formation of a white emulsion, which was left to stir for 3 hr. The white emulsion was then filtered to yield 16 g (58% yield) of the pure diol, compound 18. .sup.1H NMR (CDCl.sub.3, 200 MHz) δ 1.26 (br s, 24H), 1.41-1.42 (m, 4H), 1.51-1.68 (m, 4H), 3.65 (t, 4H, J=6.5 Hz).
[0292] Compound 19.
[0293] The symmetrical diol, compound 18 (11 g, 38.5 mmol), was added to a dry Schlenk flask under nitrogen, then dry THF (700 mL) distilled from sodium was added. The system was degassed and the flask put in an ice bath. Diisopropylethylamine (6.82 mL, 42.3 mmol) was added via syringe, followed by MSCl (3.96 g, 34.4 mmol) added slowly, and the mixture was left to stir for 1 hr. The reaction was quenched with saturated NaH.sub.2PO.sub.4 solution (300 mL), and then extracted with EtOAc (3×300 mL). The organic layers were then combined, dried with MgSO.sub.4, and concentrated to yield 14 g of a mixture of the diol, monomesylate, and dimesylate. NMR showed a 1:0.8 mixture of CH.sub.2OH:CH.sub.2OMs protons. The mixture was then dissolved in dry THF (500 mL), deoxygenated, and to it was added LiBr (3.5 g, 40.23 mmol). This mixture was allowed reflux overnight, upon which the reaction was quenched with water (150 mL), and extracted with EtOAc (3×250 mL). The organic layer was then dried with MgSO.sub.4, and concentrated to yield a mixture of brominated products that were then purified by flash chromatography (DCM) to yield compound 19 (3.1 g, 25% yield) as a white powder. .sup.1H NMR (CDCl.sub.3, 500 MHz) δ 1.26 (br s, 26H), 1.38-1.46 (m, 2H), 1.55 (quintet, 2H, J=7.5 Hz), 1.85 (quintet, 2H, J=7.5 Hz), 3.403 (t, 2H, J=6.8 Hz), 3.66 (t, 2H, J=6.8 Hz).
[0294] Compound 20.
[0295] A round bottom flask was charged with compound 19 (2.01 g, 5.73 mmol) and the solid dissolved in reagent grade acetone (150 mL). Simultaneously, Jones reagent was prepared by dissolving CrO.sub.3 (2.25 g, 22 mmol) in H.sub.2SO.sub.4 (4 mL) and then slowly adding 10 mL of cold water and letting the solution stir for 10 min. The cold Jones reagent was then added to the round bottom flask slowly over 5 min., after which the solution stirred for 1 hr. The resulting orange solution turned green within several minutes. The mixture was then quenched with water (150 mL) extracted twice in ether (3×150 mL). The ether layers were then dried with magnesium sulfate, and concentrated to yield compound 20 (2.08 g, 98% yield) as a white powder. .sup.1H NMR (CDCl.sub.3, 200 MHz) δ 1.27 (br s, 26H), 1.58-1.71 (m, 2H), 1.77-1.97 (m, 2H), 2.36 (t, 2H, J=7.4 Hz), 3.42 (t, 2H, J=7 Hz).
[0296] Compound 21.
[0297] t-Butylthiol (11.32 g, 125 mmol) was added to a dry Schlenk flask and dissolved in dry THF (450 mL) distilled from sodium. The solution was deoxygenated by bubbling nitrogen through it before the flask was placed in an ice bath. n-BuLi solution in hexanes (70 mL of 1.6 M) was then added slowly via syringe over 10 min. This mixture was allowed to stir for 1 hr., then compound 20 (5.5 g, 16.2 mmol) was added and the solution was left to reflux at 60° C. overnight. The next morning an aliquot was worked up, analyzed by NMR, and the reaction deemed complete. The reaction was quenched with HCl (200 mL of 2 M) and extracted with ether (3×250 mL). The ethereal layers were then dried with magnesium sulfate, filtered, and the filtrate concentrated to yield the product, compound 21, as a white solid (5 g, 90% yield). .sup.1H NMR (CDCl.sub.3, 200 MHz) δ 1.26 (br s, 26H), 1.32 (br s, 9H), 1.48-1.70 (m, 4H), 2.35 (t, 2H, J=7.3 Hz), 2.52 (t, 2H, J=7.3 Hz). .sup.13C NMR (CDCl.sub.3, 200 MHz) δ 24.69, 28.35, 29.05, 29.21, 29.28, 29.39, 29.55, 29.89, 31.02 (3C), 33.98, 41.75, 179.60.
Example 3: Synthetic Scheme for Making a Thiolated Analog of LPA
[0298] The synthetic approach described in this example results in the preparation of thiolated LPA. The LPA analog can then be further complexed to a carrier, for example, a protein carrier, which can then be administered to an animal to elicit an immugenic response to LPA. This approach uses both organic chemistry and enzymatic reactions, the synthetic scheme for which is provided in
[0299] The starting materials were compound 15 in Example 2 and enantiomerically pure glycerophosphocholine (compound 22). These two chemicals combined to yield the di-acetylated product, compound 23, using DCC to facilitate the esterification. In one synthetic process variant, the resulting di-acylated glycerophosphocholine was treated first with phospholipase-A2 to remove the fatty acid at the sn-2 position of the glycerol backbone to produce compound 24. This substance was further treated with another enzyme, phospholipase-D, to remove the choline and form compound 26. In another synthetic process variant, the phospholipase-D treatment preceded the phospholipase-A2 treatment to yield compound 25, and treatment of compound 25 with phospholipase-D then yields compound 26. Both variants lead to the same product, the phosphatidic acid derivative, compound 26. The t-butyl protecting group in compound 26 is then removed, first using trimethyl disulfide triflate to produce compound 27, followed by a disulfide reduction to produce the desired LPA derivative, compound 28. As those in the art will appreciate, the nitrobenzyl sulfenyl reaction sequence described in Example 1 can also be used to produce compound 28.
[0300] Compound 23.
[0301] To a flame-dried Schlenk flask were added the thioether acid, compound 15 (10 g, 35.8 mmol), compound 22 (glycerophosphocholine-CdCl2 complex, 4.25 g, 8.9 mmol), DCC (7.32 g, 35.8 mmol), and DMAP (2.18 g, 17.8 mmol), after which the flask was evacuated and filled with nitrogen. A minimal amount of dry, degassed DCM was added (100 mL), resulting in a cloudy mixture. The flask was covered with foil and then left to stir until completed, as by TLC (silica, 10:5:1 DCM:MeOH:concentrated NH4OH). The insolubility of compound 16 precluded monitoring its disappearance by TLC, but the reaction was stopped when the product spot of Rf 0.1 was judged not to be increasing in intensity. This typically required 3 to 4 days, and in some cases, addition of more DCC and DMAP. Upon completion, the reaction mixture was filtered, and the filtrate concentrated to yield a yellow oil, which was purified using flash chromatography using the solvent system described above to yield 3.6 g (50% yield) of a clear wax containing a mixture of compound 23 and monoacylated products in a ratio of 5 to 1, as estimated from comparing the integrals for the peaks for the (CH3)3N—, —CH2StBu and —CH2COO— moieties. Analysis of the oil by HRMS (ESI-TOF) produced a prominent ion at m/z 820.4972, calculated for M+Na.sup.+=C.sub.40H.sub.80NNaO.sub.8PS.sub.2.sub.
[0302] A. Synthesis Variant 1—Phospholipase-A2 Treatment
[0303] Compound 24.
[0304] A mixture of compound 23 and monoacetylated products as described above (3.1 g, 3.9 mmol) was dissolved in Et.sub.2O (400 mL) and methanol (30 mL). Borate buffer (100 mL, pH 7.4 0.1M, 0.072 mM in CaCl2) was added, followed by phospholipase-A2 (from bee venom, 130 units, Sigma). The resulting mixture was left to stir for 10 hr., at which point TLC (silica, MeOH:water 4:1—the previous solvent system 10:5:1 DCM:MeOH:concentrated NH4OH proved ineffective) showed the absence of the starting material (Rf=0.7) and the appearance of a new spot (Rf=0.2). The organic and aqueous layers were separated and the aqueous layer was washed with ether (2×250 mL). The product was extracted from the aqueous layer with a mixture of DCM:MeOH (2:1, 2×50 mL). The organic layers were then concentrated by rotary evaporation to yield product as a white wax (1.9 g, 86% yield) that NMR showed to be a pure product (compound 24). .sup.1H NMR (CDCl3, 500 MHz) δ 1.25-1.27 (br s, 12H), 1.31 (s, 9H), 1.35-1.45 (m, 2H), 1.52-1.60 (m, 4H), 2.31 (t, 2H, J=7.5 Hz), 2.51 (t, 2H, J=7.5 Hz), 3.28 (br s, 9H) 3.25-3.33 (br s, 2H), 3.78-3.86 (m, 1H), 3.88- 3.96 (m, 2H), 4.04-4.10 (m, 2H), 4.26-4.34 (m, 2H). Analysis of the wax by HRMS (ESI-TOF) produced a prominent ion at m/z 550.2936, calculated for M+Na.sup.+ 550.2943 (C.sub.24H.sub.50NNaO.sub.7PS.sub.2.sub.
