HK1168362B - Antibodies that recognize sulphatides and sulphated proteoglycans and the use thereof - Google Patents

Description

Anti-sulfatide and anti-sulfated proteoglycan antibodies and uses thereof

Technical Field

The present invention relates to novel monoclonal antibodies (MAbs) that specifically and with high affinity recognize sulfatides and sulfated proteoglycans. The invention also relates to pharmaceutical compositions comprising the monoclonal antibodies of the invention or fragments derived from these antibodies. In addition, the present invention relates to a kit for diagnosing cardiovascular diseases, comprising the antibody or fragment thereof of the present invention.

Background

After the hybridoma technology (Koehler y Milstein Nature, 256: 495-497, 1975) for obtaining murine MAbs developed for more than 30 years, it has proved very useful in disease diagnosis and basic research, but only 20 antibodies for human therapy have been registered (Pharmavitae, Monoclonal Abs Update, 6-363, 2008). This is largely due to their short half-life in blood, poor recognition of murine effector functions by the human immune system, and the immune response elicited by these antibodies when injected into patients due to their murine origin (HAMA response, acronym of "human anti-mouse antibody"). Several studies have demonstrated that: following administration of the foreign antibody, the immune response generated in the patient may be quite strong and can substantially eliminate the therapeutic utility of the antibody following the initial treatment. Furthermore, according to Khazaeli, m.b.y col.journal of Immunotherapy 15: 42-52(1994), that subsequent treatment with irrelevant murine antibodies after administration of murine MAbs to patients may be ineffective or even dangerous due to cross-reactive HAMA responses. From the above information it can be seen that there is a need to obtain therapeutic antibodies in a form that is less immunogenic in humans, that can be easily and economically obtained, and that are suitable for the preparation of therapeutic formulations and other uses. Morrison s.l.y col.adv immunol., 44: 65-92(1989).

Several methods have been developed to humanize antibodies from mice or rats, thus reducing the xenogenic response to these foreign proteins when injected into humans. One of the initial attempts to reduce immunogenicity was to generate "chimeric" antibodies in which the variable domains of murine proteins were linked to constant domains of human molecules, achieving not only reduced immunogenicity, but also activation of immune effector functions. Morrison s.l.y col.pnas USA, 81: 6851-6855(1984). These chimeric molecules retain the characteristics of the original antibody associated with antigen binding, while their constant regions are non-immunogenic.

Atherosclerosis and its consequences have had a significant impact on people in the world, which since several years ago was the major cause of morbidity and mortality in developed countries (Meli a n, A.y col. am. j. pathol., 155: 775, 1999) and cuba (OMS, 2004, Anuario Estad ustico, MINSAP, 2007). Atherosclerosis is a chronic inflammatory disease of a multifactorial nature that contributes greatly to the onset of myocardial and cerebral infarction, gangrene and loss of limb function. Great, d.r.y col.trends Immunol 22: 180-181 (2001); ross, R.y col. am Heart J138 (5Pt 2): s419-20 (1999). One of the major causes of atherosclerosis is hypercholesterolemia. By interacting with proteoglycans, the transport of low molecular weight lipoproteins (LDL) through the arterial wall is blocked in the extracellular matrix of the intima of the artery and undergoes oxidative modification. Lipoproteins that bind to proteoglycans of the intima of arteries are more prone to changes in the lipid and protein moieties, such as oxidation and enzymatic hydrolysis, which increase their atherogenic potential. ApoB-100 contains several regions through which it can bind to glycosaminoglycan chains of proteoglycans, which are common in that a plurality of basic amino acids are present. Camejo, g., E.y col. 205-22, (1998); chang, t.y.col.curr Opinllipdol 12: 289-96 (2001); camejo, g., U.y col. atheroscler Suppl 3: 3-9(2002). The density of negative charges on glycosaminoglycans affects their interaction with LDL, and the degree of sulfation affects the interaction of LDL with proteoglycans. Sambandam T.y col. 561-568(1991).

In addition, oxidized LDL can be internalized by macrophages through scavenger receptors on the macrophage surface, which results in the accumulation of intracellular cholesterol, followed by the formation of foam cells. These events represent the major steps in the initiation of an immune response, involving monocytes/macrophages, mast cells, dendritic cells, T cells and NKTs. Camejo, G.y col. atherosclerosis139 (2): 205-22 (1998); Hurt-Camejo, E.y col. invest Clin 42Suppl 1: 43-73 (2001); skalen, k., M.y col. nature 417: 750-754(2002).

