Methods and Compositions for Treating Diseases or Disorders

Abstract

The present application provides agents that specifically inhibit the IGFBP7 / CD93 signaling pathway, such as agents that specifically block the interaction between CD93 and IGFBP7, methods of using the agents, and methods for identifying the agents.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 62 / 906,282, filed September 26, 2019, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy, created on September 22, 2020, is designated 251609_000034_SL.txt and is 11,372 bytes in size. [Technical Field]

[0003] The present invention relates to methods and compositions comprising agents that interrupt (or block) the CD93 / IGFBP7 signaling pathway. [Background technology]

[0004] Pathological angiogenesis, driven by an imbalance between pro- and anti-angiogenic signaling, is a hallmark of many diseases, both malignant and benign. Unlike healthy adults, where angiogenesis is tightly regulated, such diseases are characterized by uncontrolled new blood vessel formation, resulting in microvascular networks characterized by vascular immaturity with severe structural and functional abnormalities. The result of these abnormalities is further modification of the microenvironment, which often serves to promote disease progression and attenuate responses to conventional therapies.

[0005] Therefore, there is a need to develop methods or compositions for normalizing or promoting the maturation of the vasculature in these diseases (such as cancer). Summary of the Invention [Means for solving the problem]

[0006] The present application provides methods of treating a tumor (such as cancer) in a subject in need thereof, comprising administering to the subject an effective amount of a CD93 / IGFBP7 blocking agent that specifically inhibits the IGFBP7 / CD93 signaling pathway. In some embodiments, the CD93 / IGFBP7 blocking agent blocks the interaction between CD93 and IGFBP7.

[0007] In some embodiments, the CD93 / IGFBP7 blocking agent comprises an anti-CD93 antibody that specifically recognizes CD93. In some embodiments, the anti-CD93 antibody binds to CD93 competitively with mAb MM01 or mAb 7C10. In some embodiments, the anti-CD93 antibody binds to an epitope that overlaps or substantially overlaps with the epitope of mAb MM01 or mAb 7C10. In some embodiments, the anti-CD93 antibody also blocks the interaction between CD93 and multimerin 2 (MMRN2). In some embodiments, the anti-CD93 antibody does not block the interaction between CD93 and MMRN2. In some embodiments, the anti-CD93 antibody binds to the IGFBP7-binding site on CD93. In some embodiments, the anti-CD93 antibody binds to a region on CD93 outside the IGFBP7-binding site. In some embodiments, the anti-CD93 antibody binds to the extracellular region of CD93. In some embodiments, the extracellular region of CD93 comprises residues 22-580 of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the anti-CD93 antibody binds to the EGF-like region of CD93. In some embodiments, the EGF-like region of CD93 consists of residues 257-469 and / or 260-468 of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the anti-CD93 antibody binds to the C-type lectin domain of CD93. In some embodiments, the C-type lectin domain of CD93 comprises residues 22-174 of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the anti-CD93 antibody binds to the long loop region of CD93. In some embodiments, the long loop region of CD93 comprises residues 96-141 of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the anti-CD93 antibody is an anti-human CD93 antibody. In some embodiments, the anti-human CD93 antibody is mAb MM01 or a humanized version thereof. In some embodiments, the anti-CD93 antibody is a full-length antibody, a single-chain Fv (scFv), a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), a (dsFv)2, a V HH, Fv-Fc fusion, scFv-Fc fusion, scFv-Fv fusion, diabody, tribody, or tetrabody. In some embodiments, the anti-CD93 is comprised in a fusion protein.

[0008] In some embodiments, the CD93 / IGFBP7 blocking agent is a polypeptide. In some embodiments, the polypeptide is an inhibitory CD93 polypeptide. In some embodiments, the inhibitory CD93 polypeptide is a fragment of CD93 or a variant of CD93 comprising the extracellular domain of CD93. In some embodiments, the polypeptide is a soluble polypeptide. In some embodiments, the polypeptide is membrane-bound. In some embodiments, the inhibitory CD93 polypeptide comprises a variant of the extracellular domain of CD93. In some embodiments, the polypeptide binds to IGFBP7 with a higher affinity than to MMNR2. In some embodiments, the polypeptide does not bind to MMNR2. In some embodiments, the polypeptide binds to IGFBP7 with a higher affinity than CD93. In some embodiments, the inhibitory CD93 polypeptide comprises an F238 residue, and amino acid numbering is based on SEQ ID NO: 1. In some embodiments, the inhibitory CD93 polypeptide further comprises a stabilization domain. In some embodiments, the stabilization domain is an Fc domain. In some embodiments, the polypeptide is about 50 to about 200 amino acids in length.

[0009] In some embodiments, the CD93 / IGFBP7 blocker comprises an anti-IGFBP7 antibody that specifically recognizes IGFBP7. In some embodiments, the anti-IGFBP7 antibody binds to IGFBP7 competitively with mAb R003 or mAb 2C6. In some embodiments, the anti-IGFBP7 antibody binds to an epitope that overlaps with the epitope of mAb R003 or mAb 2C6. In some embodiments, the anti-IGFBP7 antibody also blocks the interaction between IGFBP7 and IGF-1, IGF-2, and / or IGF1R. In some embodiments, the anti-IGFBP7 antibody does not block the interaction between IGFBP7 and IGF-1, IGF-2, and / or IGF1R. In some embodiments, the anti-IGFBP7 antibody binds to the CD93-binding site on IGFBP7. In some embodiments, the anti-IGFBP7 antibody binds to a region on IGFBP7 that is outside the CD93-binding site. In some embodiments, the anti-IGFBP7 antibody binds to the N-terminal domain (residues 28-106) of IGFBP7. In some embodiments, the N-terminal domain of IGFBP7 consists of residues 28-106 of the amino acid sequence of SEQ ID NO:2. In some embodiments, the anti-IGFBP7 antibody binds to the Kazal-like domain of IGFBP7. In some embodiments, the Kazal-like domain of IGFBP7 consists of residues 105-158 of the amino acid sequence of SEQ ID NO:2. In some embodiments, the anti-IGFBP7 antibody binds to the Ig-like C2-type domain of IGFBP7. In some embodiments, the Ig-like C2-type domain of IGFBP7 consists of residues 160-264 of the amino acid sequence of SEQ ID NO:2. In some embodiments, the anti-IGFBP7 antibody binds to the insulin-binding (IB) domain of IGFBP7. In some embodiments, the anti-IGFBP7 antibody is an anti-human IGFBP7 antibody. In some embodiments, the anti-human IGFBP7 antibody is mAb R003 or a humanized version thereof. In some embodiments, the anti-IGFBP7 antibody is a full-length antibody, a single-chain Fv (scFv), a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), a (dsFv)2, a V HH, Fv-Fc fusion, scFv-Fc fusion, scFv-Fv fusion, diabody, tribody, or tetrabody. In some embodiments, the anti-IGFBP7 antibody is comprised in a fusion protein.

[0010] In some embodiments, the CD93 / IGFBP7 blocking agent is a polypeptide, and the polypeptide is an inhibitory IGFBP7 polypeptide comprising an IGFBP7 variant. In some embodiments, the inhibitory IGFBP7 polypeptide binds to CD93 but does not activate CD93. In some embodiments, the inhibitory IGFBP7 polypeptide binds to CD93 with higher affinity than to IGF-1, IGF-2, and / or IGF1R. In some aspects, the polypeptide binds to CD93 with higher affinity than IGFBP7. In some embodiments, the inhibitory IGFBP7 polypeptide comprises the IB domain of IGFBP7. In some embodiments, the inhibitory IGFBP7 polypeptide further comprises a stabilization domain. In some embodiments, the stabilization domain is an Fc domain. In some embodiments, the inhibitory IGFBP7 polypeptide is about 50 to about 200 amino acids in length.

[0011] In some embodiments, the CD93 / IGFBP7 blocking agent comprises a fusion protein, a peptide analog, an aptamer, an avimer, an anticalin, a speigelmer, or a small molecule compound.

[0012] In some embodiments of any of the above methods, the CD93 / IGFBP7 blocking agent reduces expression of CD93 or IGFBP7. In some embodiments, the CD93 / IGFBP7 blocking agent comprises an siRNA, shRNA, miRNA, antisense RNA, or a gene editing system.

[0013] In some embodiments of any of the above methods, the method further comprises administering a second agent to the subject. In some embodiments, the second agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of an anti-PD1 antibody, an anti-PD-L1 antibody, and an anti-CTLA4 antibody. In some embodiments, the second agent is a chemotherapeutic agent. In some embodiments, the second agent is an immune cell. In some embodiments, the second agent is an anti-angiogenesis inhibitor. In some embodiments, the anti-angiogenesis inhibitor is an anti-VEGF inhibitor.

[0014] In some embodiments of any of the above methods, the cancer is characterized by abnormal tumor vasculature.

[0015] In some embodiments of any of the above methods, the cancer is characterized by high expression of VEGF.

[0016] In some embodiments of any of the above methods, the cancer is characterized by high expression of CD93.

[0017] In some embodiments of any of the above methods, the cancer is characterized by high expression of IGFBP7.

[0018] In some embodiments of any of the above methods, the cancer is a solid tumor. In some embodiments, the cancer is colorectal cancer, non-small cell lung cancer, glioblastoma, renal cell carcinoma, cervical cancer, ovarian cancer, fallopian tube cancer, peritoneal cancer, breast cancer, prostate cancer, bladder cancer, oral squamous cell carcinoma, head and neck squamous cell carcinoma, brain tumor, bone cancer, or melanoma. In some embodiments, the cancer is highly vascular. In some embodiments, the cancer is triple-negative breast cancer (TNBC). In some embodiments, the cancer is melanoma. In some embodiments, the patient is resistant to (or resistant to) a previous treatment, including administration of an immune checkpoint inhibitor, e.g., an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, or a combination thereof. In some embodiments, "enriched" as used herein refers to a greater amount or density of blood vessels (e.g., at least 10%, 20%, 30%, 40%, or 50% or more) in tumor tissue compared to the amount or density of blood vessels in the corresponding tissue in a subject without cancer.

[0019] In some embodiments, methods are provided for determining whether a candidate agent is useful for treating cancer, comprising determining whether the candidate agent disrupts CD93 / IGFBP7 interaction, wherein the candidate agent is useful for treating cancer if shown to specifically disrupt CD93 / IGFBP7 interaction. In some embodiments, the method comprises determining whether the candidate agent disrupts the interaction of CD93 and IGFBP7 on the cell surface. In some embodiments, the method comprises determining whether the candidate agent specifically disrupts the interaction of CD93 and IGFB7 in an in vitro assay system. In some embodiments, the in vitro system is a yeast two-hybrid system. In some embodiments, the in vitro system is an ELISA-based assay. In some embodiments, the in vitro system is a FACS-based assay. In some embodiments, the candidate agent is an antibody, peptide, fusion peptide, peptide analog, polypeptide, aptamer, avimer, anticalin, spiegelmer, or small molecule compound. In some embodiments, the method comprises contacting the candidate agent with a CD93 / IGFBP7 complex. In some embodiments, an agent identified by any of the above methods is provided.

[0020] In some embodiments, non-naturally occurring polypeptides are also provided, where the non-naturally occurring polypeptide is a variant inhibitory CD93 polypeptide comprising the extracellular domain of CD93, and the polypeptide blocks the interaction between CD93 and IGFBP7. In some embodiments, the variant inhibitory CD93 polypeptide is membrane-bound. In some embodiments, the variant inhibitory CD93 polypeptide is soluble. In some embodiments, the variant inhibitory CD93 polypeptide binds to IGFBP7 with a higher affinity than to MMNR2. In some embodiments, the variant inhibitory CD93 polypeptide binds to IGFBP7 with a higher affinity than CD93. In some embodiments, the inhibitory CD93 polypeptide comprises an F238 residue, where the amino acid numbering is based on SEQ ID NO: 1. In some embodiments, the inhibitory CD93 polypeptide further comprises a stabilization domain. In some embodiments, the stabilization domain is an Fc domain. In some embodiments, the inhibitory polypeptide is about 50 to about 200 amino acids in length.

[0021] In some embodiments, non-naturally occurring variant inhibitory IGFBP7 polypeptides comprising variants of IGFBP7 are also provided, wherein the polypeptides block the interaction between CD93 and IGFBP7. In some embodiments, the variant inhibitory IGFBP7 polypeptides bind to CD93 but do not activate CD93. In some embodiments, the variant inhibitory IGFBP7 polypeptides bind to CD93 with higher affinity than to IGF-1, IGF-2, and / or IGF1R. In some embodiments, the variant inhibitory IGFBP7 polypeptides bind to CD93 with higher affinity than IGFBP7. In some embodiments, the variant inhibitory IGFBP7 polypeptides comprise the IB domain of IGFBP7. In some embodiments, the variant inhibitory IGFBP7 polypeptides further comprise a stabilization domain. In some embodiments, the stabilization domain is an Fc domain. In some embodiments, the variant inhibitory IGFBP7 polypeptides are about 50 to about 200 amino acids in length.

[0022] In some embodiments, pharmaceutical compositions are also provided that include the above-described agent, non-naturally occurring polypeptide, or non-naturally occurring variant inhibitory IGFBP7 polypeptide and a pharmaceutically acceptable carrier and / or excipient. [Brief explanation of the drawings]

[0023] [Figure 1A] Figures 1A-1G show the identification of CD93 as a receptor protein in tumor vasculature regulated by VEGF signaling. Figure 1A shows a Venn diagram depicting the overlap of tumor vascular genes significantly reduced (Log2 fold change < -0.5) by VEGF inhibitors from four different published RNA-Seq datasets. CD93 was the only gene found to be downregulated in all datasets, with 10 additional genes (list, right) downregulated in three of the four datasets. [Figure 1B]FIG. 1B shows tube formation in HUVEC cells when the indicated genes were knocked down. [Figure 1C] FIG. 1C shows analysis of TCGA normal and GTEx datasets for CD93 transcription. [Figure 1D] Figure ID shows representative IHC staining of human pancreatic, PDA, and PNET tumors for CD93 expression. [Figure 1E] FIG. 1E shows immunofluorescence (“IF”) staining of surface CD93 on mouse aortic endothelial cells (MAECs) cultured with or without VEGF. [Figure 1F] FIG. 1F shows immunofluorescence staining of specimens from normal pancreatic and orthotopic KPC tumor tissues stained for CD93 and CD31. [Figure 1G] Figure 1G shows immunofluorescence staining of specimens from normal skin and subcutaneously implanted B16 mouse tumors stained for CD93 and CD31. Scale bar = 50 μm. [Figure 2A] Figures 2A-2E show that blocking IGFBP7 / CD93 interaction inhibits tumor growth and promotes vascular maturation. Figure 2A shows the change in tumor volume after treatment with control or mouse CD93 monoclonal antibody ("mAb"). B6 mice were implanted with KPC tumor cells and initiated with twice-weekly treatment with control or mouse CD93 mAb. Tumor growth was monitored over time. [Figure 2B] Figure 2B shows IF staining of CD31 in tumor sections from control and mCD93 mAb 7C10-treated mice. Blood vessel density, percentage of circular vessels, and total vessel length were compared between groups. Arrows indicate circular vessels. Scale bar = 50 μm. [Figure 2C] Figure 2C shows that frozen tumor sections were co-stained for CD31 and αSMA, and the percentage of αSMA+ vessels in each field was quantified. Scale bar = 50 μm. [Figure 2D]Figure 2D shows tumor sections co-stained for CD31 and NG2, and the percentage of NG2+ blood vessels in each field was quantified. Each dot represents the average value from one animal, of which at least five random fields were analyzed. Scale bar = 50 μm. [Figure 2E] Figure 2E shows that KPC tumor-bearing mice were treated twice weekly with control or CD93 mAb, followed by intravenous injection of lectin-FITC to assess tumor perfusion. Overlay of CD31+ vessels with lectin-FITC clearly demonstrates perfused and nonperfused tumor vessels. Quantification of perfused tumor vessels is shown on the right. Each dot represents the mean value for one animal, with at least five random fields taken for each animal (n = 5). *P < 0.05, **P < 0.01; p values ​​were determined by unpaired Student's t-test. All data represent the mean ± SEM. [Figure 3A] Figures 3A-3F show that CD93 blockade promotes immune cell infiltration in tumors. Figure 3A shows representative images of CD3 and CD31 immunostaining and DAPI nuclear staining in transplanted KPC tumors on days 8 and 15 after the start of control or anti-CD93 treatment. [Figure 3B] Figure 3B shows quantification of CD3+ T cells in control or anti-CD93-treated tumor tissues. Each dot represents the average value for one animal, with at least five random fields taken for each animal. [Figure 3C] Flow cytometry analysis after 15 days of antibody treatment is shown. Flow cytometry analysis was performed to determine the percentage of infiltrating CD45+ leukocytes. Each dot represents one tumor. [Figure 3D] Flow cytometry analysis after 15 days of antibody treatment is shown. Flow cytometry analysis was performed to determine the number of CD45+ leukocytes, CD3+ T cells, CD4+ and CD8+ T cell subsets. Each dot represents one tumor. [Figure 3E]Flow cytometry analysis after 15 days of antibody treatment was performed to determine the percentage of granulocytic (CD3-CD11c-CD11b+Ly6G+Ly6C-) and monocytic (CD3-CD11c-CD11b+Ly6G-Ly6C+) MDSCs among CD45+ leukocytes in the tumor. Each dot represents one tumor. [Figure 3F] Figure 3F shows a representative image of CD3 and CD31 immunostaining in subcutaneous B16 mouse tumors 14 days after antibody treatment. Each dot represents the average value from one animal, and at least five random fields were analyzed. *P<0.05, **P<0.01, ***P<0.001. p values ​​were determined by unpaired Student's t-test. All data represent the mean ± SEM. [Figure 4A] Figures 4A-4G show that IGFBP7 was identified as a binding partner of CD93. Figure 4A shows a graphic view of a target well with a positive hit (IGFBP7) for CD93-Ig in a human genome-scale receptor array (GSRA) screening system. Wells containing an expression construct for Fc receptor (FcR) were used as a positive control. [Figure 4B] Figure 4B shows HEK293T cells transduced with control or CD93 genes stained with IGFBP7-Ig for binding in the presence of control, anti-CD93, or anti-IGFBP7 mAb as indicated. [Figure 4C] FIG. 4C shows HUVEC cells stained with control or IGFBP7-Ig in the presence or absence of mAb against hCD93. [Figure 4D] FIG. 4D shows that HUVEC cell lysates were immunoprecipitated with control IgG or CD93 mAb and blotted with CD93 and IGFBP7 antibodies. [Figure 4E] Figure 4E shows the microscale thermophoresis (MST) binding curve of human IGFBP7 to CD93. Kd values ​​are shown. [Figure 4F]Figure 4F shows control or mouse CD93 transduced HEK293T cells stained with mouse IGFBP7-Ig for binding. Monoclonal antibodies against mouse CD93 and IGFBP7 were added to assess their blocking ability. [Figure 4G] Figure 4G is a schematic diagram showing the structure of a series of chimeric proteins created by replacing each domain of IGFBP7 (BP7) with the corresponding portion from IGFBPL1 (BPL1). The binding of each chimeric protein to CD93 transfectants was tested by flow cytometry. The binding index refers to the mean fluorescence intensity (MFI) of the CD93 transfectant divided by the MFI of the control. [Figure 5A] Figures 5A-5E show IGFBP7 expression on tumor vascular endothelium. Figure 5A shows H&E staining and IF co-staining of IGFBP7 and CD31 in human pancreatic and PDA cancers. The percentage of IGFBP7-positive vessels in pancreatic ductal adenocarcinoma (PDAC) and normal pancreas was quantified. Each dot represents the average value from one tissue, and at least five random fields were analyzed. I: Pancreatic islet. Scale bar = 50 µm. [Figure 5B] Figure 5B shows implanted KPC tumor tissue co-stained for IGFBP7 and CD31. The dashed line separates the central region (C) from the tumor edge (E). Scale bar 100 µm. [Figure 5C] Figure 5C shows a representative Western blot for HIF-1α and IGFBP7 expression in HUVEC cells treated with DMOG (0, 10 and 24 hours). L: Protein ladder. [Figure 5D] Figure 5D shows IGFBP7 expression on mouse aortic endothelial cells (MAEC) detected by immunofluorescence. MAEC cells were incubated with dimethyloxaloylglycine (DMOG) with or without a mouse VEGFR-blocking mAb to induce hypoxia. The percentage of IGFBP7-expressing cells was quantified. Dots represent values ​​from randomly acquired fields. Scale bar = 50 μm. [Figure 5E]Figure 5E shows a violin plot showing IGFBP7 expression in tumor endothelial cells from a xenograft colon cancer model (Zhao Q., Cancer Research 2018;78(9):2370-82) 24 hours after aflibercept treatment. *P<0.05, ***P<0.001. p-values ​​were determined by unpaired Student's t-test. All data represent the mean ± SEM. [Figure 6A] Figures 6A-6D show that targeting the IGFBP7 / CD93 pathway improves drug delivery and facilitates chemotherapy. Figure 6A shows immunofluorescence staining of doxorubicin and hypoxia (hypoxyprobe) in control or CD93 mAb-treated KPC tumor-bearing mice. Control or CD93 mAb-treated KPC tumor-bearing mice were injected with doxorubicin and pimonidazole twice weekly to assess drug delivery and hypoxia, respectively. Doxorubicin entry and hypoxic (hypoxyprobe) areas within the tumor were quantified. Each dot represents one animal, and the entire tumor tissue was analyzed. [Figure 6B] Figure 6B shows tumor volume curves for the groups treated with control, mCD93 mAb alone, 5-FU alone, and the combination of mCD93 mAb and 5-FU. n=7. *P=0.045; **P=0.0163. B6 mice were subcutaneously implanted with 2x105 B16 mouse melanoma cells, and antibody and 5-FU treatment was initiated on day 6 when tumors became palpable. [Figure 6C] Figure 6C shows Kaplan-Meier survival analysis of the control, mCD93 mAb alone, 5-FU alone, and mCD93 mAb plus 5-FU treatment groups. n=7. *P=0.045**; P=0.0163. B6 mice were subcutaneously implanted with 2 x 10 B16 mouse melanoma cells, and antibody and 5-FU treatment was initiated on day 6 when tumors became palpable. [Figure 6D]Figure 6D shows immunofluorescence staining of Ki-67 and cleaved caspase 3 (CC3) in B16 mouse tumor tissues treated with 5-FU alone and the combination of 5-FU and mCD93 mAb. The percentage of Ki-67- and CC3-positive cells in tumor tissues was quantified. Each dot represents one animal, and the entire tumor tissue was analyzed. Scale bar = 50 μm. *P < 0.05, **P < 0.01. p values ​​were determined by unpaired Student's t-test. All data represent the mean ± SEM. [Figure 7A] Figures 7A-7G show that CD93 blockade sensitizes tumors to anti-PD-1 therapy. Figure 7A shows tumor weights after 14 days of antibody treatment. KPC tumor-bearing mice began treatment with control or anti-CD93. In some groups, CD4+ or CD8+ T cells were depleted with the respective antibodies before treatment with anti-CD93. *P<0.05, **P<0.01. p values ​​were determined by unpaired Student's t-test. All data represent the mean ± SEM. Each dot represents one tumor. [Figure 7B] Figure 7B shows representative images of immunostaining for B7-H1 and CD31 in subcutaneous KPC mouse tumors. [Figure 7C] Figure 7C shows flow cytometry analysis of single-cell suspensions of tumor tissue for B7-H1 expression. The percentages of B7-H1-positive cells among tumor cells, CD45+ leukocytes, and CD31+ ECs were determined. *P<0.05, **P<0.01. p values ​​were determined by unpaired Student's t-test. All data represent the mean ± SEM. Each dot represents one tumor. [Figure 7D] Figure 7D shows tumor growth curves in KPC tumor-bearing mice. Treatment began 7 days after KPC tumor inoculation. *P<0.05, **P<0.01. p values ​​were determined by unpaired Student's t-test. All data represent the mean ± SEM. [Figure 7E]Figure 7E shows tumor weights after 16 days of treatment with antibodies in KPC tumor-bearing mice. Treatment began 7 days after KPC tumor inoculation. *P<0.05, **P<0.01. p values ​​were determined by unpaired Student's t-test. All data represent the mean ± SEM. Each dot represents one tumor. [Figure 7F] Figure 7F shows the number of immune cells in tumors as determined by flow cytometry. *P<0.05, **P<0.01. p-values ​​were determined by unpaired Student's t-test. All data represent the mean ± SEM. Each dot represents one tumor. [Figure 7G] Figure 7G shows the composition of immune cells within tumors as determined by flow cytometry. *P<0.05, **P<0.01. p-values ​​were determined by unpaired Student's t-test. All data represent the mean ± SEM. Each dot represents one tumor. [Figure 8A] Figures 8A and 8B show that anti-CD93 treatment does not affect the proportions of T cell subsets within the tumor. Figure 8A shows FACS analysis of tumor-infiltrating T cell subsets upon 15 days of antibody treatment. [Figure 8B] FIG. 8B shows analysis of intracellular cytokines IFN-γ and TNF-α in CD8+ T cell subsets from freshly isolated tumor-infiltrating lymphocytes (TILs) upon 4 hours of PMA+ionomycin stimulation. [Figure 9A] Figures 9A and 9B show that anti-CD93 increases ICAM-1 expression on tumor vessels. Figure 9A shows representative images of ICAM-1 and CD31 immunostaining in tumor tissue from subcutaneous KPC mouse tumors after 14 days of antibody treatment. [Figure 9B] FIG. 9B shows representative images of immunostaining for CD45, CD31, and ICAM-1 in tumor tissue from subcutaneous B16 mouse tumors after 14 days of antibody treatment. [Figure 10A]Figures 10A and 10B show the identification of binding domains on IGFBP7 for CD93. Each extracellular domain of IGFBP7, including insulin-binding (IB), Kazal, and Ig, was swapped with the corresponding domain on IGFBP1 using PCR cloning and fused to the C-terminal Ig. These chimeric mutants were transiently expressed in HEK293T cells, and the supernatant was used to stain CD93 transfectants. Figure 10A shows whether various chimeric IGFBP7 mutants bind to CD93. [Figure 10B] Figure 10B shows various human genes containing IB domains assembled on expression vectors containing Fc-tags. The constructs were transiently transduced into HEK293T cells to produce Fc-tagged fusion proteins in the supernatant. The supernatant was used to stain CD93 transfectants by flow cytometry. The binding index represents the ratio of the binding MFI of the CD93 transfectants to that of control cells. [Figure 11A] Figures 11A and 11B show IGFBP7 transcription in human PDA cancer. Figure 11A shows increased IGFBP7 transcripts in human PDA compared to normal pancreas. [Figure 11B] FIG. 11B shows FACS analysis of the TCGA PDA dataset showing that transcription of IGFBP7 correlates with known endothelial cell markers, including PECAM1, CD34, VWF, and KDR (VEGFR2). [Figure 12A] Figures 12A and 12B show selective expression of IGFBP7 on mouse tumor vasculature. Figure 11A shows IF staining of IGFBP7 and CD31 in specimens from normal pancreas of naive B6 mice and tissue from orthotopic KPC mouse tumors. I refers to pancreatic islets. [Figure 12B] Figure 12B shows IF staining of IGFBP7 and CD31 in specimens from normal skin of naive B6 mice and tissue from subcutaneously implanted KPC and B16 mouse tumors. Scale bar = 50 μm. [Figure 13A]Figures 13A-13C show that blocking IGFBP7 / CD93 interaction inhibits angiogenesis and tumor growth. Figure 13A shows the results of a tube formation assay performed on IGFBP7 knockdown and control HUVEC cells. [Figure 13B] FIG. 13B shows the results of a tube formation assay performed with or without exogenous IGFBP7 protein in WT or CD93 knockdown HUVEC cells. [Figure 13C] FIG. 13C shows the results of a transwell migration assay performed on WT or CD93 knockdown HUVEC cells with or without exogenous IGFBP7 protein. [Figure 14A] Figures 14A-14F show that IGFBP7 blockade retards tumor growth and promotes tumor vascular maturation. Figure 14A shows that mouse IGFBP7 binds to MAEC cells and the interaction can be blocked by IGFBP7 mAb (clone 2C6). [Figure 14B] Figure 14B shows the change in tumor volume after treatment with IGFBP7 antibody. C57BL / 6 mice bearing palpable KPC tumors were treated twice a week with control or mIGFBP7 mAb (clone 2C6). Tumor growth was monitored over time (n = 10 mice / group). [Figure 14C] Figure 14C shows IF staining of CD31 on frozen tumor sections. Blood vessel density, percentage of round vessels, and total vessel length were compared between groups. Arrows indicate round vessels. Scale bar = 50 μm. [Figure 14D] Figure 14D shows representative images of IF staining of CD31 and αSMA on frozen KPC mouse tumor sections. Each dot represents a random field from three animals, with at least three random fields taken from each animal. [Figure 14E] Figure 14E shows representative images of IF staining of CD31 and NG2 on frozen KPC mouse tumor sections. Each dot represents a random field from three animals, with at least three random fields taken from each animal. [Figure 14F] Figure 14F shows representative images of IF staining of CD31 and activated integrin β1 (9EG7) in KPC mouse tumor sections, as well as quantification of 9EG7+ vessels (% of total vessels). Each dot represents the average value for one animal, with at least five random fields taken for each animal. Scale bar = 50 μm. [Figure 15] Figure 15 shows that human IGFBP7 does not bind to human IGF1R transfectants. Wild-type CHO and IGF1R-transfected CHO cells were stained with human IGF1R staining antibody to confirm surface IGF1R expression. At the same time, the cells were incubated with IGFBP7-Ig for analysis of possible interactions by flow cytometry analysis. [Figure 16] FIG. 16 shows the ability of various commercially available anti-human IGFBP7 mAbs and anti-CD93 mAbs to block the CD93 / IGFBP7 interaction. [Figure 17A] Figures 17A and 17B show that CD93 on non-hematopoietic cells mediates the anti-tumor effect of blocking CD93. Figure 17A shows a representative image of IF staining of a B16 tumor that detected injected anti-CD93 on the tumor vasculature (CD31+). [Figure 17B] Figure 17B shows tumor growth in CD93 chimeric mice after treatment with anti-CD93 antibody. WT B6 mice reconstituted with bone marrow (BM) cells from WT or CD93KO mice were inoculated with B16 tumor cells followed by antibody treatment. ***p<0.001. [Figure 18A] 18A-18C show that CD93 blockade inhibits mouse tumor growth. Both CD93 (FIG. 18A) and IGFBP7 (FIG. 18B) were upregulated in the tumor vasculature of subcutaneous B16 tumors. [Figure 18B] Both CD93 (FIG. 18A) and IGFBP7 (FIG. 18B) were upregulated in the tumor vasculature of subcutaneous B16 tumors. [Figure 18C]Figure 18C shows tumor growth after anti-CD93 antibody treatment. Mice bearing palpable B16 tumors were treated with control or anti-CD93 (7C10). n=10. *p<0.01. [Figure 19A] Figures 19A-19G show that CD93 blockade promotes a favorable tumor immune microenvironment. Figure 19A shows representative images of CD3 and CD31 immunostaining of B16 tumors after 2 weeks of antibody treatment. [Figure 19B] FIG. 19B shows flow cytometry analysis of infiltrating CD45+ leukocytes in B16 tumors. [Figure 19C] FIG. 19C shows immune cell subsets in B16 tumors. [Figure 19D] Anti-CD93 increased the percentage of TEM (CD44hiCD62L-), PD1+ and granzyme B+ cells in CD8+ TILs. [Figure 19E] Anti-CD93 increased the percentage of cytokine-producing cells in CD8+ TILs. [Figure 19F] Figure 19F shows the effect of anti-CD93 treatment on PD1+ cells, TEM cells, and Treg cells. The same treatment caused an increase in PD1+ and TEM cells, accompanied by a decrease in Treg cells in the CD4+ T cell compartment. [Figure 19G] Figure 19G shows a representative image of IF staining of B16 tumor tissue. IF staining revealed reduced hypoxic areas and fewer CD11b+ suppressors in anti-CD93-treated tumors. *p<0.05, **p<0.001, ***p<0.001. [Figure 20A] Figures 20A-20E show that CD93 blockade sensitizes B16 melanoma to immune checkpoint blockade (ICB) therapy. Figure 20A shows representative images of antibody-treated B16 tumors stained for PD-L1, CD31, and CD45. [Figure 20B] Figure 20B shows flow cytometry analysis of PD-L1 in different cell types. B6 mice bearing palpable B16 tumors were treated twice weekly with the indicated antibodies. [Figure 20C] FIG. 20C shows tumor growth and survival curves. [Figure 20D] FIG. 20D shows quantification of intratumoral immune cells. [Figure 20E] Figure 20E shows the quantification of TEM cells (CD44hiCD62L-) in different T cell subsets. *p<0.05, **p<0.01, ***p<0.001. [Figure 21A] Figures 21A-21D show that the IGFBP7 / CD93 pathway is upregulated in the vasculature of triple-negative breast cancer (TNBC). Figure 21A shows a representative image of IF staining of CD93 in human TNBC tumors. CD31 is used to stain blood vessels. [Figure 21B] Figure 21B shows a representative image of IF staining of IGFBP7 in human TNBC tumors. CD31 is used to stain blood vessels. [Figure 21C] Figure 21C shows a representative image of IF staining of CD93 in mouse 4T1 tumors. CD31 is used to stain blood vessels. [Figure 21D] Figure 21D shows a representative image of IF staining of IGFBP7 in mouse 4T1 tumors. CD31 is used to stain blood vessels. [Figure 22] FIG. 22 shows that IGFBP7 expression is associated with poor prognosis in TNBC. [Figure 23A] Figures 23A and 23B show that anti-CD93 inhibits orthotopic BC tumor growth in vivo. Mice were orthotopically implanted with 4T1 tumors (Figure 23A). Once palpable, mice were treated twice weekly with control or anti-CD93 mAb (clone 7C10, 10 mg / kg). [Figure 23B] Mice were orthotopically implanted with PY8119 (Figure 23B). Once palpable, mice were treated twice weekly with control or anti-CD93 mAb (clone 7C10, 10 mg / kg). [Figure 24]Figures 24A-24C show that blocking CD93 signaling promotes tumor vascular maturation in orthotopic 4T1 tumors. Ten days after anti-CD93 treatment, 4T1 tumor tissues were stained for αSMA (Figure 24A) and NG2 (Figure 24B) to examine pericyte coverage on CD31+ vessels. Blood vessels were counted by CD31 staining. Figure 24C shows that anti-CD93 treatment significantly attenuates tumor hypoxia and increases perfusion, as indicated by pimonidazole and lectin-FITC staining, respectively. [Figure 25] Figures 25A-25C show that CD93 blockade promotes a favorable TME. 4T1 tumors treated with anti-CD93 showed more CD3+ T cell infiltration with fewer intratumoral MDSCs based on IF (Figures 25A, 25B) and FACS analysis (Figure 25C). [Figure 26A] Figures 26A-26C show that IGFBP7 and CD93 are upregulated in the vasculature in human cancers. IGFBP7 is upregulated in the vasculature in human cancers, including kidney, head and neck, and colon. [Figure 26B] CD93 is upregulated in the vasculature in human cancers, including kidney, head and neck, and colon. [Figure 26C] FIG. 26C shows that both CD93 and IGFBP7 are upregulated in melanoma-associated endothelium. [Figure 27A] Figures 27A and 27B show the abundance of the IGFBP7 / CD93 pathway in human cancers resistant to anti-PD therapy. Figure 27A shows the expression levels of IGFBP7 and CD93 in patients with metastatic urothelial carcinoma. In a phase II study of patients with metastatic urothelial carcinoma treated with anti-PD-L1 (77), the expression levels of IGFBP7 and CD93 were compared between non-responders (SD / PD) and responders (CR / PR). Statistical analysis was performed using the Wilcoxon rank-sum test. [Figure 27B]Figure 27B shows the expression levels of IGFBP7 and CD93 in melanoma patients. In a cohort of melanoma patients receiving anti-PD-1 therapy (78), the expression of IGFBP7 and CD93 was determined in responders and non-responders. Statistical analysis was performed using an unpaired Student's t-test. [Figure 28A] Figures 28A-28E show that IGFBP7 and MMRN2 bind to different motifs on CD93. In Figure 28A, HEK293T cells transfected to express group 14 C-type lectin molecules were stained for IGFBP7-Ig and MMRN2-Ig binding. [Figure 28B] In Figure 28B, CHO cells stably expressing CD93 were stained with control or MMRN2-Ig in the presence or absence of IGFBP7-His protein. [Figure 28C] In Figure 28C, wells coated with IGFBP7-His protein were incubated with CD93-His protein and then examined for MMRN2-Ig binding by ELISA. CD93-His protein-coated wells served as a positive control. [Figure 28D] In Figure 28D, HEK293T cells transfected with control or CD93 constructs were stained with MMRN-Ig for binding in the presence or absence of anti-mCD93 (7C10). [Figure 28E] In Figure 28E, HEK293T cells transfected to express different mouse CD93 variants were stained with anti-CD93 (7C10), IGFBP7-Ig, and MMRN2-Ig. DETAILED DESCRIPTION OF THE INVENTION

