METHODS OF USING MONOCLONAL ANTIBODIES TARGETING EPITOPES OF ASPH

Abstract

Monoclonal antibodies (MAbs) targeting one or more specific epitopes of aspartyl (asparaginyl) -hydroxylase (ASPH), including humanized, bi-specific, and other chimeric MAb variants, and fragments thereof (collectively ASPH epitope-specific MAbs, or simply ASPH MAbs), are disclosed. Methods of production, purification, and use of the ASPH epitope-specific MAbs, and compositions comprising them, as agents in therapeutic and diagnostic applications to interact with target molecules in cell-free samples, cell- and tissue-based assays, animal models, and in a subject are also disclosed. Other aspects of the invention relate to use of the molecules disclosed herein to diagnose, ameliorate, or treat cell proliferation disorders and related diseases.

Claims

1. A method of treating cancer in a mammalian subject, comprising administering to a subject in need thereof a composition comprising an antibody or fragment or variant thereof, which binds to one or more peptide epitopes of human aspartyl (asparaginyl) -hydroxylase (ASPH) in an amount sufficient to treat cancer, wherein said antibody comprises: a recombinant heavy chain and a recombinant light chain, each heavy and each light chain comprising 3 complementarity-determining regions (CDRs), or fragment or variant thereof, which binds to one or more peptide epitopes of human aspartyl (asparaginyl) -hydroxylase (ASPH), wherein at least one of said peptide epitopes comprises at least 4 consecutive amino acid residues located within or adjacent to a position in the catalytic domain of ASPH that is within 30 amino acids of the C-terminus of human ASPH, corresponding to the sequence QDASSFRLIFIVDVWHPELTPQQRRSLPAI represented by positions 729-758 of SEQ ID NO: 1; wherein said antibody or fragment or variant thereof contains one or more conservative amino acid substitutions in which the functional activity relating to binding of the antibody or fragment thereof to an epitope of ASPH is retained; wherein said antibody comprises a recombinant heavy chain comprising a CDR1 comprising a sequence selected from the group consisting of NFMC represented by SEQ ID NO: 31, and NAMC represented by SEQ ID NO: 32; a CDR2 comprising a sequence selected from the group consisting of CIYF represented by SEQ ID NO: 33, and CIDN represented by SEQ ID NO: 34; and a CDR3 comprising a sequence selected from the group consisting of DGPGSISWKI represented by SEQ ID NO: 35, and NFNI represented by SEQ ID NO: 36; wherein said antibody comprises a recombinant light chain comprising a CDR1 comprising a sequence selected from the group consisting of SVYSKNR represented by SEQ ID NO: 37, and SVYDNNR represented by SEQ ID NO: 38; a CDR2 comprising the sequence LAS represented by SEQ ID NO: 39; and a CDR3 comprising a sequence selected from the group consisting of QGTYDSSGWYWA represented by SEQ ID NO: 40, and LGSYSGYIYI represented by SEQ ID NO: 41.

2. The method of claim 1, wherein said antibody or fragment or variant binds to one or more peptides selected from the group consisting of (a) a peptide comprising 29 amino acids with Cysteine at its amino terminus, plus 28 amino acids corresponding to positions 731-758 at the C-terminal end of human ASPH represented by SEQ ID NO: 1, with the Threonine at relative position 19, corresponding to position 748 of human ASPH, phosphorylated, as CASSFRLIFIVDVWHPEL-T(PO.sub.3H.sub.2)-PQQRRSLPAI represented by SEQ ID NO: 19; and (b) a peptide comprising 29 amino acids with Cysteine at its amino terminus, plus 28 amino acids corresponding to positions 731-758 at the C-terminal end of human ASPH represented by SEQ ID NO: 1, as CASSFRLIFIVDVWHPELTPQQRRSLPAI, represented by SEQ ID NO: 20.

3. The method of claim 1, wherein said antibody comprising a recombinant heavy chain and a recombinant light chain, or a fragment thereof, which binds to one or more peptide epitopes of human aspartyl (asparaginyl) -hydroxylase (ASPH), wherein at least one of said peptide epitopes is located within or adjacent to a position in the catalytic domain of ASPH that is within 30 amino acids of the C-terminus of human ASPH, corresponding to the sequence QDASSFRLIFIVDVWHPELTPQQRRSLPAI represented by positions 729-758 of SEQ ID NO: 1; wherein said antibody binds to an epitope comprising at least 4 consecutive amino acid residues located within 30 amino acids from the C-terminal end of human ASPH represented by SEQ ID NO: 1; wherein said epitope comprising at least 4 consecutive amino acid residues located within 30 amino acids from the C-terminal end of human ASPH comprises the consecutive amino acid residues selected from the group consisting of PELT represented by SEQ ID NO: 42, ELTP represented by SEQ ID NO: 43, LTPQ represented by SEQ ID NO: 44, TPQQ represented by SEQ ID NO: 45, PQQR represented by SEQ ID NO: 46, QQRR represented by SEQ ID NO: 47, QRRS represented by SEQ ID NO: 48, RRSL represented by SEQ ID NO: 49, RSLP represented by SEQ ID NO: 50, SLPA represented by SEQ ID NO: 51, and LPAI represented by SEQ ID NO: 52.

4. The method of claim 3, wherein said peptide epitope comprises a phosphorylated threonine, T(PO.sub.3H.sub.2) at relative amino acid position 4 of PELT represented by SEQ ID NO: 42; at relative amino acid position 3 of ELTP represented by SEQ ID NO: 43; at relative amino acid position 2 of LTPQ represented by SEQ ID NO: 44; and at relative amino acid position 1 of TPQQ represented by SEQ ID NO: 45.

5. A method of treating cancer in a mammalian subject comprising administering to a subject in need thereof a composition comprising a monoclonal antibody comprising a recombinant heavy chain and a recombinant light chain, or a fragment thereof, which binds to one or more peptide epitopes of human aspartyl (asparaginyl) -hydroxylase (ASPH), wherein at least one of said peptide epitopes is located within or adjacent to a position in the catalytic domain of ASPH that is within 30 amino acids of the C-terminus of human ASPH, corresponding to the sequence QDASSFRLIFIVDVWHPELTPQQRRSLPAI represented by positions 729-758 of SEQ ID NO: 1; wherein said antibody binds to an epitope comprising at least 4 consecutive amino acid residues located within 30 amino acids from the C-terminal end of human ASPH represented by SEQ ID NO: 1; wherein said antibody comprises a recombinant heavy chain and a recombinant light chain, wherein said recombinant heavy chain comprises a polypeptide sequence selected from the group consisting of SEQ ID NOS 21-25; and wherein said recombinant light chain comprises a polypeptide sequence selected from the group consisting of SEQ ID NOS 26-30.

6. The method of claim 5, wherein said antibody is selected from the group consisting of 5H4/5K3 and 9H2/9K1, wherein antibody 5H4/5K3 comprises a heavy chain designated 5H4, represented by the sequence SEQ ID NO: 25, and a light chain 5K3, represented by the sequence SEQ ID NO: 27; and wherein antibody 9H2/9K1 comprises a heavy chain designated 9H2, represented by the sequence SEQ ID NO: 29, and a light chain 9K1 represented by the sequence SEQ ID NO: 30.

7. The method of claim 1, wherein said mammalian subject is a selected from the group consisting of a human, non-human primate, canine, feline, bovine, equine, and a porcine subject.

8. The method of claim 2, wherein said mammalian subject is a human subject.

9. The method of claim 1, wherein said cancer is selected from the group consisting of cancers of the liver, hepatocellular carcinoma and cholangiocarcinoma, pancreatic cancer, gastric cancer, colon cancer, kidney cancer, non-small cell lung cancer, breast cancer, ovarian cancer, cervical cancer, head-and-neck cancers secondary to human papilloma virus infection, prostate cancer, brain cancer, glioblastoma multiform, neuroblastoma, retinoblastoma, and medulloblastoma, and osteosarcoma.

10. The method of claim 1, wherein said antibody or fragment or variant thereof is humanized and comprises one or more complementarity determining regions (CDRs) derived from a non-human source, and one or more portions of the constant regions of a human antibody.

11. The method of claim 1, wherein said antibody or fragment or variant thereof is bispecific and comprises one or more complementarity determining regions (CDRs) derived from a non-human source targeting one or more peptide epitopes located within or adjacent to the catalytic domain of ASPH, and an antibody targeting other epitopes selected from the group consisting of the T-cell redirector class, comprising an antibody targeting one or more ASPH CDRs and an antibody targeting CD3; the NK-cell redirector class, comprising an antibody targeting one or more ASPH CDRs and an antibody targeting CD16A; the tumor targeting immunomodular class, comprising an antibody targeting one or more ASPH CDRs and an antibody targeting CD40 or 4-1BB; and the dual immunomodular class, comprising an antibody targeting one or more ASPH CDRs and an antibody targeting PD-L1, PD-1, CTLA-4, TGF-, LAG-3, TIM-3, or OX40.

12. A method of inhibiting the proliferation of tumor cell or tissue samples grown in culture, comprising administering to the locus of the tumor cells a composition comprising an antibody or fragment or variant thereof which binds to one or more peptide epitopes of human aspartyl (asparaginyl) -hydroxylase (ASPH) in an amount sufficient to inhibit proliferation, wherein said antibody comprises a recombinant heavy chain and a recombinant light chain, each heavy and each light chain comprising 3 complementarity-determining regions (CDRs), or fragment or variant thereof, which binds to one or more peptide epitopes of human aspartyl (asparaginyl) -hydroxylase (ASPH), wherein at least one of said peptide epitopes comprises at least 4 consecutive amino acid residues located within or adjacent to a position in the catalytic domain of ASPH that is within 30 amino acids of the C-terminus of human ASPH, corresponding to the sequence QDASSFRLIFIVDVWHPELTPQQRRSLPAI represented by positions 729-758 of SEQ ID NO: 1; wherein said antibody or fragment or variant thereof contains one or more conservative amino acid substitutions in which the functional activity relating to binding of the antibody or fragment thereof to an epitope of ASPH is retained; wherein said antibody comprises a recombinant heavy chain comprising a CDR1 comprising a sequence selected from the group consisting of NFMC represented by SEQ ID NO: 31, and NAMC represented by SEQ ID NO: 32; a CDR2 comprising a sequence selected from the group consisting of CIYF represented by SEQ ID NO: 33, and CIDN represented by SEQ ID NO: 34; and a CDR3 comprising a sequence selected from the group consisting of DGPGSISWKI represented by SEQ ID NO: 35, and NFNI represented by SEQ ID NO: 36; wherein said antibody comprises a recombinant light chain comprising a CDR1 comprising a sequence selected from the group consisting of SVYSKNR represented by SEQ ID NO: 37, and SVYDNNR represented by SEQ ID NO: 38; a CDR2 comprising the sequence LAS represented by SEQ ID NO: 39; and a CDR3 comprising a sequence selected from the group consisting of QGTYDSSGWYWA represented by SEQ ID NO: 40, and LGSYSGYIYI represented by SEQ ID NO: 41.

13. The method of claim 12, comprising inhibiting the proliferation of isolated tumor cells in tissue samples grown in culture.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

Statement Concerning Drawings Executed in Color

[0022] The provisional and non-provisional US patent or application files contain at least one drawing executed in color. Copies of these color drawing(s) associated with patent application files, published applications, or issued patents will be provided by the United States Patent and Trademark Office upon request and payment of the necessary fee. Non-Provisional application Ser. No. 16/444,617, filed Jun. 18, 2019, and Provisional Application No. 62/686,107, filed Jun. 18, 2018, both contain 28 pages of color drawings, which are incorporated by reference, as noted above.

Statement Concerning Aspects of the Invention Understood by Reference to the Drawings

[0023] The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

[0024] FIG. 1 sets forth an illustration showing the Activation of Notch Signaling Pathway by ASPH. Aspartyl(asparaginyl)--hydroxylase (ASPH) is an iron-dependent dioxygenase that catalyzes the hydroxylation of 13 carbons of aspartic acid and asparagine residues in domains of a variety of proteins, including Notch and Notch ligand homologs. Intense activation of the Notch pathway by ASPH is observed in tumor tissues. Inhibition of ASPH allows normal activation of the Notch Pathway.

[0025] FIG. 2A sets forth an illustration showing the Locations of Epitopes of Interest on Human ASPH (3D Structure). FIG. 2B lists peptide domain sequences.

[0026] FIG. 3 sets forth an illustration showing the Locations of Epitopes of Interest on the Sequence of ASPH.

