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US-12617825-B2 - Methods and compositions for reducing the immunogenicity of chimeric notch receptors

US12617825B2US 12617825 B2US12617825 B2US 12617825B2US-12617825-B2

Abstract

The present invention relates to methods and compositions for reducing the immunogenicity of chimeric Notch receptors, and specifically to transcription factors useful for controlling gene expression delivered to tissues by such chimeric Notch receptors.

Inventors

  • Peter Emtage
  • Amy E. Gilbert
  • Anselm Levskaya
  • Spencer Scott
  • Vladimir Slepushkin

Assignees

  • CELL DESIGN LABS, INC.

Dates

Publication Date
20260505
Application Date
20220408

Claims (20)

  1. 1 . A method of treating a CD19-expressing cancer in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of a T cell expressing a chimeric Notch polypeptide comprising from N-terminal to C-terminal and in covalent linkage: a) an extracellular domain comprising a binding agent that specifically binds to human CD19; b) a human Notch 2 or human Notch 3 core domain comprising one or more proteolytic cleavage sites; and c) an intracellular domain comprising a transcriptional regulator comprising a DNA binding domain of human origin and a transactivation domain of human origin; and wherein said T cell also comprises a nucleic acid construct comprising a cognate DNA binding sequence for the DNA binding domain of human origin, a promoter sequence, and a nucleic acid sequence encoding either a cytokine that locally induces and recruits immune cells to the CD19-expressing cancer or a chimeric antigen receptor (CAR) that targets an antigen other than CD19 on cancer cells in the human subject.
  2. 2 . The method of claim 1 , wherein the DNA binding domain is from human Hepatocyte Nuclear Factor 1 α (HNF1α) or human Early Growth Response 1 α (EGR1 α).
  3. 3 . The method of claim 2 , wherein the DNA binding domain is from human HNF1α.
  4. 4 . The method of claim 3 , wherein the human HNF1α DNA-binding domain of the chimeric Notch polypeptide comprises the DNA-binding domain of any one of the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7.
  5. 5 . The method of claim 3 , wherein the human HNF1α DNA-binding domain of the chimeric Notch polypeptide consists of the amino acid sequence of amino acids 1-283 of SEQ ID NO: 5.
  6. 6 . The method of claim 1 , wherein the polypeptide comprises a human Notch 3 core domain.
  7. 7 . The method of claim 6 , wherein the human Notch 3 core domain of the chimeric Notch polypeptide comprises the amino acid sequence of amino acids 1374-1734 of SEQ ID NO: 27.
  8. 8 . The method of claim 1 , wherein the transactivation domain is from human RelA (p65), human WWTR1 (TAZ), or human CREB3 (LZIP).
  9. 9 . The method of claim 1 , wherein the transactivation domain is from human RelA (p65).
  10. 10 . The method of claim 9 , wherein the transactivation domain of the chimeric Notch polypeptides comprises the transactivation domain from any one of SEQ ID NOs: 12-17.
  11. 11 . The method of claim 1 , wherein the binding agent is an scFv, a VHH, a bispecific antibody, or a BiTE.
  12. 12 . The method of claim 1 , wherein the intracellular domain further comprises a Nuclear Localization Sequence (NLS) upstream of the transcriptional regulator.
  13. 13 . The method of claim 12 , wherein the NLS is a native human Notch 3 NLS.
  14. 14 . The method of claim 1 , wherein the chimeric Notch polypeptide further comprises one or more linkers.
  15. 15 . The method of claim 14 , wherein the one or more linkers comprise the amino acid sequence of one or more of SEQ ID NO: 3 and SEQ ID NO: 4.
  16. 16 . The method of claim 1 , wherein the human Notch 2 or human Notch 3 core domain comprises three Lin-12 repeat regions (LNRs) and a transmembrane domain.
  17. 17 . The method of claim 1 , wherein the chimeric Notch polypeptide comprises from N-terminal to C-terminal and in covalent linkage: a) an extracellular domain comprising a scFv that specifically binds to human CD19; b) a human Notch 3 core domain comprising one or more proteolytic cleavage sites; c) a first glycine serine linker; and d) an intracellular domain comprising a transcriptional regulator comprising a DNA binding domain of human HNF1α, a second glycine serine linker, and a human RelA (p65) transactivation domain.
  18. 18 . The method of claim 17 , wherein: the human Notch 3 core domain of the chimeric Notch polypeptide comprises the amino acid sequence of amino acids 1374-1734 or 1374-1738 of SEQ ID NO: 27; the first glycine serine linker comprises the sequence set forth in SEQ ID NO:3; the DNA binding domain of human HNF1α comprises the DNA-binding domain of any one of the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7; the second glycine serine linker comprises the sequence set forth in SEQ ID NO:4; and wherein the human RelA transactivation domain is a transactivation domain from any one of the sequences of SEQ ID NOs: 12-17.
  19. 19 . The method of claim 17 , wherein the chimeric Notch polypeptide comprises a human CD8 alpha signal peptide immediately upstream of the scFv that specifically binds to human CD19.
