NOTCH1 and NOTCH4 decoys and how to use them

Abstract

Fusion proteins are described that consist of EGF-like repeats 10-14 of Notch1 or Notch4 fused to human IgG Fc. Also provided are pharmaceutical compositions containing the fusion proteins and uses of the fusion proteins in methods for inhibiting angiogenesis and treating diseases or disorders.

Description

[Technical field]

[0001] Introduction This application claims the benefit of priority to U.S. Provisional Application No. 63 / 326,316, filed April 1, 2022, the contents of which are incorporated by reference in their entirety herein.

[0002] This invention was made with Government support under Grant No. W81XWH-18-1-0019 awarded by the Department of Defense and Grant No. HL112626 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] Electronic Sequence Listing Reference The contents of the electronic sequence listing (name: UIC0103WO_ST26.xml, size: 27,516 bytes, creation date: March 9, 2023) are incorporated by reference in their entirety into this specification. [Background technology]

[0004] background Angiogenesis is a tightly regulated, multi-step process that defines the development of new blood vessels arising from pre-existing vessels. Under physiological conditions, this action is restricted to fetal and postnatal development, as well as to tissue growth and wound healing in adulthood. Angiogenesis can be triggered by hypoxia, inflammation, and other physiological and pathophysiological signals. Abnormal angiogenesis has been implicated in several pathological conditions, such as cancer, vascular malformations, and retinopathies. Understanding how angiogenesis contributes to tumor growth and inflammation has long been a key area of ​​interest for therapeutic intervention. Without vascular support, tumors can grow to a size of 2 mm. 3 Tumors rarely grow beyond 100 nm, demonstrating the critical role of angiogenesis in tumor growth. This vulnerability prompts tumors to upregulate pro-angiogenic factors and recruit nearby endothelial cells to sustain their growth and ultimately lead to metastatic spread.

[0005] Notch signaling functions in angiogenesis through the regulation of endothelial cell fate decisions, often controlling alternative angiogenic pathways such as vascular endothelial growth factor receptor (VEGFR) signaling to carry out this function. Mammals express four homologous Notch receptors (Notch1-4) and five ligands (Jagged (JAG)1, 2 and delta-like ligand (DLL)1, 3, 4). Ligand-receptor interaction leads to cleavage of the Notch receptor, translocation of the Notch intracellular domain (NICD) to the nucleus, and transcription of downstream target genes. It is well documented that the Notch ligand DLL4 acts as a negative regulator of VEGF receptors through endothelial Notch to limit angiogenesis and generate an adequate number of functional blood vessels. The Notch ligand, JAG1, plays a proangiogenic role, but the mechanism of JAG1-Notch signaling in endothelial cells is not fully understood. Tumor vasculature regularly utilizes angiogenic pathways responsive to VEGF, which are regulated by hypoxia, resulting in upregulation of DLL4 and activation of Notch signaling. Thus, the involvement of Notch signaling in pathological angiogenesis intersects with VEGFR signaling, highlighting it as a potential target for controlling this process. Furthermore, Notch ligands may promote metastatic spread of tumor cells through both blood and lymphatic vasculature.

[0006] Approaches that globally inhibit Notch pathway activation have raised safety concerns due to toxicity. The most prominent class of Notch inhibitors are those that target γ-secretase. γ-secretase inhibitors (GSIs) block the cleavage of Notch and the subsequent translocation of the Notch intracellular domain (ICN) to the nucleus, inhibiting Notch signaling. Aberrant activation of Notch1 in patients with T-cell acute lymphoblastic leukemia (T-ALL) led to the introduction of GSIs for use in clinical trials. However, most patients suffered from gastrointestinal symptoms such as diarrhea in a dose-dependent manner, and treatment was suboptimal. Animal studies have further confirmed that systemic inhibition of Notch signaling leads to the accumulation of secretory goblet cells in the intestine, resulting in gastrointestinal toxicity. The development of DLL4 neutralizing antibodies was the next promising step to target the endothelial Notch1 signaling axis without the toxicity issues associated with global Notch blockade. The role of DLL4 / Notch1 signaling in tumor vascular development has been extensively studied, and the first cellular step of angiogenesis, the formation of tip cells, has been shown to be inhibited by DLL4-Notch signaling. Thus, the rationale for inhibiting DLL4-Notch1 signaling seems paradoxical, as its inhibition would stimulate excess but poorly perfused tumor vasculature, resulting in reduced tumor growth. Despite being promising, anti-DLL4 therapies evaluated in preclinical animal models resulted in liver pathological changes and severe vascular neoplasia.

[0007] Given that limiting functional vasculature may inhibit several types of malignant tumors, the development of new and safe approaches to target the Notch pathway remains an important clinical issue. Notch decoys, including: Notch1 (N1 1-36 ), Repeat 1-24 (N1 1-24 ), or repeat 1-13 (N1 1-13 ) fused in frame to human IgG constant fragment (Fc), and the epidermal growth factor (EGF)-like repeats 10-36 of human Notch1 (N1 10-36 ), Repeat 14-36 (N1 14-36 ), Repeat 10-24 (N110-24 ), or repeat 14-24 (N1 14-24 The Notch decoys, comprising human IgG Fc fused in frame to Notch IgG1, have been reported and shown to effectively inhibit tumor growth while minimizing the gastrointestinal toxicity associated with Notch inhibition (Kangsamaksin et al. (2015) Cancer Discovery 5(2):182-197). Adenoviral administration of the decoy-generating vector was used to generate and evaluate the activity of these Notch decoys in mice. Although adenoviral vectors have been approved for some human clinical trials, purified proteins allow for controlled dosage and carry a lower risk of inappropriate immune responses than viral administration.

[0008] EGF repeats of the Notch4 receptor protein operably attached to a half-life extending moiety are described in US 7,662,919. Notch1 decoy fusion proteins consisting of EGF-like repeats 10-18 or 10-20 conjugated to the Fc portion of a human antibody are described in US 11,026,996. However, these fusions are difficult to produce in sufficient quantities for use as therapeutic agents due to the large number of complex post-translational modifications. Thus, there is a continuing need to develop therapeutically useful Notch decoys that can be easily produced on a practical scale. DISCLOSURE OF THEINVENTION

[0009] SUMMARY OF THE PRESENT APPLICATION The present invention provides a fusion protein, the sequence of which, starting from the N-terminus of the fusion protein, is as follows: (a) The extracellular domain of the human Notch1 or Notch4 receptor protein, followed by (b) the Fc portion of an antibody; is identical to the amino acid sequence in wherein the extracellular domain of the human Notch1 or Notch4 receptor protein is (i) beginning with an amino acid present at the N-terminus of EGF-like repeat 10; (ii) extends to and includes the C-terminal amino acid of EGF-like repeat 14 as the C-terminal amino acid of the extracellular domain. In some aspects, the fusion protein further comprises a signal sequence. Nucleic acid molecules encoding the fusion proteins and host cells harboring the nucleic acid molecules are also provided, as are pharmaceutical compositions comprising the fusion proteins in admixture with a pharma- ceutically acceptable excipient.

