Synthetic notch agonist constructs and methods of use

Abstract

Disclosed herein are methods and compositions for synthetic Notch agonist constructs that activate Notch signaling. The constructs provide targeted cell-cell communication for therapeutic and diagnostic applications.

Description

[0001] Attorney Docket Number 10110-473WO1 SYNTHETIC NOTCH AGONIST CONSTRUCTS AND METHODS OF USE CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 701,952, filed October 1, 2024, which is expressly incorporated herein by reference in its entirety. REFERENCE TO SEQUENCE LISTING The sequence listing submitted on October 1, 2025, as an .XML entitled “10110- 473WO1_ST26.xml” created on October 1, 2025, and having a file size of 17,286 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5). STATEMENT OF GOVERNMENT SUPPORT This invention was made with government support under Grant No. CA241169 awarded by NIH. The government has certain rights in the invention. BACKGROUND 1. The Notch pathway is a conserved signaling system that regulates cell fate decisions, tissue homeostasis, and immune cell development. Notch receptors are massive (~290kD) transmembrane proteins that are activated by a distinctive, mechanical force-driven mechanism. Notch signaling is initiated when a Delta-like (DLL) or Jagged (JAG) ligand forms a trans interaction with a Notch receptor on the surface of an adjacent cell. Endocytosis of the ligand then generates a “pulling” force that propagates to the negative regulatory region (NRR) of Notch. This pulling destabilizes the NRR, which exposes internal cleavage sites for processing by the intramembrane proteases ADAM10 (S2 cleavage) and ^-secretase (S3 cleavage). Following these proteolytic events, the Notch intracellular domain (NICD) translocates to the nucleus to function as a transcriptional co-activator. 2. Dysfunctional Notch signaling causes numerous inherited and acquired diseases. Loss- of-function mutations in Notch receptors and ligands are linked to the development of aortic valve disease (Notch1), Alagille syndrome (Notch2, Jagged1), CADASIL (Notch3), spondylocostal dysostosis (DLL3), and other congenital disorders. In cancer, Notch functions as a tumor suppressor depending on the cell type, and both loss-of-function and hyperactivating mutations influence tumorigenesis and disease progression. Notch activation may stimulate either proliferation or differentiation in different stem cell populations. Attorney Docket Number 10110-473WO1 3. Although Notch inhibitors are widely available, the requirement for mechanical force in Notch activation has precluded the development of soluble agonists. Specifically, these agents are challenging to engineer because they must somehow “pull” on the Notch receptor despite lacking a method of force generation. Several strategies have been developed to activate Notch receptors in vitro through mimicry of the physiological activation process. Notch signaling may be induced through co-culture of Notch-expressing cells and ligand-expressing cells, by culturing Notch-expressing cells on plates coated with ligands or antibodies, or by administration of ligand- coated microbeads. Unfortunately, this NRR unfolding approach has been ineffective for receptor subtypes with stable NRRs (e.g., Notch1 / 2 / 4), and the lack of soluble agonists remains a significant void in our biochemical toolkit for manipulating the Notch pathway. 4. The role of Notch agonists in Notch activation for the treatment of Notch related diseases or development syndromes will lead to the expansion of several Notch-based strategies for enhancing or re-activating Notch signaling in patients lacking the regulation of Notch pathway and subsequent lack of cell fate decisions, tissue homeostasis, and immune cell development. In addition, Notch signaling is important for several natural stages of T cell maturation, and ex vivo Notch activation is required for the differentiation of T cells from hematopoietic stem cells (HSCs). Therefore, there is a potential to use Notch agonists to enhance / upregulate or reactivate Notch activation. SUMMARY 5. Disclosed are methods and compositions related to multimeric costimulatory agonists. 6. In one aspect, disclosed herein are multimeric costimulatory synthetic Notch agonist (SNAG) construct (including, but not limited to bispecific protein agonist) comprising a JAG1 agonist construct (such as, for example, JAG1 or JAG2 monomers, a JAG1 or JAG2 antibody or fragment thereof.) and an affinity matured Notch ligand (including, but not limited to DeltaMAXmonomers, tetramers of biotinylated DeltaMAXwith streptavidin, monomer of DeltaMAXjoined to glycine-serine linker, or dimers of DeltaMAXjoined to dimeric human IgG1 Fc domain); wherein said affinity matured Notch ligand is connected to said JAG1 agonist construct. In one aspect, the affinity matured Notch ligand monomer (such as, for example, a DeltaMAXmonomer) can comprise SEQ ID NO: 1. In one aspect, the JAG1 monomer can comprise SEQ ID NO: 3. In some aspects, the JAG1 and / or the affinity matured Notch ligand can be a soluble protein such as, for example soluble JAG1 or soluble SNAG. 7. Also disclosed herein are multimeric costimulatory synthetic Notch agonist (SNAG) constructs of any preceding aspect; wherein said Affinity matured Notch ligand is connected to Attorney Docket Number 10110-473WO1 said JAG1 construct by a multimerization motif (such as, for example an immunoglobulin Fc domain (such as for example, SEQ ID NO: 2), C-terminal domain of T4 fibritin (FOLDON) motif, a streptavidin-biotin complex, or a leucine zipper (including, but not limited to a trimeric leucine zipper). In some aspects, the Affinity matured Notch ligand is fused at the N- terminus of the human IgG1 Fc domain via a first linker (such as, for example, a glycine-serine linker) and JAG1 construct is fused at the C-terminus of a human IgG1 Fc domain via a second linker (such as, for example, a glycine-serine linker). In one aspect, the multimeric costimulatory synthetic Notch agonist (SNAG) construct is a dimer comprising two Affinity matured Notch ligands fused at N- terminus of the human IgG1 Fc domain and two JAG1 constructs fused at C-terminus of a human IgG1 Fc domain. 8. In some aspects, disclosed herein are multimeric costimulatory synthetic Notch agonist (SNAG) constructs of any preceding aspect, wherein the JAG1 construct comprises JAG1 scFv, JAG1 diabody, JAG1 nanobody, or a JAG1 antibody fragment thereof. In one aspect, the JAG1 construct comprises JAG1 scFv, wherein the JAG1 scFv comprises a H286Q substitution. 9. In one aspect, disclosed herein are vectors encoding the multimeric costimulatory agonist of any preceding aspects and cells encoding said vector or the multimeric costimulatory agonist of any preceding aspect. In some aspects, the cells can comprise a cell lacking Notch signaling, a Notch receptor or ligand expressing cell, a tumor infiltrating lymphocyte, ^^ T cells, ^^ T cells, feeder cell, B cell, natural killer cell, chimeric antigen receptor (CAR) T cell, CAR NK cell CAR macrophage (CARMA), or dendritic cell. 10. Also disclosed herein, in one aspect is a method of activating Notch expression in a cell in vitro or ex vivo comprising obtaining cells lacking Notch signaling and culturing the cells lacking Notch signaling in media comprising one or more of the multimeric costimulatory agonists. 11. In one aspect, disclosed herein are methods of treating, inhibiting, decreasing, reducing, ameliorating, and / or preventing a cancer and / or a Notch signaling associated disorder in a subject comprising administering to the subject the multimeric costimulatory SNAGs of any preceding aspect. Also, disclosed herein are methods of treating, decreasing, inhibiting, reducing, ameliorating and / or preventing a Notch signaling associated disorder in a subject comprising obtaining cells lacking Notch signaling and culturing the cells lacking Notch signaling in media comprising one or more of the multimeric costimulatory agonists, vector, or cells of any preceding aspect; and administering the cultured cells to the subject. 12. In some aspects, disclosed herein the Notch signaling associated disorder comprises Adams-Oliver syndrome, Alagille syndrome, autosomal recessive spondylocostal dysostosis, Attorney Docket Number 10110-473WO1 Hajdu-Cheney syndrome, or cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy. 13. Also disclosed herein are synthetic Notch agonist (SNAG) constructs comprising an affinity matured Notch ligand (such as, for example, DeltaMAXpolypeptide as set forth in SEQ ID NO: 1), a linker or multimerization motif, and a target-binding domain specific to a biomarker other than Notch. Such constructs are configured to induce Notch activation between a Notch receptor-expressing cell and a biomarker-expressing cell. 14. In certain embodiments, the linker or multimerization motif comprises a human IgG1 Fc domain (such as for example, SEQ ID NO: 2), a leucine zipper (including, but not limited to a trimeric leucine zipper), C-terminal domain of T4 fibritin (FOLDON) motif, a streptavidin-biotin complex, or a flexible glycine-serine linker, thereby providing structural stability and controlled multimerization. In related embodiments, the target-binding domain may include a single-chain variable fragment, an antibody fragment, a nanobody, or a receptor-binding domain to enable precise recognition of selected targets. 15. The biomarker, disclosed herein bound by the SNAG construct can be selected from diverse categories, including Notch ligands such as DLL4 (including, but not limited to DLL4 variants that lack C2 and DSL domains) or JAG1, tumor-associated antigens such as PD-L1, HER2, or CD19, or immunostimulatory receptors such as CD40. This modularity allows the SNAG platform to be adapted for a wide range of therapeutic applications. 16. In some embodiments, the SNAG construct is provided as a dimer comprising two DeltaMAXdomains fused to the N-termini of an Fc domain and two target-binding domains fused to the C-termini of the Fc domain. In alternative embodiments, the construct is trimeric and comprises DeltaMAXtogether with a CD40 ligand extracellular domain fused to a trimeric leucine zipper, thereby facilitating cooperative activation of Notch signaling. 17. In one aspect, the target-binding domain comprises a nanobody specific for a BC2 epitope tag. Such constructs can restore Notch signaling in DLL4 variants that lack C2 and DSL domains, compensating for deficiencies in endogenous ligand interactions. In other embodiments, the target-binding domain comprises an antibody fragment specific for JAG1, including JAG1 molecules carrying an H268Q substitution. 18. In some embodiments, the constructs in which the target-binding domain recognizes PD-L1, can be arranged as dimers comprising DeltaMAXand the PD-L1-binding domain fused to both N- and C-termini of an Fc domain. Similarly, in some aspects, the target-binding domain recognizes HER2, and such constructs are capable of activating Notch signaling in mixed populations of HER2-positive tumor cells and Notch receptor-expressing immune cells. In related Attorney Docket Number 10110-473WO1 embodiments, the target-binding domain binds CD19, permitting activation of Notch signaling in mixed populations of B cells and Notch receptor-expressing cells. 19. In one aspect, the target-binding domain comprises a CD40 ligand extracellular domain. In some such embodiments, DeltaMAXand the CD40 ligand extracellular domain are fused to a trimeric leucine zipper scaffold, resulting in constructs that activate Notch signaling between CD40-positive B cells and CD40-negative T cells. 20. Also disclosed herein are SNAG constructs comprising defined amino acid sequences. In some embodiments, the construct comprises BC2-SNAGFc, comprising the amino acid sequence as set forth in SEQ ID NO: 6. In some embodiments, the construct comprises BC2- SNAG, comprising the amino acid sequence as set forth in SEQ ID NO: 5. In some embodiments, the construct comprises PDL1-SNAG, comprising the amino acid sequence as set forth in SEQ ID NO: 7. In some embodiments, the construct comprises PDL1-SNAGFc, comprising the amino acid sequence as set forth in SEQ ID NO: 8. In some embodiments, the construct comprises HER2- SNAGFc, comprising the amino acid sequence as set forth in SEQ ID NO: 9. In some embodiments, the construct comprises CD19-SNAGFc, comprising the amino acid sequence as set forth in SEQ ID NO: 10. In some embodiments, the construct comprises CD40-SNAG, comprising the amino acid sequence as set forth in SEQ ID NO: 11. These sequence-defined variants provide representative examples of engineered SNAG constructs described herein. BRIEF DESCRIPTION OF THE DRAWINGS 21. 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. 22. FIG.1 shows the construct design and structure of JAG1-SNAGFcbispecific protein. 23. FIGS. 2A and 2B show fluorescent reporter assays and schematics depicting JAG1- SNAGFcin HEK293T cells expressing JAG1 or JAG1H268Qmutant. FIG.2A shows a fluorescent reporter assay to evaluate SNAG-mediated activation of Notch1. Increasing concentrations (1 nM, 10 nM, or 100 nM) of DeltaMAX- Fc, or JAG1-SNAGFcwere added to Notch1-Gal4 mCitrine reporter cells alone or a 1:1 mixture of Notch1 reporter cells and HEK293T cells expressing JAG1 and fluorescence was measured by flow cytometry. A representative experiment from three biological replicates is shown. Mean fluorescence intensity (MFI) was normalized to the mean MFI of Notch1 reporter cells alone. Error bars represent the standard deviation of three technical replicates with the P value by Student’s t test shown above each comparison. Further FIG. 2A shows a schematic depicting the multivalent binding of a dimeric Fc-tagged SNAG (JAG1- Attorney Docket Number 10110-473WO1 SNAGFc) to Notch1 and JAG1 overexpressing HEK293T cell. FIG. 2B shows a fluorescent reporter assay to evaluate SNAG-mediated activation of Notch1. Increasing concentrations (1 nM, 10 nM, or 100 nM) of DeltaMAX- Fc, or JAG1-SNAGFcwere added to Notch1-Gal4 mCitrine reporter cells alone or a 1:1 mixture of Notch1 reporter cells and HEK293T cells expressing JAG1H268Qmutant and fluorescence was measured by flow cytometry. A representative experiment from three biological replicates is shown. Mean fluorescence intensity (MFI) was normalized to the mean MFI of Notch1 reporter cells alone. Error bars represent the standard deviation of three technical replicates with the P value by Student’s t test shown above each comparison. FIG. 2B, additionally depicts a cartoon schematic depicting the multivalent binding of a dimeric Fc-tagged SNAG (JAG1-SNAGFc) to Notch1 and JAG1H268Qmutant overexpressing HEK293T cell. 24. FIGS. 3A-3I show a design concept for synthetic Notch agonists. FIG. 3A shows a flow cytometry histogram overlay of Notch1 reporter cells stimulated by soluble or plated (non- specifically adsorbed) DeltaMAX. A cartoon depicts the site-specifically biotinylated DeltaMAX(N- EGF5) construct. FIG. 3B shows a histogram overlay of Notch1 reporter cells stimulated with soluble or plated DeltaMAX-Fc protein. FIG. 3C shows a histogram overlay of Notch1 reporter cells stimulated with plated or soluble DeltaMAX-SA tetramers. FIG. 3D shows a schematic depicting the extracellular domains (ECDs) of Notch1 and DLL4 interacting during canonical Notch activation, with the negative regulatory region (NRR) and ligand-binding domains (LBD) of Notch1 (EGF domains 8-12) shaded. FIG. 3E shows a schematic of a generalized SNAG construct alongside a cartoon depicting SNAG-mediated Notch activation. FIG. 3F shows a representation of the modular design of SNAGs. FIG. 3G shows a schematic highlighting targets of SNAGs against canonical Notch ligands. FIG. 3H shows a schematic highlighting targets of SNAGs against tumor biomarkers. FIG. 3I shows a schematic highlighting a target of SNAGs against a B cell biomarker. Below each panel is the corresponding endocytic mechanism. 25. FIGS.4A-4E show SNAGs rescue the signaling of binding-deficient DLL4 and JAG1 mutants. FIG.4A shows a cartoon schematic depicting a SNAG binding to Notch1 and a loss-of- function DLL4 mutant. The “headless” loss-of-function DLL4 protein (DLL4HL) was generated by replacing the Notch-binding C2-DSL region with a BC2 peptide epitope recognized by the anti-BC2 nanobody. FIG. 4B shows a cartoon schematic depicting the multivalent binding of a dimeric Fc-tagged SNAG (BC2-SNAGFc) to Notch1 and “headless” DLL4. FIGS. 4C-4E show fluorescent reporter assays used to evaluate SNAG-mediated activation of Notch1 in cocultures with HEK293T cells expressing DLL4HL (FIG.4C), JAG1 (FIG.4D), or JAG1H268Q (FIG.4E). Increasing concentrations (1 nM, 10 nM, or 100 nM) of the indicated SNAGs were added to Attorney Docket Number 10110-473WO1 Notch1-Gal4 Citrine reporter cells alone, or to a 1:1 mixture of Notch1 reporter cells and HEK293 cells expressing DLL4HL, and fluorescence was measured by flow cytometry. A representative experiment from three biological replicates is shown. Mean fluorescence intensity (MFI) was normalized to the mean MFI of Notch1 reporter cells alone. Error bars represent the standard deviation of n = 3 technical replicates, with the P value by a two-sided Student’s t test shown above each comparison. 26. FIGS. 5A-5F show SNAGs targeting tumor antigens activate Notch in mixed cell populations. FIG.5A shows increasing concentrations (1 nM, 10 nM, or 100 nM) of DeltaMAX-Fc, PDL1-SNAG, or PDL1-SNAGFc added to Notch1-Gal4 mCitrine reporter cells alone or in a 1:1 co-culture with PDL1-expressing MDA-MB-231 cells, with fluorescence measured by flow cytometry. FIG. 5B shows MDA-MB-231 cells incubated with PDL1-SNAGFc, soluble DeltaMAX-Fc, or plated DeltaMAX-Fc, with Notch1 activation assessed by Western Blot using an antibody against the cleaved NICD. Data represent a single experiment (n = 1). FIG. 5C shows increasing concentrations (1 nM, 10 nM, or 100 nM) of DeltaMAX-Fc or CD19-SNAGFc added to Notch1 reporter cells or 1:1 mixtures of reporter cells and CD19-expressing 3T3 cells. FIG. 5D shows increasing concentrations (1 nM, 10 nM, or 100 nM) of DeltaMAX-Fc or HER2-SNAGFc added to Notch1 reporter cells or 1:1 mixtures of reporter cells and HER2-expressing SK-BR-3 cells. FIG. 5E shows increasing concentrations (1 nM, 10 nM, or 100 nM) of trimeric CD40- SNAG or DeltaMAX-Fc added to reporter cells alone, or to a 1:2 mixture of Notch1 reporter cells and CD40-expressing OCI-Ly3 cells. FIG.5F shows Notch1 reporter cells stimulated with plated DeltaMAX-Fc, DLL4-Fc, EDTA, DLL4-expressing HEK293T cells, and PDL1-SNAGFc in the presence of MDA-MB-231 cells, with fluorescence measured using flow cytometry. The dashed line denotes the activation level induced by cellular activation with HEK293T-DLL4 cells. Ligands were adsorbed at 100 nM concentrations, EDTA was added to a concentration of 1 mM, and soluble PDL1-SNAGFc was added to a concentration of 100 nM. For FIGS.5A, 5C, 5D, 5E, and 5F, a representative experiment from three biological replicates is shown. Mean fluorescence intensity (MFI) was normalized to the mean MFI of Notch1 reporter cells alone. Error bars represent the standard deviation of n = 3 technical replicates, with the P value by a two-sided Student’s t test shown above each comparison (P value is 6.467E-7 in comparison of DeltaMAX- Fc to CD40-SNAG at 10 nM). 27. FIGS. 6A-6G show that SNAG-mediated Notch activation requires endocytosis. FIG. 6A shows representative immunofluorescence images of fluorescently labeled CD19-SNAGs (magenta) used to stain the surface of CD19-expressing 3T3 cells that were kept on ice. CD19- SNAGs were visualized by Alexa Fluor anti-Fc 647. To visualize the contours of the cells, the Attorney Docket Number 10110-473WO1 actin cytoskeleton was stained using phalloidin-488 (green). Nuclei were counterstained by Hoechst 33342 (blue). Scale, 20 µm. FIGS. 6B-6C show representative immunofluorescence images of fluorescently labeled CD19-SNAGs used to stain the surface of CD19-expressing 3T3 cells in the absence (FIG. 6B) or presence of Dynasore (FIG. 6C), followed by washing away unbound SNAGs and subjecting the cells to a 15 min incubation in a 37 ºC incubator to resume cellular processes, including endocytosis. After 15 min the cells were fixed and stained in parallel with the no endocytosis samples. Scale, 20 µm. A representative staining is shown for each experimental condition from one of two biological replicates. FIGS.6D-6F show flow cytometry histogram overlays depicting Notch1 reporter activity induced by soluble CD19-SNAGFc, BC2- SNAG, or BC2-SNAGFc in the presence or absence of Dynasore. FIG.6D shows Notch1 reporter cells co-cultured with CD19-overexpressing 3T3 cells. FIGS. 6E-6F show Notch1 reporter cells co-cultured with HEK293 cells expressing DLL4HL. FIG.6G shows a flow cytometry histogram overlay depicting Notch1 reporter activity induced by immobilized BC2-SNAG in the presence or absence of Dynasore. A representative histogram is shown for each experimental condition from one biological replicate for CD19-SNAGFc (soluble), two biological replicates for BC2- SNAG (soluble), or three biological replicates for both BC2-SNAGFc (soluble) and BC2-SNAG (plated). 28. FIGS. 7A-7C show that SNAGs increase activation markers of CD8^ T cells. Peripheral blood mononuclear cells (PBMCs) were used to isolate naïve CD8^ T cells that were treated with 100 nM of DeltaMAX-Fc, PDL1-SNAGFc, HER2-SNAGFc, or without treatment (control) in the presence of PD-L1^ MDA-MB-231 cells or HER2^ SK-BR-3 cells, followed by human CD3 / CD28 stimulation for 48 hours prior to harvesting. FIG. 7A shows a schematic depicting a SNAG engaging the target biomarkers expressed on a tumor cell and Notch receptors of a CD8^ T cell. FIGS.7B-7C show bar charts. FIG.7B shows the percentage of CD8^ and GrB^ T cells. FIG. 7C shows relative mRNA levels for GZMB, IFNG, and HES4 normalized to the control, where each bar represents the fold-change. Error bars represent the standard deviation of n = 3 technical replicates, with the P value by a two-sided Student’s t test shown above each comparison. 29. FIGS. 8A-8G show the expression of SNAG target biomarkers on various cell lines. Cell lines used in the study were analyzed by flow cytometry to measure surface expression of the indicated biomarker. In each panel, flow cytometry histograms show the staining of each cell line with the indicated antibody. FIG. 8A shows BC2-DLL4HL HEK293T cells stained with a fluorescently labeled anti-DLL4 antibody, FIG. 8B shows JAG1 HEK293T cells stained with an anti-JAG1 antibody, FIG. 8C shows JAG1H268Q HEK293T cells stained with an anti-JAG1 Attorney Docket Number 10110-473WO1 antibody, FIG.8D shows MDA-MB-231 cells stained with an anti-PDL1 antibody, FIG.8E shows CD19-expressing 3T3 cells stained with an anti-CD19 antibody, FIG. 8F shows SK-BR-3 cells stained with an anti-HER2 antibody, and FIG.8G shows OCI-Ly3 cells stained with an anti-CD40 antibody. 30. FIGS. 9A-9E show SDS-PAGE analysis of purified SNAG proteins. SDS-PAGE was used to evaluate the purity of the indicated SNAGs following nickel and size exclusion chromatography. FIG. 9A shows SDS-PAGE analysis of BC2-SNAGs, PDL1-SNAGs, and the CD19-SNAG. FIG. 9B shows SDS-PAGE analysis of the JAG1-SNAG. FIG. 9C shows SDS- PAGE analysis of the HER2-SNAG. FIG.9D shows SDS-PAGE analysis of the DLL4 variant of PDL1-SNAG. FIG.9E shows SDS-PAGE analysis of the CD40-SNAG. 31. FIGS.10A-10C show the gating schemes used to separate Notch1 reporter CHO cells and activated cells. Flow cytometry gating strategies were applied to select Notch1 CHO reporter cells alone (FIG.10A) or in coculture with BC2-DLL4HL over-expressing HEK293T cells (FIG. 10B). FIG. 10C shows the gating strategy to determine the population of non-activated and activated Notch1 CHO reporter cells in coculture with BC2-DLL4HL over-expressing HEK293T cells without SNAG (left) and with BC2-SNAGFc (right). 32. FIG. 11 shows the functional comparison of SNAGs incorporating DLL4 versus DeltaMAX. Increasing concentrations (1 nM, 10 nM, or 100 nM) of SNAGs were added to a 1:1 mixture of Notch1 reporter cells and MDA-MB-231 cells. To calculate the percent activation, the mean MFI of 100 nM PDL1-SNAGFcadded to the coculture was used as theMAXimum, and the mean MFI of Notch1 reporter cells alone as the minimum (shown as the control is the coculture without SNAG added). A representative experiment from three biological replicates is shown. Mean fluorescence intensity (MFI) was normalized to the mean MFI of Notch1 reporter cells alone. Error bars represent the standard deviation of n = 3 technical replicates. 