Antibody and use thereof
View Patent ↗An antibody provided by the present invention has a low reactivity with amyloid precursor proteins, and has a higher reactivity with amylospheroids than with amyloid β fibrils or monomeric amyloid β-proteins. According to the present invention, an antibody is provided that has a higher reactivity with amylospheroids than with amyloid precursor proteins, and has any one or more of the following properties: (i) a higher activity with amylospheroids than with amyloid β fibrils; (ii) a higher reactivity with amylospheroids than with monomeric amyloid β-proteins; and (iii) an activity of inhibiting neuronal cell death induced by amylospheroids.
1. An isolated monoclonal antibody obtained from a hamster or obtained by humanization of hamster monoclonal antibody having a higher reactivity with amylospheroid than with amyloid precursor proteins, and having any one or more of the following properties:
(i) a higher activity with amylospheroid than with amyloid β fibrils;
(ii) a higher reactivity with amylospheroid than with monomeric amyloid β-proteins; and
(iii) an activity of inhibiting the neuronal cell death induced by amylospheroid.
2. The antibody according to claim 1 , exhibiting a reactivity with amylospheroid at least 3 times higher than its reactivity with amyloid β fibrils, in a system wherein a reactivity of an antibody with amylospheroid is compared with a reactivity of the antibody with amyloid β fibrils at the same antibody concentration, antibody amount, antigen protein concentration, and antigen protein amount.
3. The antibody according to claim 1 , exhibiting a reactivity with amylospheroid at least 5 times higher than its reactivity with amyloid β fibrils, in a system wherein a reactivity of an antibody with amylospheroid is compared with a reactivity of the antibody with amyloid β fibrils at the same antibody concentration, antibody amount, antigen protein concentration, and antigen protein amount.
4. The antibody according to claim 1 , exhibiting a reactivity with amylospheroid at least 50 times higher than its reactivity with monomeric amyloid β-proteins, in a system wherein a reactivity of an antibody with amylospheroid is compared with a reactivity of the antibody with monomeric amyloid β-proteins at the same antibody concentration, antibody amount, antigen protein concentration, and antigen protein amount.
5. The antibody according to claim 1 , exhibiting a reactivity with amylospheroid at least 500 times higher than its reactivity with monomeric amyloid β-proteins, in a system wherein a reactivity of an antibody with amylospheroid is compared with a reactivity of the antibody with monomeric amyloid β-proteins at the same antibody concentration, antibody amount, antigen protein concentration, and antigen protein amount.
6. The antibody according to claim 1 , being obtained using amylospheroid as an antigen.
7. The antibody according to claim 1 , having a dissociation constant with amylospheroid of not more than 10 −9 .
8. The antibody according to claim 1 , reacting specifically with Alzheimer's disease brains while not exhibiting a significant cross-reactivity with human normal tissues.
9. The antibody according to claim 1 , recognizing an epitope specific to a tertiary structure of amylospheroid.
10. The antibody according to claim 1 , being a monoclonal antibody produced from a hybridoma having an accession number of either FERM BP-10871 or FERM BP-10872.
11. A neuron protector comprising the antibody according to claim 1 .
12. A reagent for detecting Alzheimer's disease, comprising the antibody according to claim 1 .
13. A medicine comprising the antibody according to claim 1 .
14. A therapeutic agent for Alzheimer's disease, comprising the antibody according to claim 1 .
15. A hybridoma for producing the antibody according to claim 1 .
16. A humanized antibody obtained by humanization of a hamster monoclonal antibody produced from a hybridoma having an accession number of either FERM BP-10871 or FERM BP-10872.
