RU2791022C2 - Therapeutic agents and methods for the treatment of tlr2-mediated diseases and disorders - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology. The use of a polypeptide specifically binding to oxidized phospholipid (OxPL) in the treatment of Kawasaki disease, where the polypeptide inhibits the biological activity of OxPL, is disclosed. In this case, the polypeptide contains: a variable heavy chain domain (VH) containing complementarity-determining regions (CDR) of the VH amino acid sequence represented in SEQ ID NO: 2, and a variable light chain domain (VL) containing the CDR of the VL amino acid sequence represented in SEQ ID NO: 2 or 4.

EFFECT: invention makes it possible to use it for the effective treatment of Kawasaki disease in a patient.

15 cl, 22 dwg, 1 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] According to this application, in accordance with 35 U.S.C. §119 claims priority on U.S. Provisional Application No. 62/623276, filed January 29, 2018, the disclosure of which is incorporated herein by reference.

GOVERNMENT RIGHTS

[0002] The present invention was made with government support under Grant No. HL088093 awarded by the US National Institutes of Health. The government has certain rights to the present invention.

FIELD OF TECHNOLOGY

[003] The present invention relates to methods and treatment of TLR2-mediated diseases and disorders, including the introduction of an antibody, antibody fragment or polypeptide that binds and inhibits the biological activity of oxidized phospholipids.

INCLUDING A SEQUENCE LIST AS A REFERENCE

[0004] Filed with the application is a sequence listing named "Sequence-Listing_ST25.txt", created on January 29, 2019, and having a size of 34203 bytes and a machine-readable format for an IBM-PC compatible MS-Windows operating system. Thus, the sequence listing is incorporated herein by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

[0005] Phospholipids containing polyunsaturated fatty acids are very prone to modification by reactive oxygen species. Such phospholipids are susceptible to peroxidation to form oxidized phospholipids (OxPL), which induce cytotoxicity and apoptosis and play a significant role in inflammation. It has been shown that OxPL play a role in the transcription of interleukins, smooth muscle cell phenotype switching, and apoptotic mechanisms of modified phospholipids. Thus, peroxidation significantly alters the physicochemical properties of membrane lipid bilayers and thus induces signal transduction depending on the formation or recognition of membrane domains or molecular binding. Different types of OxPL can interact with specific binding sites and receptors resulting in the activation of individual signal transduction pathways.

[0006] Human coronary atherosclerosis is a chronic inflammatory disease resulting from lipid abnormalities. Pro-inflammatory oxidized low-density lipoprotein (OxLDL) is thought to be the link between lipid accumulation and vascular wall inflammation. Moreover, elevated levels of phospholipid oxidation products have been detected in various organs and pathological conditions, including atherosclerotic vessels, inflamed lungs, non-alcoholic steatohepatosis, plasma from coronary heart disease patients, as well as in apoptotic cells, virus-infected cells, and cells stimulated by inflammatory agonists. Two HDL-associated enzymes have been studied: serum paraoxonase (PON1) and PAF-acetylhydrolase (PAF-AH); both of which are responsible for the hydrolysis of oxidized plasma phospholipids, thus providing evidence for their role in atherosclerosis. Another important marker of oxidative stress is the binding of OxPL to the apolipoprotein B-100 (OxPL/apoB) LDL particle. Elevated levels of OxPL/apoB are associated with coronary heart disease, progression of carotid and femoral artery atherosclerosis, and prognosis of cardiovascular events.

SUMMARY OF THE INVENTION

[0007] The present invention relates to methods and treatment of TLR2-mediated diseases and disorders, including the introduction of an antibody, antibody fragment or polypeptide that binds and inhibits the biological activity of an oxidized phospholipid. As shown in the studies presented herein, neutralization of OxPL by endogenous in vivo expression of the EO6 antibody (using an EO6-scFv transgenic mouse) significantly inhibits the development of atherosclerosis induced by TLR2 agonism. Injections of the TLR2 agonist PAM3CSK4 into Ldlr ^-/- mice on a high cholesterol diet resulted in a significant increase in atherosclerosis. A similar set of injections of EO6-scFv-transgenic mice (with background Ldlr ^-/- ) resulted in a significant inhibition of the formation of lesions. Other studies presented herein show that OxPL neutralization may protect against disease progression in a TLR2-driven mouse model of Kawasaki disease. It has been shown that administration of the pathogen Lactobaccilus casei causes a Kawasaki disease-like disease in mice leading to increased atherosclerosis, coronary arteritis, and abdominal aortic aneurysms. It depends on TLR2, because administration of L. casei to mice deficient in TLR-2 does not result in disease. IL-1 expression is also strongly associated with Kawasaki disease. OxPL are also potent inducers of IL-1 release and inflammation. Injection of L. casei EO6 transgenic mice (with Ldlr ^-/- background) following an identical protocol resulted in a significant reduction not only in atherosclerosis, but also, importantly, in coronary artery arteritis, compared with injections in Ldlr ^-/- mice. The EO6 antibody does not bind directly to L. casei and thus likely neutralizes the inflammatory effects of OxPL caused by the inflammatory effects associated with TLR2-mediated agonism.

[0008] The development of coronary arteritis and later coronary artery aneurysms is a fatal complication in children with Kawasaki disease, occurring in an estimated 25% of children despite existing therapy, mainly intravenous immunoglobulin (IVIg), derived from mixed and purified human plasma, and aspirin, which is a general, non-specific anti-inflammatory therapeutic agent. Due to the ability of any anti-OxPL antibody to reduce inflammatory processes, including the reduction of IL-1B production, as well as their ability to provide protection in a TLR2-mediated mouse model of Kawasaki syndrome, injections of a humanized or human equivalent of an anti-OxPL antibody modified to increase its biological effectiveness may provide protection against TLR2-associated diseases, including Kawasaki disease. Because such anti-OxPL antibodies are present in the human B cell repertoire, a targeted recombinant therapeutic agent may provide clinical benefit in such a disease without the side effects of immunosuppression or plasma-derived therapeutics.

[0009] Thus, experimental evidence suggests that atherosclerosis and inflammatory arteritis caused by TLR2-mediated agonism in vivo in mice can be prevented by neutralizing OxPL. Of course, TLR2 agonism is associated with numerous bacterial diseases, but also with various so-called autoimmune-mediated diseases, such as systemic lupus erythematosus, rheumatoid arthritis, etc. Overall, the evidence suggests that through neutralization of OxPL using antibodies, fragments of antibodies or other binding domains against PC OxPL can improve or prevent the development of many diseases aggravated or worsened by activation of TLR2-mediated signaling pathways.

In some embodiments, the present invention relates to a method of treating an individual with a Toll-like receptor 2 (TLR2) mediated disease or disorder, comprising administering a therapeutically effective amount of an antibody, antibody fragment, or polypeptide that specifically binds to an oxidized phospholipid (OxPL), wherein the antibody, fragment the antibody or polypeptide inhibits the biological activity of OxPL. In an additional embodiment, the method further comprises administering to the individual an additional therapeutic agent that can be used to treat a TLR2-mediated disease or disorder. Non-limiting examples of TLR2-mediated diseases or disorders include Kawasaki disease, type 2 diabetes, rheumatoid arthritis, dermatological diseases, multiple sclerosis, systemic lupus erythematosus, ulcerative colitis, Graves' disease, Sjögren's syndrome, autoimmune thyroid disease, or vasculitis. In a particular embodiment, TLR2 -mediated disease or disorder is Kawasaki disease. In a further embodiment, the method further comprises administering intravenous immunoglobulin (IVIG) and/or salicylates to the individual. In a further embodiment, the subject is a human less than five years of age. In another embodiment, the biological activity of OxPL comprises activation of the CD36-TLR2 apoptosis pathway. In yet another embodiment, the antibody, antibody fragment or polypeptide is a single chain variable fragment (ScFv). In some embodiments, the antibody or antibody fragment recognizes and binds the phosphocholine end group of an oxidized phospholipid, wherein the antibody or antibody fragment comprises a heavy chain variable domain (V _H ) and/or a light chain variable domain (V _L ), and where (a) the V domain _H contains an amino acid sequence comprising one, two or three complementarity determining regions (CDRs) selected from the group consisting of: SEQ ID NO: 6 and a sequence that is at least 95%, 96%, 97%, 98%, 99% or 99.9% identical to SEQ ID NO: 6; SEQ ID NO: 7 and a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 99.9% identical to SEQ ID NO: 7; and SEQ ID NO: 8 and a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 99.9% identical to SEQ ID NO: 8; and (b) the V _L domain contains an amino acid sequence comprising one, two or three complementarity determining regions (CDRs) selected from the group consisting of: SEQ ID NO: 9 or 12 and a sequence that is at least 95% 96 %, 97%, 98%, 99% or 99.9% identical to SEQ ID NO: 9 or 12; SEQ ID NO: 10 and a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 99.9% identical to SEQ ID NO: 10; and SEQ ID NO: 11 and a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 99.9% identical to SEQ ID NO: 11. In a further embodiment, an antibody, antibody fragment, or polypeptide administered intravascularly. In a further embodiment, the V _H domain comprises an amino acid sequence comprising CDRs comprising SEQ ID NOs: 6, 7, and 8 and/or the V _L domain comprises an amino acid sequence comprising CDRs comprising SEQ ID NOs: 9, 10, and 11, or SEQ ID NOs: 10, 11 and 12. In another embodiment, the antibody or antibody fragment is selected from the group consisting of: (a) an antibody or scFv with heavy and light chain domains containing complementarity determining regions of SEQ ID NOs: 6, 7, 8, 9, 10 and 11; and (b) antibodies or scFvs with heavy and light chain domains comprising complementarity determining regions of SEQ ID NOs: 6, 7, 8, 10, 11, and 12. In another embodiment, the heavy and light chain domains are fused to an Fc or FC2 region. In another embodiment, the antibody fragment contains a single chain variable fragment ("scFv") that recognizes the phosphocholine end group of the oxidized phospholipid. In a specific embodiment, the scFv is physiologically soluble. You can use other OxPL-binding agents that inhibit the biological activity of OxPL (see, for example, international publication No. WO 2013/020995, incorporated herein by reference for all purposes).

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1A-B shows ( A ) a diagram of a method that can be used to generate a single chain variable fragment ("scFv"). As indicated, site-directed mutagenesis can be used to mutagenesis the heavy chain variable domain ("V _H ") of a double-chain immunoglobulin antibody to increase scFv solubility (left). The linker, leader and effector regions of the scFv are also indicated (right). ( B ) An overall diagram showing a blueprint of the genetic components encoding the scFv EO6 antibody fragment (top); and a general vector diagram showing the coding sequence for the EO6-scFv antibody fragment relative to other vector elements used to generate transgenic mice (bottom).

[0011] Figure 2 shows the nucleotide (SEQ ID NO: 1) and amino acid (SEQ ID NO: 2) sequences and scFv annotation.

[0012] Figure 3 shows diagrams of an in vivo mouse model for investigating the functions of scFvs of the invention in mice with pro-inflammatory conditions on a high cholesterol diet when the mice were exposed to the exogenous Toll-like receptor (TLR2) agonist Pam3CSK ₄ (PAM3).

[0013] Figures 4A-C show the effects on consumption and body weight of mice using the in vivo model shown in Figure 3 . (A) No significant difference was observed in food intake in Ldlr ^−/− mice compared to Ldlr ^−/− EO6scFv ^+/+ mice upon vehicle administration. (B) When mice were injected with Pam3, Ldlr ^-/- mice consumed less food compared to Ldlr ^-/- EO6scFv ^+/+ mice. (C) After 12 weeks, the body weight of Ldlr ^-/- EO6scFv ^+/+ mice was significantly higher than that of Ldlr ^-/- mice. At week 12, no significant difference in body weight was observed between Ldlr ^-/- EO6scFv ^+/+ mice and Ldlr ^-/- mice upon vehicle administration.

[0014] Figure 5A-B shows that no significant differences were observed in plasma (A) cholesterol or (B) triglycerides in EO6scTg Ldlr ^-/- (LDLrKO (knockout)) mice compared to Ldlr ^-/- mice.

[0015] Figure 6A-B shows that no significant difference was observed in (A) lipoprotein cholesterol profile or (B) plasma lipoprotein triglyceride profile in Ldlr ^-/- EO6scFv ^+/+ mice compared to Ldlr ^-/- mice, e.g. , lipoprotein levels were similar in both strains of mice.

[0016] Onfigure 7A-E shown to have observed less measurable atherosclerosis in miceLdlr ^-/- EO6scFv^+/+compared to miceLdlr ^-/- .(A)The degree of total aortic atherosclerosis was higher in miceLdlr ^-/- compared to miceLdlr ^-/- EO6scFv^+/+.(B)In particular, a higher degree of atherosclerosis was determined in the abdominal aorta (below the diaphragm) in mice.Ldlr ^-/- compared to miceLdlr ^-/- EO6scFv^+/+.(C) On figures (A) and (B) above show a two-dimensional analysis of the degree of atherosclerosis by planimetry. The exact mass of the excised and cleaned aortas is the best indicator of total atherosclerosis, because. it includes thickness measurement. Mouse aortic weightLdlr ^-/- was significantly higher than in mice.Ldlr ^-/- EO6scFv^+/+.(D)When controlling body weight, the ratio of the mass of the aorta and the body of miceLdlr ^-/- was significantly higher than the ratio of the mass of the aorta and the body of miceLdlr ^-/- EO6scFv^+/+.(E)The degree of atherosclerosis in the aortic root did not differ between the two groups.

[0017] Figures 8A-H show the results of quantitative PCR (qPCR) analysis of inflammatory gene expression in adipose tissue of Ldlr ^-/- EO6scFv ^+/+ mice compared to Ldlr ^-/- mice. In particular, expression of (A) IL-1b, (B) IL-6, (C) TNFα, (E) MCP1, (F) MIP1α, (G) MIP1β, and (H) IL-10 tended to be more low in Ldlr ^-/- EO6scFv ^+/+ mice compared to Ldlr ^-/- mice, while IL-12 expression was slightly higher ( D ).

[0018] Figures 9A-G show the results of an enzyme-linked immunosorbent assay (ELISA) to measure cytokine levels in adipose tissue extracts from Ldlr ^-/- EO6scFv ^+/+ mice compared to Ldlr ^-/- mice. In particular, measured levels of cytokines (A) IL-1b, (B) IL-6, (C) TNFα, (D) MCP1, (E) MIP1α, (F) MIP1β and (G) IL-10 reflected the results of the expression analysis genes shown in Fig.8 .

[0019] Figure 10A-H shows that bone marrow-derived Ldlr ^-/- EO6scFv ^+/+ cells, when differentiated into M1 or M2 macrophage cells and stimulated with PAM3, showed lower expression of (A) IL-1β, (B) IL -6, (C) IL-12, (D) TNFα, (E) MCP1, (F) MIP1α, (G) MIP1β, and (H) RANTES compared with differentiated Ldlr ^-/- mouse macrophages. Data shown is a comparison of M1 cells derived from Ldlr-/- EO6scFv ^+/+ or Ldlr ^-/- mice. Similar data were obtained in the case of M2 cells, for example, lower expression in M2 cells of Ldlr ^-/- EO6scFv ^+/+ compared to Ldlr ^-/- , except that the absolute levels of cytokine expression were lower.

[0020] Figure 11 shows sustained expression of EO6-scFv mRNA in various adipose tissue derived from Ldlr ^-/- EO6scFv ^+/+ mice. This is the result of infiltration by macrophages. Macrophages express the apoE promoter and thus express the EO6-scFv transgene.

