Monoclonal antibodies against ELTD1 and uses thereof

Abstract

The present disclosure is directed to novel monoclonal antibodies that bind to ELTD1 and methods of use thereof, including the detection and treatment of cancer, multiple sclerosis, retinopathy, or promoting tissue regeneration.

Description

[Technical Field]

[0001] The present invention relates generally to the field of antibodies to ELTD1 that may be used in the treatment of multiple sclerosis, retinopathy, tissue regeneration, and in the detection and treatment of cancer.

[0002] Statement Regarding Federally Sponsored Research Not applicable

[0003] Incorporation by Reference of Material Filed on a Compact Disc This application contains a Sequence Listing that has been submitted via EFS-Web in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy above, created in 2019, is named OMRF2013WO_Seq_Listing.txt and is bytes in size. [Background technology]

[0004] Without limiting the scope of the invention, its background is described in relation to EGF, latrophilin, and seven transmembrane domain-containing protein 1 (ELTD1).

[0005] ELTD1 is a latrophilin-like orphan receptor of the adhesion G protein-coupled receptor family. In humans, the ELTD1 gene encodes ELTD1. ELTD1 appears to play a role in angiogenesis (both physiological and pathological) and glioblastoma. However, its role in other diseases has been little investigated. Summary of the Invention

[0006] In one embodiment, the invention includes a method of treating a subject having or suspected of having multiple sclerosis, comprising delivering to the subject an antibody or antibody fragment having binding affinity for EGF, latrophilin, and seven-transmembrane domain-containing protein 1 (ELTD1). In one aspect, the antibody is a recombinant bivalent scFv Fc fusion antibody. In another aspect, the antibody fragment is a recombinant scFv (single-chain variable fragment) antibody, Fab fragment, F(ab')2 fragment, or Fv fragment. In another aspect, the antibody or antibody fragment is chimeric, humanized, fully human, or bispecific. In another aspect, the antibody or antibody fragment comprises an Fc portion mutated to alter (eliminate or enhance) FcR interactions to increase half-life and / or increase or decrease effector function, such as antibody-dependent cellular cytotoxicity or complement activation. In another embodiment, the antibody light chain variable region and heavy chain variable region have the amino acid sequences of SEQ ID NOs: 2 and 4, 6 and 8, 10 and 12, 14 and 16, 18 and 20, or 22 and 24, respectively. In another embodiment, the antibody has light chain variable region complementarity determining regions (CDRs) CDR1, CDR2, and CDR3 of SEQ ID NOs: 25, 26, and 27; 31, 32, and 33; 37, 38, and 39; 43, 44, and 45; 49, 50, and 51; 55, 56, and 57; 61, 62, and 63; 67, 68, and 69; 73, 74, and 75; 79, 80, and 81; 85, 86, and 87; 91, 92, and 93; 97, 98, and 99; 103, 104, and 105; 109, 110, and 111; 115, 116, and 117; 121, 122, and 123; or 127, 128, 129, respectively.In another embodiment, the antibody has heavy chain variable region CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 28, 29, and 30; 34, 35, and 36; 40, 41, and 42; 46, 47, and 48; 52, 53, and 54; 58, 59, and 60; 64, 65, and 66; 70, 71, and 72; 76, 77, and 78; 82, 83, and 84; 88, 89, and 90; 94, 95, and 96; 100, 101, and 102; 106, 107, and 108; 112, 113, and 114; 118, 119, and 120; 124, 125, and 126; or 130, 131, and 132, respectively. In another embodiment, the antibody or antibody fragment is adapted for administration or genetic delivery using an RNA or DNA sequence or vector encoding the antibody or antibody fragment. In another embodiment, the method further comprises providing an active agent for treating or alleviating a symptom of multiple sclerosis selected from corticosteroids, plasma exchange (plasmapheresis), ocrelizumab (Ocrevus), beta-interferon, glatiramer acetate (Copaxone, Glatopa), fingolimod (Gilenya), dimethyl fumarate (Tecfidera), teriflunomide (Aubagio), siponimod (Mayzent), ocrelizumab (Ocrevus), natalizumab (Tysabri), alemtuzumab (Campath, Lemtrada), mitoxantrone, or a functional derivative thereof. In another embodiment, the method further comprises attaching a probe to the antibody or antibody fragment for non-invasive in vivo detection of ELTD1.

[0007] In another embodiment, the invention includes a method of treating a subject having or suspected of having retinopathy, comprising delivering to the subject an antibody or antibody fragment having binding affinity for EGF, latrophilin, and seven-transmembrane domain-containing protein 1 (ELTD1). In one aspect, the antibody is a recombinant bivalent scFv Fc fusion antibody. In another aspect, the antibody fragment is a recombinant scFv (single-chain variable fragment) antibody, Fab fragment, F(ab')2 fragment, or Fv fragment. In another aspect, the antibody or antibody fragment is chimeric, humanized, fully human, or bispecific. In another aspect, the antibody or antibody fragment comprises an Fc portion mutated to alter (eliminate or enhance) FcR interactions to increase half-life and / or increase or decrease effector function, such as antibody-dependent cellular cytotoxicity or complement activation. In another embodiment, the antibody light chain variable region and heavy chain variable region have the amino acid sequences of SEQ ID NOs: 2 and 4, 6 and 8, 10 and 12, 14 and 16, 18 and 20, or 22 and 24, respectively. In another embodiment, the antibody has light chain variable region CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 25, 26, and 27; 31, 32, and 33; 37, 38, and 39; 43, 44, and 45; 49, 50, and 51; 55, 56, and 57; 61, 62, and 63; 67, 68, and 69; 73, 74, and 75; 79, 80, and 81; 85, 86, and 87; 91, 92, and 93; 97, 98, and 99; 103, 104, and 105; 109, 110, and 111; 115, 116, and 117; 121, 122, and 123; or 127, 128, and 129, respectively. In another embodiment, the antibody has heavy chain variable region CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 28, 29, and 30; 34, 35, and 36; 40, 41, and 42; 46, 47, and 48; 52, 53, and 54; 58, 59, and 60; 64, 65, and 66; 70, 71, and 72; 76, 77, and 78; 82, 83, and 84; 88, 89, and 90; 94, 95, and 96; 100, 101, and 102; 106, 107, and 108; 112, 113, and 114; 118, 119, and 120; 124, 125, and 126; or 130, 131, and 132, respectively.In another embodiment, the antibody or antibody fragment is adapted for administration or gene delivery using an RNA or DNA sequence or vector encoding the antibody or antibody fragment. In another embodiment, the method further comprises providing an active agent that treats or alleviates a symptom of retinopathy selected from a corticosteroid, a VEGF inhibitor, a PKC inhibitor, or a growth hormone inhibitor.

[0008] In another embodiment, the invention includes a pharmaceutical formulation comprising a polyclonal antiserum or one or more monoclonal antibodies or antibody fragments that bind to EGF, latrophilin, and seven-transmembrane domain-containing protein 1 (ELTD1). In one aspect, the antibody is a recombinant bivalent scFv-Fc fusion antibody. In another aspect, at least one of the antibody fragments is a recombinant scFv (single-chain variable fragment) antibody, Fab fragment, F(ab')2 fragment, or Fv fragment. In another aspect, the antibody or antibody fragment is chimeric, humanized, fully human, or bispecific. In another aspect, the antibody or antibody fragment comprises an Fc portion that has been mutated to alter (eliminate or enhance) FcR interactions, increase half-life, and / or increase or decrease effector functions such as antibody-dependent cellular cytotoxicity or complement activation. In another aspect, at least one of the light chain variable region and the heavy chain variable region has the amino acid sequence of SEQ ID NOs: 2 and 4, 6 and 8, 10 and 12, 14 and 16, 18 and 20, or 22 and 24, respectively. In another embodiment, the antibody has light chain variable region complementarity determining regions (CDRs) CDR1, CDR2, and CDR3 of SEQ ID NOs: 25, 26, and 27; 31, 32, and 33; 37, 38, and 39; 43, 44, and 45; 49, 50, and 51; 55, 56, and 57; 61, 62, and 63; 67, 68, and 69; 73, 74, and 75; 79, 80, and 81; 85, 86, and 87; 91, 92, and 93; 97, 98, and 99; 103, 104, and 105; 109, 110, and 111; 115, 116, and 117; 121, 122, and 123; or 127, 128, and 129, respectively. In another embodiment, the antibody has heavy chain variable region CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 28, 29, and 30; 34, 35, and 36; 40, 41, and 42; 46, 47, and 48; 52, 53, and 54; 58, 59, and 60; 64, 65, and 66; 70, 71, and 72; 76, 77, and 78; 82, 83, and 84; 88, 89, and 90; 94, 95, and 96; 100, 101, and 102; 106, 107, and 108; 112, 113, and 114; 118, 119, and 120; 124, 125, and 126; or 130, 131, and 132, respectively.In another embodiment, the antibody or antibody fragment is adapted for administration or gene delivery using an RNA or DNA sequence or vector encoding the antibody or antibody fragment, hi another embodiment, at least one of the antibodies or antibody fragments further comprises a cell penetrating peptide and / or is an intrabody.

[0009] In another embodiment, the present invention includes a method of promoting tissue regeneration, comprising contacting a cell or tissue with a polyclonal antiserum or an antibody or antibody fragment having binding affinity for ELTD1. In one aspect, the antibody is a recombinant bivalent scFv Fc fusion antibody. In another aspect, the antibody fragment is a recombinant scFv (single-chain variable fragment) antibody, Fab fragment, F(ab')2 fragment, or Fv fragment. In another aspect, the antibody or antibody fragment is chimeric, humanized, fully human, or bispecific. In another aspect, the antibody or antibody fragment comprises an Fc portion mutated to alter (eliminate or enhance) FcR interactions, increase half-life, and / or increase or decrease effector functions such as antibody-dependent cellular cytotoxicity or complement activation. In another aspect, the light chain variable region and heavy chain variable region of the antibody have the amino acid sequences of SEQ ID NOs: 2 and 4, 6 and 8, 10 and 12, 14 and 16, 18 and 20, or 22 and 24. In another embodiment, the antibody has light chain variable region CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 25, 26, and 27; 31, 32, and 33; 37, 38, and 39; 43, 44, and 45; 49, 50, and 51; 55, 56, and 57; 61, 62, and 63; 67, 68, and 69; 73, 74, and 75; 79, 80, and 81; 85, 86, and 87; 91, 92, and 93; 97, 98, and 99; 103, 104, and 105; 109, 110, and 111; 115, 116, and 117; 121, 122, and 123; or 127, 128, and 129, respectively. In another embodiment, the antibody has heavy chain variable region CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 28, 29, and 30; 34, 35, and 36; 40, 41, and 42; 46, 47, and 48; 52, 53, and 54; 58, 59, and 60; 64, 65, and 66; 70, 71, and 72; 76, 77, and 78; 82, 83, and 84; 88, 89, and 90; 94, 95, and 96; 100, 101, and 102; 106, 107, and 108; 112, 113, and 114; 118, 119, and 120; 124, 125, and 126; or 130, 131, and 132, respectively.In another embodiment, the antibody or antibody fragment is adapted for administration or gene delivery using an RNA or DNA sequence or vector encoding the antibody or antibody fragment. In another embodiment, the cells are contacted in vitro. In another embodiment, the cells are contacted in vivo. In another embodiment, the cells are cardiac cells, liver cells, muscle cells, kidney cells, skin cells, lung cells, bladder cells, intestinal cells, hair follicle cells, retinal cells, corneal cells, stomach cells, nerve cells, endothelial cells, pancreatic cells, thyroid cells, epidermal cells, neuronal cells, microglial cells, astrocytes, or breast cells. In another embodiment, the tissue regeneration includes at least one of nerve regeneration, vascular regeneration, or bone regeneration.

[0010] In another embodiment, the present invention includes a method of treating a subject having or suspected of having cancer, comprising delivering to the subject an antibody or antibody fragment having binding affinity for ELTD1. In another aspect, the antibody is a recombinant bivalent scFv-Fc fusion antibody. In another aspect, the antibody fragment is a recombinant scFv (single-chain variable fragment) antibody, Fab fragment, F(ab')2 fragment, or Fv fragment. In another aspect, the antibody or antibody fragment is chimeric, humanized, fully human, or bispecific. In another aspect, the antibody or antibody fragment comprises an Fc portion mutated to alter (eliminate or enhance) FcR interactions, increase half-life, and / or increase or decrease effector functions such as antibody-dependent cellular cytotoxicity or complement activation. In another aspect, the light chain variable region and heavy chain variable region of the antibody have the amino acid sequences of SEQ ID NOs: 2 and 4, 6 and 8, 10 and 12, 14 and 16, 18 and 20, or 22 and 24, respectively. In another embodiment, the antibody has light chain variable region CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 25, 26, and 27; 31, 32, and 33; 37, 38, and 39; 43, 44, and 45; 49, 50, and 51; 55, 56, and 57; 61, 62, and 63; 67, 68, and 69; 73, 74, and 75; 79, 80, and 81; 85, 86, and 87; 91, 92, and 93; 97, 98, and 99; 103, 104, and 105; 109, 110, and 111; 115, 116, and 117; 121, 122, and 123; or 127, 128, and 129, respectively. In another embodiment, the antibody has heavy chain variable region CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 28, 29, and 30; 34, 35, and 36; 40, 41, and 42; 46, 47, and 48; 52, 53, and 54; 58, 59, and 60; 64, 65, and 66; 70, 71, and 72; 76, 77, and 78; 82, 83, and 84; 88, 89, and 90; 94, 95, and 96; 100, 101, and 102; 106, 107, and 108; 112, 113, and 114; 118, 119, and 120; 124, 125, and 126; or 130, 131, and 132, respectively.In another embodiment, the antibody or antibody fragment is adapted for administration or gene delivery using an RNA or DNA sequence or vector encoding the antibody or antibody fragment. In another embodiment, the subject has metastatic colorectal cancer, first-line non-squamous non-small cell lung cancer, recurrent glioblastoma multiforme, metastatic renal cell carcinoma, persistent, recurrent, or metastatic cervical cancer, and epithelial ovarian cancer, fallopian tube cancer, or primary peritoneal cancer. In another embodiment, the method further comprises treating the subject with a second cancer therapy, such as radiation, chemotherapy, immunotherapy, toxin therapy, or surgery. In another embodiment, the antibody or antibody fragment is adapted for administration by gene delivery using an RNA or DNA sequence or vector encoding the antibody or antibody fragment. In another embodiment, the antibody or antibody fragment exhibits reduced or no bleeding during administration. In another aspect, the cancer is metastatic colorectal cancer, first-line non-squamous non-small cell lung cancer, recurrent glioblastoma multiforme, metastatic renal cell carcinoma, persistent, recurrent, or metastatic cervical cancer, and epithelial ovarian, fallopian tube, or primary peritoneal cancer.

[0011] In another embodiment, the invention includes a monoclonal antibody or binding fragment thereof that specifically binds to ELTD1, comprising a light chain variable region and a heavy chain variable region having the amino acid sequences of SEQ ID NOs: 2 and 4, 6 and 8, 10 and 12, 14 and 16, 18 and 20, or 22 and 24, respectively. In one aspect, the antibody is a recombinant bivalent scFv Fc fusion antibody. In another aspect, the binding fragment is a recombinant scFv (single-chain variable fragment) antibody, Fab fragment, F(ab')2 fragment, or Fv fragment. In another aspect, the antibody or antibody fragment is chimeric, humanized, fully human, or bispecific. In another aspect, the antibody or antibody fragment comprises an Fc portion that has been mutated to alter (eliminate or enhance) FcR interactions, increase half-life, and / or increase or decrease effector function, such as antibody-dependent cellular cytotoxicity or complement activation. In another aspect, the antibody is an scFv and is encoded by at least one nucleic acid of SEQ ID NOs: 1, 5, 9, 13, 17, or 21. In another embodiment, the monoclonal antibody or binding fragment thereof has light chain variable region CDR1, CDR2, and CDR3 of SEQ ID NOs: 25, 26, and 27; 31, 32, and 33; 37, 38, and 39; 43, 44, and 45; 49, 50, and 51; 55, 56, and 57; 61, 62, and 63; 67, 68, and 69; 73, 74, and 75; 79, 80, and 81; 85, 86, and 87; 91, 92, and 93; 97, 98, and 99; 103, 104, and 105; 109, 110, and 111; 115, 116, and 117; 121, 122, and 123; or 127, 128, and 129, respectively. In another embodiment, the monoclonal antibody or binding fragment thereof has heavy chain variable region CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 28, 29, and 30; 34, 35, and 36; 40, 41, and 42; 46, 47, and 48; 52, 53, and 54; 58, 59, and 60; 64, 65, and 66; 70, 71, and 72; 76, 77, and 78; 82, 83, and 84; 88, 89, and 90; 94, 95, and 96; 100, 101, and 102; 106, 107, and 108; 112, 113, and 114; 118, 119, and 120; 124, 125, and 126; or 130, 131, and 132, respectively.In another embodiment, the monoclonal antibody or binding fragment thereof comprises light chain variable region CDR1, CDR2, and CDR3 of SEQ ID NOs: 25, 26, and 27; 31, 32, and 33; 37, 38, and 39; 43, 44, and 45; 49, 50, and 51; 55, 56, and 57; 61, 62, and 63; 67, 68, and 69; 73, 74, and 75; 79, 80, and 81; 85, 86, and 87; 91, 92, and 93; 97, 98, and 99; 103, 104, and 105; 109, 110, and 111; 115, 116, and 117; 121, 122, and 123; or 127, 128, and 129, respectively. or having at least one or both of heavy chain variable region CDR1, CDR2, and CDR3 of SEQ ID NOs: 28, 29, and 30; 34, 35, and 36; 40, 41, and 42; 46, 47, and 48; 52, 53, and 54; 58, 59, and 60; 64, 65, and 66; 70, 71, and 72; 76, 77, and 78; 82, 83, and 84; 88, 89, and 90; 94, 95, and 96; 100, 101, and 102; 106, 107, and 108; 112, 113, and 114; 118, 119, and 120; 124, 125, and 126; or 130, 131, and 132, respectively. In one embodiment, the antibody or antibody fragment is adapted for administration or gene delivery using an RNA or DNA sequence or vector encoding the antibody or antibody fragment, hi another embodiment, the monoclonal antibody further comprises a probe attached to the antibody or antibody fragment.

[0012] In another embodiment, the present invention provides a method for detecting ELTD1 in a subject, comprising obtaining a biological sample from the subject and contacting the biological sample with a monoclonal antibody or binding fragment thereof that specifically binds to ELTD1, wherein the monoclonal antibody or binding fragment thereof is selected from the group consisting of SEQ ID NOs: 25, 26, and 27; 31, 32, and 33; 37, 38, and 39; 43, 44, and 45; Light chains 49, 50, and 51; 55, 56, and 57; 61, 62, and 63; 67, 68, and 69; 73, 74, and 75; 79, 80, and 81; 85, 86, and 87; 91, 92, and 93; 97, 98, and 99; 103, 104, and 105; 109, 110, and 111; 115, 116, and 117; 121, 122, and 123; or 127, 128, and 129 are possible. The variable regions CDR1, CDR2, and CDR3 are set forth in SEQ ID NOs: 28, 29, and 30; 34, 35, and 36; 40, 41, and 42; 46, 47, and 48; 52, 53, and 54; 58, 59, and 60; 64, 65, and 66; 70, 71, and 72; 76, 77, and 78; 82, 83, and 84; 88, 89, and 90; 94, 95, and 96; 100, 101, and and detecting binding of the monoclonal antibody or binding fragment thereof to the sample. In one embodiment, the antibody is a recombinant bivalent scFv Fc fusion antibody.

