METHODS AND COMPOSITIONS FOR INDUCING NOTCH SIGNALING IN TUMOR MICROENVIRONMENTS
The disclosure provides methods for inducing Notch signaling in a targeted manner within aggregations of cells. The methods include contacting the aggregation of cells with a bi-specific molecule that facilitates trans-binding of Notch receptor. The bi-specific molecule comprising a cell-targeting domain that specifically binds to a cell-specific antigen expressed in the aggregation of cells, and a Notch-binding domain that specifically binds to Notch receptor. In some aspects, the disclosed methods and reagents provide methods of promoting pro-inflammatory states in tumor micro-environments.
1 . A method of inducing Notch signaling in an aggregation of cells comprising a first cell-type that expresses a cell-specific antigen and a second cell-type that expresses Notch, comprising:
contacting the aggregation of cells with a bi-specific molecule comprising a cell-targeting domain that specifically binds to the cell-specific antigen, and a Notch-binding domain that specifically binds to Notch,
wherein binding of the bi-specific molecule to the cell-specific antigen on a first cell of the first cell-type and trans-binding to Notch on a second cell of the second cell-type causes Notch signaling in the second cell.
2 . The method of claim 1 , wherein the first cell-type that expresses the cell-specific antigen and the second cell-type that expresses Notch are the same cell-type.
3 . The method of claim 1 , wherein the first cell-type that expresses the cell-specific antigen and the second cell-type that expresses Notch are different cell-types.
4 . The method of claim 1 , wherein the aggregation of cells is in a tumor microenvironment.
5 . The method of claim 4 , wherein the first cell-type comprises tumor cells and the second cell-type comprises non-tumor cells in the tumor microenvironment, wherein binding of the bi-specific molecule to the cell-specific antigen on a tumor cell and trans-binding to Notch on a non-tumor cell causes Notch signaling in the non-tumor cell.
6 . The method of claim 5 , wherein the non-tumor cells comprise, stromal cells, endothelial cells, and immune cells, alone or in any combination.
7 . The method of claim 6 , wherein the first cell-type comprises tumor cells and the second cell-type comprises immune cells, wherein binding of the bi-specific molecule to the cell-specific antigen on a tumor cell and trans-binding to Notch on an immune cell causes Notch signaling in the immune cell.
8 . The method of claim 7 , wherein Notch signaling in the immune cell promotes an immune-responsive state in the tumor microenvironment.
9 . The method of claim 8 , wherein the immune cell is a monocyte and trans-binding of the bi-specific molecule to Notch on the monocyte promotes differentiation of the monocyte into a dendritic cell.
10 . The method of claim 8 , wherein trans-binding of the bi-specific molecule to Notch on the immune cell promotes differentiation to M1 macrophages.
11 . The method of claim 8 , wherein trans-binding of the bi-specific molecule to Notch on the immune cell promotes conversion of immunosuppressive myeloid cells from an anti-inflammatory state to a pro-inflammatory state.
12 . The method of claim 8 , wherein trans-binding of the bi-specific molecule to Notch on the immune cell promotes an anti-tumor phenotype in a CD4 + T cell, in a CD8 + T cell, and/or in a NK cell.
13 . A method of promoting a pro-inflammatory state in a tumor microenvironment comprising a tumor cell and a non-tumor cell, the method comprising:
administering to the tumor microenvironment a bi-specific molecule that comprises a cell-targeting domain that specifically binds to a cell-specific antigen expressed by the tumor cell and a Notch-binding domain that trans-binds to Notch expressed by a non-tumor cell in the tumor micro-environment, thereby inducing Notch signaling in the non-tumor cell.
14 . The method of claim 13 , wherein the non-tumor cell is a stromal cell, an endothelial cell, or an immune cell.
15 . The method of claim 14 , wherein the immune cell is a monocyte and trans-binding of the bi-specific molecule to Notch on the monocyte promotes differentiation of the monocyte into a dendritic cell.
16 . The method of claim 14 , wherein trans-binding of the bi-specific molecule to Notch on the immune cell promotes differentiation to an M1 macrophage.
