WO2025166271A1

WO2025166271A1 - Chimeric notch activating proteins

Info

Publication number: WO2025166271A1
Application number: PCT/US2025/014172
Authority: WO
Inventors: Vince LUCA; David Perez
Original assignee: H Lee Moffitt Cancer Center and Research Institute Inc
Current assignee: H Lee Moffitt Cancer Center and Research Institute Inc
Priority date: 2024-02-01
Filing date: 2025-01-31
Publication date: 2025-08-07
Anticipated expiration: 2026-08-01

Abstract

Disclosed herein is the engineering of a new class of chimeric Notch activating proteins (NAPs) that are used as tissue- specific activators of Notch signaling. The disclosed bispecific NAPs create the tension necessary to activate Notch by crosslinking it with targeted internalized biomarkers on adjacent cells. In some embodiments, it includes loss-of-function DLL4 mutants Notch signaling rescued by NAPs.

Description

CHIMERIC NOTCH ACTIVATING PROTEINS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63/548,615, filed February 1 , 2024, and U.S. Provisional Application No. 63/663,744, filed June 25, 2024, which are hereby incorporated herein by reference in their entireties.

STATEMENT OF GOVERNMENT INTEREST

[0002] This invention was made with Government Support under Grant No. GM 133482 awarded by the National Institutes of Health. The Government has certain rights in the invention.

SEQUENCE LISTING

[0003] This application contains a sequence listing filed in ST.26 format entitled “320805-2200 Sequence Listing” created on January 30, 2025, and having 29,427 bytes. The content of the sequence listing is incorporated herein in its entirety.

BACKGROUND OF THE INVENTION

[0004] Notch signaling regulates the development and maintenance of tissues - specifically in disease, it determines the progression of tumors depending on context. A bottleneck in the development of drugs capable of activating Notch is the requisite for a “pulling” force that is provided by ligand trans-endocytosis. Soluble drugs are unable to produce the mechanical force necessary and are typically unable to agonize Notch. SUMMARY OF THE INVENTION

[0005] Disclosed herein is the engineering of a new class of chimeric Notch activating proteins (NAPs) that are used as tissue-specific activators of Notch signaling. The disclosed bispecific NAPs create the tension necessary to activate Notch by crosslinking it with targeted internalized biomarkers on adjacent cells. As shown herein, loss-of-function DLL4 mutants Notch signaling can be rescued by NAPs. To demonstrate the wider utility of this approach, the NAP design was adapted to target biomarkers known to undergo endocytosis when engaged by therapeutic antibodies. Notch was successfully activated with NAPs targeting CD19 and HER2 confirming the applicability with other targets. Also disclosed is a NAP that targets Jag1 to recover Notch activation in the presence of a loss-of- function mutation resulting in Alagille syndrome. Applications of the disclosed NAP technology range from promoting stem cell differentiation in vivo to enhancing the proliferation and efficacy of adoptively transferred T cells. The ability of NAPs to activate Notch as soluble proteins will facilitate the scaling-up of the technology.

[0006] For example, disclosed herein is a NAP containing a cell-targeting domain that specifically binds to a cell-surface receptor on a first target cell that does not express a Notch receptor; and an affinity matured Delta-like 4 Notch-binding domain (Delta^MAX) that binds a Notch receptor on a second target cell. In some embodiments, the Delta^MAX has the amino acid sequence SEQ ID NO:1 , or a conservative variant thereof having at least 90% sequence identity to SEQ ID NO:1.

[0007] In some embodiments, the cell-targeting domain comprises an antibody fragment, an antibody derivative, a DARpin, an aptamer, or a functional domain thereof, that specifically binds the cell-surface receptor on the target cell. For example, the antibody fragment can be a single-chain fragment variable (scFv) antibody, a bispecific antibody, an Fab fragment, an F(ab)₂ fragment, a _HH fragment, a VNAR fragment, or a nanobody. In some embodiments, the cell-targeting domain specifically binds to the cell surface receptor with an affinity characterized by a dissociation constant (Kd) of 50 nM or less.

[0008] In some embodiments, the target cell is a cancer cell or cancer progenitor/stem cell. For example, the cancer cell can be a leukemic cell or a progenitor thereof.

[0009] In some embodiments, the cell-targeting domain and the Notch-binding domain are joined by an intervening flexible linker domain. In some embodiments, the celltargeting domain and the Notch-binding domain are joined by a linker with a dimerization domain. For example, the linker can be a fragment crystallizable (Fc) domain of an antibody.

[0010] In some embodiments, the cell-surface receptor is CD19, and wherein the cell-targeting domain is an antibody fragment of Loncastuximab. For example, the celltargeting domain can have the amino acid sequence SEQ ID NO:2, and the NAP can have the amino acid sequence SEQ ID NO:3.

[0011] In some embodiments, the cell-surface receptor is HER2, and wherein the cell-targeting domain is an antibody fragment of Trastuzamab. For example, the celltargeting domain can have the amino acid sequence SEQ ID NO:4, and the NAP can have the amino acid sequence SEQ ID NO:5.

[0012] In some embodiments, the cell-surface receptor is PDL1 , and wherein the cell-targeting domain is an antibody fragment of Pembrolizumab or Atezolizumab. For example, the cell-targeting domain can have the amino acid sequence SEQ ID NO:25, and the NAP can have the amino acid sequence SEQ ID NO:26.

[0013] In some embodiments, the cell-surface receptor is CD33, and wherein the cell-targeting domain is an antibody fragment of Gemtuzumab. In some embodiments, the cell-surface receptor is TROP2, and wherein the cell-targeting domain is an antibody fragment of Sacituzumab. In some embodiments, the cell-surface receptor is Tissue Factor, and wherein the cell-targeting domain is an antibody fragment of Tisotumab. In some embodiments, the cell-surface receptor is CD24 or ASGR1. [0014] Also disclosed are polynucleotides with nucleic acid seqeucnes encoding the NAPs disclosed herein. Also disclosed is a vector containing these polynucleotides. Also disclosed are cultured cells containing these vectors.

[0015] Also disclosed are pharmaceutical compositions containing a NAP disclosed herein and a pharmaceutically acceptable carrier.

[0016] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF FIGURES

[0017] FIG. 1 shows an embodiment of a Notch activating protein (NAP) using a nanobody specific for DLL4 and (GS)₅ linker (NAP^DLL4).

[0018] FIG. 2 shows an embodiment of a NAP using a nanobody specific for DLL4 and an Fc linker (NAP^DLL4-Fc).

[0019] FIGs. 3A and 3B show embodiments of a NAP using a nanobody or scFv and a (GS)₅ or Fc linker (NAP^DLL4-Fc) as ways to increase avidity.

[0020] FIG. 4A and 4B show that Delta^MAX alone acts as an inhibitor in the co-culture of DLL4 expressing HEK293T cells with Notch 1 expressing reporter CHO cells, in contrast, the NAP^DLL4 acts as an activator despite containing Delta^MAX which is an inhibitor.

[0021] FIGs. 5A and 5B show that in the co-culture of a loss-of-function DLL4 (HL) expressed in HEK293T cells with Notchl expressing reporter CHO cells in the absence of NAPDLL4 there is no appreciable Notchl activation, and as the NAP^DLL4 concentration is increased there is an increase in Notchl activation.

[0022] FIGs. 6A and 6B show avidity enhancement with NAP^DLL4-Fc.

[0023] FIG. 7 shows a modular plasmid was generated such that sequences of nanobodies or scFv could be easily inserted between the Nhel/Notl restriction sites and streamline the targeting of different receptors while not having to manipulate the framework of DeltaMAX-Fc. The plasmid is pAcGP67A which is used for insect cell production of proteins.

[0024] FIGs. 8A and 8B show Notch activation (fold change) using Delta^MAX-Fc, NAP^CD19-Fc, CD19, Delta^MAX-Fc and CD19, or NAP^CD19-Fc and CD19 in 3T3 mouse fibroblasts overexpressing CD19 (FIG. 8A) or CD19+ OCI-Ly3 lymphoma cancer cells.

[0025] FIG. 9 shows Notch activation (fold change) using Delta^MAX-Fc, NAP^CD19-Fc, HER2, Delta^MAX-Fc and HER2, or NAP^CD19-Fc and HER2 in SK-BR-3 HER2+ breast cancer cells.

[0026] FIG. 10 illustrates a Jagged 1^Ndr/Ndr (Nodder) mice that carry a single base-pair mutation in the extracellular part of jagged 1 . The name Nodder reflects the nodding behaviour and balance defects in the heterozygous state. [0027] FIG. 11 shows a NAP^Jag1-Fc using an scFv derived from an antibody called Jag1 b70 that binds outside of the region where the H268Q mutation is found and its use in activating Notch signaling in subjects with a Jag1 mutation.

[0028] FIG. 12 shows notch activation after treatment with Delta^MAX-Fc, NAP^PDL1-Fc with or without PDL1.

[0029]

[0030] FIGs. 13A to 13E show a design concept for synthetic Notch agonists. FIG. 13A shows a flow cytometry histogram overlay of Notch 1 reporter cells stimulated by soluble or plated (non-specifically adsorbed) DeltaMAX. The cartoon depicts the site-specifically biotinylated DeltaMAX(N-EGF5) construct. FIG. 13b is a histogram overlay of Notchl reporter cells stimulated with soluble or plated DeltaMAX-Fc protein. FIG. 13C is a histogram overlay of Notchl reporter cells stimulated with plated or soluble DeltaMAX-SA tetramers. FIG. 13D is a cartoon schematic depicting the ECDs of Notchl and DLL4 interacting during canonical Notch activation. The NRR and ligand-binding domains (LBD) of Notchl (EGF domains 8-12) are shaded. FIG. 13E is a schematic of a generalized SNAG construct alongside a cartoon depicting SNAG-mediated Notch activation.