[0013] In another embodiment, the present invention includes a bifunctional monoclonal antibody or antibody fragment thereof or polyclonal serum having binding affinity for ELTD1, which downregulates both Notch1 and VEGFR2 upon contact with target cells. In another aspect, the antibody is a recombinant bivalent scFv-Fc fusion antibody. In another aspect, the antibody fragment is a recombinant scFv (single-chain variable fragment) antibody, Fab fragment, F(ab')2 fragment, or Fv fragment. In another aspect, the antibody or antibody fragment is chimeric, humanized, fully human, or bispecific. In another aspect, the antibody or antibody fragment comprises an Fc portion mutated to alter (eliminate or enhance) FcR interactions, increase half-life, and / or increase or decrease effector functions such as antibody-dependent cellular cytotoxicity or complement activation. In another aspect, the antibody is an scFv and is encoded by at least one nucleic acid of SEQ ID NO: 1, 5, 9, 13, 17, or 21. In another embodiment, the monoclonal antibody has the amino acid sequence of SEQ ID NOs: 2 and 4, 6 and 8, 10 and 12, 14 and 16, 18 and 20, or 22 and 24. In another embodiment, the monoclonal antibody or binding fragment thereof has light chain variable region CDR1, CDR2, and CDR3 of SEQ ID NOs: 25, 26, and 27; 31, 32, and 33; 37, 38, and 39; 43, 44, and 45; 49, 50, and 51; 55, 56, and 57; 61, 62, and 63; 67, 68, and 69; 73, 74, and 75; 79, 80, and 81; 85, 86, and 87; 91, 92, and 93; 97, 98, and 99; 103, 104, and 105; 109, 110, and 111; 115, 116, and 117; 121, 122, and 123; or 127, 128, and 129, respectively.In another embodiment, the monoclonal antibody or binding fragment thereof has heavy chain variable region CDR1, CDR2, and CDR3 of SEQ ID NOs: 28, 29, and 30; 34, 35, and 36; 40, 41, and 42; 46, 47, and 48; 52, 53, and 54; 58, 59, and 60; 64, 65, and 66; 70, 71, and 72; 76, 77, and 78; 82, 83, and 84; 88, 89, and 90; 94, 95, and 96; 100, 101, and 102; 106, 107, and 108; 112, 113, and 114; 118, 119, and 120; 124, 125, and 126; or 130, 131, and 132. In another embodiment, the monoclonal antibody or binding fragment thereof comprises the light chain variable region CDR1, CDR2, and CDR3 of SEQ ID NOs: 25, 26, and 27; 31, 32, and 33; 37, 38, and 39; 43, 44, and 45; 49, 50, and 51; 55, 56, and 57; 61, 62, and 63; 67, 68, and 69; 73, 74, and 75; 79, 80, and 81; 85, 86, and 87; 91, 92, and 93; 97, 98, and 99; 103, 104, and 105; 109, 110, and 111; 115, 116, and 117; 121, 122, and 123; or 127, 128, and 129, or , and have at least one or both of heavy chain variable region CDR1, CDR2, and CDR3 of SEQ ID NOs: 28, 29, and 30; 34, 35, and 36; 40, 41, and 42; 46, 47, and 48; 52, 53, and 54; 58, 59, and 60; 64, 65, and 66; 70, 71, and 72; 76, 77, and 78; 82, 83, and 84; 88, 89, and 90; 94, 95, and 96; 100, 101, and 102; 106, 107, and 108; 112, 113, and 114; 118, 119, and 120; 124, 125, and 126; or 130, 131, and 132, respectively. In another embodiment, the antibody or antibody fragment is adapted for administration or gene delivery using an RNA or DNA sequence or vector encoding the antibody or antibody fragment, hi another embodiment, the monoclonal antibody or antibody fragment thereof inhibits proliferation or initiates cell death, or both, in vitro.In another embodiment, when the antibody binds to cancer cells, it reduces the expression of at least one of sodium channel protein type 5 subunit alpha or L1 cell adhesion molecule. In another embodiment, when the antibody binds to cancer cells, it increases the expression of at least one of ADA, SCN5A, L1CAM, BMP2, alkaline phosphatase (ALPL), and TRPM8 in all tumors that express ELTD1. In another embodiment, when the antibody binds to cancer cells, it reduces the expression of SELENBP1. In another embodiment, when the antibody binds to hepatocellular carcinoma, it reduces VWA1. In another embodiment, when the antibody binds to lung cancer cells, it reduces the expression of SCUBE3, increases the expression of PLCH1, increases the expression of CHRNA1, or increases the expression of CDH2. In another embodiment, when the antibody binds to breast cancer cells, it reduces IFITM10, increases DCDC2, increases CHST9, and increases CDH2. In another embodiment, when the antibody binds to cancer cells, it increases the expression of CD74. In another embodiment, when the antibody binds to a retinal cell, the antibody decreases the expression of at least one of adenosine deaminase or apelin. In another embodiment, when the antibody binds to a retinal cell, the antibody increases the expression of bone gamma-carboxyglutamic acid protein, matrilin 2, or von Willebrand factor A domain-containing 1. In another embodiment, when the antibody is provided to a subject with multiple sclerosis, the antibody decreases the expression of at least one of sodium channel protein type 5 subunit alpha, adenosine deaminase, apelin, spinster homolog 2, or bone morphogenetic protein 2. In another embodiment, when the antibody is provided to a subject with multiple sclerosis, the antibody increases the expression of at least one of CD74 or IKAROS family zinc finger 1 (Ikaros). In another embodiment, when the antibody is provided to a subject at a site in need of tissue regeneration, the antibody increases the expression of at least one of bone gamma-carboxyglutamic acid (gla) protein, matrilin 2, or von Willebrand factor A domain-containing 1.In another aspect, the antibody is provided in an amount sufficient to treat a cancer selected from metastatic colorectal cancer, first-line non-squamous non-small cell lung cancer, recurrent glioblastoma multiforme, metastatic renal cell carcinoma, persistent, recurrent, or metastatic cervical cancer, and epithelial ovarian, fallopian tube, or primary peritoneal cancer.

[0014] In another embodiment, the invention includes a method of treating a disease or condition in a subject with an anti-EGF, latrophilin, and seven transmembrane domain-containing protein 1 (ELTD1) antibody or fragment thereof, comprising identifying a subject in need of disease or condition treatment and providing an effective amount of an antibody or binding fragment thereof that specifically binds to ELTD1 sufficient to alleviate the symptoms of or treat the disease or condition, wherein the disease or condition is selected from multiple sclerosis, retinopathy, cancer, or tissue regeneration. In one aspect, the antibody is a polyclonal or monoclonal antibody, or fragment thereof. In another aspect, the antibody is a recombinant bivalent scFv Fc fusion antibody. In another aspect, the monoclonal antibody or binding fragment thereof is a recombinant scFv (single-chain variable fragment) antibody, Fab fragment, F(ab')2 fragment, or Fv fragment. In another aspect, the monoclonal antibody or antibody fragment is chimeric, humanized, fully human, or bispecific. In another embodiment, the antibody or antibody fragment comprises an Fc portion that has been mutated to alter (eliminate or enhance) FcR interactions to increase half-life and / or increase or decrease effector function, such as antibody-dependent cellular cytotoxicity or complement activation. In another embodiment, the antibody is an scFv and is encoded by at least one nucleic acid of SEQ ID NOs: 1, 5, 9, 13, 17, or 21. In another embodiment, the monoclonal antibody has the amino acid sequence of SEQ ID NOs: 2 and 4, 6 and 8, 10 and 12, 14 and 16, 18 and 20, or 22 and 24. In another embodiment, the monoclonal antibody or binding fragment thereof has light chain variable region CDR1, CDR2, and CDR3 of SEQ ID NOs: 25, 26, and 27; 31, 32, and 33; 37, 38, and 39; 43, 44, and 45; 49, 50, and 51; 55, 56, and 57; 61, 62, and 63; 67, 68, and 69; 73, 74, and 75; 79, 80, and 81; 85, 86, and 87; 91, 92, and 93; 97, 98, and 99; 103, 104, and 105; 109, 110, and 111; 115, 116, and 117; 121, 122, and 123; or 127, 128, and 129, respectively.In another embodiment, the monoclonal antibody or binding fragment thereof has heavy chain variable region CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 28, 29, and 30; 34, 35, and 36; 40, 41, and 42; 46, 47, and 48; 52, 53, and 54; 58, 59, and 60; 64, 65, and 66; 70, 71, and 72; 76, 77, and 78; 82, 83, and 84; 88, 89, and 90; 94, 95, and 96; 100, 101, and 102; 106, 107, and 108; 112, 113, and 114; 118, 119, and 120; 124, 125, and 126; or 130, 131, and 132, respectively. In another embodiment, the monoclonal antibody or binding fragment thereof comprises light chain variable region CDR1, CDR2, and CDR3 of SEQ ID NOs: 25, 26, and 27; 31, 32, and 33; 37, 38, and 39; 43, 44, and 45; 49, 50, and 51; 55, 56, and 57; 61, 62, and 63; 67, 68, and 69; 73, 74, and 75; 79, 80, and 81; 85, 86, and 87; 91, 92, and 93; 97, 98, and 99; 103, 104, and 105; 109, 110, and 111; 115, 116, and 117; 121, 122, and 123; or 127, 128, and 129, respectively. have either or both of the heavy chain variable region CDR1, CDR2, and CDR3 of SEQ ID NOs: 28, 29, and 30; 34, 35, and 36; 40, 41, and 42; 46, 47, and 48; 52, 53, and 54; 58, 59, and 60; 64, 65, and 66; 70, 71, and 72; 76, 77, and 78; 82, 83, and 84; 88, 89, and 90; 94, 95, and 96; 100, 101, and 102; 106, 107, and 108; 112, 113, and 114; 118, 119, and 120; 124, 125, and 126; or 130, 131, and 132, respectively. In another embodiment, the antibody or antibody fragment is adapted for administration or gene delivery using an RNA or DNA sequence or vector encoding the antibody or antibody fragment. In another embodiment, upon binding, the antibody downregulates nestin. In another embodiment, the bifunctional monoclonal antibody further comprises a probe.In another embodiment, the bifunctional monoclonal antibody further comprises a probe detectable by MRI. In another embodiment, when the antibody binds to cancer cells, the antibody reduces the expression of at least one of sodium channel protein type 5 subunit alpha or L1 cell adhesion molecule. In another embodiment, when the antibody binds to cancer cells, the antibody increases the expression of CD74. In another embodiment, when the antibody binds to retinal cells, the antibody reduces the expression of at least one of adenosine deaminase or apelin. In another embodiment, when the antibody binds to retinal cells, the antibody increases the expression of bone gamma-carboxyglutamic acid protein, matrilin 2, or von Willebrand factor A domain-containing 1. In another embodiment, when the antibody is provided to a subject with multiple sclerosis, the antibody reduces the expression of at least one of sodium channel protein type 5 subunit alpha, adenosine deaminase, apelin, spinster homolog 2, or bone morphogenetic protein 2. In another embodiment, when the antibody is provided to a subject with multiple sclerosis, the antibody increases the expression of at least one of CD74 or IKAROS family zinc finger 1 (Ikaros). In another embodiment, when the antibody is provided to a subject at a site in need of tissue regeneration, the antibody increases the expression of at least one of bone gamma-carboxyglutamic acid (gla) protein, matrilin 2, or von Willebrand factor A domain-containing 1. In another embodiment, when the antibody downregulates nestin. In another embodiment, the antibody further comprises a probe. In another embodiment, the antibody further comprises a probe detectable by MRI.

[0015] In another embodiment, the invention includes a method of diagnosing a disease or condition in a subject using an anti-EGF, Latrophilin, and seven transmembrane domain-containing protein 1 (ELTD1) antibody or a fragment thereof, comprising: identifying a subject in need of treatment for a disease or condition; obtaining a biological sample from the subject; and contacting the sample with an anti-EGF, Latrophilin, and seven transmembrane domain-containing protein 1 (ELTD1) antibody or a binding fragment thereof comprising a detectable marker, wherein the disease or condition is selected from multiple sclerosis, retinopathy, cancer, or tissue in need of regeneration.

