text:
MULTI-SPECIFIC ANTIBODIES
The present invention relates to antigen binding sites, compositions and uses thereof in the treatment of conditions associated with pathogenic proteins. The present invention provides an antigen binding site that binds to or specifically binds to CD33 and a pathogenic protein.
MILES, Luke Anthony (c/- St Vincent's Institute of Medical Research, 9 Princes StreetFitzroy, Victoria 3065, 3065, AU)
AU2019/050204
ST VINCENT'S INSTITUTE OF MEDICAL RESEARCH (9 Princes Street, Fitzroy, Victoria 3065, 3065, AU)
C07K16/28; A61K39/395; A61P25/16; A61P25/28; C07K19/00
Domestic Patent References:
WO2016201389A2 2016-12-15
US9066928B1 2015-06-30
JIANG, T. ET AL.: "CD 33 in Alzheimer's disease", MOLECULAR NEUROBIOLOGY, vol. 49, no. 1, February 2014 (2014-02-01), pages 529 - 535, XP055637206
FPA PATENT ATTORNEYS PTY LTD (Level 43, 101 Collins StreetMelbourne, Victoria 3000, 3000, AU)
1. An antigen binding site that binds to or specifically binds to CD33 and a
pathogenic protein.
2. An antigen binding site according to claim 1 , wherein the antigen binding site comprises an antigen binding domain of an antibody that binds to or specifically binds to CD33 and a pathogenic protein.
pathogenic protein, the antigen binding site comprises:
- a first antigen binding domain of an antibody that binds to or specifically binds to CD33; and
- a second antigen binding domain of an antibody that binds to or specifically binds to a pathogenic protein.
4. An antigen binding site according to claim 3, wherein the first antigen binding domain comprises, consists essentially of or consists of a heavy chain variable domain (VH) and a light chain variable domain (VL).
5. An antigen binding site according to claim 3 or 4, wherein the second antigen binding domain comprises, consists essentially of or consists of a VH and a VL.
6. An antigen binding site according to any one of claims 1 to 5, wherein the
pathogenic protein is associated with a neurodegenerative disease.
7. An antigen binding site according to claim 6, wherein the pathogenic protein is a protein that aggregates or is misfolded.
8. An antigen binding site according to claim 7, wherein the pathogenic protein is selected from the group consisting of amyloid beta, amyloid fragments, amyloid precursor protein, amyloid precursor protein fragments, British peptide, tau (preferably phosphorylated tau) or a (alpha)-synuclein.
9. An antigen binding site according to any one of claims 1 to 8, wherein binding of the antigen binding site to CD33 does not result in activation of CD33.
10. An antigen binding site according to any one of claims 1 to 9, wherein binding to CD33 on the surface of a cell results in CD33-mediated endocytosis.
1 1. An antigen binding site according to any one of claims 1 to 10, wherein the
antigen binding site binds to or specifically binds to an epitope on human CD33 molecule comprising, consisting essentially of or consisting of an amino acid sequence as shown in SEQ ID NO:4.
12. An antigen binding site according to any one of claims 1 to 1 1 , wherein the
pathogenic protein is human amyloid-b.
13. An antigen binding site according to any one of claims 1 to 12, wherein the
antigen binding site binds to or specifically binds to a human amyloid-b molecule comprising, consisting essentially of or consisting of an amino acid sequence as shown in SEQ ID NO: 1.
14. An antigen binding site according to claim 12 or 13, wherein the antigen binding site binds CD33 and amyloid-b simultaneously.
15. An antigen binding site according to any one of claims 1 to 11 , wherein the
pathogenic protein is human tau.
16. An antigen binding site according to claim 15, wherein the tau is phosphorylated, preferably phosphorylated on S422.
17. An antigen binding site according to claim 15 or 16, wherein the antigen binding site binds to or specifically binds to a human tau molecule comprising, consisting essentially of or consisting of an amino acid sequence as shown in SEQ ID NO:2.
18. An antigen binding site according to any one of claims 15 to 17, wherein the antigen binding site binds CD33 and tau simultaneously.
pathogenic protein is human a-synuclein.
20. An antigen binding site according to claim 19, wherein the antigen binding site binds to or specifically binds to a human a-synuclein molecule comprising, consisting essentially of or consisting of an amino acid sequence as shown in SEQ ID NO:3.
21. An antigen binding site according to claim 20, wherein the antigen binding site binds CD33 and a-synuclein simultaneously.
antigen binding site does not significantly increase phagocytic activity of a microglial cell.
antigen binding site does not suppress the phagocytic activity of a microglial cell.
24. An antigen binding site that binds to or specifically binds to CD33 and a
- a first antigen binding domain of an antibody that binds to or specifically binds to CD33, wherein the first antigen binding domain comprises one or more complementarity determining regions;
- a second antigen binding domain of an antibody that binds to or specifically binds to a pathogenic protein, wherein the second antigen binding domain comprises complementarity determining regions wherein the sequence of any of the complementarity determining regions of the first or second antigen binding domains are as described herein.
25. An antigen binding site that binds to or specifically binds to CD33 and amyloid beta, the antigen binding site comprises:
- a first antigen binding domain of an antibody that binds to or specifically binds to CD33, wherein the first antigen binding domain comprises CDR L1 , CDR L2 and CDR L3 from SEQ ID NO: 38; and CDR H1 , CDR H2 and CDR H3 from SEQ ID NO: 37;
- a second antigen binding domain of an antibody that binds to or specifically binds to amyloid beta, wherein the second antigen binding domain comprises CDR L1 , CDR L2 and CDR L3 from SEQ ID NO: 20; and CDR H1 , CDR H2 and CDR H3 from SEQ ID NO: 19.
27. An antigen binding site that binds to or specifically binds to CD33 and tau the antigen binding site comprises:
- a second antigen binding domain of an antibody that binds to or specifically binds to tau, wherein the second antigen binding domain comprises CDR L1 , CDR L2 and CDR L3 from SEQ ID NO: 74; and CDR H1 , CDR H2 and CDR H3 from SEQ ID NO: 73.
28. An antigen binding site that binds to or specifically binds to CD33 and a- synuclein, the antigen binding site comprises:
- a second antigen binding domain of an antibody that binds to or specifically binds to a-synuclein, wherein the second antigen binding domain comprises CDR L1 , CDR L2 and CDR L3 from SEQ ID NO: 92; and CDR H1 , CDR H2 and CDR H3 from SEQ ID NO: 91.
29. An antigen binding site comprising a first antigen binding domain of an antibody and second antigen binding domain of an antibody, wherein the first antigen binding domain binds to or specifically binds to CD33, wherein the first antigen binding domain comprises:
(i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO:23, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set in SEQ ID NO:24 and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 25;
(ii) a VH comprising a sequence at least about 95% or 96% or 97% or 98% or 99% identical to a sequence set forth in SEQ ID NO: 37; (iii) a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO:23, a CDR2 comprising a sequence set forth between in SEQ ID NO: 24 and a CDR3 comprising a sequence set forth in SEQ ID NO: 25; or
(iv) a VH comprising a sequence set forth in SEQ ID NO: 37; and
(v) a VL comprising a CDR1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 26, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 27 and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 28;
(vi) a VL comprising a sequence at least about 95% identical to a sequence set forth in SEQ ID NO: 38;
(vii) a VL comprising a CDR1 comprising a sequence set SEQ ID NO: 26, a CDR2 comprising a sequence set forth in SEQ ID NO: 27 and a CDR3
comprising a sequence set forth in SEQ ID NO: 28; or
(viii) a VL comprising a sequence set forth in SEQ ID NO: 38; wherein the second antigen binding domain binds to or specifically binds to amyloid-beta, wherein the second antigen binding domain comprises:
(i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 5, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set in SEQ ID NO: 6 and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 7; (ii) a VH comprising a sequence at least about 95% or 96% or 97% or 98% or 99% identical to a sequence set forth in SEQ ID NO: 19;
(iii) a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 5, a CDR2 comprising a sequence set forth between in SEQ ID NO: 6 and a CDR3 comprising a sequence set forth in SEQ ID NO: 7; or
(v) a VL comprising a CDR1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 8, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO:
9 and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 10;
(vii) a VL comprising a CDR1 comprising a sequence set SEQ ID NO: 8, a CDR2 comprising a sequence set forth in SEQ ID NO: 9 and a CDR3 comprising a sequence set forth in SEQ ID NO: 10; or
(viii) a VL comprising a sequence set forth in SEQ ID NO: 20.
30. An antigen binding site according to claim 29, wherein the first antigen binding domain further comprises:
(i) a VH comprising a framework region (FR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 29, a FR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set in SEQ ID NO: 30, a FR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 31 , and a FR4 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 32; or
(ii) a VH comprising a FR1 comprising a sequence set forth in SEQ ID NO:
29, a FR2 comprising a sequence set forth between in SEQ ID NO: 30, a FR3 comprising a sequence set forth in SEQ ID NO: 31 , and a FR4 comprising a sequence set forth in SEQ ID NO: 32; and
(iii) a VL comprising a FR1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 33, a FR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 34, a FR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 35, and a FR4 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 36; or
(iv) a VL comprising a FR1 comprising a sequence set forth in SEQ ID NO:
33, a FR2 comprising a sequence set forth between in SEQ ID NO: 34, a FR3 comprising a sequence set forth in SEQ ID NO: 35, and a FR4 comprising a sequence set forth in SEQ ID NO: 36.
31. An antigen binding site according to claim 29 or 30, wherein the second antigen binding domain further comprises:
(i) a VH comprising a framework region (FR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 1 1 , a FR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set in SEQ ID NO: 12, a FR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 13, and a FR4 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 14; or
1 1 , a FR2 comprising a sequence set forth between in SEQ ID NO: 12, a FR3 comprising a sequence set forth in SEQ ID NO: 13, and a FR4 comprising a sequence set forth in SEQ ID NO: 14; and
32. An antigen binding site comprising a first antigen binding domain of an antibody and second antigen binding domain of an antibody, wherein the first antigen binding domain binds to or specifically binds to CD33, wherein the first antigen binding domain comprises: (i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO:23, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set in SEQ ID NO:24 and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 25;
(ii) a VH comprising a sequence at least about 95% or 96% or 97% or 98% or 99% identical to a sequence set forth in SEQ ID NO: 37;
(iii) a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO:23, a CDR2 comprising a sequence set forth between in SEQ ID NO: 24 and a CDR3 comprising a sequence set forth in SEQ ID NO: 25; or
(vii) a VL comprising a CDR1 comprising a sequence set SEQ ID NO: 26, a CDR2 comprising a sequence set forth in SEQ ID NO: 27 and a CDR3 comprising a sequence set forth in SEQ ID NO: 28; or (viii) a VL comprising a sequence set forth in SEQ ID NO: 38; wherein the second antigen binding domain binds to or specifically binds to amyloid-beta, wherein the second antigen binding domain comprises:
(i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 41 , a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set in SEQ ID NO: 42 and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 43;
(iii) a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 41 , a CDR2 comprising a sequence set forth between in SEQ ID NO: 42 and a CDR3 comprising a sequence set forth in SEQ ID NO: 43; or
(vi) a VL comprising a sequence at least about 95% identical to a sequence set forth in SEQ ID NO: 56; (vii) a VL comprising a CDR1 comprising a sequence set SEQ ID NO: 44, a CDR2 comprising a sequence set forth in SEQ ID NO: 45 and a CDR3 comprising a sequence set forth in SEQ ID NO: 46; or
(i) a VH comprising a framework region (FR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 47, a FR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set in SEQ ID NO: 48, a FR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 49, and a FR4 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 50; or
47, a FR2 comprising a sequence set forth between in SEQ ID NO: 48, a FR3 comprising a sequence set forth in SEQ ID NO: 49, and a FR4 comprising a sequence set forth in SEQ ID NO: 50; and
(iii) a VL comprising a FR1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 51 , a FR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 52, a FR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 53, and a FR4 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 54; or
51 , a FR2 comprising a sequence set forth between in SEQ ID NO: 52, a FR3 comprising a sequence set forth in SEQ ID NO: 53, and a FR4 comprising a sequence set forth in SEQ ID NO: 54.
(iv) a VH comprising a sequence set forth in SEQ ID NO: 37; and (v) a VL comprising a CDR1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 26, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 27 and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 28;
(vii) a VL comprising a CDR1 comprising a sequence set SEQ ID NO: 26, a CDR2 comprising a sequence set forth in SEQ ID NO: 27 and a CDR3 comprising a sequence set forth in SEQ ID NO: 28; or
(viii) a VL comprising a sequence set forth in SEQ ID NO: 38; wherein the second antigen binding domain binds to or specifically binds to tau, wherein the second antigen binding domain comprises:
(i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 59, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set in SEQ ID NO: 60 and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 61 ;
(ii) a VH comprising a sequence at least about 95% or 96% or 97% or 98% or 99% identical to a sequence set forth in SEQ ID NO: 73; (iii) a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 59, a CDR2 comprising a sequence set forth between in SEQ ID NO: 60 and a CDR3 comprising a sequence set forth in SEQ ID NO: 61 ; or
(vii) a VL comprising a CDR1 comprising a sequence set SEQ ID NO:62, a CDR2 comprising a sequence set forth in SEQ ID NO: 63 and a CDR3 comprising a sequence set forth in SEQ ID NO: 64; or
(iii) a VL comprising a FR1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 69, a FR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 70, a FR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 71 , and a FR4 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 72; or
69, a FR2 comprising a sequence set forth between in SEQ ID NO: 70, a FR3 comprising a sequence set forth in SEQ ID NO: 71 , and a FR4 comprising a sequence set forth in SEQ ID NO: 72.
(viii) a VL comprising a sequence set forth in SEQ ID NO: 38; wherein the second antigen binding domain binds to or specifically binds to a- synuclein, wherein the second antigen binding domain comprises:
(i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 77, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set in SEQ ID NO: 78 and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 79;
(ii) a VH comprising a sequence at least about 95% or 96% or 97% or 98% or 99% identical to a sequence set forth in SEQ ID NO: 91 ;
(iii) a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 77, a CDR2 comprising a sequence set forth between in SEQ ID NO: 78 and a CDR3 comprising a sequence set forth in SEQ ID NO: 79; or
(iv) a VH comprising a sequence set forth in SEQ ID NO: 91 ; and
(vi) a VL comprising a sequence at least about 95% identical to a sequence set forth in SEQ ID NO: 92; (vii) a VL comprising a CDR1 comprising a sequence set SEQ ID NO: 80, a CDR2 comprising a sequence set forth in SEQ ID NO: 81 and a CDR3
40. An antigen binding site according to claim 38 or 39 wherein the second antigen binding domain further comprises:
41. A bispecific antibody comprising: a first light chain variable region and a first heavy chain variable region, wherein said first light chain variable region comprises: a CDR L1 as set forth in SEQ ID NO: 26, a CDR L2 as set forth in SEQ ID NO:27 and a CDR L3 as set forth in SEQ ID NO: 28; and wherein said first heavy chain variable region comprises: a CDR H1 as set forth in SEQ ID NO:23, a CDR H2 as set forth in SEQ ID NO: 24, and a CDR H3 as set forth in SEQ ID NO: 25; a second light chain variable region and a second heavy chain variable region, wherein said second light chain variable region comprises: a CDR L1 as set forth in SEQ ID NO: 8, a CDR L2 as set forth in SEQ ID NO: 9 and a CDR L3 as set forth in SEQ ID NO: 10; and wherein said second heavy chain variable region comprises: a CDR H1 as set forth in SEQ ID NO: 5, a CDR H2 as set forth in SEQ ID NO: 6, and a CDR H3 as set forth in SEQ ID NO: 7.