17 . The method of claim 14 , wherein binding of the bi-specific molecule to Notch on the immune cell promotes conversion of immunosuppressive myeloid cells from an anti-inflammatory state to a pro-inflammatory state.
18 . The method of one of claims 1 - 17 , wherein the Notch-binding domain comprises a Notch-binding domain of a mammalian Notch receptor ligand.
19 . The method of claim 18 , wherein the mammalian Notch receptor ligand is a ligand to a mammalian Notch1, Notch2, Notch3, or Notch4 receptor.
20 . The method of claim 18 , wherein the Notch receptor ligand is a Delta protein or Jagged protein, or a derivative thereof.
21 . The method of claim 20 , wherein the Delta protein is Delta Like Ligand 1 (DLL1).
22 . The method of claim 20 , wherein the Delta protein is DLL3.
23 . The method of claim 20 , wherein the Delta protein is DLL4.
24 . The method of claim 20 , wherein the Jagged protein is Jagged 1.
25 . The method of claim 20 , wherein the Jagged protein is Jagged 2.
26 . The method of claim 18 , wherein the Notch receptor ligand is Dlk1, Dlk2, DNER, EGFL 7, and F3/contactin.
27 . The method of claim 18 , wherein the Notch-binding domain comprises an extracellular domain of a Delta protein or a Jagged protein, or a derivative thereof.
28 . The method of claim 27 , wherein the extracellular domain contains one or more mutations from wild-type resulting in enhanced affinity or specificity of the extracellular domain to the Notch receptor as compared to the wild-type extracellular domain.
29 . The method of one of claim 18 or 27 , wherein the Delta protein is a human Delta protein and/or wherein the Jagged protein is a human Jagged protein, or a derivative thereof.
30 . The method of one of claim 18 or 27 , wherein the Delta protein is a rat Delta protein and/or wherein the Jagged protein is a rat Jagged protein, or a derivative thereof.
31 . The method of one of claims 1 - 17 , wherein the Notch-binding domain comprises an antibody, an antibody-like molecule, a DARPin, an aptamer, other engineered binding modules or scaffolds, and the like, or a functional domain thereof, that binds to Notch with an affinity (K d ) of about 100 nM to less than 1 nM.
32 . The method of one of claims 1 - 17 , wherein the cell-targeting domain specifically binds to cell-specific antigen with an affinity (K d ) greater than about 100 nM.
33 . The method of one of claims 1 - 17 , wherein the cell-targeting domain comprises an antibody, an antibody-like molecule, a receptor, a DARPin, an aptamer, other engineered binding modules or scaffolds, and the like, or a functional antigen-binding domain thereof, that specifically binds to the antigen characteristic of the cell-type of interest.
34 . The method of claim 31 or claim 33 , wherein the antibody-like molecule is an antibody fragment and/or antibody derivative.
35 . The method of claim 31 or claim 33 , wherein the antibody-like molecule is a single-chain antibody, a bispecific antibody, an Fab fragment, an F(ab) 2 fragment, a V H H fragment, a V NAR fragment, or a nanobody.
36 . The method of claim 35 , wherein the single-chain antibody is a single chain variable fragment (scFv), or a single-chain Fab fragment (scFab).
37 . The method of one of claims 1 - 17 , wherein the antigen is a cell surface marker for a tumor cell.
38 . The method of one of claim 5 , 12 - 17 , or 37 , wherein the cancer cell or cancer progenitor cell is selected from T (leukemic) cell, breast cancer cell, prostate cell, lung cancer cell, glioblastoma, colorectal cancer cell, cervical cancer cell, melanoma cancer cell, pancreatic cancer cell, esophageal cancer cell, and the like, or a progenitor of any of the foregoing.
39 . The method of claim 37 , wherein the cell surface marker is CD33.
40 . The method of claim 37 , wherein the cell surface marker is mesothelin.
41 . The method of one of claims 1 - 17 , wherein the cell-targeting domain and the Notch-binding domain are joined by an intervening flexible linker domain.
42 . The method of one of claims 1 - 17 , wherein the molecule is a fusion polypeptide wherein the cell-targeting domain and the Notch-binding domain are polypeptides that do not naturally occur together.