There is experimental evidence to prove that: proteoglycans present on the surface of macrophages are involved in the binding of oxidized LDL to these cells and the internalization or incorporation of these particles, which ultimately leads to the formation of foam cells. Halvorsen B.y col. biochem j.331: 743-752(1998).

There is no doubt that adopting a healthier lifestyle and using anti-thrombotic and lipid-lowering agents has had an impact on reducing the risk of cardiovascular events, but these strategies are still insufficient to completely eliminate these risks.

As mentioned above, atherosclerosis is a multifactorial inflammatory disease in which multiple antigens are of great significance for its development, so different strategies for active and passive immunotherapy are being developed which aim at achieving greater therapeutic effects on the disease.

One of these strategies is to increase HDL, as HDL-cholesterol has an inverse relationship to cardiovascular disease. CETP is a key enzyme in HDL metabolism and is considered a potential target for therapy, as a reduction in its activity increases HDL levels. Strategies using vaccines that induce antibodies capable of binding to and inhibiting CETP function have been described in WO1997/041227 and WO 2006/133196. However, recent studies have shown that phase III clinical trials using the CETP inhibitor Torcetrapib have failed, which has raised doubt about this strategy. Nichols s.j.y col.circulation.9; 118: 2506-14 (2008); hermann M.y col. curr Hypertens Rep, 11: 76-80(2009).

Some authors have described vaccines using oxidized LDL as an immunogen, aimed at inhibiting the formation of atherosclerotic plaques. Palinski W.y col, proc.natl.acad.sci.usa 92: 821-25 (1995); ameli S y col, arterioscler, thromb, vasc, biol, 16: 1074-79 (1996); freigang S.y col, arterioscl, thromb, vasc, biol, 18: 1972-829 (1998); zhou X.y col, arterioscler, thromb, vasc, biol.21: 108-14 (2001); george J.y col. 147-52 (1998); fredrikson g.n.y col.arterioscler.thromb.vasc.biol.23: 879-84 (2003); US 2008/0070265a 1. Another strategy is to develop prophylactic and therapeutic vaccines based on specific fragments of the oxidized apolipoprotein C-III aimed at inducing an immune response capable of preventing or reducing the formation of atherosclerotic lesions (WO 2001/064008, WO 2003/020765, WO 2004/080375 and WO 2004/081045).

Another vaccine approach described is based on peptides conjugated to aldehydes such as MDA or 4-HNE to induce antibodies that interact with the alpha/beta receptor of T cells to prevent the formation of atherosclerotic lesions (WO 2001/068119).

Several authors support the importance of vaccines against pathogens in atherosclerosis to prevent the formation of atherosclerotic plaques (WO1998/033510, US 006291437B1, US6471965B1, US006808713B 1).

Another proposed strategy for delaying or reducing the severity of atherosclerosis due to dietary cholesterol intake is the use of vaccines against sterols (US 2002/0018808a 1).

Passive immunotherapy as a therapeutic tool may also play an important role in atherosclerosis. Passive immunization for the treatment or prevention of atherosclerosis by using human antibodies directed against oxidized or modified fragments of Apo B100 has been described (US 005196324a, US2007/0098725a1, US 2008/0075716a 1).

In addition, passive immunization using specific antibodies against phosphorylcholine has been proposed as a therapeutic combination for the treatment or prevention of atherosclerosis (US 2007/0286868a1, US2007/0122419a 1).

Another strategy described is the use of antibodies or antigen-binding fragments that specifically bind to human M-CSF (US 2007326414B 2).

Another treatment for this disease is: mabs that prevent adhesion of monocytes to the vascular endothelium and thus invasion of the endothelium and surrounding tissue were used (US 005541296 a).

The use of monoclonal antibodies as glycoprotein IIb/IIIa receptor inhibitors and thus as inhibitors of platelet aggregation has been described (WO 1999/052551, US 005976532A).

In addition, human monoclonal antibodies directed against protective peptide epitopes of apolipoprotein CIII were obtained for use in passive immunotherapy (WO 2004/081046).

In addition, the use of intravenous immunoglobulin (IVIG) may have an effect of preventing arteriosclerosis undi N y col. 445-452(2008).

