Anti-podoplanin antibody
The object of the present invention is to provide a humanized or mouse-human chimeric anti-podoplanin antibody or an antibody fragment containing the antigen-binding region thereof, and the object is achieved by providing an isolated humanized or mouse-human chimeric anti-podoplanin antibody which comprises a predetermined amino acid sequence, or an antibody fragment containing the antigen-binding region thereof.
1. An isolated humanized anti-podoplanin antibody, or an antibody fragment thereof, comprising an antigen-binding region, which comprises a variable region comprising the amino acid sequence I or II below,
the amino acid sequences of the heavy-chain FRs 1 to 4 and the light-chain FRs 1 to 4 in the following amino acid sequence I optionally have a sequence identity of not less than 90% to the amino acid sequences represented by SEQ ID NOs: 7 to 14 below, respectively, and
the amino acid sequences of the heavy-chain FRs 1 to 4 and the light-chain FRs 1 to 4 in the following amino acid sequence II optionally have a sequence identity of not less than 90% to the amino acid sequences represented by SEQ ID NOs: 7, 15, 16, 10 to 14 below, respectively;
wherein, in the amino acid sequence I,
the amino acid sequence of the heavy-chain CDR1 is the amino acid sequence represented by SEQ ID NO: 1,
the amino acid sequence of the heavy-chain CDR2 is the amino acid sequence represented by SEQ ID NO: 2,
the amino acid sequence of the heavy-chain CDR3 is the amino acid sequence represented by SEQ ID NO: 3,
the amino acid sequence of the light-chain CDR1 is the amino acid sequence represented by SEQ ID NO: 4,
the amino acid sequence of the light-chain CDR2 is the amino acid sequence represented by SEQ ID NO: 5,
the amino acid sequence of the light-chain CDR3 is the amino acid sequence represented by SEQ ID NO: 6,
the amino acid sequence of the heavy-chain FR1 is the amino acid sequence represented by SEQ ID NO: 7,
the amino acid sequence of the heavy-chain FR2 is the amino acid sequence represented by SEQ ID NO: 8,
the amino acid sequence of the heavy-chain FR3 is the amino acid sequence represented by SEQ ID NO: 9,
the amino acid sequence of the heavy-chain FR4 is the amino acid sequence represented by SEQ ID NO: 10,
the amino acid sequence of the light-chain FR1 is the amino acid sequence represented by SEQ ID NO: 11,
the amino acid sequence of the light-chain FR2 is the amino acid sequence represented by SEQ ID NO: 12,
the amino acid sequence of the light-chain FR3 is the amino acid sequence represented by SEQ ID NO: 13, and
the amino acid sequence of the light-chain FR4 is the amino acid sequence represented by SEQ ID NO: 14; and
wherein, in the amino acid sequence II
the amino acid sequence of the heavy-chain CDR1 is the amino acid sequence represented by SEQ ID NO: 1,
the amino acid sequence of the heavy-chain CDR2 is the amino acid sequence represented by SEQ ID NO: 2,
the amino acid sequence of the heavy-chain CDR3 is the amino acid sequence represented by SEQ ID NO: 3,
the amino acid sequence of the light-chain CDR1 is the amino acid sequence represented by SEQ ID NO: 4,
the amino acid sequence of the light-chain CDR2 is the amino acid sequence represented by SEQ ID NO: 5,
the amino acid sequence of the light-chain CDR3 is the amino acid sequence represented by SEQ ID NO: 6,
the amino acid sequence of the heavy-chain FR1 is the amino acid sequence represented by SEQ ID NO: 7,
the amino acid sequence of the heavy-chain FR2 is the amino acid sequence represented by SEQ ID NO: 15,
the amino acid sequence of the heavy-chain FR3 is the amino acid sequence represented by SEQ ID NO: 16,
the amino acid sequence of the heavy-chain FR4 is the amino acid sequence represented by SEQ ID NO: 10,
the amino acid sequence of the light-chain FR1 is the amino acid sequence represented by SEQ ID NO: 11,
the amino acid sequence of the light-chain FR2 is the amino acid sequence represented by SEQ ID NO: 12,
the amino acid sequence of the light-chain FR3 is the amino acid sequence represented by SEQ ID NO: 13, and
the amino acid sequence of the light-chain FR4 is the amino acid sequence represented by SEQ ID NO: 14,
wherein said anti-podoplanin antibody and antigen-binding region thereof specifically bind to the PLAG4 domain of human podoplanin.