42. A bispecific antibody comprising: a first light chain variable region and a first heavy chain variable region, wherein said first light chain variable region comprises: a CDR L1 as set forth in SEQ ID NO: 26, a CDR L2 as set forth in SEQ ID NO:27 and a CDR L3 as set forth in SEQ ID NO: 28; and wherein said first heavy chain variable region comprises: a CDR H1 as set forth in SEQ ID NO:23, a CDR H2 as set forth in SEQ ID NO: 24, and a CDR H3 as set forth in SEQ ID NO: 25; a second light chain variable region and a second heavy chain variable region, wherein said second light chain variable region comprises: a CDR L1 as set forth in SEQ ID NO: 44, a CDR L2 as set forth in SEQ ID NO:
45 and a CDR L3 as set forth in SEQ ID NO: 46; and wherein said second heavy chain variable region comprises: a CDR H1 as set forth in SEQ ID NO: 41 , a CDR H2 as set forth in SEQ ID NO: 42, and a CDR H3 as set forth in SEQ ID NO: 43.
63 and a CDR L3 as set forth in SEQ ID NO: 64; and wherein said second heavy chain variable region comprises: a CDR H1 as set forth in SEQ ID NO: 59, a CDR H2 as set forth in SEQ ID NO: 60, and a CDR H3 as set forth in SEQ ID NO: 61.
44. A bispecific antibody comprising: a first light chain variable region and a first heavy chain variable region, wherein said first light chain variable region comprises: a CDR L1 as set forth in SEQ ID NO: 26, a CDR L2 as set forth in SEQ ID NO:27 and a CDR L3 as set forth in SEQ ID NO: 28; and wherein said first heavy chain variable region comprises: a CDR H1 as set forth in SEQ ID NO:23, a CDR H2 as set forth in SEQ ID NO:
24, and a CDR H3 as set forth in SEQ ID NO: 25; a second light chain variable region and a second heavy chain variable region, wherein said second light chain variable region comprises: a CDR L1 as set forth in SEQ ID NO: 80, a CDR L2 as set forth in SEQ ID NO: 81 and a CDR L3 as set forth in SEQ ID NO: 82; and wherein said second heavy chain variable region comprises: a CDR H1 as set forth in SEQ ID NO: 77, a CDR H2 as set forth in SEQ ID NO: 78, and a CDR H3 as set forth in SEQ ID NO: 79.
antigen binding site is in the form of:
(i) a minibody;
(ii) a dimeric scFv (di-scFv);
(iii) one of (i) or (ii) linked to a constant region of an antibody, Fc or a heavy chain constant domain (CH) 2 and/or CFI3; or (iv) one of (i) or (ii) linked to a protein that binds to an immune effector cell.
46. An antigen binding site according to claim 45, wherein the antigen binding site is in the form of:
(i) a diabody;
(ii) a triabody;
(iii) a tetrabody;
(iv) a F(ab')2;
(v) one of (i) to (iv) linked to a constant region of an antibody, Fc or a heavy chain constant domain (CH) 2 and/or CFI3; or
(vi) one of (i) to (iv) linked to a protein that binds to an immune effector cell.
47. A fusion protein comprising an antigen binding site according to any one of claims 1 to 46.
48. A conjugate in the form of an antigen binding site according to any one of claims 1 to 47, conjugated to a label or a cytotoxic agent.
49. A nucleic acid encoding an antigen binding site according to any one of claims 1 to 48.
50. A nucleic acid according to claim 49, wherein the nucleic acid comprises,
consists essentially of or consists of the nucleotide sequence of SEQ ID Nos: 39, 40, 21 and 22.
52. A nucleic acid according to claim 49, wherein the nucleic acid comprises, consists essentially of or consists of the nucleotide sequence of SEQ ID Nos: 39, 40, 75 and 76.
53. A nucleic according to claim 49, wherein the nucleic acid comprises, consists essentially of or consists of the nucleotide sequence of SEQ ID Nos: 39, 40, 93 and 94.
54. A vector comprising a nucleic acid according to any one of claims 49 to 53.
55. An vector according to claim 54, wherein the vector is a bicistronic expression construct, e.g., comprising the following operably linked components in 5' to 3' order:
(i) a promoter
(ii) a nucleic acid encoding a first polypeptide;
(iii) an internal ribosome entry site; and
(iv) a nucleic acid encoding a second polypeptide, wherein the first polypeptide comprises a first VH and first VL and the second polypeptide comprises a second VH and second VL.
56. A combination of vectors comprising:
(i) a first expression construct comprising a nucleic acid encoding a polypeptide comprising a first VH and a first VL operably linked to a promoter; and
(ii) a second expression construct comprising a nucleic acid encoding a polypeptide comprising a second VH and a second VL operably linked to a promoter, wherein the first VH, first VL, second VH and second are selected from the group consisting of SEQ ID Nos: 21 , 22, 39, 40, 57, 58, 75, 76 93 and 94, and wherein the combination of vectors when present in a cell allow formation of an antigen binding site according to any one of claims 1 to 46.
57. A cell comprising a nucleic acid, vector or combination of vectors according to any one of claims 49 to 56.
58. A pharmaceutical composition comprising an antigen binding site according to any one of claims 1 to 46 and a pharmaceutically acceptable carrier, diluent or excipient.
59. A method of treating a neurodegenerative disease associated with a pathogenic protein in a subject, the method comprising administering to the subject an antigen binding site according to any one of claims 1 to 46, a fusion protein according to claim 47, a conjugate according to claim 48, or a pharmaceutical composition according to claim 58, thereby treating the neurodegenerative disease in the subject.
60. A method of treating a neurodegenerative disease associated with a pathogenic protein in a subject, the method comprising the steps of:
- providing a subject identified as having a neurodegenerative disease associated with a pathogenic protein;
- administering to the subject an antigen binding site according to any one of claims 1 to 46, a fusion protein according to claim 47, a conjugate according to claim 48, or a pharmaceutical composition according to claim 58; thereby treating the neurodegenerative disease in the subject.
61. Use of an antigen binding site according to any one of claims 1 to 46, a fusion protein according to claim 47, a conjugate according to claim 48, or a
pharmaceutical composition according to claim 58 in the preparation of a medicament for treating a neurodegenerative disease associated with a pathogenic protein in a subject.
62. Use of an antigen binding site according to any one of claims 1 to 46, a fusion protein according to claim 47, a conjugate according to claim 48, or a pharmaceutical composition according to claim 58 for treating a
neurodegenerative disease associated with a pathogenic protein in a subject.
63. The use of claim 61 or 62, wherein the subject is identified as having a
neurodegenerative disease associated with a pathogenic protein.
64. A method according to claim 59 or 60, or the use according to any one of claims 61 to 63, wherein the neurodegenerative disease associated with a pathogenic protein is a neurodegenerative disease associated with accumulation of an extracellular pathogenic protein.
Cross-reference to earlier applications
This application claims priority to Australian provisional application AU 2018900778, the entire contents of which is herein incorporated by reference in its entirety.
The present invention relates to antigen binding sites, compositions and uses thereof in the treatment of conditions associated with pathogenic proteins, particularly extracellular pathogenic proteins.
Various neurodegenerative diseases are associated with or caused by the aggregation and/or deposition of proteins in the brain. One such disease is Alzheimer's disease (AD) which is characterized by the presence of monomers of the Ab peptide that first form soluble oligomers and then aggregate into extracellular fibrillar deposits known as amyloid plaques. Levels of Ab are elevated in the AD brain because of its increased production and/or impaired removal, with recent therapeutic strategies targeting both processes. This includes the inhibition of secretases to reduce Ab production, as well as active and, in particular, passive immunization approaches for clearance. These strategies, however, are challenging; secretase inhibition affects additional substrates with potential off-target effects, whereas passive immunization may be costly once effectiveness is demonstrated in clinical trials.
To date there are no effective treatments for individuals having Alzheimer's disease or any other disease associated with or caused by the presence of pathogenic protein, such as protein oligomers, aggregates and/or deposits.
Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and/or combined with other pieces of prior art by a skilled person in the art. Summary of the invention
The present invention provides an antigen binding site that binds to or specifically binds to CD33 and a pathogenic protein. Preferably, the antigen binding site comprises an antigen binding domain of an antibody, the antigen binding domain binds to or specifically binds to CD33 and a pathogenic protein.
The invention provides an antigen binding site that binds to or specifically binds to CD33 and a pathogenic protein, the antigen binding site comprises:
- a second antigen binding domain of an antibody that binds to or specifically binds to a pathogenic protein. Preferably, the first antigen binding domain comprises, consists essentially of or consists of a heavy chain variable domain (VH) and a light chain variable domain (VL). Preferably, the second antigen binding domain comprises, consists essentially of or consists of a VH and a VL.
In any aspect, the pathogenic protein is extracellular or predominantly extracellular. The extracellular pathogenic protein may be associated with a neurodegenerative disease. Typically the extracellular pathogenic protein is a protein that aggregates or is misfolded. The extracellular pathogenic protein may be amyloid beta, amyloid fragments, amyloid precursor protein, amyloid precursor protein fragments, British peptide, tau (preferably phosphorylated tau) or a (alpha)-synuclein.
Preferably, the antigen binding site of the invention binds to or specifically binds to human CD33. More preferably, the antigen binding site of the invention does not bind to mouse CD33. Typically, the binding of an antigen binding site of the invention does not result in activation of CD33, preferably it does not cause phosphorylation of the immunoreceptor tyrosine-based inhibition motif of CD33 or induce an increase in MCP-1 mRNA. In one embodiment, binding of the antigen binding site of the invention to CD33 on the surface of a cell results in CD33-mediated endocytosis. The capacity for an antigen binding site of the invention to induce CD33-mediated endocytosis can be determined by any method described herein. Typically, binding of the antigen binding site of the invention to CD33 results in internalisation of CD33 and the bound antigen binding site. Preferably, the antigen binding site binds to or specifically binds to an epitope on human CD33 molecule comprising, consisting essentially of or consisting of an amino acid sequence as shown in SEQ ID NO:4.
In one embodiment, the antigen binding site binds to human CD33, specifically the human CD33 extracellular domain, with a binding affinity (e.g KD) of less than 100nM, preferably less than 75 nM, preferably less than 50nM, preferably less than 25nM. An exemplary method to determine binding affinity is any one described herein, including Microscale thermophoresis (MST) as described in Example 1.
In one aspect, an antigen binding site of the invention binds to or specifically binds to CD33 and human amyloid-b. Preferably, the antigen binding site binds to or specifically binds to a human amyloid-b molecule comprising, consisting essentially of or consisting of an amino acid sequence as shown in SEQ ID NO: 1. In this embodiment, the antigen binding site may be capable of binding CD33 and amyloid-b, preferably the antigen binding site is capable of binding CD33 and amyloid-b simultaneously.
In another aspect, an antigen binding site of the invention binds to or specifically binds to CD33 and human tau. Preferably the tau is phosphorylated, even more preferably phosphorylated on S422. Preferably, the antigen binding site binds to or specifically binds to a human tau molecule comprising, consisting essentially of or consisting of an amino acid sequence as shown in SEQ ID NO:2. In this embodiment, the antigen binding site may be capable of binding CD33 and tau, preferably the antigen binding site is capable of binding CD33 and tau simultaneously.
In another aspect, an antigen binding site of the invention binds to or specifically binds to CD33 and human a-synuclein. Preferably, the antigen binding site binds to or specifically binds to a human a-synuclein molecule comprising, consisting essentially of or consisting of an amino acid sequence as shown in SEQ ID NO:3. In this embodiment, the antigen binding site may be capable of binding CD33 and a-synuclein, preferably the antigen binding site is capable of binding CD33 and a-synuclein simultaneously.
In one aspect, an antigen binding site of the invention that binds to CD33 has an affinity that is not statistically different to the antibody M195 (lintuzumab). Typically, the antigen binding site of the invention that binds to CD33 binds to the same epitope on CD33 as M195 (lintuzumab). Preferably, an antigen binding site of the invention competes with M195 (lintuzumab) for binding to CD33.
In another aspect, an antigen binding site of the invention that binds to amyloid-b has an affinity that is not statistically different to the antibody BIIB037 (aducanumab). Typically, the antigen binding site of the invention that binds to amyloid-b binds to the same epitope on amyloid-b as BIIB037 (aducanumab). Preferably, an antigen binding site of the invention competes with BIIB037 (aducanumab) for binding to amyloid-b.
In another aspect, an antigen binding site of the invention that binds to tau has an affinity that is not statistically different to the antibody pS422TAU. Typically, the antigen binding site of the invention that binds to tau binds to the same epitope on tau as pS422TAU. Preferably, an antigen binding site of the invention competes with pS422TAU for binding to tau.
In another aspect, an antigen binding site of the invention that binds to a- synuclein has an affinity that is not statistically different to the antibody NI-202.12F4. Typically, the antigen binding site of the invention that binds to a-synuclein binds to the same epitope on a-synuclein as NI-202.12F4. Preferably, an antigen binding site of the invention competes with NI-202.12F4 for binding to a-synuclein.
In an embodiment of any aspect, the antigen binding site of the invention does not significantly increase phagocytic activity of a microglial cell. In one embodiment, the antigen binding site of the invention does not suppress the phagocytic activity of a microglial cell. In one embodiment of the invention, the antigen binding site of the invention does not activate or increase the activity of microglial cells. In one embodiment, the antigen binding site of the invention does not suppress the activity of, or activate, a microglial cell.
An antigen binding site of the invention may bind to human CD33 and not detectably bind to or bind significantly to murine CD33. The binding of an antigen binding site to human CD33, and lack of binding to murine CD33, may be determined by any method described herein, particularly flow cytometry as described in Example 1.
The invention provides an antigen binding site that binds to or specifically binds to CD33 and a pathogenic protein, the antigen binding site comprises: - a first antigen binding domain of an antibody that binds to or specifically binds to CD33, wherein the first antigen binding domain comprises one or more complementarity determining regions;
The invention provides an antigen binding site that binds to or specifically binds to CD33 and amyloid beta, the antigen binding site comprises:
- a first antigen binding domain of an antibody that binds to or specifically binds to CD33, wherein the first antigen binding domain comprises CDR L1 , CDR L2 and CDR L3 from SEQ ID NO: 38; and CDR H1 , CDR H2 and CDR H3 from SEQ ID NO:
- a second antigen binding domain of an antibody that binds to or specifically binds to amyloid beta, wherein the second antigen binding domain comprises CDR L1 , CDR L2 and CDR L3 from SEQ ID NO: 56; and CDR H1 , CDR H2 and CDR H3 from SEQ ID NO: 55. The invention provides an antigen binding site that binds to or specifically binds to CD33 and tau, the antigen binding site comprises:
The invention provides an antigen binding site that binds to or specifically binds to CD33 and a-synuclein, the antigen binding site comprises:
The present invention also provides an antigen binding site comprising a first antigen binding domain of an antibody and second antigen binding domain of an antibody, wherein the first antigen binding domain binds to or specifically binds to CD33, wherein the first antigen binding domain comprises:
(i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 5, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set in SEQ ID NO: 6 and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 7;
(v) a VL comprising a CDR1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 8, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 9 and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 10;
In this aspect of the invention, the first antigen binding domain further comprises: (i) a VH comprising a framework region (FR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 29, a FR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set in SEQ ID NO: 30, a FR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 31 , and a FR4 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 32; or
(ii) a VH comprising a FR1 comprising a sequence set forth in SEQ ID NO: 29, a FR2 comprising a sequence set forth between in SEQ ID NO: 30, a FR3 comprising a sequence set forth in SEQ ID NO: 31 , and a FR4 comprising a sequence set forth in SEQ ID NO: 32; and
(ii) a VL comprising a FR1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 33, a FR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 34, a FR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 35, and a FR4 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 36; or
(iv) a VL comprising a FR1 comprising a sequence set forth in SEQ ID NO: 33, a FR2 comprising a sequence set forth between in SEQ ID NO: 34, a FR3 comprising a sequence set forth in SEQ ID NO: 35, and a FR4 comprising a sequence set forth in SEQ ID NO: 36.
