Diagnosis, prognosis and treatment of malignant tumors

Abstract

The present disclosure relates to antibodies and derivatives that bind to the full-length subtype of aspartate beta-hydroxylase (ASPH) and uses thereof. These antibodies and derivatives are useful in compositions and methods for detecting and / or treating a malignant tumor in a subject, as well as in compositions and methods for predicting subsequent complications of progression of an untreated malignant tumor in a subject.

Description

Technical Field

[0001] The present disclosure generally relates to methods for diagnosing and treating mammalian malignancies by contacting malignant cells or body fluids containing components derived from mammalian malignant cells with antibodies that recognize the full-length isoform of human aspartate beta-hydroxylase (ASPH) (Gene ID: 444). Methods for treating mammalian malignancies with monoclonal antibodies (mAbs) or derivatives thereof (such as single chain variable fragments (scFvs) and nanobodies) that recognize the full-length isoform of ASPH linked to a cytotoxic agent are also within the present invention. Background Art

[0002] Cancer or malignancy is a multifactorial disease that was responsible for approximately 9.6 million deaths in 2018. Worldwide, it is the second leading cause of death (Bray et al. (2018)). Complex modifications of the genome and epigenome, influenced by interactions between the host and the environment, lead to the development and progression of cancer. Despite advances in characterizing the molecular mechanisms of tumorigenesis, tumor progression, and metastasis (Coyle et al. (2017)), delayed cancer detection, limited surgical options, treatment resistance, and tumor recurrence are serious obstacles to reducing the morbidity and mortality of cancer. As metastasis is the leading cause of cancer mortality, early detection and design of therapeutics targeting the multistep / multifaceted metastatic mechanisms are critical.

[0003] Improved materials and methods for early detection as well as improved treatments targeting the mechanisms of metastasis are needed. Summary of the invention

[0004] Several pathways have been shown to contribute to the metastasis of malignant tumors. ASPH is a key factor in malignant transformation of solid tumors by enhancing cell proliferation, migration, invasion, cell stemness, angiogenesis, lymphogenesis, migration, dormancy-reactivation-overgrowth at a distance. ASPH also promotes tumor growth by inducing immunosuppression. These effects are partially achieved via activation of Notch, TGFβ, HGF-cMET, HIF and SRC signaling pathways. ASPH expression is necessary for early embryonic development and differentiation, but is silenced after birth and is not detected in normal adult tissues (except placenta) in adulthood. When ASPH reappears and is upregulated by growth factors and hypoxia, tumorigenesis is induced and a malignant phenotype is maintained in a wide range of human tumors. Compared with no or low levels, moderate to high levels of ASPH expression lead to recurrence / recurrence, early progression, metastasis and shortened survival in cancer patients. Therefore, targeting ASPH can have a general and far-reaching clinical impact on cancer diagnosis, prognosis and treatment. This article provides a new ASPH-based diagnosis and anti-metastasis strategy to improve cancer treatment results.

[0005] Embodiments of the present invention provide an LRC1 antibody that binds to the full-length isoform of ASPH. Although hundreds of monoclonal antibodies against ASPH proteins have been generated, the LRC1 antibodies of the present invention provide several important advantages over these prior art techniques. The LRC1 antibodies provide improved sensitivity and specificity for assays designed to detect early tumor formation using liquid biopsies. This is due to the unique and unexpected properties of the LRC1 antibody, including: (1) recognition of the full-length isoform of ASPH that has transforming activity when overexpressed in cells; (2) recognition of the full-length isoform of ASPH in tumors derived from the mesoderm (such as sarcomas, leukemias, and lymphomas); (3) detection of as little as 25 pg of the full-length isoform of ASPH when bound to a solid support; (4) detection of the full-length isoform of ASPH present in biological fluids containing ectosomes and exosomes as a companion diagnostic test for malignant tumors; (5) the described LRC1 mAb-based immunoassay involves the use of only a single LRC1 by using 15-25 μL of peripheral blood from a finger prick. mAbs and lateral flow and microfluidic assays using digital and optical control and automation provide a highly sensitive technology for early cancer diagnosis; (6) the exact epitope (i.e., NPVEDS) to which LRC1 binds on the full-length isoform of ASPH has been determined, which is an oncofetal and oncogenic protein that is specifically and highly expressed on the surface of tumor cells and tumor cell-derived exosomes and ectosomes; and (7) the unique amino acid and nucleic acid sequences of LRC1 mAbs have been determined. Embodiments of the present invention greatly improve their ability to detect cancer at an early stage. The use of a single antibody to have this degree of immunoreactivity against tumors derived from the endoderm, mesoderm, and ectoderm is unprecedented and is associated with the development, progression, and metastasis of human tumors.