[0010] The present invention also provides the use of the fusion protein in a method for inhibiting angiogenesis and treating angiogenic disease or disorder in a subject.In some aspects, the angiogenic disease or disorder comprises an ocular disease or disorder, such as age-related macular degeneration (AMD) or diabetic retinopathy.In other aspects, the angiogenic disease or disorder is cancer or vascular inflammation. [Brief description of the drawings]

[0011] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Figure 1 shows the construction and expression of Notch decoys. Notch decoys are composed of specific EGF-like repeats (10-14) of the Notch1 or Notch4 extracellular domain fused to a human Fc domain. The resulting fusion proteins are referred to herein as "N110-14Fc" and "N410-14Fc".

[0012] [Diagram 2] Figure 2 shows that N110-14Fc inhibits DLL4-induced cleavage of the Notch1 receptor. HUVECs were plated on 1 μg / mL recombinant hDLL4 in the presence of N110-14Fc or an IgG Fc isotype control for 24 hours, and cleaved Notch1 was quantified by Western blot analysis.

[0013] [Diagram 3]Figure 3 shows quantitative real-time polymerase chain reaction (qRT-PCR) analysis of targets of canonical Notch signaling in HUVECs treated with N110-14Fc. Cells were treated with the indicated concentrations of Notch peptibody or IgG Fc for 24 hours. ***P value << 0.001, **P value < 0.01, *P value < 0.05.

[0014] [Figure 4] Figure 4 shows qRT-PCR analysis of canonical Notch signaling targets in HUVECs treated with N410-14Fc. Cells were treated with the indicated concentrations of Notch peptibody or IgG Fc for 24 hours. *P value < 0.05.

[0015] [Diagram 5] Figure 5 shows the average number of sprouts for HUVEC-coated beads embedded in fibrin gel and treated for 12 days with increasing doses of human IgG Fc, N110-14Fc, or N410-14Fc. After 12 days, N110-14Fc significantly reduced the number of sprouts at dosages of 5 and 10 μg / ml. Box plots show median, minimum, and maximum values.

[0016] [Figure 6] Figure 6 shows the average sprout length of HUVEC-coated beads embedded in fibrin gel and treated for 12 days with increasing doses of human IgG Fc, N110-14Fc, or N410-14Fc. After 12 days, N110-14Fc significantly reduced sprout length at dosages of 5 and 10 μg / ml. Box plots show median, minimum, and maximum values. **P value < 0.01, *P value < 0.05.

[0017] [Figure 7]Figure 7 shows that N110-14Fc inhibits retinal angiogenesis in mouse neonates. C57BL / 6 mice were intragastrically injected with 12.5 mg / kg recombinant N110-14Fc peptibody or IgG Fc for 3 postnatal days (P1-P3). Quantification of retinal vessels at postnatal day 5 (P5) stained with isolectin B4 is shown. Radial growth and vascular coverage near the angiogenic front were reduced in N110-14Fc-treated mice (N=7-8), whereas tip cell density and vascular coverage of the mature plexus were not statistically altered. Box plots show median, minimum, and maximum values. **P value < 0.01, *P value < 0.05. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Detailed Description of the Invention The present invention provides peptibody-based Notch inhibitors composed of the core binding domains EGF-like repeats 10-14 of either Notch1 or Notch4. These Notch peptibodies showed high secretion properties and production yields compared to previous Notch decoys. Using surface plasmon resonance spectroscopy combined with co-immunoprecipitation assays, it was observed that Notch1 and Notch4 peptibodies exhibited strong yet distinct binding properties to the Notch ligands DLL4 and JAG1. Both Notch1 and Notch4 peptibodies disrupt Notch signaling in endothelial cells and reduce the expression of canonical Notch targets after treatment. While conventional DLL4 inhibitors cause excessive sprouting, Notch1 peptibodies reduced angiogenesis in a three-dimensional in vitro sprouting assay. Administration of Notch1 peptibodies to newborn mice reduced radial growth of retinal blood vessels, confirming their antiangiogenic properties. The present invention therefore provides a fusion protein (herein referred to as N1) composed of human Notch1 and Notch4 extracellular domains, containing EGF-like repeats 10-14, fused to human IgG Fc. 10-14 Fc and N4 10-14The present invention provides a method for inhibiting angiogenesis and treating angiogenic diseases or disorders, comprising administering to the subject angiogenesis-inhibiting antibody or antibody complex ....

[0019] Specifically, the present invention provides a fusion protein, the sequence of which, starting from the N-terminus of the fusion protein, is identical to an amino acid sequence in (a) the extracellular domain of a human Notch1 or Notch4 receptor protein, followed by (b) the Fc portion of an antibody, wherein the extracellular domain of the human Notch1 or Notch4 receptor protein (i) begins with the amino acid present at the N-terminus of EGF-like repeat 10 and (ii) extends to and includes the C-terminal amino acid of EGF-like repeat 14 as the C-terminal amino acid of the extracellular domain.

[0020] As used herein, "fusion protein" or "chimeric protein" refers to a protein made by combining two or more proteins to result in a single protein having the functional properties of the two or more proteins. The fusion proteins of the invention comprise or consist of EGF-like repeats 10-14 of Notch1 or Notch4 fused in frame to the Fc portion of an antibody. In one aspect, the fusion protein is set forth in SEQ ID NO:1 or SEQ ID NO:3.

[0021] As used herein, "Notch", "Notch protein" and "Notch receptor protein" are synonymous. Furthermore, the terms "Notch-based fusion protein" and "Notch decoy" are synonymous. The following Notch amino acid sequences are known and readily available: Notch1 (GENBANK Accession No. S18188 (rat)); Notch2 (GENBANK Accession No. NP_077334 (rat)); Notch3 (GENBANK Accession No. Q61982 (mouse)); and Notch4 (GENBANK Accession No. T09059 (mouse)). The following Notch nucleic acid sequences are known and readily available: Notch1 (GENBANK Accession Nos. XM_342392 (rat) and NM_017617 (human)); Notch2 (GENBANK Accession Nos. NM_024358 (rat), M99437 (human and AF308601 (human)); Notch3 (GENBANK Accession Nos. NM_008716 (mouse) and XM_009303 (human)); and Notch4 (GENBANK Accession Nos. NM_010929 (mouse) and NM_004557 (human)). In some aspects, the fusion proteins of the invention include a mammalian Notch1 or a mammalian Notch containing EGF-like repeats 10-14. In another aspect, the fusion protein of the invention comprises a human Notch1 or human Notch4 extracellular domain containing EGF-like repeats 10-14, or more preferably a human Notch1 extracellular domain containing EGF-like repeats 10-14. In a further aspect, the fusion protein of the invention comprises a human Notch1 extracellular domain containing EGF-like repeats 10-14 comprising or consisting of SEQ ID NO:5, or a human Notch4 extracellular domain containing EGF-like repeats 10-14 comprising or consisting of SEQ ID NO:6.

[0022] In some aspects, the Notch1 extracellular domain containing EGF-like repeats 10-14 or the Notch4 extracellular domain containing EGF-like repeats 10-14 does not contain a signal sequence. Exemplary Notch1 and Notch4 extracellular domains containing EGF-like repeats 10-14 without a signal sequence are set forth in SEQ ID NO:24 and SEQ ID NO:25, respectively. In other aspects, the Notch1 or Notch4 extracellular domain containing EGF-like repeats 10-14 includes a signal sequence. Exemplary Notch1 and Notch4 proteins with signal sequences are set forth in SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0023] As used herein, the Fc portion of an antibody encompasses the domain derived from the constant region of an immunoglobulin, preferably a human immunoglobulin, including fragments, analogs, variants, mutants or derivatives of the constant region. Suitable immunoglobulins include IgG1, IgG2, IgG3, IgG4, as well as other classes such as IgA, IgD, IgE, and IgM. IgG subclasses are well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4.