2. The isolated humanized anti-podoplanin antibody or antibody fragment thereof, containing the antigen-binding region thereof according to claim 1 , wherein the anti-podoplanin antibody comprises a constant region of a human antibody of the IgG class.
3. An isolated mouse-human chimeric anti-podoplanin antibody, or an antibody fragment thereof, comprising an antigen-binding region, which comprises variable regions comprising the following amino acid sequences,
the amino acid sequences of the heavy-chain FRs 1 to 4 and the light-chain FRs 1 to 4 optionally have a sequence identity of not less than 90% to the amino acid sequences represented by SEQ ID NO: 17 to 24 below, respectively,
wherein
the amino acid sequence of the heavy-chain CDR1 is the amino acid sequence represented by SEQ ID NO: 1,
the amino acid sequence of the heavy-chain CDR2 is the amino acid sequence represented by SEQ ID NO: 2,
the amino acid sequence of the heavy-chain CDR3 is the amino acid sequence represented by SEQ ID NO: 3,
the amino acid sequence of the light-chain CDR1 is the amino acid sequence represented by SEQ ID NO: 4,
the amino acid sequence of the light-chain CDR2 is the amino acid sequence represented by SEQ ID NO: 5,
the amino acid sequence of the light-chain CDR3 is the amino acid sequence represented by SEQ ID NO: 6,
the amino acid sequence of the heavy-chain FR1 is the amino acid sequence represented by SEQ ID NO: 17,
the amino acid sequence of the heavy-chain FR2 is the amino acid sequence represented by SEQ ID NO: 18,
the amino acid sequence of the heavy-chain FR3 is the amino acid sequence represented by SEQ ID NO: 19,
the amino acid sequence of the heavy-chain FR4 is the amino acid sequence represented by SEQ ID NO: 20,
the amino acid sequence of the light-chain FR1 is the amino acid sequence represented by SEQ ID NO: 21,
the amino acid sequence of the light-chain FR2 is the amino acid sequence represented by SEQ ID NO: 22,
the amino acid sequence of the light-chain FR3 is the amino acid sequence represented by SEQ ID NO: 23, and
the amino acid sequence of the light-chain FR4 is the amino acid sequence represented by SEQ ID NO: 24,
wherein said anti-podoplanin antibody and antigen-binding region thereof specifically bind to the PLAG4 domain of human podoplanin.
4. The isolated mouse-human chimeric anti-podoplanin antibody or antibody fragment thereof, comprising an antigen-binding region thereof according to claim 3 , wherein the anti-podoplanin antibody comprises a constant region of a human antibody of the IgG class.
5. A method of inhibiting the binding between podoplanin and CLEC-2, the method comprising the step of administering
an isolated humanized anti-podoplanin antibody, or an antibody fragment thereof, comprising an antigen-binding region which comprises a variable region comprising the amino acid sequence I or II below,
the amino acid sequences of the heavy-chain FRs 1 to 4 and the light-chain FRs 1 to 4 in the following amino acid sequence I optionally have a sequence identity of not less than 90% to the amino acid sequences represented by SEQ ID NOs: 7 to 14 below, respectively, and
the amino acid sequences of the heavy-chain FRs 1 to 4 and the light-chain FRs 1 to 4 in the following amino acid sequence II optionally have a sequence identity of not less than 90% to the amino acid sequences represented by SEQ ID NOs: 7, 15, 16, 10 to 14 below, respectively;
wherein in the amino acid sequence I,
the amino acid sequence of the heavy-chain CDR1 is the amino acid sequence represented by SEQ ID NO: 1,
the amino acid sequence of the heavy-chain CDR2 is the amino acid sequence represented by SEQ ID NO: 2,