[0021] Figure 12 shows fluorescence microscopy images showing that PAM3 treatment of macrophages (RAW264.7) induced OxPL production. Because macrophages of mice Ldlr ^-/- EO6scFv ^+/+ responded less to TLR2 stimulation, and since macrophages express and secrete EO6-scFv into culture suggest that TLR2-stimulated macrophages produce OxPL and that this in turn activates inflammatory gene expression in an autocrine manner. To test this hypothesis, macrophages were stimulated with PAM3: left panel, vehicle treatment; right panel, treatment with TLR2 agonist PAM3. RAW264.7 cultures were incubated with Pam3 (1 μg/ml) or vehicle control for 18 hours and surface stained for OxPL with EO6 IgM antibody and goat anti-msIgM conjugate with FITC. This indicates that TLR2-stimulated macrophages produced OxPL.

[0022] Onfigure 13A-Fshown to have observed less measurable atherosclerosis in miceLdlr ^-/- EO6scFv^+/+ RAG1 ^-/- compared to miceLdlr ^-/- RAG1 ^-/- . Because RAG1 KO mice do not have B or T cells, all atherosclerotic events associated with immune cells are directly attributed to the action of macrophages. Notably, the absolute rate of atherosclerosis in miceLdlr ^-/- RAG1^-/- was reduced by half compared to miceLdlr ^-/- .(A)Abdominal aorta isolated from miceLdlr ^-/- RAG1 ^-/- (left) and miceLdlr ^-/- EO6scFv^+/+ RAG1 ^-/- (on right). Abdominal aortas of miceLdlr ^-/- EO6scFv^+/+ RAG1 ^-/- apparently had less atherosclerotic lesions than miceLdlr-/-RAG1 ^-/- .(B)The size of lesions in the aortic sinus was significantly less in miceLdlr ^-/- EO6scFv^+/+ RAG1 ^-/- compared to miceLdlr ^-/- RAG1 ^-/- .(C) Mice Ldlr ^-/- RAG1^-/- had a higher percentage of total body damage compared to miceLdlr ^-/- EO6scFv^+/+ RAG1 ^-/- .(D) Mice Ldlr ^-/- RAG1 ^-/- had a higher percentage of abdominal aortic lesions compared to miceLdlr ^-/- EO6scFv^+/+ RAG1 ^-/- .(E)Mouse aortic weightLdlr ^-/- RAG1 ^-/- was significantly higher than in mice.Ldlr ^-/- EO6scFv^+/+ RAG1 ^-/- .(F) When controlling body weight, the ratio of the mass of the aorta and the body of miceLdlr ^-/- RAG1 ^-/- was significantly higher than the ratio of the mass of the aorta and the body of miceLdlr ^-/- EO6scFv^+/+ RAG1 ^-/- . Because RAG1 KO mice lack B- and T-cells, the main type of immune cells contributing to the development of atherosclerosis in these mice is macrophage. Thus, these data suggest that the main mechanism by which macrophages contribute to the development of atherosclerosis in this The TLR2-induced model is the answer to OxPL.

[0023] Figure 14 shows images of children with Kawasaki disease and, in addition, aneurysms of the coronary and abdominal arteries associated with Kawasaki disease.

[0024] Figure 15 shows a mouse model for investigating the effect of LCWE and HFC nutrition on atherosclerosis in Ldlr ^-/- mice, Ldlr ^-/- EO6Tg mice, Ldlr ^-/- IK17Tg mice, TLR2 ^-/- Ldlr ^-/- mice.

[0025] Figure 16 shows analysis of full face images of aortic lesions in mice fed HFC for 12 weeks and TLR2 activated by LCWE. EO6scFv-Tg significantly reduced anterior aortic lesions.

[0026] Figure 17 shows an area analysis of abdominal aortic lesions in various Ldlr ^−/− mice injected with LCWE and fed with HFC for 12 weeks. Data are expressed as percentages of atherosclerotic lesion measured in the abdominal aorta by Sudan IV staining. EO6Tg and IK17Tg were found to have a statistically significant protective effect. TLR2 was also found to be statistically significantly reduced compared to Ldlr ^-/- mice.

[0027] Figure 18 shows transverse sections of the aortic roots of Ldlr ^−/− mice and EO6-Tg Ldlr ^−/− mice treated with LCWE. EO6scFv-Tg reduced aortic root injury, necrotic nucleus size and, most importantly in relation to the manifestation of Kawasaki disease, coronary arteritis. The arrows indicate the coronary arteries in cross section. Extensive arteritis (large cell mass) was present in Ldlr ^−/− mice but absent in EO6-Tg Ldlr ^−/− mice.

[0028] Figure 19 shows additional transverse sections of the aortic roots of Ldlr ^−/− mice and EO6-Tg Ldlr ^−/− mice treated with LCWE. The arrows indicate the coronary arteries in cross section. Extensive arteritis (large cell mass) was present in Ldlr ^−/− mice but absent in EO6-Tg Ldlr ^−/− mice.

[0029] Figure 20 shows transverse sections of aortic roots from Ldlr ^-/- TLR2 ^-/- and IK17-Tg ^+/+ Ldlr ^-/- mice treated with LCWE. The arrows indicate the coronary arteries in cross section. Both EO6scFv and IK17scFv reduced coronary arteritis. EO6 (anti-OxPL antibody) but not IK17 (anti-MDA antibody) reduced aortic root damage.

[0030] Figure 21 shows additional transverse sections of the aortic roots of Ldlr ^-/- TLR2 ^-/- and IK17-Tg ^+/+ Ldlr ^-/- mice treated with LCWE. The arrows indicate the coronary arteries in cross section. The absence of coronary arteritis in Ldlr ^-/- TLR2 ^-/- mice indicates the importance of TLR2 activation in a mouse model of Kawasaki disease.

[0031] Figure 22 shows plasma levels of inflammatory cytokines in control LDLr ^−/− mice and EO6-Tg LDLr ^−/− mice treated with LCWE and fed with HFC for 12 weeks. Plasma cytokines were measured simultaneously by multiplex cytokine assays using the Bio-Plex Pro mouse cytokine assay kit (Bio-Rad Laboratories, USA). A significant decrease (p<0.04) in serum levels of TNFα, RANTES, MCP-1, CXCL1, IL-6 and IL-12 proteins was observed in EO6scFv-Tg LDLr ^-/- mice compared to control mice.

DETAILED DESCRIPTION

[0032] Within the scope of the invention and the claims, terms in the singular include reference to the plural unless the context clearly indicates otherwise. Thus, for example, reference to "single chain variable fragment" or "scFv" includes a plurality of single chain variable fragments, and reference to "oxidized phospholipid" includes reference to one or more oxidized phospholipids and their equivalents known to those skilled in the art, etc. d.

[0033] Unless otherwise indicated, all technical and scientific terms used in the present description have the meaning generally understood by a person skilled in the art to which the invention belongs. While any methods and reagents similar or equivalent to those described herein may be used in the practice of the described methods and compositions, examples of methods and materials are described below.

[0034] All publications mentioned in the present description are incorporated herein by reference in their entirety for the purpose of describing the methodology presented in the publications, which can be used in conjunction with the present description. In addition, with respect to any term presented in one or more publications that is similar or identical to the term specifically defined in the present description, the definition of the term specifically presented in the present description will take precedence in all senses.

[0035] In addition, the use of the union "and" means "and/or", unless otherwise indicated. Likewise, the terms "comprise", "comprises", "comprising", "include", "includes" and "comprising" are used interchangeably and are not limiting.

[0036] If the term "comprising" is used in the description of various embodiments, those skilled in the art should also understand that, in some specific cases, an embodiment may be described alternatively using the terms "consisting essentially of" or "consisting of".

[0037] The terms "antibody" and "immunoglobulin" are used interchangeably in the broadest sense and include monoclonal antibodies (e.g., full length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies, provided that they exhibit the desired biological activity) and may also include some antibody fragments. The antibody may be human, humanized and/or affinity matured.

[0038] Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and some of them can be further divided into subclasses (isotypes), for example, IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ and IgA ₂ . The heavy chain constant domains corresponding to different classes of immunoglobulins are named α, δ, ε, γ and μ, respectively. The structures and three-dimensional configurations of subunits of different classes of immunoglobulins are well known.

[0039] "Antibody fragments" comprise only a portion of an intact antibody, where the portion typically retains at least one, but generally most or all of the functions normally associated with that portion when present in the intact antibody. Examples of antibody fragments include Fab-, Fab'-, F(ab')₂- and Fv-fragments; diabody; linear antibodies; single chain antibody molecules; and multispecific antibodies formed from antibody fragments. In one embodiment, the antibody fragment contains the antigen-binding site of an intact antibody and thus retains the ability to bind to an antigen. In another embodiment, an antibody fragment, e.g., a fragment containing an Fc region, retains at least one of the biological functions normally associated with an Fc region when present in an intact antibody, such as FcRn binding, antibody half-life modulation, ADCC function and antibody binding. In one embodiment, the antibody fragment is a monovalent antibody having a half-lifein vivo, essentially similar to that of the intact antibody. For example, such an antibody fragment may contain an antigen-binding arm linked to an Fc sequence capable of conferring stabilityin vivo. However, it should be understood that a long half-life of an antibody is not necessary in some cases (eg, in the treatment of acute ischemia/reperfusion injury).

[0040] An "antigen" is a predefined antigen to which an antibody can selectively bind. The target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other natural or synthetic compound. In one embodiment of the present invention, the antigen is OxPL.

[0041] The term "anti-OxPL antibody" or "antibody that binds to OxPL" refers to an antibody that can bind to OxPL with sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic agent to target OxPL. In some embodiments, an anti-OxPL antibody has the same or similar binding specificity and K _d as an EO6 antibody or a QX5 antibody. In yet another embodiment, the anti-OxPL antibody binds to the PC end group of OxPL.

[0042] A "blocking" antibody or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. Some blocking antibodies or antagonistic antibodies substantially or completely inhibit the biological activity of an antigen.

[0043] The term "binding affinity" generally refers to the strength of the total amount of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise indicated, within the meaning of the invention, the term "binding affinity" refers to a characteristic binding affinity, reflecting a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (K _d ). Affinity can be measured by methods known in the art, including those described herein. Low affinity antibodies tend to bind antigen slowly and tend to dissociate quickly, while high affinity antibodies tend to bind antigen faster and tend to stay bound longer. Many methods are known in the art for measuring binding affinity, any of which can be used for the purposes of the present invention.

[0044] The term "biological sample" includes various types of samples obtained from an individual, and they can be used in diagnostic analysis or monitoring. The definition includes blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures or cells derived therefrom, and their progeny. The definition also includes samples processed in any way after they have been obtained, for example, by treatment with reagents, solubilization or enrichment with certain components, such as proteins or polynucleotides, or immersion in a semi-solid or solid matrix to obtain sections. The term "biological sample" includes a clinical sample, as well as cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluid, and tissue samples. The source of the biological sample may be solid tissue in the form of a fresh, frozen and/or preserved organ or tissue sample, or a biopsy or aspirate; blood or any blood component; physiological fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid; cells from any stage of embryonic or individual development. In some embodiments, the biological sample is obtained from a primary or metastatic tumor. The biological sample may contain compounds not naturally mixed with tissue, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.

[0045] The term "diabodies" refers to small antibody fragments with two antigen-binding sites, where the fragments contain a heavy chain variable domain (V _H ) connected to a light chain variable domain (V _L ) in one polypeptide chain (V _H -V _L ) . Using a linker too short to pair two domains on the same strand, the domains are forced to pair with complementary domains on the other strand and two antigen-binding sites are obtained. Diabodies are described in more detail, for example, in EP 404,097; WO 93/11161; and Hollinger et al. , Proc. Natl. Acad. sci. USA 90:6444-6448 (1993). Triatebodies and tetrabodies are also described in Hudson et al. , Nat. Med. 9:129-134 (2003).

[0046] A "disorder" or "disease" is any condition that would benefit from treatment with a substance/molecule or method of the present invention. This term includes a TLR2-mediated disease or disorder such as Kawasaki disease.

[0047] The term "effective amount" refers to an amount, in the required doses and for the necessary periods of time, effective to achieve the desired therapeutic or prophylactic result.

[0048] As used herein, the term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain, including a native sequence Fc region and a variant Fc region.

[0049] A "functional Fc region" has the effector function of a native sequence Fc region. Such effector functions typically require a combination of an Fc region and binding to a domain (eg, an antibody variable domain) and can be assessed using various assays, as described, for example, in the definitions herein.

[0050] A "native Fc region sequence" contains an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native human Fc region sequences include a native human IgG1 Fc region sequence (non-A and A allotypes), a native human IgG2 Fc region sequence, a native human IgG3 Fc region sequence, and a native human IgG4 Fc region sequence, as well as their natural options.

[0051] The term "Fc receptor" or "FcR" means a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native human FcR. In other embodiments, the FcR is an IgG antibody-binding receptor (gamma receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ mainly in their cytoplasmic domains. The FcγRIIA activating receptor contains an immunoreceptor tyrosine activating motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine inhibitory motif (ITIM) in its cytoplasmic domain (see, for example, Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). An overview of FcR is provided, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al. , Immunomethods 4:25-34 (1994); and de Haas et al. , J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including FcRs that will be identified in the future, are included in the term "FcR" in the present description.

[0052] The Fc receptor also includes the neonatal FcRn receptor responsible for the transport of maternal IgG to the fetus (Guyer et al. , J. Immunol. 117:587 (1976) and Kim et al. , J. Immunol. 24:249 (1994 )) and regulation of homeostasis by immunoglobulins. Methods for measuring binding to FcRn are known (see, for example, Ghetie and Ward., Immunol. Today 18(12):592-598 (1997); Ghetie et al. , Nature Biotechnology , 15(7):637-640 (1997 ); Hinton et al. , J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004/92219 (Hinton et al. ).

[0053] In vivo human FcRn binding and serum half-life of high-affinity human FcRn-binding polypeptides can be analyzed, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered polypeptides with a variant Fc region. WO 2000/42072 (Presta) describes antibody variants with improved or reduced FcR binding. See also, for example, Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).

[0054] "Fv" is the minimum antibody fragment containing a complete antigen recognition and antigen binding site. In double-chain Fv species, this region consists of a dimer of tightly, non-covalently linked one heavy chain variable domain and one light chain variable domain. In single chain Fv species, one heavy chain variable domain and one light chain variable domain can be covalently linked via a flexible peptide linker such that light and heavy chains can be linked in a "dimeric" structure similar to that of double chain Fv species. They are in such a configuration that the three HVRs of each variable domain interact to form an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six HVRs confer antigen-binding specificity on the antibody. However, even a single variable domain (or half of the Fv containing only three antigen-specific HVRs) has the ability to recognize and bind an antigen, albeit at a lower affinity than the entire binding site.

[0055] The Fab fragment also contains a light chain constant domain and a heavy chain first constant domain (CH1). Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy-terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. As used herein, "Fab'-SH" is the designation for a Fab' in which the cysteine residues of the constant domains contain a thiol group. F(ab') ₂ antibody fragments were initially generated as pairs of Fab' fragments containing hinge cysteines to each other. Other chemical compounds of antibody fragments are also known.

[0056] Cleavage of antibodies with papain results in two identical antigen-binding fragments, called "Fab" fragments, each containing one antigen-binding site, and a residual "Fc" fragment, the name of which reflects its ability to easily crystallize. Treatment with pepsin results in an F(ab') ₂ fragment containing two antigen-binding sites and still able to cross-link the antigen.

[0057] "Framework" residues or "FR" are variable domain residues other than hypervariable region residues as defined herein.