33. FIGS. 12A-12E show immunofluorescent endocytosis assays in 3T3 cells expressing CD19. FIG.12A shows negative control staining utilizing anti-Fc 647 alone (zoom in), with anti- Fc 647 (magenta) on the left and merged with actin (green) and nuclei (blue) on the right. FIGS. 12B-12C show surface staining of CD19-SNAGFc-647 (magenta) with multiple cells visualized, where the highlighted rectangle in FIG. 12B was used as the zoomed-in “no endocytosis” panel for FIG. 6A. FIGS. 12D-12E show immunofluorescence images of CD19-SNAGFc-647 (magenta) allowed to endocytose for 15 minutes with multiple cells visualized, where the highlighted rectangle in FIG.12D was used as the zoomed-in “15 min endocytosis” panel for FIG. 6A. Data represent a single experiment (n = 1). Attorney Docket Number 10110-473WO1 34. FIGS. 13A-13B show the analysis of protein expression levels of biomarkers associated with a cytotoxic phenotype. PBMCs were used to isolate naïve CD8+ T cells that were treated with 100 nM of DeltaMAX-Fc, PDL1-SNAGFc, HER2-SNAGFc, or without treatment (control) in the presence of PD-L1+ MDA-MB-231 cells or HER2+ SK-BR-3 cells, followed by human CD3 / CD28 stimulation for 48 hours prior to harvesting. FIGS. 13A-13B show bar charts of flow cytometry analysis for IFN^ and GrB expression. Error bars represent the standard deviation of n = 3 technical replicates, with the P value by a two-sided Student’s t test shown above each comparison. DETAILED DESCRIPTION 35. Before the present compounds, compositions, articles, devices, and / or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. A. Definitions 36. As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like. 37. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10”as well as “greater than or equal to 10” is also disclosed. It is also understood that the throughout the application, data is Attorney Docket Number 10110-473WO1 provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed. 38. In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings: 39. “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. 40. An "increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase so long as the increase is statistically significant. 41. A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant. 42. "Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels. 43. By “reduce” or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always Attorney Docket Number 10110-473WO1 necessary for the standard or relative value to be referred to. For example, “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control. 44. By “prevent” or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed. 45. The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline. The subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician. 46. The term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination. 47. The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. 48. "Biocompatible" generally refers to a material and any metabolites or degradation products thereof that are generally non-toxic to the recipient and do not cause significant adverse effects to the subject. Attorney Docket Number 10110-473WO1 49. "Comprising" is intended to mean that the compositions, methods, etc. include the recited elements, but do not exclude others. "Consisting essentially of'' when used to define compositions and methods, shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. "Consisting of'' shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and / or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure. 50. A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative." 51. “Effective amount” of an agent refers to a sufficient amount of an agent to provide a desired effect. The amount of agent that is “effective” will vary from subject to subject, depending on many factors such as the age and general condition of the subject, the particular agent or agents, and the like. Thus, it is not always possible to specify a quantified “effective amount.” However, an appropriate “effective amount” in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of an agent can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts. An “effective amount” of an agent necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. 52. A "pharmaceutically acceptable" component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation provided by the disclosure and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. When used in reference to administration to a human, the term generally implies the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration. 53. "Pharmaceutically acceptable carrier" (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is Attorney Docket Number 10110-473WO1 generally safe and non-toxic and includes a carrier that is acceptable for veterinary and / or human pharmaceutical or therapeutic use. The terms "carrier" or "pharmaceutically acceptable carrier" can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil / water or water / oil emulsion) and / or various types of wetting agents. As used herein, the term "carrier" encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further herein. 54. “Pharmacologically active” (or simply “active”), as in a “pharmacologically active” derivative or analog, can refer to a derivative or analog (e.g., a salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) having the same type of pharmacological activity as the parent compound and approximately equivalent in degree. 55. “Therapeutic agent” refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., a non-immunogenic cancer). The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the terms “therapeutic agent” is used, then, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc. 56. “Therapeutically effective amount” or “therapeutically effective dose” of a composition (e.g. a composition comprising an agent) refers to an amount that is effective to achieve a desired therapeutic result. In some embodiments, a desired therapeutic result is the control of type I diabetes. In some embodiments, a desired therapeutic result is the control of obesity. Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect, such as pain relief. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and / or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art. In some instances, a desired biological or medical response is achieved following Attorney Docket Number 10110-473WO1 administration of multiple dosages of the composition to the subject over a period of days, weeks, or years. 57. The term “JAG1-SNAGFc” refers to a soluble bispecific format that enables the SNAG- bound antibodies or fragments thereof to “pull” on Notch receptors, triggering Notch activation in mixed populations of JAG-expressing and non-expressing cells. The "SNAG" in JAG1- SNAGFctypically denotes high-affinity variant of the Notch ligand Delta-like 4 (DLL4) to antibody fragments that induce target internalization. 58. “PBMC” means a diverse population of immune cells derived from peripheral blood, which includes lymphocytes (T cells, B cells, and NK cells) and monocytes. These cells are often used in vitro to study immune responses or to expand specific T cell subsets, including ^^ T cells. 59. In some embodiments, the JAG1 construct includes an antibody or antigen-binding fragment thereof that specifically binds Jagged 1, as recited by the appended claims. In some embodiments, the antibody or immunologically active fragment thereof that binds Jagged 1 is a monoclonal antibody, domain antibody, single chain, Fab fragment, a F(ab')2 fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, or a single domain light chain antibody. In some embodiments, such an antibody or immunologically active fragment thereof that binds Jagged 1 is a rodent (e.g., mouse or rat), chimeric, humanized or fully human monoclonal antibody. 60. “DeltaMAX” refers to high affinity synthetic Notch ligand. It contains ten mutations that increase its affinity for human Notch receptors by 500- to 1000-fold, making it a more potent activator than DLL4 in co-culture and plate-bound formats. 61. Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon B. Compositions 62. Disclosed are the components to be used to prepare the disclosed compositions as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular multimeric costimulatory synthetic Notch agonist (SNAG) construct (including, but not limited to JAG1- Attorney Docket Number 10110-473WO1 SNAGFcbispecific protein agonists) is disclosed and discussed and a number of modifications that can be made to a number of molecules including the multimeric costimulatory synthetic Notch agonist (SNAG) construct are discussed, specifically contemplated is each and every combination and permutation of multimeric costimulatory synthetic Notch agonist (SNAG) construct and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C- D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods. Multimeric costimulatory agonists 63. In one aspect, disclosed herein are multimeric costimulatory synthetic Notch agonist (SNAG) construct (including, but not limited to bi-specific protein agonist) comprising a JAG1 construct (such as, for example, a JAG1 antibody, or fragment thereof) and a Affinity matured Notch ligand (DeltaMAXcomprises tetramers of biotinylated DeltaMAXwith streptavidin, monomers of DeltaMAX, three monomers of DeltaMAXjoined by glycine-serine linker, or dimers of DeltaMAXjoined by dimeric human IgG1 Fc domain); wherein said JAG1 construct is connected to said Affinity matured Notch ligand . In one aspect, the JAG1 monomer can comprise SEQ ID NO: 3. In some aspects, the JAG1 and / or the SNAG can be a soluble protein such as, for example soluble JAG1 or soluble SNAG. 64. As noted above, the costimulatory agonist construct can comprise one or more JAG1 monomer agonists linked to one or more affinity matured Notch ligand. Also disclosed herein are multimeric costimulatory synthetic Notch agonist (SNAG) construct of any preceding aspect; wherein said Affinity matured Notch ligand is connected to said JAG1 construct by a multimerization motif (such as, for example an immunoglobulin Fc domain (such as for example, SEQ ID NO: 2), C-terminal domain of T4 fibritin (FOLDON) motif, a streptavidin-biotin complex, or a leucine zipper (including, but not limited to a trimeric leucine zipper). 65. In some aspects, the JAG1 construct can comprise JAG1 antibody or antibody fragment (such as for example, a scFv, nanobody, or SNAG heavy and light chains). Attorney Docket Number 10110-473WO1 66. The Fc domain can comprise any mammalian Fc domain, including, but not limited to a human IgA, IgG1, IgG2A, IgG2B, or IgG3 Fc domain. In one aspect, the multimeric JAG1 can dimerize through the presence of cysteine bonds. For example, disclosed herein are multimeric costimulatory agonists, wherein the multimeric costimulatory agonist is linked to a second costimulatory agonist construct comprising an Fc dimer forming a tetramer of dimeric costimulatory constructs. 67. It is understood and herein contemplated that the disclosed JAG1-SNAGFcbispecific protein, and / or multimeric costimulatory synthetic Notch agonist (SNAG) constructs can be encoded on a vector and either expressed by the vector or integrated into a cell genome and expressed by a cell or a vector in said cell. Accordingly, disclosed herein are vectors encoding any of the multimeric costimulatory SNAGs disclosed herein as well as cells (such as, for examples, cells lacking Notch signaling, a Notch receptor or ligand expressing cell, a tumor infiltrating lymphocyte, ^^ T cells, ^^ T cells, feeder cell, B cell, natural killer cell, chimeric antigen receptor (CAR) T cell, CAR NK cell CAR macrophage (CARMA), or dendritic cell) comprising said vectors or multimeric costimulatory agonists. 68. In some aspects, disclosed herein are synthetic Notch agonist (SNAG) constructs comprising a DeltaMAXpolypeptide, a linker or multimerization motif, and a target-binding domain specific to a biomarker other than Notch. Such constructs are configured to induce Notch activation between a Notch receptor-expressing cell and a biomarker-expressing cell. 69. In certain embodiments, the linker or multimerization motif comprises a human IgG1 Fc domain, a trimeric zipper, a streptavidin-biotin complex, or a flexible glycine-serine linker, thereby providing structural stability and controlled multimerization. In related embodiments, the target-binding domain may include a single-chain variable fragment, an antibody fragment, a nanobody, or a receptor-binding domain to enable precise recognition of selected targets. 70. The biomarker, disclosed herein bound by the SNAG construct can be selected from diverse categories, including Notch ligands such as DLL4 or JAG1, tumor-associated antigens such as PD-L1, HER2, or CD19, or immunostimulatory receptors such as CD40. This modularity allows the SNAG platform to be adapted for a wide range of therapeutic applications. 71. In some embodiments, the SNAG construct is provided as a dimer comprising two DeltaMAXdomains fused to the N-termini of an Fc domain and two target-binding domains fused to the C-termini of the Fc domain. In alternative embodiments, the construct is trimeric and comprises DeltaMAXtogether with a CD40 ligand extracellular domain fused to a trimeric leucine zipper, thereby facilitating cooperative activation of Notch signaling. Attorney Docket Number 10110-473WO1 72. In another aspect, the target-binding domain comprises a nanobody specific for a BC2 epitope tag. Such constructs can restore Notch signaling in DLL4 variants that lack C2 and DSL domains, compensating for deficiencies in endogenous ligand interactions. In other embodiments, the target-binding domain comprises an antibody fragment specific for JAG1, including JAG1 molecules carrying an H268Q substitution. 73. In some embodiments, the constructs in which the target-binding domain recognizes PD-L1, can be arranged as dimers comprising DeltaMAXand the PD-L1-binding domain fused to both N- and C-termini of an Fc domain. Similarly, in some aspects, the target-binding domain recognizes HER2, and such constructs are capable of activating Notch signaling in mixed populations of HER2-positive tumor cells and Notch receptor-expressing immune cells. In related embodiments, the target-binding domain binds CD19, permitting activation of Notch signaling in mixed populations of B cells and Notch receptor-expressing cells. 74. In one aspect, the target-binding domain comprises a CD40 ligand extracellular domain. In some such embodiments, DeltaMAXand the CD40 ligand extracellular domain are fused to a trimeric leucine zipper scaffold, resulting in constructs that activate Notch signaling between CD40-positive B cells and CD40-negative T cells. 75. Also disclosed herein are SNAG constructs comprising defined amino acid sequences. In some embodiments, the construct comprises BC2-SNAGFc, comprising the amino acid sequence as set forth in SEQ ID NO: 6. In some embodiments, the construct comprises BC2- SNAG, comprising the amino acid sequence as set forth in SEQ ID NO: 5. In some embodiments, the construct comprises PDL1-SNAG, comprising the amino acid sequence as set forth in SEQ ID NO: 7. In some embodiments, the construct comprises PDL1-SNAGFc, comprising the amino acid sequence as set forth in SEQ ID NO: 8. In some embodiments, the construct comprises HER2- SNAGFc, comprising the amino acid sequence as set forth in SEQ ID NO: 9. In some embodiments, the construct comprises CD19-SNAGFc, comprising the amino acid sequence as set forth in SEQ ID NO: 10. In some embodiments, the construct comprises CD40-SNAG, comprising the amino acid sequence as set forth in SEQ ID NO: 11. These sequence-defined variants provide representative examples of engineered SNAG constructs described herein. Immunoassays and fluorochromes 76. The steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Maggio et al., Enzyme-Immunoassay, (1987) and Nakamura, et al., Enzyme Immunoassays: Heterogeneous and Homogeneous Systems, Handbook of Experimental Immunology, Vol. 1: Immunochemistry, 27.1-27.20 (1986), each of which is incorporated herein by reference in its entirety and specifically for its teaching regarding Attorney Docket Number 10110-473WO1 immunodetection methods. Immunoassays, in their most simple and direct sense, are binding assays involving binding between antibodies and antigen. Many types and formats of immunoassays are known and all are suitable for detecting the disclosed biomarkers. Examples of immunoassays are enzyme linked immunosorbent assays (ELISAs), radioimmunoassay (RIA), radioimmune precipitation assays (RIPA), immunobead capture assays, Western blotting, dot blotting, gel-shift assays, Flow cytometry, protein arrays, multiplexed bead arrays, magnetic capture, in vivo imaging, fluorescence resonance energy transfer (FRET), and fluorescence recovery / localization after photobleaching (FRAP / FLAP). 77. In general, immunoassays involve contacting a sample suspected of containing a molecule of interest (such as the disclosed biomarkers) with an antibody to the molecule of interest or contacting an antibody to a molecule of interest (such as antibodies to the disclosed biomarkers) with a molecule that can be bound by the antibody, as the case may be, under conditions effective to allow the formation of immunocomplexes. Contacting a sample with the antibody to the molecule of interest or with the molecule that can be bound by an antibody to the molecule of interest under conditions effective and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) is generally a matter of simply bringing into contact the molecule or antibody and the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e., to bind to, any molecules (e.g., antigens) present to which the antibodies can bind. In many forms of immunoassay, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot or Western blot, can then be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected. 78. Immunoassays can include methods for detecting or quantifying the amount of a molecule of interest (such as the disclosed biomarkers or their antibodies) in a sample, which methods generally involve the detection or quantitation of any immune complexes formed during the binding process. In general, the detection of immunocomplex formation is well known in the art and can be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any radioactive, fluorescent, biological or enzymatic tags or any other known label. 79. As used herein, a label can include a fluorescent dye, a member of a binding pair, such as biotin / streptavidin, a metal (e.g., gold), or an epitope tag that can specifically interact with a molecule that can be detected, such as by producing a colored substrate or fluorescence. Substances suitable for detectably labeling proteins include fluorescent dyes (also known herein as fluorochromes and fluorophores) and enzymes that react with colorimetric substrates (e.g., Attorney Docket Number 10110-473WO1 horseradish peroxidase). The use of fluorescent dyes is generally preferred in the practice of the invention as they can be detected at very low amounts. Furthermore, in the case where multiple antigens are reacted with a single array, each antigen can be labeled with a distinct fluorescent compound for simultaneous detection. Labeled spots on the array are detected using a fluorimeter, the presence of a signal indicating an antigen bound to a specific antibody. 80. Fluorophores are compounds or molecules that luminesce. Typically fluorophores absorb electromagnetic energy at one wavelength and emit electromagnetic energy at a second wavelength. Representative fluorophores include, but are not limited to, 1,5 IAEDANS; 1,8-ANS; 4- Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM); 5- Carboxynapthofluorescein; 5-Carboxytetramethylrhodamine (5-TAMRA); 5-Hydroxy Tryptamine (5-HAT); 5-ROX (carboxy-X-rhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6- JOE; 7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD); 7-Hydroxy-4- I methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine (ACMA); ABQ; Acid Fuchsin; Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Aequorin (Photoprotein); AFPs - AutoFluorescent Protein - (Quantum Biotechnologies) see sgGFP, sgBFP; Alexa Fluor 350^; Alexa Fluor 430^; Alexa Fluor 488^; Alexa Fluor 532^; Alexa Fluor 546^; Alexa Fluor 568^; Alexa Fluor 594^; Alexa Fluor 633^; Alexa Fluor 647^; Alexa Fluor 660^; Alexa Fluor 680^; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA- S; Aminomethylcoumarin (AMCA); AMCA-X; Aminoactinomycin D; Aminocoumarin; Anilin Blue; Anthrocyl stearate; APC-Cy7; APTRA-BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO- TAG^ CBQCA; ATTO- TAG^ FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); Berberine Sulphate; Beta Lactamase; BFP blue shifted GFP (Y66H); Blue Fluorescent Protein; BFP / GFP FRET; Bimane; Bisbenzemide; Bisbenzimide (Hoechst); bis- BTC; Blancophor FFG; Blancophor SV; BOBO^ -1; BOBO^-3; Bodipy492 / 515; Bodipy493 / 503; Bodipy500 / 510; Bodipy; 505 / 515; Bodipy 530 / 550; Bodipy 542 / 563; Bodipy 558 / 568; Bodipy 564 / 570; Bodipy 576 / 589; Bodipy 581 / 591; Bodipy 630 / 650- X; Bodipy 650 / 665-X; Bodipy 665 / 676; Bodipy Fl; Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO^ -1; BO-PRO^ -3; Brilliant Sulphoflavin FF; BTC; BTC-5N; Calcein; Calcein Blue; Calcium Crimson - ; Calcium Green; Calcium Green-1 Ca2+Dye; Calcium Green-2 Ca2+; Calcium Green-5N Ca2+; Calcium Green-C18 Ca2+; Calcium Orange; Calcofluor White; Carboxy-X-rhodamine (5-ROX); Cascade Blue^; Cascade Yellow; Attorney Docket Number 10110-473WO1 Catecholamine; CCF2 (GeneBlazer); CFDA; CFP (Cyan Fluorescent Protein); CFP / YFP FRET; Chlorophyll; Chromomycin A; Chromomycin A; CL-NERF; CMFDA; Coelenterazine; Coelenterazine cp; Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazine hcp; Coelenterazine ip; Coelenterazine n; Coelenterazine O; Coumarin Phalloidin; C-phycocyanine; CPM I Methylcoumarin; CTC; CTC Formazan; Cy2^; Cy3.1 8; Cy3.5^; Cy3^; Cy5.1 8; Cy5.5^; Cy5^; Cy7^; Cyan GFP; cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3’DCFDA; DCFH (Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di 16-ASP); Dichlorodihydrofluorescein Diacetate (DCFH); DiD- Lipophilic Tracer; DiD (DilC18(5)); DIDS; Dihydorhodamine 123 (DHR); Dil (DilC18(3)); I Dinitrophenol; DiO (DiOC18(3)); DiR; DiR (DilC18(7)); DM-NERF (high pH); DNP; Dopamine; DsRed; DTAF; DY-630-NHS; DY-635- NHS; EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium Bromide; Ethidium homodimer-1 (EthD-1); Euchrysin; EukoLight; Europium (111) chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FIF (Formaldehyd Induced Fluorescence); FITC; Flazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43^; FM 4-46; Fura Red^ (high pH); Fura Red^ / Fluo-3; Fura-2; Fura-2 / BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GeneBlazer; (CCF2); GFP (S65T); GFP red shifted (rsGFP); GFP wild type’ non-UV excitation (wtGFP); GFP wild type, UV excitation (wtGFP); GFPuv; Gloxalic Acid; Granular blue; Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine (FluoroGold); Hydroxytryptamine; Indo-1, high calcium; Indo-1 low calcium; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO JO-1; JO-PRO-1; LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B; Calcein / Ethidium homodimer; LOLO-1; LO-PRO-1; ; Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker Blue-White; Lyso Tracker Green; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue; LysoSensor Green; LysoSensor Yellow / Blue; Mag Green; Magdala Red (Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-lndo-1; Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; IMAXilon Brilliant Flavin 10 GFF;MAXilon Brilliant Flavin 8 GFF; Merocyanin; Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxedidole; Noradrenaline; Nuclear Fast Red; i Nuclear Yellow; Nylosan Attorney Docket Number 10110-473WO1 Brilliant lavin E8G; Oregon Green^; Oregon Green^ 488; Oregon Green^ 500; Oregon Green^ 514; Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP- Cy5.5; PE-TexasRed (Red 613); Phloxin B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO- 1; PO- I PRO-3; Primuline; Procion Yellow; Propidium lodid (Pl); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufin; RH 414; Rhod- 2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine: Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; R-phycocyanine; R-phycoerythrin (PE); rsGFP; S65A; S65C; S65L; S65T; Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron I Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP^ (super glow BFP); sgGFP^ (super glow GFP); SITS (Primuline; Stilbene Isothiosulphonic Acid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARF1; Sodium Green; SpectrumAqua; SpectrumGreen; SpectrumOrange; Spectrum Red; SPQ (6-methoxy- N-(3 sulfopropyl) quinolinium); Stilbene; Sulphorhodamine B and C; Sulphorhodamine Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO 15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange; Tetracycline; Tetramethylrhodamine (TRITC); Texas Red^; Texas Red-X^ conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TON; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TIER; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC TetramethylRodaminelsoThioCyanate; True Blue; Tru Red; Ultralite; Uranine B; Uvitex SFC; wt GFP; WW 781; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO- PRO 3; YOYO-1;YOYO-3; Sybr Green; Thiazole orange (interchelating dyes); semiconductor nanoparticles such as quantum dots; or caged fluorophore (which can be activated with light or other electromagnetic energy source), or a combination thereof. 