[0305] Anal. Calculated. for C24H50NO7PS+2 H2O (563.73): C, 51.13; H, 9.66; N, 2.48. Found: C, 50.90; H, 9.37; N, 2.76.
[0306] Compound 26.
[0307] The lyso compound 24 (1.5 g, 2.7 mmol) was dissolved in a mixture of sec-butanol (5 mL) and Et.sub.2O (200 mL), and the resulting cloudy mixture was sonicated until the cloudiness dissipated. Buffer (200 mL, pH 5.8, 0.2 M NaOAc, 0.08 M CaCl2) was added, followed by cabbage extract (80 mL of extract from savoy cabbage (which contains phospholipase-D), containing 9 mg of protein/mL). The reaction was stirred for 1 day and monitored by TLC (C18 RP SiO2, 5:1 ACN:water), Rf of starting material and product=0.3 and 0.05, respectively. In order to push the reaction to completion, as needed an additional portion of cabbage extract (50 mL) was added and the reaction stirred a further day. This process was repeated twice more, as needed to complete the conversion. When the reaction was complete, the mixture was concentrated on the rotary evaporator to remove the ether, and then EDTA solution (0.5 M, 25 mL) was added and the product extracted into a 5:4 mixture of MeOH:DCM (300 mL). Concentration of the organic layer followed by recrystallization of the residue from DCM and acetone afforded pure product (0.9 g, 75% yield). .sup.1H NMR (CDCl3, 200 MHz) δ 1.25-1.27 (br s, 12H), 1.33 (s, 9H), 1.52-1.60 (m, 4H), 2.34 (t, 2H, J=7.5 Hz), 2.52 (t, 2H, J=7.5 Hz), 3.6-3.8 (br s, 1H), 3.85-3.97 (br s, 2H), 4.02-4.18 (m, 2H).
[0308] Compound 27.
[0309] The protected sample LPA, compound 26 (0.150 g, 0.34 mmol), was methanol washed and added to a vial in the glove box. This was then suspended in a mixture of AcOH:THF (1:1, 10 mL), which never fully dissolved even after 1 hr. of sonication. Solid [Me.sub.2SSMe]OTf (0.114 g, 0.44 mmol) was then added. This was left to stir for 18 hr. The reaction was monitored by removing an aliquot, concentrating it to dryness under vacuum, and re-dissolving or suspending the residue in CD.sub.3OD for observing the .sup.1H NMR shift of the CH.sub.2 peak closest to the sulfur. The starting material had a peak at 2.52 ppm, whereas the unsymmetrical disulfide formed at this juncture had a peak at around 2.7 ppm. This material (compound 27) was not further isolated or characterized.
[0310] Compound 28.
[0311] The mixture containing compound 27 was treated with water (100 μL) immediately followed by PMe3 (0.11 g, 1.4 mmol). After stirring for 3 hr. the solvent was removed by vacuum to yield an insoluble white solid. Methanol (5 mL) was added, the mixture centrifuged, and the mother liquor decanted. Vacuum concentration yielded 120 mg (91% yield) of compound 28, a beige solid. Compound 28 is a thiolated LPA hapten that can be conjugated to a carrier, for example, albumin or KLH, via disulfide bond formation. Characterization of compound 28: .sup.1H NMR (1:1 CD.sub.3OD:CD.sub.3CO.sub.2D, 500 MHz) δ 1.25-1.35 (br s, 12H), 1.32-1.4 (m, 2H), 1.55-1.6 (m, 4H), 2.34 (t, 2H, J=7), 2.47 (t, 2H, J=8.5), 3.89-3.97 (br s, 2H), 3.98-4.15 (m, 2H), 4.21 (m, 1H). Negative ion ES of the sample dissolved in methanol produced a predominant ion at m/z=385.1.
Example 4: Monoclonal Antibodies to LPA
[0312] Antibody Production
[0313] Using an approach employing a derivatized lipid as described in previous examples, a C-12 thio-LPA analog (compound 28 in Example 3) as the key component of a hapten formed by the cross-linking of the analog via the reactive SH group to a protein carrier (KLH) via standard chemical cross-linking using either IOA or SMCC as the cross-linking agent, monoclonal antibodies against LPA were generated. To do this, mice were immunized with the thio-LPA-KLH hapten (in this case, thiolated-LPA:SMCC:KLH). Of the 80 mice immunized against the LPA analog, the five animals that showed the highest titers against LPA (determined using an ELISA in which the same LPA analog (compound 28) as used in the hapten was conjugated to BSA using SMCC and laid down on the ELISA plates) were chosen for moving to the hybridoma phase of development.
[0314] The spleens from these five mice were harvested and hybridomas were generated by standard techniques. Briefly, one mouse yielded hybridoma cell lines (designated 504A). Of all the plated hybridomas of the 504A series, 66 showed positive antibody production as measured by the previously-described screening ELISA.
[0315] Table 1, below, shows the antibody titers in cell supernatants of hybridomas created from the spleens of two of mice that responded to an LPA analog hapten in which the thiolated LPA analog was cross-linked to KLH using heterobifunctional cross-linking agents. These data demonstrate that the anti-LPA antibodies do not react either to the crosslinker or to the protein carrier. Importantly, the data show that the hybridomas produce antibodies against LPA, and not against S1P.
TABLE-US-00001 TABLE 1 LPA hybridomas LPA S1P 3rd bleed binding binding Cross mouse titer OD at Supernatants OD at OD at reactivity # 1:312,500 from 24 well 1:20 1:20 w/S1P* 1 1.242 1.A.63 1.197 0.231 low 1.A.65 1.545 0.176 none 2 0.709 2.B.7 2.357 0.302 low 2.B.63 2.302 0.229 low 2.B.83 2.712 0.175 none 2.B.104 2.57 0.164 none 2.B.IB7 2.387 0.163 none 2.B.3A6 2.227 0.134 none *Cross reactivity with S1P from 24 well supernatants: high = OD >1.0-2.0 at [1:20]; mid = OD 0.4-1.0 at [1:20]; low = OD 0.4-0.2 at [1:20]; none = OD <0.2 OD at [1:20].
[0316] The development of anti-LPA mAbs in mice was monitored by ELISA (direct binding to 12:0 and 18:1 LPA and competition ELISA). A significant immunological response was observed in at least half of the immunized mice and five mice with the highest antibody titer were selected to initiate hybridoma cell line development following spleen fusion.
[0317] After the initial screening of over 2000 hybridoma cell lines generated from these 5 fusions, a total of 29 anti-LPA secreting hybridoma cell lines exhibited high binding to 18:1 LPA. Of these hybridoma cell lines, 24 were further subcloned and characterized in a panel of ELISA assays. From the 24 clones that remained positive, six hybridoma clones were selected for further characterization. Their selection was based on their superior biochemical and biological properties. Mouse hybridoma cell lines 504B-6C2, 504B7.1, 504B58/3F8, 504A63.1 and 504B3A6 (corresponding to clones referred to herein as B3, B7, B58, A63, and B3A6, respectively) were received on May 8, 2007 by the American Type Culture Collection (ATCC Patent Depository, 10801 University Blvd., Manassas, Va. 20110) for patent deposit purposes on behalf of LPath Inc. and were granted deposit numbers PTA-8417, PTA-8420, PTA-8418, PTA-8419 and PTA-8416, respectively.
[0318] All anti-LPA antibodies and portions thereof referred to herein were derived from these cell lines.
[0319] Direct Binding Kinetics
[0320] The binding of 6 anti-LPA mAbs (B3, B7, B58, A63, B3A6, D22) to 12:0 and 18:0 LPA (0.1 uM) was measured by ELISA. EC.sub.50 values were calculated from titration curves using 6 increasing concentrations of purified mAbs (0 to 0.4 ug/ml). EC.sub.50 represents the effective antibody concentration with 50% of the maximum binding. Max denotes the maximal binding (expressed as OD450). Results are shown in Table 2.
TABLE-US-00002 TABLE 2 Direct Binding Kinetics of Anti-LPA mAbs B3 B7 B58 D22 A63 B3A6 12:0 LPA EC.sub.50 (nM) 1.420 0.413 0.554 1.307 0.280 0.344 Max (OD450) 1.809 1.395 1.352 0.449 1.269 1.316 18:1 LPA EC.sub.50 (nM) 1.067 0.274 0.245 0.176 0.298 0.469 Max (OD450) 1.264 0.973 0.847 0.353 1.302 1.027
[0321] The kinetics parameters k.sub.a (association rate constant), k.sub.d (disassociation rate constant) and K.sub.D (association equilibrium constant) were determined for the 6 lead candidates using the BIAcore 3000 Biosensor machine. In this study, LPA was immobilized on the sensor surface and the anti-LPA mAbs were flowed in solution across the surface. As shown, all six mAbs bound LPA with similar K.sub.D values ranging from 0.34 to 3.8 pM and similar kinetic parameters.
