CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation application that claims priority to and the benefit of U.S. Non-Provisional application Ser. No. 17/036,337, filed Sep. 29, 2020, issued as U.S. Pat. No. 11,236,178 on Feb. 1, 2022, U.S. Non-Provisional application Ser. No. 16/444,617, filed Jun. 18, 2019, issued as U.S. Pat. No. 10,961,320 on Mar. 30, 2021 and U.S. Provisional Application No. 62/686,107, filed Jun. 18, 2018, the entire contents of which are incorporated by reference in their entirety.

INCORPORATION-BY-REFERENCE OF A SEQUENCE LISTING

The Sequence Listing contained in the files “761_190_045_US_01_Sequence_Listing_ST25.txt”, created on 2022-01-25, modified on 2022-01-25, file size 44,400 bytes, containing SEQ ID NOS: 1-52, “761_190_033_US_01_Sequence_Listing_ST25.txt”, created on 2020-09-01, modified on 2020-09-01, file size 44,370 bytes, containing SEQ ID NOS: 1-52, “761_190_026_US_Sequence_Listing_ST25.txt”, created on 2019-05-21, modified on 2019-05-21, file size 35,033 bytes, containing SEQ ID NOS: 1-30, and “761_190_025_US_Sequence_Listing_ST25.txt”, created on 2018-06-17, modified on 2018-06-17, file size 34,990 bytes, containing SEQ ID NOS: 1-30, are incorporated by reference in its entirety herein.

FIELD OF THE INVENTION

Monoclonal antibodies (MAbs) targeting one or more specific epitopes of aspartyl (asparaginyl) β-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.

BACKGROUND OF THE INVENTION

Aspartyl(asparaginyl)-β-hydroxylase (ASPH) is an iron-dependent dioxygenase that catalyzes the hydroxylation of β carbons of aspartic acid and asparagine residues in calcium binding Epidermal Growth Factor (cbEGF)-like domains of a variety of proteins, including Notch and Notch ligand homologs (Dinchuk, Focht et al. 2002) extracellular matrix proteins, and low density lipoprotein (LDL) receptors. ASPH was first observed to be involved in the hydroxylation of a specific aspartic acid residue in the blood coagulation cascade proteins (Drakenberg, Fernlund et al. 1983) where the hydroxylated residue is underlined in the consensus sequence CX[D/N]X₄[Y/F]XC. The role of hydroxylated residue is presently unknown, but the sole known crystal structure with a beta-hydroxylated asparagine (PDB ID 5JZZ: McDonough, M. A., Pfeffer, I., and Munzel (2016) Aspartyl/Asparaginyl beta-hydroxylase (AspH)oxygenase and TPR domains in complex with manganese, N-oxalylglycine and cyclic peptide substrate mimic of factor X. DOI: 10.2210/pdb5JZZ/pdb).

ASPH is generally classified as a peptide-aspartate beta-dioxygenase (EC 1.14.11.16), a member of the alpha-ketoglutarate-dependent hydroxylases superfamily, which catalyzes the following chemical reaction, facilitated by iron as a cofactor.

peptide-L-aspartate+2-oxoglutarate+O₂→peptide-3-hydroxy-L-aspartate+succinate+CO₂ (Reaction 1)

ASPH is not normally expressed in adult cells (Lavaissiere, Jia et al. 1996), but is expressed during invasion of the uterine wall by trophoblasts during development of the placenta (Gundogan, Elwood et al. 2007). ASPH is overexpressed in a variety of tumors, including hepatocellular, cholangiocarcinoma, gastric cancer, pancreatic cancer, non-small cell lung cancer, glioblastoma multiform, osteosarcoma, cervical cancer, ovarian cancer and breast cancer (Yang, Song et al. 2010), and enhances signaling in the Notch pathway (Cantarini, de la Monte et al. 2006).

FIG. 1 sets forth an illustration showing the Activation of Notch Signaling Pathway by ASPH. FIGS. 2 and 3 set forth illustrations showing the Locations of Epitopes of Interest on ASPH.

Known and computationally predicted ASPH substrates are illustrated in FIG. 4 and FIG. 5. Prediction of ASPH substrates is based upon the protein possessing A) a cbEGF domain and B) the consensus sequence CX[D/N]X₄[Y/F]XC. Of particular interest are nearly all of the Notch signaling proteins, not only including the receptors Notch1-4, but many of the known ligands such as Jagged1&2 and DII1&4, but also known Notch pathway modulator human homologues of Crumbs from Drosophila. ASPH is known to hydroxylate lipid receptor proteins, including Lrp1. Lrp1 is known to have an interaction with Wnt5a of the canonical Wnt signaling pathway (El Asmar, Terrand et al. 2016). ASPH substrate Gas6 is the ligand of the Tyro3, Axel and Mer (TAM) kinases, which have been implicated in cancer (Wu, Ma et al. 2018). Known ASPH substrates including the fibrillins are involved in the release of TGF-beta, which is implicated in cancer (Furler, Nixon et al. 2018). In addition to cancer, ASPH hydroxylated substrates are found in nearly all of the blood coagulation proteins involved in thrombosis (see panel C in FIG. 4), and many of the proteins involved in lipid uptake including LDLR, VLDLR and Lrp1 (see panel B in FIG. 4) and cholesterol homeostasis. Thus, ASPH expression is expected to have a cascade of effects, but may have particular value in the treatment of cancer, as well as thrombosis and lipid/cholesterol associated cardiovascular diseases.

ASPH is known to contain multiple phosphorylation sites (Tong, Gao et al. 2013), including T748. Phosphorylation of ASPH is known to alter the expression and function of ASPH (Borgas, Gao et al. 2015), and plays a potential role in migration and tissue invasion of hepatocellular carcinoma (Borgas, Gao et al. 2015). Antibodies selective for ASPH phosphorylation state should be useful in the diagnosis of cancer and distinguishing normally expressed ASPH from tumor expressed ASPH.