In this aspect of the invention, the second antigen binding domain further comprises: (i) a VH comprising a framework region (FR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 1 1 , a FR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set in SEQ ID NO: 12, a FR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 13, and a FR4 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 14; or
(ii) a VH comprising a FR1 comprising a sequence set forth in SEQ ID NO: 1 1 , a FR2 comprising a sequence set forth between in SEQ ID NO: 12, a FR3 comprising a sequence set forth in SEQ ID NO: 13, and a FR4 comprising a sequence set forth in SEQ ID NO: 14; and
(iv) a VL comprising a FR1 comprising a sequence set forth in SEQ ID NO: 15, a FR2 comprising a sequence set forth between in SEQ ID NO: 16, a FR3 comprising a sequence set forth in SEQ ID NO: 17, and a FR4 comprising a sequence set forth in SEQ ID NO: 18. The present invention also provides an antigen binding site comprising a first antigen binding domain of an antibody and second antigen binding domain of an antibody, wherein the first antigen binding domain binds to or specifically binds to CD33, wherein the first antigen binding domain comprises:
(v) a VL comprising a CDR1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 44, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 45 and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 46; (vi) a VL comprising a sequence at least about 95% identical to a sequence set forth in SEQ ID NO: 56;
In this aspect of the invention, the first antigen binding domain further comprises:
In this aspect of the invention, the second antigen binding domain further comprises:
(ii) a VH comprising a FR1 comprising a sequence set forth in SEQ ID NO: 47, a FR2 comprising a sequence set forth between in SEQ ID NO: 48, a FR3 comprising a sequence set forth in SEQ ID NO: 49, and a FR4 comprising a sequence set forth in SEQ ID NO: 50; and
(iv) a VL comprising a FR1 comprising a sequence set forth in SEQ ID NO: 51 , a FR2 comprising a sequence set forth between in SEQ ID NO: 52, a FR3 comprising a sequence set forth in SEQ ID NO: 53, and a FR4 comprising a sequence set forth in SEQ ID NO: 54.
(iii) a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 59, a CDR2 comprising a sequence set forth between in SEQ ID NO: 60 and a CDR3 comprising a sequence set forth in SEQ ID NO: 61 ; or
(ii) a VH comprising a FR1 comprising a sequence set forth in SEQ ID NO: 29, a FR2 comprising a sequence set forth between in SEQ ID NO: 30, a FR3 comprising a sequence set forth in SEQ ID NO: 31 , and a FR4 comprising a sequence set forth in SEQ ID NO: 32; and (iii) a VL comprising a FR1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 33, a FR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 34, a FR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 35, and a FR4 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 36; or
(ii) a VH comprising a FR1 comprising a sequence set forth in SEQ ID NO: 65, a FR2 comprising a sequence set forth between in SEQ ID NO: 66, a FR3 comprising a sequence set forth in SEQ ID NO: 67, and a FR4 comprising a sequence set forth in SEQ ID NO: 68; and (ili) a VL comprising a FR1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 69, a FR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 70, a FR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 71 , and a FR4 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 72; or
(iv) a VL comprising a FR1 comprising a sequence set forth in SEQ ID NO: 69, a FR2 comprising a sequence set forth between in SEQ ID NO: 70, a FR3 comprising a sequence set forth in SEQ ID NO: 71 , and a FR4 comprising a sequence set forth in SEQ ID NO: 72.
(i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 77, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set in SEQ ID NO: 78 and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to a sequence set forth in SEQ ID NO: 79; (ii) a VH comprising a sequence at least about 95% or 96% or 97% or 98% or 99% identical to a sequence set forth in SEQ ID NO: 91 ;
The present invention also provides a bispecific antibody comprising:
a first light chain variable region and a first heavy chain variable region, wherein said first light chain variable region comprises:
a CDR L1 as set forth in SEQ ID NO: 26, a CDR L2 as set forth in SEQ ID NO:27 and a CDR L3 as set forth in SEQ ID NO: 28; and
wherein said first heavy chain variable region comprises:
a CDR H1 as set forth in SEQ ID NO:23, a CDR H2 as set forth in SEQ ID NO: 24, and a CDR H3 as set forth in SEQ ID NO: 25; a second light chain variable region and a second heavy chain variable region, wherein said second light chain variable region comprises:
a CDR L1 as set forth in SEQ ID NO: 8, a CDR L2 as set forth in SEQ ID NO: 9 and a CDR L3 as set forth in SEQ ID NO: 10; and
wherein said second heavy chain variable region comprises:
a CDR H1 as set forth in SEQ ID NO: 5, a CDR H2 as set forth in SEQ ID NO: 6, and a CDR H3 as set forth in SEQ ID NO: 7.
a CDR L1 as set forth in SEQ ID NO: 44, a CDR L2 as set forth in SEQ ID NO: 45 and a CDR L3 as set forth in SEQ ID NO: 46; and
a CDR H1 as set forth in SEQ ID NO: 41 , a CDR H2 as set forth in SEQ ID NO: 42, and a CDR H3 as set forth in SEQ ID NO: 43.
wherein said first heavy chain variable region comprises: a CDR H1 as set forth in SEQ ID NO:23, a CDR H2 as set forth in SEQ ID NO: 24, and a CDR H3 as set forth in SEQ ID NO: 25; a second light chain variable region and a second heavy chain variable region, wherein said second light chain variable region comprises:
a CDR H1 as set forth in SEQ ID NO: 59, a CDR H2 as set forth in SEQ ID NO: 60, and a CDR H3 as set forth in SEQ ID NO: 61 .
a CDR H1 as set forth in SEQ ID NO: 77, a CDR H2 as set forth in SEQ ID NO: 78, and a CDR H3 as set forth in SEQ ID NO: 79.
In any aspect of the present invention, the antigen binding site or bispecific antibody as described herein, may further comprise a linker between a VH and VL.
As described herein, the antigen binding site may be in the form of: (i) a minibody;
(iii) one of (i) or (ii) linked to a constant region of an antibody, Fc or a heavy chain constant domain (CH) 2 and/or CH3; or
(iv) one of (i) or (ii) linked to a protein that binds to an immune effector cell.
Further, as described herein, the antigen binding site may be in the form of:
(v) one of (i) to (iv) linked to a constant region of an antibody, Fc or a heavy chain constant domain (CH) 2 and/or CH3; or
In any aspect of the present invention, the antigen binding site or bispecific antibody is in the form of a diabody. Preferably, the diabody comprises, consists essentially of or consists of a first chain comprising from N to C terminus VH1 - linker - VL2 and a second chain comprising from N to C terminus VH2 - linker - VL1 , wherein VH1 and VL1 form a first binding domain and VH2 and VL1 form a second binding domain, wherein the first binding domain binds to CD33 and the second binding domain binds to a pathogenic protein, for example any pathogenic protein associated with a neurodegenerative disease (e.g. amyloid beta, amyloid fragments, amyloid precursor protein, amyloid precursor protein fragments, British peptide, TDP43, C90RF72, Prion protein, apolipoproteins, tau (preferably phosphorylated tau) or a (alpha)-synuclein). Preferably, the VL1 and VH1 comprise, consist essentially of or consist of SEQ ID Nos: 38 and 37, and VL2 and VH2 comprising consist essentially of or consist of SEQ ID Nos: selected from the group consisting of SEQ ID Nos: 20 and 19, 56 and 55, 74 and 73, and 92 and 91. Typically, the linker comprises, consists essentially of or consists of SEQ ID Nos: 95 or 96.
As described herein an antigen binding site, or bispecific antibody as described herein, may be in the form of a bispecific IgG, appended IgG, bispecific antibody fragment, bispecific fusion protein or bispecific conjugate. A bispecific IgG includes CrossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-lgG, Knobs-in-holes common LC, Knobs-in-holes assembly, Charge pair, Fab-arm exchange, SEEDbody, Triomab, LUZ-Y, Fcab, kl-body, and Orthogonal Fab. Appended IgG includes DVD-lgG, lgG(H)scFv, scFv-(H)lgG, lgG(L)-scFv, scFv-(L)lgG, lgG(L,H)-Fv, lgG(H)-V, V(H)-lgG, lgG(L)-V, V(L)-lgG, KIH IgG-scFab, 2scFv-lgG, lgG-2scFv, scFv4-lg, Zybody, and DVI- IgG (four-in-one). Bispecific antibody fragments include a nanobody, nanobody-FISA, BiTE, diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, Triple Body, Miniantibody, Minibody, TriBi minibody, scFV-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFV-KIH, Fab-scFv-Fc, Tetravalent HCAb, scDiabody-Fc, Diabody-Fc, Tandem scFv-Fc, and Intrabody. Bispecific fusion proteins include dock and lock, ImmTAC, HSAbody, scDiabody-HSA, andTandem scFv-Toxin. Bispecific antibody conjugates include IgG-lgG, Cov-X-Body and scFv1 -PEG-scFv2.
As used herein, the foregoing antigen binding sites can also be referred to as antigen binding domains of antibodies or antigen binding proteins. Alternatively, the foregoing antigen binding sites may also be referred to as bispecific polypeptides.
Preferably, an antigen binding site as described herein is an antibody or antigen binding fragment thereof. Typically, the antigen binding site is an antibody, for example, a monoclonal antibody.
In any aspect or embodiment, the antibody is a naked antibody. Specifically, the antibody is in a non-conjugated form and is not adapted to form a conjugate.
In one example, the complementarity determining region sequences (CDRs) of an antigen binding site of the invention are defined according to the Kabat numbering system.
In another example, the CDRs are defined according to the IMGT numbering system. The present invention also provides antigen binding domains or antigen binding fragments of the foregoing antibodies.
The invention also provides a fusion protein comprising an antigen binding site, antibody, di-scFv, F(ab')2, minibody, diabody, triabody, tetrabody, single-chain antibody molecule, or multispecific antibody as described herein.
The invention also provides a conjugate in the form of an antigen binding site, antibody, di-scFv, F(ab')2, minibody, diabody, triabody, tetrabody, single-chain antibody molecule, or multispecific antibody or fusion protein as described herein conjugated to a label or a cytotoxic agent.
The invention also provides an antibody for binding to an antigen binding site, antibody, di-scFv, F(ab')2, minibody, diabody, triabody, tetrabody, single-chain antibody molecule, or multispecific antibody, fusion protein, or conjugate as described herein.
The invention also provides a nucleic acid encoding an antigen binding site, antibody, di-scFv, F(ab')2, minibody, diabody, triabody, tetrabody, single-chain antibody molecule, or multispecific antibody, fusion protein or conjugate as described herein. A nucleic acid may encode any one or more first or second antigen binding domains as described herein. More specifically, the nucleic acid may encode a VH or VL as described herein, particular those shown in Table 1.
In one aspect, the nucleic acid comprises, consists essentially of or consists of one or more nucleotide sequences as shown in Table 1. For example, the nucleic acid may comprise, consist essentially of or consist of the nucleotide sequence of SEQ ID Nos: 39, 40, 21 and 22. In another example, the nucleic acid may comprise, consist essentially of or consist of the nucleotide sequence of SEQ ID Nos: 39, 40, 57 and 58. In another example, the nucleic acid may comprise, consist essentially of or consist of the nucleotide sequence of SEQ ID Nos: 39, 40, 75 and 76. In another example, the nucleic acid may comprise, consist essentially of or consist of the nucleotide sequence of SEQ ID Nos: 39, 40, 93 and 94.
In one embodiment, such a nucleic acid is included in an expression construct in which the nucleic acid is operably linked to a promoter. Such an expression construct can be in a vector, e.g., a plasmid. In examples of the invention directed to single polypeptide chain antigen binding sites, the expression construct may comprise a promoter linked to a nucleic acid encoding that polypeptide chain.
In examples directed to multiple polypeptide chains that form an antigen binding site, an expression construct comprises a nucleic acid encoding a polypeptide comprising, e.g., a VH, or VH and VL, operably linked to a promoter and a nucleic acid encoding a polypeptide comprising, e.g., a VL, or VH and VL, operably linked to a promoter.
In another example, the expression construct is a bicistronic expression construct, e.g., comprising the following operably linked components in 5' to 3' order:
The present invention also contemplates separate expression constructs one of which encodes a first polypeptide comprising a first VH and first VL and another of which encodes a second polypeptide comprising a second VH and second VL. For example, the present invention also provides a composition comprising:
(ii) a second expression construct comprising a nucleic acid encoding a polypeptide comprising a second VH and a second VL operably linked to a promoter.
In any aspect of a nucleic acid of the invention, the first VH and first VL when formed into an antigen binding site of the present invention may bind the same or different antigens. Similarly, in any aspect of the invention, the second VH and second VL when formed into an antigen binding site of the present invention may bind the same or different antigens. For example, the first VH may form part of a binding domain that binds to CD33 whereas the first VL may form part of a binding domain that binds to a pathogenic protein. Similarly, the second VH may form part of a binding domain that binds to a pathogenic protein, whereas the second VL may form part of a binding domain that binds to CD33.
The invention provides a cell comprising a vector or nucleic acid described herein. Preferably, the cell is isolated, substantially purified or recombinant. In one example, the cell comprises the expression construct of the invention or:
(i) a first expression construct comprising a nucleic acid encoding a polypeptide comprising a first VH and first VL operably linked to a promoter; and
(ii) a second expression construct comprising a nucleic acid encoding a polypeptide comprising a second VH and a second VL operably linked to a promoter, wherein the first and second polypeptides associate to form an antigen binding site of the present invention.
Examples of cells of the present invention include bacterial cells, yeast cells, insect cells or mammalian cells.
The invention also provides a pharmaceutical composition comprising an antigen binding site, or comprising a CDR and/or FR sequence as described herein, or an antibody, di-scFv, F(ab')2, minibody, diabody, triabody, tetrabody, single-chain antibody molecule, or multispecific antibody, fusion protein, or conjugate as described herein and a pharmaceutically acceptable carrier, diluent or excipient.
The invention also provides a diagnostic composition comprising an antigen binding site, or comprising a CDR and/or FR sequence as described herein, or antigen binding site, antibody, di-scFv, F(ab')2, minibody, diabody, triabody, tetrabody, single- chain antibody molecule, or multispecific antibody, fusion protein or conjugate as described herein, a diluent and optionally a label.
The invention also provides a kit or article of manufacture comprising an antigen binding site, or comprising a CDR and/or FR sequence as described herein or an antibody, di-scFv, F(ab')2, minibody, diabody, triabody, tetrabody, single-chain antibody molecule, or multispecific antibody, fusion protein or conjugate as described herein.
An antigen binding site, a protein or antibody as described herein may comprise a human constant region, e.g., an IgG constant region, such as an lgG1 , lgG2, lgG3 or lgG4 constant region or mixtures thereof. In the case of an antibody or protein comprising a VH and a VL, the VH can be linked to a heavy chain constant region and the VL can be linked to a light chain constant region.
In one example, a protein or antibody as described herein comprises a constant region of an lgG4 antibody or a stabilized constant region of an lgG4 antibody. In one example, the protein or antibody comprises an lgG4 constant region with a proline at position 241 (according to the numbering system of Kabat (Kabat et al., Sequences of Proteins of Immunological Interest Washington DC United States Department of Health and Human Services, 1987 and/or 1991 )).
In one example a protein or antibody as described herein or a composition of a protein or antibody as described herein, comprises a heavy chain constant region, comprising a stabilized heavy chain constant region, comprising a mixture of sequences fully or partially with or without the C-terminal lysine residue.
In one example, an antibody of the invention comprises a VH disclosed herein linked or fused to an lgG4 constant region or stabilized lgG4 constant region (e.g., as discussed above) and the VL is linked to or fused to a kappa light chain constant region.
The functional characteristics of an antigen binding site of the invention will be taken to apply mutatis mutandis to an antibody of the invention.