[0322] The Anti-LPA Murine mAbs Exhibit High Affinity to LPA
[0323] LPA was immobilized to the sensor chip at densities ranging 150 resonance units. Dilutions of each mAb were passed over the immobilized LPA and kinetic constants were obtained by nonlinear regression of association/dissociation phases. Errors are given as the standard deviation using at least three determinations in duplicate runs. Results are shown in Table 3. Apparent affinities were determined by K.sub.D=k.sub.a/k.sub.d.
[0324] k.sub.a=Association rate constant in M.sup.−1s.sup.−1k.sub.d=Dissociation rate constant in s.sup.−1
TABLE-US-00003 TABLE 3 Affinity of anti-LPA mAb for LPA mAbs k.sub.a (M.sup.−1 s.sup.−1) k.sub.d (s.sup.−1) K.sub.D (pM) A63 4.4 ± 1.0 × 10.sup.5 1 × 10.sup.−6 2.3 ± 0.5 B3 7.0 ± 1.5 × 10.sup.5 1 × 10.sup.−6 1.4 ± 0.3 B7 6.2 ± 0.1 × 10.sup.5 1 × 10.sup.−6 1.6 ± 0.1 D22 3.0 ± 0.9 × 10.sup.4 1 × 10.sup.−6 33 ± 10 B3A6 1.2 ± 0.9 × 10.sup.6 1.9 ± 0.4 × 10.sup.−5 16 ± 1.2
[0325] Specificity Profile of Six Anti-LPA mAbs.
[0326] Many isoforms of LPA have been identified to be biologically active and it is preferable that the mAb recognize all of them to some extent to be of therapeutic relevance. The specificity of the anti-LPA mAbs was evaluated utilizing a competition assay in which the competitor lipid was added to the antibody-immobilized lipid mixture.
[0327] Competition ELISA assays were performed with the anti-LPA mAbs to assess their specificity. Thiolated 18:1 LPA-BSA conjugate was captured on ELISA plates. Each competitor lipid (up to 10 uM) was serially diluted in BSA (1 mg/ml)-PBS and then incubated with the mAbs (3 nM). Mixtures were then transferred to LPA coated wells and the amount of bound antibody was measured with a secondary antibody. Data are normalized to maximum signal (A.sub.450) and are expressed as percent inhibition. Assays were performed in triplicate. IC.sub.50: Half maximum inhibition concentration; MI: Maximum inhibition (% of binding in the absence of inhibitor); ---: not estimated because of weak inhibition. A high inhibition result indicates recognition of the competitor lipid by the antibody. As shown in Table 4, all the anti-LPA mAbs recognized the different LPA isoforms.
TABLE-US-00004 TABLE 4 Specificity profile of anti-LPA mAbs. 14:0 LPA 16:0 LPA 18:1 LPA 18:2 LPA 20:4 LPA IC.sub.50 MI IC.sub.50 MI IC.sub.50 MI IC.sub.50 MI IC.sub.50 MI uM % uM % uM % uM % uM % B3 0.02 72.3 0.05 70.3 0.287 83 0.064 72.5 0.02 67.1 B7 0.105 61.3 0.483 62.9 >2.0 100 1.487 100 0.161 67 B58 0.26 63.9 5.698 >100 1.5 79.3 1.240 92.6 0.304 79.8 B104 0.32 23.1 1.557 26.5 28.648 >100 1.591 36 0.32 20.1 D22 0.164 34.9 0.543 31 1.489 47.7 0.331 31.4 0.164 29.5 A63 1.147 31.9 5.994 45.7 — — — — 0.119 14.5 B3A6 0.108 59.9 1.151 81.1 1.897 87.6 — — 0.131 44.9
[0328] Interestingly, the anti-LPA mAbs were able to discriminate between 12:0 (lauroyl), 14:0 (myristoyl), 16:0 (palmitoyl), 18:1 (oleoyl), 18:2 (linoleoyl) and 20:4 (arachidonoyl) LPAs. A desirable EC.sub.50 rank order for ultimate drug development is 18:2>18:1>20:4 for unsaturated lipids and 14:0>16:0>18:0 for the saturated lipids, along with high specificity. The specificity of the anti-LPA mAbs was assessed for their binding to LPA related biolipids such as distearoyl-phosphatidic acid, lysophosphatidylcholine, S1P, ceramide and ceramide-1-phosphate. None of the antibodies demonstrated cross-reactivity to distearoyl PA and LPC, the immediate metabolic precursor of LPA.
Example 5: Cloning of the Murine Anti-LPA Antibodies—Overview
[0329] Chimeric antibodies to LPA were generated using the variable domains (Fv) containing the active LPA binding regions of one of three murine antibodies from hybridomas with the Fc region of a human IgG1 immunoglobulin. The Fc regions contained the CH1, CH2, and CH3 domains of the human antibody. Without being limited to a particular method, chimeric antibodies could also have been generated from Fc regions of human IgG1, IgG2, IgG3, IgG4, IgA, or IgM. As those in the art will appreciate, “humanized” antibodies can be generated by grafting the complementarity determining regions (CDRs, e.g., CDR1-4) of the murine anti-LPA mAbs with a human antibody framework regions (e.g., Fr1, Fr4, etc.) such as the framework regions of an IgG1.
[0330] The overall strategy for cloning of the murine mAb against LPA consisted of cloning the murine variable domains of both the light chain (VL) and the heavy chain (VH) from each antibody. The consensus sequences of the genes show that the constant region fragment is consistent with a gamma isotype and that the light chain is consistent with a kappa isotype. The murine variable domains were cloned together with the constant domain of the human antibody light chain (CL) and with the constant domain of the human heavy chain (CH1, CH2, and CH3), resulting in a chimeric antibody construct.
[0331] The variable domains of the light chain and the heavy chain were amplified by PCR. The amplified fragments were cloned into an intermediate vector (pTOPO). After verification of the sequences, the variable domains were then assembled together with their respective constant domains. The variable domain of the light chain was cloned into pCONkappa2 and the variable domain of the heavy chain was cloned into pCONgamma1f. The cloning procedure included the design of an upstream primer to include a signal peptide sequence, a consensus Kozak sequence preceding the ATG start codon to enhance translation initiation, and the 5′ cut site, HindIII. The downstream primer was designed to include the 3′ cut site Apal for the heavy chain and BsiWl for the light chain.
[0332] The vectors containing the variable domains together with their respective constant domains were transfected into mammalian cells. Three days after transfections, supernatants were collected and analyzed by ELISA for binding to LPA. Detailed methods for cloning, expression and characterization of the anti-LPA antibody variable domains are shown in US Patent Application Publication Nos: 20090136483, commonly owned with the instant invention and incorporated herein by reference in its entirety.
[0333] Binding characteristics for the chimeric antibodies are shown in Table 5. “HC” and “LC” indicate the identities of the heavy chain and light chain, respectively.
TABLE-US-00005 TABLE 5 Binding characteristics of the chimeric anti-LPA antibodies B3, B7, and B58. Titer EC50 Max HC x LC (ug/ml) (ng/ml) OD 1 B7 B7 3.54 43.24 2.237 2 B7 B58 1.84 25.79 1.998 3 B7 B3 2.58 24.44 2.234 4 B58 B7 3.80 38.99 2.099 5 B58 B58 3.42 41.3 2.531 6 B58 B3 2.87 29.7 2.399 7 B3 B7 4.18 49.84 2.339 8 B3 B58 0.80 20.27 2.282 9 B3 B3 4.65 42.53 2.402
[0334] It can be seen from Table 5 that it is possible to optimize antibody binding to LPA by recombining light chains and heavy chains from different hybridomas (i.e., different clones) into chimeric molecules.
Example 6 Lpath's Lead Murine Antibody, Lpathomab™ (LT3000)—Overview
[0335] Murine antibody clone B7 was chosen as the lead compound and renamed Lpathomab™, also known as LT3000. As described above, this murine anti-LPA mAb, was derived from a hybridoma cell line following immunization of mice with a protein-derivatized LPA immunogen. A hybridoma cell line with favorable properties was identified and used to produce a monoclonal antibody using standard hybridoma culture techniques.