81. A modifier unit such as a radionuclide can be incorporated into or attached directly to any of the compounds described herein by halogenation. Examples of radionuclides useful in this embodiment include, but are not limited to, tritium, iodine-125, iodine-131, iodine-123, iodine- 124, astatine-210, carbon-11, carbon-14, nitrogen-13, fluorine-18. In another aspect, the Attorney Docket Number 10110-473WO1 radionuclide can be attached to a linking group or bound by a chelating group, which is then attached to the compound directly or by means of a linker. Examples of radionuclides useful in the apset include, but are not limited to, Tc-99m, Re-186, Ga-68, Re-188, Y-90, Sm-153, Bi-212, Cu-67, Cu-64, and Cu-62. Radiolabeling techniques such as these are routinely used in the radiopharmaceutical industry. 82. The radiolabeled compounds are useful as imaging agents to diagnose cancers and / or neurological disease (e.g., a neurodegenerative disease) or a mental condition or to follow the progression or treatment of such a disease or condition in a mammal (e.g., a human). The radiolabeled compounds described herein can be conveniently used in conjunction with imaging techniques such as positron emission tomography (PET) or single photon emission computerized tomography (SPECT). 83. Labeling can be either direct or indirect. In direct labeling, the detecting antibody (the antibody for the molecule of interest) or detecting molecule (the molecule that can be bound by an antibody to the molecule of interest) include a label. Detection of the label indicates the presence of the detecting antibody or detecting molecule, which in turn indicates the presence of the molecule of interest or of an antibody to the molecule of interest, respectively. In indirect labeling, an additional molecule or moiety is brought into contact with, or generated at the site of, the immunocomplex. For example, a signal-generating molecule or moiety such as an enzyme can be attached to or associated with the detecting antibody or detecting molecule. The signal- generating molecule can then generate a detectable signal at the site of the immunocomplex. For example, an enzyme, when supplied with suitable substrate, can produce a visible or detectable product at the site of the immunocomplex. ELISAs use this type of indirect labeling. 84. As another example of indirect labeling, an additional molecule (which can be referred to as a binding agent) that can bind to either the molecule of interest or to the antibody (primary antibody) to the molecule of interest, such as a second antibody to the primary antibody, can be contacted with the immunocomplex. The additional molecule can have a label or signal-generating molecule or moiety. The additional molecule can be an antibody, which can thus be termed a secondary antibody. Binding of a secondary antibody to the primary antibody can form a so-called sandwich with the first (or primary) antibody and the molecule of interest. The immune complexes can be contacted with the labeled, secondary antibody under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes can then be generally washed to remove any non-specifically bound labeled secondary antibodies, and the remaining label in the secondary immune complexes can then be detected. The additional molecule can also be or include one of a pair of molecules or moieties that can bind to Attorney Docket Number 10110-473WO1 each other, such as the biotin / avadin pair. In this mode, the detecting antibody or detecting molecule should include the other member of the pair. 85. Other modes of indirect labeling include the detection of primary immune complexes by a two step approach. For example, a molecule (which can be referred to as a first binding agent), such as an antibody, that has binding affinity for the molecule of interest or corresponding antibody can be used to form secondary immune complexes, as described above. After washing, the secondary immune complexes can be contacted with another molecule (which can be referred to as a second binding agent) that has binding affinity for the first binding agent, again under conditions effective and for a period of time sufficient to allow the formation of immune complexes (thus forming tertiary immune complexes). The second binding agent can be linked to a detectable label or signal-genrating molecule or moiety, allowing detection of the tertiary immune complexes thus formed. This system can provide for signal amplification. 86. Immunoassays that involve the detection of as substance, such as a protein or an antibody to a specific protein, include label-free assays, protein separation methods (i.e., electrophoresis), solid support capture assays, or in vivo detection. Label-free assays are generally diagnostic means of determining the presence or absence of a specific protein, or an antibody to a specific protein, in a sample. Protein separation methods are additionally useful for evaluating physical properties of the protein, such as size or net charge. Capture assays are generally more useful for quantitatively evaluating the concentration of a specific protein, or antibody to a specific protein, in a sample. Finally, in vivo detection is useful for evaluating the spatial expression patterns of the substance, i.e., where the substance can be found in a subject, tissue or cell. 87. Provided that the concentrations are sufficient, the molecular complexes ([Ab-Ag]n) generated by antibody-antigen interaction are visible to the naked eye, but smaller amounts may also be detected and measured due to their ability to scatter a beam of light. The formation of complexes indicates that both reactants are present, and in immunoprecipitation assays a constant concentration of a reagent antibody is used to measure specific antigen ([Ab-Ag]n), and reagent antigens are used to detect specific antibody ([Ab-Ag]n). If the reagent species is previously coated onto cells (as in hemagglutination assay) or very small particles (as in latex agglutination assay), “clumping” of the coated particles is visible at much lower concentrations. A variety of assays based on these elementary principles are in common use, including Ouchterlony immunodiffusion assay, rocket immunoelectrophoresis, and immunoturbidometric and nephelometric assays. The main limitations of such assays are restricted sensitivity (lower detection limits) in comparison to assays employing labels and, in some cases, the fact that very high concentrations of analyte can actually inhibit complex formation, necessitating safeguards Attorney Docket Number 10110-473WO1 that make the procedures more complex. Some of these Group 1 assays date right back to the discovery of antibodies and none of them have an actual “label” (e.g. Ag-enz). Other kinds of immunoassays that are label free depend on immunosensors, and a variety of instruments that can directly detect antibody-antigen interactions are now commercially available. Most depend on generating an evanescent wave on a sensor surface with immobilized ligand, which allows continuous monitoring of binding to the ligand. Immunosensors allow the easy investigation of kinetic interactions and, with the advent of lower-cost specialized instruments, may in the future find wide application in immunoanalysis. 88. The use of immunoassays to detect a specific protein can involve the separation of the proteins by electophoresis. Electrophoresis is the migration of charged molecules in solution in response to an electric field. Their rate of migration depends on the strength of the field; on the net charge, size and shape of the molecules and also on the ionic strength, viscosity and temperature of the medium in which the molecules are moving. As an analytical tool, electrophoresis is simple, rapid and highly sensitive. It is used analytically to study the properties of a single charged species, and as a separation technique. 89. Generally the sample is run in a support matrix such as paper, cellulose acetate, starch gel, agarose or polyacrylamide gel. The matrix inhibits convective mixing caused by heating and provides a record of the electrophoretic run: at the end of the run, the matrix can be stained and used for scanning, autoradiography or storage. In addition, the most commonly used support matrices - agarose and polyacrylamide - provide a means of separating molecules by size, in that they are porous gels. A porous gel may act as a sieve by retarding, or in some cases completely obstructing, the movement of large macromolecules while allowing smaller molecules to migrate freely. Because dilute agarose gels are generally more rigid and easy to handle than polyacrylamide of the same concentration, agarose is used to separate larger macromolecules such as nucleic acids, large proteins and protein complexes. Polyacrylamide, which is easy to handle and to make at higher concentrations, is used to separate most proteins and small oligonucleotides that require a small gel pore size for retardation. 90. Proteins are amphoteric compounds; their net charge therefore is determined by the pH of the medium in which they are suspended. In a solution with a pH above its isoelectric point, a protein has a net negative charge and migrates towards the anode in an electrical field. Below its isoelectric point, the protein is positively charged and migrates towards the cathode. The net charge carried by a protein is in addition independent of its size - i.e., the charge carried per unit mass (or length, given proteins and nucleic acids are linear macromolecules) of molecule differs Attorney Docket Number 10110-473WO1 from protein to protein. At a given pH therefore, and under non-denaturing conditions, the electrophoretic separation of proteins is determined by both size and charge of the molecules. 91. Sodium dodecyl sulphate (SDS) is an anionic detergent which denatures proteins by “wrapping around” the polypeptide backbone - and SDS binds to proteins fairly specifically in a mass ratio of 1.4:1. In so doing, SDS confers a negative charge to the polypeptide in proportion to its length. Further, it is usually necessary to reduce disulphide bridges in proteins (denature) before they adopt the random-coil configuration necessary for separation by size; this is done with 2-mercaptoethanol or dithiothreitol (DTT). In denaturing SDS-PAGE separations therefore, migration is determined not by intrinsic electrical charge of the polypeptide, but by molecular weight. 92. Determination of molecular weight is done by SDS-PAGE of proteins of known molecular weight along with the protein to be characterized. A linear relationship exists between the logarithm of the molecular weight of an SDS-denatured polypeptide, or native nucleic acid, and its Rf. The Rf is calculated as the ratio of the distance migrated by the molecule to that migrated by a marker dye-front. A simple way of determining relative molecular weight by electrophoresis (Mr) is to plot a standard curve of distance migrated vs. log10MW for known samples, and read off the logMr of the sample after measuring distance migrated on the same gel. 93. In two-dimensional electrophoresis, proteins are fractionated first on the basis of one physical property, and, in a second step, on the basis of another. For example, isoelectric focusing can be used for the first dimension, conveniently carried out in a tube gel, and SDS electrophoresis in a slab gel can be used for the second dimension. One example of a procedure is that of O’Farrell, P.H., High Resolution Two-dimensional Electrophoresis of Proteins, J. Biol. Chem. 250:4007- 4021 (1975), herein incorporated by reference in its entirety for its teaching regarding two- dimensional electrophoresis methods. Other examples include but are not limited to, those found in Anderson, L and Anderson, NG, High resolution two-dimensional electrophoresis of human plasma proteins, Proc. Natl. Acad. Sci. 74:5421-5425 (1977), Ornstein, L., Disc electrophoresis, L. Ann. N.Y. Acad. Sci.121:321349 (1964), each of which is herein incorporated by reference in its entirety for teachings regarding electrophoresis methods. Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227:680 (1970), which is herein incorporated by reference in its entirety for teachings regarding electrophoresis methods, discloses a discontinuous system for resolving proteins denatured with SDS. The leading ion in the Laemmli buffer system is chloride, and the trailing ion is glycine. Accordingly, the resolving gel and the stacking gel are made up in Tris-HCl buffers (of different concentration and pH), while the tank buffer is Tris-glycine. All buffers contain 0.1% SDS. Attorney Docket Number 10110-473WO1 94. One example of an immunoassay that uses electrophoresis that is contemplated in the current methods is Western blot analysis. Western blotting or immunoblotting allows the determination of the molecular mass of a protein and the measurement of relative amounts of the protein present in different samples. Detection methods include chemiluminescence and chromagenic detection. Standard methods for Western blot analysis can be found in, for example, D.M. Bollag et al., Protein Methods (2d edition 1996) and E. Harlow & D. Lane, Antibodies, a Laboratory Manual (1988), U.S. Patent 4,452,901, each of which is herein incorporated by reference in their entirety for teachings regarding Western blot methods. Generally, proteins are separated by gel electrophoresis, usually SDS-PAGE. The proteins are transferred to a sheet of special blotting paper, e.g., nitrocellulose, though other types of paper, or membranes, can be used. The proteins retain the same pattern of separation they had on the gel. The blot is incubated with a generic protein (such as milk proteins) to bind to any remaining sticky places on the nitrocellulose. An antibody is then added to the solution which is able to bind to its specific protein. 95. The attachment of specific antibodies to specific immobilized antigens can be readily visualized by indirect enzyme immunoassay techniques, usually using a chromogenic substrate (e.g. alkaline phosphatase or horseradish peroxidase) or chemiluminescent substrates. Other possibilities for probing include the use of fluorescent or radioisotope labels (e.g., fluorescein,125I). Probes for the detection of antibody binding can be conjugated anti-immunoglobulins, conjugated staphylococcal Protein A (binds IgG), or probes to biotinylated primary antibodies (e.g., conjugated avidin / streptavidin). 96. The power of the technique lies in the simultaneous detection of a specific protein by means of its antigenicity, and its molecular mass. Proteins are first separated by mass in the SDS- PAGE, then specifically detected in the immunoassay step. Thus, protein standards (ladders) can be run simultaneously in order to approximate molecular mass of the protein of interest in a heterogeneous sample. 97. The gel shift assay or electrophoretic mobility shift assay (EMSA) can be used to detect the interactions between DNA binding proteins and their cognate DNA recognition sequences, in both a qualitative and quantitative manner. Exemplary techniques are described in Ornstein L., Disc electrophoresis - I: Background and theory, Ann. NY Acad. Sci. 121:321-349 (1964), and Matsudiara, PT and DR Burgess, SDS microslab linear gradient polyacrylamide gel electrophoresis, Anal. Biochem. 87:386-396 (1987), each of which is herein incorporated by reference in its entirety for teachings regarding gel-shift assays. Attorney Docket Number 10110-473WO1 98. In a general gel-shift assay, purified proteins or crude cell extracts can be incubated with a labeled (e.g.,32P-radiolabeled) DNA or RNA probe, followed by separation of the complexes from the free probe through a nondenaturing polyacrylamide gel. The complexes migrate more slowly through the gel than unbound probe. Depending on the activity of the binding protein, a labeled probe can be either double-stranded or single-stranded. For the detection of DNA binding proteins such as transcription factors, either purified or partially purified proteins, or nuclear cell extracts can be used. For detection of RNA binding proteins, either purified or partially purified proteins, or nuclear or cytoplasmic cell extracts can be used. The specificity of the DNA or RNA binding protein for the putative binding site is established by competition experiments using DNA or RNA fragments or oligonucleotides containing a binding site for the protein of interest, or other unrelated sequence. The differences in the nature and intensity of the complex formed in the presence of specific and nonspecific competitor allows identification of specific interactions. Refer to Promega, Gel Shift Assay FAQ, available at <http: / / www.promega.com / faq / gelshfaq.html> (last visited March 25, 2005), which is herein incorporated by reference in its entirety for teachings regarding gel shift methods. 99. Gel shift methods can include using, for example, colloidal forms of COOMASSIE (Imperial Chemicals Industries, Ltd) blue stain to detect proteins in gels such as polyacrylamide electrophoresis gels. Such methods are described, for example, in Neuhoff et al., Electrophoresis 6:427-448 (1985), and Neuhoff et al., Electrophoresis 9:255-262 (1988), each of which is herein incorporated by reference in its entirety for teachings regarding gel shift methods. In addition to the conventional protein assay methods referenced above, a combination cleaning and protein staining composition is described in U.S. Patent 5,424,000, herein incorporated by reference in its entirety for its teaching regarding gel shift methods. The solutions can include phosphoric, sulfuric, and nitric acids, and Acid Violet dye. 100. Radioimmune Precipitation Assay (RIPA) is a sensitive assay using radiolabeled antigens to detect specific antibodies in serum. The antigens are allowed to react with the serum and then precipitated using a special reagent such as, for example, protein A sepharose beads. The bound radiolabeled immunoprecipitate is then commonly analyzed by gel electrophoresis. Radioimmunoprecipitation assay (RIPA) is often used as a confirmatory test for diagnosing the presence of HIV antibodies. RIPA is also referred to in the art as Farr Assay, Precipitin Assay, Radioimmune Precipitin Assay; Radioimmunoprecipitation Analysis; Radioimmunoprecipitation Analysis, and Radioimmunoprecipitation Analysis. 101. While the above immunoassays that utilize electrophoresis to separate and detect the specific proteins of interest allow for evaluation of protein size, they are not very sensitive for Attorney Docket Number 10110-473WO1 evaluating protein concentration. However, also contemplated are immunoassays wherein the protein or antibody specific for the protein is bound to a solid support (e.g., tube, well, bead, or cell) to capture the antibody or protein of interest, respectively, from a sample, combined with a method of detecting the protein or antibody specific for the protein on the support. Examples of such immunoassays include Radioimmunoassay (RIA), Enzyme-Linked Immunosorbent Assay (ELISA), Flow cytometry, protein array, multiplexed bead assay, and magnetic capture. 102. Radioimmunoassay (RIA) is a classic quantitative assay for detection of antigen- antibody reactions using a radioactively labeled substance (radioligand), either directly or indirectly, to measure the binding of the unlabeled substance to a specific antibody or other receptor system. Radioimmunoassay is used, for example, to test hormone levels in the blood without the need to use a bioassay. Non-immunogenic substances (e.g., haptens) can also be measured if coupled to larger carrier proteins (e.g., bovine gamma-globulin or human serum albumin) capable of inducing antibody formation. RIA involves mixing a radioactive antigen (because of the ease with which iodine atoms can be introduced into tyrosine residues in a protein, the radioactive isotopes125I or131I are often used) with antibody to that antigen. The antibody is generally linked to a solid support, such as a tube or beads. Unlabeled or “cold” antigen is then adding in known quantities and measuring the amount of labeled antigen displaced. Initially, the radioactive antigen is bound to the antibodies. When cold antigen is added, the two compete for antibody binding sites - and at higher concentrations of cold antigen, more binds to the antibody, displacing the radioactive variant. The bound antigens are separated from the unbound ones in solution and the radioactivity of each used to plot a binding curve. The technique is both extremely sensitive, and specific. 103. Enzyme-Linked Immunosorbent Assay (ELISA), or more generically termed EIA (Enzyme ImmunoAssay), is an immunoassay that can detect an antibody specific for a protein. In such an assay, a detectable label bound to either an antibody-binding or antigen-binding reagent is an enzyme. When exposed to its substrate, this enzyme reacts in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or visual means. Enzymes which can be used to detectably label reagents useful for detection include, but are not limited to, horseradish peroxidase, alkaline phosphatase, glucose oxidase, ^- galactosidase, ribonuclease, urease, catalase, malate dehydrogenase, staphylococcal nuclease, asparaginase, yeast alcohol dehydrogenase, alpha.-glycerophosphate dehydrogenase, triose phosphate isomerase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Attorney Docket Number 10110-473WO1 104. Variations of ELISA techniques are know to those of skill in the art. In one variation, antibodies that can bind to proteins can be immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing a marker antigen can be added to the wells. After binding and washing to remove non-specifically bound immunocomplexes, the bound antigen can be detected. Detection can be achieved by the addition of a second antibody specific for the target protein, which is linked to a detectable label. This type of ELISA is a simple “sandwich ELISA.” Detection also can be achieved by the addition of a second antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label. 105. Another variation is a competition ELISA. In competition ELISA’s, test samples compete for binding with known amounts of labeled antigens or antibodies. The amount of reactive species in the sample can be determined by mixing the sample with the known labeled species before or during incubation with coated wells. The presence of reactive species in the sample acts to reduce the amount of labeled species available for binding to the well and thus reduces the ultimate signal. 106. Regardless of the format employed, ELISAs have certain features in common, such as coating, incubating or binding, washing to remove non-specifically bound species, and detecting the bound immunecomplexes. Antigen or antibodies can be linked to a solid support, such as in the form of plate, beads, dipstick, membrane or column matrix, and the sample to be analyzed applied to the immobilized antigen or antibody. In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate can then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells can then be “coated” with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein and solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface. 107. In ELISAs, a secondary or tertiary detection means rather than a direct procedure can also be used. Thus, after binding of a protein or antibody to the well, coating with a non- reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the control clinical or biological sample to be tested under conditions effective to allow immunecomplex (antigen / antibody) formation. Detection of the Attorney Docket Number 10110-473WO1 immunecomplex then requires a labeled secondary binding agent or a secondary binding agent in conjunction with a labeled third binding agent. 