SIGLEC-6 ANTIBODIES, DERIVATIVE COMPOUNDS AND RELATED USES
The invention provides antibodies, antibody-based fragments or antibody fragments (antigen-binding fragments), as well as derivative molecules such as bispecific T-cell engagers, antibody-drug conjugates (ADCs) and chimeric antigen receptors (CARs), that specifically recognize Siglec-6 and related compositions. Also provided in the invention are methods of using such antibodies in various diagnostic and therapeutic applications.
1 . An isolated antibody that binds to the same epitope on sialic acid-binding immunoglobulin-like lectin-6 (Siglec-6) as antibody RC-1, RC-2, or ARN-1.
2 . The antibody of claim 1 ,
comprising three heavy chain CDRs and three light chain CDRs of antibody RC-1, wherein RC-1 is characterized by a mature heavy chain variable region having an amino acid sequence comprising SEQ ID NO:1 and a mature light chain variable region having an amino acid sequence comprising SEQ ID NO:2;
comprising three heavy chain CDRs and threw light chain CDRs of antibody RC-2, wherein RC-2 is characterized by a mature heavy chain variable region having an amino acid sequence comprising SEQ ID NO:3 and a mature light chain variable region having an amino acid sequence comprising SEQ ID NO:4; or comprising three heavy chain CDRs and three light chain CDRs of antibody ARN-1, wherein ARN-1 is characterized by a mature heavy chain variable region having an amino acid sequence comprising SEQ ID NO:5 and a mature light chain variable region having an amino acid sequence comprising SEQ ID NO:6.
3 . The antibody of claim 2 , wherein the CDRs are of a definition selected from the group consisting of Kabat, Chothia, Kabat/Chothia Composite, AbM, Contact, and IMGT.
4 . The antibody of claim 3 ,
wherein the mature heavy chain variable region comprises the three Kabat heavy chain CDRs of SEQ ID NOs:7-9 and the mature light chain variable region comprises the three Kabat light chain CDRs of SEQ ID NOs: 10-12;
wherein the mature heavy chain variable region comprises the three Kabat heavy chain CDRs of SEQ ID NOs:13-15 and the mature light chain variable region comprises the three Kabat light chain CDRs of SEQ ID NOs:16-18; or
wherein the mature heavy chain variable region comprises the three Kabat heavy chain CDRs of SEQ ID NOs:19-21 and the mature light chain variable region comprises the three Kabat light chain CDRs of SEQ ID NOs:22-24.
5 . The antibody of any one of claims 1-4 ,
comprising a mature heavy chain variable region having an amino acid sequence at least 90% identical to SEQ ID NO: 1, and a mature light chain variable region having an amino acid sequence at least 95% identical to SEQ ID NO:2;
comprising a mature heavy chain variable region having an amino acid sequence at least 90% identical to SEQ ID NO:3, and a mature light chain variable region having an amino acid sequence at least 95% identical to SEQ ID NO:4; or
comprising a mature heavy chain variable region having an amino acid sequence at least 90% identical to SEQ ID NO: 5, and a mature light chain variable region having an amino acid sequence at least 90% identical to SEQ ID NO:6.
6 . The antibody of claim 5 ,
comprising a mature heavy chain variable region having an amino acid sequence at least 95% identical to SEQ ID NO: 1, and a mature light chain variable region having an amino acid sequence at least 95% identical to SEQ ID NO:2;
comprising a mature heavy chain variable region having an amino acid sequence at least 95% identical to SEQ ID NO:3, and a mature light chain variable region having an amino acid sequence at least 95% identical to SEQ ID NO:4; or
comprising a mature heavy chain variable region having an amino acid sequence at least 95% identical to SEQ ID NO:5, and a mature light chain variable region having an amino acid sequence at least 95% identical to SEQ ID NO:6.
7 . The antibody of claim 6 ,
comprising a mature heavy chain variable region having an amino acid sequence of SEQ ID NO: 1, and a mature light chain variable region having an amino acid sequence of SEQ ID NO:2;
comprising a mature heavy chain variable region having an amino acid sequence of SEQ ID NO:3, and a mature light chain variable region having an amino acid sequence of SEQ ID NO:4; or
comprising a mature heave chain variable region having an amino acid sequence of SEQ ID NO:5, and a mature light chain variable region having an amino acid sequence of SEQ ID NO:6.
8 . The antibody of one of claims 1-7 , wherein the antibody is chimeric, humanized, or human.
9 . The antibody of claim 8 , wherein the antibody is human.
10 . The antibody of any preceding claim , wherein the antibody is IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, synthetic IgG, IgM, F(ab)2, Fv, scFv, IgGACH2, F(ab′)2, scFv2CH3, Fab, VL, VH, scFv4, scFv3, scFv2, dsFv, Fv, scFv-Fc, (scFv)2, a non-depleting IgG, a diabody, a dual-affinity re-targeting (DART) antibody, or a bivalent antibody.