Previously designed antibodies to ASPH did not result in direct suppression of tumor cell proliferation (Yeung, Finney et al. 2007). Despite the high affinity of these antibodies, the targeted epitope did not sufficiently disrupt catalytic activity of ASPH. Consequently, while the antibodies were internalized into the cancer cells expressing ASPH, there was no direct antibody activity leading to cellular senescence or cytotoxicity. To address this issue, radioisotopes have been conjugated to previously described high affinity ASPH antibodies, leading to modest activity (Revskaya, Jiang et al. 2017). Other previous anti-ASPH strategies include small molecule inhibitors of ASPH (Aihara, Huang et al. 2014), a dendritic cell approach (Noda, Shimoda et al. 2012), and a vaccine approach (Iwagami, Casulli et al. 2017).

This application describes the epitope selection for phospho-selective ASPH antibodies, as well as antibodies for ASPH catalytic activity inhibition, including epitopes on both the catalytic and non-catalytic domains, demonstration of high affinity for ASPH, strong IHC staining of cancerous but not normal tissue, and direct activity against cancer cells.

SUMMARY OF THE INVENTION

The present invention relates to monoclonal antibodies (MAbs) targeting one or more specific epitopes of aspartyl (asparaginyl) β-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.

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) β-hydroxylase (ASPH), wherein at least one of said peptide epitopes is located within or adjacent to the catalytic domain of ASPH.

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.

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.

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.

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.

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.

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 the antibodies noted above, and one or more portions of the constant regions of a human antibody, and fragments thereof.

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 the antibodies noted above, 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-0, LAG-3, TIM-3, or OX40.

A better understanding of the invention will be obtained from the following detailed descriptions and accompanying drawings, which set forth illustrative embodiments that are indicative of the various ways in which the principals of the invention may be employed.

BRIEF DESCRIPTION OF THE DRAWINGS
Statement Concerning Aspects of the Invention Understood by Reference to the Drawings

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:

FIG. 1 sets forth an illustration showing the Activation of Notch Signaling Pathway by ASPH. Aspartyl(asparaginyl)-β-hydroxylase (ASPH) is an iron-dependent dioxygenase that catalyzes the hydroxylation of β 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.

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

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

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.

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.

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

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

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 μm.

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 μm.

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 μm.

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 μm.

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).

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).

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).

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).

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, Al and B1).

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).

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.

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).

FIG. 20 sets forth an illustration demonstrating Interaction of ASPH with 2H4/2K5. Concentrations are 500 nM (dark red, 1^stline from top), 250 nM (light green, 2^ndand 3^rdlines from top), 125 nM (blue, 4^thand 5^thlines from top), and 62.5 nM (dark green, 6^thand 7^thlines from top).

FIG. 21 Interaction of ASPH with 5H1/5K1. Concentrations are 500 nM (dark red, 1^stline from top), 250 nM (light green, 2^ndand 3^rdlines from top), 125 nM (blue, 4^thand 5^thlines from top), 62.5 nM (dark green, 6^thand 7^thlines from top), 31.2 nM (orange, 8^thand 9^thlines from top), and 15.6 nM (red, 10^thand 11^thlines from top).

FIG. 22 sets forth an illustration demonstrating Interaction of ASPH with 5H4/5K3. Concentrations are 500 nM (dark red, 1^stand 2^ndlines from top), 250 nM (light green, 3^rdand 4^thlines from top), 125 nM (blue, 5^thand 6^thlines from top), 62.5 nM (dark green, 7^thand 8^thlines from top), 31.2 nM (orange, 9^thand 10^thlines from top), and 15.6 nM (red, 11^thand 12^thlines from top).

FIG. 23 sets forth an illustration demonstrating Interaction of ASPH with 9H2/9K1. Concentrations are 500 nM (dark red, 1^stand 2^ndlines from top), 250 nM (light green, 3^rdand 4^thlines from top), 125 nM (blue, 5^thand 6^thlines from top), 62.5 nM (dark green, 7^thand 8^thlines from top), 31.2 nM (orange, 9^thand 10^thlines from top), and 15.6 nM (red, 11^thand 12^thlines from top).

FIG. 24 sets forth an illustration demonstrating Interaction of ASPH with 9H2/9K3. Concentrations are 500 nM (dark red, 1^stline from top), 250 nM (light green, 2^ndand 3^rdlines from top), 125 nM (blue, 4^thand 5^thlines from top), 62.5 nM (dark green, 6^thline from top), and 31.2 nM (orange, 7^thline from top).

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).

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

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

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

TABLE #T0

Summary of Staining Patterns in Panels of Photographic Images of Cell Samples (from +++ to −)*

Top/

Bottom/

Top
Top
Bottom
Bottom

FIG.
Description
Left
Right
Left
Right

6
Positive 5H4/5K3 staining visualized with DAB (brown) on
+
++
+++
−

Human Hepatocellular Carcinoma at 4 μg/ml, 8 μg/ml, and

10 μg/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 μg/ml, 8 μg/ml, and

10 μg/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, AS 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

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

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, μg=micro gram(s); ul, μl=micro liter(s); uM, μM=micromolar.

Specific abbreviations and their corresponding meanings include:

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.

The term “amino acid” encompasses both naturally occurring and non-naturally occurring amino acids unless otherwise designated.

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.

The term “paratope” refers to a set of CDRs.

DETAILED DESCRIPTION OF THE INVENTION

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.

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.

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

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.

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

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) β-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.

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.

- 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;
- 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;
- 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.

- 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,
- 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;
- 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 [[AND]] LGSYSGYIYI (SEQ ID NO: 41) corresponding to residues 113-122 of SEQ ID NOS: 26, 27, 28, and 30.

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.

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.

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.

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.

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.

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.

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.

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-β, LAG-3, TIM-3, or OX40.