[0336] Applicant has performed a comprehensive series of pre-clinical efficacy studies to confirm the potential therapeutic utility of an anti-LPA-antibody-based approach. It is believed that antibody neutralization (e.g., reduction in effective concentration) of extracellular LPA could result in a marked decrease in disease progression in humans. For cancer, LPA neutralization could result in inhibition of tumor proliferation and the growing vasculature needed to support tumor growth. Furthermore, recent research suggests that many angiogenesis inhibitors may also act as anti-invasive and anti-metastatic compounds that could also mitigate the spread of cancer to sites distant from the initial tumor. For fibrosis, LPA neutralization could result in a reduction of the inflammation and fibrosis associated with the aberrant wound-healing response following tissue injury. Thus, Lpathomab™ could have several mechanisms of action, including: [0337] A direct effect on tumor cell growth, migration and susceptibility to chemotherapeutic agents [0338] An indirect effect on tumors through anti-angiogenic effects [0339] An additional indirect effect on tumors by preventing the release and neutralization of synergistic pro-angiogenic growth factors [0340] A direct effect on proliferation, migration, and transformation of fibroblasts to the myofibroblast phenotype and collagen production by myofibroblasts [0341] An indirect effect on tissue fibrosis by preventing the expression and release of synergistic pro-angiogenic, pro-inflammatory and pro-fibrotic growth factors
Example 7: Biophysical Properties of Lpathomab/LT3000
[0342] Lpathomab/LT3000 has high affinity for the signaling lipid LPA (K.sub.D of 1-50 pM as demonstrated by surface plasmon resonance in the BiaCore assay, and in a direct binding ELISA assay); in addition, LT3000 demonstrates high specificity for LPA, having shown no binding affinity for over 100 different bioactive lipids and proteins, including over 20 bioactive lipids, some of which are structurally similar to LPA. The murine antibody is a full-length IgG1k isotype antibody composed of two identical light chains and two identical heavy chains with a total molecular weight of 155.5 kDa. The biophysical properties are summarized in Table 6.
TABLE-US-00006 TABLE 6 General Properties of Lpathomab (LT3000) Identity LT3000 Antibody isotype Murine IgG1k Specificity Lysophosphatidic acid (LPA) Molecular weight 155.5 kDa OD of 1 mg/mL 1.35 (solution at 280 nm) K.sub.D 1-50 pM Apparent Tm 67° C. at pH 7.4 Appearance Clear if dissolved in 1x PBS buffer (6.6 mM phosphate, 154 mM sodium chloride, pH 7.4) Solubility >40 mg/mL in 6.6 mM phosphate, 154 mM sodium chloride, pH 7.4
[0343] Lpathomab has also shown biological activity in preliminary cell based assays such as cytokine release, migration and invasion; these are summarized in Table 7 along with data showing specificity of LT3000 for LPA isoforms and other bioactive lipids, and in vitro biological effects of LT3000.
TABLE-US-00007 TABLE 7 LT3000 (Lpathomab, B7 antibody) A. Competitor Lipid 14:0 16:0 18:1 18:2 20:4 LPA LPA LPA LPA LPA IC.sub.50 (□M) 0.105 0.483 >2.0 1.487 0.161 MI (%) 61.3 62.9 100 100 67 B. Competitor Lipid LPC S1P C1P Cer DSPA MI (%) 0 2.7 1.0 1 0 C. Cell based assay LPA isoform % Inhibition (over LPA taken as 100) Migration 18:1 35* Invasion 14:0 95* IL-8 Release 18:1 20 IL-6 Release 18:1 23* % Induction (over LPA + TAXOL taken as 100) Apoptosis 18:1 79 A. Competition ELISA assay was performed with Lpathomab and 5 LPA isoforms. 18:1 LPA was captured on ELISA plates. Each competitor lipid (up to 10□M) was serially diluted in BSA/PBS and incubated with 3 nM Lpathomab. Mixtures were then transferred to LPA coated wells and the amount of bound antibody was measured. B. Competition ELISA was performed to assess specificity of Lpathomab. Data were normalized to maximum signal (A.sub.450) and were expressed as percent inhibition (n = 3). IC.sub.50: half maximum inhibition concentration; MI %: maximum inhibition (% of binding in the absence of inhibitor). C. Migration assay: Lpathomab (150□g/mL) reduced SKOV3 cell migration triggered by 1□M LPA (n = 3); Invasion assay: Lpathomab (15 mg/mL) blocked SKOV3 cell invasion triggered by 2□M LPA (n = 2); Cytokine release of human IL-8 and IL-6: Lpathomab (300-600 □g/mL, respectively) reduced 1□M LPA-induced release of pro-angiogenic and metastatic IL-8 and IL-6 in SKOV3 conditioned media (n = 3). Apoptosis: SKOV3 cells were treated with 1□M Taxol; 1□M LPA blocked Taxol induced caspase-3 activation. The addition to Lpathomab (150□g/mL) blocked LPA-induced protection from apoptosis (n = 1). Data Analysis: Student-t test, *denotes p < 0.05.
[0344] The potent and specific binding of Lpathomab/LT3000 to LPA results in reduced availability of extracellular LPA with potentially therapeutic effects against cancer-, angiogenic- and fibrotic-related disorders.
[0345] A second murine anti-LPA antibody, B3, was also subjected to binding analysis as shown in Table 8.
TABLE-US-00008 TABLE 8 Biochemical characteristics of B3 antibody A. BIACORE High density surface Low density surface Lipid Chip 12:0 LPA 18:0 LPA K.sub.D (pM), site 1 (site2) 61(32) 1.6 (0.3) B. Competition Lipid Cocktail (C.sub.16:C.sub.18:C.sub.18:1:C.sub.18:2:C.sub.20:4, ratio 3:2:5:11:2) (μM) IC.sub.50 0.263 C. Neutralization Assay B3 antibody (nmol) LPA (nmol) 0 0.16 0.5 0.0428 1 0.0148 2 under limit of detection A. Biacore analysis for B3 antibody. 12:0 and 18:0 isoforms of LPA were immobilized onto GLC sensor chips; solutions of B3 were passed over the chips and sensograms were obtained for both 12:0 and 18:0 LPA chips. Resulted sensograms showed complex binding kinetics of the antibody due to monovalent and bivalent antibody binding capacities. K.sub.D values were calculated approximately for both LPA 12 and LPA 18. B. Competition ELISA assay was performed with B3 and a cocktail of LPA isoforms (C.sub.16:C.sub.18:C.sub.18:1:C.sub.18:2:C.sub.20:4 in ratio 3:2:5:11:2). Competitor/Cocktail lipid (up to 10 μM) was serially diluted in BSA/PBS and incubated with 0.5 μg/mL B3. Mixtures were then transferred to a LPA coated well plate and the amount of bound antibody was measured. Data were normalized to maximum signal (A.sub.450) and were expressed as IC.sub.50 (half maximum inhibition concentration). C. Neutralization assay: Increasing concentrations of B3 were conjugated to a gel. Mouse plasma was then activated to increase endogenous levels of LPA. Activated plasma samples were then incubated with the increasing concentrations of the antibody-gel complex. LPA leftover which did not complex to the antibody was then determined by ELISA. LPA was sponged up by B3 in an antibody concentration dependent way.
[0346] Selected studies conducted with Lpathomab/LT3000/B7 and B3 are described in Lpath's patent applications e.g., US Patent Application Publication Nos: 20090136483, 20080145360, 20100034814 and 20110076269, all of which are commonly owned with the instant invention and are incorporated herein by reference in their entirety. Briefly, in cancer and angiogenesis models, B7/LT3000 demonstrated:
[0347] Inhibition of tumor growth in human tumor xenograft models in vivo;
[0348] Reduction in LPA-dependent cell proliferation and invasion of human tumor in vitro;
[0349] Reduction in angiogenesis, together with reductions in circulating levels of tumorigenic/angiogenic growth factors including IL6, IL8, GM-CSF, MMP2 in vivo;
[0350] Reduction in metastatic potential; and
[0351] Neutralization of LPA-induced protection against tumor-cell death.
[0352] In In Vitro Models:
[0353] Reduction of proliferation of OVCAR3 ovarian cancer cells;
[0354] Neutralization of LPA-induced release of IL-8 from Caki-1, IL-8 and IL-6 from SKOV3 (ovarian) tumor cells in vitro;
[0355] Mitigation of LPA's effects in protecting SKOV3 tumor cells from apoptosis (which suggests enhanced efficacy when used in combination with standard chemotherapeutic agents);
[0356] Inhibition of LPA-induced tumor cell migration and invasion from chemotherapeutic agents.
[0357] In In Vivo Models:
[0358] Inhibition of metastasis and progression of orthotopic, intraperitoneal and subcutaneous human tumors implanted in nude mice;
[0359] Reduction of tumor-associated angiogenesis in subcutaneous SKOV3 xenograft models and in prostate DU145 cancer cells;
[0360] Neutralization of bFGF- and VEGF-induced angiogenesis in the murine Matrigel plug assay; and
[0361] Reduced choroidal neovascularization in a model of laser-induced injury of Bruch's membrane in the eye.