[0006] In some embodiments, the LRC1 antibody comprises a light chain and a heavy chain, wherein the light chain comprises a light chain variable region (LCVR), and the heavy chain comprises a heavy chain variable region (HCVR). The LCVR comprises a complementary determining region LCDR1 consisting of the amino acid sequence SEQ ID NO: 5, a LCDR2 consisting of the amino acid sequence SEQ ID NO: 6, and a LCDR3 consisting of the amino acid sequence SEQ ID NO: 7. The HCVR comprises a complementary determining region HCDR1 consisting of the amino acid sequence SEQ ID NO: 2, a HCDR2 consisting of the amino acid sequence SEQ ID NO: 3, and a HCDR3 consisting of the amino acid sequence SEQ ID NO: 4.

[0007] In an alternative embodiment, the LRC1 antibody that binds to the full-length isoform of ASPH comprises a light chain comprising a LCVR having the amino acid sequence of SEQ ID NO: 9 and a heavy chain comprising a HCVR having the amino acid sequence of SEQ ID NO: 8. In another alternative embodiment, the antibody that binds to the full-length isoform of ASPH comprises a light chain comprising the amino acid sequence of SEQ ID NO: 11 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 10. In yet another alternative embodiment, the antibody that binds to the full-length isoform of ASPH comprises two light chains and two heavy chains, each light chain comprising the amino acid sequence of SEQ ID NO: 11 and each heavy chain comprising the amino acid sequence of SEQ ID NO: 10.

[0008] In another aspect, an embodiment of the present invention provides a DNA molecule comprising a polynucleotide sequence encoding a light chain polypeptide having the amino acid sequence of SEQ ID NO: 11. In one embodiment, the DNA molecule encoding the light chain polypeptide comprises the nucleic acid sequence of SEQ ID NO:21.

[0009] In another aspect, an embodiment of the present invention provides a DNA molecule comprising a polynucleotide sequence encoding a heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 10. In one embodiment, the DNA molecule encoding the heavy chain polypeptide comprises the nucleic acid sequence of SEQ ID NO:20.

[0010] In another aspect, an embodiment of the present invention provides a recombinant host cell comprising the above DNA molecule, which can express an antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 10 and a light chain having the amino acid sequence of SEQ ID NO: 11.

[0011] In another aspect, an embodiment of the present invention provides a method for producing an antibody that binds to a full-length isoform of human ASPH, the antibody comprising a heavy chain and a light chain, the heavy chain comprising the amino acid sequence SEQ ID NO: 10, and the light chain comprising the amino acid sequence SEQ ID NO: 11. The method comprises the following steps: (i) culturing the above-mentioned recombinant host cell under conditions such that ASPH mAb is expressed; and (ii) recovering the expressed antibody from the host cell.

[0012] In another aspect, an embodiment of the present invention provides a pharmaceutical composition comprising the above-mentioned antibody and one or more pharmaceutically acceptable carriers, diluents, fillers, adjuvants, conjugates or excipients.