Therapeutic Uses of Compositions Comprising Compounds of the Invention

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

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.

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

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

The foregoing discussion may be better understood in connection with the following representative examples which are presented for purposes of illustrating the principal 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

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#T1

Summary of Sequence IDNumbers

Name
Description
Length
Type
SEQ ID NO:

Human ASPH
Polypeptide corresponding to Human ASPH
758
AA
01

deposited as GenBank Accession No

Q12797, starting at the N-terminus with

MAQRKNAKSSand ending at the C-terminus

with PQQRRSLPAI

Canine ASPH
Polypeptide corresponding to Canine
798
AA
02

ASPH deposited as GenBank Accession No

XP_022267901, starting at the N-

terminus with MAEETKHGGH and ending at

the C-terminus with PQQRHSLPAI

Peptide #H1
KRRSNEVLR corresponding to residues
9
AA
03

391-399 of human ASPH

Peptide #H2
DRQQFLGHM corresponding to residues
9
AA
04

428-436 of human ASPH

Peptide #H3
GYLLIGDNDN corresponding to residues
10
AA
05

463-470 of human ASPH

Peptide #H4
RSLYNVNG corresponding to residues
8
AA
06

562-569 of human ASPH

Peptide #H5
PQQRRSLPAIcorresponding to residues
10
AA
07

749-758 of human ASPH

Peptide #H6
FLPEDENLREcorresponding to residues
10
AA
08

612-621 of human ASPH

Peptide #H7
VWPHTGPTNCcorresponding to residues
10
AA
09

676-685 of human ASPH

Peptide #H8
LWQQGRRNEcorresponding to residues
9
AA
10

630-638 of human ASPH

Peptide #C1
KRRSNEVLR corresponding to residues
9
AA
11

427-435 of canine ASPH

Peptide #C2
DRQQFLGHM corresponding to residues
9
AA
12

464-472 of canine ASPH

Peptide #C3
GYLLIGDNNN corresponding to residues
10
AA
13

499-508 of canine ASPH

Peptide #C4
RSLYNVHG corresponding to residues
8
AA
14

598-605 of canine ASPH

Peptide #C5
PQQRHSLPAIcorresponding to residues
10
AA
15

785-794 of canine ASPH

Peptide #C6
FLPEDENLREcorresponding to residues
10
AA
16

648-657 of canine ASPH

Peptide #C7
VWPHIGPINCcorresponding to residues
10
AA
17

712-721 of canine ASPH

Peptide #C8
LWQQGRKNEcorresponding to residues
9
AA
18

666-674 of canine ASPH

Peptide #1
Synthetic peptide comprising 29 amino
29
AA
19

(CASSF-
acids with Cysteine at its amino

PO3H2)
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.

CASSFRLIFIVDVWHPEL-T(PO3H2)-PQQRRSLPAI

Peptide #2
Synthetic peptide comprising 29 amino
29
AA
20

acids with Cysteine at its amino

terminus, plus 28 amino acids

corresponding to positions 731-758 at

the C-terminal end of human ASPH.

CASSFRLIFIVDVWHPELTPQQRRSLPAI

Clone 1H2
Translated variable region of Clone ID
150
AA
21

#1H2 comprising a GQPK sequence at the

start of the constant region for a

heavy chain sequence.

METGLRWLLLVAVLKGVQCQSLEESGGDLVKPGASLTLT

CTASGLSFSDNFMCWVRQAPGKGLEWIACIYFDSSGITY

YASWAKGRFTISKTSSPTVTLQMTSLTAADTATYFCARD

GPGSISWDLWGQGTLVTVSSGQPKAPSVFPLAP

Clone 1K6
Translated variable region of Clone ID
148
AA
22

#1K6 comprising a GDPV sequence at the

start of the constant region for a

kappa sequence.

MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSPVSAAVGG

TVTISCQSSKSVYSKNRLAWYQQKPGQPPKLLIYEASKL

ASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCQGTYD

SSGWYWAFGGGTEVVVK [Image Omitted]

APTVLIFPPA

Clone 5H1
Translated variable region of Clone ID
142
AA
23

#5H1.

METGLRWLLLVAVLKGVQCQSLEESGGDLVKPGASLTLT

CKASGFDFSSNAMCWVRQAPGKGPEWIACIDNGDGSTDY

ATWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCTRNF

NLWGPGHPGHRLERTAESPVGVSTG

Clone 5H3
Translated variable region of Clone ID
143
AA
24

#5H3 comprising a GQPK sequence at the

start of the constant region for a

heavy chain sequence.

METGLRWLLLVAVLKGVQCQSLEESGGDLVKPGASLTLT

CKASGFDFSSNAMCWVRQAPGKGPEWIACIDNGDGSTDY

ATWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCTRNF

NLWGQGTLVTVSSGQPKAPSVFPLAP

Clone 5H4
Translated variable region of Clone ID
143
AA
25

#5H4 comprising a GQPK sequence at the

start of the constant region for a

heavy chain sequence.

METGLRWLLLVAVLKGVQCQSLEESGGDLVKPGASLTLT

CKASGFDFSSNAMCWVRQAPGKGPEWIACIDNGDGSTDY

ATWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCTRNF

NLWGQGTLVTVSSGQPKAPSVFPLAP

Clone 5K1
1 Translated variable region of Clone
146
AA
26

ID#5K1 comprising a GDPV sequence at

the start of the constant region for a

kappa sequence.

MDTRAPTQLLGLLLLWLPGATFAQVLTQTASSVSAAVGG

TVTISCQSSQSVYDNNRLAWFQQKPGQPPKLLIYETSKL

ASGVPLRFKGSGSGTQFTLTISDLECDDAATYYCLGSYS

GYIYTFGGGTEVVVK [Image Omitted]

APTVLIFPPA

Clone 5K3
Translated variable region of Clone ID
146
AA
27

#5K3 comprising a GDPV sequence at the

start of the constant region for a

kappa sequence.