[0024] This application provides methods and compositions useful for promoting a favorable tumor microenvironment for therapeutic intervention. Leaky and irregular vascular networks within solid tumors pose significant obstacles to drug delivery and impair immune cell infiltration. The inventors of this application have newly discovered that insulin-like growth factor binding protein 7 (IGFBP7) signals through CD93, which is central to this abnormality. The expression of CD93 and IGFBP7, both of which are controlled by VEGF signaling, is upregulated in tumor tissue. Surprisingly, disruption of the interaction between IGFBP7 and CD93 with either an IGFBP7 monoclonal antibody or a CD93 monoclonal antibody was found to attenuate tumor growth and promote vascular maturation. Blockade of CD93 increases tumor perfusion, alleviates hypoxia, and facilitates chemotherapy. Furthermore, targeting CD93 promotes intratumoral T cell infiltration, thereby sensitizing tumors to anti-PD1 antibody therapy. Thus, the present application identifies novel molecular interactions that contribute to aberrant tumor angiogenesis and provides a novel approach to cancer therapy.

[0025] The present application provides agents that specifically inhibit the IGFBP7 / CD93 signaling pathway, for example, agents that specifically block the interaction between CD93 and IGFBP7. Suitable agents include blocking antibodies that specifically recognize CD93, blocking antibodies that specifically recognize IGFBP7, inhibitory CD93 polypeptides comprising at least a portion of the extracellular domain of CD93 or a variant thereof, inhibitory polypeptides comprising a variant of IGFBP7, and other agents such as peptides, peptide analogs, fusion peptides, aptamers, avimers, anticalins, spiegelmers, small molecule compounds, siRNAs, shRNAs, miRNAs, antisense RNAs, and gene editing systems. These agents are useful for treating cancer or for contributing to one or more aspects of cancer treatment, such as blocking abnormal tumor angiogenesis, normalizing immature and leaky blood vessels, promoting the formation of functional vascular network in tumors, promoting vascular maturation, promoting favorable tumor microenvironment, increasing immune cell infiltration in tumors, increasing tumor perfusion, and reducing hypoxia in tumors.The agents described herein are also useful for enhancing tumor sensitivity to second therapy or facilitating the delivery of second therapeutic agents.Therefore, the agents described herein are particularly useful in combination therapy, for example, in combination with chemotherapeutic agents and immunomodulatory agents.

[0026] Thus, in one aspect, a method of treating cancer or one or more aspects of cancer treatment in a subject is provided, comprising administering to the subject an effective amount of an agent that specifically inhibits the IGFBP7 / CD93 signaling pathway (e.g., an agent that specifically blocks the interaction between CD93 and IGFBP7).

[0027] In another aspect, novel agents (eg, anti-CD93, anti-IGFBP7, inhibitory CD93 polypeptides, and inhibitory IGFBP7 polypeptides) that specifically block the interaction between CD93 and IGFBP7 are provided.

[0028] In another aspect, methods are provided for identifying agents useful in cancer treatment (eg, agents that specifically block the interaction between CD93 and IGFBP7), eg, in the context of high-throughput screening.

[0029] Kits, agents (such as any of the agents described herein), polynucleotides encoding agents (such as any of the agents described herein), and reagents (such as isolated CD93 / IGFBP7 complexes) useful in the methods described herein are also provided.

[0030] I. Definition Unless otherwise defined, 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 application belongs. In addition, any methods or materials similar or equivalent to those described herein can be used in the practice of this application. For purposes of this application, the following terms are defined:

[0031] It is understood that embodiments of the present application described herein with the term "comprising" include embodiments "consisting of" and / or "consisting essentially of."

[0032] An agent that inhibits the interaction between CD93 and IGFBP7 refers to any agent that reduces the level of binding between CD93 and IGFBP7 compared to the level of binding between CD93 and IGFBP7 in the absence of the agent. In some embodiments, the agent reduces the level of binding between CD93 and IGFBP7 by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some embodiments, the agent reduces the level of binding between CD93 and IGFBP7 to an undetectable level or eliminates the binding between CD93 and IGFBP7. Suitable methods for detecting and / or measuring (quantifying) the binding of CD93 to IGFBP7 are well known to those skilled in the art and include those described herein.

[0033] "Angiogenesis" refers to the process by which new blood vessels sprout from pre-existing blood vessels.

[0034] The term "antibody" is used in its broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, chimeric antibodies, full-length antibodies and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity. Antibodies and / or antibody fragments can be derived from murine antibodies, rabbit antibodies, human antibodies, fully humanized antibodies, camel antibody variable domains and humanized versions, shark antibody variable domains and humanized versions, and camelized antibody variable domains.

[0035] An "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This fragment consists of a dimer of one heavy-chain variable region domain and one light-chain variable region domain in tight, non-covalent association. The folding of these two domains results in six hypervariable loops (three loops each from the heavy and light chains) that contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind to antigen, although with lower affinity than the entire binding site.

[0036] A "single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment comprising the VH and VL antibody domains linked into a single polypeptide chain. In some embodiments, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a general review of scFvs, see Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994) (incorporated herein by reference in its entirety for all purposes).

[0037] As used herein, "diabody(s)" refers to a complex comprising two scFv polypeptides. In some embodiments, interchain pairing of the VH and VL domains is achieved, but not intrachain pairing, resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites.

[0038] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (HVR) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired antibody specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can contain residues that are not found in either the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. Generally, humanized antibodies comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992) (each of which is incorporated by reference in its entirety for all purposes).

[0039] As used herein, a first antibody "competes" with a second antibody for binding to a target (e.g., CD93 or IGFBP7) if it inhibits target binding of the second antibody by at least about 50% (e.g., at least about any of 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) in the presence of an equimolar concentration of the first antibody, or vice versa. A high-throughput process for "binning" antibodies based on these cross-competitions is described in WO 03 / 48731, which is incorporated herein by reference in its entirety for all purposes.

[0040] "Percent (%) amino acid sequence identity" or "homology" for the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the polypeptide to be compared, after aligning the sequences and taking into account any conservative substitutions as part of the sequence identity.Alignment for determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software.Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the entire length of the sequences to be compared. However, for purposes herein, % amino acid sequence identity values ​​are generated using the sequence comparison computer program MUSCLE (Edgar, R.C., Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5(1):113, 2004; each of which is incorporated herein by reference in its entirety for all purposes).

[0041] "Homology" refers to the sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. If both positions in two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if each position in two DNA molecules is occupied by adenine, the molecules are homologous at that position. The percent homology between two sequences is a function of 100 times the number of matching or homologous positions shared by the two sequences divided by the number of positions compared. For example, if 6 out of 10 positions in two sequences are matched or homologous, the two sequences are 60% homologous. As an example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, comparisons are performed when the two sequences are aligned to maximize homology.

[0042] As used herein, the term "epitope" refers to the specific group of atoms or amino acids on an antigen to which an antibody or diabody binds. Two antibodies or antibody portions may bind to the same epitope within an antigen if they exhibit competitive binding to the antigen.

[0043] As used herein, a first antibody (such as a diabody) "competes" with a second antibody (such as a diabody) for binding to a target antigen if it inhibits target antigen binding of the second antibody by at least about 50% (e.g., at least about any of 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) in the presence of an equimolar concentration of the first antibody. A high-throughput process for "binning" antibodies based on these cross-competitions is described in WO 03 / 48731, which is incorporated herein by reference in its entirety for all purposes.

[0044] The terms "polypeptide" or "peptide" are used herein to encompass all types of naturally occurring and synthetic proteins, including protein fragments of any length, fusion proteins, and modified proteins, including, but not limited to, glycoproteins, as well as any other type of modified protein (e.g., proteins resulting from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosylation, pegylation, biotinylation, etc.).

[0045] As used herein, the terms "specifically bind," "specifically recognize," and "specific for" refer to a measurable and reproducible interaction, such as binding between a target and an antibody (such as a diabody). In certain embodiments, specific binding determines the presence of a target in the presence of a heterogeneous population of molecules, including biomolecules (e.g., cell surface receptors). For example, an antibody that specifically recognizes a target (which may be an epitope) is an antibody (such as a diabody) that binds to this target with higher affinity, avidity, more readily, and / or with a longer duration than its binding to other molecules. In some embodiments, the extent of binding of the antibody to unrelated molecules is less than about 10% of the antibody's binding to the target, as measured, for example, by radioimmunoassay (RIA). In some embodiments, an antibody that specifically binds to a target has a binding affinity of ≦10 -5 M, ≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M, or ≤ 10 -12 Dissociation constant of M (K D) In some embodiments, the antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In some embodiments, specific binding can include, but does not require, exclusive binding. The binding specificity of an antibody or antigen-binding domain can be determined experimentally by methods known in the art. Such methods include, but are not limited to, Western blot, ELISA, RIA, ECL, IRMA, EIA, BIACORE™, and peptide scan.

[0046] As used herein, "composition(s)" includes and is applicable to the compositions of the present application. The present application also provides pharmaceutical compositions containing the components described herein.

[0047] As used herein, "treatment" or "treating" is an approach to obtaining beneficial or desired results, including clinical results. For purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from a disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or slowing the worsening of the disease), preventing or slowing the spread of the disease (e.g., metastasis), preventing or slowing the recurrence of the disease, slowing or slowing the progression of the disease, improving the disease state, causing remission (partial or total) of the disease, reducing the dose of one or more other drugs required to treat the disease, slowing the progression of the disease, improving quality of life, and / or prolonging survival. "Treatment" also encompasses the reduction of pathological consequences of hyperplasia, such as tumors (e.g., cancer), restenosis, or pulmonary hypertension. The methods of the present application contemplate any one or more of these aspects of treatment. The benefit to a treated subject is statistically significant, or at least perceptible to the patient or to the physician.

[0048] As used herein, the term "effective amount" refers to an amount of an agent or composition sufficient to treat a particular condition, disorder, state, or disease, e.g., ameliorate, alleviate, relieve, and / or delay one or more of its symptoms (e.g., clinical or subclinical symptoms). With respect to therapeutic use, beneficial or desired results include, for example, reducing one or more symptoms (biochemical, histological, and / or behavioral) resulting from the disease (including its complications and intermediate pathological phenotypes manifested during the development of the disease), improving the quality of life of a person suffering from the disease, reducing the dose of other drugs required to treat the disease, enhancing the effectiveness of another drug, slowing disease progression, and / or prolonging patient survival. With respect to hyperplasia (e.g., cancer, restenosis, or pulmonary hypertension), an effective amount includes an amount sufficient to shrink the hyperplastic tissue (e.g., a tumor) and / or reduce the rate of growth of the hyperplastic tissue (e.g., inhibiting hyperplasia or tumor growth), or prevent or delay other undesirable cell proliferation in the hyperplasia. In some embodiments, an effective amount is an amount sufficient to delay the onset of hyperplasia (e.g., cancer, restenosis, or pulmonary hypertension). In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. An effective amount can be administered in one or more doses. In the case of cancer, an effective amount of a drug or composition is: (i) reducing the number of tumor cells; (ii) reducing tumor size; (iii) inhibiting, delaying, partially delaying, and preferably stopping tumor cell invasion into peripheral organs; (iv) inhibiting (i.e., partially delaying, and preferably stopping) tumor metastasis; (v) inhibiting tumor growth; (vi) preventing or delaying tumor development and / or recurrence; and / or (vii) alleviating to some extent one or more symptoms associated with cancer. It should be noted that when a combination of active ingredients is administered, the effective amount of the combination may or may not include the amount of each ingredient that would be effective when administered individually. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug(s) used, the method of administration, and the like.

[0049] As used herein, the term "simultaneous administration" means that the first and second therapies in a combination therapy are administered at a time interval of about 15 minutes or less, e.g., about 10 minutes or less, about 5 minutes or less, or about 1 minute or less. When the first and second therapies are administered simultaneously, the first and second therapies can be contained in the same composition (e.g., a composition containing both the first and second therapies) or can be contained in separate compositions (e.g., the first therapy is contained in one composition and the two therapies are contained in another composition).

[0050] As used herein, the term "sequential administration" means that the first and second therapies in a combination therapy are administered at a time interval of more than about 15 minutes, for example, more than about 20 minutes, more than 30 minutes, more than 40 minutes, more than 50 minutes, more than 60 minutes, or more. Either the first or second therapy can be administered first. The first and second therapies are contained in separate compositions, which can be included in the same or different packages or kits.

[0051] As used herein, the term "concurrent administration" means that the administration of a first therapy and a second therapy in a combination therapy overlap with each other.

[0052] As used herein, "pharmaceutically acceptable" or "pharmacologically compatible" means a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. A pharmaceutically acceptable carrier or excipient preferably meets required toxicological and manufacturing testing standards and / or is included in the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration or other state / federal government, or is listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeias for use in mammals, more particularly humans.

[0053] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water or aqueous solutions, saline solutions, and aqueous dextrose and glycerol solutions are preferably used as carriers, particularly for injectable solutions. Alternatively, the carrier can be a solid dosage form carrier, including, but not limited to, one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavoring agent, and a coloring agent. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin, which is incorporated by reference in its entirety for all purposes.

[0054] The term "tumor" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth and includes benign or malignant abnormal growth of tissue. The term "tumor" includes cancer.

[0055] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a mammal, including, but not limited to, a human, bovine, equine, feline, canine, rodent, or primate. In some embodiments, the subject is a human. In preferred embodiments, the subject is a human.

[0056] Reference herein to "about" a value or parameter includes (and accounts for) a variation that is directed to the value or parameter itself. For example, a statement that refers to "about X" includes a statement of "X." In certain embodiments, a range may be within an order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5%. The acceptable variation encompassed by the term "about" or "approximately" depends on the particular system being studied and can be readily understood by one of ordinary skill in the art.

[0057] As used herein, the term "about X to Y" has the same meaning as "about X to about Y."

[0058] As used in this specification and claims, the singular forms "a," "an," or "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "method" includes one or more methods, and / or steps of the type described herein and / or that would be apparent to one of ordinary skill in the art upon reading this disclosure. As will be apparent to one of ordinary skill in the art, a subject being evaluated, selected for treatment, and / or treated is a subject in need of such activity.

[0059] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of statistical analysis, molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of the art. Such tools and techniques are available, for example, in Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York; Ausubel et al. eds. (2005) Current Protocols in Molecular Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Bonifacino et al. eds. (2005) Current Protocols in Cell Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Immunology, John Wiley and Sons, Inc.: Hoboken, NJ; Coico et al. eds. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Protein Science, John Wiley and Sons, Inc.: Hoboken, NJ; and Enna et al. eds. (2005) Current Protocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken, NJ. Additional techniques are described, for example, in U.S. Pat. No. 7,912,698 and U.S. Patent Application Publication Nos. 2011 / 0202322 and 2011 / 0307437, each of which is incorporated by reference in its entirety for all purposes.

[0060] The terms and expressions employed are used as terms of description rather than of limitation, and the use of such terms and expressions does not exclude any equivalents of the features or portions thereof shown and described, and various modifications are possible within the scope of the claimed technology.

[0061] II. Treatment method In one embodiment, the present application provides a method for treating a tumor (e.g., cancer) in a subject or one or more aspects of tumor (e.g., cancer) treatment, comprising administering to the subject an effective amount of an agent that specifically inhibits the IGFBP7 / CD93 signaling pathway (e.g., an agent that blocks the interaction between CD93 and IGFBP7). An agent "blocks the interaction between CD93 and IGFBP7" if the agent reduces the binding between CD93 and IGFBP7 compared to the level of binding between CD93 and IGFBP7 in the absence of the agent. In some embodiments, the agent reduces the binding between CD93 and IGFBP7 by at least about 10%, 20%, 30%, 40%, or 50%. In some embodiments, the agent reduces the binding between CD93 and IGFBP7 by at least about 60%, 70%, 80%, 90%, or more. In some embodiments, the agent blocks the CD93 / IGFBP7 interaction to undetectable levels or eliminates the binding between CD93 and IGFBP7.

[0062] Suitable methods for determining the binding of CD93 and IGFBP7 are known in the art and can include, for example, ELISA, pull-down assays, surface plasmon resonance assays, chip-based assays, FACS, yeast two-hybrid systems, phage display, and FRET.

[0063] The agents described herein can be administered directly or in the form of a polynucleotide encoding the agent. Thus, as used herein, the term "administering to a subject" encompasses both administering an agent directly to a subject and administering a polynucleotide encoding the agent, for example, via a vector.

[0064] In some embodiments, methods are provided for treating a tumor (e.g., cancer) in a subject, comprising administering to the subject an effective amount of an agent that specifically inhibits the IGFBP7 / CD93 signaling pathway (e.g., an agent that blocks the interaction between CD93 and IGFBP7). In some embodiments, the agent is an antibody, peptide, polypeptide, peptide analog, fusion peptide, aptamer, avimer, anticalin, spiegelmer, small molecule compound, siRNA, shRNA, miRNA, antisense RNA, or gene editing system. In some embodiments, the agent is a blocking antibody that specifically recognizes CD93. In some embodiments, the agent is a blocking antibody that specifically recognizes IGFBP7. In some embodiments, the agent is an inhibitory CD93 polypeptide comprising at least a portion of the extracellular domain of CD93 or a variant thereof. In some embodiments, the agent is an inhibitory polypeptide comprising a variant of IGFBP7. In some embodiments, the method further comprises administering to the subject a second therapeutic agent (e.g., a chemotherapeutic agent, an immunomodulatory agent, or immune cells).

[0065] In some embodiments, methods are provided for blocking aberrant tumor angiogenesis in a subject, comprising administering to the subject an effective amount of an agent that specifically inhibits the IGFBP7 / CD93 signaling pathway (e.g., an agent that blocks the interaction between CD93 and IGFBP7). In some embodiments, the agent is selected from the group consisting of an antibody, a peptide, a polypeptide, a peptide analog, a fusion peptide, an aptamer, an avimer, an anticalin, a spiegelmer, a small molecule compound, an siRNA, an shRNA, an miRNA, an antisense RNA, and a gene editing system. In some embodiments, the agent is a blocking antibody that specifically recognizes CD93. In some embodiments, the agent is a blocking antibody that specifically recognizes IGFBP7. In some embodiments, the agent is an inhibitory CD93 polypeptide comprising at least a portion of the extracellular domain of CD93 or a variant thereof. In some embodiments, the agent is an inhibitory polypeptide comprising a variant of IGFBP7. In some embodiments, the method further comprises administering to the subject a second therapeutic agent (e.g., a chemotherapeutic agent, an immunomodulatory agent, or an immune cell).

[0066] In some embodiments, a method for normalizing immature and leaky blood vessels in a subject is provided, comprising administering to the subject an effective amount of an agent that specifically inhibits the IGFBP7 / CD93 signaling pathway (e.g., an agent that blocks the interaction between CD93 and IGFBP7). In some embodiments, the agent is selected from the group consisting of an antibody, a peptide, a polypeptide, a peptide analog, a fusion peptide, an aptamer, an avimer, an anticalin, a spiegelmer, a small molecule compound, an siRNA, an shRNA, an miRNA, an antisense RNA, and a gene editing system. In some embodiments, the agent is a blocking antibody that specifically recognizes CD93. In some embodiments, the agent is a blocking antibody that specifically recognizes IGFBP7. In some embodiments, the agent is an inhibitory CD93 polypeptide comprising at least a portion of the extracellular domain of CD93 or a variant thereof. In some embodiments, the agent is an inhibitory polypeptide comprising a variant of IGFBP7. In some embodiments, the method further comprises administering to the subject a second therapeutic agent (e.g., a chemotherapeutic agent, an immunomodulatory agent, or an immune cell).

[0067] In some embodiments, a method is provided for promoting the formation of a functional vascular network in a tumor in a subject, comprising administering to the subject an effective amount of an agent that specifically inhibits the IGFBP7 / CD93 signaling pathway (e.g., an agent that blocks the interaction between CD93 and IGFBP7). In some embodiments, the agent is selected from the group consisting of an antibody, a peptide, a polypeptide, a peptide analog, a fusion peptide, an aptamer, an avimer, an anticalin, a spiegelmer, a small molecule compound, an siRNA, an shRNA, an miRNA, an antisense RNA, and a gene editing system. In some embodiments, the agent is a blocking antibody that specifically recognizes CD93. In some embodiments, the agent is a blocking antibody that specifically recognizes IGFBP7. In some embodiments, the agent is an inhibitory CD93 polypeptide comprising at least a portion of the extracellular domain of CD93 or a variant thereof. In some embodiments, the agent is an inhibitory polypeptide comprising a variant of IGFBP7. In some embodiments, the method further comprises administering to the subject a second therapeutic agent (e.g., a chemotherapeutic agent, an immunomodulatory agent, or an immune cell).

[0068] In some embodiments, a method is provided for promoting vascular maturation in a tumor in a subject, comprising administering to the subject an effective amount of an agent that specifically inhibits the IGFBP7 / CD93 signaling pathway (e.g., an agent that blocks the interaction between CD93 and IGFBP7). In some embodiments, a method is provided for promoting vascular normalization in a tumor in a subject, comprising administering to the subject an effective amount of an agent that specifically inhibits the IGFBP7 / CD93 signaling pathway (e.g., an agent that blocks the interaction between CD93 and IGFBP7). In some embodiments, the agent is selected from the group consisting of an antibody, a peptide, a polypeptide, a peptide analog, a fusion peptide, an aptamer, an avimer, an anticalin, a spiegelmer, a small molecule compound, an siRNA, an shRNA, an miRNA, an antisense RNA, and a gene editing system. In some embodiments, the agent is a blocking antibody that specifically recognizes CD93. In some embodiments, the agent is a blocking antibody that specifically recognizes IGFBP7. In some embodiments, the agent is an inhibitory CD93 polypeptide comprising at least a portion of the extracellular domain of CD93 or a variant thereof. In some embodiments, the agent is an inhibitory polypeptide comprising a variant of IGFBP7. In some embodiments, the method further comprises administering to the subject a second therapeutic agent (e.g., a chemotherapeutic agent, an immunomodulatory agent, or immune cells, etc.). In some embodiments, vascular normalization is characterized by increased association of pericytes and / or smooth muscle cells with endothelial cells lining the vascular wall, formation of a more normal (e.g., having a more physiological thickness) basement membrane, and / or closer association of blood vessels with the basement membrane. In some embodiments, vascular normalization described herein does not include a decrease in the number of blood vessels (e.g., a less dense network).

[0069] In some embodiments, a method for promoting a favorable tumor microenvironment in a subject is provided, comprising administering to the subject an effective amount of an agent that specifically inhibits the IGFBP7 / CD93 signaling pathway (e.g., an agent that blocks the interaction between CD93 and IGFBP7). In some embodiments, the agent is selected from the group consisting of an antibody, a peptide, a polypeptide, a peptide analog, a fusion peptide, an aptamer, an avimer, an anticalin, a spiegelmer, a small molecule compound, an siRNA, an shRNA, an miRNA, an antisense RNA, and a gene editing system. In some embodiments, the agent is a blocking antibody that specifically recognizes CD93. In some embodiments, the agent is a blocking antibody that specifically recognizes IGFBP7. In some embodiments, the agent is an inhibitory CD93 polypeptide comprising at least a portion of the extracellular domain of CD93 or a variant thereof. In some embodiments, the agent is an inhibitory polypeptide comprising a variant of IGFBP7. In some embodiments, the method further comprises administering to the subject a second therapeutic agent (e.g., a chemotherapeutic agent, an immunomodulatory agent, or an immune cell).

[0070] In some embodiments, a method for increasing immune cell infiltration into a tumor in a subject is provided, comprising administering to the subject an effective amount of an agent that specifically inhibits the IGFBP7 / CD93 signaling pathway (e.g., an agent that blocks the interaction between CD93 and IGFBP7). In some embodiments, the method increases the infiltration of CD3+ cells (e.g., tumor-infiltrating leukocytes ("TILs")). In some embodiments, the method increases the infiltration of CD45+ cells (e.g., TILs). In some embodiments, the method increases the infiltration of CD8+ cells (e.g., NK cells or T cells). In some embodiments, the method increases immune cell infiltration into the tumor by at least about 20%, 30%, 40%, 50%, or more. In some embodiments, the agent is selected from the group consisting of an antibody, a peptide, a polypeptide, a peptide analog, a fusion peptide, an aptamer, an avimer, an anticalin, a spiegelmer, a small molecule compound, an siRNA, an shRNA, an miRNA, an antisense RNA, and a gene editing system. In some embodiments, the agent is a blocking antibody that specifically recognizes CD93. In some embodiments, the agent is a blocking antibody that specifically recognizes IFGBP7. In some embodiments, the agent is an inhibitory CD93 polypeptide comprising at least a portion of the extracellular domain of CD93 or a variant thereof. In some embodiments, the agent is an inhibitory polypeptide comprising a variant of IGFBP7. In some embodiments, the method further comprises administering to the subject a second therapeutic agent (e.g., a chemotherapeutic agent, an immunomodulatory agent, or immune cells).

[0071] In some embodiments, a method for increasing tumor perfusion in a subject is provided, comprising administering to the subject an effective amount of an agent that specifically inhibits the IGFBP7 / CD93 signaling pathway (e.g., an agent that blocks the interaction between CD93 and IGFBP7). In some embodiments, tumor perfusion is increased by at least about 20%, 30%, 40%, 50%, or more. In some embodiments, the agent is selected from the group consisting of antibodies, peptides, polypeptides, peptide analogs, fusion peptides, aptamers, avimers, anticalins, spiegelmers, small molecule compounds, siRNAs, shRNAs, miRNAs, antisense RNAs, and gene editing systems. In some embodiments, the agent is a blocking antibody that specifically recognizes CD93. In some embodiments, the agent is a blocking antibody that specifically recognizes IGFBP7. In some embodiments, the agent is an inhibitory CD93 polypeptide comprising at least a portion of the extracellular domain of CD93 or a variant thereof. In some embodiments, the agent is an inhibitory polypeptide comprising a variant of IGFBP7. In some embodiments, the method further comprises administering to the subject a second therapeutic agent (e.g., a chemotherapeutic agent, an immunomodulatory agent, or immune cells, etc.).

[0072] In some embodiments, a method for reducing hypoxia in a tumor in a subject is provided, comprising administering to the subject an effective amount of an agent that specifically inhibits the IGFBP7 / CD93 signaling pathway (e.g., an agent that blocks the interaction between CD93 and IGFBP7). In some embodiments, tumor hypoxia is reduced by at least about 20%, 30%, 40%, 50%, or more. In some embodiments, the agent is selected from the group consisting of an antibody, a peptide, a polypeptide, a peptide analog, a fusion peptide, an aptamer, an avimer, an anticalin, a spiegelmer, a small molecule compound, an siRNA, an shRNA, an miRNA, an antisense RNA, and a gene editing system. In some embodiments, the agent is a blocking antibody that specifically recognizes CD93. In some embodiments, the agent is a blocking antibody that specifically recognizes IGFBP7. In some embodiments, the agent is an inhibitory CD93 polypeptide comprising at least a portion of the extracellular domain of CD93 or a variant thereof. In some embodiments, the agent is an inhibitory polypeptide comprising a variant of IGFBP7. In some embodiments, the method further comprises administering to the subject a second therapeutic agent (e.g., a chemotherapeutic agent, an immunomodulatory agent, or immune cells, etc.).