[0016] For a more complete understanding of the features and advantages of the present invention, reference should be made to the detailed description of the invention taken in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0017] [Figure 1A] These results demonstrate that monoclonal anti-ELTD1 treatment is more effective than polyclonal anti-ELTD1 treatment in increasing animal survival and reducing tumor volume (TV) in mice bearing glioblastoma (GBM). (Figure 1A) Percent survival curves for all treatment groups. Anti-ELTD1 polyclonal antibody (pAb) and monoclonal antibody (mAb) treatment significantly increased overall survival after tumor detection compared with untreated (UT) controls. (Figure 1B) Tumor volume (TV) for each treatment group 9 days after tumor detection. Anti-ELTD1 pAb and mAb treatment significantly reduced TV compared with UT (*p=0.0384, **p=0.0067). Representative morphological MR images of UT controls (Figure 1C), anti-ELTD1 pAb-treated (Figure 1D), and anti-ELTD1 mAb-treated (Figure 1E) 9 days after tumor detection are outlined in yellow. [Figure 1B]These results demonstrate that monoclonal anti-ELTD1 treatment is more effective than polyclonal anti-ELTD1 treatment in increasing animal survival and reducing tumor volume (TV) in mice bearing glioblastoma (GBM). (Figure 1A) Percent survival curves for all treatment groups. Anti-ELTD1 polyclonal antibody (pAb) and monoclonal antibody (mAb) treatment significantly increased overall survival after tumor detection compared with untreated (UT) controls. (Figure 1B) Tumor volume (TV) for each treatment group 9 days after tumor detection. Anti-ELTD1 pAb and mAb treatment significantly reduced TV compared with UT (*p=0.0384, **p=0.0067). Representative morphological MR images of UT controls (Figure 1C), anti-ELTD1 pAb-treated (Figure 1D), and anti-ELTD1 mAb-treated (Figure 1E) 9 days after tumor detection are outlined in yellow. [Figure 1C] These results demonstrate that monoclonal anti-ELTD1 treatment is more effective than polyclonal anti-ELTD1 treatment in increasing animal survival and reducing tumor volume (TV) in mice bearing glioblastoma (GBM). (Figure 1A) Percent survival curves for all treatment groups. Anti-ELTD1 polyclonal antibody (pAb) and monoclonal antibody (mAb) treatment significantly increased overall survival after tumor detection compared with untreated (UT) controls. (Figure 1B) Tumor volume (TV) for each treatment group 9 days after tumor detection. Anti-ELTD1 pAb and mAb treatment significantly reduced TV compared with UT (*p=0.0384, **p=0.0067). Representative morphological MR images of UT controls (Figure 1C), anti-ELTD1 pAb-treated (Figure 1D), and anti-ELTD1 mAb-treated (Figure 1E) 9 days after tumor detection are outlined in yellow. [Figure 1D]These results demonstrate that monoclonal anti-ELTD1 treatment is more effective than polyclonal anti-ELTD1 treatment in increasing animal survival and reducing tumor volume (TV) in mice bearing glioblastoma (GBM). (Figure 1A) Percent survival curves for all treatment groups. Anti-ELTD1 polyclonal antibody (pAb) and monoclonal antibody (mAb) treatment significantly increased overall survival after tumor detection compared with untreated (UT) controls. (Figure 1B) Tumor volume (TV) for each treatment group 9 days after tumor detection. Anti-ELTD1 pAb and mAb treatment significantly reduced TV compared with UT (*p=0.0384, **p=0.0067). Representative morphological MR images of UT controls (Figure 1C), anti-ELTD1 pAb-treated (Figure 1D), and anti-ELTD1 mAb-treated (Figure 1E) 9 days after tumor detection are outlined in yellow. [Figure 1E] These results demonstrate that monoclonal anti-ELTD1 treatment is more effective than polyclonal anti-ELTD1 treatment in increasing animal survival and reducing tumor volume (TV) in mice bearing glioblastoma (GBM). (Figure 1A) Percent survival curves for all treatment groups. Anti-ELTD1 polyclonal antibody (pAb) and monoclonal antibody (mAb) treatment significantly increased overall survival after tumor detection compared with untreated (UT) controls. (Figure 1B) Tumor volume (TV) for each treatment group 9 days after tumor detection. Anti-ELTD1 pAb and mAb treatment significantly reduced TV compared with UT (*p=0.0384, **p=0.0067). Representative morphological MR images of UT controls (Figure 1C), anti-ELTD1 pAb-treated (Figure 1D), and anti-ELTD1 mAb-treated (Figure 1E) 9 days after tumor detection are outlined in yellow. [Figure 2A]This study demonstrates that monoclonal anti-ELTD1 treatment of GBM-bearing mice normalizes intratumoral vasculature. Representative morphological images with MR perfusion maps are shown for each treatment group: UT control (Figure 2A-B), anti-ELTD1 pAb-treated animals (Figure 2C-D), and anti-ELTD1 mAb-treated animals (Figure 2E-F). (Figure 2G) Quantitative analysis of tumor-relative cerebral blood flow (rCBF) differences. rCBF perfusion levels were significantly increased with both anti-ELTD1 treatments, with anti-ELTD1 mAb significantly superior to ELTD1 pAb. mAb treatment also normalized perfusion levels (***p=0.0001 UT vs. pAb, ****p<0.0001 UT vs. mAb). [Figure 2B] This study demonstrates that monoclonal anti-ELTD1 treatment of GBM-bearing mice normalizes intratumoral vasculature. Representative morphological images with MR perfusion maps are shown for each treatment group: UT control (Figure 2A-B), anti-ELTD1 pAb-treated animals (Figure 2C-D), and anti-ELTD1 mAb-treated animals (Figure 2E-F). (Figure 2G) Quantitative analysis of tumor-relative cerebral blood flow (rCBF) differences. rCBF perfusion levels were significantly increased with both anti-ELTD1 treatments, with anti-ELTD1 mAb significantly superior to ELTD1 pAb. mAb treatment also normalized perfusion levels (***p=0.0001 UT vs. pAb, ****p<0.0001 UT vs. mAb). [Figure 2C] This study demonstrates that monoclonal anti-ELTD1 treatment of GBM-bearing mice normalizes intratumoral vasculature. Representative morphological images with MR perfusion maps are shown for each treatment group: UT control (Figure 2A-B), anti-ELTD1 pAb-treated animals (Figure 2C-D), and anti-ELTD1 mAb-treated animals (Figure 2E-F). (Figure 2G) Quantitative analysis of tumor-relative cerebral blood flow (rCBF) differences. rCBF perfusion levels were significantly increased with both anti-ELTD1 treatments, with anti-ELTD1 mAb significantly superior to ELTD1 pAb. mAb treatment also normalized perfusion levels (***p=0.0001 UT vs. pAb, ****p<0.0001 UT vs. mAb). [Figure 2D]This study demonstrates that monoclonal anti-ELTD1 treatment of GBM-bearing mice normalizes intratumoral vasculature. Representative morphological images with MR perfusion maps are shown for each treatment group: UT control (Figure 2A-B), anti-ELTD1 pAb-treated animals (Figure 2C-D), and anti-ELTD1 mAb-treated animals (Figure 2E-F). (Figure 2G) Quantitative analysis of tumor-relative cerebral blood flow (rCBF) differences. rCBF perfusion levels were significantly increased with both anti-ELTD1 treatments, with anti-ELTD1 mAb significantly superior to ELTD1 pAb. mAb treatment also normalized perfusion levels (***p=0.0001 UT vs. pAb, ****p<0.0001 UT vs. mAb). [Figure 2E] This study demonstrates that monoclonal anti-ELTD1 treatment of GBM-bearing mice normalizes intratumoral vasculature. Representative morphological images with MR perfusion maps are shown for each treatment group: UT control (Figure 2A-B), anti-ELTD1 pAb-treated animals (Figure 2C-D), and anti-ELTD1 mAb-treated animals (Figure 2E-F). (Figure 2G) Quantitative analysis of tumor-relative cerebral blood flow (rCBF) differences. rCBF perfusion levels were significantly increased with both anti-ELTD1 treatments, with anti-ELTD1 mAb significantly superior to ELTD1 pAb. mAb treatment also normalized perfusion levels (***p=0.0001 UT vs. pAb, ****p<0.0001 UT vs. mAb). [Figure 2F] This study demonstrates that monoclonal anti-ELTD1 treatment of GBM-bearing mice normalizes intratumoral vasculature. Representative morphological images with MR perfusion maps are shown for each treatment group: UT control (Figure 2A-B), anti-ELTD1 pAb-treated animals (Figure 2C-D), and anti-ELTD1 mAb-treated animals (Figure 2E-F). (Figure 2G) Quantitative analysis of tumor-relative cerebral blood flow (rCBF) differences. rCBF perfusion levels were significantly increased with both anti-ELTD1 treatments, with anti-ELTD1 mAb significantly superior to ELTD1 pAb. mAb treatment also normalized perfusion levels (***p=0.0001 UT vs. pAb, ****p<0.0001 UT vs. mAb). [Figure 2G]This study demonstrates that monoclonal anti-ELTD1 treatment of GBM-bearing mice normalizes intratumoral vasculature. Representative morphological images with MR perfusion maps are shown for each treatment group: UT control (Figure 2A-B), anti-ELTD1 pAb-treated animals (Figure 2C-D), and anti-ELTD1 mAb-treated animals (Figure 2E-F). (Figure 2G) Quantitative analysis of tumor-relative cerebral blood flow (rCBF) differences. rCBF perfusion levels were significantly increased with both anti-ELTD1 treatments, with anti-ELTD1 mAb significantly superior to ELTD1 pAb. mAb treatment also normalized perfusion levels (***p=0.0001 UT vs. pAb, ****p<0.0001 UT vs. mAb). [Figure 3] Anti-ELTD1 antibody therapy was effective in reducing microvascular density (MVD) in GBM-bearing mice. Representative IHC images (20x magnification) of CD34 from untreated (Figure 3A), anti-ELTD1 pAb-treated (Figure 3B), and anti-ELTD1 mAb-treated (Figure 3C) animals. The dark reddish-brown staining in the slides represents blood vessels in the tumor area, highlighted by arrows. (Figure 3D) MVD analysis of all treatment groups. pAb and mAb treatments significantly reduced MVD (both ****p<0.0001). Furthermore, mAb significantly reduced MVD compared to pAb (**p<0.01). [Figure 4A]The anti-ELTD1 mAb probe exhibited significantly higher binding specificity to GBM tumors. (Figure 4A) Molecular probe construct. Gd-DTPA signal was used to detect the probe by MR imaging, while biotin labeling enabled localization within tumor tissue after termination. (Figure 4B) The relative expression percentage of the molecular probe indicates changes in either T1 relaxation or SI due to the presence of the Gd-DTPA component. The mAb-conjugated probe had significantly higher signal intensity and T1 relaxation time than the IgG control. T1: *p=0.0307 (IgG vs. pAb ELTD1 probe), ***p=0.0002 (IgG vs. mAb ELTD1 probe), SI: **p=0.008 (IgG vs. mAb ELTD1 probe). (Figures 4C and 4D) Localization and clustering of the anti-ELTD1 monoclonal-conjugated molecular probe (Figure 4C) and the nonspecific IgG-conjugated molecular probe (Figure 4D). (Figure 4E) Kinetics of antibody-bound probes: nonspecific IgG control, pAb against ELTD1, and mAb against ELTD1. (Figures 4F-H) Representative images (20x magnification) of nonspecific IgG-bound probe (Figure 4F), anti-ELTD1 pAb-bound probe (Figure 4G), and anti-ELTD1 mAb-bound probe (Figure 4H) stained with SA-HRP to localize them. The brown staining seen in the pAb-bound and mAb-bound probes represents the localized probes. [Figure 4B]The anti-ELTD1 mAb probe exhibited significantly higher binding specificity to GBM tumors. (Figure 4A) Molecular probe construct. Gd-DTPA signal was used to detect the probe by MR imaging, while biotin labeling enabled localization within tumor tissue after termination. (Figure 4B) The relative expression percentage of the molecular probe indicates changes in either T1 relaxation or SI due to the presence of the Gd-DTPA component. The mAb-conjugated probe had significantly higher signal intensity and T1 relaxation time than the IgG control. T1: *p=0.0307 (IgG vs. pAb ELTD1 probe), ***p=0.0002 (IgG vs. mAb ELTD1 probe), SI: **p=0.008 (IgG vs. mAb ELTD1 probe). (Figures 4C and 4D) Localization and clustering of the anti-ELTD1 monoclonal-conjugated molecular probe (Figure 4C) and the nonspecific IgG-conjugated molecular probe (Figure 4D). (Figure 4E) Kinetics of antibody-bound probes: nonspecific IgG control, pAb against ELTD1, and mAb against ELTD1. (Figures 4F-H) Representative images (20x magnification) of nonspecific IgG-bound probe (Figure 4F), anti-ELTD1 pAb-bound probe (Figure 4G), and anti-ELTD1 mAb-bound probe (Figure 4H) stained with SA-HRP to localize them. The brown staining seen in the pAb-bound and mAb-bound probes represents the localized probes. [Figure 4C]The anti-ELTD1 mAb probe exhibited significantly higher binding specificity to GBM tumors. (Figure 4A) Molecular probe construct. Gd-DTPA signal was used to detect the probe by MR imaging, while biotin labeling enabled localization within tumor tissue after termination. (Figure 4B) The relative expression percentage of the molecular probe indicates changes in either T1 relaxation or SI due to the presence of the Gd-DTPA component. The mAb-conjugated probe had significantly higher signal intensity and T1 relaxation time than the IgG control. T1: *p=0.0307 (IgG vs. pAb ELTD1 probe), ***p=0.0002 (IgG vs. mAb ELTD1 probe), SI: **p=0.008 (IgG vs. mAb ELTD1 probe). (Figures 4C and 4D) Localization and clustering of the anti-ELTD1 monoclonal-conjugated molecular probe (Figure 4C) and the nonspecific IgG-conjugated molecular probe (Figure 4D). (Figure 4E) Kinetics of antibody-bound probes: nonspecific IgG control, pAb against ELTD1, and mAb against ELTD1. (Figures 4F-H) Representative images (20x magnification) of nonspecific IgG-bound probe (Figure 4F), anti-ELTD1 pAb-bound probe (Figure 4G), and anti-ELTD1 mAb-bound probe (Figure 4H) stained with SA-HRP to localize them. The brown staining seen in the pAb-bound and mAb-bound probes represents the localized probes. [Figure 4D]The anti-ELTD1 mAb probe exhibited significantly higher binding specificity to GBM tumors. (Figure 4A) Molecular probe construct. Gd-DTPA signal was used to detect the probe by MR imaging, while biotin labeling enabled localization within tumor tissue after termination. (Figure 4B) The relative expression percentage of the molecular probe indicates changes in either T1 relaxation or SI due to the presence of the Gd-DTPA component. The mAb-conjugated probe had significantly higher signal intensity and T1 relaxation time than the IgG control. T1: *p=0.0307 (IgG vs. pAb ELTD1 probe), ***p=0.0002 (IgG vs. mAb ELTD1 probe), SI: **p=0.008 (IgG vs. mAb ELTD1 probe). (Figures 4C and 4D) Localization and clustering of the anti-ELTD1 monoclonal-conjugated molecular probe (Figure 4C) and the nonspecific IgG-conjugated molecular probe (Figure 4D). (Figure 4E) Kinetics of antibody-bound probes: nonspecific IgG control, pAb against ELTD1, and mAb against ELTD1. (Figures 4F-H) Representative images (20x magnification) of nonspecific IgG-bound probe (Figure 4F), anti-ELTD1 pAb-bound probe (Figure 4G), and anti-ELTD1 mAb-bound probe (Figure 4H) stained with SA-HRP to localize them. The brown staining seen in the pAb-bound and mAb-bound probes represents the localized probes. [Figure 4E]The anti-ELTD1 mAb probe exhibited significantly higher binding specificity to GBM tumors. (Figure 4A) Molecular probe construct. Gd-DTPA signal was used to detect the probe by MR imaging, while biotin labeling enabled localization within tumor tissue after termination. (Figure 4B) The relative expression percentage of the molecular probe indicates changes in either T1 relaxation or SI due to the presence of the Gd-DTPA component. The mAb-conjugated probe had significantly higher signal intensity and T1 relaxation time than the IgG control. T1: *p=0.0307 (IgG vs. pAb ELTD1 probe), ***p=0.0002 (IgG vs. mAb ELTD1 probe), SI: **p=0.008 (IgG vs. mAb ELTD1 probe). (Figures 4C and 4D) Localization and clustering of the anti-ELTD1 monoclonal-conjugated molecular probe (Figure 4C) and the nonspecific IgG-conjugated molecular probe (Figure 4D). (Figure 4E) Kinetics of antibody-bound probes: nonspecific IgG control, pAb against ELTD1, and mAb against ELTD1. (Figures 4F-H) Representative images (20x magnification) of nonspecific IgG-bound probe (Figure 4F), anti-ELTD1 pAb-bound probe (Figure 4G), and anti-ELTD1 mAb-bound probe (Figure 4H) stained with SA-HRP to localize them. The brown staining seen in the pAb-bound and mAb-bound probes represents the localized probes. [Figure 4F]The anti-ELTD1 mAb probe exhibited significantly higher binding specificity to GBM tumors. (Figure 4A) Molecular probe construct. Gd-DTPA signal was used to detect the probe by MR imaging, while biotin labeling enabled localization within tumor tissue after termination. (Figure 4B) The relative expression percentage of the molecular probe indicates changes in either T1 relaxation or SI due to the presence of the Gd-DTPA component. The mAb-conjugated probe had significantly higher signal intensity and T1 relaxation time than the IgG control. T1: *p=0.0307 (IgG vs. pAb ELTD1 probe), ***p=0.0002 (IgG vs. mAb ELTD1 probe), SI: **p=0.008 (IgG vs. mAb ELTD1 probe). (Figures 4C and 4D) Localization and clustering of the anti-ELTD1 monoclonal-conjugated molecular probe (Figure 4C) and the nonspecific IgG-conjugated molecular probe (Figure 4D). (Figure 4E) Kinetics of antibody-bound probes: nonspecific IgG control, pAb against ELTD1, and mAb against ELTD1. (Figures 4F-H) Representative images (20x magnification) of nonspecific IgG-bound probe (Figure 4F), anti-ELTD1 pAb-bound probe (Figure 4G), and anti-ELTD1 mAb-bound probe (Figure 4H) stained with SA-HRP to localize them. The brown staining seen in the pAb-bound and mAb-bound probes represents the localized probes. [Figure 4G]The anti-ELTD1 mAb probe exhibited significantly higher binding specificity to GBM tumors. (Figure 4A) Molecular probe construct. Gd-DTPA signal was used to detect the probe by MR imaging, while biotin labeling enabled localization within tumor tissue after termination. (Figure 4B) The relative expression percentage of the molecular probe indicates changes in either T1 relaxation or SI due to the presence of the Gd-DTPA component. The mAb-conjugated probe had significantly higher signal intensity and T1 relaxation time than the IgG control. T1: *p=0.0307 (IgG vs. pAb ELTD1 probe), ***p=0.0002 (IgG vs. mAb ELTD1 probe), SI: **p=0.008 (IgG vs. mAb ELTD1 probe). (Figures 4C and 4D) Localization and clustering of the anti-ELTD1 monoclonal-conjugated molecular probe (Figure 4C) and the nonspecific IgG-conjugated molecular probe (Figure 4D). (Figure 4E) Kinetics of antibody-bound probes: nonspecific IgG control, pAb against ELTD1, and mAb against ELTD1. (Figures 4F-H) Representative images (20x magnification) of nonspecific IgG-bound probe (Figure 4F), anti-ELTD1 pAb-bound probe (Figure 4G), and anti-ELTD1 mAb-bound probe (Figure 4H) stained with SA-HRP to localize them. The brown staining seen in the pAb-bound and mAb-bound probes represents the localized probes. [Figure 4H]The anti-ELTD1 mAb probe exhibited significantly higher binding specificity to GBM tumors. (Figure 4A) Molecular probe construct. Gd-DTPA signal was used to detect the probe by MR imaging, while biotin labeling enabled localization within tumor tissue after termination. (Figure 4B) The relative expression percentage of the molecular probe indicates changes in either T1 relaxation or SI due to the presence of the Gd-DTPA component. The mAb-conjugated probe had significantly higher signal intensity and T1 relaxation time than the IgG control. T1: *p=0.0307 (IgG vs. pAb ELTD1 probe), ***p=0.0002 (IgG vs. mAb ELTD1 probe), SI: **p=0.008 (IgG vs. mAb ELTD1 probe). (Figures 4C and 4D) Localization and clustering of the anti-ELTD1 monoclonal-conjugated molecular probe (Figure 4C) and the nonspecific IgG-conjugated molecular probe (Figure 4D). (Figure 4E) Kinetics of antibody-bound probes: nonspecific IgG control, pAb against ELTD1, and mAb against ELTD1. (Figures 4F-H) Representative images (20x magnification) of nonspecific IgG-bound probe (Figure 4F), anti-ELTD1 pAb-bound probe (Figure 4G), and anti-ELTD1 mAb-bound probe (Figure 4H) stained with SA-HRP to localize them. The brown staining seen in the pAb-bound and mAb-bound probes represents the localized probes. [Figure 5A]These results show that anti-ELTD1 mAb treatment of GBM-bearing mice significantly reduces Notch1 levels. (Figures 5A-D) Representative images (20x magnification) of IHC-stained tumors with anti-Notch1 in untreated animals (Figure 5A), anti-ELTD1 pAb-treated animals (Figure 5B), anti-ELTD1 mAb-treated animals (Figure 5C), and contralateral control-stained tissue (Figure 5D). (Figure 5E) Quantitative positive Notch staining of samples. Mice treated with anti-ELTD1 mAb had significantly reduced Notch levels compared with both untreated and anti-ELTD1 pAb-treated animals. There were no significant differences between untreated and pAb-treated animals, or between mAb-treated animals and contralateral (healthy control) tissue. Contralateral (control) tissue Notch levels were significantly lower than in untreated and pAb-treated animals (*p=0.0357 (mAb vs. pAb), **p=0.0015 (control vs. pAb), ***p=0.0006 (UT vs. mAb), ****p<0.0001 (UT vs. control)). [Figure 5B] These results show that anti-ELTD1 mAb treatment of GBM-bearing mice significantly reduces Notch1 levels. (Figures 5A-D) Representative images (20x magnification) of IHC-stained tumors with anti-Notch1 in untreated animals (Figure 5A), anti-ELTD1 pAb-treated animals (Figure 5B), anti-ELTD1 mAb-treated animals (Figure 5C), and contralateral control-stained tissue (Figure 5D). (Figure 5E) Quantitative positive Notch staining of samples. Mice treated with anti-ELTD1 mAb had significantly reduced Notch levels compared with both untreated and anti-ELTD1 pAb-treated animals. There were no significant differences between untreated and pAb-treated animals, or between mAb-treated animals and contralateral (healthy control) tissue. Contralateral (control) tissue Notch levels were significantly lower than in untreated and pAb-treated animals (*p=0.0357 (mAb vs. pAb), **p=0.0015 (control vs. pAb), ***p=0.0006 (UT vs. mAb), ****p<0.0001 (UT vs. control)). [Figure 5C]These results show that anti-ELTD1 mAb treatment of GBM-bearing mice significantly reduces Notch1 levels. (Figures 5A-D) Representative images (20x magnification) of IHC-stained tumors with anti-Notch1 in untreated animals (Figure 5A), anti-ELTD1 pAb-treated animals (Figure 5B), anti-ELTD1 mAb-treated animals (Figure 5C), and contralateral control-stained tissue (Figure 5D). (Figure 5E) Quantitative positive Notch staining of samples. Mice treated with anti-ELTD1 mAb had significantly reduced Notch levels compared with both untreated and anti-ELTD1 pAb-treated animals. There were no significant differences between untreated and pAb-treated animals, or between mAb-treated animals and contralateral (healthy control) tissue. Contralateral (control) tissue Notch levels were significantly lower than in untreated and pAb-treated animals (*p=0.0357 (mAb vs. pAb), **p=0.0015 (control vs. pAb), ***p=0.0006 (UT vs. mAb), ****p<0.0001 (UT vs. control)). [Figure 5D] These results show that anti-ELTD1 mAb treatment of GBM-bearing mice significantly reduces Notch1 levels. (Figures 5A-D) Representative images (20x magnification) of IHC-stained tumors with anti-Notch1 in untreated animals (Figure 5A), anti-ELTD1 pAb-treated animals (Figure 5B), anti-ELTD1 mAb-treated animals (Figure 5C), and contralateral control-stained tissue (Figure 5D). (Figure 5E) Quantitative positive Notch staining of samples. Mice treated with anti-ELTD1 mAb had significantly reduced Notch levels compared with both untreated and anti-ELTD1 pAb-treated animals. There were no significant differences between untreated and pAb-treated animals, or between mAb-treated animals and contralateral (healthy control) tissue. Contralateral (control) tissue Notch levels were significantly lower than in untreated and pAb-treated animals (*p=0.0357 (mAb vs. pAb), **p=0.0015 (control vs. pAb), ***p=0.0006 (UT vs. mAb), ****p<0.0001 (UT vs. control)). [Figure 5E]These results show that anti-ELTD1 mAb treatment of GBM-bearing mice significantly reduces Notch1 levels. (Figures 5A-D) Representative images (20x magnification) of IHC-stained tumors with anti-Notch1 in untreated animals (Figure 5A), anti-ELTD1 pAb-treated animals (Figure 5B), anti-ELTD1 mAb-treated animals (Figure 5C), and contralateral control-stained tissue (Figure 5D). (Figure 5E) Quantitative positive Notch staining of samples. Mice treated with anti-ELTD1 mAb had significantly reduced Notch levels compared with both untreated and anti-ELTD1 pAb-treated animals. There were no significant differences between untreated and pAb-treated animals, or between mAb-treated animals and contralateral (healthy control) tissue. Contralateral (control) tissue Notch levels were significantly lower than in untreated and pAb-treated animals (*p=0.0357 (mAb vs. pAb), **p=0.0015 (control vs. pAb), ***p=0.0006 (UT vs. mAb), ****p<0.0001 (UT vs. control)). [Figure 6A] Gene fold change from upregulation (red) to downregulation (blue) obtained from RNA-seq analysis is shown in ELTD1 mAb-treated mice with GBM compared to UT mice with GBM. [Figure 6B] Gene-gene correlations of genes suppressed after anti-ELT1 mAb treatment of GBM-bearing mice. Red = positive correlation, green = negative correlation. Using literature analysis software to classify gene groups in terms of published commonalities, they fall roughly into four categories: developmental genes, nestin-related, cell proliferation / angiogenesis, and astrocyte / microglial inflammation. [Figure 7A]Anti-ELTD1 treatment successfully increased the percent survival rate after GMB tumor detection and reduced tumor volume (Figure 7A). Both ELTD1-specific antibody treatments, mAb (**p=0.0058) and scFv fragment (***p=0.0001), significantly increased the percent tumor survival rate compared to UT control animals, as shown in the percent survival curves (Figure 7B). Tumor volume in GBM-bearing mice was also found to be significantly lower with anti-ELTD1 mAb (*p=0.0009) and scFv fragment (*p=0.017) compared to untreated animals (Figure 7C). Representative morphological tumor images of untreated (Figure 7D), anti-ELTD1 mAb-treated (Figure 7D), and anti-ELTD1 scFv fragment-treated (Figure 7E) mice were also shown. [Figure 7B] Anti-ELTD1 treatment successfully increased the percent survival rate after GMB tumor detection and reduced tumor volume (Figure 7A). Both ELTD1-specific antibody treatments, mAb (**p=0.0058) and scFv fragment (***p=0.0001), significantly increased the percent tumor survival rate compared to UT control animals, as shown in the percent survival curves (Figure 7B). Tumor volume in GBM-bearing mice was also found to be significantly lower with anti-ELTD1 mAb (*p=0.0009) and scFv fragment (*p=0.017) compared to untreated animals (Figure 7C). Representative morphological tumor images of untreated (Figure 7D), anti-ELTD1 mAb-treated (Figure 7D), and anti-ELTD1 scFv fragment-treated (Figure 7E) mice were also shown. [Figure 7C]Anti-ELTD1 treatment successfully increased the percent survival rate after GMB tumor detection and reduced tumor volume (Figure 7A). Both ELTD1-specific antibody treatments, mAb (**p=0.0058) and scFv fragment (***p=0.0001), significantly increased the percent tumor survival rate compared to UT control animals, as shown in the percent survival curves (Figure 7B). Tumor volume in GBM-bearing mice was also found to be significantly lower with anti-ELTD1 mAb (*p=0.0009) and scFv fragment (*p=0.017) compared to untreated animals (Figure 7C). Representative morphological tumor images of untreated (Figure 7D), anti-ELTD1 mAb-treated (Figure 7D), and anti-ELTD1 scFv fragment-treated (Figure 7E) mice were also shown. [Figure 7D] Anti-ELTD1 treatment successfully increased the percent survival rate after GMB tumor detection and reduced tumor volume (Figure 7A). Both ELTD1-specific antibody treatments, mAb (**p=0.0058) and scFv fragment (***p=0.0001), significantly increased the percent tumor survival rate compared to UT control animals, as shown in the percent survival curves (Figure 7B). Tumor volume in GBM-bearing mice was also found to be significantly lower with anti-ELTD1 mAb (*p=0.0009) and scFv fragment (*p=0.017) compared to untreated animals (Figure 7C). Representative morphological tumor images of untreated (Figure 7D), anti-ELTD1 mAb-treated (Figure 7D), and anti-ELTD1 scFv fragment-treated (Figure 7E) mice were also shown. [Figure 7E]Anti-ELTD1 treatment successfully increased the percent survival rate after GMB tumor detection and reduced tumor volume (Figure 7A). Both ELTD1-specific antibody treatments, mAb (**p=0.0058) and scFv fragment (***p=0.0001), significantly increased the percent tumor survival rate compared to UT control animals, as shown in the percent survival curves (Figure 7B). Tumor volume in GBM-bearing mice was also found to be significantly lower with anti-ELTD1 mAb (*p=0.0009) and scFv fragment (*p=0.017) compared to untreated animals (Figure 7C). Representative morphological tumor images of untreated (Figure 7D), anti-ELTD1 mAb-treated (Figure 7D), and anti-ELTD1 scFv fragment-treated (Figure 7E) mice were also shown. [Figure 8A] Figure 8 shows that relative cerebral blood flow (rCBF) was normalized with anti-ELTD1 Ab treatment in GBM-bearing mice (Figure 8A). Anti-ELTD1 mAb treatment and scFv fragment treatment were significantly more effective in minimizing the decrease in rCBF compared with untreated mice (****p<0.0001). Representative morphological images and perfusion maps are shown for untreated (Figures 8B and 8C), anti-ELTD1 mAb-treated (Figures 8D and 8E), and anti-ELTD1 scFv fragment-treated (Figures 8F and 8G) animals. [Figure 8B] Figure 8 shows that relative cerebral blood flow (rCBF) was normalized with anti-ELTD1 Ab treatment in GBM-bearing mice (Figure 8A). Anti-ELTD1 mAb treatment and scFv fragment treatment were significantly more effective in minimizing the decrease in rCBF compared with untreated mice (****p<0.0001). Representative morphological images and perfusion maps are shown for untreated (Figures 8B and 8C), anti-ELTD1 mAb-treated (Figures 8D and 8E), and anti-ELTD1 scFv fragment-treated (Figures 8F and 8G) animals. [Figure 8C]Figure 8 shows that relative cerebral blood flow (rCBF) was normalized with anti-ELTD1 Ab treatment in GBM-bearing mice (Figure 8A). Anti-ELTD1 mAb treatment and scFv fragment treatment were significantly more effective in minimizing the decrease in rCBF compared with untreated mice (****p<0.0001). Representative morphological images and perfusion maps are shown for untreated (Figures 8B and 8C), anti-ELTD1 mAb-treated (Figures 8D and 8E), and anti-ELTD1 scFv fragment-treated (Figures 8F and 8G) animals. [Figure 8D] Figure 8 shows that relative cerebral blood flow (rCBF) was normalized with anti-ELTD1 Ab treatment in GBM-bearing mice (Figure 8A). Anti-ELTD1 mAb treatment and scFv fragment treatment were significantly more effective in minimizing the decrease in rCBF compared with untreated mice (****p<0.0001). Representative morphological images and perfusion maps are shown for untreated (Figures 8B and 8C), anti-ELTD1 mAb-treated (Figures 8D and 8E), and anti-ELTD1 scFv fragment-treated (Figures 8F and 8G) animals. [Figure 8E] Figure 8 shows that relative cerebral blood flow (rCBF) was normalized with anti-ELTD1 Ab treatment in GBM-bearing mice (Figure 8A). Anti-ELTD1 mAb treatment and scFv fragment treatment were significantly more effective in minimizing the decrease in rCBF compared with untreated mice (****p<0.0001). Representative morphological images and perfusion maps are shown for untreated (Figures 8B and 8C), anti-ELTD1 mAb-treated (Figures 8D and 8E), and anti-ELTD1 scFv fragment-treated (Figures 8F and 8G) animals. [Figure 8F] Figure 8 shows that relative cerebral blood flow (rCBF) was normalized with anti-ELTD1 Ab treatment in GBM-bearing mice (Figure 8A). Anti-ELTD1 mAb treatment and scFv fragment treatment were significantly more effective in minimizing the decrease in rCBF compared with untreated mice (****p<0.0001). Representative morphological images and perfusion maps are shown for untreated (Figures 8B and 8C), anti-ELTD1 mAb-treated (Figures 8D and 8E), and anti-ELTD1 scFv fragment-treated (Figures 8F and 8G) animals. [Figure 8G]Figure 8 shows that relative cerebral blood flow (rCBF) was normalized with anti-ELTD1 Ab treatment in GBM-bearing mice (Figure 8A). Anti-ELTD1 mAb treatment and scFv fragment treatment were significantly more effective in minimizing the decrease in rCBF compared with untreated mice (****p<0.0001). Representative morphological images and perfusion maps are shown for untreated (Figures 8B and 8C), anti-ELTD1 mAb-treated (Figures 8D and 8E), and anti-ELTD1 scFv fragment-treated (Figures 8F and 8G) animals. [Figure 9A] Figure 9 shows that anti-ELTD1 Ab treatment of GBM-bearing mice significantly reduced tumor-associated vasculature. MVD was analyzed for each treatment group using an Aperio ImageScope. Representative IHC images (20x magnification) of CD34 staining from untreated (Figure 9A), anti-ELTD1 mAb-treated (Figure 9B), and scFv fragment-treated (Figure 9C) animals are shown. Arrows point to blood vessels found in the tumor area. (Figure 9D) Both mAb treatment against ELTD1 and scFv fragment treatment significantly reduced microvessel density within the tumor area (****p<0.0001). [Figure 9B] Figure 9 shows that anti-ELTD1 Ab treatment of GBM-bearing mice significantly reduced tumor-associated vasculature. MVD was analyzed for each treatment group using an Aperio ImageScope. Representative IHC images (20x magnification) of CD34 staining from untreated (Figure 9A), anti-ELTD1 mAb-treated (Figure 9B), and scFv fragment-treated (Figure 9C) animals are shown. Arrows point to blood vessels found in the tumor area. (Figure 9D) Both mAb treatment against ELTD1 and scFv fragment treatment significantly reduced microvessel density within the tumor area (****p<0.0001). [Figure 9C]Figure 9 shows that anti-ELTD1 Ab treatment of GBM-bearing mice significantly reduced tumor-associated vasculature. MVD was analyzed for each treatment group using an Aperio ImageScope. Representative IHC images (20x magnification) of CD34 staining from untreated (Figure 9A), anti-ELTD1 mAb-treated (Figure 9B), and scFv fragment-treated (Figure 9C) animals are shown. Arrows point to blood vessels found in the tumor area. (Figure 9D) Both mAb treatment against ELTD1 and scFv fragment treatment significantly reduced microvessel density within the tumor area (****p<0.0001). [Figure 9D] Figure 9 shows that anti-ELTD1 Ab treatment of GBM-bearing mice significantly reduced tumor-associated vasculature. MVD was analyzed for each treatment group using an Aperio ImageScope. Representative IHC images (20x magnification) of CD34 staining from untreated (Figure 9A), anti-ELTD1 mAb-treated (Figure 9B), and scFv fragment-treated (Figure 9C) animals are shown. Arrows point to blood vessels found in the tumor area. (Figure 9D) Both mAb treatment against ELTD1 and scFv fragment treatment significantly reduced microvessel density within the tumor area (****p<0.0001). [Figure 10A] Figure 10 shows that anti-ELTD1 Ab treatment of GBM-bearing mice reduced Notch1 levels. Notch1 positivity was analyzed using an Aperio ImageScope for each treatment group (Figure 10A), and all treatment groups were stained for Notch1 positivity. Contralateral normal tissue showed significantly reduced Notch1 staining compared to UT animals. Both anti-ELTD1 treatments successfully reduced Notch1 levels, normalizing them to those seen in contralateral tissue. Representative IHC images (20x magnification) of Notch1 staining in tumor tissue from untreated mice (Figure 10B), anti-ELTD1 mAb-treated mice (Figure 10C), scFv fragment-treated mice (Figure 10D), and contralateral control tissue (Figure 10E) (***p=0.0001, ****p<0.0001). [Figure 10B]Figure 10 shows that anti-ELTD1 Ab treatment of GBM-bearing mice reduced Notch1 levels. Notch1 positivity was analyzed using an Aperio ImageScope for each treatment group (Figure 10A), and all treatment groups were stained for Notch1 positivity. Contralateral normal tissue showed significantly reduced Notch1 staining compared to UT animals. Both anti-ELTD1 treatments successfully reduced Notch1 levels, normalizing them to those seen in contralateral tissue. Representative IHC images (20x magnification) of Notch1 staining in tumor tissue from untreated mice (Figure 10B), anti-ELTD1 mAb-treated mice (Figure 10C), scFv fragment-treated mice (Figure 10D), and contralateral control tissue (Figure 10E) (***p=0.0001, ****p<0.0001). [Figure 10C] Figure 10 shows that anti-ELTD1 Ab treatment of GBM-bearing mice reduced Notch1 levels. Notch1 positivity was analyzed using an Aperio ImageScope for each treatment group (Figure 10A), and all treatment groups were stained for Notch1 positivity. Contralateral normal tissue showed significantly reduced Notch1 staining compared to UT animals. Both anti-ELTD1 treatments successfully reduced Notch1 levels, normalizing them to those seen in contralateral tissue. Representative IHC images (20x magnification) of Notch1 staining in tumor tissue from untreated mice (Figure 10B), anti-ELTD1 mAb-treated mice (Figure 10C), scFv fragment-treated mice (Figure 10D), and contralateral control tissue (Figure 10E) (***p=0.0001, ****p<0.0001). [Figure 10D]Figure 10 shows that anti-ELTD1 Ab treatment of GBM-bearing mice reduced Notch1 levels. Notch1 positivity was analyzed using an Aperio ImageScope for each treatment group (Figure 10A), and all treatment groups were stained for Notch1 positivity. Contralateral normal tissue showed significantly reduced Notch1 staining compared to UT animals. Both anti-ELTD1 treatments successfully reduced Notch1 levels, normalizing them to those seen in contralateral tissue. Representative IHC images (20x magnification) of Notch1 staining in tumor tissue from untreated mice (Figure 10B), anti-ELTD1 mAb-treated mice (Figure 10C), scFv fragment-treated mice (Figure 10D), and contralateral control tissue (Figure 10E) (***p=0.0001, ****p<0.0001). [Figure 10E] Figure 10 shows that anti-ELTD1 Ab treatment of GBM-bearing mice reduced Notch1 levels. Notch1 positivity was analyzed using an Aperio ImageScope for each treatment group (Figure 10A), and all treatment groups were stained for Notch1 positivity. Contralateral normal tissue showed significantly reduced Notch1 staining compared to UT animals. Both anti-ELTD1 treatments successfully reduced Notch1 levels, normalizing them to those seen in contralateral tissue. Representative IHC images (20x magnification) of Notch1 staining in tumor tissue from untreated mice (Figure 10B), anti-ELTD1 mAb-treated mice (Figure 10C), scFv fragment-treated mice (Figure 10D), and contralateral control tissue (Figure 10E) (***p=0.0001, ****p<0.0001). [Figure 11A] Figure 11A shows that anti-ELTD1 Ab-conjugated probes successfully reached and infiltrated the tumor area. (Figure 11A) Constructs of molecular target probes conjugated to either nonspecific IgG, anti-ELTD1 mAb, or anti-ELTD1 scFv fragment. (Figure 11C) 90-minute binding of mAb-conjugated probes, and (Figure 11D) 180-minute binding of scFv fragment-conjugated probes. (Figure 11B) Signal intensity was significantly increased by monoclonal and fragment anti-ELTD1 conjugated probes (**p=0.0038, ***p=0.0007). [Figure 11B]Figure 11A shows that anti-ELTD1 Ab-conjugated probes successfully reached and infiltrated the tumor area. (Figure 11A) Constructs of molecular target probes conjugated to either nonspecific IgG, anti-ELTD1 mAb, or anti-ELTD1 scFv fragment. (Figure 11C) 90-minute binding of mAb-conjugated probes, and (Figure 11D) 180-minute binding of scFv fragment-conjugated probes. (Figure 11B) Signal intensity was significantly increased by monoclonal and fragment anti-ELTD1 conjugated probes (**p=0.0038, ***p=0.0007). [Figure 11C] Figure 11A shows that anti-ELTD1 Ab-conjugated probes successfully reached and infiltrated the tumor area. (Figure 11A) Constructs of molecular target probes conjugated to either nonspecific IgG, anti-ELTD1 mAb, or anti-ELTD1 scFv fragment. (Figure 11C) 90-minute binding of mAb-conjugated probes, and (Figure 11D) 180-minute binding of scFv fragment-conjugated probes. (Figure 11B) Signal intensity was significantly increased by monoclonal and fragment anti-ELTD1 conjugated probes (**p=0.0038, ***p=0.0007). [Figure 11D] Figure 11A shows that anti-ELTD1 Ab-conjugated probes successfully reached and infiltrated the tumor area. (Figure 11A) Constructs of molecular target probes conjugated to either nonspecific IgG, anti-ELTD1 mAb, or anti-ELTD1 scFv fragment. (Figure 11C) 90-minute binding of mAb-conjugated probes, and (Figure 11D) 180-minute binding of scFv fragment-conjugated probes. (Figure 11B) Signal intensity was significantly increased by monoclonal and fragment anti-ELTD1 conjugated probes (**p=0.0038, ***p=0.0007). [Figure 12]This shows that the anti-ELTD1 scFv fragment-binding molecular probe successfully reached and targeted diffuse tumor regions not seen by magnetic resonance imaging (MRI). (A, B) Upper voxel: The bulk tumor (verified by H&E, A) had traces of the anti-ELTD1 scFv fragment-binding molecular probe, as shown by SA-HRP (B). (C, D) Lower voxel: The anti-ELTD1 scFv fragment-binding molecular probe successfully reached diffuse tumor regions, as shown by H&E image (C) and SA-HRP image (D). [Figure 13] Figure 1 shows that anti-ELTD1 mAb treatment is associated with less bleeding (measured as percent positive) than anti-VEGF mAb (Avastin) treatment in G55 gliomas. Histology iron-stained tissue sections from G55 tumors in either untreated mice or mice treated with either anti-ELTD1 pAb, anti-ELTD1 mAb, or Avastin. Note the higher levels of bleeding (increased levels of iron staining) in Avastin-treated mice. There was significantly increased bleeding in tissues from Avastin-treated mice compared to other groups (all p<0.01). [Figure 14] Figure 1 shows that anti-ELTD1 mAb and scFv fragment treatments have lower Notch levels (measured as percent positive) than anti-VEGF mAb (Avastin) treatment in G55 gliomas. Notch-stained tissue sections from G55 tumors from either untreated mice or mice treated with either anti-ELTD1 pAb, anti-ELTD1 mAb, anti-ELTD1 scFv fragment, or Avastin. Note the higher Notch levels in Avastin-treated mice. Contralateral tissue from untreated (contralateral) or Avastin-treated (Avastin contralateral) mice was included for comparison. Notch levels were significantly increased in Avastin-treated tissue compared to the other groups (all p<0.05). Notch levels in untreated (UT) mice were also significantly higher compared to the other groups (all p<0.01). [Figure 15] 1 shows T2 MRI scans showing changes in the cervical spinal cord of experimental autoimmune encephalomyelitis (EAE) mice 27 days after disease progression compared to control animals. [Figure 16] Shown are contrast-enhanced images and T1-weighted MRI scans obtained using Gd-DTPA MRI contrast agent acquired early on day 10 (EAE-E) and late on day 26 (EAE-L) after disease progression compared with control animals. [Figure 17] Anti-ELTD1 mAb probe: A monoclonal antibody (mAb) against the extracellular domain of ELTD1 was conjugated to an albumin-linked Gd-DTPA and biotin construct. [Figure 18A] Figure 1 shows that the mAb anti-ELTD1 MRI probe binds more efficiently to various brain regions in the EAE mouse model of multiple sclerosis (MS) compared to a non-selective control MRI contrast agent. p<0.0001. Percent change in T1 relaxivity when the anti-ELTD1 probe is compared to a control IgG contrast agent. [Figure 18B] Figure 1 shows that the mAb anti-ELTD1 MRI probe binds more efficiently to various brain regions in the EAE mouse model of multiple sclerosis (MS) compared to a non-selective control MRI contrast agent. p<0.0001. Percent change in T1 relaxivity when the anti-ELTD1 probe is compared to a control IgG contrast agent. [Figure 18C] Figure 1 shows that the mAb anti-ELTD1 MRI probe binds more efficiently to various brain regions in the EAE mouse model of multiple sclerosis (MS) compared to a non-selective control MRI contrast agent. p<0.0001. Percent change in T1 relaxivity when the anti-ELTD1 probe is compared to a control IgG contrast agent. [Figure 19] (Figure 19A) mAb anti-ELTD1 MRI probe efficiently binds to endothelial cells in the brain of an EAE mouse model of MS. Molecularly targeted MR imaging of ELTD1 (streptavidin-HRP (horseradish peroxidase) binds to the biotin moiety of the anti-ELTD1 probe). (Figure 19B) Immunohistochemical staining of ELTD1 shows high levels in endothelial cells of EAE mice. DETAILED DESCRIPTION OF THE INVENTION