[0362] In fibrosis models, LT3000 reduced inflammation and fibrosis following bleomycin model of pulmonary fibrosis in mice, and was effective both prophylactically and interventionally in this well accepted model. In a diagnostic context, a noninvasive method for detecting fibrosis is a patient sample by correlating LPA levels with levels of one or more fibrogenic markers (e.g., cytokines or growth factors) is believed to be useful for monitoring fibrosis in the clinical setting. It has now been demonstrated that that mice with bleomycin lung injury demonstrated a decrease of IL-13 and TIMP-1 levels, as well as reduction in other relevant growth factors, after treatment with the anti-LPA antibody Lpathomab (LT3000) and consequent reduction in lung fibrosis.
[0363] These findings demonstrate a profound role for the bioactive lipid LPA in the extracellular matrix production and tissue remodeling following injury. Furthermore these studies identify LPA as a novel clinical target in treating fibrosis associated with a number of diseases and organ systems. Monoclonal antibodies to LPA are believed to have great clinical potential for treatment of fibrosis.
Example 8: Humanization of Lpathomab (LT3000)
[0364] Humanization of LT3000
[0365] The variable domains of the murine anti-LPA monoclonal antibody, LT3000 (Lpathomab) were humanized by grafting the murine CDRs into human framework regions (FR). Lefranc, M. P, (2003). Nucleic Acids Res, 31: 307-10; Martin, A. C. and J. M. Thornton, (1996) J Mol Biol, 1996. 263: 800-15; Morea, V., A. M. Lesk, and A. Tramontano (2000) Methods, 20: 267-79; Foote, J. and G. Winter, (1992) J Mol Biol, 224: 487-99; Chothia, C., et al., (1985). J Mol Biol, 186:651-63. Details of the humanization process are described in US Patent Application Publication 20090136483.
[0366] Suitable acceptor human FR sequences were selected from the IMGT and Kabat databases based on a homology to LT3000 using a sequence alignment and analysis program (SR v7.6). Lefranc, M. P. (2003) Nucl. Acids Res. 31:307-310; Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, NIH National Techn. Inform. Service, pp. 1-3242. Sequences with high identity at FR, vernier, canonical and VH-VL interface residues (VCI) were initially selected. From this subset, sequences with the most non-conservative VCI substitutions, unusual proline or cysteine residues and somatic mutations were excluded. AJ002773 was thus selected as the human framework on which to base the humanized version of LT3000 heavy chain variable domain and DQ187679 was thus selected as the human framework on which to base the humanized version of LT3000 light chain variable domain.
[0367] A three-dimensional (3D) model containing the humanized VL and VH sequences was constructed to identify FR residues juxtaposed to residues that form the CDRs. These FR residues potentially influence the CDR loop structure and the ability of the antibody to retain high affinity and specificity for the antigen. Based on this analysis, 6 residues in AJ002773 and 3 residues in DQ187679 were identified, deemed significantly different from LT3000, and considered for mutation back to the murine sequence.
[0368] Antibody Expression and Production in Mammalian Cells
[0369] The murine antibody genes were cloned from hybridomas. Synthetic genes containing the human framework sequences and the murine CDRs were assembled from synthetic oligonucleotides and cloned into pCR4Blunt-TOPO using blunt restriction sites. After sequencing and observing 100% sequence congruence, the heavy and light chains were cloned and expressed as a full length IgG1 chimeric antibody using the pConGamma vector for the heavy chain gene and pConKappa vector for the light chain gene (Lonza Biologics, Portsmouth N.H.). The expression cassette for each of these genes contained a promoter, a kozak sequence, and a terminator. These plasmids were transformed into E. coli (One Shot Top 10 chemically competent E. coli cells, Invitrogen, Cat No. C4040-10), grown in LB media and stocked in glycerol. Large scale plasmid DNA was prepared as described by the manufacturer (Qiagen, endotoxin-free MAXIPREP™ kit, Cat. No 12362). Plasmids were transfected into the human embryonic kidney cell line 293F using 293fectin and using 293F-FreeStyle Media for culture. The transfected cultures expressed approximately 2-12 mg/L of humanized antibody.
[0370] Antibody Purification
[0371] Monoclonal antibodies were purified from culture supernatants using protein A affinity chromatography. Aliquots containing 0.5 ml of ProSep-vA-Ultra resin (Millipore, Cat. No 115115827) were added to gravity-flow disposable columns (Pierce, Cat. No 29924) and equilibrated with 10-15 ml of binding buffer (Pierce, Cat. No 21001). Culture supernatants containing transiently expressed humanized antibody were diluted 1:1 with binding buffer and passed over the resin. The antibody retained on the column was washed with 15 ml of binding buffer, eluted with low pH elution buffer (Pierce, Cat. No 21004) and collected in 1 ml fractions containing 100 ul of binding buffer to neutralize the pH. Fractions with absorbance (280 nm)>0.1 were dialyzed overnight (Slide-A-Lyzer Cassettes, 3500 MWCO, Pierce, Cat. No 66382) against 1 liter of PBS buffer (Cellgro, Cat. No 021-030). The dialyzed samples were concentrated using centricon-YM50 (Amicon, Cat. No 4225) concentrators and filtered through 0.22 uM cellulose acetate membranes (Costar, Cat. No 8160). The purity of each preparation was accessed using SDS-PAGE.
[0372] SDS-PAGE Electrophoresis
[0373] Each antibody sample was diluted to 0.5 ug/ul using gel loading buffer with (reduced) or without (non-reduced) 2-mercaptoethanol (Sigma, Cat. No M-3148). The reduced samples were heated at 95° C. for 5 min while the non-reduced samples were incubated at room temperature. A 4-12% gradient gel (Invitrogen, Cat. No NP0322) was loaded with 2 ug of antibody per lane and ran at 170 volts for 1 hour at room temperature in 1× NuPAGE MOPS SDS running buffer (Invitrogen, Cat. No NP0001). After electrophoresis, the antibodies were fixed by soaking the gel in 50% methanol, 10% acetic acid for ˜10 min. The gel was then washed with 3×200 ml distilled water. Finally, the bands were visualized by staining the gel overnight in GelCode® Blue Stain (Pierce, Cat. No 2490) and destaining with water.
[0374] Quantitative ELISA
[0375] The antibody titer was determined using a quantitative ELISA. Goat-anti human IgG-Fc antibody (Bethyl A80-104A, 1 mg/ml) was diluted 1:100 in carbonate buffer (100 mM NaHCO.sub.3, 33.6 mM Na.sub.2CO.sub.3, pH 9.5). Plates were coated by incubating 100 ul/well of coating solution (thiolated LPA-BSA conjugate) at 37° C. for 1 hour. The plates were washed 4× with TBS-T (50 mM Tris, 0.14 M NaCl, 0.05% tween-20, pH 8.0) and blocked with 200 ul/well TBS/BSA (50 mM Tris, 0.14 M NaCl, 1% BSA, pH 8.0) for 1 hour at 37° C. Samples and standard were prepared on non-binding plates with enough volume to run in duplicate. The standard was prepared by diluting human reference serum (Bethyl RS10-110; 4 mg/ml) in TBS-T/BSA (50 mM Tris, 0.14 NaCl, 1% BSA, 0.05% Tween-20, pH 8.0) to the following concentrations: 500 ng/ml, 250 ng/ml, 125 ng/ml, 62.5 ng/ml, 31.25 ng/ml, 15.625 ng/ml, 7.8125 ng/ml, and 0.0 ng/ml. Samples were prepared by making appropriate dilutions in TBS-T/BSA, such that the optical density (OD) of the samples fell within the range of the standard; the most linear range being from 125 ng/ml 15.625 ng/ml. After washing the plates 4× with TBS-T, 100 ul of the standard/samples preparation was added to each well and incubated at 37° C. for 1 hour. Next the plates were washed 4× with TBS-T and incubated for 1 hour at 37° C. with 100 ul/well of HRP-goat anti-human IgG antibody (Bethyl A80-104P, 1 mg/ml) diluted 1:150,000 in TBS-T/BSA. The plates were washed 4× with TBS-T and developed using 100 ul/well of TMB substrate chilled to 4° C. After 7 minutes, the reaction was stopped with 1M H.sub.2SO.sub.4 (100 ul/well). The OD was measured at 450 nm, and the data was analyzed using Graphpad Prizm software. The standard curve was fit using a four parameter equation and used to calculate the human IgG content in the samples.