108. Enzyme-Linked Immunospot Assay (ELISPOT) is an immunoassay that can detect an antibody specific for a protein or antigen. In such an assay, a detectable label bound to either an antibody-binding or antigen-binding reagent is an enzyme. When exposed to its substrate, this enzyme reacts in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or visual means. Enzymes which can be used to detectably label reagents useful for detection include, but are not limited to, horseradish peroxidase, alkaline phosphatase, glucose oxidase, ^-galactosidase, ribonuclease, urease, catalase, malate dehydrogenase, staphylococcal nuclease, asparaginase, yeast alcohol dehydrogenase, alpha.-glycerophosphate dehydrogenase, triose phosphate isomerase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. In this assay a nitrocellulose microtiter plate is coated with antigen. The test sample is exposed to the antigen and then reacted similarly to an ELISA assay. Detection differs from a traditional ELISA in that detection is determined by the enumeration of spots on the nitrocellulose plate. The presence of a spot indicates that the sample reacted to the antigen. The spots can be counted and the number of cells in the sample specific for the antigen determined. 109. “Under conditions effective to allow immunecomplex (antigen / antibody) formation” means that the conditions include diluting the antigens and antibodies with solutions such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS) / Tween so as to reduce non-specific binding and to promote a reasonable signal to noise ratio. 110. The suitable conditions also mean that the incubation is at a temperature and for a period of time sufficient to allow effective binding. Incubation steps can typically be from about 1 minute to twelve hours, at temperatures of about 20º to 30º C, or can be incubated overnight at about 0º C to about 10º C. 111. Following all incubation steps in an ELISA, the contacted surface can be washed so as to remove non-complexed material. A washing procedure can include washing with a solution such as PBS / Tween or borate buffer. Following the formation of specific immunecomplexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immunecomplexes can be determined. 112. To provide a detecting means, the second or third antibody can have an associated label to allow detection, as described above. This can be an enzyme that can generate color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one can contact and incubate the first or second immunecomplex with a labeled antibody for a period Attorney Docket Number 10110-473WO1 of time and under conditions that favor the development of further immunecomplex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS- Tween). 113. After incubation with the labeled antibody, and subsequent to washing to remove unbound material, the amount of label can be quantified, e.g., by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2’-azido-di-(3-ethyl-benzthiazoline-6-sulfonic acid [ABTS] and H2O2, in the case of peroxidase as the enzyme label. Quantitation can then be achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer. 114. Protein arrays are solid-phase ligand binding assay systems using immobilized proteins on surfaces which include glass, membranes, microtiter wells, mass spectrometer plates, and beads or other particles. The assays are highly parallel (multiplexed) and often miniaturized (microarrays, protein chips). Their advantages include being rapid and automatable, capable of high sensitivity, economical on reagents, and giving an abundance of data for a single experiment. Bioinformatics support is important; the data handling demands sophisticated software and data comparison analysis. However, the software can be adapted from that used for DNA arrays, as can much of the hardware and detection systems. 115. One of the chief formats is the capture array, in which ligand-binding reagents, which are usually antibodies but can also be alternative protein scaffolds, peptides or nucleic acid aptamers, are used to detect target molecules in mixtures such as plasma or tissue extracts. In diagnostics, capture arrays can be used to carry out multiple immunoassays in parallel, both testing for several analytes in individual sera for example and testing many serum samples simultaneously. In proteomics, capture arrays are used to quantitate and compare the levels of proteins in different samples in health and disease, i.e. protein expression profiling. Proteins other than specific ligand binders are used in the array format for in vitro functional interaction screens such as protein-protein, protein-DNA, protein-drug, receptor-ligand, enzyme-substrate, etc. The capture reagents themselves are selected and screened against many proteins, which can also be done in a multiplex array format against multiple protein targets. 116. For construction of arrays, sources of proteins include cell-based expression systems for recombinant proteins, purification from natural sources, production in vitro by cell- free translation systems, and synthetic methods for peptides. Many of these methods can be automated for high throughput production. For capture arrays and protein function analysis, it is important that proteins should be correctly folded and functional; this is not always the case, e.g. where recombinant proteins are extracted from bacteria under denaturing conditions. Attorney Docket Number 10110-473WO1 Nevertheless, arrays of denatured proteins are useful in screening antibodies for cross-reactivity, identifying autoantibodies and selecting ligand binding proteins. 117. Protein arrays have been designed as a miniaturization of familiar immunoassay methods such as ELISA and dot blotting, often utilizing fluorescent readout, and facilitated by robotics and high throughput detection systems to enable multiple assays to be carried out in parallel. Commonly used physical supports include glass slides, silicon, microwells, nitrocellulose or PVDF membranes, and magnetic and other microbeads. While microdrops of protein delivered onto planar surfaces are the most familiar format, alternative architectures include CD centrifugation devices based on developments in microfluidics (Gyros, Monmouth Junction, NJ) and specialised chip designs, such as engineered microchannels in a plate (e.g., The Living Chip™, Biotrove, Woburn, MA) and tiny 3D posts on a silicon surface (Zyomyx, Hayward CA). Particles in suspension can also be used as the basis of arrays, providing they are coded for identification; systems include colour coding for microbeads (Luminex, Austin, TX; Bio-Rad Laboratories) and semiconductor nanocrystals (e.g., QDots™, Quantum Dot, Hayward, CA), and barcoding for beads (UltraPlex™, SmartBead Technologies Ltd, Babraham, Cambridge, UK) and multimetal microrods (e.g., Nanobarcodes™ particles, Nanoplex Technologies, Mountain View, CA). Beads can also be assembled into planar arrays on semiconductor chips (LEAPS technology, BioArray Solutions, Warren, NJ). 118. Immobilization of proteins involves both the coupling reagent and the nature of the surface being coupled to. A good protein array support surface is chemically stable before and after the coupling procedures, allows good spot morphology, displays minimal nonspecific binding, does not contribute a background in detection systems, and is compatible with different detection systems. The immobilization method used are reproducible, applicable to proteins of different properties (size, hydrophilic, hydrophobic), amenable to high throughput and automation, and compatible with retention of fully functional protein activity. Orientation of the surface-bound protein is recognized as an important factor in presenting it to ligand or substrate in an active state; for capture arrays the most efficient binding results are obtained with orientated capture reagents, which generally require site-specific labeling of the protein. 119. Both covalent and noncovalent methods of protein immobilization are used and have various pros and cons. Passive adsorption to surfaces is methodologically simple, but allows little quantitative or orientational control; it may or may not alter the functional properties of the protein, and reproducibility and efficiency are variable. Covalent coupling methods provide a stable linkage, can be applied to a range of proteins and have good reproducibility; however, orientation may be variable, chemical derivatization may alter the function of the protein and Attorney Docket Number 10110-473WO1 requires a stable interactive surface. Biological capture methods utilizing a tag on the protein provide a stable linkage and bind the protein specifically and in reproducible orientation, but the biological reagent must first be immobilized adequately and the array may require special handling and have variable stability. 120. Several immobilization chemistries and tags have been described for fabrication of protein arrays. Substrates for covalent attachment include glass slides coated with amino- or aldehyde-containing silane reagents. In the Versalinx™ system (Prolinx, Bothell, WA) reversible covalent coupling is achieved by interaction between the protein derivatised with phenyldiboronic acid, and salicylhydroxamic acid immobilized on the support surface. This also has low background binding and low intrinsic fluorescence and allows the immobilized proteins to retain function. Noncovalent binding of unmodified protein occurs within porous structures such as HydroGel™ (PerkinElmer, Wellesley, MA), based on a 3-dimensional polyacrylamide gel; this substrate is reported to give a particularly low background on glass microarrays, with a high capacity and retention of protein function. Widely used biological coupling methods are through biotin / streptavidin or hexahistidine / Ni interactions, having modified the protein appropriately. Biotin may be conjugated to a poly-lysine backbone immobilised on a surface such as titanium dioxide (Zyomyx) or tantalum pentoxide (Zeptosens, Witterswil, Switzerland). 121. Array fabrication methods include robotic contact printing, ink-jetting, piezoelectric spotting and photolithography. A number of commercial arrayers are available [e.g. Packard Biosciences] as well as manual equipment [V & P Scientific]. Bacterial colonies can be robotically gridded onto PVDF membranes for induction of protein expression in situ. 122. At the limit of spot size and density are nanoarrays, with spots on the nanometer spatial scale, enabling thousands of reactions to be performed on a single chip less than 1mm square. BioForce Laboratories have developed nanoarrays with 1521 protein spots in 85sq microns, equivalent to 25 million spots per sq cm, at the limit for optical detection; their readout methods are fluorescence and atomic force microscopy (AFM). 123. Fluorescence labeling and detection methods are widely used. The same instrumentation as used for reading DNA microarrays is applicable to protein arrays. For differential display, capture (e.g., antibody) arrays can be probed with fluorescently labeled proteins from two different cell states, in which cell lysates are directly conjugated with different fluorophores (e.g. Cy-3, Cy-5) and mixed, such that the color acts as a readout for changes in target abundance. Fluorescent readout sensitivity can be amplified 10-100 fold by tyramide signal amplification (TSA) (PerkinElmer Lifesciences). Planar waveguide technology (Zeptosens) enables ultrasensitive fluorescence detection, with the additional advantage of no intervening Attorney Docket Number 10110-473WO1 washing procedures. High sensitivity can also be achieved with suspension beads and particles, using phycoerythrin as label (Luminex) or the properties of semiconductor nanocrystals (Quantum Dot). A number of novel alternative readouts have been developed, especially in the commercial biotech arena. These include adaptations of surface plasmon resonance (HTS Biosystems, Intrinsic Bioprobes, Tempe, AZ), rolling circle DNA amplification (Molecular Staging, New Haven CT), mass spectrometry (Intrinsic Bioprobes; Ciphergen, Fremont, CA), resonance light scattering (Genicon Sciences, San Diego, CA) and atomic force microscopy [BioForce Laboratories]. 124. Capture arrays form the basis of diagnostic chips and arrays for expression profiling. They employ high affinity capture reagents, such as conventional antibodies, single domains, engineered scaffolds, peptides or nucleic acid aptamers, to bind and detect specific target ligands in high throughput manner. 125. Antibody arrays have the required properties of specificity and acceptable background, and some are available commercially (BD Biosciences, San Jose, CA; Clontech, Mountain View, CA; BioRad; Sigma, St. Louis, MO). Antibodies for capture arrays are made either by conventional immunization (polyclonal sera and hybridomas), or as recombinant fragments, usually expressed in E. coli, after selection from phage or ribosome display libraries (Cambridge Antibody Technology, Cambridge, UK; BioInvent, Lund, Sweden; Affitech, Walnut Creek, CA; Biosite, San Diego, CA). In addition to the conventional antibodies, Fab and scFv fragments, single V-domains from camelids (VHH) or engineered human equivalents (Domantis, Waltham, MA) may also be useful in arrays. 126. The term “scaffold” refers to ligand-binding domains of proteins, which are engineered into multiple variants capable of binding diverse target molecules with antibody-like properties of specificity and affinity. The variants can be produced in a genetic library format and selected against individual targets by phage, bacterial or ribosome display. Such ligand-binding scaffolds or frameworks include ‘Affibodies’ based on Staph. aureus protein A (Affibody, Bromma, Sweden), ‘Trinectins’ based on fibronectins (Phylos, Lexington, MA) and ‘Anticalins’ based on the lipocalin structure (Pieris Proteolab, Freising-Weihenstephan, Germany). These can be used on capture arrays in a similar fashion to antibodies and may have advantages of robustness and ease of production. 127. Nonprotein capture molecules, notably the single-stranded nucleic acid aptamers which bind protein ligands with high specificity and affinity, are also used in arrays (SomaLogic, Boulder, CO). Aptamers are selected from libraries of oligonucleotides by the Selex™ procedure and their interaction with protein can be enhanced by covalent attachment, through incorporation of brominated deoxyuridine and UV-activated crosslinking (photoaptamers). Photocrosslinking to Attorney Docket Number 10110-473WO1 ligand reduces the crossreactivity of aptamers due to the specific steric requirements. Aptamers have the advantages of ease of production by automated oligonucleotide synthesis and the stability and robustness of DNA; on photoaptamer arrays, universal fluorescent protein stains can be used to detect binding. 128. Protein analytes binding to antibody arrays may be detected directly or via a secondary antibody in a sandwich assay. Direct labelling is used for comparison of different samples with different colours. Where pairs of antibodies directed at the same protein ligand are available, sandwich immunoassays provide high specificity and sensitivity and are therefore the method of choice for low abundance proteins such as cytokines; they also give the possibility of detection of protein modifications. Label- free detection methods, including mass spectrometry, surface plasmon resonance and atomic force microscopy, avoid alteration of ligand. What is required from any method is optimal sensitivity and specificity, with low background to give high signal to noise. Since analyte concentrations cover a wide range, sensitivity has to be tailored appropriately; serial dilution of the sample or use of antibodies of different affinities are solutions to this problem. Proteins of interest are frequently those in low concentration in body fluids and extracts, requiring detection in the pg range or lower, such as cytokines or the low expression products in cells. 129. An alternative to an array of capture molecules is one made through ‘molecular imprinting’ technology, in which peptides (e.g., from the C-terminal regions of proteins) are used as templates to generate structurally complementary, sequence-specific cavities in a polymerizable matrix; the cavities can then specifically capture (denatured) proteins that have the appropriate primary amino acid sequence (ProteinPrint™, Aspira Biosystems, Burlingame, CA). 130. Another methodology which can be used diagnostically and in expression profiling is the ProteinChip® array (Ciphergen, Fremont, CA), in which solid phase chromatographic surfaces bind proteins with similar characteristics of charge or hydrophobicity from mixtures such as plasma or tumour extracts, and SELDI-TOF mass spectrometry is used to detection the retained proteins. 131. Large-scale functional chips have been constructed by immobilizing large numbers of purified proteins and used to assay a wide range of biochemical functions, such as protein interactions with other proteins, drug-target interactions, enzyme-substrates, etc. Generally they require an expression library, cloned into E. coli, yeast or similar from which the expressed proteins are then purified, e.g. via a His tag, and immobilized. Cell free protein transcription / translation is a viable alternative for synthesis of proteins which do not express well in bacterial or other in vivo systems. Attorney Docket Number 10110-473WO1 132. For detecting protein-protein interactions, protein arrays can be in vitro alternatives to the cell-based yeast two-hybrid system and may be useful where the latter is deficient, such as interactions involving secreted proteins or proteins with disulphide bridges. High-throughput analysis of biochemical activities on arrays has been described for yeast protein kinases and for various functions (protein-protein and protein-lipid interactions) of the yeast proteome, where a large proportion of all yeast open-reading frames was expressed and immobilised on a microarray. Large-scale ‘proteome chips’ promise to be very useful in identification of functional interactions, drug screening, etc. (Proteometrix, Branford, CT). 133. As a two-dimensional display of individual elements, a protein array can be used to screen phage or ribosome display libraries, in order to select specific binding partners, including antibodies, synthetic scaffolds, peptides and aptamers. In this way, ‘library against library’ screening can be carried out. Screening of drug candidates in combinatorial chemical libraries against an array of protein targets identified from genome projects is another application of the approach. 134. A multiplexed bead assay, such as, for example, the BD™ Cytometric Bead Array, is a series of spectrally discrete particles that can be used to capture and quantitate soluble analytes. The analyte is then measured by detection of a fluorescence-based emission and flow cytometric analysis. Multiplexed bead assay generates data that is comparable to ELISA based assays, but in a “multiplexed” or simultaneous fashion. Concentration of unknowns is calculated for the cytometric bead array as with any sandwich format assay, i.e. through the use of known standards and plotting unknowns against a standard curve. Further, multiplexed bead assay allows quantification of soluble analytes in samples never previously considered due to sample volume limitations. In addition to the quantitative data, powerful visual images can be generated revealing unique profiles or signatures that provide the user with additional information at a glance. 1. Antibodies (a) Antibodies Generally 135. The term “antibodies” is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, also included in the term “antibodies” are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules or fragments thereof, as long as they are chosen for their ability to interact with DLL4 such that DLL4 is inhibited from interacting with Notch1. The antibodies can be tested for their desired activity using the in vitro assays described herein, or by analogous methods, after which their in vivo therapeutic and / or prophylactic activities are tested according to known clinical testing methods. There are five major classes of human Attorney Docket Number 10110-473WO1 immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. One skilled in the art would recognize the comparable classes for mouse. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. 136. The term “monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules. The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity. 137. The disclosed monoclonal antibodies can be made using any procedure which produces mono clonal antibodies. For example, disclosed monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro. 138. The monoclonal antibodies may also be made by recombinant DNA methods. DNA encoding the disclosed monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques, e.g., as described in U.S. Patent No. 5,804,440 to Burton et al. and U.S. Patent No. 6,096,441 to Barbas et al. 139. In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94 / 29348 published Dec. 22, 1994 and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typically produces two identical antigen Attorney Docket Number 10110-473WO1 binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment that has two antigen combining sites and is still capable of cross-linking antigen. 140. As used herein, the term “antibody or fragments thereof” encompasses chimeric antibodies and hybrid antibodies, with dual or multiple antigen or epitope specificities, and fragments, such as F(ab’)2, Fab’, Fab, Fv, scFv, VHH, and the like, including hybrid fragments. Thus, fragments of the antibodies that retain the ability to bind their specific antigens are provided. For example, fragments of antibodies which maintain DLL4, JAG1, JAG2 and or Notch1 binding activity are included within the meaning of the term “antibody or fragment thereof.” Such antibodies and fragments can be made by techniques known in the art and can be screened for specificity and activity according to the methods set forth in the Examples and in general methods for producing antibodies and screening antibodies for specificity and activity (See Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988)). 141. Also included within the meaning of “antibody or fragments thereof” are conjugates of antibody fragments and antigen binding proteins (single chain antibodies). 142. The fragments, whether attached to other sequences or not, can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove / add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc. In any case, the antibody or antibody fragment must possess a bioactive property, such as specific binding to its cognate antigen. Functional or active regions of the antibody or antibody fragment may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to a skilled practitioner in the art and can include site-specific mutagenesis of the nucleic acid encoding the antibody or antibody fragment. (Zoller, M.J. Curr. Opin. Biotechnol.3:348-354, 1992). 143. As used herein, the term “antibody” or “antibodies” can also refer to a human antibody and / or a humanized antibody. Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans, and thus can give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods serves to lessen the chance that an antibody administered to a human will evoke an undesirable immune response. Attorney Docket Number 10110-473WO1 (b) Human antibodies 144. The disclosed human antibodies can be prepared using any technique. The disclosed human antibodies can also be obtained from transgenic animals. For example, transgenic, mutant mice that are capable of producing a full repertoire of human antibodies, in response to immunization, have been described (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551-255 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993)). Specifically, the homozygous deletion of the antibody heavy chain joining region (J(H)) gene in these chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production, and the successful transfer of the human germ-line antibody gene array into such germ-line mutant mice results in the production of human antibodies upon antigen challenge. Antibodies having the desired activity are selected using Env-CD4-co- receptor complexes as described herein. (c) Humanized antibodies 145. Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule. Accordingly, a humanized form of a non-human antibody (or a fragment thereof) is a chimeric antibody or antibody chain (or a fragment thereof, such as an sFv, Fv, Fab, Fab’, F(ab’)2, or other antigen-binding portion of an antibody) which contains a portion of an antigen binding site from a non-human (donor) antibody integrated into the framework of a human (recipient) antibody. 