[0031] FIGs 14A to 14D show SNAGs rescue the signaling of binding-deficient DLL4 and JAG1 mutants. FIG. 14A is a cartoon schematic depicting a SNAG binding to Notchl 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. 14B is a cartoon schematic depicting the multivalent binding of a dimeric Fc-tagged SNAG (BC2-SNAGFc) to Notchl and “headless” DLL4. FIGs. 14C to 14E show fluorescent reporter assays used to evaluate SNAG-mediated activation of Notchl in cocultures with HEK293T cells expressing DLL4HL (FIG. 14C), JAG1 (FIG. 14D), or JAG1 H268Q (FIG. 14E). Increasing concentrations (1 nM, 10 nM, or 100 nM) of the indicated SNAGs were added to Notch1-Gal4 Citrine reporter cells alone, or to a 1 :1 mixture of Notchl 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 Notchl 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.

[0032] FIGs. 15A to 15E show SNAGs targeting tumor antigens activate Notch in mixed cell populations. FIG. 15A shows cartoon schematics of PDLl , CD19, and HER2 SNAGs. FIG. 15B shows PDL1 -SNAG-mediated activation of Notchl evaluated in a fluorescent reporter assay. Increasing concentrations (1 nM, 10 nM, or 100 nM) of DeltaMAX-Fc, PDL1-SNAG, or PDLI-SNAGFc were added to Notch1-Gal4 mCitrine reporter cells alone, or a 1 :1 mixture of Notchl reporter cells and MDA-MB-231 cells. FIG. 15C shows activation of Notchl in MDA-MB-231 cells, which express both PDL1 and Notch"! , was assessed by Western Blot using an antibody against the activated NICD. FIGs. 15D ad 15E shows Notchl activation by CD19-SNAGs and HER2-SNAGs, was evaluated using a fluorescent reporter assay. Increasing concentrations (1 nM, 10 nM, or 100 nM) of DeltaMAX-Fc or each SNAGFc were added to Notch1-Gal4 mCitrine reporter cells alone, or a 1 :1 mixture of Notchl reporter cells and CD19-overexpressing 3T3 cells (FIG. 15D) or HER2-expressing SK-BR-3 cells (FIG. 15E). FIGs. 15B, 15D, and 15E involve a representative experiment from three biological replicates. Mean fluorescence intensity (MFI) was normalized to the mean MFI of Notchl 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.

[0033] FIGs. 16A to 16F show SNAG-mediated Notch activation requires endocytosis. FIIG. 17A contain 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 visualized by Alexa Fluor anti-Fc 647. To visualize the contours of the cells, the actin cytoskeleton was stained using phalloidin-488. Nuclei counterstained by Hoechst 33342. Scale, 20 mm. FIG. 16B contain representative immunofluorescence images of fluorescently labeled CD19-SNAGs used to stain the surface of CD19 expressing 3T3 cells, followed by washing away unbound SNAGs and subjecting the cells to a 15 min incubation in a 37 °C 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 mm. FIGs. 16A to 16E show flow cytometry histogram overlays depicting Notchl reporter activity induced by soluble CD19-SNAGFc, BC2-SNAG, or BC2-SNAGFc in the presence or absence of Dynasore. FIG. 16C shows Notchl reporter cells co-cultured with CD19-overexpressing 3T3 cells. In FIG. 16D and 16E show Notchl reporter cells cocultured with HEK293 cells expressing DLL4HL. FIG. 16F show a flow cytometry histogram overlay depicting Notchl 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 of three biological replicates.

[0034] FIGs. 17A to 17C show trimeric CD40-SNAGs activate Notchl signaling. FIG. 17A is a cartoon schematic depicting a SNAG binding to Notchl and CD40 receptor on adjacent cell. The CD40-SNAG was generated by using a GCN4 leucine zipper to trigger dimerization of both the Notchl - and CD40-targeting domains. FIG. 17B is a fluorescent reporter assay used to evaluate SNAG-mediated activation of Notchl . Increasing concentrations (1 nM, 10 nM, or 100 nM) of DeltaMAX-Fc or CD40-SNAG were added to Notch 1-Gal4 mCitrine reporter cells alone or a 1 :2 mixture of Notchl reporter cells and OCI- Ly3 cells that have high expression of the CD40 receptor 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 Notchl 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. 17C contains flow cytometry plots showing the percentage of B cells and T cells on a healthy donor sample, gated on lymphoid, single, live cells. The percentage of CD40+ cells in the B cell subpopulation (CD19+) are shown in the top panel, and the T cell subpopulation (CD3+) on shown in the bottom panel.

[0035] FIGs. 18A to 18D show CD40-SNAGs improve the expansion and phenotype of y0 T cells. FIG. 18A is a bar chart showing the percentage of live cells in culture with different concentrations of CD40-SNAG or controls; gated on lymphoid, single cells. FIG. 18B shows percentage of yb TOR expression in T cells cultured with CD40-SNAG vs controls. The histogram to the right shows data from a representative biological replicate; gated on lymphoid, single, viable, CD3+ cells. FIG. 18C is a bar graph showing the total number of cells in culture by each condition (left) and the relative number of y6 TCR+ cells (right). FIG. 18D contains bar charts representing the distribution of different differentiation phenotypes in y6 TCR+ T cells cultured with CD40-SNAG or controls. A representative flow cytometry zebra plot from one healthy donor is shown to the right. Cells were analyzed and counted on day seven, in the bar charts each symbol correspond to an independent donor (biological replicate), p values over the bars were calculated using paired t-tests comparing to the control.

[0036] FIGs. 19A to 19G show 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 (FIGs. 19A-19F), flow cytometry histograms show the staining of each cell line with the indicated antibody. FIG. 19A shows BC2-DLL4^HL HEK293T cells stained with a fluorescently labeled anti-DLL4 antibody, FIGs. 19B to 19C show JAG1 HEK293T cells or JAG1^H268Q HEK293T cells stained with an anti- JAG1 antibody, FIG. 19D shows MDA-MB-231 cells stained with an anti-PDL1 antibody, FIG. 19E shows CD19-expressing 3T3 cells stained with an anti-CD19 antibody, FIG. 19F shows SK-BR-3 cells stained with an anti-HER2 antibody, and FIG. 19G shows OCI-Ly3 cells stained with an anti-CD40 antibody.

[0037] FIGs. 20A to 20D 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. 20A shows SDS-PAGE analysis of BC2-SNAGs, PDL1- SNAGs, and the CD19-SNAG. FIG. 20B shows SDS-PAGE analysis of the of the JAG1- SNAG. FIG. 20C shows SDS-PAGE analysis of the HER2-SNAG. FIG. 20D shows SDS- PAGE analysis of the CD40-SNAG. [0038] FIGs. 21 A to 21 C show gating schemes used to separate Notchl reporter CHO cells and activated cells. Flow cytometry gating strategies for selecting Notchl CHO reporter cells alone (FIG. 21 A) or in a coculture with BC2-DLL4^HL over-expressing HEK293T cells (FIG. 21 B). Gating strategy to determine the population of non-activated and activated Notchl CHO reporter cells in coculture with BC2-DLL4^HL over-expressing HEK293T cells without SNAG (FIG. 21 C, left) and with BC2-SNAG^Fc (FIG. 21 C, right).

[0039] FIGs. 22A to 22E show immunofluorescent endocytosis assays in 3T3 cells expressing CD19. FIG. 22A shows negative control staining utilizing anti-Fc 647 alone (zoom in). Anti-Fc 647 left and merged with actin and nuclei on the right. FIG. 22B to 22C show surface stainings of CD19-SNAG^Fc-647 with multiple cells visualized. The highlighted rectangle in FIG. 22B was used as the zoomed-in “no endocytosis’’ panel for Figure 16A. FIGs. 22D to 22E contain immunofluorescence images of CD19-SNAG^Fc-647 allowed to endocytose for 15 min with multiple cells visualized. The highlighted rectangle in FIG. 22D was used as the zoomed-in “15 min endocytosis” panel for Figure 16A.

DETAILED DESCRIPTION

[0040] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and 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, since the scope of the present disclosure will be limited only by the appended claims.

[0041] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0042] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0043] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0044] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0045] Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of chemistry, biology, and the like, which are within the skill of the art.

[0046] 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 to perform the methods and use the probes disclosed and claimed herein. 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, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

[0047] Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.

Definitions

[0048] It must be noted that, 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.

[0049] 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. 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.

[0050] 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.

[0051] The term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.

[0052] The term “carrier” means a compound, composition, substance, or structure that, when in combination with a compound or composition, aids or facilitates preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature of the compound or composition for its intended use or purpose. For example, a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.

[0053] 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.

[0054] The term “antibody” refers to natural or synthetic antibodies that selectively bind a target antigen. The term includes 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 that selectively bind the target antigen.

[0055] The term “specifically binds”, as used herein, when referring to a polypeptide (including antibodies) or receptor, refers to a binding reaction which is determinative of the presence of the protein or polypeptide or receptor in a heterogeneous population of proteins and other biologies. Thus, under designated conditions (e.g. immunoassay conditions in the case of an antibody), a specified ligand or antibody “specifically binds” to its particular “target” (e.g. an antibody specifically binds to an endothelial antigen) when it does not bind in a significant amount to other proteins present in the sample or to other proteins to which the ligand or antibody may come in contact in an organism. Generally, a first molecule that “specifically binds” a second molecule has an affinity constant (Ka) greater than about 10⁵ M-¹ (e.g., 10⁶ M-¹, 10⁷ M^-1, 10⁸ M~¹, 10⁹ M~¹, 10¹° M~¹, 10¹¹ M~¹, and 10¹² M~¹ or more) with that second molecule.

[0056] A “chimeric molecule” is a single molecule created by joining two or more molecules that exist separately in their native state. The single, chimeric molecule has the desired functionality of all of its constituent molecules. Frequently, one of the constituent molecules of a chimeric molecule is a “targeting molecule” or “targeting moiety.” The targeting molecule is a molecule such as a ligand or an antibody that specifically binds to its corresponding target, for example a receptor on a cell surface.

[0057] A “fusion protein” refers to a polypeptide formed by the joining of two or more polypeptides through a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide. The fusion protein can be formed by the chemical coupling of the constituent polypeptides or it can be expressed as a single polypeptide from nucleic acid sequence encoding the single contiguous fusion protein. A single chain fusion protein is a fusion protein having a single contiguous polypeptide backbone. Fusion proteins can be prepared using conventional techniques in molecular biology to join the two genes in frame into a single nucleic acid, and then expressing the nucleic acid in an appropriate host cell under conditions in which the fusion protein is produced.