[0376] Direct Binding ELISA
[0377] The LPA-binding affinities of the humanized antibodies were determined using a direct binding ELISA assay. Microtiter ELISA plates (Costar) were coated overnight with 1.0 ug/ml thiolated C12:0 LPA conjugated to Imject malieimide activated bovine serum albumin (BSA) (Pierce Co.) diluted in 0.1 M carbonate buffer (pH 9.5) at 37° C. for 1 h. Plates were washed with PBS (137 mM NaCl, 2.68 mM KCl, 10.1 mM Na.sub.2HPO.sub.4, 1.76 mM KH.sub.2PO.sub.4; pH 7.4) and blocked with PBS/BSA/tween-20 for 1 hr at room temp or overnight at 4° C. For the primary incubation (1 hr at room temperature), a dilution series of the anti-LPA antibodies (0.4 ug/mL, 0.2 ug/mL, 0.1 ug/mL, 0.05 ug/mL, 0.0125 ug/mL, and 0 ug/mL) was added to the microplate (100 ml per well). Plates were washed and incubated with 100 ul per well of HRP conjugated goat anti-human (H+L) diluted 1:20,000 (Jackson, cat#109-035-003) for 1 hr at room temperature. After washing, the peroxidase was developed with tetramethylbenzidine substrate (Sigma, cat No T0440) and stopped by adding 1 M H.sub.2SO.sub.4. The optical density (OD) was measured at 450 nm using a Thermo Multiskan EX. The EC.sub.50 (half-maximal binding concentration) was determined by a least-squares fit of the dose-response curves with a four parameter equation using the Graphpad Prism software.
[0378] LPA Competition ELISA
[0379] The specificity of the humanized antibody was determined by competition ELISA. Thiolated C18:0 LPA-BSA conjugate coating material was diluted to 0.33 ug/ml with carbonate buffer (100 mM NaHCO3, 33.6 mM Na2CO3, pH 9.5). Plates were coated with 100 ul/well of coating solution and incubated at 37° C. for 1 hour. The plates were washed 4 times with PBS (100 mM Na2HPO4, 20 mM KH2PO4, 27 mM KCl, 1.37 mM NaCl, pH 7.4) and blocked with 150 ul/well of PBS, 1% BSA, 0.1% tween-20 for 1 h at room temperature. The humanized, anti-LPA antibodies were tested against lipid competitors (14:0 LPA (Avanti, Cat. No 857120), 18:1 LPA (Avanti, Cat. No 857130), 18:1 LPC (Avanti, Cat. No 845875), cLPA (Avanti, Cat. No 857328), 18:1 PA (Avanti, Cat. No 840875), PC (Avanti, Cat. No 850454) at 5 uM, 2.5 uM, 1.25 uM, 0.625 uM, and 0.0 uM. The antibody was diluted to 0.5 ug/ml in PBS, 0.1% tween-20 and combined with the lipid samples at a 1:3 ratio of antibody to sample on a non-binding plate. The plates were washed 4 times with PBS and incubated for 1 hour at room temperature with 100 ul/well of the primary antibody/lipid complex. Next the plates were washed 4 times with PBS and incubated for 1 h at room temperature with 100 ul/well of HRP-conjugated goat anti-human antibody diluted 1:20,000 in PBS, 1% BSA, 0.1% tween-20. Again the plates were washed 4 times with PBS and developed using TMB substrate (100 ul/well) at 4° C. After 8 minutes, the reaction was stopped with 100 ul/well of 1M H2SO4. The optical density (OD) was measured at 450 nm using a Thermo Multiskan EX. Raw data were transferred to GraphPad software for analysis.
[0380] Thermostability
[0381] The thermostability of the humanized antibodies were studied by measuring their LPA-binding affinity (EC50) after heating using the direct binding ELISA. Antibodies dissolved in PBS (Cellgo, Cat. No 021-040) were diluted to 25 ug/ml and incubated at 60° C., 65° C., 70° C., 75° C. and 80° C. for 10 min. Prior to increasing the temperature, 10 ul of each sample was removed and diluted with 90 ul of PBS and stored on ice. The samples were then vortexed briefly and the insoluble material was removed by centrifugation for 1 min at 13,000 rpm. The binding activity of the supernatant was determined using the direct LPA-binding ELISA and compared to a control, which consisted of the same sample without heat treatment.
[0382] Surface Plasmon Resonance
[0383] All binding data were collected on a ProteOn optical biosensor (BioRad, Hercules Calif.). 12:0 LPA-thiol and 18:0 LPA-thiol were coupled to a maleimide modified GLC sensor chip (Cat. No 176-5011). First, the GLC chip was activated with an equal mixture of sulfo-NHS/EDC for seven minutes followed by a 7 minute blocking step with ethyldiamine. Next sulfo-MBS (Pierce Co., cat #22312) was passed over the surfaces at a concentration of 0.5 mM in HBS running buffer (10 mM HEPES, 150 mM NaCl, 0.005% tween-20, pH 7.4). LPA-thiol was diluted into the HBS running buffer to a concentration of 10, 1 and 0.1 uM and injected for 7 minutes producing 3 different density LPA surfaces (˜100, ˜300 and ˜1400 RU). Next, binding data for the humanized antibodies was collected using a 3-fold dilution series starting with 25 nM as the highest concentration (original stocks were each diluted 1 to 100). Surfaces were regenerated with a 10 second pulse of 100 mM HCl. All data were collected at 25° C. Controls were processed using a reference surface as well as blank injections. The response data from each surface showed complex binding behavior which a likely caused by various degrees of multivalent binding. In order to extract estimates of the binding constants, data from the varying antibody concentrations were globally fit using 1-site and 2-site models. This produced estimates of the affinity for the bivalent (site 1) and monovalent site (site 2).
[0384] LPA Molar Binding Capacity
[0385] The molar ratio of LPA:mAb was determined using a displacement assay. Borosilicate tubes (Fisherbrand, Cat. No 14-961-26) were coated with 5 nanomoles of biotinylated LPA (50 ug of lipid (Echelon Bioscienes, Cat. No L-012B, Lot No F-66-136 were suspended in 705 ul of 1:1 chloroform:methanol yielding a 100 uM solution) using a dry nitrogen stream. The coated tubes were incubated with 75 ul (125 pmoles) of antibody dissolved in PBS (Cellgro, Cat. No 021-030) at room temperature. After 3 hours of incubation, the LPA:mAb complexes were separated from free lipid using protein desalting columns (Pierce, Cat, No 89849), and the molar concentration of bound biotinylated LPA was determined using the HABA/Avidin displacement assay (Pierce, Cat. No 28010) according to the manufacturer's instructions.
[0386] Engineering of the Humanized Variants
[0387] The murine anti-LPA antibody was humanized by grafting of the Kabat CDRs from LT3000 V.sub.H and V.sub.L into acceptor human frameworks. Seven humanized variants were transiently expressed in HEK 293 cells in serum-free conditions, purified and then characterized in a panel of assays. Plasmids containing sequences of each light chain and heavy chain were transfected into mammalian cells for production. After 5 days of culture, the mAb titer was determined using quantitative ELISA. All combinations of the heavy and light chains yielded between 2-12 ug of antibody per ml of cell culture.
[0388] Characterization of the Humanized Variants
[0389] All the humanized anti-LPA mAb variants exhibited binding affinity in the low picomolar range similar to the chimeric anti-LPA antibody (also known as LT3010) and the murine antibody LT3000. All of the humanized variants exhibited a T.sub.M similar to or higher than that of LT3000. With regard to specificity, the humanized variants demonstrated similar specificity profiles to that of LT3000. For example, LT3000 demonstrated no cross-reactivity to lysophosphatidyl choline (LPC), phosphatidic acid (PA), various isoforms of lysophosphatidic acid (14:0 and 18:1 LPA, cyclic phosphatidic acid (cPA), and phosphatidylcholine (PC).
[0390] Activity of the Humanized Variants
[0391] Five humanized variants were further assessed in in vitro cell assays. LPA is known to play an important role in eliciting the release of interleukin-8 (IL-8) from cancer cells. LT3000 reduced IL-8 release from ovarian cancer cells in a concentration-dependent manner. The humanized variants exhibited a similar reduction of IL-8 release compared to LT3000.
[0392] Two humanized variants were also tested for their effect on microvessel density (MVD) in a Matrigel tube formation assay for neovascularization. Both were shown to decrease MVD formation.
Example 9: Preliminary Animal Pharmacokinetics of Lpathomab
[0393] Preliminary PK studies were conducted with Lpathomab. For IV dosed groups, mice were injected with a single 30 mg/kg dose and sacrificed at time points up to 15 days. Antibody was also given via i.p. administration and animals were sacrificed during the first 24 hrs to compare levels of mAb in the blood over this period of time for different routes of delivery. Pharmacokinetic parameters were assessed by WinNonlin. Three mice were sacrificed at each time point and plasma samples were collected and analyzed for mAb levels by ELISA. The half-life of Lpathomab in mice was determined to be 102 hrs (4.25 days) by i.v. administration. Moreover, the antibody is fully distributed to the blood within 6-12 hrs when given i.p., suggesting that the i.p. administration is suitable for xenografts and other studies.
