KR102207221B1 - Methods of inhibiting pathological angiogenesis with doppel-targeting molecules - Google Patents

Abstract

The invention described herein inhibits the generation of pathological neovascularization, tumors, cancer, atherosclerosis (atherosclerosis), tuberculosis (tuberculosis), asthma (asthma), pulmonary arterial hypertension (PAH), neoplasms (neoplasms). ) And neoplasm-related conditions. All related compositions and methods are described herein.

Description

Method for inhibiting pathologic angiogenesis using Doppel-targeting molecules {METHODS OF INHIBITING PATHOLOGICAL ANGIOGENESIS WITH DOPPEL-TARGETING MOLECULES}

The invention described herein inhibits pathological angiogenesis, tumors, cancer, atherosclerosis (atherosclerosis), tuberculosis (tuberculosis), asthma (asthma), pulmonary arterial hypertension (PAH), neoplasms and The present invention relates to a doppel-targeting molecule useful for treating diseases associated with pathological angiogenesis, such as neoplasm-related conditions, or for detecting Doppel expression in a patient. Related compositions and methods are also described.

Cancer is a diverse set of diseases that differ greatly in genotype and phenotype. Nevertheless, angiogenesis (the formation of new blood vessels) plays an important role in the proliferation of cancer cells across cancer types. Indeed, the independence of solid tumors to neovascular growth has made tumor vessels an attractive target of cancer targets. Therefore, cancer therapy that interferes with angiogenesis is being developed. For example, bevacizumab, a humanized monoclonal antibody against vascular endothelial growth factor (VEGF), has been used to potentiate the effects of different active chemotherapeutic regimens.

To date, various agents that inhibit angiogenesis (eg, endostatin, angiostatin, and thrombospondin) have been described. However, angiogenesis is important for normal physiological processes such as growth and development or wound healing. Current anti-angiogenic therapies, such as anti-VEGF monoclonal antibodies and tyrosine kinase receptor (TKR, also known as receptor tyrosine kinase (TKR)) inhibitors, which do not differentiate between pathological and normal angiogenesis, are systemic in cellular signaling. By inducing a decline in function, it inhibits not only pathological angiogenesis, but also beneficial angiogenesis. For example, considering the vast biological role of VEGF in thyroid function, bone marrow function, immunomodulation, renal function, vascular homeostasis, initiation of aggregation, and physiological angiogenesis, systemic inhibition of VEGF can interfere with all of these essential functions. .

Therefore, there is a need for an anti-angiogenic therapy that distinguishes between normal and pathological angiogenesis.

The present application is used to inhibit pathological angiogenesis in a subject in need of inhibition of pathological angiogenesis, or used to treat a tumor in a subject in need of tumor treatment, or in a subject in need of inhibition of tumorigenesis In a subject in need of inhibiting tumorigenesis or reducing the vasculature of the tumor, reducing the vasculature of the tumor, or of a disease or condition selected from cancer, atherosclerosis, tuberculosis, asthma, and pulmonary arterial hypertension (PAH). Treating a disease or condition selected from cancer, atherosclerosis, tuberculosis, asthma, and pulmonary hypertension in a subject in need thereof, or a neoplastic or neoplastic-related condition such as breast carcinoma, lung carcinoma, gastric carcinoma, Esophageal carcinoma, direct colon carcinoma, liver carcinoma, ovarian carcinoma, androblastoma, cervical carcinoma, endometrial carcinoma, endometrial hyperplasia, endometriosis, fibrosarcoma, sarcoid carcinoma, head and neck cancer, nasopharyngeal carcinoma, laryngeal carcinoma, Hepatoblastoma, Kaposi's sarcoma, melanoma, skin carcinoma, hemangioma, cavernous hemangioma, hemangioblastoma, pancreatic carcinoma, retinoblastoma, astrocytoma, glioblastoma, schwannoma, oligodendrogliomas, glioblastoma, neuroblastoma, rhabdomyosarcoma, osteogenic sarcoma, smooth muscle Use to treat sarcoma, urinary tract carcinoma, thyroid carcinoma, Wilm's tumor, renal cell carcinoma, prostate carcinoma, nevus-associated dysvascular hyperplasia, brain tumor-associated edema, and/or Meigs syndrome, or to detect Doppel expression in a subject. For the Doppel-targeting molecules are described.

In some embodiments, the dopel-targeting molecule binds to dopel and inhibits dopel-tyrosine kinase receptor signaling, such as inhibiting or interfering with the interaction between a dopel and a tyrosine kinase receptor selected from VEGFR2, VEGFR1, VEGFR3, bFGFR, and PDGFR. do.

In some embodiments, the Doppel-targeting molecule is selected from an anti-Doppel antibody, a Doppel-binding fragment of an anti-Doppel antibody, and a heparin-containing conjugate.

In some embodiments, the Doppel-targeting molecule is

(i) an antibody produced by the hybridoma cell line clone 4D6 or a Doppel-binding fragment or derivative thereof;

(ii) Antibody produced by hybridoma cell line clone 5C7, or a Doppel-binding fragment or derivative thereof

(iii) an antibody produced by the hybridoma cell line clone 7D9 or a Doppel-binding fragment or derivative thereof;

(iv) a Doppel-binding fragment or derivative thereof produced by or by hybridoma cell line clone 1B12;

(v) an antibody produced by the hybridoma cell line clone 1C8, or a Doppel-binding fragment or derivative thereof; And

(vi) Antibodies produced by hybridoma cell line clone 6F11 or a Doppel-binding fragment or derivative thereof

It is an anti-Doppel antibody selected from.

In some embodiments, the Doppel-targeting molecule is a heparin-containing conjugate comprising a heparin moiety conjugated to a bile acid moiety or a sterol moiety. In some embodiments, the Doppel-targeting molecule is an LMWH- oligo DOCA conjugate. In some embodiments, the Doppel-targeting molecule is an LMWH- selected from LHbisD4, LHmonoD1, LHmonoD2, LHmonoD4, LHbisD1, LHbisD2, LHtriD1, LHtriD3, and LHtetraD1. Oligo is a DOCA conjugate.

In some embodiments, the Doppel-targeting molecule is intravenous injection, oral administration, subcutaneous injection, intraocular injection, intradermal injection, intramuscular injection, intraperitoneal injection, intratracheal administration, inhalation, intranasal administration, sublingual administration, oral administration, Formulated for rectal administration, vaginal administration, or topical administration.

In some embodiments, the Doppel-targeting molecule is formulated into a composition for intravenous administration.

In some embodiments, the Doppel-targeting molecule is formulated into a composition for oral administration. In some embodiments, the Doppel-targeting molecule is formulated into a composition for oral administration and selective absorption in the intestine or ileum.

The present application also provides a method for inhibiting pathological angiogenesis in a subject in need thereof, a method for treating a tumor in a subject in need thereof, a method for inhibiting tumor development in a subject in need thereof, and blood in need thereof. A method of reducing the vascular structure of a tumor in a subject, a method of treating a disease or condition selected from cancer, atherosclerosis, tuberculosis, asthma, and pulmonary arterial hypertension (PAH) in a subject in need thereof, and a subject in need thereof In neoplasms or neoplasm-related conditions such as breast carcinoma, lung carcinoma, gastric carcinoma, esophageal carcinoma, colorectal carcinoma, liver carcinoma, ovarian carcinoma, androblastoma, cervical carcinoma, endometrial carcinoma, endometrial hyperplasia, Endometriosis, fibrosarcoma, sarcoid carcinoma, head and neck cancer, nasopharyngeal carcinoma, laryngeal carcinoma, hepatoblastoma, Kaposi's sarcoma, melanoma, skin carcinoma, hemangioma, cavernous hemangioma, hemangioblastoma, pancreatic carcinoma, retinoblastoma, astrocytoma, glioblastoma Blastoma, schwannoma, oligodendroglioma, glioblastoma, neuroblastoma, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcoma, urinary tract carcinoma, thyroid carcinoma, Wilm's tumor, renal cell carcinoma, prostate carcinoma, nevus-associated abnormal angioproliferative disorder, brain tumor-associated edema, and /Or a method of treating Meigs' syndrome, and/or detecting Dopel expression in a subject, wherein the method comprises administering to the subject an effective amount of a Doppel-targeting molecule as described herein. Includes.

In some embodiments, the Doppel-targeting molecule is oral administration, intravenous injection, subcutaneous injection, intraocular injection, intradermal injection, intramuscular injection, intraperitoneal injection, intratracheal administration, inhalation, intranasal administration, sublingual administration, oral administration, It is administered by rectal administration, vaginal administration, or topical administration. In some embodiments, the method comprises orally administering a Doppel-targeting molecule. In some embodiments, the method comprises administering the Doppel-targeting molecule by intravenous injection.

In some embodiments, the anti-Doppel antibody or a Doppel-binding fragment thereof specifically binds SEQ ID NO: 11, SEQ ID NO:12, or SEQ ID NO: 61.

According to any embodiment, the subject can be a human. According to any embodiment, the subject is cancer, atherosclerosis, tuberculosis, asthma, and pulmonary arterial hypertension (PAH), or a neoplasm or neoplasm-related condition such as breast carcinoma, lung carcinoma, gastric carcinoma, esophageal carcinoma, Direct colon carcinoma, liver carcinoma, ovarian carcinoma, androblastoma, cervical carcinoma, endometrial carcinoma, endometrial hyperplasia, endometriosis, fibrosarcoma, sarcoid carcinoma, head and neck cancer, nasopharyngeal carcinoma, laryngeal carcinoma, hepatoblastoma, Kaposi's sarcoma, melanoma, skin carcinoma, hemangioma, cavernous hemangioma, hemangioblastoma, pancreatic carcinoma, retinoblastoma, astrocytoma, glioblastoma, schwannoma, oligodendrogliomas, glioblastoma, neuroblastoma, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcoma, urinary tract A disease or condition selected from carcinoma, thyroid carcinoma, Wilm's tumor, renal cell carcinoma, prostate carcinoma, nevus-associated dysvascular hyperplasia, brain tumor-associated edema, and/or Meigs syndrome, a tumor or at risk of developing it.

In some embodiments, the Dopel-targeting molecule is used or administered in an effective amount effective to interfere with the interaction of Dopel with a tyrosine kinase receptor such as VEGFR2, VEGFR1, VEGFR3, bFGFR, and/or PDGFR. In some embodiments, the effective amount is effective to inhibit angiogenesis. In some embodiments, an effective amount is effective in inhibiting tumorigenesis. In some embodiments, the effective amount is effective to reduce the vasculature of the tumor. In some embodiments, the effective amount is effective to reduce the pathological vasculature associated with cancer, atherosclerosis, tuberculosis, asthma, or pulmonary arterial hypertension (PAH), respectively.

With respect to methods and uses for detecting Dopel expression in a subject, the Dopel-targeting molecule can be administered to a subject or to a physiological sample obtained from a subject. The subject is any as described above, and as described above, for example, has a risk of developing a tumor and/or a disease selected from cancer, atherosclerosis, tuberculosis, asthma, and pulmonary arterial hypertension (PAH). Or there may be a risk of developing a condition and/or a risk of developing a neoplasm or neoplasm-related condition.

In addition, there is provided a hybridoma cell line that produces a Doppel-binding antibody as described herein, comprising the following (i) to (vi):

(i) Hybridoma cell line clone 4D6 deposited on November 23, 2016 as mPrnd# 4D6 with the Korea Cell Line Research Foundation under accession number KCLRE-BP-00384;

(ii) Hybridoma cell line clone 5C7 deposited on November 23, 2016 as mPrnd# 5C7 with the Korea Cell Line Research Foundation under accession number KCLRE-BP-00385;

(iii) Hybridoma cell line clone 7D9 deposited on November 23, 2016 as mPrnd# 7D9 with the Korea Cell Line Research Foundation under accession number KCLRE-BP-00387;

(iv) Hybridoma cell line clone 1B12 deposited on November 23, 2016 as mPrnd# 1B12 with the Korea Cell Line Research Foundation under accession number KCLRE-BP-00383;

(v) Hybridoma cell line clone 1C8 deposited on November 23, 2016 as mPrnd# 1C8 with the Korea Cell Line Research Foundation under accession number KCLRE-BP-00382; And

(vi) Hybridoma cell line clone 6F11 deposited on November 23, 2016 as mPrnd# 6F11 with the Korea Cell Line Research Foundation under accession number KCLRE-BP-00386.

In addition, there is provided a composition comprising a monoclonal antibody produced by these hybridoma cell lines, and a pharmaceutically acceptable carrier or diluent therewith.

The present application inhibits pathological angiogenesis and treats diseases and conditions associated with pathological angiogenesis, such as tumors, cancer, atherosclerosis, tuberculosis, asthma, pulmonary arterial hypertension (PAH), neoplasms and neoplasm-related conditions, and blood Doppel-targeting molecules useful for detecting Dopel expression in a subject are described.