MDTRAPTQLLGLLLLWLPGATFAQVLTQTASSVSAAVGG

TVTISCQSSQSVYDNNRLAWFQQKPGQPPKLLIYETSKL

ASGVPLRFKGSGSGTQFTLTISDLECDDAATYYCLGSYS

GYIYTFGGGTEVVVK [Image Omitted]

APTVLIFPPA

Clone 5K6
Translated variable region of Clone ID
146
AA
28

#5K6 comprising a GDPV sequence at the

start of the constant region for a

kappa sequence.

MDTRAPTQLLGLLLLWLPGATFAQVLTQTASSVSAAVGG

TVTISCQSSQSVYDNNRLAWFQQKPGQPPKLLIYETSKL

ASGVPLRFKGSGSGTQFTLTISDLECDDAATYYCLGSYS

GYIYTFGGGTEVVVK [Image Omitted]

APTVLIFPPA

Clone 9H2
Translated variable region of Clone ID
142
AA
29

#9H2 comprising a GQPK sequence at the

start of the constant region for a

heavy chain sequence.

METGLRWLLLVAVLKGVQCQSLEESGGDLVKPGASLTLT

CKASGFDFISNAMCWVRQAPGKGPEWIACIDNGDGSTDY

ATWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCTRNF

NLWGQGTL?TVSSGQPKAPSVFPLAP

Clone 9K1
Translated variable region of Clone ID
146
AA
30

#9K1 comprising a GDPV sequence at the

start of the constant region for a

kappa sequence.

MDTRAPTQLLGLLLLWLPGATFAQVLTQTASSVSAAVGG

TVTISCQSSQSVYDNNRLAWFQQKSGQPPKLLIYETSKL

ASGVPLRFKGSGSGTQFTLTISDLECDDAATYYCLGSYS

GYIYTFGGGTEVVVK [Image Omitted]

APTVLIFPPA

Clone 1H2
CDR1 region of clone 1H2 corresponding
4
AA
31

CDR1
to corresponding to residues 50-53 of

SEQ ID NO: 21.

NFMC

Clones 5H1,
CDR1 region of Clones 5H1, 9H2, 5H3,
4
AA
32

9H2, 5H3,
5H4, corresponding to residues 50-53 of

5H4 CDR1
SEQ ID NOS: 23, 29, 24, and 25.

NAMC

Clone 1H2
The CDR2 regions from the heavy chain
4
AA
33

CDR2
clone 1H2 corresponding to residues

68-71 of SEQ ID NO: 21.

CIYF

Clones 5H1,
The CDR2 regions from the heavy chain
4
AA
34

9H2, 5H3,
clones 5H1, 9H2, 5H3, 5H4 corresponding

5H4 CDR2
to residues 68-71 of SEQ ID NO: 23, 29,

24, and 25.

CIDN

Clone 1H2
CDR3 regions from the heavy chain clone
10
AA
35

CDR3
1H2 corresponding to residues 117-126

of SEQ ID NO: 21.

DGPGSISWDI

Clones 5H1,
CDR3 regions from the heavy chain
4
AA
36

9H2, 5H3,
clones 5H1, 9H2, 5H3, 5H4 corresponding

5H4 CDR3
to residues 116-119 of SEQ ID NOS: 23,

29, 24, and 25.

NFNI

Clone 1K6
The CDR1 regions from the kappa chain
7
AA
37

CDR1
clone 1K6, corresponding to residues

50-56 of SEQ ID NO: 22.

SVYSKNR

Clones 5K1,
The CDR1 regions from the kappa chain
7
AA
38

5K3, 5K6,
clones 5K1, 5K3, 5K6, and 9K1

and 9K1
corresponding to residues 50-56 of SEQ

CDR1
ID NOS: 26, 27, 28, and

30.

SVYDNNR

Clones 1K6,
The CDR2 regions from the kappa chain
3
AA
39

5K1, 5K3,
clones 1K6, 5K1, 5K3, 5K6, and 9K1

5K6, and
corresponding to residues 78-80 of SEQ

9K1 CDR2
ID NOS: 22, 26, 27, 28, and 30.

LAS

Clone 1K6
The CDR3 regions from the kappa chain
12
AA
40

CDR3
clone 1K6 corresponding to residues

113-124 of SEQ ID NO: 22.

QGTYDSSGWYWA

Clones 5K1,
The CDR3 regions from the kappa chain
10
AA
41

5K3, 5K6,
clones 5K1, 5K3, 5K6, and 9K1

and 9K1
corresponding to residues 113-122 of

CDR3
SEQ ID NOS: 26, 27, 28, and 30.

LGSYSGYIYI

Peptide
Four aa peptide corresponding to aa
4
AA
42

PELT
745-748 near the carboxy terminus of

human ASPH.

PELT

Peptide
Four aa peptide corresponding to aa
4
AA
43

ELTP
746-749 near the carboxy terminus of

human ASPH.

ELTP

Peptide
Four aa peptide corresponding to aa
4
AA
44

LTPQ
747-750 near the carboxy terminus of

human ASPH.

LTPQ

Peptide
Four aa peptide corresponding to aa
4
AA
45

TPQQ
748-751 near the carboxy terminus of

human ASPH.

TPQQ

Peptide
Four aa peptide corresponding to aa
4
AA
46

PQRR
749-752 near the carboxy terminus of

human ASPH.

PQQR

Peptide
Four aa peptide corresponding to aa
4
AA
47

QQRR
750-753 near the carboxy terminus of

human ASPH.

QQRR

Peptide
Four aa peptide corresponding to aa
4
AA
48

QRSS
751-754 near the carboxy terminus of

human ASPH.

QHRS

Peptide
Four aa peptide corresponding to aa
4
AA
49

RSSL
752-755 near the carboxy terminus of

human ASPH.

RRSL

Peptide
Four aa peptide corresponding to aa
4
AA
50

RSLP
753-756 near the carboxy terminus of

human ASPH.

