Antibody immunization therapy for treatment of atherosclerosis

an antibody and atherosclerosis technology, applied in the field of new recombinant human antibodies, can solve the problems of efficacy and plasma half-lives reduction of murine antibodies, and achieve the effect of reducing the risk of allergic reactions and long plasma half-life of antibodies

Inactive Publication Date: 2008-01-10
BIOINVENT INT AB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0028] Recently a novel technology for generation of variability in antibody libraries was presented (WO98 / 32845, Soderlind et al., 2000, Nature BioTechnol. 18:852-856). Antibody fragments derived from this library all have the same framework regions and only differ in their CDRs. Since the framework regions are of germline sequence the immunogenicity of antibodies derived from the library, or similar libraries produced using the same technology, are expected to be particularly low (Soderlind et al., 2000, Nature BioTechnol. 18:852-856). This property is expected to be of great value for therapeutic antibodies reducing the risk for the patient to form antibodies to the administered antibody thereby reducing risks for allergic reactions, the occurrence of blocking antibodies, and allowing a long plasma half-life of the antibody. Several antibodies derived from recombinant libraries have now reached into the clinic and are expected to provide therapeutic drugs in the near future.

Problems solved by technology

As a consequence the efficacy and plasma half-lives of the murine antibodies were decreased, and often side effects from allergic reactions, caused by the foreign antibody, prevented successful treatment.
Thus, using such libraries the problem becomes the one to find the specific binder already existing in the library, and not to generate it through immunisations.

Method used

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  • Antibody immunization therapy for treatment of atherosclerosis
  • Antibody immunization therapy for treatment of atherosclerosis
  • Antibody immunization therapy for treatment of atherosclerosis

Examples

Experimental program
Comparison scheme
Effect test

example 1

Selection of scFv Against MDA Modified Peptides IEIGL EGKGF EPTLE ALFGK (SEQ.ID NO: 70) (P45, Table 1) and KTTKQ SFDLS VKAQY KKNKH (SEQ. ID NO: 52) (P210, Table 1).

[0050] The target antigens were chemically modified to carry Malone-di-aldehyde (MDA) groups on lysines and histidines. The modified peptides were denoted IEI (P45) and KTT (P210).

[0051] Selections were performed using BioInvent's n-CoDeR™ scFv library for which the principle of construction and production have been described in Soderlind et al. 2000, Nature BioTechnology. 18, 852-856. The library contains approximately 2×1010 independent clones and a 2000 fold excess of clones were used as input for each selection. Selections were performed in three rounds. In selection round 1, Immunotubes (NUNC maxisorb™ 444202) were coated with 1.2 ml of 20 μg / ml MDA-modified target peptides in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4) with end over end agitation at +4° C. over night. The tubes were then blocked w...

example 2

Transfer of Genes Encoding the Variable Parts of Selected scFv to Full Length Human IgG1 Vestors

[0062] Bacteria containing scFv clones to be converted to Ig-format were grown over night in LB supplemented with 100 μg / ml ampicillin. Plasmid DNA was prepared from over night cultures using the Quantum Prep, plasmid miniprep kit from Biorad (# 732-6100). The DNA concentration was estimated by measuring absorbance at 260 nm, and the DNA was diluted to a concentration of 2 ng / μl. VH and VL from the different scFv-plasmids were PCR amplified in order to supply these segments with restriction sites compatible with the expression vectors (see below). 5′ primers contain a BsmI and 3′ primers contain a BsiWI restriction enzyme cleavage site (shown in italics). 3′ primers also contained a splice donor site (shown in bold).

Primers for amplification of VH-segments:(SEQ. ID. NO: 13)5′VH:5′-GGTGTGCATTCCGAGGTGCAGCTGTTGGAG(SEQ. ID. NO: 14)3′VH:5′-GACGTACGACTCACCTGAGCTCACGGTGACCAGPrimers for amplif...

example 3

Stable Transfection of NSO Cells Expressing Antibodies Against MDA Modified Peptides from Apolipoprotein B-100

[0076] NSO cells (ECACC no. 85110503) were cultured in DMEM (cat nr 31966-021, Invitrogen) supplemented with 10% Fetal Bovine Serum (cat no. 12476-024, lot: 1128016, Invitrogen) and 1×NEAA (non-essential amino acids, cat no. 11140-053, Invitrogen). Cell cultures are maintained at 37° C. with 5% CO2 in humidified environment.

[0077] DNA constructs to be transfected were four constructs of IEI specific antibodies (IEI-A8, IEI-D8, IEI-E3, IEI-G8), two of KTT specific antibodies (KTT-B8, KTT-D6) and one control antibody (JFPA12). The day before transfection, the cells were trypsinized and counted, before plating them in a T-75 flask at 12×106 cells / flask. On the day of transfection, when the cells were 85-90% confluent, the cells were plated in 15 ml DMEM+1×NEAA+10% FBS (as above). For each flask of cells to be transfected, 35-40 μg of DNA were diluted into 1.9 ml of OPTI-MEM® ...

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Abstract

The present invention relates to passive immunization for treating or preventing atherosclerosis using an isolated human antibody directed towards at least one oxidized fragment of apolipoprotein B in the manufacture of a pharmaceutical composition for therapeutical or prophylactical treatment of atherosclerosis by means of passive immunization, as well as method for preparing such antibodies, and a method for treating a mammal, preferably a human using such an antibody to provide for passive immunization.

Description

PRIORITY INFORMATION [0001] This application is a continuation of U.S. patent application Ser. No. 10 / 679,032 filed on Oct. 3, 2003 which claims priority to U.S. Provisional Patent Appln. No. 60 / 421,067, filed Oct. 25, 2002, Swedish Patent Application No. 0302312-4, filed Sep. 27, 2003 and Swedish Patent Application No. 0202959-3, filed Oct. 4, 2002.BACKGROUND OF THE INVENTION [0002] 1. Field of Invention [0003] The present invention relates to new recombinant human antibodies raised against peptides being derivatives of apolipoprotein B, in particular antibodies to be used for immunization therapy for treatment of atherosclerosis, method for their preparation, and method for passive immunization using said antibodies. [0004] In particular the invention includes: [0005] The use of any isolated recombinant antibody raised against an oxidized form of the peptides listed in table 1, in particular MDA-modified peptides, preferably together with a suitable carrier and adjuvant as an immu...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/395A61P9/10C07K16/00C07K14/775C07K16/18
CPCA61K2039/505C07K14/775C07K2317/622C07K2317/21C07K16/18A61P37/04A61P9/00A61P9/10
Inventor CARLSSON, ROLANDBENGSSTON, JENNYSTRANDBURG, LEIF
Owner BIOINVENT INT AB
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