[0073] In some embodiments, a method is provided for reducing immunosuppressive cells (such as Treg cells, granulocytic myeloid-derived suppressor cells (gMDSCs), and tumor-associated macrophages (Macs)) in a subject, comprising administering to the subject an effective amount of an agent that specifically inhibits the IGFBP7 / CD93 signaling pathway (such as an agent that blocks the interaction between CD93 and IGFBP7). In some embodiments, the method reduces immunosuppressive cells in the tumor microenvironment. In some embodiments, the immunosuppressive cells are reduced by at least about 20%, 30%, 40%, 50%, or more. In some embodiments, the agent is selected from the group consisting of an antibody, a peptide, a polypeptide, a peptide analog, a fusion peptide, an aptamer, an avimer, an anticalin, a spiegelmer, a small molecule compound, an siRNA, an shRNA, an miRNA, an antisense RNA, and a gene editing system. In some embodiments, the agent is a blocking antibody that specifically recognizes CD93. In some embodiments, the agent is a blocking antibody that specifically recognizes IGFBP7. In some embodiments, the agent is an inhibitory CD93 polypeptide comprising at least a portion of the extracellular domain of CD93 or a variant thereof. In some embodiments, the agent is an inhibitory polypeptide comprising a variant of IGFBP7. In some embodiments, the method further comprises administering to the subject a second therapeutic agent (e.g., a chemotherapeutic agent, an immunomodulatory agent, or immune cells, etc.).

[0074] In some embodiments, a method for enhancing the sensitivity of a tumor to a second therapy is provided, comprising administering to the subject an effective amount of an agent that specifically inhibits the IGFBP7 / CD93 signaling pathway (e.g., an agent that blocks the interaction between CD93 and IGFBP7). In some embodiments, the agent is selected from the group consisting of an antibody, a peptide, a polypeptide, a peptide analog, a fusion peptide, an aptamer, an avimer, an anticalin, a spiegelmer, a small molecule compound, an siRNA, an shRNA, an miRNA, an antisense RNA, and a gene editing system. In some embodiments, the agent is a blocking antibody that specifically recognizes CD93. In some embodiments, the agent is a blocking antibody that specifically recognizes IGFBP7. In some embodiments, the agent is an inhibitory CD93 polypeptide comprising at least a portion of the extracellular domain of CD93 or a variant thereof. In some embodiments, the agent is an inhibitory polypeptide comprising a variant of IGFBP7. In some embodiments, the method further comprises administering to the subject a second therapy (e.g., chemotherapy, immunotherapy, cell therapy, radiation therapy, etc.). In some embodiments, the second therapy is immunotherapy. In some embodiments, the second therapy comprises administration of an immune checkpoint inhibitor, including, for example, an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, or a combination thereof, such as an anti-PD1 antibody and an anti-CTLA4 antibody.

[0075] In some embodiments, a method is provided for facilitating delivery of a second therapeutic agent (e.g., a chemotherapeutic agent or an immunomodulatory agent), comprising administering to a subject an effective amount of an agent that specifically inhibits the IGFBP7 / CD93 signaling pathway (e.g., an agent that blocks the interaction between CD93 and IGFBP7). In some embodiments, a method is provided for improving the efficacy of a second therapeutic agent (e.g., a chemotherapeutic agent or an immunomodulatory agent), comprising administering to a subject an effective amount of an agent that specifically inhibits the IGFBP7 / CD93 signaling pathway (e.g., an agent that blocks the interaction between CD93 and IGFBP7). In some embodiments, the agent is selected from the group consisting of an antibody, a peptide, a polypeptide, a peptide analog, a fusion peptide, an aptamer, an avimer, an anticalin, a spiegelmer, a small molecule compound, an siRNA, an shRNA, an miRNA, an antisense RNA, and a gene editing system. In some embodiments, the agent is a blocking antibody that specifically recognizes CD93. In some embodiments, the agent is a blocking antibody that specifically recognizes IGFBP7. In some embodiments, the agent is an inhibitory CD93 polypeptide comprising at least a portion of the extracellular domain of CD93 or a variant thereof. In some embodiments, the agent is an inhibitory polypeptide comprising a variant of IGFBP7. In some embodiments, the method further comprises sequentially, simultaneously, and / or concurrently administering to the subject a second therapeutic agent (such as, for example, a chemotherapeutic agent, an immunomodulatory agent, or an immune cell). In some embodiments, the second therapeutic agent is an immune checkpoint inhibitor, including, for example, an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, or a combination thereof, such as an anti-PD1 antibody and an anti-CTLA4 antibody.

[0076] The agents described herein are also useful for one or more of the following: 1) increasing the number of blood vessels covered with pericytes; 2) increasing the vessel length of blood vessels with a circular shape; 3) increasing blood vessel-associated α-smooth muscle actin (α-SMA)-positive cells; and 4) reducing the activation of β integrins. In some embodiments, a method for increasing blood vessels covered with pericytes is provided, comprising administering to a subject an effective amount of an agent that specifically inhibits the IGFBP7 / CD93 signaling pathway (e.g., an agent that blocks the interaction between CD93 and IGFBP7). In some embodiments, a method for increasing blood vessel length of blood vessels with a circular shape is provided, comprising administering to a subject an effective amount of an agent that specifically inhibits the IGFBP7 / CD93 signaling pathway (e.g., an agent that blocks the interaction between CD93 and IGFBP7). In some embodiments, a method for increasing blood vessel-associated α-smooth muscle actin (α-SMA)-positive cells is provided, comprising administering to a subject an effective amount of an agent that specifically inhibits the IGFBP7 / CD93 signaling pathway (e.g., an agent that blocks the interaction between CD93 and IGFBP7). In some embodiments, a method for reducing β1 integrin activation is provided, comprising administering to a subject an effective amount of an agent that specifically inhibits the IGFBP7 / CD93 signaling pathway (e.g., an agent that blocks the interaction between CD93 and IGFBP7). In some embodiments, the agent is selected from the group consisting of an antibody, a peptide, a polypeptide, a peptide analog, a fusion peptide, an aptamer, an avimer, an anticalin, a spiegelmer, a small molecule compound, an siRNA, an shRNA, an miRNA, an antisense RNA, and a gene editing system. In some embodiments, the agent is a blocking antibody that specifically recognizes CD93. In some embodiments, the agent is a blocking antibody that specifically recognizes IGFBP7. In some embodiments, the agent is an inhibitory CD93 polypeptide comprising at least a portion of the extracellular domain of CD93 or a variant thereof. In some embodiments, the agent is an inhibitory polypeptide comprising a variant of IGFBP7.In some embodiments, the method further comprises administering to the subject a second therapeutic agent (e.g., a chemotherapeutic agent, an immunomodulatory agent, or immune cells, etc.) sequentially, simultaneously, and / or concurrently.

[0077] In some embodiments, the methods described herein comprise administering to a subject an effective amount of an anti-CD93 antibody that specifically recognizes CD93 and blocks the interaction between CD93 and IGFBP7. In some embodiments, the anti-CD93 antibody further blocks the interaction between CD93 and MMNR2. In some embodiments, the anti-CD93 antibody does not block the interaction between CD93 and MMNR2. In some embodiments, the anti-CD93 antibody binds to the IGFBP7-binding site on CD93. In some embodiments, the anti-CD93 antibody binds to a region of CD93 that is outside the IGFBP7-binding site, e.g., outside the site required for stable interaction, and thus its binding indirectly affects binding to IGFBP7. In some embodiments, the anti-CD93 antibody binds to CD93 competitively with mAb MM01 or mAb 7C10. In some embodiments, the anti-CD93 antibody binds to an epitope that overlaps or substantially overlaps with the epitope of mAb MM01 or mAb 7C10. In some embodiments, the anti-CD93 antibody binds to an epitope that does not substantially overlap with the epitope of mAb MM01 or mAb 7C10. In some embodiments, "substantially overlapping" above refers to a scenario in which at least about 50%, 60%, 70%, 80%, or 90% of the residues on CD93 to which the anti-CD93 antibody binds overlap with the residues to which mAb MM01 or mAb 7C10 binds. In some embodiments, the anti-CD93 antibody is mAb MM01 or a humanized version thereof. In some embodiments, the method further comprises administering to the subject a second therapeutic agent (e.g., a chemotherapeutic agent, an immunomodulatory agent, or immune cells, etc.). In some embodiments, the second therapeutic agent is an immune checkpoint inhibitor (e.g., an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, or a combination thereof, such as a combination of an anti-PD1 antibody and an anti-CTLA4 antibody).

[0078] In some embodiments, the methods described herein comprise administering to a subject an effective amount of a polypeptide comprising at least a portion of the extracellular domain of CD93 or a variant thereof (an inhibitory CD93 polypeptide) that specifically blocks the interaction between CD93 and IGFBP7. In some embodiments, the method further comprises administering to the subject a second therapeutic agent (e.g., a chemotherapeutic agent, an immunomodulatory agent, or immune cells, etc.). In some embodiments, the second therapeutic agent is an immune checkpoint inhibitor (an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, or a combination thereof, such as a combination of an anti-PD1 antibody and an anti-CTLA-4 antibody). In some embodiments, the inhibitory CD93 polypeptide further comprises a stabilizing domain (e.g., an Fc). In some embodiments, the inhibitory CD93 polypeptide is about 50 to about 100 amino acids in length. In some aspects, the CD93 portion of the inhibitory CD93 polypeptide, i.e., the portion corresponding to the extracellular domain of CD93 or a portion thereof and carrying out the function of blocking binding of CD93 to IGFBP7, is about 50 to about 100 amino acids in length. In some embodiments, the inhibitory CD93 polypeptide comprises an F238 residue, where the amino acid numbering is based on SEQ ID NO:1.

[0079] In some embodiments, the inhibitory CD93 polypeptide is a soluble polypeptide. In some embodiments, the inhibitory CD93 polypeptide is membrane-bound, for example, via a GPI linkage. In certain embodiments, the membrane-bound inhibitory CD93 polypeptide is cleaved from the membrane before administration. These inhibitory CD93 polypeptides can be administered to a subject via any administration route, such as intravenous. Alternatively, the inhibitory polypeptide can be administered to a subject via administration of a polynucleotide encoding the inhibitory CD93 polypeptide, for example, via a vector platform.

[0080] In some embodiments, the inhibitory CD93 polypeptide is membrane-bound via a transmembrane domain. Such an inhibitory CD93 polypeptide can be introduced into a subject by introducing a polynucleotide (such as cDNA or mRNA) encoding the inhibitory polypeptide into the subject's cells, causing the inhibitory CD93 polypeptide to be expressed on the cell surface. For example, the membrane-bound inhibitory CD93 polypeptide can be a dominant-negative form of CD93 that binds to IGFBP7 but cannot transmit downstream signals. The dominant-negative form of CD93 can contain one or more mutations that inactivate the intracellular signaling domain of CD93. Alternatively, the dominant-negative form of CD93 lacks the intracellular domain of CD93.

[0081] Also contemplated herein are inhibitory CD93 polypeptides comprising one or more mutations in the extracellular domain, such as mutations that enable the inhibitory CD93 polypeptide to exhibit preferential binding to IGFBP7 over other binding partners of CD93, such as MMNR2. In some embodiments, the inhibitory CD93 polypeptide binds to IGFBP7 with higher affinity than to MMNR2. In some embodiments, the inhibitory CD93 polypeptide binds to IGFBP7 with higher affinity compared to wild-type CD93.

[0082] In some embodiments, the methods described herein comprise administering to a subject an effective amount of an anti-IGFBP7 antibody that specifically recognizes IGFBP7 and blocks the interaction between CD93 and IGFBP7. In some embodiments, the anti-IGFBP7 antibody further blocks the interaction between IGFBP7 and one or more of its other binding partners, such as IGF-1, IGF-2, and IGF1R. In some embodiments, the anti-IGFBP7 antibody does not block the interaction between IGFBP7 and one or more of its binding partners. In some embodiments, the anti-IGFBP7 antibody binds to the CD93-binding site on IGFBP7. In some embodiments, the anti-IGFBP7 antibody binds to a region of IGFBP7 outside the CD93-binding site, e.g., outside the site required for stable interaction, and thus its binding indirectly affects binding to CD93. In some embodiments, the anti-IGFBP7 antibody binds to the insulin-binding (IB) domain of IGFBP7. In some embodiments, the anti-IGFBP7 antibody binds to IGFBP7 competitively with mAb R003 or mAb 2C6. In some embodiments, the anti-IGFBP7 antibody binds to an epitope that overlaps or substantially overlaps with the epitope of mAb R003 or mAb 2C6. In some embodiments, the term "substantially overlaps" refers to a scenario in which at least about 50%, 60%, 70%, 80%, or 90% of the residues on IGFBP7 to which the anti-IGFBP7 antibody binds overlap with the residues to which mAb R003 or mAb 2C6 binds. In some embodiments, the anti-IGFBP7 antibody is mAb R003 or a humanized version thereof. In some embodiments, the method further comprises administering to the subject a second therapeutic agent (e.g., a chemotherapeutic agent, an immunomodulatory agent, or immune cells, etc.). In some embodiments, the second therapeutic agent is an immune checkpoint inhibitor (e.g., an anti-PD1 antibody or an anti-PD-L1 antibody, etc.).

[0083] In some embodiments, the methods described herein comprise administering to a subject an effective amount of a polypeptide comprising a variant of IGFBP7 (an inhibitory IGFBP7 polypeptide) that specifically blocks the interaction between CD93 and IGFBP7. Such variants include, but are not limited to, mutant forms of IGFBP7 and fragments (portions) of IGFBP7. In some embodiments, the methods further comprise administering to the subject a second therapeutic agent (e.g., a chemotherapeutic agent, an immunomodulatory agent, or immune cells). In some embodiments, the second therapeutic agent is an immune checkpoint inhibitor (e.g., an anti-PD1 antibody or an anti-PD-L1 antibody). In some embodiments, the inhibitory IGFBP7 polypeptide further comprises a stabilization domain (e.g., Fc). In some embodiments, the inhibitory IGFBP polypeptide is about 50 to about 100 amino acids in length. In some embodiments, the IGFBP portion of the inhibitory IGFBP7 polypeptide, i.e., the portion corresponding to IGFBP7 or a portion thereof and carrying out the function of blocking binding of IGFBP7 to CD93, is about 50 to about 100 amino acids in length. In some embodiments, the inhibitory IGFBP7 polypeptide comprises the IB domain of IGFBP7. In some embodiments, the inhibitory IGFBP7 polypeptide does not comprise any domain of IGFBP7 other than the IB domain.

[0084] The inhibitory IGFBP7 polypeptide can be administered to a subject via any route of administration, such as intravenously. Alternatively, the inhibitory polypeptide can be administered to a subject via administration of a polynucleotide encoding the inhibitory IGFBP7 polypeptide.

[0085] Also contemplated herein are inhibitory IGFBP7 polypeptides comprising one or more mutations that enable the inhibitory IGFBP7 polypeptide to exhibit preferential binding to CD93 over one or more other binding partners of IGFBP7, such as IGF-1, IGF-2, and IGF1R. In some embodiments, the inhibitory IGFBP7 polypeptide binds to CD93 with higher affinity than to one or more other binding partners of IGFBP7, such as IGF-1, IGF-2, and IGF1R. In some embodiments, the inhibitory IGFBP7 polypeptide binds to CD93 with higher affinity compared to wild-type IGFBP7.

[0086] In some embodiments, the methods described herein comprise administering to a subject an effective amount of an agent that reduces expression of CD93 or IGFBP7. In some embodiments, the agent is selected from the group consisting of siRNA, shRNA, miRNA, antisense RNA, and a gene editing system.

[0087] In some embodiments, the subject suitable for the methods described herein is a human. In some embodiments, the subject is characterized by abnormal tumor vasculature. In some embodiments, the subject is characterized by dense or abundant blood vessels. In some embodiments, the subject has received a prior treatment, such as a prior treatment comprising administering an inhibitor of the VEGF signaling pathway, including an anti-VEGF antibody or an inhibitory polypeptide comprising one or more VEGFR domains. In some embodiments, the subject is characterized by high expression of CD93. In embodiments, the subject is characterized by high expression of IGFBP7. In some embodiments, the subject is characterized by high expression of VEGF. In some embodiments, the tumors discussed herein are solid tumors, e.g., the solid tumor may be colon (or colorectal) cancer, non-small cell lung cancer, glioblastoma, renal cell carcinoma, cervical cancer, ovarian cancer, fallopian tube cancer, peritoneal cancer, breast cancer, prostate cancer, bladder cancer, oral squamous cell carcinoma, head and neck squamous cell carcinoma, brain tumor, bone cancer, or melanoma.

[0088] In some embodiments, before administering a CD93 / IGFBP7 blocking agent, the presence and distribution of CD93 or IGFBP7 on blood vessels in a subject's tissue (such as tumor blood vessels) are evaluated, for example, to determine the relative level and activity of CD93 or IGFBP7 on the subject's blood vessels. Subjects whose tissue blood vessels (such as tumor blood vessels) express CD93 or IGFBP7 (such as those that express CD93 or IGFBP7 or express them at high levels) can be candidates for treatment with a CD93 / IGFBP7 blocking agent. This can be achieved by obtaining a sample tissue (such as tumor tissue) and testing it, for example, using an immunoassay to determine the relative prominence of CD93 or IGFBP7, and optionally additional markers on cells. In vivo imaging can also be used to detect CD93 or IGFBP7 expression. Other methods that can be used to detect CD93 and IGFBP7 expression include RNA-based methods, such as RT-PCR or Northern blotting.

[0089] The method can include multiple administrations of CD93 / IGFBP7 blocking agent.In some embodiments, after the first round of administration, the level and / or activity of CD93 or IGFBP7 in the subject can be remeasured, and if still elevated, further rounds of administration can be carried out.In this way, multiple administrations of CD93 / IGFBP7 blocking agent can be carried out.

[0090] Drugs that inhibit the IGFBP7 / CD93 signaling pathway The agent can be an antibody, polypeptide, peptide, polynucleotide, peptidomimetic, natural product, carbohydrate, aptamer, avimer, anticalin, spiegelmer, or small molecule. Specific examples of what the agent can be are described below, and methods for identifying suitable agents are characterized in subsequent aspects of this application. In some embodiments, the agent is a fusion protein (e.g., a fusion protein comprising a half-life-extending domain (e.g., an Fc domain)).

[0091] CD93 CD93 is a type I transmembrane protein belonging to the C-type lectin gene family and is known as the complement C1q receptor (ClqRp). CD93 consists of a C-type lectin-like domain (D1), five EGF-like repeats (D2), a mucin-like domain (D3), a transmembrane domain (D4), a cytoplasmic domain (D5), and a 79-amino acid DX domain located between D1 and D2 [9]. CD93 is primarily expressed on endothelial cells (ECs) and is involved in promoting angiogenesis as a soluble growth factor and EC adhesion molecule. Previous studies have shown that multimerin 2 (MMRN2) interacts with CD93 to promote EC adhesion, migration, and in vitro angiogenesis. MMRN2, also known as EndoGlyx-1, is an endothelial-specific member of the EDEN protein family and a component of the ECM. In tumor tissue, MMRN2 has been found to be expressed along tumor capillaries and co-expressed with CD93 in tumor neovasculature. See Galvagni et al., Matrix Biol. (2017) 64, 112-127 (incorporated herein by reference in its entirety for all purposes).

[0092] The human CD93 gene is located at 20p11.21 and encodes a polypeptide of 652 amino acid residues. The term "CD93 polypeptide" encompasses the gene product of human CD93, including naturally occurring variants thereof. Human CD93 polypeptide includes the amino acid sequence found in GenBank Accession No. NP_036204.2 and naturally occurring variants thereof. "Natural variants" include, for example, allelic variants. Typically, these differ from a given sequence by only one, two, or three amino acid residues, typically by no more than 10 or 20 amino acid residues. Typically, variants have conservative substitutions. The CD93 polypeptide sequence from NP_036204.2 is set forth as SEQ ID NO: 1. Natural variants of human CD93 include those with the A220V, V318A, or P541 mutations.

[0093] CD93 as described herein includes any naturally occurring CD93 or variant thereof that has the function of CD93, including CD93 orthologs found in other species, such as horses, oxen, chimpanzees, chickens, zebrafish, dogs, pigs, cattle, sheep, rats, mice, guinea pigs, or primates.

[0094] IGFBP7 Insulin-like growth factor (IGF)-binding protein (IGFBP) 7, also known as Mac25, IGFBP-rp1, tumor-derived adhesion factor (TAF), prostacyclin-stimulating factor (PSF), and angiomodulin (AGM), is a secreted extracellular matrix (ECM) protein belonging to the IGFBP family (57, 58). Members of the IGFBP family contain an IGF-binding (IB) domain at their N-terminus, which binds to IGF1 and helps regulate its bioavailability in the blood. IGFBP7 lacks the C-terminal domain that functions to stabilize IGF1 binding, and therefore its affinity for IGF-1 is significantly lower than that of IGFBPs 1-6 (59). IGFBP7 has been found to be expressed in many normal tissues and cancer cells; however, the exact role of IGFBP7 in cancer has been controversial. On the other hand, IGFBP7 has been shown to be released from cancer cells and act as a tumor suppressor, inducing tumor apoptosis and suppressing angiogenesis (60); IGF1R was proposed as a receptor, and IGFBP7 binding blocked the interaction between IGF-1 and IGF1R, inhibiting the expansion and aggressiveness of cancer stem-like cells (61, 62). Administration of IGFBP7 inhibited tumor growth in vivo, and IGFBP7- / - mice were susceptible to diethylnitrosamine-induced liver carcinoma (55, 63). On the other hand, IGFBP7 has been shown to be upregulated in blood vessels of cancer tissues and could promote angiogenesis (48, 64). IGFBP7 is strongly induced by VEGF in vascular ECs (48), and a synergistic effect between IGFBP7 and VEGF in angiogenesis has been reported (50). Each of the above references is incorporated by reference in its entirety for all purposes.

[0095] The human IGFBP7 gene is located on 4q12 and encodes a polypeptide. One isoform of the polypeptide has 264 amino acid residues (SEQ ID NO:2), including a signal peptide domain (residues 1-26 of SEQ ID NO:2), an insulin-binding domain (IB domain, residues 28-106 of SEQ ID NO:2), a Kazal-like domain (residues 105-158 of SEQ ID NO:2), and an Ig-like C2-type domain (residues 160-264 of SEQ ID NO:2).

[0096] The IGFBP7 described in this application includes any naturally occurring IGFBP7 or variant thereof that has the function of IGFBP7, including IGFBP7 orthologs found in other species, such as horses, bulls, chimpanzees, chickens, zebrafish, dogs, pigs, cows, sheep, rats, mice, guinea pigs, or primates.

[0097] Anti-CD93 or anti-IGFBP antibodies The methods described herein, in some embodiments, comprise the use of an anti-CD93 antibody that specifically recognizes CD93 and specifically blocks the interaction between CD93 and IGFBP7. The application also provides, in one aspect, any of the novel anti-CD93 antibodies described herein.

[0098] In some embodiments, the CD93 recognized by the anti-CD93 antibody is human CD93. In some embodiments, the human CD93 comprises or has the amino acid sequence of SEQ ID NO: 1 or a naturally occurring variant of human CD93. In some embodiments, the naturally occurring variant of human CD93 is derived from tumor tissue.

[0099] In some embodiments, the anti-CD93 antibody binds to the IGFBP7 binding site on CD93. In some embodiments, the anti-CD93 antibody binds to a region of CD93 outside the IGFBP7 binding site.

[0100] In some embodiments, the anti-CD93 antibody binds to the extracellular region of CD93. In some embodiments, the anti-CD93 antibody binds to the extracellular region of human CD93 (e.g., residues A24 to K580 according to SEQ ID NO: 1).

[0101] In some embodiments, the anti-CD93 antibody binds to the C-type lectin domain of CD93. In some embodiments, the anti-CD93 antibody binds to the C-type lectin domain of human CD93 (such as, for example, residues T22 to N174 according to SEQ ID NO: 1).

[0102] In some embodiments, the anti-CD93 antibody binds to the long loop region in the C-type lectin domain of CD93. In some embodiments, the anti-CD93 antibody binds to the long loop region in the C-type lectin domain of human CD93 (such as, for example, residues G96-C141 according to SEQ ID NO: 1). In some embodiments, the anti-CD93 antibody binds to less conserved residues within the C-type lectin domain of CD93 or within the long loop region in the C-type lectin domain. For example, the anti-CD93 antibody binds to any one or more (such as about 2, 3, 4, 5, 6, 7, 8, 9, or 10) of residues selected from G96, Q98, R99, E100, K101, G102, K103, C104, L105, D106, P107, S108, L109, K112, S115, V117, G118, G120, E121, D122, T123, P124, Y125, S126, N127, H129, K130, E131, L132, R133, N134, S135, C136, I137, S138, K139, and R140 according to SEQ ID NO:1. In some embodiments, the anti-CD93 antibody binds to a region of human CD93 comprising or consisting of residues F182 to Y262 according to SEQ ID NO: 1. In some embodiments, the anti-CD93 antibody binds to F238 according to SEQ ID NO: 1.

[0103] In some embodiments, the anti-CD93 antibody binds to the DX domain between the C-type lectin-like domain (D1 domain) and the EGF-like domain (D2 domain). In some embodiments, the anti-CD93 antibody binds to the DX domain of human CD93 (e.g., residues I175 to L256 or I175 to S259 according to SEQ ID NO: 1). In some embodiments, the anti-CD93 antibody binds to F238 according to SEQ ID NO: 1.

[0104] In some embodiments, the anti-CD93 antibody binds to both the DX domain and the C-type lectin domain of CD93. In some embodiments, the anti-CD93 antibody binds to both F238 and the C-type lectin domain (e.g., residues T22 to N174 according to SEQ ID NO: 1) of human CD93. In some embodiments, the anti-CD93 antibody binds to both F238 and the long loop region in the C-type lectin domain (e.g., residues G96 to C141 according to SEQ ID NO: 1) of human CD93. In some embodiments, the anti-CD93 antibody binds to both F238 and any one or more (such as about 2, 3, 4, 5, 6, 7, 8, 9, or 10) residues selected from G96, Q98, R99, E100, K101, G102, K103, C104, L105, D106, P107, S108, L109, K112, S115, V117, G118, G120, E121, D122, T123, P124, Y125, S126, N127, H129, K130, E131, L132, R133, N134, S135, C136, I137, S138, K139, and R140 according to SEQ ID NO:1.

[0105] In some embodiments, the anti-CD93 antibody binds to an EGF-like region of CD93, such as residues C257 to M469 or P260 to T468 according to SEQ ID NO: 1.

[0106] In some embodiments, the anti-CD93 antibody also blocks the interaction between CD93 and MMNR2. In some embodiments, the anti-CD93 antibody binds to the same epitope on CD93 as that to which MMNR2 binds. In some embodiments, the anti-CD93 antibody binds to an epitope on CD93 that is different from that to which MMNR2 binds.

[0107] In some embodiments, the anti-CD93 antibody does not block the interaction between CD93 and MMNR2.

[0108] In some embodiments, the anti-CD93 antibody is a polyclonal antibody. In some embodiments, the anti-CD93 antibody is a monoclonal antibody.

[0109] In some embodiments, the anti-CD93 antibody is an anti-human CD93 antibody.

[0110] In some embodiments, the anti-CD93 antibody is humanized or chimeric.

[0111] In some embodiments, the anti-CD93 antibody binds to CD93 competitively with mAb MM01 (Sino Biological), R3 (Sino Biological), or 273107 (Sino Biological). In some embodiments, the anti-CD93 antibody binds to an epitope that overlaps or substantially overlaps with the epitope of mAb MM01 (Sino Biological), R3 (Sino Biological), or 273107 (Sino Biological). In some embodiments, the anti-CD93 antibody does not bind to an epitope that substantially overlaps with the epitope of mAb MM01 (Sino Biological), R3 (Sino Biological), or 273107 (Sino Biological). In some aspects, "substantially overlaps" refers to a scenario in which at least about 50%, 60%, 70%, 80%, or 90% of the residues on CD93 to which the anti-CD93 antibody binds overlap with the residues to which MMO1 (Sino Biological), R3 (Sino Biological), or 273107 (Sino Biological) bind. In some embodiments, the anti-CD93 antibody binds to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the residues on CD93 to which MMO1 (Sino Biological), R3 (Sino Biological), or 273107 (Sino Biological) bind.

[0112] In some embodiments, the anti-CD93 antibody does not bind to CD93 competitively with mAb MM02 (Sino Biological). In some embodiments, the anti-CD93 antibody does not bind to CD93 competitively with mAb R004 (Sino Biological).

[0113] In some embodiments, the anti-CD93 antibody binds to CD93 competitively with mAb 7C10. In some embodiments, the anti-CD93 antibody binds to an epitope that overlaps or substantially overlaps with the epitope of 7C10. In some embodiments, the anti-CD93 antibody does not bind to an epitope that substantially overlaps with the epitope of 7C10. In some embodiments, the anti-CD93 antibody binds to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the residues on CD93 to which 7C10 binds.

[0114] In some embodiments, the anti-CD93 antibody is an anti-human CD93 monoclonal antibody selected from the group consisting of EPR5386 (abcam), 3D12 (sigma-aldrich), 1A4 (sigma-aldrich), 1A10E10, 2F7D11, R139, R3, mNI-11, X-2, and MM01.

[0115] In some embodiments, the anti-human CD93 antibody is mAb MM01 or a humanized version thereof.

[0116] In some embodiments, the anti-CD93 antibody is a full-length antibody or an immunoglobulin derivative. In some embodiments, the anti-CD93 antibody is an antigen-binding fragment, such as a single-chain Fv (scFv), Fab, Fab', F(ab'), Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv), V HThe anti-CD93 antibody is an antigen-binding fragment selected from the group consisting of H, Fv-Fc fusion, scFv-Fc fusion, scFv-Fv fusion, diabody, tribody, and tetrabody. In some embodiments, the anti-CD93 antibody is an scFv. In some embodiments, the anti-CD93 antibody is Fab or Fab'. In some embodiments, the anti-CD93 antibody is a chimeric antibody, a human antibody, a partially humanized antibody, a fully humanized antibody, or a semi-synthetic antibody. The antibody and / or antibody fragment may be derived from a mouse antibody, a rabbit antibody, a human antibody, a fully humanized antibody, a camel antibody variable domain and humanized version, a shark antibody variable domain and humanized version, and a camel antibody variable domain.

[0117] In some embodiments, the anti-CD93 antibody comprises an Fc fragment. In some embodiments, the Fc fragment is selected from the group consisting of Fc fragments derived from IgG, IgA, IgD, IgE, IgM, and combinations and hybrids thereof. In some embodiments, the Fc fragment is derived from human IgG. In some embodiments, the Fc fragment comprises the Fc region of human IgG1, IgG2, IgG3, IgG4, or a combination or hybrid IgG.

[0118] B. Anti-IGFBP7 antibody In some embodiments, the methods described herein comprise the use of an anti-IGFBP7 antibody that specifically recognizes IGFBP7 and specifically blocks the interaction between CD93 and IGFBP7. The present application also provides, in one aspect, any of the novel anti-IGFBP7 antibodies described herein.

[0119] In some embodiments, the IGFBP7 recognized by the anti-IGFBP7 antibody is human IGFBP7. In some embodiments, the IGFBP7 is mouse IGFBP7.

[0120] In some embodiments, the anti-IGFBP7 antibody binds to the CD93 (such as human CD93) binding site on IGFBP7. In some embodiments, the anti-IGFBP7 antibody binds to a region of IGFBP7 that is outside the CD93 binding site.

[0121] In some embodiments, the anti-IGFBP7 antibody binds to the insulin-binding domain ("IB domain") of IGFBP7. In some embodiments, the anti-IGFBP7 antibody binds to the IB domain of human IGFBP7 (e.g., residues S28 to G106 according to SEQ ID NO:2).

[0122] In some aspects, the anti-IGFBP7 antibody binds to the Kazal-like domain of IGFBP7. In some embodiments, the anti-IGFBP7 antibody binds to the Kazal-like domain of human IGFBP7 (e.g., residues P105 to Q158 according to SEQ ID NO: 2).

[0123] In some embodiments, the anti-IGFBP7 antibody binds to the Ig-like C2 domain of IGFBP7. In some embodiments, the anti-IGFBP7 antibody binds to the Ig-like C2 domain of human IGFBP7 (e.g., residues P160 to T264 according to SEQ ID NO: 2).

[0124] In some embodiments, the anti-IGFBP7 antibody does not specifically bind to any one or more of IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP6, IGFBPL1, KAZALD1, HTRA1, WISP1, WISP3, NOV, CYR61, CTGF, and ESM1. In some embodiments, the anti-IGFBP7 antibody does not specifically bind to any molecule selected from the group consisting of IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP6, IGFBPL1, KAZALD1, HTRA1, WISP1, WISP3, NOV, CYR61, CTGF, and ESM1.