[0018] While the making and use of various embodiments of the invention are discussed in detail below, it should be understood that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not limit the scope of the invention.

[0019] To facilitate understanding of the present invention, several terms are defined below. Terms defined herein have meanings commonly understood by one of ordinary skill in the art relevant to the present invention. Terms such as "a," "an," and "the" are not intended to refer to only a single entity, but include general classes of which specific examples may be used to describe. While the terminology herein is used to describe particular embodiments of the present invention, their use does not limit the present invention except as outlined in the claims.

[0020] As discussed above, ELTD1 is known to be involved in angiogenesis and has been associated with glioblastoma. Little else is known about its role in biology and disease. Here, we have shown that ELTD1 may be useful in the diagnosis and treatment of other diseases, including multiple sclerosis and retinopathies, and may be important more generally in tissue regeneration. We used the antibodies taught herein against ELTD1.

[0021] As used herein, the terms "antibody" or "antibody peptide(s)" refer to an intact antibody or a binding fragment thereof that competes with the intact antibody for specific binding. Binding fragments are produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Binding fragments include Fab, Fab', F(ab')2, Fv, and single-chain variable fragment (scFv) antibodies. An antibody other than a "bispecific" or "bifunctional" antibody is understood to have each of its binding sites identical. An antibody substantially inhibits the adhesion of a receptor to a counter-receptor when excess antibody reduces the amount of receptor bound to the counter-receptor by at least about 20%, 40%, 60%, or 80%, more usually by more than about 85% (as measured in an in vitro competitive binding assay).

[0022] As used herein, the term "antibody" is used in the broadest sense and specifically encompasses monoclonal antibodies (including full-length antibodies or other bivalent Fc region-containing antibodies, e.g., bivalent scFv Fc fusion antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, scFv), so long as they exhibit the desired biological activity. Antibodies (Ab) and immunoglobulins (Ig) are glycoproteins with the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules that lack antigen specificity. The latter type of polypeptide is produced, for example, at low levels by the lymphatic system and at increased levels by myelomas. The present invention includes monoclonal antibodies (and binding fragments thereof) that are fully recombinant, i.e., in which the complementarity-determining regions (CDRs) are genetically spliced ​​into a human antibody framework, often referred to as antibody veneering. Thus, in certain aspects, monoclonal antibodies are completely synthetic antibodies. In certain embodiments, monoclonal antibodies (and binding fragments thereof) may be produced in bacterial cells or eukaryotic cells, including plant cells.

[0023] As used herein, the term "antibody fragment" refers to a portion of a full-length antibody, generally the antigen-binding or variable region, and includes Fab, Fab', F(ab')2, Fv, and scFv fragments. Papain digestion of antibodies yields two identical antigen-binding fragments called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a name reflecting its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment with two antigen-binding fragments capable of cross-linking antigen, and another residual fragment (called pFc'). As used herein, "functional fragment" with respect to antibodies refers to Fv, F(ab), and F(ab')2 fragments.

[0024] As used herein, an "Fv" fragment is the minimum antibody fragment which contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association (V H -V L In this configuration, the three CDRs of each variable domain interact to form a V H -V L The six CDRs define an antigen-binding site on the surface of the dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

[0025] Fab fragments, also designated F(ab), also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments only by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. F(ab') fragments are generated by cleavage of the disulfide bond at the hinge cysteines of the F(ab')2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those skilled in the art.

[0026] Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain contains a variable domain (V) at one end. H ) followed by several constant domains. Each light chain has a variable domain (V L ) and a constant domain at its other end. The light chain constant domain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the heavy chain variable domain. Particular amino acid residues are thought to form an interface between the light chain variable domain and the heavy chain variable domain (Clothia et al., J. Mol. Biol. 186, 651-66, 1985); Novotny and Haber, Proc. Natl. Acad. Sci. USA 82 4592-4596 (1985) (relevant portions are incorporated herein by reference).

[0027] As used herein, an "isolated" antibody is one that has been identified and separated and / or recovered from components of its production environment. Contaminating components of its production environment are materials that would interfere with diagnostic or therapeutic uses of the antibody, and these may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, the antibody is purified as measurable by at least three different methods: 1) to greater than 50% by weight, e.g., greater than 75%, or greater than 85%, or greater than 95%, or greater than 99% by weight, of the antibody as determined by the Lowry method; 2) to a degree sufficient to obtain at least 10 residues of N-terminal or internal amino acid sequence, e.g., at least 15 residues of sequence, by use of a rotating cup sequencer; and 3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver staining. Isolated antibodies include antibodies in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

[0028] As used herein, the term "antibody mutant" refers to an amino acid sequence variant of an antibody in which one or more amino acid residues have been modified. Such variants necessarily have less than 100% sequence identity or similarity with an amino acid sequence having at least 75%, e.g., at least 80%, or at least 85%, or at least 90%, or at least 95, 96, 97, 98, or 99% amino acid sequence identity or similarity with the amino acid sequence of either the heavy or light chain variable domain of the antibody.

