Anti-ALK2 antibody
This application provides: an antibody which specifically binds to an ALK2 protein and has an activity of inhibiting BMP signal transduction mediated by ALK2; a method for producing the antibody; and a pharmaceutical composition comprising the antibody, for treating and/or preventing ectopic ossification and/or bone dysplasia, anemia, or diffuse intrinsic pontine glioma (DIPG).
1. A monoclonal antibody or an antigen-binding fragment thereof which inhibits activin like kinase 2 (ALK2)-mediated bone morphogenetic proteins (BMP) signal transduction and specifically binds to a polypeptide sequence consisting of at least 7 amino acids present in any one of the following amino acid sequences (a) to (d):
(a) the amino acid sequence of SEQ ID NO: 84;
(b) the amino acid sequence consisting of amino acid position numbers 21 to 123 of the amino acid sequence of SEQ ID NO: 84;
(c) the amino acid sequence of SEQ ID NO: 86; and
(d) the amino acid sequence consisting of amino acid position numbers 21 to 123 of the amino acid sequence of SEQ ID NO: 86,
wherein the monoclonal antibody or antigen binding fragment comprises a heavy chain variable region having complementarity determining region (CDR) H1, CDRH2, and CDRH3 and a light chain variable region having CDRL1, CDRL2, and CDRL3, wherein a set of the amino acid sequences of CDRH1, CDRH2, and CDRH3 and the amino acid sequences of CDRL1, CDRL2, and CDRL3 is selected from the group consisting of:
(1) SEQ ID NO: 72 for CDRH1, SEQ ID NO: 73 for CDRH2, and SEQ ID NO: 74 for CDRH3, and SEQ ID NO: 75 for CDRL1, SEQ ID NO: 76 for CDRL2, and SEQ ID NO: 77 for CDRL3;
(2) SEQ ID NO: 59 for CDRH1, SEQ ID NO: 60 for CDRH2, and SEQ ID NO: 61 for CDRH3, and SEQ ID NO: 62 for CDRL1, SEQ ID NO: 63 or 71 for CDRL2, and SEQ ID NO: 64 for CDRL3;
(3) SEQ ID NO: 78 for CDRH1, SEQ ID NO: 79 for CDRH2, and SEQ ID NO: 80 for CDRH3, and SEQ ID NO: 81 for CDRL1, SEQ ID NO: 82 for CDRL2, and SEQ ID NO: 83 for CDRL3; and
(4) SEQ ID NO: 65 for CDRH1, SEQ ID NO: 66 for CDRH2, and SEQ ID NO: 67 for CDRH3, and SEQ ID NO: 68 for CDRL1, SEQ ID NO: 69 for CDRL2, and SEQ ID NO: 70 for CDRL3.
2. The monoclonal antibody or the antigen-binding fragment thereof according to claim 1 , wherein the antibody binds to a wild-type ALK2 protein and a mutant ALK2 protein, and optionally binds to an ALK2 extracellular region.
3. The monoclonal antibody or the antigen-binding fragment thereof according to claim 1 , wherein the antibody or the antigen-binding fragment is a chimeric antibody, a humanized antibody, a human antibody, single-chain Fv, a bispecific antibody, or a multispecific antibody.
4. The monoclonal antibody or the antigen-binding fragment thereof according to claim 1 , wherein the antibody comprises a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence of
SEQ ID NO: 2 and SEQ ID NO: 4;
SEQ ID NO: 6 and SEQ ID NO: 8;
SEQ ID NO: 6 and amino acid residues 21 to 133 of SEQ ID NO: 36;
SEQ ID NO: 10 and SEQ ID NO: 12; or
SEQ ID NO: 14 and SEQ ID NO: 16 respectively.
5. The antigen-binding fragment according to claim 1 , wherein the antigen-binding fragment is selected from the group consisting of Fab, F(ab′) 2 , Fab′, and Fv.
6. The monoclonal antibody according to claim 1 , wherein the antibody comprises a heavy chain constant region selected from the group consisting of a human immunoglobulin G1 heavy chain, a human immunoglobulin G2 heavy chain, and a human immunoglobulin G4 heavy chain, and a light chain constant region, wherein the light chain constant region is a human immunoglobulin κ light chain constant region.