1 shows the expression of Doppel in blood vessels of mouse xenograft tumor tissues by whole tissue fixation (top) and immunofluorescence (bottom) staining.
Figure 2 shows the relative mRNA expression of Doppel in mouse normal endothelial cells (EC) and tumor ECs (mean ± sem).
Figure 3 shows the dose-dependent inhibition of TEC budding by anti-Doppel antibodies upon stimulation with 10% fetal bovine serum, or mouse VEGF. Results are mean±sem, and scale bars represent 50 μm.
Figure 4 shows the schematic structure of the LMWH-based conjugates synthesized to enhance binding affinity for dopel. Structures depict different oligomers of deoxycholic acid ( oligo DOCA), designated mono DOCA, bis DOCA, tri DOCA, and tetra DOCA. The structure and conjugation ratio of the LMWH- oligo DOCA conjugate conjugated with the -COOH group of the saccharide unit varies from 1 to 4 molecules per molecule of LMWH.
Figure 5 shows in vitro doppel binding of LMWH-oligoDOCA conjugates using SPR. LHbisD4, in which four molecules of the dimer deoxycholic acid were conjugated to one molecule of LMWH, showed the highest reaction unit after binding to Doppel among all conjugates screened.
6 shows the SPR analysis between Prnp -LH bis D4 and Prnd -LH bis D4. The dissociation rate constant is calculated from the response curve.
7 shows LHbisD4 bound to TEC and TEC ^-/-dpl.
8 shows a molecular dynamic simulation between the spherical domain of Doppel and the LHbisD4 fragment. Fragment-based structural studies using computer simulations have shown that Doppel's globular domain contains a hydrophobic groove to accommodate the large hydrophobic side chain of LHbisD4, so that the Doppel's globular domain plays a key role in the binding of LHbisD4. Showed.
9 shows the plasma concentrations of LMWH and LHbisD4 after oral delivery to rats. Results are mean±sem. LHbisD4 is absorbed orally.
Figure 10 shows the distribution of Cy5.5-labeled LHbisD4 in ^{TEC and TEC -/-dpl} plugs in vivo and ex vivo after its oral delivery.
Figure 11 is the total fluorescence measurements (mean ± sem) of LHbisD4 administered orally and LMWH injected intravenously in TEC and TEC ^{-/-dpl plugs.}
12 is a tumor growth inhibition experiment of LHbisD4 orally administered to MDA-MB-231 tumors once a day at a dose of 2.5, 5, and 10 mg/kg or twice a day at a dose of 5 and 10 mg/kg. Shows. Results are mean±sem. *** p <0.001 between each group and control group. Between 10 mg/kg once daily and 10 mg/kg twice daily, p <0.01.
Figure 13 shows the difference in MDA-MB-231 tumor weight when treated with different doses of the tested LHbisD4 (mean ± sem, * p <0.05).
14 shows an experiment for inhibiting tumor growth in SCC7 tumors of an anti-Doppel antibody injected intravenously at a dose of 1 mg/kg once for 4 days. Results are mean±sem.
Figures 15A-15D show that the absence of doppel in mice was wild-type (WT), and doppel heterozygous (Dpl ^+/- ), and homozygous (Dpl ^-/- ) knockout C57BL/6 mice (n = 3-4). It has been shown to delay tumor growth and angiogenesis of melanoma (B16f10). Tumor volume was monitored for 21 days after inoculation (FIG. 15A). A picture of the isolated tumor at the end of the experiment is shown (FIG. 15B). Mean tumor weight was also evaluated (FIG. 15C ). Data are mean±sem; For WT *** P <0.001 and between ^{Dpl +/-} and Dpl ^-/- ** P <0.01. 15D shows immunofluorescence images of Doppel (green), CD31 (red), and DAPI (blue) at B16f10 tumor incisions isolated from ^{WT, Dpl +/-} , and Dpl ^{-/- mice.}
Figures 16A-16D depict the volume of thymoma (EL4) in wild type (WT), Doppel heterozygote (Dpl ^+/- ), and Doppel homozygous (Dpl ^-/- ) knockout C57BL/6 mice (n = 3). do. Tumor volume was monitored for 11 days after inoculation (Figure 16A). Pictures of isolated tumors at the end of the experiment (FIG. 16B) and average tumor weight (FIG. 16C) are also shown. Data are presented as mean ± sem, *** P <0.001 for WT, and ** P <0.01 ^{between Dpl +/-} and Dpl ^-/-. Figure 16D shows immunofluorescence images of Doppel (green), CD31 (red), and DAPI (blue) in EL4 tumor incisions isolated from ^{WT, Dpl +/-} , and Dpl ^{-/- mice.}
17A-17D depict the volume of colon cancer (CT26) in wild-type (WT) and Doppel heterozygote (Dpl ^+/- ) knockout C57BL/6 mice (n = 3). Tumor volume was monitored for 21 days after inoculation (FIG. 17A ). Pictures of the isolated tumors at the end of the experiment (FIG. 17B) and average tumor weight (FIG. 17C) are shown. Data are expressed as mean±sem. 17D shows immunofluorescence images of Doppel (green), CD31 (red), and DAPI (blue) at EL4 tumor incisions isolated from ^{WT, and Dpl +/- mice.}
18A-18D are CD31 immunofluorescence images of wound-healing vessels in wild-type (WT), Doppel heterozygote (Dpl ^+/- ) and homozygous (Dpl ^-/- ) knockout C57BL/6 mice (FIG. 18A) and The wound-healing rate (FIG. 18B) is shown (n = 3, mean ± sem). Wounds were created in tumor-bearing mice. Matrigel ^® -fibrin matrix was implanted subcutaneously in the presence of VEGF in WT, Dpl ^+/- , and Dpl ^-/- knockout C57BL/6 mice (n = 3). ^{Figure 18C shows hemoglobin levels in Matrigel ®} -fibrin plugs 1 week after implantation and Figure 18D shows H&E staining.
19A-19G show that anti-Doppel mAb retards tumor growth and angiogenesis. Figures 19A and 19B show intravenous IgG (10 mg/kg, once weekly), 5C7 antidoffel mAb (5 and 10 mg/kg, once weekly), and 4D6 antidoffel mAb (5 and 10 mg/kg weekly). It shows the change in volume and weight of colon cancer (CT26) in mice (n = 5) treated once and 10 mg/kg twice a week). Data are mean ± sem, ** p <0.01, *** p <0.001 compared to control. Between groups 10 mg/kg once a week and 10 mg/kg twice a week *** p <0.001. 19C and 19D show changes in volume and weight of human colon cancer (HCT116) treated with saline (control) and intravenous 4D6 antidoffel mAb (5 and 10 mg/kg once a week and 10 mg/kg twice a week). Is shown (n = 5). Figure 19E shows immunofluorescence images of Doppel (red), CD31 (green), and DAPI (blue) of tumor incisions isolated from HCT116 tumor-bearing mice treated with saline and test antibodies. Figure 19F shows immunohistochemical staining for CD31 and Figure 19G shows the amount of mean vascular density of tumor incisions isolated from HCT116 tumor-bearing mice treated with saline and test antibody.
Figures 20A-20D show the generation of anti-Doffel mAb. Figure 20A shows the inhibition of TEC germination after incubation with the cell supernatant of the different clones (7D9, 1B12, 1C8, 5C7, 4D6, 6F11) of the Doppel-blocking mAb, and the Doppel-blocking mAb in the presence or absence of mouse VEGF. Figure 20B shows the total number of germinations from each TEC spheroid (n = 50, mean ± sem). Figure 20C shows immunoblot of phosphorylation-VEGFR2, total VEGFR2, and actin in TEC treated with different concentrations of purified anti-Doppel mAb clones (5C7 and 4D6) upon stimulation with VEGF (50 ng/mL). Figure 20D shows the inhibition of TEC germination after incubation with different concentrations of purified 4D6 anti-Doppel mAb.
21A-21D relate to CT26 tumor-bearing mice treated with control and test antibodies. 21A shows a picture of a CT26 tumor isolated at the end of the experiment. Figure 21B shows immunofluorescence images of Doppel (red), CD31 (green), and DAPI (blue) in tumor incisions isolated from CT26 tumor-bearing mice treated with control and test antibodies. Figure 21C depicts immunohistochemical staining for CD31 and Figure 21D depicts the amount of mean vascular density in tumor incisions isolated from CT26 tumor-bearing mice treated with saline and test antibody.
Figures 22A-22I show that Doppel maintains VEGFR2 on the cell surface and promotes VEGF-signaling. Figure 22A shows VEGFR2 (green) in HCT116 tumor incisions treated with saline and 4D6 anti-Doppel mAb (10 mg/kg twice a week) ^{and B16f10 tumor incisions from WT, Dpl +/-} , and Dpl ^{-/- mice.} , CD31 (red), and DAPI (blue) are shown. Figure 22B shows the whole lung from B16f10 tumor-bearing WT, Dpl ^+/- , and Dpl ^-/- mice and treated with saline and 4D6 anti-Doppel mAb (10 mg/kg twice weekly) from HCT116 tumor-bearing mice and Immunoblots of total VEGFR2, Doppel, and Actin present in the tumor are shown. Figures 22C-22E show blots of protein and mRNA levels of VEGFR2 and Doppel after treatment with scrambled siRNA and Doppel siRNA in TEC isolated from squamous (TEC-SCC7) and colon (TEC-CT26) cancers. 22F shows blots showing surface and total VEGFR2 and Doppel levels in unstimulated and VEGF-stimulated (5 min; 100 ng/mL) HUVECs and Hu.dpl. The rate of VEGFR2 after incubation with VEGF (25 ng/mL) in impermeable HUVEC and Hu.dpl is shown in Fig.22G, where the amount of VEGFR2 on the cell membrane over time is shown in the case of HUVEC and Hu.dpl, respectively. Figure 22H shows the immunoblot of phosphorylation-VEGFR2, total VEGFR2, total doppel, and GAPDH in HUVEC and Hu.dpl when treated with different concentrations of VEGF. Figure 22I illustrates the proposed mechanism of action, wherein healthy endothelial cells express VEGFR2 but not doppel, and tumor endothelial cells express VEGFR2 with doppel, acting as a support pillar for VEGFR, and the proposed In the mechanism of action, the Doppel monoclonal antibody (Doppel mAb) targets Doppel, degrading the Doppel-VEGFR2 complex, inhibiting VEGF signaling, and stopping tumor angiogenesis.
Figures 23A-23B show VEGFR2 in EL4 tumor incisions isolated from ^{WT, Dpl +/-} , and Dpl ^{-/- mice (Figure 23A) and CT26 tumor incisions from WT and Dpl +/-} mice (Figure 23B). Immunofluorescence images of (green), CD31 (red), and DAPI (blue) are shown.
Figures 24A-24B show VEGFR2 (green), CD31 (red), and DAPI (blue) in mouse CT26 colon cancer (Figure 24A) and human HCT116 colon tumor incisions (Figure 24B) isolated from mice treated with saline and test compounds. The immunofluorescence image of is shown.
Figure 25A shows the inhibition of Doppel expression by Doppel siRNA in Doppel-transfected HUVECs (Hu.dpl). 25B and 25C show the mRNA levels in Doppel (FIG. 25B) and VEGFR2 (FIG. 25C) after treatment of Hu.dpl with scrambled and Doppel esiRNA or siRNA.
Figures 26A-26C show flow cytometric analysis of VEGFR2 after incubation with VEGF (25 ng/mL) in impermeable HUVEC and Hu.dpl (Figure 26A); Blot showing membrane and cytoplasmic VEGFR2 and Doppel levels in unstimulated and VEGF-stimulated (5 min; 100 ng/mL) HUVEC and Hu.dpl (FIG. 26B ); And blots showing total VEGFR2 and Doppel levels in unstimulated and different concentrations of VEGF-stimulated (5 min) HUVEC and Hu.dpl (FIG. 26C ).
Figures 27A-27G show that recombinant doppel promotes angiogenesis. Figure 27A shows the induction of germination in HUVEC spheroids after incubation with different concentrations of recombinant Doppel (rDP l ) in the presence or absence of VEGF, and Figure 27B shows the total number of germinations in each HUVEC spheroid (n = 50, mean ± sem). Doppel transfected HUVEC (Hu.dpl) spheroids were used for comparison in the presence of VEGF. HUVEC and Hu.dpl spheroids, dispersed in a matrigel-fibrin matrix, were implanted subcutaneously into female SCID mice with 10 μg of rDP l in the presence or absence of VEGF. It was stained 1 week after transplantation. Figure 27C shows the 3D structure of the vascular network formed in HUVEC and Hu.dpl spheroids, stained with anti-human CD34 (red) and stained with confocal microscopy, and Figure 27D is in the HUVEC and Hu.dpl spheroid plugs. Shows the hemoglobin level. Figure 27E- Figure 27G is treated with intravenous rDP l (1 mg/kg, once every 2 days after 4 days of cancer cell inoculation) or Doppel heterozygote (Dpl ^+/- ) knockout C57BL/6 mice (n = 5 ) Shows the changes in volume and weight of thymoma cancer (EL4) inoculated with 1 and 10 μg of rDP l in ). Tumor volume was monitored for 10 days after inoculation (FIG. 27E ). An image of the explanted tumor was taken (FIG. 27F), and the tumor weight was measured (FIG. 27G). Data are mean±sem.
Figures 28A-28E show the expression of Doppel in human skin microvascular endothelial cells (HDMECs) 1 day after transfection with Doppel cDNA (Figure 28A). Figure 28B shows the immunoblot of phosphorylation-VEGFR2, total VEGFR2, total Doppel, and GAPDH in transient Doppel-transfected HDMECs upon treatment with VEGF. Figure 28C depicts cell proliferation and Figures 28D and 28E depict the emergence of transient Doppel-transfected HDMECs.
29A-29B show that Doppel interacts with VEGFR2. Figure 29A shows a blot by immunoprecipitation (IP) of VEGFR2 and Doppel in recombinant Doppel (rDp 1) and Doppel cDNA (Dpl cDNA) transfected cells. Figure 29B shows the phosphorylation in the process when, HUVEC with different concentrations of VEGF in the presence or absence of rDp l (1 ㎍ / mL) -VEGFR2, total VEGFR2, immunoblot of whole dopel, and GAPDH.
FIG. 30 shows the colocalization between the injected rDp 1 -Alexa 488 and blood vessels (FIG. 30A) and the injected rDp 1 -Alexa 488 and VEGFR2 (FIG. 30B) in the tumor incision.
31A-C show the annotated amino acid sequence for Doppel. Figure 31A shows an amino acid sequence comprising a signal peptide at the N-terminus and a poly-His tail at the C-terminus (SEQ ID NO: 9). Figure 31B shows the amino acid sequence with a poly-His tail at the C-terminus (SEQ ID NO: 10). Figure 31C shows the domain map of the dopples represented by the signal peptide and poly-His tail as in Figure 31A.
Figure 32 shows the results of an epitope mapping experiment for mouse antibody 4D6, showing that 4D6 binds to the epitope YWQFPD (SEQ ID NO: 11).
FIG. 33 shows the results of an epitope mapping experiment for mouse antibody 5C7, indicating that 5C7 binds to the epitope FPDGIY of Doppel (SEQ ID NO: 12). )

The uses, compositions, and methods described herein are due to the discovery that the prion-like protein, Doppel, is specific for tumor endothelial cells (TEC), ie, tumor endothelial cells (TEC) surface markers. The present inventors have found that high expression of doppel on TEC correlates with high blood vessel formation. Without wishing to be bound by theory, it is believed that Doppel plays a role in angiogenesis by interacting with tyrosine kinase receptors such as VEGFR2. In this regard, the present inventors confirmed that Dopel co-localized with the tyrosine kinase receptor to form a complex, and that inhibition of Dopel depleted the membrane tyrosine kinase receptor.

Thus, in accordance with some embodiments, a method based on the discovery that inhibits the activity of the doppel-targeting molecule used to inhibit pathological angiogenesis in a subject in need thereof, and, for example, by binding to the doppel, is optionally By inhibiting pathological Doppel-tyrosine kinase receptor signaling, pathological blood vessels, including pathologic angiogenesis and tumor angiogenesis associated with asthma, tuberculosis, atherosclerosis, pulmonary arterial hypertension (PAH), and neoplastic and neoplastic-related conditions. It can provide selective inhibition of production.

Thus, in some embodiments, it is used to inhibit pathological angiogenesis in a subject in need thereof, e.g., a molecule that inhibits Doppel activity, such as interfering with Dopel expression (e.g., Doppel antisense nucleic acid) or Doppel and tyrosine kinase receptors. Use in a method for inhibiting pathologic angiogenesis comprising administering a molecule that inhibits the interaction of (e.g., binds to a dopel, or blocks a binding site of the doppel-tyrosine kinase receptor signaling pathway). Doppel-targeting molecules for

In some embodiments, a method for inhibiting pathologic angiogenesis is provided by administering a dopel-targeting molecule that binds to dopel, inhibits dopel-tyrosine kinase receptor signaling, and inhibits angiogenesis. The present application also describes doppel-targeting molecules (eg, molecules that bind to doppel) and pharmaceutical compositions comprising them.

Endothelial cells (ECs) lining the blood vessels are advantageous targets for cancer therapy as they may be more accessible to circulating pharmaceuticals. Thus, targeting tumor endothelial cell surface markers such as Doppel provides a more predictable and more effective approach to cancer therapy than targeting cancer cells.

Angiogenesis-based tumor therapy has several theoretical advantages over traditional cancer therapy (such as radiation and chemotherapy). For example, (1) EC is more genetically stable than cancer cells and does not show gene mutations, (2) phenotypic expression of tumor endothelium is different from normal endothelial cells, and (3) EC is Because it is not part of it, the tendency to develop resistance to chemotherapy is low, and (4) the location of the endothelium at the border between blood and tumor cells makes the targeting of highly expressed or upregulated surface receptors or proteins attractive.

Justice

Unless otherwise defined, technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which the present invention belongs. Reference is made to various methodologies known to those skilled in the art. Publications and other documents describing such known methodologies with reference are incorporated herein by reference in their entirety as fully described. Any suitable materials and/or methods known to those skilled in the art can be used to carry out the present invention. However, specific materials and methods are described. Materials, reagents, etc. referenced in the following description and examples can be obtained from commercial sources unless otherwise noted.

The following terms are used throughout as defined below.

As used herein and in the appended claims, singular forms such as "a" and "a" and "the" and similar references in the context describing the components (especially the claims below) are not otherwise specified herein or explicitly contradicted by context. , Interpreted as encompassing both the singular and the plural. The recitation of numerical ranges herein is only intended to be provided in shorthand, referring individually to each distinct value falling within that range, unless otherwise specified herein, each distinct value being used herein to the extent that it is individually recited herein. Is incorporated into. All methods described herein can be performed in any suitable order unless otherwise specified herein or otherwise clearly contradicted by context. The use of any and all examples, or illustrative language ("for example" or "for example") herein, unless otherwise stated, is merely intended to better illustrate the embodiment and not to limit the scope of the claims. The language of this specification should not be construed as necessarily referring to any non-claimed component.

As used herein, “about” is understood by those of skill in the art and will vary to some extent depending on the context in which it is used. When there is use of a term that is not clear to those skilled in the art in the context in which it is used, “about” means adding or subtracting up to 10% of the particular term.

As used herein, “subject” refers to any animal in need of anti-tumor or anti-cancer or anti-angiogenic therapy, including any mammal, including humans, cats, rats, dogs, horses, monkeys, or other species. it means. In some embodiments, the subject is a human. For example, the subject may have or are at risk of developing a tumor or cancer.

As used herein, the phrase “effective amount” refers to an amount that provides a particular effect to which a molecule, agent, or composition is administered. It is emphasized that an effective amount is not always effective in treating a target condition in a given subject, although such an amount is considered to be an effective amount by one of skill in the art. One of skill in the art can determine and adjust such amounts according to standard practice necessary to treat a particular subject and/or condition.

As used herein, the term “angiogenesis” refers to the creation of new blood vessels. As used herein, the term "oncogenic" refers to the growth of a tumor. As used herein, the term “pathological angiogenesis” refers to angiogenesis associated with cancer, tumor, or other disease or condition, such as a disease or condition associated with increased vasculature, and is distinguished from physiological angiogenesis, such as growth, wound Occurs during healing, and formation of granulation tissue.

As used herein, the term "angiogenic factor" refers to molecules that promote angiogenesis, such as VEGF, FGF, PDGFB, EGF, LPA, HGF, PD-ECF, IL-8, angiogenesis, TNF-alpha, TGF-beta, TGF-alpha, proliferin, and PLGF.