[0058] A “spacer” as used herein refers to a peptide that joins the proteins comprising a fusion protein. Generally a spacer has no specific biological activity other than to join the proteins or to preserve some minimum distance or other spatial relationship between them. However, the constituent amino acids of a spacer may be selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity of the molecule.

[0059] A “ligand”, as used herein, refers generally to all molecules capable of reacting with or otherwise recognizing or binding to a receptor on a target cell.

[0060] The term “vector” refers to a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. The vectors can be expression vectors.

[0061] The term “operably linked to” refers to the functional relationship of a nucleic acid with another nucleic acid sequence. Promoters, enhancers, transcriptional and translational stop sites, and other signal sequences are examples of nucleic acid sequences operably linked to other sequences. For example, operable linkage of DNA to a transcriptional control element refers to the physical and functional relationship between the DNA and promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA.

[0062] The terms “transformation” and “transfection” mean the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell including introduction of a nucleic acid to the chromosomal DNA of said cell.

[0063] The term “percent (%) sequence identity” or “homology” is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical with the nucleotides or amino acids in a reference nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.

Notch activating proteins (NAPs)

[0064] Disclosed herein is a chimeric Notch activating protein (NAP) comprising a cell-targeting domain that specifically binds to a cell-surface receptor on a target cell and an affinity matured Delta-like 4 Notch-binding domain (Delta^MAX).

[0065] In some embodiments, the Delta^MAX has the amino acid sequence: SSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAWSPGPCTFGTVSTPVLGTN SFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKFAIQGSL AVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNPSCL PGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHPGCRHGTCSTP WQCLCDEGWGGLYCDQDLNYCTHHSPCKNGATCRNSGPRSYTCTCRPGYTGVDCELEL SECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQG ANYACECPPNFTGSNCE (SEQ ID NO:1), or a conservative variant thereof having at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:1.

Cell Targeting Domains

[0066] As indicated above, the cell-targeting domain specifically binds to an antigen characteristic of the cell-type of interest. The cell-targeting domain typically binds to the antigen characteristic of the cell-type of interest with an affinity that is at least greater than the binding affinity of the Notch-binding domain for the Notch receptor, as described above. In some cases, cell-targeting domain typically binds to the antigen characteristic of the celltype of interest with an affinity that is at least about 2 times, 3 times, 4 times, 5 times, 6 times, or 7 times greater than the binding affinity of the Notch-binding domain for the Notch receptor. In some instances, the binding affinity of the cell-targeting domain for the antigen characteristic of the cell-type of interest is at least an order of magnitude greater than the binding affinity of the Notch binding domain for a Notch receptor. For example, the dissociation constant (Kd) characterizing the affinity of the cell-targeting domain for the antigen characteristic of the cell-type of interest can be about 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 1 nM, 0.75 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM, 0.005 nM, and 0.001 nM, or even smaller. Typical (K_d) ranges characterizing the binding affinity of the cell-targeting domain for the antigen characteristic of the cell-type of interest include from about 30 nM to about 10 nM, from about 20 nM to about 1 nM, from about 10 nM to about 0.1 nM, from about 0.5 nM to about 0.05 nM, and from about 0.1 nM to about 0.001 nM, or even lower, or any subrange therein.

[0067] In preferred embodiments, the target receptor is internalized by the celltargeting domain when engaged since Notch requires a tension force that is generated by ligand trans-endocytosis.

[0068] The cell-targeting domain comprises an affinity reagent designed to specifically bind to an antigen characteristic of the cell-type of interest. In this context, the term “affinity reagent” refers to any molecule that can bind the antigen characteristic of the cell-type of interest with a specific affinity (i.e., detectable over background). As with the above description with respect to the Notch-binding domain, exemplary, non-limiting categories of affinity reagent include antibodies, an antibody-like molecule (including antibody derivatives and antigen (i.e., cell-specific antigen)-binding fragments thereof), peptides that specifically interact with a particular antigen (e.g., peptibodies), antigen-binding scaffolds (e.g., DARPins, HEAT repeat proteins, ARM repeat proteins, tetratricopeptide repeat proteins, and other scaffolds based on naturally occurring repeat proteins, etc., [see, e.g., Boersma and Pluckthun, Curr. Opin. Biotechnol. 22:849-857, 2011 , and references cited therein, each incorporated herein by reference in its entirety]), aptamers, or a functional Notch-binding domain or fragment thereof. Again, these affinity reagents are described in more detail below in the “Additional definitions” section.

[0069] In some embodiments, the antigen is a cell surface biomarker for a cancer cell or a cancer progenitor cell. As used herein, the term “cancer” refer to cells which exhibit autonomous, unregulated growth, such that they exhibit an aberrant growth phenotype characterized by a significant loss of control over cell proliferation. Cells of interest for detection, analysis, or treatment in the present application include precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and non-metastatic cells. The term “cancer progenitor cell” is interchangeable with terms such as “cancer stem cell,” “tumor propagating cells,” and “tumor-initiating cells,” all of which refer to pluripotent cells that themselves may be benign but give rise to cancer cells through a process of aberrant differentiation. These progenitor cells exhibit indefinite self-replication through asymmetric cell division, often have very slow proliferation rates, and are often resistant to toxic agents due in part to high-level expression. Many types of cancers are known to those of skill in the art, including solid tumors such as carcinomas, sarcomas, glioblastomas, melanomas, lymphomas, myelomas, etc., and circulating cancers such as leukemias. Cancers of virtually every tissue are known, and functional roles of Notch have been established in many cancers, such as influencing tumor initiation, tumor progression, tumor maintenance, drug resistance, and the like. For example, relevant discussions of Notch signaling as a target in cancer intervention are provided in Rizzo, P„ et al., “Rational targeting of Notch signaling in cancer,” Oncogene 27:5124-5131 (2008); Ranganathan, P., et al., “Notch signaling in solid tumors: a little bit of everything but not all the time,” Nature Reviews Cancer 11 :338-351 (2011); Espinoza, I. and L. Miele, “Notch inhibitors for cancer treatment,” Pharmacology & Therapeutics 139:95-110 (2013); and Yuan, X., et al., “Notch signaling: An emerging therapeutic target for cancer treatment,” Cancer Letters 369:20-27 (2015), each of which is incorporated herein by reference in its entirety. Illustrative cancers or cancer cell types encompassed by the present disclosure include but are not limited to ovarian cancer, breast cancer, colon cancer, lung cancer, prostate cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, head and neck cancer, and brain cancer. In some embodiments, the cancer cell is selected from T (leukemic) cell, breast cancer cell, prostate cell, lung cancer cell, glioblastoma, colo-rectal cancer cell, cervical cancer cell, melanoma cancer cell, pancreatic cancer cell, esophageal cancer cell, and the like, or a progenitor of any of the foregoing.

[0070] Cancer antigens can be, for example, tumor specific or tumor associated antigens that are known in the art. Exemplary antigens that are characteristic of various cancers and their qualifications as determinants of cancer cells are discussed widely in the literature. For example, see Cheever, Martin A., et al., “The prioritization of cancer antigens: a national cancer institute pilot project for the acceleration of translational research,” Clinical Cancer Research 15(17):5323-5337 (2009), incorporated herein by reference in its entirety. The role of Notch signaling, and thus its potential as a therapeutic target, in a wide variety of cancer cell-types is reviewed by Rizzo, P„ et al., “Rational targeting of Notch signaling in cancer,” Oncogene 27:5124-5131 (2008) and Nowell and Radtke, “Notch as a tumour suppressor,” Nature Reviews Cancer 17:145-159 (2017), each incorporated herein by reference in its entirety. In some embodiments, the antigen characteristic of a cell-type of interest can be a cell surface marker of any cancer or tumor type of interest. In a few illustrative, non-limiting embodiments the antigen characteristic of a cell-type of interest is CD33, CD326, or CD133.

[0071] Relevant antigens that are characteristic of the cancer cells of interest are known and domains that specifically bind to such antigens are available or can be readily produced for incorporation into the disclosed NAP. An illustrative, non-limiting example of an antigen characteristic of a target cell-type is the cell-surface marker CD33, which is an antigen that is characteristic of some leukemic cells. As described in Walter, R. B., et al., “Acute myeloid leukemia stem cells and CD33-targeted immunotherapy,’’ Blood 119(26)6198-6208 (2012), incorporated herein by reference in its entirety, the cell-surface marker CD33 is characteristic of a group of myeloid precursor cells and is an attractive antigen used in targeted immunotherapy for acute myeloid leukemias (AMLs). As described, some AMLs involve the development of a diverse population of cell lineages from the progenitor leukemic stem cells (LSCs). The several lineages of leukemic cells from such AMLs are predominantly or exclusively characterized by expression of CD33 on the cell surface at sufficient levels that it can be used as to target specific immunotherapeutic therapies for these AMLs.

[0072] In some embodiments, the cell-surface receptor is CD33 and the celltargeting domain is an antibody fragment of Gemtuzumab.

[0073] In some embodiments, the cell-surface receptor is CD19 and the celltargeting domain is an antibody fragment of Loncastuximab. Therefore, in some embodiments, the cell-targeting domain is an scFv having the amino acid sequence: QVQLVQPGAEWKPGASVKLSCKTSGYTFTSNWMHWVKQAPGQGLEWIGEIDPSDSYTN YNQNFQGKAKLTVDKSTSTAYMEVSSLRSDDTAVYYCARGSNPYYYAMDYWGQGTSVTV SSGGGGSGGGGSGGGGSEIVLTQSPAIMSASPGERVTMTCSASSGVNYMHWYQQKPGT SPRRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEPEDAATYYCHQRGSYTFGGGTKLE IK (SEQ ID NO:2). Therefore, in some embodiments, the NAP has the amino acid sequence: SSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAWSPGPCTFGTVSTPVLGTN SFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKFAIQGSL AVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNPSCL PG TGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHPGCRHGTCSTP WQCLCDEGWGGLYCDQDLNYCTHHSPCKNGATCRNSGPRSYTCTCRPGYTGVDCELEL SECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQG ANYACECPPNFTGSNCEGSGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC WVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEGLHNHYTQKSL SLSPGKGSGASQVQLVQPGAEWKPGASVKLSCKTSGYTFTSNWMHWVKQAPGQGLEWI GEIDPSDSYTNYNQNFQGKAKLTVDKSTSTAYMEVSSLRSDDTAVYYCARGSNPYYYAMD YWGQGTSVTVSSGGGGSGGGGSGGGGSEIVLTQSPAIMSASPGERVTMTCSASSGVNY MHWYQQKPGTSPRRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEPEDAATYYCHQR GSYTFGGGTKLEIK (SEQ ID N0:3).