[0027] FIG. 4 (Panels A-H, plus a polypeptide domain key) sets forth an illustration showing experimentally confirmed and computationally predicted substrates of ASPH, including those found in the following types of proteins: A. Notch signaling pathway, B. Lipid receptors, C. Blood coagulation cascade proteins, D. Thrombospondins, E. Complement cascade proteins, F. FAT cadherin domain proteins, G. Bone associated proteins, and H. 7-transmembrane domain containing proteins.

[0028] FIG. 5 (Panels A-G, plus a polypeptide domain key) sets forth an illustration showing experimentally confirmed and computationally predicted substrates of ASPH (continued), including those found in the following types of proteins: A. TGF-b containing proteins, B. Platelet associated proteins, C. Eye/retina associated proteins, D. Mammary cancer metastasis proteins, E. Slit proteins, F. Miscellaneous proteins, and G. Drosophila homologues.

[0029] FIG. 6 sets forth an illustration demonstrating positive 5H4/5K3 staining visualized with DAB (brown) on Human Hepatocellular Carcinoma at 4 g/ml, 8 g/ml, and 10 g/ml (3 images). No-primary negative control was performed to identify nonspecific secondary binding (Neg, bottom right image). The scale bar represents 20 m.

[0030] FIG. 7 sets forth an illustration demonstrating positive 9H2/9K1 staining visualized with DAB (brown) on Human Hepatocellular Carcinoma at 4 g/ml, 8 g/ml, and 10 g/ml (3 images). No-primary negative control was performed to identify nonspecific secondary binding (Neg, bottom right image). The scale bar represents 20 m.

[0031] FIG. 8 sets forth an illustration demonstrating 5H4/5K3 Phase III on TMAs for samples labeled as LV12 Core F4 (top image), and LV12 Core F4-Isotype (bottom image). Positive 5H4/5K3 staining was visualized with DAB (brown) on TMAs (LV12 Core F4, top image). Isotype negative control was performed with Rabbit IgG (bottom image). The scale bar represents 20 m.

[0032] FIG. 9 sets forth an illustration demonstrating 5H4/5K3 Phase III on TMAs for samples labeled as PCO2 Core A6 (top image), and PCO2 Core A6-Isotype (bottom image). Positive 5H4/5K3 staining was visualized with DAB (brown, top image). Isotype negative control was performed with Rabbit IgG (bottom image). The scale bar represents 20 m.

[0033] FIG. 10 sets forth an illustration demonstrating 5H4/5K3 Phase III on TMAs for samples labeled as OV03 Core C5 (top image), and OV03 Core C5-Isotype (bottom image). Positive 5H4/5K3 staining was visualized with DAB (brown, top image). Isotype negative control was performed with Rabbit IgG (bottom image). The scale bar represents 20 m.

[0034] FIG. 11 sets forth an illustration demonstrating 5H4/5K3 Phase III on TMAs for samples labeled as OV01 Core D2 (top image), and OV01 Core D2-Isotype (bottom image). Positive 5H4/5K3 staining was visualized with DAB (brown, top image). Isotype negative control was performed with Rabbit IgG (bottom image). The scale bar represents 20 m.

[0035] FIG. 12 sets forth an illustration demonstrating activity of 5H4/5K3 Against Granulosa Cell Tumor Samples (A11 and B11). Positive 5H4/5K3 staining was visualized with DAB (brown) Against Granulosa Cell Tumor (top images, A11 and B11) Isotype negative control was performed with Rabbit IgG (bottom images, A11 and B11).

[0036] FIG. 13 sets forth an illustration demonstrating activity of 5H4/5K3 Against Serrous Cystadenocarcinoma Stage III Samples (C5 and D5). Positive 5H4/5K3 staining was visualized with DAB (brown) against Serrous Cystadenocarcinoma Stage III (top images, C5 and D5). Isotype negative control was performed with Rabbit IgG (bottom images, C5 and D5).

[0037] FIG. 14 sets forth an illustration demonstrating activity of 5H4/5K3 Against Serrous Cystadenocarcinoma Stage III Samples (C8 and D8). Positive 5H4/5K3 staining was visualized with DAB (brown) against Serrous Cystadenocarcinoma Stage III (top images, C8 and D8). Isotype negative control was performed with Rabbit IgG (bottom images, C8 and D8).

[0038] FIG. 15 sets forth an illustration demonstrating activity of 5H4/5K3 Against Endometrioid Adenocarcinoma Stage III Samples (E8 and F8). Positive 5H4/5K3 staining was visualized with DAB (brown) against Endometrioid Adenocarcinoma Stage III (top images, E8 and F8). Isotype negative control was performed with Rabbit IgG (bottom images, E8 and F8).

[0039] FIG. 16 sets forth an illustration demonstrating reaction of 5H4/5K3 Against Normal Ovarian Tissue Samples (A1 and B1). Lack of 5H4/5K3 staining by DAB against Normal Ovarian Tissue is shown in top images, A1 and B1. Isotype negative control was performed with Rabbit IgG (bottom images, A1 and B1).

[0040] FIG. 17 sets forth an illustration demonstrating reaction of 5H4/5K3 against Thecoma (Theca Cell) Tumor Tissue (A5 and B5). 5H4/5K3 staining was visualized with DAB (brown, top images) against Thecoma (Theca Cell) Tumor Tissue (A5 and B5). Isotype negative control was performed with Rabbit IgG (bottom images, A5 and B5).

[0041] FIG. 18 sets forth an illustration demonstrating Immobilization of Protein G on Channels 1 (Red, top line) and 2 (Blue, bottom line) followed by Capture of Antibody on Channel 1.

[0042] FIG. 19 sets forth an illustration demonstrating Interaction of ASPH with Mock sample. Concentrations are 500 nM (dark red, top line on right portion of the graph), 250 nM (light green), 125 nM (blue), 62.5 nM (dark green) 31.2 nM (orange), 15.6 nM (red).

[0043] FIG. 20 sets forth an illustration demonstrating Interaction of ASPH with 2H4/2K5. Concentrations are 500 nM (dark red, 1.sup.st line from top), 250 nM (light green, 2.sup.nd and 3.sup.rd lines from top), 125 nM (blue, 4.sup.th and 5.sup.th lines from top), and 62.5 nM (dark green, 6.sup.th and 7.sup.th lines from top).

[0044] FIG. 21 Interaction of ASPH with 5H1/5K1. Concentrations are 500 nM (dark red, 1.sup.st line from top), 250 nM (light green, 2.sup.nd and 3.sup.rd lines from top), 125 nM (blue, 4.sup.th and 5.sup.th lines from top), 62.5 nM (dark green, 6.sup.th and 7.sup.th lines from top), 31.2 nM (orange, 8.sup.th and 9.sup.th lines from top), and 15.6 nM (red, 10.sup.th and 11.sup.th lines from top).

[0045] FIG. 22 sets forth an illustration demonstrating Interaction of ASPH with 5H4/5K3. Concentrations are 500 nM (dark red, 1.sup.st and 2.sup.nd lines from top), 250 nM (light green, 3.sup.rd and 4.sup.th lines from top), 125 nM (blue, 5.sup.th and 6.sup.th lines from top), 62.5 nM (dark green, 7.sup.th and 8.sup.th lines from top), 31.2 nM (orange, 9.sup.th and 10.sup.th lines from top), and 15.6 nM (red, 11.sup.th and 12.sup.th lines from top).

[0046] FIG. 23 sets forth an illustration demonstrating Interaction of ASPH with 9H2/9K1. Concentrations are 500 nM (dark red, 1.sup.st and 2.sup.nd lines from top), 250 nM (light green, 3.sup.rd and 4.sup.th lines from top), 125 nM (blue, 5.sup.th and 6.sup.th lines from top), 62.5 nM (dark green, 7.sup.th and 8.sup.th lines from top), 31.2 nM (orange, 9.sup.th and 10.sup.th lines from top), and 15.6 nM (red, 11.sup.th and 12.sup.th lines from top).

[0047] FIG. 24 sets forth an illustration demonstrating Interaction of ASPH with 9H2/9K3. Concentrations are 500 nM (dark red, 1.sup.st line from top), 250 nM (light green, 2.sup.nd and 3.sup.rd lines from top), 125 nM (blue, 4.sup.th and 5.sup.th lines from top), 62.5 nM (dark green, 6.sup.th line from top), and 31.2 nM (orange, 7.sup.th line from top).

[0048] FIG. 25 sets forth an illustration demonstrating Interaction of ASPH with 8H1/8K1. Concentrations are 500 nM (dark red, first and second lines from the top), 250 nM (light green), 125 nM (blue), 62.5 nM (dark green) 31.2 nM (orange), 15.6 nM (red).

[0049] FIG. 26 sets forth an illustration demonstrating IC50 Curves for 5H4/5K3, 9H2/9K1 and Mock Antibody Samples Carried Out in 4T1 Cells.

[0050] FIG. 27 sets forth an illustration demonstrating IC50 Curves for 5H4/5K3, 9H2/9K1 and Mock Antibody Samples Carried Out in MCF7 Cells.

[0051] FIG. 28 sets forth an illustration demonstrating IC50 Curves for 5H4/5K3, 9H2/9K1 and Mock Antibody Samples Carried Out in MV411 Cells.

[0052]

TABLE-US-00001 TABLE #T0 Summary of Staining Patterns in Panels of Photographic Images of Cell Samples (from +++ to )* Top/Top Top Bottom/ Bottom Figure Description Left Right Bottom Left Right 6 Positive 5H4/5K3 staining visualized + ++ +++ with DAB (brown) on Human Hepatocellular Carcinoma at 4 g/ml, 8 g/ml, and 10 g/ml (3 images). No-primary negative control was performed to identify nonspecific secondary binding (Neg, bottom right image). 7 Positive 9H2/9K1 staining visualized (+) + ++ with DAB (brown) on Human Hepatocellular Carcinoma at 4 g/ml, 8 g/ml, and 10 g/ml (3 images). No-primary negative control was performed to identify nonspecific secondary binding (Neg, bottom right image). 8 5H4/5K3 Phase III on TMAs for samples +++ labeled as LV12 Core F4 (top image), and LV12 Core F4 - Isotype (bottom image). 9 5H4/5K3 Phase III on TMAs for samples +++ labeled as PC02 Core A6 (top image), and PC02 Core A6 - Isotype (bottom image). Positive 5H4/5K3 staining was visualized with DAB (brown,, top image). Isotype negative control was performed with Rabbit IgG (bottom image). 10 5H4/5K3 Phase III on TMAs for samples +++ labeled as OV03 Core C5 (top image), and OV03 Core C5 - Isotype (bottom image). Positive 5H4/5K3 staining was visualized with DAB (brown, top image). Isotype negative control was performed with Rabbit IgG (bottom image). 11 5H4/5K3 Phase III on TMAs for samples +++ labeled as OV01 Core D2 (top image), and OV01 Core D2 - Isotype (bottom image). Positive 5H4/5K3 staining was visualized with DAB (brown, top image). Isotype negative control was performed with Rabbit IgG (bottom image). 12 Activity of 5H4/5K3 Against Granulosa ++ ++ Cell Tumor Samples (A11 and B11). Positive 5H4/5K3 staining was visualized with DAB (brown) Against Granulosa Cell Tumor (top images, A11 and B11) Isotype negative control was performed with Rabbit IgG (bottom images, A11 and B11). 13 Activity of 5H4/5K3 Against Serrous +++ +++ Cystadenocarcinoma Stage III Samples (C5 and D5). Positive 5H4/5K3 staining was visualized with DAB (brown) Against Serrous Cystadenocarcinoma Stage III (top images, C5 and D5) Isotype negative control was performed with Rabbit IgG(bottom images, C5 and D5). 14 Activity of 5H4/5K3 Against +++ +++ Serrous Cystadenocarcinoma Stage III Samples (C8 and D8). Positive 5H4/5K3 staining was visualized with DAB (brown) Against Serrous Cystadenocarcinoma Stage III (top images, C8 and D8) Isotype negative control was performed with Rabbit IgG (bottom images, C8 and D8). 15 Activity of 5H4/5K3 Against +++ +++ () () Endometrioid Adenocarcinoma Stage III Samples (E8 and F8). Positive 5H4/5K3 staining was visualized with DAB (brown) Against Endometrioid Adenocarcinoma Stage III (top images, E8 and F8). Isotype negative control was performed with Rabbit IgG (bottom images, E8 and F8). 16 Reaction of 5H4/5K3 Against Normal Ovarian Tissue Samples (A1 and B1). Lack of 5H4/5K3 staining by DAB Against Normal Ovarian Tissue (top images, A1 and B1). Isotype negative control was performed with Rabbit IgG (bottom images, A1 and B1). 17 Reaction of 5H4/5K3 Against Thecoma (+) (+) (Theca Cell) Tumor Tissue (A5 and B5). 5H4/5K3 staining was visualized with DAB (brown, top images) Against Thecoma (Theca Cell) Tumor Tissue (A5 and B5). Isotype negative control was performed with Rabbit IgG (bottom images, A5 and B5). *Staining intensities of different panels for each sample were evaluated on a scale from +++, ++, +, (+), (), and where reaction with DAB to produce an intense brown color after reaction with cells was designated as +++, to , where all cells were mostly blue or white.