As used herein, the term “tyrosine kinase receptor” refers to a class of cell surface receptors having an extracellular domain that binds a ligand and an intracellular domain that phosphorylates a tyrosine amino acid. Most tyrosine kinase receptors have high affinity for certain growth factors, cytokines, or hormones. Tyrosine kinase receptors are families based on structural similarity, such as EGF receptor family, insulin receptor family, PDGF receptor (PDGFR) family, FGF receptor (FGFR) family, VEGF receptor (VEGFR) family, HGF receptor family, Trk receptor family, Eph receptor family, LTK receptor family, TIE receptor family, ROR receptor family, DDR receptor family, RET receptor family, KLG receptor family, RYK receptor family, and MuSK receptor family. Non-limiting examples of tyrosine kinase receptors related to the methods described herein include VEGFR2, VEGFR1, VEGFR3, bFGFR, and PDGFR.

As used herein, the term “VEGF receptor” or “VEGFR” refers to a cell receptor for VEGF, usually a cell-surface receptor present on vascular endothelial cells, as well as variants thereof that retain the ability to bind to VEGF. An example of a VEGF receptor is the fms-like tyrosine kinase (flt) (also known as VEGFR1), a transmembrane receptor of the tyrosine kinase family. The flt receptor includes an extracellular domain, a transmembrane domain, and an intracellular domain with tyrosine kinase activity. The extracellular domain is involved in the binding of VEGF while the intracellular domain is involved in signal transduction. Another example of a VEGF receptor is the flk-1 receptor (also known as KDR or VEGFR2). VEGFR2 exhibits potent tyrosine kinase receptor activity and plays a major role in angiogenesis.

As used herein, the phrases “composition for inhibiting tumor-associated angiogenesis” and “composition for inhibiting tumor-associated tumor development” refer to therapeutic agents for inhibiting tumor-associated angiogenesis or tumorigenesis by reducing tumor vasculature ( Molecules).

In the following content, for purposes of explanation and not limitation, detailed and specific embodiments are set forth in order to provide a thorough understanding of the present invention. It will be apparent to those skilled in the art that the present invention can be implemented in other embodiments that deviate from the details and specific embodiments. In addition, while the present invention is described in terms of specific embodiments, and specific combinations of properties, the present invention provides for the various options described herein to all possible modifications and combinations, such as Doppel-targeting molecules, agents, routes of administration, target subjects. It should be understood that all possible variations and combinations, such as, effective amounts, etc. are included.

All publications and other references described herein are incorporated herein by reference.

Certain aspects of the invention described herein are described in [Al-Hilal, et. al., J. Clin. Invest. 126 (4): 1251-66 (Apr. 2016), the entire contents of which are incorporated herein by reference.

Doppel

Dpl is a prion-like protein encoded by a gene named PRNL located near the PRNP (prion protein coding gene) locus. See, eg, Golaniska et al. , Folia Neuropathol. 42 (Supp. A) 47-54 (2004). Doppel is evolutionarily conserved from humans to sheep and cattle to rats, suggesting essential functions (Behrens et al ., EMBO Reports 2:347 (2001); Behrens et al, EMBO J. 21:3652 (2002)). . Human Doppel is a 179 amino acid residue protein with a molecular weight of about 14 KDa (nonglycosylated). The amino acid sequence of the peptide is shown in Figure 31. The glycosylated form of Doppel with a band size of -42 KDa has been reported in the literature. See, for example, Al-Hilal et al. , J. Clinic. Invest. 126:1251-1266 (2016)] and Peoc'h et al. , J Biol Chem. 277(45):43071-43078 (2002). Doppel has also been reported to exist in dimer form (~28 KDa).

As used herein, “dopel” means any dopel protein. In some embodiments, the Doppel protein is glycosylated. In some embodiments, the Doppel protein is not glycosylated. In some embodiments, the doppel protein is in monomeric form. In some embodiments, the Doppel protein is in a dimeric form.

As mentioned above, the uses, compositions and methods described herein allow doppel to play a role in pathological angiogenesis, such as inhibiting interactions with tyrosine kinase receptors (e.g., VEGFR2), such as by binding to doppels. Tumor endothelial cell (TEC) surface marker that inhibits Doppel angiogenesis, or otherwise, tumor angiogenesis and pathological vessels associated with such as asthma, tuberculosis, atherosclerosis, pulmonary hypertension (PAH), neoplasms, and neoplastic-related conditions. It is due to the inventor's discovery that pathological angiogenesis, including production, can be selectively inhibited.

For example, Doppel expression on TEC may increase during pathologic angiogenesis relative to its expression during normal or physiological angiogenesis conditions, or Doppel expression on TEC may be associated with pathological tumor-associated angiogenesis or tumorigenesis.

Doppel-targeting molecule

Thus, according to some embodiments, a molecule for inhibiting pathologic angiogenesis and methods of use thereof are provided, wherein the molecule inhibits doppel activity, such as the molecule inhibits dopel expression or Interfere with the interaction (eg, inhibit Doppel-VEGFR2 signaling). Such molecules are referred to herein as "Doppel-targeting" molecules.

In some embodiments, the dopel-targeting molecule binds to the dopel and interferes with the interaction of dopel with one or more of the tyrosine kinase receptors, such as VEGFR2, VEGFR1, VEGFR3, bFGFR, PDGFR. In some embodiments, the molecule comprises a molecule that binds to a dopel, preferentially binds to a dopel, and/or inhibits interaction with one or more of tyrosine kinase receptors such as VEGFR2, VEGFR1, VEGFR3, bFGFR, and PDGFR. .

In some embodiments, the Doppel-targeting molecule is an anti-Doppel antibody, or a Doppel-binding fragment thereof, as described below.

In some embodiments, the Doppel-targeting molecule is a heparin conjugate, as described in detail below.

In some embodiments, the Doppel-targeting molecule targets the Doppel-VEGFR2 axis and induces its internalization and degradation.

In some embodiments, the dopel-targeting molecule binds to a dopel expressed on the TEC surface. In some embodiments, the Dopel-targeting molecule targets tumor endothelial cells that express Dopel.

In some embodiments, the Doppel-targeting molecule elicits a therapeutic response in tumor endothelial cells.

In some embodiments, the Doppel-targeting molecule is useful for detecting tumor endothelial cells, such as when the Doppel-targeting molecule comprises or is conjugated to a detectable label.

Heparin-containing conjugate

In some embodiments, the Doppel-targeting molecule is a heparin-containing conjugate. As used herein, “heparin-containing conjugate” refers to a conjugate comprising heparin conjugated to another moiety, such as another functional moiety, directly or through a linker.

As used herein, “heparin” refers to any heparin species, including, without limitation, unfractionated heparin, low molecular weight heparin, very low molecular weight (vLMWH) heparin, and heparin sulfate.

In some embodiments, the functional moiety of the heparin-containing conjugate is a bile acid, which can be conjugated to heparin to provide an increased or decreased doppel-targeting efficacy, or to target delivery to a specific site of action, such as the intestine or ileum. Or a monomer, a dimer, or an oligomer of a sterol.

Examples of suitable bile acids include, without limitation, cholic acid, deoxycholic acid, kenodeoxycholic acid, lysocholic acid, ursocholic acid, isoursodeoxycholic acid, ragodeoxycholic acid, glycocholic acid, taurocholic acid, glycodeoxycholic acid, glycosyl Kenodeoxycholic acid, dihydrocholic acid, hyocholic acid, hyodeoxycholic acid, and any mixture of two or more thereof.

Examples of suitable sterols include, without limitation, cholesterol, coprostanol, cholesterol, epicholesterol, ergosterol, ergocalciphenol, and any mixture of two or more thereof.

An exemplary heparin conjugate useful as a Doppel-targeting molecule is an extremely low molecular weight (vLMWH) heparin conjugated to one or more deoxycholic acid (DOCA) to form an oligomer of deoxycholic acid (oligo DOCA). These conjugates are called LMWH-oligo DOCA conjugates. Different oligo DOCA conjugates include mono DOCA, bis DOCA, tri DOCA, and tetra DOCA. The LMWH-oligoDOCA conjugate can be conjugated with the -COOH group of the saccharide unit. In an exemplary embodiment, the conjugation rate may vary from 1 to 4 molecules per molecule of LMWH. In certain embodiments, the conjugate is LHbisD4, an LMWH-oligoDOCA conjugate comprising low molecular weight heparin conjugated to 4 repeat units of bis DOCA. See, for example, FIG. 4, and see also Al-Hilal et al. , J. Clinic. Invest. 126:1251-1266 (2016) .

Heparin-containing conjugates are effective as Doppel-targeting molecules when administered orally. Thus, in some embodiments, the heparin-conjugate (e.g., LMWH-oligoDOCA conjugate) is formulated for oral administration and used as a dopel-targeting molecule to inhibit pathological angiogenesis, wherein the heparin-containing conjugate is a dopel-targeted It is administered orally for efficacy as a molecule.

Anti-doppel antibody

In some embodiments, the Doppel-targeting molecule is an anti-Doppel antibody, or an antibody fragment or peptide that binds to a related species, such as Dopel, and inhibits the interaction of Dopel and tyrosine kinase receptors such as VEGFR2, VEGFR1, VEGFR3, bFGFR, and PDGFR. It is a Doppel-binding antibody fragment comprising a. In certain embodiments, the anti-dopel antibody or related species binds to dopel and inhibits the interaction of dopel with VEGFR2.

According to any of these embodiments, the antibody is any antibody or polyclonal antibody or monoclonal antibody, or a derivative of an antibody, such as a single chain antibody, chimeric antibody, humanized antibody (or other species-specific modified for use in other target species). Antibodies), veneer antibodies, and the like, and may be antibody-like molecules. Antibodies can be conjugated to other moieties. Antibodies can be glycosylated or non-glycosylated, or have a modified glycosylation pattern.

Antibodies and antibody-like molecules suitable for use in the methods described herein can be prepared using methodologies known in the art. An exemplary method is illustrated in the Examples below.

For example, an antibody is produced in a host (e.g., a mammalian host) using a Doppel protein or fragment thereof, such as an antigen comprising its N-terminal or globular domain, and a tyrosine kinase receptor (e.g., VEGFR2) and its Screened for their ability to inhibit interaction. For example, a polyclonal antibody against Dopel can be prepared by collecting blood from a mammal immunized with Dopelle, investigating for an increase in the antibody of interest in the serum, and isolating the serum from the blood by any conventional method. . In some embodiments, fractions containing polyclonal antibodies can be isolated, including serum containing polyclonal antibodies.

The general structure of antibodies is known in the art and is summarized only briefly herein. Immunoglobulin monomers comprise two heavy chains and two light chains linked by disulfide bonds. Each heavy chain is paired with one of the light chains directly attached through a disulfide bond. Each heavy chain contains a constant region (varies depending on the antibody isotype) and a variable region. The variable region comprises three hypervariable regions (or complementarity determining regions) designated CDRH1, CDRH2 and CDRH3 and supported within the framework region. Each light chain contains a constant region and a variable region, and the variable region contains three hypervariable regions (designated CDRL1, CDRL2 and CDRL3) supported by the framework in a manner similar to the variable region of the heavy chain.

The hypervariable regions of each pair of heavy and light chains cooperate with each other to provide an antigen binding site capable of binding to the target antigen. The binding specificity of the heavy and light chain pairs is defined by their respective CDR sequences. Thus, when a set of CDR sequences generated for a particular binding specificity (i.e., a sequence of three CDRs for a heavy and light chain) is established, that set of CDR sequences is in principle any Any other antibody framework region linked to the antibody constant region can be inserted at an appropriate location.

As used herein, "monoclonal antibody" refers to an antibody obtained from a single clone of B-lymphocytes or from cells transfected with the light and heavy chain genes of a single antibody. Monoclonal antibodies are directed against a single antigenic site and are therefore highly specific. In contrast to conventional (polyclonal) antibody preparations, typically comprising different antibodies, directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies for use in the methods described herein are methods known to those of skill in the art, for example, recovering immune cells from antigen-immunizing mammals, examining high levels of the antibody of interest in serum, and performing cell fusion. Can be produced. Immune cells used for cell fusion are typically obtained from the spleen. Other parental cells suitable to be fused with the immune cells include, for example, myeloma cells of mammals, such as myeloma cells having acquired properties for selection of cells fused by drugs. The immune cells and myeloma cells are prepared by known methods, for example, Milstein et al., Methods Enzymol. 73:3-46 (1981)]. Final hybridomas obtained by cell fusion can be selected by culturing them in standard selection media, such as HAT media (media containing hypoxanthine, aminopterin and thymidine). Cell culture is typically continuous in HAT medium for several days to several weeks, the time sufficient for all other cells to be killed, except for the desired hybridoma (non-fused cells). Next, standard limiting dilutions are performed to screen and clone hybridoma cells producing the desired antibody.

In addition to the above method of immunizing non-human animals with antibodies for producing hybridomas, human lymphocytes such as EB virus-infected can be immunized in vitro with antigens, antigen-expressing cells, or their lysates. The immunized lymphocytes can then be fused with infinitely dividing human-derived myeloma cells, such as U266, to yield hybridomas that produce the desired human antibody capable of binding to the antigen. See, for example, Japanese Published Patent Application (JP-A) Sho 63-17688, which has been requested for non-examination.

The obtained hybridoma can be transplanted into the abdominal cavity of a mouse, and ascites can be extracted. The obtained monoclonal antibody can be purified by, for example, an ammonium sulfate precipitation method, a protein A or protein G column, a DEAE ion exchange chromatography, or an affinity column to which the protein of the present invention is coupled.

"Humanized" forms of non-human (eg, murine) antibodies can be obtained as chimeric antibodies, containing minimal sequences derived from non-human immunoglobulins. In general, humanized antibodies have variable regions derived from non-human immunoglobulins and framework regions (FR) comprising at least one or two variable domains corresponding to human immunoglobulin sequences. Thus, in some embodiments, the anti-Doppel antibody comprises a human antibody framework region. Such antibodies can be prepared by known methods. The humanized antibody may optionally contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See, eg, Jones et al., Nature 321:522-525 (1986); Reichmann et al., Nature 332:323-329 (1988); Presta , Curr. Op. Struct. Biol. 2:593-596 (1992).

As another method for obtaining antibodies useful in the methods described herein, transgenic animals of human antibody genes can be immunized with Doppel protein, Doppel protein-expressing cells, or lysates thereof. The obtained antibody-producing cells are collected and fused with myeloma cells to obtain hybridomas, from which a human antibody against Doppel can be prepared. Alternatively, immune cells that produce antibodies, such as immunized lymphocytes, can be immortalized with oncogenes and used to make monoclonal antibodies.

Monoclonal antibodies against Doppel can also be made using recombinant genetic engineering techniques. See, for example, Borrebaeck CAK and Larrick JW, Therapeutic Monoclonal Antibodies (MacMillan Publishers Ltd. (1990)) For example, DNA encoding an antibody against Doppel is an immune cell, such as an antibody producing antibody. Recombinant Doppel antibodies are prepared by cloning in hybridomas or immunized lymphocytes, inserting into an appropriate vector and introducing into host cells.

As mentioned above, according to any of these embodiments, the Dopel-targeting molecule may be an antibody fragment that binds to the Dopel. As used herein, the term “antibody fragment” or “Doppel-binding fragment” refers to antibodies, including, without limitation, F _ab fragments, F _ab' fragments, F( _ab >) ₂ fragments, and smaller fragments, diabodies, etc. Or any Doppel-binding fragment of an antibody-like molecule. Antibody “fragments” can be prepared from full-length antibodies, or can be synthesized into “fragments”, for example using recombinant techniques.

Antibody fragments useful as Doppel-targeting molecules may comprise a portion of a full length antibody, such as an antigen-binding domain or variable region domain thereof. Examples of suitable antibody fragments are Fab, F(ab')2, Fv, or single chain Fv (scFv), wherein the heavy and light chain derived Fv fragments are ligated by an appropriate linker (e.g., See Huston et al. , Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)); Diamond body; Linear antibodies; Single chain antibody molecules; And multispecific antibodies formed from antibody fragments.

Doppel-binding antibody fragments can be produced by treatment of a doppel-binding antibody with an enzyme such as papain or pepsin. Alternatively, a gene encoding a Doppel antibody fragment can be constructed, inserted into an expression vector, and expressed in an appropriate host cell (Co et al. , J. Immunol. 152:2968-2976 (1994); Better and Horwitz, Methods Enzymol . 178:476-496 (1989); Pluckthun and Skerra, Methods Enzymol . 178:497-515 (1989); Lamoyi, Methods Enzymol. 121:652-663 (1986); Rousseaux et al ., Methods Enzymol. 121:663-669 (1986); Bird and Walker, Trends Biotechnol. 9:132-137 (1991)).