Chimeric mabs that react with sulfatides and sulfated proteoglycans, or recognize macrophages and atherosclerotic lesions, have the ability to inhibit the formation of atherosclerotic lesions when administered at low doses, and induce antibody responses against these sulfated molecules have not been described.

Disclosure of Invention

The present invention relates to monoclonal antibodies characterized by recognizing sulfatide and sulfated proteoglycan or fragments derived therefrom.

The anti-sulfatide and anti-sulfated proteoglycan antibodies of the invention are preferably monoclonal. Also included within the scope of the invention are antibody fragments, such as the Fab fragments, Fab '-SH, and F (ab') 2 of the anti-sulfatide and anti-sulfation proteoglycan antibodies provided in the specification. These antibody fragments may be produced by conventional means, such as enzymatic digestion, or may be produced by recombinant techniques. These antibody fragments may be chimeric or humanized. These fragments are used for diagnostic and therapeutic purposes as described in the present specification. The invention also includes embodiments of substantially pure antibodies and fragments.

In particular embodiments, the antibodies of the invention are characterized by the following heavy and light chain variable region sequences.

Heavy chain:

HCDR1 RYSVH

HCDR2 MIWGGGSTDYNSALKS

HCDR3 SGVRRGRAQAWFAY

HFR1 QVQLKESGPGLVAPSQSLSITCTVSGFSLS

HFR2 WVRQPPGKGLEWLG

HFR3 RLSISKDNSKSQVFLKMNSLQTDDTAMYYCAR

HFR4 WGQGTLVTVSA

light chain:

LCDR1 KASQDVSTAVA

LCDR2 SASYRYT

LCDR3 QQHYSTPWT

LFR1 DIVMTQSHKFMSTSVGDRVSITC

LFR3 GVPDRFTGSGSGTDFTFTISSVQAEDLAVYYC

LFR4 FGGGTKLELK

in addition, the antibodies of the invention include: for the heavy chain is the human IgG1 constant region; for the light chain, human ck.

The present invention includes compositions, including pharmaceutical compositions, comprising the anti-sulfatide and anti-sulfated proteoglycan antibodies of the present invention or fragments derived therefrom. The compositions as used in the present specification include one or more antibodies that bind to sulfatides and sulfated proteoglycans.

These compositions may also comprise suitable carriers, such as pharmaceutically acceptable excipients, including buffers or adjuvants, which are well known in the art.

In another embodiment, the invention relates to a pharmaceutical composition comprising a MAb whose heavy and light chain variable region sequences are shown below.

Heavy chain:

HCDR1 RYSVH

HCDR2 MIWGGGSTDYNSALKS

HCDR3 SGVRRGRAQAWFAY

HFR1 QVQLKESGPGLVAPSQSLSITCTVSGFSLS

HFR2 WVRQPPGKGLEWLG

HFR3 RLSISKDNSKSQVFLKMNSLQTDDTAMYYCAR

HFR4 WGQGTLVTVSA

light chain:

LCDR1 KASQDVSTAVA

LCDR2 SASYRYT

LCDR3 QQHYSTPWT

LFR1 DIVMTQSHKFMSTSVGDRVSITC

LFR2 WYQQKPGQSPKLLIY

LFR3 GVPDRFTGSGSGTDFTFTISSVQAEDLAVYYC

LFR4 FGGGTKLELK

in a third aspect, the invention relates to a kit for diagnosing atherosclerotic lesions comprising one of the antibodies of the invention or a fragment derived therefrom. More specifically, the reagent set comprises mabs having the variable region sequences of the heavy and light chains as described above.

In another aspect, the invention relates to the use of the antibodies of the invention for the treatment of cardiovascular diseases, especially those that show signs of atherosclerotic lesions.

The term antibody refers generally to a MAb, and more specifically to a murine MAb or a chimeric antibody.

Obtaining of the antibody:

in general, the anti-sulfatide and anti-sulfated proteoglycan mabs of the present invention can be obtained by hybridoma method from mice immunized with glycolipid extracts obtained from natural or synthetic sources, the method first described in Kohler et al, Nature, 256: 495(1975). Splenocytes from immunized mice were fused with myeloma cells p3.x63ag86.5.3, cultured on selective media as described, and productive clones were selected by detecting immunoglobulins in culture supernatants by ELISA.