[0024] "Fc portion of an antibody", "Fc region", and "Fc domain" refer interchangeably to a crystallizable fragment obtained by papain digestion of an immunoglobulin, including all or substantially all of the C-terminal half of the heavy chain. For example, the Fc portion may include a CH2 domain, a CH3 domain, a CH4 domain, a CH2-CH3 domain, a CH2-CH4 domain, a CH2-CH3-CH4 domain, a hinge-CH2 domain, a hinge-CH2-CH3 domain, a hinge-CH2-CH4 domain, or a hinge-CH2-CH3-CH4 domain. The Fc domain may be derived from an antibody belonging to any of the immunoglobulin classes (i.e., IgA, IgD, IgE, IgG, or IgM), or an antibody belonging to any of the IgG antibody subclasses (i.e., IgG1, IgG2, IgG3, or IgG4). The Fc domain may be a naturally occurring Fc sequence, including naturally occurring allelic or splice variants. Alternatively, the Fc domain may be a hybrid domain that includes portions of the Fc domains of two or more different Ig isotypes, such as an IgG2 / IgG4 hybrid Fc domain. In a specific aspect, the Fc domain is derived from a human immunoglobulin molecule. Alternatively, the Fc domain may be a humanized or deimmunized version of an Fc domain from a non-human animal, including, but not limited to, mouse, rat, rabbit, camel, llama, dromedary, and monkey.

[0025] The sequence of the human IgG1 immunoglobulin constant region is shown below. ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7), Here, the core hinge sequence is underlined. It should be understood that the C-terminal lysine is optional. Thus, the C-terminal lysine of the IgG sequence can be removed or replaced with a non-lysine amino acid, such as alanine, to further extend the serum half-life of the Fc fusion protein.

[0026] In one aspect, the hinge sequence can include substitutions that confer desirable pharmacokinetic, biophysical, and / or biological properties. Some exemplary hinge sequences are shown in Table 1. Table 1 [Table 1] *Core hinge area underlined.

[0027] In some aspects, the Fc domain is a variant Fc sequence, e.g., an Fc sequence that has been modified (e.g., by amino acid substitution, deletion, and / or insertion) relative to a parent Fc sequence (e.g., an unmodified Fc polypeptide that is then modified to generate the variant) to provide a desired structural feature and / or biological activity. For example, modifications can be made to the Fc region to generate an Fc variant that has (a) increased or decreased antibody-dependent cell-mediated cytotoxicity (ADCC), (b) increased or decreased complement-mediated cytotoxicity (CDC), (c) increased or decreased affinity for C1q, and / or (d) increased or decreased affinity for Fc receptors compared to the parent Fc. Such Fc region variants typically contain at least one amino acid modification in the Fc region. Combining amino acid modifications may be particularly desirable.

[0028] In another aspect, an Fc fusion protein described herein comprises the CH2 and CH3 regions of human IgG1, as shown below. VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (sequence number 13).

[0029] In some aspects, the fusion proteins described herein comprise an Fc domain that is at least 50%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13. In other aspects, the fusion proteins described herein comprise an Fc domain of SEQ ID NO: 13 with 1-5, 1-10, 1-15, 1-20, or 1-25 substitutions or conservative substitutions. Fc variants can include those described below, which are based on the numbering scheme of the EU index according to Kabat et al. (1991) NIH Publication 91-3242, National Technical Information Service, Springfield, VA. Such variants include, but are not limited to, Fc regions in which one or more of amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 have been replaced with a different amino acid residue such that the variant Fc region has an altered affinity for an effector ligand (e.g., an Fc receptor or the C1 component of complement), as described in U.S. Pat. Nos. 5,624,821 and 5,648,260, which are incorporated herein by reference; Fc regions in which one or more of amino acid residues 329, 331, and 322 have been replaced with a different amino acid residue such that the variant Fc region has altered affinity for an effector ligand (e.g., an Fc receptor or the C1 component of complement), as described in U.S. Pat. Nos. 5,624,821 and 5,648,260, which are incorporated herein by reference; No. 6,194,551, incorporated herein by reference; an Fc region having one or more mutations at amino acid residues 252, 254, 256, 433, 435, and 436 to extend the serum half-life of the fusion protein, as described in U.S. Pat. No. 6,277,375, incorporated herein by reference; and / or an Fc region having substitutions at positions 259, 308, 428, and 434 to increase binding to FcRn and / or improve pharmacokinetic properties.

[0030] In some aspects, hybrid IgG isotypes with specific biological characteristics can be used. For example, IgG1 / IgG3 or IgG1 / IgG2 hybrid variants can be constructed by substituting IgG1 positions in the CH2 and / or CH3 regions with amino acids from IgG2 or IgG3 at positions where the two isotypes differ.

[0031] In other aspects, glycosylation of Fc is modified. The oligosaccharides covalently attached to the Fc region can be altered, for example, by engineering or otherwise expressing IgG in different organisms or cell lines (e.g., Lec-13 CHO cells or rat hybridoma YB2 / 0 cells); by modulating enzymes involved in the glycosylation pathway (e.g., FUT8 [α1,6-fucosyltransferase] and / or β1-4-N-acetylglucosaminyltransferase III [GnTIII]); by modifying the carbohydrate(s) after IgG is expressed; or by expressing the fusion protein in the presence of a fucose analog as an enzyme inhibitor. Other methods of glycoform modification of Fc-containing fusion proteins include using glycoengineered strains of yeast (Li et al. (2006) Nature Biotechnology 24(2):210-215) or plants (Cox et al. (2006) Nat. Biotechnol. 24(12):1591-7).

[0032] In some aspects, the fusion protein does not contain a linker between the Notch1 or Notch4 extracellular domain containing EGF-like repeats 10-14 and the Fc portion of the antibody. For example, the fusion protein can contain an Fc domain directly linked to a Notch1 or Notch4 extracellular domain containing EGF-like repeats 10-14 without any intervening sequences.

[0033] In other aspects, a linker or adapter molecule consisting of 1-5 or 1-20 amino acids may be inserted between the Fc domain and the Notch1 or Notch4 extracellular domain containing EGF-like repeats 10-14. Exemplary linkers include, but are not limited to, DLGPG (SEQ ID NO: 14), GSGSGSGSGSGS (SEQ ID NO: 15), AGGGGGSG (SEQ ID NO: 16), AGGGGSGG (SEQ ID NO: 17), QPDEPGGS (SEQ ID NO: 18), ELQLEESAAEAQDGELD (SEQ ID NO: 19), TVAAPS (SEQ ID NO: 20), QPDEPGGSG (SEQ ID NO: 21), or ELQLEESAAEAQDGELDG (SEQ ID NO: 22), TVAAPSG (SEQ ID NO: 23). In some cases, the linker comprises, consists essentially of, or consists of an amino acid sequence that is at least 50%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 14-23. In some aspects, the linker sequence does not contain cysteine ​​residues. The linker or adapter molecule may also be designed with a protease cleavage site to allow for separation of the fused moieties.