7. The monoclonal antibody according to claim 6 , wherein the heavy chain constant region is a human immunoglobulin G1 heavy chain constant region comprising a leucine (Leu) to alanine (Ala) substitution at position 234, and a leucine (Leu) to alanine (Ala) substitution at position 235.
8. The monoclonal antibody according to claim 6 , wherein the heavy chain constant region is a human immunoglobulin G4 heavy chain constant region comprising a serine (Ser) to proline (Pro) substitution at position 241.
9. The monoclonal antibody or the antigen-binding fragment thereof according to claim 1 , wherein the antibody comprises
(a) a heavy chain variable region sequence selected from the group consisting of:
a1) the amino acid sequence consisting of amino acid position numbers 20 to 142 of the amino acid sequence of SEQ ID NO: 28;
a2) the amino acid sequence consisting of amino acid position numbers 20 to 142 of the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 105;
a3) the amino acid sequence consisting of amino acid position numbers 20 to 140 of the amino acid sequence of SEQ ID NO: 40;
a4) the amino acid sequence consisting of amino acid position numbers 20 to 140 of the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 107;
a5) the amino acid sequence consisting of amino acid position numbers 20 to 140 of the amino acid sequence of SEQ ID NO: 44 or SEQ ID NO: 109;
a6) the amino acid sequence consisting of amino acid position numbers 20 to 140 of the amino acid sequence of SEQ ID NO: 46;
a7) the amino acid sequence consisting of amino acid position numbers 20 to 140 of the amino acid sequence of SEQ ID NO: 48;
a8) the amino acid sequence consisting of amino acid position numbers 20 to 137 of the amino acid sequence of SEQ ID NO: 111;
a9) the amino acid sequence consisting of amino acid position numbers 20 to 137 of the amino acid sequence of SEQ ID NO: 113;
a10) the amino acid sequence consisting of amino acid position numbers 20 to 137 of the amino acid sequence of SEQ ID NO: 121;
a11) the amino acid sequence consisting of amino acid position numbers 20 to 137 of the amino acid sequence of SEQ ID NO: 123;
a12) an amino acid sequence having at least 95% identity to any one amino acid sequence selected from the amino acid sequences a1) to a11); and
a13) an amino acid sequence having at least 99% identity to any one amino acid sequence selected from the amino acid sequences a1) to a11); and
(b) a light chain variable region sequence selected from the group consisting of:
b1) the amino acid sequence consisting of amino acid position numbers 21 to 133 of the amino acid sequence of SEQ ID NO: 32;
b2) the amino acid sequence consisting of amino acid position numbers 21 to 133 of the amino acid sequence of SEQ ID NO: 34;
b3) the amino acid sequence consisting of amino acid position numbers 21 to 133 of the amino acid sequence of SEQ ID NO: 36;
b4) the amino acid sequence consisting of amino acid position numbers 21 to 133 of the amino acid sequence of SEQ ID NO: 38;
b5) the amino acid sequence consisting of amino acid position numbers 21 to 129 of the amino acid sequence of SEQ ID NO: 50;
b6) the amino acid sequence consisting of amino acid position numbers 21 to 129 of the amino acid sequence of SEQ ID NO: 52;
b7) the amino acid sequence consisting of amino acid position numbers 21 to 129 of the amino acid sequence of SEQ ID NO: 54;
b8) the amino acid sequence consisting of amino acid position numbers 21 to 129 of the amino acid sequence of SEQ ID NO: 56;
b9) the amino acid sequence consisting of amino acid position numbers 21 to 129 of the amino acid sequence of SEQ ID NO: 58;
b10) the amino acid sequence consisting of amino acid position numbers 21 to 128 of the amino acid sequence of SEQ ID NO: 115;
b11) the amino acid sequence consisting of amino acid position numbers 21 to 128 of the amino acid sequence of SEQ ID NO: 117;
b12) the amino acid sequence consisting of amino acid position numbers 21 to 128 of the amino acid sequence of SEQ ID NO: 119;
b13) the amino acid sequence consisting of amino acid position numbers 21 to 129 of the amino acid sequence of SEQ ID NO: 125;
b14) the amino acid sequence consisting of amino acid position numbers 21 to 129 of the amino acid sequence of SEQ ID NO: 127;
b15) the amino acid sequence consisting of amino acid position numbers 21 to 129 of the amino acid sequence of SEQ ID NO: 129;
b16) an amino acid sequence having at least 95% identity to any one amino acid sequence selected from the amino acid sequences b1) to b15); and
b17) an amino acid sequence having at least 99% identity to any one amino acid sequence selected from the amino acid sequences b1) to b15),
wherein a combination of a heavy chain variable region sequence in (a) and a light chain variable region sequence in (b) that the monoclonal antibody comprises is selected from the group consisting of the combinations of: a1) and b1); a1) and b2); a2) and b1); a2) and b2); a2) and b3); a2) and b4); a3) and b3); a4) and b5); a4) and b6); a4) and b7); a5) and b5); a5) and b6); a5) and b7); a6) and b5); a6) and b6); a6) and b7); a6) and b8); a7) and b7); a7) and b8); a7) and b9); a8) and b8); a9) and b6); a10) and b8); a11) and b10); a11) and b11); a11) and b12); a12) and b10); a12) and b11); a12) and b12); a13) and b13); a13) and b14); a13) and b15); a14) and b13); a14) and b14); and a14) and b15).