Terms and Definitions

[0053] The following is a list of abbreviations, plus terms and their definitions, used throughout the specification and the claims:

[0054] General abbreviations and their corresponding meanings include: aa or AA=amino acid; mg=milligram(s); ml or mL=milliliter(s); mm=millimeter(s); mM=millimolar; nmol=nanomole(s); pmol=picomole(s); ppm=parts per million; RT=room temperature; U=units; ug, g=micro gram(s); ul, l=micro liter(s); uM, M=micromolar.

Specific Abbreviations and their Corresponding Meanings Include

[0055] The terms cell and cells, which are meant to be inclusive, refer to one or more cells which can be in an isolated or cultured state, as in a cell line comprising a homogeneous or heterogeneous population of cells, or in a tissue sample, or as part of an organism, such as a transgenic animal.

[0056] The term amino acid encompasses both naturally occurring and non-naturally occurring amino acids unless otherwise designated.

[0057] The term complementarity-determining regions or CDRs are defined by Wikipedia, as part of the variable chains in immunoglobulins (antibodies) and T cell receptors, generated by B-cells and T-cells respectively, where these molecules bind to their specific antigen. CDRs, which comprise the most variable parts of antibodies, are crucial to the diversity of antigen specificities generated by lymphocytes.

[0058] The term paratope refers to a set of CDRs.

Detailed Description of the Invention

[0059] The present invention relates to monoclonal antibodies (MAbs) targeting one or more specific epitopes of aspartyl (asparaginyl) -hydroxylase (ASPH), including chimeric and humanized MAb variants, and fragments thereof (collectively ASPH epitope-specific MAbs, or simply ASPH MAbs), are disclosed. Methods of production, purification, and use of the ASPH epitope-specific MAbs, and compositions comprising them, as agents in therapeutic and diagnostic applications to interact with target molecules in cell-free samples, cell- and tissue-based assays, animal models, and in a subject are also disclosed. Other aspects of the invention relate to use of the molecules disclosed herein to diagnose, ameliorate, or treat cell proliferation disorders and related diseases.

[0060] One aspect relates to an isolated monoclonal antibody, or a fragment thereof, which binds to a one or more peptide epitopes of human aspartyl (asparaginyl) -hydroxylase (ASPH), wherein at least one of said peptide epitopes is located within or adjacent to the catalytic domain of ASPH.

[0061] Another aspect relates to an antibody, or a fragment thereof, as noted above, wherein at least one of said peptide epitopes located within or adjacent to the catalytic domain of ASPH is located within 30 amino acids of the C-terminus of ASPH.

[0062] Another aspect relates to an antibody, which binds to one or more synthetic peptides selected from the group consisting of (a) a synthetic peptide comprising 29 amino acids with Cysteine at its amino terminus, plus 28 amino acids corresponding to positions 731-758 at the C-terminal end of human ASPH, with the Threonine at 19 (corresponding to 748 of ASPH) phosphorylated, as CASSFRLIFIVDVWHPEL-T(PO3H2)-PQQRRSLPAI represented by SEQ ID NO: 19; and (b) a synthetic peptide comprising 29 amino acids with Cysteine at its amino terminus, plus 28 amino acids corresponding to positions 731-758 at the C-terminal end of human ASPH, as CASSFRLIFIVDVWHPELTPQQRRSLPAI represented by SEQ ID NO: 20.

[0063] Related aspects include an antibody, which binds to an epitope comprising at least 4 consecutive amino acid residues located within 30 amino acids from the C-terminal end of human ASPH, including an antibody wherein said epitope comprising at least 4 consecutive amino acid residues located within 30 amino acids from the C-terminal end of human ASPH comprises the consecutive amino acid selected from the group consisting of

[0064] PELT represented by SEQ ID NO: 42,

[0065] ELTP represented by SEQ ID NO: 43,

[0066] LTPQ represented by SEQ ID NO: 44,

[0067] TPQQ represented by SEQ ID NO: 45,

[0068] PQQR represented by SEQ ID NO: 46,

[0069] QQRR represented by SEQ ID NO: 47,

[0070] QRRS represented by SEQ ID NO: 48,

[0071] RRSL represented by SEQ ID NO: 49,

[0072] RSLP represented by SEQ ID NO: 50,

[0073] SLPA represented by SEQ ID NO: 51, and

[0074] LPAI represented by SEQ ID NO: 52.

[0075] Related aspects also include an antibody, wherein said peptide epitope comprises a phosphorylated threonine, T(PO3H2).

[0076] Another aspect relates to an isolated monoclonal antibody, or a fragment thereof, which binds to a one or more peptide epitopes of human aspartyl (asparaginyl) -hydroxylase (ASPH), wherein said antibody comprises a recombinant heavy chain and a recombinant light chain, wherein said recombinant heavy chain comprises a polypeptide sequence selected from the group consisting of SEQ ID NOS 21-25; and wherein said recombinant light chain comprises a polypeptide sequence selected from the group consisting of SEQ ID NOS 26-30.

[0077] Another aspect relates to an antibody selected from the group consisting of 5H4/5K3 and 9H2/9K1, wherein antibody 5H4/5K3 comprises a heavy chain designated 5H4, represented by the sequence SEQ ID NO: 25, and a light chain 5K3 represented by the sequence SEQ ID NO: 27; and wherein antibody 9H2/9K1 comprises a heavy chain designated 9H2, represented by the sequence SEQ ID NO: 29, and a light chain 9K1 represented by the sequence SEQ ID NO: 30.

[0078] Another aspect relates to an isolated monoclonal antibody, or a fragment thereof, which binds to a one or more peptide epitopes of human aspartyl (asparaginyl) -hydroxylase (ASPH), wherein said antibody comprises a recombinant heavy chain comprising: [0079] a CDR1 comprising a sequence selected from the group consisting of NFMC (SEQ ID NO: 31), corresponding to residues 50-53 of SEQ ID NO: 21, and NAMC (SEQ ID NO: 32), corresponding to residues 50-53 of SEQ ID NOS: 23, 29, 24, and 25; [0080] a CDR2 comprising a sequence selected from the group consisting of CIYF (SEQ ID NO: 33) corresponding to residues 68-71 of SEQ ID NO: 21 and CIDN (SEQ ID NO: 34) corresponding to residues 68-71 of SEQ ID NO: 23, 29, 24, and 25; [0081] a CDR3 comprising a sequence selected from the group consisting of DGPGSISWKI (SEQ ID NO: 35) corresponding to residues 117-126 of SEQ ID NO: 21, and NFNI (SEQ ID NO: 36) corresponding to residues 116-119 of SEQ ID NOS: 23, 29, 24, and 25.

[0082] Another aspect relates to an isolated monoclonal antibody, or a fragment thereof, which binds to a one or more peptide epitopes of human aspartyl (asparaginyl) -hydroxylase (ASPH), wherein said antibody comprises a recombinant light chain comprising [0083] a CDR1 comprising a sequence selected from the group consisting of SVYSKNR (SEQ ID NO: 37) corresponding to residues 50-56 of SEQ ID NO: 22, and SVYDNNR SEQ ID NO: 38) corresponding to residues 50-56 of SEQ ID NOS: 26, 27, 28, and 30, [0084] a CDR2 comprising the sequence LAS (SEQ ID NO: 39) corresponding to residues 78-80 of SEQ ID NOS: 22, 26, 27, 28, and 30; [0085] a CDR3 comprising a sequence selected from the group consisting of QGTYDSSGWYWA (SEQ ID NO: 40) corresponding to residues 113-124 of SEQ ID NO: 22, and LGSYSGYIYI (SEQ ID NO: 41) corresponding to residues 113-122 of SEQ ID NOS: 26, 27, 28, and 30.

[0086] Related aspects include variants of the monoclonal antibodies or fragments thereof, that contain one or more conservative amino acid substitutions in which the functional activity relating to binding of the antibody or fragment thereof to an epitope of ASPH is retained. Related aspects also include truncated or fusion variants of the monoclonal antibodies comprising one or more insertions or deletions of amino acids in which the in which the functional activity relating to binding of the antibody or fragment thereof to an epitope of ASPH is retained. Related aspects also include variants comprising one or more combinations of conservative amino acid substitutions, insertions, and deletions, particularly where the number of residues that are altered by substitution, insertion, or deletion is small, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 11-15, 16-20, and 21-25 residues compared to the parent antibody molecule. Related aspects also include molecules having one or more larger insertions or deletions of amino acid residues or polypeptide domains that do not alter the functional binding activity of the antibody to a desired epitope in a target molecule.

[0087] Another aspect relates to a composition comprising any of the antibodies noted above, including compositions comprising at least one antibody that targets ASPH and one or more pharmaceutical excipients.

[0088] Another aspect relates to a method of using any of the antibodies noted above, to inhibit the proliferation of isolated tumor cell samples grown in culture.

[0089] Another aspect relates to a method of using any of the antibodies noted above, to inhibit the proliferation of tumor cells in tissue samples grown in culture.

[0090] Another aspect relates to a method of treating cancer in a mammalian subject, comprising administering to a subject in need thereof an antibody as noted above in an amount sufficient to treat cancer. Related aspects include methods wherein said mammalian subject is a selected from the group consisting of a human, non-human primate, canine, feline, bovine, equine, and a porcine subject. A preferred aspect relates to a method, wherein said mammalian subject is a human subject.

[0091] Related aspects also include methods noted above wherein said cancer is selected from the group consisting of cancers of the liver, hepatocellular carcinoma and cholangiocarcinoma, pancreatic cancer, gastric cancer, colon cancer, kidney cancer, non-small cell lung cancer, breast cancer, ovarian cancer, cervical cancer, head-and-neck cancers secondary to human papilloma virus infection, prostate cancer, brain cancer, glioblastoma multiform, neuroblastoma, retinoblastoma, and medulloblastoma, and osteosarcoma.

[0092] Another aspect relates to a kit for diagnosis of cancer in a mammalian subject, wherein said kit comprises an antibody, or a fragment thereof, of any of any of the antibodies noted above.

[0093] Another aspect relates to a humanized antibody comprising one or more complementarity determining regions (CDRs) derived from a non-human source targeting one or more peptide epitopes located within or adjacent to the catalytic domain of ASPH of any of claims 1-10, and one or more portions of the constant regions of a human antibody, and fragments thereof.

[0094] Another aspect relates to a bispecific antibody comprising one or more complementarity determining regions (CDRs) derived from a non-human source targeting one or more peptide epitopes located within or adjacent to the catalytic domain of ASPH of any of claims 1-10, and an antibody targeting other epitopes selected from the group consisting of the T-cell redirector class, comprising an antibody targeting one or more ASPH CDRs and an antibody targeting CD3; the NK-cell redirector class, comprising an antibody targeting one or more ASPH CDRs and an antibody targeting CD16A; the tumor targeting immunomodular class, comprising an antibody targeting one or more ASPH CDRs and an antibody targeting CD40 or 4-1BB; and the dual immunomodular class, comprising an antibody targeting one or more ASPH CDRs and an antibody targeting PD-L1, PD-1, CTLA-4, TGF-, LAG-3, TIM-3, or OX40.

Therapeutic Uses of Compositions Comprising Compounds of the Invention

[0095] Antibodies with direct activity against ASPH antibodies should be useful in the discovery and development of therapeutic drug products intended for use in the treatment of a variety of cancers. These include cancers of the liver, such as hepatocellular carcinoma and cholangiocarcinoma, pancreatic cancer, gastric cancer, colon cancer, kidney cancer, non-small cell lung cancer, breast cancer, ovarian cancer, cervical cancer, head-and-neck cancers secondary to human papilloma virus infection, prostate cancer, brain cancers of various types, including glioblastoma multiform, neuroblastoma, retinoblastoma, and medulloblastoma, and osteosarcoma.

