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Agents and compositions and methods utilizing same useful in diagnosing and/or treating or preventing plaque forming diseases

a technology applied in the field of agents and compositions and methods of utilizing same for treating plaque forming diseases, can solve the problems of teaching not providing means with which to enable, abnormal physiochemical properties, and more severe and widespread abnormalities

Inactive Publication Date: 2005-04-28
RAMOT AT TEL AVIV UNIV LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This approach induces high antibody titers capable of disrupting and preventing amyloid plaque formation, offering rapid onset and long-lasting protection with minimal adverse effects, and is amenable to large-scale production.

Problems solved by technology

This rearrangement is associated with abnormal physiochemical properties, including insolubility in non-denaturing detergents and partial resistance to proteolysis.
In both types of the disease, the pathology is similar, but the abnormalities tend to be more severe and widespread in cases beginning at an earlier age.
In addition, these teachings do not provide means with which to enable the penetration of such antibodies into the brain through the blood brain barrier (BBB).
Furthermore, this patent fails to teach the use of phage display technology as a delivery method for antigens or antibodies.
Yet furthermore, no experimental results demonstrating the in vivo effectiveness of such antibodies are demonstrated by U.S. Pat. No. 5,688,561.
Because this proposed dosage would barely alter the level of endogenous circulating beta-amyloid and because EP 526511 does not recommend the use of an adjuvant, it seems implausible that any therapeutic benefit would result therefrom.
These findings suggest that the teachings of Schenk and colleagues are inefficient at best.
Since, as has already been mentioned hereinabove, the normal concentration of beta-amyloid in human serum is 50-200 pg / ml, immunization with that peptide could be expected to produce either low antibody titers or high toxicity if strong adjuvants are used and as such it is not applicable for therapy.
However, they fail to teach use of an antigen so displayed to effect immunization.
Collectively, the prior art fails to teach means with which an effective titer of anti-aggregation antibodies can be generated in vivo in a short time and / or be introduced into the brains of patients suffering a plaque-forming disease.
In addition, the persistence of titers generated via prior art teachings has not been established.

Method used

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  • Agents and compositions and methods utilizing same useful in diagnosing and/or treating or preventing plaque forming diseases
  • Agents and compositions and methods utilizing same useful in diagnosing and/or treating or preventing plaque forming diseases
  • Agents and compositions and methods utilizing same useful in diagnosing and/or treating or preventing plaque forming diseases

Examples

Experimental program
Comparison scheme
Effect test

example 1

Generation of an IgM Hybridoma 508

[0239] Immunization of a mouse with a 16 amino acid peptide of beta-amyloid (acids 1-16 of SEQ ID NO:3) conjugated to KLH (SEQ ID NO:9) was carried out as described hereinabove. Repetitious immunization eventually produced a low but measurable antibody titer against beta-amyloid. Subsequent splenectomy of the immunized mouse facilitated preparation of IgM hybridoma 508 expressing scFvAb with specificity to beta-amyloid. RNA was subsequently extracted from this hybridoma. The IgM 508 hybridoma showed specific activity to Aβ in preventing its toxic affects on PC12 cells (Anavi S, 1998, M. Sc. thesis from the department of Molecular Microbiology and Biotechnology of the Tel-Aviv University, I).

example 2

Cloning of the Variable Domains of the 508 IgM Hybridoma as a scFv

[0240] MAb 508 showed specific recognition of β-amyloid and prevented its toxic affects on PC12 cells (Anavi S, 1998, ibid). For cloning the 508 antibody as a scFv in a phage display vector, RNA was extracted from 108 508 hybridoma cells and was used as a source for antibody variable region coding sequences. RT-PCR was used to amplify the variable domains that were cloned into the phage display vector pCC-Gal6(Fv), as described in Materials and Methods. When hybridoma derived antibodies are cloned as scFvs, some of the clones may contain aberrant sequences that are not functional. Therefore, to identify phagemid clones carrying functional β-amyloid binders from the generated clones, 10 individual clones were picked at random and soluble scFv-CBD fusion protein was produced thereby. FIG. 2 shows a physical map of plasmid pCC-508 which was used to express the 508-scFv. The CBD domain serves as an immunological detectio...

example 3

Site Directed Mutagenesis of 508-(Fv) Antibody

[0241] The DNA sequencing analysis of pCC508-(Fv) revealed the unusual appearance of a cysteine residue at the position 96 of VL CDR3 (Kabat et al, Sequences of proteins of immunological interest, 5th Ed., 1991). The deduced amino acid sequence of VL CDR3 is: H89QRSSYPCT97 (SEQ ID NO:15). The presence of an unpaired cysteine residue in a scFv may reduce its folding yield and also decrease its stability in solution and its storage half life. Therefore, 508(Fv) was subcloned into an expression vector and produced in E. coli as described in Materials and Methods. FIG. 6 summarizes the production process of 508(Fv)-CBD by the cellulose-assisted refolding method (Berdichevsky et al, Protein Expr Purif, 17 (2):249-59 (1999)). Although 508(Fv)-CBD could be purified to near homogeneity (FIG. 6 lane 7) by this method, it refolded relatively poorly and was unstable upon storage at 4° C. (FIG. 7). It was assumed that substitution of the cysteine w...

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Abstract

A method of immunizing against plaque forming diseases using display technology is provided. The method utilize novel agents, or pharmaceutical compositions for vaccination against plaque forming diseases which rely upon presentation of an antigen or epitope on a display vehicle. The method further includes agents, or pharmaceutical compositions for vaccination against plaque forming diseases, which rely upon presentation of an antibody, or an active portion thereof, on a display vehicle. Whether antigens or antibodies are employed, disaggregation of plaques results from the immunization.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of U.S. application Ser. No. 10 / 162,889, filed Jun. 6, 2002, now abandoned, which is a continuation of U.S. application Ser. No. 09 / 629,971, filed Jul. 31, 2000, now abandoned, which is a continuation-in-part of U.S. application Ser. No. 09 / 473,653, filed Dec. 29, 1999, which is a continuation-in-part of U.S. provisional application No. 60 / 152,417, filed Sep. 3, 1999, now abandoned, the entire contents of each being incorporated herein by reference.BACKGROUND OF THE INVENTION [0002] The present invention relates to agents and compositions and to methods of utilizing same for treating plaque-forming diseases. More particularly, the methods according to the present invention involve the use of (i) plaque derived antigens cloned and displayed on the surface of a display vehicle for in vivo elicitation of antibodies capable of preventing plaque formation and of disaggregating existing plaques; and (ii) ant...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K35/74G01N33/53A61K35/76A61K38/00A61K38/17A61K39/00A61K39/395A61K49/00A61P19/08A61P25/28A61P35/00A61P43/00C07K14/47C07K16/18C12N7/01C12N15/02
CPCA61K38/1709A61K39/0007A61K49/0004A61K2039/6075C07K2319/00C07K14/4711C07K16/18C07K2317/622C07K2317/74C07K14/47A61P19/08A61P25/28A61P35/00A61P43/00
Inventor SOLOMON, BEKAHANAN, EILATFRENKEL, DAN
Owner RAMOT AT TEL AVIV UNIV LTD