[0125] In some embodiments, the anti-IGFBP7 antibody also blocks the interaction between IGFBP7 and IGF-1, IGF-2, and / or IGF1R.

[0126] In some embodiments, the anti-IGFBP7 antibody does not block the interaction between IGFBP7 and IGF-1, IGF-2, and / or IGF1R.

[0127] In some embodiments, the anti-IGFBP7 antibody is a polyclonal antibody. In some embodiments, the anti-IGFBP7 antibody is a monoclonal antibody.

[0128] In some embodiments, the anti-IGFBP7 antibody is an anti-human IGFBP7 antibody.

[0129] In some embodiments, the anti-IGFBP7 antibody is humanized or chimeric.

[0130] In some embodiments, the anti-IGFBP7 antibody binds to IGFBP7 competitively with mAb R003 (Sino Biological), MMO1 (Sino Biological), R065 (Sino Biological), or R115 (Sino Biological). In some embodiments, the anti-IGFBP7 antibody binds to an epitope that overlaps with the epitope of mAb R003 (Sino Biological), MMO1 (Sino Biological), R065 (Sino Biological), or R115 (Sino Biological). In some embodiments, the anti-IGFBP7 antibody binds to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues on IGFBP7 to which R003 (Sino Biological), MMO1 (Sino Biological), R065 (Sino Biological), or R115 (Sino Biological) binds.

[0131] In some embodiments, the anti-IGFBP7 antibody binds to IGFBP7 competitively with mAb 2C6. In some embodiments, the anti-IGFBP7 antibody binds to an epitope that overlaps with the epitope of mAb 2C6. In some embodiments, the anti-IGFBP7 antibody binds to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the residues on IGFBP7 to which 2C6 binds.

[0132] In some embodiments, the anti-IGFBP7 antibody is an anti-human IGFBP7 monoclonal antibody selected from the group consisting of mAb AEDO-9 (clone name, same for the following antibodies) (Bosterbio), 1D9E7 (LifeSpan BioSciences), 5A4A9 (LifeSpan BioSciences), 192520 (R&D systems), H3 (Santa Cruz Biotechnology), 40012B (R&D Systems), EPR11912(B) (Abcam), MM0346-3N37 (Abcam), 01 (i.e., MM01, Sino Biological), 003 (i.e., R003, Sino Biological). In some embodiments, the anti-human IGFBP7 monoclonal antibody is mAb 003 (i.e., R003, Sino Biological) or a humanized version thereof.

[0133] In some embodiments, the anti-IGFBP antibody is a full-length antibody or an immunoglobulin derivative. In some embodiments, the anti-IGFBP antibody is an antigen-binding fragment, such as a single-chain Fv (scFv), Fab, Fab', F(ab'), Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv), V HThe antigen-binding fragment is selected from the group consisting of H, Fv-Fc fusion, scFv-Fc fusion, scFv-Fv fusion, diabody, tribody, and tetrabody. In some embodiments, the anti-IGFBP antibody is an scFv. In some embodiments, the anti-IGFBP antibody is Fab or Fab'. In some embodiments, the anti-IGFBP antibody is a chimeric antibody, a human antibody, a partially humanized antibody, a fully humanized antibody, or a semi-synthetic antibody. The antibody and / or antibody fragment can be derived from a mouse antibody, a rabbit antibody, a human antibody, a fully humanized antibody, a camel antibody variable domain and humanized version, a shark antibody variable domain and humanized version, and a camel antibody variable domain.

[0134] In some embodiments, the anti-IGFBP antibody comprises an Fc fragment. In some embodiments, the Fc fragment is selected from the group consisting of Fc fragments derived from IgG, IgA, IgD, IgE, IgM, and combinations and hybrids thereof. In some embodiments, the Fc fragment is derived from human IgG. In some embodiments, the Fc fragment comprises the Fc region of human IgG1, IgG2, IgG3, IgG4, or a combination or hybrid IgG.

[0135] Competition assays and epitope mapping The following description of competition assays and epitope mapping uses anti-IGFBP7 antibodies as an example for demonstration purposes, and applies equally to the anti-CD93 antibodies described above.

[0136] Competition can be evaluated, for example, by flow cytometry test.In this test, the cells with IGFBP7 polypeptide that have IGFBP7 can be first incubated with antibody (for example, mAb 2C6), and then incubated with the test antibody labeled with fluorescent dye or biotin.An antibody is said to compete with 2C6 or bind to IGFBP7 competitively with 2C6 when the binding obtained after pre-incubation with saturating amount of 2C6 is about 80% or less, preferably 50% or less, 40% or less (for example, about 30%, 20% or 10%) of the binding obtained by the antibody without pre-incubation with 2C6 (measured by fluorescence). Alternatively, an antibody is said to compete with 2C6 if the binding obtained using 2C6 antibody labeled (with a fluorescent dye or biotin) on cells preincubated with a saturating amount of the test antibody is less than about 80%, preferably less than 50%, less than 40% (e.g., about 30%, 20%, or 10%) of the binding obtained without preincubation with the test antibody.

[0137] A simple competitive assay can also be used, in which a test antibody is pre-adsorbed and applied at a saturating concentration to a surface on which IGFBP7 has been immobilized. In a simple competitive assay, the surface is preferably a BIACORE chip (or other medium suitable for surface plasmon resonance analysis). A control antibody (e.g., 2C6) is then contacted with the surface at a concentration that saturates IGFBP7, and the binding of the control antibody to IGFBP7 and the surface is measured. This binding of the control antibody is compared to the binding of the control antibody to the IGFBP7-containing surface in the absence of the test antibody. A significant reduction in the binding of the control antibody to the IGFBP7-containing surface in the presence of the test antibody in the test assay indicates that the test antibody recognizes substantially the same epitope as the control antibody, such that the test antibody "cross-reacts" with the control antibody. Any test antibody that reduces the binding of the control (e.g., 2C6) antibody to IGFBP7 by at least about 30% or more, preferably about 40%, can be considered to be an antibody that binds to substantially the same epitope or determinant as the control (e.g., 2C6). Preferably, such a test antibody reduces the binding of the control antibody (e.g., 2C6) to IGFBP7 by at least about 50% (e.g., at least about 60%, at least about 70%, or more). It should be understood that the order of the control antibody and the test antibody can be reversed: i.e., the control antibody can be bound to the surface first, and then the test antibody can be contacted with the surface in a competition assay. Preferably, the antibody with a higher affinity for IGFBP7 is bound to the surface first, as this would be expected to result in a larger reduction in binding (assuming the antibodies are cross-reactive) for the second antibody. Further examples of such assays are provided, for example, in Saunal (1995) J. Immunol. Methods 183: 33-41, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0138] Preferably, monoclonal antibodies that recognize an IGFBP7 epitope react with an epitope that is present on a substantial percentage or all of the relevant IGFBP7 alleles.

[0139] In a preferred embodiment, the antibody binds to IGFBP7-expressing cells from one or more subjects with a disease characterized by the presence of IGFBP7-positive cells, i.e., subjects who are candidates for treatment by one of the methods described herein using the anti-IGFBP7 antibodies of the present application. Thus, once an antibody that specifically recognizes IGFBP7 on cells is obtained, it can be tested for its ability to bind to IGFBP7-positive cells (e.g., cancer cells). In particular, before treating a patient with one of the antibodies of the present invention, it is beneficial to test the antibody's ability to bind to malignant cells obtained from the patient, e.g., in a blood sample or tumor biopsy, to maximize the likelihood that the treatment will be beneficial to the patient. In one embodiment, the antibodies of the present application are tested in an immunoassay to test their ability to bind to IGFBP7-expressing cells (e.g., malignant cells). For example, a tumor biopsy is performed and tumor cells are collected. The ability of a given antibody to bind to cells is then evaluated using standard methods well known to those skilled in the art. Antibodies found to bind to a substantial proportion (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80% or more) of cells known to express IGFBP7 (e.g., tumor cells) from a significant proportion (e.g., 5%, 10%, 20%, 30%, 40%, 50% or more) of a subject or patient are suitable for use in the present invention, both for diagnostic purposes to determine the presence or level of malignant cells in a patient, and for use in the therapeutic methods described herein (e.g., for increasing or decreasing the number or activity of malignant cells). To assess antibody binding to cells, the antibody can be directly or indirectly labeled. If indirectly labeled, a secondary labeled antibody is typically added.

[0140] Determining whether an antibody binds within the epitope region can be performed by methods known to those skilled in the art. As an example of such a mapping / characterization method, the epitope region of an anti-IGFBP7 antibody can be determined by epitope "footprinting," which uses chemical modification of exposed amines / carboxyls in the IGFBP7 protein. One specific example of such footprinting technology is the use of HXMS (hydrogen-deuterium exchange detected by mass spectrometry), in which hydrogen / deuterium exchange, binding, and back-exchange of amide protons in receptor and ligand proteins occur, while backbone amide groups involved in protein binding are protected from back-exchange and therefore remain deuterated. The relevant regions can then be identified by digestive proteolysis, fast microbore high-performance liquid chromatography separation, and / or electrospray ionization mass spectrometry. See, for example, Ehring H, Analytical Biochemistry, Vol. 267 (2) pp. 252-259 (1999); Engen, JR and Smith, DL (2001) Anal. Chem. 73, 256A-265A (each of which is incorporated herein by reference in its entirety for all purposes). Another example of a suitable epitope identification technique is nuclear magnetic resonance epitope mapping (NMR), which typically compares the positions of signals in two-dimensional NMR spectra of free antigen and antigen complexed with an antigen-binding peptide such as an antibody. Typically, the antigen is selectively isotopically labeled with 15N so that only signals corresponding to the antigen are seen in the NMR spectrum, and signals from the antigen-binding peptide are not seen. Antigen signals derived from amino acids involved in interaction with the antigen-binding peptide typically shift in position in the spectrum of the complex compared to the spectrum of the free antigen, thus allowing the identification of amino acids involved in binding.See, e.g., Ernst Schering Res Found Workshop. 2004; (44): 149-67; Huang et al., Journal of Molecular Biology, Vol. 281 (1) pp. 61-67 (1998); and Saito and Patterson, Methods. 1996 Jun; 9 (3): 516-24 (each of which is incorporated by reference in its entirety for all purposes).

[0141] Epitope mapping / characterization can also be performed using mass spectrometry. See, for example, Downard, J Mass Spectrom. 2000 Apr; 35 (4): 493-503 and Kiselar and Downard, Anal Chem. 1999 May 1; 71 (9): 1792-1801 (each of which is incorporated herein by reference in its entirety for all purposes). Protease digestion techniques can also be useful in the context of epitope mapping and identification. Regions / sequences associated with antigenic determinants can be determined by protease digestion, for example, by using trypsin at a ratio of about 1:50 relative to IGFBP7, or by overnight digestion at pH 7-8 followed by mass spectrometry (MS) analysis for peptide identification. Subsequent comparison of the sample subjected to trypsin digestion with a sample incubated with the antibody and then subjected to digestion, e.g., with trypsin, can identify peptides protected from trypsin cleavage by the anti-IGFBP7 binder (thereby revealing the footprint of the binder). Other enzymes, such as chymotrypsin, pepsin, etc., can also, or alternatively, be used in similar epitope characterization methods. Furthermore, enzyme digestion can provide a rapid method for analyzing whether potential antigenic determinant sequences lie within regions of the IGFBP7 polypeptide that are not surface-exposed and therefore most likely not relevant in terms of immunogenicity / antigenicity.

[0142] Site-directed mutagenesis is another technique useful for elucidating binding epitopes. For example, in "alanine scanning," each residue in a protein segment is replaced with an alanine residue, and the resulting binding affinity is measured. If the mutation results in a significant decrease in binding affinity, it is highly likely to be involved in binding. Monoclonal antibodies specific for structural epitopes (i.e., antibodies that do not bind to unfolded proteins) can be used to verify that the alanine substitution does not affect the overall folding of the protein. See, for example, Clackson and Wells, Science 1995; 267:383-386; and Wells, Proc Natl Acad Sci USA 1996; 93:1-6.

[0143] Electron microscopy can also be used for epitope "footprinting." For example, Wang et al., Nature 1992; 355:275-278 used the combined application of cryo-electron microscopy, three-dimensional image reconstruction, and X-ray crystallography to determine the physical footprint of Fab fragments on the capsid surface of native cowpea mosaic virus.

[0144] Other forms of " label-free " assay for epitope evaluation include surface plasmon resonance (SPR, BIACORE) and reflectance interference spectroscopy (RifS).See, for example, Fagerstam et al., Journal of Molecular Recognition 1990;3:208-14; Nice et al., J. Chroma-togr. 1993;646:159-168; Leipert et al., Angew. Chem. Int. Ed. 1998;37:3308-3311; Kroger et al., Biosensors and Bioelectronics 2002;17:937-944.

[0145] It should also be noted that an antibody (first antibody) that binds to the same or substantially the same epitope as an antibody of the present application (second antibody) can be identified in one or more of the exemplary competition assays described herein. In some embodiments, a first antibody that binds to substantially the same epitope as a second antibody refers to a scenario in which the residues to which the first antibody binds have at least about 50%, 60%, 70%, 80%, or 90% overlap with the residues to which the second antibody binds.

[0146] Drugs containing anti-CD93 or anti-IGFBP7 antibodies A. Anti-CD93 or anti-IGFBP7 Fc fusion proteins In some embodiments, the agent comprising an anti-CD93 antibody or anti-IGFBP7 antibody described herein is a fusion protein. In some embodiments, the anti-CD93 and / or anti-IGFBP7 antibody (e.g., an anti-CD93 and / or anti-IGFBP7 antibody fragment) is fused to an Fc fragment via a linker (such as a peptide linker). Any of the anti-CD93 or anti-IGFBP7 antibodies described in the "Anti-CD93 or Anti-IGFBP7 Antibodies" section can be used in an anti-CD93 or anti-IGFBP7-Fc fusion protein.

[0147] 1. Fc fragment The terms "Fc region," "Fc domain," or "Fc" refer to the C-terminal non-antigen-binding region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes native Fc regions and variant Fc regions. In some embodiments, a human IgG heavy chain Fc region extends from Cys226 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may be present or absent without affecting the structure or stability of the Fc region. Unless otherwise specified herein, the numbering of amino acid residues in an IgG or Fc region follows the EU numbering system for antibodies, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0148] In some embodiments, the Fc fragment comprises an immunoglobulin heavy chain constant region, including a hinge region, a CH2 domain, and / or a CH3 domain. As used herein, the term "hinge region" or "hinge sequence" refers to the amino acid sequence located between the linker and the CH2 domain. In some embodiments, the fusion protein comprises an Fc fragment including a hinge region. In some embodiments, the hinge region comprises the amino acid sequence CPPCP (SEQ ID NO: 3), which is the sequence found in a natural IgG1 hinge region, to facilitate dimerization. In some embodiments, the Fc fragment of the fusion protein begins with the hinge region and extends to the C-terminus of the IgG heavy chain. In some embodiments, the fusion protein comprises an Fc fragment without a hinge region. In some embodiments, the Fc fragment comprises a human IgG heavy chain hinge region (starting at Cys226), an IgG CH2 domain, and / or an IgG CH3 domain.

[0149] In some embodiments, the fusion protein comprises an Fc fragment selected from the group consisting of Fc fragments derived from IgG, IgA, IgD, IgE, IgM, and combinations and hybrids thereof. In some embodiments, the Fc fragment is derived from human IgG. In some embodiments, the Fc fragment comprises the Fc region of human IgG1, IgG2, IgG3, IgG4, or combination or hybrid IgG. In some embodiments, the Fc fragment is an IgG1 Fc fragment. In some embodiments, the Fc fragment comprises the CH2 and CH3 domains of IgG1. In some embodiments, the Fc fragment is an IgG4 Fc fragment. In some embodiments, the Fc fragment comprises the CH2 and CH3 domains of IgG4. IgG4 Fc is known to exhibit lower effector activity than IgG1 Fc and may therefore be desirable for some applications. In some embodiments, the Fc fragment is derived from a mouse immunoglobulin.

[0150] In some embodiments, the IgG CH2 domain begins at Ala231. In some embodiments, the IgG CH3 domain begins at Gly341. In some embodiments, the C-terminal Lys residue of human IgG is absent. In some embodiments, conservative amino acid substitutions are made in the Fc region without affecting the desired structure and / or stability of the Fc.

[0151] Additionally, anti-CD93 or anti-IGFBP7-Fc fusion proteins comprising any of the Fc variants described below or combinations thereof are contemplated. In some embodiments, the Fc fragment comprises a sequence that has been modified or otherwise altered to have enhanced antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) effector function.

[0152] Heterodimerization of non-identical polypeptides in anti-CD93 or anti-IGFBP7-Fc fusion proteins can be promoted by methods known in the art, including, but not limited to, heterodimerization via the knob-into-hole technique. The structure and assembly methods for the knob-into-hole technique can be found, for example, in U.S. Patent Nos. 5,821,333, 7,642,228, U.S. Patent Application Publication No. 2011 / 0287009, and PCT / US2012 / 059810, which are incorporated by reference in their entireties for all purposes. This technique was developed by replacing small amino acid residues in one Fc CH3 domain with larger ones to introduce a "knob" (or protuberance) and replacing one or more large amino acid residues with smaller ones to introduce a "hole" (or cavity) in the other Fc CH3 domain. In some embodiments, one chain of the Fc fragment in the fusion protein comprises the knob and the second chain of the Fc fragment comprises the hole.

[0153] Preferred residues for forming the knob are generally naturally occurring amino acid residues, preferably selected from arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Most preferred are tryptophan and tyrosine. In one embodiment, the original residues for forming the knob have small side chain volumes, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine. Exemplary amino acid substitutions in the CH3 domain of IgG for forming the knob include, but are not limited to, T366W, T366Y, or F405W substitutions.

[0154] Preferred residues for hole formation are typically naturally occurring amino acid residues, preferably selected from alanine (A), serine (S), threonine (T), and valine (V). In one embodiment, the original residue for hole formation has a large side chain volume, such as tyrosine, arginine, phenylalanine, or tryptophan. Exemplary amino acid substitutions in the CH3 domain of an IgG to create a hole include, but are not limited to, T366S, L368A, F405A, Y407A, Y407T, and Y407V substitutions. In a specific embodiment, the knob comprises a T366W substitution, and the hole comprises a T366S / L368A / Y407V substitution. It is understood that other modifications to the Fc region known in the art that promote heterodimerization are also contemplated and encompassed by the present application.

[0155] Methods are contemplated that include agents such as isolated variants of anti-CD93 or anti-IGFBP7-Fc fusion proteins (e.g., full-length anti-CD93 or anti-IGFBP7 antibody variants) that include any of the variants described herein (e.g., Fc variants, effector function variants, glycosylation variants, cysteine ​​engineered variants), or combinations thereof.

[0156] 2. Linker In some embodiments, an anti-CD93 or anti-IGFBP7-Fc fusion protein described herein comprises an anti-CD93 or anti-IGFBP7 antibody described herein fused to an Fc fragment via a linker.

[0157] The length, degree of flexibility, and / or other characteristics of the linker used in an anti-CD93 or anti-IGFBP7-Fc fusion protein can have some effect on properties including, but not limited to, the affinity, specificity, or avidity of the anti-CD93 or anti-IGFBP7 antibody and / or its affinity, specificity, or avidity for one or more particular antigens or epitopes present on CD93 and / or IGFBP7. For example, a longer linker can be selected to ensure that two adjacent antibody moieties do not sterically interfere with each other. In some embodiments, the linker (such as a peptide linker) contains flexible residues (such as glycine and serine) to allow adjacent antibody moieties to move freely relative to each other. For example, a glycine-serine doublet can be a suitable peptide linker. In some embodiments, the linker is a non-peptide linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker is a cleavable linker.

[0158] Other linker considerations include the effect on the physical or pharmacokinetic properties of the resulting anti-CD93 or anti-IGFBP7-Fc fusion protein, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (more or less stable as well as programmed degradation), rigidity, flexibility, immunogenicity, modulation of antibody binding, ability to be incorporated into micelles or liposomes, etc.

[0159] a. Non-peptide linker Any one or all of the linkers described herein can be achieved by any chemical reaction that links two molecules, so long as the components or fragments retain their respective activities, i.e., binding to target CD93 or IGFBP7, binding to FcR, and / or ADCC / CDC. This linkage can involve many chemical mechanisms, such as covalent bonding, affinity binding, intercalation, coordinate binding, and complex formation. In some embodiments, the linkage is covalent. Covalent bonding can be achieved by direct condensation of existing side chains or by incorporation of an external crosslinking molecule. Many bivalent or multivalent linking agents are useful for coupling protein molecules, such as Fc fragments, to anti-CD93 or anti-IGFBP7 antibodies of the present invention. For example, representative coupling agents can include organic compounds, such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene, and hexamethylenediamine. This list is not intended to be exhaustive of the various classes of coupling agents known in the art, but rather is illustrative of the more common coupling agents (see Killen and Lindstrom, Jour. Immun. 133:1335-2549 (1984); Jansen et al., Immunological Reviews 62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987); each incorporated by reference in its entirety for all purposes).

[0160] Linkers that can be applied in the present application have been described in the literature (see, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984), which describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester), incorporated by reference in its entirety for all purposes. In some embodiments, non-peptide linkers used herein include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride); (ii) SMPT (4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyl-dithio)-toluene (Pierce Chem. Co., Cat. No. (21558G)); (iii) SPDP (succinimidyl 6-[3-(2-pyridyldithio)propionamido]hexanoate) (Pierce Chem. Co., catalog number 21651G); (iv) sulfo-LC-SPDP (6[3-(2-pyridyldithio)-propianamido]hexanoate sulfosuccinimidyl (Pierce Chem., Co. catalog number 2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem., catalog number 24510) conjugated to EDC.

[0161] The above linkers contain components with different properties, thus resulting in anti-CD93 or anti-IGFBP7-Fc fusion proteins with different physiochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS-ester-containing linkers are less soluble than sulfo-NHS esters. Furthermore, the linker SMPT contains a sterically hindered disulfide bond, which can form fusion proteins with improved stability. Disulfide bonds are generally less stable than other linkages because they are cleaved in vitro, resulting in less available fusion protein. In particular, sulfo-NHS can enhance the stability of carbodiimide coupling. Carbodiimide coupling (such as EDC) when used in conjunction with sulfo-NHS forms esters that are more resistant to hydrolysis than carbodiimide coupling alone.

[0162] b. Peptide linker Any one or all of the linkers described herein can be peptide linkers. The peptide linker can have a naturally occurring sequence or a non-naturally occurring sequence. For example, a sequence derived from the hinge region of a heavy chain-only antibody can be used as a linker. See, for example, WO 1996 / 34103 (incorporated by reference in its entirety for all purposes). In some embodiments, the peptide linker comprises the amino acid sequence of CPPCP (SEQ ID NO: 3), a sequence found in a natural IgG1 hinge region.

[0163] The peptide linker may be of any suitable length, in some embodiments, the length of the peptide linker may be about 1 aa to about 10 aa, about 1 aa to about 20 aa, about 1 aa to about 30 aa, about 5 aa to about 15 aa, about 10 aa to about 25 aa, about 5 aa to about 30 aa, about 10 aa to about 30 aa, about 30 aa to about 50 aa, about 50 aa to about 100 aa, or about 1 aa to about 100 aa.

[0164] An important technical feature of such peptide linkers is that they do not contain any polymerization activity. The properties of peptide linkers, including the absence of promotion of secondary structure, are known in the art and are described, for example, in Dall'Acqua et al. (Biochem. (1998) 37, 9266-9273), Cheadle et al. (Mol Immunol (1992) 29, 21-30), and Raag and Whitlow (FASEB (1995) 9(1), 73-80, each of which is incorporated by reference in its entirety for all purposes. A particularly preferred amino acid in the context of a "peptide linker" is Gly. Furthermore, peptide linkers that do not promote any secondary structure are preferred. Linkage of the molecules to each other can be provided, for example, by genetic engineering. Methods for preparing fused and operably linked antibody constructs and expressing them in mammalian cells or bacteria are well known in the art (see, for example, WO 99 / 54440; Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY 1989 and 1994, or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001; each of which is incorporated herein by reference in its entirety for all purposes).

[0165] In some embodiments, the peptide linker is a stable linker that is not cleavable by proteases, such as matrix metalloproteinases (MMPs).

[0166] In some embodiments, the peptide linker does not tend to adopt a rigid three-dimensional structure, but rather provides flexibility to the polypeptide (e.g., the first and / or second components), such as providing flexibility between the anti-CD93 or anti-IGFBP7 antibody and the Fc fragment. In some embodiments, the peptide linker is a flexible linker. Exemplary flexible linkers include glycine polymers (G) and n (SEQ ID NO: 4), glycine-serine polymers (e.g., (GS) n (SEQ ID NO: 5), (GSGGS) n (SEQ ID NO: 6), (GGGGS) n (SEQ ID NO: 7), and (GGGS) n (SEQ ID NO: 8) (where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and may therefore be able to function as neutral tethers between components. Glycine has access to significantly more phi-psi space than alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11 173-142 (1992)). Those skilled in the art will recognize that the design of anti-CD93 or anti-IGFBP7-Fc fusion proteins can include linkers that are fully or partially flexible, and the linker can include a flexible linker portion as well as one or more portions that confer a less flexible structure to provide the desired fusion protein structure.

[0167] In some embodiments, the anti-CD93 or anti-IGFBP7 antibody (e.g., anti-CD93 or anti-IGFBP7 antibody fragment) and the Fc fragment are linked to each other by a linker of sufficient length to allow the anti-CD93 or anti-IGFBP7-Fc fusion protein to fold in a manner that allows binding to target CD93 or IGFBP7 as well as FcR. In some embodiments, the linker comprises the amino acid sequence of SRGGGGSGGGGSGGGGSLEMA (SEQ ID NO: 9). In some embodiments, the linker comprises the amino acid sequence of (GGGGS) n (SEQ ID NO: 13), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. In some embodiments, the linker comprises the amino acid sequence of TSGGGGS (SEQ ID NO: 10). In some embodiments, the linker comprises the amino acid sequence of GEGTSTGSGGSGGSGGAD (SEQ ID NO: 11).

[0168] Natural linkers perform their functions by adopting various conformations in secondary structures, such as helix, β-strand, coil / bend, and turn. An α-helical linker can act as a rigid spacer that effectively separates protein domains, thus reducing their unfavorable interactions. A non-helical linker with a Pro-rich sequence can increase the rigidity of the linker and reduce interdomain interference. In some embodiments, an anti-CD93 or anti-IGFBP7 antibody (e.g., antibody fragment) and an Fc fragment (or an antibody containing an Fc fragment) are linked to each other by an α-helical linker having the amino acid sequence A(EAAAK)4A (SEQ ID NO: 12).

[0169] B. Multispecific anti-CD93 or anti-IGFBP7 molecules A multispecific molecule is a molecule that has binding specificities for at least two different antigens or epitopes (e.g., a bispecific antibody has binding specificities for two antigens or epitopes). Multispecific molecules with more than two valencies and / or specificities are also contemplated. For example, trispecific antibodies can be prepared (Tutt et al. J. Immunol. 147: 60 (1991)). It will be understood that one skilled in the art can select and combine appropriate features of the subject multispecific molecules described herein to form the multispecific anti-CD93 or anti-IGFBP7 molecules of the present application.

[0170] In some embodiments, the agent that blocks the interaction between CD93 and IGFBP7 comprises a multispecific (e.g., bispecific) anti-CD93 or anti-IGFBP7 molecule comprising an anti-CD93 or anti-IGFBP7 antibody according to any one of the anti-CD93 or anti-IGFBP7 antibodies described herein and a second binding moiety (such as a second antibody) that specifically recognizes a second antigen. In some embodiments, the multispecific anti-CD93 or anti-IGFBP7 molecule comprises an anti-CD93 or anti-IGFBP7 antibody and a second antibody that specifically recognizes a second antigen.

[0171] In some embodiments, the multispecific anti-CD93 or anti-IGFBP7 molecule can be, for example, a diabody (Db), single-chain diabody (scDb), tandem scDb (Tandab), linear dimeric scDb (LD-scDb), cyclic dimeric scDb (CD-scDb), didiabody, tandem scFv, tandem di-scFv (e.g., bispecific T cell engager), tandem tri-scFv, tri(a)body, bispecific Fab2, di-miniantibody, tetrabody, scFv-Fc-scFv fusion, dual-affinity retargeting (DART) antibody, dual variable region (DVD) antibody, IgG-scFab, scFab-ds-scFv, Fv2-Fc, IgG-scFv fusion, dock and lock (DLOCK) antibody, or any combination thereof. These antibodies are: DNL (Dimer Lock) antibodies, Knob-into-Hole (KiH) antibodies (bispecific IgG prepared by KiH technology), DuoBody (bispecific IgG prepared by DuoBody technology), heteromultimeric antibodies, or heteroconjugate antibodies.

[0172] In some embodiments, the agent comprises an anti-CD93 and an anti-IGFBP7 antibody. In some embodiments, the agent is a bispecific antibody.

[0173] In some embodiments, the agent that blocks the interaction between CD93 and IGFBP7 comprises a multispecific (e.g., bispecific) anti-CD93 molecule comprising a first anti-CD93 antibody that specifically binds to a first epitope on CD93 and a second anti-CD93 antibody that specifically binds to a second epitope on CD93. In some embodiments, one or both of the first and second epitopes overlap or substantially overlap with the epitope of mAb MM01 or mAb 7C10. In some embodiments, one or both of the first and second antibodies bind to CD93 competitively with mAb MM01 or mAb 7C10. In some embodiments, one or both of the first and second antibodies also block the interaction between CD93 and MMRN2. In some embodiments, one or both of the first and second antibodies do not block the interaction between CD93 and MMRN2. In some embodiments, one or both of the first antibody and the second antibody bind to a region on CD93 outside the IGFBP7 binding site.

[0174] In some embodiments, the agent that blocks the interaction between CD93 and IGFBP7 comprises a multispecific (e.g., bispecific) anti-IGFBP7 molecule comprising a first anti-IGFBP7 antibody that specifically binds to a first epitope on IGFBP7 and a second anti-IGFBP7 antibody that specifically binds to a second epitope on IGFBP7. In some embodiments, one or both of the first and second epitopes overlap or substantially overlap with the epitope of mAb R003 or mAb 2C6. In some embodiments, one or both of the first and second antibodies bind to IGFBP7 competitively with mAb R003 or mAb 2C6.

[0175] Inhibitory CD93 or IGFBP7 polypeptides A. Inhibitory CD93 Polypeptides The methods described herein, in some embodiments, involve the use of a polypeptide that blocks the interaction between CD93 and IGFBP7 (an "inhibitory CD93 polypeptide"), comprising the extracellular domain of CD93 or a variant thereof. The present application, in certain aspects, provides novel and non-naturally occurring inhibitory CD93 polypeptides described herein. In some embodiments, the inhibitory CD93 polypeptide is a soluble polypeptide.

[0176] In some embodiments, the inhibitory CD93 polypeptide is membrane-bound. In some aspects, the membrane-bound inhibitory CD93 polypeptide binds to IGFBP7 but does not induce CD93 / IGFBP7 signaling. In some embodiments, the membrane-bound inhibitory CD93 polypeptide binds to IGFBP7 and attenuates CD93 / IGFBP7 signaling. In some embodiments, the membrane-bound inhibitory CD93 polypeptide is introduced by a gene editing system or an mRNA delivery vehicle.

[0177] In some embodiments, the inhibitory CD93 polypeptide comprises the extracellular domain of CD93 (e.g., human CD93) or a variant thereof. In some embodiments, the inhibitory CD93 polypeptide comprises the amino acid sequence of residues A24 to K580 of SEQ ID NO: 1, or a variant thereof having at least about 80% (e.g., about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to residues A24 to K580 of SEQ ID NO: 1. In some embodiments, the inhibitory CD93 polypeptide further comprises residue F238, where amino acid numbering is based on SEQ ID NO: 1.