[0034] The fusion proteins of the invention can be chemically synthesized or prepared by recombinant DNA technology. Thus, in another aspect, the invention provides a nucleic acid molecule encoding the fusion protein of the invention. Exemplary nucleic acid molecules are set forth herein as SEQ ID NO:2 and SEQ ID NO:4. In a related aspect, the invention provides a vector, particularly an expression vector, comprising a nucleic acid molecule encoding the fusion protein. In some aspects, the vector provides replication, transcription and / or translation regulatory sequences that facilitate recombinant synthesis of the fusion protein in eukaryotic or prokaryotic cells. Thus, the invention also provides a host cell for recombinant expression of the fusion protein, and a method for harvesting and purifying the fusion protein produced by the host cell. Recombinant protein production and purification are routinely performed by those skilled in the art. The fusion protein can be purified by any suitable method known in the art, including but not limited to gel filtration and / or affinity purification. In some aspects, the fusion protein further comprises a tag to facilitate purification, such as an epitope tag, where the tag can be optionally cleaved using a protease prior to further analysis.

[0035] Alternatively, the fusion proteins of the invention can be synthesized by any of the chemical synthesis techniques known in the art, in particular solid phase synthesis techniques, for example using a commercially available automated peptide synthesizer. See, for example, Stewart & Young (1984) Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co.; Tam et al. (1983) J. Am. Chem. Soc. 105:6442; Merrifield (1986) Science 232:341-347; Barany et al. (1987) Int. J. Peptide Protein Res. 30:705-739; and US 5,424,398.

[0036] Angiogenesis is thought to involve a complex interplay of molecules that stimulate and inhibit the growth of endothelial cells, the primary cells of capillaries. Under normal conditions, these molecules appear to maintain microvessels in a quiescent state (i.e., in the absence of capillary growth) for long periods of time. However, when necessary (such as during wound repair), these cells can rapidly proliferate and be replaced for short periods of time. Although angiogenesis is a highly regulated process under normal conditions, many conditions (characterized as "angiogenic diseases") are caused by persistent unregulated angiogenesis. In other words, unregulated angiogenesis can directly cause certain pathological conditions or exacerbate existing pathological conditions. In vivo evidence, including clinical observations, indicates that abnormal angiogenesis is associated with many pathological conditions, including rheumatoid arthritis, inflammation, cancer, psoriasis, degenerative eye diseases, and others.

[0037] As demonstrated herein, the fusion proteins of the present invention exhibit anti-angiogenic activity. Thus, in one aspect, the present invention further provides a method of inhibiting angiogenesis by contacting a cell or tissue with an effective amount of the fusion protein of the present invention. According to this aspect, the cell is an endothelial cell and the tissue can be a tissue containing endothelial cells and / or a tissue exhibiting excessive vascularity. Examples of such tissues include, but are not limited to, ocular tissue, tumor tissue, and joint tissue. An effective amount of the fusion protein is an amount that provides a measurable reduction in angiogenesis (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction) compared to the same cell or tissue not contacted with the fusion protein. Inhibition or reduction of angiogenesis can be assessed by measuring cleavage of endogenous Notch1 expressed on the surface of endothelial cells, determining expression of Notch target genes NRARP, HEY2, RND1, and / or DLL4, and / or assessing the formation and / or length of endothelial sprouts, as demonstrated herein.

[0038] The fusion protein of the present invention also finds application in the treatment of angiogenic diseases or disorders by inhibiting Notch signaling. Accordingly, another aspect of the present invention provides a method of treating angiogenic diseases or disorders in a subject by administering to the subject an effective amount of the fusion protein of the present invention. As used herein, "treating", "treatment" or "treat" can mean to temporarily or permanently alleviate, suppress, inhibit, eliminate or delay the appearance of symptoms, clinical signs, or underlying pathology of a disease or disorder. Suppressing a disease or disorder includes administering a fusion protein of the present invention to a subject after the disease or disorder is induced but before it is clinically manifested. Suppressing a disease or disorder includes administering a fusion protein of the present invention to a subject after the disease or disorder is clinically manifested.

[0039] For the purposes of the present invention, an "angiogenic disease or disorder" is characterized by persistent, unregulated or abnormal angiogenesis that results in excessive blood vessel formation. Angiogenic diseases are also characterized by having high levels of vascular inflammation or in response to high levels of tissue inflammation. For example, neovascularization of the eye is considered the most common cause of blindness. In certain pre-existing conditions, such as arthritis, newly formed capillaries invade the joints and destroy cartilage. In diabetes, new capillaries formed in the retina invade the vitreous, bleeding and causing blindness. The growth and metastasis of solid tumors also depend on angiogenesis (Folkman (1986) Cancer Res. 46:467-473; Folkman (1989) J. Natl. Cancer Inst. 82:4-6). For example, it has been shown that when tumors expand larger than 2 mm, they need to obtain their own blood supply and therefore induce the growth of new capillaries. These new blood vessels, when embedded within tumors, provide a means for tumor cells to enter the circulation and metastasize to distant sites such as the liver, lungs, and bone (Weidner et. al. (1991) N. Engl. J. Med. 324(1):1-8).

[0040] Thus, the fusion proteins of the present invention can be used to treat angiogenic diseases or disorders, including, but not limited to, atherosclerosis, wound healing, eye diseases or disorders, pre-eclampsia, ischemia, stroke, cardiovascular disease, psoriasis, lymphedema, vascular inflammation or cancer.

[0041] In some aspects, the present invention provides a method for treating ocular diseases or conditions.In particular, the fusion protein is useful for treating ocular diseases or disorders such as: retinopathy of prematurity, corneal graft rejection, retrolental fibroplasia, neovascular glaucoma, rubeosis, proliferative diabetic retinopathy, ischemic retinopathy, intraocular neovascularization, corneal neovascularization, retinal neovascularization, choroidal neovascularization, diabetic macular edema, diabetic retinal ischemia, diabetic retinal edema, neovascular age-related macular degeneration (AMD), central retinal vein occlusion, branch retinal vein occlusion, retinitis pigmentosa, rubeosis iridis, angiogenesis-related visual impairment or vision loss (blindness), retinoblastoma, uveitis and corneal graft neovascularization, ocular angiogenesis associated with infection or surgical intervention, and other abnormal neovascularization conditions of the eye.In other aspects, the ocular disease or disorder is AMD or diabetic retinopathy.

[0042] In certain aspects, AMD is wet or dry AMD. Wet macular degeneration occurs when abnormal blood vessels grow behind the macula. These blood vessels are fragile and can leak fluid and blood, resulting in scarring of the macula and can cause rapid and severe damage. Bruch's membrane is destroyed, usually near the drusen deposits. This is where new blood vessel growth, or neovascularization, occurs. Central vision can become distorted or lost completely within a short period of time, sometimes within a few days. Thus, treatment according to the methods of the present invention will improve or reduce blood vessel growth or neovascularization, reducing vision loss.

[0043] In some embodiments, the present invention provides a method for treating cancer.Cancer that can be treated can include, but is not limited to, primary and metastatic solid tumors, including, for example: cancer of the following sites: breast, colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach, pancreas, liver, gallbladder and bile duct, small intestine, urinary tract (including kidney, bladder and urothelium), female reproductive organs (including cervix, uterus and ovary, and choriocarcinoma and gestational trophoblastic disease), male reproductive organs (including prostate, seminal vesicle, testis and germ cell tumors), endocrine glands (including thyroid, adrenal and pituitary gland), and skin, as well as hemangiomas, melanomas, sarcomas (including those originating from bone and soft tissue, and Kaposi's sarcoma) and brain, nerve, eye and meningeal tumors (including astrocytoma, glioma, glioblastoma, retinoblastoma, neuroma, neuroblastoma, neurinoma and meningioma). The fusion proteins are also useful for the treatment of solid tumors arising from hematopoietic malignancies such as leukemias (i.e., chloromas, plasmacytomas, and mycosis fungoides plaques and tumors, and cutaneous T-cell lymphoma / leukemia), as well as for the treatment of lymphomas (both Hodgkin's and non-Hodgkin's lymphomas). Additionally, the fusion proteins may be used alone or in combination with radiation therapy and / or other chemotherapeutic agents.