After identification of hybridoma cells that produce antibodies with the desired specificity, affinity, and/or activity, the antibody-producing clones can be subcloned by limiting dilution procedures and grown by standard methods of cell culture growth (Goding, Monoclonal Abs: Principles and Practice, p. lines.59-103 (Academic Press, 1986)). Suitable media for this purpose include, for example, the media D-MEM or RPMI-1640. In addition, hybridoma cells can be grown in animals as ascites tumors.

The mabs secreted by the subclones are suitably isolated from the culture medium, ascites fluid or serum by conventional procedures for purification of the immunoglobulins, e.g. by a-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

The antibody of the present invention can also be obtained by properly manipulating the mouse immunoglobulin gene by genetic engineering techniques. For example, the nucleic acid can be obtained by conventional genetic manipulation techniques such as those described in the art, particularly, for example, amplification, Cloning, gene sequencing and digestion, such as those described in Sambrook et al, Molecular Cloning: a Laboratory Manual 3rd. edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, n.y.current Protocols in molecular biology (f.m. ausubel, et al. edition, (2003)); la series Methods in envimology (Academic Press, Inc.): and (3) PCR 2: a practical proproach (M.J. MacPherson, B.D. Hames yG.R.Taylor eds. (1995)), Harlow Lane, eds. (1988) ABS, antibody mechanical, y Animal cell culture (R.I. Freshney, ed. (1987)), chimeric antibodies of the invention were obtained from RNA purified from cells producing murine monoclonal antibodies.

cDNA synthesis and PCR (acronym for polymerase chain reaction) amplification of antibody variable regions can be performed from RNA encoding murine antibodies, cDNA is synthesized, and VK and VH variable regions are amplified by PCR, which can be performed by conventional techniques described in the art for this purpose.

The PCR products of each of the heavy and light chains were cloned separately into vectors for gene sequencing. The resulting clones are sequenced using any of the methods described for this purpose, e.g., using T7DNA polymerase, using the dideoxynucleotide method, according to the manufacturer's instructions.

The variable region genes for the heavy chain VH and light chain VK were obtained by enzymatic restriction digestion of intermediate constructs and cloned into respective expression vectors according to conventional techniques for constructing chimeric genes. Any of the vectors described can be used for this purpose for efficient expression of recombinant proteins, in particular MAbs.

For expression of the chimeric antibody, NS0 cells can be used, which are electroporated with the DNA construct in each expression vector containing the antibody gene. These cells were grown in selective media. Immunoglobulin-producing clones were detected by assaying culture supernatants using ELISA (enzyme-linked immunosorbent assay).

Selection of antibodies with the desired specificity and function:

in some embodiments, the antibodies of the invention can be detected by a variety of techniques described in the art for this purpose, such as ELISA.

In some embodiments of the invention, the antibodies produced are assayed for biological activity. In some embodiments, antibodies of the invention are tested for their activity in binding to an antigen.

Antigen binding assays known in the art and useful in the present specification include, inter alia: direct or competitive binding assays using techniques such as Western blotting, radioimmunoassay, ELISA, double-antibody immunoassays (sandwich), immunoprecipitation assays, fluorescent immunoassays, and protein a immunoassays. Exemplary assays for antigen binding are included in the examples section that follows.

In addition, in the present invention, clones producing antibodies capable of identifying atherosclerotic plaques in human aortic tissue sections can be identified, which can be performed using conventional immunohistochemical techniques described in the art.

In another aspect, the ability of an antibody of the invention to induce an anti-heparin response in a mouse can be determined. To this end, different groups of animals are immunized with the antibodies of the invention and serum samples of these animals are tested for the presence of anti-heparin antibodies.

In a further aspect, the anti-atherosclerotic effect of the antibodies of the invention may be determined, for which a model of the generation of atherosclerotic lesions in rabbits induced by lipocalin (Lipofundin) may be used (Tak & cs E, H & rs J, F zesi S, Jellinek H.1986Arterio scleroderma in admixture. Morp Igazsaggy Orv Sz.26: 99-105; Noa M & A S R (1992) Aerosol y leisi.

Pharmaceutical composition

In one embodiment, the invention provides a pharmaceutical composition comprising one or more antibodies of the invention. In one embodiment, the composition comprising the antibody further comprises a pharmaceutically acceptable excipient.

In one aspect, the invention provides a kit comprising one or more antibodies of the invention, and may further comprise a buffer solution. In one embodiment, the buffered solution is pharmaceutically acceptable. In one embodiment, the composition comprising an antibody further comprises a carrier molecule, which in some embodiments is pharmaceutically acceptable. In one embodiment, the kit further comprises instructions for administering or using the composition, e.g., antibody, to a subject.