[0029] The term "variable" in the context of antibody variable domains refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs), also known as hypervariable regions, in both the light-chain and heavy-chain variable domains. There are at least two techniques for determining CDRs: (1) an approach based on interspecies sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. 1987)); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Chothia, C. et al. (1989), Nature 342:877), or both, Chothia and Kabat. The more highly conserved portions of variable domains are called framework regions (FRs). Naturally occurring heavy and light chain variable domains each contain four FR regions, which primarily adopt a β-sheet configuration and are connected by three CDRs, which form loops that connect, and in some cases form part of, the β-sheet structure. The CDRs within each chain are held together in close proximity by the FR regions and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (Kabat et al. (See, e.g., J. Immunol. 1999, 10:149-150, 2001). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

[0030] The light chains of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.

[0031] Depending on the amino acid sequence of the constant domain of their heavy chains, "immunoglobulins" can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG4; IgA-1 and IgA-2. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0032] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture and are uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the disclosed and claimed invention may be made by the hybridoma method first described by Kohler and Milstein, Nature 256, 495 (1975), the relevant portions of which are incorporated herein by reference.

[0033] All monoclonal antibodies utilized in accordance with the disclosed and claimed invention are either (1) the result of a deliberate immunization protocol, as described in more detail herein below, or (2) the result of an immune response that naturally leads to the production of antibodies during the course of disease or cancer.

[0034] Use of the disclosed and claimed monoclonal antibodies of the present invention may require administration of such or similar monoclonal antibodies to a subject, such as a human. However, when monoclonal antibodies are produced in non-human animals, such as rodents or chickens, administration of such antibodies to a human patient typically elicits an immune response, which is directed against the antibody itself. Such a response limits the duration and effectiveness of such therapy. To overcome this problem, the disclosed and claimed monoclonal antibodies of the present invention can be "humanized," i.e., engineered to remove their antigenic portions and thus replace similar portions in a human antibody, while retaining the antibody's affinity for a particular ELTD1. This engineering may require only a few amino acids or may involve the entire framework region of the antibody, leaving only the antibody's complementarity-determining regions intact. Several methods for humanizing antibodies are known in the art and are disclosed in U.S. Pat. No. 6,180,370, issued January 30, 2001 to Queen et al., U.S. Pat. No. 6,054,927, issued April 25, 2000 to Brickell, U.S. Pat. No. 5,869,619, issued February 9, 1999 to Studnicka, U.S. Pat. No. 5,861,155, issued January 19, 1999 to Lin, U.S. Pat. No. 5,712,120, issued January 27, 1998 to Rodriquez et al., and U.S. Pat. No. 4,816,567, issued March 28, 1989 to Cabilly et al. (relevant portions of which are incorporated herein by reference).

[0035] Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fab, Fab', F(ab')2, Fv, scFv, or other antigen-binding subsequences of antibodies) that are composed primarily of human immunoglobulin sequences and contain minimal sequence derived from non-human immunoglobulins. Humanization can be performed by substituting non-human (i.e., rodent, chicken) CDRs or CDR sequences for the corresponding sequences of a human antibody, according to the method of Winter and colleagues (Jones et al., 1986; Riechmann et al., 1988; Verhoeyen et al., 1988). (See also U.S. Patent No. 5,225,539.) In some cases, the F of a human immunoglobulin is v Framework residues are replaced by corresponding non-human residues from the donor antibody. Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. A humanized antibody optimally also comprises at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region (Fc).

[0036] The disclosed and claimed invention further includes the use of fully human monoclonal antibodies against ELTD1. Fully human antibodies essentially relate to antibody molecules in which the entire sequences of both the light and heavy chains, including the CDRs, arise from human genes. Such antibodies are referred to herein as "human antibodies" or "fully human antibodies." Human monoclonal antibodies can be prepared by trioma technology, human B-cell hybridoma technology (see Kozbor, et al., Hybridoma, 2:7 (1983)), and EBV hybridoma technology for producing human monoclonal antibodies (see Cole, et al., PNAS 82:859 (1985)). Human monoclonal antibodies may be utilized in the practice of the disclosed and claimed invention and may be produced using human hybridomas (see Cote, et al., PNAS 80:2026 (1983)) or by transforming human B cells in vitro with Epstein-Barr virus (see Cole, et al., 1985) (relevant portions incorporated herein by reference).

[0037] Additionally, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon raising, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, but not limited to, U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, as well as Marks et al., J. Biol. Chem. 267:16007, (1992), Lonberg et al., Nature, 368:856 (1994), Morrison, 1994, Fishwild et al., Nature Biotechnol. 14:845 (1996), Neuberger, Nat. Biotechnol. 14:826 (1996), and Lonberg and Huszar, Int. Rev. Immunol. 13:65 (1995) (relevant portions of which are incorporated herein by reference).

[0038] A method for producing a desired antibody, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771, issued June 29, 1999 to Hori et al., and is incorporated herein by reference. The method comprises introducing an expression vector containing a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses the antibody containing the heavy and light chains.

[0039] As used herein, the term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in whom the disorder is to be prevented.

[0040] As used herein, the term "disorder" refers to any condition that would benefit from treatment with a polypeptide, including chronic and acute disorders or diseases, including infectious or pathological conditions that predispose a mammal to the disorder in question.

[0041] Antibodies or antibody fragments can be produced with engineered sequences or glycosylation states to confer desired levels of activity in antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), or antibody-dependent complement deposition (ADCD) function, as measured by bead- or cell-based assays or in vivo tests in animal models.

[0042] Alternatively, or in addition, it may be useful to combine an amino acid modification with one or more additional amino acid modifications that alter the complement component Clq binding and / or complement-dependent cytotoxicity (CDC) function of the Fc region of an IL-23p19-binding molecule. Particularly interesting binding polypeptides may be those that bind Clq and exhibit complement-dependent cytotoxicity. Polypeptides with existing Clq-binding activity, and optionally the additional ability to mediate CDC, may be modified to enhance one or both of these activities. Amino acid modifications that alter Clq and / or modify its complement-dependent cytotoxicity function are described, for example, in WO / 0042072, incorporated herein by reference.

[0043] The Fc region of an antibody can be engineered to alter effector function, for example, by modifying Clq binding and / or FcγR binding, thereby altering complement-dependent cytotoxicity (CDC) and / or antibody-dependent cell-mediated cytotoxicity (ADCC) activity. These "effector functions" are involved in activating or reducing biological activity (e.g., in a subject). Examples of effector functions include Clq binding, CDC, Fc receptor binding, ADCC, phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors; BCRs), and the like. Such effector functions may require that the Fc region be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays (e.g., Fc binding assays, ADCC assays, CDC assays, etc.).

[0044] For example, antibody variant Fc regions can be generated that have improved Clq binding and improved FcγRIII binding (e.g., both improved ADCC activity and improved CDC activity). Alternatively, where reduced or eliminated effector function is desired, variant Fc regions can be engineered with reduced CDC activity and / or reduced ADCC activity. In other embodiments, only one of these activities can be increased, and optionally, the other activity can be decreased (e.g., to generate Fc region variants with improved ADCC activity but reduced CDC activity, and Fc region variants with improved CDC activity but reduced ADCC activity).

[0045] Single-chain variable fragments (scFvs) are fusions of the heavy and light chain variable regions of an immunoglobulin, linked together by a short (usually serine or glycine) linker. This chimeric molecule retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of a linker peptide. This modification usually leaves the specificity unchanged. These molecules were historically generated to facilitate phage display, which allows for the convenient expression of antigen-binding domains as single peptides. Alternatively, scFvs can be generated directly from subcloned heavy and light chains derived from hybridomas or B cells. Single-chain variable fragments lack the constant Fc region found in intact antibody molecules and therefore lack the consensus binding sites (e.g., protein A / G) used to purify antibodies. These fragments can often be purified / immobilized using protein L, as protein L interacts with the variable region of the kappa light chain.

[0046] Flexible linkers are generally composed of amino acid residues that promote helices and turns, such as alanine, serine, and glycine. However, other residues can also function. Phage display can be used to rapidly select tailored linkers for single-chain antibodies (scFv) from protein linker libraries. A random linker library was constructed in which genes for heavy and light chain variable domains were linked by a segment encoding an 18-amino acid polypeptide of variable composition. The scFv repertoire (approximately 5 × 10 6The resulting scFvs (1054 distinct members) were displayed on filamentous phage and subjected to affinity selection with a hapten. The population of selected variants showed significantly increased binding activity while retaining considerable sequence diversity. 1054 individual variants were then screened to obtain catalytically active scFvs that were efficiently produced in soluble form. Sequence analysis revealed a conserved proline in the linker two residues after the VH C-terminus, as well as abundant arginine and proline at other positions as the only common feature of the selected tethers. In certain embodiments, the antibody fragments are further modified to increase their serum half-life by using altered Fc regions or mutations to various constant regions, as known in the art.

[0047] Multiple Sclerosis. Multiple sclerosis (MS) is one of the most common diseases of the central nervous system (brain and spinal cord). It is an inflammatory condition associated with demyelination, or loss of the myelin sheath. Myelin, the fatty substance that covers nerves, acts as an insulator, allowing nerves to transmit impulses from one point to another. In MS, the loss of myelin is accompanied by a breakdown in the nerve's ability to conduct electrical impulses to and from the brain, which leads to the various symptoms of MS, such as visual impairment, loss of muscle coordination, strength loss, sensory impairment, speech and swallowing disorders, bladder control disorders, sexual dysfunction, and cognitive impairment. Plaques or lesions with myelin loss appear as hardened, scar-like areas. Because these scars appear in different areas of the brain and spinal cord at different times, the term "multiple" sclerosis literally means "many scars."

[0048] Currently, no laboratory tests, symptoms, or physical findings provide a definitive diagnosis of MS. Complicating matters, MS symptoms can easily be confused with a wide variety of other conditions, including acute disseminated encephalomyelitis, Lyme disease, HIV-associated myelopathy, HTLV-I-associated myelopathy, neurosyphilis, progressive multifocal leukoencephalopathy, systemic lupus erythematosus, polyarteritis nodosa, Sjögren's syndrome, Behçet's disease, sarcoidosis, paraneoplastic syndromes, subacute combined spinal cord degeneration, subacute myelooptic neuropathy, adrenomyeloneuropathy, spinocerebellar syndrome, hereditary spastic paraplegia / primary lateral sclerosis, stroke, tumor, arteriovenous malformation, arachnoid cyst, Arnold-Chiari malformation, and cervical spondylosis. Therefore, the diagnosis of MS must be made by a process that demonstrates findings consistent with MS and excludes other causes.

[0049] Retinopathy. Retinopathy is any damage to the retina of the eye that can lead to vision loss. Retinopathy often refers to retinal vascular disease, or damage to the retina caused by abnormal blood flow. Age-related macular degeneration, while technically included under the umbrella term retinopathy, is often considered a separate entity. Retinopathy, or retinal vascular disease, can be broadly classified as proliferative and nonproliferative. Retinopathy is often an ocular manifestation of systemic disease, such as that seen in diabetes or hypertension. Diabetes was the most common cause of retinopathy in the United States as of 2008. Diabetic retinopathy is the leading cause of blindness among working-age people. It accounts for approximately 5% of blindness worldwide and has been designated a priority eye disease by the World Health Organization.

[0050] Many people often have no symptoms until quite late in the disease process. Once irreversible damage has occurred, patients often become symptomatic. Symptoms are usually painless and may include vitreous hemorrhage, "floaters" or small objects floating in the field of vision, decreased vision, and a "curtain falling" over the eye.

[0051] The development of retinopathy can be classified as proliferative or non-proliferative. Both types cause disease by altering normal blood flow to the retina through different mechanisms. The retina is supplied by small blood vessels branching from the central retinal artery. Proliferative retinopathy refers to damage caused by abnormal blood vessel growth. Normally, angiogenesis is part of natural tissue growth and formation. When the rate of angiogenesis is abnormally high or rapid, abnormal growth of blood vessels, called neovascularization, occurs. In non-proliferative retinopathy, abnormal blood flow to the retina occurs due to direct damage and impairment of the blood vessels themselves. Many causes of retinopathy can lead to both proliferative and non-proliferative forms, but some causes are more closely related to one type.

[0052] Tissue regeneration. Regeneration in humans is the regeneration of lost tissue or organs in response to injury. This contrasts with wound healing, which involves closing the injury site with a scar. Some tissues, such as skin and large organs including the liver, regenerate quite easily, while other tissues are thought to have little or no regenerative capacity. However, ongoing research, particularly in the heart and lung, suggests there is hope that various tissues and organs may eventually be able to regenerate.

[0053] There are two types of tissue regeneration: spontaneous regeneration and guided regeneration. The former involves tissues that exhibit the ability to regenerate naturally without external intervention, such as the heart, endometrium, fingers (fingers), kidneys, liver, and toes. The latter involves external influences to bring about or enhance regeneration, such as dedifferentiation of cells at the site of injury, transplantation of stem cells, transplantation of laboratory-grown tissues, and transplantation of bioartificial tissues.

[0054] Cancer. Cancer is a group of diseases involving abnormal cell growth that can invade or spread to other parts of the body. These contrast with benign tumors, which do not spread to other parts of the body. Possible signs and symptoms include a lump, abnormal bleeding, persistent cough, unexplained weight loss, and changes in bowel movements. These symptoms may indicate cancer, but there may be other causes. Over 100 types of cancer affect humans.

[0055] Antibodies against non-ELTD1 angiogenesis targets have been approved for use in humans for the treatment of a variety of cancers, including metastatic colorectal cancer, first-line non-squamous non-small cell lung cancer, recurrent glioblastoma multiforme, metastatic renal cell carcinoma, persistent, recurrent, or metastatic cervical cancer, and epithelial ovarian, fallopian tube, or primary peritoneal cancer.

[0056] Illustratively, the antibodies, fragments, and scFv antibodies taught herein can be used in cancer therapy by blocking tumor angiogenesis. The antibodies (polyclonal, monoclonal, and binding fragments thereof) of the present invention inhibit angiogenesis by reducing neovascularization in tumors (or cancers) to levels at least 10% lower than the angiogenesis level of corresponding control tissue, and in some cases to levels at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% lower than the angiogenesis level of comparable control tissue. A reduction in angiogenesis does not mean a complete absence of angiogenesis, but rather a sufficient reduction in the extent or level of neovascularization. Angiogenesis can be determined using methods known to those skilled in the art, including, for example, in vitro and in vivo models, including counting the number of blood vessels and / or the number of vascular branch points, as discussed herein and in the Examples. Thus, novel anti-ELTD1 humanized antibodies with enhanced therapeutic efficacy would benefit cancer patients.

[0057] Cancer can spread from its original site by local spread, known as metastasis, lymphatic spread to regional lymph nodes, or hematogenous spread via the blood to distant sites. When cancer spreads via the hematogenous route, it usually spreads throughout the body. However, cancer "seeds" grow only in certain selected sites ("soil"), as postulated in the soil-and-seed hypothesis of cancer metastasis. Symptoms of metastatic cancer vary depending on the location of the tumor and may include enlarged lymph nodes (which can be felt or sometimes seen subcutaneously and are usually firm), an enlarged liver or spleen that can be felt in the abdomen, pain or fractures in affected bones, and neurological symptoms.

[0058] There are many treatment options for cancer. The main ones include surgery, chemotherapy, radiation therapy, hormone therapy, targeted therapy, and palliative care. The treatment used depends on the type, location, and grade of the cancer, as well as the patient's health and preferences. The intent of treatment may or may not be curative.

[0059] Therapeutic methods (including prophylactic treatments) of the present disclosure generally involve the administration of a therapeutically effective amount of a composition described herein to a subject in need thereof, including a mammal, particularly a human. Such treatments will preferably be administered to subjects, particularly humans, who are suffering from, have, are susceptible to, or are at risk for, or have symptoms of, cancer. The determination of a subject "at risk" can be made by any objective or subjective determination, such as by diagnostic testing or the opinion of the subject or a health care provider (e.g., genetic testing, enzyme or protein markers, Markers (as defined herein), family history, etc.).

[0060] The cancer may be carcinoma, sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple myeloma, or seminoma. In some embodiments, the cancer is bladder cancer, blood cancer, bone cancer, brain cancer, breast cancer, central nervous system cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, gallbladder cancer, gastrointestinal tract cancer, reproductive organ cancer, genitourinary tract cancer, head cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, muscle tissue cancer, cervical cancer, oral or nasal mucosa cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, spleen cancer, small intestine cancer, large intestine cancer, stomach cancer, testicular cancer, or thyroid cancer. In some embodiments, the cancer is leukemia, non-small cell lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, kidney cancer, breast cancer, or prostate cancer.

[0061] In one embodiment, the present disclosure provides a method for monitoring the progress of a treatment. The method includes determining the level of changes in hematological parameters and / or cancer stem cell (CSC) analysis or diagnostic measurements (e.g., screening, assay) using cell surface proteins as diagnostic markers (e.g., CD34, CD38, CD90, and CD117) in a subject suffering from or susceptible to a cancer (e.g., leukemia)-related disorder or symptom thereof, wherein the subject has been administered a therapeutic dose of a composition described herein. The marker levels determined in this manner can be compared to known marker levels in either healthy normal controls or other affected patients to establish the subject's disease status. In preferred embodiments, a second marker level in the subject is determined at a time later than the determination of the first level, and the two levels are compared to monitor the course of the disease or the effectiveness of the treatment. In certain embodiments, a pre-treatment marker level in the subject is determined before the initiation of treatment according to the methods described herein. This pre-treatment marker level can then be compared to the marker level in the subject after the initiation of treatment to determine the effectiveness of the treatment.

[0062] To treat cancer using the methods and compositions of the present disclosure, tumor cells or a subject will generally be contacted with a compound and at least one other therapy, provided in combined amounts effective to achieve a reduction in one or more disease parameters. This process may involve, for example, contacting the cells / subject with both agents / therapies simultaneously using a single composition or pharmacological formulation containing both agents, or contacting the cells / subject with two separate compositions or formulations simultaneously, where one composition contains the compound and the other contains the other agent.

[0063] Alternatively, the antibody may precede or follow the other treatment by intervals ranging from minutes to weeks. Generally, one will ensure that no significant period of time passes between deliveries, allowing the therapies to still exert their beneficial combined effect on the cell / subject. In such cases, it is contemplated that the cells will be contacted with both modalities within about 12-24 hours of each other, within about 6-12 hours of each other, or with a delay of only about 12 hours. In some situations, it may be desirable to extend the treatment period significantly, where several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks) pass between each administration.

[0064] Antibody conjugates. The antibodies of the present disclosure can be linked to at least one agent to form an antibody conjugate. To increase the effectiveness of antibody molecules as diagnostic or therapeutic agents, it is customary to link, covalently bond, or complex them to at least one desired molecule or moiety. Such molecules or moieties can be, but are not limited to, at least one effector molecule or reporter molecule. Effector molecules include molecules with desired activity, such as cytotoxic activity. Non-limiting examples of effector molecules bound to antibodies include toxins, antitumor agents, therapeutic enzymes, radionuclides, antiviral agents, chelators, cytokines, growth factors, and oligonucleotides or polynucleotides. In contrast, a reporter molecule is defined as any moiety that can be detected using an assay. Non-limiting examples of reporter molecules conjugated to antibodies include ligands such as enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, photoaffinity molecules, colored particles, or biotin.

[0065] Antibody conjugates are often used as diagnostic agents. Antibody diagnostics are generally divided into two classes: those for use in in vitro diagnostics, such as various immunoassays, and those for use in in vivo diagnostic protocols, commonly known as "antibody-directed imaging." Many suitable contrast agents are known in the art, as are methods for binding contrast agents to antibodies (see, for example, U.S. Patent Nos. 5,021,236, 4,938,948, and 4,472,509). The contrast moieties used can be paramagnetic ions, radioisotopes, fluorescent dyes, NMR-detectable substances, and X-ray contrast agents.

[0066] Example 1 First, we successfully amplified the extracellular domain of the mouse ELTD1 gene, encoding residues Glu20 to Leu455 (574 aa), by reverse transcription-polymerase chain reaction (RT-PCR) using RNA derived from bEnd3 (mouse brain endothelial polyoma middle T antigen-transformed cells). The extracellular domain of the human ELTD1 gene was amplified by RT-PCR using RNA prepared from HUVECs. The sequences of these two genes were confirmed by Sanger sequencing. We cloned these two genes into mammalian expression vectors and expressed the ELTD1 fragment Glu20-Leu455 (574 aa) (Favara et al., "A review of ELTD1, a pro-angiogenic adhesion GPCR", Biochem Soc Trans (2014) 42(6):1658-1664) and C. K We successfully expressed a murine recombinant protein of the human ELTD1 fragment fused with the human immunoglobulin kappa light chain constant domain (ELTD1). After transfection into the human embryonic kidney (HEK) 293F overexpression system, the recombinant protein was purified by affinity chromatography using KappaSelect resin (GE Healthcare).

[0067] The protein was used to immunize chickens. The immunized chickens were then used to generate a chicken scFv (single-chain Fv) library as described. Antibodies specific to the extracellular domain of mouse ELTD1 were then selected from the chicken scFv library as well as from human naive and synthetic libraries (Barbas, CF, 2001). mRNA was prepared from B cells, and libraries were constructed from two chickens from each group of selected clones.