Example 10: Spinal Cord Injury and Immunohistochemical Staining of LPA Using Monoclonal Antibody to LPA
[0394] Immunohistochemical methods can be used to determine the presence and location of LPA in cells. Spinal cords (adult (3 months old) male C57BL/6 mice) from animals with and without spinal cord injury were immunostained 4 days after injury. Adult C57BL/6 mice (20-30 g) were anaesthetized with a mixture of ketamine and xylazine (100 mg/kg and 16 mg/kg, respectively) in phosphate buffered saline (PBS) injected intraperitoneally. The spinal cord was exposed at the low thoracic to high lumbar area, at level T12, corresponding to the level of the lumbar enlargement. Fine forceps were used to remove the spinous process and lamina of the vertebrae and a left hemisection was made at T12. A fine scalpel was used to cut the spinal cord, which was cut a second time to ensure that the lesion was complete, on the left side of the spinal cord, and the overlying muscle and skin were then sutured. This resulted in paralysis of the left hindlimb. After 2 or 4 days the animals were re-anaesthetized as above and then perfused with PBS through the left ventricle of the heart, followed by 4% paraformaldehyde (PFA). After perfusion, the spinal cords were gently removed using fine forceps and post-fixed for 1 hour in cold 4% PFA followed by paraffin embedding or cryo-preserving in 20% sucrose in PBS overnight at 40 C for frozen sections. Tissues for taken from n=3 uninjured mice and n=3 injured mice at 2 and 4 days post-injury. As described in Goldshmit Y, Galea M P, Wise G, Bartlett P F, Turnley A M: Axonal regeneration and lack of astrocytic gliosis in EphA4-deficient mice. J Neurosci 2004, 24(45):10064-10073.
[0395] IHC frozen spinal cord sagittal sections (10 μm) were examined using standard immunohistochemical procedures to determine expression and localization of the different LPA receptors. Frozen sections were postfixed for 10 min with 4% PFA and washed 3 times with PBS before blocking for 1 hour at room temperature (RT) in blocking solution containing 5% goat serum (Millipore) and 0.1% Triton-X in PBS in order to block non-specific antisera interactions. Primary antibodies used were B3 (0.1 mg/ml) rabbit anti-LPA1 (1:100, Cayman Chemical, USA), rabbit anti-LPA2 (1:100, Abcam, UK) and mouse anti-GFAP (1:500, Dako, Denmark). Primary antibodies were added in blocking solution and sections incubated over night at 40 C. They were then washed and incubated in secondary antibody for 1 hr at RT, followed by Dapi counterstain. Sections were coverslipped in Fluoromount (Dako) and examined using an Olympus BX60 microscope with a Zeiss Axiocam HRc digital camera and Zeiss Axiovision 3.1 software capture digital images. Some double labeled sections were also examined using a Biorad MRC1024 confocal scanning laser system installed on a Zeiss Axioplan 2 microscope. All images were collated and multi-colored panels produced using Adobe Photoshop 6.0.
[0396] After injury, non-neuronal glial cells in the CNS called astrocytes respond to many damage and disease states resulting in a “glial response”. Glial Fibrillary Acidic Protein (GFAP) antibodies are widely used to see the reactive astrocytes which form part of this response, since reactive astrocytes stain much more strongly with GFAP antibodies than normal astrocytes. LPA was revealed by immunohistochemistry using antibody B3 (0.1 mg/ml overnight). Fluorescence microscopy showed that reactive astrocytes are present in spinal cords 4 days after injury, and these cells stain positively for LPA. In contrast, uninjured (control) spinal cords have little to no staining for astrocytes or LPA. Thus LPA is present in reactive astrocytes of the spinal cord. In both injured and control animals, the central canal (hypothesized to be a stem cell niche) does not stain for LPA.
Example 11: Functional Recovery in Anti-LPA Antibody-Treated Mice Following Spinal Cord Injury (SCI)
[0397] Wildtype mice were given spinal cord hemisection injury as described in Example 10 above. Administration of anti-LPA antibody B3 for two weeks following SCI was found to result in significant functional recovery as determined by open field locomotor test (mBBB) and grid walking test (Goldshmit, et al. (2008), J. Neurotrauma 25(5): 449-465). mBBB is an assessment of hindlimb functional deficits, using a scale ranging from 0, indicating complete paralysis, to 14, indicating normal movement of the hindlimbs. Results are presented as mean+/−SEM.
Example 12: Antibody to LPA Improves Axonal Regeneration and Neuronal Survival Following Spinal Cord Injury (SCI)
[0398] In addition to the functional improvement described in the preceding examples following administration of B3 mAb to wildtype mice for 2 weeks following SCI, anti-LPA antibody treatment also resulted in axonal regeneration through the lesion site and a significant increase in traced neuronal cells that project their processes towards the brain. Tetramethylrhodamine dextran (TMRD) was used to label descending axons that reached the lesion site in isotype controls (n=6) compared to axons that managed to regenerate through the lesion site in B3-treated mice (n=7). Hematoxylin staining was used to reveal the lesion site. Labeled axons also belong to neuronal cells that accumulate label in their cells bodies upstream from the lesion site. Quantitation of number of labeled neuronal cells rostral to lesion site is significantly higher in B3 treated mice (
Example 13: Neuroprotective Effects of Anti-LPA Antibody Following SCI
[0399] Following SCI as described above, treatment with anti-LPA antibody B3 (0.5 mg/mouse, subcutaneous, twice weekly) for one or two weeks significantly reduces astrocytic gliosis and glial scar formation, as well as neuronal apoptosis. B3 treatment reduces GFAP expression (
Example 14: Anti-LPA Antibody in Murine Cortical Impact Model of Traumatic Brain Injury (TBI)—Preventive
[0400] The mouse is an ideal model organism for TBI studies because there is an accepted model of human TBI, the type I IFN system in the mouse is similar to that in human, and the ability to generate gene-targeted mice helps to clarify cause and effect rather than mere correlations. Adult mice were anaesthetised with a single ip injection of Ketamine/Xylazine and the scalp above the parietal bones shaved with clippers. Each scalp was disinfected with chlorhexideine solution and an incision made to expose the right parietal bone. A dentist's drill with a fine burr tip was then used to make a 3 mm diameter circular trench of thinned bone centred on the centre of the right parietal bone. Fine forceps were then used to twist and remove the 3 mm plate of parietal bone to expose the parietal cortex underneath. The plate of bone removed was placed into sterile saline and retained. The mouse was mounted in a stereotaxic head frame and the tip of the impactor (2 mm diameter) positioned in the centre of the burr hole at right angles to the surface of the cortex and lowered until it just touches the dura mater membrane covering the cortex. A single impact injury (1.5 mm depth) was applied using the computer controller. The mouse was removed from the head frame and the plate of bone replaced. Bone wax was applied around the edges of the plate to seal and hold the plate in position. The skin incision was then closed with fine silk sutures and the area sprayed with chlorhexidine solution. The mouse was then returned to a holding box underneath a heat lamp and allowed to regain consciousness (total time anaesthetised=30-40 minutes).
Treatments:
[0401] Treatments or isotype controls were injected at various time points. Anti-LPA antibody (B3 or other) was injected by tail-IV (0.5 mg). Following 24-48 hours, the animals were sacrificed and their brains analysed.
Analysis:
[0402] Neuronal death/survival (TUNEL analysis), reactive astrogliosis (revealed by Ki67 positive cells co-labelled with GFAP) and NS/PC responses (proliferation by CD133/Ki67, migration to the injury site by CD133 and differentiation) are analysed. The immune response is assessed by CD11b immunostaining. Quantification is performed by density measurement using ImageJ (NIH).
[0403] Results:
[0404] Data from this model show that anti-LPA antibody treatment (B3) administered before injury reduces the degree of hemorrhage normally seen in the mouse brain following TBI in this cortical impact model (
Example 15: Anti-LPA Antibodies in Murine Cortical Impact Model of Traumatic Brain Injury (TBI)
[0405] Based on the results of the study described in Example 14, a larger double-blinded prevention study using the same murine cortical impact model was undertaken. Mice were subjected to TBI using Controlled Cortical Impact (CCI) and treated with either isotype control monoclonal antibody or anti-LPA antibody B3 given as a single intravenous dose of 0.5 mg antibody (approx. 25 mg/kg) prior to injury. Mice were sacrificed 24 hours later, at which time the infarct size was photographed and its volume quantified.
Example 16: Anti-LPA Antibodies in Murine Cortical Impact Model of Traumatic Brain Injury (TBI)—Interventional Study #1
[0406] Based on the results of the studies described above, a larger double-blinded interventional treatment study was undertaken using the same clinically relevant murine cortical impact model. Mice (8 animals per group) were subjected to TBI using Controlled Cortical Impact (CCI) and treated with either isotype control monoclonal antibody or anti-LPA antibody B3 given as a single intravenous dose of 0.5 mg antibody (approx. 25 mg/kg) 30 minutes after surgery. Mice were sacrificed 48 hours later, at which time the infarct size was photographed and quantified histologically using image analysis.