An antigen binding site as described herein may be purified, substantially purified, isolated and/or recombinant.
An antigen binding site of the invention may be part of a supernatant taken from media in which a hybridoma expressing an antigen binding site of the invention has been grown.
The present invention provides a method of treating a neurodegenerative disease associated with a pathogenic protein in a subject, the method comprising administering to the subject an antigen binding site or antibody, composition fusion protein, or conjugate as described herein; thereby treating the neurodegenerative disease in the subject.
The present invention provides for use of an antigen binding site or antibody, composition fusion protein, or conjugate as described herein in the preparation of a medicament for treating a neurodegenerative disease associated with a pathogenic protein in a subject.
The present invention provides for use of an antigen binding site or antibody, composition fusion protein, or conjugate as described herein for treating a neurodegenerative disease associated with a pathogenic protein in a subject.
The present invention provides a method of treating a neurodegenerative disease associated with a pathogenic protein in a subject, the method comprising the steps of:
- administering to the subject an antigen binding site or antibody, composition fusion protein, or conjugate as described herein; thereby treating the neurodegenerative disease in the subject.
The present invention provides for use of an antigen binding site or antibody, composition fusion protein, or conjugate as described herein in the preparation of a medicament for treating a neurodegenerative disease associated with a pathogenic protein in a subject identified as having a neurodegenerative disease associated with a pathogenic protein.
The present invention provides for use of an antigen binding site or antibody, composition fusion protein, or conjugate as described herein for treating a neurodegenerative disease associated with a pathogenic protein in a subject identified as having a neurodegenerative disease associated with a pathogenic protein. Preferably, the neurodegenerative disease associated with a pathogenic protein is a neurodegenerative disease associated with accumulation of a pathogenic protein. In any aspect, the pathogenic protein may be amyloid beta, amyloid fragments, amyloid precursor protein, amyloid precursor protein fragments, British peptide, TDP43, C90RF72, Prion protein, apolipoproteins, tau (preferably phosphorylated tau) or a (alpha)-synuclein. The invention also provides an animal or tissue derived therefrom comprising a cell described herein.
As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.
Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.
Figure 1. Diabody chain format. V Hi -V L 2 (upper) and V H 2-V Li (lower) for the CD33:A diabody.
Figure 2. Fluorescence intensity shifts (Ab uptake) by BV2 cells (left) transfected with hCD33 (human CD33), and (right) untransfected cells. Flow cytometry histograms (top) show that diabody treatment of hCD33 positive cells drives most of the cells into a higher fluorescence intensity population after 1 hour incubation. This represents an extremely significant (P<0.0001 , n=3) increase in the uptake of Ab compared to cells treated with buffer matched controls. For untransfected cells there is a significant (P<0.019) but smaller lowering of Ab uptake for cells treated with the diabody.
Figure 3. The addition of the Oϋ33-Ab (1 pg/ml) or buffer control did not significantly alter mRNA expression of neuroinflam matory markers (a) Mcp1 (b) TNF a (c) 1L-6 (d) 1L-1B (e) NFkB (f) FLT-1 in differentiated human THP-1 microglia. Two-way analysis of variance (ANOVA). Data is Mean ± SEM, n=3. Figure 4. Cells were transfected with CD33. The CD33-A diabody labelled with pHrodo (in the absence of Ab) was added to free serum media for 30 min or 1 hour. This demonstrates that the diabody is internalised by CD33 positive cells. The dominant mechanism of action is receptor-mediated endocytosis of the CD33-directed diabody with its payload .
Figure 5. The diabody lacks the Fc portion and does not enhance microglial uptake of fluorescent latex beads by phagocytosis, which suggests that the diabody is not regulating phagocytosis when it engages CD33. One-way analysis of variance (ANOVA) yields (P= 0.1596, n=3).
Figure 6. (A) Effect of CD33-pTau diabody on phosphorylated Tau peptide uptake by microglial cells as measured by mean fluorescence intensity (left) and the percentage of cells internalising pTau peptide (right). (B) Effect of CD33-a-Syn diabody on human a-synuclein uptake by microglial cells as measured the by mean fluorescence intensity (left) and by the percentage of cells internalising a-synuclein (right) n=6.
Figure 7. Oϋ33-Ab diabody binding to increasing concentrations of (A) recombinant hCD33 extracellular domain and synthetic Ab 1-42 freshly dissolved (B) and aged for 4 hours (C), respectively. Data were normalized to fraction bound (AFnorm/amplitude).
Detailed description of the embodiments
It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.
Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims.
The present inventors have developed antigen binding sites, for example antibodies, that bind to and induce the endocytosis of extracellular pathogenic proteins. The antigen binding sites as described herein have the capacity remove pathogenic proteins from the extracellular space, thereby being useful to treat various neurodegenerative diseases associated with extracellular pathogenic proteins
Neuroinflammation is a pathological hallmark of neurodegeneration. Antibodies like crenezumab have been designed on a backbone that only weakly stimulates immune cells by Fc mediated action to try and limit the safety concerns with overstimulating the immune system implicated in safety issues with previous and current immunotherapies. Without being bound by any theory or mode of action, the antigen binding sites described herein have the advantage of not activating the immune system, yet effectively providing for a mechanism of clearing pathological protein aggregates by microglial-mediated endocytosis.
It was expected that the diabody (a large ligand) with high affinity for CD33 would most likely bind to, and activate CD33 signalling resulting in reduced phagocytosis. The genes of CD33 that are protective for Alzheimer's disease have reduced expression of full length CD33. That is, inhibitors of CD33 are sought to confer protection from Alzheimer's disease. Here, the inventors surprisingly found that CD33 is not activated and phagocytosis is not supressed by CD33-AP diabody treatment. This is important because this would have the exact opposite desirable effect of CD33 inhibitors and would likely exacerbate the disease by hindering the ability of microglia to clean the brain of debris. Diabody treatment enhances microglial consumption of pathological proteins without the negative consequences of off-target phagocytosis, supressed phagocytosis or promotion of neuroinflammation. This approach thus provides for a therapeutic advantage not previously identified. General
Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter. Thus, as used herein, the singular forms"a","an" and"the" include plural aspects, and vice versa, unless the context clearly dictates otherwise. For example, reference to"a" includes a single as well as two or more; reference to"an" includes a single as well as two or more; reference to"the" includes a single as well as two or more and so forth.
Those skilled in the art will appreciate that the present invention is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.
One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.
All of the patents and publications referred to herein are incorporated by reference in their entirety.
The present invention is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the present invention.
Any example or embodiment of the present invention herein shall be taken to apply mutatis mutandis to any other example or embodiment of the invention unless specifically stated otherwise. Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for example, in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).
Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991 ), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1- 4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-lnterscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).
The description and definitions of variable regions and parts thereof, immunoglobulins, antibodies and fragments thereof herein may be further clarified by the discussion in Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 , Bork et al., J Mol. Biol. 242, 309- 320, 1994, Chothia and Lesk J. Mol Biol. 196:901 -917, 1987, Chothia et al. Nature 342, 877-883, 1989 and/or or Al-Lazikani et al., J Mol Biol 273, 927-948, 1997.
The term"and/or", e.g.,"X and/or Y" shall be understood to mean either"X and Y" or"X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.
As used herein the term "derived from" shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source. Reference herein to a range of, e.g., residues, will be understood to be inclusive. For example, reference to"a region comprising amino acids 56 to 65" will be understood in an inclusive manner, i.e., the region comprises a sequence of amino acids as numbered 56, 57, 58, 59, 60, 61 , 62, 63, 64 and 65 in a specified sequence.
Selected Definitions
CD33 is a transmembrane receptor expressed on cells of myeloid lineage including hematopoietic and phagocytic cells, macrophages, monocytes, dendritic cells, and microglial cells. In the brain, CD33 is mainly expressed on microglial cells. CD33 is also known as sialic acid binding Ig-like lectin 3, Siglec-3, gp67, p67 and is able to bind to high-affinity sialoglycans on target cells, mediating cell-cell interaction.
The term "CD33" as provided herein includes homologues or variants that maintain the activity of CD33 (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the native protein). In some embodiments, variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring form.
In some embodiments, the CD33 protein is the protein as identified by the UniProt sequence reference UniProtKB - P20138 (CD33_HUMAN), homolog or functional fragment thereof.
As used herein, reference to CD33 is to a molecule that has at least one biochemical or biophysical activity of CD33. CD33 key biochemical or biophysical activities include adhesion processes of human primary immune cells. CD33 is able to bind to high-affinity sialoglycans on target cells, mediating cell-cell interaction. Similarly, CD33 can interact with sialylated pathogens and viruses, which are decorated on their surface with host-derived or self-synthesized sialoglycans.
Tau proteins are also known as T proteins, MAPT, DDPAC, FTDP-17, MAPTL, MSTD, MTBT1 , MTBT2, PPND, PPP1 R103, or microtubule associated protein tau. Tau protein is a highly soluble microtubule-associated scaffolding protein that is expressed predominantly in neurons and is abundant in axons although it can be found in the soma, dendrites and nucleus.
The term"tau" as provided herein includes the microtubule-associated proteins MAP2 and MAP4, homologues or variants that, in a non-disease form, maintain the activity of tau (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the native protein). In some embodiments, variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring form. In some embodiments, the tau proteins may include any of the family of six isoforms which range from 352 to 441 amino acids. Their molecular weight ranges from 45 to 65 kDa when run on polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate (SDS- PAGE). The tau variants differ from each other by the presence of either three or four repeat-regions in the carboxy-terminal (C-terminal) part of the molecule and the absence or presence of one or two inserts (29 or 58 amino acids) in the amino-terminal (N-terminal) part.
In some embodiments, the tau protein is the protein as identified by the UniProt sequence reference UniProtKB - P10636 (TAU_HUMAN), homolog or functional fragment thereof.
For the purposes of nomenclature only and not a limitation, an exemplary amino acid sequence of tau is SEQ ID NO:2.
As used herein, reference to tau is to a molecule that has at least one biochemical or biophysical activity of tau. Tau key biochemical or biophysical activities include the stabilization of microtubules and promotion of tubulin assembly into microtubules. Microtubule stability is generally achieved through phosphorylation of tau or through the dedicated activity of the isoform.
Amyloid beta (Ab) denotes peptides of 36-43 amino acids and is the main component of amyloid plaques found in the brains of Alzheimer patients. Amyloid beta is also present in pre-amyloid parenchymal deposits and within the walls of leptomeningeal and cerebral vessels. Amyloid beta is composed of 1 1 -15 amino acids of the transmembrane domain and 28 amino acids of the extracellular domain of amyloid precursor protein (APP). The functions of APP in the nervous system include mediating adhesion and in the growth of neuronal and non-neuronal cells.
APP is cleaved by beta secretase and gamma secretase to yield Ab. APP occurs as several Ab-containing isoforms of 695, 751 , and 770 amino acids, with the latter two APP containing a domain that shares structural and functional homologies with Kunitz serine protease inhibitors. Amyloid beta molecules can aggregate to form flexible soluble oligomers which may exist in several forms. It is believed that certain misfolded oligomers can induce other Ab molecules to also take the misfolded oligomeric form. These oligomers, as well as some folded forms, are toxic to nerve cells.
The term "amyloid beta" or"Ab" as provided herein includes homologues or variants that, in a non-disease form, maintain the activity of amyloid beta (e g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the native protein). In some embodiments, variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring form.
In some embodiments, the amyloid beta protein and peptides derived from amyloid precursor protein may be as identified by the UniProt sequence reference UniProtKB - P05067 (A4_HUMAN), homolog or functional fragment thereof.
For the purposes of nomenclature only and not a limitation, an exemplary amino acid sequence of amyloid beta is SEQ ID NO: 1 .
Alpha-synuclein is a protein that is also known as SNCA, NACP, PARK1 , PARK4, PD1. Alpha-synuclein is abundant in the human brain in presynaptic terminals of neurons. It can also be found in neuroglial cells.
At least three isoforms of alpha-synuclein are produced through alternative splicing. The majority form of the protein is the full-length protein of 140 amino acids. Other isoforms are alpha-synuclein-126, which lacks residues 41 -54 due to loss of exon 3; and alpha-synuclein-1 12, which lacks residue 103-130 due to loss of exon 5. The term "alpha-synuclein" as provided herein includes the three isoforms mentioned above, homologues or variants that, in a non-disease form, maintain the activity of alpha-synuclein (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the native protein). In some embodiments, variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring form.
In some embodiments, alpha-synuclein is the protein as identified by the UniProt sequence reference UniProtKB - P37840 (SYUA_HUMAN), homolog or functional fragment thereof.
For the purposes of nomenclature only and not a limitation, an exemplary amino acid sequence of alpha-synuclein is SEQ ID NO:3.
As used herein, reference to alpha-synuclein is to a molecule that has at least one biochemical or biophysical activity of alpha-synuclein. Alpha-synuclein interacts with phospholipids and proteins and may play roles in maintaining a supply of synaptic vesicles in presynaptic terminals by clustering synaptic vesicle and it may also help regulate the release of the neurotransmitter dopamine.
The term "isolated protein" or "isolated polypeptide" is a protein or polypeptide that by virtue of its origin or source of derivation is not associated with naturally- associated components that accompany it in its native state; is substantially free of other proteins from the same source. A protein may be rendered substantially free of naturally associated components or substantially purified by isolation, using protein purification techniques known in the art. By"substantially purified" is meant the protein is substantially free of contaminating agents, e.g., at least about 70% or 75% or 80% or 85% or 90% or 95% or 96% or 97% or 98% or 99% free of contaminating agents.
The term "recombinant" shall be understood to mean the product of artificial genetic recombination. Accordingly, in the context of a recombinant protein comprising an antibody antigen binding domain, this term does not encompass an antibody naturally-occurring within a subject's body that is the product of natural recombination that occurs during B cell maturation. However, if such an antibody is isolated, it is to be considered an isolated protein comprising an antibody antigen binding domain. Similarly, if nucleic acid encoding the protein is isolated and expressed using recombinant means, the resulting protein is a recombinant protein comprising an antibody antigen binding domain. A recombinant protein also encompasses a protein expressed by artificial recombinant means when it is within a cell, tissue or subject, e.g., in which it is expressed.
The term"protein" shall be taken to include a single polypeptide chain, i.e., a series of contiguous amino acids linked by peptide bonds or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex). For example, the series of polypeptide chains can be covalently linked using a suitable chemical or a disulphide bond. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions.
The term "polypeptide" or "polypeptide chain" will be understood from the foregoing paragraph to mean a series of contiguous amino acids linked by peptide bonds.
As used herein, the term "antigen binding site'' is used interchangeably with "antigen binding domain" and shall be taken to mean a region of an antibody that is capable of specifically binding to an antigen, i.e. , a VH or a VL or an Fv comprising both a VH and a VL. The antigen binding domain need not be in the context of an entire antibody, e.g., it can be in isolation (e.g., a domain antibody) or in another form, e.g., as described herein, such as a scFv.