As used herein, the term "diabody" has two antigen-binding sites, wherein the fragment comprises a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL) by a linker. It means a small antibody fragment. If the linker is too short, it cannot pair between the two domains on the same chain, and the domain is forced to pair with the complementary domain of the other chain, resulting in two antigen-binding sites. Diabodies are described in more detail, for example in EP 404 097; WO 93/11161; And Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

As exemplified in the examples, antibodies and antibody fragments can be screened for Doppel-binding activity using conventional techniques. For example, use of absorbance measurement, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blot assay, and/or immunofluorescence assay to determine Dopel-binding activity. can do. For example, in the case of ELISA, a known anti-dopel antibody is immobilized on a plate, and a dopel is applied to this plate, so that a sample containing a test antibody, such as a culture supernatant of an antibody-producing cell or a purified antibody can be applied. have. Next, the primary antibody is recognized and a secondary antibody labeled with an enzyme such as alkaline phosphatase is applied, and the plate is incubated. Next, it is washed, and an enzyme substrate such as nitrophenyl phosphate is added to the plate and the absorbance is measured to evaluate the antigen binding activity of the sample. The C-terminal or N-terminal fragment of the Doppel protein can be used as an antigen. In another example, surface plasmon resonance assays can be used to assess the activity of antibodies according to the invention.

In some embodiments, the anti-Doppel antibody binds to one or more forms of Doppel, such as one or more of the monomeric, dimer, glycosylated and non-glycosylated forms described above. In some embodiments, the anti-Doppel antibody binds to one or more forms of human Dopel, such as one or more of the monomeric, dimer, glycosylated and nonglycosylated forms described above. In some embodiments, the anti-Doppel antibody binds to one or more forms of a non-human species of Doppel, such as one or more of the monomeric, dimer, glycosylated and non-glycosylated forms described above.

In some embodiments, the anti-dopel antibody preferentially binds to one or more forms of dopel described above, such as preferentially to one or more forms of dopel described above as compared to one or more other forms. In some embodiments, the anti-dopel antibody preferentially binds to one or more forms of human doppel. In some embodiments, the anti-Doppel antibody preferentially binds to one or more forms of a non-human species of Doppel.

In some embodiments, the anti-dopel antibody preferentially binds to one of the doppel forms described above, and preferentially binds to one of the doppel forms described above as compared to the other forms. In some embodiments, the anti-dopel antibody preferentially binds to a form of human doppel. In some embodiments, the anti-Doppel antibody preferentially binds to a form of a non-human species of Doppel.

In some embodiments, the anti-Doppel antibody binds to the glycosylated form of human Doppel (described above) having a molecular weight of about 42 KDa with greater affinity than the non-glycosylated form of Doppel. The anti-Doppel antibody produced by the hybridoma cell line clone 4D6 is a specific embodiment of this type of antibody.

In some embodiments, the anti-Doppel antibody binds to the non-glycosylated form of human Doppel (-14 KDa) with an affinity greater than the glycosylated form of Doppel (-42 KDa). Anti-Doppel antibodies produced by hybridoma cell line clones 5C7, 7D9 and 1C8 are specific embodiments of this type of antibody.

In some embodiments, the anti-Doppel antibody binds to the dimeric form of human Doppel protein (~28 KDa). Anti-Doppel antibodies produced by hybridoma cell line clones 6F11 and 1B12 are specific embodiments of this type of antibody.

According to any antibody embodiment, the anti-Doppel antibody is capable of binding to an isolated form of a Doppel, a recombinant form of a human Doppel, and/or a doppel from a Doppel-transfected Huvec cell lysate, and the like.

In some embodiments, the anti-Doppel antibody is an antibody produced in a hybridoma cell line selected from any one of clones 7D9, 1B12, 1C8, 5C7, 4D6, and 6F11, or a derivative of such an antibody, such as of the types of antibody derivatives described above. One or more, or a fragment or derivative thereof, of such an antibody that binds to a dopel, such as any one or more of the types of antibody fragments described above.

The hybridoma cell lines 7D9, 1B12, 1C8, 5C7, 4D6 and 6F11 are the Korean Cell Line Research Foundation, (110- 744), 28 Yeongeon-dong, Jongno-gu, Seoul, Korea, Cancer Research Institute, College of Medicine, Seoul National University).

Table 1 Clone Registered name Accession number 7D9 mPrnd# 7D9 KCLRF-BP-00387 1B12 mPrnd# 1B12 KCLRF-BP-00383 1C8 mPrnd# 1C8 KCLRF-BP-00382 5C7 mPrnd# 5C7 KCLRF-BP-00385 4D6 mPrnd# 4D6 KCLRF-BP-00384 6F11 mPrnd# 6F11 KCLRF-BP-00386

The sequences of the complementarity determining regions (CDRs) and variable chains of exemplary anti-Doppel antibodies are provided in Tables 2 to 5 below.

Table 2-Light chain CDR sequences Antibody CDRL1 CDRL2 CDRL3 4D6 RSSQSIVHSNGNTYLE (SEQ ID NO: 13) KVSNRFS
(SEQ ID NO: 14) FQGSHVPLT
(SEQ ID NO: 15) 1B12 RASESVDSHGNSFMH (SEQ ID NO: 16) LASSLES
(SEQ ID NO: 17) QQNNEDPLT
(SEQ ID NO: 18) 1C8 RSSQSIVHSNGNTYLE (SEQ ID NO: 19) KVSNRFS
(SEQ ID NO: 20) FQGSHVPWT (SEQ ID NO: 21) 7D9 RSSQSIVHSNGNTYLE (SEQ ID NO: 22) KVSNRFS
(SEQ ID NO: 23) FQGSHVPLT
(SEQ ID NO: 24) 5C7 RASQEISGYLS
(SEQ ID NO: 25) AASILDS
(SEQ ID NO: 26) LQYASYPFM
(SEQ ID NO: 27) 6F11 RASQEISGYLS
(SEQ ID NO: 28) AASILDS
(SEQ ID NO: 29) LQYASYPFM
(SEQ ID NO: 30)

Table 3-Heavy chain CDR sequences Antibody CDRH1 CDRH2 CDRH3 4D6 NYWMQ
(SEQ ID NO: 31) AIYPGDGNTRYSQKFKG
(SEQ ID NO: 32) RDYGSSYWYFDV
(SEQ ID NO: 33) 1B12 TSGMGVG
(SEQ ID NO: 34) HIWWDDDKYYNPSLKS (SEQ ID NO: 35) RADGYGFAY (SEQ ID NO: 36) 1C8 NYGMS
(SEQ ID NO: 37) TISSGGRYIYYPDSVKG (SEQ ID NO: 38) DSSDYGFAY
(SEQ ID NO: 39) 7D9 TSGMGVS
(SEQ ID NO: 40) HIYWDDDKRYNPSLKS (SEQ ID NO: 41) TSMMVPPWFAY (SEQ ID NO: 42) 5C7 SHWMN
(SEQ ID NO: 43) QIYPRNGDTNYNGKFKG (SEQ ID NO: 44) STTIVTTGAY (SEQ ID NO: 45) 6F11 SHWMN
(SEQ ID NO: 46) QIYPRNGDTNYNGKFKG (SEQ ID NO: 47) STTIVTTGAY (SEQ ID NO: 48)

Table 4-Light chain variable region sequence Antibody Variable light chain sequence 4D6 DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGAGTKLELKR
(SEQ ID NO: 49) 1B12 NIVLTQSPASLAVSLGQRATISCRASESVDSHGNSFMHWYQQKPGQPPKLLIYLASSLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNNEDPLTFGGGTKLELKR
(SEQ ID NO: 50) 1C8 DVLMTQIPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLEIKR
(SEQ ID NO: 51) 7D9 DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGAGTKLELKR
(SEQ ID NO: 52) 5C7 DIQMTQSPSSLSASLGERVSLTCRASQEISGYLSWLQQKPDGTIKRLIYAASILDSGVPKRFSGSRSGSDYSLTISSLESEDFADYYCLQYASYPFMFGAGTKLELKR (SEQ ID NO: 53) 6F11 DIQMTQSPSSLSASLGERVSLTCRASQEISGYLSWLQQKPDGTIKRLIYAASILDSGVPKRFSGSRSGSDYSLTISSLESEDFADYYCLQYASYPFMFGAGTKLELKR (SEQ ID NO: 54)

Table 5-Heavy chain variable region sequence Antibody Variable heavy chain sequence 4D6 QVQLHQSGSELARPGASVKLSCKASGYTFTNYWMQWVKQRPGQGLEWIGAIYPGDGNTRYSQKFKGKATLTADKSSSTAYMQLSSLASEDSAVYYCARRDYGSSYWYFDVWGAGTTVTVSS (SEQ ID NO: 55) 1B12 QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNPSLKSQLTISKDTSRNQVFLKITSVDTADTATYYCARRADGYGFAYWGQGTLVTVSA (SEQ ID NO: 56) 1C8 EVQLVESGGDLVKPGGSLKLSCAASGFTFSNYGMSWVRQTPDKRLEWVATISSGGRYIYYPDSVKGRFTVSRDNAKNTLYLQMSSLKSEDTAMYYCARDSSDYGFAYWGQGTLVTVSA (SEQ ID NO: 57) 7D9 QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWLAHIYWDDDKRYNPSLKSRLTISKDTSRNQVFLKITSVDTADTATYYCARTSMMVPPWFAYWGQGTLVTVSA (SEQ ID NO: 58) 5C7 QVQLQQSGTELVRPGSSVKISCKASGYAFSSHWMNWVKQRPGQGLEWIGQIYPRNGDTNYNGKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCSRSTTIVTTGAYWGQGTLVTVSA (SEQ ID NO: 59) 6F11 QVQLQQSGTELVRPGSSVKISCKASGYAFSSHWMNWVKQRPGQGLEWIGQIYPRNGDTNYNGKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCSRSTTIVTTGAYWGQGTLVTVSA (SEQ ID NO: 60)

In some embodiments, the anti-Doppel antibody is at least 85%, 90%, 95%, 99% with an antibody produced by a hybridoma cell line selected from any one of clones 7D9, 1B12, 1C8, 5C7, 4D6, and 6F11, Or 100% identical amino acid sequence.

In some embodiments, the complementarity determining region (CDR) of the anti-Doppel antibody or a Doppel-binding fragment thereof is of an antibody produced by a hybridoma cell line selected from any one of clones 7D9, 1B12, 1C8, 5C7, 4D6, and 6F11. CDR and at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % same.

In some embodiments, the anti-Doppel antibody or a Doppel binding fragment thereof is at least 85%, 86%, 87 with one or more or all of the CDR sequences shown in Table 2 for antibodies 7D9, 1B12, 1C8, 5C7, 4D6, and 6F11, respectively. %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical light chain CDR sequences. For example, an anti-Doppel antibody or a dopel-binding fragment thereof has the CDRs for 4D6 (SEQ ID NOs: 13, 14 and 15) and at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical light chain CDRs.

In some embodiments, the anti-Doppel antibody or Dopel binding fragment thereof is at least 85%, 86%, 87 with one or more or all of the CDR sequences shown in Table 3 for antibodies 7D9, 1B12, 1C8, 5C7, 4D6, and 6F11, respectively. %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% comprise the same heavy chain CDR sequences. For example, an anti-Doppel antibody or a Dopel-binding fragment thereof has the CDRs for 4D6 (SEQ ID NOs: 31, 32 and 33) and at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical heavy chain CDRs.

In some embodiments, the heavy chain variable domain sequence of the anti-Doppel antibody or a Doppel-binding fragment thereof is a heavy chain variable of an antibody produced by a hybridoma cell line selected from any one of clones 7D9, 1B12, 1C8, 5C7, 4D6, and 6F11. At least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

In some embodiments, the anti-Doppel antibody or Dopel binding fragment thereof is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, with any one of the variable domain sequences shown in Table 5, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical heavy chain variable domain sequences.

In some embodiments, the light chain variable domain sequence of the anti-Doppel antibody or a Doppel-binding fragment thereof is a light chain variable of an antibody produced by a hybridoma cell line selected from any one of clones 7D9, 1B12, 1C8, 5C7, 4D6, and 6F11. At least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

In some embodiments, the anti-Doppel antibody or a doppel binding fragment thereof is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, with any one of the variable domain sequences shown in Table 4, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical light chain variable domain sequences.

In some embodiments, the heavy chain variable domain sequence and light chain variable domain sequence of the anti-Doppel antibody or a Doppel-binding fragment thereof are produced by a hybridoma cell line selected from any one of clones 7D9, 1B12, 1C8, 5C7, 4D6, and 6F11. At least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, respectively, of the heavy chain variable domain sequence and the light chain variable domain sequence of the antibody , 97%, 98%, 99%, or 100% the same.

In some embodiments, the anti-Doppel antibody or Dopel binding fragment thereof is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% with the light chain variable domain sequence shown in Table 4. , 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical light chain variable domain sequences and at least 85%, 86%, 87%, 88% of the corresponding heavy chain variable domains shown in Table 5 , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical corresponding heavy chain variable domain sequences. For example, the anti-Doppel antibody or a Dopel binding fragment thereof is at least 85%, 86%, 87%, 88%, 89%, 90%, 91 with the light and heavy chain variable domain sequences for 4D6, i.e. SEQ ID NOs: 49 and 55. %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical light and heavy chain variable domain sequences.

In some embodiments, the framework region sequence of the anti-Doppel antibody or a Doppel-binding fragment thereof is a framework of an antibody produced by a hybridoma cell line selected from any one of clones 7D9, 1B12, 1C8, 5C7, 4D6, and 6F11. At least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

In some embodiments, the heavy chain variable region of the anti-Doppel antibody or a Doppel binding fragment thereof is at least 85 with the CDRH1 of an antibody produced by a hybridoma cell line selected from any of clones 7D9, 1B12, 1C8, 5C7, 4D6, and 6F11. %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences It includes CDRH1 including. In some embodiments, the heavy chain variable region of the anti-Doppel antibody or a Doppel binding fragment thereof is at least 85%, 86%, 87%, 88%, 89%, 90 with SEQ ID NO: 31, 34, 37, 40, 43, or 46. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences.

In some embodiments, the heavy chain variable region of the anti-Doppel antibody or a Doppel binding fragment thereof is at least with the CDRH2 sequence of an antibody produced by a hybridoma cell line selected from any of clones 7D9, 1B12, 1C8, 5C7, 4D6, and 6F11. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acids It includes a CDRH2 sequence comprising the sequence. In some embodiments, the heavy chain variable region of the anti-Doppel antibody or a Doppel binding fragment thereof is at least 85%, 86%, 87%, 88%, 89%, 90 with SEQ ID NO: 32, 35, 38, 41, 44, or 47. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences.

In some embodiments, the heavy chain variable region of the anti-Doppel antibody or a Doppel binding fragment thereof is at least with the CDRH3 sequence of an antibody produced by a hybridoma cell line selected from any of clones 7D9, 1B12, 1C8, 5C7, 4D6, and 6F11. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acids It includes a CDRH3 sequence comprising the sequence. In some embodiments, the heavy chain variable region of the anti-Doppel antibody or a Doppel binding fragment thereof is at least 85%, 86%, 87%, 88%, 89%, 90 with SEQ ID NO: 33, 36, 39, 42, 45, or 48. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences.

In some embodiments, the light chain variable region of the anti-Doppel antibody or a Doppel binding fragment thereof is at least with the CDRL1 sequence of an antibody produced by a hybridoma cell line selected from any of clones 7D9, 1B12, 1C8, 5C7, 4D6, and 6F11. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acids It includes a CDRL1 sequence comprising the sequence. In some embodiments, the heavy chain variable region of the anti-Doppel antibody or Dopel binding fragment thereof is at least 85%, 86%, 87%, 88%, 89%, 90 with SEQ ID NO: 13, 16, 19, 22, 25, or 28. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences.

In some embodiments, the light chain variable region of the anti-Doppel antibody or a Doppel binding fragment thereof is at least with the CDRL2 sequence of an antibody produced by a hybridoma cell line selected from any of clones 7D9, 1B12, 1C8, 5C7, 4D6, and 6F11. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acids It includes a CDRL2 sequence comprising the sequence. In some embodiments, the heavy chain variable region of the anti-Doppel antibody or a Doppel binding fragment thereof is at least 85%, 86%, 87%, 88%, 89%, 90 with SEQ ID NO: 14, 17, 20, 23, 26, or 29. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences.

In some embodiments, the light chain variable region of the anti-Doppel antibody or a Doppel binding fragment thereof is at least with the CDRL3 sequence of an antibody produced by a hybridoma cell line selected from any of clones 7D9, 1B12, 1C8, 5C7, 4D6, and 6F11. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acids It includes a CDRL3 sequence comprising the sequence. In some embodiments, the heavy chain variable region of the anti-Doppel antibody or Dopel binding fragment thereof is at least 85%, 86%, 87%, 88%, 89%, 90 with SEQ ID NO: 15, 18, 21, 24, 27, or 30. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences.