[0013] In another aspect, an embodiment of the present invention provides a method for diagnosing a malignant tumor in a mammal by contacting a body fluid (biological fluid) from the mammal with an antibody that binds to the full-length isoform of ASPH (SEQ ID NO: 1) under conditions sufficient to form an antigen-antibody complex and detecting the antigen-antibody complex. Malignancies detected in this manner include those arising from: (1) endodermal tissues, such as head and neck cancer, thyroid cancer, thymic cancer, non-small cell lung cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, gallbladder cancer, bile duct cancer, pancreatic cancer, colon and rectal cancer, kidney cancer, prostate cancer, breast cancer, ovarian cancer, fallopian tube cancer, and cervical cancer; (2) mesodermal tissues, such as soft tissue sarcomas, osteosarcomas, chondrosarcomas, malignant mesotheliomas, myeloid leukemias, lymphoid leukemias, multiple myeloma, and lymphomas; (3) ectodermal tissues, such as skin cancer, melanoma, central nervous system (CNS) tumors, such as primary malignant CNS tumors of neuronal and glial cell origin, and metastatic CNS tumors (e.g., glioblastoma multiforme (GBM)) are also detected. Patient-derived tissue samples, such as biopsies of solid tumors, and body fluids (biological fluids), such as CNS-derived cerebrospinal fluid (CSF), blood (serum), urine, saliva, sputum, lung effusions, and ascites fluids are contacted with the ASPH-specific antibodies of the invention.

[0014] In another aspect, an embodiment of the present invention provides a pharmaceutical composition comprising the above-mentioned antibody and one or more pharmaceutically acceptable carriers, diluents, fillers, adjuvants, conjugates or excipients.

[0015] The assays described herein can be used to predict the prognosis of (pre)malignant diseases. The method for the prognosis of a mammalian malignant tumor is performed by: (a) contacting a body fluid from a mammal with an ASPH-specific antibody of the present invention under conditions sufficient to form an antigen-antibody complex, and detecting the antigen-antibody complex; (b) quantifying the amount of the complex to determine the ASPH level in the fluid; and (c) comparing the ASPH level in the fluid with a normal tissue (which has a trace or undetectable ASPH level). An increase in ASPH levels over time indicates a gradual worsening of the disease and, therefore, indicates a poor prognosis. If the ASPH level is above a reference level, the subject is identified as having a malignant tumor. When the biological sample is serum, the detected ASPH level is compared to a reference level. When the ASPH level is above the reference level, the subject is further provided with a confirmed diagnosis and subsequent treatment suitable for the malignant tumor. In some embodiments, treatment comprises administering to the subject a composition comprising a therapeutically effective amount of an ASPH-specific antibody of the invention linked to a cytotoxic agent (a substance that kills cells, including cancer cells, which can prevent cancer cells from dividing and growing and can cause a reduction in tumor size), including conventional chemotherapeutic drugs (e.g., doxorubicin, paclitaxel), tubulin inhibitors (maytansinoid alkaloids [DM1 and DM4] or auristatins), DNA damaging agents (e.g., calicheamicin), auristatins (e.g., monomethyl auristatin E (MMAE) and F (MMAF)), derutecan, duocarmycin, SN-38, PE38 (a 38 kDa fragment of Pseudomonas exotoxin A), PBD (pyrrolobenzodiazepine ), radioactive isotopes (e.g., alpha particles, protons), and their derivatives. Malignancies that can be diagnosed using this assay include, but are not limited to, head and neck cancer, thyroid cancer, thymic cancer, non-small cell lung cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, bile duct cancer, gallbladder cancer, pancreatic cancer, colon and rectal cancer, kidney cancer, prostate cancer, breast cancer, ovarian cancer, fallopian tube cancer and cervical cancer, soft tissue sarcoma, osteosarcoma, chondrosarcoma, malignant mesothelioma, myeloid leukemia, lymphoid leukemia, multiple myeloma, lymphoma, skin cancer, melanoma, and glioblastoma.