[0058] "Humanized" forms of non-human (eg, murine) antibodies are chimeric antibodies containing a minimal sequence derived from a non-human immunoglobulin. In one embodiment, the humanized antibody is a human immunoglobulin (recipient antibody) in which HVR residues of the recipient are replaced with residues from a non-human HVR species (donor antibody) such as a mouse, rat, rabbit or non-human primate having the desired specificity, affinity and/or capacity. In some cases, human immunoglobulin framework residues are replaced with corresponding non-human residues. In addition, humanized antibodies may contain residues not found in the recipient antibody or donor antibody. These modifications can be made to further improve the properties of the antibody. In general, a humanized antibody will contain substantially all of at least one, and typically two, variable domains in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or essentially, all of the FRs are human immunoglobulin FR sequences. The humanized antibody will optionally also contain at least a portion of an immunoglobulin constant region (Fc), typically from a human immunoglobulin. For more details, see, for example, Jones et al. , Nature 321:522-525 (1986); Riechmann et al. , Nature 332:323-329 (1988); and Presta, Curr. Op.Struct. Biol. 2:593-596 (1992). See also, for example, Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. soc. Transactions 23:1035-1038 (1995); Hurle and Gross, C urr. Op.Biotech , 5:428-433 (1994); and U.S. Patent Nos. 6,982,321 and 7,087,409.

[0059] A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and/or obtained by any of the methods for producing human antibodies described herein. This definition of a human antibody specifically excludes a humanized antibody containing non-human antigen-binding residues. Human antibodies can be obtained by a variety of methods known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol. , 227:381 (1991); Marks et al. , J. Mol. Biol. , 222:581 (1991). Also available for the preparation of human monoclonal antibodies are the methods described in Cole et al. , Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al. , J. Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol. , 5:368-74 (2001). Human antibodies can be generated by administering an antigen to a transgenic animal modified to produce such antibodies in response to antigen challenge, but whose endogenous loci are inactivated, such as immunized xeno mice (see, for example, US Pat. Nos. 6,075,181 and 6,150,584). See also, for example, Li et al. , Proc. Natl. Acad. sci. USA, 103:3557-3562 (2006) concerning human antibodies produced by human B-cell hybridoma technology. It should be noted that the term "human antibody" does not include naturally occurring human-produced antibodies, rather it refers to antibodies that do not contain any epitope or antigenic fragment that a human would not recognize as "foreign".

[0060] "Human effector cells" are leukocytes that express one or more FcRs and perform effector functions. In some embodiments, the cells express at least FcγRIII and carry out ADCC effector functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK), monocytes, cytotoxic T cells, and neutrophils. Effector cells can be isolated from a native source such as blood.

[0061] The term "hypervariable region", "HVR" or "HV" as used herein refers to regions of an antibody variable domain that are hypervariable in sequence and/or form structurally defined loops. Typically, antibodies contain six HVRs: three in the V _H domain (H1, H2, H3) and three in the V _L domain (L1, L2, L3). In native antibodies, H3 and L3 show the highest diversity among the six HVRs, and in particular, H3 is considered to play a unique role in conferring precise specificity on antibodies. See, for example, Xu et al. , Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003). In fact, Camelid's natural heavy chain antibodies are functional and stable in the absence of a light chain. See, for example, Hamers-Casterman et al. , Nature 363:446-448 (1993); Sheriff et al. , Nature Struct. Biol. 3:733-736 (1996).

[0062] "Individual" or "patient" is a vertebrate. In some embodiments, the implementation of the vertebrate is a mammal. Mammals include, but are not limited to, farm animals (such as cows), sport animals, pets (such as cats, dogs, and horses), primates, mice, and rats. In some embodiments, the mammal is a human.

[0063] An "isolated" antibody or antibody fragment is an antibody or antibody fragment that has been identified and separated from a component of its natural environment. Contaminant components of the natural environment are materials that interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody will be purified (1) to greater than 95% by weight of the antibody as determined by the Lowry method, and typically greater than 99% by weight, (2) to an extent sufficient to produce at least 15 residues N-terminal or internal amino acid sequence using a rotating beaker sequencer, or (3) to homogeneity by electrophoresis in SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver staining. An isolated antibody includes the antibody in situ in recombinant cells, since at least one component of the antibody's natural environment will be absent. Typically, however, isolated antibody will be obtained by at least one purification step.

[0064] An "isolated" nucleic acid molecule is a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule with which it is typically associated in the natural source of an antibody nucleic acid. An isolated nucleic acid molecule is in a form or condition other than those found in nature. Thus, the isolated nucleic acid molecules are different from the nucleic acid molecule that exists in natural cells. However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells typically expressing the antibody, where, for example, the nucleic acid molecule is located in a different chromosomal location than in natural cells.

[0065] As used herein, the term "label" refers to a compound or composition conjugated or fused directly or indirectly to a reagent, such as a nucleic acid probe or antibody, and facilitating detection of the reagent to which it is conjugated or fused. The label itself may be detectable (eg, radioactive labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze a detectable chemical change in the substrate compound or composition.

[0066] "Light chains" of antibodies (immunoglobulins) of any vertebrate species can be assigned to one of two distinct types, named kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0067] As used herein, the term "monoclonal antibody" refers to an antibody derived from a population of substantially homogeneous antibodies, i. the individual antibodies that make up a population are identical except for possible mutations, such as naturally occurring mutations, which may be present in minor amounts. Thus, the term "monoclonal" refers to the nature of an antibody as not being a mixture of different antibodies. In some embodiments, such a monoclonal antibody typically comprises an antibody comprising a target-binding polypeptide sequence, wherein the target-binding polypeptide sequence is obtained by a method comprising selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection method may be the selection of a unique clone from a plurality of clones, such as a collection of hybridoma clones, phage clones, or recombinant DNA clones. It should be understood that the selected target-binding sequence can be further modified, for example, to improve the affinity for the target, to humanize the target-binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo , to obtain a multispecific antibody, etc. , and that an antibody containing an altered target-binding sequence is also a monoclonal antibody for the purposes of the present invention. Unlike polyclonal antibody preparations, which typically include different antibodies against different determinants (epitopes), each monoclonal antibody from a monoclonal antibody preparation is directed against the same determinant on the antigen. In addition to their specificity, monoclonal antibody preparations are preferred because they are usually not contaminated with other immunoglobulins.

[0068] The term "monoclonal" is indicative of the nature of an antibody as derived from a substantially homogeneous population of antibodies, and should not be construed as requiring the production of an antibody by any particular method. For example, monoclonal antibodies for use in the present invention can be obtained by various methods, including, for example, the hybridoma method (for example, Kohler and Milstein, Nature , 256:495-97 (1975); Hongo et al. , Hybridoma, 14 (3): 253-260 (1995), Harlow et al. , Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al. , in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier , NY, 1981)), recombinant DNA methods (see, for example, US Pat. No. 4,816,567), phage display technologies (see, for example, Clackson et al . , J. Mol. Biol 222: 581-597 (1992); Sidhu et al. , J. Mol. Biol. 338(2): 299-310 (2004); Lee et al. , J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al. , J. Immunol. Methods 284(1 -2): 119-132 (2004) and technologies for producing human or similar antibodies in animals containing parts or all from human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, for example, WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jacobovits et al. , Proc. Natl. Acad. sci. USA 90: 2551 (1993); Jacobovits et al. , Nature 362: 255-258 (1993); Bruggemann et al. , Year in Immunol . 7:33 (1993); US patents No. 5545807; 5545806; 5569825; 5625126; 5633425 and 5661016; Marks et al. , Bio/Technology 10: 779-783 (1992); Longberg et al. , Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al. , Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

[0069] Monoclonal antibodies as used herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from particular species or belonging to a particular class or subclass of antibodies, while the rest of the chains are identical or homologous to the corresponding sequences in antibodies derived from other biological species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies, provided that they exhibit the desired biological activity (see, for example, US patent No. 4816567 and Morrison et al. , Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies include antibodies in which the antigen-binding region of the antibody is derived from an antibody obtained, for example, by immunizing monkeys with an antigen of interest.

[0070] As used herein, the terms "polynucleotide" or "nucleic acid" refer to polymers of nucleotides of any length, and include DNA and RNA. Nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or analogs thereof, which may be incorporated into a polymer by the action of a DNA or RNA polymerase, or by a synthetic reaction. The polynucleotide may contain modified nucleotides such as methylated nucleotides and their analogs. If present, the modification to the nucleotide structure can be made before or after polymer assembly. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide can be further modified after synthesis, for example by conjugation with a label. Other types of modifications include, for example, "capping", replacement of one or more naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, modifications with uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged bonds (for example, phosphothioates, phosphodithioates, etc.), modifications that include "suspended" molecules, such as, for example, proteins (for example, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc. ), modifications with intercalators (e.g. acridine, psoralen, etc.), modifications including chelators (e.g., metals, radioactive metals, boron, oxidizing metals, etc.), modifications including alkylators, modifications with modified bonds (for example, alpha-anomeric nucleic acids, etc.), as well as unmodified forms of polynucleotides. In addition, any of the hydroxyl groups typically present in sugars may be replaced, for example, with phosphonate groups, phosphate groups protected by standard protecting groups or activated to provide further bonds to additional nucleotides, or may be conjugated to solid or semi-solid supports. The 5' and 3' OH can be phosphorylated or replaced with amines or organic capping groups of 1-20 carbons. Other hydroxyls can also be derivatized to standard protecting groups. Polynucleotides can also contain similar forms of ribose or deoxyribose sugars generally known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl-, 2 '-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, alpha-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and basic nucleoside analogs such as methyl riboside . One or more phosphodiester bonds can be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments where phosphate is replaced by P(O)S("thioatom"), P(S)S("dithioatom"),"(O)NR ₂ ("amidate") , P(O)R, P(O)OR', CO or CH ₂ ("formacetal"), in which each R or R' independently represents H or substituted or unsubstituted alkyl (1-20 C), optionally containing an ester bond (--O--), aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl.Not all linkages in a polynucleotide need be identical.The foregoing description applies to all polynucleotides described herein, including RNA and DNA.

[0071] "Single chain Fv" or "scFv" antibody fragments contain the V _H and V _L domains of the antibody, where these domains are in the same polypeptide chain. Typically, the scFv polypeptide further comprises a polypeptide linker between the V _H and V _L domains to allow the scFv to form the desired structure for antigen binding. Figure 1 shows the structure of the antibody and scFv. For a review of scFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0072] In the context of the invention, the term "substantially similar" or "substantially the same" means a sufficiently high degree of similarity between two numerical values (for example, one of which is associated with an antibody of the present invention, and the other is associated with a reference/comparator antibody) so that one of skill in the art will appreciate that the differences between the two values are small or have no biological and/or statistical significance in the context of the biological characteristic measured by the values (eg, K _d values). The difference between the two values is, for example, less than about 50%, less than about 40%, less than about 30%, less than about 20%, and/or less than about 10% as a function of the reference/comparator value.

[0073] As used herein, the phrase "significantly reduced" or "significantly different" means a significantly high degree of difference between two numerical values (typically one associated with a molecule and the other associated with a reference/comparator molecule) such that one skilled in the art will appreciate that the difference between the two values is statistically significant in the context of the biological characteristic measured by the indicated values (eg, K _d values). The difference between the two values is, for example, more than about 10%, more than about 20%, more than about 30%, more than about 40% and/or more than about 50% as a function of the value for the reference/comparator molecule.

[0074] "TLR2-related disease and disorders" includes, but is not limited to, autoimmune diseases including rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, Sjögren's syndrome, psoriasis, multiple sclerosis, and autoimmune diabetes. TLR-related conditions (e.g., directly and/or indirectly associated with TLRs, such as TLR2, etc.) may include any one or more of: diabetes, obesity, sepsis, inflammatory disease (e.g., Crohn's disease), immune disorders, metabolic disease (e.g., conditions associated with the metabolic syndrome), endocrine disease, atherosclerosis, asthma, cardiovascular disease, immunological conditions, and/or any other suitable conditions. For example, the TLR2-mediated disease or disorder may be selected from the group consisting of Kawasaki disease, type 2 diabetes, rheumatoid arthritis, dermatological disease, multiple sclerosis, systemic lupus erythematosus, ulcerative colitis, Graves' disease, Sjögren's syndrome, autoimmune thyroid diseases, vasculitis; and any combination thereof.

[0075] As used herein, the term "treatment" or "treatment" refers to a clinical intervention in an attempt to change the natural state of the individual being treated or the cell being treated, and this can be done for prophylaxis or during the development of clinical pathology. The desired effects of treatment include preventing the onset or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, reducing the rate of disease progression, improving or temporarily alleviating the condition of the disease, and remission or improving prognosis. In some embodiments, an antibody (humanized or non-humanized), antibody fragment or polypeptide of the present invention, or a humanized antibody of the present invention is used to delay the development of a disease or disorder.

[0076] The term "variable" refers to the fact that parts of the variable domains differ significantly in their sequence among antibodies and contribute to the binding and specificity of each particular antibody for a particular antigen. However, the variability is distributed unevenly across the variable domains of antibodies. It is concentrated in three segments called complementarity determining regions or hypervariable regions (CDR or HVR as used interchangeably herein) in the light and heavy chain variable domains. The more conservative parts of the variable domains are called framework (FR). Each of the native heavy and light chain variable domains contains four FR regions, largely in β-sheet configuration, connected by three HVRs forming loops connecting, and in some cases forming part of, β-sheet structures. The HVRs in each strand are held in close proximity by FR regions and, together with the HVRs of the other strand, are involved in the formation of the antigen-binding site of antibodies (see Kabat et al. , Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in the binding of an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

[0077] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is associated. One type of vector is a "plasmid", which is a closed circular double-stranded DNA into which additional DNA segments can be ligated. Another type of vector is the phage vector. Another type of vector is the viral vector, where additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication in the host cell into which they are inserted (eg, bacterial vectors containing a bacterial origin of replication and mammalian episomal vectors). Other vectors (eg, non-episomal mammalian vectors) can integrate into the genome of a host cell upon introduction into the host cell and replicate along with the host genome. In addition, some vectors can direct the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors suitable for recombinant DNA methods are in the form of plasmids.

[0078] An "Fc region variant" comprises an amino acid sequence that differs from the native Fc region sequence by at least one amino acid modification, typically one or more amino acid substitutions. Typically, an Fc region variant contains at least one an amino acid substitution relative to the native sequence Fc region or the Fc region of the parent polypeptide, e.g., from about one to about ten amino acid substitutions, and typically from about one to about five amino acid substitutions in the native sequence of the Fc region or in the Fc regions of the parent polypeptide. The Fc region variant of the present invention has at least about 80% homology to the native sequence of the Fc region and/or the Fc region of the parent polypeptide, at least about 90% homology, and typically at least about 95% homology. .

[0079] The term "oxidized phospholipids" (OxPL) refers to phospholipids with a phosphocholine (PC) end group. OxPL are highly proinflammatory and proatherogenic. Phosphorylcholine, the polar end group on some phospholipids, has been strongly associated with the development of cardiovascular disease. Reactive oxygen species produced during coronary inflammation cause low-density lipoprotein (LDL) oxidation to form oxidized LDL (oxLDL). In fact, cardiovascular disease (CVD) such as atherosclerosis, unstable angina or acute coronary syndrome has been shown to be associated with elevated plasma levels of oxLDL. LDL is a circulating lipoprotein particle containing lipids with a polar PC end group and proteins, in particular the apoB100 protein.

[0080] During LDL oxidation, PC is formed containing neoepitopes not present in unmodified LDL. The resulting oxLDL-exposed PC is recognized by scavenger receptors on macrophages such as CD36, and the resulting macrophage-engulfed oxLDL promotes the formation of pro-inflammatory foam cells in the vessel wall. Oxidized LDL is also recognized by receptors on the endothelial cell surface and has been shown to stimulate a range of responses including endothelial dysfunction, apoptosis, and a response to unfolded proteins. PC neoepitopes are also exposed to LDL after modification by phospholipase A2 or disease-associated amino-reactive metabolites such as aldehydes formed from the oxidation of glycated proteins. These alternatively modified LDL particles are also pro-inflammatory factors in CVD. Phosphorylcholine (PC) antibodies have been shown to bind to oxidized or otherwise modified LDL and block the pro-inflammatory activity of oxLDL in in vivo models or in vitro studies.