In some embodiments, the anti-Doppel antibody or Dopel-binding fragment thereof comprises one or more of the following characteristics:

(a) the light chain immunoglobulin variable domain sequence is at least 85%, 86% of the CDRs of the light chain variable domain of the antibody produced by the hybridoma cell line selected from any one of clones 7D9, 1B12, 1C8, 5C7, 4D6, and 6F11. , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more CDRs, and /Or (shown in Table 2);

(b) the heavy chain immunoglobulin variable domain sequence is at least 85%, 86% of the CDRs of the heavy chain variable domain of the antibody produced by the hybridoma cell line selected from any one of clones 7D9, 1B12, 1C8, 5C7, 4D6, and 6F11. , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more CDRs, and /Or (shown in Table 3);

(c) the light chain immunoglobulin variable domain sequence is at least 85%, 86%, 87 with the light chain variable domain of an antibody produced by a hybridoma cell line selected from any one of clones 7D9, 1B12, 1C8, 5C7, 4D6, and 6F11. %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical and/or (shown in Table 4 );

(d) The heavy chain immunoglobulin variable domain sequence is at least 85%, 86%, 87 with the heavy chain variable domain of an antibody produced by a hybridoma cell line selected from any one of clones 7D9, 1B12, 1C8, 5C7, 4D6, and 6F11. %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical (shown in Table 5).

In other embodiments, one or more amino acid residues of the CDRs of an antibody disclosed herein are substituted with other amino acids. Substitutions can be "conservative" in that they are substitutions within the same amino acid family, based on the following family classification:

(1) Amino acids having basic side chains: lysine, arginine, histidine.

(2) Amino acids having acidic side chains: aspartic acid, glutamic acid.

(3) Amino acids with uncharged polar side chains: asparagine, glutamine, serine, threonine, tyrosine.

(4) Amino acids having non-polar side chains: glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, cysteine.

In other embodiments, one or more amino acid residues are added to or deleted from one or more CDRs of the antibody. Such additions or deletions may occur at the N-terminus or C-terminus of the CDR or at one or more of the positions within the CDR.

Diversification of the amino acid sequence of the CDRs of an anti-Doppel antibody by addition, deletion or substitution of amino acids can result in various effects such as increased binding affinity for the target antigen.

The constant region of an anti-Doppel antibody can also be variable. For example, the antibody may be provided with the Fc region of any isotype: IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4) or IgM.

In some embodiments, the heavy chain constant domain sequence of the anti-Doppel antibody is at least 85% of the heavy chain constant domain sequence of the antibody produced by a hybridoma cell line selected from any one of clones 7D9, 1B12, 1C8, 5C7, 4D6, and 6F11. , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

In some embodiments, the light chain constant domain sequence of the anti-Doppel antibody is at least 85% of the light chain variable domain sequence of the antibody produced by a hybridoma cell line selected from any one of clones 7D9, 1B12, 1C8, 5C7, 4D6, and 6F11. , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

In some embodiments, the heavy chain constant domain sequence and the light chain constant domain sequence of the anti-Doppel antibody are each heavy chain constant of an antibody produced by a hybridoma cell line selected from any one of clones 7D9, 1B12, 1C8, 5C7, 4D6, and 6F11. Domain sequence and light chain constant domain sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99%, or 100% the same.

In some embodiments, the anti-Doppel antibody or a doppel-binding fragment thereof comprises a heavy chain variable domain sequence and a light chain variable domain sequence that together form an antigen binding site that binds to the doppel. In some embodiments, the anti-Doppel antibody or a Doppel-binding fragment thereof binds an epitope to which an antibody produced by a hybridoma cell line selected from any one of clones 7D9, 1B12, 1C8, 5C7, 4D6, and 6F11 binds. For example, in some embodiments, the anti-Doppel antibody or Doppel binding fragment is capable of binding to the amino acid sequence YWQFPD (SEQ ID NO: 11) of a mouse or human Doppel (see Figure 32). Alternatively, in some embodiments, the anti-Doppel antibody or Doppel binding fragment is the amino acid sequence FPDGIY of a murine doppel (SEQ ID NO: 12) (see Figure 33) or the corresponding sequence of a human doppel, FPDGIH (SEQ ID NO: 61). Can be combined with

In some embodiments, the anti-Doppel antibody or Dopel binding fragment thereof, such as the CDR sequences disclosed in Tables 2 and 3 and the epitope specificity of the antibody produced by the hybridoma cell line clones 7D9, 1B12, 1C8, 5C7, 4D6, or 6F11. , May possess a combination of one or more of the disclosed structural and/or functional features disclosed herein.

Methods for determining the epitope binding site of a given antibody, such as immunoassays, are known in the art and are exemplified in the Examples below.

In some embodiments, the anti-dopel antibody or dopel binding fragment thereof is a dopel with a specificity and sensitivity profile similar to one or more of the antibodies produced by a hybridoma cell line selected from clones 7D9, 1B12, 1C8, 5C7, 4D6, or 6F11. Combine.

Composition

The present application also describes compositions comprising one or more Doppel-targeting molecules. In some embodiments, the composition comprises a pharmaceutically acceptable excipient or carrier, such as a carrier suitable for the intended route of administration.

The composition can be prepared for any route of administration, including any oral, parenteral, or topical route of administration. In some embodiments, the composition is suitable for injection or infusion, such as intravenous injection or infusion, and may be prepared as a sterile composition, such as for injection or infusion. In other embodiments, the compositions are suitable for oral administration and can be prepared, for example, in liquid or solid oral dosage forms (eg, solutions, syrups, powders, granules, tablets, capsules, suspensions, emulsions, or oral sprays). In other embodiments, the pharmaceutical composition may be in the form of a solution or powder suitable for inhalation, such as for nasal or oral inhalation. In another embodiment, the pharmaceutical composition is suitable for rectal or vaginal administration, such as a suppository formulation. In other embodiments, the composition is suitable for topical or transdermal administration, such as a solution, emulsion, gel, or patch. Suitable components and excipients for such compositions are known in the art.

Thus, in some embodiments, the Doppel-targeting molecule is administered orally, subcutaneous injection, intravenous injection or infusion, intraocular injection, intradermal injection, intramuscular injection, intraperitoneal injection, intratracheal administration, inhalation, intranasal administration, sublingual administration. It is formulated for oral administration, rectal administration, vaginal administration, or topical administration.

In some embodiments, the Doppel-targeting molecule is formulated for oral administration. As mentioned above, the LMHW-oligoDOCA conjugate may be particularly suitable for oral administration. In some embodiments, the Doppel-targeting molecule is formulated for oral administration and selective absorption in the intestine or ileum. For example, the LMHW-oligoDOCA conjugate doppel-targeting molecule can be localized in the intestine or ileum. Additionally or alternatively, the formulations can provide targeted release of the Doppel-targeting molecule in the intestine or ileum, for example using pH-controlled and/or delayed release formulations known in the art.

In some embodiments, the Doppel-targeting molecule is formulated for intravenous injection. Antibodies and antibody fragments are typically administered by intravenous injection to avoid degradation in the digestive tract, but by formulating the antibodies/fragments in a manner that protects them from digestive enzymes, e.g., using formulation techniques known in the art, It can be formulated for oral delivery.

Examples of formulations for parenteral administration include sterile aqueous solutions, water-insoluble solutions, suspensions, emulsions, lyophilized formulations, and suppositories. Non-aqueous solutions and suspensions include, for example, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, or injectable esters such as ethyloleate. Bases for suppository formulations include, for example, witepsol, macrogel, Tween 61, cacao butter, laurine butter, glycerogelatin, and the like.

Formulations for oral delivery may include poloxamer, labrasol, polyethylene glycol, and mixtures thereof.

In some embodiments, the composition comprises an effective amount of a doppel-targeting molecule to interfere with the interaction of dopel and tyrosine kinase receptors, such as VEGFR2, VEGFR1, VEGFR3, bFGFR, and PDGFR. In some embodiments, the composition comprises an effective amount of a Doppel-targeting molecule to reduce pathological angiogenesis. In some embodiments, the composition comprises an effective amount of a Doppel-targeting molecule to reduce tumor angiogenesis. In some embodiments, the composition comprises an effective amount of a Doppel-targeting molecule to reduce pathological vasculature. In some embodiments, the composition comprises an effective amount of a Doppel-targeting molecule to reduce tumor vasculature. In some embodiments, the composition comprises an effective amount of a Doppel-targeting molecule to reduce tumor angiogenesis.

In some embodiments, the composition comprises an effective amount of a Doppel-targeting molecule to reduce angiogenesis associated with asthma. In some embodiments, the composition comprises an effective amount of a Doppel-targeting molecule to reduce angiogenesis associated with tuberculosis. In some embodiments, the composition comprises an effective amount of a Doppel-targeting molecule to increase angiogenesis associated with atherosclerosis. In some embodiments, the composition comprises an effective amount of a Doppel-targeting molecule to reduce angiogenesis associated with pulmonary arterial hypertension (PAH). In some embodiments, the composition comprises an effective amount of a Doppel-targeting molecule to reduce angiogenesis associated with a neoplasm or neoplasm-related condition.

In some embodiments, the composition is effective to elicit a therapeutic response in tumor endothelial cells.

In some embodiments, the composition is effective in detecting tumor endothelial cells, such as when the Doppel-targeting molecule comprises or is conjugated to a detectable label.

In some embodiments, the composition is a neoplasm or neoplasm-related condition, such as breast carcinoma, lung carcinoma, gastric carcinoma, esophageal carcinoma, colorectal carcinoma, liver carcinoma, ovarian carcinoma, androblastoma, cervical carcinoma, endometrial Carcinoma, endometrial hyperplasia, endometriosis, fibrosarcoma, sarcoma carcinoma, head and neck cancer, nasopharyngeal carcinoma, laryngeal carcinoma, hepatoblastoma, Kaposi's sarcoma, melanoma, skin carcinoma, hemangioma, cavernous hemangioma, hemangioblastoma, pancreatic carcinoma, Retinoblastoma, astrocytoma, glioblastoma, schwannoma, oligodendroma, glioblastoma, neuroblastoma, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcoma, urinary tract carcinoma, thyroid carcinoma, Wilm's tumor, renal cell carcinoma, prostate carcinoma, nevus-related abnormal blood vessels Effective in inducing a therapeutic response to hyperplasia, edema (e.g. associated with brain tumors), and/or Meigs syndrome

In some embodiments, the compositions disclosed herein are used to inhibit angiogenesis in a subject in need thereof. In some embodiments, the subject is a disease or condition in which Doppel-Tyrosine kinase receptor signaling (such as Doppel-VEGFR2 signaling) is involved, such as a tumor or cancer, or a disease or condition associated with increased vasculature, such as asthma, tuberculosis, Suffers from or is at risk of developing atherosclerosis, pulmonary arterial hypertension (PAH), or a neoplasm or neoplasm-related condition.

How to identify Doppel-targeting molecules

Exemplary Doppel-targeting molecules are described above. Additional Doppel-targeting molecules can be identified by screening methods, such as those described below and illustrated in the Examples below.

A suitable method may include evaluating the binding of a test molecule with a Doppel protein (or a fragment thereof that forms a complex with a tyrosine kinase inhibitor (eg, VEGFR2)) as illustrated in the Examples below. These methods additionally or alternatively comprise culturing endothelial cells when the dopel and tyrosine kinase inhibitors interact with each other in the presence or absence of a test agent that always binds a dopel protein or fragment thereof, and Detecting at least one of the internalization and/or degradation of the tyrosine kinase inhibitor, and inhibiting the internalization of the tyrosine kinase inhibitor and/or promoting the degradation of the tyrosine kinase inhibitor compared to the internalization and/or degradation detected in the absence of the test molecule. And selecting a test molecule to be tested. Additionally or alternatively, doppel-blocking activity can be detected, for example, by assaying for inhibition of phosphorylation and degradation of tyrosine kinase inhibitors. Additionally or alternatively, Doppel-blocking activity can be determined, for example, by assaying the number of germinations formed in endothelial cell spheroids and by assaying the inhibition of endothelial cell germination, which can be detected by comparing the number of germinations that occurred in a control. Can be detected.

Any doppel protein can be used by evaluating the binding of the test molecule. In certain embodiments, the Doppel protein or fragment thereof forms a complex with a target tyrosine kinase inhibitor, such as the VEGFR2 protein.

Any medium can be used to cultivate endothelial cells in cases where the Doppel and tyrosine kinase inhibitors constantly interact with each other in the presence or absence of the test agent. For example, cell extracts, cell culture supernatant, microbial fermentation culture products, extracts of marine organisms, plant extracts, and the like may be used.

Inhibition of pathological angiogenesis

As described above, the Doppel-targeting molecules and compositions described herein are useful for inhibiting pathological angiogenesis and treating related diseases or conditions in subjects in need thereof. Accordingly, these uses and methods are provided herein.

As mentioned above, the term “subject” as used herein refers to any mammal, including humans, cats, mice, dogs, horses, monkeys, or other species. In some embodiments, the subject is a human.

As used herein, "treating" refers to reducing, retarding, or delaying pathologic angiogenesis (or tumorigenesis), and/or some pathological increased blood vessels, even if some pathological angiogenesis (or tumorigenesis) still occurs. It includes reducing, retarding, or delaying the increased vasculature, even if the structure still occurs.

Doppel-targeting molecules and compositions described herein are useful for inhibiting pathological angiogenesis in subjects with diseases or conditions in which Doppel or Doppel-tyrosine kinase receptor signaling is involved, or diseases or conditions indicative of increased vasculature. Do. Non-limiting examples of such diseases or conditions include tumors, cancers such as, without limitation, angiogenic cancer; Atherosclerosis; Tuberculosis; Asthma, and pulmonary arterial hypertension (PAH). Thus, the disclosed methods and Doppel-targeting molecules and/or compositions may be useful for treating a variety of neoplastic and non-neoplastic diseases and conditions.

The Doppel-targeting molecules and compositions disclosed herein generate a therapeutic response against tumor endothelial cells (TECs) and are useful in the treatment of diseases or conditions characterized by the presence of these cells.

The Doppel-targeting molecules and compositions described herein are useful for treating neoplasms and neoplasm-related conditions.

As used herein, the term “neoplasm” refers to an abnormal growth of tissue, known as a tumor upon formation of a lump. Neoplasms can be benign or malignant. Malignant neoplasms are generally referred to as cancer.

Neoplasms and neoplasm-related conditions that can be treated include breast carcinoma, lung carcinoma, gastric carcinoma, esophageal carcinoma, colorectal carcinoma, liver carcinoma, ovarian carcinoma, androblastoma, cervical carcinoma, endometrial carcinoma, endometrial carcinoma. Hyperplasia, endometriosis, fibrosarcoma, sarcoid carcinoma, head and neck cancer, nasopharyngeal carcinoma, laryngeal carcinoma, hepatoblastoma, Kaposi's sarcoma, melanoma, skin carcinoma, hemangioma, cavernous hemangioma, hemangioblastoma, pancreatic carcinoma, retinoblastoma, stellate form Cytoma, glioblastoma, schwannoma, oligodendroglioma, glioblastoma, neuroblastoma, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcoma, urinary tract carcinoma, thyroid carcinoma, Wilm's tumor, renal cell carcinoma, prostate carcinoma, nevus-related abnormal angioproliferative disease, edema ( For example associated with brain tumors), and Meigs syndrome.

Accordingly, the present application is for use in a method of inhibiting pathological angiogenesis in a Doppel-targeting molecule and a subject in need thereof, such as a subject suffering or at risk of developing any one or more of the above-mentioned conditions. Such methods are provided, including compositions comprising them. The above uses and methods include administering to a subject in need thereof an effective amount of a Doppel-targeting molecule or composition described herein. In some embodiments, an effective amount is effective in interfering with the interaction of doppel and tyrosine kinase receptors, such as VEGFR2, VEGFR1, VEGFR3, bFGFR, or PDGFR. In certain embodiments, the effective amount is effective in interfering with the interaction of Doppel and VEGFR2. In some embodiments, the effective amount is effective to reduce the vasculature of the tumor. In some embodiments, the effective amount is effective to elicit a therapeutic response in tumor endothelial cells. In some embodiments, the effective amount is effective to reduce, retard, or delay pathological angiogenesis or tumorigenesis. In some embodiments, the effective amount is effective to reduce angiogenesis associated with asthma. In some embodiments, the effective amount is effective to reduce angiogenesis associated with tuberculosis. In some embodiments, the effective amount is effective to reduce angiogenesis associated with atherosclerosis. In some embodiments, the effective amount is effective to reduce angiogenesis associated with pulmonary arterial hypertension (PAH). In some embodiments, the effective amount is effective to reduce angiogenesis associated with a neoplasm or neoplasm-related disease.

The Doppel-targeting molecule can be administered via any route of administration, including any parenteral or topical route of administration. In some embodiments, the method comprises oral administration, subcutaneous injection, intravenous injection or infusion, intraocular injection, intradermal injection, intramuscular injection, intraperitoneal injection, intratracheal administration, inhalation, intranasal administration, sublingual administration, oral administration, And administering the Doppel-targeting molecule by rectal administration, vaginal administration, or topical administration.