Pharmaceutical compositions comprising The antibodies of The invention are prepared by mixing The antibodies of The desired purity with a physiologically acceptable carrier molecule, optionally an excipient or stabilizer, to preserve them (Remington: The Science and Practice of Pharmacy 20th edition (2000)) in The form of an aqueous solution, lyophilized or other lyophilized formulation. The acceptable vehicle, excipient, or stabilizer is non-toxic to the recipient at all dosages and concentrations employed.

The mabs of the present invention are present in combination in a pharmaceutical composition in an amount effective for the desired purpose.

Formulations for in vivo administration must be sterile. This is achieved by filtration through sterile filtration membranes.

In one aspect, the invention shows how to use an antibody of the invention for the preparation of a medicament for the therapeutic and/or prophylactic treatment of a disorder, such as a cardiovascular disease.

The antibodies of the invention are useful for treating, inhibiting, delaying the progression of, preventing/delaying the onset of, ameliorating, or preventing a disease, disorder or process associated with the expression and/or activity of one or more antigenic molecules.

According to the invention, the therapeutic dose of these antibodies will be from 10. mu.g to 10mg per dose, preferably from 100. mu.g to 1mg per dose.

The mabs of the present invention are administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal routes, and if local treatment is desired, by intralesional (intradivision) routes.

In another aspect, the invention provides kits for diagnosing a disorder, such as a cardiovascular disease.

Examples

The following examples are intended to illustrate the invention without limiting its scope.

In the following examples, all restriction or modification enzymes and reagents and materials used were obtained commercially, unless otherwise indicated.

Example 1 recognition of bovine cerebrosulfate by chimeric MAb anti-SO 3

PolySorp plates, Nunc, were coated with a solution of bovine brain sulfatide at a concentration of 4. mu.g/mL in methanol at 50. mu.L/well using ELISA, and the solvent was evaporated off by incubation at 37 ℃ for 90 minutes. Then, the plate was blocked with Phosphate Buffered Saline (PBS) containing 1% bovine Serum Albumin (SAB) at 200. mu.L/well for 1 hour at room temperature. Then, 50. mu.L/well of chimeric antibody anti-SO 3 at various concentrations in PBS was added and incubated for 1 hour at 37 ℃. The plates were then washed with PBS and 50 μ L/well of goat antiserum anti-human γ chain conjugated to alkaline phosphatase (Sigma) was added. The plate at 37 degrees C temperature 1 h incubation, then again washing the plate, and adding 100u L/hole substrate solution, which is pH9.8 diethanolamine buffer in 1mg/mL p-nitrophenyl phosphate. After incubation for 30 minutes at room temperature, the absorbance of the reaction product was measured at 405nm in an ELISA reader.

As a negative control, a chimeric MAb modified by replacing R in the heavy chain variable region at position 98 of the chimeric monoclonal anti-SO 3 with S was used. Figure 1 shows the reactivity of different chimeric mabs against sulfatide. The figure shows that the chimeric monoclonal anti-SO 3 recognizes sulfatide even at concentrations as low as 0.01 mg/ml. In contrast, chimeric mabs modified at position 98 did not show any reactivity.

Example 2 heparin identification test

It was subsequently determined whether the chimeric monoclonal anti-SO 3 recognized sulfated molecules that were more complex than sulfatides. Heparin was chosen for this study, a highly sulfated molecule that serves as a model for sulfated glycosaminoglycans.

The test for anti-heparin reactivity was performed based on the ELISA technique developed for biglican by Skalen, K.M.y cols (Nature 417: 750-754, 2002) with minor modifications. Maxisorp microtiter plates (Nunc) were coated with 10. mu.g/mL (100. mu.L/well) heparin (Sigma) in Hepes Buffered Saline Solution (HBSS) (20mM Hepes, 150mM NaCl, pH7.4) and incubated overnight at 4 ℃. The plate was washed 3 times with HBSS and then blocked with HBSS containing 1% SAB (HBSS-BSA) for 1 hour at room temperature. The plates were washed 3 times with 0.02% HBSS-Tween20(HBSS-T) and serial dilutions of chimeric monoclonal anti-SO 3 were added over 1 hour at room temperature: from binding buffer (10mM Hepes, 20mM NaCl, 2mM CaCl₂，2mM MgCl₂pH7.4) of the starting concentration of 40. mu.g/mL. As a negative control, a chimeric antibody modified by replacing R in the heavy chain variable region at position 98 of the chimeric monoclonal antibody-SO 3 with S was used. The plates were washed 2 times with HBSS-T and then incubated with goat antiserum anti-human gamma chain conjugated to alkaline phosphatase (Sigma-Aldrich, USA) in HBSS-T (containing 0.1% SAB) for 1 hour at room temperature. The desired wash was performed and the reaction was developed using the substrate p-nitrophenyl phosphate dissolved in diethanolamine buffer, pH 9.8. The absorbance of the product was quantified at 405nm in an ELISA reader (Organon Teknica, Austria).