the amino acid sequence of the heavy-chain CDR3 is the amino acid sequence represented by SEQ ID NO: 3,
the amino acid sequence of the light-chain CDR1 is the amino acid sequence represented by SEQ ID NO: 4,
the amino acid sequence of the light-chain CDR2 is the amino acid sequence represented by SEQ ID NO: 5,
the amino acid sequence of the light-chain CDR3 is the amino acid sequence represented by SEQ ID NO: 6,
the amino acid sequence of the heavy-chain FR1 is the amino acid sequence represented by SEQ ID NO: 7,
the amino acid sequence of the heavy-chain FR2 is the amino acid sequence represented by SEQ ID NO: 8,
the amino acid sequence of the heavy-chain FR3 is the amino acid sequence represented by SEQ ID NO: 9,
the amino acid sequence of the heavy-chain FR4 is the amino acid sequence represented by SEQ ID NO: 10,
the amino acid sequence of the light-chain FR1 is the amino acid sequence represented by SEQ ID NO: 11,
the amino acid sequence of the light-chain FR2 is the amino acid sequence represented by SEQ ID NO: 12,
the amino acid sequence of the light-chain FR3 is the amino acid sequence represented by SEQ ID NO: 13, and
the amino acid sequence of the light-chain FR4 is the amino acid sequence represented by SEQ ID NO: 14; and
wherein, in the amino acid sequence II,
the amino acid sequence of the heavy-chain CDR1 is the amino acid sequence represented by SEQ ID NO: 1,
the amino acid sequence of the heavy-chain CDR2 is the amino acid sequence represented by SEQ ID NO: 2,
the amino acid sequence of the heavy-chain CDR3 is the amino acid sequence represented by SEQ ID NO: 3,
the amino acid sequence of the light-chain CDR1 is the amino acid sequence represented by SEQ ID NO: 4,
the amino acid sequence of the light-chain CDR2 is the amino acid sequence represented by SEQ ID NO: 5,
the amino acid sequence of the light-chain CDR3 is the amino acid sequence represented by SEQ ID NO: 6,
the amino acid sequence of the heavy-chain FR1 is the amino acid sequence represented by SEQ ID NO: 7,
the amino acid sequence of the heavy-chain FR2 is the amino acid sequence represented by SEQ ID NO: 15,
the amino acid sequence of the heavy-chain FR3 is the amino acid sequence represented by SEQ ID NO: 16,
the amino acid sequence of the heavy-chain FR4 is the amino acid sequence represented by SEQ ID NO: 10,
the amino acid sequence of the light-chain FRI is the amino acid sequence represented by SEQ ID NO: 11,
the amino acid sequence of the light-chain FR2 is the amino acid sequence represented by SEQ ID NO: 12,
the amino acid sequence of the light-chain FR3 is the amino acid sequence represented by SEQ ID NO: 13, and
the amino acid sequence of the light-chain FR4 is the amino acid sequence represented by SEQ ID NO: 14,
to a human subject,
wherein said anti-podoplanin antibody and antigen-binding region thereof specifically bind to the PLAG4 domain of human podoplanin.
6. A pharmaceutical composition, comprising, an effective amount for specifically binding to podoplanin of an isolated humanized anti-podoplanin antibody or an antibody fragment thereof, containing an antigen-binding region which comprises a variable region comprising the amino acid sequence I or II below,
the amino acid sequences of the heavy-chain FRs 1 to 4 and the light-chain FRs 1 to 4 in the following amino acid sequence I optionally have a sequence identity of not less than 90% to the amino acid sequences represented by SEQ ID NOs: 7 to 14 below, respectively, and
the amino acid sequences of the heavy-chain FRs 1 to 4 and the light-chain FRs 1 to 4 in the following amino acid sequence II optionally have a sequence identity of not less than 90% to the amino acid sequences represented by SEQ ID NOs: 7, 15, 16, 10 to 14 below, respectively;