[0074] In some embodiments, the cell-surface receptor is HER2 and the celltargeting domain is an antibody fragment of Trastuzamab. Therefore, in some embodiments, the cell-targeting domain is an scFv having the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRY ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVS SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGK APKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKV EIK (SEQ ID NO:4). Therefore, in some embodiments, the NAP has the amino acid sequence: SSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAWSPGPCTFGTVSTPVLGTN SFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKFAIQGSL AVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNPSCL PGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHPGCRHGTCSTP WQCLCDEGWGGLYCDQDLNYCTHHSPCKNGATCRNSGPRSYTCTCRPGYTGVDCELEL SECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQG ANYACECPPNFTGSNCEGSGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC WVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEGLHNHYTQKSL SLSPGKGSGASEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDY WGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAV AWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTT PPTFGQGTKVEIK (SEQ ID NO:5).

[0075] In some embodiments, the cell-surface receptor is PD-L1 and the celltargeting domain is an antibody fragment of Atezolizumab. Therefore, in some embodiments, the cell-targeting domain is an scFv having the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTY YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSG GGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPK LLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR TVAAPS (SEQ ID NO:25). Therefore, in some embodiments, the NAP comprises the amino acid sequence: SSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAWSPGPCTFGTVSTPVLGTN SFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKFAIQGSL AVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNPSCL PGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHPGCRHGTCSTP

WQCLCDEGWGGLYCDQDLNYCTHHSPCKNGATCRNSGPRSYTCTCRPGYTGVDCELEL SECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQG ANYACECPPNFTGSNCEGSGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC

WVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEGLHNHYTQKSL SLSPGKGSGASEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWV

AWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYW GQGTLVTVSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPAT FGQGTKVEIKRTVAAPS (SEQ ID NO:26, NAP^PDL1-Fc = Delta^MAX-Fc-PDL1scFv).

[0076] In some embodiments, the cell-surface receptor is TROP2 and the celltargeting domain is an antibody fragment of Sacituzumab.

[0077] In some embodiments, the cell-surface receptor is Tissue Factor and the celltargeting domain is an antibody fragment of Tisotumab.

[0078] In some embodiments, the cell-surface receptor is Delta-like 4 (DLL4).

Therefore, in some embodiments, the cell-targeting domain is an scFv having the amino acid sequence:

QVQLQESGGGLVQAGGSLRLSCAASGSISGGVEMGWYRQAPGKEREFVAGINDGGNIYY ADNVEGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVSTTYPSYNESQYADWIAYWGQG AQVTVSS (SEQ ID NO:6). Therefore, in some embodiments, the NAP has the amino acid sequence:

SSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAWSPGPCTFGTVSTPVLGTN SFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKFAIQGSL AVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNPSCL PGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHPGCRHGTCSTP

WQCLCDEGWGGLYCDQDLNYCTHHSPCKNGATCRNSGPRSYTCTCRPGYTGVDCELEL SECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQG

ANYACECPPNFTGSNCEGSGSGSGSGSQVQLQESGGGLVQAGGSLRLSCAASGSISGGV EMGWYRQAPGKEREFVAGINDGGNIYYADNVEGRFTISRDNAKNTVYLQMNSLKPEDTAV YYCAVSTTYPSYNESQYADWIAYWGQGAQVTVSS (SEQ ID NO:7). In some embodiments, the NAP has the amino acid sequence:

SSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAWSPGPCTFGTVSTPVLGTN SFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKFAIQGSL AVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNPSCL PGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHPGCRHGTCSTP WQCLCDEGWGGLYCDQDLNYCTHHSPCKNGATCRNSGPRSYTCTCRPGYTGVDCELEL SECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQG ANYACECPPNFTGSNCEGSGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC WVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEGLHNHYTQKSL SLSPGKGSGASQVQLQESGGGLVQAGGSLRLSCAASGSISGGVEMGWYRQAPGKEREFV AGINDGGNIYYADNVEGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVSTTYPSYNESQY ADWIAYWGQGAQVTVSS (SEQ ID N0:8).

[0079] In some embodiments, the cell-surface receptor is Jagged 1 (Jag1) and the cell-targeting domain is an antibody fragment of Jag1b70. Therefore, in some embodiments, the cell-targeting domain is an scFv having the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGIHWVRQAPGKGLEWVGWITPDGGYTD YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARAGTLFAYWGQGTLVTVSSGGG GSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLI YSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTTATTFGQGTKVEIK (SEQ ID NO:9). Therefore, in some embodiments, the NAP has the amino acid sequence: SSVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAWSPGPCTFGTVSTPVLGTN SFAVRDDSSGGGRNPLQLPLNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKFAIQGSL AVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDYFGHYVCQPDGNPSCL PGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHPGCRHGTCSTP WQCLCDEGWGGLYCDQDLNYCTHHSPCKNGATCRNSGPRSYTCTCRPGYTGVDCELEL SECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQG ANYACECPPNFTGSNCEGSGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC WVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEGLHNHYTQKSL SLSPGKGSGASEVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGIHWVRQAPGKGLEWV GWITPDGGYTDYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARAGTLFAYWGQ GTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWY QQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTTATTF GQGTKVEIK (SEQ ID NO: 10).

Linker [0080] In one embodiment, cell-targeting domain and the Delta^MAX Notch-binding domain are disposed in consecutively, in any order or orientation, within the NAP. In an alternative embodiment, the cell-targeting domain and the Delta^MAX Notch-binding domain, in any order or orientation, are joined by at least an intervening flexible linker domain. The linker domain functions as a spacer to allow each domain sufficient space to assume its natural three-dimensional shape without requiring significant adjustment, thus allowing freedom to contact and bind their corresponding targets without mutual interference. The linker can be of sufficient length and flexibility to allow independent movement of each domain, thus maximizing their potential to locate and bind their respective targets. The linker can be a synthetic polypeptide sequence, which is typically between about four and about 40 amino acids in length (e.g., about 5, 10, 15, 20, 25, 30, 35, 40 amino acids), although it can be longer, and can be part of an expressed fusion construct. The linker is typically designed to avoid significant formation of rigid secondary structures that could reduce the flexibility or distance provided between the proximate components. Thus, the linker is designed to provide a linear or alpha-helical structure. Such linkers are commonly used and are well- understood in the art.

[0081] An illustrative example of a linker is a 15 amino acid residue linker with 3x repeats of the sequence GGGGS (SEQ ID NO:1 1), which was utilized in a specific embodiment described in more detail below. In some embodiments, the cell-targeting domain and the Notch-binding domain are joined by an intervening flexible linker domain. For example, the linker can be GSGSGSGSGS (SEQ ID NO:12), GGS, GGSGGS ((GGS)₂, SEQ ID NO:13), GGSGGSGGS ((GGS)₃, SEQ ID NO:14), GGSGGSGGSGGS ((GGS)₄, SEQ ID NO:15), GGGS (SEQ ID NO:16), GGGSGGGS ((GGGS)₂, SEQ ID NO:17), GGGSGGGSGGGS ((GGGS)₃, SEQ ID NO:18), GGGSGGGSGGGSGGGS ((GGGS)₄, SEQ ID NO:19), GGGGS (SEQ ID NQ:20), GGGGSGGGGS ((GGGGS)₂, SEQ ID NO:21), GGGGSGGGGSGGGGS ((GGGGS)₃, SEQ ID NO:22), or GGGGSGGGGSGGGGSGGGGS ((GGGGS)₄, SEQ ID NO:23).

[0082] In some embodiments, the cell-targeting domain and the Notch-binding domain are joined by an intervening linker with a dimerization domain. For example, the linker can be a fragment crystallizable (Fc) domain of an antibody. Therefore, in some embodiments, the linker has the amino acid sequence: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEGLHNHYTQKSLSLSPGK (SEQ ID NO:24), or a conservative variant thereof having at least 65%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:24.

Pharmaceutical Composition

[0083] In another aspect, the disclosure provides a pharmaceutical composition comprising the NAP described herein. The pharmaceutical composition can also comprise pharmaceutically acceptable carriers, stabilizers, excipients, and other additives to provide an appropriate formulation for the preferred route of administration, as is familiar in the art. These additional additives are typically designed to avoid affecting the biological activity or availability of the NAP.

[0084] In other embodiments, pharmaceutical compositions of the present disclosure can also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized Sepharose™, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).

[0085] Generally, the pharmaceutical composition is formulated for appropriate systemic administration, such as oral or injection (e.g., subdermal) administration. However, other routes of administration are commonly used and are also encompassed herein.

Nucleic Acids, Vectors, Cell-Expression Systems

[0086] In another aspect, the disclosure provides a nucleic acid encoding polypeptide components of the NAP described above. In embodiments where the NAP is a fusion protein (e.g., all components are polypeptide joined in a single polymer), the nucleic acid can encode the entire fusion protein.

[0087] As used herein, the term “nucleic acid” refers to any polymer molecule that comprises multiple nucleotide subunits (i.e., a polynucleotide). Nucleic acids encompassed by the present disclosure can include deoxyribonucleotide polymer (DNA), ribonucleotide polymer (RNA), cDNA or a synthetic nucleic acid known in the art.