  20. 20 . A method of treating a CD19-expressing cancer in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of a T cell expressing a chimeric Notch polypeptide comprising from N-terminal to C-terminal and in covalent linkage: a) an extracellular domain comprising an scFv that specifically binds to human CD19; b) a human Notch 3 core domain comprising one or more proteolytic cleavage sites; and c) an intracellular domain comprising a transcriptional regulator comprising a human HNF1α DNA binding domain and a human RelA (p65) transactivation domain; and wherein said T cell also comprises a nucleic acid construct comprising a cognate DNA binding sequence for the HNF1a DNA binding domain, a promoter sequence, and a nucleic acid sequence encoding either a cytokine that locally induces and recruits immune cells to the CD19-expressing cancer or a chimeric antigen receptor (CAR) that targets an antigen other than CD19 on cancer cells in the human subject.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application Ser. No. 62/603,993, filed Jun. 19, 2017, and U.S. Provisional Patent Application Ser. No. 62/556,765, filed Sep. 11, 2017, both of which are hereby incorporated by reference in their entirety. STATEMENT REGARDING SEQUENCE LISTING The Sequence Listing associated with this application is provided in text format in lieu of paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is “356829_ST25.txt.” The text file is 218,000 bytes, was created on Jul. 25, 2022, and is being submitted electronically via EFS-Web. TECHNICAL FIELD The present invention relates to molecular biology, and particularly to methods and compositions for reducing the immunogenicity of certain receptors useful for controlling selective gene expression in cells of the monocyte/macrophage lineage, and applications thereof. BACKGROUND An important problem which limits the development of gene therapy in humans is the regulation of therapeutic gene expression, such that gene expression or the vehicle used to realize expression, does not give rise to enhanced immunogenicity resulting in host rejection. One way to realize gene expression is described in U.S. Pat. No. 9,670,281, and Roybal et al., Cell, Feb. 11, 2016. There is described activation of gene expression using chimeric Notch receptors. Notch receptors are single pass transmembrane proteins that mediate cell-cell contact signaling and play a central role in development and other aspects of cell-to-cell communication between two contacting cells, in which one contacting cell has the Notch receptor, and the other contacting cell is a cell that exhibits a ligand on its surface which binds to the corresponding Notch receptor. The engagement of native Notch and Delta, it's native ligand, leads to two-step proteolysis of the Notch receptor that ultimately causes the release of the intracellular portion of the receptor from the membrane into the cytoplasm, where it moves to the nucleus. There the released domain alters cell behavior by functioning as a transcriptional regulator. Notch receptors are involved in and are required for a variety of cellular functions during development and are critical for the function of numerous cell-types across species. Described in U.S. Pat. No. 9,670,281 are chimeric Notch receptors which show that the Notch expressing cell can have one or more different binding moieties on the cell surface, for example, scFVs, nanobodies, single chain T-cell receptors, to name a few, that recognize a ligand associated with a cell ultimately causing the release of the intracellular, transcriptional regulatory portion of the receptor from the membrane into the cytoplasm resulting in transcriptional regulation. Engineered cells bearing chimeric Notch receptors that encounter their specific target antigen will then be cleaved such that their cytosolic fragment is free to translocate into the cell nucleus to regulate the transcription of any open reading frame (ORF) under the control of a synthetic promoter. The ORF expressed could be a cytokine to locally induce and recruit immune activity to the location of target antigen detection. Further, the ORF expressed could be a chimeric antigen T-cell receptor (CAR-T) that targets a separate, distinct target antigen for target cell killing, only after the priming target antigen detected by the chimeric Notch receptor has been detected. This enables highly-specific combinatorial antigen pattern recognition to allow greater discrimination between diseased or cancerous cells and healthy cells. This could greatly enable the application of engineered CAR-T cells to safely target a wider range of tumors with less side-effects on healthy tissue. To date, the transcriptional machinery used in chimeric Notch constructs has been GAL4-VP16. Since the DNA-binding fragment, GAL4, is of yeast origin, and VP16, a highly acidic portion of the herpes simplex virus protein, GAL4-VP16 is highly immunogenic, and thus limits the use of chimeric Notch receptors for treating human disease. Another major obstacle in the efficacy of many immunotherapy-based approaches for solid tumors, including cell therapy, is delivery of drugs or activation of immune cells in the solid tumor. Cells of the monocyte/macrophage lineage make up a major component of immune cells that infiltrate into solid tumors (Long et al., Oncoimmunology 2:e26860, 2013 doi:10.4161/onci26860). Because these cell types are actively recruited and retained in the solid tumor they could be an important cell type for the delivery of gene therapy. The genetic engineering of macrophages with clinically approved vectors such has HIV-1-based lentivirus has been difficult due to the inhibition of HIV-1 infection in macrophages. Hrecka et al. (“Vpx relieves the inhibition of HIV-1 infection of macrophages mediated by