For the purposes for the present disclosure, the term "antibody" includes a protein capable of specifically binding to one or a few closely related antigens (e.g., CD33) by virtue of an antigen binding domain contained within a Fv. This term includes four chain antibodies (e.g., two light chains and two heavy chains), recombinant or modified antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies, CDR-grafted antibodies, primatized antibodies, de-immunized antibodies, synhumanized antibodies, half-antibodies, bispecific antibodies). An antibody generally comprises constant domains, which can be arranged into a constant region or constant fragment or fragment crystallizable (Fc). Exemplary forms of antibodies comprise a four- chain structure as their basic unit. Full-length antibodies comprise two heavy chains (~50 to 70 kD) covalently linked and two light chains (~23 kDa each). A light chain generally comprises a variable region (if present) and a constant domain and in mammals is either a k light chain or a l light chain. A heavy chain generally comprises a variable region and one or two constant domain(s) linked by a hinge region to additional constant domain(s). Heavy chains of mammals are of one of the following types a, d, e, y, or m. Each light chain is also covalently linked to one of the heavy chains. For example, the two heavy chains and the heavy and light chains are held together by inter-chain disulfide bonds and by non-covalent interactions. The number of inter-chain disulfide bonds can vary among different types of antibodies. Each chain has an N- terminal variable region (VH or VL wherein each are -1 10 amino acids in length) and one or more constant domains at the C- terminus. The constant domain of the light chain (CL which is -1 10 amino acids in length) is aligned with and disulfide bonded to the first constant domain of the heavy chain (CH1 which is 330 to 440 amino acids in length). The light chain variable region is aligned with the variable region of the heavy chain. The antibody heavy chain can comprise 2 or more additional CH domains (such as, CH2, CH3 and the like) and can comprise a hinge region between the CH1 and CH2 constant domains. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2) or subclass. In one example, the antibody is a murine (mouse or rat) antibody or a primate (such as, human) antibody. In one example the antibody heavy chain is missing a C-terminal lysine residue. In one example, the antibody is humanized, synhumanized, chimeric, CDR- grafted or deimmunized.
The terms "full-length antibody", "intact antibody" or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antigen binding fragment of an antibody. Specifically, whole antibodies include those with heavy and light chains including an Fc region. The constant domains may be wild- type sequence constant domains (e.g., human wild-type sequence constant domains) or amino acid sequence variants thereof.
As used herein,"variable region" refers to the portions of the light and/or heavy chains of an antibody as defined herein that is capable of specifically binding to an antigen and, includes amino acid sequences of complementarity determining regions (CDRs); i.e. , CDR1 , CDR2, and CDR3, and framework regions (FRs). For example, the variable region comprises three or four FRs (e.g., FR1 , FR2, FR3 and optionally FR4) together with three CDRs. VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain.
As used herein, the term"complementarity determining regions" (syn. CDRs; i.e. , CDR1 , CDR2, and CDR3) refers to the amino acid residues of an antibody variable region the presence of which are major contributors to specific antigen binding. Each variable region domain (VH or VL) typically has three CDRs identified as CDR1 , CDR2 and CDR3. The CDRs of VH are also referred to herein as CDR H1 , CDR H2 and CDR H3, respectively, wherein CDR H1 corresponds to CDR 1 of VH, CDR H2 corresponds to CDR 2 of VH and CDR H3 corresponds to CDR 3 of VH. Likewise, the CDRs of VL are referred to herein as CDR L1 , CDR L2 and CDR L3, respectively, wherein CDR L1 corresponds to CDR 1 of VL, CDR L2 corresponds to CDR 2 of VL and CDR L3 corresponds to CDR 3 of VL. In one example, the amino acid positions assigned to CDRs and FRs are defined according to Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 (also referred to herein as"the Kabat numbering system"). In another example, the amino acid positions assigned to CDRs and FRs are defined according to the Enhanced Chothia Numbering Scheme (http://www.bioinfo.org.uk/mdex.html). The present invention is not limited to FRs and CDRs as defined by the Kabat numbering system, but includes all numbering systems, including the canonical numbering system or of Chothia and Lesk J. Mol. Biol. 196: 901 -917, 1987; Chothia et al. , Nature 342: 877-883, 1989; and/or Al-Lazikani et al. , J. Mol. Biol. 273: 927-948, 1997; the numbering system of Honnegher and PlCikthun J. Mol. Biol. 309: 657-670, 2001 ; or the IMGT system discussed in Giudicelli et al., Nucleic Acids Res. 25: 206-211 1997. In one example, the CDRs are defined according to the Kabat numbering system. Optionally, heavy chain CDR2 according to the Kabat numbering system does not comprise the five C-terminal amino acids listed herein or any one or more of those amino acids are substituted with another naturally-occurring amino acid. In this regard, Padlan et al., FASEB J., 9: 133-139, 1995 established that the five C-terminal amino acids of heavy chain CDR2 are not generally involved in antigen binding.
"Framework regions" (FRs) are those variable region residues other than the CDR residues. The FRs of VH are also referred to herein as FR H1 , FR H2, FR H3 and FR H4, respectively, wherein FR H1 corresponds to FR 1 of VH, FR H2 corresponds to FR 2 of VH, FR H3 corresponds to FR 3 of VH and FR H4 corresponds to FR 4 of VH. Likewise, the FRs of VL are referred to herein as FR L1 , FR L2, FR L3 and FR L4, respectively, wherein FR L1 corresponds to FR 1 of VL, FR L2 corresponds to FR 2 of VL, FR L3 corresponds to FR 3 of VL and FR L4 corresponds to FR 4 of VL.
As used herein, the term "Fv" shall be taken to mean any protein, whether comprised of multiple polypeptides or a single polypeptide, in which a VL and a VH associate and form a complex having an antigen binding domain, i.e., capable of specifically binding to an antigen. The VH and the VL which form the antigen binding domain can be in a single polypeptide chain or in different polypeptide chains. Furthermore, an Fv of the invention (as well as any protein of the invention) may have multiple antigen binding domains which may or may not bind the same antigen. This term shall be understood to encompass fragments directly derived from an antibody as well as proteins corresponding to such a fragment produced using recombinant means. In some examples, the VH is not linked to a heavy chain constant domain (CH) 1 and/or the VL is not linked to a light chain constant domain (CL). Exemplary Fv containing polypeptides or proteins include a Fab fragment, a Fab' fragment, a F(ab') fragment, a scFv, a diabody, a triabody, a tetrabody or higher order complex, or any of the foregoing linked to a constant region or domain thereof, e.g., CH2 or CH3 domain, e.g., a minibody. A "Fab fragment" consists of a monovalent antigen-binding fragment of an immunoglobulin, and can be produced by digestion of a whole antibody with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain or can be produced using recombinant means. A "Fab 1 fragment" of an antibody can be obtained by treating a whole antibody with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of a heavy chain comprising a VH and a single constant domain. Two Fab' fragments are obtained per antibody treated in this manner. A Fab' fragment can also be produced by recombinant means. A "F(ab')2 fragment" of an antibody consists of a dimer of two Fab' fragments held together by two disulfide bonds, and is obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction. A "Fab2" fragment is a recombinant fragment comprising two Fab fragments linked using, for example a leucine zipper or a CH3 domain. A"single chain Fv" or"scFv" is a recombinant molecule containing the variable region fragment (Fv) of an antibody in which the variable region of the light chain and the variable region of the heavy chain are covalently linked by a suitable, flexible polypeptide linker.
As used herein, the term "binds" in reference to the interaction of an antigen binding site or an antigen binding domain thereof with an antigen means that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the antigen. For example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody binds to epitope "A", the presence of a molecule containing epitope"A" (or free, unlabeled"A"), in a reaction containing labeled"A" and the protein, will reduce the amount of labeled "A" bound to the antibody.
As used herein, the term"specifically binds" or"binds specifically" shall be taken to mean that an antigen binding site of the invention reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular antigen or cell expressing same than it does with alternative antigens or cells. Generally, but not necessarily, reference to binding means specific binding, and each term shall be understood to provide explicit support for the other term.
As used herein, the term"does not detectably bind" shall be understood to mean that an antigen binding site, e.g., an antibody, binds to a candidate antigen at a level less than 10%, or 8% or 6% or 5% above background. The background can be the level of binding signal detected in the absence of the protein and/or in the presence of a negative control protein (e.g., an isotype control antibody) and/or the level of binding detected in the presence of a negative control antigen. The level of binding is detected using biosensor analysis (e.g. Biacore) in which the antigen binding site is immobilized and contacted with an antigen.
As used herein, the term "does not significantly bind" shall be understood to mean that the level of binding of an antigen binding site of the invention to a polypeptide is not statistically significantly higher than background, e.g., the level of binding signal detected in the absence of the antigen binding site and/or in the presence of a negative control protein (e.g., an isotype control antibody) and/or the level of binding detected in the presence of a negative control polypeptide. The level of binding is detected using biosensor analysis (e.g. Biacore) in which the antigen binding site is immobilized and contacted with an antigen.
As used herein, the term "epitope" (syn. "antigenic determinant") shall be understood to mean a region of an antigen (e.g. CD33, amyloid beta, tau or alpha- synuclein) to which an antigen binding site comprising an antigen binding domain of an antibody binds. Unless otherwise defined, this term is not necessarily limited to the specific residues or structure to which the antigen binding site makes contact. For example, this term includes the region spanning amino acids contacted by the antigen binding site and 5-10 (or more) or 2-5 or 1 -3 amino acids outside of this region. In some examples, the epitope comprises a series of discontinuous amino acids that are positioned close to one another when antigen binding site is folded, i.e. , a "conformational epitope". The skilled artisan will also be aware that the term "epitope" is not limited to peptides or polypeptides. For example, the term"epitope" includes chemically active surface groupings of molecules such as sugar side chains, phosphoryl side chains, or sulfonyl side chains, and, in certain examples, may have specific three- dimensional structural characteristics, and/or specific charge characteristics.
As used herein, the term"condition" refers to a disruption of or interference with normal function and is not to be limited to any specific condition, and will include diseases or disorders.
As used herein, the term"subject" shall be taken to mean any animal including humans, for example a mammal. Exemplary subjects include but are not limited to humans and non-human primates. For example, the subject is a human.
In one example, an antigen binding site as described herein according to any aspect or embodiment is an antibody, preferably a bispecific antibody.
Methods for generating antibodies are known in the art and/or described in Harlow and Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988). Generally, in such methods an antigen (e.g., hCD33) or a region thereof (e.g., an extracellular region) or immunogenic fragment or epitope thereof or a cell expressing and displaying same (i.e., an immunogen), optionally formulated with any suitable or desired carrier, adjuvant, or pharmaceutically acceptable excipient, is administered to a non-human animal, for example, a mouse, chicken, rat, rabbit, guinea pig, dog, horse, cow, goat or pig. The immunogen may be administered intranasally, intramuscularly, sub-cutaneously, intravenously, intradermally, intraperitoneally, or by other known route.
The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. One or more further immunizations may be given, if required to achieve a desired antibody titer. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal is bled and the serum isolated and stored, and/or the animal is used to generate monoclonal antibodies (mAbs).
Monoclonal antibodies are one exemplary form of antibody contemplated by the present invention. The term"monoclonal antibody" or"mAb" refers to a homogeneous antibody population capable of binding to the same antigen(s), for example, to the same epitope within the antigen. This term is not intended to be limited with regard to the source of the antibody or the manner in which it is made.
For the production of mAbs any one of a number of known techniques may be used, such as, for example, the procedure exemplified in US4196265 or Harlow and Lane (1988), supra.
For example, a suitable animal is immunized with an immunogen under conditions sufficient to stimulate antibody producing cells. Rodents such as rabbits, mice and rats are exemplary animals. Mice genetically-engineered to express human antibodies, for example, which do not express murine antibodies, can also be used to generate an antibody of the present invention (e.g., as described in W02002/066630).
Following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the mAb generating protocol. These cells may be obtained from biopsies of spleens, tonsils or lymph nodes, or from a peripheral blood sample. The B cells from the immunized animal are then fused with cells of an immortal myeloma cell, generally derived from the same species as the animal that was immunized with the immunogen.
Hybrids are amplified by culture in a selective medium comprising an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary agents are aminopterin, methotrexate and azaserine.
The amplified hybridomas are subjected to a functional selection for antibody specificity and/or titer, such as, for example, by flow cytometry and/or immunohistochemstry and/or immunoassay (e.g. radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay, dot immunoassay, and the like).
Alternatively, ABL-MYC technology (NeoClone, Madison Wl 53713, USA) is used to produce cell lines secreting MAbs (e.g., as described in Largaespada et al, J. Immunol. Methods. 197 85-95, 1996).
Antibodies can also be produced or isolated by screening a display library, e.g., a phage display library, e.g., as described in US6300064 and/or US5885793. For example, the present inventors have isolated fully human antibodies from a phage display library.
The antibody of the present invention may be a synthetic antibody. For example, the antibody is a chimeric antibody, a humanized antibody, a human antibody synhumanized antibody, primatized antibody or a de-immunized antibody.
As described herein an antigen binding site, or bispecific antibody as described herein, may be in the form of a bispecific IgG, appended IgG, bispecific antibody fragment, bispecific fusion protein or bispecific conjugate. A bispecific IgG includes CrossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-lgG, Knobs-in-holes common LC, Knobs-in-holes assembly, Charge pair, Fab-arm exchange, SEEDbody, Triomab, LUZ-Y, Fcab, kl-body, and Orthogonal Fab. Appended IgG includes DVD-lgG, lgG(H)scFv, scFv-(H)lgG, lgG(L)-scFv, scFv-(L)lgG, lgG(L,H)-Fv, lgG(H)-V, V(H)-lgG, lgG(L)-V, V(L)-lgG, KIH IgG-scFab, 2scFv-lgG, lgG-2scFv, scFv4-lg, Zybody, and DVI- IgG (four-in-one). Bispecific antibody fragments include a nanobody, nanobody-HSA, BiTE, diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, Triple Body, Miniantibody, Minibody, TriBi minibody, scFV-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFV-KIH, Fab-scFv-Fc, Tetravalent HCAb, scDiabody-Fc, Diabody-Fc, Tandem scFv-Fc, and Intrabody. Bispecific fusion proteins include dock and lock, ImmTAC, HSAbody, scDiabody-HSA, andTandem scFv-Toxin. Bispecific antibody conjugates include IgG-lgG, Cov-X-Body and scFv1 -PEG-scFv2. These formats and other are described in Speiss et al. Molecular Immunology, (2015), 95-106.
Antigen Binding Domain Containing Proteins
Diabodies, Triabodies, Tetrabodies
In some examples, an antigen binding site or protein of the invention is or comprises a diabody, triabody, tetrabody or higher order protein complex such as those described in W098/044001 and/or W094/007921 .
For example, a diabody is a protein comprising two associated polypeptide chains, each polypeptide chain comprising the structure V L -X-V H or V H -X-V L , wherein V L is an antibody light chain variable region, VH is an antibody heavy chain variable region, X is a linker comprising insufficient residues to permit the V H and V L in a single polypeptide chain to associate (or form an Fv) or is absent, and wherein the V H of one polypeptide chain binds to a V | _ of the other polypeptide chain to form an antigen binding domain, i.e. , to form a Fv molecule capable of specifically binding to one or more antigens. The V L and V H can be the same in each polypeptide chain or the V L and V H can be different in each polypeptide chain so as to form a bispecific diabody (i.e., comprising two Fvs having different specificity).
Single Chain Fv (scFv)
The present invention encompasses a dimeric scFv, i.e., a protein comprising two scFv molecules linked by a non-covalent or covalent linkage, e.g., by a leucine zipper domain (e.g., derived from Fos or Jun). Alternatively, two scFvs are linked by a peptide linker of sufficient length to permit both scFvs to form and to bind to an antigen, e.g., as described in US20060263367.
Heavy Chain Antibodies
Heavy chain antibodies differ structurally from many other forms of antibodies, in so far as they comprise a heavy chain, but do not comprise a light chain. Accordingly, these antibodies are also referred to as"heavy chain only antibodies". Heavy chain antibodies are found in, for example, camelids and cartilaginous fish (also called IgNAR).
The variable regions present in naturally occurring heavy chain antibodies are generally referred to as "VHH domains" in camelid antibodies and V-NAR in IgNAR, in order to distinguish them from the heavy chain variable regions that are present in conventional 4-chain antibodies (which are referred to as "V H domains") and from the light chain variable regions that are present in conventional 4-chain antibodies (which are referred to as "V L domains").
A general description of heavy chain antibodies from camelids and the variable regions thereof and methods for their production and/or isolation and/or use is found inter alia in the following references WO94/04678, WO97/49805 and WO 97/49805.
