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US-12624072-B2 - Engineering broadly reactive human notch ligands as novel tools for biomedical applications

US12624072B2US 12624072 B2US12624072 B2US 12624072B2US-12624072-B2

Abstract

Disclosed are compositions and methods for engineered DLL4 proteins. In one aspect, disclosed herein are engineered DLL4 proteins comprising a conservative amino acid substitution at a residue corresponding to residues 28, 107, 143, 194, and 206 as set forth in SEQ ID NO: 1 and further comprising at least one conservative amino acid substitution at residues 256, 257, 271, 280, 301, and 305 as set forth in SEQ ID NO: 1.

Inventors

  • Vincent Luca
  • David Gonzalez Perez

Assignees

  • H. LEE MOFFITT CANCER CENTER AND RESEARCH INSTITUTE, INC.

Dates

Publication Date
20260512
Application Date
20200501

Claims (20)

  1. 1 . An engineered DLL4 protein comprising a conservative amino acid substitution at a residue corresponding to residues 28, 107, 143, 194, and 206 as set forth in SEQ ID NO: 1 and further comprising at least one conservative amino acid substitution at residues 256, 257, 271, 280, 301, and 305 as set forth in SEQ ID NO: 1.
  2. 2 . The engineered DLL4 protein of claim 1 , wherein the substitution at residue 28 comprises a glycine to serine substitution (G28S).
  3. 3 . The engineered DLL4 protein of claim 1 , wherein the substitution at residue 107 comprises a phenylalanine to leucine substitution (F107L).
  4. 4 . The engineered DLL4 protein of claim 1 , wherein the substitution at residue 143 comprises an isoleucine to phenylalanine substitution (1143F).
  5. 5 . The engineered DLL4 protein of claim 1 , wherein the substitution at residue 194 comprises a histidine to tyrosine substitution (H194Y).
  6. 6 . The engineered DLL4 protein of claim 1 , wherein the substitution at residue 206 comprises a leucine to proline substitution (L206P).
  7. 7 . The engineered DLL4 protein of claim 1 , wherein the amino acid at residue 256 comprises a histidine, tyrosine, phenylalanine, leucine, asparagine, isoleucine, valine, or aspartic acid (H256, H256Y, H256F, H256L, H256N, H256I, H256V, or H256D).
  8. 8 . The engineered DLL4 protein of claim 1 , wherein the substitution at residue 256 comprises a histidine to tyrosine substitution (H256Y).
  9. 9 . The engineered DLL4 protein of claim 1 , wherein the amino acid at residue 257 comprises a proline, histidine, leucine, isoleucine, threonine, asparagine, tyrosine, serine, or phenylalanine (N257, N257P, N257H, N257L, N257I, N257T, N257Y, N257S, or N257F).
  10. 10 . The engineered DLL4 protein of claim 1 , wherein the substitution at residue 257 comprises an asparagine to proline substitution (N257P).
  11. 11 . The engineered DLL4 protein of claim 1 , wherein the amino acid at residue 271 comprises a leucine, proline, histidine, asparagine, threonine, or isoleucine (T271, T271L, T271P, T271H, T271N, or T271I).
  12. 12 . The engineered DLL4 protein of claim 1 , wherein the substitution at residue 271 comprises a threonine to leucine substitution (T271L).
  13. 13 . The engineered DLL4 protein of claim 1 , wherein the amino acid at residue 280 comprises a phenylalanine to leucine, tyrosine, or histidine substitution (F280Y, F280L, or F280H).
  14. 14 . The engineered DLL4 protein of claim 1 , wherein the amino acid at residue 301 comprises a serine, asparagine, arginine, or histidine (S301, S301H, S301N, or S301R).
  15. 15 . The engineered DLL4 protein of claim 1 , wherein the substitution at residue 301 comprises a serine to histidine substitution (S301H) or a serine to arginine substitution (S301R).
  16. 16 . The engineered DLL4 protein of claim 1 , wherein the amino acid at residue 305 comprises a glutamine to proline, arginine, or leucine substitution (Q305P, Q305R, or Q305L).
  17. 17 . The engineered DLL4 protein of claim 1 , wherein DLL4 protein comprises SEQ ID NO: 3 or SEQ ID NO: 4.
  18. 18 . An antibody that specifically binds the engineered DLL4 proteins of claim 1 .
  19. 19 . A method of treating a Notch 1-3 related cancer in a subject comprising administering to the subject the engineered DLL4 proteins of claim 1 .
  20. 20 . A method of treating a Notch 1-3 related cancer in a subject comprising administering to the subject the antibody of claim 18 .