10. The monoclonal antibody or the antigen-binding fragment thereof according to claim 9 , wherein the antibody is an antibody comprising
(a) a heavy chain comprising the heavy chain variable region sequence consisting of amino acid position numbers 20 to 142 of the amino acid sequence of SEQ ID NO: 30 and a light chain comprising the light chain variable region sequence consisting of amino acid position numbers 21 to 133 of the amino acid sequence of SEQ ID NO: 36;
(b) a heavy chain comprising the amino acid sequence consisting of amino acid position numbers 20 to 472 of the amino acid sequence of SEQ ID NO: 30 and a light chain comprising the amino acid sequence consisting of amino acid position numbers 21 to 238of the amino acid sequence of SEQ ID NO: 36;
(c) a heavy chain comprising the amino acid sequence consisting of amino acid position numbers 20 to 468 of the amino acid sequence of SEQ ID NO: 105 and a light chain comprising the amino acid sequence consisting of amino acid position numbers 21 to 238of the amino acid sequence of SEQ ID NO: 36;
(d) a heavy chain comprising the heavy chain variable region sequence consisting of amino acid position numbers 20 to 140 of the amino acid sequence of SEQ ID NO: 42 and a light chain comprising the light chain variable region sequence consisting of amino acid position numbers 21 to 129 of the amino acid sequence of SEQ ID NO: 52;
(e) a heavy chain comprising the heavy chain variable region sequence consisting of amino acid position numbers 20 to 140 of the amino acid sequence of SEQ ID NO: 44 and a light chain comprising the light chain variable region sequence consisting of amino acid position numbers 21 to 129 of the amino acid sequence of SEQ ID NO: 56;
(f) a heavy chain comprising the amino acid sequence consisting of amino acid position numbers 20 to 470 of the amino acid sequence of SEQ ID NO: 42 and a light chain comprising the amino acid sequence consisting of amino acid position numbers 21 to 234of the amino acid sequence of SEQ ID NO: 52;
(g) a heavy chain comprising the amino acid sequence consisting of amino acid position numbers 20 to 470 of the amino acid sequence of SEQ ID NO: 44 and a light chain comprising the amino acid sequence consisting of amino acid position numbers 21 to 234of the amino acid sequence of SEQ ID NO: 56;
(h) a heavy chain comprising the amino acid sequence consisting of amino acid position numbers 20 to 470 of the amino acid sequence of SEQ ID NO: 107 and a light chain comprising the amino acid sequence consisting of amino acid position numbers 21 to 234of the amino acid sequence of SEQ ID NO: 52; or
(i) a heavy chain comprising the amino acid sequence consisting of amino acid position numbers 20 to 470 of the amino acid sequence of SEQ ID NO: 109 and a light chain comprising the amino acid sequence consisting of amino acid position numbers 21 to 234of the amino acid sequence of SEQ ID NO: 56.
11. The monoclonal antibody according to claim 1 , wherein the antibody comprises a heavy chain in which one to several carboxyl-terminal amino acids are deleted and/or has a pyroglutamylated amino-terminal amino acid residue in a heavy or light chain thereof.
12. A pharmaceutical composition comprising at least one monoclonal antibody or antigen-binding fragment according to claim 1 , and optionally at least one drug selected from the group consisting of anti-inflammatory drugs, steroids, bisphosphonates, muscle relaxants, and retinoic acid receptor (RAR) γ agonists.