[0178] In some embodiments, the inhibitory CD93 polypeptide comprises a C-type lectin domain of CD93 (e.g., human CD93) or a variant thereof. In some embodiments, the inhibitory CD93 polypeptide comprises the amino acid sequence of residues T22 to N174 of SEQ ID NO: 1, or a variant thereof having at least about 80% (e.g., about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to residues T22 to N174 of SEQ ID NO: 1. In some embodiments, the inhibitory CD93 polypeptide further comprises residue F238, where amino acid numbering is based on SEQ ID NO: 1.

[0179] In some embodiments, the inhibitory CD93 polypeptide comprises a long loop region in the C-type lectin domain of CD93 (e.g., human CD93) or a variant thereof. In some embodiments, the inhibitory CD93 polypeptide comprises the amino acid sequence of residues G96 to C141 of SEQ ID NO: 1, or a variant thereof having at least about 80% (e.g., about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to residues G96 to C141 of SEQ ID NO: 1. In some embodiments, the inhibitory CD93 polypeptide further comprises at least one or more (e.g., at least about 10, 15, 20, 25, 30, 35, or all) of residues selected from G96, Q98, R99, E100, K101, G102, K103, C104, L105, D106, P107, S108, L109, K112, S115, V117, G118, G120, E121, D122, T123, P124, Y125, S126, N127, H129, K130, E131, L132, R133, N134, S135, C136, I137, S138, K139, and R140, where amino acid numbering is based on SEQ ID NO:1.

[0180] In some embodiments, an inhibitory CD93 polypeptide comprises a DX domain between the C-type lectin-like domain (D1 domain) and the EGF-like domain (D2 domain) of CD93 (such as human CD93), or a variant thereof. In some embodiments, the inhibitory CD93 polypeptide comprises the amino acid sequence of residues I175-L256 and I175-L259 of SEQ ID NO: 1, or a variant thereof having at least about 80% (e.g., about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to residues I175-L256 and I175-L259 of SEQ ID NO: 1.

[0181] In some embodiments, the inhibitory CD93 polypeptide comprises the amino acid sequence of any one of residues F182 to Y262, I175 to L256, and / or I175 to L259 of SEQ ID NO: 1, or a variant thereof having at least about 80% (e.g., about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, etc.) sequence identity to the sequence of any one of residues F182 to Y262, I175 to L256, and I175 to L259 of SEQ ID NO: 1. In some embodiments, the inhibitory CD93 polypeptide further comprises residue F238 based on SEQ ID NO: 1. In some embodiments, the inhibitory CD93 polypeptide further comprises at least one or more (e.g., at least about 10, 15, 20, 25, 30, 35, or all) of residues selected from G96, Q98, R99, E100, K101, G102, K103, C104, L105, D106, P107, S108, L109, K112, S115, V117, G118, G120, E121, D122, T123, P124, Y125, S126, N127, H129, K130, E131, L132, R133, N134, S135, C136, I137, S138, K139, and R140. Amino acid numbering is based on SEQ ID NO:1.

[0182] In some embodiments, the inhibitory CD93 polypeptide comprises the amino acid sequence of residues T22 to Y262 of SEQ ID NO: 1, or a variant thereof having at least about 80% (e.g., about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to residues T22 to Y262 of SEQ ID NO: 1. In some embodiments, the inhibitory CD93 polypeptide further comprises residue F238 based on SEQ ID NO: 1. In some embodiments, the inhibitory CD93 polypeptide further comprises at least one or more (e.g., at least about 10, 15, 20, 25, 30, 35, or all) of residues selected from G96, Q98, R99, E100, K101, G102, K103, C104, L105, D106, P107, S108, L109, K112, S115, V117, G118, G120, E121, D122, T123, P124, Y125, S126, N127, H129, K130, E131, L132, R133, N134, S135, C136, I137, S138, K139, and R140, based on SEQ ID NO:1.

[0183] In some embodiments, the inhibitory CD93 polypeptide comprises an F238 residue, where the amino acid numbering is based on SEQ ID NO:1.

[0184] In some embodiments, the inhibitory CD93 polypeptide comprises one, two, three, four, or five of the five EGF-like regions of CD93 (e.g., human CD93) or variants thereof. In some embodiments, the inhibitory CD93 polypeptide comprises the amino acid sequence of residues C257 to M469 or P260 to T468 of SEQ ID NO: 1, or a variant thereof having at least about 80% (e.g., about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to residues C257 to M469 or P260 to T468 of SEQ ID NO: 1.

[0185] In some embodiments, the variants described herein are naturally occurring variants. In some embodiments, the variants do not include non-conservative substitutions. In some embodiments, the variants include only one or more conservative substitutions. In some embodiments, the one or more conservative substitutions include or consist of the substitutions shown in Table 1 below under the heading "preferred substitutions." [Table 1]

[0186] In some embodiments, the inhibitory CD93 polypeptide binds to IGFBP7 with a higher affinity than to MMNR2. ... D At most half, 1 / 5, 1 / 10, 1 / 20, 1 / 50, 1 / 100, 1 / 1000 of K D and binds to IGFBP7.

[0187] In some embodiments, the inhibitory CD93 polypeptide binds to IGFBP7 with a higher affinity than CD93. In some aspects, the inhibitory CD93 polypeptide binds to IGFBP7 with a higher affinity than CD93. In some embodiments, the inhibitory CD93 polypeptide binds to IGFBP7 with a higher affinity than CD93. D At most half, 1 / 5, 1 / 10, 1 / 20, 1 / 50, 1 / 100, 1 / 1000 of K D It binds to IGFBP7.

[0188] In some embodiments, the inhibitory CD93 polypeptide further comprises a stabilization domain. The stabilization domain can be any domain that stabilizes the inhibitory IGFBP7 polypeptide (e.g., extends the half-life of the inhibitory IGFBP7 polypeptide in vivo). In some embodiments, the stabilization domain is an Fc domain. Exemplary Fc domains include those described in the "Fc Fragment" section.

[0189] In some embodiments, the inhibitory polypeptide is about 50 to about 1000 amino acids in length, e.g., about 50 to 800, 50 to 500, 50 to 400, 50 to 300, or 50 to 200 amino acids in length. In some embodiments, the inhibitory polypeptide is about 50 to about 100 amino acids, about 100 to about 150 amino acids, or about 150 to about 200 amino acids in length.

[0190] B. Inhibitory IGFBP Polypeptides The methods described herein, in some embodiments, involve the use of polypeptides that block the interaction between CD93 and IGFBP7 ("inhibitory IGFBP7 polypeptides"), including variants of IGFBP7. In one aspect, the present application provides novel and non-naturally occurring inhibitory IGFBP7 polypeptides described herein.

[0191] In some embodiments, the inhibitory IGFBP7 polypeptide binds to CD93 but does not activate CD93.

[0192] In some embodiments, the inhibitory IGFBP7 polypeptide binds to CD93 with a higher affinity than to IGF-1, IGF-2, and / or IGF1R. In some embodiments, the inhibitory IGFBP7 polypeptide binds to CD93 with a higher affinity than to IGF-1, IGF-2, and / or IGF1R. In some embodiments, the inhibitory IGFBP7 polypeptide binds to CD93 with a higher affinity than to IGF-1, IGF-2, and / or IGF1R. D At most half, 1 / 5, 1 / 10, 1 / 20, 1 / 50, 1 / 100, 1 / 1000 of K D It binds to IGFBP7.

[0193] In some embodiments, the inhibitory IGFBP7 polypeptide binds to CD93 with a higher affinity than IGFBP7. In some aspects, the inhibitory IGFBP7 polypeptide binds to CD93 with a higher affinity than IGFBP7. In some embodiments, the inhibitory IGFBP7 polypeptide binds to CD93 with a higher affinity than IGFBP7. In some embodiments, the inhibitory IGFBP7 polypeptide binds to CD93 with a higher affinity than IGFBP7. D At most half, 1 / 5, 1 / 10, 1 / 20, 1 / 50, 1 / 100, 1 / 1000 of K D It binds to CD93.

[0194] In some embodiments, the inhibitory IGFBP7 polypeptide comprises the IB domain of IGFBP7 (e.g., human IGFBP7) or a variant thereof. In some embodiments, the inhibitory IGFBP7 polypeptide comprises the amino acid sequence of residues S28 to G106 of SEQ ID NO:2, or a variant thereof having at least about 80% (e.g., about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to residues S28 to G106 of SEQ ID NO:2.

[0195] In some embodiments, the inhibitory IGFBP7 polypeptide comprises or further comprises the Kazal-like domain of IGFBP7 (e.g., human IGFBP7) or a variant thereof. In some embodiments, the inhibitory IGFBP7 polypeptide comprises or further comprises the amino acid sequence of residues P105-Q158 of SEQ ID NO:2, or a variant thereof having at least about 80% (e.g., about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to residues P105-Q158 of SEQ ID NO:2.

[0196] In some embodiments, the inhibitory IGFBP7 polypeptide comprises or further comprises the Ig-like C2 domain of IGFBP7 (e.g., human IGFBP7) or a variant thereof. In some embodiments, the inhibitory IGFBP7 polypeptide comprises or further comprises the amino acid sequence of residues P160 to T264 of SEQ ID NO:2, or a variant thereof having at least about 80% (e.g., about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to residues P160 to T264 of SEQ ID NO:2.

[0197] In some embodiments, the variants described herein are naturally occurring variants. In some embodiments, the variants do not include non-conservative substitutions. In some embodiments, the variants include only one or more conservative substitutions. In some embodiments, the one or more conservative substitutions include or consist of the substitutions shown in Table 1 under the heading "Preferred Substitutions."

[0198] In some embodiments, the inhibitory IGFBP7 polypeptide also blocks the interaction between CD93 and MMNR2. In some aspects, the inhibitory IGFBP7 polypeptide binds to the same epitope on CD93 as that to which MMNR2 binds. In some embodiments, the inhibitory IGFBP7 polypeptide binds to an epitope on CD93 that is different from that to which MMNR2 binds.

[0199] In some embodiments, the inhibitory IGFBP7 polypeptide does not block the interaction between CD93 and MMNR2.

[0200] In some embodiments, the inhibitory IGFBP7 polypeptide is a soluble polypeptide.

[0201] In some embodiments, the inhibitory IGFBP7 polypeptide is membrane-bound. In some embodiments, the membrane-bound inhibitory IGFBP7 polypeptide binds to CD93 but does not induce or attenuate CD93 / IGFBP7 signal transduction. In some embodiments, the membrane-bound inhibitory IGFBP7 polypeptide is introduced by gene editing system or mRNA delivery vehicle.

[0202] In some embodiments, the inhibitory IGFBP polypeptide further comprises a stabilization domain. The stabilization domain can be any domain that stabilizes the inhibitory IGFBP7 polypeptide (e.g., extends the half-life of the inhibitory IGFBP7 polypeptide in vivo). In some embodiments, the stabilization domain is an Fc domain. Exemplary Fc domains include those described in the "Fc Fragment" section.

[0203] In some embodiments, the inhibitory polypeptide is about 50 to about 1000 amino acids in length, e.g., about 50 to 800, 50 to 500, 50 to 400, 50 to 300, or 50 to 200 amino acids in length. In some embodiments, the inhibitory polypeptide is about 50 to about 100 amino acids, about 100 to about 150 amino acids, or about 150 to about 200 amino acids in length.

[0204] Other drugs that inhibit the IGFBP3 / CD93 signaling pathway Other agents capable of inhibiting IGFBP3 / CD93 other than those described above are also contemplated for use in the methods described herein. In some embodiments, the agent comprises a peptide, polypeptide, peptide analog, fusion peptide, aptamer, avimer, anticalin, spiegelmer, or small molecule compound.

[0205] In some embodiments, the agent reduces expression of CD93 (such as human CD93). In some embodiments, the agent reduces expression of CD93 (such as human CD93) by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% compared to the level of CD93 without the agent. In some embodiments, the agent reduces CD93 expression to a baseline level. In some embodiments, the baseline level is the level of CD93 expression in a non-tumor organ of the subject. In some embodiments, the baseline level is the level (or average level) of CD93 expression in a subject or group of subjects without a disease or condition or abnormal vasculature.

[0206] In some embodiments, the agent reduces the expression of IGFBP7 (such as human IGFBP7). In some embodiments, the agent reduces the expression of IGFBP7 (such as human IGFBP7) by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% compared to the level of IGFBP7 without the agent. In some embodiments, the agent reduces the expression of IGFBP7 to a reference level. In some embodiments, the reference level is the level of IGFBP7 expression in a non-tumor organ of the subject. In some embodiments, the reference level is the level (or average level) of IGFBP7 expression in a subject or group of subjects without a disease or condition or abnormal vasculature.

[0207] In some embodiments, the agent comprises an siRNA, shRNA, miRNA, or antisense RNA that targets CD93 (such as human CD93), or an siRNA, shRNA, miRNA, or antisense RNA that specifically targets IGFBP7 (such as human IGFBP7).

[0208] In some embodiments, the agent comprises a genome editing system targeting CD93 or IGFBP7. In some embodiments, the genome editing system comprises a DNA nuclease, such as a DNA nuclease engineered (e.g., programmable or targetable) to induce genome editing of a target DNA sequence of CD93 or IGFBP7. Any suitable DNA nuclease can be used, including, but not limited to, CRISPR-associated protein (Cas) nuclease, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), meganuclease, other endonucleases or exonucleases, variants thereof, fragments thereof, and combinations thereof. In some embodiments, the genome editing comprises modifying CD93 so that the modified CD93 no longer binds to IGFBP7 or binds to IGFBP7 to a lesser extent than wild-type CD93. In some embodiments, the modification comprises inserting a transgene comprising a CD93 variant. In some embodiments, the variant CD93 has a mutation at F238 based on SEQ ID NO: 1. In some embodiments, the variant CD93 has a F238T mutation based on SEQ ID NO: 1.

[0209] In some embodiments, the genome editing comprises modifying IGFBP7 so that the modified IGFBP7 no longer binds to CD93 or binds to CD93 to a lesser extent than wild-type IGFBP7. In some embodiments, the modification comprises inserting a transgene comprising a variant of IGFBP7. In some embodiments, the variant of IGFBP7 has a C-type lectin domain, and the C-type lectin domain of IGFBP7 is not derived from IGFBP7.

[0210] Vascular maturation / normalization The successful function of all tissues depends on the establishment of a hierarchically structured, mature vascular network. In contrast to healthy conditions, many human diseases exhibit dysregulated and excessive new blood vessel formation. Solid tumors are one characteristic example. Far from being a mass of proliferating cancer cells, solid tumors are aggregates of cancer cells, vascular networks, lymphatic vessels, and various other cells, all of which contribute to the local microenvironment. Angiogenesis within solid tumors is largely driven by hypoxia, a hallmark of the tumor microenvironment that directly leads to the production of proangiogenic factors such as VEGF via the regulation of oxygen-sensing molecules. See Goel et al., Cold Spring Harb Perspect Med 2012;2:a006486.

[0211] The microenvironmental abundance of VEGF and other pro-angiogenic factors drives continued angiogenesis and the creation of abnormal vascular networks. Structurally, blood vessels are often dilated, follow tortuous paths, and exhibit heterogeneous distribution, with certain areas within tumors being hypovascular and others hypervascular. At the cellular level, pro-angiogenic factors induce weakening of VE-cadherin-mediated endothelial cell (EC) junctions and EC migration, altering vessel wall architecture. Similarly, perivascular cells (composed of PVCs, pericytes, and vascular smooth muscle cells (VSMCs)) are often only loosely attached to ECs and are reduced in number. Finally, the perivascular basement membrane (BM) is also structurally abnormal in tumors, being excessively thin or absent in some areas and abnormally thick in others. See Goel et al., Cold Spring Harb Perspect Med 2012;2:a006486.

[0212] A direct consequence of these structural abnormalities is a marked abnormality in tumor vascular function. The haphazard and heterogeneous distribution of blood vessels results in heterogeneous blood flow, sluggish in some areas and excessive in others. Furthermore, reduced PVC coverage, EC dissociation, and excessive vesiculo-vaculor organelles (VVO) result in significant tumor vascular permeability, accompanied by excessive extravasation of fluid and proteins into the extracellular compartment. This leakage, along with the relative lack of functional intratumoral lymphatics, leads to a significant increase in tumor interstitial fluid pressure (IFP) to a level that balances intravascular pressure, resulting in reduced transvascular flow. Furthermore, compressive forces exerted by the proliferating mass of cancer cells can cause vascular compression and collapse. The net result is a heterogeneous blood supply and resulting hypoxia and acidosis. The described physiological changes have a direct impact on the behavior of solid tumors. Hypoxic tumor cells often exhibit a more aggressive phenotype, activate oncogenes, and undergo "epithelial-mesenchymal transition" (EMT), which enhances their metastatic potential. Furthermore, the adverse microenvironment impairs the function of antitumor immune cells and their delivery to tumors. Importantly, tumor response to therapy is also affected. Hypoxia is known to reduce the sensitivity of tumor cells to radiation and chemotherapy, and delivery of systemically administered cytotoxic agents to tumors is dramatically hindered, especially in areas of low blood flow and elevated tumor IFP. See Goel et al., Cold Spring Harb Perspect Med 2012;2:a006486.

[0213] The present application provides methods and compositions useful for normalizing blood vessels (i.e., promoting the maturation of abnormal vasculature) in diseases or conditions (e.g., cancers, such as solid tumors). In some embodiments, the abnormal blood vessels are associated with hypoxic conditions.

[0214] "Normalization of the vasculature," "normalizing immature and leaky blood vessels," "vascular maturation," or "promoting the formation of a functional vascular network," and "promoting a favorable tumor microenvironment" generally refer to or include the conversion of a network of leaky, tortuous, and disorganized blood vessels (e.g., tumor blood vessels) to a more organized network of less permeable, less dilated, and / or less tortuous blood vessels. In some embodiments, vascular normalization is characterized by more mature blood vessels (e.g., longer blood vessels, rounder blood vessels). In some embodiments, vascular normalization is characterized by increased association of pericytes and / or smooth muscle cells with endothelial cells lining the blood vessel wall, the formation of a more normal basement membrane (e.g., with a more physiological thickness), and / or a closer association of blood vessels with the basement membrane. Normalization of the vasculature may also involve the pruning of immature blood vessels, along with increased integrity and stability of the residual vasculature. In some embodiments, the vascular normalization described herein is characterized by the maintenance of vascular density.

[0215] In some embodiments, vascular maturation (or vascular normalization) can be characterized by vascular morphology. In some embodiments, vascular normalization is characterized by an increase in the length of blood vessels in tissue. Blood vessel length can be measured in units of total blood vessel length (e.g., mm) per field of view, as described in the Examples (see, e.g., Figure 2B). In some embodiments, blood vessel length (e.g., total length per field of view) increases by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% after administration of an IGFBP7 / CD93 blocking agent. In some embodiments, blood vessels are identified by CD31 expression.

[0216] In some embodiments, vascular normalization is characterized by an increase in the percentage of round vessels in the tissue (% of round vessels / total vessels). The percentage of round vessels can be measured by dividing the number of round vessels by the total number of vessels, as described in the Examples (see, e.g., Figure 2B). In some embodiments, the percentage of round vessels increases by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% after administration of an IGFBP7 / CD93 blocking agent. In some embodiments, blood vessels are identified by CD31 expression.

[0217] In some embodiments, vascular normalization is characterized by the maintenance of vascular density of blood vessels in tissue. Vascular density can be measured in units of blood vessels per field of view, as described in the Examples (see, e.g., Figure 2B). In some embodiments, vascular density does not decrease by more than about 30%, 20%, 10%, or 5% after administration of an IGFBP7 / CD93 blocking agent. In some embodiments, vascular density does not increase by more than about 30%, 20%, 10%, or 5% after administration of an IGFBP7 / CD93 blocking agent. In some embodiments, vascular density does not increase or decrease by more than about 30%, 20%, 10%, or 5% after administration of an IGFBP7 / CD93 blocking agent. In some embodiments, blood vessels are identified by CD31 expression.

[0218] In some embodiments, vascular maturation (or vascular normalization) can be characterized by a higher density of pericytes (e.g., NG2+ pericytes) and / or a higher density of smooth muscle cells (e.g., α-SMA+ smooth muscle cells). In some embodiments, vascular normalization is characterized by an increase in NG2 expression on the blood vessels. In some embodiments, NG2 expression on the blood vessels is increased by at least about 25%, 50%, 75%, 100%, 125%, 150%, 175%, or 200% after administration of an IGFBP7 / CD93 blocking agent. In some embodiments, vascular normalization is characterized by an increase in α-SMA+ expression on the blood vessels. In some embodiments, α-SMA+ expression on the blood vessels is increased by at least about 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, or 250% after administration of an IGFBP7 / CD93 blocking agent. In some embodiments, vascular normalization is characterized by an increase in ICAM expression on blood vessels. In some embodiments, ICAM+ expression on blood vessels is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, or 70% after administration of an IGFBP7 / CD93 blocking agent. In some embodiments, vascular normalization is characterized by a decrease in activated integrin β1 expression on blood vessels. In some embodiments, activated integrin expression on blood vessels is decreased by at least about 10%, 20%, 30%, 40%, or 50% after administration of an IGFBP7 / CD93 blocking agent. In some embodiments, blood vessels are identified by CD31 expression.

[0219] In some embodiments, vascular maturation (or vascular normalization) can be characterized by vascular perfusion and / or permeability. In some embodiments, vascular normalization is characterized by an increase in vascular permeability or perfusion. Permeability or perfusion can be assessed by assessing the distribution of an administered drug (such as a lectin) in blood vessels, for example, as described in the Examples (e.g., FIG. 2E). In some embodiments, vascular perfusion increases by at least about 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% after administration of an IGFBP7 / CD93 blocking agent.

[0220] In some embodiments, vascular normalization is characterized by a reduction in hypoxia in the tissue. Tumor hypoxia can be assessed, for example, as described in the Examples (e.g., FIG. 6A). In some embodiments, tumor hypoxia is assessed by the percentage (%) of pimonidazole positivity (i.e., pimonidazole-positive area divided by total tumor area, etc.). In some embodiments, tumor hypoxia is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% after administration of an IGFBP7 / CD93 blocking agent.

[0221] In some embodiments, vascular normalization is characterized by more effective drug delivery. The effectiveness of drug delivery can be determined, for example, by evaluating the distribution of the drug in a tissue (such as a tumor tissue) after drug delivery (e.g., as described in the Examples (e.g., Figure 6A)). In some embodiments, the presence / distribution of the drug (such as a chemotherapeutic drug) in the tissue after delivery is increased by at least about 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% after administration of an IGFBP7 / CD93 blocking agent.

[0222] In some embodiments, vascular normalization is characterized by increased infiltration of immune cells in tissue (e.g., tumor tissue). Immune cell infiltration in tissue can be measured by assessing the percentage of immune cells in tissue (e.g., tumor tissue) (e.g., by measuring the number of immune cells in tissue divided by tumor weight units (e.g., mg) or by measuring the number of immune cells in tissue divided by field of view, as described in Figures 3A and 3D). In some embodiments, the immune cells are tumor-infiltrating lymphocytes. In some embodiments, the immune cells comprise CD45+ leukocytes. In some embodiments, the immune cells comprise CD3+ T cells. In some embodiments, the immune cells comprise CD4+ T cells. In some embodiments, the immune cells comprise CD8+ T cells. In some embodiments, the immune cells are endogenous immune cells. In some embodiments, the immune cells are exogenous immune cells. In some embodiments, the immune cells are engineered immune cells (e.g., CART cells) derived from a subject. In some embodiments, the percentage of immune cells in a tissue (e.g., tumor tissue) is increased by at least about 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% after administration of an IGFBP7 / CD93 blocking agent.

[0223] In some embodiments, the ratio of suppressor immune cells among infiltrating immune cells is reduced after administration of an IGFBP7 / CD93 blocking agent. In some embodiments, the suppressor immune cells comprise myeloid-derived suppressor cells (MDSCs). In some embodiments, the MDSCs comprise granulocytic MDSCs (e.g., CD3-CD11c-CD11b+Ly6G+Ly6C-CD45+ leukocytes). In some embodiments, the MDSCs comprise monocytic MDSCs (e.g., CD3-CD11c-CD11b+Ly6G-Ly6C+CD45+ leukocytes). In some embodiments, the MDSCs comprise both granulocytic and monocytic MDSCs. In some embodiments, the ratio of suppressor immune cells among infiltrating immune cells is reduced by at least 10%, 20%, 30%, 40%, or 50% after administration of an IGFBP7 / CD93 blocking agent.

[0224] The various parameters described in the sections above (such as vessel length, morphology, hypoxia, perfusion, immune cell infiltration, drug delivery, etc.) can be assessed at various time points after administration of one or more IGFBP7 / CD93 blocking agents. In some embodiments, the parameters are assessed 14 days after administration of an IGFBP7 / CD93 blocking agent, where the agent is administered about twice a week for two weeks.

[0225] endpoint Any of the parameters described in the "Vascular Maturation / Normalization" section (such as vessel length, morphology, hypoxia, perfusion, immune cell infiltration, drug delivery, etc.) can be used as a feature of the method (e.g., a method of treating cancer). The "Vascular Maturation / Normalization" section is incorporated herein in its entirety for discussion of features of various embodiments of the method.

[0226] In some embodiments, the subject has decreased tumor cell proliferation and / or increased tumor cell apoptosis. Tumor cell proliferation and apoptosis can be assessed by proliferation or apoptosis markers (such as Ki-67 and cleaved caspase 3 (CC3) described in the Examples). In some embodiments, tumor cell proliferation is characterized by Ki-67-positive cells in the tumor. In some embodiments, Ki-67-positive cells in the tumor are reduced by at least about 10%, 20%, 30%, 40%, 50%, or 60% after administration of an IGFBP7 / CD93 blocker. In some embodiments, tumor cell apoptosis is characterized by CC3-positive cells in the tumor tissue. In some embodiments, CD3-positive cells in the tumor tissue are increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% after administration of an IGFBP7 / CD93 blocker.

[0227] In some embodiments, a subject has at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% reduction in tumor size, number of cancer cells, or tumor growth rate compared to the corresponding tumor size, number of cancer cells, or tumor growth rate in the same subject before treatment, or compared to the corresponding activity in another subject not receiving treatment. The magnitude of these effects can be measured using standard methods, such as in vitro assays using purified enzymes, cell-based assays, animal models, or human studies.

[0228] disease or condition The methods described herein are applicable to any disease or condition associated with abnormal vasculature. In some embodiments, the disease or condition is age-related macular degeneration (ARMD). In some embodiments, the disease or condition is psoriasis. In some embodiments, the disease or condition is a benign tumor. In some embodiments, the disease or condition is cancer.

[0229] cancer In some embodiments, the disease or condition described herein is cancer.Cancer that can be treated using any of the methods described herein includes any type of cancer.The types of cancer that can be treated with the agents described in this application include, but are not limited to, carcinoma, blastoma, sarcoma, benign and malignant tumors, and malignancies (malignant tumors), such as sarcoma, carcinoma, and melanoma.Also included are adult tumors / cancers and pediatric tumors / cancers.

[0230] In various embodiments, the cancer is an early stage cancer, a non-metastatic cancer, a primary cancer, an advanced cancer, a locally advanced cancer, a metastatic cancer, a cancer in remission, a recurrent cancer, a cancer in an adjuvant setting, a cancer in a neoadjuvant setting, or a cancer that is substantially refractory to treatment.

[0231] In some embodiments, the cancer is a solid tumor.

[0232] In some embodiments, the cancer comprises CD93+ tumor endothelial cells. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the endothelial cells in the tumor are CD93-positive. In some embodiments, the cancer comprises at least 20%, 40%, 60%, 80%, or 100% more CD93+ endothelial cells than CD93+ endothelial cells in normal tissues of the subject. In some embodiments, the cancer comprises at least 20%, 40%, 60%, 80%, or 100% more CD93+ endothelial cells than CD93+ endothelial cells in the corresponding organ of a subject or group of subjects without cancer.

[0233] In some embodiments, the cancer comprises IGFBP7+ blood vessels. In some embodiments, the cancer comprises at least 20%, 40%, 60%, 80%, or 100% more IGFBP7+ blood vessels than blood vessels in normal tissues of the subject. In some embodiments, the cancer comprises at least 20%, 40%, 60%, 80%, or 100% more IGFBP7+ blood vessels than blood vessels in a corresponding organ in a subject or group of subjects without cancer.

[0234] In some embodiments, the cancer (e.g., a solid tumor) is characterized by tumor hypoxia. Tumor hypoxia can be assessed, for example, as described in the Examples (e.g., FIG. 6A). In some embodiments, the cancer is characterized by a pimonidazole-positive percentage (i.e., pimonidazole-positive area divided by total tumor area) of at least about 1%, 2%, 3%, 4%, or 5%.

[0235] Examples of cancers that may be treated by the methods of the present application include anal cancer, astrocytoma (e.g., cerebellum and brain), basal cell carcinoma, bladder cancer, bone cancer (e.g., osteosarcoma and malignant fibrous histiocytoma), brain tumors (e.g., glioma, brainstem glioma, cerebellar or cerebral astrocytoma (e.g., astrocytoma, malignant glioma, medulloblastoma, and glioblastoma)), breast cancer (e.g., TNBC), cervical cancer, colon cancer, colorectal cancer, endometrial cancer (e.g., uterine cancer), esophageal cancer, eye cancer (e.g., intraocular melanoma and retinoblastoma), stomach cancer, gastrointestinal stromal tumor (GIST), head and neck cancer, hepatocellular carcinoma (HCC), and hepatocellular carcinoma (HCC). These include, but are not limited to, alveolar (liver) cancer (e.g., hepatocarcinoma and hepatoma), liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), medulloblastoma, melanoma, mesothelioma, myelodysplastic syndrome, nasopharyngeal carcinoma, neuroblastoma, ovarian cancer, pancreatic cancer, parathyroid cancer, peritoneal cancer, pituitary tumor, rectal cancer, kidney cancer, renal pelvis and ureter cancer (transitional cell carcinoma), rhabdomyosarcoma, skin cancer (e.g., non-melanoma (e.g., squamous cell carcinoma), melanoma, and Merkel cell carcinoma), small intestine cancer, squamous cell carcinoma, testicular cancer, thyroid cancer, and tuberous sclerosis.Further examples of cancers can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, § on Hematology and Oncology, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); The Merck Manual of Diagnosis and Therapy, 20th Edition, § on Hematology and Oncology, published by Merck Sharp & Dohme Corp., 2018 (ISBN 978-0-911-91042-1) (2018 digital online edition at the internet website of Merck Manuals); and the SEER Program Coding and Staging Manual 2016, each of which is incorporated by reference in its entirety for all purposes.

[0236] In some embodiments, the cancer is triple-negative breast cancer (TNBC, e.g., TNBC with high IGFBP or CD93 expression). In some embodiments, the cancer is melanoma. In some embodiments, the patient is refractory to previous treatments including administration of an immune checkpoint inhibitor, e.g., an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, or a combination thereof.

[0237] subject In some embodiments, the subject is a mammal (such as a human).

[0238] In some embodiments, the subject has tissue containing abnormal blood vessels that contain CD93+ endothelial cells. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the endothelial cells in the tissue with abnormal blood vessels are CD93-positive. In some embodiments, the tissue with abnormal blood vessels contains at least 20%, 40%, 60%, 80%, or 100% more CD93+ endothelial cells than CD93+ endothelial cells in normal tissue of the subject. In some embodiments, the tissue with abnormal blood vessels contains at least 20%, 40%, 60%, 80%, or 100% more CD93+ endothelial cells than CD93+ endothelial cells in the corresponding organ in a subject or group of subjects without abnormal blood vessels.

[0239] In some embodiments, the subject has tissue containing abnormal blood vessels, including IGFBP7+ blood vessels. In some embodiments, the tissue contains at least 20%, 40%, 60%, 80%, or 100% more IGFBP7+ blood vessels than normal tissue in the subject. In some embodiments, the tissue contains at least 20%, 40%, 60%, 80%, or 100% more IGFBP7+ blood vessels than blood vessels in a corresponding organ in a subject or group of subjects without abnormal blood vessels.

[0240] In some embodiments, subjects are selected for treatment based on abnormal vasculature. In some embodiments, the abnormal vasculature is characterized by CD93+ endothelial cells (e.g., by measuring CD93+CD31+ cells). In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of endothelial cells in tissue with abnormal vasculature are CD93-positive. In some embodiments, tissue with abnormal vasculature contains at least 20%, 40%, 60%, 80%, or 100% more CD93+ endothelial cells than CD93+ endothelial cells in normal tissue of the subject. In some embodiments, tissue with abnormal vasculature contains at least 20%, 40%, 60%, 80%, or 100% more CD93+ endothelial cells than CD93+ endothelial cells in the corresponding organ in a subject or group of subjects without abnormal vasculature.