wherein in the amino acid sequence I,
the amino acid sequence of the heavy-chain CDR1 is the amino acid sequence represented by SEQ ID NO: 1,
the amino acid sequence of the heavy-chain CDR2 is the amino acid sequence represented by SEQ ID NO: 2,
the amino acid sequence of the heavy-chain CDR3 is the amino acid sequence represented by SEQ ID NO: 3,
the amino acid sequence of the light-chain CDR1 is the amino acid sequence represented by SEQ ID NO: 4,
the amino acid sequence of the light-chain CDR2 is the amino acid sequence represented by SEQ ID NO: 5 ,
the amino acid sequence of the light-chain CDR3 is the amino acid sequence represented by SEQ ID NO: 6,
the amino acid sequence of the heavy-chain FR1 is the amino acid sequence represented by SEQ ID NO: 7,
the amino acid sequence of the heavy-chain FR2 is the amino acid sequence represented by SEQ ID NO: 8,
the amino acid sequence of the heavy-chain FR3 is the amino acid sequence represented by SEQ ID NO: 9,
the amino acid sequence of the heavy-chain FR4 is the amino acid sequence represented by SEQ ID NO: 10,
the amino acid sequence of the light-chain FRI is the amino acid sequence represented by SEQ ID NO: 11,
the amino acid sequence of the light-chain FR2 is the amino acid sequence represented by SEQ ID NO: 12,
the amino acid sequence of the light-chain FR3 is the amino acid sequence represented by SEQ ID NO: 13, and
the amino acid sequence of the light-chain FR4 is the amino acid sequence represented by SEQ ID NO: 14; and
wherein, in the amino acid sequence II,
the amino acid sequence of the heavy-chain CDR1 is the amino acid sequence represented by SEQ ID NO: 1,
the amino acid sequence of the heavy-chain CDR2 is the amino acid sequence represented by SEQ ID NO: 2,
the amino acid sequence of the heavy-chain CDR3 is the amino acid sequence represented by SEQ ID NO: 3,
the amino acid sequence of the light-chain CDR1 is the amino acid sequence represented by SEQ ID NO: 4,
the amino acid sequence of the light-chain CDR2 is the amino acid sequence represented by SEQ ID NO: 5,
the amino acid sequence of the light-chain CDR3 is the amino acid sequence represented by SEQ ID NO: 6,
the amino acid sequence of the heavy-chain FR1 is the amino acid sequence represented by SEQ ID NO: 7,
the amino acid sequence of the heavy-chain FR2 is the amino acid sequence represented by SEQ ID NO: 15,
the amino acid sequence of the heavy-chain FR3 is the amino acid sequence represented by SEQ ID NO: 16,
the amino acid sequence of the heavy-chain FR4 is the amino acid sequence represented by SEQ ID NO: 10,
the amino acid sequence of the light-chain FRI is the amino acid sequence represented by SEQ ID NO: 11,
the amino acid sequence of the light-chain FR2 is the amino acid sequence represented by SEQ ID NO: 12,
the amino acid sequence of the light-chain FR3 is the amino acid sequence represented by SEQ ID NO: 13, and
the amino acid sequence of the light-chain FR4 is the amino acid sequence represented by SEQ ID NO: 14;
and a pharmaceutically acceptable carrier,
wherein said anti-podoplanin antibody and antigen-binding region thereof specifically bind to the PLAG4 domain of human podoplanin.
7. The pharmaceutical composition according to claim 6 , wherein said isolated humanized anti-podoplanin antibody, or an antibody fragment thereof is present in a therapeutically effective amount effective to inhibit the binding between podoplanin and CLEC-2.
8. The pharmaceutical composition according to claim 7 , wherein said isolated humanized anti-podoplanin antibody, or an antibody fragment thereof is present in a therapeutically effective amount effective to inhibit platelet aggregation.