Pharmaceutical Compositions

[0096] Related aspects of the invention are directed to compositions, including pharmaceutical compositions, comprising the compounds of the invention, noted above. One aspect of the invention is directed to a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient and a therapeutically effective amount of the compound or salt disclosed above. Still another aspect of the invention relates to a method for pharmaceutical formulation of previously described compounds for use in oral and intravenous applications, and in implantable materials.

[0097] Another aspect of the present invention relates to a pharmaceutical composition including a pharmaceutically acceptable carrier and a compound according to the aspects of the present invention. The pharmaceutical composition can contain one or more of the above-identified compounds of the present invention.

Various Modifications and Alternatives, Generally

[0098] While specific aspects of the invention have been described in detail, it will be appreciated by those skilled in the art that various modifications and alternatives to those details could be developed in light of the overall teachings of the disclosure. Accordingly, the particular arrangements disclosed are meant to be illustrative only, and not limiting as to the scope of the invention, which is to be given the full breadth of the appended claims, and any equivalent, thereof.

EXAMPLES

[0099] The foregoing discussion may be better understood in connection with the following representative examples which are presented for purposes of illustrating the principle methods and compositions of the invention, and not by way of limitation. Various other examples will be apparent to the person skilled in the art after reading the present disclosure without departing from the spirit and scope of the invention. It is intended that all such other examples be included within the scope of the appended claims.

General Materials and Methods

[0100] All parts are by weight (e.g., % w/w), and temperatures are in degrees centigrade ( C.), unless otherwise indicated. Table #T1 presents a summary of the nucleotide and amino acid sequences described in this application.

TABLE-US-00002 TABLE#T1 SummaryofSequenceIDNumbers Name Description Length Type SEQIDNO: HumanASPH PolypeptidecorrespondingtoHumanASPH 758 AA 01 depositedasGenBankAccessionNo Q12797,startingattheN-terminuswith MAQRKNAKSSandendingattheC-terminus withPQQRRSLPAI CanineASPH PolypeptidecorrespondingtoCanine 798 AA 02 ASPHdepositedasGenBankAccessionNo XP_022267901,startingattheN- terminuswithMAEETKHGGHandendingat theC-terminuswithPQQRHSLPAI Peptide#H1 KRRSNEVLRcorrespondingtoresidues 9 AA 03 391-399ofhumanASPH Peptide#H2 DRQQFLGHMcorrespondingtoresidues 9 AA 04 428-436ofhumanASPH Peptide#H3 GYLLIGDNDNcorrespondingtoresidues 10 AA 05 463-470ofhumanASPH Peptide#H4 RSLYNVNGcorrespondingtoresidues 8 AA 06 562-569ofhumanASPH Peptide#H5 PQQRRSLPAIcorrespondingtoresidues 10 AA 07 749-758ofhumanASPH Peptide#H6 FLPEDENLREcorrespondingtoresidues 10 AA 08 612-621ofhumanASPH Peptide#H7 VWPHTGPTNCcorrespondingtoresidues 10 AA 09 676-685ofhumanASPH Peptide#H8 LWQQGRRNEcorrespondingtoresidues 9 AA 10 630-638ofhumanASPH Peptide#C1 KRRSNEVLRcorrespondingtoresidues 9 AA 11 427-435ofcanineASPH Peptide#C2 DRQQFLGHMcorrespondingtoresidues 9 AA 12 464-472ofcanineASPH Peptide#C3 GYLLIGDNNNcorrespondingtoresidues 10 AA 13 499-508ofcanineASPH Peptide#C4 RSLYNVHGcorrespondingtoresidues 8 AA 14 598-605ofcanineASPH Peptide#C5 PQQRHSLPAIcorrespondingtoresidues 10 AA 15 785-794ofcanineASPH Peptide#C6 FLPEDENLREcorrespondingtoresidues 10 AA 16 648-657ofcanineASPH Peptide#C7 VWPHTGPTNCcorrespondingtoresidues 10 AA 17 712-721ofcanineASPH Peptide#C8 LWQQGRKINEcorrespondingtoresidues 9 AA 18 666-674ofcanineASPH Peptide#1 Syntheticpeptidecomprising29amino 29 AA 19 (CASSF- acidswithCysteineatitsamino PO3H2) terminus,plus28aminoacids correspondingtopositions731-758at theC-terminalendofhumanASPH,with theThreonineat19(correspondingto 748ofASPH)phosphorylated. CASSFRLIFIVDVWHPEL-T(PO3H2)-PQQRRSLPAI Peptide#2 Syntheticpeptidecomprising29amino 29 AA 20 acidswithCysteineatitsamino terminus,plus28aminoacids correspondingtopositions731-758at theC-terminalendofhumanASPH. CASSFRLIFIVDVWHPELTPQQRRSLPAI Clone1H2 TranslatedvariableregionofCloneID 150 AA 21 #1H2comprisingaGQPKsequenceatthe startoftheconstantregionfora heavychainsequence. METGLRWLLLVAVLKGVQCQSLEESGGDLVKPGASLTLT CTASGLSFSDNFMCWVRQAPGKGLEWIACIYFDSSGITY YASWAKGRFTISKTSSPTVTLQMTSLTAADTATYFCARD GPGSISWDLWGQGTLVTVSSGQPKAPSVFPLAP Clone1K6 TranslatedvariableregionofCloneID 148 AA 22 #1K6comprisingaGDPVsequenceatthe startoftheconstantregionfora kappasequence. MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSPVSAAVGG TVTISCQSSKSVYSKNRLAWYQQKPGQPPKLLIYEASKL ASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCQGTYD SSGWYWAFGGGTEVVVK APTVLIFPPA Clone5H1 TranslatedvariableregionofCloneID 142 AA 23 #5H1. METGLRWLLLVAVLKGVQCQSLEESGGDLVKPGASLTLT CKASGFDFSSNAMCWVRQAPGKGPEWIACIDNGDGSTDY ATWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCTRNF NLWGPGHPGHRLERTAESPVGVSTG Clone5H3 TranslatedvariableregionofCloneID 143 AA 24 #5H3comprisingaGQPKsequenceatthe startoftheconstantregionfora heavychainsequence. METGLRWLLLVAVLKGVQCQSLEESGGDLVKPGASLTLT CKASGFDFSSNAMCWVRQAPGKGPEWIACIDNGDGSTDY ATWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCTRNF NLWGQGTLVTVSSGQPKAPSVFPLAP Clone5H4 TranslatedvariableregionofCloneID 143 AA 25 #5H4comprisingaGQPKsequenceatthe startoftheconstantregionfora heavychainsequence. METGLRWLLLVAVLKGVQCQSLEESGGDLVKPGASLTLT CKASGFDFSSNAMCWVRQAPGKGPEWIACIDNGDGSTDY ATWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCTRNF NLWGQGTLVTVSSGQPKAPSVFPLAP Clone5K1 TranslatedvariableregionofCloneID 146 AA 26 #5K1comprisingaGDPVsequenceatthe startoftheconstantregionfora kappasequence. MDTRAPTQLLGLLLLWLPGATFAQVLTQTASSVSAAVGG TVTISCQSSQSVYDNNRLAWFQQKPGQPPKLLIYETSKL ASGVPLRFKGSGSGTQFTLTISDLECDDAATYYCLGSYS GYIYTFGGGTEVVVKGDPVAPTVLIFPPA Clone5K3 TranslatedvariableregionofCloneID 146 AA 27 #5K3comprisingaGDPVsequenceatthe startoftheconstantregionfora kappasequence. MDTRAPTQLLGLLLLWLPGATFAQVLTQTASSVSAAVGG TVTISCQSSQSVYDNNRLAWFQQKPGQPPKLLIYETSKL ASGVPLRFKGSGSGTQFTLTISDLECDDAATYYCLGSYS GYIYTFGGGTEVVVK APTVLIFPPA Clone5K6 TranslatedvariableregionofCloneID 146 AA 28 #5K6comprisingaGDPVsequenceatthe startoftheconstantregionfora kappasequence. MDTRAPTQLLGLLLLWLPGATFAQVLTQTASSVSAAVGG TVTISCQSSQSVYDNNRLAWFQQKPGQPPKLLIYETSKL ASGVPLRFKGSGSGTQFTLTISDLECDDAATYYCLGSYS GYIYTFGGGTEVVVK APTVLIFPPA Clone9H2 TranslatedvariableregionofCloneID 142 AA 29 #9H2comprisingaGQPKsequenceatthe startoftheconstantregionfora heavychainsequence. METGLRWLLLVAVMKGVQCQSLEESGGDLVKPGASLTLT CKASGFDFISNAMCWVRQAPGKGPEWIACIDNGDGSTDY ATWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCTRNF NLWGQGTL?TVSSGQPKAPSVFPLAP Clone9K1 TranslatedvariableregionofCloneID 146 AA 30 #9K1comprisingaGDPVsequenceatthe startoftheconstantregionfora kappasequence. MDTRAPTQLLGLLLLWLPGATFAQVLTQTASSVSAAVGG TVTISCQSSQSVYDNNRLAWFQQKSGQPPKLLIYETSKL ASGVPLRFKGSGSGTQFTLTISDLECDDAATYYCLGSYS GYIYTFGGGTEVVVK APTVLIFPPA Clone1H2 CDR1regionofclone1H2 4 AA 31 CDR1 correspondingtocorrespondingto residues50-53ofSEQIDNO:21. NFMC Clones5H1, CDR1regionofClones5H1,9H2,5H3, 4 AA 32 9H2,5H3, 5H4,correspondingtoresidues50-53 5H4CDR1 ofSEQIDNOS:23,29,24,and25. NAMC Clone1H2 TheCDR2regionsfromtheheavychain 4 AA 33 CDR2 clone1H2correspondingtoresidues 68-71ofSEQIDNO:21. CIYF Clones5H1, TheCDR2regionsfromtheheavychain 4 AA 34 9H2,5H3, clones5H1,9H2,5H3,5H4 5H4CDR2 correspondingtoresidues68-71of SEQIDNO:23,29,24,and25. CIDN Clone1H2 CDR3regionsfromtheheavychainclone 10 AA 35 CDR3 1H2correspondingtoresidues117-126 ofSEQIDNO:21. DGPGSISWDI Clones5H1, CDR3regionsfromtheheavychain 4 AA 36 9H2,5H3, clones5H1,9H2,5H3,5H4corresponding 5H4CDR3 toresidues116-119ofSEQIDNOS:23, 29,24,and25. NFNI Clone1K6 TheCDR1regionsfromthekappachain 7 AA 37 CDR1 clone1K6,correspondingtoresidues 50-56ofSEQIDNO:22. SVYSKNR Clones5K1, TheCDR1regionsfromthekappachain 7 AA 38 5K3,5K6, clones5K1,5K3,5K6,and9K1 and9K1 correspondingtoresidues50-56ofSEQ CDR1 IDNOS:26,27,28,and30. SVYDNNR Clones1K6, TheCDR2regionsfromthekappachain 3 AA 39 5K1,5K3, clones1K6,5K1,5K3,5K6,and9K1 5K6,and correspondingtoresidues78-80ofSEQ 9K1CDR2 IDNOS:22,26,27,28,and30. LAS Clone1K6 TheCDR3regionsfromthekappachain 12 AA 40 CDR3 clone1K6correspondingtoresidues 113-124ofSEQIDNO:22. QGTYDSSGWYWA Clones5K1, TheCDR3regionsfromthekappachain 10 AA 41 5K3,5K6, clones5K1,5K3,5K6,and9K1 and9K1 correspondingtoresidues113-122of CDR3 SEQIDNOS:26,27,28,and30. LGSYSGYIYI Peptide Fouraapeptidecorrespondingtoaa 4 AA 42 PELT 745-748nearthecarboxyterminusof humanASPH. PELT Peptide Fouraapeptidecorrespondingtoaa 4 AA 43 ELTP 746-749nearthecarboxyterminusof humanASPH. ELTP Peptide Fouraapeptidecorrespondingtoaa 4 AA 44 LTPQ 747-750nearthecarboxyterminusof humanASPH. LTPQ Peptide Fouraapeptidecorrespondingtoaa 4 AA 45 TPQQ 748-751nearthecarboxyterminusof humanASPH. TPQQ Peptide Fouraapeptidecorrespondingtoaa 4 AA 46 PQRR 749-752nearthecarboxyterminusof humanASPH. PQQR Peptide Fouraapeptidecorrespondingtoaa 4 AA 47 QQRR 750-753nearthecarboxyterminusof humanASPH. QQRR Peptide Fouraapeptidecorrespondingtoaa 4 AA 48 QRSS 751-754nearthecarboxyterminusof humanASPH. QRRS Peptide Fouraapeptidecorrespondingtoaa 4 AA 49 RSSL 752-755nearthecarboxyterminusof humanASPH. RRSL Peptide Fouraapeptidecorrespondingtoaa 4 AA 50 RSLP 753-756nearthecarboxyterminusof humanASPH. RSLP Peptide Fouraapeptidecorrespondingtoaa 4 AA 51 SLPA 754-757nearthecarboxyterminusof humanASPH. SLPA Peptide Fouraapeptidecorrespondingtoaa 4 AA 52 LPAI 746-758nearthecarboxyterminusof humanASPH. LPAI