Example 17: Anti-LPA Antibodies in Murine Cortical Impact Model of Traumatic Brain Injury (TBI)—Interventional Study #2
[0407] In this double-blinded study, mice (8 per group) were subjected to TBI and treated with an anti-LPA antibody as described in Example 16, but here the mice were sacrificed 7 days after injury. Infarct size was measured by MRI on day 1 and day 7 post-injury, and the results are shown in
Example 18: LPA in Patients' Cerebrospinal Fluid is a Marker for TBI
[0408] CSF samples from five TBI patients were obtained from the Neurotrauma Tissue and Fluid Bank, located at the National Trauma Research institute, The Alfred Hospital, Melbourne, Australia, which is part of the Australian Brain Bank Network. CSF was collected from five TBI patients for 5 consecutive days starting at 24 hours after injury (Day 1). Each sample has 2×10 μl and 2×100 μl aliquots=4 tubes per patient. 3× control samples provided, collected at the time of elective neurosurgery. Each control sample has 2×10 μl and 2×100 μl aliquots=4 tubes per subject. Sample information is in Table 9.
TABLE-US-00009 TABLE 9 CSF sample information Sample name Day 1 Day 2 Day 3 Day 4 Day 5 04 04_1 04_2 04_3 04_4 04_5 02 02_1 02_2 02_3 02_4 02_5 03 03_1.sup.# 03_2 03_3 03_4 03_5 01 01_1.sup.## 01_2 01_3 01_4 01_5 05 05_1 05_2 05_3 05_4 05_5 Control 1 Control 2 Control 3 .sup.#samples 03_1, 03_2, 03_3, 03_4, and 03_5 were slightly pale yellow in color .sup.##samples 01_1 were noticeably reddish-pink in color
[0409] Patient information is in Table 10. “Admission date” refers to hospital admission. “GCS” refers to Glasgow Coma Score. ISS refers to the Injury Severity Scale. GOSE refers to Extended Glasgow Outcome Scale. Under mechanism of injury, “MVA” refers to motor vehicle accident, “Ped” refers to pedestrian accident, “Pen” refers to penetrating injury.
TABLE-US-00010 TABLE 10 TBI patient information Patient Admission Oxygen Focal/ Mech of code Age Sex Date Saturation Diffuse injury GCS ISS GOSE 01 23 M 5 Mar. 2004 Non-hypoxic Focal Ped 7 33 4 02 19 M 12 Apr. 2004 Non-hypoxic Diffuse MVA 8 30 5 03 50 M 15 Apr. 2004 Hypoxic Diffuse MVA 5 41 4 04 33 M 26 May 2004 Non-hypoxic Focal Ped 4 38 1 05 50 M 4 Jul. 2005 Normoxic Pen 10 20 8 Control 1 42 M 27 May 2008 Non-Hypoxic Control 3 80 M 28 Nov. 2007 Non-Hypoxic Control 4 56 M 17 May 2005 Non-Hypoxic
[0410] The GCS is used to quantitate the severity of coma in a patient who has suffered traumatic brain injury. Mental alertness varies from fully alert to lethargic and stuporous all the way to deep coma, where a patient is minimally responsive or unresponsive to external stimuli. The GCS grades this level of consciousness on a scale from 3 (worst, deep coma) to 15 (normal, alert). A Coma Score of 13 or higher indicates a mild brain injury, 9 to 12 a moderate injury and 8 or less a severe brain injury.
[0411] The GOSE is a practical index of outcome or recovery following head injury designed to complement the Glasgow Coma Scale. The eight levels of recovery are: 1) Dead; 2) Vegetative State; 3) Lower Severe Disability; 4) Upper Severe Disability; 5) Lower Moderate Disability; 6) Upper Moderate Disability; 7) Lower Good Recovery; 8) Upper Good Recovery.
[0412] The ISS is an anatomical scoring system that provides an overall score for patients with multiple injuries. Each injury is assigned an Abbreviated Injury Scale (AIS) score (from 1 to 6, with 1 being minor, 5 severe and 6 an unsurvivable injury) and is allocated to one of six body regions (Head, Face, Chest, Abdomen, Extremities (including Pelvis), External). Only the highest AIS score in each body region is used. The 3 most severely injured body regions have their score squared and added together to produce the ISS score.
[0413] Levels of LPA (multiple lipid species) in CSF samples were measured by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) by Professor Andrew Morris at the University of Kentucky under contract with Lpath. The various LPA species in control and injured CSF were determined using published methods. Gellett et al. (2012) BBRC 422:758-763; Federico et al. (2012) Mol. Endocrinol. 26:786-797. While LPA has been detected in CSF (Sato et al., 2005), a detailed analysis by LC-MS identifying the key molecular species of LPA in CSF has not previously been achieved. Numerous LPA species [14:0, 16:1, 16:0, 18:3, 18:2, 18:1, 18:0, 20:4 and 22:6 acyl (ester-linked) LPA] were measured and all physiologically relevant species (16; 0, 18:2, 18:1, 18:0 and 20:4) were detected in the CSF.
[0414]
[0415]
[0416]
[0417] These preliminary data are the first evidence that LPA levels increase in CSF following neurotrauma. This novel observation that LPA is a biomarker for neurotrauma is the basis for the methods and kits described and claimed herein.
Example 19 LPA Metabolites in CSF from Patients with TBI
[0418] The LPA precursors lyso-PAF and LPC were measured in CSF of TBI patients at 1, 2, 3, 4 and 5 days after injury. Zhao Z, Yu M, Crabb D, Xu Y, Liangpunsakul S. Ethanol-induced alterations in fatty acid-related lipids in serum and tissues in mice. Alcohol Clin Exp Res 2010; 35:229-34. The results are shown in
Example 20 LPA Levels in CSF Samples from Additional Neurotrauma Patients
[0419] Further to Example 18, CSF samples from an additional eight TBI patients were obtained. Patient data are shown in Table 11, where available (“-” indicates data not available).
TABLE-US-00011 TABLE 11 TBI patient information - second cohort Focal (F) or Patient Mechanism Diffuse (D) Hypoxic (Hx) or Number Age Sex of injury GCS ISS GOSE injury Normoxic (Nx) 06 40 M fall/jump 7 21 6 D Nx 07 33 M motor bicycle accident 3 43 4 F&D Hx 08 33 M fall/jump 7 17 6 F Nx 09 21 F motor vehicle accident 7 21 3 D Nx 10 26 F fall/jump 3 45 3 F Hx 11 22 M penetrating injury 7 30 5 F Nx 12 35 M fall/jump 4 45 5 F Hx 13 25 M motor vehicle accident 4 41 — — Hx
[0420] CSF samples were collected over five days post-injury, but collection began earlier after injury than for the five patients in Example 18.
Example 21 Time Course of LPA Levels in CSF from Neurotrauma Patients
[0421] Total acyl-LPA levels were measured in CSF from eleven neurotrauma patients at times up to approximately six days after injury (of the data from the five patients described in Example 18 and the eight patients described in Example 20, data for two patients had to be omitted from the time course due to insufficient information as to time of CSF sampling). Measurements were made by liquid chromatography-mass spectrometry (LC-MS) as described in previous examples. LPA levels were graphed over time in hours after injury as a scatter plot shown in
[0422]
Example 22 LPA Isoforms in CSF Samples from 13 Neurotrauma Patients Vs. Controls
[0423]
Example 23 Correlation of LPA Measurement with Severity of Injury
[0424] Because the data above indicate that LPA levels are typically highest in the first 24 hours after neurotrauma, the disease severity scores for the eight patients described in Example 20 were graphed against the LPA levels in the CSF samples taken from these patients within 24 hours post-injury.
[0425]
[0426]
[0427] Unlike the GCS and GOSE scoring systems, the Injury Severity Scale(ISS) is an anatomical scoring system that provides an overall score for patients with multiple injuries (polytrauma). Each injury is assigned an Abbreviated Injury Scale (AIS) score (from 1 to 6, with 1 being minor, 5 severe, and 6 an unsurvivable injury) and is allocated to one of six body regions (Head, Face, Chest, Abdomen, Extremities (including Pelvis), External). Only the highest AIS score in each body region is used. The three most severely injured body regions have their scores squared and added together to produce the ISS score. Thus, in contrast to the GCS and GOSE, a low score on the ISS is the most favorable, and a high score is the most severe. As can be seen in
[0428] Thus, it can be seen that, in all three standard scoring methods for neurotrauma and polytrauma, higher levels of LPA in the CSF are correlated with increasing severity of injury, indicating that LPA serves both qualitatively and quantitatively as a biomarker for serious injury such as TBI.
[0429] All of the compositions and methods described and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit and scope of the invention as defined by the appended claims.
[0430] All patents, patent applications, and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the invention pertains. All patents, patent applications, and publications, including those to which priority or another benefit is claimed, are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.
[0431] The invention illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of”, and “consisting of” may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.