As shown in fig. 2, the chimeric monoclonal anti-SO 3 has high reactivity against heparin. In contrast, the modified chimeric antibody used as an isotype control showed no reactivity at any of the concentrations studied.

Example 3: determination of the identification of J774 cell lines by flow cytometry

Monocytes and macrophages are of importance in inflammatory processes, such as atherosclerosis: (BPhysiol Rev 83: 1069-1112, 2003). These cells synthesize proteoglycans, which has been confirmedIt is clear that some pathways for incorporation of oxidized LDL in macrophages involve cell membrane proteoglycans when foam cells are formed (Halvorsen B, et al. biochem J.331: 743- -752, 1998).

To determine whether the anti-SO 3-chimeric antibody was able to recognize macrophages, we performed flow cytometry experiments using the murine macrophage line J774 cultured in DMEM-F12(Gibco BRL, Paisley, Scotland) supplemented with 8% inactivated fetal bovine serum (SFT; Gibco), 2mM L-glutamine, 100U/mL penicillin, 100. mu.g/mL streptomycin.

Cells (0.5X 10)⁶Per tube) was incubated with 20 μ L/tube of inactivated rabbit serum at 37 ℃ for 10 minutes to block Fc- γ receptors. Then, anti-SO 3-chimeric MAb and isotype control-modified chimeric antibody, both biotinylated and at a concentration of 10. mu.g/mL, in PBS containing 1% bovine serum albumin (Sigma, St. Louis, Mo.) and 0.01% sodium azide at pH7.4, were added and placed in an ice bath for 30 minutes. After washing the cells, they were incubated with streptavidin-fluorescein isothiocyanate complex (Jackson immunoresearch laboratories, West Grove, PA) for 30 minutes in an ice bath as diluted at 1/200. Cells were washed, resuspended in PBS containing 1% sodium azide and analyzed on a flow cytometer (Becton-Dickinson, san jose, CA).

As shown in fig. 3, the chimeric antibody used as an isotype control did not recognize cell line J774. In contrast, the anti-SO 3-chimeric antibody recognized 93.7% of the cells.

Example 4: identification of atherosclerotic plaques in human aorta

Immunohistochemical determination of the recognition of the chimeric antibody anti-SO 3 was performed on human aortic fragments fixed in formalin and embedded in paraffin. 4 μm tissue sections were used, mounted on silanized glass slides and incubated at 68 ℃ for 12 hours. Tissue sections were deparaffinized in xylene and hydrated in decreasing concentrations of ethanol. They were then washed in distillation for 5 minutes and in PBS. Antigen exposure was performed using a thermostatic water bath set at a temperature of 100 ℃. The plate was immersed in citric acid at pH6.0In saline buffer, kept in a water bath for 30 minutes, and then boiled in citrate buffer of pH6.8 for 10 minutes using a microwave oven. The sections were cooled for 20 minutes and then washed with distilled water and PBS. With 3% of H₂O₂The solution was inhibited endogenous peroxidase at room temperature for 10 minutes, washed with PBS, and added with biotinylated chimeric antibody anti-SO 3 and isotype control antibody at a concentration of 50. mu.g/mL and allowed to stand at room temperature for 30 minutes. Then, the sections were washed with PBS and streptavidin-peroxidase complex (Anacrom Diagnostics) was added at the same temperature and left for the same time. Finally, the tissue sections were incubated with a fresh mixture solution of 3, 3' -Diaminobenzidine (DAB) in 1mL of substrate buffer for 3-5 minutes. The samples were dehydrated in increasing concentrations of alcohol using Mayer's hematoxylin for contrast, clarified in xylene, and finally plated in permanent medium Eukitt (Kinder GmbH)&Co.) middle seal. Evaluation was performed using a white light microscope (Leica).