[0016] The assay formats described herein can also be used to generate temporal data for the prognosis of malignant diseases. The method of monitoring the progress of treatment of a mammalian subject with a malignant tumor is performed by: (a) contacting a first biological sample from a mammalian subject with an antibody that binds to a full-length isoform of ASPH under conditions sufficient to form an antigen-antibody complex, and then detecting and measuring the antigen-antibody complex to determine the level of ASPH in the first sample; (b) administering a therapeutic agent for treating a malignant tumor to the subject; (c) contacting a second post-treatment biological sample from the mammalian subject with an antibody that binds to a full-length isoform of ASPH under conditions sufficient to form an antigen-antibody complex, and then detecting and measuring the antigen-antibody complex to determine the level of ASPH in the second sample; and (c) comparing the levels of ASPH in the two samples. A second level measurement that is significantly lower than the first level measurement indicates that the therapeutic agent is effective, and vice versa. In addition, the therapeutic agent can be a therapeutically effective amount of an ASPH-specific antibody of the present invention linked to a cytotoxic agent. Malignancies that may be monitored using this assay include, but are not limited to, head and neck cancer, thyroid cancer, thymic cancer, non-small cell lung cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, bile duct cancer, gallbladder cancer, pancreatic cancer, colon and rectal cancer, kidney cancer, prostate cancer, breast cancer, ovarian cancer, fallopian tube cancer and cervical cancer, soft tissue sarcoma, osteosarcoma, chondrosarcoma, malignant mesothelioma, myeloid leukemia, lymphoid leukemia, multiple myeloma, lymphoma, skin cancer, melanoma and glioblastoma.

[0017] The present invention also includes diagnostic kits. A kit for detecting tumor cells comprises an antibody or a functionally active fragment thereof that binds to a full-length isoform of ASPH, wherein the antibody or functionally active fragment binds to residues 286-291 of SEQ ID NO: 1. The kit optionally comprises means for detecting binding of the antibody to tumor cells. For example, the kit comprises a detectable marker, such as a non-radioactive marker, such as Gd +++ or Fe ++or radioactive compounds. The kit may also include instructions for use, standard reagents for determining positive antibody binding and / or negative controls for determining lack of antibody binding. These components are packaged together in a kit. In some embodiments, the kit is a diagnostic assay formulated in a standard double antibody sandwich binding format, in which an antibody that binds to the full-length isoform of ASPH captures the ASPH antigen in a patient sample, and another ASPH-specific antibody is used to detect the captured ASPH. For example, the capture antibody is immobilized on a solid phase, such as a component of an assay plate, an assay well, a nitrocellulose membrane, a bead, a dipstick, or an elution column. The second antibody, i.e., the detection antibody, is typically labeled with a detectable label (such as a calorimetric agent or a radioisotope). These components are packaged together in a kit. Malignant tumors that can be diagnosed using the kit include, but are not limited to, head and neck cancer, thyroid cancer, thymic cancer, non-small cell lung cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, bile duct cancer, gallbladder cancer, pancreatic cancer, colon and rectal cancer, kidney cancer, prostate cancer, breast cancer, ovarian cancer, fallopian tube cancer and cervical cancer, soft tissue sarcoma, osteosarcoma, chondrosarcoma, malignant mesothelioma, myeloid leukemia, lymphoid leukemia, multiple myeloma, lymphoma, skin cancer, melanoma and glioblastoma.

[0018] Other implementations are also described and documented herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] For the purpose of illustration, certain embodiments of the invention are shown in the following drawings. It should be understood, however, that the invention is not limited to the precise arrangements, dimensions and instruments shown. In the drawings:

[0020] Figure 1 Shown is a representative example of ASPH overexpression in human chondrosarcoma as shown by immunohistochemistry (IHC) using LRC1 (highlighted by arrows). Magnification = 400×.

[0021] Figure 2 Shown are representative examples of ASPH expression in liposarcoma as shown by IHC using LRC1.

[0022] Figure 3 Shown are representative examples of ASPH expression in retroperitoneal sarcomas as shown by IHC using LRC1.