146. To generate a humanized antibody, residues from one or more complementarity determining regions (CDRs) of a recipient (human) antibody molecule are replaced by residues from one or more CDRs of a donor (non-human) antibody molecule that is known to have desired antigen binding characteristics (e.g., a certain level of specificity and affinity for the target antigen). In some instances, Fv framework (FR) residues of the human antibody are replaced by corresponding non-human residues. Humanized antibodies may also contain residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. Humanized antibodies generally contain at least a portion of an antibody constant region (Fc), typically that of a human antibody (Jones et al., Nature, 321:522-525 (1986), Reichmann et al., Nature, 332:323-327 (1988), and Presta, Curr. Opin. Struct. Biol., 2:593-596 (1992)). Attorney Docket Number 10110-473WO1 147. Methods for humanizing non-human antibodies are well known in the art. For example, humanized antibodies can be generated according to the methods of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986), Riechmann et al., Nature, 332:323-327 (1988), Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Methods that can be used to produce humanized antibodies are also described in U.S. Patent No. 4,816,567 (Cabilly et al.), U.S. Patent No. 5,565,332 (Hoogenboom et al.), U.S. Patent No. 5,721,367 (Kay et al.), U.S. Patent No. 5,837,243 (Deo et al.), U.S. Patent No. 5, 939,598 (Kucherlapati et al.), U.S. Patent No.6,130,364 (Jakobovits et al.), and U.S. Patent No.6,180,377 (Morgan et al.). (d) Administration of binding molecules 148. Administration of the binding molecules can be done as disclosed herein. Nucleic acid approaches for binding molecule delivery also exist. The binding molecules can also be administered to patients or subjects as a nucleic acid preparation (e.g., DNA or RNA) that encodes the antibody or antibody fragment, such that the patient's or subject's own cells take up the nucleic acid and produce and secrete the encoded antibody or antibody fragment. The delivery of the nucleic acid can be by any means, as disclosed herein, for example. 2. Pharmaceutical carriers / Delivery of pharmaceutical products 149. As described above, the compositions can also be administered in vivo in a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art. 150. The compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like, including topical intranasal administration or administration by inhalant. As used herein, "topical intranasal administration" means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector. Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation. The exact amount of the compositions required will vary from Attorney Docket Number 10110-473WO1 subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. 151. Parenteral administration of the composition, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No.3,610,795, which is incorporated by reference herein. 152. The materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as "stealth" and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor- level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Attorney Docket Number 10110-473WO1 Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)). (a) Pharmaceutically Acceptable Carriers 153. The compositions, including antibodies, can be used therapeutically in combination with a pharmaceutically acceptable carrier. 154. Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered. 155. Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art. 156. Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like. 157. The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally. 158. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl Attorney Docket Number 10110-473WO1 oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. 159. Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. 160. Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.. 161. Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines. (b) Therapeutic Uses 162. Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms of the disorder are effected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. For example, guidance in selecting appropriate doses for antibodies can be found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Attorney Docket Number 10110-473WO1 Park Ridge, N.J., (1985) ch.22 and pp.303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp.365-389. A typical daily dosage of the antibody used alone might range from about 1 µg / kg to up to 100 mg / kg of body weight or more per day, depending on the factors mentioned above. EXAMPLES 163. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and / or methods claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric. Example 1: Evaluate Notch1 activation in HEK293T cells overexpressing JAG1 and JAG1 H268Q and the binding properties of the JAG1.b70 antibody 164. To evaluate Notch1 activation in HEK293T cells overexpressing JAG1 and JAG1 H268Q : HEK293T cells were engineered to overexpress either wild-type JAG1 or the JAG1 H268Q mutant. These cells were co-cultured with Notch1-expressing cells to assess the fold change in Notch1 activation. A luciferase-based reporter assay was employed to quantify Notch1 signaling in different experimental groups: (1) HEK293T cells overexpressing wild-type JAG1, (2) HEK293T cells overexpressing JAG1 H268Q mutant, and (3) a control group of Notch1 cells cultured alone. 165. In another experiment binding affinity of JAG1.b70 antibody was evaluated: To investigate the binding properties of the JAG1.b70 antibody, HEK293T cells overexpressing JAG1 were used. Surface plasmon resonance (SPR) was performed to measure the affinity and specificity of the JAG1.b70 antibody for JAG1. The control group consisted of HEK293T cells without JAG1 expression, serving as a baseline to confirm the antibody’s selectivity. Materials and Methods: Protein expression and purification 166. All SNAG sequences were cloned into a pAcGP67A vector for insect cell production containing an N-terminal gp67 signal peptide and C-terminal 8xHis-tag. Monomeric SNAGs were generated by fusing a truncated version of the DeltaMAXprotein spanning from the N-terminus to EGF5 (N- EGF5) fused to a biomarker-targeting scFv or nanobody using a flexible Attorney Docket Number 10110-473WO1 (GS)5 linker. Dimeric SNAGFcconstructs were generated by fusing DeltaMAX(N-EGF5) and the JAG1 scFV to the N- and C-termini of a human IgG1 Fc domain, respectively. All SNAGFcconstructs contained short GSG-linkers between the Fc sequence and DeltaMAXor the JAG1 scFV. Each scFv was generated by fusing the C-terminus of the variable heavy (VH) domain to the N- terminus of the variable light (VL) domain with a (GGGGS)3linker. 167. All SNAG constructs in this study were expressed for by infecting Trichoplusia ni insect cell cultures (Expression Systems) at a density of 2 ×106cells ml−1with recombinant Baculovirus. Culture supernatants were harvested after 48h, and proteins were purified by nickel and size- exclusion chromatography. Biotinylated proteins were site-specifically modified using BirA ligase and excess biotin was removed by purifying the proteins on a size-exclusion column. Protein purity was assessed by SDS-PAGE using TGX 12% Precast gels (Bio-Rad). All proteins were flash-frozen in liquid nitrogen and stored at −80 °C following purification. Cell culture and generation of cell lines 168. Mammalian cells were cultured at 37 °C, with a humidified atmosphere of 5% CO2, washed with Dulbecco’s PBS (DPBS, Corning), and detached with trypsin-EDTA 0.25% (Gibco) for subculturing or cell-based assays. Briefly, transfections of HEK293T cells were carried out with packaging vectors VSV-G and d8.9 in the presence of polyethyleneimine at a ratio of 4:1 (DNA:polyethyleneimine). HEK293T cells were cultured in high-glucose DMEM (Cytiva) supplemented with 10% FBS (peak serum) and 2% penicillin / streptomycin (Gibco). Puromycin was added to HEK293T cell cultures to maintain homogeneous populations of receptor-expressing cells. Expression of receptors on the cell surface was confirmed by flow cytometry (BD Accuri C6 plus) staining the cell lines with anti- hJAG1 PE in DMEM supplemented with 10% FBS for 1 h at 4 °C. Notch activation with DeltaMAXmultimers 169. On day one, DeltaMAX-Fc were reconstituted in DPBS and adsorbed to tissue culture 96-well plates (Coastar) for 1 h at 37 °C. The wells were then washed three times with 200 ^l of DPBS to remove unbound proteins. Next, CHO-K1 N1-Gal4 cells were detached with trypsin-EDTA 0.25% (Gibco) and manually counted. Appropriate dilutions were prepared in ^- MEM media to ensure 30,000 CHO-K1 N1- Gal4 cells per well in a volume of 50 ^L. Cells were transferred to the ligand-coated plates and cultured for 24 h at 37 °C in 5% CO2. On day two, CHO-K1 N1-Gal4 cells were washed with 200 µl DPBS, detached with 30 µL of trypsin-EDTA 0.25%, and quenched with 170 ^L of ^-MEM media. Finally, cells were resuspended, and the H2B-mCitrine signal was measured by flow cytometry (BD Accuri C6 plus). CHO-K1 N1-Gal4 cells alone were used as the control. The measurements represent the mean fluorescent intensity Attorney Docket Number 10110-473WO1 as fold-change of Notch activation ± s.d. of three technical replicates. Notch activation was normalized to wells containing CHO-K1 N1-Gal4 cells alone. Notch activation with SNAGs in coculture of cells expressing the JAG1 170. On day one, cells expressing the JAG1 of the SNAG (signal-sending cells) were detached with trypsin-EDTA, counted manually, and dilutions prepared such that 50 ^L of DMEM containing 15,000 signal-sender cells were added to wells of a tissue culture 96-well plate. The next day, CHO-K1 N1-Gal4 reporter cells (signal-receiver cells) were detached with trypsin- EDTA, and 50 µl of ^-MEM media containing 30,000 cells were added to the tissue culture 96- well plate containing the signal-sending cells after combining with the indicated DeltaMAXor SNAG protein. Wells without signal-sending cells were used to determine background activation of Notch by DeltaMAXand SNAGs. When testing inhibition of endocytosis, 80 µM of the Dynamin inhibitor I (Dynasore, Sigma) was added to the mixture of Notch reporter cells with protein and added to the tissue culture 96-well plate containing the signal-sending cells. Notch activation was measured as previously described. Results: 171. In the first experiment, co-culturing Notch1 cells with HEK293T cells overexpressing wild-type JAG1 led to a robust increase in Notch1 activation, resulting in a 6- to 8-fold enhancement compared to the control group of Notch1 cells alone. However, the introduction of the JAG1 H268Q mutant showed a marked reduction in activation, with only a 2- to 4-fold increase in Notch1 activity. These results demonstrate that the JAG1 H268Q mutation impairs the ability of JAG1 to activate Notch1 signaling. 172. In the second experiment, SPR analysis revealed that the JAG1.b70 antibody exhibited high affinity for the JAG1 protein, with a dissociation constant (Kd) in the nanomolar range. The antibody demonstrated remarkable selectivity, binding exclusively to HEK293T cells overexpressing JAG1, while no significant binding was observed in the control group of HEK293T cells without JAG1 expression. This indicates the high specificity of JAG1.b70 towards the JAG1 protein. Discussion: 173. The results of these experiments provide key insights into the functional differences between wild-type JAG1 and the JAG1H268Q mutant, as well as the therapeutic potential of the JAG1-SNAG bispecific construct. The impaired Notch1 activation observed in the presence of the JAG1 H268Q mutant suggests that this mutation disrupts the ability of JAG1 to effectively engage Notch1, which is critical in the pathophysiology of diseases such as Alagille Attorney Docket Number 10110-473WO1 syndrome. Reduced signaling in the JAG1 H268Q mutant group shows that mutations in JAG1 contribute to defective Notch signaling, a hallmark of several genetic disorders. 174. Furthermore, the high affinity and selectivity of the JAG1.b70 antibody for JAG1 highlights its potential as a therapeutic tool for modulating JAG1-mediated signaling pathways. The specificity of JAG1.b70 reduces the likelihood of off-target effects, making it an attractive candidate for therapies aimed at conditions where aberrant JAG1-Notch interactions play a role. 175. In conclusion, the data not only underscore the importance of JAG1 in mediating Notch1 signaling but also demonstrate the potential of selective bispecific constructs like JAG1- SNAG in therapeutic applications. Example 2: Engineering synthetic agonists for targeted activation of Notch signaling 176. The Notch pathway is a cell-to-cell communication system that regulates embryonic development, tissue homeostasis, and immune cell differentiation. The signaling of mechanosensitive Notch receptors is tightly regulated and aberrant Notch activity causes various human diseases. For example, loss-of-function mutations in Notch components are linked to the development of aortic valve disease (Notch1), Alagille syndrome (Notch2, Jagged1), CADASIL (Notch3), spondylocostal dysostosis (DLL3). In cancer, Notch functions as a tumor suppressor or oncogene depending on the cell type, and both loss-of-function and hyperactivating mutations influence tumorigenesis and disease progression. Notch is also pleiotropic with respect to its guidance of cell fate decisions, in that Notch activation stimulates either proliferation or differentiation in different stem cell populations. These diverse functions suggest that Notch agonists and antagonists are viable therapeutics in certain biomedical contexts. 177. The role of Notch in T cell biology has led to the development of Notch-based strategies for enhancing cancer immunotherapy. Notch signaling is important for several natural stages of T cell maturation, and ex vivo Notch activation is required for the differentiation of T cells from hematopoietic stem cells (HSCs). This function is used to generate allogeneic T cells for off-the-shelf adoptive T cell or chimeric antigen receptor (CAR) T cell therapies. Notch activation enhances the antitumor function of fully mature, activated T cells. Genetic overexpression of an activated form of Notch, as well as culturing T cells in the presence of Notch1-specific antibodies or ligand-expressing cells, are associated with improved tumor clearance in various animal models of cancer. Detailed analysis of the T cells used in these studies reveals that these phenotypes are due to the Notch-stimulated induction of exhaustion-resistant or stem-like phenotypes. Attorney Docket Number 10110-473WO1 178. At the molecular level, Notch receptors are massive (~290kD) transmembrane proteins that are activated by a distinctive, mechanical force-driven mechanism. Notch signaling is initiated when a Delta-like (DLL) or Jagged (JAG) ligand forms a trans-interaction with a Notch receptor on the surface of an adjacent cell. Endocytosis of the ligand then generates a pulling force that propagates to the negative regulatory region (NRR) of Notch. This pulling destabilizes the NRR, which exposes internal cleavage sites for processing by the intramembrane proteases ADAM10 (S2 cleavage) and ^-secretase (S3 cleavage). Following these proteolytic events, the Notch intracellular domain (NICD) translocates to the nucleus to function as a transcriptional co- activator. 179. Notch inhibitors are widely available, but soluble agonists are challenging engineers because they must pull on the Notch receptor despite lacking a method of force generation. Multiple strategies have been developed to activate Notch receptors in vitro through mimicry of the physiological activation process. Notch signaling is induced through co-culture of Notch-expressing cells and ligand-expressing cells, by culturing Notch-expressing cells on plates coated with ligands or antibodies, or by administration of ligand-coated microbeads. The only Notch agonist antibody, A13, functions by destabilizing the metastable NRR domain of Notch3. This NRR unfolding approach is ineffective for receptor subtypes with stable NRRs (Notch1 / 2 / 4). Oligomerized ligands also show limited promise as agonists. For example, pre-clustering DLL1- Fc fusions with anti-Fc antibodies, JAG1-coated DNA-origami structures, and DLL4 proteins fused to designed trimeric scaffolds (C3-DLL4) stimulate low levels of Notch activation in vitro. 180. In this study, bispecific proteins are engineered that stimulate activation of Notch signaling in desired cellular contexts. SNAGs are developed that enhance the signaling of weakly activating JAG1 ligands, as well as those that selectively activate Notch in the presence of several immunologically-relevant cell surface biomarkers. SNAGs stimulate increased expression of Notch target genes and T cell activation markers in primary human T cells. The modularity and versatility of this SNAG platform provide a blueprint for the development of a diverse repertoire of Notch-based biologics. Results: Soluble DLL4 ligand multimers do not activate Notch signaling. 181. As an initial attempt to generate Notch agonists, an investigation is conducted to determine whether soluble oligomers of an affinity-matured DLL4 ligand (DeltaMAX) activate Notch signaling. DeltaMAXcontains ten mutations that increase its affinity for human Notch receptors by 500- to 1000-fold (KD 24 to 54 nM), making it a more potent activator than DLL4 (KD > 10 µM) in co-culture and plate-bound formats. The increased affinity, coupled with Attorney Docket Number 10110-473WO1 receptor crosslinking through multimerization, introduces tension in the absence of an endocytic pulling force. To test this hypothesis, Notch1-Gal4 mCitrine reporter cells are incubated with soluble and immobilized DeltaMAXmultimers (FIGS. 3A-3C). DeltaMAXdimers are generated through the C-terminal addition of a dimeric human IgG1 Fc domain (FIG.3B), and tetramers are generated by pre-mixing a 4:1 molar ratio of biotinylated DeltaMAXwith streptavidin (FIG. 3C). Neither the monomers nor the multimers induce reporter activity. By contrast, the plated DeltaMAXligands potently stimulate Notch1 activation (FIGS. 3A-3C). This indicates that the receptor crosslinking by DeltaMAX-Fc dimers and DeltaMAX-SA tetramers is insufficient for signaling activation. Design of synthetic Notch agonists. 182. To develop soluble Notch agonists, bispecific proteins are engineered that recapitulate the endocytosis-linked activation mechanism of DLL and JAG ligands (FIG. 3D). SNAGs are created by fusing DeltaMAXto the N-terminus of biomarker-targeting antibody fragments via a flexible (GS)5 linker, or by fusing DeltaMAXand antibody fragments to the N- and C-termini of a dimeric IgG1 Fc domain (FIG.3E). These design concepts form a molecular bridge between Notch-expressing cells and cells that express a given surface protein. SNAGs then activate Notch if the enforced interactions induce endocytic or tensile force capable of unfolding the NRR. To this end, a modular SNAG platform (FIG. 3F) is developed to have exchangeable modules for biomarker-specific SNAGs against canonical Notch ligands (FIG. 3G), biomarkers upregulated in tumors (FIG.3H), and an immunostimulatory biomarker (FIG.3I). SNAGs rescue the signaling of a signaling-deficient DLL4 mutant. 183. To demonstrate proof-of-concept, SNAGs are tested to determine whether they rescue the activity of a signaling-deficient DLL4 mutant. Loss-of-function DLL4 cells are generated by expressing a headless DLL4 truncation where the Notch-binding C2 and DSL domains are replaced with a BC2 epitope tag (BC2-DLL4HL) (FIG. 6A and FIG. 8A), BC2- SNAGs are then generated by fusing DeltaMAXto a BC2-specific nanobody (FIGS. 4A-4B and FIG.9A). BC2-DLL4HL cells alone do not activate signaling in a Notch1-Gal4 mCitrine reporter assay, whereas the addition of 1 nM to 100 nM concentrations of SNAGs stimulates a dose- dependent increase in reporter activity (FIG. 6C and FIGS. 10A-10C). Monomeric BC2-SNAGs containing the (GS)5 linker (BC2-SNAG) stimulate a ~6-fold increase in Notch1 signaling, whereas dimeric BC2-SNAG Fc fusion proteins (BC2-SNAGFc) are more effective and induce a ~10-fold increase (FIG.6C). Administration of the monomeric or dimeric BC2-SNAGs alone does not substantially increase Notch1 reporter activity, indicating that a mixture of target-expressing and non-expressing cells is required for SNAG-mediated activation (FIG.6C). Attorney Docket Number 10110-473WO1 SNAGs bolster the activity of weakly-signaling JAG1 ligands. 184. DLL or JAG ligands preferentially signal through certain Notch receptor subtypes, and JAG1 is a particularly weak activator of Notch1. A JAG1-targeting SNAG (JAG1-SNAGFc) is tested to determine whether it potentiates JAG1-Notch1 signaling. In the JAG1-SNAGFc construct, DeltaMAXand an scFv derived from the JAG1-targeting antibody B70 are fused to the N- and C-termini of an IgG1 Fc domain as described above (FIG. 9B). A signaling assay is performed to measure the activation of Notch1 reporter cells by JAG1-overexpressing HEK293 cells (JAG1-293 cells) in the presence or absence of the SNAG. Addition of the JAG1-SNAGFc increases Notch1 reporter activity by ~4-fold compared to JAG1-293 cells alone, and JAG1-293 cells do not stimulate a significant increase in reporter activity (FIG.6D and FIG.8B). The JAG1- SNAGFc is also evaluated with a JAG1 H268Q Nodder mutant that causes Alagille syndrome- like symptoms in mice by decreasing JAG1-Notch1 binding (FIG. 8C). Addition of the JAG1- SNAGFc to cocultures of Notch1 and JAG1H268Q cells increases JAG1 signaling by up to 7-fold compared to Notch1 cells alone (FIG.6E). These data indicate that SNAGs function as signaling enhancers by potentiating the activity of endogenous or mutated ligands. SNAGs targeting tumor antigens activate Notch in mixed cell populations. 185. SNAGs targeting the tumor antigens PD-L1, CD19, or HER2 (FIG.3H) are tested to determine whether they stimulate Notch activation. Notch signaling enhances the function of activated T cells, and SNAGs localized to the tumor microenvironment have the potential to stimulate localized activation of tumor-associated lymphocytes. For these SNAGs, the targeting arms are derived from antibody-drug conjugates (ADCs) that are pre-selected for their ability to induce target internalization. SNAGs incorporating ADC antibodies mimic the physiological endocytosis mechanism of DLL or JAG ligands. 186. Monomeric and dimeric PDL1-SNAGs are generated by fusing DeltaMAXto a single-chain variable fragment (scFv) derived from the ADC antibody Atezolizumab. In the monomeric PDL1-SNAG, DeltaMAXand the scFv are connected using a (GS)5 linker, and in the dimeric PDL1-SNAG (PDL1-SNAGFc), DeltaMAXand the scFv are fused to the N- and C-termini of an IgG1 Fc domain (FIG.3H and FIG. 9A). Addition of the monomeric PDL1-SNAG to a 1:1 mixture of Notch1 reporter cells and PDL1-expressing MDA-MB-231 cells does not activate Notch1 (FIG.5A and FIG.8D). The dimeric PDL1-SNAGFc protein stimulates a ~7-fold increase in Notch1 signaling in the coculture, suggesting that multimerization or avidity-enhancement is required for SNAGs to effectively target biomarkers other than Notch ligands (FIG.5A). Neither the PDL1-SNAG nor the PDL1-SNAGFc substantially increased Notch1 reporter activity in the absence of MDA-MB-231 cells. To test the importance of Notch-binding affinity in SNAG design, Attorney Docket Number 10110-473WO1 a PDL1-SNAGFc incorporating wild type DLL4 (FIG. 9D) is generated, which binds to Notch1 with ~1000-fold decreased affinity (Kd 24.7 µM) compared to DeltaMAX(Kd 24 nM). The wild type DLL4 SNAG is unable to activate Notch1 at all concentrations tested, indicating that the enhanced binding of the DeltaMAXvariant is essential for SNAG function (FIG.11). SNAGs do not activate signaling on cells expressing both Notch1 and PDL1. 187. Given the ubiquitous expression of Notch1 in mammalian cells, SNAGs could activate signaling when Notch1 and the target protein are both present on the cell surface. To test this possibility, MDA-MB-231 cells are cultured in the presence of soluble DeltaMAX-Fc, PDL1- SNAGFc, or immobilized DeltaMAX-Fc and the levels of activated Notch1 are monitored by Western Blot (FIG. 5B). The plated DeltaMAX-Fc protein stimulates high levels of Notch1 activation, whereas the PDL1-SNAGFc does not induce signaling over the background levels observed for soluble DeltaMAX-Fc alone (FIG.5B). The inability of SNAGs to activate Notch1 in MDA-MB-231 cells shows that the present design does not enable sufficient intercellular crosslinking in cultures of cells expressing both Notch1 and the biomarker. Development of SNAGs targeting CD19, HER2, and CD40. 