[0081] Glycerophospholipids are a common class of lipids important for the integrity of cell membranes. Oxidation of esterified unsaturated fatty acids significantly alters the biological activity of phospholipids. In addition to disrupting structural function, oxidation renders oxidized phospholipids (OxPL) "self-modified" type markers recognized by soluble and cell-associated innate immune receptors, including scavenger receptors, natural (germline-encoded) antibodies, and C-reactive protein, thus targeting removal of senescent and apoptotic cells or oxidized lipoproteins. In addition, OxPLs acquire new types of biological activities uncharacteristic of their non-oxidized precursors, including the ability to regulate innate and adaptive immune responses. The effects of OxPL, described in vitro and in vivo, suggest their potential importance in various pathologies, including atherosclerosis, acute inflammation, lung injury, and many other conditions.

[0082] Glycerophospholipids contain a broad class of lipids consisting of a glycerol backbone, a phosphate-containing polar end group, and two fatty acid residues. PL-linked polyunsaturated fatty acids (PUFAs) are the main target of non-enzymatic or enzymatic oxidation, not associated with the formation of metabolic energy. Oxidative fragmentation of the PL molecule results in the formation of several biologically active products, including small reactive PUFA moieties such as non-esterified oxidized fatty acids (eg hydroperoxides and isoprostanes) and lysophospholipids. These products exhibit many types of biological activity. The available evidence suggests that the non-enzymatic oxidation of PL-PUFA occurs according to the same basic mechanisms for the oxidation of free (non-esterified) PUFA. This assumption is confirmed by the definition of similar classes of molecules formed during the oxidation of free and PL-bound PUFA, presented in the present description. In contrast to non-enzymatic oxidation, enzymatic oxidation of PL-PUFA differs significantly from that of non-esterified PUFAs. While free PUFAs can be oxidized by a variety of enzymes belonging to different protein families and inserting different oxidized groups, only one group of lipoxygenases (12/15 lipoxygenases) uses PL-PUFAs as substrates, producing PL-hydroperoxides. Further oxidation and rearrangement proceed without the participation of enzymes, and thus oxidation initiated by enzymatic and non-enzymatic mechanisms leads to the formation of many similar advanced PL oxidation products.

[0083] Toll-like receptor 2, also known as TLR2, is a protein encoded in humans by the TLR2 gene. TLR2 is also referred to as CD282 (282 differentiation cluster). TLR2 plays a role in the immune system. TLR2 is a membrane receptor expressed on the surface of some cells that recognizes foreign substances and transmits appropriate signals to cells of the immune system. TLR2 plays a fundamental role in pathogen recognition and activation of innate immunity. Toll-like receptors (TLRs) are highly conserved from Drosophila to humans and share structural and functional similarities. They recognize pathogen-associated molecular patterns (PAMPs) expressed on the infectious agent and mediate the production of cytokines necessary for the development of effective immunity. Different TLRs have different expression profiles. This gene is most expressed in peripheral blood leukocytes and mediates the host response to Gram-positive bacteria and yeast through NF-κB stimulation. TLR2 detects a wide range of microbial components such as Gram-positive bacterial lipoteichoic acid, bacterial lipoproteins, and zymosan. Of the 11 TLRs characterized, TLR2 is unique in its ability to heterodimerize with TLR1 or TLR6, resulting in a relatively broad ligand specificity.

[0084] CD36, being a TLR2 co-receptor, suggests that there is a pro-inflammatory pathway between endogenously produced lipids and innate immune activation. Studies have also shown that increased endothelial expression and activation of TLR2 occurs in areas of impaired blood flow, such as damage-prone areas in the aortic trunk and heart. Thus, TLR2 expression may promote atherosclerosis in non-BM-derived cells, such as endothelial cells, and thus may contribute to the pro-inflammatory phenotype of activated endothelial cells.

[0085] In atherosclerosis-prone mice deficient in lipoprotein receptors low density (Ldlr ^-/- ) complete deficiency of TLR2 leads to a decrease in atherosclerosis. However, loss of TLR2 expression in BM-derived cells has no effect on disease progression. The data suggest that an unknown endogenous TLR2 agonist influenced the progression of lesions through TLR2 activation in non-BM-derived cells. As shown herein, intraperitoneal administration of the synthetic TLR2/TLR1 agonist Pam3CSK4 significantly increased disease burden in mice.Ldlr ^-/- . Complete TLR2 deficiency in miceLdlr ^-/- , as well as TLR2 deficiency only in BM-derived cells in miceLdlr ^-/- reduced Pam3CSK4-mediated atherosclerosis, suggesting a role for TLR2 expression by BM-derived cells in transducing the effects of an exogenous TLR2 agonist.

[0086] OxPLs can activate TLR2-mediated signaling, resulting in pro-inflammatory signaling in the cell. In addition, OxPL-mediated activation of TLR2 can lead to apoptosis and cell death if it acts in conjunction with signal transduction pathways that promote ER stress. OxPL induces IL-8 signaling from endothelial cells and induces IL-1β and TNFα signaling in macrophages via a TLR2-dependent signaling pathway. As shown herein, activation of macrophages by the synthetic TLR2 agonist PAM3CSK4 directly stimulates macrophages to produce OxPL. It has also been shown that TLR4 activation by agonists such as LPS will also lead to the production of OxPL by macrophages (Popat et al ., JCI, 2017). Overall, the data presented herein suggest that OxPL can directly activate macrophages via TLR2 (or TLR4) to induce a pro-inflammatory signal and/or apoptosis, and conversely, activate macrophages via TLR2 or TLR4 signaling, in turn , will cause macrophages to form OxPL. In the latter case, if macrophages are stimulated with TLR2/4 agonists, locally formed OxPLs can potentially amplify and enhance the inflammatory pathway through autocrine effects. Thus, the reported studies suggest that OxPLs can directly stimulate TLR2 pathways as well as act in a paracrine manner to amplify pro-inflammatory TLR2/4 agonist signaling. These insights explain why in vivo neutralization of OxPL with an anti-OxPL antibody under various inflammatory conditions provides such pronounced anti-inflammatory effects that are manifested in the reduction of disease progression.

[0087] The data suggest that antibodies or fragments thereof that bind to OxPL, including EO6, or other antibodies designed to bind to the phosphocholine (PC) end groups of PC-containing oxidized phospholipids (OxPL), can be used to improve the adverse effects of agonism TLR2, which occurs in various diseases. These include atherosclerosis, autoimmune disorders, and in particular Kawasaki disease, a childhood disorder of unknown etiology in which TLR2-mediated agonism is thought to contribute to coronary arteritis leading to coronary artery aneurysms, severe coronary artery calcification, impaired coronary blood flow, acute thrombosis and significant morbidity and death. The disease can also affect young people when asymptomatic coronary artery aneurysms transform into acute thrombosis, causing acute myocardial infarction. Kawasaki disease may also be associated with myocarditis, heart failure, and the need for a heart transplant.

[0088] Innate natural antibodies (NAb) represent the host's first line of defense against common oxidation-specific epitopes (OSE) on endogenous neoepitopes (OxLDL and apoptotic cells) and exogenous pathogen epitopes and maintain host homeostasis. OSEs are ubiquitous, occur in many inflammatory tissues, including atherosclerotic lesions, and are a major target of IgM NAb. Typical IgM NAb EO6 binds to the phosphocholine (PC) end group in oxidized phospholipids (OxPL) and blocks the uptake of OxLDL by macrophages. A natural mouse IgM anti-OxPL antibody was cloned and characterized that binds to the phosphorylcholine ("PC") end group of OxPL but not to native non-oxidized phospholipids ("PL"). However, antibodies like IgM Nab EO6 have limited solubility and are not easily synthesized.

[0089] The parental EO6 antibody is a mouse IgM antibody cloned, characterized and is the subject of US Pat. No. 6,225,070, incorporated herein by reference. US Patent Publication No. 20150376268 A1 describes a fully functional single chain antibody and humanized antibodies that bind to OxPL. It describes numerous unique molecular changes in the DNA sequence of the parent antibody framework regions, heavy and light chains and linker sequences, which were determined using repeated rounds of experiments, which led to the development of a fully functional EO6-scFv. When this sequence is inserted into an appropriate vector, the resulting scFc is expressed in dissolved form and has all of the immunological binding properties of the parent antibody for its identified target antigens, including the ability to bind to the unique anti-idiotypic antibody AB1-2, whose epitopes consist of heavy and light chains of the parent antibody. antibodies. The description of that application also relates to single chain variable antibody fragments ("scFv") V _H , V _L and complementarity determining regions selectively binding to oxidized phospholipids. The ScFvs of the invention are soluble and easy to synthesize. The description of US Patent Publication No. 20150376268 A1 is incorporated herein by reference for all purposes.

[0090] In the studies presented herein, neutralization of OxPL by endogenous in vivo expression of the EO6 antibody (using an EO6-scFv transgenic mouse) significantly inhibited the formation of atherosclerosis induced by TLR2 agonism. In particular, injection of the TLR2 agonist PAM3CSK4 into Ldlr ^-/- mice fed a high cholesterol diet resulted in a significant increase in atherosclerosis. A similar set of injections of EO6-scFv-transgenic mice (against the background of Ldlr ^-/- ) resulted in a significant inhibition of the formation of lesions.

[0091] In other studies presented herein, OxPL neutralization may protect in a mouse model of Kawasaki disease. It has been shown that administration of the pathogen Lactobaccilus casei eliminates Kawasaki disease-like disease in mice with resulting increase in atherosclerosis, coronary arteritis, and abdominal aortic aneurysms. It depends on TLR2, as administration of L. casei to TLR2-deficient mice does not cause disease. Importantly, IL-1 has been shown to be involved, and OxPL is also noted to be a potent inducer of IL-1 release. Injection of L. casei EO6 transgenic mice (with Ldlr ^-/- background) following an identical protocol resulted in a significant reduction in not only atherosclerosis, but, more importantly, coronary arteritis, compared with injections of Ldlr ^-/- mice. The EO6 antibody does not bind directly to L. casei and thus neutralizes OxPL, the formation of which is caused by the inflammatory effects associated with TLR2-mediated agonism. The development of coronary arteritis and then coronary artery aneurysms is a major and very dangerous complication in children who develop Kawasaki disease and is estimated to occur in up to 25% of children despite existing therapy. Typically, Kawasaki disease is treated with intravenous immunoglobulin (IVIG) derived from mixed and purified human plasma and aspirin (a common but non-specific anti-inflammatory therapeutic agent). Injections of high titers of humanized or human-equivalent anti-OxPL antibodies modified for increased biological efficacy may then provide protection without any expected side effects, as such anti-OxPL antibodies are present in the repertoire of human B cells.

[0092] Thus, experimental data suggest that atherosclerosis and inflammatory arteritis caused by TLR2 mediated agonism in vivo in mice can be prevented by neutralization of OxPL. Of course, TLR2 agonism contributes to many bacterial diseases, but also to various so-called autoimmune-mediated diseases such as systemic lupus erythematosus, rheumatoid arthritis, and others. Evidence suggests that OxPL neutralization with anti-PC OxPL antibodies can improve or prevent many diseases enhanced or affected by activation of TLR-mediated signaling pathways.

[0093] The present invention relates to the use of antibodies, antibody fragments and humanized antibodies that bind to OxPL and, in some cases, have the same or similar binding specificity with an EO6 antibody. Antibody fragments can be produced by conventional methods, such as enzymatic digestion, or by recombinant methods. In some cases, there are advantages to using antibody fragments rather than whole antibodies. The smaller size of the fragments allows for faster clearance and may result in better tissue access. Under acute conditions, the half-life of antibody fragments is not critical. For a review of some antibody fragments, see Hudson et al. (2003) Nat. Med. 9:129-134.

[0094] Although the present specification provides specific antibody sequences and sequences of antibody fragments having biological activity, it further describes that these sequences can be used to generate improved variants. Thus, in some cases, an antibody or antibody fragment may have a percent identity with respect to the sequence of the present invention.

[0095] In some embodiments, modifications to the amino acid sequence of the antibodies provided herein are provided. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Variants of the amino acid sequence of an antibody can be obtained by making appropriate changes to the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues in the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be performed to obtain the final product, provided that the final construct has the desired characteristics. Amino acid changes can be made to the amino acid sequence of an antibody during sequencing.

[0096] The present invention provides an antibody or antibody fragment capable of binding to OxPL or phosphorylcholine and/or a phosphorylcholine conjugate, wherein the antibody or antibody fragment comprises a heavy chain variable domain (V _H ) and/or a light chain variable domain (V _L ), and where

(a) the V _H domain contains an amino acid sequence comprising one, two or three complementarity determining regions (CDRs) selected from the group consisting of:

SEQ ID NO: 6 and a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 99.9% identical to SEQ ID NO: 6;

SEQ ID NO: 7 and a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 99.9% identical to SEQ ID NO: 7; And

SEQ ID NO: 8 and a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 99.9% identical to SEQ ID NO: 8;

(b) the V _L domain contains an amino acid sequence comprising one, two or three complementarity determining regions (CDRs) selected from the group consisting of:

SEQ ID NO: 9 or 12 and a sequence that is at least 95%, 96%, 97%, 98%, 99% or 99.9% identical to SEQ ID NO: 9 or 12;

SEQ ID NO: 10 and a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 99.9% identical to SEQ ID NO: 10; And

SEQ ID NO: 11 and a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 99.9% identical to SEQ ID NO: 11.

[0097] In one embodiment, the antibody or antibody fragment comprises a V _H domain containing an amino acid sequence comprising CDRs containing SEQ ID NOs: 6, 7, and 8, and/or a V _L domain containing an amino acid sequence comprising CDRs containing SEQ ID NOs: 9, 10 and 11 or SEQ ID NOs: 10, 11 and 12.

[0098] In one embodiment, the present invention provides an antibody or scFv selected from the group consisting of: (a) an antibody or scFv with heavy and light chain domains comprising complementarity determining regions of SEQ ID NOs: 6, 7, 8, 9, 10 and 11; and (b) an antibody or scFv with heavy and light chain domains comprising complementarity determining regions of SEQ ID NOs: 6, 7, 8, 10, 11, and 12. In one embodiment, any of (a) or (b) is linked to Fc region.

[0099] In one embodiment, the present invention provides an antibody comprising a light chain variable region as set forth in SEQ ID NO: 2 from amino acid 1 to about amino acid 146. In another embodiment, the present invention provides an antibody with a humanized light chain variable region , comprising the sequence of SEQ ID NO: 4 from amino acid 1 to approximately amino acid 135. In another embodiment, the present invention provides an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 2 from approximately amino acid 162 to approximately amino acid 269. In another embodiment, In an embodiment, the present invention relates to an antibody comprising a humanized heavy chain variable region comprising the sequence of SEQ ID NO: 4 from about amino acid 152 to about amino acid 258.

[00100] In another embodiment, the present invention provides a chimeric antibody comprising, for example, a mouse VH and/or VL and a human Fc region. For example, SEQ ID NO: 14 includes the sequence of a chimeric antibody of the present invention. In SEQ ID NO: 14 amino acids 1-33 contain the Ig kappa chain leader for antibody secretion; amino acids 34-146 contain the EO6 light chain variable region; amino acids 147-161 include a flexible linker sequence; amino acids 162-284 contain an EO6 heavy chain variable region with a P201A, S224A, and A225D triple mutation relative to wild-type mouse EO6 antibody; amino acids 285-517 contain an Fc region, in SEQ ID NO: 14 the Fc region is a human IgG1-Fc modified with C290S and H294Y to increase ADCC activity. SEQ ID NO: 14 also includes an additional linker and His tag sequence, which one of ordinary skill in the art will recognize are optional (eg, SEQ ID NO: 14 amino acid 518 to amino acid 528). The present invention also includes a coding sequence of SEQ ID NO: 14 containing SEQ ID NO: 13. A person skilled in the art can easily determine the nucleic acid sequence corresponding to the various domains defined above. The present invention also includes a chimeric antibody sequence, ie. at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical to SEQ ID NO: 14 amino acid 1 to amino acid 284 associated with the Fc region of another immunoglobulin (e.g., IgA , IgD, IgE, IgG and IgM or any of the subclasses (isotypes), for example, IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ and IgA ₂ ).