TABLE-US-00003 Sequence#SQ1:LocationsofPeptides#H1-#H8AlongHumanASPH(758aa) ID ASPH_HUMANReviewed;758AA. AC Q12797;A0A0A0MSK8;A6NDF4;A6NHI2;B4DIC9;B4E2K4;B7ZM95;E5RGP5; AC F5H667;Q6NXR7;Q8TB28;Q9H291;Q9H2C4;Q9NRI0;Q9NRI1;Q9Y4J0; DT 01-NOV-1997,integratedintoUniProtKB/Swiss-Prot. DT 17-APR-2007,sequenceversion3. DT 25-APR-2018,entryversion181. [...Textomitted...] SQ SEQUENCE758AA;85863MW;4AE56D1D8DF0AF0CCRC64; MAQRKNAKSSGNSSSSGSGSGSTSAGSSSPGARRETKHGGHKNGRKGGLSGTSFFTWFMV60 IALLGVWTSVAVVWFDLVDYEEVLGKLGIYDADGDGDFDVDDAKVLLGLKERSTSEPAVP120 PEEAEPHTEPEEQVPVEAEPQNIEDEAKEQIQSLLHEMVHAEHVEGEDLQQEDGPTGEPQ180 QEDDEFLMATDVDDRFETLEPEVSHEETEHSYHVEETVSQDCNQDMEEMMSEQENPDSSE240 PVVEDERLHHDTDDVTYQVYEEQAVYEPLENEGIEITEVTAPPEDNPVEDSQVIVEEVSI300 FPVEEQQEVPPETNRKTDDPEQKAKVKKKKPKLLNKFDKTIKAELDAAEKLRKRGKIEEA360 Peptide#H1<391...399> VNAFKELVRKYPQSPRARYGKAQCEDDLAEKRRSNEVLRGAIETYQEVASLPDVPADLLK420 Peptide#H2<428...436>Peptide#H3<463...470> LSLKRRSDRQQFLGHMRGSLLTLQRLVQLFPNDTSLKNDLGVGYLLIGDNDNAKKVYEEV480 LSVTPNDGFAKVHYGFILKAQNKIAESIPYLKEGIESGDPGTDDGRFYFHLGDAMQRVGN540 Peptide#H4<562569> KEAYKWYELGHKRGHFASVWQRSLYNVNGLKAQPWWTPKETGYTELVKSLERNWKLIRDE600 Peptide#H6<612...621>#H8<630...638> GLAVMDKAKGLFLPEDENLREKGDWSQFTLWQQGRRNENACKGAPKTCTLLEKFPETTGC660 Peptide#H7<676...685> RRGQIKYSIMHPGTHVWPHTGPTNCRLRMHLGLVIPKEGCKIRCANETKTWEEGKVLIFD720 Peptide#H5<749...758> DSFEHEVWQDASSFRLIFIVDVWHPELTPQQRRSLPAI758 // Sequence#SQ2:LocationsofPeptides#C1-#C8AlongCanineASPH, isoformX1(794aa) LOCUS XP_022267901794aalinearMAM05Sep.2017 DEFINITION aspartyl/asparaginylbeta-hydroxylaseisoformX1[Canislupus familiaris]. ACCESSION XP_022267901 VERSION XP_022267901.1 [...Textomitted...] ORIGIN 1MAEETKHGGHKNGRKGGLSGSSFFTWFMVIALLGVWTSVAVVWFDLVDYEEVLAKAKDFR 61YNLSEVLQGKLGVYDADGDGDFDVDDAKVLLGLTKDGSNENIDSLEEVLNILAEESSDWF 121YGFLSFLYDIMTPFEMLEEEEEESETADGVDGLKERSASKPTVPPEEAEPYPWLEEQVIE 181DSGPQNTEDEVQEVQIESLLHEAVYTEHGDDVQQEEDGQVREPQPEDDFLVGSDTDDRYE 241PLETGTFHEETEDSYHIEETASQAYNQDMEEMMYEQDNPDSMEPIVGDDARTYHEADDLT 301YQDYDEPVYEPPENEGLESSDNAGEDSNIILEEVYMPPAEEQQEVPPETNRKTDDPEIKE 361KVKKKKPKLLNKFDKTIKAELDAAEKLRKRGKIEEALSAFQELVRKYPQSPRARYGKAQC Peptide#C1<427...435>Peptide#C2<464...472> 421EDDLAEKRRSNEVLRGAIETYQEVASLPNVPTDLLKLTLKRRSDRQQFLGHMRGSLITLQ Peptide#C3<499...508> 481KLVQLFPDDMSLKNDLGVGYLLIGDNNNAQKVYEEVLNVTPNDGFAKVHYGFILKAQNKI Peptide#C4<598 541AESIPYLKEGIESGDPGTDDGRFYFHLGDAMQRVGNKEAYKWYELGHKRGHFASVWQRSL .605>Peptide#C6<648...657> 601YNVHGLKAQPWWTPKETGYTELVKSLERNWKLIRDEGLAVMDKAKGLFLPEDENLREKGD Peptide#C8<666...674>Peptide#C7<712... 661WSQFTLWQQGRKNENACKGAPKTCSLLDKFPETTGCRRGQIKYSIMHPGTHVWPHTGPTN .721> 721CRLRMHLGLVIPKEGCKIRCANETKTWEEGKVLIFDDSFEHEVWQDATSFRLIFIVDVWH Peptide#C5<785...794> 781PELTPQQRHSLPAI // Sequence#SQ3:AlignedHumanASPH(758aa)andCanineASPH, IsoformX1(794aa)Sequences Query IDXP_022267901.1 Description aspartyl/asparaginylbeta-hydroxylaseisoformX1[Canislupus familiaris] Moleculetypeaminoacid QueryLength794 Subject IDQ12797.3 Description RecName:Full=Aspartyl/asparaginylbeta-hydroxylase;AltName: Full=Aspartatebeta-hydroxylase;Short=ASPbeta-hydroxylase; AltName:Full=Peptide-aspartatebeta-dioxygenase Moleculetypeaminoacid SubjectLength758 Query 1MAE-------------------------------ETKHGGHKNGRKGGLSGSSFFTWFMV29 MA+ETKHGGHKNGRKGGLSG+SFFTWFMV Sbjct 1MAQRKNAKSSGNSSSSGSGSGSTSAGSSSPGARRETKHGGHKNGRKGGLSGTSFFTWFMV60 Query 30IALLGVWTSVAVVWFDLVDYEEVLAKAKDFRYNLSEVLQGKLGVYDADGDGDFDVDDAKV89 IALLGVWTSVAVVWFDLVDYEEVLGKLG+YDADGDGDFDVDDAKV Sbjct 61IALLGVWTSVAVVWFDLVDYEEVL---------------GKLGIYDADGDGDFDVDDAKV105 Query 90LLGLTKDGSNENIDSLEEVLNILAEESSDWFYGFLSFLYDIMTPFEMLEEEEEESETADG149 LLGL Sbjct 106LLGL-------------------------------------------------------- Query 150VDGLKERSASKPTVPPEEAEPYPWLEEQVIEDSGPQNTEDEVQEVQIESLLHEAVYTEH-208 KERSS+PVPPEEAEP+EEQV++PQNEDE+EQI+SLLHEV+EH Sbjct 110----KERSTSEPAVPPEEAEPHTEPEEQVPVEAEPQNIEDEAKE-QIQSLLHEMVHAEHV164 Query 209-GDDVQQEEDGQVREPQPEDD-FLVGSDTDDRYEPLETGTFHEETEDSYHIEETASQAYN266 G+D+QQEDGEPQEDDFL++DDDR+ELEHEETESYH+EETSQN Sbjct 165EGEDLQQE-DGPTGEPQQEDDEFLMATDVDDRFETLEPEVSHEETEHSYHVEETVSQDCN223 Query 267QDMEEMMYEQDNPDSMEPIVGDDARTYHEADDLTYQDYDEP-VYEPPENEGLESS-----320 QDMEEMMEQ+NPDSEP+V+DR+H+DD+TYQY+EVYEPENEG+E+ Sbjct QDMEEMMSEQENPDSSEPVV-EDERLHHDTDDVTYQVYEEQAVYEPLENEGIEITEVTAP Query 321--DNAGEDSNIILEEVYMPPAEEQQEVPPETNRKTDDPEIKEKVKKKKPKLLNKFDKTIK378 DNEDS+I+EEV+PEEQQEVPPETNRKTDDPEKKVKKKKPKLLNKFDKTIK Sbjct 283PEDNPVEDSQVIVEEVSIFPVEEQQEVPPETNRKTDDPEQKAKVKKKKPKLLNKFDKTIK342 Peptide#C1<427...435> Query 379AELDAAEKLRKRGKIEEALSAFQELVRKYPQSPRARYGKAQCEDDLAEKRRSNEVLRGAI438 AELDAAEKLRKRGKIEEA++AF+ELVRKYPQSPRARYGKAQCEDDLAEKRRSNEVLRGAI Sbjct 343AELDAAEKLRKRGKIEEAVNAFKELVRKYPQSPRARYGKAQCEDDLAEKRRSNEVLRGAI402 Peptide#H1<391...399> Peptide#C2<464...472> Query 439ETYQEVASLPNVPTDLLKLTLKRRSDRQQFLGHMRGSLITLQKLVQLFPDDMSLKNDLGV498 ETYQEVASLP+VPDLLKL+LKRRSDRQQFLGHMRGSL+TLQ+LVQLFP+DSLKNDLGV Sbjct 403ETYQEVASLPDVPADLLKLSLKRRSDRQQFLGHMRGSLLTLQRLVQLFPNDTSLKNDLGV462 Peptide#H2<428...436> Peptide#C3<499...508> Query 499GYLLIGDNNNAQKVYEEVLNVTPNDGFAKVHYGFILKAQNKIAESIPYLKEGIESGDPGT558 GYLLIGDN+NA+KVYEEVL+VTPNDGFAKVHYGFILKAQNKIAESIPYLKEGIESGDPGT * Sbjct 463GYLLIGDNDNAKKVYEEVLSVTPNDGFAKVHYGFILKAQNKIAESIPYLKEGIESGDPGT552 Peptide#H3<463...470> Peptide#C4<598605> Query 559DDGRFYFHLGDAMQRVGNKEAYKWYELGHKRGHFASVWQRSLYNVHGLKAQPWWTPKETG618 DDGRFYFHLGDAMQRVGNKEAYKWYELGHKRGHFASVWQRSLYNV+GLKAQPWWTPKETG Sbjct * 523DDGRFYFHLGDAMQRVGNKEAYKWYELGHKRGHFASVWQRSLYNVNGLKAQPWWTPKETG582 Peptide#H4<562569> Peptide#C6<648...657>#C8<666...674> Query 619YTELVKSLERNWKLIRDEGLAVMDKAKGLFLPEDENLREKGDWSQFTLWQQGRKNENACK678 YTELVKSLERNWKLIRDEGALVMDKAKGLFLPEDENLREKGDWSQFTLWQQGR+NENACK Sbjct * 583YTELVKSLERNWKLIRDEGLAVMDKAKGLFLPEDENLREKGDWSQFTLWQQGRRNENACK642 Peptide#H6<612...621>#H8<630...638> Peptide#C7<712...721> Query 679GAPKTCSLLDKFPETTGCRRGQIKYSIMHPGTHVWPHTGPTNCRLRMHLGLVIPEKGCKI738 GAPKTC+LL+KFPETTGCRRGQIKYSIMHPGTHVWPHTGPTNCRLRMHLVLVIPKEGCKI Sbjct 643GAPKTCTLLEKFPETTGCRRGQIKYSIMHPGHTVWPHTGPTNCRLRMHLGLVIPKEGCKI702 Peptide#H7<676...685> Peptide#C5<785...794> Query 739RCANETKTWEEGKVLIFDDSFEHEVWQDATSFRLIFIVDVWHPELTPQQRHSLPAI794 RCANETKTWEEGKVLIFDDSFEHEVWQDA+SFRLIFIVDVWHPELTPQQR_SLPAI Sbjct * 703RCANETKTWEEGKVLIFDDSFEHEVWQDASSFRLIFIVDVWHPELTPQQRRSLPAI758 Peptide#H5<749...758>

Example 1Design and Synthesis of Synthetic Peptides Corresponding to Epitopes of ASPH

Synthesis of Exemplary Compounds

[0101] Synthetic peptides derived from human and/or canine ASPH were designed that correspond to eight domain regions (#1-#8, as #H1-#H8 and #C1-#C8), as penultimate domain epitopes of the full length polypeptide, as illustrated in FIG. 2A, and shown in FIG. 2B and below in Table #T2. These peptides were rationally selected based upon the spatial distance from the substrate, as found in crystal structure 5JZZ deposited at the RCSB Protein Databank. Peptide epitope domain regions #1-#3 are from the non-catalytic domain, while peptide epitope domain regions #4 & #5 are from the C-terminal catalytic domain, but outside of residues 650-700. Peptide epitope domain regions #6, #7, and #8 are within or near the C-terminal catalytic domain of ASPH.