[0088] Nucleotide subunits of the nucleic acid polymers can be naturally occurring or artificial or modified. A nucleotide typically contains a nucleobase, a sugar, and at least one phosphate group. The nucleobase is typically heterocyclic. Canonical nucleobases include purines and pyrimidines and more specifically adenine (A), guanine (G), thymine (T) (or typically in RNA, uracil (U) instead of thymine (T)), and cytosine (C). The sugar is typically a pentose sugar. Suitable sugars include, but are not limited to, ribose and deoxyribose. The nucleotide is typically a ribonucleotide or deoxyribonucleotide. The nucleotide typically contains a monophosphate, diphosphate, or triphosphate. These are generally referred to herein as nucleotides or nucleotide residues to indicate the subunit. Without specific identification, the general terms nucleotides, nucleotide residues, and the like, are not intended to imply any specific structure or identity. The nucleotides can also be synthetic or modified.

[0089] In another aspect, the disclosure provides vectors comprising the nucleic acid sequences described herein, such as a vector comprising a nucleic acid sequence encoding the polypeptide described above. Such vectors are useful for the recombinant expression of the fusion protein in a cell-based expression system. Such expression systems are well- known in the art, and include cell strains optimized for recombinant expression of genes associated with specific vector parameters. For example, any vector described herein can further comprise a promoter sequence to facilitate expression of the nucleic acid encoding the fusion protein in the intended cellular expression system. Any appropriate promoter can be used, such as a constitutive promoter or inducible promoter, appropriate for the expression system to be used, as known in the art. For example, an inducible promoter can comprise an acetamide-inducible promoter. Additionally, the vector can also include selectable markers, such as antibiotic or toxin resistance genes, that will confer protection against such applied agents. In this manner, cells that are successfully transformed with the operational vector can be retained in culture and the non-transformed cells in the system can be removed.

[0090] Also provided are cultured cells transfected with any vector described herein, or progeny thereof, wherein the cell is capable of expressing a protein, e.g., fusion protein, as described above. The cell can be prokaryotic, such as E. coli, or eukaryotic, such as yeast, arthropod, or mammalian.

Methods

[0091] The disclosed NAP has a variety of applications. As described, a significant advantage is the ability to confer target-cell specificity in the modulation (i.e., inhibition) of Notch signaling. The NAP is specifically conferred by the choice of a high affinity celltargeting domain that specifically binds antigen that is characteristic of the target cell. Thus, the NAP can be administered to a heterogeneous population of cells, such as in vivo in a complex organism or in vitro in a culture.

[0092] Accordingly, in one aspect, the disclosure provides a method for modulating (e.g., agonizing) Notch signaling in a cell-type of interest, the method comprising contacting a population of cells comprising the cell-type of interest with an effective amount of the disclosed NAP. Similarly, in another aspect, the disclosure provides a method for enhancing Notch-dependent development in a cell type of interest, comprising contacting a heterogeneous population of cells comprising the cell-type of interest with an effective amount of the disclosed NAP. The heterogeneous population of cells can be in vivo in a living organism or in vitro/ex vivo in a culture. In some scenarios, the heterogeneous population of cells comprises a plurality of similar cells (i.e., derived from the same origin or source) but which are at different stages of development and differentiation. The application of the amount of the disclosed NAP can provide a homogenizing influence on the population of cell-type of interest, which all express the same characteristic antigen, but may be at different stages of development or differentiation. This allows the members of this population reset to the same phase of (non)differentiation to provide a more homogenized population.

[0093] The methods of these aspects can be useful, for example, for expansion and manipulation of a population of cells, such as stem cells or progeny cells with some degree of differentiation along a developmental lineage, as obtained from a subject. For example, in some instances, the disclosed methods and compositions can be applied to ex vivo stem cell (or progeny cell) production and/or engineering. In this regard, administration of stem cells or progeny cells with some degree of differentiation can be therapeutically beneficial for a variety of medical conditions where the extant population of functional cells is deficient in some way. For example, donor stem cells, such as from umbilical cord blood, can be cultured for eventual administration. However, the initially obtained population of stem cells, while reflecting a “cell-type of interest,” may still be rather heterogeneous, reflecting various stages of quiescence and differentiation. Because it is desirable to confer desired characteristics on the ex vivo population en masse and/or expand the relevant ex vivo subpopulation of isolated cells to sufficient numbers for administration, the application of the disclosed methods and compositions to the initial ex vivo population can prevent premature development and differentiation of the cells that are further advanced towards certain endpoints. Accordingly, the resulting population exhibits greater homogeneity in its quiescence, or stages of differentiation, which makes it amenable to more uniform expansion and/or potential manipulation into a preferred developmental lineage. Persons of ordinary skill in the art can readily apply this approach as part of a method to produce expanded populations of desired stem or other progenitor cells in ex vivo cultures. The culture can then be rationally and more uniformly guided along a desired developmental lineage for various therapeutic applications using known culturing conditions and growth factors for that purpose. For example, the disclosed compositions and methods can be applied as part of an approach to homogenize and expand ex vivo cultures of progenitor cells, e.g., hematopoietic stem cells (HSC), which can be rationally differentiated various desired progeny lineages, such as T- cell precursors, T-cell subsets, dendritic cells, NK cells, and the like, using known growth/developmental factors. For example, see Delaney, C., et al., “Notch-mediated expansion of human cord blood progenitor cells capable of rapid myeloid reconstitution,” Nature Medicine 16(2):232-237 (2010), incorporated herein by reference in its entirety, which describes a similar approach to generating an ex vivo population of CD34+ progenitor cells with enhanced myeloid engraftment characteristics. See also WO/2015/187815, WO/2013/086436, which address expanding and selectively differentiating progenitor cells using modulation of Notch signaling, each of which is incorporated herein by reference in its entirety. The role of Notch signaling in development and regulation of the immune system, including T-cell development is the focus of investigations, see, e.g., Radtke F., et al., “Regulation of innate and adaptive immunity by Notch,” Nature Review Immunology 13:427-437 (2013), and Taghon, T., et al., (2012), “Notch signaling during human T cell development,” Radtke, F. (Ed.), Chapter 4 in Notch Regulation of the Immune System, Vol. 360 of the series Current Topics in Microbiology and Immunology, pgs. 75-97, Springer Berlin Heidelberg, each incorporated herein by reference in its entirety. As indicated above, it will be appreciated that such approaches are not necessarily limited to just stem cells or stem cells of a certain type, but can be applied to obtaining, homogenizing, expanding, and/or further differentiating any type of stem cell or cell already differentiated to some degree along a defined developmental path or lineage.

[0094] Because the cell-type of interest is specifically (or preferentially) targeted, it will be appreciated that the above applications can also be modified for in vivo methods for modulating (e.g., inhibiting) Notch signaling in an entire cell-population of interest. This can be applied in efforts to homogenize, expand, and ultimately differentiate a progenitor cell type of interest to produce higher levels of progeny cells in a particular developmental lineage.

[0095] Determination of Notch modulation can be performed according to any established method indicative of Notch signaling. For example, determination of Notch modulation, either increased or decreased signaling, can be performed by monitoring relative or absolute levels of downstream gene products resulting from Notch activation. An illustrative, non-limiting example of a relevant downstream product is Hes1. Descriptions of monitoring downstream Hes1 levels to assess Notch signaling are provided in more detail below. Alternatively, reporter systems are available to indicate Notch signaling, such as the CHO-K1 Notch reporter system. See, e.g., Sprinzak, D., et al., “Cis-interactions between Notch and Delta generate mutually exclusive signalling states,” Nature 465(7294) :86-90 (2010), incorporated herein by reference in its entirety. Thus, in some embodiments, Notch modulation, e.g., inhibition, is determined by a reporter CHO-K1 Notch reporter system or by assessing a change in a downstream signaling factor, such as Hes1 . In some embodiments, the change assessed is significant compared to control.

[0096] In another aspect, the disclosure provides a method for inhibiting the development of a cancer cell or cancer progenitor cell, comprising contacting the cancer cell or cancer progenitor cell with the disclosed NAP. In the context of the NAP as described above, the cancer cell or cancer progenitor cell in this method is the cell-type of interest and the cell-targeting domain specifically binds to an antigen characteristic of the cancer cell or cancer progenitor cell.

[0097] The term “inhibiting the development of a cancer or cancer progenitor cell” refers to slowing, suspending, or stopping the transformation, reproduction, or differentiation of the cancer cell or cancer progenitor cell relative to similar conditions where the NAP are not contacted to the cell.

[0098] The method of this aspect is applicable to any cancer cell where Notch plays a functional role in initiation, maintenance, resistance, and/or progression of the cancer. In many case, the role of Notch is resultant of its enhanced, or dysregulated, signaling in the cells. Such cancers are described in more detail above, although some non-limiting examples of the cancer cell include T (leukemic) cell, breast cancer cell, prostate cell, lung cancer cell, glioblastoma, colo-rectal cancer cell, cervical cancer cell, melanoma cancer cell, pancreatic cancer cell, esophageal cancer cell, and the like, or a progenitor of any of the foregoing.

[0099] In some embodiments, the disclosed NAP is used to treat a squamous cell cancers where Notch1-3 is inactivated, a neuroendocrine tumors with overall low Notch expression, or pancreatic cancers where Notchl may inhibit tumor growth.

[0100] The method of this aspect can be applied in vitro, for example, in a biopsy sample obtained from a subject with cancer. In vitro applications can include scenarios where the sample is being tested for the presence of cancer or tested for the responsiveness to Notch-based intervention. In other embodiments, the cancer cell is in vivo in a subject with the cancer and the amount of NAP is administered to the subject.

[0101] The NAP can also be applied in a method of treating or inhibiting cancer in a subject in need thereof. Such method comprises administering a therapeutically effective amount of the NAP, such as in a pharmaceutical composition as described above, to the subject. Again, the cancer cell or cancer progenitor cell in the subject is the cell-type of interest and the cell targeting domain of the molecule specifically binds to an antigen characteristic of the cancer cell or cancer progenitor cell.

[0102] The NAP can be designed for any particular cell-type of interest by the selection of the antigen characteristic of the cell-type of interest and the corresponding celltargeting domain to be incorporated into the NAP. Cancer cells applicable in this method are described elsewhere herein. In a specific and illustrative embodiment, the subject has leukemia and the cell targeting domain of the NAP specifically binds to CD33.