[0044] Other uses of the fusion protein include the treatment of autoimmune diseases, such as rheumatism, immune-mediated arthritis, osteoarthritis, and the like; skin diseases, such as psoriasis; vascular diseases, such as hemangiomas, capillary proliferation within atherosclerotic plaques, and the like; pulmonary fibrosis; Osler-Weber syndrome; myocardial angiogenesis; asthma; plaque neovascularization; telangiectasia; hemophilic joints; angiofibromas; eye diseases, and wound granulation. Other uses include the treatment of diseases characterized by excessive or abnormal stimulation of endothelial cells, including, but not limited to, intestinal adhesions, Crohn's disease, atherosclerosis, scleroderma, and hypertrophic scars (i.e., keloids). Another use is the treatment of neuroinflammatory diseases, including infection-induced neuroinflammation and Alzheimer's disease (AD). Another use is as a contraceptive agent by inhibiting ovulation and placenta formation. The fusion proteins of the invention may also be useful in the treatment of diseases that involve angiogenesis as a pathological consequence, such as cat scratch disease and ulcers (Helicobacter pylori).

[0045] The fusion protein may also be useful in treating metabolic syndrome, a combination of medical disorders that increase the risk of cardiovascular disease and diabetes. Other well-known names for such syndromes include syndrome X, insulin resistance syndrome, and Reaven's syndrome. Some features of this syndrome include fasting hyperglycemia, high blood pressure, central obesity (also known as visceral obesity), reduced high-density lipoprotein (LDL), elevated triglycerides, elevated uric acid levels, etc. The above fasting hyperglycemia includes type 2 diabetes or impaired fasting glucose, and impaired glucose tolerance or insulin resistance. In addition to metabolic syndrome, Notch decoys may indicate signs of pre-diabetic conditions.

[0046] The treatment method according to the present invention is carried out by administering an effective amount of the fusion protein or a pharmaceutical composition containing it to a subject having a disease or disorder characterized by excessive or abnormal angiogenesis. As used herein, the term "administration" or "administering" refers to the process of delivering an agent to a patient. The process of administration can vary depending on the agent(s) and the desired effect.

[0047] The term "effective amount" or "therapeutically effective amount" depends on the condition of the subject and the particular fusion protein administered. The term refers to an amount effective to achieve a desired clinical effect. The effective amount will vary depending on the nature of the condition being treated, the length of time activity is desired, and the age and condition of the subject, and is ultimately determined by the healthcare provider. In general, however, the dose used for adult human treatment typically ranges from 0.001 mg / kg to about 200 mg / kg per day. The dose may be from about 0.05 mg / kg to about 10 g / kg per day. The desired dose may be conveniently administered in a single dose, or may be administered as multiple doses administered at appropriate intervals, for example, as two, three, four or more divided doses per day. Multiple doses may be desired or required.

[0048] The subject to be treated is a mammal, which may be a human.Before diagnosis, the subject may be at risk for angiogenic disease or disorder because of exposure to one or more risk factors for developing angiogenic disease or disorder or genetic risk.Illustratively, one or more risk factors for cancer include, for example, subject has family history of cancer, age, smoking, exposure to sunlight, consumption of alcoholic beverages, lack of physical activity, obesity, and / or dietary deficiency.Illustrative examples of risk factors for developing macular degeneration include, for example, subject has family history of macular degeneration, age, smoking, continuous exposure to sunlight, high-fat diet, dietary deficiency, high blood pressure, obesity, and / or light-colored eyes.

[0049] Suitable methods of administering fusion proteins, such as pharmaceutical compositions comprising the fusion proteins described herein, are well known in the art. Multiple routes can be used to administer fusion proteins, but certain routes can provide more rapid and effective responses than others. Depending on the situation, pharmaceutical compositions comprising fusion proteins are applied or injected into body cavities, absorbed through the skin or mucous membranes, ingested, inhaled, and / or introduced into the circulation. For example, in some situations, it is desirable to deliver pharmaceutical compositions orally; intravenously, intratumorally, intraperitoneally, intracerebrally (intracemally), intraventricularly, intramuscularly, intraocularly, intraarterially, intraportally, intralesional, intramedullary, intrathecal, intraventricularly, transdermally, subcutaneously, intraperitoneally, intranasally, enterally, topically, sublingually, urethrally, vaginally, or rectally; by controlled, delayed, sustained, or other modified release systems; or by implanted devices. In one aspect, drug exposure can be optimized by maintaining a constant drug plasma concentration over time. Such a steady state is generally achieved in clinical practice by continuous infusion of the drug at a dose that depends on the clearance of the drug and the plasma concentration to be maintained. Optionally, the composition is administered locally by intratumoral, intrathecal, intracerebral (intraparenchymal), intraventricular, or intraarterial or intravenous administration targeted to the region of interest. Alternatively, the fusion protein is administered locally via implantation of a matrix, membrane, sponge, or other suitable material in which the desired compound is absorbed or encapsulated. When an implantation device is used, the device is, in one aspect, implanted in any suitable tissue or organ, and delivery of the fusion protein is via, for example, diffusion, time-release bolus, or continuous administration.

[0050] Intraocular administration of the fusion protein can be performed using intraocular implants, intravitreal injections, systemic administration, topical application, nanoparticles, microparticles, eye drops, bioadhesive gels or fibrin sealants, polysaccharides that modulate the permeability of the epithelial cell barrier complex, peptides that enhance corneal drug delivery, mucosal administration such as administration using biovector polymers, aqueous ophthalmic sprays, and electrodynamic ophthalmic spray treatments. In a particular aspect, the fusion protein can be administered by intravitreal injection or locally, such as in the form of eye drops.

[0051] The fusion protein may be administered as a single agent therapy or simultaneously or metronomically with other treatments such as surgery or tumor removal. As used herein, the term "concurrent" or "concurrently" means that the fusion protein and other treatments are administered within 48 hours, preferably within 24 hours, more preferably within 12 hours, even more preferably within 6 hours, and most preferably within 3 hours of each other. As used herein, the term "metronomically" means that the fusion protein is administered at a different time than the other treatments and at a regular frequency for repeated administration. For example, the fusion protein of the present invention may be administered with one or more VEGF inhibitors. For example, the fusion protein of the present invention may be administered with one or more VEGF inhibitors or in combination with laser treatment for vision loss.

[0052] The fusion protein provided herein may be in the form of a pharmaceutical composition comprising the fusion protein and a pharma- ceutically acceptable excipient. The fusion protein may be formulated in the form of a tablet or lozenge formulated in a conventional manner. For example, tablets and capsules for oral administration may contain conventional excipients, such as binders, fillers, lubricants, disintegrants, and wetting agents. Binders include, but are not limited to, syrup, acacia, gelatin, sorbitol, tragacanth, mucilage of starch, and polyvinylpyrrolidone. Fillers may be lactose, sugar, microcrystalline cellulose, corn starch, calcium phosphate, and sorbitol. Lubricants include, but are not limited to, magnesium stearate, stearic acid, talc, polyethylene glycol, and silica. Disintegrants may be potato starch and sodium starch glycolate. Wetting agents may be sodium lauryl sulfate. Tablets may be coated according to methods well known in the art.