[00101] In one embodiment, the present invention provides an scFv comprising a linker between a light chain variable region and a heavy chain variable region. The linker may be any number of commonly used peptide linkers. In one embodiment, the linker contains a repeating unit GGGS (SEQ ID NO: 5). GGGS (SEQ ID NO: 5) may be repeated 2, 3, 4 or more times.

[00102] In another embodiment, the present invention includes an scFv comprising the light chain variable region of SEQ ID NO: 2 from amino acid 1 to amino acid 146 linked by a peptide linker to the heavy chain variable region of SEQ ID NO: 2 from amino acid 162 to about amino acid 269. In a specific embodiment, the scFv comprises the sequence of SEQ ID NO: 2 from amino acid 1 to amino acid 269. In another embodiment, the present invention provides a scFv having the polypeptide sequence of SEQ ID NO: 2 from amino acid 1 to about amino acid 269, or from amino acid 1 to about amino acids 303. In a further embodiment, the present invention provides an scFv having a polypeptide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least at least 98% or at least 99% sequence identity with respect to SEQ ID NO : 2 from amino acid 1 to approximately amino acid 303, and selectively binds to an oxidized phospholipid.

[00103] In additional embodiments, the fusion constructs comprise a first domain comprising SEQ ID NO: 2 amino acid 1 to approximately amino acid 269, or amino acid 1 to approximately amino acid 303, or a variant thereof, operably linked to a second domain comprising (i) a detectable label or (ii) a polypeptide of interest. One of skill in the art will appreciate that such fusion constructs can be prepared by chemical or molecular biology techniques that combine a coding sequence containing the sequence of SEQ ID NO: 1 or a variant thereof with a coding sequence for, for example, a polypeptide of interest. Coding sequences and domains can be separated by a linker or connected directly.

[00104] In yet another embodiment, the present invention includes an scFv comprising a light chain variable region of SEQ ID NO: 4 from amino acid 1 to amino acid 135 linked via a peptide linker to a heavy chain variable region of SEQ ID NO: 4 from amino acid 152 to approximately amino acid 258. In a specific embodiment, the scFv comprises the sequence of SEQ ID NO: 4 from amino acid 1 to amino acid 258. In another embodiment, the present invention provides an scFv having the polypeptide sequence of SEQ ID NO: 4 from amino acid 1 to about amino acid 258, or from amino acid 1 to about amino acid 263. In a further embodiment, the present invention provides a scFv having a polypeptide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with respect to ranging from SEQ ID NO: 4 from amino acid 1 to about amino acid 258, and selectively binding to an oxidized phospholipid.

[00105] In further embodiments, the fusion constructs comprise a first domain comprising SEQ ID NO: 4 amino acid 1 to approximately amino acid 258, or amino acid 1 to approximately amino acid 264, or a variant thereof, operably linked to a second domain comprising (i) a detectable label or (ii) a polypeptide of interest. One of skill in the art will appreciate that such fusion constructs can be prepared by chemical or molecular biology techniques that combine a coding sequence containing the sequence of SEQ ID NO: 3, or a variant thereof, with a coding sequence for, for example, a polypeptide of interest. Coding sequences and domains can be separated by a linker or connected directly.

[00106] Nucleic acid molecules encoding the amino acid sequences of antibodies, antibody fragments, and antibody variants are prepared by various methods known in the art. In the case of obtaining variants, such methods include, as non-limiting examples, isolation from a natural source (in the case of variants of the natural amino acid sequence) or obtaining by oligonucleotide-mediated (or site-specific) mutagenesis, PCR mutagenesis and cassette mutagenesis of a previously obtained variant or non-variant version of the antibody.

[00107] In a specific embodiment, the present invention relates to a mouse scFv encoded by the polynucleotide sequence of SEQ ID NO: 1. In a further embodiment, the present invention relates to a mouse scFv encoded by a polynucleotide sequence having at least 75%, at least 80% , at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect to SEQ ID NO: 1 and resulting in the formation of a polypeptide that selectively binds to oxidized phospholipids.

[00108] In another embodiment, the present invention relates to scFv containing a linker between the variable region of the light chain and the variable region of the heavy chain. The linker may be any number of commonly used peptide linkers. In one embodiment, the implementation of the linker contains a repeating unit GGGS (SEQ ID NO: 5). GGGS (SEQ ID NO: 5) may be repeated 2, 3, 4 or more times.

[00109] The present invention also includes humanized antibodies. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody may have one or more amino acid residues inserted into it from a non-human source. These non-human amino acid residues are often referred to as "import" residues, typically taken from an "import" variable domain. Humanization can essentially be carried out by the method of Winter et al. (Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239 :1534-1536) by replacing the hypervariable region sequences with the corresponding human antibody sequences. Thus, such "humanized" antibodies are chimeric antibodies (US Pat. No. 4,816,567) wherein a substantially smaller than intact human variable domain is replaced with a corresponding non-human species sequence. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are replaced with residues from similar regions in rodent antibodies.

[00110] When selecting human light and heavy chain variable domains for use in making humanized antibodies, it may be important to reduce antigenicity. According to the so-called "best fit" method, the variable domain sequence of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence closest to the rodent sequence is then adopted as the human backbone for the humanized antibody. See, for example, Sims et al. (1993) J. Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901. Another method uses a particular framework derived from the consensus sequence of all human antibodies from a particular subgroup of light or heavy chains. The same framework can be used for several other humanized antibodies. See, for example, Carter et al. (1992) Proc. Natl. Acad. sci. USA 89:4285; Presta et al. (1993) J. Immunol. , 151:2623.

[00111] It is also generally desirable to humanize antibodies while maintaining high antigen affinity and other beneficial biological properties. To achieve this goal, in one way, humanized antibodies are generated by analyzing parental sequences and various conceptual humanized products using 3D models of parental and humanized sequences. Three-dimensional immunoglobulin models are publicly available and well known to those skilled in the art. Computer programs are available that illustrate and display possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. The study of these mappings allows one to analyze the possible role of the residues in the functioning of the candidate immunoglobulin sequence, i.e. analysis of residues that affect the ability of a candidate immunoglobulin to bind to its antigen. Thus, FR residues can be selected and combined from the recipient and imported sequences so as to achieve the desired antibody characteristic, such as increased affinity for target antigens. In general, hypervariable region residues directly and most significantly affect antigen binding.

[00112] In a specific embodiment, the present invention relates to a humanized scFv encoded by the polynucleotide sequence of SEQ ID NO: 3. In a further embodiment, the present invention relates to a humanized scFv encoded by a polynucleotide sequence having at least 75%, at least 80% , at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect to SEQ ID NO: 3 and resulting in a polypeptide that selectively binds to oxidized phospholipids.

[00113] The present invention also relates to scFv, presented in the present description, additionally containing a crystallizable fragment ("Fc") of the antibody. In a specific embodiment, the Fc region is derived from a human antibody or a humanized antibody. The Fc region is the tail region of an antibody that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. This property allows antibodies to activate the immune system. In the IgG, IgA, and IgD antibody isotypes, the Fc region consists of two identical protein fragments derived from the second and third constant domains of the two antibody heavy chains; The IgM and IgE Fc regions contain three heavy chain constant domains (CH 2-4 domains) in each polypeptide chain. The IgG Fc regions carry highly conserved N-glycosylation sites. Glycosylation of the Fc fragment is required for Fc receptor mediated activity. The N-glycans attached to this site are predominantly core-fucosylated biantennary structures of the complex type. In addition, small amounts of these N-glycans also bear bisected GlcNAc and α-2,6-linked sialic acid residues. The other part of an antibody, called the Fab region, contains variable sections that define a specific target that the antibody can bind to. The scFvs of the present invention are composed of Fab region elements. In contrast, the Fc regions of all antibodies in a class are the same for each species; they are constant, not variable. Thus, the Fc region is sometimes referred to as the "fragment-constant region". Thus, polynucleotide and polypeptide sequences encoding Fc regions for an infinite number of biological species have already been identified and known to the person skilled in the art. In particular, in an embodiment, the present invention provides an scFv polynucleotide sequence as provided herein, further comprising a polynucleotide sequence encoding an Fc region from an IgG antibody (eg, a human IgG antibody). In a further embodiment, the present invention provides an scFv polypeptide sequence as provided herein, further comprising an IgG antibody Fc region polypeptide sequence.

[00114] In particular, in an embodiment, the present invention provides an scFv polynucleotide sequence as provided herein, further comprising a polynucleotide sequence encoding the Fc region of an IgG antibody (eg, human IgG antibody). In a further embodiment, the present invention provides an scFv polypeptide sequence as provided herein, further comprising an Fc region polypeptide sequence from an IgG antibody. In one embodiment, the Fc region coding sequence comprises the sequence shown in SEQ ID NO: 3 from about nucleotide 790 to about nucleotide 1518.

[001115] In an additional embodiment, the present invention relates to a vector containing a polynucleotide sequence encoding an scFv given above with reference to SEQ ID NOs: 1 and 3, or sequences having a sequence identity of at least 99%, at least 95% , at least 90%, at least 85%, at least 80%, at least 75%, or at least 70% identity with respect to SEQ ID NO: 1 or SEQ ID NO: 3.

[00116] The present invention also provides a humanized antibody having an EO6 antibody binding specificity. The humanized antibody contains (i) a sequence shown in SEQ ID NO: 4 from amino acid 1 to approximately amino acid 506, or (ii) a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99%, 99.5% or 99.8% identical with respect to SEQ ID NO: 4 from amino acid 1 to approximately amino acid 506.

[00117] The present invention also relates to a polynucleotide encoding a humanized antibody of the present invention. The polynucleotide contains a sequence selected from the group consisting of (i) a polynucleotide encoding SEQ ID NO: 4, (ii) a polynucleotide hybridizing under stringent conditions with a polynucleotide consisting of SEQ ID NO: 3 and encoding a humanized antibody that binds to OxPL with specificity sufficiently similar to an EO6 antibody, (iii) a polynucleotide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99, 5% or 99.8% identical to SEQ ID NO: 3 and encoding an antibody that binds OxPL with a specificity quite similar to the EO6 antibody; (iv) a polynucleotide shown in SEQ ID NO: 3; (v) a polynucleotide according to any one of paragraphs. (i)-(iv), where the polynucleotide contains RNA.

[00118] Polynucleotide sequences encoding the polypeptide components of the antibody or antibody fragments of the present invention can be obtained by standard recombinant methods. Desired polynucleotide sequences can be isolated and sequenced from antibody-producing cells, such as hybridoma cells. Alternatively, polynucleotides can be synthesized using a nucleotide synthesizer or PCR methods. Once obtained, the sequences encoding the polypeptides are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in prokaryotic host cells. Many vectors available and known in the art can be used for the purposes of the present invention. The choice of a suitable vector will depend primarily on the size of the nucleic acids to be inserted into the vector and the particular host cell to be transformed using the vector. Each vector contains different components depending on its function (amplification or expression of a heterologous polynucleotide, or both) and compatibility with the particular host cell in which it resides. Vector components typically include, but are not limited to, an origin of replication, a selectable marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, a heterologous nucleic acid insert, and a transcription termination sequence.

[00119] In general, plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are used in conjunction with these hosts. The vector, as a rule, carries the site of the origin of replication, as well as marker sequences with which it is possible to carry out phenotypic selection of transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from the E. coli species. pBR322 contains the genes encoding ampicillin (Amp) and tetracycline (Tet) resistance and thus allows easy identification of transformed cells. pBR322, its derivatives, or other microbial plasmids or bacteriophages may also contain, or be modified to contain, promoters that the microorganism can use to express endogenous proteins. Examples of pBR322 derivatives used to express specific antibodies are detailed in Carter et al. , US Pat. No. 5,648,237.

[00120] In addition, phage vectors containing replicon and control sequences compatible with the host microorganism can be used as transforming vectors with these hosts. For example, bacteriophage vectors can be used in the production of a recombinant vector that can be used to transform susceptible host cells such as E. coli LE392.

[00121] The expression vector of the present invention may contain two or more promoter-cistron pairs encoding each of the polypeptide components. The promoter is a non-translated regulatory sequence located upstream (5') of the cistron and modulating its expression. Prokaryotic promoters generally fall into two classes, inducible and constitutive. An inducible promoter is a promoter that initiates increased levels of cistron transcription under its control in response to changes in culture conditions, such as the presence or absence of a nutrient, or a change in temperature.

[00122] A large number of promoters are well known and recognized by various potential host cells. The selected promoter can be operably linked to a DNA cistron encoding a light or heavy chain by removing the promoter from the DNA source by restriction enzyme digestion and inserting the promoter sequence into the vector of the invention. A native promoter sequence and many heterologous promoters can be used to regulate the amplification and/or expression of target genes. In some embodiments, heterologous promoters are used because they generally allow higher transcription and higher yields of the expressed target gene compared to the native promoter of the target polypeptide.

[00123] Promoters suitable for use with prokaryotic hosts include the PhoA promoter, the β-galactamase and lactose promoter systems, the tryptophan (trp) promoter system, and hybrid promoters such as the tac or trc promoter. However, other promoters operable in bacteria (such as other known bacterial or phage promoters) are also suitable. Their nucleotide sequences have been published, allowing one of skill in the art to ligate them operably to cistrons encoding target light and heavy chains (Siebenlist et al. , (1980) Cell 20:269) using linkers or adapters to provide any necessary restriction sites.

[00124] In another embodiment, the production of the immunoglobulins of the present invention can occur in the cytoplasm of the host cell and thus does not require secretory signal sequences in each cistron. As such, immunoglobulin light and heavy chains are expressed, folded, and assembled to form functional immunoglobulins in the cytoplasm. Some strains of host cells (for example, strains trxBE.coli) provide cytoplasmic conditions favorable for disulfide bond formation, thus allowing proper folding and assembly of expressed protein subunits. Proba and PluckthunGene, 159:203 (1995).

[00125] Prokaryotic host cells suitable for expression of the antibodies of the invention include Archaebacteria and Eubacteria , such as Gram-negative or Gram-positive organisms. Examples of bacteria that can be used include Escherichia (e.g. E. coli ), Bacilli (e.g. B. subtilis ), Enterobacteria , Pseudomonas species (e.g. P. aeruginosa ), Salmonella typhimurium , Serratia marcescans , Klebsiella , Proteus , Shigella , Rhizobia , Vitreoscilla or Paracoccus . In one embodiment, Gram-negative cells are used. In one embodiment, E. coli cells are used as host cells of the present invention. Examples of E. coli strains include strain W3110 (Bachmann, Cellular and Molecular biology, vol. 2 (Washington, DC: American Society for Microbiology, 1987), pp.1190-1219; ATCC deposit No. 27,325) and their derivatives, including strain 33D3 (US patent No. 5639635). Other strains and their derivatives are also suitable, such as E. coli 294 (ATCC 31.446), E. coli B, E. coli X 1776 (ATCC 31.537) and E. coli RV308. These examples are illustrative and not restrictive. Methods for constructing derivatives of any of the above bacteria having certain genotypes are known in the art and are described, for example, in Bass et al. , Proteins 8:309-314 (1990). As a rule, it is necessary to select suitable bacteria, taking into account the replication of the replicon in the bacterial cells. For example, E. coli , Serratia spp. or Salmonella spp. can be suitably used as a host when well-known plasmids such as pBR322, pBR325, pACYC177 or pKN410 are used to deliver the replicon. Generally, the host cell should secrete minimal amounts of proteolytic enzymes, and it may be desirable to include additional protease inhibitors in the cell culture.