A general description of heavy chain antibodies from cartilaginous fish and the variable regions thereof and methods for their production and/or isolation and/or use is found inter alia in W02005/1 18629.
Other Antibodies and Proteins Comprising Antigen Binding Domains Thereof
The present invention also contemplates other antibodies and proteins comprising antigen-binding domains thereof, such as:
(i) "key and hole" bispecific proteins as described in US5731 168;
(ii) heteroconjugate proteins, e.g., as described in US4676980;
(iii) heteroconjugate proteins produced using a chemical cross-linker, e.g., as described in US4676980; and
(iv) Fab 3 (e.g., as described in EP19930302894).
Antigen binding proteins also contemplated as those where CDRs or equivalent are engineered in or grafted onto a protein scaffold eg. DARPins. In other words, non- immunoglobulin scaffolds are also contemplated such as ABD, Adhiron, Adnectin/Monobody, Affibody, Affilin, Affimer, Affitin/Nanofitin, Alphabody, Anticalin, Armadillo repeat proteins, Atrimer/ Tetranectin, Avimer/ Maxibody, Centyrin, DARPin, Fynomer, Kunitz domain, Obody/OB-fold, Pronectin, Repebody as referred to in Trends in Biotechnology, July 2015, Vol. 33, No. 7. p408-418.
Mutations to Proteins
The present invention also provides an antigen binding site or a nucleic acid encoding same having at least 80% identity to a sequence disclosed herein. In one example, an antigen binding site or nucleic acid of the invention comprises sequence at least about 85% or 90% or 95% or 97% or 98% or 99% identical to a sequence disclosed herein.
Alternatively, or additionally, the antigen binding site comprises a CDR (e.g., three CDRs) at least about 80% or 85% or 90% or 95% or 97% or 98% or 99% identical to CDR(s) of a VH or VL as described herein according to any aspect or embodiment.
In another example, a nucleic acid of the invention comprises a sequence at least about 80% or 85% or 90% or 95% or 97% or 98% or 99% identical to a sequence encoding an antigen binding site having a function as described herein according to any aspect or embodiment. The present invention also encompasses nucleic acids encoding an antigen binding site of the invention, which differs from a sequence exemplified herein as a result of degeneracy of the genetic code.
The % identity of a nucleic acid or polypeptide may be determined by GAP (Needleman and Wunsch. Mol. Biol. 48, 443-453, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. The query sequence is at least 50 residues in length, and the GAP analysis aligns the two sequences over a region of at least 50 residues. For example, the query sequence is at least 100 residues in length and the GAP analysis aligns the two sequences over a region of at least 100 residues. For example, the two sequences are aligned over their entire length.
The present invention also contemplates a nucleic acid that hybridizes under stringent hybridization conditions to a nucleic acid encoding an antigen binding site described herein. A"moderate stringency" is defined herein as being a hybridization and/or washing carried out in 2 x SSC buffer, 0.1 % (w/v) SDS at a temperature in the range 45°C to 65°C, or equivalent conditions. A"high stringency" is defined herein as being a hybridization and/or wash carried out in 0.1 x SSC buffer, 0.1 % (w/v) SDS, or lower salt concentration, and at a temperature of at least 65°C, or equivalent conditions. Reference herein to a particular level of stringency encompasses equivalent conditions using wash/hybridization solutions other than SSC known to those skilled in the art. For example, methods for calculating the temperature at which the strands of a double stranded nucleic acid will dissociate (also known as melting temperature, or Tm) are known in the art. A temperature that is similar to (e.g., within 5°C or within 10°C) or equal to the Tm of a nucleic acid is considered to be high stringency. Medium stringency is to be considered to be within 10°C to 20°C or 10°C to 15°C of the calculated Tm of the nucleic acid.
The present invention also contemplates mutant forms of an antigen binding site of the invention comprising one or more conservative amino acid substitutions compared to a sequence set forth herein. In some examples, the antigen binding site comprises 10 or fewer, e.g., 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 conservative amino acid substitutions. A"conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain and/or hydropathicity and/or hydrophilicity.
Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), b- branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Hydropathic indices are described, for example in Kyte and Doolittle J. Mol. Biol., 157\ 105-132, 1982 and hydrophylic indices are described in, e.g., US4554101 .
The present invention also contemplates non-conservative amino acid changes. For example, of particular interest are substitutions of charged amino acids with another charged amino acid and with neutral or positively charged amino acids. In some examples, the antigen binding site comprises 10 or fewer, e.g., 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 non-conservative amino acid substitutions. In one example, the mutation(s) occur within a FR of an antigen binding domain of an antigen binding site of the invention. In another example, the mutation(s) occur within a CDR of an antigen binding site of the invention.
Exemplary methods for producing mutant forms of an antigen binding site include:
• mutagenesis of DNA (Thie et al., Methods Mol. Biol. 525 309-322, 2009) or RNA (Kopsidas et al., Immunol. Lett. 107\ 163-168, 2006; Kopsidas et al. BMC Biotechnology, 7: 18, 2007; and W01999/058661 );
• introducing a nucleic acid encoding the polypeptide into a mutator cell, e.g., XL- 1 Red, XL-mutS and XL-mutS-Kanr bacterial cells (Stratagene);
• DNA shuffling, e.g., as disclosed in Stemmer, Nature 370 389-91 , 1994; and
• site directed mutagenesis, e.g., as described in Dieffenbach (ed) and Dveksler (ed) (ln PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratories, NY, 1995).
Exemplary methods for determining biological activity of the mutant antigen binding sites of the invention will be apparent to the skilled artisan and/or described herein, e.g., antigen binding. For example, methods for determining antigen binding, competitive inhibition of binding, affinity, association, dissociation and therapeutic efficacy are described herein.
Constant Regions
The present invention encompasses antigen binding sites and/or antibodies described herein comprising a constant region of an antibody. This includes antigen binding fragments of an antibody fused to an Fc.
Sequences of constant regions useful for producing the proteins of the present invention may be obtained from a number of different sources. In some examples, the constant region or portion thereof of the protein is derived from a human antibody. The constant region or portion thereof may be derived from any antibody class, including IgM, IgG, IgD, IgA and IgE, and any antibody isotype, including lgG1 , lgG2, lgG3 and lgG4. In one example, the constant region is human isotype lgG4 or a stabilized lgG4 constant region.
In one example, the Fc region of the constant region has a reduced ability to induce effector function, e.g., compared to a native or wild-type human lgG1 or lgG3 Fc region. In one example, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC) and/or antibody-dependent cell-mediated phagocytosis (ADCP) and/or complement-dependent cytotoxicity (CDC). Methods for assessing the level of effector function of an Fc region containing protein are known in the art and/or described herein.
In one example, the Fc region is an lgG4 Fc region (i.e. , from an lgG4 constant region), e.g., a human lgG4 Fc region. Sequences of suitable lgG4 Fc regions will be apparent to the skilled person and/or available in publically available databases (e.g., available from National Center for Biotechnology Information).
In one example, the constant region is a stabilized lgG4 constant region. The term"stabilized lgG4 constant region" will be understood to mean an lgG4 constant region that has been modified to reduce Fab arm exchange or the propensity to undergo Fab arm exchange or formation of a half-antibody or a propensity to form a half antibody. "Fab arm exchange" refers to a type of protein modification for human lgG4, in which an lgG4 heavy chain and attached light chain (half-molecule) is swapped for a heavy-light chain pair from another lgG4 molecule. Thus, lgG4 molecules may acquire two distinct Fab arms recognizing two distinct antigens (resulting in bispecific molecules). Fab arm exchange occurs naturally in vivo and can be induced in vitro by purified blood cells or reducing agents such as reduced glutathione. A"half antibody" forms when an lgG4 antibody dissociates to form two molecules each containing a single heavy chain and a single light chain.
In one example, a stabilized lgG4 constant region comprises a proline at position 241 of the hinge region according to the system of Kabat (Kabat et al., Sequences of Proteins of Immunological Interest Washington DC United States Department of Health and Human Services, 1987 and/or 1991 ). This position corresponds to position 228 of the hinge region according to the EU numbering system (Kabat et al., Sequences of Proteins of Immunological Interest Washington DC United States Department of Health and Human Services, 2001 and Edelman et at, Proc. Natl. Acad. USA, 63, 78-85, 1969). In human lgG4, this residue is generally a serine. Following substitution of the serine for proline, the lgG4 hinge region comprises a sequence CPPC. In this regard, the skilled person will be aware that the"hinge region" is a proline-rich portion of an antibody heavy chain constant region that links the Fc and Fab regions that confers mobility on the two Fab arms of an antibody. The hinge region includes cysteine residues which are involved in inter-heavy chain disulfide bonds. It is generally defined as stretching from Glu226 to Pro243 of human lgG1 according to the numbering system of Kabat. Hinge regions of other IgG isotypes may be aligned with the lgG1 sequence by placing the first and last cysteine residues forming inter-heavy chain disulphide (S-S) bonds in the same positions (see for example WO2010/080538).
Additional examples of stabilized lgG4 antibodies are antibodies in which arginine at position 409 in a heavy chain constant region of human lgG4 (according to the EU numbering system) is substituted with lysine, threonine, methionine, or leucine (e.g., as described in W02006/033386). The Fc region of the constant region may additionally or alternatively comprise a residue selected from the group consisting of: alanine, valine, glycine, isoleucine and leucine at the position corresponding to 405 (according to the EU numbering system). Optionally, the hinge region comprises a proline at position 241 (i.e., a CPPC sequence) (as described above).
In another example, the Fc region is a region modified to have reduced effector function, i.e., a"non-immunostimulatory Fc region". For example, the Fc region is an lgG1 Fc region comprising a substitution at one or more positions selected from the group consisting of 268, 309, 330 and 331 . In another example, the Fc region is an lgG1 Fc region comprising one or more of the following changes E233P, L234V, L235A and deletion of G236 and/or one or more of the following changes A327G, A330S and P331 S (Armour et at, Eur J Immunol. 29 2613-2624, 1999; Shields et at, J Biol Chem. 276(9) 6591 -604, 2001 ). Additional examples of non-immunostimulatory Fc regions are described, for example, in Dall'Acqua et at, J Immunol. 177 : 1 129-1 138 2006; and/or Hezareh J Virol ;75\ 12161 -12168, 2001 ).
In another example, the Fc region is a chimeric Fc region, e.g., comprising at least one CH2 domain from an lgG4 antibody and at least one CH3 domain from an lgG1 antibody, wherein the Fc region comprises a substitution at one or more amino acid positions selected from the group consisting of 240, 262, 264, 266, 297, 299, 307, 309, 323, 399, 409 and 427 (EU numbering) (e.g., as described in WO2010/085682). Exemplary substitutions include 240F, 262L, 264T, 266F, 297Q, 299A, 299K, 307P, 309K, 309M, 309P, 323F, 399S, and 427F.
Additional Modifications
The present invention also contemplates additional modifications to an antibody or antigen binding site comprising an Fc region or constant region.
For example, the antibody comprises one or more amino acid substitutions that increase the half-life of the protein. For example, the antibody comprises a Fc region comprising one or more amino acid substitutions that increase the affinity of the Fc region for the neonatal Fc region (FcRn). For example, the Fc region has increased affinity for FcRn at lower pH, e.g., about pH 6.0, to facilitate Fc/FcRn binding in an endosome. In one example, the Fc region has increased affinity for FcRn at about pH 6 compared to its affinity at about pH 7.4, which facilitates the re-release of Fc into blood following cellular recycling. These amino acid substitutions are useful for extending the half life of a protein, by reducing clearance from the blood.
Exemplary amino acid substitutions include T250Q and/or M428L or T252A, T254S and T266F or M252Y, S254T and T256E or H433K and N434F according to the EU numbering system. Additional or alternative amino acid substitutions are described, for example, in US20070135620 or US7083784.
Protein Production
In one example, an antigen binding site described herein according to any example is produced by culturing a hybridoma under conditions sufficient to produce the protein, e.g., as described herein and/or as is known in the art.
Recombinant Expression
In another example, an antigen binding site described herein according to any example is recombinant. In the case of a recombinant protein, nucleic acid encoding same can be cloned into expression constructs or vectors, which are then transfected into host cells, such as E. coli cells, yeast cells, insect cells, or mammalian cells, such as simian COS cells, Chinese Hamster Ovary (CHO) cells, human embryonic kidney (HEK) cells, or myeloma cells that do not otherwise produce the protein. Exemplary cells used for expressing a protein are CHO cells, myeloma cells or HEK cells. Molecular cloning techniques to achieve these ends are known in the art and described, for example in Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley- Interscience (1988, including all updates until present) or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989). A wide variety of cloning and in vitro amplification methods are suitable for the construction of recombinant nucleic acids. Methods of producing recombinant antibodies are also known in the art, see, e g., US4816567 or US5530101.
Following isolation, the nucleic acid is inserted operably linked to a promoter in an expression construct or expression vector for further cloning (amplification of the DNA) or for expression in a cell-free system or in cells.
As used herein, the term"promoter" is to be taken in its broadest context and includes the transcriptional regulatory sequences of a genomic gene, including the TATA box or initiator element, which is required for accurate transcription initiation, with or without additional regulatory elements (e.g., upstream activating sequences, transcription factor binding sites, enhancers and silencers) that alter expression of a nucleic acid, e.g., in response to a developmental and/or external stimulus, or in a tissue specific manner. In the present context, the term "promoter" is also used to describe a recombinant, synthetic or fusion nucleic acid, or derivative which confers, activates or enhances the expression of a nucleic acid to which it is operably linked. Exemplary promoters can contain additional copies of one or more specific regulatory elements to further enhance expression and/or alter the spatial expression and/or temporal expression of said nucleic acid.
As used herein, the term "operably linked to" means positioning a promoter relative to a nucleic acid such that expression of the nucleic acid is controlled by the promoter. Many vectors for expression in cells are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, a sequence encoding a protein (e.g., derived from the information provided herein), an enhancer element, a promoter, and a transcription termination sequence. The skilled artisan will be aware of suitable sequences for expression of a protein. Exemplary signal sequences include prokaryotic secretion signals (e.g., pelB, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II), yeast secretion signals (e.g., invertase leader, a factor leader, or acid phosphatase leader) or mammalian secretion signals (e.g., herpes simplex gD signal).
Exemplary promoters active in mammalian cells include cytomegalovirus immediate early promoter (CMV-IE), human elongation factor 1 -a promoter (EF1 ), small nuclear RNA promoters (U1 a and U1 b), a-myosin heavy chain promoter, Simian virus 40 promoter (SV40), Rous sarcoma virus promoter (RSV), Adenovirus major late promoter, b-actin promoter; hybrid regulatory element comprising a CMV enhancer/ b- actin promoter or an immunoglobulin promoter or active fragment thereof. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651 ); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture; baby hamster kidney cells (BHK, ATCC CCL 10); or Chinese hamster ovary cells (CHO).
Typical promoters suitable for expression in yeast cells such as for example a yeast cell selected from the group comprising Pichia pastoris, Saccharomyces cerevisiae and S. pombe, include, but are not limited to, the ADH1 promoter, the GAL1 promoter, the GAL4 promoter, the CUP1 promoter, the PH05 promoter, the nmt promoter, the RPR1 promoter, or the TEF1 promoter.
Means for introducing the isolated nucleic acid or expression construct comprising same into a cell for expression are known to those skilled in the art. The technique used for a given cell depends on the known successful techniques. Means for introducing recombinant DNA into cells include microinjection, transfection mediated by DEAE-dextran, transfection mediated by liposomes such as by using lipofectamine (Gibco, MD, USA) and/or cellfectin (Gibco, MD, USA), PEG-mediated DNA uptake, electroporation and microparticle bombardment such as by using DNA-coated tungsten or gold particles (Agracetus Inc., Wl, USA) amongst others. The host cells used to produce the protein may be cultured in a variety of media, depending on the cell type used. Commercially available media such as Ham's FI0 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing mammalian cells. Media for culturing other cell types discussed herein are known in the art.