[0053] The fusion proteins provided herein may be in liquid formulations, such as aqueous or oily suspensions, solutions, emulsions, syrups, and elixirs. The fusion proteins may also be formulated as a dry product for constitution with water or other suitable vehicle before use. Such liquid formulations may contain additives, such as suspending agents, emulsifying agents, non-aqueous vehicles, and preservatives. Suspending agents may be sorbitol syrup, methylcellulose, glucose / sugar syrup, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, and hydrogenated edible fats. Emulsifying agents may be lecithin, sorbitan monooleate, and acacia. Non-aqueous vehicles may be edible oils, almond oil, fractionated coconut oil, oily esters, propylene glycol, and ethyl alcohol. Preservatives may be methyl or propyl p-hydroxybenzoates and sorbic acid. In particular, the fusion proteins of the present invention may be in aqueous formulations for topical administration, such as in the form of eye drops.

[0054] The fusion proteins provided herein can also be formulated as suppositories, which can contain suppository bases such as cocoa butter or glycerides. The fusion proteins provided herein can also be formulated for inhalation, such as in the form of a solution, suspension, or emulsion that can be administered as a dry powder, or in the form of an aerosol using a propellant such as dichlorodifluoromethane or trichlorofluoromethane. The fusion proteins provided herein can also be formulated as transdermal formulations, such as creams, ointments, lotions, pastes, medicated plasters, patches, or membranes, containing aqueous or non-aqueous vehicles. The fusion proteins provided herein can also be formulated for parenteral administration, such as injections, intratumoral injections, or continuous infusions. The injectable formulations can be in the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain compounding agents, including but not limited to suspending agents, stabilizing agents, and dispersing agents. The fusion proteins can also be provided in powder form for reconstitution with a suitable vehicle, including but not limited to sterile pyrogen-free water.

[0055] The fusion protein provided herein can also be formulated as a depot preparation, which can be administered by implantation or intramuscular injection.The fusion protein can be formulated with a suitable polymer or hydrophobic material (e.g., as an emulsion in an acceptable oil), an ion exchange resin, or a sparingly soluble derivative (e.g., as a sparingly soluble salt).

[0056] The foregoing can be better understood with reference to the following discussion and results, which are presented for illustrative purposes and are not intended to limit the scope of the invention.

[0057] Example 1: Materials and Methods Expression and purification of Notch decoy. N1 10-14 Fc and N4 10-14The expression vector of Fc was transfected into HEK EXPI293™ cells using the EXPI293™ Expression System (ThermoFisher Scientific). The Notch decoy was then purified from the culture medium by HITRAP® rProtein A FF (GE Healthcare) affinity chromatography. The eluted fraction was collected and immediately dialyzed to exchange the buffer into phosphate-buffered saline. The protein was concentrated with a VIVASPIN® 20 10,000 MWCO centrifugal concentrator (Sartorius).

[0058] Cell lines. All cell cultures were maintained at 37 °C in a mixture of 5% CO2 and 95% humidified air. HUVECs isolated from human umbilical veins (Lonza) were cultured in EGM-2 medium (Lonza) on culture plates coated with rat tail type I collagen (BD Biosciences). HEK293T cells were purchased from ATCC and maintained in Dulbecco's modified Eagle's medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS). Normal human lung fibroblasts (NHLFB) were purchased from Lonza and cultured in Fibroblast Growth Medium (Lonza).

[0059] Western blot analysis. Cells were lysed in ice-cold cell RIPA buffer (Cell Signaling) containing 1x protease inhibitor (ThermoFisher Scientific), 1x phosphatase inhibitor (ThermoFisher Scientific), and 1 mM DDT, and Western blot analysis was performed. Primary antibodies against cleaved Notch, a protein tag sold under the trade name FLAG®, MYC, and actin were obtained from Cell Signaling Technology and incubated in blocking buffer (5% BSA, and 1x TBST (0.1% polysorbate 20)). Gel images were acquired using a CHEMIDOC™ MP imaging system (Bio-Rad), and quantification was performed using ImageJ.

[0060] Co-immunoprecipitation assay. N1 10-14 Fc or N4 10-14 Fc and full-length DLL4-MYC or JAG1-FLAG® were transiently co-transfected into HEK-293T cells using a transfection reagent sold under the trade name LIPOFECTAMINE® 2000. The cross-linking agent disuccinimidyl glutarate (ThermoFisher Scientific) was added to the cultures at a final concentration of 20 nmol / ml 24 hours after transfection and incubated for 30 minutes. Cells were then lysed in 100 μl of 1x cell lysis buffer from Cell Signaling. Lysates were pulled down by 20 μl of Protein A / G magnetic beads (ThermoFisher Scientific). To reverse cross-linking before Western blot analysis, immune complexes were treated with 50 μmol / ml dithiothreitol (DTT) and boiled for 4 minutes before electrophoresis.

[0061] Affinity analysis. N1 10-14 Fc and N4 10-14 The binding kinetics of Fc was analyzed on a BIACORE™ T200 system (GE Healthcare) using a surface plasmon resonance (SPR)-based assay. Human IgG Fc (Sino Biologics) was first immobilized on a CM5 biosensor chip. Appropriate concentrations of hDLL4-Fc (Sino Biologics) and hJAG1-Fc (Sino Biologics) were then captured on the surface with up to 20,000 response units (RU). Various concentrations of N1 10-14 Fc and N4 10-14Fc was passed over the chip together with running buffer (1x HBS-N (10 mM HEPES, 150 mM NaCl) and 0.005% polysorbate 20, 1 mM CaCl2, 2 mM MgCl2, pH 7.4). After each reaction, the captured ligand and analyte were removed by regeneration buffer (1x HBS-N (10 mM HEPES, 150 mM NaCl) and 0.005% polysorbate 20, 1 mM CaCl2, 2 mM MgCl2, pH 7.4). The entire reaction was carried out at 25°C with a flow rate of 25 μl / min. Sensorgrams for each concentration were acquired and analyzed with BIACORE™ evaluation software (GE Healthcare). The equilibrium constant K D is the dissociation rate constant k d and the binding rate constant k a The ratio (k d / k a ) was calculated.

[0062] Quantitative real-time polymerase chain reaction (qRT-PCR). Human IgG Fc (Sino Biologics), N1 10-14 Fc, or N4 10-14 Total RNA from HUVECs treated with either Fc was collected after 24 hours using Qiagen RNEASY® according to the manufacturer's recommendations. Complementary DNA (cDNA) synthesis was performed using a cDNA reverse transcription kit (ThermoFisher Scientific) with approximately 1 μg of RNA per 20 μl. Real-time PCR was performed on a VIIA™ 7 Real-time PCR System (Life Technologies) using a cyanine dye sold under the trade name SYBR™ Green.

[0063] Cell viability assay. 4 × 10 cells in a 96-well plate 3 HUVEC cells (Lonza) were seeded and cultured at N1 10-14 Fc or N4 10-14Increasing concentrations of either Fc or human IgG Fc (Sino Biologics) were added, with each concentration replicated six times. Human IgG Fc (Sino Biologics) was added as a control. After 72 h of incubation, cell viability was determined by XTT assay (Biotium).