[00126] Host cells are transformed using the expression vectors described above and cultured in conventional nutrient media modified as necessary to induce promoters, select transformants, or amplify genes encoding the desired sequences.

[00127] The term "transformation" means the insertion of DNA into a prokaryotic host such that the DNA is replicated as an extrachromosomal element or by insertion into a chromosome. Depending on the host cell used, transformation is carried out by standard methods suitable for such cells. Calcium treatment with calcium chloride is generally used for bacterial cells containing significant cell walls. Another method uses polyethylene glycol/DMSO for transformation. Another method used is electroporation.

[00128] Prokaryotic cells used to obtain the polypeptides of the present invention are grown in media known in this field and suitable for culturing selected host cells. Examples of suitable media include Luria Broth (LB) with the necessary nutrient supplements. In some embodiments, the media also contain a selection agent, selected based on the design of the expression vector, to selectively grow prokaryotic cells containing the expression vector. For example, ampicillin is added to a growth medium for cells expressing an ampicillin resistance gene.

[00129] Any necessary additional substances, in addition to sources of carbon, nitrogen and inorganic phosphate, can also be included in suitable concentrations alone or mixed with another additional substance or environment, such as a complex source of nitrogen. Optionally, the culture medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, dithioerythritol, and dithiothreitol. Prokaryotic host cells are cultured at suitable temperatures.

[00130] In one embodiment, the expressed polypeptides are secreted into the periplasm of the host cells and isolated from it. Isolation of proteins typically involves disruption of the microorganism, typically by osmotic shock, sonication, or lysis. After cell destruction, cell debris or whole cells can be removed by centrifugation or filtration. Proteins can be further purified, for example with an affinity chromatography resin. Alternatively, the proteins can be transported to the culture media and isolated therefrom. Cells may be removed from the culture and the culture supernatant may be filtered and concentrated to further purify the protein produced. Expressed polypeptides can be further isolated and identified using well known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blotting. One skilled in the art can use and optimize large scale or small scale fermentation.

[00131] Standard protein purification methods known in the art can be used. The following methods are examples of suitable purification methods: fractionation on immunoaffinity or ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or a cation exchange resin such as DEAE, chromatofocusing, SDS-PAGE electrophoresis, ammonium sulfate precipitation and gel- filtration.

[00132] The present invention also relates to an expression vector encoding an antibody, antibody fragment, or polypeptide as described herein, transferred to a suitable host organism. A suitable host organism is a microorganism, a yeast, or a mammalian cell system. Generally, the mammalian cell system is composed of cells of monocytic origin (eg, macrophages, monocytes, and neutrophils), cells of lymphocytic origin (eg, myeloma, hybridoma, and normal immortalized B cell), parenchymal (eg, hepatocytes), and non-parenchymal cells (eg, stellate cells).

[00133] The present invention also relates to pharmaceutical compositions or compositions containing a therapeutically effective amount of an antibody, antibody fragment or polypeptide of the present invention. The pharmaceutical compositions or formulations may further contain carriers, excipients, diluents, solubilizers, stabilizers, buffers, tonicity modifiers, bulking agents, viscosity increasing/lowering agents, surfactants, chelating agents and adjuvants.

[00134] The "therapeutically effective amount" of the substance/molecule of the present invention, agonist or antagonist may vary depending on such factors as the disease state, age, sex and weight of the individual, and the ability of the substance/molecule, agonist or antagonist to elicit the desired response in individual. A therapeutically effective amount is also one in which any toxic or adverse effects of the substance/molecule, agonist or antagonist are outweighed by the therapeutically beneficial effects.

[00135] The term "prophylactically effective amount" refers to an amount effective at the required doses and for the required periods of time in achieving the desired prophylactic result. As a rule, but not necessarily, because a prophylactic dose is used in individuals before the development of the disease or in its early stages, the prophylactically effective amount will be less than the therapeutically effective amount.

[00136] Therapeutic formulations containing an antibody or fragment thereof of the present invention are prepared for storage by mixing the antibody or fragment having the desired purity with optional physiologically acceptable carriers, excipients, or stabilizers (Remington: The Science and Practice of Pharmacy 20th edition ( 2000)) in the form of aqueous solutions, lyophilized or other dried formulations. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the doses and concentrations employed and include buffers such as phosphate, citrate, histidine, 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 counterions such as sodium; complex compounds with metals (for example, Zn-protein complexes); and/or non-ionic surfactants or polyethylene glycol (PEG).

[00137] the Pharmaceutical compositions presented in the present description, you can enter parenterally by injection, infusion or implantation, for local or systemic administration. Within the scope of the invention, parenteral administration includes intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, intrasynovial and subcutaneous administration.

[00138] The pharmaceutical compositions provided herein may be formulated in any dosage form suitable for parenteral administration, including solutions, suspensions, emulsions, and solid forms suitable for preparing solutions or suspensions in liquid prior to injection. Such dosage forms can be prepared by conventional methods known to those skilled in the art of pharmacy (see Remington: The Science and Practice of Pharmacy , supra).

[00139] Pharmaceutical compositions intended for parenteral administration may include one or more pharmaceutically acceptable carriers and excipients, including, but not limited to, aqueous carriers, water-miscible carriers, non-aqueous carriers, antimicrobial agents or preservatives against the growth of microorganisms, stabilizers, solubility enhancers, isotonic agents, buffering agents, antioxidants, local anesthetics, suspending and dispersing agents, humectants or emulsifiers, complexing agents, sequestrants or chelating agents, cryoprotectants, lyoprotectants, thickeners, pH adjusters and inert gases.

[00140] Suitable aqueous vehicles include, as non-limiting examples, water, saline, saline or phosphate buffered saline (PBS), sodium chloride injection, Ringer's solution for injection, isotonic dextrose solution for injection, sterile water for injection, dextrose solution and Ringer's lactate for injection. Non-aqueous carriers include, but are not limited to, vegetable fatty oils, castor oil, corn oil, cottonseed oil, olive oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, hydrogenated vegetable oils, hydrogenated soybean oil, coconut oil medium chain triglycerides and palm oil. Water-miscible carriers include, but are not limited to, ethanol, 1,3-butanediol, liquid polyethylene glycol (eg, polyethylene glycol 300 and polyethylene glycol 400), propylene glycol, glycerin, N -methyl-2-pyrrolidone, dimethylacetamide, and dimethyl sulfoxide.

[00141] In one embodiment, the pharmaceutical compositions provided herein are formulated as sterile solutions ready for use. In another embodiment, the pharmaceutical compositions provided herein are formulated as sterile dry soluble products, including powders and hypodermic tablets, which, if necessary, can be reconstituted with a carrier prior to use. In yet another embodiment, the pharmaceutical compositions are described as sterile suspensions ready for use. In yet another embodiment, the pharmaceutical compositions are described as sterile dry insoluble products for reconstitution with a carrier prior to use. In another embodiment, the pharmaceutical compositions are described as sterile emulsions ready for use.

[00142] Pharmaceutical compositions can be formulated as a suspension, solid, semi-solid, or thixotropic liquid for administration as an implantable depot formulation. In one embodiment, the pharmaceutical compositions provided herein are dispersed in a solid internal matrix surrounded by an outer polymeric membrane that is insoluble in bodily fluids but allows the active ingredient in the pharmaceutical compositions to diffuse through it.

[00143] Suitable internal matrices include polymethyl methacrylate, polybutyl methacrylate, plasticized or unplasticized polyvinyl chloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene vinyl acetate copolymers, silicone rubber, polydimethylsiloxane, silicon carbonate copolymers, hydrophilic polymers such as hydrogels of esters of acrylic and methacrylic acid, collagen, cross-linked polyvinyl alcohol and cross-linked, partially hydrolyzed polyvinyl acetate.

[00144] Suitable outer polymeric membranes include polyethylene, polypropylene, ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers, ethylene/vinyl acetate copolymers, silicone rubber, polydimethylsiloxanes, neoprene rubber, chlorinated polyethylene, polyvinyl chloride, vinyl chloride/vinyl acetate copolymers, vinylidene chloride, ethylene, and propylene. , polyethylene terephthalate ionomer, butyl rubber, epichlorohydrin rubber, ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcohol terpolymer, and ethylene/vinyloxyethanol copolymer.

[00145] The composition of the present invention may also contain more than one active compound, if necessary, for the particular indication being treated, preferably with complementary activities that do not adversely affect each other. Such molecules are present in combination in amounts effective for the intended purpose.

[00146] The active ingredients can also be encapsulated, for example, by coacervation or interfacial polymerization methods, for example, a hydroxymethylcellulose or gelatin microcapsule and a polymethyl methacrylate microcapsule, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions). , nanoparticles and nanocapsules) or macroemulsions. Such methods are described in Remington: The Science and Practice of Pharmacy 20th edition (2000).

[00147] Formulations for use for in vivo administration must be sterile. This is easily achieved by filtration through sterile filtration membranes.

[00148] Sustained release formulations can be obtained. Suitable examples of sustained release formulations include semi-permeable matrices of solid hydrophobic polymers containing the immunoglobulin of the invention, where the matrices are in the form of shaped articles such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactides (US Pat. No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate , non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT (injectable microspheres consisting of a copolymer of lactic acid-glycolic acid and leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid. Although polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow molecules to be released for over 100 days, some hydrogels release proteins for a shorter period of time. When encapsulated immunoglobulins remain in the body for an extended period of time, they can denature or aggregate upon exposure to moisture at 37° C., resulting in loss of biological activity and possible changes in immunogenicity. Rational strategies for stabilization can be developed depending on the mechanism involved. For example, if the mechanism of aggregation is found to be intermolecular S--S bonding via thiodisulfide exchange, stabilization can be achieved through modification of sulfhydryl residues, lyophilization from acidic solutions, moisture control, use of suitable additives, and development of special polymer matrix compositions.

[00149] The antibodies, antibody fragments and polypeptides provided herein bind to OxPL and block their pro-inflammatory effects. The in vivo use of an antibody, antibody fragment or polypeptide of the present invention is expected to block the biological effects of OxPL in a variety of different situations. For example, OxPL has been shown to be produced by macrophages and other cells via a TLR2-mediated mechanism. These released OxPLs contribute to unwanted vasoactive effects throughout the patient's body. Thus, injection/infusion of an antibody, antibody fragment, or polypeptide of the present invention, if used urgently and/or chronically, can block these adverse effects and/or, alternatively, block or reduce similar inflammatory phenomena resulting from TLR2 activation. Similarly, an antibody, antibody fragment, or polypeptide of the present invention can also be infused into an individual to block OxPL-mediated pro-inflammatory effects resulting from various pathological conditions such as viral or bacterial infections or autoimmune disorders. Thus, an antibody, antibody fragment, or polypeptide of the present invention will be effective as anti-inflammatory agents in other systemic inflammatory conditions mediated by TLR2 activation, such as rheumatoid arthritis. Thus, the antibody, antibody fragment or polypeptide of the present invention can be used in many clinical circumstances or conditions where anti-inflammatory and/or anti-atherosclerotic agents need to be administered temporarily and/or chronically.

[00150] The present invention relates to methods of treatment using an antibody, an antibody fragment, and a polypeptide of the present invention to treat an individual with a TLR2-mediated disease or disorder. In a specific embodiment, the present invention relates to the treatment of a TLR2-mediated disease or disorder with a therapeutically effective amount of an antibody, antibody fragment, or polypeptide of the present invention. Non-limiting examples of TLR2-mediated diseases or disorders include Kawasaki disease (Kang et al. , Korean J Pediatr 60(7):208-215 (2017)), type 2 diabetes (Sepehri et al. , Cell MolBiol Lett 21:2 (2016) ) ) ; _ multiple sclerosis (Hossain et al. , Oncotarget 6(34):35131-35132 (2015)), systemic lupus erythematosus (Liu et al. , European Journal of Immunology , 45(9):2683-2693 (2015)), ulcerative colitis (Folova et al. , Journal of Histochemistry & Cytochemistry 56(3):267-274 (2008), Graves' disease (Peng et al. , Front Immunol , 7:578 (2016)), Sjögren's syndrome (Sisto et al. , ClinExp Med 17(3):341-350 (2017), autoimmune thyroid disease (Peng et al. , Front Immunol 7:547 (2016) and vasculitis (Summers et al. , Arthritis Rheum 63(4):1124- 35 (2011)).In a specific embodiment, the present and The invention relates to the treatment of an individual with Kawasaki disease with a therapeutically effective amount of an antibody, antibody fragment or polypeptide of the present invention.

[00151] For the therapeutic use described herein, kits and articles of manufacture are also presented herein. Such kits may contain a carrier, package, or container compartmentalized to contain one or more containers, such as vials, test tubes, and the like, where each of the containers contains one of the individual elements for use in the method presented in the present description. Suitable containers include, for example, bottles, vials, syringes and test tubes. Containers can be made from various materials such as glass or plastic.

[00152] For example, containers may contain one or more of the antibodies, antibody fragments, or polypeptides described herein, optionally in a composition or in combination with another agent as described herein. The containers optionally have a sterile inlet (eg, the container may be an intravenous solution bag or a vial with a hypodermic stopper). Such kits, optionally, contain a description, or a label, or instructions relating to use in the methods presented in the present description.

[00153] A kit will typically contain one or more additional containers, each containing one or more of various materials (such as reagents, optionally in concentrated form, and/or devices) that are commercially and user to use the connection presented in the present description. Non-limiting examples of such materials include buffers, diluents, filters, needles, syringes; carrier, packaging, container, vial and/or vial label indicating contents and/or instructions for use, and package inserts with instructions for use. Typically will also include a set of instructions.

[00154] The label may be on the container or associated with the container. The label may be on the container when the letters, numbers or other indicia forming the label are affixed, pressed or engraved on the container itself, the label may be associated with the container when it is in a receptacle or carrier also holding the container, such as an insert in packaging. The label can be used to indicate that the content will be used for a particular therapeutic application. The label may also include instructions for using the content, such as in the methods presented herein. These other therapeutic agents can be used, for example, in the amount indicated in the Physician's Desk Reference (PDR) or otherwise determined by a person skilled in the art.

[00155] The following examples are presented for illustrative purposes and not to limit the invention. While typical methods may be used, other methods known to those skilled in the art may alternatively be used.

EXAMPLES

[00156] Materials . Synthetic standards sn-glycero-3-phosphocholine (PGPC), 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PAzPC), 1-palmitoyl-2-(9-oxo)nanoyl-sn-glycero-3-phosphocholine (PONPC) and LPS-free mouse IgM EO6 natural antibody were obtained from Avanti Polar Lipids (Alabaster, AL). 1-(palmitoyl)-2-(5-keto-6-octen-dioyl)-3-phosphocholine (KOdiAPC) and 1-palmitoyl-2-(4-keto-dodec-3-en-dioyl)-sn-glycero -3-phosphocholine (KDdiAPC) was purchased from Cayman Chemicals (Ann Arbor, MI). All solvents were HPLC grade.

[00157] Generation and characterization of EO6 scFv transgenic mice. Generation of C57BL/6 transgenic mice expressing the T15/EO6 idiotype as a single chain variable antibody fragment designated EO6-scFv-Tg. Briefly, the cDNA encoding the heavy and light chain EO6 variable regions was fused to the 15-amino acid peptide by overlap extension PCR and cloned into the expression vector pSecTag2A (Invitrogen) containing the mouse Ig kappa chain leader for secretion and c- myc and poly-His as epitope tags. HEK293 cells were transfected and the binding properties of EO6-scFv secreted into the culture supernatant were found to resemble those of intact EO6. The same construct was then cloned into the pLiv7 liver-specific expression vector and used to generate transgenic (Tg) mice with a C57BL/6 background expressing the apoE promoter-regulated EO6-scFv transgene. The progeny were screened for plasma EO6-scFv titer and transgene insertion by DNA tail PCR amplification. The original EO6-scFv transgenic lines were bred with each other to produce "homozygous" transgenic mice and were in turn bred to produce Ldl ^-/- mice with a C57BL/6 background. All animals were genotyped for EO6-scFv and Ldlr ^-/- , respectively, and plasma was analyzed to confirm EO6-scFv expression by immunoassay. EO6-scFv mRNA was strongly expressed in the liver, peritoneal macrophages, and spleen and, to a lesser extent, in the heart. In this study, plasma levels of EO6-scFv averaged 20-30 µg/mL.