In some embodiments, the method comprises administering the Dopel-targeting molecule by oral administration. In some embodiments, the method comprises administering the Doppel-targeting molecule by intravenous injection.

In some embodiments, the method comprises treating the subject with an additional therapy. For example, a method of treatment may include administering a chemotherapeutic agent to the subject, or providing radiation therapy. As used herein, the term “chemotherapeutic agent” refers to a molecule useful for treating cancer, such as a small chemical compound used to treat cancer. Non-limiting examples of chemotherapeutic agents include, but are not limited to, alkylating agents, anti-metabolites, anti-tumor antibiotics, plant alkaloid/microtubule inhibitors, DNA linking agents, biologics, bisphosphonates, hormones, and other drugs known to be useful in treating cancer. Includes. Non-limiting examples of chemotherapeutic agents include aminoglutethimide, amsacrine, anastrozole, asparaginase, beg, bicalutamide, bleomycin, burelin, busulfan, camptothecin, capecitabine, carboplatin. , Carmustine, chlorambucil, cisplatin, cladribine, cladronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, dieestrol, diethyl Stilvestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, fluta Mid, Gemcitabine, Genistein, Goserelin, Hydroxyurea, Idarubicin, Ifosfamide, Imatinib, Interferon, Irinotecan, Ironotecan, Letrozole, Leucovorin, Leuprolide, Rebamisol, Lomustine, Meclo Retamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotan, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pami Dronate, Pentostatin, Plicamycin, Polpimer, Procarbazine, Lalitrexed, Rituximab, Streptozosine, Suramine, Tamoxifen, Temozolomide, Teniposide, Testosterone, Thioguanine, Thiotepa, Titano Sen dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, and vinorelbine.

How to detect doppel

Doppel-targeting molecules and compositions described herein also detect the presence of a doppel, such as determining whether a dopel is associated with a particular disease or condition, or whether it is expressed in a subject, or is expressed by a subject's TEC. It is useful as an agent for doing so.

For this use, the Doppel-targeting molecule is administered to a subject and/or a biological sample obtained from a subject and then detects any binding between the Doppel-targeting molecule and any doppel present in the subject or sample. In some embodiments, the Doppel-targeting molecule comprises or is conjugated to a detectable label. In another embodiment, the binding between the Doppel-targeting molecule and any Doppel present in the subject or sample is detected using an antibody specific for the Doppel-targeting molecule and/or the Doppel-targeting molecule/Doppel complex.

If it is determined that Dopel is associated with a disease or condition, or is determined to be expressed by the subject's TEC, the subject can be treated by the methods described herein.

In certain embodiments, the subject is at any risk of developing a tumor, such as, as described above, and/or cancer, atherosclerosis, tuberculosis, asthma, and pulmonary arterial hypertension (PAH). There is a risk of developing a disease or condition selected from and/or a risk of developing a neoplasm or neoplasm-related condition.

Example

The following examples illustrate specific embodiments, but are merely illustrative.

Example 1: Classification of tumor endothelial cells (TEC) to evaluate Dopel expression

This example describes a method of purifying endothelial cells from different tumor tissue types.

Tumor endothelial cells were isolated using dual markers according to some modified published procedures. Briefly, tumors were _{grown subcutaneously in the flanks of C 3} H/HeN mice, excised, minced using two surgical knives, and digested in 9 mL collagenase and 1 mL dispase solution per gram of tissue. The tissue was incubated for 30 minutes in a 37° C. water bath under continuous stirring. Subsequently, 75 μl of DNaseI solution per 10 mL cell suspension was added and incubated for an additional 30 minutes at 37° C. with continuous stirring. The degraded tissue was sieved through a 100 μm cell strainer and single cells were isolated. Cells were collected by centrifugation at 400×g for 7 minutes at room temperature. To remove red blood cells, granulocytes, deactivated cells and cell debris, resuspend the cells in 10 mL Ficoll separation medium (per gram of starting material) and carefully as a suspension on 7.5 mL Ficoll-plaques (pre-warmed to room temperature). Layered. The interlayer-containing viable cells were transferred to a new tube. Cells were collected in FACS tubes and incubated with anti-mouse CD31-PE and anti-mouse CD34-FITC antibodies (final concentration of 2 μg/mL). The FACS machine was made by adjusting conditions with a sheet fluid pressure of 29.9 psi, a fractionation frequency of 44 kHz, and a plate voltage of 3,500 V. The collected cells were suspended in 10 mL ECGM containing 10% FBS and centrifuged. Next, the cells were plated on 0.2% gelatin-coated 100 mm dishes and incubated overnight, supplemented with normal ECGM. The next day, the medium was mated with supplemented ECGM containing 10% FBS. Cells were grown to the point of fusion.

Example 2: Immunofluorescence and whole tissue fixation staining for Dopel expression

The tumor was dissected and cut into approximately 2 mm x 2 mm tissue (plug) using a scalpel. The plug was fixed in methanol containing 25% DMSO for 24 hours at 4° C. in a 50 mL reaction tube. The plug was washed 3 times (at least 30 minutes each time) with sterile PBS at 4° C. with gentle agitation. The plug was blocked at 4° C. under mild agitation with 5% BSA for 3 hours. Tumor plugs were incubated in blocking buffer containing primary antibody overnight at 4° C. under mild agitation. The next day, the plug was washed 3 times with TBST for 3 hours at 4° C. under mild agitation. The plugs were incubated in blocking buffer containing a fluorescence-labeled secondary antibody (5% BSA in TBST) at 4° C. under mild agitation. After washing by gently stirring at 4° C. for 3 hours, the plug was dyed with Chekst dye. The plug was inserted into the fixing medium and the slide was sealed with Pertex to prevent drying. The three-dimensional structure of the capillary network was analyzed with a confocal microscope (Carl Zeiss LSM710, Leica DM IRB/E; Leica Co., Germany). For Doppel and CD31 detection, the incisions were simultaneously stained with goat anti-mouse doppel followed by Alexa488-linked donkey anti-goat and PE-linked rat anti-mouse CD31.

To assess Dopel expression at the molecular level, TEC was isolated from highly angiogenic squamous cell carcinoma (SCC7) grown in mice. Whole-tissue-fixed and immunofluorescent staining revealed that most of the vasculature expressed Doppel at high levels in the primary tumor (FIG. 1).

Example 3: Quantitative PCR for Doppel expression

The mRNA was purified using the Quick Prep Micro mRNA purification kit (Amersham, Piscataway, NJ). Single stranded cDNA was generated using the Superscript III primary strand synthesis system (Invitrogen) according to the manufacturer's instructions. Quantitative PCR was performed using the Brilliant SYBR Green QPCR master mix and MX4000, and the limiting cycle number was obtained using MX4000 software v.4.20 (Stratagene, La Jolla, CA). The amplification conditions were 1 cycle at 95° C. for 10 minutes, followed by 40 cycles at 95° C. for 20 seconds each; 56°C, 30 seconds and 72°C, 30 seconds. Quantitative PCR was performed in triplicate and the number of limiting cycles was averaged. Gene expression was normalized to 70 Kd U1 small nuclear ribonucleoprotein polypeptide A (Snrnp70 ), a gene that is uniformly expressed in all ECs as assessed by SAGE. Relative expression was ^{calculated using the equation of 2 (Rt-Et)} /2 ^(Rn-En) , where Rt is the limiting number of cycles observed in the experimental sample for Snrnp70, and Et is the experiment for the gene of interest (GOI). Is the number of limiting cycles observed in the sample, Rn is the average limiting cycles observed for Srnp70 in B.End.3 or HUVEC samples, and En is the average limiting cycles observed for GOI in B.End.3 or HUVEC samples It's a number. The primers used were as follows:

Mouse doppel :

Omnidirectional: GGACATCGACTTTGGAGCAGAG (SEQ ID NO: 1);

Backward: CAGCATCTCCTTGGTCACGTTG (SEQ ID NO: 2),

Human doppel :

Omnidirectional: GATGGCATCCACTACAACGGCT (SEQ ID NO: 3);

Backward: GTTGTCTGGCTTCTGGAACTCC (SEQ ID NO: 4),

Mouse Snrnp70 :

Omnidirectional: GCCTTCAAGACTCTGTTCGTGG (SEQ ID NO: 5);

Backward: CTCGATGAAGGCATAACCACGG (SEQ ID NO: 6),

Human Snrnp70 :

Omnidirectional: TCAAGACTCTCTTCGTGGCGAG (SEQ ID NO: 7),

Backward: GGCTTTCCTGACCGCTTACTGT (SEQ ID NO: 8).

Quantitative RT-PCR analysis revealed that transcription levels were approximately 38 times higher in TEC purified in SCC7 than in normal mouse endothelium ( FIG. 2 ). Taken together, these data suggest that, at both the transcript and protein levels, Doppel expression increases ectopically during tumorigenesis and is specific for TEC in tumors.

Example 4: Generation of TEC spheroids and subcutaneous implantation in mice

The globule-based in vivo angiogenesis assay was performed according to published procedures. Briefly, 8×10 ⁶ TEC or TEC ^-/-dpl was suspended in 200 mL medium containing 20% methanol solution and 80% culture medium. Cells were distributed in 25 μl for each spheroid, in non-adherent plastic square Petri dishes. Spheroids formed by TEC or TEC ^-/-dpl were harvested and mixed in a Matrigel/Fibrin mixture containing mouse VEGF and bFGF. TEC or TEC ^-/-dpl spheroids (1000 spheroids per plug) of each female SCID mouse (5-6 weeks old, Jungang Animal Lab, Korea) or female Balb/c nude mice (Orinet bio, Seongnam, Korea) It was inoculated subcutaneously on the flank. The spheroids were grown for 3 weeks. At a dose of 2.5 kg/kg, Cy5.5-labeled LMWH was injected intravenously, and 2 hours after injection, its accumulation at the TEC implantation site was examined using Optimax-MX3 (GE Medical Systems, Milwaukee, WI). . In another experiment, at a dose of 10 mg/kg, Cy5.5-labeled LHbisD4 was administered orally, and its distribution was also reviewed as previously described. The matrix plug was removed and a portion of the plug was analyzed for microvessel density and drug accumulation via immunofluorescence staining.

Handoffel eliminated FBS or mVEGF-induced germination of TEC spheroids in a dose-dependent manner ( FIG. 3 ). These data suggest that Doppel regulates VEGFR2 signaling in endothelial cells.

Example 5: Synthesis of Doppel-binding LMWH-DOCA conjugate

This example describes the synthesis of several bile acid (deoxycholic acid)-based heparin conjugates useful as Doppel-targeting molecules, as described herein.

Deoxycholic acid (DOCA) derivatives were synthesized by chemical conjugation of deoxycholic acid and lysine, or lysine substituents containing 2-4 amine groups, using a building block method. They were referred to as monomer, dimer, trimer, and tetramer deoxycholic acid ( mono DOCA, bis DOCA, tri DOCA, and tetra DOCA, respectively), respectively ( FIG. 4 ). Prior to conjugation with LMWH, the DOCA derivative was reacted with ethylenediamine (EDA) to introduce a central primary amine into each DOCA derivative. The LMWH-DOCA conjugate was synthesized by chemically coupling a hydrophilic LMWH backbone with an amine-conjugated DOCA derivative ( mono DOCA-NH ₂ , bis DOCA-NH ₂ , tri DOCA-NH ₂ , and tetra DOCA-NH ₂ ). LMWH (100 mg) was dissolved in 3.125 mL FA at accelerated temperature. The DOCA derivative was also dissolved in the co-solvent system of DMF/FA. The carboxylic acid of LMWH was activated using EDAC/NHS and reacted with the DOCA derivative at 4° C. for 12 hours at different feed model ratios ranging from 1:12. The reacted materials were then precipitated in an excess of cold ethanol and dried under vacuum before centrifugation. The dried heparin conjugate was dissolved in water before lyophilization. The synthesis of the LMWH-DOCA conjugate was ^{confirmed using 1} H NMR, and specific amide binding and bile acid peaks were identified in a specific ppm range. The relative anticoagulant activity of the LMWH-DOCA conjugate was determined by a kinetic method using an anti-factor Xa (anti-FXa) development assay kit and a chromogenic substrate sensitive to FXa (S2222). Conjugation rates were determined as described in Fini et al. with some modifications.

Example 6: Measurement of binding affinity between Doppel and LMWH-DOCA conjugate

This example describes a method of assessing the dissociation rate constant between Doppel and the LMWH-DOCA conjugate.

The affinity measurement of the LMWH and LMWH-DOCA conjugates and Prnp and Prnd (Doppel) was performed by surface plasmon resonance on BIAcore T100 (GE Healthcare). Using standard EDAC-NHS coupling method, recombinant proteins were immobilized on a sensor chip (GE Healthcare) at a density of 4000-7000. Measurements were performed at a flow rate of 20 μl/min, and the chip surface was regenerated after each assay cycle using 50 mM NaOH. Each concentration was analyzed in duplicate. Kinetic analysis of the obtained data was performed using BIAcore T100 evaluation software (GE Healthcare).

A novel heparin-based compound (conjugate of deoxycholic acid and heparin) was designed. LHbisD4, a chemical conjugate of four dimeric DOCA molecules to one LMWH molecule, was selected for subsequent experiments. _{LHbisD4 exhibited a higher binding affinity (K D} = 7.8 nM) to Doppel than LMWH or other bile acid-based conjugates (FIG. 5 ). In addition, unlike LMWH, when compared to LMWH, LHbisD4 showed anticoagulant activity or did not show a change in binding affinity to the cellular prion protein, Prnp (FIG. 6). LHbisD4 binding was also significantly reduced in ^{TEC -/-dpl} than in TEC (FIG. 7 ). Fragment-based structural studies using computer simulations showed that Doppel's globular domain plays a crucial role in binding to LHbisD4, as this region contains hydrophobic grooves to capture the macrohydrophobic side chains of LHbisD4. Is ( Fig. 8 ).

Example 7: Pharmacokinetics and biodistribution studies of LMWH-DOCA conjugate, LHbisD4

This example demonstrates that the Doppel-targeting molecule LHbisD4 can be administered orally for tumor-specific vascular targeting.

For kinetic experiments, Sprague Dory (SD) rats (male, body weight 250-260 g) were randomly classified into 2 groups (n = 4-5 in each group) for LMWH and LHbisD4. Samples treated at a dose of 10 mg/kg were administered orally after mixing with Labrasol and Poloxamer 188 (BASF Aktiengesellschaft, Germany). Blood was collected 15 minutes, 30 minutes, and 1, 2, 3, 4, 6, and 8 hours after drug administration. Plasma was isolated by centrifugation at 4,000×g for 20 minutes. The amount of drug in plasma was measured with a heparin-orange chemosensor kit. The heparin orange chemsense kit (Heparin Orange G26; MW 675.57) obtained from Professor Tae-Hyang Jung of the Lumino Genomics Laboratory at the National University of Singapore was used to detect the plasma concentration of the sample by the method previously described. Briefly, heparin orange, a chemosensor material that reacts with the heparin conjugate, has a reaction pattern that is usually a charge-charge interaction, and the final complex emits luminescence.

Biodistribution of LHbisD4 in tumor tissue was performed in individual groups of SCC7-tumor-bearing mice (n=3-4 at each time point) by the method described previously. In all biodistribution experiments, we used a fluorescent marker to track LHbisD4. Mice were administered LHbisD4-Cy5.5 by oral gavage (10 mg/kg). Mice were perfused with PBS buffer (30 mL) at predetermined time points (0.5, 2, 4, 8 and 24 hours). Fluorescent Cy5.5 was extracted from the tissue by incubating with formamide at 25°C for 48 hours. The sample was centrifuged and the fluorescence intensity of the supernatant was detected by excitation/absorption of 670 nm/720 nm using a Wallac 1420 VICTOR plate reader (Perkin-Elmer Life Sciences). Results were normalized to protein levels in the corresponding tissues. Tissue autofluorescence was corrected by subtracting the fluorescence signal of untreated tumors from individual readings in treated mice. The amount of LHbisD4 was calculated per gram of tissue. Similarly, fluorescence signal levels in mouse serum were at different time points (15 minutes, 30 minutes, 1, 2, 3, 4, 6, 8, 10 and 24 hours) using excitation/luminescence readings at 670 nm/720 nm. Extracted.

LHbisD4 administered orally showed a maximum plasma concentration of 9.3 ± 1.4 μg/mL in rats ( FIG. 9 ). When administered orally, the targeting ability of LHbisD4 was first ^{evaluated using TEC and TEC -/-dpl} in an in vivo, globular-based angiogenesis model. The fluorescence signal in the TEC plug was significantly higher than that ^{of the TEC -/-dpl} plug for Cy5.5-labeled LHbisD4 administered orally. ^{TEC/TEC -/-dpl} ratio 6.5 ± 0.8 times, p <0.001; 10 and 11 ).

Example 8: Inhibition of Doppel to treat tumors

This example illustrates a method of treating tumors by targeting the Doppel and Doppel-VEGFR2 axes to inhibit pathological angiogenesis.