FIG. 4 shows how the anti-SO 3 chimeric antibody reacts strongly with atherosclerotic lesion samples present in the aorta. Reactivity with lipid-filled macrophages or foam cells and with diseased lipid nuclei was observed (reactivity shown in dark grey). The figure shows that the antibody used as isotype control does not recognize human aortic sections.

Example 5 ability of anti-SO 3 chimeric antibodies to induce an anti-heparin response in mice

BALB/c female mice were used; they received 50. mu.g of anti-SO 3-chimeric antibody subcutaneously (in 200. mu.L). Immunizations were performed every 14 days to complete a total of 4 doses. anti-SO 3-chimeric antibodies were administered without the addition of adjuvants or carrier proteins. Serum samples were taken on days 0 and 49 (7 days after dose 4).

The presence of anti-heparin antibodies in the serum of the immunized animals was determined using the ELISA technique described in example 2: heparin (10. mu.g/mL, 100. mu.L/well) coated Maxisorp plates were used. Mouse serum in binding buffer was tested at a dilution of 1/100, 100. mu.L/well. As secondary antibodies, goat anti-mouse IgG and IgM antisera conjugated to alkaline phosphatase (Jackson) were used.

Figure 5 shows the results of tests performed using mouse sera taken on days 0 and 49. The presence of anti-heparin antibodies was not detected in the preimmune serum (day 0) of any animal. In contrast, the presence of these serum antibodies was detected after immunization of mice with anti-SO 3-chimeric antibodies. The results show that: the anti-SO 3-chimeric antibody not only strongly recognizes heparin, but also has a surprising ability to induce a response against this molecule (vaccine effect).

Example 6 anti-SO 3 anti-atherosclerotic Effect of chimeric antibodies

To determine whether anti-SO 3-chimeric antibodies could produce a biological effect in vivo, we used the previously described model: lipoin was used to induce atherosclerotic lesions in rabbits (Tak cs E and cols. Morphol Igazs a. g. yi Orv Sz.26: 99-105, 1998, Noa M & R. more Progress in Medical Sciences, 6: 14-19, 1992).

15 New Zealand rabbits were used, which were divided into 3 groups of 5 rabbits each. Group 1 received no treatment (negative control). Group 2 received 2mL/kg of 20% lipocalin (Braun) daily intravenously for 8 days. Group 3 received 3 doses of 100 μ g anti-SO 3-chimeric antibody in PBS subcutaneously at 7 day intervals, beginning daily administration of lipocalin the day of the last immunization, following the same protocol as used in the animals of group 2. All rabbits were sacrificed under anesthesia 1 day after receiving the last dose of lipocalin, and the negative control animals of group 1 were sacrificed on the same day. Aortas were harvested from animals and physiological studies were performed to determine the presence of macroscopic and microscopic atherosclerotic lesions.

The aorta from untreated rabbits from group 1 showed no macroscopic lesions. Macroscopic lesions were observed in all the aortas from group 2 rabbits receiving 2mL of lipocalin/kg for 8 days. There were no macroscopic lesions in the aorta of rabbits that received 3 doses of anti-SO 3-chimeric antibody prior to lipocalin administration.

For microscopic lesion studies, segments of the aorta were fixed in formalin and embedded in paraffin. Tissue sections of 4 μm were used, mounted on silanized slides, and stained with hematoxylin-eosin. Evaluation was performed using a white light microscope (Leica).

When tissue sections of the aorta of untreated rabbits were evaluated, all showed normal structure of the arteries without change, as shown in fig. 6. In aortic sections from all rabbits from the group receiving lipocalin, characteristic lesions were observed: intimal thickening, deposition of extracellular material between muscle, elastic and collagen fibers, and tissue architecture distortion occur. In contrast, no microscopic lesions were observed in samples from 3 rabbits receiving 3 doses of anti-SO 3-chimeric antibody followed by application of lipocalin. In samples from the remaining 2 rabbits, tissue changes were observed: there is some discontinuous thickening in some regions of the arterial wall, with deposition of extracellular material between the fibres. There is no thickening of the intima.