[00158] OxPL Mass Spectrometry . PC-containing phospholipids were isolated from NNCM. The cell media were removed and the cells were washed with PBS. The contents of each well were scraped into 1 ml methanol/acetic acid solution (3% v/v) containing 0.01% BHT, transferred to a 10 ml glass conical tube and capped under N ₂ (gas). Ten nanograms of DNPC was added to each sample for quantitation purposes as an internal standard. Two milliliters of hexane containing BHT was added to the tube, capped under N ₂ (gas), vortexed for five seconds, and then centrifuged for 5 minutes at 3500 rpm. and 4°C. The top hexane layer was then siphoned off using a glass Pasteur pipette and discarded. The hexane/BHT wash was repeated three times, capped under N ₂ (gas), vortexed for five seconds, and centrifuged after each wash. After a final wash with hexane/BHT, 2 ml of chloroform containing BHT and 750 µl of PBS were added to the tube, and then vortexed and centrifuged as described above. The lower organic layer was removed using a glass Pasteur pipette and transferred to a clean 15 ml glass conical tube where the solution was aspirated using a liquid nitrogen vaporizer and then re-diluted in 300 µl chloroform/methanol (2:1 v/v) for storage at -80°C.

[00159] OxPL separation was performed using reverse phase (RP) chromatography. The heart extracts were re-diluted in RP eluent consisting of 60:40 acetonitrile:water, 10 mM ammonium formate and 0.1% formic acid just prior to injection. Thirty microliters of sample was injected onto an Ascentis Express C18 HPLC column (15 cm x 2.1 mm, 2.7 µm; Supelco Analytical, Bellefonte, Pennsylvania, USA) separated by a Prominence UFLC system from Shimadzu Corporation (Canby, Oregon, USA) . Elution was performed using a linear gradient of solvent A (acetonitrile/water, 60:40 v/v) and solvent B (isopropanol/acetonitrile, 90:10, v/v), both solvents containing 10 mM ammonium formate and 0.1% formic acid. The composition of the mobile phase used was as follows: initial solvent B at 32% to 4.00 min; transition to 45% B; 5.00 min 52% B; 8.00 min 58% B; 11.00 min 66% B; 14.00 min 70% B; 18.00 min 75% B; 21.00 min 97% B; 25.00 min 97% B; 25.10 min 32% B. A flow rate of 260 μl/min was used for the assay, and the sample tray and column oven were kept at 4 and 45°C, respectively.

[00160] OxPL detection was performed by mass spectrometry in positive polarity mode. An MRM scan was performed with 6 transitions using the 184.3 m/z ionic product corresponding to the digested phosphocholine residue. Six commercially available standards PONPC, POVPC, PGPC, PAzPC, KOdiAPC and KDdiAPC were injected and accurate peak attribution depended on retention time and mass transitions. Mass spectrometry conditions were as follows: gas curtain, 26 lb/in ² ; impact gas, medium; ion spray voltage, 5500 V; temperature, 500.0°C; ion source gas 1, 40.0 ^psi ; ion source gas 2, 30.0 ^psi ; declustering potential, 125 V, arrival potential, 10 V; impact energy, 53 V; potential at the output of the collision cell, 9 V; and exposure time, 50 ms. External mass calibration was carried out at regular intervals. For quantitation, multiple reaction monitoring (MRM) calibration curves were generated for each of the 6 commercially available OxPL standards and the peaks were normalized to their relative responses. During extraction, ten nanograms of internal standard was added to all samples. A 4000 QTRAP® triple quadrupole mass spectrometer system with a Turbo V electrospray ion source from AB Sciex (Framingham, Massachusetts, USA) was connected to a liquid chromatography system.

[00161] Development of abdominal aortic aneurysms and dilation in a LCWE-induced vasculitis KD mouse model. Kawasaki disease causes persistent coronary arteritis (CA) in young children and is now considered the leading cause of acquired heart disease in children in economically developed countries. In an animal model, CA induced by Lactobacillus casei cell wall extract (LCWE) in mice closely mimics the pathogenesis of KD in humans. Group B L. casei (ATCC 11578) were grown in MRS Lactobacillus broth, harvested by centrifugation during the exponential growth phase, and washed with PBS at pH 7.40. After harvest, cells were treated with 4% SDS overnight and then sequentially incubated with 250 μg/ml RNase, DNase I and trypsin. The final pellet was then sonicated (5 g compacted wet weight in 15 ml PBS) for 2 hours under pulsed conditions of 9 sec pulse/5 sec pause at 20 kHz (Vibra Cell, Sonics & Materials Inc., Newtown, CT). After centrifugation for 1 hour at 20,000×g, the concentration of the supernatant was determined taking into account the content of rhamnose by colorimetric analysis using phenol-sulfuric acid (Dubois et al. 1956). The endotoxin concentration in this preparation was <1.5 pg/μg as determined by reaction with horseshoe crab amoebocyte lysate (Associates of Cape Cod Inc., East Falmouth, MA).

[00162] Four week old C57/BL6 mice were injected with 250 μg of LCWE in PBS or saline alone. Mice were sacrificed at 4 time points - 7, 14, 21 and 28 days. Abdominal and coronary arteries were identified on serial sections (7 μm), fixed with formalin and stained with hematoxylin and eosin. For immunohistochemical analysis, sections were pretreated with 0.3% hydrogen peroxide in PBS for 30 min. Anti-inflammatory marker antibodies or isotype control antibodies were coated in 0.5% bovine serum albumin in PBS at 1:100 for 1 hour. The slides were then washed and a biotinylated horseradish peroxidase-conjugated secondary antibody (Vector Lab, Burlingame, CA) was applied at 1 :500 for 30 min, washed and stained with streptavidin-conjugated horseradish peroxidase at 1:1000 for 30 min. Immunohistochemical staining was determined using the SK-4100 DAB kit according to the manufacturer's instructions (Vector Lab). The data indicate that the formation of LCWE-induced AAA and the histological pattern of intense inflammation in EO6-Tg mice were significantly reduced compared to control mice (Fig.17-21).

[00163] Serum inflammatory cytokine assay. For the simultaneous detection of various cytokines in the plasma of Ldlr ^-/- or Ldlr ^-/- /EO6scFv-Tg mice, which were injected with LCWE and maintained on a HFC diet for 12 weeks, a Bio-Plex Pro 23-plex mouse cytokine immunoassay (Bio- Rad Laboratories, Inc.). Measurements and data analysis of all assays were performed based on the protocol of the Bio-Plex system in combination with the Bio-Plex Manager software. The results are shown in Fig.10. Compared to Ldlr ^-/- mice (n=7-10), it was observed in Ldlr ^-/- EO6-scFv-Tg mice (n=7-10) that plasma levels of several pro-inflammatory cytokines/chemokines (TNFα, CCL2, CCL5 , CXCL1, IL-6, and IL-12) were significantly reduced by Bio-Plex (Bio-Rad) multiplex assays, indicating a generalized reduction in systemic inflammation in EO6scFv-TG Ldlr ^-/- mice.

[00164] In the present description presents a number of embodiments of the invention. Despite this, it should be understood that various modifications can be made without deviating from the scope and essence of the present invention. Thus, other embodiments are within the scope of the following claims.

--->

SEQUENCE LIST

<110>The Regents of the University of California

<120>THERAPEUTIC AGENTS AND METHODS FOR THE TREATMENT OF TLR2-MEDIATED

DISEASES AND DISORDERS

<130>00015-348WO1

<140>Not yet assigned

<141>2019-01-29

<150>US 62/623,276

<151>2018-01-29

<160>14

<170>PatentIn version 3.5

<210>1

<211>930

<212>DNA

<213>Artificial falseness

<220>

<223>Designed domain of scFV antibody E06

<220>

<221>CDS

<222>(1)..(930)

<400>1

atg gag aca gac aca ctc ctg cta tgg gta ctg ctg ctc tgg gtt cca 48

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

ggt tcc act ggt gac gcg gcc cag ccg gcc agg cgc gcc gta cga agc 96

Gly Ser Thr Gly Asp Ala Ala Gln Pro Ala Arg Arg Ala Val Arg Ser

20 25 30

tta gac att gtg atg act cag tct cca tct tcc ctt tct gtg tca gca 144

Leu Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Ala

35 40 45

ggt aag aag gtc acc att agt tgc acg gcc agt gag agc ctt tat tca 192

Gly Lys Lys Val Thr Ile Ser Cys Thr Ala Ser Glu Ser Leu Tyr Ser

50 55 60

agc aaa cac aag gtg cac tac ttg gct tgg tac cag aag aaa cca gag 240

Ser Lys His Lys Val His Tyr Leu Ala Trp Tyr Gln Lys Lys Pro Glu

65 70 75 80

caa tct cct aaa ctg ctg ata tac ggg gca tcc aac cga tac att ggg 288

Gln Ser Pro Lys Leu Leu Ile Tyr Gly Ala Ser Asn Arg Tyr Ile Gly

85 90 95

gtc cct gat cgc ttc aca ggc agt gga tct ggg aca gat ttc act ctg 336

Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

100 105 110

acc atc agc agt gta cag gtt gaa gac ctc aca cat tat tac tgt gca 384

Thr Ile Ser Ser Val Gln Val Glu Asp Leu Thr His Tyr Tyr Cys Ala

115 120 125

cag ttt tac agc tat ccg ctc acg ttc ggt gct ggg acc aag ctg gaa 432

Gln Phe Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu

130 135 140

atc aaa ggt ggt gga gga tca ggt gga ggt ggt tca gga ggt ggc gga 480

Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

tcc gag gtg aag ctg gtg gag tct gga gga ggc ttg gta cag cct ggg 528

Ser Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

165 170 175

ggt tct ctg aga ctc tcc tgt gca act tct ggg ttc acc ttc agt gat 576

Gly Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp

180 185 190

ttc tac atg gag tgg gtc cgc cag gct cca ggg aag aga ctg gag tgg 624

Phe Tyr Met Glu Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp

195 200 205

att gct gca agt aga aac aaa gct aat gat tat aca aca gag tac gct 672

Ile Ala Ala Ser Arg Asn Lys Ala Asn Asp Tyr Thr Thr Glu Tyr Ala

210 215 220

gac tct gtg aag ggt cgg ttc atc gtc tcc aga gac act tcc caa agc 720

Asp Ser Val Lys Gly Arg Phe Ile Val Ser Arg Asp Thr Ser Gln Ser

225 230 235 240

atc ctc tac ctt cag atg aat gcc ctg aga gcc gag gac act gcc att 768

Ile Leu Tyr Leu Gln Met Asn Ala Leu Arg Ala Glu Asp Thr Ala Ile

245 250 255

tat tac tgt gca aga gat tac tac ggt agt agc tac tgg tac ttc gat 816

Tyr Tyr Cys Ala Arg Asp Tyr Tyr Gly Ser Ser Tyr Trp Tyr Phe Asp

260 265 270

gtc tgg ggc gca ggg acc acg gtc acc gtc tcc tct cga gga ggg ccc 864

Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Arg Gly Gly Pro

275 280 285

gaa caa aaa ctc atc tca gaa gag gat ctg aat agc gcc gtc gac cat 912

Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser Ala Val Asp His

290 295 300

cat cat cat cat cat tga 930

His His His His His His

305

<210>2

<211>309

<212>PROTEIN

<213>Artificial poignancy

<220>

<223>Synthetic construction

<400>2

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Ala Ala Gln Pro Ala Arg Arg Ala Val Arg Ser

20 25 30

Leu Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Ala

35 40 45

Gly Lys Lys Val Thr Ile Ser Cys Thr Ala Ser Glu Ser Leu Tyr Ser

50 55 60

Ser Lys His Lys Val His Tyr Leu Ala Trp Tyr Gln Lys Lys Pro Glu

65 70 75 80

Gln Ser Pro Lys Leu Leu Ile Tyr Gly Ala Ser Asn Arg Tyr Ile Gly

85 90 95

Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

100 105 110

Thr Ile Ser Ser Val Gln Val Glu Asp Leu Thr His Tyr Tyr Cys Ala

115 120 125

Gln Phe Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu

130 135 140

Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

165 170 175

Gly Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp

180 185 190

Phe Tyr Met Glu Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp

195 200 205

Ile Ala Ala Ser Arg Asn Lys Ala Asn Asp Tyr Thr Thr Glu Tyr Ala

210 215 220

Asp Ser Val Lys Gly Arg Phe Ile Val Ser Arg Asp Thr Ser Gln Ser

225 230 235 240

Ile Leu Tyr Leu Gln Met Asn Ala Leu Arg Ala Glu Asp Thr Ala Ile

245 250 255

Tyr Tyr Cys Ala Arg Asp Tyr Tyr Gly Ser Ser Tyr Trp Tyr Phe Asp

260 265 270

Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Arg Gly Gly Pro

275 280 285

Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser Ala Val Asp His

290 295 300

His His His His His His

305

<210>3

<211>1554

<212>DNA

<213>Artificial poignancy

<220>

<223>E06 humanized single chain antibody with IgG1-Fc (E06scFv-Fc)

<220>

<221>CDS

<222>(1)..(1554)