[0103] The NAP can be formulated and dosed for any appropriate route of administration. Furthermore, the administration of the NAP, or a pharmaceutical composition containing the same, can also be administered in combination with other therapeutic interventions, including other anti-cancer therapeutics. In certain embodiments, at least one additional therapeutic and the disclosed NAP as disclosed herein are administered concurrently to a subject. When administered in combination, each component can be administered at the same time or sequentially in any order at different points in time. Thus, each component can be administered separately but sufficiently closely in time so as to provide the desired therapeutic effect. Such additional therapeutic agents can be cytotoxic agents that further inhibit or treat the cancer. Many such agents are known. Nonlimiting examples include aldesleukin, altretamine, amifostine, asparaginase, bleomycin, capecitabine, carboplatin, carmustine, cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, docetaxel, doxorubicin, dronabinol, duocarmycin, etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron, hydroxyurea, idarubicin, ifosfamide, interferon alpha, irinotecan, lansoprazole, levamisole, leucovorin, megestrol, mesna, methotrexate, metoclopramide, mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron, paclitaxel (Taxol™), pilocarpine, prochloroperazine, rituximab, saproin, tamoxifen, taxol, topotecan hydrochloride, trastuzumab, vinblastine, vincristine, vinorelbine tartrate, and the like.

[0104] While much of this disclosure addresses the dysregulation of Notch in cancers, it will be appreciated that dysregulation of Notch plays a role in other, non- cancerous diseases. Thus, it is also desirable to address dysregulation in specific cell-types for such non-cancerous diseases. Accordingly, in yet another aspect, the disclosure provides a method of treating a disease treatable by inhibiting Notch signaling in a cell-type of interest, comprising administering a therapeutically effective amount of the NAP, or a pharmaceutical composition containing the same, as disclosed herein. Illustrative, nonlimiting examples of such other diseases include spondylocostal dysostoses, Alagille syndrome, Hajdu-Cheney syndrome, Alzheimer disease, cerebral autosomal dominant arteriopathy with subcortical infarcts, aortic valve disease, and leukoencephalopathy. As described herein, the specific targeting of the NAP requires the incorporation of cell-targeting domain that specifically binds to an antigen characteristic of the altered or diseased cell type. Many characteristic antigens are known or readily discoverable, and are thus encompassed by the present disclosure through the application of ordinary skill in the art.

[0105] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

EXAMPLES

Example 1:

[0106] FIG. 1 shows an embodiment of a Notch activating protein (NAP) using a nanobody specific for DLL4 and (GS)₅ linker (NAP^DLL4). [0107] FIG. 2 shows an embodiment of a NAP using a nanobody specific for DLL4 and an Fc linker (NAP^DLL4-Fc).

[0108] FIGs. 3A and 3B show embodiments of a NAP using a nanobody or scFv and a (GS)₅ or Fc linker (NAP^DLL4-Fc) as ways to increase avidity.

[0109] FIG. 4A and 4B show that Delta^MAX alone acts as an inhibitor in the co-culture of DLL4 expressing HEK293T cells with Notch 1 expressing reporter CHO cells, in contrast, the NAP^DLL4 acts as an activator despite containing Delta^MAX which is an inhibitor.

[0110] FIGs. 5A and 5B show that in the co-culture of a loss-of-function DLL4 (HL) expressed in HEK293T cells with Notchl expressing reporter CHO cells in the absence of NAPDLL4 there is no appreciable Notchl activation, and as the NAP^DLL4 concentration is increased there is an increase in Notchl activation.

[0111] FIGs. 6A and 6B show avidity enhancement with NAP^DLL4-Fc.

[0112] Table 1 is a list of therapeutic antibodies that engage the respective receptor and result in its internalization - the goal was to exploit this feature since Notch requires a tension force that is generated by ligand trans-endocytosis.

[0113] FIG. 7 shows a modular plasmid was generated such that sequences of nanobodies or scFv could be easily inserted between the Nhel/Notl restriction sites and streamline the targeting of different receptors while not having to manipulate the framework of DeltaMAX-Fc. The plasmid is pAcGP67A which is used for insect cell production of proteins.

[0114] FIGs. 8A and 8B show Notch activation (fold change) using Delta^MAX-Fc, NAP^CD19-Fc, CD19, Delta^MAX-Fc and CD19, or NAP^CD19-Fc and CD19 in 3T3 mouse fibroblasts overexpressing CD19 (FIG. 8A) or CD19+ OCI-Ly3 lymphoma cancer cells.

[0115] FIG. 9 shows Notch activation (fold change) using Delta^MAX-Fc, NAP^CD19-Fc, HER2, Delta^MAX-Fc and HER2, or NAP^CD19-Fc and HER2 in SK-BR-3 HER2+ breast cancer cells.

[0116] FIG. 10 illustrates a Jagged 1^Ndr/Ndr (Nodder) mice that carry a single base-pair mutation in the extracellular part of jagged 1 . The name Nodder reflects the nodding behaviour and balance defects in the heterozygous state. [0117] FIG. 1 1 shows a NAP^Jag1-Fc using an scFv derived from an antibody called Jag1 b70 that binds outside of the region where the H268Q mutation is found and its use in activating Notch signaling in subjects with a Jag1 mutation.

Example 2:

Results

Soluble DLL4 ligand multimers do not activate Notch signaling

[0118] As an initial attempt to generate Notch agonists, it was investigated whether soluble oligomers of an affinity-matured DLL4 ligand (Delta^MAX) activate Notch signaling (Gonzalez-Perez, D. et al. Nat Chem Biol 2022 1-9). Delta^MAX 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 (Gonzalez-Perez, D. et al. Nat Chem Biol 2022 1-9). It was hypothesized that this increased affinity, coupled with receptor crosslinking through multimerization, could introduce tension in the absence of an endocytic pulling force. To test this hypothesis, Notch1-Gal4 mCitrine reporter cells (Sprinzak, D. et al. Nature 2010 465:86-90) were incubated with soluble and immobilized Delta^MAX multimers (Fig. 13A-13C). Delta^MAX dimers were generated through the C-terminal addition of a dimeric human IgG 1 Fc domain (Fig. 13B), and tetramers were generated by pre-mixing a 4:1 molar ratio of biotinylated Delta^MAX with streptavidin (SA, Fig. 13C). Neither the monomers nor the multimers induced reporter activity. By contrast, the plated Delta^MAX ligands potently stimulated Notchl activation (Fig. 13A-13C). This indicates that the receptor crosslinking by Delta^MAX-Fc dimers and Delta^MAX-SA tetramers is insufficient for signaling activation.

Design of synthetic Notch agonists.

[0119] To develop soluble Notch agonists, bispecific proteins were engineered that recapitulate the endocytosis-linked activation mechanism of DLL and JAG ligands (Fig. 13D). SNAGs were created by fusing Delta^MAX to the N-terminus of biomarker-targeting antibody fragments via a flexible (GS)₅ linker, or by fusing Delta^MAX and antibody fragments to the N- and C-termini of a dimeric IgG 1 Fc domain (Fig. 13E). These design concepts are intended to form a “molecular bridge” between Notch-expressing cells and cells that express a given surface protein. Conceptually, SNAGs should then activate Notch if the enforced interactions induce endocytic or tensile force capable of unfolding the NRR.

SNAGs rescue the signaling of a signaling-deficient DLL4 mutant.

[0120] To demonstrate proof-of-concept, whether SNAGs could rescue the activity of a signaling-deficient DLL4 mutant was tested. Loss-of-function DLL4 cells were generated by expressing a “headless” DLL4 truncation where the Notch-binding C2 and DSL domains (Luca, V. C. et al. Science 2015 347:847-853; Cordle, J. et al. Nat Struct Mol Biol 2008 15:849-857) were replaced with a BC2 epitope tag (BC2-DLL4^HL) (Figs. 14A, 19A) (Braun, M. B. et al. Sci Rep 2016 6). BC2-SNAGs were then generated by fusing Delta^MAX to a BC2- specific nanobody (Figs. 14A-14B, 20A). BC2-DLL4^HL cells alone did not activate signaling in a Notch 1-Gal4 mCitrine reporter assay, whereas the addition of 1 nM to 100 nM concentrations of SNAGs stimulated a dose-dependent increase in reporter activity (Figs. 14C, 21A-21 C). Monomeric BC2-SNAGs containing the (GS)s linker (BC2-SNAG) stimulated a ~6-fold increase in Notch 1 signaling, whereas dimeric BC2-SNAG Fc fusion proteins (BC2- SNAG^Fc) were more effective and induced a ~10-fold increase (Fig. 14C). Importantly, administration of the monomeric or dimeric BC2-SNAGs alone did not substantially increase Notch 1 reporter activity, indicating that a mixture of target-expressing and non-expressing cells is required for SNAG-mediated activation (Fig. 14C).

SNAGs bolster the activity of weakly-signaling JAG1 ligands.

[0121] DLL or JAG ligands preferentially signal through certain Notch receptor subtypes, and JAG1 is a particularly weak activator of Notchl (Andersson, E. R. et al. Gastroenterology 2018 154:1080-1095). Therefore, whether a JAG 1 -targeting SNAG (JAG1-SNAG^Fc) could potentiate JAG1-Notch1 signaling was tested. In the JAG1-SNAG^Fc construct, Delta^MAX and an scFv derived from the JAG 1 -targeting antibody B70 (Lafkas, D. et al. Nature 2015 528:127-131) were fused to the N- and C-termini of an lgG1 Fc domain as described above (Fig. 20B). A signaling assay was then performed to measure the activation of Notchl reporter cells by JAG 1 -overexpressing HEK293 cells (JAG 1-293 cells) in the presence or absence of the SNAG. Addition of the JAG1-SNAG^Fc increased Notchl reporter activity by ~4-fold compared to JAG1-293 cells alone, and that JAG1-293 cells did not stimulate a significant increase in reporter activity (Figs. 14D, 19B). The JAG1-SNAG^Fc was also evaluated with a JAG1 H268Q “Nodder” mutant that causes Alagille syndrome-like symptoms in mice by decreasing JAG1-Notch1 binding (Fig. 19C). Addition of the JAG1- SNAG^Fc to cocultures of Notchl and JAG1 ^H268Q cells increased JAG1 signaling by up to 7- fold compared to JAG1 ^H268Q cells alone (Fig. 14E). These data indicate that SNAGs can function as “signaling enhancers” by potentiating the activity of endogenous or mutated ligands.