[0068] Targeting ELTD1 with a monoclonal antibody affects several genes associated with either multiple sclerosis (MS), retinopathy, or tissue regeneration. RNA-seq data were first acquired from a human glioblastoma (GBM) xenograft nude mouse model derived from either untreated tumor tissue or tumor tissue treated with a monoclonal antibody against the extracellular domain of ELTD1.

[0069] The antibody binding procedure was as follows: Ag coating on microtiter plates: nM N-hCk-control: 0.5 μg / mL N-hCk-control (25 kDa); 15 nM N-hCk-mELTDl: 1 μg / mL N-hCk-mouse ELTDl (60 kDa); 15 nM N-hCk-hELTDl: 1 μg / mL N-hCk-human ELTDl (65 kDa); Blocked with 3% BSA in PBS antibody diluent: 100 nM to 0.001 nM, 1 / 10 serial dilution, 6 points. Second antibody: a-rabbit Fc-HRP. Substrate: ABTS. Read at 405 nm.

[0070] The N17 clone was selected to generate a monoclonal antibody against human ELTD1 ECD recombinant protein (Figure 3). The N17 clone binds to both human and mouse ELTD1, but with higher affinity to human ELTD1 (Figure 4).

[0071] Preparation of recombinant extracellular domains of ELTD1-human Cκ fusion proteins. To construct the extracellular domains of human ELTD1 and mouse ELTD1 expression vectors, genes encoding human ELTD1 (Glu20-Leu406) and mouse ELTD1 (Glu20-Leu455) were chemically synthesized (Genscript, Picataway, NJ, USA). As previously reported, the genes were transformed with a C -terminated ... κThe antibody was subcloned into a modified pCEP4 vector encoding the human immunoglobulin kappa light chain constant domain (HIL-K) [Lee Y, Kim H, Chung J. An antibody reactive to the Gly63-Lys68 epitope of NT-proBNP exhibits O-glycosylation-independent binding. Exp Mol Med. 2014;46:e114].

[0072] Expression vectors encoding the extracellular domains of human ELTD1 and mouse ELTD1 were transfected into HEK293F cells (Invitrogen, Carlsbad, CA, USA) using 25 kDa linear polyethylenimine (Polyscience, Warrington, PA, USA) as previously reported [Boussif O, Lezoualc'h F, Zanta MA, Mergny MD, Scherman D, Demeneix B, et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci US A. 1995;92(16):7297-301]. κ The fusion protein was purified from the culture supernatant by affinity chromatography using KappaSelect resin (GE Healthcare) according to the manufacturer's instructions.

[0073] Generation of anti-ELDT1 antibodies. Human ELTD1 C was transfected into White Leghorn chickens. κThe fusion protein was then immunized. A phage-displayed chicken single-chain variable fragment (scFv) library was then constructed using total RNA isolated from the bone marrow, spleen, and bursa of Fabricius of the immunized chickens, as previously described [Andris-Widhopf J, Rader C, Steinberger P, Fuller R, Barbas CF, 3rd. Methods for the generation of chicken monoclonal antibody fragments by phage display. J Immunol Methods. 2000;242(1-2):159-81]. Positive clones were enriched by biopanning and screened by phage enzyme immunoassay, as previously described [Barbas, CF2001. Phage Display: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY]. Phage clones showing cross-reactivity to human ELTD1 and mouse ELTD1 were selected, and their nucleotide sequences were determined by Sanger sequencing.

[0074] The genes of selected scFv clones were subcloned into a modified mammalian expression vector encoding the hinge region of human IgG1 and the 3' region of the CH2-CH3 domains of rabbit IgG, as previously reported [Han J, Lee JH, Park S, Yoon S, Yoon A, Hwang DB, et al. A phosphorylation pattern-recognizing antibody specifically reacts to RNA polymerase II bound to exons. Exp Mol Med. 2016;48(11):e271]. An expression vector encoding an anti-ELTD1 bivalent scFv Fc fusion with two scFv molecules dimerized by disulfide bonds was transfected into HEK293F cells (Invitrogen) as described above. The bivalent scFv Fc fusion protein was purified from the culture supernatant of transiently transfected HEK293F cells using a Protein A Sepharose column (Repligen, Waltham, MA, USA) according to the manufacturer's instructions. In one example, the monoclonal antibody is a bivalent scFv Fc fusion antibody, where the scFvs can be the same or different.

[0075] Enzyme-linked immunosorbent assay. Human ELTD1 or mouse ELTD1 C in coating buffer (0.1 M NaHCO3, pH 8.6) was added to the wells of a 96-well microtiter plate (Corning Inc., Corning, NY, USA). κThe fusion protein was coated and then blocked with 3% (w / v) BSA in phosphate-buffered saline (PBS). After incubation with serial 10-fold diluted anti-ELTD1 scFv-rFc fusion protein (0.01–100 nM), horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Fc-specific) (Jackson Immuno Research, Inc., West Grove, PA, USA) was added to each well. After washing with 0.05% (v / v) Tween 20 in PBS (PBST), ABTS HRP substrate solution (Thermo Scientific Pierce, Rockford, IL, USA) was added, and absorbance was measured at 405 nm using a Multiskan Ascent microplate reader (LabSystems, Helsinki, Finland).

[0076] The following sequences include the nucleic acid and amino acid sequences of various light and heavy chains of monoclonal antibodies, in these particular cases, resulting in an scFv. Additionally, the complementarity determining regions (CDRs) were determined using the Kabat rules using software available at IMGT.org. The following table includes alternative CDR sequences comparing the Kabat, Clothia, and IMGT rules, all of which can be used in conjunction with the present invention to modify antibody scaffolds (e.g., human antibodies) with the CDRs disclosed herein.

[0077] Anti-ELTD1 scFv N5, nucleotide sequence, SEQ ID NO: 1, variable light chain sequence, linker (bold), and variable heavy chain sequence (underlined).

[0078] TIFF0007818219000001.tif81170

[0079] Anti-ELTD1 scFv N5, amino acid sequences SEQ ID NOS: 2, 3, and 4, respectively, including the variable light chain sequence, linker (bold), and variable heavy chain sequence (italics). CDR1, CDR2, and CDR3 are double underlined and listed from amino-terminus to carboxy-terminus.

[0080] TIFF0007818219000002.tif26170

[0081] GQSSRSSSGGGSSGGGGS

[0082] TIFF0007818219000003.tif32170

[0083] Anti-ELTD1 scFv N7, nucleotide sequence, SEQ ID NO:5, variable light chain sequence, linker (bold), and variable heavy chain sequence (underlined).

[0084] TIFF0007818219000004.tif72170

[0085] Anti-ELTD1 scFv N7, amino acid sequences SEQ ID NOs: 6, 7, 8, respectively, including the variable light chain sequence, linker (bold), and variable heavy chain sequence (italics). CDR1, CDR2, and CDR3 are double underlined.

[0086] JPEG0007818219000005.jpg25166

[0087] GQSSRSSGGGGSSGGGGS

[0088] TIFF0007818219000006.tif30170

[0089] Anti-ELTD1 scFv N11, nucleotide sequence, SEQ ID NO: 9, variable light chain sequence, linker (bold), and variable heavy chain sequence (underlined).

[0090] TIFF0007818219000007.tif73170

[0091] Anti-ELTD1 scFv N11, amino acid sequence, SEQ ID NOs: 10, 11, 12, variable light chain sequence, linker (bold), and variable heavy chain sequence (italics), respectively. CDR1, CDR2, and CDR3 are double underlined.

[0092] TIFF0007818219000008.tif24170

[0093] GQSSRSSGGGGSSGGGGS

[0094] TIFF0007818219000009.tif36170

[0095] Anti-ELTD1 scFv N15, nucleotide sequence, SEQ ID NO: 13, variable light chain sequence, linker (bold), and variable heavy chain sequence (underlined).

[0096] TIFF0007818219000010.tif73170

[0097] Anti-ELTD1 scFv N15, amino acid sequence, SEQ ID NOs: 14, 15, 16, variable light chain sequence, linker (bold), and variable heavy chain sequence (italics), respectively. CDR1, CDR2, and CDR3 are double underlined.

[0098] TIFF0007818219000011.tif22170

[0099] GQSSRSSGGGGSSGGGGS

[0100] TIFF0007818219000012.tif29170

[0101] Anti-ELTD1 scFv N16, nucleotide sequence, SEQ ID NO: 17, variable light chain sequence, linker (bold), and variable heavy chain sequence (underlined).

[0102] TIFF0007818219000013.tif72170

[0103] Anti-ELTD1 scFv N16, amino acid sequence, SEQ ID NOs: 18, 19, 20, variable light chain sequence, linker (bold), and variable heavy chain sequence (italics), respectively. CDR1, CDR2, and CDR3 are double underlined.

[0104] TIFF0007818219000014.tif24170

[0105] GQSSRSSSGGGSSGGGGS

[0106] TIFF0007818219000015.tif31170

[0107] Anti-ELTD1 scFv N17, nucleotide sequence, SEQ ID NO: 21, variable light chain sequence, linker (bold), and variable heavy chain sequence (underlined).

[0108] TIFF0007818219000016.tif72170

[0109] Anti-ELTD1 scFv N17, amino acid sequence, SEQ ID NOs: 22, 23, 24, variable light chain sequence, linker (bold), and variable heavy chain sequence (italics), respectively. CDR1, CDR2, and CDR3 are double underlined.

[0110] TIFF0007818219000017.tif27170

[0111] GQSSRSSSGGGSSGGGGS

[0112] TIFF0007818219000018.tif28170

[0113] [Table 1]

[0114] [Table 2]

[0115] [Table 3]

[0116] [Table 4]

[0117] [Table 5]

[0118] [Table 6]

[0119] [Table 7]

[0120] [Table 8]

[0121] [Table 9]

[0122] [Table 10]

[0123] [Table 11]

[0124] [Table 12]

[0125] Example 2. Optimized monoclonal antibody treatment against ELTD1 of glioblastoma (GBM) in the G55 xenograft mouse model.

[0126] Glioblastoma is an aggressive brain tumor found in adults, and available therapeutic approaches have not significantly improved patient survival. Recently, we discovered that ELTD1, an angiogenesis biomarker, is highly expressed in human gliomas. While polyclonal anti-ELTD1 treatment was effective in preclinical glioma models, pAb binding is potentially fortuitous, difficult to manufacture, and not a preferred molecule for drug development and approval. Therefore, the purpose of this example was to determine the effects of optimized monoclonal anti-ELTD1 treatment in a G55 xenograft glioma model. Magnetic resonance imaging (MRI) was used to assess the effects of treatment on animal survival, tumor volume, perfusion rate, and binding specificity. Immunohistochemistry and histology were performed to confirm and characterize microvessel density and Notch1 levels and to identify molecular probes. RNA sequencing was used to analyze the effects of mAb treatment. Monoclonal anti-ELTD1 treatment significantly increased animal survival, reduced tumor volume, normalized vasculature, and demonstrated higher binding specificity within tumors compared to both control and polyclonal-treated mice. Notch1-positive staining and RNA-seq results suggest that ELTD1 has the ability to interact with and disrupt Notch1 signaling. Although little is known about ELTD1, particularly its ligands and pathways, these data indicate that monoclonal anti-ELTD1 antibodies can be used for antiangiogenic therapy in glioblastoma.

[0127] In the United States, 82% of all malignant gliomas diagnosed in adults are characterized as glioblastoma (GBM), with an incidence of 3.19 per 100,000 [1, 2]. This high-grade glioma is characterized by disorganized angiogenesis, invasiveness, high vascularity, and resistance to apoptosis [3]. Current treatment regimens involve surgical resection followed by radiation therapy and chemotherapy with temozolomide or the antiangiogenic mAb bevacizumab [4, 5]. However, even with treatment, the median patient survival is only 12–14 months after detection, and fewer than 5% of patients survive more than 5 years after diagnosis [2, 6]. GBMs exhibit gene amplification and / or mutations in the epidermal growth factor (EGF) receptor, and higher EGFR levels have been shown to promote migration, tumor growth, and angiogenesis [7]. Gliomas are highly dependent on angiogenesis for tumor growth, and new blood vessels are key to delivering oxygen and nutrients to the tumor site. For many years, the primary focus among pro-angiogenic factors has been vascular endothelial growth factor (VEGF) for its role in increasing angiogenesis in cancer. During tumor development, pro-angiogenic cytokines that further increase VEGF-A are upregulated, along with other microvascular growth factors such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) [8]. Upon upregulation, VEGF-A binds to VEGF receptor 2 (VEGFR2) on endothelial cells, initiating a cascade of signaling pathways that promote the formation of new blood vessels [9]. Bevacizumab is a monoclonal therapy directed against VEGF-A that has been approved for the treatment of GBM in addition to multiple other cancers. However, this chemotherapy drug has not significantly increased the survival rate of patients with GBM. Furthermore, bevacizumab has serious side effects, including severe / fatal bleeding, which occurs up to five times more frequently

[10] . Because bevacizumab failed to increase patient survival, it was crucial to shift the focus from VEGF to other angiogenic factors present in GBM.In certain embodiments, the present invention can be used to treat one or more cancers selected from glioma, breast cancer, hepatocellular carcinoma, lung cancer, ovarian cancer, ovarian cancer, metastatic colorectal cancer, cervical cancer, renal cell carcinoma, glioblastoma, or non-squamous non-small cell lung cancer.

[0128] Epidermal growth factor, latrophilin, and seven-transmembrane domain-containing protein 1 (ELTD1), also known as adhesion G protein-coupled receptor L4 (ADGRL4), was first discovered in developing cardiomyocytes

[11] . ELTD1, a novel regulator of cerebral angiogenesis, was found to promote tumor growth and metastasis

[12] . We previously reported that ELTD1 was highly expressed in high-grade gliomas and was expressed in both endothelial cells and tumor cells

[11] . Furthermore, ELTD1 expression was shown to be regulated by two major angiogenic pathways: VEGF increases ELTD1 expression, and DLL4-Notch signaling decreases ELTD1 expression in normal vasculature

[12] . Further investigation of ELTD1 showed that increased signaling from VEGF-A led to increased ELTD1 expression in endothelial cells, and targeting ELTD1 reduced VEGFR2 expression in glioma models [13, 14].

[0129] Approximately 17,000 new cases of GBM are diagnosed each year, spurring the need for new, more effective cancer therapeutics [1]. Polyclonal antibody (pAb) treatment against ELTD1 in orthotopic GL261 and human G55 xenograft glioma preclinical models was previously found to successfully reduce tumor volume (TV), increase survival, and decrease microvascular density (MVD) levels compared to untreated (UT) controls

[15] . However, batch-to-batch variability and the lack of selectivity of pAbs raised concerns about specificity as a long-term treatment for patients. Monoclonal antibodies (mAbs), produced from single B-cell clones, allow for the creation of homogenous antibodies and have been established as a successful class of targeted therapy for various cancers and chronic inflammatory diseases

[16] . Novel mAb treatments bind to growth factors overexpressed in tumors, disrupting downstream signaling effects and reducing tumor cell growth, proliferation, and migration

[17] .

[0130] Previous studies have shown that pAb treatment against ELTD1 is an effective treatment in preclinical GBM models. In this example, we used an optimized monoclonal antibody (mAb) against ELTD1, which has higher specificity by binding only to the outer region of the receptor (430AA), overcoming the limitations imposed by pAb treatment, in the hope of obtaining a more specific and pronounced effect in the G55 glioma preclinical model.

[0131] Preparation of the recombinant extracellular domain of ELTD1 human Cκ fusion protein, generation of monoclonal antibodies, and immunoassays are as described above.

[0132] G55 Xenograft Model and Treatment. All animal experiments were performed with the approval of the Oklahoma Medical Research Foundation Institutional Animal Care and Use Committee (Protocols 17–48) in accordance with NIH guidelines. Human G55 xenograft cells were intracerebrally implanted into 2-month-old male mice (Hsd: athymic nude Foxn1 mice, Harlan Inc., Indianapolis, IN) as previously described [13, 15]. Animals were divided into three groups: UT, pAb, and mAb anti-ELTD1 treatment. When tumors reached 6–7 mm3 (measured by MRI), mice were either left UT or treated every 3–4 days (Monday / Thursday, Tuesday / Friday, Wednesday / Saturday) with 2 mg / kg of either polyclonal anti-ELTD1 (Bioss, ETL / ELTD1 Polyclonal Antibody, bs-13111R) or an optimized mAb against ELTD1. All mice were euthanized when tumors reached 150 mm3 or greater.

[0133] In vivo magnetic resonance (MR) techniques. Morphological imaging. Mice were anesthetized and placed in a cradle. A 30 cm Bruker Biospin horizontal bore magnet operating at 7 Tesla (Bruker BioSpin GmbH, Karlsruhe, Germany) was used. All MRI experiments were performed using a BA6 gradient set and mouse head coil as previously described

[15] . All animals were imaged every 2–3 days starting 10 days after G55 implantation surgery until the end of the study.

[0134] Perfusion imaging. Arterial spin labeling, a perfusion imaging technique, was used as previously described

[23] . Perfusion maps were obtained in a single axial slice of the brain located at the point on the rostral-caudal axis where the tumor had its largest cross-section. Five regions of interest (ROIs) were manually outlined around the tumor, and appropriate ROIs were taken from the contralateral side of the brain for comparison. To calculate the difference in rCBF values, tumor rCBF values ​​were obtained at late (before culling) and early (at the time of tumor detection) tumor stages and normalized to the rCBF values ​​of the contralateral brain region of the corresponding animal.

[0135] Molecularly targeted MR imaging (mt-MRI). The contrast agent, biotin-BSA (bovine serum albumin)-Gd (gadolinium)-DTPA, was prepared as previously described by the present inventors

[15] , based on a modification of the method developed by Dafni et al. [24, 25]. pAb anti-ELTD1 (Bioss) or mAb anti-ELTD1 was conjugated to the albumin moiety via sulfo-NHS-EDC conjugation according to the Hermanson protocol

[26] . mt-MRI was performed when tumor volumes reached approximately 130–180 mm3. A molecular probe consisting of a biotin-albumin-Gd-DTPA construct conjugated to an anti-ELTD1 antibody was injected into mice via the tail vein catheter. A nonspecific mouse immunoglobulin IgG Ab (Alpha Diagnostics) was used together with the biotin-albumin-Gd-DTPA construct as a negative control. MRI was performed as previously described [13, 24]. Relative probe concentrations were calculated to assess ETLD1 and nonspecific IgG contrast agent levels in each animal. Subtraction images were generated from the pre-contrast and post-contrast (90 min) datasets of the slices of interest by computing the difference in T1 relaxation times between the post-contrast and pre-contrast images on a pixel-by-pixel basis. From the subtraction images, tumors of equal size (0.05 cm) were identified within the region with the highest T1 relaxation at TR 800 ms in the tumor parenchyma and contralateral brain of each animal after anti-ETLD1 probe injection. 2Ten ROIs (1000 x 1000 x 1000) were acquired. T1 values ​​obtained from ROIs within the tumor region were normalized to the corresponding contralateral side. The T1 relaxation value of a designated ROI was computed from all pixels within the ROI using the following formula (processed by ParaVision 5.0, Bruker): S(TR) = S0(1-e-TR / T1), where TR is the repetition time, S0 is the signal intensity at TR (in integer machine units), T1 and TE = 0, and T1 is the longitudinal relaxation time constant

[27] . Overlays of subtraction and T1-weighted images were generated using Photoshop software (version CS 6).

[0136] Immunohistochemistry and standard staining. After the final MRI examination, all mice were euthanized. The brain of each animal was removed, preserved in 10% neutral buffered formalin, and processed routinely. Hematoxylin-eosin staining: Tissues were fixed in 10% neutral buffered formalin, dehydrated, and embedded in paraffin. Sections were deparaffinized, rehydrated, and stained according to standard protocols. Some reagents were manufactured by Vector Labs Inc. (VLI, Burlingame, CA).

[0137] Paraffin-embedded tissue sections (5 μm) mounted on HistoBond® Plus slides (Statlab Medical Products, Lewisville, TX) were rehydrated and washed in phosphate-buffered saline (PBS). Sections were treated with ImmPRESS™ VR Reagent Anti-Rabbit IgG Peroxidase (VLI catalog number MP-6401). Antigen retrieval (pH 6 citrate antigen unmasking solution (VLI catalog no. H-3300) was achieved by 20 minutes in a steamer, followed by 30 minutes at room temperature. Sections were treated with peroxidase blocking reagent (Bloxall, VLI catalog no. SP-6000) followed by 2.5% normal horse serum to block nonspecific binding. Rabbit anti-CD34 antibody (abcam81289, 5.28 μg / mL, Cambridge, MA) or rabbit anti-NOTCH 1 (abcam52627, 11 μg / mL, Cambridge, MA) was applied to each section and incubated overnight (4°C) in a humidified chamber. After incubation, sections were washed in PBS and ImmPRESS VR reagent was applied according to the manufacturer's instructions.

