Multispecific binding molecules having specificity to dystroglycan and laminin-2
Provided herein multispecific (e.g., bispecific) binding molecules comprising a first binding domain that binds an extracellular portion of dystroglycan and a second binding domain that binds laminin-2. Further provided herein are methods for making such binding molecules and uses of such binding molecules for treating and/or preventing alpha-dystroglycanopathies.
1. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a bispecific binding molecule comprising a first binding domain that binds an extracellular portion of dystroglycan and a second binding domain that binds laminin-2, wherein the bispecific binding molecule is a bispecific binding protein comprising one or more polypeptide chains, and wherein the bispecific binding molecule comprises two light chains comprising a structure represented by the formula:
V L1 -L 5 -V L2 -L 6 -C L [III]
and two heavy chains comprising a structure represented by the formula:
V H1 -L 7 -V H2 -L 8 -C H1 -hinge-C H2 -C H3 [IV]
wherein:
V L1 is a first immunoglobulin light chain variable domain;
V L2 is a second immunoglobulin light chain variable domain;
V H1 is a first immunoglobulin heavy chain variable domain;
V H2 is a second immunoglobulin heavy chain variable domain;
C L is an immunoglobulin light chain constant domain;
C H1 is an immunoglobulin C H1 heavy chain constant domain;
C H2 is an immunoglobulin C H2 heavy chain constant domain;
C H3 is an immunoglobulin C H3 heavy chain constant domain;
hinge is an immunoglobulin hinge region connecting the C H1 and C H2 domains; and
L 5 , L 6 , L 7 , and L 8 are amino acid linkers;
wherein the V H1 and V L1 domains form a V H1 /V L1 binding pair, and wherein the V H2 and V L2 domains form a V H2 /V L2 binding pair, and wherein
(a) the V H1 domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:9, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:18; the V L1 domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:28, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:38, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:43; the V H2 domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:52, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:57, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:62; and the V L2 domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:67, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:72, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:77; or
(b) the V H2 domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:9, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:18; the V L2 domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:28, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:38, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:43; the V H1 domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:52, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:57, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:62; and the V L1 domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:67, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:72, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:77.
2. The nucleic acid molecule of claim 1 , wherein L 5 , L 6 , L 7 , and L 8 are each 0 to 50 amino acid residues in length.
3. The nucleic acid molecule of claim 1 , wherein L 5 , L 6 , L 7 , and L 8 are each 0 to 25 amino acid residues in length.
4. The nucleic acid molecule of claim 1 , wherein L 5 , L 6 , L 7 , and L 8 are each 0 to 14 amino acid residues in length.
5. The nucleic acid molecule of claim 1 , wherein the L 5 and L 7 linkers comprise the amino acid sequence of GGGGSGGGGS (SEQ ID NO:294), and wherein the L 6 and L 8 linkers are each 0 amino acid residues in length.
6. The nucleic acid molecule of claim 1 , wherein one or both of the variable domains of the polypeptides of formula III and formula IV are humanized or mouse variable domains.
7. The nucleic acid molecule of claim 1 , wherein the V H1 domain comprises the amino acid sequence of SEQ ID NO:170.
8. The nucleic acid molecule of claim 1 , wherein the V L1 domain comprises the amino acid sequence of SEQ ID NO:171.
9. The nucleic acid molecule of claim 1 , wherein the V H2 domain comprises the amino acid sequence of SEQ ID NO:192.
10. The nucleic acid molecule of claim 1 , wherein the V L2 domain comprises the amino acid sequence of SEQ ID NO:193.
11. The nucleic acid molecule of claim 1 , wherein the V H1 domain comprises the amino acid sequence of SEQ ID NO:170, the V L1 domain comprises the amino acid sequence of SEQ ID NO:171, the V H2 domain comprises the amino acid sequence of SEQ ID NO:192, and the V L2 domain comprises the amino acid sequence of SEQ ID NO:193.
12. The nucleic acid molecule of claim 1 , wherein the V H1 domain comprises the amino acid sequence of SEQ ID NO:192.
13. The nucleic acid molecule of claim 1 , wherein the V L1 domain comprises the amino acid sequence of SEQ ID NO:193.
14. The nucleic acid molecule of claim 1 , wherein the V H2 domain comprises the amino acid sequence of SEQ ID NO:170.
15. The nucleic acid molecule of claim 1 , wherein the V L2 domain comprises the amino acid sequence of SEQ ID NO:171.
16. The nucleic acid molecule of claim 1 , wherein the V H1 domain comprises the amino acid sequence of SEQ ID NO:192, the V L1 domain comprises the amino acid sequence of SEQ ID NO:193, the V H2 domain comprises the amino acid sequence of SEQ ID NO:170, and the V L2 domain comprises the amino acid sequence of SEQ ID NO:171.