SCC7 (1×10 ⁶ cells) cells were inoculated subcutaneously on the dorsal flank of C3H/HeN mice (male, 6-7 weeks old, n = 6-8, Orient Bio Inc., Seongnam, Korea). When tumors reached 50-70 mm 3, mice were treated with LHbisD4 (10 mg/kg) orally every day for 3 weeks. Mice were sacrificed when the tumor reached a volume greater than 2.5 cm 3.

MDA-MB-231 cells were injected into Balb/c nude mice (male, 6-7 weeks old, n = 6-8, Orient Bio Inc., Seongnam, Korea). LHbisD4 (2.5, 5, and 10 mg/kg) was administered daily by oral gavage.

For other groups of mice, LHbisD4 was administered twice a day at doses of 5 and 10 mg/kg. Mice were sacrificed when the tumor reached a volume greater than 1.8 cm 3.

In the case of other mouse groups, anti-Doppel antibody was administered once per 4 days at a dose of 1 mg/kg. Mice were sacrificed when the tumor reached a volume greater than 3.0 cm 3.

The anti-tumor effect of LHbisD4 was also observed in the MDAMB-231 human breast carcinoma xenograft model ( FIGS. 12 and 13 ). The results suggest that LHbisD4 showed a dose-dependent effect up to 10 mg/kg, in other words, tumor growth was inhibited to a significant extent when the dose of LHbisD4 was increased.

The anti-tumor effect of the anti-Doppel antibody was also observed in the SCC7 xenograft model and the tumor volume was significantly reduced compared to the control group ( FIG. 14 ). These results highlight the success of Doppel targeting, obtaining selective angiogenesis inhibition.

Example 9: Development of anti-Doppel mAb selectively stops tumor angiogenesis and eliminates the off-tumor effect of anti-angiogenic therapy.

A complete Doppel knockout (KO) mouse model was generated from immunogenic C57BL/6 mice (Behrens A, Brandner S, Genoud N, Aguzzi A. Normal neurogenesis and scrapie pathogenesis in neural grafts lacking the prion protein homologue Doppel. EMBO Rep. 2001;2:347-352). The effect of Doppel deletion on tumor growth in mice was tested. Nocturnal (WT), heterozygous (Dpl ^+/- ) and homozygous (Dpl ^-/- ) Doppel KO mice of both sexes were spawned and grown without any developmental problems, demonstrating that the absence of Doppels did not cause any congenital damage. Suggests. Syngeneic B16F10 melanoma, EL4 thymoma, and CT26 colon cancer cells were inoculated into WT, Dpl ^+/- , and Dpl ^-/- KO litters (n = 3-4), and tumors were measured for 3 weeks after inoculation, and tumors The volume was calculated. Tumor volume and mass were significantly smaller in Doppel KO mice compared to WT litters ( FIGS. 15 AC and 16 AC and 17 AC ). Gene dose effects were also observed in tumorigenesis: the heterozygous Dpl ^+/- mice had slightly larger tumors than the homozygous Dpl ^{-/- mice.} Whole-tissue-fixed immunofluorescence (IF) staining revealed that tumors in WT mice had a large vascular layer, whereas tumors in Dpl ^+/- and Dpl ^-/- KO mice had a rare few blood vessels in the vascular network. ( FIGS. 15D and 16D and 17D ). Doppel was not expressed in tumor vessels ^{of Dpl -/- KO mice, and Dpl +/-} KO mice showed fewer Doppel-positive tumor vessels than WT. However, the Doppel deletion in mice did not interfere with the healing rate of normal wounds in the body involved in physiological angiogenesis ( FIGS. 18A and B ). A 6-mm-diameter wound was created in the contralateral flank of tumor-bearing Doppel WT and KO mice. The wound healing rate of Dpl ^+/- and Dpl ^-/- KO mice did not differ from that of WT litters. The density of vascular structures in wound tissue healing in WT mice was the same as in KO mice. Matrigel-induced vasculature was also normal in WT and KO mice ( Fig. 18C, D ). CD31-staining showed uniform vascular network and hemoglobin content in Matrigel plugs explanted from WT and KO mice. Thus, the absence of Doppel retards tumor growth in mice without interfering with physiological angiogenesis.

Custom-made anti-Doppel mAbs were obtained and tested for recognition of mouse dopels present in the host-derived blood vessels of tumors, the ability to block doppel function in vitro, and the ability to stop tumor growth. Selection strategies include screening for binding to mouse dopel using ELISA and testing for the ability to detect dopel protein in TEC lysates using western blot. Six independent clones (7D9, 1B12, 1C8, 5C7, 4D6, and 6F11) were identified. Of the six clones, 5C7 and 4D6 eliminated mouse VEGF (mVEGF)-induced germination of TEC spheroids ( FIG. 20A, B ). The two most potent clones (5C7 and 4D6) were purified by Protein A affinity chromatography and tested for their inhibitory effect on Doppel/VEGFR2 signaling. The purified 5C7 and 4D6 clones prevented mVEGF-induced VEGFR2 phosphorylation and germination of TEC spheroids in TEC in a dose-dependent manner ( FIGS. 20C, D ).

These initial experiments demonstrated that anti-Doppel mAb clones antagonize Doppel-mediated angiogenic activity.

Based on in vitro data, we selected clones 4D6 and 5C7 to study the antitumor and antiangiogenic effects in the murine-derived CT26 colon cancer model. When tumors reached an average size of 50 mm 3, we treated mice with intravenous doses of 5 and 10 mg/kg of 4D6 and 5C7 mAb clones. 5C7 and 4D6 mAb significantly regressed tumor volume and body weight compared to saline and control IgG-treated groups, and 4D6 showed excellent activity ( FIGS. 19A, B and 21A ). When administered once or twice a week at a dose of 10 mg/kg, 4D6 mAb reduced tumor growth by 71.1 ± 4.2% or 87.9 ± 3.9%, respectively. In mAb-treated tumors, the number of Doppel-(red) and CD31-positive blood vessels (green) were significantly lowered compared to saline-treated or IgG-treated tumors ( FIG. 21B ); Mean vascular density also decreased with tumor volume ( FIG. 21C ).

Preliminary experiments with 4D6 and 5C7-treated tumor-bearing mice, upon review and histological analysis, showed no body weight changes, no obvious organ lesions, and no clinical chemistry or hematologic blood profile changes.

The anti-tumor effect of 4D6 mAb was also observed in HCT116 human colon cancer mouse xenografts. 4D6 mAb inhibited tumor growth ( FIG. 19C, D ), Doppel- and CD31-positive tumor vessels ( FIG. 19E ), and mean tumor vessel density in proportion to dose and dosing frequency ( FIG. 19F, G ).

In summary, in a therapeutic setting, anti-Doppel mAb treatment limits tumor progression.

The influence of Doppel on angiogenesis and VEGF2 expression visualizes vascular networks and VEGFR2 levels in explanted tumors and normal lungs from ^{WT, Dpl +/-} and Dpl ^{-/- KO mice and 4D6 anti-Doppel mAb treated mice.} It was measured and studied. ^{Doppel's gene deletion reduced VEGFR2 expression in tumors of Dpl +/-} and Dpl ^-/- KO mice compared to WT mice, but VEGFR2 expression was not changed in lungs of tumor-bearing mice ( FIGS. 22A, B and FIG. 23 and 24 ).

In order to investigate the role of Doppel in VEGFR2 signaling, the Doppel gene was derived from two different TECs isolated from squamous (TEC-SCC7) and colon cancer (TEC-CT26) and Doppel-transfected HUVECs (Hu.dpl). silenced using siRNA; Cells transfected with scrambled siRNA were used as controls. Doppel silencing reduced the protein levels of both Doppel and VEGFR2 in TEC-SCC7, TEC-CT26 ( FIG. 22C ) and Hu.dpl ( FIG. 25A ). In contrast, Doppel silencing decreased the mRNA levels of Doppel ( FIGS. 22D and 25B ), but did not change VEGFR2 mRNA levels in these cells ( FIGS. 22E and 25C ). Based on the observation that the protein level, not the mRNA level of VEGFR2, changed in the Doppel-silent EC, it was hypothesized that Doppel enlarged and stabilized the cell surface retention of VEGFR2 and made it more reactive to VEGF.

VEGF triggers VEGFR2 internalization and rearranges signaling clusters on the EC surface. Therefore, the membrane distribution of VEGFR2 in HUVEC and Hu.dpl cells was evaluated by stimulating the cells with VEGF. Surface receptors of cells were labeled with sulfo-NHS-biotin, cells were lysed, lysates were precipitated with streptavidin-conjugated magnetic beads, and the precipitates were analyzed by immunoblot using anti-VEGFR2 and anti-Doppel antibodies. I did. VEGF-stimulated HUVECs internalized VEGFR2 more than in VEGF-stimulated Hu.dpl cells ( FIG. 22F ); Receptor internalization was proportional to the amount of surface doppel. As determined by FACS analysis, at a higher rate than in Hu.dpl cells, HUVEC internalized VEGFR2 was due to VEGF stimulation ( FIGS. 22G and 26A ). Short exposure of VEGF depleted VEGFR2 from the membrane and increased the intracellular receptor pool of HUVECs, but the membrane content of VEGFR2 was similar to Hu.dpl in the presence or absence of VEGF ( FIG. 26B ). Total VEGFR2 was not altered in Hu.dpl by increasing VEGF concentration ( FIG. 26C ).

The effect of Doppel on VEGFR2 dimerization in the absence of VEGF was investigated. VEGFR2 homodimers/clusters were identified in intact cells using an orthotopic proximal ligation assay (PLA) ( FIG. 22G, H ). Hu.dpl cells had a higher VEGFR2 homodimer than HUVECs, and VEGFR2 dimerization/clustering was severely attenuated after Doppel siRNA or 5C7 anti-Doppel mAb treatment ( FIG. 22G, H ). Western blot was used to observe the dynamics between VEGFR2 monomer-dimer ( FIG. 28A ). In HUVECs, a prominent 125 kDa and relatively weaker 250 kDa bands appeared as VEGFR2 monomers and dimers, respectively. In Hu.dpl cells, VEGFR2 was detected in the 250 kDa band at the time of immunoblot. However, in Hu.dpl cells treated with Doppel siRNA or 5C7 anti-Doppel mAb, the band appeared at 125 kDa ( FIG. 28B ), suggesting that VEGFR2 promotes dimerization/clustering in the presence of Doppel.

Correspondingly, the same VEGF concentration dose-dependently reduced the total level of VEGFR2 in HUVECs. Upon stimulation with elevated concentrations of VEGF, the phosphorylation of VEGFR2 in Hu.dpl cells was much greater in HUVECs ( FIG. 28C ). Thus, Doppel controls VEGFR2 distribution, maintains high levels of VEGFR2 on the cell surface, and promotes and sustains VEGFR2 signaling.

The role of Doppel in tumor angiogenesis was evaluated in a rescue model involving re-expression of Doppel using cDNA transfection or treatment with a recombinant Doppel (rDP 1) protein. Human skin microvascular endothelial cells (HDMEC) were transfected for these experiments. VEGF-mediated VEGFR2 phosphorylation, cell proliferation, and germination of HDMEC spheroids were significantly increased in Doppel cDNA-transfected cells compared to mock or control cells ( FIGS. 29A-D ). Co-immunoprecipitation experiments using Doppel cDNA-transfected and rDP 1 -treated cells showed a physical interaction between VEGFR2 and Doppel or VEGFR2 and rDP 1 (FIG. 30A ). In the presence of rDP I , VEGF robustly and dose-dependently increased VEGFR2 phosphorylation ( FIG. 30B ). A similar phenomenon was observed in Doppel transfected HDMEC and Hu.dpl. These in vitro experiments suggest that soluble rDP l acts as a membrane bound doppel and helps in inducing angiogenesis.

To test this hypothesis, HUVEC spheroids were treated with VEGF with or without rDP l. VEGF promoted the germination of HUVEC spheroids, and germination was increased when co-treated with different concentrations of rDP l and in Hu.dpl spheroids (FIG. 27A ).

HUVEC spheroids were implanted in vivo on matrigel-fibrin gels in the presence of Doppel and VEGF according to a proven protocol (Alajati et al. , Nat Methods. 5:439-445 (2008) and Laib et al. , Nat Protoc . 4: 1202-1215 (2009)) , Hu.dpl nine compared with the upper body. HUVEC spheroids failed to grow any vascular network 1 week after transplantation in the absence of VEGF and rDP l , and the presence of VEGF stimulated angiogenesis (FIG. 27B ). In contrast, the presence of VEGF and rDP 1 resulted in a completely sparged vascular layer in the Matrigel-Fibrin gel. The hemoglobin content of VEGF and rDP l -treated HUVEC spheroids, which are indicators of the sparged blood vessels, was higher than that of the control and VEGF-treated spheroids and was similar to the Hu.dpl spheroids ( FIG. 27C ). These findings support that rDP l enhances angiogenesis in tumors.

In order to remedy this angiogenesis and tumor growth, Dpl ^+/- KO mouse was treated 3 times with intravenous injection of the at 4 days after EL4 tumor inoculation, rDP l 1 mg / kg of the rDP l. EL4 cells were also inoculated with 1 to 10 μg of rDP l ^{in a mixture with 50 μl of Matrigel in Dpl+/−} KO mice and grown for 10 days. rDP l mixture ^{dose-dependently rescued tumor growth in Dpl +/-} KO mice, and tumor size and weight were 10 μg of rDP l -mixed and WT control litter or 1 μg of rDP l -mixed and intravenous rDP l -Similar between treated mice ( FIG. 27D-F ). In tumor tissue, intravenous injected rDP l was concentrated and co-localized with VEGFR2- and CD31-positive vessels ( FIG. 30 ). These data demonstrated that Doppel's proangiogenic effects corresponded to clear and improved tumor growth in vivo.

Most modulators that control the spatial distribution of tyrosine kinase receptors are equally important in the mechanisms of development and resistance to tumor angiogenesis, metastasis or treatment response. This experiment confirmed the Doppel-VEGFR2 interaction as a key driver of VEGFR2 trafficking and signaling for selective control of tumor angiogenesis. Without wishing to be bound by theory, we believe that Doppel tilts the angiogenic balance towards the'on' mode, delays cell surface retention of VEGFR2, and helps to continue VEGF-signaling. Doppel's gene deletion and pharmacological inhibition inhibits VEGF/VEGFR2- signaling in tumors, reduces VEGFR2 expression in TEC, retards tumor vessel growth, and contracts tumors, but preserves VEGF-function in normal cells. . These data support the central assumption that Doppel is a pro-angiogenic factor and that Doppel-targeting molecules such as anti-Doppel mAb are practical anti-angiogenic agents. Again, without being bound by theory, it is believed that Doppel-targeting molecules, including the novel anti-Doppel mAbs described herein, block Doppel, whose role in normal (non-pathological) physiological angiogenesis is unknown.

Example 10: Epitope Mapping and Antibody Specificity

Methods for determining the epitope binding site of a given antibody are known in the art. For example, the following methodology can be applied to characterize anti-Doppel antibodies:

Various peptide fragments of Doppel were generated. For example, a series of polypeptides having the appropriate length obtained by sequentially shortening the doppel from the C-terminus or N-terminus was produced. Then, the binding affinity of antibodies against these polypeptides was investigated and the recognition site was roughly determined. Next, a series of shorter peptide fragments were generated and the binding affinity of antibodies against these peptides was investigated to determine the epitope.

The binding affinity of the second anti-Doppel antibody could be compared with the first antibody. For example, when the second antibody binds to the peptide fragment of the Dopel to which the first anti-Doppel antibody binds, it could be determined that the first antibody and the second antibody recognize the same epitope. Additionally or alternatively, if the second anti-Doppel antibody competes with the first anti-Doppel antibody for binding to Dopel (i.e., if the second antibody inhibits binding between the Dopel and the first antibody), the first antibody and 2 The antibody was able to determine that the first antibody and the second antibody recognized the same epitope (or overlapping epitope) even if the specific epitope sequence was not determined. In general, if the first antibody has a specific effect such as antigen neutralizing activity, and the first antibody and the second antibody bind to the same epitope, it could be predicted that the second antibody will have the same activity.