[0064] Wound healing assay. HUVECs (Lonza) treated with CellTracker™ CMFDA dye (ThermoFisher) were seeded onto 24-well plates coated with rat tail collagen type I (BD Biosciences) and a "scratch wound" was made using a 200 μl pipette tip. After wounding, the cells were transfected with IgG Fc, N1 10-14 Fc, or N4 10-14 The cells were treated with either different concentrations of Fc (5 μ / μl or 10 μ / μl). After about 14 hours, the migration of cells into the scratch wound was imaged using a microscope.

[0065] Fibrin bead assay (FiBA). To evaluate the angiogenic potential of Notch peptibodies, 6 × 10 4 HUVEC cells (Lonza) were used to coat 150 beads sold under the trade name CYTODEX® (Sigma) in endothelial growth medium (EGM, Lonza). The endothelial cell-coated beads were coated with human IgG Fc (Sino Biologics), N1 10-14 Fc, or N4 10-14 The cells were embedded in fibrin gel (3mg / ml) with either Fc or 5x10 4 NHLFB were seeded onto the fibrin gel of EGM. The medium was changed every other day until day 12. The number and length of sprouts were analyzed by Image J (NIH).

[0066] Mice. All mice used in this study were maintained on a pure C57BL / 6J background. Male and female pups were used randomly in these studies.

[0067] Retinal analysis. Postnatal day 1 (P1) pups of C57BL / 6 mice were treated with 12.5 mg / kg recombinant N1.10-14 Fc decoy or Fc was injected intragastrically for 3 days (P1-P3). Eyes were isolated at P5 and fixed in 4% paraformaldehyde (ThermoFisher Scientific) on a nutator for 1 h at 4°C. After fixation, eyes were washed with 1x PBS solution. Retinas were dissected and permeabilized overnight on a nutator with 1x PBS containing 1% bovine serum albumin (BSA, Fisher Bioreagents) and 0.5% TRITON™ X-100 (Fisher Bioreagents) at 4°C. Samples were then immunostained overnight at 4°C with biotinylated IB4 (1:50, Vector Laboratories) and anti-α-SMA-FITC (1:200, MilliporeSigma) in PBLEC (5% TRITON™ X-100, 1M MgCl2, 1M CaCl2, and 1M MnCl2 in 1x PBS). IB4 was detected with streptavidin conjugated to a fluorescent dye sold under the trade name ALEXA FLUOR® 647 (Invitrogen). Immunostained retinas were post-fixed with 4% formaldehyde and mounted in mounting medium sold under the trade name VECTASHIELD® (Vector Laboratories). Whole mount retinal images were acquired using a Leica Dmi8 platform. All images were analyzed using ImageJ (NIH).

[0068] Statistics. For qRT-PCR analysis, relative expression was calculated using the ΔΔCt method with the following steps: (1) Normalization to a reference gene: ΔCt GOI = Ct GOI -Ct BA , and (2) the relative expression between conditions: ΔΔCt GOI = ΔCt EXP -ΔCt CNTUnless otherwise stated, t-test analysis was performed on all quantified data and significant differences between groups were determined using GraphPad Prism 9. P values ​​less than 0.05 were considered statistically significant. p values ​​< 0.05 are indicated with one star (*), p values ​​< 0.01 are indicated with two stars (**), and p values ​​< 0.001 are indicated with three stars (***). All experiments shown were repeated at least three times.

[0069] Example 2: Design and characterization of Notch peptibodies The extracellular domains of the four Notch proteins are composed of various numbers of EGF-like repeats. For Notch1, a combination of structure-function analysis and high-resolution crystal structure revealed that EGF-like repeats 11-12 are important for receptor-ligand interactions. In contrast to the Notch1 receptor, the core ligand-binding domain of the endothelial-specific Notch gene, Notch4, has not been fully characterized. Notch4 is the most divergent of the four mammalian Notch receptors, whereas EGF11-12 is highly homologous between Notch1 and Notch4. Of the four human Notch receptors, Notch1, 2, and 4 show a high level of conservation between EGF-like repeats 10-14, whereas Notch3 shows a notable difference in conservation, with EGF7-10 of the Notch3 receptor most closely corresponding to EGF11-13 of Notch1. Furthermore, protein alignments of human and mouse Notch receptors across EGF10-14 demonstrate high identity and similarity across species. Here, recombinant fragments of the human Notch1 and Notch4 extracellular domains containing the sequences of Notch1 and Notch4 EGF-like repeats 10-14 were fused to human IgG Fc. The resulting fusion protein is referred to herein as "N1 10-14 Fc" and "N4 10-14 This is called "Fc" (Figure 1).

[0070] N1 10-14 Fc and N4 10-14The Fc proteins were produced in HEK293F cells by transfection with the corresponding expression constructs. The secreted proteins were subsequently column purified and separated on Coomassie-stained 4-20% SDS-PAGE gels, revealing the N1 Fc protein with the expected band at approximately 50 kDa under reducing conditions. 10-14 Fc and N4 10-14 The approximate molecular weight (MW) of the Fc is shown. To confirm the identity of the protein, Western blot analysis was performed under both non-reducing and reducing conditions using an antibody specific for human IgG Fc, showing a specific band at approximately 100 kDa under non-reducing conditions and approximately 50 kDa under reducing conditions. No evidence of cleavage between the Notch EGF-like repeats and the Fc domain, or any other form of degradation, was evident. As expected, detection of native protein under non-reducing conditions was twice the predicted molecular weight, indicating dimerization of IgG Fc. To accurately assess the oligomeric state, mass photometry was used to determine the N1 10-14 Fc and N4 10-14 The Fc proteins were evaluated and both Notch decoys showed a mixture of oligomers with the 100 kDa dimer being the predominant component.

[0071] Example 3: N1 10-14 Fc and N4 10-14 Both Fc bind to DLL4 and JAG1 We subsequently determined whether the homologous ligand-binding domain of Notch4 is sufficient to bind the Notch ligands DLL4 and JAG1. In the case of Notch1, EGF-like repeats 11 and 12 correspond to the core binding domain and have been shown to be sufficient for ligand interaction alone. There is little evidence that Notch4 binds to the ligands DLL4 and JAG1. To confirm the binding specificity, we used N1 10-14 Fc, N4 10-14 Fc, full-length FLAG®-tagged JAG1, and full-length Myc-tagged DLL4 were co-expressed in 293T cells and co-immunoprecipitation was performed with a Notch decoy, which served as a "bait" protein. 10-14 Fc and N4 10-14Fc co-immunoprecipitated with both DLL4 and JAG1, verifying pan-ligand binding.

[0072] Surface plasmon resonance (SPR) spectroscopy was used to characterize and quantify the interactions between these proteins. Recombinant Fc-tagged hDLL4 and hJAG1 were immobilized on a sensor chip using amine coupling, and multi-cycle kinetic experiments were performed using N1 10-14 Fc or N4 10-14 As a control, recombinant IgG Fc was immobilized on the sensor chip. 、 N1 10-14 Fc and N4 10-14 Fc and the dissociation constants (K D ) interacted. Table 2 [Table 2]

[0073] As expected, N1 10-14 Fc bound to DLL4 and JAG1. As demonstrated here, there is a conserved binding domain within Notch4 that facilitates the interaction with DLL4 and JAG1. A notable feature of the sensorgrams between the two decoys is the lack of N1 binding to DLL4. 10-14 Fc is N4 10-14 The SPR-based binding assay showed that hJAG1 and N1 bound to hDLL4 with faster on- and off-rates than Fc, indicating a different binding mechanism. 10-14 Fc or N4 10-14 Although no interaction was measured between the Notch1 extracellular domain and Fc, the interaction between that of JAG1 was weak, suggesting that a pulling force is required to stabilize JAG1 in the state required for the interaction. It is concluded that the Notch decoys described herein can readily bind members of the Delta-like or Jagged / Serrate classes of Notch ligands.