17. An expression vector comprising the nucleic acid molecule of claim 1 .
18. An isolated host cell comprising the expression vector of claim 17 .
19. An isolated host cell comprising the nucleic acid molecule of claim 1 .
20. A method of producing a bispecific binding molecule, the method comprising:
a) culturing the host cell of claim 19 under conditions such that the host cell expresses the bispecific binding molecule; and
b) isolating the bispecific binding molecule from the host cell.
21. A vector system comprising one or more vectors encoding two light chains and two heavy chains of a bispecific binding molecule comprising a first binding domain that binds an extracellular portion of dystroglycan and a second binding domain that binds laminin-2, wherein the bispecific binding molecule is a bispecific binding protein comprising one or more polypeptide chains, and wherein the bispecific binding molecule comprises two light chains comprising a structure represented by the formula:
V L1 -L 5 -V L2 -L 6 -C L [III]
and two heavy chains comprising a structure represented by the formula:
V H1 -L 7 -V H2 -L 8 -C H1 -hinge-C H2 -C H3 [IV]
wherein:
V L1 is a first immunoglobulin light chain variable domain;
V L2 is a second immunoglobulin light chain variable domain;
V H1 is a first immunoglobulin heavy chain variable domain;
V H2 is a second immunoglobulin heavy chain variable domain;
C L is an immunoglobulin light chain constant domain;
C H1 is an immunoglobulin C H1 heavy chain constant domain;
C H2 is an immunoglobulin C H2 heavy chain constant domain;
C H3 is an immunoglobulin C H3 heavy chain constant domain;
hinge is an immunoglobulin hinge region connecting the C H1 and C H2 domains; and
L 5 , L 6 , L 7 , and L 8 are amino acid linkers;
wherein the V H1 and V L1 domains form a V H1 /V L1 binding pair, and wherein the V H2 and V L2 domains form a V H2 /V L2 binding pair, and wherein
(a) the V H1 domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:9, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:18; the V L1 domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:28, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:38, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:43; the V H2 domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:52, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:57, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:62; and the V L2 domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:67, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:72, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:77; or
(b) the V H2 domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:9, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:18; the V L2 domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:28, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:38, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:43; the V H1 domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:52, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:57, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:62; and the V L1 domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:67, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:72, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:77.
22. The vector system of claim 21 , wherein L 5 , L 6 , L 7 , and L 8 are each 0 to 50 amino acid residues in length.
23. The vector system of claim 21 , wherein L 5 , L 6 , L 7 , and L 8 are each 0 to 25 amino acid residues in length.
24. The vector system of claim 21 , wherein L 5 , L 6 , L 7 , and L 8 are each 0 to 14 amino acid residues in length.
25. The vector system of claim 21 , wherein the L 5 and L 7 linkers comprise the amino acid sequence of GGGGSGGGGS (SEQ ID NO:294), and wherein the L 6 and L 8 linkers are each 0 amino acid residues in length.
26. The vector system of claim 21 , wherein one or both of the variable domains of the polypeptides of formula III and formula IV are humanized or mouse variable domains.
27. The vector system of claim 21 , wherein the V H1 domain comprises the amino acid sequence of SEQ ID NO:170.
28. The vector system of claim 21 , wherein the V L1 domain comprises the amino acid sequence of SEQ ID NO:171.
29. The vector system of claim 21 , wherein the V H2 domain comprises the amino acid sequence of SEQ ID NO:192.
30. The vector system of claim 21 , wherein the V L2 domain comprises the amino acid sequence of SEQ ID NO:193.
31. The vector system of claim 21 , wherein the V H1 domain comprises the amino acid sequence of SEQ ID NO:170, the V L1 domain comprises the amino acid sequence of SEQ ID NO:171, the V H2 domain comprises the amino acid sequence of SEQ ID NO:192, and the V L2 domain comprises the amino acid sequence of SEQ ID NO:193.
32. The vector system of claim 21 , wherein the V H1 domain comprises the amino acid sequence of SEQ ID NO:192.
33. The vector system of claim 21 , wherein the V L1 domain comprises the amino acid sequence of SEQ ID NO:193.
34. The vector system of claim 21 , wherein the V H2 domain comprises the amino acid sequence of SEQ ID NO:170.
35. The vector system of claim 21 , wherein the V L2 domain comprises the amino acid sequence of SEQ ID NO:171.
36. The vector system of claim 21 , wherein the V H1 domain comprises the amino acid sequence of SEQ ID NO:192, the V L1 domain comprises the amino acid sequence of SEQ ID NO:193, the V H2 domain comprises the amino acid sequence of SEQ ID NO:170, and the V L2 domain comprises the amino acid sequence of SEQ ID NO:171.
37. An isolated host cell comprising the vector system of claim 21 .
38. A method of producing a bispecific binding molecule, the method comprising:
a) culturing the host cell of claim 37 under conditions such that the host cell expresses the bispecific binding molecule; and
b) isolating the bispecific binding molecule from the host cell.