RSLP

Peptide
Four aa peptide corresponding to aa
4
AA
51

SLPA
754-757 near the carboxy terminus of

human ASPH.

SLPA

Peptide
Four aa peptide corresponding to aa
4
AA
52

LPAI
746-758 near the carboxy terminus of

human ASPH.

LPAI

Locations of Peptides #H1-#H8 Along Human ASPH (758 aa)

ID ASPH_HUMAN Reviewed; 758 AA.

AC Q12797; A0A0A0MSK8; A6NDF4; A6NHI2; B4DIC9; B4E2K4; B7ZM95; E5RGP5;

AC F5H667; Q6NXR7; Q8TB28; Q9H291; Q9H2C4; Q9NRI0; Q9NRI1; Q9Y4J0;

DT 1 Nov. 1997, integrated into UniProtKB/Swiss-Prot.

DT 17 Apr. 2007, sequence version 3.

DT 25 Apr. 2018, entry version 181.

[...Text omitted...]

SQ SEQUENCE 758 AA; 85863 MW; 4AE56D1D8DF0AF0CCRC64;

MAQRKNAKSS GNSSSSGSGS GSTSAGSSSP GARRETKHGG HKNGRKGGLS GTSFFTWFMV 60

IALLGVWTSV AVVWFDLVDY EEVLGKLGIY DADGDGDFDV DDAKVLLGLK ERSTSEPAVP 120

PEEAEPHTEP EEQVPVEAEP QNIEDEAKEQ IQSLLHEMVH AEHVEGEDLQ QEDGPTGEPQ 180

QEDDEFLMAT DVDDRFETLE PEVSHEETEH SYHVEETVSQ DCNQDMEEMM SEQENPDSSE 240

PVVEDERLHH DTDDVTYQVY EEQAVYEPLE NEGIEITEVT APPEDNPVED SQVIVEEVSI 300

FPVEEQQEVP PETNRKTDDP EQKAKVKKKK PKLLNKFDKT IKAELDAAEK LRKRGKIEEA 360

Peptide #H1<391.399>

VNAFKELVRK YPQSPRARYG KAQCEDDLAE KRRSNEVLRG AIETYQEVAS LPDVPADLLK 420

Peptide #H2<428..436> Peptide #H3<463...470>

LSLKRRSDRQ QFLGHMRGSL LTLQRLVQLF PNDTSLKNDL GVGYLLIGDN DNAKKVYEEV 480

LSVTPNDGFA KVHYGFILKA QNKIAESIPY LKEGIESGDP GTDDGRFYFH LGDAMQRVGN 540

Peptide #H4<562569>

KEAYKWYELG HKRGHFASVW QRSLYNVNGL KAQPWWTPKE TGYTELVKSL ERNWKLIRDE 600

Peptide #H6<612...621> #H8<630..638>

GLAVMDKAKG LFLPEDENLR EKGDWSQFTL WQQGRRNENA CKGAPKTCTL LEKFPETTGC 660

Peptide #H7<676...685>

RRGQIKYSIM HPGTHVWPHT GPTNCRLRMH LGLVIPKEGC KIRCANETKT WEEGKVLIFD 720

Peptide #H5<749...758>

DSFEHEVWQD ASSFRLIFIV DVWHPELTPQ QRRSLPAI 758

//

Sequence #5Q2: Locations of Peptides #C1-#C8 Along

Canine ASPH, isoform X1 (794 aa)

LOCUS XP_022267901 794 aa linear MAM 5 Sep. 2017

DEFINITION aspartyl/asparaginyl beta-hydroxylase isoform X1 [Canis lupus

familiaris].

ACCESSION XP_022267901

VERSION XP_022267901.1

[...Text omitted...]

ORIGIN

1 MAEETKHGGH KNGRKGGLSG SSFFTWFMVIA LLGVWTSVA VVWFDLVDYE EVLAKAKDFR

61 YNLSEVLQGK LGVYDADGDG DFDVDDAKVL LGLTKDGSNE NIDSLEEVLN ILAEESSDWF

121 YGFLSFLYDI MTPFEMLEEE EEESETADGV DGLKERSASK PTVPPEEAEP YPWLEEQVIE

181 DSGPQNTEDE VQEVQIESLL HEAVYTEHGD DVQQEEDGQV REPQPEDDFL VGSDTDDRYE

241 PLETGTFHEE TEDSYHIEET ASQAYNQDME EMMYEQDNPD SMEPIVGDDA RTYHEADDLT

301 YQDYDEPVYE PPENEGLESS DNAGEDSNII LEEVYMPPAE EQQEVPPETN RKTDDPEIKE

361 KVKKKKPKLL NKFDKTIKAE LDAAEKLRKR GKIEEALSAF QELVRKYPQS PRARYGKAQC

Peptide #C1<427..435> Peptide #C2<464..472>

421 EDDLAEKRRS NEVLRGAIETYQEVASLPNV PTDLLKLTLK RRSDRQQFLG HMRGSLITLQ

Peptide #C3<499...508>

481 KLVQLFPDDM SLKNDLGVGY LLIGDNNNAQ KVYEEVLNVT PNDGFAKVHY GFILKAQNKI

Peptide #C4<598

541 AESIPYLKEG IESGDPGTDD GRFYFHLGDA MQRVGNKEAY KWYELGHKRG HFASVWQRSL

.605> Peptide #C6<648...657>

601 YNVHGLKAQP WWTPKETGYT ELVKSLERNW KLIRDEGLAV MDKAKGLFLP EDENLREKGD

Peptide #C8<666..674> Peptide #C7<712.....