188. To generate a SNAG targeting the B-lymphocyte antigen CD19, an scFv derived from the CD19-targeting ADC loncastuximab is fused to the C-terminus of DeltaMAX-Fc (CD19- SNAGFc, FIG.9A). The CD19-SNAG is then added to Notch1 reporter cells or to co-cultures of Notch1 reporter cells and CD19-overexpressing 3T3 fibroblast cells (FIG. 8E). The CD19- SNAGFc protein stimulates up to a 6-fold increase in reporter activity in the co-culture compared to untreated Notch1 cells (FIG. 5C). To generate a SNAG targeting the breast cancer antigen HER2 (HER2-SNAGFc), the CD19-targeting arm is replaced with an scFv derived from the HER2-targeting ADC trastuzumab (FIG.9C). Addition of the HER2-SNAGFc to a mixed culture of Notch1 reporter cells and HER2-expressing SK-BR-3 breast cancer cells induces a 6-fold increase in reporter activity (FIG. 5D and FIG. 8F) at the highest concentration tested (100 nM), which is similar to the level of activation observed for the PDL1-SNAGFc and the CD19-SNAGFc constructs (FIGS. 5A and 5C). In the absence of biomarker-expressing cells, neither the CD19- SNAGFc nor the HER2-SNAGFc stimulated a significant increase in signaling compared to DeltaMAX-Fc alone (FIGS. 5C-5D). The development of PD-L1, CD19, and HER2 SNAGs demonstrates that SNAGs facilitate signaling by engaging cell surface proteins beyond endogenous ligands. 189. A SNAG targeting CD40 (CD40-SNAG) is also generated, an immunostimulatory receptor that undergoes endocytosis upon binding to CD40 ligand (CD40L). A CD40-SNAG is used to activate Notch in CD40- T cells in the presence of CD40+ B cells, as these cell types Attorney Docket Number 10110-473WO1 colocalize in germinal centers, the tumor microenvironment, and peripheral blood. To generate a CD40-SNAG, DeltaMAXand the ECD of CD40L are fused to the N- and C-termini of a trimeric leucine zipper (FIG.3I and FIG.9E), respectively. This trimeric scaffold is selected instead of an Fc domain for the CD40-SNAG because CD40L is naturally a homotrimer. Addition of the CD40- SNAG robustly activates Notch in mixed cultures of Notch1 reporter cells and CD40-expressing OCI-Ly3 cells, but only weakly in Notch1 reporter cells alone (FIG.5E). The OCI-Ly3 cells used in this assay are non-adherent, showing that SNAGs also facilitate Notch activation between adherent and suspension cells. Comparison of SNAGs to conventional methods of Notch activation. 190. To assess the relative effectiveness of SNAGs, Notch activation levels of the soluble PDL1-SNAGFc and various conventional methods of Notch agonism are compared. In this assay, Notch reporter cells are stimulated with plated DeltaMAX-Fc, plated DLL4-Fc, EDTA, DLL4-expressing HEK293T cells, and PDL1-SNAGFc in the presence of PDL1-expressing cells (FIG. 5F). In the plate-bound format, 100 nM concentrations of each ligand are non-specifically adsorbed to the surfaces, and 100 nM concentrations of SNAG are added to the co-culture. In the presence of PDL1-expressing cells, the PDL1-SNAGFc induces higher levels of reporter activity than all methods of activation except for the plated DeltaMAX-Fc protein, which exhibits superagonist activity. The PDL1-SNAGFc stimulates higher levels of signaling than DLL4- overexpressing cells that typically serve as a benchmark for Notch activation. The DLL4-Fc protein does not detectably activate Notch1 in this format, which is due to the lack of C-terminally anchored coupling required for optimal signaling with wild type ligands. Endocytosis is required for SNAG-mediated Notch activation. 191. Ligand endocytosis is important for Notch activation, and this process is regulated by ubiquitination of DLL or JAG ICDs by the E3 ligase Mindbomb1. To determine whether endocytosis occurs with a SNAG targeting a surface protein other than a natural Notch ligand, an immunofluorescent endocytosis assay is performed utilizing CD19-SNAGFc in CD19-expressing cells. CD19-SNAGFc coupled with a fluorescent secondary antibody (Alexa Fluor 647-labeled anti-Fc) binds strongly to the surface of the CD19-expressing cells when the mixture is incubated on ice (FIG. 6A and FIGS. 12A-12C). Following CD19-SNAGFc binding, incubation at 37 °C permits the resumption of cellular activity, including endocytosis. Visualizing the cells after a 15 min incubation at 37 °C shows that the majority of bound CD19-SNAGFc is internalized (FIG. 6B and FIGS. 12D-12E). Performing the assay in the presence of the dynamin-dependent endocytosis inhibitor Dynasore blocks SNAG uptake, showing that SNAGs are internalized (FIG. 6C). Attorney Docket Number 10110-473WO1 192. To test whether endocytosis is necessary for SNAG signaling, SNAGs are co- administered with Dynasore. Dynasore completely ablates the activity of CD19-SNAGFc in co- cultures of Notch1- and CD19-expressing cells, showing that endocytosis is required for SNAG- mediated activation utilizing CD19 as a biomarker (FIG. 6D). BC2-SNAGs targeting BC2- DLL4HL are unable to activate Notch1 in co-cultures of Notch1 and BC2-DLL4HL cells in the presence of Dynasore, confirming that endocytosis is also required for SNAG-mediated rescue of DLL4 signaling (FIGS. 6E-6F). Immobilized SNAGs are also unable to activate Notch1 in the presence of Dynasore, showing that endocytosis in the Notch-receptor cell is essential for Notch activation by plated ligands (FIG. 6G). Notch activation by plated ligands, SNAGs targeting a DLL4 loss-of-function mutant, and SNAGs targeting tumor antigens each depend on endocytosis. SNAGs stimulate the expression of T cell activation markers. 193. To investigate whether bispecific SNAGs influence the behavior of activated T cells, in vitro co-cultures of primary human CD8^ T cells with tumor cells expressing the SNAG targets PD-L1 or HER2 are established (FIG. 7A). The co-cultures are treated with PDL1- SNAGFc or HER2-SNAGFc and activated with anti-CD28 / anti-CD3-coated beads. Addition of the PDL1-SNAGFc or HER2-SNAGFc, but not DeltaMAX-Fc alone, increases the proportion of Granzyme B (GrB)-expressing CD8^ T cells, indicative of a cytotoxic phenotype. At the transcriptional level, SNAG-treated T cells show elevated expression of GZMB and IFNG, key markers of effector function, as well as HES4, a canonical Notch target gene (FIGS. 7B-7C). In the PDL1-SNAGFc-treated co-cultures, GZMB, IFNG, and HES4 are upregulated by 18-, 37-, and 3-fold, respectively, relative to untreated controls. Treatment with HER2-SNAGFc results in 4-fold, 52-fold, and 16-fold increases in GZMB, IFNG, and HES4 expression, respectively. This increased expression is also found at the protein level, as intracellular staining followed by flow cytometry reveals increased GrB and IFN^ expression in SNAG-treated T cells (FIGS. 13A and 13B). Discussion: 194. The development of soluble agonists has been a longstanding challenge in the Notch field. The SNAG platform described here provides a solution to this problem and a framework for the development of a diverse array of Notch activating biologics. Such agents have a wide range of translational applications, particularly in cancers where Notch functions as a tumor suppressor, T cell manufacturing, T cell immunotherapy, wound healing, and other areas of regenerative medicine. These first-generation SNAGs are engineered using an Fc-fusion format used in clinically viable protein drugs, which may also help to accelerate in vivo translation. Attorney Docket Number 10110-473WO1 195. SNAGs facilitate potent activation of Notch signaling in mixed populations of cells. These designs are further affinity-tuned to improve SNAG function. The DeltaMAXarm (KD of 24 nM) and the JAG1-, PDL1-, and HER2-targeting arms (KD of 0.9 nM, 1.8 nM, and 5 nM, respectively) bind with similar affinities. Lowering the affinity of the Notch-binding arm improves specificity and tissue distribution as observed for bispecific inhibitory antibodies and T cell engagers. Wild-type DLL4 is ineffective when incorporated into SNAGs, showing that there is a lower limit for the allowable affinity range. Higher-order oligomers beyond the dimeric and trimeric SNAG scaffolds tested here may lead to increased signaling potency. Future studies focus on optimizing affinity and multimerization to maximize signaling while maintaining favorable biochemical properties. 196. A surprising observation is that PDL1-SNAGs do not activate signaling on cells expressing both PDL1 and Notch1. These SNAGs engage the two targets in cis on the surface of a single cell, as opposed to bridging PDL1 and Notch1 proteins between cells, and cis interactions do not introduce sufficient tension to unfold the NRR. This is attributed to the restricted diffusion of SNAGs in the two-dimensional environment of the membrane, which promotes preferential cis interactions by increasing the local concentration. Cis inhibition of Notch signaling occurs when ligands and receptors are expressed on the same cell, and SNAGs are similarly unable to activate Notch in this context. The ability of SNAGs to mediate unidirectional signaling enables highly selective targeting, which could minimize risks of toxicity from global Notch agonism. 197. There is longstanding interest in using Notch to manufacture off-the-shelf CD4+ or CD8+ T cells for immunotherapy. By contrast, the use of Notch agonists to bolster the effector function of mature or activated T cells has emerged more recently. Addition of PDL1 or HER2 SNAGs stimulates Notch signaling in CD8+ T cells cocultured with tumor cells expressing the target biomarkers. T cells stimulated in this fashion also have increased expression of the T cell activation markers GrB and IFN^. These findings show that injected SNAGs have the potential to enhance the function of adoptively transferred T cells in vivo similarly to what is achieved ex vivo with immobilized agonists. 198. An important consideration in SNAG design, especially in the context of immunotherapy, is the ubiquitous expression of Notch receptors in nearly all cell types. In addition to effector T cells, regulatory T cells, myeloid-derived suppressor cells, macrophages, and other immune cells are present in the tumor microenvironment. Activation of Notch is not desirable in all of these populations and induces a range of immunostimulatory and immunosuppressive phenotypes. The ubiquitous expression of Notch proteins also hinders the tissue distribution of Attorney Docket Number 10110-473WO1 SNAGs or results in Notch inhibition in the absence of a relevant biomarker. Further modifications are necessary to maximize on-target Notch activation with SNAGs or alternative agonist scaffolds. 199. While SNAGs are effective in mixed cell populations, the development of unconditional agonists, those that do not rely on a secondary target, remains a challenging problem. The NRR of Notch3 appears uniquely susceptible to antibody-mediated destabilization, whereas the engineering of agonists targeting other Notch receptors with more stable NRRs requires alternative solutions. In vitro activation of Notch1 with ligands fused to DNA origami structures, Fc-clustered DLL1 proteins, and trimerization scaffolds shows that induced receptor clustering is an effective strategy. The development of the trimeric C3-DLL4 agonists is the most promising among these approaches, as it stimulates Notch in T cell bioreactors and induced pluripotent stem cell-derived ameloblasts. In vitro use of this method requires multiple rounds of doxycycline induction of Notch expression over a prolonged 96-hour incubation period to activate Notch. The development of SNAGs and the related technologies above represents a first step towards the widespread implementation of Notch agonists for basic and translational research. Methods: 200. Protein expression and purification. All SNAG sequences were cloned into a pAcGP67A vector for insect cell production containing an N-terminal gp67 signal peptide and C- terminal 8xHis-tag. Monomeric SNAGs were generated by fusing a truncated version of the DeltaMAXprotein spanning from the N-terminus to EGF5 (N-EGF5) fused to a biomarker-targeting scFv or nanobody using a flexible (GS)5 linker. Dimeric SNAGFcconstructs were generated by fusing DeltaMAX(N-EGF5) and the biomarker targeting module to the N- and C-termini of a human IgG1 Fc domain, respectively. All SNAGFcconstructs contained short GSG-linkers between the Fc sequence and DeltaMAXor the targeting module. Published sequences of atezolizumab, trastuzumab, and loncastuximab were converted into a scFv format prior to being incorporated into SNAGs, and the sequence of the BC2-specific nanobody was obtained from the Protein Data Bank (PDB ID 5VIN). Each scFv was generated by fusing the C-terminus of the variable heavy (VH) domain to the N-terminus of the variable light (VL) domain with a (GGGGS)3linker. Biotinylated DeltaMAX(N-EGF5) protein was generated through enzymatic modification of a C-terminal biotin acceptor peptide (BirA tag) as previously described. The “headless” loss-of- function DLL4HLmutant was generated by replacing the C2 and DSL domains of human DLL4 with the BC2-peptide sequence, which was connected to the N-terminus of EGF1 by a short GSG- linker. The DLL4HLconstruct was cloned into a pLenti-IRES-Puro vector for mammalian expression. Attorney Docket Number 10110-473WO1 201. All SNAG constructs in this study were expressed for by infecting Trichoplusia ni insect cell cultures (Expression Systems) at a density of 2^×^106cells^ml−1with recombinant Baculovirus. Culture supernatants were harvested after 48h, and proteins were purified by nickel and size-exclusion chromatography. Biotinylated proteins were site-specifically modified using BirA ligase and excess biotin was removed by purifying the proteins on a size-exclusion column. Protein purity was assessed by SDS-PAGE using TGX 12% Precast gels (Bio-Rad). All proteins were flash-frozen in liquid nitrogen and stored at ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ 202. Cell culture and generation of cell lines. Mammalian cells were cultured at 37^°C, with a humidified atmosphere of 5% CO2, washed with Dulbecco’s PBS (DPBS, Corning), and detached with trypsin-EDTA 0.25% (Gibco) for subculturing or cell-based assays. Notch reporter cell lines CHO-K1 N1-Gal4 were a gift from Dr. Michael Elowitz (California Institute of Technology). Briefly, transfections of HEK293T cells were carried out with packaging vectors VSV-G and d8.9 in the presence of polyethyleneimine at a ratio of 4:1 (DNA:polyethyleneimine). HER2+SK-BR-3 cells, human CD19-overexpressing 3T3 cells, PD-L1+MDA-MB-231, and CD40+OCI-Ly3 cells were gifts from Drs. Brian Czerniecki, Fred Locke, Eric Lau, and John Cleveland, respectively (Moffit Cancer Center). HEK293T, SK-BR-3, 3T3 mouse fibroblast, and MDA-MB-231 cells were cultured in high-glucose DMEM (Cytiva) supplemented with 10% FBS (peak serum) and 2% penicillin / streptomycin (Gibco). Puromycin 5^µg^ml−1was added to HEK293T cell cultures to maintain homogeneous populations of receptor-expressing cells. CHO- K1 N1-Gal4 cells were cultured in minimum essential medium Eagle-alpha modification (^- MEM, Cytiva) supplemented with 10% FBS (peak serum), 2% penicillin / streptomycin (Gibco), 400^µg^ml−1of zeocin (Alfa aesar) and 600^µg^ml−1of geneticin (Gibco). Expression of receptors on the cell surface was confirmed by flow cytometry (BD Accuri C6 plus) staining the cell lines with anti-hDLL4 PE (Biolegend, 1:100), anti-hJAG1 APC (Biolegend, 1:100), anti-hPDL1 FITC (Biolgend, 1:100), anti-hHER2 (Cell Signaling Technologies, 1:100) followed by anti-IgG Alexa Fluor 488 (Biolegend, 1:200), anti-hCD19 FITC (Biolegend, 1:100), and anti-hCD40 PE (Biolegend, 1:100) in DMEM supplemented with 10% FBS for 1^h at 4^°C. 203. Notch stimulation with DeltaMAXmultimers. On day one, biotinylated DeltaMAX, DeltaMAXtetramers formed with streptavidin, or DeltaMAX-Fc were reconstituted in DPBS and adsorbed to tissue culture 96-well plates (Costar) for 1^h at 37^°C. The wells were then washed three times with 200^^l of DPBS to remove unbound proteins. Next, CHO-K1 N1-Gal4 cells were detached with trypsin-EDTA 0.25% (Gibco) and manually counted. Appropriate dilutions were prepared in ^-MEM media to ensure 30,000 CHO-K1 N1-Gal4 cells per well in a volume of 50^µL. Cells were transferred to the ligand-coated plates and cultured for 24^h at 37^°C in 5% CO2. On Attorney Docket Number 10110-473WO1 day two, CHO-K1 N1-Gal4 cells were washed with 200^µl DPBS, detached with 30^µL of trypsin- EDTA 0.25%, and quenched with 170^µL of ^-MEM media. Finally, cells were resuspended, and the H2B-mCitrine signal was measured by flow cytometry (BD Accuri C6 plus). CHO-K1 N1- Gal4 cells alone were used as the control. The measurements represent the mean fluorescent intensity as fold-change of Notch activation^±^s.d. of three technical replicates. Notch activation was normalized to wells containing CHO-K1 N1-Gal4 cells alone. 204. Notch activation with SNAGs in coculture of cells expressing the target biomarker. On day one, cells expressing the target receptor of the SNAG (signal-sending cells) were detached with trypsin-EDTA, counted manually, and dilutions prepared such that 50 µL of DMEM containing 15,000 signal-sender cells were added to wells of a tissue culture 96-well plate. The next day, CHO-K1 N1-Gal4 reporter cells (signal-receiver cells) were detached with trypsin- EDTA, and 50^µl of ^-MEM media containing 30,000 cells were added to the tissue culture 96- well plate containing the signal-sending cells after combining with the indicated DeltaMAXor SNAG protein. For the CD40-SNAG experiments, the difference was that an equal amount of signal-sending and signal-receiving cells were added same day. Wells without signal-sending cells were used to determine background activation of Notch by DeltaMAXand SNAGs. When testing inhibition of endocytosis, 80 µM of the Dynamin inhibitor I (Dynasore, Sigma) was added to the mixture of Notch reporter cells with protein and added to the tissue culture 96-well plate containing the signal-sending cells. Notch activation was measured as previously described. 205. Comparison of SNAGs to conventional methods of Notch activation. On day one, cells expressing the canonical Notch ligand, DLL4, and PD-L1+MDA-MB-231 cells expressing the target receptor of the SNAG were detached with trypsin-EDTA, counted manually, and dilutions prepared such that 50 µL of DMEM containing 15,000 signal-sender cells were added to wells of a tissue culture 96-well plate. The next day, DeltaMAX-Fc and DLL4-Fc in DPBS were adsorbed to tissue culture 96-well plates (Costar) for 1^h at 37^°C. The wells were then washed three times with 200^^l of DPBS to remove unbound proteins. Next, CHO-K1 N1-Gal4 cells were detached with trypsin-EDTA 0.25% (Gibco) and manually counted. Appropriate dilutions were prepared in ^-MEM media to ensure 30,000 CHO-K1 N1-Gal4 cells per well in a volume of 50^µL. Cells were transferred to wells with adsorbed ligands, signal-sending cells only, and after combining with DeltaMAX-Fc, EDTA (1.0 mM final concentration), or in coculture with PDL1-SNAGFcwith or without DAPT (3 µM final concentration), and cultured for 24^h at 37^°C in 5% CO2. On day two, CHO-K1 N1-Gal4 cells were washed with 200^µl DPBS, detached with 30^µL of trypsin-EDTA 0.25%, and quenched with 170^µL of ^-MEM media. Finally, cells were resuspended, and the H2B-mCitrine signal was measured by flow cytometry (BD Accuri C6 plus). Attorney Docket Number 10110-473WO1 CHO-K1 N1-Gal4 cells alone were used as the control. The measurements represent the mean fluorescent intensity as fold-change of Notch activation^±^s.d. of three technical replicates. Notch activation was normalized to wells containing CHO-K1 N1-Gal4 cells alone. 206. Western blot detection of Notch1 activation by the PDL1-SNAGFcin MDA- MB-231 cells. DeltaMAX(100 nM protein in 600 µL of DPBS) was non-specifically adsorbed to a single well of a 12-well plate for 1 hour at 37 ºC as a positive control for Notch1 activation. The positive control well and three additional wells were then seeded with 200 x 103cells with MDA- MB-231 cells. The plate was centrifuged at 400 x g for 4 min to ensure cells were retained at the bottom of each well, and then the media of all wells was discarded. In the first uncoated well, 600 µL of DMEM was added as a negative control. The second well was filled with 600 µL of media containing 100 nM of DeltaMAX-Fc to monitor Notch1 activation by soluble ligand. The third was filled with 600 µL of media containing 100 nM PDL1-SNAGFc. The following day, the media was aspirated from all four wells, and the samples were resuspended in 60 µL of Laemli sample buffer with 5% b-mercaptoethanol to lyse cells, followed by boiling at 100 °C for 4 min. Lastly, the samples were analyzed by western blotting using equal protein amounts of cell lysates separated by SDS-PAGE (12% Mini-PROTEAN TGX Precast Protein Gels, Bio-Rad) and transferred to PVDF membranes using an iBlot2 Gel Transfer Device (Thermo Fisher Scientific). The membranes were blocked in 3% BSA^+^0.1% TBS-Tween. Primary antibodies were anti-Notch1 (D1E11 rabbit mAb, Cell Signaling Technology, 1:1,000), anti-cleaved Notch1 (Val1744 rabbit mAb, Cell Signaling Technology, 1:1,000), and b-actin (rabbit polyclonal Ab, Cell Signaling Technology, 1:1,000). Secondary antibody anti-Rabbit IgG conjugated to HRP (Goat polyclonal Ab, Vector Laboratories, 1:8,000) was used for detection of proteins using SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific). Images were acquired using a Chemidoc Imaging System and analyzed with Image-Lab v.6 software (Bio-Rad). 207. Immunofluorescent cell staining. For endocytosis assays, cells were grown on glass-like polymer bottoms in 24 well black frame plates (Cellvis). For visualization of CD19- SNAGFcprotein binding, 500 nM protein was preincubated with anti-Fc 647 (Alexa Fluor) at 1:200 dilution for 1h on rotation in +4°C. The CD19-SNAGFc-647 solution was added to cells on ice that were further kept in +4°C for 1 h. For endocytosis, the incubation was followed by washing away non-bound CD19-SNAGFc-647 with PBS, and 37°C DMEM added to the cells followed by a 15 min incubation in a 37°C incubator. After incubation of CD19-SNAGFc-647 with or without endocytosis, the cells were fixed in 3% paraformaldehyde and permeabilized with 0.15% Triton X-100 in PBS for 10 min at RT. Nonspecific binding was blocked by incubation in 3% BSA in PBS with 0.05% Triton X-100 and 0.1M glycine for 60 min at RT. Cells were further stained for Attorney Docket Number 10110-473WO1 filamentous actin with Alexa 488 conjugated to phalloidin (Invitrogen) for 45 min to visualize contours of the individual cells. Hoechst 33342 (Invitrogen) was used to counterstain nuclei. Images were acquired using a Keyence BZ-X710 microscope using a Nikon Plan Apo 20x objective. The far-red channel (magenta) was processed with the de-haze function in the BZ- X710LE analyzer software. A minimum of 100 cells were imaged for each condition. 208. Treatment of CD8+T cells with HER2-SNAGFcand PDL1-SNAGFc. Human CD8^ T cells were purified from peripheral blood mononuclear cells (PBMCs) using the MagniSort™ Human CD8+T Cell Enrichment Kit (Invitrogen #8804-6812-74) according to the manufacturer's protocol. Naïve CD8^ T cells were then co-cultured at a 1:1 ratio with PD-L1^ MDA-MB-231 cells or HER2^ SK-BR-3 cells, with or without DeltaMAX-Fc or SNAGFc. To enhance cell-cell contact, cultures were subjected to a short centrifugation at 300 × g for 1 minute. T cells were activated by adding Human CD3 / CD28 Dynabeads (Gibco #11161D). After 48 hours of stimulation, cells were treated with BD GolgiStop™ Protein Transport Inhibitor (BD Biosciences #554724) for 4 hours. Fixation and permeabilization were performed using the BD Cytofix / Cytoperm™ Fixation / Permeabilization Kit (BD Biosciences #554714) according to the manufacturer's protocol. Cells were then stained with human anti-IFN^ (APC), anti-granzyme B (FITC), anti-CD8 (PE) antibodies and DAPI followed by flow cytometry analysis. Data was quantified by FlowJo 10 software. To quantify relative mRNA levels, the total RNA was extracted using TRIzol™ Reagent (Invitrogen #15596026). cDNA synthesis was performed using 500 ng of purified RNA with the Verso cDNA Synthesis Kit (Thermo Fisher Scientific #AB1453B). Quantitative PCR (qPCR) was conducted using primers specific for IFN^, GrB, HES4, and B2M. Gene expression levels were normalized to B2M as an internal control, and fold changes were calculated relative to the control condition.