[0241] In some embodiments, the abnormal vasculature is characterized by an abnormal level of IGFBP7+ blood vessels. In some embodiments, the tissue contains at least 20%, 40%, 60%, 80%, or 100% more IGFBP7+ blood vessels than normal tissue in the subject. In some embodiments, the tissue contains at least 20%, 40%, 60%, 80%, or 100% more IGFBP7+ blood vessels than blood vessels in a corresponding organ in a subject or group of subjects without abnormal blood vessels.

[0242] In some embodiments, the subject has had at least one prior therapy. In some embodiments, the prior therapy includes radiation therapy, chemotherapy, and / or immunotherapy. In some embodiments, the subject is resistant, refractory, or relapsed to the prior therapy. In some embodiments, the prior therapy includes administration of an immune checkpoint inhibitor, such as an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, or a combination thereof.

[0243] Combination therapy The present application also provides methods of administering an agent that inhibits the IGFBP7 / CD93 signaling pathway (an "IGFBP7 / CD93 blocker") described herein to a subject to treat a disease or condition (such as cancer), the method further comprising administering a second agent or therapy. In some embodiments, the second agent or therapy is a standard or commonly used agent or therapy for treating the disease or condition. In some embodiments, the second agent or therapy comprises a chemotherapeutic agent. In some embodiments, the second agent or therapy comprises surgery. In some embodiments, the second agent or therapy comprises radiation therapy. In some embodiments, the second agent or therapy comprises immunotherapy. In some embodiments, the second agent or therapy comprises cell therapy (such as cell therapy comprising immune cells (e.g., CART cells)). In some embodiments, the second agent or therapy comprises an angiogenesis inhibitor.

[0244] In some embodiments, the second agent is a chemotherapeutic agent. In some embodiments, the second agent is an antimetabolite. In some embodiments, the antimetabolite is 5-FU.

[0245] In some embodiments, the second agent is an immune checkpoint modulator. In some embodiments, the immune checkpoint modulator is an inhibitor of an immune checkpoint protein selected from the group consisting of PD-L1, PD-L2, CTLA4, PD-L2, PD-1, CD47, TIGIT, GITR, TIM3, LAG3, CD27, 4-1BB, and B7H4. In some embodiments, the immune checkpoint protein is PD-1. In some embodiments, the second agent is an anti-PD-1 antibody or fragment thereof. In some embodiments, the second agent is an anti-CTLA4 antibody or fragment thereof. In some embodiments, the second agent is a combination of an anti-PD1 antibody or fragment thereof and an anti-CTLA4 antibody or fragment thereof.

[0246] In some embodiments, the IGFBP7 / CD93 blocking agent is administered simultaneously with a second agent or therapy. In some embodiments, the IGFBP7 / CD93 blocking agent that inhibits the IGFBP7 / CD93 signaling pathway is administered concurrently with the second agent or therapy. In some embodiments, the IGFBP7 / CD93 blocking agent is administered sequentially with the second agent or therapy. In some embodiments, the IGFBP7 / CD93 blocking agent is administered in the same unit dosage form as the second agent or therapy. In some embodiments, the IGFBP7 / CD93 blocking agent is administered in a different unit dosage form from the second agent or therapy.

[0247] Dosage regimen and route of administration The dose of an IGFBP7 / CD93 blocking agent, and in some embodiments, a second agent described herein, administered to a subject (such as a human) can vary depending on the particular composition, method of administration, and the particular type and stage of the disease or condition (such as cancer) being treated. The amount should be sufficient to produce a desired response, such as a therapeutic response to the disease or condition (such as cancer). In some embodiments, the amount of the IGFBP7 / CD93 blocking agent and / or second agent is a therapeutically effective amount.

[0248] In some embodiments, the amount of IGFBP7 / CD93 blocking agent is sufficient to promote vascular normalization (e.g., increased vascular length, increased number of round blood vessels, maintained vascular density, and / or increased pericytes and / or smooth muscle cells), increased perfusion of tissue (such as tumor tissue), reduced hypoxia, increased amount of drug delivered to tissue, increased immune cell infiltration in tissue, and / or inhibited tumor cell proliferation.

[0249] In some embodiments, the amount of the IGFBP7 / CD93 blocking agent is sufficient to increase the length of blood vessels in the tissue (e.g., total length per field of view) by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% after administration of the IGFBP7 / CD93 blocking agent. In some embodiments, the amount of the IGFBP7 / CD93 blocking agent is sufficient to increase the percentage of round blood vessels in the tissue (% of round blood vessels / total blood vessels) by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% after administration of the IGFBP7 / CD93 blocking agent. In some embodiments, the amount of the IGFBP7 / CD93 blocking agent is sufficient to maintain the density of blood vessels in the tissue after administration of the IGFBP7 / CD93 blocking agent.

[0250] In some embodiments, the amount of IGFBP7 / CD93 blocking agent is sufficient to increase pericytes in the tissue (e.g., NG2-positive expression on blood vessels) by at least about 25%, 50%, 75%, 100%, 125%, 150%, 175%, or 200% after administration of the IGFBP7 / CD93 blocking agent. In some embodiments, the amount of IGFBP7 / CD93 blocking agent is sufficient to increase smooth muscle cells in the tissue (e.g., α-SMA+ expression on blood vessels) by at least about 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, or 250% after administration of the IGFBP7 / CD93 blocking agent. In some embodiments, the amount of IGFBP7 / CD93 blocking agent is sufficient to increase ICAM+ expression by at least about 10%, 20%, 30%, 40%, 50%, 60%, or 70% after administration of the IGFBP7 / CD93 blocking agent, hi some embodiments, the amount of IGFBP7 / CD93 blocking agent is sufficient to decrease expression of activated integrins by at least about 10%, 20%, 30%, 40%, or 50% after administration of the IGFBP7 / CD93 blocking agent.

[0251] In some embodiments, the amount of the IGFBP7 / CD93 blocking agent is sufficient to increase vascular permeability or perfusion in a tissue by at least about 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% following administration of the IGFBP7 / CD93 blocking agent.

[0252] In some embodiments, the amount of the IGFBP7 / CD93 blocking agent is sufficient to reduce hypoxia in the tissue by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% following administration of the IGFBP7 / CD93 blocking agent.

[0253] In some embodiments, the amount of IGFBP7 / CD93 blocking agent is sufficient to increase the presence / distribution of a drug (such as a chemotherapeutic drug) in a tissue after delivery by at least about 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% following administration of the IGFBP7 / CD93 blocking agent.

[0254] In some embodiments, the amount of IGFBP7 / CD93 blocking agent is sufficient to increase immune cell infiltration in a tissue (e.g., the percentage of immune cells in a tissue) by at least about 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% after administration of the IGFBP7 / CD93 blocking agent. In some embodiments, the amount of IGFBP7 / CD93 blocking agent is sufficient to decrease the proportion of suppressor immune cells among infiltrating immune cells in a tissue by at least about 10%, 20%, 30%, 40%, or 50% after administration of the IGFBP7 / CD93 blocking agent.

[0255] In some embodiments, the amount of the IGFBP7 / CD93 blocking agent is sufficient to reduce proliferation of cells (e.g., tumor cells) in the tissue by at least about 10%, 20%, 30%, 40%, 50%, or 60% after administration of the IGFBP7 / CD93 blocking agent. In some embodiments, the amount of the IGFBP7 / CD93 blocking agent is sufficient to increase apoptosis of cells (e.g., tumor cells) in the tissue by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% after administration of the IGFBP7 / CD93 blocking agent.

[0256] In some embodiments, the amount of IGFBP7 / CD93 blocking agent is sufficient to result in at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% reduction in tumor size, number of cancer cells, or rate of tumor growth compared to the corresponding tumor size, number of cancer cells, or rate of tumor growth in the same subject before treatment, or compared to the corresponding activity in another subject not receiving treatment.

[0257] In some embodiments, the IGFBP7 / CD93 blocking agent comprises an anti-CD93 antibody. In some aspects, the subject is a human, and the amount of anti-CD93 antibody for each administration is equivalent to a dose of about 300 μg for a mouse. In some embodiments, the subject is a human, and the amount of anti-CD93 antibody for each administration is about 2 g or less (e.g., about 50-75 mg). In some embodiments, the subject is a human, and the amount of anti-CD93 antibody for each administration is about 30 mg / kg or less (e.g., about 0.8 mg / kg to about 1.2 mg / kg, etc.). In some embodiments, the subject is a human, and the amount of anti-CD93 antibody for each administration is 30-45 mg / m 2 In some embodiments, the subject is a human and the amount of anti-CD93 antibody for each administration is about 75 mg (or about 1.25 mg / kg, or about 45 mg / m 2 ) is as follows.

[0258] In some embodiments, the IGFBP7 / CD93 blocker comprises an anti-IGFBP7 antibody. In some embodiments, the subject is a human, and the amount of anti-IGFBP7 antibody for each administration is equivalent to a dose of about 300 μg for a mouse. In some embodiments, the subject is a human, and the amount of anti-IGFBP7 antibody for each administration is about 2 g or less (e.g., about 50-75 mg). In some embodiments, the subject is a human, and the amount of anti-IGFBP7 antibody for each administration is about 30 mg / kg or less (e.g., about 0.8 mg / kg to about 1.2 mg / kg, etc.). In some embodiments, the subject is a human, and the amount of anti-IGFBP7 antibody for each administration is 30-45 mg / m2 In some embodiments, the subject is a human and the amount of anti-IGFBP7 antibody for each administration is about 75 mg (or about 1.25 mg / kg, or about 45 mg / m 2 ) is as follows.

[0259] In some embodiments, the anti-IGFBP7 antibody or anti-CD93 antibody is administered for at least about 1, 3, 7, 10, 12, or 14 days. In some embodiments, the anti-IGFBP7 antibody or anti-CD93 antibody is administered at a frequency of at least about twice per week.

[0260] In some embodiments, the method includes administering a second agent, wherein the second agent is 5-FU. In some embodiments, the subject is a human and the amount of 5-FU antibody for each administration corresponds to a dose of about 3 mg to about 4 mg for a mouse.

[0261] In some embodiments of any of the methods described herein, the IGFBP7 / CD93 blocking agent and / or second agent composition is administered intravenously, intraarterially, intraperitoneally, intravesically, subcutaneously, intrathecally, intrapulmonary, intramuscularly, intratracheally, intraocularly, transdermally, orally, or by inhalation. In some embodiments, the IGFBP7 / CD93 blocking agent and / or second agent is administered intravenously.

[0262] III. Diagnostic and Prognostic Methods Also provided herein are methods for diagnosing or prognosing a subject, comprising determining the subject's suitability for a treatment such as described in Section II or a different therapy, determining the subject's likelihood of responsiveness to a method such as described in Section II or a different therapy, and determining the maturation state of blood vessels in a tissue of the subject.

[0263] In some embodiments, methods are provided for determining a subject's suitability for a treatment, comprising measuring the level of CD93 expression in the subject's tissue. In some embodiments, methods are provided for determining a subject's suitability for a treatment, comprising measuring the level of IGFBP7 expression in the subject's tissue. In some embodiments, the subject has cancer and the tissue is tumor tissue. In some embodiments, the treatment comprises a CD93 / IGFBP7 blocking agent. In some embodiments, the treatment comprises a cancer therapy (e.g., a cell therapy such as a chemotherapeutic agent). In some embodiments, a higher CD93 or IGFBP7 expression level compared to a reference level indicates a lower suitability for the treatment.

[0264] In some embodiments, methods of prognosis in a subject with cancer (such as a solid tumor) are provided, comprising measuring the level of CD93 expression in a tumor sample in vitro or in vivo, wherein a higher CD93 expression level compared to a reference level indicates a high likelihood of non-response or poor response to treatment. In some embodiments, the reference level is the level of CD93 expression (such as the mean CD93 expression) in a non-tumor sample from the subject or in a corresponding tissue from a different subject (or group of subjects) who does not have cancer.

[0265] In some embodiments, provided is a method for prognosis in subjects with cancer (for example, solid tumor), comprising measuring the level of IGFBP7 expression in tumor samples in vitro or in vivo, and comparing with reference level, a higher IGFBP7 expression level indicates that the subject is likely to be unresponsive or poorly responsive to treatment.In some embodiments, the reference level is the level of IGFBP7 expression (such as the average IGFBP7 expression) in the non-tumor sample of the subject or in the corresponding tissue of different subjects (or a group of subjects) that do not have cancer.

[0266] In some embodiments, the therapy comprises a cell therapy. In some embodiments, the therapy comprises an agent selected from a chemotherapeutic agent (such as an antimetabolite, such as an immune checkpoint modulator), a radiation agent, or an immunotherapeutic agent. In some embodiments, the agent has a size of 1 μm, 0.5 μm, 0.2 μm, or 0.1 μm or less.

[0267] In some embodiments, a method for determining the maturation state of blood vessels in a subject's tissue (such as a cancer tissue) is provided, comprising administering an imaging agent comprising an anti-CD93 antibody labeled with an imaging molecule. In some embodiments, the imaging molecule is a radionuclide.

[0268] In some embodiments, a method for determining the maturity state of blood vessels in a target tissue (such as cancer tissue) is provided, comprising administering an imaging agent comprising an anti-IGFBP7 antibody labeled with an imaging molecule.In some embodiments, the imaging molecule is a radionuclide.

[0269] IV. Methods for Identifying Agents that Disrupt the Interaction Between CD93 and IGFBP7 The agent described herein can be identified by evaluating the ability of the agent to disrupt the interaction between CD93 and IGFBP7.Provided herein is a method for identifying an agent (such as antibody, peptide, polypeptide, peptide analog, fusion peptide, aptamer, avimer, anticalin, spiegelmer, and small molecule compound) that is useful for treating cancer or one or more aspects of cancer treatment, including but not limited to blocking abnormal tumor angiogenesis, normalizing immature and leaky tumor blood vessels, promoting functional vascular network in tumor, promoting vascular maturation, promoting favorable tumor microenvironment, increasing immune cell infiltration in tumor, increasing tumor perfusion, reducing hyperplasia in tumor, enhancing tumor sensitivity to second therapy, and promoting the delivery of second drug. The methods generally involve determining whether a candidate agent specifically disrupts the CD93 / IGFBP7 interaction, and if the candidate agent is shown to specifically disrupt the CD93 / IGFBP7 interaction, it is useful for treating cancer and in aspects of cancer therapy.

[0270] The agent can be an antibody, antibody-like scaffold, small molecule, fusion protein, peptide, mimetic, or inhibitory nucleotide (e.g., RNAi) directed against (i) CD93; (ii) IGFBP7; (iii) a novel site (e.g., a newly created epitope determinant) created by the CD93 / IGFBP7 interaction, or (iv) a protein complex containing any of these.

[0271] Thus, for example, in some embodiments, a method for determining whether a candidate agent is useful for treating cancer is provided, the method comprising determining whether the candidate agent specifically disrupts the CD93 / IGFBP7 interaction, and wherein if the candidate agent is shown to specifically disrupt the CD93 / IGFBP interaction, the candidate agent is useful for treating cancer. In some embodiments, the method further comprises determining whether the candidate agent specifically disrupts the CD93 / MMRN2 interaction. In some embodiments, the method further comprises determining whether the candidate agent preferentially disrupts the binding of CD93 / IGFBP7 over CD93 / MMRN2. In some embodiments, the method further comprises determining whether the candidate agent specifically disrupts the binding of the interaction between IGFBP7 and IGF-1, IGF-2, and / or IGF1R. In some embodiments, the method further comprises determining whether the candidate agent preferentially disrupts binding of CD93 / IGFBP7 over IGFBP7 / IGF-1, IGFBP-7 / IGF-2, and / or IGFBP-7 / IGF1R.

[0272] In some embodiments, methods are provided for screening for agents useful for treating cancer, comprising: a) providing a plurality of candidate agents; and b) identifying candidate agents that specifically disrupt the CD93 / IGFBP7 interaction, thereby obtaining agents useful for treating cancer.

[0273] In some embodiments, methods are provided for identifying an agent that specifically disrupts the CD93 / IGFBP7 interaction, comprising: a) contacting a candidate agent with a CD93 / IGFBP7 complex; and b) evaluating the effect of the candidate agent on the CD93 / IGFBP7 complex, thereby identifying an agent that specifically disrupts the CD93 / IGFBP7 interaction. In some embodiments, the method further comprises providing a CD93 / IGFBP7 complex. In some embodiments, the method further comprises forming a CD93 / IGFBP7 complex. In some embodiments, the CD93 / IGFBP7 complex is present on the cell surface. In some embodiments, the CD93 / IGFBP7 complex is present in an in vitro system.

[0274] In some embodiments, the CD93 / IGFBP7 complex does not occur in nature. For example, the complex may include a CD93 variant and / or an IGFBP7 variant. In some embodiments, the variant CD93 has a higher binding affinity for IGFBP7 than wild-type CD93. In some embodiments, the variant IGFBP7 has a higher binding affinity for CD93 than wild-type IGFBP7. Suitable CD93 variants and IGFBP7 variants include those described in the sections above. In some embodiments, the present application also provides a non-naturally occurring CD93 / IGFBP7 complex comprising any of the CD93 and / or IGFBP7 variants described herein. Such a complex is useful for identifying candidate agents that disrupt the interaction between CD93 and IGFBP7.

[0275] In some embodiments, a method for identifying an agent that specifically disrupts the CD93 / IGFBP7 interaction is provided, comprising: a) contacting a candidate agent with CD93; and b) evaluating the interaction between IGFBP7 and CD93; wherein reduced interaction compared to when CD93 is not contacted with the candidate agent indicates that the agent specifically disrupts the CD93 / IGFBP7 interaction. In some embodiments, the method further comprises providing CD93. In some embodiments, the method further comprises providing IGFBP7. Suitable CD93s include wild-type CD93 and variants thereof. Suitable IGFBP7s include wild-type IGFBP93 and variants thereof. Any of the CD93 variants and / or IGFBP7 variants described herein can be used in the identification method.

[0276] In some embodiments, a method is provided for identifying an agent that specifically disrupts CD93 / IGFBP7 interaction, comprising: a) contacting a candidate agent with IGFBP7; and b) assessing the interaction between IGFBP7 and CD93; wherein reduced interaction compared to when IGFBP7 is not contacted with the candidate agent indicates that the agent specifically disrupts CD93 / IGFBP7 interaction. In some embodiments, the method further comprises providing IGFBP7. In some embodiments, the method further comprises providing CD93. In some embodiments, the method further comprises providing IGFBP7. Suitable CD93s include wild-type CD93 and variants thereof. Suitable IGFBP7s include wild-type IGFBP93 and variants thereof. Any of the CD93 variants and / or IGFBP7 variants described herein can be used in the identification method.

[0277] Disruption of CD93 / IGFBP7 binding activity and / or CD93 / IGFBP7 pathway activity can be measured by PCR, TaqMan PCR, phage display systems, gel electrophoresis, reporter gene assays, yeast two-hybrid assays, Northern or Western analysis, immunohistochemistry, conventional scintillation cameras, gamma cameras, linear scanners, PET scanners, SPECT scanners, MRI scanners, NMR scanners, or X-ray instruments. Disruption can also be measured using a method selected from label displacement, surface plasmon resonance, fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET), fluorescence quenching, and fluorescence polarization.

[0278] Changes in CD93 / IGFBP7 binding activity and / or CD93 / IGFBP7 pathway activity can be detected by detecting changes in the interaction between CD93 and IGFBP7, by detecting changes in the levels of CD93 and / or IGFBP7, or by detecting changes in the levels of one or more proteins in the CD93 / IGFBP7 pathway. The cells in which the above can be detected can be of tumor origin, cultured cells, or obtained from or within transgenic organisms. Such transgenic organisms include, but are not limited to, mice, rats, rabbits, sheep, cattle, or primates.

[0279] The screening assays of the present application can include methods suitable for high-throughput screening of chemical libraries, making the assays particularly suitable for identifying small molecule drug candidates. The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays, and cell-based assays, which are well characterized in the art. For in vitro screening, agents can be identified, for example, by phage display, GST pull-down, FRET (fluorescence resonance energy transfer), or BIAcore (surface plasmon resonance; BIAcore AB, Uppsala, Sweden) analysis. For in vivo screening, agents can be identified, for example, by yeast two-hybrid analysis, co-immunoprecipitation, co-localization by immunofluorescence, or FRET.

[0280] In screening experiments involving disruption of the CD93 / IGFBP7 interaction, cells expressing CD93 or IGFBP7 can be incubated in a binding buffer containing labeled IGFBP7 or CD93, respectively, in the presence or absence of increasing concentrations of a candidate drug. To validate and calibrate the assay, control competition reactions using increasing concentrations of unlabeled IGFBP7 or CD93, respectively, can be performed. After incubation, a washing step is performed to remove unbound IGFBP7 or CD93. Bound labeled CD93 or IGFBP7 is measured appropriately for a given label (e.g., by scintillation counting, fluorescence, antibody dye, etc.). A decrease of at least 10% (e.g., at least 20%, 30%, 40%, 50%, or 60%) of the amount of bound labeled CD93 or IGFBP7 in the presence of the candidate drug indicates displacement of binding by the candidate drug.

[0281] In some embodiments, a candidate agent is considered to specifically bind in this or other assays described herein if it displaces at least 10%, 20%, 30%, 40%, 50%, or preferably 60%, 70%, 80%, 90% or more of labeled CD93 or IGFBP7 at a concentration of 1 mM or less. Of course, the roles of CD93 and IGFBP7 can be swapped; one skilled in the art can adapt the method to add CD93 to IGFBP7 in the presence of various concentrations of the candidate agent to determine disruption of the CD93 / IGFBP7 interaction.

[0282] Disruption of the CD93 / IGFBP7 interaction can be monitored by surface plasmon resonance (SPR). Surface plasmon resonance assays can be used as a quantitative method to measure binding between two molecules by measuring the change in mass near the immobilized sensor caused by the binding or loss of IGFBP7 from the aqueous phase to CD93 immobilized on the sensor (or vice versa). This change in mass is measured as resonance units versus time after injection or removal of IGFBP7 or the candidate drug, and is measured using a Biacore Biosensor (Biacore AB). CD93 can be immobilized on a sensor chip (e.g., a research-grade CM5 chip; Biacore AB) according to the method described by Salamon et al. ((Salamon et al., 1996, Biophys J. 71: 283-294; Salamon et al., 2001, Biophys. J. 80: 1557-1567; Salamon et al., 1999, Trends Biochem. Sci. 24: 213-219; each incorporated herein by reference for all purposes). Sarrio et al. demonstrated that SPR can be used to detect ligand binding to the GPCR A(1) adenosine receptor immobilized on a lipid layer on the chip (Sarrio et al., 2000, Mol. Cell. Biol. 20: 5164-5174; incorporated herein by reference for all purposes). Conditions for IGFBP7 binding to CD93 in an SPR assay can be fine-tuned by one of skill in the art using the conditions reported by Sarrio et al. as a starting point.

[0283] SPR can be used to assay for inhibitors of binding in at least two ways. First, IGFBP7 can be pre-bound to immobilized CD93, followed by injection of a candidate drug at concentrations ranging from 0.1 nM to 1 pM. Displacement of bound IGFBP7 can be quantified, allowing for detection of inhibitor binding. Alternatively, chip-bound CD93 can be pre-incubated with a candidate drug and then challenged with IGFBP7. The difference in IGFBP7 binding to inhibitor-exposed CD93 compared to binding on chips not pre-exposed to the inhibitor indicates binding or displacement of IGFBP7 in the presence of CD93. In either assay, a 10% (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%) or greater decrease in the amount of IGFBP7 bound in the presence of the candidate drug compared to the amount of IGFBP7 bound in the absence of the candidate drug indicates that the candidate drug inhibits the interaction between CD93 and IGFBP7. Although CD93 is immobilized above, one skilled in the art can easily adapt the method so that IGFBP7 is the immobilized component.

[0284] Another method for detecting agents that inhibit the CD93 / IGFBP7 interaction uses fluorescence resonance energy transfer (FRET). FRET is a quantum mechanical phenomenon that occurs between a fluorescent donor (D) and a fluorescent acceptor (A) that are in close proximity (usually separated by <100 angstroms) when the emission spectrum of the fluorescent donor (D) overlaps with the excitation spectrum of the fluorescent acceptor (A). The molecules to be tested, such as CD93 and IGFBP7, are labeled with a complementary pair of donor and acceptor fluorophores. When tightly bound by the CD93 / IGFBP7 interaction, the fluorescence emitted upon excitation of the donor fluorophore has a wavelength that differs from that emitted in response to that excitation wavelength when CD93 and IGFBP7 are unbound, and measuring the emission intensity at each wavelength provides quantification of bound versus unbound molecules. Donor fluorophores for labeling CD93 or IGFBP7 are well known in the art. Examples include variants of Aequorea victoria GFP known as Cyan FP (CFP, donor (D)) and Yellow FP (YFP, acceptor (A)).

[0285] In some embodiments, the addition of a candidate agent to a mixture of labeled IGFBP7 and YFP-CD93 results in inhibition of energy transfer, as evidenced, for example, by a decrease in YFP fluorescence compared to a sample without the candidate agent. In assays using FRET to detect CD93 / IGFBP7 interaction, a decrease of 10% or more (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more) in the intensity of fluorescence emission at the acceptor wavelength in a sample containing the candidate agent compared to a sample without the candidate agent indicates that the candidate agent inhibits CD93 / IGFBP7 interaction. Conversely, an increase of 10% or more (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more) in the intensity of fluorescence emission at the acceptor wavelength in a sample containing the candidate agent compared to a sample without the candidate agent indicates that the candidate agent induces a conformational change and enhances CD93 / IGFBP7 interaction.

[0286] A variation of FRET uses fluorescence quenching to monitor molecular interactions. One molecule in the interaction pair can be labeled with a fluorophore, and the other molecule can be labeled with a molecule that quenches the fluorophore's fluorescence when placed in close juxtaposition. A change in fluorescence upon excitation indicates a change in the association of the molecules tagged with the fluorophore:quencher pair. Generally, an increase in fluorescence of labeled CD93 indicates the displacement of the quencher-bearing IGFBP7 molecule. Of course, a similar effect occurs when IGFBP7 is fluorescently labeled and CD93 bears a quencher. For quenching assays, a 10% or greater (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or greater) increase in the intensity of fluorescence emission in samples containing the candidate agent compared to samples without the agent indicates that the candidate agent inhibits the CD93 / IGFBP7 interaction. Conversely, a decrease in the intensity of fluorescence emission of 10% or more (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more) in a sample containing the candidate agent compared to a sample without the candidate agent indicates that the candidate agent induces a conformational change and enhances CD93 / IGFBP7 interaction.

[0287] In addition to surface plasmon resonance and FRET techniques, fluorescence polarization measurements are useful for quantifying binding. The fluorescence polarization value of a fluorescently labeled molecule depends on its rotational correlation time, or tumbling rate. Complexes such as those formed by fluorescently labeled IGFBP7 or CD93 associated with CD93 or IGFBP7, respectively, have higher polarization values ​​than uncomplexed labeled IGFBP7 or CD93. If a candidate drug interferes with or inhibits the CD93 / IGFBP7 interaction, inclusion of the candidate drug in the CD93 / IGFBP7 interaction results in a decrease in fluorescence polarization compared to a mixture without the candidate drug. Fluorescence polarization is well suited to identifying small molecules that disrupt complex formation. A decrease of 10% or more (e.g., 20%, 30%, 40%, 50%, 60% or more) in fluorescence polarization in samples containing the candidate drug compared to fluorescence polarization in samples without the candidate drug indicates that the candidate drug inhibits the CD93 / IGFBP7 interaction.

[0288] Another detection system is bioluminescence resonance energy transfer (BRET), which uses light transfer between fusion proteins containing a bioluminescent luciferase and a fluorescent acceptor. Generally, one molecule of the CD93 / IGFBP7 interaction pair is fused to a luciferase (e.g., Renilla luciferase (Rluc))—a donor that emits light at a wavelength of approximately 395 nm in the presence of a luciferase substrate (e.g., DeepBlueC). The other molecule of the pair is fused to an acceptor fluorescent protein that can absorb light from the donor and emit light at a different wavelength. An example of a fluorescent protein is GFP (green fluorescent protein), which emits light at approximately 510 nm. Addition of a candidate drug to a mixture of donor-fused IGFBP7 and acceptor-fused CD93 (or vice versa) results in inhibition of energy transfer, as evidenced by a decrease in acceptor fluorescence, for example, compared to a sample without the candidate drug. In assays using BRET to detect CD93 / IGFBP7 interaction, a 10% or greater (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or greater) decrease in the intensity of fluorescence emission at the acceptor wavelength in samples containing the candidate agent compared to samples without the candidate agent indicates that the candidate agent inhibits CD93 / IGFBP7 interaction. Conversely, a 10% or greater (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or greater) increase in the intensity of fluorescence emission at the acceptor wavelength in samples containing the candidate agent compared to samples without the candidate agent indicates that the candidate agent induces a conformational change and enhances CD93 / IGFBP7 interaction.

[0289] It is understood that any of the binding assays described herein can be performed with any ligand other than CD93 and IGFBP7 that binds to CD93 or IGFBP7 (e.g., agonist, antagonist, etc.), such as a small molecule identified as described herein, or a CD93 or IGFBP7 mimetic, including, but not limited to, any natural or synthetic peptide, polypeptide, antibody or antigen-binding fragment thereof, lipid, carbohydrate, and small organic molecule.

[0290] Any of the binding assays described can be used to determine the presence of an inhibitor in a sample (e.g., a tissue sample) that binds to CD93 or IGFBP7 or affects the binding of CD93 to IGFBP7. To do so, CD93 is reacted with IGFBP7 in the presence or absence of the sample, and binding is measured as appropriate for the binding assay being used. A decrease of 10% or more (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more) in CD93 / IGFBP7 binding indicates that the sample contains an inhibitor that blocks the CD93 / IGFBP7 interaction.

[0291] Any of the binding assays described can also be used to determine the presence of inhibitors in a library of compounds. Such screening techniques, using, for example, high-throughput screening, are well known in the art.

[0292] The present application also provides methods for identifying an agent capable of inhibiting the CD93 / IGFBP7 signaling pathway, comprising measuring a signaling response induced by CD93 / IGFBP7 interaction in the presence of the agent and comparing it to a signaling response induced by CD93 / IGFBP7 interaction in the absence of the agent. In some embodiments, the method comprises: a) contacting CD93 with IGFBP7 in the presence and absence of a test agent under conditions that allow interaction between CD93 and IGFBP7; and b) measuring the signaling response induced by CD93 / IGFBP7 interaction, wherein a change in the response in the presence of the test agent of at least about 10% (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) compared to the response in the absence of the test agent indicates that the test agent is identified as capable of inhibiting CD93 / IGFBP7 interaction.

[0293] The present application provides methods for identifying CD93 or IGFBP7 mimetics that have the same, similar, or improved functional effect as CD93 or IGFBP7 in interacting with IGFBP7 or CD93, the method comprising measuring the interaction of the candidate mimetic with IGFBP7 or CD93. In some embodiments, the method comprises: a) contacting CD93 or IGFBP7 with the candidate mimetic under conditions that allow the candidate mimetic to interact with CD93 or IGFBP7; and b) measuring the interaction of the mimetic with CD93 or IGFBP7; wherein the candidate mimetic is distinguished as a CD93 or IGFBP7 mimetic of the present application if the interaction is at least about 10% (e.g., about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) of the interaction observed for CD93 / IGFBP7 interaction.

[0294] Furthermore, the present application also provides a method for identifying a CD93 or IGFBP7 mimetic that has the same, similar, or improved functional effect as CD93 or IGFBP7 upon interaction with IGFBP7 or CD93, respectively, comprising measuring the signaling response induced by the interaction of CD93 or IGFBP7 with the mimetic and comparing it to the signaling response induced by CD93 / IGFBP7 interaction. In some embodiments, the method comprises: a) contacting CD93 or IGFBP7 with a candidate mimetic under conditions that allow the candidate mimetic to interact with CD93 or IGFBP7; and b) measuring the signaling response induced by the interaction of CD93 or IGFBP7 with the mimetic; wherein the candidate mimetic is distinguished as a CD93 or IGFBP7 mimetic of the present application if the signaling response is at least about 10% (e.g., about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) of the signaling response observed for CD93 / IGFBP7 interaction.