[0138] Five digitally captured ROIs (20x magnification) were identified in each case to characterize microvascular density (MVD) and Notch expression levels. Only areas containing tumor tissue were analyzed, excluding areas with necrosis and / or significant artifacts. The number of positive pixels was divided by the total number of pixels (negative and positive) within the analyzed area. ROIs were analyzed and imaged using an Aperio ImageScope (Leica Biosystems, Buffalo Grove, IL).

[0139] Streptavidin-horseradish peroxidase (SA-HRP) sections were processed as above, except for overnight incubation with ready-to-use Strp-HRP (VLI Catalog No. SA-5704). Appropriate washes were performed in PBS. Slides were incubated with NovaRed® (VLI Catalog No. SK-4805) chromogen for visualization. Counterstaining was performed with Hematoxylin QS Nuclear Counterstain (VLI). Appropriate positive and negative tissue controls were used.

[0140] RNA isolation and preparation. After the final MRI examination, mice were euthanized. Brains were removed, flash-frozen, and stored at -80°C. Total RNA from tumor tissues from all groups was purified using the RNeasy Mini Kit (Qiagen) and quantified by spectrophotometry (Nanodrop).

[0141] The RNA concentration was confirmed and the overall quality of the RNA was verified. Sequencing libraries were generated according to the manufacturer's protocol (Lexogen Quantseq FWD library prep kit). Briefly, the first strand of cDNA was generated using a 5'-labeled poly-T oligomer primer, and after RNase digestion, the second strand was generated using a 5'-labeled random primer. A subsequent PCR step using additional primers added a complete adapter sequence to the initial 5' tag, added a unique index for sample demultiplexing, and amplified the library. The final library for each sample was analyzed for appropriate size and quantity (Agilent Tapestation). These libraries were pooled in equimolar amounts (fluorimetric analysis). The final pool was fully quantified using qPCR (Roche LightCycler 480 instrument with Kapa Biosystems Illumina Library Quantification reagents). Sequencing was performed using high-throughput chemistry and 75-bp single-end reads (Illumina Nextseq 500 instrument).

[0142] Bioinformatics analysis. Paired-end fastq files were checked for quality using multiQC

[28] , with the following mean (standard deviation) descriptive values: 54 million reads (12), 68.8% (2.8%) duplicated reads, and 51.3 (0.7%) GC content. Indexing and alignment were performed using kallisto

[29] against build 38 of the human reference genome (GRCh38) from the Genome Reference Consortium. Count assignment to exon features and normalization were performed along with the alignment using the biojupies pipeline 4 to obtain a count matrix. Significant differential genes were determined by DESeq2

[30] for genes with both a Benjamini-Hochberg adjusted p-value <0.05 and an absolute log fold change >1.3. Gene set enrichment analysis was performed on the identified differential genes using enrichR

[31] (gseapy API).

[0143] Statistical analysis. Survival curves were analyzed using Kaplan-Meier curves. Tumor volume, perfusion changes, and immunohistochemical protein levels, as well as molecular-targeted MRI data, were analyzed and compared by one-way or two-way ANOVA with multiple comparisons (Tukey or Sidak, respectively). Data are expressed as mean ± standard deviation, and a p-value of *<0.05, **<0.01, ***<0.001, or ****<0.0001 was considered statistically significant.

[0144] Previous studies have shown that nonspecific IgG antibody treatment as a control group did not differ from untreated animals; therefore, in this example, we utilized only untreated animals as the control group

[15] . G55 glioma-bearing mice were treated with Abs against ELTD1. As shown in Figure 1A, both anti-ELTD1 pAb (p = 0.0207) and anti-ELTD1 mAb (p = 0.0024) significantly increased survival compared with untreated mice (the mean survival time of untreated animals was approximately 9 days after tumor detection). Morphological imaging and analysis showed that tumor volume (as evidenced by MRI) 9 days after tumor detection was significantly lower in mAb ELTD1-treated (p = 0.0067) and pAb ELTD1-treated (p = 0.0384) compared with controls (Figure 1B). Representative images of tumor-bearing mice from all treatment groups are shown in Figure 1C.

[0145] MRI perfusion measures relative cerebral blood flow (rCBF) and can be used to assess changes in the microvasculature associated with tumor angiogenesis. Healthy normal tissue has a set rCBF, but as tumors grow, they disrupt the vasculature and therefore reduce perfusion rates. Differences in rCBF indicated decreased rCBF and increased angiogenesis within the tumor region in untreated mice, while perfusion values ​​normalized in anti-ELTD1-treated animals. Representative morphological MRIs are shown in Figures 2A-2F along with corresponding cerebral perfusion maps. The tumor, outlined by a yellow dashed line in Figures 2B, 2D, and 2F, shows the resulting decreased perfusion (shown as a decrease in normalized rCBF) as a result of the tumor. Anti-ELTD1 mAb treatment and anti-ELTD1 pAb treatment successfully reduced angiogenesis and, therefore, increased perfusion within the tumor region. Anti-ELTD1 pAb treatment minimized the decrease in rCBF compared to UT mice (p<0.0001). Anti-ELTD1 mAb treatment was significantly more effective in reducing rCBF compared with both pAb-treated (p=0.0001) and UT animals (p<0.0001) (Fig. 2G), normalizing rCBF within the tumor region to contralateral levels.

[0146] ELTD1 has been associated with pathological angiogenesis. Therefore, we analyzed microvessel density (MVD) to determine whether anti-ELTD1 Ab treatment altered tumor vasculature. Representative CD34 IHC images from each treatment group are shown in Figures 3A-3C. CD34 analysis showed that anti-ELTD1 treatment significantly reduced MVD levels (p<0.0001) compared with untreated animals (Figure 3D). Anti-ELTD1 mAb treatment further reduced MVD levels (p=0.0013) compared with polyclonal anti-ELTD1 treatment, restoring MVD to near-normal levels.

[0147] To determine where the Abs were localized in vivo, we synthesized biotin-BSA (bovine serum albumin)-Gd-DTPA probes conjugated to either nonspecific IgG, anti-ELTD1 pAb, or anti-ELTD1 mAb (Figure 4A). The molecular probes were injected into untreated G55 glioma-bearing mice via a tail vein catheter, and T1 relaxation times and signal intensities were calculated by MRI. T1 relaxation is an MRI imaging parameter that decreases in the presence of the molecular probes of the present invention. The results, shown in Figure 4B, show the presence of the molecular probes as a relative expression percentage due to their effect on T1 relaxation. Both T1 (p = 0.0002) and signal intensity (p = 0.008) were significantly increased with the anti-ELTD1 mAb-conjugated probe compared to the nonspecific IgG-conjugated probe. The anti-ELTD1 pAb-conjugated probe significantly increased T1 relaxation (p = 0.0307) but had no significant effect on signal intensity (p = 0.0602).

[0148] Representative molecular-targeted MRI data of anti-ELTD1 mAb and nonspecific IgG probes were overlaid on morphological images of untreated G55 tumor-bearing animals. Figure 4C shows that the anti-ELTD1 mAb-conjugated probes had increased binding specificity to tumor regions. Background binding of the nonspecific IgG probe was primarily clustered around blood vessels (Figure 4D). After monitoring the expression of the molecular probes, we found that the anti-ELTD1 mAb-conjugated probes had a more pronounced and sustained effect compared with both the nonspecific IgG-conjugated and anti-ELTD1 pAb-conjugated probes (Figure 4E).

[0149] The molecular probe had a biotin label attached to it to further localize it within the tissue. At the end of molecular targeting, the animals were sacrificed and tissues were collected for histology. By staining tumor tissue with SA-HRP, we confirmed the molecular targeting results. The anti-ELTD1 mAb- and pAb-conjugated probes were localized within the tumor tissue after sacrifice, while there was no trace of the nonspecific IgG-conjugated probe within the tissue (Figure 4F-H). This data indicates that the anti-ELTD1 mAb-conjugated probe has significantly higher binding specificity to the tumor region than both the pAb-conjugated probe and the IgG probe.

[0150] Notch1 signaling is important for cell differentiation, proliferation, and tumor angiogenesis, and normal vasculature has been shown to decrease ELTD1 expression

[32] . Therefore, we investigated whether anti-ELTD1 Ab treatment altered Notch1 levels in tissues from mice with GBM. Positive analysis of stained samples indicated that untreated glioma tumor samples had the highest amount of Notch1. Anti-ELTD1 mAb treatment significantly reduced Notch1 expression levels compared to anti-ELTD1 pAb treatment (p = 0.0357) and untreated control animals (p = 0.0006), lowering expression to contralateral levels (Figure 5E).

[0151] Because in vivo data showed that anti-ELTD1 mAb treatment against ELTD1 was more effective in the G55 xenograft model, we investigated the effects of anti-ELTD1 mAb treatment (compared to untreated treatment) on genes within the tumor area. Among all genes shown in Figure 6A, ADA, SCN5A, L1CAM, BMP2, ALPL, and TRPM8 (all increased in tumors), and SELENBP1 (decreased in tumors) were directly associated with glioma. Meanwhile, other genes were associated with various other cancers, such as hepatocellular carcinoma (VWA1-decreased

[33] ), lung cancer (SCUBE3-decreased

[34] , PLCH1-increased

[35] , CHRNA1-increased

[36] , CDH2-increased

[37] ), and breast cancer (IFITM10-decreased

[38] , DCDC2-increased

[39] , CHST9-increased

[40] , CDH2-increased

[41] ). To confirm whether some of the genes downregulated upon anti-ELTD1 mAb treatment were similarly co-regulated in other experiments, we first calculated gene-gene Pearson correlations using experiments from the publicly available microarray platform GPL570, part of NCBI's GEO database. Figure 6B shows the clustered gene-gene correlations of downregulated genes using the GPL570 data. Broadly, four clusters (developmental genes, nestin-related genes, cell proliferation / angiogenesis, and astrocyte-microglia inflammation) are evident, indicating that the groups of genes found to be differentially expressed in these studies are consistent with those observed in other experiments.

[0152] The table below summarizes the expression changes induced in different diseases or conditions using the novel antibodies of the present invention.

[0153] [Table 13]

[0154] [Table 14]

[0155] Through a global microarray meta-analysis (GAMMA)

[42] , we identified that the angiogenesis marker ELTD1 is highly expressed in high-grade gliomas, and other groups have suggested that high ELTD1 expression levels may correlate with glioma aggressiveness [32, 43]. Previous studies have shown that anti-ELTD1 pAb treatment is effective in mouse GL261 and human G55 xenograft glioma models

[15] . Other groups have also found that microRNA-139-5p directly binds and targets ELTD1, inhibiting cell proliferation in gliomas

[44] .

[0156] This example focuses on optimized mAb therapy against ELTD1 in an aggressive human G55 xenograft glioma mouse model. The data show that repeated IV treatment with both anti-ELTD1 pAb and anti-ELTD1 mAb resulted in significant reductions in tumor volume and increased survival. While previous studies by the inventors demonstrated a 7-10 day increase in survival with anti-ELTD1 pAb treatment, this current study demonstrated only an average increase of 5 days

[15] . The discrepancy between studies is due to differences in doubling times between G55 cells. The 2017 study used high-passage G55 cells, which had a doubling time of 2.5 days and a mean survival time of 18 days in untreated mice. However, this example used low-passage G55 cells, which appear more aggressive due to a faster doubling time of 2 days and a mean survival time of 10 days. However, anti-ELTD1 mAb treatment was able to extend the doubling time to approximately 2.7 days, even in this more aggressive glioma model.

[0157] The optimized anti-ELTD1 mAb treatment not only restored perfusion levels to normal, indicating normalization of vasculature within the tumor region, but was also effective in significantly reducing microvascular density (MVD) levels. Collectively, these data indicate that the mAb of the present invention and its use in mAb therapy against ELTD1 had a more pronounced impact on tumor-associated microvasculature compared to both untreated and anti-ELTD1 pAb-treated animals.

[0158] Previous studies using human G55 cells have shown increased bleeding when anti-VEGF therapy was used in mice and patients receiving Avastin treatment [10, 15]. We did not observe any bleeding within the tumor areas treated with anti-ELTD1 pAb or anti-ELTD1 mAb, either on MR images or when stained with Prussian blue. This indicates that anti-ELTD1 Ab treatment may be safer for use in the clinic.

[0159] Collectively, these data demonstrated that ELTD1 is an important angiogenesis marker in high-grade gliomas. Additionally, by using the optimized mAb against ELTD1 for GBM in mice, we were able to significantly increase survival rates, reduce tumor volume, and normalize tumor-associated vasculature. These data demonstrate that the optimized mAb against ELTD1 had higher binding specificity compared to the anti-ELTD1 pAb, as confirmed by molecular targeting and histology.

[0160] While previous studies have shown that ELTD1 expression is increased by VEGF in normal vasculature but suppressed by Notch / DLL4 interactions, we demonstrate that this relationship may be more complex in the tumor environment

[12] . We found that anti-ELTD1 mAb treatment significantly reduced Notch1 protein levels in tumors to levels similar to those in contralateral normal tissue. Furthermore, RNA sequencing data showed that three of the genes affected by anti-ELTD1 mAb treatment (SCN5A, L1CAM, and BMP2) are directly associated with glioma and are known to affect and interact with Notch signaling. Thus, ELTD1 has a more complex relationship with Notch1 than previously understood.

[0161] Aside from the possible relationship with Notch, RNA-seq data provided further insight into which pathways anti-ELTD1 Ab treatment targets. ADA and BMP2 expression correlate with poor prognosis in glioma patients [45, 46], and both were downregulated by anti-ELTD1 treatment. Furthermore, anti-ELTD1 treatment downregulated SCN5A, TRPM8, and BMP2, all of which have been shown to increase glioma cell proliferation, migration, and invasion [47-49]. Alkaline phosphatase (ALPL) is a stem cell marker highly expressed around necrotic areas within tumors, and high expression of ALPL and CD133 (another stem cell marker) has been associated with poor patient prognosis

[50] . Anti-ELTD1 mAb therapy successfully downregulated ALPL. CD133+ glioma cells exhibit neurosphere-like growth, promote tumor formation, and are resistant to standard therapy

[51] . L1CAM, a gene downregulated by anti-ELTD1 treatment, is overexpressed in GBM and CD133+ glioma cells and regulates the growth, migration, and survival of developing neurons [52, 53]. Furthermore, targeting and inhibiting L1CAM in CD133+ glioma cells suppressed tumor growth and increased survival in a glioma xenograft model

[54] . Glioma stem cells (GSCs), the primary cause of GBM recurrence after treatment, are characterized by CD133

[55] . However, recent reports have shown that CD133 may not be a strong marker for GSCs, and CD133- cells with GSC characteristics can initiate invasive tumors [55, 56]. Nestin was initially considered a neural stem cell marker and was identified in CD133-positive cells and It is found on the surface of both CD133-negative cells and may function as a more efficient GSC marker in GBM [55, 56]. Furthermore, nestin has been shown to play an important role in the proliferation, migration, and survival of GBM and other cancers [57-59]. Interestingly, gene-gene clustering analysis of genes downregulated by anti-ELTD1 treatment suggests a downregulation of nestin-related pathways in response.

[0162] These data suggest that the optimized anti-ELTD1 mAb is a potential antiangiogenic therapy for GBM. Although discovered in 2001, ELTD1 has many unknowns, including its mechanism of action and its ligands.

[0163] Example 3 In this example, we generated an anti-ELTD1 single-chain variable fragment (scFv) and demonstrated that it maintained binding of the 430AA external domain of ELTD1 in a G55 glioma xenograft preclinical model. We assessed tumor volume using morphological MRI and measured vascular changes within the tumor region after anti-ELTD1 scFv treatment compared with anti-ELTD1 mAb treatment using perfusion imaging. Animal survival was also determined after anti-ELTD1 scFv or anti-ELTD1 mAb treatment. Using mt-MR imaging, we also evaluated the binding affinity and specificity of the anti-ELTD1 scFv fragment as a probe.

[0164] Preparation of recombinant extracellular domain of ELTD1 human Cκ fusion protein was as described in Example 2 above. Generation of anti-ELDT1 Ab was as described above. Enzyme-linked immunosorbent assay was as described above. G55 xenograft model and treatments were as described above. In vivo magnetic resonance (MR) techniques were as described above. Immunohistochemistry and standard staining were as described above. Statistical analysis was as described above.

[0165] We implanted human G55 cells intracerebrally into 2-month-old male athymic nude mice. Tumor growth was monitored by morphological MR imaging, and tumors were detected at 6-7 mm. 3) were treated with either monoclonal anti-ELTD1 mAb or anti-ELTD1 scFv (also referred to herein as fragment) administered via the tail vein every 3–4 days. The percent survival rate of G55 glioma-bearing mice after tumor detection was significantly higher with both anti-ELTD1 mAb treatment (*p=0.0058) and anti-ELTD1 scFv treatment (***p=0.0001), as shown in Figure 7A. Because the mean survival time in the untreated group was 9 days, we compared tumor volume 9 days after tumor detection. Tumor volume 9 days after tumor detection was significantly lower in anti-ELTD1 Ab-treated mice (mAb) compared with untreated controls (mAb***p=0.0009, scFv fragment*p=0.017) (Figure 7B). Representative MRI images of G55 tumor-bearing mice from all treatment groups are shown in Figures 7C–7E.

[0166] Because anti-ELTD1 treatment targets angiogenesis, we investigated whether these treatments affected the microvasculature. Changes in tumor microvasculature associated with tumor angiogenesis can be measured by a decrease in relative cerebral blood flow (rCBF). As the vasculature within the tumor region grows, it becomes exponentially more disorganized, thus resulting in a decrease in perfusion rate. Perfusion scans performed using MRI demonstrated a characteristic decrease in rCBF within the tumor region of untreated animals (Figure 8A). Perfusion values ​​in mice treated with anti-ELTD1 Ab treatment were significantly improved compared to untreated animals (all p<0.0001). Anti-ELTD1 mAb treatment normalized perfusion values, whereas anti-ELTD1 scFv fragment-treated animals showed a slight increase in perfusion (Figure 8A). Figures 8C, 8E, and 8G show representative perfusion scans from each group. The untreated perfusion scan (Figure 8C) has a distinct dark area (outlined by a yellow dashed line) only within the tumor region, indicating reduced perfusion within that area. However, the tumor region in the perfusion scans of the anti-ELTD1 mAb-treated mice (Figure 8E) and the anti-ELTD1 scFv fragment-treated mice (Figure 8G) is homologous to the contralateral tissue. Furthermore, we determined the effect of anti-ELTD1 Ab treatment on tumor-associated vasculature. Both anti-ELTD1 Ab treatments significantly reduced microvessel density (MVD) levels within the tumor region compared to untreated animals (p<0.0001) (Figure 9D). Representative CD34 IHC images of all treatment groups are shown in Figures 9A-9C.

[0167] In normal vasculature, ELTD1 expression has been shown to be upregulated by VEGF and downregulated by Notch / DLL4

[33] . In a previous study, we investigated the relationship between VEGF and ELTD1 and found that targeting ELTD1 reduced VEGFR2 (VEGF receptor) levels in a glioma model

[31] . Therefore, in this example, we again determined whether anti-ELTD1 Ab treatment affected Notch1. Tissues from glioma-bearing mice from each group were stained for Notch1 and analyzed for positivity. We sought to characterize the difference in Notch1 positivity levels between tissue within the tumor area and contralateral normal tissue. Figure 10A shows that the positivity levels in the contralateral tissue were significantly lower (p<0.0001) compared with the levels within the tumor area of ​​untreated animals. Furthermore, our anti-ELTD1 mAb and anti-ELTD1 scFv fragment treatments successfully reduced Notch1 levels in the tumor area (p = 0.0001 and p < 0.0001, respectively), bringing them to levels similar to those in the contralateral normal tissue (Figure 10A). Representative histological images shown in Figures 10B–10E demonstrate reduced Notch1 staining in the tumor area treated with anti-ELTD1 Ab.

[0168] To determine whether antibody treatment crossed the blood-brain barrier (BBB) ​​and contributed to the previous results shown above, we conjugated either nonspecific IgG, anti-ELTD1 mAb, or anti-ELTD1 scFv fragment to the molecular probe described above (albumin-biotin-Gd-DTPA) and shown in Figure 11A

[28] . The molecular probe was injected via the tail vein into untreated glioma-bearing animals and monitored by MR molecular targeting imaging. The Gd-DTPA conjugated to the molecular probe allowed for the determination of the location of probe binding and the measurement of signal intensity within the tumor region. Figure 10B shows that the nonspecific IgG-conjugated probe was a suitable control because the signal intensity was at baseline after 90 minutes. However, the difference in signal intensity was significantly higher for the anti-ELTD1 mAb-conjugated molecular probe and the anti-ELTD1 scFv fragment-conjugated molecular probe (p = 0.0007 and p = 0.0038, respectively), as shown in Figure 11B. Furthermore, Figure 11C shows how the anti-ELTD1 mAb-conjugated probe localized within the tumor region over a 90-minute period. Because the signal intensity of the anti-ELTD1 scFv fragment-conjugated probe decreased slightly 90 minutes after injection, we examined the binding of the molecular probe for up to 180 minutes. Figure 11D shows that the fragment-conjugated probe required a longer time to bind to and localize within the tumor region.