Description

This application is a United States National Phase Patent Application of International Patent Application Number PCT/US2020/030977, filed on May 1, 2020, which claims the benefit of U.S. Provisional Application No. 62/841,460, filed on May 1, 2019, applications which are incorporated herein by reference in their entireties. This invention was made with government support under Grant No. CA204738 awarded by the National Institutes of Health. The government has certain right in the invention. I. BACKGROUND The Notch pathway plays a central role in cellular homeostasis, embryonic development and stem cell renewal. Despite Notch receptors and ligands being major emerging targets for cancer therapeutics, the intrinsically low binding affinity between Delta-like (DLL) or Jagged (Jag) ligands and Notch receptors limits the utility of these molecules in biomedical applications. What are needed are new therapeutics and ligands that have enhanced affinity for multiple human Notch receptors. II. SUMMARY Disclosed are methods and compositions related to engineered DLL4 proteins. In one aspect, disclosed herein are engineered DLL4 proteins comprising a conservative amino acid substitution at a residue corresponding to residues 28, 107, 143, 194, and 206 as set forth in SEQ ID NO: 1 and further comprising at least one conservative amino acid substitution at residues 256, 257, 271, 280, 301, and 305 as set forth in SEQ ID NO: 1. Also disclosed herein are engineered DLL4 protein of any preceding aspect, wherein the substitution at residue 28 comprises a glysine to serine substitution (G28S). In one aspect, disclosed herein are engineered DLL4 protein of any preceding aspect, wherein the substitution at residue 107 comprises a phenylalanine to leucine substitution (F107L). Also disclosed herein are engineered DLL4 protein of any preceding aspect, wherein the substitution at residue 143 comprises a isoleucine to phenylalanine substitution (I143F). In one aspect, disclosed herein are engineered DLL4 protein of any preceding aspect, wherein the substitution at residue 194 comprises a histidine to tyrosine substitution (H194Y). Also disclosed herein are engineered DLL4 protein of any preceding aspect, wherein wherein the substitution at residue 206 comprises a leucine to proline substitution (L206P). In one aspect, disclosed herein are engineered DLL4 proteins of any preceding aspect, wherein the amino acid at residue 256 comprises a histidine, tyrosine, phenylalanine, leucine, asparagine, isoleucine, valine, or aspartic acid (such as, for example, a H256Y substitution, H256F substitution, H256L substitution. H256N substitution, H256I substitution, H256V substitution, or H256D substitution). Also disclosed herein are engineered DLL4 proteins of any preceding aspect, wherein the amino acid at residue 257 comprises a proline, histidine, leucine, isoleucine, threonine, asparagine, tyrosine, serine, or phenylalanine (such as, for example, a N257P substitution, N257H substitution, N257L substitution, N257I substitution, N257T substitution, N257Y substitution, N257S substitution, or N257F substitution). In one aspect, disclosed herein are engineered DLL4 proteins of any preceding aspect, wherein the amino acid at residue 271 comprises a leucine, proline, histidine, asparagine, threonine, or isoleucine (such as, for example, a T271L substitution, T271P substitution, T271H substitution, T271N substitution, or T271I substitution). Also disclosed herein are engineered DLL4 proteins of any preceding aspect, wherein the amino acid at residue 280 comprises a phenylalanine, leucine, tyrosine, or histidine (F280Y substitution, F280L substitution, or F280H substitution). In one aspect, disclosed herein are engineered DLL4 proteins of any preceding aspect, wherein the amino acid at residue 301 comprises a serine, asparagine, arginine, or histidine (S301H substitution, S301N substitution, or S301R substitution). Also disclosed herein are engineered DLL4 proteins of any preceding aspect, wherein the amino acid at residue 305 comprises a glutamine, proline, arginine, or leucine (Q305P substitution, Q305R substitution, or Q305L substitution). In one aspect, disclosed herein are engineered DLL4 proteins of any preceding aspect, wherein DLL4 protein comprises SEQ ID NO: 3 or SEQ ID NO: 4. Also disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating, and/or preventing a cancer and/or metastasis comprising in a subject administering to the subject any of the engineered DLL4 proteins of any preceding aspect. In one aspect, disclosed herein are antibodies that bind to any of the engineered DLL4 proteins of any preceding aspect. III. BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description illustrate the disclosed compositions and methods. FIG. 1 shows the canonical NOTCH signal mechan