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Antibodies that recognize sulphatides and sulphated proteoglycans and the use thereof

A proteoglycan, sulfatide technology, applied in the field of monoclonal antibodies, can solve problems such as doubts, failure of phase III clinical trials, etc.

Active Publication Date: 2014-07-16
CENT DE INMUNOLOGIA MOLECULAR CENT DE INMUNOLO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, recent studies have shown that a phase III clinical trial with the CETP inhibitor torcetrapib failed, casting doubt on this strategy

Method used

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  • Antibodies that recognize sulphatides and sulphated proteoglycans and the use thereof
  • Antibodies that recognize sulphatides and sulphated proteoglycans and the use thereof
  • Antibodies that recognize sulphatides and sulphated proteoglycans and the use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Example 1. Recognition of chimeric MAb anti-SO3 for bovine sulfatide

[0094] Using ELISA, PolySorp plates, Nunc, were coated with sulfatide solution at a concentration of 4 μg / mL in methanol at 50 μL / well, and the solvent was evaporated by incubation at 37° C. for 90 minutes. Then, the plate was blocked with phosphate buffered saline (PBS) containing 1% bovine serum albumin (SAB) at 200 μL / well at room temperature for 1 hour. Then, 50 μL / well of different concentrations of chimeric antibody anti-SO3 in PBS was added and incubated at 37° C. for 1 hour. Plates were then washed with PBS and 50 μL / well of goat antiserum anti-human gamma chain conjugated to alkaline phosphatase (Sigma) was added. The plates were incubated at 37°C for 1 hour, after which the plates were washed again and 100 μL / well of substrate solution consisting of 1 mg / mL p-nitrophenyl phosphate in diethanolamine buffer pH 9.8 was added. After incubation for 30 minutes at room temperature, the absorbanc...

Embodiment 2

[0096] Example 2. Heparin recognition test

[0097] It was then determined whether the chimeric monoclonal anti-SO3 recognized sulfated molecules more complex than sulfatides. Heparin, a highly sulfated molecule used as a model for sulfated glycosaminoglycans, was chosen for this study.

[0098] The test for anti-heparin reactivity was performed based on the ELISA technique for biglican developed by Skalen, K.M.y cols (Nature 417:750-754, 2002) with slight modifications. Maxisorp microtiter plates (Nunc) were coated with 10 μg / mL (100 μL / well) of heparin (Sigma) in Hepes buffered saline solution (HBSS) (20 mM Hepes, 150 mM NaCl, pH 7.4) and incubated overnight at 4°C . Plates were washed 3 times with HBSS and then blocked with HBSS containing 1% SAB (HBSS-BSA) for 1 hour at room temperature. Wash the plate 3 times with 0.02% HBSS-Tween20 (HBSS-T), and add serial dilutions of chimeric monoclonal anti-SO3 within 1 hour at room temperature: from binding buffer (10mM Hepes, 20m...

Embodiment 3

[0100] Example 3: Determination of recognition of the J774 cell line by flow cytometry

[0101] Monocytes and macrophages are important in inflammatory processes such as atherosclerosis ( B E. Physiol Rev 83:1069-1112, 2003). These cells synthesize proteoglycans, and it has been demonstrated that certain pathways for the incorporation of oxidized LDL in macrophages involve cell membrane proteoglycans in the formation of foam cells (Halvorsen B, et al. Biochem J. 331:743--752, 1998).

[0102] To determine whether anti-SO3-chimeric antibodies could recognize macrophages, we performed flow cytometry experiments using the murine macrophage cell line J774 cultured in cells supplemented with 8% inactivated fetal calf serum (SFT; Gibco), 2 mM L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin in DMEM-F12 (Gibco BRL, Paisley, Scotland).

[0103] Cells (0.5x10 6 / tube) was incubated with 20 μL / tube of inactivated rabbit serum at 37° C. for 10 minutes to block Fc-γ receptor...

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Abstract

The invention relates to the field of biotechnology, especially new products for human health. The present invention provides novel specific monoclonal antibodies that recognize sulfatides and sulfated proteoglycans specifically and with high affinity. The anti-sulfatide and anti-sulfated proteoglycan antibodies described in this specification constitute important diagnostic and therapeutic reagents acting on the pathological processes associated with the development of atherosclerotic plaques. Accordingly, the present invention provides pharmaceutical compositions comprising the monoclonal antibodies of the present invention or fragments derived from these antibodies for therapeutic and diagnostic use in relation to cardiovascular diseases. In addition, the present invention relates to fragments of monoclonal antibodies recognizing sulfatides and sulfated proteoglycans, which fragments are useful in the treatment or diagnosis of this condition.

Description

technical field [0001] The present invention relates to novel monoclonal antibodies (MAbs) that recognize sulfatides and sulfated proteoglycans specifically and with high affinity. 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, which comprises the antibody or fragment thereof of the present invention. Background technique [0002] After more than 30 years of development of hybridoma technology (Koehler y Milstein Nature, 256:495-497, 1975) for obtaining murine MAbs, it has been proved to be very useful in disease diagnosis and basic research, but only 20 An antibody for human therapy (Pharma Vitae, Monoclonal Abs Update, 6-363, 2008). This is largely due to their short half-life in the blood, poor recognition of murine effector functions by the human immune system, and, when ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/00C07K16/18A61P9/10
CPCC07K2317/24G01N2800/323C07K16/44C07K16/18G01N33/6893A61K2039/505A61P9/00A61P9/10A61K39/395
Inventor C·马蒂奥德阿卡斯塔德尔瑞奥A·M·维兹克兹洛普兹A·洛普兹瑞克纳Y·费尔南德兹玛瑞奥Y·索托洛普兹V·布瑞托纳维罗
Owner CENT DE INMUNOLOGIA MOLECULAR CENT DE INMUNOLO
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