Isolation of Proteins
Methods for isolating a protein are known in the art and/or described herein.
Where an antigen binding site is secreted into culture medium, supernatants from such expression systems can be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants. Alternatively, or additionally, supernatants can be filtered and/or separated from cells expressing the protein, e.g., using continuous centrifugation.
The antigen binding site prepared from the cells can be purified using, for example, ion exchange, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, affinity chromatography (e.g., protein A affinity chromatography or protein G chromatography), or any combination of the foregoing. These methods are known in the art and described, for example in W099/57134 or Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988).
The skilled artisan will also be aware that a protein can be modified to include a tag to facilitate purification or detection, e.g., a poly-histidine tag, e.g., a hexa-histidine tag, or a influenza virus hemagglutinin (HA) tag, or a Simian Virus 5 (V5) tag, or a FLAG tag, or a glutathione S-transferase (GST) tag. The resulting protein is then purified using methods known in the art, such as, affinity purification. For example, a protein comprising a hexa-his tag is purified by contacting a sample comprising the protein with nickel-nitrilotriacetic acid (Ni-NTA) that specifically binds a hexa-his tag immobilized on a solid or semi-solid support, washing the sample to remove unbound protein, and subsequently eluting the bound protein. Alternatively, or in addition a ligand or antibody that binds to a tag is used in an affinity purification method.
Assaying Activity of an Antigen Binding Site
Binding to antigen and Mutants Thereof
It will be apparent to the skilled artisan from the disclosure herein that antigen binding sites of the present invention bind to CD33 and any one of amyloid beta, tau or alpha-synuclein. Methods for assessing binding to a protein are known in the art, e.g., as described in Scopes (In: Protein purification: principles and practice, Third Edition, Springer Verlag, 1994). Such a method generally involves immobilizing the antigen binding site and contacting it with labelled antigen (e.g. CD33 or pathogenic protein). Following washing to remove non-specific bound protein, the amount of label and, as a consequence, bound antigen is detected. Of course, the antigen binding site can be labeled and the antigen immobilized. Panning-type assays can also be used. Alternatively, or additionally, surface plasmon resonance assays can be used.
Optionally, the dissociation constant (Kd), association constant (Ka) and/or affinity constant (K D ) of an immobilized antigen binding site for an antigen or an epitope thereof is determined. The "Kd" or "Ka" or"K D " for an antigen binding protein is in one example measured by a radiolabeled or fluorescently-labeled ligand binding assay. In the case of a "Kd", this assay equilibrates the antigen binding site with a minimal concentration of labeled ligand or epitope thereof in the presence of a titration series of unlabeled ligand. Following washing to remove unbound ligand or epitope thereof, the amount of label is determined, which is indicative of the Kd of the protein.
According to another example the Kd, Ka or KD is measured by using surface plasmon resonance assays, e.g., using BIAcore surface plasmon resonance (BIAcore, Inc., Piscataway, NJ) with immobilized antigen or a region thereof or immobilized antigen binding site. Determining endocytic activity
Methods for determining the endocytosis inducing activity of an antigen binding site as described herein are known to the skilled person. However, an exemplary method includes that described in the Examples.
Conditions to be Treated
The antigen binding sites of the present invention are useful in the treatment or prevention of any condition associated, or caused by, the presence or over-expression of a pathogenic protein, preferably a pathogenic protein that is, or is predominantly, extracellular.
A subject in need of treatment may be one that exhibits impaired memory function, cognitive function or subclinical or clinical symptoms of a neurodegenerative disease. The selection of an individual for treatment may involve a screening step for identifying whether the individual is displaying impaired cognitive function, memory function or a clinical manifestation of a neurodegenerative disease. A subject in need of treatment may be one that is identified as having early, intermediate or late stage disease and in the case of Alzheimer's disease may be identified as having either diffuse Ab oligomers or plaques.
In Alzheimer's disease there is a significant cognitive decline from a previous level of performance in one or more areas of cognitive domains, preferably documented by standardised neuropsychological testing. The cognitive domains that are affected in Alzheimer's disease include learning and memory, complex attention, executive function, perceptual - motor, social cognition, and language. This list of domains is not exhaustive
In addition, other neurodegenerative diseases that could be treated by the invention are characterised by deficits in the listed cognitive domains as well as motor function.
A decline of memory and learning is documented and at least one other cognitive domain. The decline in cognition is progressive and gradual. Standardised neuropsychological tests of cognition that could be administered to identify an individual in need of treatment or to determine the effectiveness of the treatment include any of the following tests or one or more of its components: Neuropsychological Test Battery, Alzheimer's Disease Assessment Scale-cognitive subscale (ADAS-cog), Mini-Mental State Examination, Severe Impairment Battery, Disability Assessment Scale for Dementia, Clinical Dementia Rating Scale Sum of Boxes, Alzheimer's Disease Cooperative Study Clinical Global Impression of Change, Wechsler Memory Scale Visual Immediate, Wechsler Memory Scale Verbal Immediate, Rey Auditory Verbal Learning Test, Wechsler Memory Digit Span, Controlled Word Association Test, Category Fluency Test, Wechsler Memory Scale Visual Delayed, Wechsler Memory Scale Verbal Delayed, RAVLT delayed, Wechsler Memory Scale, Stroop Task, Wisconsin Card Sorting Task, or other tests of memory and executive function.
A patient with cognitive dysfunction caused by a neurodegenerative disease may have one or more of the following impairments in the highlighted domains, for example:
- Learning and memory: Cannot keep track of plans, repeats themselves in conversation, needs frequent reminders to perform tasks;
- Complex attention: Difficulty in environments with multiple stimuli, difficulty holding new information in mind;
- Executive function: Inability to perform complex projects, inability to make decisions;
- Language: Difficulties with expressive or receptive language, use of general terms instead of correct word, may not recall names of friends and family;
- Perceptual-motor: Difficulty with previously familiar motor tasks and activities, navigation; and
- Social cognition: Changes in behaviour, digression from social norms, makes reckless decisions, shows poor insight into these decisions.
A patient with frontotemporal dementia may show impairments in one or more of the domains of language, social cognition, perceptual-motor, executive function and complex attention without learning and memory impairment or learning and memory impairment may be present. In Parkinson's disease motor deficits may be present with or without deficits in other domains of cognition, or deficits may be present. In Huntington's disease, motor deficits may be present without deficits in other domains of cognition, or deficits may be present. In Amyotrophic Lateral Sclerosis motor deficits may be present without deficits in other domains of cognition, or deficits may be present.
The neurodegenerative diseases to which the invention can be applied are those where pathogenic protein is extracellular and cause or contribute to the disease or a symptom thereof. The pathogenic protein may be in a pathogenic form when in an altered structure such as an oligomer, an aggregate or a deposit. Alzheimer's disease, dementia with Lewy bodies, Parkinson's disease, frontotemporal lobar degeneration and British and Danish familial dementia are non-limiting examples of diseases associated with extracellular pathogenic protein. Alzheimer's disease is the most common example of these diseases in which oligomers or plaques composed of amyloid beta are formed in the brain. Other neurodegenerative diseases are caused by the pathological aggregation of one or more of the proteins: Amyloid beta, amyloid fragments, amyloid precursor protein, amyloid precursor protein fragments or British peptide.
In a preferable embodiment the condition, disease or syndrome is Alzheimer's disease. In these embodiments the individual to be treated may display impairment in the following cognitive domains including learning and memory, complex attention, executive function, perceptual - motor, social cognition, and language. Alternatively, the individual may display one or more of the following symptoms: Age-associated cognitive impairment, Age-associated neuronal dysfunction not restricted to cognitive impairment, short term memory loss, inability to acquire new information, semantic memory impairments, apathy, mild cognitive impairment, language, executive or visuoconstructional problems or apraxia, long term memory impairment, irritability and aggression, and exhaustion.
Treatment as used herein refers to therapeutic treatment and also involves ameliorating a symptom associated with a disease. Therapeutic treatment can be measured by an increase or recovery in any one or more of the group consisting of cognitive function; short term memory; ability to acquire new information; semantic memory; apathy; language, executive or visuoconstructional problems or apraxia; long term memory; irritability and aggression; or exhaustion. Treatment can also be measured via reduction in the presence of pathogenic protein or a reduction in the particular forms of pathogenic protein such as protein aggregates or deposits. The presence and reduction of the pathogenic protein that can be visualised or detected by imaging techniques or biochemical techniques described herein. For example, in relation to Alzheimer's disease, treatment may relate to a reduction in a soluble or insoluble isoforms of amyloid-b peptide or a reduction in the number of amyloid-b plaques. Alternatively, the outcome of the treatment may be determined by neuropsychological or cognitive testing. Improving memory may be determined by memory tests, typically a test administered by a clinical professional. Standardised neuropsychological tests of cognition that could be administered to test the effectiveness of the treatment include any of the following tests or one or more of its components: Neuropsychological Test Battery, Alzheimer's Disease Assessment Scale- cognitive subscale (ADAS-cog), Mini-Mental State Examination, Severe Impairment Battery, Disability Assessment Scale for Dementia, Clinical Dementia Rating Scale Sum of Boxes, Alzheimer's Disease Cooperative Study Clinical Global Impression of Change, Wechsler Memory Scale Visual Immediate, Wechsler Memory Scale Verbal Immediate, Rey Auditory Verbal Learning Test, Wechsler Memory Digit Span, Controlled Word Association Test, Category Fluency Test, Wechsler Memory Scale Visual Delayed, Wechsler Memory Scale Verbal Delayed, Rey Auditory Verbal Learning Test, Wechsler Memory Scale, Stroop Task, Wisconsin Card Sorting Task, Trail Making Test, or any other tests of memory and executive function alone or in combination.
Accordingly, the antigen binding sites of the present invention find application in treating such disorders.
In some examples, an antigen binding site as described herein can be administered orally, parenterally, by inhalation spray, adsorption, absorption, topically, rectally, nasally, bucally, vaginally, intraventricularly, via an implanted reservoir in dosage formulations containing conventional non-toxic pharmaceutically-acceptable carriers, or by any other convenient dosage form. The term"parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, and intracranial injection or infusion techniques.
Methods for preparing an antigen binding site into a suitable form for administration to a subject (e.g. a pharmaceutical composition) are known in the art and include, for example, methods as described in Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Co., Easton, Pa., 1990) and U.S. Pharmacopeia: National Formulary (Mack Publishing Company, Easton, Pa., 1984).
The pharmaceutical compositions of this invention are particularly useful for parenteral administration, such as intravenous administration or administration into a body cavity or lumen of an organ or joint. The compositions for administration will commonly comprise a solution of an antigen binding site dissolved in a pharmaceutically acceptable carrier, for example an aqueous carrier. A variety of aqueous carriers can be used, e.g., buffered saline and the like. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of an antigen binding site of the present invention in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs. Exemplary carriers include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as mixed oils and ethyl oleate may also be used. Liposomes may also be used as carriers. The vehicles may contain minor amounts of additives that enhance isotonicity and chemical stability, e.g., buffers and preservatives.
Upon formulation, an antigen binding site of the present invention will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically/prophylactically effective. Formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but other pharmaceutically acceptable forms are also contemplated, e.g., tablets, pills, capsules or other solids for oral administration, suppositories, pessaries, nasal solutions or sprays, aerosols, inhalants, liposomal forms and the like. Pharmaceutical "slow release" capsules or compositions may also be used. Slow release formulations are generally designed to give a constant drug level over an extended period and may be used to deliver an antigen binding site of the present invention.
W02002/080967 describes compositions and methods for administering aerosolized compositions comprising antibodies for the treatment of, e.g., asthma, which are also suitable for administration of an antigen binding site of the present invention.
Dosages and Timing of Administration
Suitable dosages of an antigen binding site of the present invention will vary depending on the specific an antigen binding site, the condition to be treated and/or the subject being treated. It is within the ability of a skilled physician to determine a suitable dosage, e.g., by commencing with a sub-optimal dosage and incrementally modifying the dosage to determine an optimal or useful dosage. Alternatively, to determine an appropriate dosage for treatment/prophylaxis, data from the cell culture assays or animal studies are used, wherein a suitable dose is within a range of circulating concentrations that include the ED50 of the active compound with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. A therapeutically/prophylactically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration or amount of the compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma maybe measured, for example, by high performance liquid chromatography.
In some examples, a method of the present invention comprises administering a prophylactically or therapeutically effective amount of a protein described herein.
The term "therapeutically effective amount" is the quantity which, when administered to a subject in need of treatment, improves the prognosis and/or state of the subject and/or that reduces or inhibits one or more symptoms of a clinical condition described herein to a level that is below that observed and accepted as clinically diagnostic or clinically characteristic of that condition. The amount to be administered to a subject will depend on the particular characteristics of the condition to be treated, the type and stage of condition being treated, the mode of administration, and the characteristics of the subject, such as general health, other diseases, age, sex, genotype, and body weight. A person skilled in the art will be able to determine appropriate dosages depending on these and other factors. Accordingly, this term is not to be construed to limit the present invention to a specific quantity, e.g., weight or amount of protein(s), rather the present invention encompasses any amount of the antigen binding site(s) sufficient to achieve the stated result in a subject.
As used herein, the term "prophylactically effective amount" shall be taken to mean a sufficient quantity of a protein to prevent or inhibit or delay the onset of one or more detectable symptoms of a clinical condition. The skilled artisan will be aware that such an amount will vary depending on, for example, the specific antigen binding site(s) administered and/or the particular subject and/or the type or severity or level of condition and/or predisposition (genetic or otherwise) to the condition. Accordingly, this term is not to be construed to limit the present invention to a specific quantity, e.g., weight or amount of antigen binding site(s), rather the present invention encompasses any amount of the antigen binding site(s) sufficient to achieve the stated result in a subject.
The present invention additionally comprises a kit comprising one or more of the following:
(i) an antigen binding site of the invention or expression construct(s) encoding same;
(ii) a cell of the invention;
(iii) a complex of the invention; or
(iii) a pharmaceutical composition of the invention.
In the case of a kit for detecting CD33 or a pathogenic protein, the kit can additionally comprise a detection means, e.g., linked to a antigen binding site of the invention. In the case of a kit for therapeutic/prophylactic use, the kit can additionally comprise a pharmaceutically acceptable carrier.
Optionally a kit of the invention is packaged with instructions for use in a method described herein according to any example. Table 1 : The following table describes amino acid and nucleotide sequences as described herein and referred to herein.
V Hi -Vi_2 and V H 2-V Li chain constructs were co-transfected into FreeStyleTM 293- F cells (Invitrogen). Cell culture supernatants were harvested by centrifugation and concentrated by tangential flow filtration (Millipore, Proflux M12). Diabodies were purified with Ni-NTA resin (Qiagen) followed by size exclusion chromatography, dialyzed extensively against Buffer A (20 mM HEPES pH 7.5 and 50 mM NaCI), and finally concentrated to 5 mg/mL (measured by absorbance at 280 nm) and stored in small aliquots at -80°C until required.
Ab1-42 labelled with pHrodo.
Ab1 -42 was briefly dissolved in a small volume of 60 mM NaOH then diluted in PBS to 1 mg/mL, as determined by UV spectroscopy (e=94,526) (Gunn et al, 2012) and 200 pg aliquots (pH 10) were stored at -80°C. A pHrodo™Red Microscale Labeling Kit (life technology) was used to label 200 pg of Ab according to the manufacturer's instructions. To adjust the pH of the Ab stock to pH 8.0, 2 pL of 0.5M NaH 2 P0 4 was added to each 200 pg Ab sample. 4 pL of pHrodo dye was added to Ab solution and incubated for 15 minutes at room temperature. Excess dye was separated from peptides using resin (Bio-Gel P-6 fine resin suspended in PBS). 1 pM of Ab-PHrodo was added to the DMEM media (no serum) for 3 hours. After incubation, cells were extensively washed with PBS. Ab was quantified using flow cytometry.
