Cancer cell specific anti-podocalyxin antibody and method for producing same
The present invention provides a method for producing an antibody against podocalyxin expressed specifically in cancer cells. The method includes a step of introducing a nucleic acid encoding all or a portion of podocalyxin into cells expressing a cancer cell-specific sugar chain structure to cause cancer cell-specific podocalyxin or a portion thereof to be expressed therein, a step of immunizing a non-human mammal with the cancer cell-specific podocalyxin or portion thereof to obtain antibodies, and a step of purifying the antibodies by primary screening using purified cancer cell-specific podocalyxin or a portion thereof.
1. A cancer cell-specific anti-podocalyxin antibody or antigen-binding fragment thereof, comprising
(i) a first amino acid sequence comprising the following CDRs:
heavy chain CDR1:
(SEQ ID NO: 2)
GFNFNTNAMN,
heavy chain CDR2:
(SEQ ID NO: 3)
LIRSKSNNYATYYADSVKD,
and
heavy chain CDR3:
(SEQ ID NO: 4)
GYGSY;
and
(ii) a second amino acid sequence comprising the following CDRs:
light chain CDR1:
(SEQ ID NO: 5)
KASQSVNNDVA,
light chain CDR2:
(SEQ ID NO: 6)
FASNRYT,
and
light chain CDR3:
(SEQ ID NO: 7)
QLDYNSTWT.
2. The cancer cell-specific anti-podocalyxin antibody or antigen-binding fragment thereof according to claim 1 , wherein the first amino acid sequence comprises SEQ ID NO: 10, the second amino acid sequence comprises SEQ ID NO: 8, or both.
3. The cancer cell-specific anti-podocalyxin antibody or antigen-binding fragment thereof according to claim 1 , which has one or more N-linked sugar chains bound to a Fc region of the antibody and has no fucose bound to N-acetylglucosamine at a reducing end of the N-linked sugar chains.
4. A nucleic acid encoding the first amino acid sequence and/or the second amino acid sequence as claimed in claim 1 .
5. An expression vector comprising the nucleic acid as claimed in claim 4 .
6. A transformant comprising the expression vector as claimed in claim 5 .
7. A method for producing a cancer-cell specific anti-podocalyxin antibody comprising the steps of:
expressing an antibody in the transformant as claimed in claim 6 ; and
collecting the antibody.
8. A pharmaceutical composition, comprising, as an active ingredient, the cancer cell-specific anti-podocalyxin antibody or antigen-binding fragment thereof as claimed in claim 1 .
9. A pharmaceutical composition, comprising, as an active ingredient, the cancer cell-specific anti-podocalyxin antibody or antigen-binding fragment thereof as claimed in claim 1 to which a substance having an anti-cancer activity has been bound.
10. The pharmaceutical composition according to claim 8 , which is a preventive or therapeutic agent for cancer.
11. A cancer testing method, comprising:
a step of measuring cancer cell-specific podocalyxin in a sample collected from a subject by using a cancer cell-specific anti-podocalyxin antibody or antigen-binding fragment thereof as claimed in claim 1 to measure the cancer cell-specific podocalyxin.
12. A cancer testing kit, comprising a cancer cell-specific anti-podocalyxin antibody or antigen-binding fragment thereof as claimed in claim 1 .
13. A cancer cell-specific anti-podocalyxin antibody or antigen-binding fragment thereof, comprising
(i) a first amino acid sequence comprising the following CDRs:
heavy chain CDR1:
(SEQ ID NO: 2)
GFNFNTNAMN,
heavy chain CDR2:
(SEQ ID NO: 3)
LIRSKSNNYATYYADSVKD,
and
heavy chain CDR3:
(SEQ ID NO: 4)
GYGSY;
and
(ii) a second amino acid sequence comprising the following CDRs:
light chain CDR1:
(SEQ ID NO: 5)
KASQSVNNDVA,
light chain CDR2:
(SEQ ID NO: 6)
FASNRYT,
and
light chain CDR3:
(SEQ ID NO: 7)
QLDYNSTWT;
wherein one or more of the CDRs have one amino acid addition, substitution, or deletion.
14. The cancer cell-specific anti-podocalyxin antibody or antigen-binding fragment thereof according to claim 13 , which has one or more N-linked sugar chains bound to a Fc region of the antibody and has no fucose bound to N-acetylglucosamine at a reducing end of the N-linked sugar chains.
15. A nucleic acid encoding the first amino acid sequence and/or the second amino acid sequence as claimed in claim 14 .
16. An expression vector comprising the nucleic acid as claimed in claim 15 .
17. A transformant comprising the expression vector as claimed in claim 16 .
18. A method for producing a cancer-cell specific anti-podocalyxin antibody comprising the steps of:
expressing an antibody in the transformant as claimed in claim 17 ; and
collecting the antibody.
19. A pharmaceutical composition, comprising, as an active ingredient, the cancer cell-specific anti-podocalyxin antibody or antigen-binding fragment thereof as claimed in claim 13 .
20. A pharmaceutical composition, comprising, as an active ingredient, the cancer cell-specific anti-podocalyxin antibody or antigen-binding fragment thereof as claimed in claim 13 to which a substance having an anti-cancer activity has been bound.
21. The pharmaceutical composition according to claim 19 , which is a preventive or therapeutic agent for cancer.
22. A cancer testing method, comprising:
a step of measuring cancer cell-specific podocalyxin in a sample collected from a subject by using a cancer cell-specific anti-podocalyxin antibody or antigen-binding fragment thereof as claimed in claim 13 to measure the cancer cell-specific podocalyxin.
23. A cancer testing kit, comprising a cancer cell-specific anti-podocalyxin antibody or antigen-binding fragment thereof as claimed in claim 13 .