[0295] The measurement of the mimic signaling activity of interaction with CD93 or IGFBP7 can be carried out by the methods described herein for other assays, such as SPR and FRET. Any of the binding assays described can be used to determine the presence of a mimic in a sample (e.g., a tissue sample that binds to CD93 or IGFBP7). To do so, CD93 or IGFBP7 is reacted with or without the sample, and signaling is measured as appropriate for the assay being used. An increase of about 10% or more (e.g., about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more) in the signaling of CD93 or IGFBP7 indicates that the sample contains a mimic that binds to CD93 or IGFBP7.

[0296] Any of the signaling assays described can also be used to determine the presence of a mimetic in a library of compounds. Such screening techniques are well known in the art, for example, using high-throughput screening.

[0297] Candidate or test compounds or agents of or used by the present application can be obtained using any of a number of approaches to combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; "one-bead one-compound" library methods; and synthetic library methods using affinity chromatography selection. While the biological library approach is limited to peptide libraries, the other four approaches are applicable to peptide, non-peptide oligomer, or small molecule libraries of compounds (Lam et al. (1997) Anticancer Drug Des. 12: 145; incorporated by reference in its entirety for all purposes).

[0298] Examples of methods for the synthesis of molecular libraries are known in the art, for example, in DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91: 11422; Zuckermann et al. (1994) J. Med. Chem. 37: 2678; Cho et al. (1993) Science 261: 1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2061; and Gallop et al. (1994) J. Med. Chem. 37: 1233, each of which is incorporated by reference in its entirety for all purposes. Libraries of compounds can be displayed in solution (e.g., Houghten (1992) Biotechniques 13: 412), or on beads (Lam (1991) Nature 354: 82), on chips (Fodor (1993) Nature 364: 555), on bacteria (Ladner, U.S. Pat. No. 5,223,409), on spores (Ladner '409), on plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89: 1865), or on phage (Scott and Smith (1990) Science 249: 386); (Devlin (1990) Science 249: 404); (Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87: 6378); (Felici (1991) J. Mol. Biol. 222: 301); (Ladner, supra); each of which is incorporated by reference in its entirety for all purposes.

[0299] In some embodiments, a cell-based assay is provided, comprising contacting a cell expressing CD93 or IGFBP7 with a candidate or test compound or agent, and determining the ability of the test compound to inhibit the activity of said CD93 or IGFBP7. Determining the ability of the test compound to inhibit CD93 / IGFBP7 interaction can be achieved, for example, by determining the ability of the candidate or test compound or agent to inhibit CD93 / IGFBP7 interaction.

[0300] Determining the ability of a candidate or test compound or agent to inhibit the CD93 / IGFBP7 signaling pathway can be achieved by determining direct binding. These determinations can be achieved, for example, by coupling CD93 or IGFBP7 with a radioisotope or enzyme label, so that the binding of the protein to the candidate or test compound or agent can be determined by detecting the labeled protein in the complex. For example, a molecule, such as a protein, can be directly or indirectly bound to the CD93 or IGFBP7. 125 I, 35 S, 14 C, or 3 H, and radioactive isotopes can be detected by direct counting of radioemission or by scintillation counting. Alternatively, molecules can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label can be detected by determination of conversion of an appropriate substrate to product.

[0301] It is also within the scope of this application to determine the ability of candidate or test compound or drug to inhibit CD93 / IGFBP7 interaction without labeling any of the interactants.For example, a microphysiometer can be used to detect the interaction between a test compound and CD93 or IGFBP7 without labeling any of the interactants (McConnell et al. (1992) Science 257: 1906; the entirety of which is incorporated by reference for all purposes).As used herein, a "microphysiometer" (e.g., Cytosensor) is an analytical instrument that uses a light-addressable potentiometric sensor (LAPS) to measure the rate at which cells acidify their environment.The change in this acidification rate can be used as an indicator of the interaction between a compound and receptor.

[0302] In some embodiments, cell-free assays are provided in which a protein or biologically active portion thereof is contacted with a candidate or test compound or agent (e.g., or a compound being tested for its ability to inhibit CD93 / IGFBP7 interaction) and the ability of the test compound to bind to CD93 or IGFBP7, or a biologically active portion thereof, is determined. Binding of the test compound to CD93 or IGFBP7 can be determined directly or indirectly, as described above.

[0303] Such determination can be achieved using techniques such as real-time biomolecular interaction analysis (BIA). Sjolander et al., 1991 Anal. Chem. 63:2338-2345 and Szabo et al., 1995 Curr. Opin. Struct. Biol. 5:699-705 (each of which is incorporated by reference in its entirety for all purposes). As used herein, "BIA" refers to a technique for studying biospecific interactions in real time without labeling any of the interactants (e.g., BIAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indicator of real-time reactions between biomolecules.

[0304] In some embodiments of the above-described assay methods of the present application, it may be desirable to immobilize CD93 or IGFBP7 to facilitate separation of uncomplexed from complexed forms of the protein and to adapt the assay to automation. Binding of test compounds to CD93 or IGFBP7 can be achieved in any vessel suitable for containing reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes. In some embodiments, fusion proteins can be provided that add a domain that allows the protein to bind to a matrix. For example, glutathione-S-transferase / kinase fusion proteins or glutathione-S-transferase / target fusion proteins can be adsorbed onto glutathione Sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione-derivatized microtiter plates, which are then combined with the test compound, or the test compound and unadsorbed CD93 or IGFBP7, and the mixture is incubated under conditions that allow complex formation (e.g., physiological conditions of salt and pH). After incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix is ​​immobilized in the case of beads, and the complexes are measured, e.g., directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the matrix and the level of binding determined using standard techniques.

[0305] Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the present application. For example, CD93 or IGFBP7 can be immobilized using conjugation of biotin and streptavidin. Biotinylated CD93 or IGFBP7 or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Illinois) and immobilized in the wells of a streptavidin-coated 96-well plate (Pierce Chemical). Alternatively, antibodies reactive with CD93 or IGFBP7 or target molecules can be coated on the wells of the plate (derivatized), and unbound CD93 or IGFBP7 can be captured in the wells by antibody conjugation. Methods for detecting such complexes include immunodetection of the complex using antibodies reactive with CD93 or IGFBP7 or target molecules, in addition to those described above for GST-immobilized complexes.

[0306] In some embodiments, CD93 or IGFBP7 can be used as a "bait protein" in a two-hybrid or three-hybrid assay to identify other proteins that bind to CD93 or IGFBP7 (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al., 1993 Cell 72:223-232; Madura et al., 1993 J. Biol. Chem. 268:12046-12054; Bartel et al., 1993 Biotechniques 14:920-924; Iwabuchi et al., 1993 Oncogene 8:1693-1696; and Brent, WO 94 / 10300; each of which is incorporated by reference in its entirety for all purposes).

[0307] The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, a gene encoding CD93 or IGFBP7 is fused to a gene encoding the DNA-binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence encoding an unidentified protein (the "prey" or "sample") from a library of DNA sequences is fused to a gene encoding the activation domain of a known transcription factor. If the "bait" and "prey" proteins are able to interact in vivo to form a kinase-dependent complex, the DNA-binding and activation domains of the transcription factor will be brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected, and cell colonies containing functional transcription factors can be isolated and used to obtain cloned genes encoding proteins that interact with CD93 or IGFBP7.

[0308] It should be understood that the protein-protein interaction assays described herein may also be useful for determining whether an agent blocks interactions between CD93 or IGFBP7 and other binding partners (e.g., the interaction between CD93 and MMNR2 and the interaction between IGFBP7 and IGF-1, IGF-2, or IGF1R).

[0309] Also provided are agents identified by any of the methods described herein.Therefore, it is within the scope of this application to further use the agents identified as described herein in appropriate animal models.For example, the agents identified as described herein (for example, agents that can block CD93 / IGFBP7 interaction) can be used in animal models to determine the efficacy, toxicity, or side effects of treatment with such agents.Alternatively, the agents identified as described herein can be used in animal models to determine the mechanism of action of such agents.Furthermore, this application relates to the use of novel agents identified by the above screening assays for the treatment described herein.

[0310] V. Methods of Preparation, Nucleic Acids, Vectors, Host Cells, and Media In some embodiments, methods for preparing CD93 / IGFBP7 blocking agents (such as anti-CD93 antibodies, anti-IGFBP7 antibodies, inhibitory CD93 polypeptides, inhibitory IGFBP7 polypeptides described herein), and compositions comprising the agents, nucleic acid constructs, vectors, host cells, or media produced during preparation of the agents are provided.

[0311] Polypeptide Expression and Production The polypeptides described herein (e.g., anti-CD93 or anti-IGFBP7 antibodies, e.g., inhibitory CD93 or IGFBP7 polypeptides) can be prepared using any method known in the art, including those described below and in the Examples.

[0312] Monoclonal antibodies Monoclonal antibodies are obtained from a substantially homogeneous population of antibodies, i.e., the antibodies comprising the population are identical except for naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of distinct antibodies. For example, monoclonal antibodies can be produced using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be produced by recombinant DNA methods (U.S. Pat. No. 4,816,567). In the hybridoma method, a mouse or other suitable host animal, such as a hamster or llama, is immunized as described above to elicit lymphocytes that produce, or are capable of producing, antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0313] The immunizing agent typically contains an antigenic protein or a fusion variant thereof. Generally, if cells of human origin are desired, peripheral blood lymphocytes ("PBLs") are used, or if non-human mammalian sources are desired, spleen cells or lymph node cells are used. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell. Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103; incorporated by reference in its entirety for all purposes.

[0314] Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. Rat or mouse myeloma cell lines are commonly used. The hybridoma cells thus prepared are seeded and grown in an appropriate medium, preferably containing one or more substances that inhibit the growth or survival of unfused parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the medium for the hybridoma will typically contain hypoxanthine, aminopterin, and thymidine, substances that prevent the growth of HGPRT-deficient cells (HAT medium).

[0315] Preferred immortalized myeloma cells are those that fuse efficiently, support stable high-level production of antibody by selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, mouse myeloma lines are preferred, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center (San Diego, CA, USA) and SP-2 cells (and their derivatives, e.g., X63-Ag8-653) available from the American Type Culture Collection (Manassas, VA, USA). Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); each of which is incorporated herein by reference in its entirety for all purposes).

[0316] Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0317] The medium in which hybridoma cells are cultured can be assayed for the presence of monoclonal antibodies against desired antigens.Preferably, the binding affinity and specificity of monoclonal antibodies can be determined by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).Such techniques and assays are known in the art.For example, binding affinity can be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0318] After identifying hybridoma cells that produce antibodies of the desired specificity, affinity, and / or activity, the clones can be subcloned by limiting dilution and grown by standard methods (Goding, supra). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. Furthermore, hybridoma cells can be grown in vivo as tumors in mammals.

[0319] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0320] Monoclonal antibodies can also be produced by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567 and described above. DNA encoding monoclonal antibodies can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of mouse antibodies). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector and then transfected into host cells, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, that do not otherwise produce immunoglobulin proteins, and the monoclonal antibody can be synthesized in such recombinant host cells. Review articles on recombinant bacterial expression of antibody-encoding DNA include Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Plueckthun, Immunol. Revs. 130:151-188 (1992).

[0321] In a further embodiment, antibodies can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990); Clackson et al., Nature, 352:624-628 (1991); and Marks et al., J. Mol. Biol., 222:581-597 (1991), each of which is incorporated by reference in its entirety for all purposes, and which describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications described the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as strategies for constructing very large phage libraries (Waterhouse et al., Nucl. Acids Res., 21:2265-2266 (1993). These techniques are therefore viable alternatives to traditional monoclonal antibody hybridoma techniques for the isolation of monoclonal antibodies.

[0322] For example, the DNA may be modified by substituting the coding sequence for human heavy and light chain constant domains for the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, et al., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Typically, such a non-immunoglobulin polypeptide replaces the constant domains of an antibody or replaces the variable domains of one antigen-binding site of an antibody, creating a chimeric bivalent antibody containing one antigen-binding site with specificity for an antigen and another antigen-binding site with specificity for a different antigen.

[0323] The monoclonal antibodies described herein can be monovalent, and their preparation is well known in the art. For example, one method involves recombinant expression of an immunoglobulin light chain and a modified heavy chain. The heavy chain is generally truncated at any point within the Fc region to prevent heavy chain cross-linking. Alternatively, to prevent cross-linking, the relevant cysteine ​​residues can be replaced with other amino acid residues or deleted. In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to generate antibody fragments, particularly Fab fragments, can be achieved using conventional techniques known in the art.

[0324] Chimeric or hybrid antibodies can also be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

[0325] Nucleic acid molecules encoding polypeptides In some embodiments, a polynucleotide encoding any one of the antibodies (such as an anti-CD93 or anti-IGFBP7 antibody) or polypeptides (such as an inhibitory CD93 or IGFBP7 polypeptide) described herein is provided. In some embodiments, a polynucleotide prepared using any one of the methods described herein is provided. In some embodiments, a nucleic acid molecule comprises a polynucleotide encoding a heavy chain or a light chain of an antibody (e.g., an anti-CD93 or anti-IGFBP7 antibody). In some embodiments, a nucleic acid molecule comprises a polynucleotide encoding an inhibitory CD93 polypeptide or an inhibitory IGFBP7 polypeptide. In some embodiments, a nucleic acid molecule comprises both a polynucleotide encoding a heavy chain and a polynucleotide encoding a light chain of an antibody (e.g., an anti-CD93 or anti-IGFBP7 antibody). In some embodiments, a first nucleic acid molecule comprises a first polynucleotide encoding the heavy chain and a second nucleic acid molecule comprises a second polynucleotide encoding the light chain. In some embodiments, a nucleic acid molecule encoding an scFv (e.g., an anti-CD93 or anti-IGFBP7 scFv) is provided. In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding an inhibitory CD93 polypeptide or an inhibitory IGFBP7 polypeptide.

[0326] In some such embodiments, the heavy and light chains of an antibody (e.g., an anti-CD93 or anti-IGFBP7 antibody) are expressed as two separate polypeptides, either from one nucleic acid molecule or from two separate nucleic acid molecules. In some embodiments, for example, when the antibody is an scFv, a single polynucleotide encodes a single polypeptide comprising both the heavy and light chains linked together.

[0327] In some embodiments, a polynucleotide encoding a heavy or light chain of an antibody (e.g., an anti-CD93 or anti-IGFBP7 antibody) comprises a nucleotide sequence encoding a leader sequence that, when translated, is located at the N-terminus of the heavy or light chain. As noted above, the leader sequence may be the native heavy or light chain leader sequence or may be another heterologous leader sequence.

[0328] In some embodiments, the polynucleotide is DNA. In some embodiments, the polynucleotide is RNA. In some embodiments, the RNA is mRNA.

[0329] The nucleic acid molecule can be constructed using recombinant DNA techniques routine in the art. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in a selected host cell.

[0330] nucleic acid construct In some embodiments, a nucleic acid construct is provided that includes any one of the polynucleotides described herein. In some embodiments, a nucleic acid construct is provided that is prepared using any of the methods described herein.

[0331] In some embodiments, the nucleic acid construct further comprises a promoter operably linked to the polynucleotide, hi some embodiments, the polynucleotide corresponds to a gene and the promoter is the wild-type promoter of the gene.

[0332] vector The terms "vector," "cloning vector," and "expression vector" refer to a vehicle by which DNA or RNA sequences (e.g., foreign genes) can be introduced into a host cell in order to genetically modify the host and promote expression (e.g., transcription and translation) of the introduced sequences. Vectors include plasmids, synthetic RNA and DNA molecules, phages, viruses, and the like. In certain embodiments, the vector is a viral vector, such as, but not limited to, an adenovirus, adeno-associated, alphavirus, herpes, lentivirus, retrovirus, or vaccinia vector.

[0333] In some embodiments, vectors are provided comprising any polynucleotide encoding the heavy and / or light chain of any one of the antibodies described herein (e.g., anti-CD93 or anti-IGFBP7 antibodies). In some embodiments, vectors are provided comprising any polynucleotide encoding a polypeptide described herein (e.g., an inhibitory CD93 or IGFBP7 polypeptide). In some embodiments, vectors are provided comprising any nucleic acid construct described herein. In some embodiments, vectors prepared using any method described herein are provided. Also provided are vectors comprising a polynucleotide encoding any of the polypeptides (e.g., an anti-CD93 or anti-IGFBP7 antibody or an inhibitory CD93 or IGFBP7 polypeptide). Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc. In some embodiments, the vector comprises a first polynucleotide sequence encoding a heavy chain and a second polynucleotide sequence encoding a light chain. In some embodiments, the heavy and light chains are expressed from the vector as two separate polypeptides.

[0334] In some embodiments, a first vector comprises a polynucleotide encoding a heavy chain of an antibody (e.g., an anti-CD93 or anti-IGFBP7 antibody), and a second vector comprises a polynucleotide encoding a light chain of an antibody (e.g., an anti-CD93 or anti-IGFBP7 antibody). In some embodiments, the first vector and the second vector are transfected into host cells in similar amounts (e.g., similar molar amounts or similar masses). In some embodiments, a molar or mass ratio of the first vector to the second vector of 5:1 to 1:5 is transfected into host cells. In some embodiments, a mass ratio of 1:1 to 1:5 is used for the vector encoding the heavy chain to the vector encoding the light chain. In some embodiments, a mass ratio of 1:2 is used for the vector encoding the heavy chain to the vector encoding the light chain.

[0335] In some embodiments, a vector optimized for expression of a polypeptide in CHO or CHO-derived cells or NSO cells is selected. Exemplary such vectors are described, for example, in Running Deer et al., Biotechnol. Prog. 20:880-889 (2004).

[0336] In certain embodiments, the vector is a viral vector.In certain embodiments, the viral vector can be, but is not limited to, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, an alphavirus vector, a herpesvirus vector, and a vaccinia virus vector.In some embodiments, the viral vector is a lentivirus vector.

[0337] In some embodiments, the vector is a non-viral vector. The non-viral vector can be a plasmid or a transposon (such as a PiggyBac- or Sleeping Beauty transposon).

[0338] host cell In some embodiments, a host cell is provided that comprises any of the polypeptides, nucleic acid constructs, and / or vectors described herein. In some embodiments, a host cell is provided that is prepared using any of the methods described herein. In some embodiments, the host cell is capable of producing any of the polypeptides (such as antibodies or inhibitory polypeptides) described herein under fermentation conditions.

[0339] In some embodiments, the polypeptides described herein (e.g., anti-CD93 or anti-IGFBP7 antibodies or inhibitory CD93 or IGFBP7 polypeptides) can be expressed in prokaryotic cells, such as bacterial cells; or eukaryotic cells, such as fungal cells (e.g., yeast), plant cells, insect cells, and mammalian cells. Such expression can be carried out, for example, according to procedures known in the art. Exemplary eukaryotic cells that can be used to express polypeptides include, but are not limited to, COS cells, including COS7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44, Lec13, and FUT8 CHO cells; PER.C6® cells (Crucell); and HSO cells. In some embodiments, the polypeptides described herein (e.g., anti-CD93 or anti-IGFBP7 antibodies or inhibitory CD93 or IGFBP7 polypeptides) can be expressed in yeast. See, for example, U.S. Patent Application Publication No. 2006 / 0270045. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the heavy and / or light chains of the desired antibody. For example, in some embodiments, CHO cells produce polypeptides with higher levels of sialylation than the same polypeptides produced in 293 cells.

[0340] Introduction of one or more nucleic acids into desired host cells can be achieved by any method, including, but not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, etc. Non-limiting exemplary methods are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press (2001), which is incorporated by reference in its entirety for all purposes. Nucleic acids can be transiently or stably transfected into desired host cells according to any suitable method.

[0341] The present invention also provides host cells containing any of the polynucleotides or vectors described herein. In some embodiments, the present invention provides host cells containing anti-CD93 or anti-IGFBP7 antibodies. Any host cell capable of overexpressing heterologous DNA can be used for the purpose of isolating genes encoding antibodies, polypeptides, or proteins of interest. Non-limiting examples of mammalian host cells include, but are not limited to, COS, HeLa, and CHO cells. See also WO 87 / 04462. Suitable non-mammalian host cells include prokaryotes (such as Escherichia coli or B. subtilis) and yeast (Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Kluyveromyces lactis).

[0342] In some embodiments, the polypeptide is produced in a cell-free system. Non-limiting exemplary cell-free systems are described, for example, in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009); Spirin, Trends Biotechnol. 22: 538-45 (2004); Endo et al., Biotechnol. Adv. 21: 695-713 (2003).

[0343] Culture medium In some embodiments, a medium is provided that comprises any of the polypeptides, polynucleotides, nucleic acid constructs, vectors, and / or host cells described herein. In some embodiments, a medium is provided that is prepared using any of the methods described herein.

[0344] In some embodiments, the medium contains hypoxanthine, aminopterin, and / or thymidine (e.g., HAT medium). In some embodiments, the medium does not contain serum. In some embodiments, the medium contains serum. In some embodiments, the medium is D-MEM or RPMI-1640 medium.

[0345] Polypeptide purification Polypeptides (e.g., anti-CD93 or anti-IGFBP7 antibodies, e.g., inhibitory CD93 or IGFBP7 polypeptides) can be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrix or hydrophobic interaction chromatography. Suitable affinity ligands include ligands that bind to the ROR1 ECD and antibody constant regions. In some embodiments, Protein A, Protein G, Protein A / G, or antibody affinity columns can be used to purify antibodies that bind constant regions and contain Fc fragments. Hydrophobic interaction chromatography, e.g., butyl or phenyl columns, can also be suitable for purifying some polypeptides, such as antibodies. Ion exchange chromatography (e.g., anion exchange chromatography and / or cation exchange chromatography) can also be suitable for purifying some polypeptides, such as antibodies. Mixed-mode chromatography (e.g., reversed-phase / anion exchange, reversed-phase / cation exchange, hydrophilic interaction / anion exchange, hydrophilic interaction / cation exchange, etc.) can also be suitable for purifying some polypeptides, such as antibodies. Many methods for purifying polypeptides are known in the art.

[0346] VI. Compositions, Kits, and Articles of Manufacture The present application also provides compositions, kits, medicaments, and unit dosage forms for use in any of the methods described herein.

[0347] composition Any of the CD93 / IGFBP7 blocking agents described herein can be present in a composition (eg, a formulation) that includes other agents, excipients, or stabilizers.

[0348] In some embodiments, the composition further comprises a targeting agent or carrier that facilitates delivery of the CD93 / IGFBP7 blocking agent to tumor tissue or tissue associated with abnormal vascularity or hypoxia. Exemplary carriers include liposomes, micelles, nanodispersed albumin and modifications thereof, polymeric nanoparticles, dendrimers, and inorganic nanoparticles of different compositions.

[0349] In some embodiments, the compositions are suitable for administration to humans. In some embodiments, the compositions are suitable for administration to mammals, such as domestic pets and agricultural animals, in veterinary settings. There are a wide variety of suitable formulations of compositions containing CD93 / IGFBP7 blocking agents. The following formulations and methods are merely exemplary and in no way limiting. Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the compound dissolved in a diluent such as water, saline, or orange juice; (b) capsules, sachets, or tablets, each containing a predetermined amount of the active ingredient as a solid or granules; (c) suspensions in a suitable liquid; and (d) suitable emulsions. Tablet forms can contain one or more of lactose, mannitol, cornstarch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffers, wetting agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms can include pastilles containing the active ingredient in a flavor, usually sucrose and acacia or tragacanth, and an inert base such as gelatin and glycerin, or sucrose and acacia; emulsions, gels, and the like containing the active ingredient plus excipients known in the art.

[0350] Suitable carrier, excipient and diluent examples include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline, syrup, methylcellulose, methyl and propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.In some embodiments, the composition comprising CD93 / IGFBP7 blocker and carrier as discussed herein is present in a dry formulation (for example, lyophilized composition).Preparation can further comprise lubricant, wetting agent, emulsifier and suspending agent, preservative, sweetener or flavoring agent.

[0351] Preparations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostatic agents, and solutes that make the preparation compatible with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives.The preparations can be provided in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a lyophilized state, which only requires the addition of a sterile liquid excipient for injection, such as water, immediately before use.Extemporaneous injection solutions and suspensions can be prepared from the above-mentioned types of sterile powders, granules, and tablets.Injectable preparations are preferred.

[0352] In some embodiments, the composition is formulated to have a pH range of about 4.5 to about 9.0 (including, for example, any of the pH ranges of about 5.0 to about 8.0, about 6.5 to about 7.5, and about 6.5 to about 7.0). In some embodiments, the composition is formulated to have a pH of about 6 or greater (including, for example, any of about 6.5, 7, or 8 (e.g., about 8) or greater). The composition can also be made isotonic with blood by the addition of a suitable tonicity modifier, such as glycerol.

[0353] kit The kits provided herein include one or more containers containing a CD93 / IGFBP7 blocking agent or a pharmaceutical composition comprising a CD93 / IGFBP7 blocking agent and / or other agent described herein, and in some embodiments, further include instructions for use according to any of the methods described herein. The kit may further include instructions for selecting a suitable subject for treatment. The instructions provided with the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0354] In some embodiments, the kit comprises: a) a composition comprising a CD93 / IGFBP7 blocking agent, comprising an anti-CD93 antibody or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier; and optionally, b) instructions for administering the CD93 / IGFBP7 blocking agent for the treatment of a disease or condition. In some embodiments, the kit comprises: a) a composition comprising a CD93 / IGFBP7 blocking agent, comprising an anti-IGFBP7 antibody or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier; and optionally, b) instructions for administering the CD93 / IGFBP7 blocking agent for the treatment of a disease or condition. In some embodiments, the kit comprises: a) a composition comprising a CD93 / IGFBP7 blocking agent, comprising an inhibitory CD93 polypeptide or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier; and optionally, b) instructions for administering the CD93 / IGFBP7 blocking agent for the treatment of a disease or condition. In some embodiments, the kit comprises: a) a composition comprising a CD93 / IGFBP7 blocking agent, comprising an inhibitory IGFBP7 polypeptide or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier; and optionally b) instructions for administering the CD93 / IGFBP7 blocking agent for the treatment of a disease or condition.

[0355] The kit of the present invention is in a suitable package. Suitable packages include, but are not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), etc. The kit can optionally provide additional components such as buffers and interpretive information. Thus, the present application also provides products that include vials (e.g., sealed vials), bottles, jars, flexible packaging, etc.

[0356] In some embodiments, the kit includes one or more components that facilitate delivery of a CD93 / IGFBP7 blocking agent, or a composition comprising the agent, and / or an additional therapeutic agent to a subject. In some embodiments, the kit includes, for example, a syringe and needle suitable for delivering cells to a subject. In such embodiments, the CD93 / IGFBP7 blocking agent, or a composition comprising the agent, may be included in the kit in a bag or in one or more vials. In some embodiments, the kit includes components that facilitate intravenous or intra-arterial delivery of a CD93 / IGFBP7 blocking agent, or a composition comprising the agent, to a subject. In some embodiments, the CD93 / IGFBP7 blocking agent, or a composition comprising the agent, may be included in, for example, a bottle or bag (e.g., a blood bag or similar bag that can hold up to about 1.5 L of solution containing cells), and the kit further includes tubing and needles suitable for delivering the CD93 / IGFBP7 blocking agent, or a composition comprising the agent, to a subject.

[0357] The instructions for use of the composition generally include information about the dosage, administration schedule, and administration route for the intended treatment.The container can be a unit dose, bulk package (e.g., a multi-dose package), or sub-unit dose.For example, a kit can be provided that contains zinc as described herein in a dosage amount sufficient to provide effective treatment for a subject over a long period of time, such as 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 7 months, 8 months, 9 months, or more.The kit can also include multiple unit doses of the pharmaceutical composition and instructions for use, and can be packaged in a quantity sufficient for storage and use in pharmacies, such as hospital pharmacies and compounding pharmacies. [Example]

[0358] The following examples are intended purely to be illustrative of the present application and should not be construed as limiting the invention in any way. The following examples and detailed description are offered by way of illustration and not by way of limitation.

[0359] Example 1 To identify novel targets that may be involved in VEGF inhibitor-induced vascular normalization, we studied gene expression profiles in tumor ECs under in vivo VEGF inhibitor treatment from four recently published RNA-Seq datasets (28-31). Three datasets were derived from xenograft tumor models treated with VEGF inhibitors, and one was derived from a human neuroendocrine tumor. Genes that were consistently decreased across multiple datasets were selected using a log2 fold change cutoff of <-0.5. We identified 11 genes whose expression was significantly decreased by VEGF inhibitors in at least three datasets (Figure 1A). Most of these are transmembrane or extracellular matrix proteins (see Table 2). Five candidate genes upregulated in tumor ECs were selected, and their functions were tested in a tube formation assay using freshly isolated human endothelial cells (HUVECs) from blood vessels. Among them, knockdown of the CD93 gene resulted in a significant decrease in tube formation in HUVECs (Figure 1B). [Table 2]

[0360] Analysis of the Cancer Genome Atlas (TCGA) database for pancreatic cancer revealed that CD93 transcription was significantly higher in pancreatic ductal adenocarcinoma (PDA) than in normal pancreas (Figure 1C). Furthermore, CD93 protein was significantly upregulated in blood vessels within PDA and pancreatic neuroendocrine tumors (PNETs), the two major tumor types in the pancreas (Figure 1D).

[0361] CD93 expression was also assessed in normal tissues and tumors in mice. Freshly isolated aortic endothelial cells (MAECs) expressed negligible levels of CD93, but were upregulated by incubation with VEGF, confirming that VEGF signaling directly regulates CD93 expression (Figure 1E). As revealed by co-immunofluorescence staining of CD93 and CD31, blood vessels in normal mouse pancreas and skin express very low levels of CD93. Interestingly, CD93 expression in tumor vasculature was dramatically increased in orthotopic KPC and B16 melanoma models (Figures 1F and 1G). These results indicate that CD93 is selectively upregulated in tumor vasculature, which may be due to exposure to VEGF in the tumor microenvironment ("TME").

[0362] Example 2 To evaluate the possible effects of CD93 in vivo, we generated a mAb (clone 7C10, rat IgG) specific for mouse CD93 by immunizing rats with a mouse CD93 fusion protein. C57BL / 6 mice were implanted with the KPC tumor line derived from KPC transgenic mice (36). Once tumors became palpable, the mice were treated with 7C10 twice weekly for 2 weeks. 7C10 alone was able to delay KPC tumor growth by approximately 60% (Figure 2A). IF staining of tumor tissues showed no obvious changes in CD31+ microvessel density upon 7C10 treatment. However, there was a significant increase in vessel length by more than 1.8-fold, and the proportion of vessels with a round shape increased threefold in 7C10-treated tumors (Figure 2B). Furthermore, after treatment, there was a 3.5-fold increase in pericyte-covered vessels compared to controls, based on co-staining for NG2 and CD31 (Figure 2C). Consistent with this observation, there were more than two-fold more α-smooth muscle actin (α-SMA)-positive cells associated with blood vessels in 7C10-treated tumors (FIG. 2D).

[0363] To determine whether structural changes in tumor vasculature could lead to functional improvements, we examined tumor vascular perfusion in response to CD93 blockade. The tumor-bearing mice described above, receiving 1 week of antibody treatment, were intravenously injected with lectin-FITC before sacrifice. In control tumors, few vessels located at the tumor edge were FITC-positive, whereas in 7C10-treated tumors, the majority of vessels in both the center and edge of the tumor were found to be stained with FITC-lectin (Figure 2E). There were significantly more FITC-positive microvessels in 7C10-treated tumors than in controls (75% vs. 20%). In summary, these results support that targeting CD93 can normalize tumor vasculature and promote vascular maturation and perfusion in tumors.