17. The humanized antibody according to claim 16 , or a fragment of the humanized antibody according to claim 16 , comprising a humanized heavy chain and a humanized light chain,
the humanized heavy chain including:
three heavy chain complementarity-determining regions (CDRs) obtained from a hamster monoclonal antibody produced from the hybridoma having the accession number of FERM BP-10872, the three heavy chain CDRs being heavy chain CDRs 1 to 3; and
a heavy chain variable domain framework sequence obtained from a human immunoglobulin heavy chain; and
the humanized light chain including:
three light chain complementarity-determining regions (CDRs) obtained from a hamster monoclonal antibody produced from the hybridoma having the accession number of FERM BP-10872, the three light chain CDRs being light chain CDRs 1 to 3; and
a light chain variable domain framework sequence obtained from a human immunoglobulin light chain,
wherein the three heavy chain CDRs 1 to 3 have the following amino acid sequences, respectively:
(SEQ ID NO: 11)
heavy chain CDR1: Asp Tyr Phe Met Ser;
(SEQ ID NO 12)
heavy chain CDR2: Gly Ile Glu Ile Lys Ser Tyr Phe
Tyr Ala Thr Tyr Tyr Phe Gly Ser Val Lys Gly;
and
(SEQ ID NO: 13)
heavy chain CDR3: Asn Arg Glu Val Gly Gly Leu Asp
Asn,
and
the three light chain CDRs 1 to 3 have the following amino acid sequences, respectively:
(SEQ ID NO: 14)
light chain CDR1: Thr Leu Arg Ser Gly Ile Ser Val
Gly Gly Lys Asn Ile Tyr;
(SEQ ID NO: 15)
light chain CDR2: Tyr Ser Ser Tyr Ser Asn Lys Gln
Leu Gly Pro;
and
(SEQ ID NO: 16)
light chain CDR3: Ser Ile His Glu Ser Asn Ala Tyr
Val.
18. The humanized antibody according to claim 16 , or a fragment of the humanized antibody according to claim 16 , comprising
a humanized heavy chain variable domain having an amino acid sequence according to SEQ ID NO: 17; and
a light chain variable domain having an amino acid sequence according to SEQ ID NO: 18,
wherein the amino acid sequence according to SEQ ID NO: 17 is as follows:
[Chemical Formula 1]
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
20 25 30
Phe Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Xaa Gly Ile Glu Ile Lys Ser Tyr Phe Tyr Ala Thr Tyr Tyr Phe Gly
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
65 70 75 80
Xaa Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Thr Xaa Asn Arg Glu Val Gly Gly Leu Asp Asn Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
where “Xaa” at position 49 is Gly or Ala, “Xaa” at position 81 is Leu or Val, and “Xaa” at position 100 is Thr or Arg, and
the amino acid sequence according to SEQ ID NO. 18 is as follows:
[Chemical Formula 2]
Gln Xaa Val Leu Thr Gln Pro Xaa Ser Leu Ser Ala Ser Pro Gly Ala
1 5 10 15
Ser Ala Ser Leu Thr Cys Thr Leu Arg Ser Gly Ile Ser Val Gly Gly
20 25 30
Lys Asn Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Pro Pro Gln Xaa
35 40 45
Xaa Leu Xaa Tyr Ser Ser Tyr Ser Asn Lys Gln Leu Gly Pro Gly Val
50 55 60
Pro Ser Arg Phe Ser Gly Ser Lys Asp Xaa Ser Ala Asn Ala Xaa Ile
65 70 75 80
Leu Leu Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys
85 90 95
Ser Ile His Glu Ser Asn Ala Tyr Val Phe Gly Gly Gly Thr Lys Leu
100 105 110
Thr Val Leu Gly
115
where “Xaa” at position 2 is Ser or Ala, “Xaa” at position 8 is Ser or Ala, “Xaa” at position 48 is Tyr or Phe, “Xaa” at position 49 is Leu or Phe, “Xaa” at position 51 is Lys, Phe, or Arg, “Xaa” at position 74 is Ala or Thr, and “Xaa” at position 79 is Gly or Ala.
19. The humanized antibody or the fragment thereof according to claim 18 ,
wherein the humanized heavy chain variable domain has an amino acid sequence according to SEQ ID NO:5, and
the light chain variable domain has an amino acid sequence according to SEQ ID NO. 7.
20. A method for detecting an individual with Alzheimer's disease, the method comprising:
bringing a biological sample obtained from an individual suspected of Alzheimer's disease into contact with the antibody according to claim 1 ; and
determining whether or not a substance that reacts with the antibody exists in the sample.
21. A hybridoma having an accession number of either FERM BP-10871 or FERM BP-10872.