<400>3

atg gag acc gac aca ctg ttg ttg tgg gtg ttg ctg ctc tgg gtg cca 48

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

gga agc aca ggt gac gct gct gac atc gtc atg acc cag agc ccc gac 96

Gly Ser Thr Gly Asp Ala Ala Asp Ile Val Met Thr Gln Ser Pro Asp

20 25 30

tct ctc gcg gtt tct ctg gga gag cgg gca aca atc aac tgc aca gca 144

Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Thr Ala

35 40 45

agc gaa tcc ctg tac tca tcc aag cac gtg cat tac ctc gct tgg tac 192

Ser Glu Ser Leu Tyr Ser Ser Lys His Val His Tyr Leu Ala Trp Tyr

50 55 60

cag cag aaa cca ggg caa cca cca aag ctc ctc att tat ggg gcc agc 240

Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser

65 70 75 80

aac aga tat att gga gtc cca gat cga ttc agc ggt tcc ggc tcc gga 288

Asn Arg Tyr Ile Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly

85 90 95

aca gac ttt acc ctc acg ata agc agc ctg cag gcg gaa gat gtg gcc 336

Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala

100 105 110

gtg tat tac tgc gca caa ttc tac agc tat cct ctg acc ttc gga gga 384

Val Tyr Tyr Cys Ala Gln Phe Tyr Ser Tyr Pro Leu Thr Phe Gly Gly

115 120 125

gga aca aaa gtg gag atc aaa ggc gga ggt gga tcc gga ggg ggt gga 432

Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

tct gga ggt ggc ggt agt gaa gtg cag ctg gtg gaa agt gga ggc ggc 480

Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly

145 150 155 160

ctg gtg caa cca ggt ggc tct ctg agg ctg tca tgc gct gcc tct gga 528

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

165 170 175

ttt acc ttc tca gat ttc tac atg gaa tgg gtc aga caa gcc cct gga 576

Phe Thr Phe Ser Asp Phe Tyr Met Glu Trp Val Arg Gln Ala Pro Gly

180 185 190

aag ggg ctc gag tgg gtg gcc gct tcc agg aac aag gct aat gac tac 624

Lys Gly Leu Glu Trp Val Ala Ala Ser Arg Asn Lys Ala Asn Asp Tyr

195 200 205

acc aca gag tac gcc gca agt gtt aaa ggc cgc ttt ata atc tcc cgc 672

Thr Thr Glu Tyr Ala Ala Ser Val Lys Gly Arg Phe Ile Ile Ser Arg

210 215 220

gat gac tct aag aac tcc ttg tac ctt caa atg aat agt ctc aag aca 720

Asp Asp Ser Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr

225 230 235 240

gaa gat aca gcg gta tac tac tgc gcc cgc gac tac tac gga tca agt 768

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Tyr Tyr Gly Ser Ser

245 250 255

tat tgg tac ttc gat gtt tgg aga gct ggc aca ctt gtg act gtc agc 816

Tyr Trp Tyr Phe Asp Val Trp Arg Ala Gly Thr Leu Val Thr Val Ser

260 265 270

agt ctt gat cct aaa tcc tct gac aag acc tat acc tgc cca cct tgt 864

Ser Leu Asp Pro Lys Ser Ser Asp Lys Thr Tyr Thr Cys Pro Pro Cys

275 280 285

ccc gcc cca gaa ctt ctg ggt ggc cca tcc gtg ttt ctg ttc cca cca 912

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

290 295 300

aag cca aag gat aca ctc atg atc tct cgc act ccg gaa gtc acg tgc 960

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

305 310 315 320

gtc gtg gtt gat gtg tca cac gag gac ccg gag gtc aaa ttc aat tgg 1008

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

325 330 335

tac gtg gac gga gtc gag gtg cac aac gcc aag aca aag cca cgc gaa 1056

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

340 345 350

gag cag tac aac agc acg tat aga gta gtg agc gtg ctg aca gtg ctc 1104

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

355 360 365

cac cag gat tgg ctt aac ggt aag gaa tac aag tgt aag gtc tcc aac 1152

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

370 375 380

aaa gct ctt cct gct cca ata gaa aag acc att tca aag gcc aag ggg 1200

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

385 390 395 400

caa cct cga gaa ccc cag gtg tac acg ctg cct ccc agc cga gag gag 1248

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

405 410 415

atg acc aag aac caa gta agt ctg aca tgc ctt gtc aaa ggg ttc tac 1296

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

420 425 430

ccc tca gac atc gcc gtg gaa tgg gaa agc aac ggt caa ccc gaa aac 1344

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

435 440 445

aat tac aag aca acg cca ccg gta ctc gat tcc gat ggt tcc ttt ttt 1392

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

450 455 460

ctg tac tcc aaa ctc acg gtg gac aag agt cga tgg cag cag gga aac 1440

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

465 470 475 480

gtt ttc tcc tgt tcc gtg atg cac gaa gca ctg cac aat cac tat acc 1488

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

485 490 495

cag aag tca ctg agt ttg agc cct ggc aaa gga ggg ggc gga tca cat 1536

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser His

500 505 510

cat cac cat cac cat taa 1554

His His His His His His

515

<210>4

<211>517

<212>PROTEIN

<213>Artificial poignancy

<220>

<223>Synthetic construction

<400>4

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Ala Ala Asp Ile Val Met Thr Gln Ser Pro Asp

20 25 30

Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Thr Ala

35 40 45

Ser Glu Ser Leu Tyr Ser Ser Lys His Val His Tyr Leu Ala Trp Tyr

50 55 60

Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser

65 70 75 80

Asn Arg Tyr Ile Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly

85 90 95

Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala

100 105 110

Val Tyr Tyr Cys Ala Gln Phe Tyr Ser Tyr Pro Leu Thr Phe Gly Gly

115 120 125

Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly

145 150 155 160

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

165 170 175

Phe Thr Phe Ser Asp Phe Tyr Met Glu Trp Val Arg Gln Ala Pro Gly

180 185 190

Lys Gly Leu Glu Trp Val Ala Ala Ser Arg Asn Lys Ala Asn Asp Tyr

195 200 205

Thr Thr Glu Tyr Ala Ala Ser Val Lys Gly Arg Phe Ile Ile Ser Arg

210 215 220

Asp Asp Ser Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr

225 230 235 240

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Tyr Tyr Gly Ser Ser

245 250 255

Tyr Trp Tyr Phe Asp Val Trp Arg Ala Gly Thr Leu Val Thr Val Ser

260 265 270

Ser Leu Asp Pro Lys Ser Ser Asp Lys Thr Tyr Thr Cys Pro Pro Cys

275 280 285

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

290 295 300

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

305 310 315 320

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

325 330 335

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

340 345 350

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

355 360 365

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

370 375 380

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

385 390 395 400

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

405 410 415

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

420 425 430

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

435 440 445

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

450 455 460

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

465 470 475 480

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

485 490 495

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser His

500 505 510

His His His His His His

515

<210>5

<211>5

<212>PROTEIN

<213>Artificial poignancy

<220>

<223>Peptide linker

<400>5

Gly Gly Gly Gly Ser

15

<210>6

<211>7

<212>PROTEIN

<213>Artificial poignancy

<220>

<223>CDR1 VH

<400>6

Gly Phe Thr Phe Ser Asp Phe

15

<210>7

<211>8

<212>PROTEIN

<213>Artificial poignancy

<220>

<223>CDR2 VH

<400>7

Arg Asn Lys Ala Asn Asp Tyr Thr

15

<210>8

<211>16

<212>PROTEIN

<213>Artificial poignancy

<220>

<223>CDR3 VH

<400>8

Cys Ala Arg Asp Tyr Tyr Gly Ser Ser Tyr Trp Tyr Phe Asp Val Trp

1 5 10 15

<210>9

<211>17

<212>PROTEIN

<213>Artificial poignancy

<220>

<223>CDR1 VL

<400>9

Thr Ala Ser Glu Ser Leu Tyr Ser Ser Lys His Lys Val His Tyr Leu

1 5 10 15

Ala

<210>10

<211>7

<212>PROTEIN

<213>Artificial poignancy

<220>

<223>CDR2 VL

<400>10

Gly Ala Ser Asn Arg Tyr Ile

15

<210>11

<211>10

<212>PROTEIN

<213>Artificial poignancy

<220>

<223>CDR3 VL

<400>11

Cys Ala Gln Phe Tyr Ser Tyr Pro Leu Thr

1 5 10

<210>12

<211>16

<212>PROTEIN

<213>Artificial poignancy

<220>

<223>CDR1 VL-humanized

<400>12

Thr Ala Ser Glu Ser Leu Tyr Ser Ser Lys His Val His Tyr Leu Ala

1 5 10 15

<210>13

<211>1587

<212>DNA

<213>Artificial poignancy

<220>

<223>Sequence of chimeric antibody E06scFv with human IgG1-Fc

<220>

<221>CDS

<222>(1)..(1587)

<400>13

atg gag aca gac aca ctc ctg cta tgg gta ctg ctg ctc tgg gtt cca 48

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

ggt tcc act ggt gac gcg gcc cag ccg gcc agg cgc gcc gta cga agc 96

Gly Ser Thr Gly Asp Ala Ala Gln Pro Ala Arg Arg Ala Val Arg Ser

20 25 30

tta gac att gtg atg act cag tct cca tct tcc ctt tct gtg tca gca 144

Leu Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Ala

35 40 45

ggt aag aag gtc acc att agt tgc acg gcc agt gag agc ctt tat tca 192

Gly Lys Lys Val Thr Ile Ser Cys Thr Ala Ser Glu Ser Leu Tyr Ser

50 55 60

agc aaa cac aag gtg cac tac ttg gct tgg tac cag aag aaa cca gag 240

Ser Lys His Lys Val His Tyr Leu Ala Trp Tyr Gln Lys Lys Pro Glu

65 70 75 80

caa tct cct aaa ctg ctg ata tac ggg gca tcc aac cga tac att ggg 288

Gln Ser Pro Lys Leu Leu Ile Tyr Gly Ala Ser Asn Arg Tyr Ile Gly

85 90 95

gtc cct gat cgc ttc aca ggc agt gga tct ggg aca gat ttc act ctg 336

Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

100 105 110

acc atc agc agt gta cag gtt gaa gac ctc aca cat tat tac tgt gca 384

Thr Ile Ser Ser Val Gln Val Glu Asp Leu Thr His Tyr Tyr Cys Ala

115 120 125

cag ttt tac agc tat ccg ctc acg ttc ggt gct ggg acc aag ctg gaa 432

Gln Phe Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu

130 135 140

atc aaa ggt ggt gga gga tca ggt gga ggt ggt tca gga ggt ggc gga 480

Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

tcc gag gtg aag ctg gtg gag tct gga gga ggc ttg gta cag cct ggg 528

Ser Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

165 170 175

ggt tct ctg aga ctc tcc tgt gca act tct ggg ttc acc ttc agt gat 576

Gly Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp

180 185 190

ttc tac atg gag tgg gtc cgc cag gct cca ggg aag aga ctg gag tgg 624

Phe Tyr Met Glu Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp

195 200 205

att gct gca agt aga aac aaa gct aat gat tat aca aca gag tac gct 672

Ile Ala Ala Ser Arg Asn Lys Ala Asn Asp Tyr Thr Thr Glu Tyr Ala

210 215 220

gac tct gtg aag ggt cgg ttc atc gtc tcc aga gac act tcc caa agc 720

Asp Ser Val Lys Gly Arg Phe Ile Val Ser Arg Asp Thr Ser Gln Ser

225 230 235 240

atc ctc tac ctt cag atg aat gcc ctg aga gcc gag gac act gcc att 768

Ile Leu Tyr Leu Gln Met Asn Ala Leu Arg Ala Glu Asp Thr Ala Ile

245 250 255

tat tac tgt gca aga gat tac tac ggt agt agc tac tgg tac ttc gat 816

Tyr Tyr Cys Ala Arg Asp Tyr Tyr Gly Ser Ser Tyr Trp Tyr Phe Asp

260 265 270

gtc tgg ggc gca ggg acc acg gtc acc gtc tcc tct ctg gac ccg aag 864

Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Leu Asp Pro Lys

275 280 285

tct tct gac aaa act tac aca tgc cca ccg tgc cca gca cct gaa ctc 912

Ser Ser Asp Lys Thr Tyr Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

290 295 300

ctg ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc 960

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

305 310 315 320

ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg 1008

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

325 330 335

agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg 1056

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

340 345 350

gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac aac agc 1104

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

355 360 365

acg tac cgt gtg gtc agc gtc ctg acc gtc ctg cac cag gac tgg ctg 1152

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

370 375 380

aat ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc 1200

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

385 390 395 400

ccc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca 1248

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

405 410 415

cag gtg tac acc ctg ccc cca tcc cgg gag gag atg acc aag aac cag 1296

Gln Val Tyr Thr Leu Pro Ser Arg Glu Glu Met Thr Lys Asn Gln

420 425 430

gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc 1344

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

435 440 445

gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc acg 1392

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

450 455 460

cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc 1440

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

465 470 475 480

acc gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc 1488

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

485 490 495

gtg atg cac gag gct ctg cac aac cac tac acg cag aag agc ctc tcc 1536

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

500 505 510

ctg tct ccg ggt aaa ggt gga ggt gga tca cat cat cat cat cat cat cat 1584

Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser His His His His His His

515 520 525

taa 1587

<210>14

<211>528

<212>PROTEIN

<213>Artificial poignancy

<220>

<223>Synthetic construction

<400>14

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Ala Ala Gln Pro Ala Arg Arg Ala Val Arg Ser

20 25 30

Leu Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Ala

35 40 45

Gly Lys Lys Val Thr Ile Ser Cys Thr Ala Ser Glu Ser Leu Tyr Ser

50 55 60

Ser Lys His Lys Val His Tyr Leu Ala Trp Tyr Gln Lys Lys Pro Glu

65 70 75 80

Gln Ser Pro Lys Leu Leu Ile Tyr Gly Ala Ser Asn Arg Tyr Ile Gly

85 90 95

Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

100 105 110

Thr Ile Ser Ser Val Gln Val Glu Asp Leu Thr His Tyr Tyr Cys Ala

115 120 125

Gln Phe Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu

130 135 140

Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

165 170 175

Gly Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp

180 185 190

Phe Tyr Met Glu Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp

195 200 205

Ile Ala Ala Ser Arg Asn Lys Ala Asn Asp Tyr Thr Thr Glu Tyr Ala

210 215 220

Asp Ser Val Lys Gly Arg Phe Ile Val Ser Arg Asp Thr Ser Gln Ser

225 230 235 240

Ile Leu Tyr Leu Gln Met Asn Ala Leu Arg Ala Glu Asp Thr Ala Ile

245 250 255

Tyr Tyr Cys Ala Arg Asp Tyr Tyr Gly Ser Ser Tyr Trp Tyr Phe Asp

260 265 270

Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Leu Asp Pro Lys

275 280 285

Ser Ser Asp Lys Thr Tyr Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

290 295 300

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

305 310 315 320

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

325 330 335

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

340 345 350

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

355 360 365

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

370 375 380

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

385 390 395 400

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

405 410 415

Gln Val Tyr Thr Leu Pro Ser Arg Glu Glu Met Thr Lys Asn Gln

420 425 430

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

435 440 445

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

450 455 460

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

465 470 475 480

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

485 490 495

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

500 505 510

Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser His His His His His His

515 520 525

<---

Claims (17)

1. The use of an oxidized phospholipid (OxPL) specific binding polypeptide in the treatment of Kawasaki disease, where the polypeptide inhibits the biological activity of OxPL and where the polypeptide contains: a heavy chain variable domain (VH) containing the complementarity determining regions (CDRs) of the VH amino acid sequence shown in SEQ ID NO: 2, and a light chain variable domain (VL) containing the CDRs of the VL amino acid sequence shown in SEQ ID NO: 2 or 4. 2. The use of claim 1 further comprising administering to the subject an additional therapeutic agent useful in the treatment of Kawasaki disease. 3. Use according to claim 1, further comprising administering intravenous immunoglobulin and/or salicylate to the individual. 4. Use according to any one of claims 1 to 3, wherein the subject is a human less than five years of age. 5. Use according to any one of claims 1 to 4, wherein the biological activity of OxPL comprises activation of the CD36-TLR2 apoptosis pathway. 6. Use according to any one of claims 1 to 5, wherein the polypeptide comprises a single chain variable fragment (ScFv). 7. Use according to any one of claims 1 to 6, wherein the polypeptide is administered intravascularly. 8. Use according to any one of claims 1 to 7, wherein VH and VL are connected to an Fc or FC2 region. 9. Use according to claim 6, wherein the scFv binds to the phosphocholine end group of the oxidized phospholipid and/or where the scFv is physiologically soluble. 10. Use according to claim 1, wherein VH contains the CDR amino acid sequences shown in SEQ ID NOs: 6, 7 and 8 and VL contains the CDR amino acid sequences shown in SEQ ID NOs: 9, 10 and 11. 11. Use according to claim 10, wherein VH contains an amino acid sequence that is at least 95% identical to the amino acid sequence of VH shown in SEQ ID NO: 2 and VL contains an amino acid sequence that is at least 95% identical to the amino acid sequence VL shown in SEQ ID NO: 2. 12. Use according to claim 10, wherein VH contains the VH amino acid sequence shown in SEQ ID NO: 2 and VL contains the VL amino acid sequence shown in SEQ ID NO: 2. 13. Use according to claim 1, wherein VH contains the CDR amino acid sequences shown in SEQ ID NOs: 6, 7 and 8 and VL contains the CDR amino acid sequences shown in SEQ ID NOs: 12, 10 and 11. 14. Use according to claim 13, wherein VH contains an amino acid sequence that is at least 95% identical to the amino acid sequence of VH shown in SEQ ID NO: 4 and VL contains an amino acid sequence that is at least 95% identical to the amino acid sequence VL shown in SEQ ID NO: 4. 15. Use according to claim 13, wherein VH contains the VH amino acid sequence shown in SEQ ID NO: 4 and VL contains the VL amino acid sequence shown in SEQ ID NO: 4.

Images