[0364] Example 3 We used a human genome-scale receptor array (GSRA) to search for counter-receptors of CD93. IGFBP7, a secreted protein of the insulin growth factor binding protein (IGFBP) family, was the only positive hit among approximately 6,600 human transmembrane and secreted proteins in the library (Figure 4A). Addition of human CD93 mAb (clone MM01) or IGFBP7 mAb (clone R003) significantly reduced the binding of IGFBP7 protein to CD93-transfected 293 cells (Figure 4B). Recombinant IGFBP7 protein clearly bound to HUVEC cells, while CD93 mAb MM01 completely eliminated this binding activity (Figure 4C), demonstrating that CD93 mediates the binding of IGFBP7 protein to HUVEC cells. Furthermore, we were able to immunoprecipitate IGFBP7 from HUVEC cell lysates using CD93 mAb, indicating that the CD93-IGFBP7 interaction occurs naturally in endothelial cells (ECs) (Figure 4D). The affinity of the IGFBP7 / CD93 interaction measured by microscale thermophoresis (MST) showed a K of 53.13 ± 20.19 nM. DThe values ​​were shown (Figure 4E). The interaction between CD93 and IGFBP7 is also conserved in mice, and this interaction can be blocked by the anti-mouse IGFBP7 mAb (clone 2C6) (Figure 4F) or the anti-mouse CD93 mAb (clone 7C10) (Figure 4F) used in the in vivo functional studies described above. The results suggest that CD93 mAb 7C10 mediates its function in tumor vascular normalization by blocking the IGFBP7 / CD93 interaction.

[0365] We generated chimeric CD93 proteins by replacing the C-lectin domain (approximately 1-190 aa) with that of its family members. None of the chimeric proteins could bind to IGFBP7 (data not shown). This suggests that the IGFBP7-binding site on CD93 is an uncharacterized sequence (e.g., F182-Y262 in SEQ ID NO: 1) between the C-lectin and EGF-like domains.

[0366] Various commercially available anti-human CD93 and anti-human IGFBP7 monoclonal antibodies were tested for their ability to block the CD93 / IGFBP7 interaction, and the results are shown in Figure 16.

[0367] Example 4 IGFBP7 contains an IGF-binding (IB) domain at its N-terminus, a Kazal-type serine proteinase inhibitor domain (Kazal) in its central region, and an immunoglobulin-like C2 (IgC2) domain at its C-terminus (43). To further investigate the binding interaction between IGFBP7 and CD93, we generated a series of chimeric proteins for analysis by replacing each domain of IGFBP7 with the corresponding portion from IGFBPL1 (44), an IGFBP-related protein that does not bind to CD93 (Figure 4G). As expected, IGFBP7 strongly binds to CD93+ 293 cells, whereas IGFBPL1 does not. Chimeric proteins with the IB domain replaced lose their ability to bind to CD93+ 293 cells, while replacement of the Kazal or IgC2 domains has no or minimal effect (Figure 4G and Figure S10A). To exclude the possibility that other IB domain-containing human proteins may also interact with CD93, we constructed and generated mouse Fc-tagged fusion proteins from most of the human IB domain-containing genes (n = 15). With the exception of IGFBP7, no significant binding of these recombinant proteins to CD93 was detected (Figure 10B). Thus, the IB domain on IGFBP7 is highly specific for its interaction with CD93.

[0368] Example 5 IGFBP7 expression in tissue samples from PDAC patients was analyzed by IF. In adjacent normal pancreatic tissue, IGFBP7 protein was primarily present in islet cells, with few blood vessels possessing detectable IGFBP7 protein. CD31 staining was also scarce in human PDAC tissue. However, there were more than twice as many IGFBP7-positive blood vessels compared with adjacent normal pancreas (Figure 5A). Consistent with this, analysis of the TCGA pancreatic cancer dataset revealed that IGFBP7 was significantly upregulated in human PDAC compared with normal pancreas (Figure 11A). IGFBP7 gene expression correlated well with EC signature genes, such as PECAM1 (CD31), CD34, and von Willebrand factor (VWF), in PDAC, further supporting IGFBP7 as an abundant gene in tumor ECs (Figure 11B). A similar expression pattern of IGFBP7 was observed in mouse cancer tissue. In tumor vessels, IGFBP7 expression was significantly upregulated in orthotopically implanted KPC (pancreatic adenocarcinoma) tumors compared with normal pancreas (Figure 12A). IGFBP7 expression was virtually undetectable in the blood vessels of normal mouse skin tissue, but IGFBP7 was highly expressed in CD31+ ECs in subcutaneously implanted mouse KPC and B16 tumors (Figure 12B).

[0369] Microvessels within the center of implanted mouse tumors were found to express significantly higher levels of IGFBP7 compared with those around the tumor margin (Figure 5B), suggesting that IGFBP7 upregulation may be induced by hypoxia within the tumor. To test this, ECs were cultured in dimethyloxalylglycine (DMOG) to mimic hypoxic conditions and examined for IGFBP7 expression by Western blot. Indeed, HUVEC cells cultured in DMOG were found to have elevated HIF-1α levels, accompanied by higher expression of IGFBP7 (Figure 5C).

[0370] Because the IGFBP7 gene does not contain a consensus hypoxia-responsive element (HRE, 5'-RCGTG-3' motif) (47) in its promoter region, its upregulation in ECs may not be directly induced by hypoxia. We hypothesized that hypoxia-induced VEGF (48), a potent inducer of IGFBP7 in ECs, may be involved in IGFBP7 upregulation. This hypothesis was tested in mouse endothelial cells. Similar to HUVEC cells, IGFBP7 expression was upregulated in mouse ECs in the presence of DMOG to mimic hypoxia. Inclusion of a VEGFR-blocking mAb in the culture completely prevented hypoxia-induced IGFBP7 expression in mouse ECs (Figure 5D), suggesting that hypoxia-induced IGFBP7 is completely dependent on VEGF signaling in this system. Interestingly, analysis of RNA-Seq data (GSE110501) from a xenograft colon cancer mouse model (49) showed that IGFBP7 was also significantly inhibited in tumor ECs by the VEGF inhibitor aflibercept (Figure 5E). Collectively, these results support IGFBP7 as a hypoxia-induced ECM protein in tumor-associated vasculature via VEGF signaling.

[0371] Example 6 IGFBP7 protein was constitutively expressed in HUVEC cells and was further upregulated by DMOG, accompanied by the induction of HIF-1α (Figure 5C). Knockdown of IGFBP7 gene expression significantly inhibited tube formation in HUVEC cells (Figure 13A). To determine whether IGFBP7 mediates angiogenesis through CD93, we transfected HUVEC cells with CD93 siRNA to knockdown CD93 expression as an in vitro model for testing the effects of IGFBP7 protein. Addition of exogenous IGFBP7 protein increased tube formation and proliferation of wild-type HUVEC cells. However, in CD93-knockdown HUVEC cells, IGFBP7 protein lost its effect on tube formation or EC migration in a transwell migration assay (Figures 13B and 13C). These studies indicate that CD93 mediates the pro-angiogenic effect of IGFBP7 protein on ECs.

[0372] We utilized an IGFBP7 mAb (clone 2C6, Figure 14A) that blocks IGFBP7 binding to CD93 to test its effects on tumor growth and tumor vascular maturation in vivo. Administration of 2C6 significantly inhibited KPC tumor growth by more than 40% compared to controls, as described above (Figure 14B). IF staining of tumor tissue revealed that blockade of the IGFBP7 / CD93 interaction with 2C6 significantly increased the length of round vessels and tumor microvessels but did not affect the density of CD31+ tumor vessels (Figure 14C). Similar to the effect of CD93 mAb on vascular maturation, IGFBP7 mAb improved the coverage of NG2+ pericytes along tumor vessels (Figure 14D) and increased α-SMA coverage over tumor vessels (Figure 14E). Tumor tissues from mice treated with 2C6 mAb showed a clear reduction of >50% in β1 integrin activation (Figure S4F), further supporting that anti-IGFBP7 affects integrins to normalize tumor vasculature (51). These results support that blocking IGFBP7 / CD93 interactions promotes vascular normalization and attenuates tumor growth.

[0373] Furthermore, high doses of IGFBP7 mAb and CD93 mAb (15 mg / kg or 300 μg) did not reduce tumor vascular density in vivo. These results suggest that the primary effect of altering CD93 / IGFBP7 in the TME is on vascular abnormalities, not on increased angiogenesis. This indicates that the IGFBP7 / CD93 axis may be a better therapeutic target for vascular normalization. Both IGFBP7 and CD93 are selectively upregulated in tumor vessels in mouse and human tumors. This restricted expression pattern contrasts with the widespread expression of VEGFR-1, -2, and -3 in the microvasculature of normal tissues.

[0374] Example 7 Because the CD93 / IGFBP7 interaction plays a major role in abnormal tumor angiogenesis, we further tested whether blocking this interaction with mAb could promote tumor perfusion, thereby facilitating drug delivery through vascular normalization. In the KPC model, we tested the delivery effect of doxorubicin, an anthracycline chemotherapy drug with inherent autofluorescence. Mice were intravenously injected with doxorubicin 20 minutes before sacrifice. Simultaneously, mice were treated with pimonidazole as a hypoxia probe to assess possible changes in tumor hypoxia. Greater penetration of doxorubicin into tumors was observed in CD93 mAb-treated mice; meanwhile, hypoxia was also significantly alleviated in tumors (Figure 6A). We also evaluated the antitumor effect of anti-CD93 in a B16 tumor model treated with 5-fluorouracil (5-FU). Mice were subcutaneously (sc) implanted with B16 melanoma tumors and began treatment with CD93 mAb twice weekly. Once tumors became palpable, two doses of 5-FU were administered. As expected, treatment with CD93 mAb or 5-FU alone only slightly inhibited tumor growth, and tumors eventually enlarged in both groups. Combined treatment with 5-FU and CD93 mAb dramatically inhibited tumor growth (Figure 6B) and extended the survival of a significant portion of mice (approximately 40%) beyond 20 days (Figure 6C). Histological staining showed that CD93 blockade enhanced 5-FU-induced suppression of tumor growth based on Ki-67 staining of implanted B16 melanoma tumors (Figure 6D). Collectively, these experiments demonstrate that blocking the CD93 / IGFBP7 interaction alleviates hypoxia, promotes drug delivery, and thus facilitates chemotherapy in cancer.

[0375] Example 8 Normalization of tumor vasculature can enhance immune cell trafficking to tumors, which may be due to upregulated adhesion molecules (16, 40, 41). Anti-CD93 treatment was found to increase ICAM1 expression on tumor vessels in both subcutaneous (sc) KPC and B16 tumor models (Figures 9A and 9B). In line with this, IF staining for CD3 revealed a roughly three-fold increase in TILs in KPC tumor tissues from anti-CD93-treated mice compared with those from controls at days 8 and 15 (Figures 3A and 3B). Further analysis of TIL composition by flow cytometry reveals that anti-CD93 significantly increased the percentage and absolute number of CD45+ leukocytes in tumors: approximately three-fold more CD4+ and CD8+ T cells in CD93 mAb-treated tumors than in controls (Figures 3C and 3D). Anti-CD93 did not alter the proportion of CD8+ or CD4+ TIL subsets within the CD45+ hematopoietic cell compartment (Figure 8A), nor did it alter TIL function, as indicated by similar levels of IFN-γ and TNF-α (Figure 8B). However, anti-CD93 significantly reduced the proportion of myeloid-derived suppressor cells (MDSCs) within the tumor (Figure 3E), further supporting a favorable inflammatory TME. A similar effect of anti-CD93 on promoting TILs was observed in B16 melanoma, although fewer TILs were generally present within the tumor in this model (Figure 3F). Collectively, these results support the idea that blocking the CD93 / IGFBP7 interaction conditions the inflammatory TME by improving T cell infiltration.

[0376] Example 9 We tested whether CD93 / IGFBP7 blockade could facilitate cancer immunotherapy based on immune normalization of the tumor microenvironment. We first determined whether the effect of anti-CD93 on tumor growth inhibition was dependent on T cell-mediated immune responses. Depletion of CD8+ T cells with mAb at the start of anti-CD3 treatment completely abolished the antitumor effect, whereas depletion of CD4+ T cells had only a minor effect (Figure 7A), supporting the primary role of CD8+ T cells in anti-CD93-mediated tumor suppression in this model.

[0377] We hypothesized that B7-H1 induction might be responsible for the limited antitumor effect of anti-CD93. Indeed, upregulation of B7-H1 expression in tumor tissue was observed upon anti-CD93 treatment (Figure 7B). In addition to increased B7-H1 expression in CD31+ tumor ECs, we also observed a significant increase in B7-H1 expression on both tumor cells and CD45+ leukocytes in anti-CD93-treated tumors compared to controls (Figure 7C). Thus, anti-CD93-induced upregulation of B7-H1 in the TME may limit antitumor immunity. These findings justify the combination of anti-CD93 and anti-PD-1 / PD-L1 therapy, which we subsequently tested in a KPC model. While treatment with anti-CD93 or anti-PD-1 mAb alone partially delayed tumor growth, the combination of anti-CD93 / PD-L1 mAb significantly inhibited tumor growth in this model (Figure 7D). As a result, tumor weight in the combination group was reduced to approximately 20% of that in the control group (Figure 7E). Consistent with the better antitumor effect, analysis of immune cells within tumors treated with the combination therapy showed a significant increase in the absolute numbers of both CD8+ and CD4+ T cells (Figure 7F). Concomitantly, the ratio of CD8+ T cells significantly increased and tumor-associated macrophages (TAMs) significantly decreased in the combination therapy group (Figure 7G). These results indicate that CD93 / IGFBP7 blockade can normalize tumor vasculature, which can amplify the effects of anti-PD-1 / PD-L1 cancer targeted therapy.

[0378] Example 10 This example demonstrates that CD93 on nonhematopoietic cells mediates the antitumor immunity exhibited by anti-CD93. Anti-CD93 mAb B16 was found to accumulate on the tumor vasculature of tumors upon injection (Figure 17A). In addition to ECs, CD93 is known to be expressed on several hematopoietic cell types, including monocytes, macrophages, and immature B cells (71). To fully clarify the cellular source of CD93 responsible for the antitumor effect of anti-CD93 treatment, CD93 chimeric mice were generated by reconstituting lethally irradiated wild-type (WT) B6 mice with bone marrow (BM) from WT or CD93KO mice. As expected, anti-CD93 treatment inhibited tumor growth in chimeric mice, regardless of the source of BM (Figure 17B). Because ECs are the only cellular source of CD93 on nonhematopoietic cells, this result confirmed that anti-CD93 is a blocking mAb that targets tumor vasculature.

[0379] Example 11 This example demonstrates that CD93 blockade inhibits B16 melanoma tumor growth. CD93 overexpression in tumor vasculature has been observed in many solid tumors (32-34). Similarly, both CD93 (Figure 18A) and IGFBP7 (Figure 18B) in tumor vasculature are significantly upregulated in subcutaneous B16 melanoma. When tumor-bearing mice were treated with a blocking mCD93 mAb (clone 7C10), CD93 blockade significantly inhibited tumor growth and reduced tumor weight in B16 tumors (Figure 18C). Treatment with anti-CD93 Fab remained effective in inhibiting B16 tumor growth, excluding the possibility of Fc-mediated depletion (data not shown). These data are consistent with the delayed tumor growth seen in CD93- / - mice.

[0380] Example 12 This example demonstrates that CD93 blockade significantly increases T cell infiltration and function in mouse melanoma. Normalization of tumor vasculature enhances immune cell trafficking into tumors (16, 74). Anti-CD93 treatment was found to increase CD3+ TILs approximately threefold in B16 tumors (Figure 19A). Flow cytometry analysis revealed that anti-CD93 significantly increased both the percentage and density of CD45+ immune cells in tumors (Figure 19B). Detailed analysis of immune cell composition showed that NK and T cells, particularly CD8+ T cells, were the major cell types increased in anti-CD93-treated B16 tumors (Figure 19C). Anti-CD93 significantly increased the percentage of effector memory T cells (TEMs) in the CD8+ T cell subset, as further confirmed by increased expression of PD1 and Granzyme B (Figure 19D); consistently, CD8+ TILs in CD93-treated tumors produced significantly more effector cytokines, including IFN-γ and TNF (Figure 19E). Although CD93 blockade did not affect the density of CD4+ TILs, there were relatively more effector T cells (TEMs and PD1 positive) and fewer Treg cells in anti-CD93-treated tumors (Figure 19F). Analysis also revealed that many immune suppressive cells, including Tregs, granulocytic myeloid-derived suppressor cells (gMDSCs), and tumor-associated macrophages (Macs), were significantly reduced in anti-CD93-treated tumors (Figure 19C). MDSCs and macrophages (CD11b+) preferentially localized to hypoxic regions; because MDSCs and macrophages do not express CD93 themselves, their reduction in anti-CD93-treated tumors may be caused by the alleviation of hypoxic conditions (Figure 19G). Taken together, the results support that blocking the CD93 pathway creates an immune-favorable TME in B16 melanoma.

[0381] Example 13 This example demonstrates that CD93 blockade enhances the sensitivity of B16 melanoma to immunotherapy. PD-L1 is often upregulated in tumor tissue in response to IFN-γ, often resulting in an increase in TILs (52). Indeed, upregulation of PD-L1 expression was observed in tumor tissue upon treatment with anti-CD93 (Figure 20A). In addition to CD31+ ECs, a significant increase in PD-L1 expression was observed on both tumor cells and CD45+ leukocytes with anti-CD93 (Figure 20B). Furthermore, PD1-positive TILs were more abundant in B16 tumors under anti-CD93 treatment (Figures 19E and 19G). This observed upregulation of the PD1 / PD-L1 pathway in the TME may limit anti-CD93-mediated anti-tumor immunity. In the B16 melanoma model, treatment with anti-CD93 or ICB (PD1+CTLA4-blocking mAb) alone moderately delayed tumor growth. However, the anti-CD93 / ICB combination significantly inhibited tumor growth in this model; over 80% of mice in the combination group survived for 20 days, whereas all mice in the control group died before 15 days (Figure 20C). Consistent with the better anti-tumor effect, analysis of immune cells within tumors of the combination therapy showed a significant increase in the number of CD45+ immune cells, including both CD4+ and CD8+ T cells (Figure 20D). At the same time, the effector memory phenotype (TEM, CD44 hi The number of T cells bearing CD62L- was significantly increased in both CD4+ and CD8+ T cells in the combination group (Figure 20E). Collectively, the results support that blocking CD93 signaling sensitizes tumors to ICB therapy.

[0382] Example 14 This example demonstrates that expression of the IGFBP7 / CD93 pathway is upregulated in TNBC vasculature. CD93 is one of the top genes in a previously reported human primary tumor angiogenesis gene signature (45), and CD93 overexpression in tumor vasculature has been observed in many solid tumors (30, 74-76). CD93 was found to be significantly upregulated in blood vessels within human TNBC compared with adjacent normal breast tissue (n = 5) (Figure 21A). IGFBP7 protein was barely detectable in blood vessels of adjacent normal breast tissue, whereas its expression in human TNBC vasculature was significantly increased (Figure 21B). Similarly, in an orthotopic 4T1 mouse mammary tumor model, both CD93 (Figure 21C) and IGFBP7 (Figure 21D) expression in tumor vasculature was dramatically upregulated. To assess the clinical relevance of IGFBP7 in BC, we analyzed the TCGA breast cancer dataset. Interestingly, high IGFBP7 is associated with poor prognosis in TNBC but not in ER-positive breast cancer (Figure 22).

[0383] Example 15 This example demonstrates that blocking IGFBP7 / CD93 interaction inhibits TNBC tumor growth in vivo. 4T1 tumor-bearing mice were treated with a blocking mCD93 mAb (clone 7C10) when the 4T1 tumors became palpable. Tumor growth curves showed that administration of the anti-CD93 blocking mAb significantly inhibited tumor growth and thus reduced tumor weight (Figure 23A). Similarly, the same CD93 blocking mAb had comparable antitumor effects on orthotopically implanted PY8119, another mouse TNBC model (Figure 23B).

[0384] Example 16 This example demonstrates that CD93 blockade promotes vascular maturation and improves perfusion in TNBC. Blockade of the IGFBP7 / CD93 interaction with CD93 mAb did not affect vascular density (Figure 24A). The effect of CD93 mAb on tumor vascular normalization was confirmed by increased α-SMA staining in tumor vessels (Figure 24A) and pericyte coverage (NG2+ vessels, Figure 24B). Similar results were found for anti-CD93 on vascular maturation in the PY8119 tumor model (data not shown). CD93 blockade increased tumor perfusion, as there was a more than two-fold increase in FITC-lectin-positive vessels in CD93 mAb-treated tumors; concomitantly, there were significantly fewer hypoxic regions (pimonidazole+) in 4T1 tumors with anti-CD93 treatment (Figure 24C).

[0385] Example 17 This example demonstrates that CD93 blockade increased TILs and decreased MDSCs in 4T1 tumors. Two weeks after antibody treatment, infiltrating immune cells were examined in 4T1 tumors by IF staining. Significantly more CD3+ T cells were found in tumors treated with CD93 mAb (Figure 25A). CD11b+Ly6G+MDSCs were abundant in 4T1 tumors. Interestingly, treatment with anti-CD93 significantly reduced their numbers in tumors (Figure 25B). The results of IF staining of tumor cell suspensions were further confirmed by FACS analysis (Figure 25C). Therefore, CD93 blockade can create a favorable TME for immunotherapy in TNBC.

[0386] Example 18 This example demonstrates that IGFBP7 and CD93 are upregulated in the vasculature within human cancer. IGFBP7 expression is upregulated in human cancer compared to adjacent normal tissue (Figure 26A). CD93 expression in human cancer is primarily present in tumor vasculature based on immunofluorescence staining (Figure 26B). Both CD93 and IGFBP7 are upregulated in blood vessels within human melanoma (Figure 26C).

[0387] Example 19 This example demonstrates enrichment of the IGFBP7 / CD93 pathway in human cancers resistant to anti-PD therapy. Tumor vascular dysfunction limits anti-tumor immunity and significantly threatens immunotherapy (19). Gene expression of IGFBP7 and CD93 was examined in cancer patients undergoing anti-PD therapy. In a phase II study of patients with metastatic urothelial carcinoma treated with atezolizumab (anti-PD-L1 mAb) (77), baseline levels of both IGFBP7 and CD93 expression were significantly higher in tumor tissue from non-responders compared with those from responders (Figure 27A). Consistently, in a small cohort of metastatic melanoma patients undergoing anti-PD1 therapy (78), baseline IGFBP7 levels tended to be lower in patients who responded to anti-PD1 therapy compared with those who did not benefit (Figure 27B). Although a trend toward increased mean CD93 expression was observed in non-responders, this association did not reach statistical significance (Figure 27B). In summary, the IGFBP7 / CD93 pathway in the TME may contribute to cancer resistance to anti-PD therapy in the clinic.

[0388] Example 20 This example demonstrates that IGFBP7 and MMRN2 bind to distinct motifs on CD93. MMRN2, an ECM protein not present in the GSRA library (42), is another known ligand for CD93. In addition to CD93, MMRN2 also interacts with two additional group 14 C-type lectin members, CLEC14A and CD248; in contrast to MMRN2, IGFBP7 bound only to CD93 and not to any other C-type lectin molecules (Figure 28A). MMRN2 and IGFBP7 did not compete with each other for CD93 binding, as addition of IGFBP7 did not interfere with CD93 binding by MMRN2, and vice versa (Figure 28B). ELISA assays confirmed that preincubation of IGFBP7-coated wells with CD93 protein resulted in MMRN2 binding (Figure 28C), suggesting that CD93 can simultaneously bind its two ligands to form a protein complex together. The anti-mouse CD93 (clone 7C10) used for in vivo studies was found to also block the interaction between CD93 and MMRN2 (Fig. 28D). When we examined the binding of these two ligands to several mouse CD93 mutants with point mutations, we found that two CD93 mutants (C103S and C135S) that lost binding to MMRN2 bound highly to IGFBP7 (Fig. 28E). All of this supported the idea that IGFBP7 and MMRN2 bind to different sites on CD93.

[0389] The following are the methods and materials used in the examples.

[0390] Cell lines, fusion proteins and antibodies KPC cells were derived from KrasLSLG12D / +;Trp53R172H;Pdx1-Cre (KPC) transgenic mice. Human IGFBP7 (Fc tag) and mouse IGFBP7 (Fc tag) were purchased from Sino Biological Co., Ltd. Rat anti-mouse CD93 mAb (clone 7C10) was generated from a hybridoma derived from the fusion of SP2 myeloma with B cells from a rat immunized with mouse CD93-Ig. Hamster anti-mouse IGFBP7 mAb (clones 2C6, 6F1) was generated from a hybridoma derived from the fusion of SP2 myeloma with B cells from an Armenian hamster immunized with mouse IGFBP7-Ig. Hybridomas were adapted and cultured in hybridoma serum-free medium (Life Technologies). Antibodies in the supernatant were purified using a HiTrap Protein G affinity column (GE Healthcare). Anti-mouse VEGFR-2 (clone DC101) was purchased from BioXcell. Anti-human IGFBP7 mAb (R003, Sino Biological) and anti-human CD93 (MM01, Sino Biological) were used to block human IGFBP7-CD93 interaction. Commercially available antibodies were purchased from Biolegend unless otherwise noted.

[0391] IGFBP7 chimera and CD93-F238L mutant IGFBP7-IGFBPL1 chimeras were generated by two-step PCR. The chimeric proteins share similar structures and contain domains from IGFBP7 and IGFBPL1 exchanged at different cleavage sites. Supernatants were collected from target-transfected HEK293T cells for downstream binding assays. The CD93-F238L mutant, containing a phenylalanine to leucine substitution, was generated by PCR using full-length CD93 as a template to change the codon sequence from TTC (phenylalanine) to ACC (leucine) (46). All constructs were verified by sequencing.

[0392] Flow cytometry Cell surface and intercellular staining and flow cytometric analysis followed a previously described protocol (71). Dead cells were excluded using the SYTOX® Blue Dead Cell Stain Kit (Thermo Fisher Scientific). Flow cytometric analysis was performed using a BD FACS Calibur or BD LSRFortessa™ cell analyzer (BD Bioscience, Franklin Lakes, NJ, USA), and data were then analyzed using FlowJo software (Tree Star Inc.).

[0393] Microscale Thermophoresis (MST) Experiments IGFBP7 protein (R&D Systems, Minneapolis, MN) was labeled with Monolith His-Tag Labeling Kit, RED-tris-NTA 2 nd Fluorescent dyes were labeled using a Generation (Nanotemper GmbH, Munich, Germany). From a 100 nM stock, samples were diluted to a concentration of 20 nM in PBS + 0.05% P20, loaded into a Premium MST capillary, and pretested for successful labeling and protein stability using a Monolith NT.115 Pico Instrument (Nanotemper GmbH, Munich, Germany). A 5.9 μM stock solution of recombinant human CD93 protein (R&D Systems, Minneapolis, MN) was diluted 2x16 times with PBS + 0.05% P20 to create a dilution series ranging from 5.9 μM to 180 pM. 20 nM IGFBP7 was added 1:1 to each concentration so that each sample contained a final concentration of 10 nM IGFBP7. Samples were loaded into MST Premium capillaries and measured for microscale thermophoresis using the aforementioned instrument. The experiment was performed using 20% ​​laser power and a PICO Red detector at Medium MST power. The experiment was repeated once with two replicates using the same procedure. Data were analyzed using MO Affinity Analysis software (Nanotemper GMBH, Munich, Germany).

[0394] EC culture Pooled human umbilical vein ECs (HUVECs) purchased from Thermo Fisher were cultured in Medium 200 containing LVES (Life Technologies). C57BL / 6 mouse primary aortic ECs and endothelial medium containing supplements were purchased from Cell Biologies. For tube formation, 2 x 10 5 HUVECs were seeded on Matrigel at 1000 cells / ml in 24-well plates. Images were recorded every 4-6 hours after incubation. Transwell 6.5 mm polycarbonate membrane inserts pre-loaded in 24-well culture plates (Come 3422, 8 μm) were used in the cell migration model. 1 x 10 in 200 μl 5 1 ml of HUVEC cells were loaded into each 24-well insert, and 500 μl of FBS-containing medium containing different reagents was placed in the lower chamber. After approximately 20 hours, the migrated cells were fixed with methanol, stained with Giemsa solution, and counted under a light microscope.

[0395] Mouse tumor model All animal care, experiments, and euthanasia were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of the University of Colorado Anschutz Medical Campus. C57BL / 6 mice were purchased from Jackson Laboratory (Bar Harbor, ME). Mice aged 6–8 weeks were used in these experiments. KPC (4x10 5 ) cells were injected subcutaneously into the right flank of C57BL / 6 mice. After tumors became palpable, the mice were placed in a 1 / 2 × (length × width) 2Mice were randomized into different treatment groups based on tumor volume, calculated as 0.01 (mean ± SD). 300 μg / mouse of therapeutic antibody was intraperitoneally injected twice weekly for a total of four doses. Tumor diameters (length and width) were measured every two or three days using a caliper. 14 days after the first treatment, mice were euthanized and sacrificed, and tumor tissues were excised for detailed analysis. Tumor tissues for FITC-lectin, doxorubicin delivery, and hydroxyprobe assays were obtained eight days ...

Claims

1. A composition for treating a tumor or cancer in a subject in need thereof, comprising a CD93 / IGFBP7 blocking agent that specifically inhibits the IGFBP7 / CD93 signaling pathway, wherein the CD93 / IGFBP7 blocking agent comprises an anti-CD93 antibody that specifically recognizes CD93, or an anti-IGFBP7 antibody that specifically recognizes IGFBP7, and wherein the anti-CD93 antibody and the anti-IGFBP7 antibody block the interaction between CD93 and IGFBP7.

2. The composition of claim 1 , wherein the CD93 / IGFBP7 blocking agent comprises an anti-CD93 antibody.

3. The composition of claim 2, wherein the anti-CD93 antibody also blocks the interaction between CD93 and MMRN2.

4. The composition described in claim 2, wherein the anti-CD93 antibody does not block the interaction between CD93 and MMRN2.

5. (a) the anti-CD93 antibody binds to CD93 competitively with mAb MM01, and / or (b) the anti-CD93 antibody binds to an epitope that overlaps with the epitope of mAb MM01.

6. (a) The anti-CD93 antibody is a full-length antibody, a single-chain Fv (scFv), a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), a (dsFv) ２ , V Ｈ H, Fv-Fc fusion, scFv-Fc fusion, scFv-Fv fusion, diabody, tribody, or tetrabody; and / or (b) the composition of any one of claims 2 to 5, wherein the anti-CD93 antibody is comprised in a fusion protein.

7. The composition of claim 1 , wherein the CD93 / IGFBP7 blocking agent comprises an anti-IGFBP7 antibody.

8. (a) the anti-IGFBP7 antibody binds to IGFBP7 competitively with mAb R003, and / or (b) the anti-IGFBP7 antibody binds to an epitope that overlaps with the epitope of mAb R003.

9. The anti-IGFBP7 antibody may be a full-length antibody, a single-chain Fv (scFv), a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), a (dsFv) ２ , V Ｈ 9. The composition of claim 7 or 8, which is an Fv-Fc fusion, an scFv-Fc fusion, an scFv-Fv fusion, a diabody, a tribody, or a tetrabody.

10. The composition of any one of claims 1 to 9, administered to the subject in combination with a second therapeutic agent.

11. (a) the second therapeutic agent is an immune checkpoint inhibitor, or (b) the second therapeutic agent is a chemotherapeutic agent, or (c) the second therapeutic agent is an immune cell, or (d) The composition of claim 10, wherein the second therapeutic agent is an anti-angiogenic inhibitor.

12. the immune checkpoint inhibitor is an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, or a combination thereof; and / or The composition of claim 11 , wherein the anti-angiogenesis inhibitor is an anti-VEGF inhibitor.

13. (a) the cancer is characterized by abnormal tumor vasculature, and / or (b) the cancer is characterized by high expression of CD93, and / or (c) the cancer is characterized by high expression of IGFBP7, and / or (d) the cancer is highly vascular.

14. The composition of any one of claims 1 to 13, wherein the cancer is a solid tumor.

15. 15. The composition of claim 14, wherein the cancer is colorectal cancer, non-small cell lung cancer, glioblastoma, renal cell carcinoma, cervical cancer, ovarian cancer, fallopian tube cancer, peritoneal cancer, breast cancer, prostate cancer, bladder cancer, oral squamous cell carcinoma, head and neck squamous cell carcinoma, brain tumor, bone cancer, or melanoma.

16. 16. The composition of claim 15, wherein the cancer is triple-negative breast cancer (TNBC).

17. A method for determining whether a candidate drug is useful for treating cancer, comprising determining whether the candidate drug inhibits CD93 / IGFBP7 interaction, and determining that the candidate drug is useful for treating cancer if it is shown to specifically inhibit CD93 / IGFBP7 interaction.

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