[0002] Attorney Docket Number 10110-473WO1 SEQUENCES SEQ ID NO:1: amino acid sequence of DeltaMAXSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGT NSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKFAIQ GSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPD GNPSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHPGCRH GTCSTPWQCLCDEGWGGLYCDQDLNYCTHHSPCKNGATCRNSGPRSYTCTCRPGYTG VDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSC RERNQGANYACECPPNFTGSNCE SEQ ID NO:2: amino acid sequence of IgG1 Fc DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEGLHNHYTQKSLSLSPGK SEQ ID NO:3: amino acid sequence of JAG1 scFv (derived from the JAG1.b70 antibody) EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGIHWVRQAPGKGLEWVGWITPDGGYT DYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARAGTLFAYWGQGTLVTVS SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGK APKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTTATTFGQGTK VEIK SEQ ID NO:4: amino acid sequence of JAG1-SNAGFcconstruct SSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGT NSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKFAIQ GSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPD GNPSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHPGCRH GTCSTPWQCLCDEGWGGLYCDQDLNYCTHHSPCKNGATCRNSGPRSYTCTCRPGYTG VDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSC RERNQGANYACECPPNFTGSNCEGSGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM Attorney Docket Number 10110-473WO1 HEGLHNHYTQKSLSLSPGKGSGASEVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGIH WVRQAPGKGLEWVGWITPDGGYTDYADSVKGRFTISADTSKNTAYLQMNSLRAEDTA VYYCARAGTLFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDR VTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSL QPEDFATYYCQQYYTTATTFGQGTKVEIK SEQ ID NO:5: amino acid sequence of BC2-SNAG SSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGT NSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKFAIQ GSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPD GNPSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHPGCRH GTCSTPWQCLCDEGWGGLYCDQDLNYCTHHSPCKNGATCRNSGPRSYTCTCRPGYTG VDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSC RERNQGANYACECPPNFTGSNCEGSGSGSGSGSQVQLVESGGGLVQPGGSLTLSCTASG FTLDHYDIGWFRQAPGKEREGVSCINNSDDDTYYADSVKGRFTIFMNNAKDTVYLQMN SLKPEDTAIYYCAEARGCKRGRYEYDFWGQGTQVTVSS SEQ ID NO:6: amino acid sequence of BC2-SNAGFcSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGT NSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKFAIQ GSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPD GNPSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHPGCRH GTCSTPWQCLCDEGWGGLYCDQDLNYCTHHSPCKNGATCRNSGPRSYTCTCRPGYTG VDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSC RERNQGANYACECPPNFTGSNCEGSGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEGLHNHYTQKSLSLSPGKGSGASQVQLVESGGGLVQPGGSLTLSCTASGFTLDHYDIG WFRQAPGKEREGVSCINNSDDDTYYADSVKGRFTIFMNNAKDTVYLQMNSLKPEDTAI YYCAEARGCKRGRYEYDFWGQGTQVTVSS SEQ ID NO:7: amino acid sequence of PDL1-SNAG SSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGT Attorney Docket Number 10110-473WO1 NSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKFAIQ GSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPD GNPSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHPGCRH GTCSTPWQCLCDEGWGGLYCDQDLNYCTHHSPCKNGATCRNSGPRSYTCTCRPGYTG VDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSC RERNQGANYACECPPNFTGSNCEAAAGSGSGSGSGSAGEVQLVESGGGLVQPGGSLRL SCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNT AYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSGGGGSGGGGSGGGGSDI QMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPS SEQ ID NO:8: amino acid sequence of PDL1-SNAGFcSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGT NSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKFAIQ GSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPD GNPSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHPGCRH GTCSTPWQCLCDEGWGGLYCDQDLNYCTHHSPCKNGATCRNSGPRSYTCTCRPGYTG VDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSC RERNQGANYACECPPNFTGSNCEGSGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEGLHNHYTQKSLSLSPGKGSGASEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIH WVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTA VYYCARRHWPGGFDYWGQGTLVTVSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGD RVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSL QPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPS SEQ ID NO:9: amino acid sequence of HER2-SNAGFcSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGT NSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKFAIQ GSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPD GNPSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHPGCRH GTCSTPWQCLCDEGWGGLYCDQDLNYCTHHSPCKNGATCRNSGPRSYTCTCRPGYTG Attorney Docket Number 10110-473WO1 VDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSC RERNQGANYACECPPNFTGSNCEGSGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEGLHNHYTQKSLSLSPGKGSGASEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTA VYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSAS VGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTL TISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK SEQ ID NO:10: amino acid sequence of CD19-SNAGFcSSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGT NSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKFAIQ GSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPD GNPSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHPGCRH GTCSTPWQCLCDEGWGGLYCDQDLNYCTHHSPCKNGATCRNSGPRSYTCTCRPGYTG VDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSC RERNQGANYACECPPNFTGSNCEGSGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEGLHNHYTQKSLSLSPGKGSGASQVQLVQPGAEVVKPGASVKLSCKTSGYTFTSNWM HWVKQAPGQGLEWIGEIDPSDSYTNYNQNFQGKAKLTVDKSTSTAYMEVSSLRSDDTA VYYCARGSNPYYYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSEIVLTQSPAIMSASP GERVTMTCSASSGVNYMHWYQQKPGTSPRRWIYDTSKLASGVPARFSGSGSGTSYSLTI SSMEPEDAATYYCHQRGSYTFGGGTKLEIK SEQ ID NO:11: amino acid sequence of CD40-SNAG SSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGT NSFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKFAIQ GSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPD GNPSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHPGCRH GTCSTPWQCLCDEGWGGLYCDQDLNYCTHHSPCKNGATCRNSGPRSYTCTCRPGYTG Attorney Docket Number 10110-473WO1 VDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSC RERNQGANYACECPPNFTGSNCEGSGSGGDRMKQIEDKIEEILSKIYHIENEIARIKKLIG ERTSGGSGGTGGSGGTGGSGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVT LENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSS AKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL

Claims

Attorney Docket Number 10110-473WO1 CLAIMS What is claimed is:

1. A multimeric costimulatory synthetic Notch agonist (SNAG) construct comprising an affinity matured Notch ligand and a JAG1 construct; wherein said affinity matured Notch ligand construct is connected to said JAG1 construct.

2. The multimeric costimulatory synthetic Notch agonist construct of claim 1, wherein said affinity matured Notch ligand is connected to said JAG1 construct by a multimerization motif.

3. The multimeric costimulatory synthetic Notch agonist construct of claim 2, wherein multimerization motif comprises a human IgG1 Fc domain, a leucine zipper, or T4 fibritin (FOLDON) motif.

4. The multimeric costimulatory synthetic Notch agonist construct of claim 1, wherein affinity matured Notch ligand is fused at the N- terminus of the human IgG1 Fc domain via a first linker and JAG1 construct is fused at the C-terminus of a human IgG1 Fc domain via a second linker.

5. The multimeric costimulatory synthetic Notch agonist construct of any one of claims 1-4, wherein the multimeric costimulatory synthetic Notch agonist (SNAG) construct is a dimer comprising two affinity matured Notch ligand fused at N- terminus of the human IgG1 Fc domain and two JAG1 constructs fused at C-terminus of a human IgG1 Fc domain.

6. The multimeric costimulatory synthetic Notch agonist construct of any one of claims 1-5, wherein the affinity matured Notch ligand comprises DeltaMAX.

7. The multimeric costimulatory synthetic Notch agonist construct of any one of claims 1-6, wherein the JAG1 construct comprises JAG1 scFv, JAG1 diabody, JAG1 nanobody, or a JAG1 antibody fragment thereof.

8. The multimeric costimulatory synthetic Notch agonist construct of claim 7, wherein the JAG1 construct comprises JAG1 scFv, wherein the JAG1 scFv comprises a H286Q substitution.

9. The multimeric costimulatory synthetic Notch agonist construct of any one of claims 7-8, wherein the JAG1 scFv comprises SEQ ID NO: 3.Attorney Docket Number 10110-473WO1 10. The multimeric costimulatory synthetic Notch agonist construct of claim 6, wherein DeltaMAXcomprises SEQ ID NO:

1.

11. The multimeric costimulatory synthetic Notch agonist construct of any one of claims 1-5, wherein the human IgG1 Fc domain comprises SEQ ID NO:

2.

12. The multimeric costimulatory synthetic Notch agonist construct of any one of claims 1-5 comprising the amino acid sequence as set forth in SEQ ID NO:

4.

13. A vector encoding the multimeric costimulatory synthetic Notch agonist construct of any one of claims 1-12.

14. A cell encoding the multimeric costimulatory synthetic Notch agonist construct of any one of claims 1-12 or comprising the vector of claim 13.

15. The cell of claim 14, wherein the cell comprises a cell lacking Notch signaling, a Notch receptor or ligand expressing cell, a tumor infiltrating lymphocyte, ^^ T cells, ^^ T cells, feeder cell, B cell, natural killer cell, chimeric antigen receptor (CAR) T cell, CAR NK cell CAR macrophage (CARMA), or dendritic cell.

16. A method of activating Notch expression in a cell in vitro or ex vivo comprising: obtaining cells lacking Notch signaling and culturing the cells lacking Notch signaling in media comprising one or more of the multimeric costimulatory synthetic Notch agonist constructs of any one of claims 1-12, the vector of claim 13, or the cell of claim 14 or 15.

17. A method of treating a cancer and / or Notch signaling associated disorder in a subject comprising: administering to the subject the multimeric costimulatory synthetic Notch agonist construct of any one of claims 1-12, the vector of claim 13, or the cell of claim 14 or 15.

18. The method of claim 17, wherein the Notch signaling associated disorder comprises Adams-Oliver syndrome, Alagille syndrome, autosomal recessive spondylocostal dysostosis, Hajdu-Cheney syndrome, or cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy.

19. A synthetic Notch agonist (SNAG) construct comprising: a DeltaMAXpolypeptide; a linker or a multimerization motif; andAttorney Docket Number 10110-473WO1 a target-binding domain specific to a biomarker other than Notch, wherein the SNAG construct induces Notch activation between a Notch receptor-expressing cell and a biomarker-expressing cell.

20. The SNAG construct of claim 19, wherein the linker or the multimerization motif comprises a human IgG1 Fc domain, a trimeric zipper, T4 fibritin (FOLDON) motif, a streptavidin-biotin complex, or a flexible glycine-serine linker.

21. The SNAG construct of claim 19, wherein the target-binding domain comprises a single- chain variable fragment (scFv), an antibody fragment, a nanobody, or a receptor-binding domain.

22. The SNAG construct of claim 19, wherein the biomarker is selected from: (a) a Notch ligand comprising DLL4 or JAG1; (b) a tumor-associated antigen comprising PD-L1, HER2, or CD19; or (c) an immunostimulatory receptor comprising CD40.

23. The SNAG construct of claim 19, wherein the SNAG construct is a dimer comprising two DeltaMAXdomains fused to N-termini of an Fc domain and two target-binding domains fused to C-termini of said Fc domain.

24. The SNAG construct of claim 19, wherein the construct is a trimer comprising DeltaMAXand a CD40 ligand extracellular domain fused to a trimeric leucine zipper.

25. The SNAG construct of any one of claims 19-24, wherein the target-binding domain comprises a nanobody specific for a BC2 epitope tag.

26. The SNAG construct of claim 25, wherein the SNAG construct restores Notch signaling in a DLL4 variant lacking C2 and DSL domains.

27. The SNAG construct of any one of claims 19-24, wherein the target-binding domain comprises an antibody fragment specific for JAG1.

28. The SNAG construct of claim 27, wherein the JAG1 comprises an H268Q substitution.

29. The SNAG construct of any one of claims 19-24, wherein the target-binding domain binds PD-L1.Attorney Docket Number 10110-473WO1 30. The SNAG construct of claim 29, wherein the SNAG construct is dimeric and comprises DeltaMAXand the PD-L1 fused to N- and C-termini of an Fc domain.

31. The SNAG construct of any one of claims 19-24, wherein the target-binding domain binds HER2.

32. The SNAG construct of claim 31, wherein the SNAG construct activates Notch signaling in a mixed population of HER2-positive tumor cells and Notch receptor-expressing immune cells.

33. The SNAG construct of any one of claims 19-24, wherein the target-binding domain binds CD19.

34. The SNAG construct of claim 33, wherein the SNAG construct activates Notch signaling in a mixed population of B cells and Notch receptor-expressing cells.

35. The SNAG construct of any one of claims 19-24, wherein the target-binding domain comprises a CD40 ligand extracellular domain.

36. The SNAG construct of claim 35, wherein the DeltaMAXpolypeptide and CD40 ligand extracellular domain are fused to a trimeric leucine zipper scaffold.

37. The SNAG construct of claim 35, wherein the SNAG construct activates Notch signaling between CD40-positive B cells and CD40-negative T cells.

38. The SNAG construct of any one of claims 19-26, comprising amino acid sequence as set forth in SEQ ID NO: 5 or SEQ ID NO:

6.

39. The SNAG construct of any one of claims 19-30, comprising amino acid sequence as set forth in SEQ ID NO: 7 or SEQ ID NO:

8.

40. The SNAG construct of any one of claims 19-32, comprising amino acid sequence as set forth in SEQ ID NO:

9.

41. The SNAG construct of any one of claims 19-34, comprising amino acid sequence as set forth in SEQ ID NO:

10.

42. The SNAG construct of any one of claims 19-37, comprising amino acid sequence as set forth in SEQ ID NO: 11.

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