SNAGs targeting tumor antigens activate Notch in mixed cell populations.

[0122] Next tested was whether SNAGs targeting the tumor antigens PD-L1 , CD19, or HER2 (Fig. 15A) can stimulate Notch activation. There is mounting evidence that Notch signaling enhances the function of activated T cells (Sierra, R. A. et al. Cancer Immunol Res 2014 2:800-811 ; Wilkens, A. B. et al. Blood 2022 140:2261-2275; Kondo, T. et al. Nat Commun 2017 8:15338), and SNAGs localized to the tumor microenvironment have the potential to stimulate localized activation of tumor-associated lymphocytes. For these SNAGs, the targeting arms were derived from antibody-drug conjugates (ADCs) that were pre-selected for their ability to induce target internalization. It was hypothesized that SNAGs incorporating ADC antibodies could thus mimic the physiological endocytosis mechanism of DLL or JAG ligands.

[0123] Monomeric and dimeric PDL1 -SNAGs were generated by fusing Delta^MAX to a single-chain variable fragment (scFv) derived from the ADC antibody Atezolizumab (Powles, T. et al. Nature 2014 515:558-562; Xiao, D. et al. Bioorganic Chemistry 2021 1 16:105366). In the monomeric PDL1-SNAG, Delta^MAX and the scFv were connecting using a (GS)5 linker, and in the dimeric PDL1-SNAG (PDL1-SNAG^Fc), Delta^MAX and the scFv were fused to the N- and C-termini of an lgG1 Fc domain (Figs. 15A, 20A). Unexpectedly, addition of the monomeric PDL1-SNAG to a 1 :1 mixture of Notchl reporter cells and PDL1 -expressing MDA-MB-231 cells did not activate Notchl (Figs. 15B, 19D). However, the dimeric PDL1- SNAG^Fc protein stimulated a ~7-fold increase in Notchl signaling in the coculture, suggesting that multimerization or avidity-enhancement may be required for SNAGs to effectively target biomarkers other than Notch ligands (Fig. 15B). Neither the PDL1-SNAG nor the PDL1-SNAG^Fc substantially increased Notchl reporter activity in the absence of MDA-MB-231 cells. Because of the increased efficacy of the dimeric SNAGs (Fig. 14C, Fig. 15B), subsequent CD19- and HER2-SNAGs were designed using only the Fc-fusion format (Fig. 15A).

SNAGs do not activate signaling on cells expressing both Notchl and PD-L1.

[0124] Given the ubiquitous expression of Notchl in mammalian cells, it is conceivable that SNAGs could activate signaling when Notchl and the target protein are both present on the cell surface. To test this possibility, MDA-MB-231 cells were cultured in the presence of soluble Delta^MAX-Fc, PDL1-SNAG^Fc, or immobilized Delta^MAX-Fc and monitored the levels activated Notchl by Western Blot (Fig. 15C). The plated Delta^MAX-Fc protein stimulated high levels of Notchl activation, whereas the PDL1-SNAG^Fc did not induce signaling over the background levels observed for soluble Delta^MAX-Fc alone (Fig. 15C). The inability of SNAGs to activate Notchl in MDA-MB-231 cells suggests that the present design does not enable sufficient intercellular crosslinking in cultures of cells expressing both Notchl and the biomarker.

Development of SNAGs targeting CD19 and HER2.

[0125] To generate a SNAG targeting the B-lymphocyte antigen CD19, an scFv derived from the CD19-targeting ADC loncastuximab (Zammarchi, F. et al. Blood 2018 131 :1094-1 105) was fused to the C-terminus of Delta^MAX-Fc (CD19-SNAG^Fo, Fig. 20A). The CD19-SNAG was then added to Notchl reporter cells or to co-cultures of Notchl reporter cells and CD19-overexpressing 3T3 fibroblast cells (Fig. 19E). The CD19-SNAG^Fc protein stimulated up to a 6-fold increase in reporter activity in the co-culture compared to untreated Notchl cells (Fig. 15D). To generate a SNAG targeting the breast cancer antigen HER2 (HER2-SNAG^Fc), the CD19-targeting arm was replaced with an scFv derived from the HER2- targeting ADC trastuzumab (Lewis Phillips, G. D. et al. Cancer Res 2008 68:9280-92900) (Fig. 20C). Addition of the HER2-SNAG^Fo to a mixed culture of Notchl reporter cells and HER2-expressing SK-BR-3 breast cancer cells induced a 6-fold increase in reporter activity (Figs. 15E, 19F) at the highest concentration tested (100 nM), which is similar to the level of activation observed for the PDL1-SNAG^Fo and the CD19-SNAG^Fc constructs (Fig. 15B, 15D). In the absence of biomarker-expressing cells, neither the CD19-SNAG^Fc nor the HER2- SNAG^Fc stimulated a significant increase in signaling compared to Delta^MAX-Fc alone (Figs. 15D-15E). Collective development of PD-L1 , CD19, and HER2 SNAGs demonstrates that SNAGs can facilitate signaling by engaging cell surface proteins beyond endogenous ligands.

Endocytosis is required for SNAG -mediated Notch activation.

[0126] Ligand endocytosis is important for Notch activation (Parks, A. L. et al. Development 2000 127:1373-1385), and this process is regulated by ubiquitination of DLL or JAG ICDs by the E3 ligase Mindbombl (Koo, B.-K. et al. PLoS One 2007 2:e1221 ; McMillan, B. J. et al. Mol Cell 2015 57:912-924; Cao, R. et al. Structure 2024 32(10): 1667- 1676. e5). To determine whether endocytosis occurs with a SNAG targeting a surface protein other than a natural Notch ligand, an immunofluorescent endocytosis assay was performed utilizing CD19-SNAG^Fc in CD19-expressing cells. CD19-SNAG^Fc coupled with a fluorescent secondary antibody (Alexafluor 647-labeled anti-Fc) bound strongly to the surface of the CD19-expressing cells when the mixture was incubated on ice (Figs. 16A, 22A-22C), and the contours of the cells were identified by staining for filamentous actin (Fig. 22A). Incubating the cells at 37 °C after attaching CD19-SNAG^Fc-647 to cells allowed for cellular functions, including endocytosis, to resume. Visualizing the cells after a 15 min incubation at 37 °C showed that the majority of CD19-SNAG^Fc is internalized (Figs. 16B, 22E-22D).

[0127] To test whether endocytosis is necessary for SNAG function, SNAGs with were co-administered the dynamin-dependent endocytosis inhibitor Dynasore. Dynasore completely ablated the activity of CD19-SNAG^Fc in co-cultures of Notchl- and CD19- expressing cells, indicating that endocytosis is required for SNAG-mediated activation utilizing CD19 as a biomarker (Fig. 16C). BC2-SNAGs targeting BC2-DLL4^HL were unable to activate Notchl in co-cultures of Notchl and BC2-DLL4^HL cells in the presence of Dynasore, confirming that endocytosis is also required for SNAG-mediated rescue of DLL4 signaling (Fig 16D-16E). Interestingly immobilized SNAGs were also unable to activate Notchl in the presence of Dynasore, suggesting that endocytosis in the Notch-receptor cell is essential for Notch activation by plated ligands (Fig. 16F). These studies demonstrate that Notch activation by plated ligands, SNAGs targeting a DLL4 loss-of-function mutant, and SNAGs targeting tumor antigens each depend on endocytosis. However, it is currently unclear whether endocytosis of the receptor, ligand, or both, is essential for SNAG function. SNAGs targeting CD40L increase the expansion ofy5 T cells

[0128] Next generated was a SNAG targeting CD40 (CD40-SNAG), an immunostimulatory receptor that undergoes endocytosis upon binding to CD40 ligand (CD40L) (Yellin, M. J. et al. J Immunol 1994 152:598-608). It was hypothesized that a CD40-SNAG could be used to activate Notch in T cells in the presence of CD40⁺ B cells, as these cell types are known to colocalize in germinal centers, peripheral blood, the tumor microenvironment, and other biological contexts (Mitchison, N. A. Nat Rev Immunol 2004 4:308-312). To generate a CD40-SNAG, Delta^MAX and the ECD of CD40L were fused to the N- and C-termini of a trimeric leucine zipper (Naito, M. et al. Cancer Immunol Immunother 201362:347-357) (Figs. 17A, 20D), respectively. This trimeric scaffold was selected instead of an Fc domain for the CD40-SNAG because CD40L is naturally a homotrimer. Addition of the CD40-SNAG robustly activated Notch in mixed cultures of Notch 1 reporter cells and CD40-expressing OCI-Ly3 cells (Fig. 19G), but only weakly in Notchl reporter cells alone (Fig. 17B). Notably, OCI-Ly3 cells used in this assay are non-adherent, indicating that SNAGs may facilitate Notch activation between adherent cells and those grown in suspension.

[0129] There has been a growing interest in y5 T cell-based cancer immunotherapies, including the adoptive transfer of unmodified y6 T cells and the development of y6 T CAR T cells (Mensurado, S. et al. Nat Rev Clin Oncol 2023 20: 178- 191 ; Ganapathy, T. et al. Cancer Immunol Immunother 2023 72:277-286; Frieling, J. S. et al. Sci Adv 2023 9:eadf0108). It was previously shown that Notchl and Notch2 are important for y6 T cell antitumor function (Gogoi, D. et al. J Immunol 2014 192:2054-2062), and that Notch stimulation generally induces memory phenotypes in mature and activated T cells (Wilkens, A. B. et al. Blood 2022 140:2261-2275; Kondo, T. et al. Nat Commun 2017 8:15338). Therefore, the goal was to determine whether the CD40-SNAG could be used to improve the expansion and phenotype of y6 T cells isolated from peripheral blood mononuclear cells (PBMCs). To determine the ratio of CD40-expressing and non-expressing cells, PBMCs were analyzed by flow cytometry. A representative PBMC culture contained 18.6% CD19+ CD40⁺ B cells and 71.9% CD3⁺ CD40 T cells (Fig. 17C), suggesting that the T cell fraction would be amenable to stimulation with the CD40-SNAG.