TABLE-US-00004 TABLE#T2 PeptideSequencesCorrespondingtoPenultimate DomainEpitopesofHumanandCanineASPH Epitope Short Positionsin Positionsin SEQ Domain Organism Sequence Name HumanASPH CanineASPH IDNOS #1 HUMAN/CANINE KRRSNEVLR #H1/#C1 391-399 427-435 03/11 #2 HUMAN/CANINE DRQQFLGHM #H2/C2 428-536 464-472 04/12 #3 HUMAN GYLLIGDNDN #H3 463-470 05 CANINE GYLLIGDNNN #C3 499-508 13 #4 HUMAN RSLYNVNG #H4 562-569 06 CANINE RSLYNVHG #C4 598-605 14 #5 HUMAN PQQRRSLPAI #H5 749-758 07 CANINE PQQRHSLPAI #C5 785-794 15 #6 HUMAN/CANINE FLPEDENLRE #H6/C6 612-621 648-657 08/16 #7 HUMAN/CANINE VWPHTGPTNC #H7/C7 676-685 712-721 10 #8 HUMAN LWQQGRRNE #H8 630-638 10 CANINE LWQQGRKNE #C8 666-674 18

Example 2Immunization of Peptide Candidates into Rabbits and Test Bleed

[0102] ImmunoPrecise Antibodies Ltd. (Victoria, British Columbia, Canada) carried out immunization of peptide candidates into rabbits, the testing of antibodies from rabbit B cells, cloning of variable regions into expression vectors, and DNA sequencing of selected rabbit MAbs (Examples 2-8) using standard procedures, under contract with principal investigators at Midwestern University (Glendale, Ariz.).

TABLE-US-00005 TABLE#T3 SyntheticPeptideSequencesUsedasImmunogensDirectedagainstASPH Positions Short inHuman Epitope SEQ Name Description/Sequence ASPH Domain IDNOS Peptide#1 Syntheticpeptidecomprising29aminoacids 731-758 #5 19 (CASSF- withCysteinatitsaminoterminus,plus PO3H2) 28aminoacidscorrespondingtopositions 731-758attheC-terminalendofhuman ASPH,withtheThreonineat19(corresponding to748ofASPH)phosphorylated. C-ASSFRLIFIVDVWHPEL-T(PO3H2)-PQQRRSLPAI Peptide#2 Syntheticpeptidecomprising29aminoacids 731-758 #5 20 withCysteineatitsaminoterminus,plus28 aminoacidscorrespondingtopositions 731-758attheC-terminalendofhumanASPH. C-ASSFRLIFIVDVWHPELTPQQRRSLPAI

[0103] Synthetic peptides #1 and #2 (1 mg each) were prepared at a purity of >95%. The N-terminal Cysteine residue on each peptide is used to facilitate conjugation of each peptide to other molecules. BSA and KLH (2 mg each) were synthesized or obtained from commercial sources.

TABLE-US-00006 Peptide#1 (SEQIDNO:19) CASSFRLIFIVDVWHPEL-T(PO3H2)-PQQRRSLPAI Peptide#2 (SEQIDNO:20) CASSFRLIFIVDVWHPELTPQQRRSLPAI

[0104] Briefly, 3-6 mg of immunizing/screening antigen were prepared and stored in a neutral pH, sterile, buffered solution, at a minimum concentration of 0.5 mg/L. Antigen (hapten) was conjugated to an appropriate carrier and emulsified in Freund's Complete adjuvant, and used to immunize two New Zealand White (NZW) rabbits by subcutaneous injections. Booster injections of antigen in Freund's Incomplete adjuvant were carried at 3 week intervals. Blood samples (test bleeds) were collected 7-10 days after the second boost and immune sera were tested for specific antibody titer by ELISA. Each rabbit was given a final boost, if required, and whole blood was used to obtain B cells to generate Monoclonal Antibodies (MAbs) by the methods noted below.

Example 3In Vitro Culture of Rabbit B Cells

[0105] Whole rabbit blood was collected after the final boost, and B cells were isolated, purified, and cultured by ImmunoPrecise Antibodies Ltd.

Example 4Screening and Analysis of Antibodies from Rabbit B Cells

[0106] Screening was performed on the immunizing antigen by an indirect ELISA performed by ImmunoPrecise Antibodies Ltd.

[0107] ELISA plates were obtained from Costar Corning (Catalog #0720039). Blocking solutions included BSA (Bovine serum albumin) and Skim milk powder (MP). Phosphate buffered saline (PBS) at pH 7.4, PBS with 0.05% Tween-20 at pH 7.4, and Carbonate coating buffer (CCB) at pH 9.6 were used in the ELISA tests. Primary antibodies being tested included the immune sera, B cell supernatants, and transfected supernatants (recombinant rabbit MAbs). Secondary antibodies included Goat Anti-Rabbit IgG-Fc-HRP, Subisotype IgG1, obtained from Jackson ImmunoResearch (Catalog #111-035-046), and AffiniPure goat anti-rabbit IgG (H+L), Subisotype IgG1, obtained from Jackson ImmunoResearch (Catalog #111-035-144). Substrate reagents included TMB (3,3,5,5-tetramethyl-benzidine buffer), TMB One Component HRP Microwell Substrate, and BioFx cat #TMBW-1000-01.

[0108] Briefly, B cell culture supernatants from 96-well plates were transferred to ELISA plates coated with antigen. An indirect ELISA was performed by probing each well with a secondary antibody that binds to rabbit IgG antibodies. Wells with cells that tested positive were retested with the immunizing antigen to confirm specificity and binding.

[0109] Samples corresponding to the top responding wells were preserved in lysis buffer.

[0110] Cell culture supernatants from positive wells (in a volume of <50 L) were also preserved.

Example 5Cloning Antibody Heavy and Light Chain Variable Regions in Mammalian Expression Vectors

[0111] Cells from selected wells of B cells were amplified and samples of mRNA prepared from those cells by ImmunoPrecise Antibodies Ltd. Complementary DNAs corresponding to rabbit IgG heavy and kappa light chain variable regions were prepared and cloned separately into mammalian expression vectors comprising rabbit heavy and light chain constant regions, respectively.

Example 6Expression of Antibody Heavy and Light Chain Variable Regions into HEK293 Cells

[0112] Two plasmids, one comprising a heavy chain variable and a constant region and one comprising a light chain variable and constant region, were co-transfected into HEK293 cells, and allowed to express both chains of the rabbit antibodies.

Example 7Analysis of Cell Culture Supernatants

[0113] The cell culture supernatants were assayed for activity by indirect ELISA against the immunizing peptide (Peptide #1, SEQ ID NO: 13). Ten clones (#1-#10) having positive activity against immunizing peptide were identified. One clone produced an antibody that reacted with the phosphorylated Peptide #1, and four clones produced antibodies that reacted against both the phosphorylated Peptide #1 (SEQ ID NO: 19) and the non-phosphorylated Peptide #2 (SEQ ID NO: 20).

Example 8DNA Sequencing of Heavy and Light Chain Regions from Selected Positive Rabbit MAbs

[0114] Ten clones were selected, five comprising heavy chains (1H2, 5H1, 5H3, 5H4 and 9H2), and five comprising kappa chains (1K6, 5K1, 5K3, 5K6 and 9K1). Purified plasmid DNA samples were prepared and sent to Macrogen USA for sequencing and analyzed by SnapGene Version 4.0.4.

[0115] The rabbit IgG heavy chain sequence is about 1200 bp in length, and can be sequenced from its 5 end to obtain a reliable full-length variable sequence. The rabbit kappa light chain is about 700 bp in length, and full-length variable sequence can be reliably obtained from sequencing in the 5 direction.

Analysis of Translation of Consensus Amino Acid Sequences

[0116] The nucleotide sequences of the variable regions of five heavy chains and five kappa chains were analyzed. Table #T4 discloses the translated variable regions encoded by the nucleotide sequences of the top 10 clones. Sequences highlighted in bold with a single underline (as GQPK) show the start of the constant region for heavy chains, and sequences highlighted in italic and double underline (as GDPV) show the start of the constant region of kappa chains.

TABLE-US-00007 TABLE#T4 Translatedvariableregionsequencesofthetopclones SEQ # CloneID DescriptionorSequence Length Type IDNO 1 1H2 METGLRWLLLVAVLKGVQCQSLEESGGDLVKPGASLTLTCTAS 150 AA 21 GLSFSDNFMCWVRQAPGKGLEWIACIYFDSSGITYYASWAKGR FTISKTSSPTVTLQMTSLTAADTATYFCARDGPGSISWDLWGQ GTLVTVSSGQPKAPSVFPLAP 2 1K6 MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSPVSAAVGGTVTI 148 AA 22 SCQSSKSVYSKNRLAWYQQKPGQPPKLLIYEASKLASGVPSRF KGSGSGTQFTLTISGVQCDDAATYYCQGTYDSSGWYWAFGGGT EVVVK APTVLIFPPA 3 5H1 METGLRWLLLVAVLKGVQCQSLEESGGDLVKPGASLTLTCKAS 142 AA 23 GFDFSSNAMCWVRQAPGKGPEWIACIDNGDGSTDYATWAKGRF TISKTSSTTVTLQMTSLTAADTATYFCTRNFNLWGPGHPGHRL ERTAESPVGVSTG 4 5H3 METGLRWLLLVAVLKGVQCQSLEESGGDLVKPGASLTLTCKAS 143 AA 24 GFDFSSNAMCWVRQAPGKGPEWIACIDNGDGSTDYATWAKGRF TISKTSSTTVTLQMTSLTAADTATYFCTRNFNLWGQGTLVTVS SGQPKAPSVFPLAP 5 5H4 METGLRWLLLVAVLKGVQCQSLEESGGDLVKPGASLTLTCKAS 143 AA 25 GFDFSSNAMCWVRQAPGKGPEWIACIDNGDGSTDYATWAKGRF TISKTSSTTVTLQMTSLTAADTATYFCTRNFNLWGQGTLVTVS SGQPKAPSVFPLAP 6 5K1 MDTRAPTQLLGLLLLWLPGATFAQVLTQTASSVSAAVGGTVTI 146 AA 26 SCQSSQSVYDNNRLAWFQQKPGQPPKLLIYETSKLASGVPLRF KGSGSGTQFTLTISDLECDDAATYYCLGSYSGYIYTFGGGTEV VVK APTVLIFPPA 7 5K3 MDTRAPTQLLGLLLLWLPGATFAQVLTQTASSVSAAVGGTVTI 146 AA 27 SCQSSQSVYDNNRLAWFQQKPGQPPKLLIYETSKLASGVPLRF KGSGSGTQFTLTISDLECDDAATYYCLGSYSGYIYTFGGGTEV VVK APTVLIFPPA 8 5K6 MDTRAPTQLLGLLLLWLPGATFAQVLTQTASSVSAAVGGTVTI 146 AA 28 SCQSSQSVYDNNRLAWFQQKPGQPPKLLIYETSKLASGVPLRF KGSGSGTQFTLTISDLECDDAATYYCLGSYSGYIYTFGGGTEV VVK APTVLIFPPA 9 9H2 METGLRWLLLVAVLKGVQCQSLEESGGDLVKPGASLTLTCKAS 142 AA 29 GFDFISNAMCWVRQAPGKGPEWIACIDNGDGSTDYATWAKGRF TISKTSSTTVTLQMTSLTAADTATYFCTRNFNLWGQGTL?TVS SGQPKAPSVFPLAP 10 9K1 MDTRAPTQLLGLLLLWLPGATFAQVLTQTASSVSAAVGGTVTI 146 AA 30 SCQSSQSVYDNNRLAWFQQKSGQPPKLLIYETSKLASGVPLRF KGSGSGTQFTLTISDLECDDAATYYCLGSYSGYIYTFGGGTEV VVK APTVLIFPPA

[0117] These results demonstrate that recombinant monoclonal antibodies derived from rabbits, were generated successfully against Peptide #1 (SEQ ID NO: 13). Recombinant Clones 5H1, 5H3, 5H4, and 9H2 have the same heavy chain sequences, and recombinant clones 5K1, 5K3 and 9K1 have the same kappa chain sequence.