661 WSQFTLWQQG RKNENACKGA PKTCSLLDKF PETTGCRRGQ IKYSIMHPGT HVWPHTGPTN

.721>

721 CRLRMHLGLV IPKEGCKIRC ANETKTWEEG KVLIFDDSFE HEVWQDATSF RLIFIVDVWH

Peptide #C5<785...794>

781 PELTPQQRHS LPAI

//

Sequence #5Q3: Aligned Human ASPH (758 aa) and Canine ASPH,

Isoform X1 (794 aa) Sequences

Query IDXP_022267901.1

Description aspartyl/asparaginyl beta-hydroxylase isoform X1 [Canis lupus

familiaris]

Molecule type amino acid

Query Length 794

Subject IDQ12797.3

Description RecName: Full = Aspartyl/asparaginyl beta-hydroxylase; AltName:

Full = Aspartate beta-hydroxylase; Short = ASP beta-hydroxylase; AltName:

Full = Peptide-aspartate beta-dioxygenase

Molecule type amino acid

Subject Length 758

Query 1 MAE-------------------------------ETKHGGHKNGRKGGLSGSSFFTWFMV 29

MA+ ETKHGGHKNGRKGGLSG+SFFTWFMV

Sbjct 1 MAQRKNAKSSGNSSSSGSGSGSTSAGSSSPGARRETKHGGHKNGRKGGLSGTSFFTWFMV 60

Query 30 IALLGVWTSVAVVWFDLVDYEEVLAKAKDFRYNLSEVLQGKLGVYDADGDGDFDVDDAKV 89

IALLGVWTSVAVVWFDLVDYEEVL GKLG+YDADGDGDFDVDDAKV

Sbjct 61 IALLGVWTSVAVVWFDLVDYEEVL---------------GKLGIYDADGDGDFDVDDAKV 105

Query 90 LLGLTKDGSNENIDSLEEVLNILAEESSDWFYGFLSFLYDIMTPFEMLEEEEEESETADG 149

LLGL

Sbjct 106 LLGL-------------------------------------------------------- 109

Query 150 VDGLKERSASKPTVPPEEAEPYPWLEEQVIEDSGPQNTEDEVQEVQIESLLHEAVYTEH- 208

KERS S+P VPPEEAEP+ EEQV ++ PQN EDE +E QI+SLLHE V+ EH

Sbjct 110 ----KERSTSEPAVPPEEAEPHTEPEEQVPVEAEPQNIEDEAKE-QIQSLLHEMVHAEHV 164

Query 209 -GDDVQQEEDGQVREPQPEDD-FLVGSDTDDRYEPLETGTFHEETEDSYHIEETASQAYN 266

G+D+QQE DG EPQ EDD FL+ +D DDR+E LE HEETE SYH+EET SQ N

Sbjct 165 EGEDLQQE-DGPTGEPQQEDDEFLMATDVDDRFETLEPEVSHEETEHSYHVEETVSQDCN 223

Query 267 QDMEEMMYEQDNPDSMEPIVGDDARTYHEADDLTYQDYDEP-VYEPPENEGLESS----- 320

QDMEEMM EQ+NPDS EP+V +D R +H+ DD+TYQ Y+E VYEP ENEG+E +

Sbjct 224 QDMEEMMSEQENPDSSEPVV-EDERLHHDTDDVTYQVYEEQAVYEPLENEGIEITEVTAP 282

Query 321 --DNAGEDSNIILEEVYMPPAEEQQEVPPETNRKTDDPEIKEKVKKKKPKLLNKFDKTIK 378

DN EDS +I+EEV + P EEQQEVPPETNRKTDDPE K KVKKKKPKLLNKFDKTIK

Sbjct 283 PEDNPVEDSQVIVEEVSIFPVEEQQEVPPETNRKTDDPEQKAKVKKKKPKLLNKFDKTIK 342

Peptide #C1<427.435>

Query 379 AELDAAEKLRKRGKIEEALSAFQELVRKYPQSPRARYGKAQCEDDLAEKRRSNEVLRGAI 438

AELDAAEKLRKRGKIEEA++AF+ELVRKYPQSPRARYGKAQCEDDLAEKRRSNEVLRGAI

Sbjct 343 AELDAAEKLRKRGKIEEAVNAFKELVRKYPQSPRARYGKAQCEDDLAEKRRSNEVLRGAI 402

Peptide #H1<391.399>

Peptide #C2<464.472>

Query 439 ETYQEVASLPNVPTDLLKLTLKRRSDRQQFLGHMRGSLITLQKLVQLFPDDMSLKNDLGV 498

ETYQEVASLP+VP DLLKL+LKRRSDRQQFLGHMRGSL+TLQ+LVQLFP+D SLKNDLGV

Sbjct 403 ETYQEVASLPDVPADLLKLSLKRRSDRQQFLGHMRGSLLTLQRLVQLFPNDTSLKNDLGV 462

Peptide #H2<428..436>

Peptide #C3<499..508>

Query 499 GYLLIGDNNNAQKVYEEVLNVTPNDGFAKVHYGFILKAQNKIAESIPYLKEGIESGDPGT 558

GYLLIGDN+NA+KVYEEVL+VTPNDGFAKVHYGFILKAQNKIAESIPYLKEGIESGDPGT

*

Sbjct 463 GYLLIGDNDNAKKVYEEVLSVTPNDGFAKVHYGFILKAQNKIAESIPYLKEGIESGDPGT 522

Peptide #H3<463..470>

Peptide #C4<598605>

Query 559 DDGRFYFHLGDAMQRVGNKEAYKWYELGHKRGHFASVWQRSLYNVHGLKAQPWWTPKETG 618

DDGRFYFHLGDAMQRVGNKEAYKWYELGHKRGHFASVWQRSLYNV+GLKAQPWWTPKETG

*

Sbjct 523 DDGRFYFHLGDAMQRVGNKEAYKWYELGHKRGHFASVWQRSLYNVNGLKAQPWWTPKETG 582

Peptide #H4<562569>

Peptide #C6<648..657> #C8<666.674>

Query 619 YTELVKSLERNWKLIRDEGLAVMDKAKGLFLPEDENLREKGDWSQFTLWQQGRKNENACK 678

YTELVKSLERNWKLIRDEGLAVMDKAKGLFLPEDENLREKGDWSQFTLWQQGR+NENACK

*

Sbjct 583 YTELVKSLERNWKLIRDEGLAVMDKAKGLFLPEDENLREKGDWSQFTLWQQGRRNENACK 642

Peptide #H6<612..621> #H8<630.638>

Peptide #C7<712..721>

Query 679 GAPKTCSLLDKFPETTGCRRGQIKYSIMHPGTHVWPHTGPTNCRLRMHLGLVIPKEGCKI 738

GAPKTC+LL+KFPETTGCRRGQIKYSIMHPGTHVWPHTGPTNCRLRMHLGLVIPKEGCKI

Sbjct 643 GAPKTCTLLEKFPETTGCRRGQIKYSIMHPGTHVWPHTGPTNCRLRMHLGLVIPKEGCKI 702

Peptide #H7<676-685>

Peptide #C5<785..794>

Query 739 RCANETKTWEEGKVLIFDDSFEHEVWQDATSFRLIFIVDVWHPELTPQQRHSLPAI 794

RCANETKTWEEGKVLIFDDSFEHEVWQDA+SFRLIFIVDVWHPELTPQQR_SLPAI

*

Sbjct 703 RCANETKTWEEGKVLIFDDSFEHEVWQDASSFRLIFIVDVWHPELTPQQRRSLPAI 758