9. A method of inhibiting the binding between podoplanin and CLEC-2, the method comprising the step of administering
an isolated mouse-human chimeric anti-podoplanin antibody, or an antibody fragment thereof, comprising an antigen-binding region, which comprises variable regions comprising the following amino acid sequences,
the amino acid sequences of the heavy-chain FRs 1 to 4 and the light-chain FRs 1 to 4 optionally have a sequence identity of not less than 90% to the amino acid sequences represented by SEQ ID NO: 17 to 24 below, respectively,
wherein
the amino acid sequence of the heavy-chain CDR1 is the amino acid sequence represented by SEQ ID NO: 1,
the amino acid sequence of the heavy-chain CDR2 is the amino acid sequence represented by SEQ ID NO: 2,
the amino acid sequence of the heavy-chain CDR3 is the amino acid sequence represented by SEQ ID NO: 3,
the amino acid sequence of the light-chain CDR1 is the amino acid sequence represented by SEQ ID NO: 4,
the amino acid sequence of the light-chain CDR2 is the amino acid sequence represented by SEQ ID NO: 5,
the amino acid sequence of the light-chain CDR3 is the amino acid sequence represented by SEQ ID NO: 6,
the amino acid sequence of the heavy-chain FR1 is the amino acid sequence represented by SEQ ID NO: 17,
the amino acid sequence of the heavy-chain FR2 is the amino acid sequence represented by SEQ ID NO: 18,
the amino acid sequence of the heavy-chain FR3 is the amino acid sequence represented by SEQ ID NO: 19,
the amino acid sequence of the heavy-chain FR4 is the amino acid sequence represented by SEQ ID NO: 20,
the amino acid sequence of the light-chain FRI is the amino acid sequence represented by SEQ ID NO: 21,
the amino acid sequence of the light-chain FR2 is the amino acid sequence represented by SEQ ID NO: 22,
the amino acid sequence of the light-chain FR3 is the amino acid sequence represented by SEQ ID NO: 23, and
the amino acid sequence of the light-chain FR4 is the amino acid sequence represented by SEQ ID NO: 24, to a human subject,
wherein said anti-podoplanin antibody and antigen-binding region thereof specifically bind to the PLAG4 domain of human podoplanin.
10. A pharmaceutical composition, comprising:
an effective amount for specifically binding to podoplanin of an isolated mouse-human chimeric anti-podoplanin antibody, or an antibody fragment thereof, comprising an antigen-binding region, which comprises variable regions comprising the following amino acid sequences,
the amino acid sequences of the heavy-chain FRs 1 to 4 and the light-chain FRs 1 to 4 optionally have a sequence identity of not less than 90% to the amino acid sequences represented by SEQ ID NO: 17 to 24 below, respectively,
wherein
the amino acid sequence of the heavy-chain CDR1 is the amino acid sequence represented by SEQ ID NO: 1,
the amino acid sequence of the heavy-chain CDR2 is the amino acid sequence represented by SEQ ID NO: 2,
the amino acid sequence of the heavy-chain CDR3 is the amino acid sequence represented by SEQ ID NO: 3,
the amino acid sequence of the light-chain CDR1 is the amino acid sequence represented by SEQ ID NO: 4,
the amino acid sequence of the light-chain CDR2 is the amino acid sequence represented by SEQ ID NO: 5,
the amino acid sequence of the light-chain CDR3 is the amino acid sequence represented by SEQ ID NO: 6,
the amino acid sequence of the heavy-chain FR1 is the amino acid sequence represented by SEQ ID NO: 17,
the amino acid sequence of the heavy-chain FR2 is the amino acid sequence represented by SEQ ID NO: 18,
the amino acid sequence of the heavy-chain FR3 is the amino acid sequence represented by SEQ ID NO: 19,
the amino acid sequence of the heavy-chain FR4 is the amino acid sequence represented by SEQ ID NO: 20,
the amino acid sequence of the light-chain FR1 is the amino acid sequence represented by SEQ ID NO: 21,
the amino acid sequence of the light-chain FR2 is the amino acid sequence represented by SEQ ID NO: 22,
the amino acid sequence of the light-chain FR3 is the amino acid sequence represented by SEQ ID NO: 23, and
the amino acid sequence of the light-chain FR4 is the amino acid sequence represented by SEQ ID NO: 24,
and a pharmaceutically acceptable carrier,
wherein said anti-podoplanin antibody and antigen-binding region thereof specifically bind to the PLAG4 domain of human podoplanin.
11. The pharmaceutical composition according to claim 10 , wherein said isolated mouse-human chimeric anti-podoplanin antibody, or an antibody fragment thereof is present in a therapeutically effective amount effective to inhibit the binding between podoplanin and CLEC-2.
12. The pharmaceutical composition according to claim 11 , wherein said isolated mouse-human chimeric anti-podoplanin antibody, or an antibody fragment thereof is present in a therapeutically effective amount effective to inhibit platelet aggregation.