Name of donation institution: Korea Cell Line Research Foundation

Accession number: KCLRFBP00382

Consignment Date: 20161123

Name of donation institution: Korea Cell Line Research Foundation

Accession number: KCLRFBP00383

Consignment Date: 20161123

Name of donation institution: Korea Cell Line Research Foundation

Accession number: KCLRFBP00384

Consignment Date: 20161123

Name of donation institution: Korea Cell Line Research Foundation

Accession number: KCLRFBP00385

Consignment Date: 20161123

Name of donation institution: Korea Cell Line Research Foundation

Accession number: KCLRFBP00386

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Name of donation institution: Korea Cell Line Research Foundation

Accession number: KCLRFBP00387

Consignment Date: 20161123

<110> SNU R&DB Foundation <120> METHODS OF INHIBITING PATHOLOGICAL ANGIOGENESIS WITH DOPPEL-TARGETING MOLECULES <130> 17P1141 <160> 61 <170> KopatentIn 2.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 1 ggacatcgac tttggagcag ag 22 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 2 cagcatctcc ttggtcacgt tg 22 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 3 gatggcatcc actacaacgg ct 22 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 4 gttgtctggc ttctggaact cc 22 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 5 gccttcaaga ctctgttcgt gg 22 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 6 ctcgatgaag gcataaccac gg 22 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 7 tcaagactct cttcgtggcg ag 22 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 8 ggctttcctg accgcttact gt 22 <210> 9 <211> 187 <212> PRT <213> Artificial Sequence <220> <223> Included Signal Peptide <400> 9 Met Lys Asn Arg Leu Gly Thr Trp Trp Val Ala Ile Leu Cys Met Leu 1 5 10 15 Leu Ala Ser His Leu Ser Thr Val Lys Ala Arg Gly Ile Lys His Arg 20 25 30 Phe Lys Trp Asn Arg Lys Val Leu Pro Ser Ser Gly Gly Gln Ile Thr 35 40 45 Glu Ala Arg Val Ala Glu Asn Arg Pro Gly Ala Phe Ile Lys Gln Gly 50 55 60 Arg Lys Leu Asp Ile Asp Phe Gly Ala Glu Gly Asn Arg Tyr Tyr Ala 65 70 75 80 Ala Asn Tyr Trp Gln Phe Pro Asp Gly Ile Tyr Tyr Glu Gly Cys Ser 85 90 95 Glu Ala Asn Val Thr Lys Glu Met Leu Val Thr Ser Cys Val Asn Ala 100 105 110 Thr Gln Ala Ala Asn Gln Ala Glu Phe Ser Arg Glu Lys Gln Asp Ser 115 120 125 Lys Leu His Gln Arg Val Leu Trp Arg Leu Ile Lys Glu Ile Cys Ser 130 135 140 Ala Lys His Cys Asp Phe Trp Leu Glu Arg Gly Ala Ala Leu Arg Val 145 150 155 160 Ala Val Asp Gln Pro Ala Met Val Cys Leu Leu Gly Phe Val Trp Phe 165 170 175 Ile Val Lys Leu Glu His His His His His His 180 185 <210> 10 <211> 161 <212> PRT <213> Artificial Sequence <220> <223> Not included Signal peptide <400> 10 Arg Gly Ile Lys His Arg Phe Lys Trp Asn Arg Lys Val Leu Pro Ser 1 5 10 15 Ser Gly Gly Gln Ile Thr Glu Ala Arg Val Ala Glu Asn Arg Pro Gly 20 25 30 Ala Phe Ile Lys Gln Gly Arg Lys Leu Asp Ile Asp Phe Gly Ala Glu 35 40 45 Gly Asn Arg Tyr Tyr Ala Ala Asn Tyr Trp Gln Phe Pro Asp Gly Ile 50 55 60 Tyr Tyr Glu Gly Cys Ser Glu Ala Asn Val Thr Lys Glu Met Leu Val 65 70 75 80 Thr Ser Cys Val Asn Ala Thr Gln Ala Ala Asn Gln Ala Glu Phe Ser 85 90 95 Arg Glu Lys Gln Asp Ser Lys Leu His Gln Arg Val Leu Trp Arg Leu 100 105 110 Ile Lys Glu Ile Cys Ser Ala Lys His Cys Asp Phe Trp Leu Glu Arg 115 120 125 Gly Ala Ala Leu Arg Val Ala Val Asp Gln Pro Ala Met Val Cys Leu 130 135 140 Leu Gly Phe Val Trp Phe Ile Val Lys Leu Glu His His His His His 145 150 155 160 His <210> 11 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> epitope <400> 11 Tyr Trp Gln Phe Pro Asp 1 5 <210> 12 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> epitope <400> 12 Phe Pro Asp Gly Ile Tyr 1 5 <210> 13 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Light Chain CDR Sequences <400> 13 Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu 1 5 10 15 <210> 14 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Light Chain CDR Sequences <400> 14 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 15 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Light Chain CDR Sequences <400> 15 Phe Gln Gly Ser His Val Pro Leu Thr 1 5 <210> 16 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Light Chain CDR Sequences <400> 16 Arg Ala Ser Glu Ser Val Asp Ser His Gly Asn Ser Phe Met His 1 5 10 15 <210> 17 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Light Chain CDR Sequences <400> 17 Leu Ala Ser Ser Leu Glu Ser 1 5 <210> 18 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Light Chain CDR Sequences <400> 18 Gln Gln Asn Asn Glu Asp Pro Leu Thr 1 5 <210> 19 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Light Chain CDR Sequences <400> 19 Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu 1 5 10 15 <210> 20 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Light Chain CDR Sequences <400> 20 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 21 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Light Chain CDR Sequences <400> 21 Phe Gln Gly Ser His Val Pro Trp Thr 1 5 <210> 22 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Light Chain CDR Sequences <400> 22 Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu 1 5 10 15 <210> 23 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Light Chain CDR Sequences <400> 23 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 24 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Light Chain CDR Sequences <400> 24 Phe Gln Gly Ser His Val Pro Leu Thr 1 5 <210> 25 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Light Chain CDR Sequences <400> 25 Arg Ala Ser Gln Glu Ile Ser Gly Tyr Leu Ser 1 5 10 <210> 26 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Light Chain CDR Sequences <400> 26 Ala Ala Ser Ile Leu Asp Ser 1 5 <210> 27 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Light Chain CDR Sequences <400> 27 Leu Gln Tyr Ala Ser Tyr Pro Phe Met 1 5 <210> 28 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Light Chain CDR Sequences <400> 28 Arg Ala Ser Gln Glu Ile Ser Gly Tyr Leu Ser 1 5 10 <210> 29 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Light Chain CDR Sequences <400> 29 Ala Ala Ser Ile Leu Asp Ser 1 5 <210> 30 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Light Chain CDR Sequences <400> 30 Leu Gln Tyr Ala Ser Tyr Pro Phe Met 1 5 <210> 31 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain CDR Sequences <400> 31 Asn Tyr Trp Met Gln 1 5 <210> 32 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain CDR Sequences <400> 32 Ala Ile Tyr Pro Gly Asp Gly Asn Thr Arg Tyr Ser Gln Lys Phe Lys 1 5 10 15 Gly <210> 33 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain CDR Sequences <400> 33 Arg Asp Tyr Gly Ser Ser Tyr Trp Tyr Phe Asp Val 1 5 10 <210> 34 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain CDR Sequences <400> 34 Thr Ser Gly Met Gly Val Gly 1 5 <210> 35 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain CDR Sequences <400> 35 His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 36 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain CDR Sequences <400> 36 Arg Ala Asp Gly Tyr Gly Phe Ala Tyr 1 5 <210> 37 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain CDR Sequences <400> 37 Asn Tyr Gly Met Ser 1 5 <210> 38 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain CDR Sequences <400> 38 Thr Ile Ser Ser Gly Gly Arg Tyr Ile Tyr Tyr Pro Asp Ser Val Lys 1 5 10 15 Gly <210> 39 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain CDR Sequences <400> 39 Asp Ser Ser Asp Tyr Gly Phe Ala Tyr 1 5 <210> 40 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain CDR Sequences <400> 40 Thr Ser Gly Met Gly Val Ser 1 5 <210> 41 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain CDR Sequences <400> 41 His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 42 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain CDR Sequences <400> 42 Thr Ser Met Met Val Pro Pro Trp Phe Ala Tyr 1 5 10 <210> 43 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain CDR Sequences <400> 43 Ser His Trp Met Asn 1 5 <210> 44 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain CDR Sequences <400> 44 Gln Ile Tyr Pro Arg Asn Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys 1 5 10 15 Gly <210> 45 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain CDR Sequences <400> 45 Ser Thr Thr Ile Val Thr Thr Gly Ala Tyr 1 5 10 <210> 46 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain CDR Sequences <400> 46 Ser His Trp Met Asn 1 5 <210> 47 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain CDR Sequences <400> 47 Gln Ile Tyr Pro Arg Asn Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys 1 5 10 15 Gly <210> 48 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain CDR Sequences <400> 48 Ser Thr Thr Ile Val Thr Thr Gly Ala Tyr 1 5 10 <210> 49 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Light Chain Variable Region Sequences <400> 49 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 Arg <210> 50 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Light Chain Variable Region Sequences <400> 50 Asn Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser His 20 25 30 Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Ser Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp 65 70 75 80 Pro Val Glu Ala Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Asn 85 90 95 Glu Asp Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg 100 105 110 <210> 51 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Light Chain Variable Region Sequences <400> 51 Asp Val Leu Met Thr Gln Ile Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 52 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Light Chain Variable Region Sequences <400> 52 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 Arg <210> 53 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Light Chain Variable Region Sequences <400> 53 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr 20 25 30 Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser Ile Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser 65 70 75 80 Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Phe 85 90 95 Met Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 100 105 <210> 54 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Light Chain Variable Region Sequences <400> 54 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr 20 25 30 Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser Ile Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser 65 70 75 80 Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Phe 85 90 95 Met Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 100 105 <210> 55 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain Variable Region Sequences <400> 55 Gln Val Gln Leu His Gln Ser Gly Ser Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Tyr Pro Gly Asp Gly Asn Thr Arg Tyr Ser Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Asp Tyr Gly Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly 100 105 110 Ala Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 56 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain Variable Region Sequences <400> 56 Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Gln Leu Thr Ile Ser Lys Asp Thr Ser Arg Asn Gln Val 65 70 75 80 Phe Leu Lys Ile Thr Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Arg Ala Asp Gly Tyr Gly Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ala 115 <210> 57 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain Variable Region Sequences <400> 57 Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Ser Gly Gly Arg Tyr Ile Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Asp Ser Ser Asp Tyr Gly Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala 115 <210> 58 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain Variable Region Sequences <400> 58 Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Gly Val Ser Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Arg Asn Gln Val 65 70 75 80 Phe Leu Lys Ile Thr Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Thr Ser Met Met Val Pro Pro Trp Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ala 115 120 <210> 59 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain Variable Region Sequences <400> 59 Gln Val Gln Leu Gln Gln Ser Gly Thr Glu Leu Val Arg Pro Gly Ser 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser His 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gln Ile Tyr Pro Arg Asn Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ser Arg Ser Thr Thr Ile Val Thr Thr Gly Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ala 115 <210> 60 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain Variable Region Sequences <400> 60 Gln Val Gln Leu Gln Gln Ser Gly Thr Glu Leu Val Arg Pro Gly Ser 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser His 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gln Ile Tyr Pro Arg Asn Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ser Arg Ser Thr Thr Ile Val Thr Thr Gly Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ala 115 <210> 61 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> the corresponding sequence of human doppel <400> 61 Phe Pro Asp Gly Ile His 1 5

Claims (23)

An antibody that binds to a doppel or a doppel binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein
(a) the heavy chain variable region is CDRH1 comprising the amino acid sequence of SEQ ID NO: 31; CDRH2 comprising the amino acid sequence of SEQ ID NO: 32; And a CDRH3 comprising the amino acid sequence of SEQ ID NO: 33; The light chain variable region is CDRL1 comprising the amino acid sequence of SEQ ID NO: 13; CDRL2 comprising the amino acid sequence of SEQ ID NO: 14; And CDRL3 comprising the amino acid sequence of SEQ ID NO: 15;
(b) the heavy chain variable region is a CDRH1 comprising the amino acid sequence of SEQ ID NO: 34; CDRH2 comprising the amino acid sequence of SEQ ID NO: 35; And a CDRH3 comprising the amino acid sequence of SEQ ID NO: 36; The light chain variable region is CDRL1 comprising the amino acid sequence of SEQ ID NO: 16; CDRL2 comprising the amino acid sequence of SEQ ID NO: 17; And CDRL3 comprising the amino acid sequence of SEQ ID NO: 18;
(c) the heavy chain variable region is a CDRH1 comprising the amino acid sequence of SEQ ID NO: 37; CDRH2 comprising the amino acid sequence of SEQ ID NO: 38; And a CDRH3 comprising the amino acid sequence of SEQ ID NO: 39; The light chain variable region is CDRL1 comprising the amino acid sequence of SEQ ID NO: 19; CDRL2 comprising the amino acid sequence of SEQ ID NO: 20; And CDRL3 comprising the amino acid sequence of SEQ ID NO: 21;
(d) the heavy chain variable region is a CDRH1 comprising the amino acid sequence of SEQ ID NO: 40; CDRH2 comprising the amino acid sequence of SEQ ID NO: 41; And a CDRH3 comprising the amino acid sequence of SEQ ID NO: 42; The light chain variable region is CDRL1 comprising the amino acid sequence of SEQ ID NO: 22; CDRL2 comprising the amino acid sequence of SEQ ID NO: 23; And CDRL3 comprising the amino acid sequence of SEQ ID NO: 24;
(e) the heavy chain variable region is a CDRH1 comprising the amino acid sequence of SEQ ID NO: 43; CDRH2 comprising the amino acid sequence of SEQ ID NO: 44; And a CDRH3 comprising the amino acid sequence of SEQ ID NO: 45; The light chain variable region is CDRL1 comprising the amino acid sequence of SEQ ID NO: 25; CDRL2 comprising the amino acid sequence of SEQ ID NO: 26; And CDRL3 comprising the amino acid sequence of SEQ ID NO: 27; or
(f) the heavy chain variable region is a CDRH1 comprising the amino acid sequence of SEQ ID NO: 46; CDRH2 comprising the amino acid sequence of SEQ ID NO: 47; And a CDRH3 comprising the amino acid sequence of SEQ ID NO: 48; The light chain variable region is CDRL1 comprising the amino acid sequence of SEQ ID NO: 28; CDRL2 comprising the amino acid sequence of SEQ ID NO: 29; And CDRL3 comprising the amino acid sequence of SEQ ID NO: 30. The method of claim 1, wherein the antibody or a doppel-binding fragment thereof specifically binds to SEQ ID NO: 11, the antibody or a doppel-binding fragment thereof. The method of claim 1, wherein the antibody or a doppel-binding fragment thereof specifically binds to SEQ ID NO: 12, the antibody or a doppel-binding fragment thereof. The antibody or doppel binding fragment thereof according to claim 1, wherein the antibody or a doppel binding fragment thereof binds to a non-glycosylated doppel with a greater affinity than a glycosylated doppel. The antibody or dopel binding fragment thereof according to claim 1, wherein the antibody or a doppel binding fragment thereof binds to a dimer form of a doppel. The method of claim 1, wherein the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO: 55, 56, 57, 58, 59, or 60; The light chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 49, 50, 51, 52, 53, or 54, an antibody or a Doppel binding fragment thereof. According to claim 1, wherein the antibody or a doppel binding fragment thereof is a monoclonal antibody, the antibody or a doppel binding fragment thereof. delete delete delete delete delete delete delete Breast carcinoma, lung carcinoma, gastric carcinoma, esophageal carcinoma, colorectal carcinoma, liver carcinoma, ovarian carcinoma, male embryo, including the anti-Doppel antibody according to claim 1 or a Dopel-binding fragment thereof, and a pharmaceutically acceptable carrier or diluent. Cytoma, cervical carcinoma, endometrial carcinoma, endometrial hyperplasia, endometriosis, fibrosarcoma, chorionic carcinoma, head and neck cancer, nasopharyngeal carcinoma, laryngeal carcinoma, hepatoblastoma, Kaposi's sarcoma, melanoma, skin carcinoma, hemangioma, cavernous hemangioma , Hemangioblastoma, pancreatic carcinoma, retinoblastoma, astrocytoma, glioblastoma, schwannoma, oligodendroglioma, glioblastoma, neuroblastoma, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcoma, urinary tract carcinoma, thyroid carcinoma, Wilm tumor, renal cell carcinoma, prostate Pharmaceutical composition for the treatment of carcinoma, nevus-related abnormal vascular hyperplasia, brain tumor-related edema, and Meigs syndrome delete delete delete delete delete As an anti-Doppel antibody or a Doppel-binding fragment thereof that specifically binds to the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 61 and inhibits Doppel-tyrosine kinase receptor signaling,
The antibody or binding fragment thereof is CDRH1 comprising the amino acid sequence of SEQ ID NO: 43; CDRH2 comprising the amino acid sequence of SEQ ID NO: 44; And a heavy chain variable region comprising a CDRH3 comprising the amino acid sequence of SEQ ID NO: 45, and a CDRL1 comprising the amino acid sequence of SEQ ID NO: 25; CDRL2 comprising the amino acid sequence of SEQ ID NO: 26; And a light chain variable region comprising CDRL3 comprising the amino acid sequence of SEQ ID NO: 27. delete delete

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