11 . The antibody of any preceding claim , wherein the mature light chain variable region is fused to a light chain constant region and the mature heavy chain variable region is fused to a heavy chain constant region.
12 . The antibody of claim 11 , wherein the heavy chain constant region is a mutant form of a natural human heavy chain constant region which has reduced binding to a Fcγ receptor relative to the natural human heavy chain constant region.
13 . The antibody of claim 1 , which is a scFv.
14 . The antibody of claim 13 , further comprising a Fc domain that is fused to the C-terminus of the scFv.
15 . The antibody of claim 14 , comprising an amino acid sequence at least 90% identical to SEQ ID NO:25, wherein the mature heavy chain variable region comprises the three Kabat heavy chain CDRs of SEQ ID NOs:7-9 and the mature light chain variable region comprises the three Kabat light chain CDRs of SEQ ID NOs:10-12.
16 . The antibody of any preceding claim , which is an antibody fragment.
17 . The antibody of any one of claims 1-15 , which is a bispecific antibody.
18 . The antibody of claim 17 , wherein the bispecific antibody is a T-cell engaging bispecific antibody.
19 . The antibody of any one of claims 1-15 , which is an antibody-based binding protein.
20 . The antibody of any preceding claim , which is conjugated to a synthetic molecule.
21 . The antibody of claim 20 , % herein the synthetic molecule is a cytotoxic agent, a label, a therapeutic radioisotope, a diagnostic radioisotope, or a liposome.
22 . The antibody of claim 21 , wherein the cytotoxic agent is a small molecule weight toxin, a peptide toxin, or a protein toxin.
23 . The antibody of any one of claims 1-19 linked to at least one cytotoxic agent as an antibody drug conjugate.
24 . The antibody of claim 23 , wherein the cytotoxic agent is a small molecular weight toxin, a peptide toxin, or a protein toxin, or a radionuclide.
25 . AT cell-engaging bispecific antibody (T-biAb), comprising (1) a V H domain and a V L domain of a T cell targeting antibody, and (2) a V H domain and a V L domain of an antibody of claim 1 (Siglec-6 targeting antibody);
wherein the V H domain of the Siglec-6 targeting antibody comprises the three Kabat heavy chain CDRs of SEQ ID NOs:7-9 and the V L domain of the Siglec-6 targeting antibody comprises the three Kabat light chain CDRs of SEQ ID NOs:10-12;
wherein the V H domain of the Siglec-6 targeting antibody comprises the three Kabat heavy chain CDRs of SEQ ID NOs: 13-15 and the V L domain of the Siglec-6 targeting antibody comprises the three Kabat light chain CDRs of SEQ ID NOs:16-18; or
wherein the V H domain of the Siglec-6 targeting antibody comprises the three Kabat heavy chain CDRs of SEQ ID NOs:19-21 and the V L domain of the Siglec-6 targeting antibody comprises the three Kabat light chain CDRs of SEQ ID NOs:22-24.
26 . The T-biAb of claim 25 , wherein the T cell targeting antibody is an anti-CD3 antibody.
27 . The T-biAb of claim 26 , wherein the T cell targeting antibody is an anti-CD3 antibody comprising a heavy variable region having an amino acid sequence of SEQ ID NO:34 and light variable region having an amino acid sequence of SEQ ID NO:35.
28 . The T-biAb of claim 26 , wherein the T cell targeting antibody is an anti-CD3 antibody comprising an amino acid sequence of SEQ ID NO:33.
29 . The T-biAb of claim 26 , wherein the T cell targeting antibody is an anti-CD3 antibody comprising an amino acid sequence of SEQ ID NO:39.
30 . The T-biAb of claim 25 , wherein the V H domain and the V L domain of the T cell targeting antibody forms a first scFv, and the V H domain and the V L domain of the Siglec-6 targeting antibody forms a second scFv.
31 . The T-biAb of claim 25 , wherein the V H domain of the T cell targeting antibody and the V L domain of the Siglec-6 targeting antibody forms a first tandem V H -V L fragment, and the V L domain of the T cell targeting antibody and the V H domain of the Siglec-6 targeting antibody forms a second tandem V H -V L fragment.
32 . The T-biAb of claim 31 , further comprising a C-terminal inter-chain disulfide bond to connect the two tandem V H -V L fragments.
33 . The T-biAb of claim 31 , wherein one or both tandem V H -V L fragments are further fused to an antibody Fc arm in either a V H -V L -Fc format or a V L V H -Fc format.
34 . The T-biAb of claim 33 , comprising two polypeptide chains each of which comprises a tandem V H -V L fragment that is fused to a Fc arm, wherein the two Fc arms respectively contain knob mutations and hole mutations to allow dimerization of the two polypeptide chains.