Drawings

FIG. 1 recognition of sulfatide by anti-SO 3-chimeric MAb

Various concentrations of anti-SO 3-chimeric MAb and isotype control chimeric MAb were added to ELISA plates coated with sulfatide at a concentration of 4. mu.g/mL in methanol. Reactivity was detected with goat anti-human gamma chain antiserum conjugated to alkaline phosphatase. (absorbance at 405nm in an ELISA reader: (^＊ p< 0.05, Mann-Whitney U test).

FIG. 2 recognition of heparin by anti-SO 3-chimeric MAb

Various concentrations of anti-SO 3 chimeric-MAb and isotype control chimeric MAb were added to heparin-coated ELISA plates at a concentration of 10. mu.g/mL in HBSS. Reactivity was detected with goat anti-human gamma chain antiserum conjugated to alkaline phosphatase. (absorbance at 405nm in an ELISA reader: (^＊ p< 0.05, Mann-Whitney U test).

FIG. 3 identification of J774 cell lines by anti-SO 3-chimeric MAb

Cells were incubated with 10. mu.g/mL biotinylated antibody. The response was shown using goat anti-human IgG antiserum conjugated to FITC and analyzed by flow cytometry.

FIG. 4 identification of human atherosclerotic plaques by anti-SO 3 chimeric MAbs

Fragments of human aorta (4 μm) fixed in formalin and embedded in paraffin were incubated with biotinylated anti-SO 3-chimeric antibody and isotype control antibody. The reaction was shown using streptavidin-peroxidase complex. Recognition of the epitope by anti-SO 3-MAb is indicated by dark grey color, staining the nuclei of the cells with hematoxylin contrast (400-fold).

FIG. 5: antibody response to heparin induced by immunization with anti-SO 3-chimeric MAb

Serum samples obtained from BALB/c mice immunized with anti-SO 3-chimeric MAb on days 0 and 49 were assayed by ELISA. Each symbol is a value obtained with mouse serum. pI and hI are preimmune and hyperimmune, respectively: (^＊p < 0.05, Mann-Whitney U test).

FIG. 6 Effect of treatment with anti-SO 3-chimeric MAb on atherosclerotic lesion development in a Rabbit Lipoin model

Histological sections of the thoracic aorta of rabbits representing different study groups. A: group 1, untreated animals, which showed normal structure of the arteries, with no change. B: group 2, animals treated with lipocalin, in which thickening of the intima of the arteries, deposition of extracellular material between muscle, elastic and collagen fibers, and distortion of the tissue architecture were observed. C and D: group 3, animals immunized with anti-SO 3-chimeric MAb and subsequently receiving lipocalin; no significant tissue damage or intimal thickening was observed. Hematoxylin-eosin staining, 180 fold.

Claims (7)

1. Monoclonal antibodies that specifically bind to sulfatides and sulfated proteoglycans, wherein the sequences of Complementarity Determining Regions (CDRs) of the variable regions of the heavy and light chains are as follows:

heavy chain:

HCDR1 RYSVH

HCDR2 MIWGGGSTDYNSALKS

HCDR3 SGVRRGRAQAWFAY

light chain:

LCDR1 KASQDVSTAVA

LCDR2 SASYRYT

LCDR3 QQHYSTPWT，

wherein the sequences of the framework regions within the variable regions of the heavy and light chains are as follows:

heavy chain:

HFR1 QVQLKESGPGLVAPSQSLSITCTVSGFSLS

HFR2 WVRQPPGKGLEWLG

HFR3 RLSISKDNSKSQVFLKMNSLQTDDTAMYYCAR

HFR4 WGQGTLVTVSA

light chain:

LFR1 DIVMTQSHKFMSTSVGDRVSITC

LFR2 WYQQKPGQSPKLLIY

LFR3 GVPDRFTGSGSGTDFTFTISSVQAEDLAVYYC

LFR4 FGGGTKLELK，

and wherein the sequence of the constant region is: human IgG1 for the heavy chain; for the light chain, Ck.

2. A pharmaceutical composition for the treatment of atherosclerosis comprising 10 μ g to 10mg per dose of the monoclonal antibody of claim 1.

3. The pharmaceutical composition of claim 2, further comprising a pharmaceutically acceptable excipient.

4. The pharmaceutical composition of claim 2, further comprising an adjuvant.

5. A kit for diagnosing atherosclerotic lesions comprising the monoclonal antibody of claim 1.

6. Use of the monoclonal antibody of claim 1 in the manufacture of a medicament for the treatment of atherosclerosis.

7. Use of the monoclonal antibody of claim 1 for the preparation of a vaccine.