Peptide #H5<749..758>

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

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 #T2

Peptide Sequences Corresponding to Penultimate

Domain Epitopes of Human and Canine ASPH

Positions
Positions

Epitope

Short
Human
in Canine
SEQ

Domain
Organism
Sequence
Name
ASPH
ASPH
ID NOS

#1
HUMAN/CANINE
KRRSNEVLR
#H1/#C1
391-399
427-435
03/11

#2
HUMAN/CANINE
DEQQFLGHM
#H2/C2
428-436
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
09/17

#8
HUMAN
LWQQGRRNE
#H8
630-638

10

CANINE
LWQQGRKNE
#C8

666-674
18

Example 2—Immunization of Peptide Candidates into Rabbits and Test Bleed

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 #T3

Synthetic Peptide Sequences Used as Immunogens Directed against ASPH

Positions

Short

in Human
Epitope
SEQ

Name
Description/Sequence
ASPH
Domain
ID NOS

Peptide
Synthetic peptide comprising 29 amino
731-758
#5
19

(CASSF-
acids with Cysteine at its amino terminus,

PO3H2)
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.

C-ASSFRLIFIV DVWHPEL-T(PO3H2)-PQ QRRSLPAI

Peptide
Synthetic peptide comprising 29 amino
731-758
#5
20

acids with Cysteine at its amino terminus,

plus 28 amino acids corresponding to

positions 731-758 at the C-terminal end of

human ASPH.

C-ASSFRLIFIV DVWHPELTPQ QRRSLPAI

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.

Peptide #1

(SEQ ID NO: 19)

CASSFRLIFIVDVWHPEL-T(PO3H2)-PQQRRSLPAI

Peptide #2

(SEQ ID NO: 20)

CASSFRLIFIVDVWHPELTPQQRRSLPAI

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 3—In Vitro Culture of Rabbit B Cells

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

Example 4—Screening and Analysis of Antibodies from Rabbit B Cells

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

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′-tetra methyl-benzidine buffer), TMB One Component HRP Microwell Substrate, and BioFx cat #TMBW-1000-01.

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.

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

Cell culture supernatants from positive wells (in a volume of <50 μL) were also preserved.

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

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

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 7—Analysis of Cell Culture Supernatants

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

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.

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

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 #T4

Translated variable region sequences of the top clones

#
Clone ID
Description and Sequence
Length
Type
SEQ ID NO

1
1H2
METGLRWLLLVAVLKGVQCQSLEESGGDLVKPGASLTLTCTAS
250
AA
21

GLSFSDNFMCWVRQAPGKGLEWIACIYFDSSGITYYASWAKGR

FTISKTSSPTVTLQMTSLTAADTATYFCARDGPGSISWDLWGQ

GTLVTVSSGQPKAPSVFPLAP

2
1K6
MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSPVSAAVGGTVTI
148
AA
22

SCQSSKSVYSKNRLAWYQQKPGQPPKLLIYEASKLASGVPSRF

KGSGSGTQFTLTISGVQCDDAATYYCQGTYDSSGWYWAFGGGT

EVVVK [Image Omitted]

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 [Image Omitted]

APTVLIFPPA

7
5K3
MDTRAPTQLLGLLLLWLPGATFAQVLTQTASSVSAAVGGTVTI
146
AA
27

SCQSSQSVYDNNRLAWFQQKPGQPPKLLIYETSKLASGVPLRF

KGSGSGTQFTLTISDLECDDAATYYCLGSYSGYIYTFGGGTEV

VVK [Image Omitted]

APTVLIFPPA

8
5K6
MDTRAPTQLLGLLLLWLPGATFAQVLTQTASSVSAAVGGTVTI
146
AA
28

SCQSSQSVYDNNRLAWFQQKPGQPPKLLIYETSKLASGVPLRF

KGSGSGTQFTLTISDLECDDAATYYCLGSYSGYIYTFGGGTEV

VVK [Image Omitted]

APTVLIFPPA

9
9H2
METGLRWLLLVAVLKGVQCQSLEESGGDLVKPGASLTLTCKAS
142
AA
29

GFDFISNAMCWVRQAPGKGPEWIACIDNGDGSTDYATWAKGRF

TISKTSSTTVTLQMTSLTAADTATYFCTRNFNLWGQGTL?TVS

SGQPKAPSVFPLAP

10
9K1
MDTRAPTQLLGLLLLWLPGATFAQVLTQTASSVSAAVGGTVTI
146
AA
30

SCQSSQSVYDNNRLAWFQQKSGQPPKLLIYETSKLASGVPLRF

KGSGSGTQFTLTISDLECDDAATYYCLGSYSGYIYTFGGGTEV

VVK [Image Omitted]

APTVLIFPPA

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.