35 . The T-biAb of claim 34 , wherein the first polypeptide chain comprises a tandem V H -V L fragment of a T cell targeting V L and a Siglec-6 targeting V H that is fused to one Fc arm, and the second polypeptide chain comprises a tandem V H -V L fragment of a Siglec-6 targeting V L and a T cell targeting V H that is fused to the other Fc arm.
36 . The T-biAb of claim 35 , where (a) the first polypeptide chain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:26, and a Siglec-6 targeting V H domain comprising the three Kabat heavy chain CDRs of SEQ ID NOs:7-9; and (b) the second polypeptide chain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:27, and a Siglec-6 targeting V L domain comprising the three Kabat light chain CDRs of SEQ ID NOs:10-12.
37 . The T-biAb of claim 34 , wherein the first polypeptide chain comprises a tandem V H -V L fragment of a Siglec-6 targeting V L and a T cell targeting V H that is fused to one Fc arm, and the second polypeptide chain comprises a tandem V H -V L fragment of a T cell targeting V L and a Siglec-6 targeting V H that is fused to the other Fc arm.
38 . The T-biAb of claim 37 , where (a) the first polypeptide chain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:28, and a Siglec-6 targeting V L domain comprising the three Kabat light chain CDRs of SEQ ID NOs:10-12; and (b) the second polypeptide chain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:29, and a Siglec-6 targeting V H domain comprising the three Kabat heavy chain CDRs of SEQ ID NOs:7-9.
39 . The T-biAb of claim 33 , comprising three polypeptide chains, wherein (a) the first polypeptide chain comprises a first tandem V H -V L fragment having a first C-terminal coil motif that is fused to a first Fc arm, (b) the second polypeptide chain comprises the other tandem V H -V L fragment having a second C-terminal coil motif, and (c) the third polypeptide chain comprises a second Fc arm; wherein one of the Fc arms contains knob mutations, and the other Fc arm contains hole mutations; and wherein one of the C-terminal coil motif is a E-coil, and the other C-terminal coil motif is an oppositely charged K-coil.
40 . The T-biAb of claim 39 , wherein (a) the first tandem V H -V L fragment comprises a Siglec-6 targeting V L and a T cell targeting V H , and the second tandem V H -V L fragment comprises a T cell targeting V L and a Siglec-6 targeting V H , or (b) the first tandem V H -V L fragment comprises a T cell targeting V L and a Siglec-6 targeting V H , and the second tandem V H -V L fragment comprises a Siglec-6 targeting V L and a T cell targeting V H .
41 . The T-biAb of claim 40 , wherein (a) the first polypeptide chain comprises (i) a Siglec-6 targeting V L domain comprising the three Kabat light chain CDRs of SEQ ID NOs:10-12 and (ii) an overall amino acid sequence that is at least 90% identical to SEQ ID NO:30; (b) the second polypeptide chain comprises (i) a Siglec-6 targeting V H domain comprising the three Kabat heavy chain CDRs of SEQ ID NOs:7-9 and (ii) an overall amino acid sequence that is at least 90% identical to SEQ ID NO:31, and (c) the third polypeptide chain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:32.
42 . A chimeric antigen receptor (CAR), comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular domain comprises the antibody of any one of claims 1-7 .
43 . An effector cell expressing the chimeric antigen receptor of claim 42 .
44 . The effector cell of claim 43 , which is a T cell or a natural killer (NK) cell.
45 . A pharmaceutical composition comprising a therapeutically effective amount of the antibody of any one of claims 1-24 , or the T cell-engaging bispecific antibody of any one of claims 25-41 and a pharmaceutically acceptable carrier.
46 . A polynucleotide encoding a heavy chain variable region and/or a light chain variable region of the antibody of any one of claims 1-24 .
47 . A vector comprising the polynucleotide of claim 46 .
48 . A host cell harboring the vector of claim 47 .
49 . A method of treating a disease or disorder associated with aberrant Siglec-6 expression in a subject, comprising administering the pharmaceutical composition of claim 45 to a subject in need thereof.
50 . The method of claim 49 , wherein the disease or disorder is a tumor, a leukemia, or a mast cell disorder.
51 . The method of claim 49 , wherein the disease or disorder is a chronic or an acute leukemia.
52 . The method of claim 49 , wherein the disease or disorder is chronic lymphocytic leukemia (CLL).
53 . The method of claim 49 , wherein the disease or disorder is a tumor containing Siglec-6 expressing myeloid-derived suppressor cells (MDSCs).
54 . The method of claim 49 , wherein the disease or disorder is an autoimmune disease or an inflammatory disease.
55 . The method of claim 54 , wherein the autoimmune disease or the inflammatory disease is multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, type 2 diabetes mellitus, cytokine release syndrome, graft-versus-host-disease, or HIV-associated immunopathogenesis.