Labelling ct-synuclein with pHrodo
Recombinant human a-synuclein protein (ab48842) was purchased from Abeam Australia Pty Ltd. The protein was dialyzed in PBS overnight at 4°C and concentrated to 0.9 mg/mL. 2 pL of pHrodo Red dye was added to the protein and incubated for 15 minutes. The excess dye was separated from the labelled protein by dialysis in PBS at 4°C for 2 days. The presence of labelled protein was determined by SDS- PAGE and UV spectroscopy using absorbance at 280 nm for the protein and 566 nm for the dye following the manufacturer's instructions.
Labelling pTau (S422) peptide with pHrodo pTau peptide was dissolved in 1x PBS at 1 mg/mL. 2 pL of pHrodo dye was added to pTau solution and incubated for 15 minutes (as per manufacturer's
instruction). The excess dye was separated from the labelled peptide using Bio gel PE resin and eluted in 1xPBS. The protein concentration was determined by absorbance at 280nm and at 566nm. Labelling 0033-Ab with pHrodo
CD33-Ap was dialysed overnight at 4°C in PBS. 1 mI_ of pHrodo dye was used to label 100 pg of the diabody (pH corrected with sodium bicarbonate as recommended by the manufacturer). The sample was incubated for 15 minutes at room temperature and excess dye was removed with Bio-Gel P-6 resin. The protein concentration was determined by absorbance at 280nm and at 566nm.
CD33 clones.
DNA encoding human CD33K7R (mutant
CD33(K283/288/309/312/313/315/352R) was synthetised and subcloned into a pcDNA 3.1 plasmid and sequenced by Genscript.
RNA transfections.
Plasmid encoding human CD33K7R was linearized with Xho1 enzyme and further purified with a PCR clean up kit (Qiagen). The in vitro synthesis of correctly capped RNA was made using mMESSAGE mMACHINE®T7 Ultra Kit (life technology). BV2 cells were seeded in a 12 well plate at 3x105 cells/ml and 24 hours later were transfected using 0.9 ug RNA and 0.75 pL Lipofectamine® MessengerMAX (Invitrogen) per well.
BV2 cells were seeded in a 12 well plate at 3x105 cells/ml and 24 hours later were transfected using 0.9 ug RNA and 0.75 uL Lipofectamine® MessengerMAX (Invitrogen) per well. The transfection efficiency was determined by Western blot or im m unof luorescence.
Cell culture.
The BV2 immortalized microglial cell line was cultured in complete media and incubated at 37 °C with 5% CO. Complete media was composed of DMEM high glucose with 10% (vol/vol) heat-inactivated FBS, and 1 % penicillin/streptomycin (pen/strep- Life Technologies). Bead uptake.
Transfected BV2 cells were treated with 0.5 uL/mL of 1 um diameter red fluorescence latex beads (Life technology) in serum-free DMEM medium for between 1 to 3 hours and washed extensively with PBS.
Polypeptide uptake assay
Typically, 1 pg-10 pg of a diabody was added to the serum free media for 30 min at 37 °C with 5% CO. After 30 minutes of incubation with CD33-Ap, CD33-aSyn or CD33-pTau, labelled Ab, a-syn or pTau, respectively, was further diluted to 1 mM in DMEM with no serum (Dulbecco's Modified Eagle Medium, Sigma-Aldrich) and applied to the cells for 1 h. Cells were intensively washed and harvested in PBS-EDTA for quantification of uptake in Flow cytometry. 10000 counts were acquired, and data were analysed using Flow Jo.
Bead uptake
Transfected BV2 cells were treated with 0.5 pL/mL of 1 um diameter red fluorescent latex beads (Life technology) in serum-free DMEM medium for between 1 to 3 hours and washed extensively with PBS.
Flow cytometry.
Following each substrate uptake protocol, cells were washed 3 times with PBS, harvested in PBS-EDTA and resuspended in 150 pL PBS-EDTA. Cells were
resuspended in 150 pL of PBS-EDTA. Ten thousand counts were acquired on an LRS Fortessa- Flow Cytometer (Beckman). Samples were analysed using FlowJo version 10.Statistical Analysis.
Statistical analysis was performed using GraphPad Prism 6/7 software.
Comparisons involving more than two groups used one-way ANOVA followed by Tuckey post hoc test and Dunnet' test post hoc test (compare with vehicle).
Comparisons of two groups used the two-tailed Students test. All differences were considered significantly different when P<0.05. Microscale thermophoresis (MST)
Solution MST binding studies between the diabody with the hCD33 extracellular domain (Speed Biosystems), as well as Ab1 -42 peptide were performed using standard protocols on a Monolith NT.1 15 (Nanotemper Technologies). Briefly, purified Oϋ33-Ab was labelled using the RED-NHS (Amine Reactive) Protein Labelling Kit (Nanotemper Technologies). Lyophilized Ab1 -42 peptide was taken up in 60 mM NaOH and diluted in PBS and 0.05%v/v Tween-20; samples were measured soon after dissolution, and again after 4 hours at 37 degrees. Labelled diabody was mixed with the individual Ab peptides with a final buffer condition of PBS and 0.05% Tween-20. Each replicate contained a 16 step 2 fold serial dilution series starting from 59.15 mM of aged Ab1 -42 or 1 18.3 mM of fresh Ab1-42. In addition, labelled diabody was mixed with the full-length extracellular domain of hCD33 with a final buffer condition of HBS (25mM HEPES pH 7.4; 5.4mM KCI; 140mM NaCI) and 0.05% Tween-20. Each replicate contained a 16 step 2 fold serial dilution series starting from 1 .0 pM of hCD33. The diabody protein concentration for all experiments was chosen such that the observed fluorescence was approximately 300 units at 50% LED power. The samples were loaded into standard capillaries and heated for 30 sec, followed by 5 sec cooling at 20% laser power. All experiments were performed with a minimum of 3 independent replicates. The affinity, KD, was quantified by analysing the change in normalized fluorescence (Fnorm = fluorescence after thermophoresis/initial fluorescence) as a function of the concentration of the titrated peptide. The fraction of diabody bound (AFnorm/amplitude) was plotted against the concentration of ligand and the curves were analysed using Graphpad Prism (Version 5, GraphPad, San Diego, CA, USA).
Lintuzumab (SGN-33, HuM195) binds the epitope FFHPIPYYDKNSPVHGYW with sub-nM affinity (0.2-0.5 nM), which constitutes the loop between B' and C strands in the core sheet structure of the N-terminal CD33 V-set domain. This loop has no sequence homology with murine CD33 accounting for the reported lack of cross reactivity of Lintuzumab to murine CD33 and eliminating background binding to murine CD33 on BV2 cells used in this work. Microscale thermophoresis (MST) measurements indicate low nM binding of the diabody to hCD33 (specifically 23 ± 10 nM), this affinity of the monovalent anti-CD33 diabody graft is lower than that reported for the intact bivalent anti-CD33 immunoglobulin as expected.
In order to test if the CD33 and Ab directed diabody could drive microglial uptake of Ab, murine microglia were either transfected with hCD33 or left untransfected and then incubated with pHrodo-Red labelled synthetic Ab peptide (pHrodo-Ab), and with diabody treatment or buffer matched controls. Figure 2 shows the results of experiments with flow Cytometry of BV2 cells transfected with human CD33 (left) and untransfected cells (right) incubated with pHrodo-labelled Ab peptide. Only CD33- positive cells show a significant increase in Ab uptake when treated with Oϋ33/Ab diabody. CD33-transfected cells show an extremely significant increase in Ab uptake when incubated with the diabody. Untransfected cells show a small but significant reduction in Ab uptake consistent with the diabody capturing Ab in solution (Ab can stimulate microglia). The antibody binds an epitope with no sequence homology between human and mouse CD33, so there is no cross reactivity with murine CD33. Importantly, the Oϋ33-Ab diabody did not significantly alter mRNA expression of neuroinflammatory markers McpJ TNF a, IL-6, IL-1B, NFkB and FLT-1 in differentiated human THP-1 microglia (Figure 3), suggesting that there is no activation of neuro- inflammatory processes that take place with diabody treatment. This is important because neuroinflammation might contribute significantly to neurodegenerative diseases.
Figure 4 shows results from cells that were transfected with CD33. Diabody labelled with pHrodo (in the absence of Ab) was added to free serum media for 30 min or 1 hour. After 1 hour of incubation a significant increase in internalization was observed. This demonstrates that the 0ϋ33/Ab diabody is internalised by CD33 positive cells.
In order to look at the effect of h CD33 expression on the phagocytic activity of microglial cells, BV2 cells were either transfected with hCD33 RNA or treated with buffer matched controls (no RNA) and incubated with CD33-AP diabody or buffer matched controls and incubated with pHrodo-labelled latex beads as the substrate. As shown in Figure 5, neither transfection with hCD33 nor diabody treatment resulted in significant change in the uptake of latex beads. One-way analysis of variance (ANOVA) yields P=0.16, n=3. These data indicate that the cell assay is not reporting CD33-mediated phagocytic activation of microglia, but rather CD33-mediated endocytosis of the diabody-amyloid complex.
The inventors tested the ability of CD33-pTau to drive its fluorescently labelled antigen into CD33 positive microglia. Figure 6A shows the results from an pTau endocytosis assay. BV2 cells were transfected or mock transfected and incubated with pTau pHrodo after the incubation with 10 ug CD33-pTau diabody or buffer (n=6). As shown in Figure 6A, dCD33-pTau was able to significantly (P=0.0006, n=6) drive uptake of its antigen containing the disease associated phosphorylated motif in Tau protein, and no difference was observed in diabody treatment of CD33 negative cells.
Likewise, as shown in Figure 6B CD33-aSyn diabody was able to significantly promote (P=0.0014, n=3 for both MFI and % internalisation) microglial clearance of recombinant a-synuclein and no difference was observed in the treatment of CD33 negative cells. Here, BV2 cells were transfected or mock transfected and incubated with a-syn pHrodo after the incubation with 10 ug CD33-a-syn diabody or buffer (n=6). The CD33/ a-syn diabody results in significant internalisation of a-syn.
The diabody lacks the Fc portion and does not enhance microglial uptake of latex beads by phagocytosis. These results demonstrate that phagocysis is not activated in the uptake of aggregated Ab, but rather the mechanism involves CD33-mediated endocytosis of the diabody-amyloid complex. The results demonstrate that the mechanism involves receptor(CD33)-mediated endocytosis of the diabody- amyloid/pTau/a-synuclein complexes.
This study was conducted to determine the affinity of the CD33:Ap diabody for the human CD33 IgV domain. Method
Microscale thermophoresis (MST)
Solution MST binding studies between the diabody with the human CD33 (hCD33) extracellular domain (Speed Biosystems), as well as Ab1-42 peptide were performed using standard protocols on a Monolith NT.1 15 (Nanotemper Technologies). Briefly, purified Oϋ33-Ab diabody was labelled using the RED-NHS (Amine Reactive) Protein Labelling Kit (Nanotemper Technologies). Lyophilized Ab1 -42 peptide was taken up in 60 mM NaOH and diluted in PBS and 0.05%v/v Tween-20; samples were measured soon after dissolution, and again after 4 hours at 37 degrees. Labelled diabody was mixed with the individual Ab peptides with a final buffer condition of PBS and 0.05% Tween-20. Each replicate contained a 16 step, 2-fold serial dilution of Ab starting from 59.15 mM of aged Ab1 -42 or 1 18.3 pM of fresh Ab1 -42. In addition, labelled diabody was mixed with the full-length extracellular domain of hCD33 with a final buffer condition of HBS (25mM HEPES pH 7.4; 5.4mM KCI; 140mM NaCI) and 0.05% Tween-20. Each replicate contained a 16 step, 2-fold serial dilution of hCD33 and diabody (Oϋ33-Ab) starting from 1 .0 pM. The diabody protein concentration for all experiments was chosen such that the observed fluorescence was approximately 300 units at 50% LED power. The samples were loaded into standard capillaries and heated for 30 sec, followed by 5 sec cooling at 20% laser power. All experiments were performed with a minimum of 3 independent replicates (Figure 7). The affinity, KD, was quantified by analysing the change in normalized fluorescence (Fnorm = fluorescence after thermophoresis/initial fluorescence) as a function of the concentration of the titrated peptide. The fraction of diabody bound (AFnorm/amplitude) was plotted against the concentration of ligand and the curves were analysed using Graphpad Prism (Version 5, GraphPad, San Diego, CA, USA).
The 0033-Ab diabody binds human CD33 with low nM affinity
Lintuzumab (SGN-33, HuM195) binds the epitope FFHPIPYYDKNSPVHGYW with sub-nM affinity (0.2-0.5 nM). This loop sequence is not conserved in murine CD33, eliminating background binding to murine CD33 on BV2 cells used in this work. As shown in Figure 7A-C., MST measurements indicate an affinity of 23 ± 10 nM of the diabody for the human CD33 IgV domain. This affinity of the monovalent anti-CD33 diabody graft is lower than that reported for the intact bivalent anti-CD33
immunoglobulin as expected.
The OW33-Ab diabody binds oligomeric synthetic Ab species and avoids monomeric Ab.
The inventors observed poor binding of the diabody to freshly dissolved Ab1 -42 (typically ~25 mM). Binding increased as the Ab1 -42 peptide was aged (~0.9 mM) for 4 hours. Longer aging led to artefacts associated with Ab sticking to capillaries used in the MST study. These observations suggest that the aducanumab-derived diabody has little or no affinity for monomeric synthetic Ab but shows affinity/specificity for oligomeric species. Because we cannot define precisely the oligomeric species of Ab recognised by the aducanumab derived diabody, we cannot know the effective concentration of the amyloid ligand, and therefore kinetic parameters are only a guide to trend and should not be considered as objective absolute values. These observations are consistent with reported properties of aducanumab.
Antibodies show extremely poor blood-brain-barrier penetration making therapies that target CNS diseases and pathologies difficult. Moreover, CD33 positive microglia are extremely sensitive to treatment with anti-CD33 antibodies and neuroinflammation and/or immune system activation is a common side effect of antibody therapy in vivo. The inventors have therefore synthesised bi-specific diabodies based on the variable regions of antibodies directed at Ab, pTau, a-synuclein coupled with variable domains from an anti-CD33 antibody targeting the epitope, but lacking the Fc portion of the full length antibody.
These diabodies specifically enhance clearance of Ab, p-Tau and a-synuclein respectively, by receptor-mediated (CD33) endocytosis by microglial cells, but do not elicit activation of CD33 or overactivation of microglial cells. In other words, the inventors have shown that these diabody-ligand complexes are internalised by receptor (CD33)-mediated endocytosis (CD33-transfected cells) and avoid excitation or activation of microglia with respect to proinflammatory cytokine release and
phagocytosis of other substrates. This is demonstrated in Figure 5 where off-target substrate phagocytosis is not promoted by diabody treatment, and in Figure 3 where no modulation of neuroinflammatory markers is observed. It was expected that the diabody (a large ligand) with high affinity for CD33 would most likely bind to, and activate CD33 signalling resulting in reduced phagocytosis. The genes of CD33 that are protective for Alzheimer's disease have reduced expression of full length CD33. That is, inhibitors of CD33 are sought to confer protection from
Alzheimer's disease. Here, the inventors surprisingly found that CD33 is not activated and phagocytosis is not supressed by CD33-A diabody treatment. This is important because this would have the exact opposite desirable effect of CD33 inhibitors and would likely exacerbate the disease by hindering the ability of microglia to clean the brain of debris. Diabody treatment enhances microglial consumption of pathological proteins without the negative consequences of off-target phagocytosis, supressed phagocytosis or promotion of neuroinflammation. This approach thus provides for a therapeutic advantage not previously identified.
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