[0074] Example 4: N1 10-14 Fc suppresses endothelial Notch signaling Interaction of endothelial Notch with Notch ligands such as DLL4 leads to cleavage of the intracellular domain of Notch1 and subsequent nuclear translocation. Notch decoys that inhibit DLL4-Notch interaction are expected to inhibit the cleavage of endogenous Notch1 expressed on the endothelial cell surface. To evaluate whether Notch decoys can block DLL4-mediated Notch1 activation, HUVECs were seeded on plates coated with DLL4 and then incubated with increasing concentrations of N1 10-14 At the highest dose tested, N1 10-14 Fc reduced DLL4-induced cleavage of endogenous Notch1 expressed in HUVECs (Fig. 2 ).

[0075] Once inside the nucleus, NICD interacts with the transcription factor RBPJ / CSL to regulate the expression of canonical Notch target proteins. To directly test whether Notch decoys affect the canonical Notch signaling pathway, a key regulator of endothelial cells, we used N1 10-14 Fc and N4 10-14 The inhibitory effect of IgG Fc, N1 on the Notch pathway in HUVEC was examined. 10-14 Fc, or N4 10-14 HUVECs were treated with increasing concentrations of either Fc (0, 0.5, 1, 5, and 10 μg / ml) for 24 h. Differences in mRNA expression of Notch target genes were then examined by qRT-PCR. Compared to the control group, N1 10-14 Fc significantly downregulated the Notch target genes NRARP, HEY2, RND1, DLL4, and Notch1 at multiple concentrations (Figure 3). 10-14 Fc decoy down-regulated Notch target genes to a lesser extent in HUVEC (Fig. 4).

[0076] Example 5:N1 10-14 Fc inhibits angiogenesis in vitro Angiogenesis is a tightly regulated, multi-step process in endothelial cells involving proliferation, cell migration, and tube formation. To evaluate the effect of Notch decoys on endothelial sprout formation, a three-dimensional in vitro assay was used. Beads sold under the trade name CYTODEX® were coated with HUVECs and then embedded in a fibrinogen matrix. To support the growth of HUVECs, fibroblasts were cultured on top of the matrix and supplied with growth factors. In this assay, endothelial sprouts grow out of the beads, mimicking the early stages of angiogenesis. IgG Fc, N1 10-14 Fc, or N4 10-14 The number of outgrowths and their corresponding length were assessed in HUVECs treated with either increasing concentrations of Fc. 10-14 After treatment with Notch decoy N1 Fc, the number of angiogenic sprouts (Fig. 5) and the length of newly formed sprouts (Fig. 6) were significantly increased by 100% sprouts per day. 10-14 It was decreased at concentrations of 5 μg / ml and 10 μg / ml of Fc.

[0077] To assess whether Notch decoys have a cytotoxic effect on HUVECs, cells were incubated with IgG Fc, N1 10-14 Fc, or N4 10-14 HUVECs were treated with increasing concentrations of either N1 or N2Fc for 72 h. 10-14 Fc dose-dependently inhibited N4 10-14 It was not inhibited by Fc.

[0078] We then determined whether the antiangiogenic effects observed in vitro were due in part to impaired migration or simply to viability. It has been previously demonstrated that inhibition of DLL4-Notch signaling induces endothelial cell migration. To understand the role of Notch decoys on endothelial cell migration, we used a scratch wound healing assay to examine the extent of cell migration into the scratched area. IgG Fc, N1 10-14 Fc, or N4 10-14We evaluated HUVECs treated with either N1 or Fc, and no migration defects were detected. 10-14 N4 instead of Fc 10-14 These results indicate that Fc biologically regulates endothelial cell behavior.

[0079] Example 6: N1 10-14 Fc inhibits retinal neovascularization in mice To better understand how peptibody-based Notch inhibitors affect angiogenesis in vivo, we investigated the effects of peptibody-based Notch inhibitors on N1 expression during postnatal mouse angiogenesis. 10-14 Fc treatments were tested. Human IgG Fc or N1 10-14 Neonatal mice were injected intragastrically with either N1 or N2Fc. 10-14 Fc showed a reduction in both the vascular area and radial vascular growth at the angiogenic front (Figure 7). Furthermore, filopodia-extending endothelial sprouts, called tip cells, were expressed in N1 10-14 Fc treated mice were less abundant than Fc. Although not the focus of this study, N1 10-14 Approximately half of the mice treated with Fc exhibited abnormally enlarged retinal veins.

[0080] In several vascular developmental settings, the Notch ligands JAG1 and DLL4 play important roles in recruiting vascular smooth muscle cells to new arteries during the angiogenic maturation process. 10-14 The vascular smooth muscle cell coverage in mice treated with N1 Fc was unchanged compared to the control group, indicating that N1 10-14 These results suggest that N1 Fc inhibited angiogenesis but did not affect vascular remodeling at this time point. 10-14 These results show that Fc can cause inhibition of angiogenesis in vivo.

Claims

1. A fusion protein whose sequence begins at the N-terminus of the fusion protein and follows: (a) The extracellular domain of the human Notch1 or Notch4 receptor protein, followed by (b) Fc portion of the antibody, The amino acid sequence is identical to that in [location]. Here, the extracellular domain of the human Notch1 or Notch4 receptor protein, (i) Beginning with an amino acid located at the N-terminus of EGF-like repeat 10, (ii) The fusion protein, which extends to the C-terminal amino acid of EGF-like repeat 14 and includes this as the C-terminal amino acid of the extracellular domain.

2. The fusion protein according to claim 1, further comprising a signal sequence.

3. A nucleic acid molecule encoding the fusion protein described in claim 1.

4. A host cell comprising the nucleic acid molecule described in claim 3.

5. A pharmaceutical composition comprising the fusion protein described in claim 1 and a pharmaceutically acceptable excipient.

6. A pharmaceutical composition according to claim 5 for use in a method for inhibiting angiogenesis, wherein the method comprises contacting cells or tissue with an effective amount of the fusion protein according to claim 1, thereby inhibiting angiogenesis.

7. A pharmaceutical composition according to claim 5 for use in a method for treating angiogenic disease or disorder in a subject, wherein the method comprises administering to the subject an effective amount of the fusion protein according to claim 1, thereby treating the angiogenic disease or disorder in the subject.

8. The pharmaceutical composition according to claim 7, wherein the neovascular disease or disorder includes an eye disease or disorder.

9. The pharmaceutical composition according to claim 8, wherein the eye disease or disorder is age-related macular degeneration (AMD) or diabetic retinopathy.

10. The pharmaceutical composition according to claim 7, wherein the angiogenic disease or disorder is cancer.

11. The pharmaceutical composition according to claim 7, wherein the neovascular disease or disorder is inflammation of the blood vessels.

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