TABLE-US-00008 Sequence#SQ4:MultipleSequenceAlignmentofHeavyChainsforClones1H2,5H1,9H2,5H3,and5H4 Amultiplesequencealignmentoffiveclonescomprisingheavychainsillustratesslight differencesintheencodedpolypeptidesequencesinregionswithinandjustflanking CDR1,CDR2,CDR3,withnotabledivergenceforsequencesafterCDR3forclone5H1. CLUSTALO(1.2.4)multiplesequencealignmentheavychains: [00001] [00002] [00003]

[0118] The CDR1 regions from the heavy chain clones include the sequences NFMC (SEQ ID NO: 31), corresponding to residues 50-53 of SEQ ID NO: 21, and NAMC (SEQ ID NO: 32), corresponding to residues 50-53 of SEQ ID NOS: 23, 29, 24, and 25. The CDR2 regions from the heavy chain clones include CIYF (SEQ ID NO: 33) corresponding to residues 68-71 of SEQ ID NO: 21, and CIDN (SEQ ID NO: 34) corresponding to residues 68-71 of SEQ ID NO: 23, 29, 24, and 25. The CDR3 regions from the heavy chain clones include DGPGSISWDI (SEQ ID NO: 35) corresponding to residues 117-126 of SEQ ID NO: 21, and NFNI (SEQ ID NO: 36) corresponding to residues 116-119 of SEQ ID NOS: 23, 29, 24, and 25.

TABLE-US-00009 Sequence#SQ5:MultipleSequenceAlignmentofKappaLightChains forClones1K6,5K1,5K3,5K6,and9K1 Amultiplesequencealignmentoffiveclonescomprisingkappalightchainsillustrates slightdifferencesintheencodedpolypeptidesequencesinregionswithinandjustflanking CDR1,CDR2,CDR3,withnotabledivergenceforsequenceswithinCDR3forclone1K6. CLUSTALO(1.2.4)multiplesequencealignmentkappachains: [00004] [00005] [00006]

[0119] The CDR1 regions from the kappa chain clones 1K6, 5K1, 5K3, 5K6, and 9K1 include SVYSKNR (SEQ ID NO: 37) corresponding to residues 50-56 of SEQ ID NO: 22, and SVYDNNR (SEQ ID NO: 38) corresponding to residues 50-56 of SEQ ID NOS: 26, 27, 28, and 30. The CDR2 regions from the kappa chain clones were all LAS (SEQ ID NO: 39) corresponding to residues 78-80 of SEQ ID NOS: 22, 26, 27, 28, and 30. The CDR3 regions from the kappa chain clones included QGTYDSSGWYWA (SEQ ID NO: 40) corresponding to residues 113-124 of SEQ ID NO: 22, and LGSYSGYIYI (SEQ ID NO: 41) corresponding to residues 113-122 of SEQ ID NOS: 26, 27, 28, and 30.

Example 9Analysis of MAbs by Immunohistochemistry (IHC)Phase IAntibody Triage

[0120] Antibody triage (Phase I) was performed by Reveal Biosciences (San Diego, Calif.) on a Leica Bond automated immunostainer, testing each antibody at 8 g/mL, in parallel with a negative control performed in absence of primary antibody. FFPE human hepatocellular carcinoma was used for antibody testing.

[0121] Heat induced antigen retrieval was performed using Leica Bond Epitope Retrieval Buffer 1 (Citrate Buffer, pH6.0) and Leica Bond Epitope Retrieval Buffer 2 (EDTA solution, pH9.0) for 20 minutes (ER2(20)). Non-specific antibody binding was blocked using 3% Normal Goat Serum in PBST. Tests for positive reactions were carried out by using Novocastra Bond Refine Polymer Detection reagent, and visualized with 33-diaminobenzidine (DAB; brown). A Hematoxylin nuclear counterstain (blue) was also applied.

[0122] When Phase I optimization slides were evaluated, only two samples, 5H4/5K3 and 9H2/9K1, showed positive staining in Epitope Retrieval Buffer, ER2(20), as noted below.

TABLE-US-00010 TABLE #T5 Results of Antibody Triage Host Antigen Groups Antibody Dilution Species Retrieval 1 1H2/1K6 8 g/mL Rabbit NONE 1H2/1K5 NONE 1H4/1K6 NONE 1H4/1K4 NONE 2 2H4/2K5 NONE 5H1/5K1 NONE 5H4/5K3 ER2(20) 9H2/9K1 ER2(20)

[0123] Two antibodies, 5H4/5K3 and 9H2/9K1, that showed positive staining in ER2(20), were selected for further testing in Phase II.

Example 10AAnalysis of MAbs by Immunohistochemistry (IHC)Phase IIIHC Optimization

[0124] Immunohistochemistry (IHC) Optimization was performed by Reveal Biosciences (San Diego, Calif.) on a Leica Bond automated immunostainer, by testing each antibody at 2 g/mL, 4 g/mL, 8 g/mL, and 10 g/mL.

[0125] Heat induced antigen retrieval was performed using Leica Bond Epitope Retrieval Buffer 2 (EDTA solution, pH9.0) for 20 minutes (ER2(20)). Non-specific antibody binding was blocked using 3% Normal Goat Serum in PBST. Tests for positive reactions were carried out by using Novocastra Bond Refine Polymer Detection and visualized with 33-diaminobenzidine (DAB; brown). A Hematoxylin nuclear counterstain (blue) was applied.

[0126] When Phase II optimization samples were evaluated, no staining was observed at 2 g/mL for 5H4/5K3 and 9H2/9K1. A strong signal was detected at both 8 g/mL and 10 g/mL for 5H4/5K3, as illustrated in FIG. 6. A strong signal was detected at both 8 g/mL, and 10 g/mL for 9H2/9K1, with a stronger intensity at 10 g/mL, as illustrated in FIG. 7.

[0127] These results demonstrate that 5H4/5K3 and 9H2/9K1 are notable as leads for the development of diagnostic agents, and also as therapeutic drug products suitable for use in mammals, such as humans, by grafting the CDRs onto a suitable antibody framework that will facilitate the targeting of one or more drug products to cancerous tissues in a human subject.

Example 10BAnalysis of MAbs by Immunohistochemistry (IHC)Phase IIIIHC on Tissue Micro Arrays

[0128] Immunohistochemistry (IHC) was performed on a Leica Bond automated immunostainer using 5H4/5K3 at 8 g/mL on TMAs (Table #T5).

[0129] Heat induced antigen retrieval was performed using Leica Bond Epitope Retrieval Buffer 2 (EDTA solution, pH9.0) for 20 minutes (ER2(20)). Non-specific antibody binding was blocked using 3% Normal Goat Serum in PBST.

[0130] Positivity was detected using Novocastra Bond Refine Polymer Detection and visualized with 33-diaminobenzidine (DAB; brown). A Hematoxylin nuclear counterstain (blue) was applied.

[0131] Isotype controls were performed on Human Hepatocellular carcinoma slide and each TMA type alongside their respective positive (with primary) slide using Rabbit IgG (Abcam ab172730, lot #GR3179509-3).

[0132] A human hepatocellular carcinoma FFPE block was sectioned at 4 um thickness and mounted onto positively charged slides for assay development.

TABLE-US-00011 TABLE #T6 Tissue Micro Arrays used for IHC staining in Phase III Array Name Tissue Type LV12 Liver cancer tissue array with progressive changes NT01 Normal Human Tissue PC02 Pancreatic cancer tissue array OV01 Ovary cancer tissue array OV03 Ovary cancer tissue array with progressive changes

[0133] FIG. 8 sets forth an illustration demonstrating 5H4/5K3 Phase III on TMAs for samples labeled as LV12 Core F4 (top panel), and LV12 Core F4-Isotype (bottom panel). Positive 5H4/5K3 staining was visualized with DAB (brown). Isotype negative control was performed with Rabbit IgG (right images). The scale bar represents 20 m.

[0134] FIG. 9 sets forth an illustration demonstrating 5H4/5K3 Phase III on TMAs for samples labeled as PCO2 Core A6 (top panel), and PCO2 Core A6-Isotype (bottom panel).

[0135] FIG. 10 sets forth an illustration demonstrating 5H4/5K3 Phase III on TMAs for samples labeled as OV03 Core C5 (top panel), and OV03 Core C5-Isotype (bottom panel).

[0136] FIG. 11 sets forth an illustration demonstrating 5H4/5K3 Phase III on TMAs for samples labeled as OV01 Core D2 (top panel), and OV01 Core D2-Isotype (bottom panel).

[0137] These results demonstrate that antibody 5H4/5K3 stains a broad range of ovarian cancer samples, from granuloma to serous to endometrioid cancers. Malignant cancers stain intensely, while benign and normal ovarian tissue samples do not stain under these conditions.

[0138] These and similar antibodies, plus fragments or derivatives thereof, should be useful as a key reagent in a kit to diagnose the presence of cancer cells in wide variety of research and clinical samples.

[0139] These and similar antibodies, plus fragments or derivatives thereof, may also be useful in the development of pharmaceutical compositions comprising a therapeutic agent when the CDRs are grafted onto an appropriate framework suitable to produce a drug product suitable for mammals, particularly non-human primate and human subjects, and livestock, and domestic pets, including dogs and cats.

Example 10CAnalysis of MAbs by Immunohistochemistry (IHC)Phase IIIIHC on Tissue Micro Arrays

[0140] FIG. 12 sets forth an illustration demonstrating activity of 5H4/5K3 Against Granulosa Cell Tumor Samples (A11 and B11). Positive 5H4/5K3 staining was visualized with DAB (brown) against Granulosa Cell Tumor (top images, A11 and B11). Isotype negative control was performed with Rabbit IgG (bottom images, A11 and B11).

[0141] FIG. 13 sets forth an illustration demonstrating activity of 5H4/5K3 Against Serrous Cystadenocarcinoma Stage III Samples (C5 and D5).

[0142] FIG. 14 sets forth an illustration demonstrating activity of 5H4/5K3 Against Serrous Cystadenocarcinoma Stage III Samples (C8 and D8).

[0143] FIG. 15 sets forth an illustration demonstrating activity of 5H4/5K3 Against Endometrioid Adenocarcinoma Stage III Samples (E8 and F8).

[0144] FIG. 16 sets forth an illustration demonstrating reaction of 5H4/5K3 Against Normal Ovarian Tissue Samples (A1 and B1).

[0145] FIG. 17 sets forth an illustration demonstrating reaction of 5H4/5K3 Against Thecoma (Theca Cell) Tumor Tissue (A5 and B5).

[0146] These results confirm activity of the 5H4/5K3 antibody against a variety of cancerous tissue samples, and a lack of activity against cells in normal tissue samples.

Example 11Interactions Between ASPH and Selected MAbs Captured Via Protein G

[0147] The interaction between ASPH and a set of 6 antibodies were characterized by Essai Sciences LLC (Stillwater, Okla.) on a SensiQ Pioneer SPR Platform. The COOH2 sensor chip, which contains a planar dextran surface, was used for target immobilization. The buffer system was 10 mM HEPES, pH 7.4, 150 mM NaCl, and 0.01% Tween-20.