[0169] The anti-ELTD1 scFv fragment-conjugated probe not only localized within the bulk tumor seen by MRI but also bound to the periphery of other areas initially thought to be non-tumor tissue, as seen in the last frame of Figure 11C. The glioma tissue was then stained with SA-HRP, which binds to the biotin label attached to the molecular probe, to further investigate the probe-bound areas. The anti-ELTD1 scFv fragment-conjugated probe successfully reached the bulk tumor, as indicated by the SA-HRP staining shown in the upper voxel of Figure 12. Furthermore, H&E analysis of the tissue revealed the presence of highly diffuse tumor areas along the outer cortical region of the brain, where the probe successfully bound, as shown in the last frame of Figure 12C. The molecular probe was also found by SA-HRP staining in the diffuse tumor area, as seen in the lower right voxel of Figure 12.

[0170] Example 4. Retinopathy Using the antibodies of the present invention in a retinopathy model, changes in the expression of the following RNAs were determined:

[0171] [Table 15]

[0172] Example 5. Tissue regeneration Using the antibodies of the present invention in a tissue regeneration model, changes in the expression of the following RNAs were determined:

[0173] [Table 16]

[0174] overview Antibodies are an important and well-established class of drugs. More recently, research has focused on single-chain variable fragments (scFv) as an alternative to larger whole antibody molecules. Therefore, scFvs against various targets for various cancers have been developed [34-37]. Furthermore, scFvs have been conjugated to molecular targeting moieties for the development of potential therapeutic and diagnostic applications using MRI and bioluminescence imaging [37-40].

[0175] As shown above, anti-ELTD1 pAb treatment reduced tumor volume and increased animal survival in both the mouse GL261 model and the human G55 xenograft glioma model.

[26] Because pAbs are not currently a viable therapy, the goal of this patent was to develop and optimize a monoclonal antibody therapy against ELTD1. We developed an anti-ELTD1 mAb and an anti-ELTD1 scFv fragment against the ectodomain of ELTD1. Production of monoclonal antibodies against ELTD1 was achieved in chickens. The choice of this host stems from the fact that chickens are more distant from humans than rodents, allowing for the generation of antibodies with higher affinity and potential cross-species reactivity.

[0176] Regarding treatment responses in GBM-bearing mice, both monoclonal antibodies against ELTD1 (anti-ELTD1 mAb and anti-ELTD1 scFv fragment) were successful in increasing survival rates and reducing tumor volume. Although anti-ELTD1 scFv fragment treatment appeared successful, there was significant variability in tumor volume and survival rates after tumor detection within groups.

[0177] Both anti-ELTD1 mAb and anti-ELTD1 scFv fragment successfully normalized perfusion levels within the tumor region. Furthermore, both anti-ELTD1 treatments successfully reduced and normalized microvascular density (MVD) levels within the tumor region. These results indicate that targeting ELTD1 can normalize tumor-associated vasculature within the tumor region. The relationship between ELTD1 and VEGFR2 in GBM has been demonstrated above. These data indicate that treatment of GBM tumor-bearing mice with anti-ELTD1 Ab reduced VEGFR2 levels in tumor tissue. In this study, we conducted experiments to elucidate the relationship between anti-ELTD1 mAb treatment and another important angiogenesis marker, Notch

[31] . Untreated G55 tumors were found to have increased Notch1 protein expression compared with contralateral normal tissue. However, repeated treatment with both anti-ELTD1 mAb and anti-ELTD1 scFv fragment reduced Notch1 protein expression levels within the tumor. It has been shown that Notch1, like VEGFR2, plays an important role in promoting angiogenesis.Therefore, the discovery that anti-ELTD1 Ab therapy increases two different molecules that are important for angiogenesis is an important discovery.In fact, in these experiments, treatment of GBM tumor-bearing mice with anti-VEGF mAb Avastin reduced VEGFR2, but not Notch1.The reduction of Notch1 in tumors can further explain and support the normalization of intratumoral vasculature, but does not limit the present invention.

[0178] By constructing biotin-albumin-Gd-DTPA molecular probes conjugated to either nonspecific IgG, anti-ELTD1 mAb, or anti-ELTD1 scFv fragment, we were able to localize the antibodies in vivo and quantify the signal intensity generated by the probes within the tumor region. This allowed us to determine that both the anti-ELTD1 monoclonal-conjugated probe and the anti-ELTD1 fragment-conjugated probe successfully localized within the tumor region. Furthermore, molecular targeting data showed that the anti-ELTD1 scFv fragment-conjugated probe was able to bind to diffuse tumor regions previously undetectable by MRI. This finding indicates that the anti-ELTD1 scFv fragment can be used to localize diffuse tumors, potentially providing a new detection tool for identifying the extent of tumor tissue.

[0179] By staining for hemoglobin iron in red blood cells, it was also possible to evaluate the effect of bleeding associated with antibody against VEGF (Avastin) therapy in G55 human GBM xenograft tissue sections compared with anti-ELTD1 pAb or mAb treatment (Figure 13). Considering that bleeding is one of the most serious, life-threatening adverse events of Avastin therapy in cancer, it was important to know whether anti-ELTD1 Ab therapy also caused bleeding. In the present invention, Avastin therapy showed bleeding, while anti-ELTD1 Ab therapy did not show bleeding.

[0180] Anti-ELTD1 was found to significantly reduce Notch protein levels, which were high in untreated G55 tumors and were shown to be unaffected by anti-VEGF (Avastin) treatment; anti-ELTD1 treatment (pAb, mAb, or scFv fragment of mAb) was found to significantly reduce Notch, while Avastin treatment had no effect (Figure 14).

[0181] In conclusion, these molecular targeting data demonstrate the diagnostic use of anti-ELTD1 mAb and anti-ELTD1 scFv fragment to differentiate diffuse tumors undetectable by MR imaging. Furthermore, both anti-ELTD1 treatments successfully increased survival rates, reduced tumor volume, and normalized tumor-associated vasculature. While ELTD1 has been shown to be downregulated via the Notch / DLL4 pathway in normal vasculature, this study demonstrates a relationship between ELTD1 and Notch1 in GBM tumors. It was also shown that anti-ELTD1 mAb or anti-ELTD1 scFv fragment treatment did not cause the bleeding commonly associated with Avastin treatment. This example demonstrates that both monoclonal antibody and scFv antibody therapy against ELTD1 are effective and can be used for antiangiogenic therapy(ies) for GBM tumors.

[0182] Example 4. ELTD1, a novel biomarker target for multiple sclerosis: Molecularly targeted MRI detection of ELTD1 in an experimental autoimmune encephalomyelitis (EAE) mouse model

[0183] Multiple sclerosis (MS) is an autoimmune disease that affects the nervous system's ability to transmit signals to and from the brain. Inflammation damages myelin, the protective covering that surrounds nerve cells, slowing and sometimes blocking nerve impulses. The disease is accompanied by a variety of symptoms, including vision loss, tremors, paralysis, painful spasms, imbalance, and cognitive changes. In glioblastoma (GBM), we detected overexpression of ELTD1 (epidermal growth factor, latrophilin, and seven-transmembrane domain-containing protein 1 on chromosome 1) and found that antibodies targeting ELTD1 could increase animal survival and reduce tumor volume. From RNA-seq analysis of tumor tissue from anti-ELTD1-treated animals, we found that some of the genes affected by the anti-ELTD1 antibody are also associated with MS. Using molecular-targeted MR imaging of ELTD1, we assessed ELTD1 levels in a mouse model of MS (EAE, or experimental autoimmune encephalomyelitis).

[0184] In vivo ELTD1 levels were assessed using molecular-targeted MRI (mtMRI) by coupling anti-ELTD1 Ab with a gadolinium (Gd)-based MRI contrast agent. Experimental autoimmune encephalomyelitis (EAE) mice (2-3 months old, female, n = 10, anti-ELTD1-albumin-Gd-DTPA-biotin (anti-ELTD1 probe) (n = 5 for anti-ELTD1 probe, n = 5 for nonspecific IgG contrast agent) were anesthetized with isoflurane (2-3%) for MRI scans. ELTD1 expression images were acquired using a targeted ELTD1 contrast agent probe (20 μg). MRI experiments were performed on a Bruker Biospec 7.0 Tesla / 30 cm horizontal bore imaging system. Spin-echo multislice imaging was used to image multiple brain regions in 1H. MR image slices were acquired (repetition time (TR) 0.8 s, echo time (TE) 23 ms, 128 × 128 matrix, 4 steps per acquisition, 3 × 4 cm field of view, 1 mm slice thickness). Rat brains were imaged at 0 min (pre-contrast) and 20 min, with intervals up to 90 min after contrast injection. T1-weighted images were acquired using a variable TR (repetition time) spin-echo sequence. Pixel-by-pixel relaxation maps were reconstructed from the series of T1-weighted images using a nonlinear two-parameter fitting procedure. T1 values ​​for a specified region of interest (ROI) were computed from all pixels within the identified ROI.

[0185] 15A-15C show T2 MRI scans showing changes in the cervical spinal cord of EAE mice 27 days after disease progression compared to controls.

[0186] 16A-16C show contrast images obtained using Gd-DTPA MRI contrast agent and T1-weighted MRI scans acquired 10 days (EAE-E) and 26 days (EAE-L) after disease progression.

[0187] Figure 17 shows an anti-ELTD1 mAb probe: a monoclonal antibody (mAb) against the extracellular domain of ELTD1 was conjugated to an albumin-linked Gd-DTPA and biotin construct.

[0188] Figures 18A-18C show that the anti-ELTD1 mAb MRI probe binds more efficiently to various brain regions in the EAE mouse model of MS compared to a non-selective control MRI contrast agent. p<0.0001. Percent change in T1 relaxivity when the anti-ELTD1 probe is compared to a control IgG contrast agent.

[0189] Figures 19A and 19B show that (Figure 19A) anti-ELTD1 mAb MRI probe efficiently binds to endothelial cells in the brain of an EAE mouse model of MS. Molecularly targeted MR imaging of ELTD1 (streptavidin-HRP (horseradish peroxidase) binds to the biotin moiety of the anti-ELTD1 probe). (Figure 19B) Immunohistochemical staining of ELTD1 shows high levels in endothelial cells of EAE mice.

[0190] Thus, using mtMRI, we were able to demonstrate significantly increased ELTD1 levels in EAE mice (a model of MS) ​​administered with an anti-ELTD1 mAb probe compared to EAE mice administered with a nonspecific IgG contrast agent. The data in Figures 15A-15C demonstrate the use of the present invention for in vivo mtMR imaging of ELTD1 levels in an EAE mouse model of MS. For the first time, mtMRI demonstrates noninvasive in vivo detection of ELTD1 in a mouse model of MS.

[0191] Table 5: RNA sequencing data showing that 7 of 23 genes altered by anti-ELTD-1 mAb therapy in GBM models are similarly associated with MS.

[0192] [Table 17]

[0193] [Table 18]

[0194] It is contemplated that any embodiment discussed herein can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, the compositions of the invention can be used to achieve the methods of the invention.

[0195] It will be understood that the specific embodiments described herein are not limiting of the invention, but are shown by way of illustration. The principal features of this invention may be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the appended claims.

[0196] All publications and patent applications mentioned in this specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0197] The use of the terms "a" or "an," when used in conjunction with the term "comprising" in the claims and / or this specification, can mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more." The use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternatives only or the alternatives are not mutually exclusive, although this disclosure supports a definition that refers to alternatives only and "and / or." Throughout this specification, the term "about" is used to indicate that a value includes the inherent variation of error for the device, method being employed to determine the value, or the variation that exists among test subjects.

[0198] As used in this specification and claims, the words "comprising" (and any form of "comprising," such as "comprise" and "comprises"), "having" (and any form of "having," such as "have" and "has"), "including" (and any form of "including," such as "includes" and "include"), or "containing" (and any form of "containing," such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other, unstated features, elements, components, groups, integers, and / or steps. In any of the embodiments of the compositions and methods provided herein, "comprising" may be replaced with "consisting essentially of" or "consisting of." As used herein, the term "consisting of" is used to indicate only the presence of a recited integer (e.g., feature, element, characteristic, property, method / process step, or limitation) or group of integers (e.g., feature(s), element(s), feature(s), property(ies), method / process step, or limitation(s)). As used herein, the phrase "consisting essentially of" requires the specified features, elements, components, groups, integers, and / or steps, but does not exclude the presence of other unstated features, elements, components, groups, integers, and / or steps, and those that do not materially affect the basic and novel feature(s) and / or function of the claimed invention.

[0199] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and, where order is important in a particular context, at least one of BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing this example, combinations including repeats of one or more items or terms, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, etc. Those of skill in the art will understand that there is typically no limit to the number of items or terms in any combination, unless otherwise clear from the context.

[0200] As used herein, approximating words, such as, but not limited to, "about," "substantially," or "substantially," when so modified, refer to conditions that are understood not to be necessarily absolute or complete, but that one of ordinary skill in the art would consider close enough to warrant specifying the condition as existing. The extent to which the description can vary depends on how large a change can be made and still allow one of ordinary skill in the art to recognize the modified feature as still possessing the required characteristics and capabilities of the unmodified feature. Generally, but in accordance with the foregoing considerations, numerical values ​​herein modified by approximating words such as "about" may vary from the stated value by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12, or 15%.

[0201] All of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations may be applied to the compositions and / or methods described herein, and in the steps or sequence of steps of the methods described herein, without departing from the concept, spirit, and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.

[0202] In order to assist the Patent Office and any readers of any patent that may issue on this application in interpreting the appended claims, applicant wishes to note that, unless the language "means for" or "step for" is expressly used in a particular claim, none of the appended claims are intended to invoke 35 U.S.C. 112, paragraph 6, 35 U.S.C. 112(f), or equivalents, as they exist on the filing date thereof.

[0203] For each claim, each dependent claim may depend on both any and all independent claims and each preceding dependent claim, so long as the preceding claim provides adequate support for a term or element of the claim.

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Claims

1. A pharmaceutical formulation comprising one or more monoclonal antibodies or antigen-binding fragments thereof that bind to EGF, latrophilin, and seven transmembrane domain-containing protein 1 (ELTD1), The monoclonal antibodies and antigen-binding fragments thereof are The pharmaceutical preparation has a light chain variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 55, CDR2 of SEQ ID NO: 56, and CDR3 of SEQ ID NO: 57, and a heavy chain variable region comprising CDR1 of SEQ ID NO: 58, CDR2 of SEQ ID NO: 59, and CDR3 of SEQ ID NO:

60.

2. At least one of the antigen-binding fragments is a Fab fragment, F(ab') ２ 2. The pharmaceutical formulation of claim 1, wherein the monoclonal antibody or antigen-binding fragment thereof is at least one of an Fc fragment, FcR fragment, or Fv fragment, or is chimeric, humanized, fully human, or bispecific, or is a single-chain variable fragment (scFv), or a monovalent antibody, or comprises an Fc portion that has been mutated to alter (eliminate or enhance) FcR interactions to increase half-life and / or increase or decrease effector function such as antibody-dependent cellular cytotoxicity or complement activation, or wherein the monoclonal antibody or antigen-binding fragment thereof is adapted for administration or gene delivery using an RNA or DNA sequence or vector encoding the monoclonal antibody or antigen-binding fragment thereof, or wherein the monoclonal antibody or antigen-binding fragment thereof further comprises a cell-penetrating peptide and / or is an intrabody.

3. the light chain variable region has the amino acid sequence of SEQ ID NO: 22; or the heavy chain variable region has the amino acid sequence of SEQ ID NO: 24; The pharmaceutical formulation of claim 1.

4. A composition for treating or detecting a subject having or suspected of having cancer, comprising an antibody or antigen-binding fragment thereof having binding affinity for ELTDl; the antibodies and antigen-binding fragments thereof The composition has a light chain variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 55, CDR2 of SEQ ID NO: 56, and CDR3 of SEQ ID NO: 57, and a heavy chain variable region comprising CDR1 of SEQ ID NO: 58, CDR2 of SEQ ID NO: 59, and CDR3 of SEQ ID NO:

60.

5. 5. The composition of claim 4, wherein the antigen-binding fragment is at least one of a Fab fragment, a F(ab')2 fragment, or an Fv fragment; is chimeric, humanized, fully human, or bispecific; is a single-chain variable fragment (scFv); is a monovalent antibody; or comprises an Fc portion mutated to alter (eliminate or enhance) FcR interaction, increase half-life, and / or increase or decrease effector function such as antibody-dependent cellular cytotoxicity or complement activation.

6. 5. The composition of claim 4, wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 22 and the heavy chain variable region has the amino acid sequence of SEQ ID NO:

24. the antibody or antigen-binding fragment thereof is adapted for administration or gene delivery using an RNA or DNA sequence or vector encoding the antibody or antigen-binding fragment thereof; the antibody or antigen-binding fragment thereof is adapted for administration by gene delivery using an RNA or DNA sequence or vector encoding the antibody or antigen-binding fragment thereof; or The composition, wherein the antibody or antigen-binding fragment thereof downregulates both Notch1 and VEGFR2 when contacted with the cancer cells.

7. 5. The composition of claim 4, wherein the cancer is metastatic colorectal cancer, first-line non-squamous non-small cell lung cancer, recurrent glioblastoma multiforme, metastatic renal cell carcinoma, persistent, recurrent, or metastatic cervical cancer, and epithelial ovarian, fallopian tube, or primary peritoneal cancer.

8. The composition of claim 4, wherein the antibody or antigen-binding fragment thereof exhibits reduced or no bleeding during administration.

9. a light chain variable region having the amino acid sequence of SEQ ID NO: 22 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 24; A monoclonal antibody or antigen-binding fragment thereof that specifically binds to EGF, latrophilin, and seven-transmembrane domain-containing protein 1 (ELTD1).

10. 10. The monoclonal antibody or antigen-binding fragment thereof of claim 9, wherein the antigen-binding fragment thereof is at least one of a Fab fragment, a F(ab')2 fragment, or a Fv fragment, or is chimeric, humanized, fully human, or bispecific, or comprises an Fc portion that has been mutated to alter (eliminate or enhance) FcR interactions to increase half-life and / or increase or decrease effector functions such as antibody-dependent cellular cytotoxicity or complement activation.

11. The monoclonal antibodies and antigen-binding fragments thereof are A monoclonal antibody or antigen-binding fragment thereof according to claim 9, having a light chain variable region comprising complementarity-determining region (CDR) 1 of SEQ ID NO: 55, CDR2 of SEQ ID NO: 56, and CDR3 of SEQ ID NO: 57, and a heavy chain variable region comprising CDR1 of SEQ ID NO: 58, CDR2 of SEQ ID NO: 59, and CDR3 of SEQ ID NO:

60.

12. The monoclonal antibody or antigen-binding fragment thereof of claim 9, wherein the monoclonal antibody or antigen-binding fragment thereof is adapted for administration or gene delivery using an RNA or DNA sequence or vector encoding the monoclonal antibody or antigen-binding fragment thereof, or further comprises a probe bound to the monoclonal antibody or antigen-binding fragment thereof.

13. 1. A method for detecting EGF, latrophilin, and seven transmembrane domain-containing protein 1 (ELTD1) in a subject, comprising: contacting a biological sample obtained from the subject with a monoclonal antibody or antigen-binding fragment thereof that specifically binds to ELTD1; The monoclonal antibodies and antigen-binding fragments thereof are said contacting comprising a light chain variable region comprising a complementarity determining region (CDR) 1 of SEQ ID NO: 55, a CDR2 of SEQ ID NO: 56, and a CDR3 of SEQ ID NO: 57, and a heavy chain variable region comprising a CDR1 of SEQ ID NO: 58, a CDR2 of SEQ ID NO: 59, and a CDR3 of SEQ ID NO: 60; detecting binding of said monoclonal antibody or said antigen-binding fragment thereof to said sample.

14. 1. A composition for treating multiple sclerosis, retinopathy, cancer, or tissue regeneration in a subject using an anti-EGF, Latrophilin, and seven transmembrane domain-containing protein 1 (ELTD1) antibody or antigen-binding fragment thereof, comprising: an effective amount of the antibody or antigen-binding fragment thereof that specifically binds to ELTD1 sufficient to alleviate symptoms of or treat the multiple sclerosis, retinopathy, cancer, or tissue regeneration, wherein the antibody or antigen-binding fragment thereof comprises: The composition has a light chain variable region comprising complementarity determining region (CDR) 1 of SEQ ID NO: 55, CDR2 of SEQ ID NO: 56, and CDR3 of SEQ ID NO: 57, and a heavy chain variable region comprising CDR1 of SEQ ID NO: 58, CDR2 of SEQ ID NO: 59, and CDR3 of SEQ ID NO: 60.

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