DLL3 binding proteins and methods of use
Provided herein are DLL3 binding proteins and DLL3 targeting multispecific proteins (e.g., DLL3 targeting trispecific protein) comprising a domain binding to CD3, a half-life extension domain, and a domain binding to DLL3 (such as a DLL3 binding protein as provided herein). Also provided are pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such DLL3 binding proteins, DLL3 targeting trispecific proteins. Also disclosed are methods of using the disclosed DLL3 binding proteins, DLL3 targeting trispecific proteins in the prevention, and/or treatment diseases, conditions and disorders.
1. A DLL3 targeting trispecific protein, wherein said protein comprises
(a) a first domain (A) which is a single chain variable fragment that specifically binds to a human CD3;
(b) a second domain (B) which is a single domain antibody that specifically binds to a human serum albumin protein; and
(c) a third domain (C) which is a single domain antibody that specifically binds to a DLL3 protein, wherein the third domain (C) comprises a CDR1 that has the amino acid sequence of SEQ ID No. 874, a CDR2 that has the amino acid sequence of SEQ ID No. 1316, and a CDR3 that has the amino acid sequence of SEQ ID No. 1758.
2. The DLL3 targeting trispecific protein of claim 1 , wherein the domains are linked in the order H2N-(A)-(B)-(C)-COOH, H2N-(A)-(C)-(B)-COOH, H2N-(B)-(A)-(C)-COOH, H2N-(B)-(C)-(A)-COOH, H2N-(C)-(B)-(A)-COOH, or H2N-(C)-(A)-(B)-COOH, or by linkers L1 and L2, in the order H2N-(A)-L1-(B)-L2-(C)-COOH, H2N-(A)-L1-(C)-L2-(B)-COOH, H2N-(B)-L1-(A)-L2-(C)-COOH, H2N-(B)-L1-(C)-L2-(A)-COOH, H2N-(C)-L1-(B)-L2-(A)-COOH, or H2N-(C)-L1-(A)-L2-(B)-COOH.
3. The DLL3 targeting trispecific protein of claim 1 , wherein the domains are linked in the order H2N-(A)-(B)-(C)-COOH or by linkers L1 and L2 in the order of H2N-(A)-L1-(B)-L2-(C)-COOH.
4. The DLL3 targeting trispecific protein of claim 1 , wherein the domains are linked in the order H2N-(C)-(B)-(A)-COOH or by linkers L1 and L2 in the order of H2N-(C)-L1-(B)-L2-(A)-COOH.
5. The DLL3 targeting trispecific protein of claim 1 , wherein the third domain (C) is an affinity matured binding molecule obtained from a parental molecule that specifically binds to the DLL3 protein.
6. The DLL3 targeting trispecific protein of claim 5 , wherein the affinity matured DLL3 binding molecule has a binding affinity toward the DLL3 protein that is about 2-fold about 50-fold greater than the binding affinity of the parental molecule toward the DLL3 protein.
7. The DLL3 targeting trispecific protein of claim 2 , wherein the linkers L1 and L2 are each independently selected from the group consisting of (GS) n (SEQ ID No.1809), (GGS) n (SEQ ID No.1810), (GGGS) n (SEQ ID No.1811), (GGSG) n (SEQ ID No.1812), (GGSGG) n (SEQ ID No.1813), or (GGGGS) n (SEQ ID No.1814), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
8. The DLL3 targeting trispecific protein of claim 2 , wherein the linkers L1 and L2 independently comprises the sequence of GGGGSGGGS (SEQ ID No. 1808).
9. The DLL3 targeting trispecific protein of claim 1 , wherein the second domain (B) comprises an amino acid sequence selected from the group consisting of SEQ ID Nos. 1769-1778.
10. The DLL3 targeting trispecific protein of claim 1 , wherein the first domain (A) comprises an amino acid sequence selected from the group consisting of SEQ ID Nos. 1793-1802 and 1897-1898.
11. The DLL3 targeting trispecific protein of claim 1 , comprising the sequence of SEQ ID No. 1890 or SEQ ID No. 1891.
12. A DLL3 targeting trispecific protein, wherein said protein comprises
(a) a first domain (A) which is a single chain variable fragment that specifically binds to a human CD3;
(b) a second domain (B) which is a single domain antibody that specifically binds to a human serum albumin protein; and
(c) a third domain (C) which is a single domain antibody that specifically binds to DLL3, wherein the third domain (C) comprises a sequence selected from the group consisting of SEQ ID Nos. 408, 425, 432, 430, 431, and 1886.
13. The A DLL3 targeting trispecific protein, comprising the sequence of SEQ ID No. 1890 or SEQ ID No. 1891.
14. A DLL3 single domain antibody comprising a CDR1 that has the amino acid sequence of SEQ ID No. 874, a CDR2 that has the amino acid sequence of SEQ ID No. 1316, and a CDR3 that has the amino acid sequence of SEQ ID No. 1758.
15. The DLL3 single domain antibody of claim 14 , wherein the DLL3 single domain antibody comprises a sequence that is at least 80% identical to the amino acid sequence of SEQ ID No. 432.
16. A method of treating a cancer overexpressing DLL3 protein in a subject, comprising administering an effective amount of a DLL3 targeting trispecific protein of claim 1 , to the subject.