Method of treating amykloidosis comprising administering an anti-HMGB-1 antibody
a technology of amyloidosis and antibody, applied in the field of amyloidosis agents, can solve the problems of high antibody affinity to hmgb-1, inflammatory response, and high stress on the animals being immunized, and achieve the effect of improving the immune system, reducing the risk of amyloidosis, and improving the immune system
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example 1
Selection of Highly Hydrophilic Amino Acid Sequences in the Amino Acid Sequence of Human HMGB-1, which Exhibit Low Homology to Human HMGB-2
[0105]Highly hydrophilic amino acid sequences that exhibit low homology to human HMGB-2 were selected from the amino acid sequence of human HMGB-1.
(1) The amino acid sequence of human HMGB-1 (SEQ ID NO: 6) is shown above as the data of Wen et al. (Wen et al., Nucleic Acids Res. (1989) 17: 1197-214).
(2) The hydrophilicity of each amino acid residue in the amino acid sequence of human HMGB-1 was estimated by the method of Hopp et al. (T. P., Hopp et al., Proc. Natl. Acad. Sci. USA (1981) 78: 3824-8).
[0106](3) Next, highly hydrophilic amino acid sequences from the amino acid sequence of human HMGB-1 were compared with the amino acid sequence of human HMGB-2 (M. Yoshida et al., J. Biol. Chem. (1992) 267: 6641-5). Then, some amino acid sequences exhibiting low homology to human HMGB-2 were selected from the highly hydrophilic amino acid sequences of h...
example 2
Peptide Synthesis
[0107]The peptide consisting of the amino acid sequence “Cys Lys Pro Asp Ala Ala Lys Lys Gly Val Val Lys Ala Glu Lys” (SEQ ID NO: 3), which has an extra cysteine at the N-terminus of the amino acid sequence selected in Example 1, was synthesized for the convenience of linking.
[0108]First, the peptide having the amino acid sequence “Cys Lys Pro Asp Ala Ala Lys Lys Gly Val Val Lys Ala Glu Lys” (SEQ ID NO: 3) was synthesized by the solid-phase synthesis method with t-butoxycarbonyl amino acids using the Applied Biosystems Model 430A peptide synthesizer according to the instruction manual.
[0109]The synthesized peptide was cleaved from the resins by the hydrogen fluoride method in the presence of dimethylsulfide, p-thiocresol, m-cresol, and anisole as scavengers to suppress the side reactions.
[0110]Then, the scavengers were extracted with dimethyl ether, and the synthesized peptide was extracted with 2N acetic acid.
[0111]The peptide was purified by anion exchange column ...
example 3
Immunogen Preparation
[0114]10 mg of a carrier, namely keyhole limpet hemocyanin (KLH) (Calbiochem) or bovine serum albumin (BSA) (Seikagaku Co.), was dissolved in 10 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 7.0), and then 150 μl of N,N-dimethylformamide solution containing 2.5% maleimidebenzoyl N-hydroxysuccinimide ester (MBS) (PIERCE) was added thereto. The mixture was incubated at room temperature for 30 minutes while stirring.
[0115]The mixture was loaded at 4° C. onto a gel filtration column (Sephadex G-25 column (Pharmacia LKB)) pre-equilibrated with 10 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 7.0). The absorbance was monitored at 280 nm to collect the MBS-carrier conjugate fraction.
[0116]The pH of the MBS-carrier conjugate fraction was adjusted to 7.0 using trisodium phosphate. The peptide “Cys Lys Pro Asp Ala Ala Lys Lys Gly Val Val Lys Ala Glu Lys” (SEQ ID NO: 3) synthesized as described in Example 2 was ad...
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