[0148] All channels of a COOH2 sensor chip were activated with a five-minute injection of 40 mM EDC and 10 mM NHS. Protein G was then injected across channels 1 and 2. 1 M ethanolamine, pH 8.0 was then injected across all three channels. Approximately 1000 response units of Protein G were captured on both channels 1 and 2 (FIG. 18). For each antibody-ASPH interaction, the antibody was injected on channel 1, leaving channels 2 and 3 as a Protein G reference and empty channel reference, respectively. After antibody capture, ASPH was injected at a single concentration. Following injection of ASPH, all three channels were injected with 10 mM NaOH for one minute to regenerate the Protein G surface. This was done twice for each antibody, at each tested concentration of ASPH.

[0149] All experimental results shown are from fixed-concentration analyses of the interactions. Given material constraints, as well as the nature of the interacting molecules, immobilization of the antibodies, and fixed-concentration injection of ASPH was the most feasible experimental setup for this study.

[0150] The response curves for each tested concentration of ASPH against each captured antibody are displayed below. FIG. 19 is the mock sample, which demonstrates no visible binding. The remaining antibodies (FIGS. 20-25) display affinity for ASPH that range from 60 nM (9H2/9K3, FIG. 24) to 920 nM (2H4/2K5, FIG. 22). We tested the phospho-selective antibody, 8H1/8K1 (FIG. 25), and observed no binding, even at the highest tested analyte concentration. The kinetics values for each interaction are listed in Table #T7.

TABLE-US-00012 TABLE #T7 Kinetics values for interaction of ASPH with antibodies. Antibody ka (M.sup.1s.sup.1) kd (s.sup.1) K.sub.D (M) Mock 2H4/2K5 1.83 1.681 920 0.04e3 0.002e3 20 nM 5H1/5K1 2.035 2.410 118.4 0.002e4 0.002e3 0.2 nM 5H4/5K3 1.683 2.426 144.2 0.002e4 0.002e3 0.2 nM 9H2/9K1 1.879 2.363 125.7 0.002e4 0.002e3 0.2 nM 9H2/9K3 2.985 1.848 61.9 0.004e4 0.003e3 0.1 nM 8H1/8K1

[0151] The interaction of the ASPH protein with a set of antibodies captured via Protein G was studied. A range of affinities from 60 nM to 920 nM for the binding antibodies was observed. A mock sample, and a phospho-selective antibody were also tested. No observable binding to the protein for the mock sample or the phospho-selective antibody was noted.

Example 12In Vitro Cell Proliferation Assay with Antibodies Against Epitopes of ASPH in Three Tumor Cell Lines

[0152] Experiments to determine the half maximal inhibitory concentration (IC.sub.50) of the potency of samples comprising selected antibodies in different types of cultured tumor cells were carried out by Translational Drug Development LLC.

[0153] FIG. 26 shows graphs illustrating IC.sub.50 curves for three samples tested in 4T1 Murine Breast Tumor cells (Panel A, 5H4/5K3; Panel B, 9H2/9K1; and Panel C, Mock Antibody).

[0154] FIG. 27 shows graphs illustrating IC.sub.50 curves for three samples tested in MCF-7 Human ER+ Breast Tumor cells (Panel A, 5H4/5K3; Panel B, 9H2/9K1; and Panel C, Mock Antibody).

[0155] FIG. 28 shows graphs illustrating IC.sub.50 curves for three samples tested in MV411 Human Mantle Cell Leukemia cells (Panel A, 5H4/5K3; Panel B, 9H2/9K1; and Panel C, Mock Antibody).

TABLE-US-00013 TABLE #T8 Summary of IC.sub.50 Results* Mean IC.sub.50 Mean IC.sub.50 Mean IC.sub.50 (g/mL) (g/mL) (g/mL) Cell Line Tissue Type 5H4/5K3 9H2/9K1 Mock Antibody 4T1 Murine Breast 0.026 0.008 0.280 Tumor MCF-7 Human ER+ 0.024 0.002 0.426 Breast Tumor MV411 Human Mantle 0.098 0.007 0.313 Cell Leukemia *Mean IC values are calculated as the average of IC.sub.50 values obtained from two trials, A and B, for each of 3 antibody experiments in 3 cell lines, as noted in Panels A-C of FIGS. 26 through 28.

[0156] These results demonstrate that the antibodies designated as 5H4/5K3 and 9H2/9K1 both affect the viability of three tumor cell lines being tested, with the Mab designated 9H2/9K1 being more potent than the Mab designated 5 H4/5 K3.

[0157] The antibody designated as 5H4/5K3 appears to be more selective for breast tumors 4T1 and MCF-7.

Example 13Generation of Humanized Chimeric Monoclonal Antibodies Targeting at Least One Epitope in the Catalytic Domain of ASPH

[0158] Humanized versions of non-human antibodies are chimeric antibodies that a minimal amount of polypeptide domains comprising amino acid sequences derived from the non-human antibody. Typically, residues from the hypervariable region of a human antibody are replaced with hypervariable residues from the non-human antibody, that have the desired specificity, affinity, and/or capacity. Humanized versions can also be prepared from non-human species, such as mouse, rat, rabbit, non-human primates, and other vertebrate species. Other regions, comprising amino acid residues that may contribute to structural integrity of the human antibody (framework region) may also be replaced by amino acid residues from the corresponding non-human residues. The humanized chimeric monoclonal antibodies may also comprise amino acid residues that are not found in the recipient human antibody or the non-human donor antibody. Generally, the humanized antibody comprises at least one, and preferably all of the variable domains of the donor antibody, and substantially all of the framework regions of the human antibody.

[0159] Variants may also comprise one or more portions of the constant region of an antibody, typically, a human antibody. Other types of variants, include fragments, and variants comprising one or more conservative substitutions, insertions, or deletions, that do not substantially alter the specificity, affinity, and/or capacity of the variant molecule compared to its parent molecule, but may offer additional advantages in terms of ease of production or purification, ability to be conjugated to other chemical moieties, which may facilitate covalent or non-covalent binding to other molecules comprising polypeptide domains or other reactive or non-reactive moieties, capable of providing a secondary reporter function, such as emission of fluorescent light, or conversion of a colorless substrate to an easily detectable, colored product, which may be useful as components in diagnostic kits for use in research and in clinical settings. Aspects of the invention also include variants that are >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >98%, >99%, or >99.5% identical to at least one of the variable regions of the donor antibody.

[0160] In the examples noted above, recombinant monoclonal antibodies were generated against Peptide #1 (SEQ ID NO: 13). Recombinant Clones 5H1, 5H3, 5H4, and 9H2 have the same heavy chain sequences, and recombinant clones 5K1, 5K3 and 9K1 have the same kappa chain sequence. The CDR1 regions from the heavy chain clones include the sequences NFMC (SEQ ID NO: 31) and NAMC (SEQ ID NO: 32). The CDR2 regions from the heavy chain clones include CIYF (SEQ ID NO: 33) and CIDN (SEQ ID NO: 34). The CDR3 regions from the heavy chain clones include DGPGSISWDI (SEQ ID NO: 35) and NFNI (SEQ ID NO: 36). The CDR1 regions from the kappa chain clones include SVYSKNR (SEQ ID NO: 37) and SVYDNNR(SEQ ID NO: 38). The CDR2 regions from the kappa chain clones were all LAS (SEQ ID NO: 39). The CDR3 regions from the kappa chain clones included QGTYDSSGWYWA (SEQ ID NO: 40) and LGSYSGYIYI (SEQ ID NO: 41).

[0161] Plasmids comprising cDNAs encoding rabbit antibodies targeting epitopes of ASPH described in Examples 5-8 are used as a source of nucleic acids comprising variable regions to generate humanized monoclonal antibodies that target at least one epitope in the catalytic domain of ASPH. One or more codons within the rabbit cDNAs may be altered to represent codons that are optimally used in the host cell expression system, to enhance expression of the encoded chimeric polypeptide under the control of operably-linked promoters and other genetic elements. Random and targeted mutagenesis of specific residues within the variable regions may result in antibodies that have increased affinity to its intended target, and/or reduced affinity to other targets.

Example 14Generation of Bispecific Antibodies Targeting at Least One Epitope in the Catalytic Domain of ASPH

[0162] Bispecific antibodies combine the structural domains of two distinct molecules into one molecule with the goal of preserving and perhaps enhancing functional properties of the chimeric molecule compared to its parent mono-specific molecules (Dahlen E. et al, Bispecific antibodies in cancer immunotherapy. Therapeutic Advances in Vaccines and Immunotherapy, 2018, 6:(1)3-17). In some cases, bispecific antibodies have superior therapeutic properties compared to compositions comprising mixtures of monospecific compounds.

[0163] Several classes of immunotherapeutic bispecific antibodies have been recognized, including T-cell redirectors, which act on malignant cells by targeting a tumor antigen and CD3; NK-cell redirectors, which act on malignant cells targeting a tumor antigen and CD16A; Tumor-targeted immunomodulators, which direct co-stimulation of tumor-infiltrating immune cells by targeting a tumor antigen and co-stimulatory molecules, such as CD40 or 4-1BB; and Dual immunomodulators, which simultaneously act on two immunomodulatory targets, resulting in blockade of inhibitory targets, depletion of suppressive cells, or activation of effector cells (See Table 1 of Dahlen et al).

[0164] A non-limiting list of exemplary tumor antigens includes CD19, EpCAM, CD20, CD23, BCMA, B7H3, and PSMA.

[0165] A non-limiting list of T-cell specific epitopes includes CD3, CD3e, OX40, CD27, ICOS and GITR.

[0166] A non-limiting list of co-stimulatory molecules includes CD40 and 4-1BB.

[0167] A non-limiting list of immunomodulating targets includes PD-L1, CTLA-4, TGF-, LAG-2, TIM-3, and OX40.

[0168] Bispecific antibodies comprising at least one complementarity-determining region (CDR) targeting one or more epitopes of ASPH selected from the group consisting of CDR1, CDR2, and CDR3 from the heavy chain or the light chain clones of Example 13 are prepared by fusing rabbit, other non-human, human, or humanized antibodies comprising these regions with an antibody targeting one or more tumor antigens, T-cell specific epitopes, co-stimulatory molecules, or immunomodulating targets, as noted above.

[0169] Exemplary bi-specific antibodies include a molecule comprising the CDRs of the 5H4/5K3 antibody disclosed herein, where the 5H4 CDR1=NAMC (SEQ ID NO: 31), CDR2=CIDN (SEQ ID NO: 34), and CDR3=NFNI (SEQ ID NO: 36), and where the 5K3 CDR1=SVYDNNR (SEQ ID NO: 38)), CDR2=LAS (SEQ ID NO: 39), CDR3=LGSYSGYIYI (SEQ ID NO: 41) or 9H2/9K1 antibody, where the 9H2 CDR1=NAMC (SEQ ID NO: 32), CDR2=CIDN (SEQ ID NO: 34), and CDR3=NFNI (SEQ ID NO: 36), and the 9K1 CDR1=SVYDNNR (SEQ ID NO: 38), CDR2=LAS (SEQ ID NO: 39), and CDR3=LGSYSGYIYI (SEQ ID NO: 41), combined with an antibody molecule comprising one or more tumor antigens, T-cell specific epitopes, co-stimulatory molecules, or immunomodulating targets, as noted above.

[0170] An exemplary bispecific antibody of the T-cell redirector class includes an antibody targeting one or more ASPH CDRs with an antibody targeting CD3.

[0171] An exemplary bispecific antibody of the NK-cell redirector class includes an antibody targeting one or more ASPH CDRs with an antibody targeting CD16A.

[0172] An exemplary bispecific antibody of the tumor targeting immunomodular class includes an antibody targeting one or more ASPH CDRs with an antibody targeting CD40 or 4-1BB.

[0173] An exemplary bispecific antibody of the dual immunomodular class includes an antibody targeting one or more ASPH CDRs with an antibody targeting PD-L1, PD-1, CTLA-4, TGF-, LAG-3, TIM-3, or OX40.

Statement Regarding Preferred Aspects are Meant to be Illustrative and not Limiting as to the Scope of the Invention

[0174] While the preferred aspects of the invention have been illustrated and described in detail, it will be appreciated by those skilled in the art that that various changes can be made therein without departing from the spirit and scope of the invention. Accordingly, the particular arrangements disclosed are meant to be illustrative only and not limiting as to the scope of the invention, which is to be given the full breadth of the appended claims and any equivalent thereof.

BIBLIOGRAPHY

Statement Regarding Incorporation by Reference of Journal Articles and Patent Documents

[0175] All references, patents, or applications cited herein are incorporated by reference in their entirety, as if written herein.

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