Sequence #SQ4: Multiple Sequence Alignment of Heavy Chains for Clones 1H2, 5H1, 9H2, 5H3, and 5H4

A multiple sequence alignment of five clones comprising heavy chains illustrates slight differences in the encoded polypeptide sequences in regions within and just flanking CDR1, CDR2, CDR3, with notable divergence for sequences after CDR3 for clone 5H1.

CLUSTAL O(1.2.4) multiple sequence alignment heavy chains:

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.

Sequence #SQ5: Multiple Sequence Alignment of Kappa Light Chains for Clones 1K6, 5K1, 5K3, 5K6, and 9K1

A multiple sequence alignment of five clones comprising kappa light chains illustrates slight differences in the encoded polypeptide sequences in regions within and just flanking CDR1, CDR2, CDR3, with notable divergence for sequences within CDR3 for clone 1K6.

CLUSTAL O(1.2.4) multiple sequence alignment kappa chains:

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 9—Analysis of MAbs by Immunohistochemistry (IHC)—Phase I—Antibody Triage

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

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 3′3-diaminobenzidine (DAB; brown). A Hematoxylin nuclear counterstain (blue) was also applied.

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 #T5

Results of Antibody Triage

Groups
Antibody
Dilution
Host Species
Antigen Retrieval

1
1H2/1K6

NONE

1H2/1K5

NONE

1H4/1K6

NONE

1H4/1K4

NONE

8 μg/mL
Rabbit

2
2H4/2K5

NONE

5H1/5K1

NONE

5H4/5K3

ER2(20)

9H2/9K1

ER2(20)

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

Example 10A—Analysis of MAbs by Immunohistochemistry (IHC)—Phase II—IHC Optimization

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

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 3′3-diaminobenzidine (DAB; brown). A Hematoxylin nuclear counterstain (blue) was applied.

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

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 10B—Analysis of MAbs by Immunohistochemistry (IHC)—Phase III—IHC on Tissue Micro Arrays

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

Positivity was detected using Novocastra Bond Refine Polymer Detection and visualized with 3′3-diaminobenzidine (DAB; brown). A Hematoxylin nuclear counterstain (blue) was applied.

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).

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

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

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 μm.

FIG. 9 sets forth an illustration demonstrating 5H4/5K3 Phase III on TMAs for samples labeled as PC02 Core A6 (top panel), and PC02 Core A6—Isotype (bottom panel).

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).

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).

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.

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.

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 10C—Analysis of MAbs by Immunohistochemistry (IHC)—Phase III—IHC on Tissue Micro Arrays

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).

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

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

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

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

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

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

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.

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.

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.

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 #T7

Kinetics values for interaction of ASPH with antibodies.

Antibody
ka (M⁻¹s⁻¹)
kd (s⁻¹)
K_D(M)

Mock
—
—
—

2H4/2K5
1.83 ± 0.04e3
1.681 ± 0.002e−3
920 ± 20 nM

5H1/5K1
2.035 ± 0.002e4
2.410 ± 0.002e−3
118.4 ± 0.2 nM

5H4/5K3
1.683 ± 0.002e4
2.426 ± 0.002e−3
144.2 ± 0.2 nM

9H2/9K1
1.879 ± 0.002e4
2.363 ± 0.002e−3
125.7 ± 0.2 nM

9H2/9K3
2.985 ± 0.004e4
1.848 ± 0.003e−3
61.9 ± 0.1 nM

8H1/8K1
—
—
—

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

Experiments to determine the half maximal inhibitory concentration (IC₅₀) of the potency of samples comprising selected antibodies in different types of cultured tumor cells were carried out by Translational Drug Development LLC.

FIG. 26 shows graphs illustrating IC₅₀curves for three samples tested in 4T1 Murine Breast Tumor cells (Panel A, 5H4/5K3; Panel B, 9H2/9K1; and Panel C, Mock Antibody).

FIG. 27 shows graphs illustrating IC₅₀curves 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).

FIG. 28 shows graphs illustrating IC₅₀curves 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 #T8

Summary of IC₅₀Results*

Mean IC₅₀

Mean IC₅₀
Mean IC₅₀
(μg/mL)

(μg/mL)
(μg/mL)
Mock

Cell Line
Tissue Type
5H4/5K3
9H2/9K1
Antibody

4T1
Murine Breast Tumor
0.026
0.008
0.280

MCF-7
Human ER+ Breast Tumor
0.024
0.002
0.426

MV411
Human Mantle Cell
0.098
0.007
0.313

Leukemia

*Mean IC values are calculated as the average of IC₅₀values 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.

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 5H4/5K3.

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

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

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.

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.

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).

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

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.

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).

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

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

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

A non-limiting list of immunomodulating targets includes PD-L1, CTLA-4, TGF-0, LAG-2, TIM-3, and OX40.

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.

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.

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

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

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.

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-β, 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

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

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

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PDB ID SJZZ: McDonough, M. A., Pfeffer, I., Munzel, M. (2016) Aspartyl/Asparaginyl beta-hydroxylase (AspH)oxygenase and TPR domains in complex with manganese, N-oxalylglycine and cyclic peptide substrate mimic of factor X. DOI: 10.2210/pdb5JZZ/pdb. Deposited as PDB ID SJZZ on 2016-05-16, Released on 2017 Jun. 6; Replaced by PDB ID 6RK9 on 2019-05-07.

Dahlen E., Veltonmaki, and Norten, P. (2018) Bispecific antibodies in cancer immunotherapy. Therapeutic Advances in Vaccines and Immunotherapy 6(1): 3-17.