[0130] To test the effect of the CD40-SNAG on y0 T cells, increasing amounts of CD40-SNAG were administered during an established y0 T cell expansion protocol. Addition of the CD40-SNAG increased both the relative amount and total amount of y0 T cells recovered at all concentrations tested (Figs. 18A-18C). The highest CD40-SNAG concentration (500 ng/mL) was associated with >4-fold increase in the total number of y0 T cells compared to untreated PBMCs or those treated with Delta^MAX-Fc or trimerized CD40L alone. Treatment with 500 ng/mL CD40-SNAG also induced a ~4-fold increase in the number of central memory cells and a significant reduction in the number of effector memory y6 T cells (Fig. 18D). This bias towards the central memory subset is a desirable outcome given that central memory T cells have increased survival and antitumor function compared with effector memory T cells and effector T cells (Liu, Q. et al. Protein Cell 2020 11 :549- 564).

Materials and Methods

Protein expression and purification

[0131] 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 Delta^MAX protein spanning from the N-terminus to EGF5 (N-EGF5) fused to a biomarker-targeting scFv or nanobody using a flexible (GS)₅ linker. Dimeric SNAG^Fc constructs were generated by fusing Delta^MAX (N-EGF5) and the biomarker targeting module to the N- and C-termini of a human IgG 1 Fc domain, respectively. All SNAG^Fc constructs contained short GSG-linkers between the Fc sequence and Delta^MAX or the targeting module. Published sequences of atezolizumab, trastuzumab, and loncastuximab (Abanades, B. et al. Nucleic Acids Research 2024 52:D545-D551) were converted into a scFv format prior to being incorporated into SNAGs, and the sequence of the BC2-specific nanobody (Braun, M. B. et al. Sci Rep 2016 6) was obtained from the Protein Data Bank (PDB ID 5VIN). Each scFv was generated by fusing the C-terminus of the variable heavy (V_H) domain to the N-terminus of the variable light (V_L) domain with a (GGGGS)₃ (SEQ ID NO:22) linker. Biotinylated Delta^MAX(N-EGF5) protein was generated through enzymatic modification of a C-terminal biotin acceptor peptide (BirA tag) as previously described (Gonzalez-Perez, D. et al. Nat Chem Biol 2022 1- 9). The “headless” loss-of-function DLL4^HL mutant 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 DLL4^HL construct was cloned into a pLenti- IRES-Puro vector for mammalian expression.

[0132] All SNAG constructs in this study were expressed for by infecting Trichoplusia ni insect cell cultures (Expression Systems) at a density of 2 * 10⁶ cells ml^-1 with 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

[0133] 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) (Sprinzak, D. et al. Nature 2010 465:86-90). 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 pg ml^-1 was 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 (a-MEM, Cytiva) supplemented with 10% FBS (peak serum), 2% penicillin/streptomycin (Gibco), 400 pg ml-1 of zeocin (Alfa aesar) and 600 pg ml-1 of 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, anti-hJAG1 APC, anti-hPDL1 FITC, anti-hHER2 (anti-IgG FITC), anti-hCD19 FITC, and anti-hCD40 PE in DMEM supplemented with 10% FBS for 1 h at 4 °C.

Notch activation with Delta^MAX multimers

[0134] On day one, biotinylated Delta^MAX, Delta^MAX tetramers formed with streptavidin, or Delta^MAX-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 pl 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 a-MEM media to ensure 30,000 CHO-K1 N1-Gal4 cells per well in a volume of 50 mL. 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 pl DPBS, detached with 30 pL of trypsin-EDTA 0.25%, and quenched with 170 mL of a-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. Notch activation with SNAGs in coculture of cells expressing the target tumor biomarker

[0135] 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 mL 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 pl of a-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 Delta^MAX or 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 Delta^MAX and SNAGs. When testing inhibition of endocytosis, 80 pM 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.

Western blot detection ofNotchl activation by the PDL1-SNAG^Fc in MDA-MB-231 cells.

[0136] Delta^MAX (100 nM protein in 600 mL of DPBS) was non-specifically adsorbed to a single well of a 12-well plate for 1 hour at 37 °C as a positive control for Notch 1 activation. The positive control well and three additional wells were then seeded with 200 x 10³ cells 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 mL of DMEM was added as a negative control. The second well was filled with 600 mL of media containing 100 nM of Delta^MAX-Fc to monitor Notchl activation by soluble ligand. The third was filled with 600 mL of media containing 100 nM PDL1-SNAG^Fo. The following day, the media was aspirated from all four wells, and the samples were resuspended in 60 mL of Laemli sample buffer with 5% -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 T ransfer Device (Thermo Fisher Scientific). The membranes were blocked in 3% BSA + 0.1% TBS-Tween. Primary antibodies were anti-Notch1 (D1 E11 rabbit mAb, Cell Signaling Technology, 1 :1 ,000), anti-cleaved Notchl (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).

Immunofluorescent cell staining

[0137] For endocytosis assays, cells were grown on glass-like polymer bottoms in 24 well black frame plates (Cellvis). For visualization of CD19-SNAG^Fc protein binding, 500 nM protein was preincubated with anti-Fc 647 (Alexa Fluor) at 1 :200 dilution for 1 h on rotation in +4°C. The CD19-SNAG^Fo-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-SNAG^Fo-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-SNAG^Fc-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 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 dehaze function in the BZ-X710LE analyzer software. A minimum of 100 cells were imaged for each condition. y expansion from PBMCs with CD40-SNAG

[0138] Peripheral blood mononuclear cells (PBMCs) were isolated from healthy- donor buffy coats using an established density gradient protocol. y6 expansion was performed using zoledronic acid in addition or not of CD40-SNAG. Briefly, ten million cells per condition were resuspended in RPMI [supplemented with 5%FBS, antibiotic, IL-2 (100 lU/ml) and zoledronic acid (4pM)] at 1x10⁶ cells/ml. The cells were spiked with CD40-SNAG at three different concentrations (50, 100 or 500ng/ml), Delta^MAX-Fc (100ng/ml), or CD40L trimer (100ng/ml), and cultured in 24-well plates (2ml/well) for three days at 37C (5% CO2). On day three, the full media was replaced by new media with zoledronic acid and CD40- SNAG, Delta^MAX-Fc, or CD40L trimer as on day one. Two days later, cells were lifted from the plates, centrifuged, and resuspended in new RMPI media [5%FBS, antibiotic, IL-2 (100 IU/ml)] without additional molecules. At day seven, T cells were counted, and their phenotype was assessed by surface staining and flow cytometry (using markers for human anti-CD3 - BV711 , anti-Vd2 TCR - FITC, anti-CD45RA - BV421 , anti-CD27 - BV605, and aqua live/dead fluorescent dye).

[0139] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0140] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1 . A Notch activating protein (NAP) comprising

1) a cell-targeting domain that specifically binds to a cell-surface receptor on a first target cell that does not express a Notch receptor; and

2) an affinity matured Delta-like 4 Notch-binding domain (Delta^MAX) that binds a Notch receptor on a second target cell, wherein the Delta^MAX has the amino acid sequence SEQ ID NO:1 , or a conservative variant thereof having at least 90% sequence identity to SEQ ID NO:1 .

2. The NAP of claim 1 , wherein the cell-targeting domain comprises an antibody fragment, an antibody derivative, a DARpin, an aptamer, or a functional domain thereof, that specifically binds the cell-surface receptor on the target cell.

3. The NAP of claim 10, wherein the antibody fragment is a single-chain fragment variable (scFv) antibody, a bispecific antibody, an Fab fragment, an F(ab)₂ fragment, a V_HH fragment, a VNAR fragment, or a nanobody.

4. The NAP of claim 1 , wherein the cell-targeting domain specifically binds to the cell surface receptor with an affinity characterized by a dissociation constant (Kd) of 50 nM or less.

5. The NAP of claim 1 , wherein the target cell is a cancer cell or cancer progenitor/stem cell.

6. The NAP of claim 5, wherein the cancer cell is a leukemic cell or a progenitor thereof.

7. The NAP of claim 1 , wherein the cell-targeting domain and the Notch-binding domain are joined by an intervening flexible linker domain.

8. The NAP of claim 1 , wherein the cell-targeting domain and the Notch-binding domain are joined by a linker with a dimerization domain.

9. The NAP of claim 8, wherein the linker is a fragment crystallizable (Fc) domain of an antibody.

10. The NAP of any one of claims 1 to 9, wherein the cell-surface receptor is CD19, and wherein the cell-targeting domain is an antibody fragment of Loncastuximab.

11 . The NAP of claim 10, wherein the cell-targeting domain comprises the amino acid sequence SEQ ID NO:2.

12. The NAP of claim 1 1 , having the amino acid sequence SEQ ID NO:3.

13. The NAP of any one of claims 1 to 9, wherein the cell-surface receptor is HER2, and wherein the cell-targeting domain is an antibody fragment of Trastuzamab.

14. The NAP of claim 13, wherein the cell-targeting domain comprises the amino acid sequence SEQ ID NO:4.

15. The NAP of claim 14, having the amino acid sequence SEQ ID NO:5.

16. The NAP of any one of claims 1 to 9, wherein the cell-surface receptor is PDL1 , and wherein the cell-targeting domain is an antibody fragment of Pembrolizumab or Atezolizumab.

17. The NAP of claim 14, having the amino acid sequence SEQ ID NO:26.

18. The NAP of any one of claims 1 to 9, wherein the cell-surface receptor is CD33, and wherein the cell-targeting domain is an antibody fragment of Gemtuzumab.

19. The NAP of any one of claims 1 to 9, wherein the cell-surface receptor is TROP2, and wherein the cell-targeting domain is an antibody fragment of Sacituzumab.

20. The NAP of any one of claims 1 to 9, wherein the cell-surface receptor is Tissue Factor, and wherein the cell-targeting domain is an antibody fragment of Tisotumab.

21 . The NAP of any one of claims 1 to 9, wherein the cell-surface receptor is CD24 or ASGR1.

22. A nucleic acid encoding the NAP of any one of claims 1 to 21.

23. A vector comprising the nucleic acid of claim 22.

24. A cultured cell comprising the vector of claim 23.

25. A pharmaceutical composition comprising the NAP of any one of claims 1 to 21 and a pharmaceutically acceptable carrier.