[0023] Figure 4A line graph depicting the binding of LRC1 to ASPH is provided, which represents the immunodetection of the full-length isoform 1 of the ASPH protein bound to a solid support. The upper line represents the protein OD value per well, and the assay can detect as little as 25 pg of ASPH in a 2-hour assay. The lower line is a control mAb of the same isotype. In this assay, the LRC1 mAb is labeled with biotin and binds to ASPH on the plate, which is detected by an ELISA format.

[0024] Figure 5 Detection of the LRC1 epitope on the cell surface of ectosomes and exosomes from tissue culture of a human hepatocellular carcinoma (HCC) cell line (Huh7) is shown. Comparison was made between detection of cell lysates in ectosomes (top of Figure A) and exosomes (bottom of Figure A) purified according to the procedure outlined in Figure B. Antibody CD9 was selected as an ectosome marker, while CD63 was used as an exosomal marker. Endogenous expression was present in the culture supernatant of Huh7 cells transfected with an empty vector, and levels increased when transfected with a full-length splice variant 1 construct of human ASPH, as shown by increased cellular levels in Western blot analysis. It should be noted that after transfection in Huh7 cells, the LRC1 ASPH epitope is expressed on both ectosomes and exosomes.

[0025] Figure 6 Shown are the measurements of LRC1 monoclonal antibody detection of the full-length isoform of ASPH (splice variant 1) in cancer sera compared to normal controls. Pooled cancer serum samples were from prostate, breast, lung and colon sources at different stages. Approximately 25 μL of serum sample was used for each test. Pooled normal serum samples were derived from five healthy subjects (25 μL per run). There were 22 cancer runs (circles), 20 normal runs (triangles) and four runs with blank buffer solution (squares).

[0026] Figure 7 Early detection of cancer in liquid biopsy is shown. The composite graph shows a clear separation of ASPH levels in cancer patients from normal controls, suggesting that it can be used as a screening assay for early detection of cancer. This is an automated platform using lateral flow and microfluidics-based approaches that include both optical and electronic sensing. DETAILED DESCRIPTION

[0027] The present innovation is now described with reference to the accompanying drawings, wherein the same reference numerals are used to refer to the same elements throughout. In the following description, for the purpose of explanation, many specific details are set forth to provide a comprehensive understanding of the present invention. However, it is apparent that the present invention can be implemented without these specific details. In other cases, well-known structures and devices are shown in block diagram form to facilitate description of the present invention. It should be understood that certain aspects, modes, embodiments, variations and features of the present invention are described below in different levels of detail to provide a full understanding of the present invention.

[0028] definition

[0029] For convenience, the meanings of some terms and phrases used in the specification, examples and appended claims are provided below. Unless otherwise specified or implied by the context, these terms and phrases include the meanings provided below. These definitions are provided to help describe specific embodiments and are not intended to limit the claimed invention, as the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the invention belongs. If there is a clear difference between the use of a term in the art and its definition provided herein, the definition provided in the specification shall prevail.

[0030] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content dictates otherwise. For example, reference to "a cell" includes a combination of two or more cells.

[0031] As used herein, the term "approximately" or "about" when referring to a value or parameter is generally considered to include numbers that fall within 5%, 10%, 15%, or 20% ranges in either direction (greater or less than) of the number, unless otherwise specified or obvious from the context (unless such a number would be less than 0% or more than 100% of the possible value). As used herein, reference to "approximately" or "about" a value or parameter includes and describes embodiments for that value or parameter. For example, a description referring to "about X" includes a description of "X".

[0032] As used herein, the term "or" means "and / or". The term "and / or" as used in phrases such as "A and / or B" herein is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0033] As used herein, the term "comprising" means that there may be additional elements in addition to the defined elements. The use of "comprising" means inclusion rather than limitation.

[0034] The term "consisting of" refers to compositions, methods, and corresponding components thereof as described herein that do not include any elements not recited in the description of the embodiment.

[0035] As used herein, the term "consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristics of that embodiment of the invention.

[0036] The term "statistically significant" or "significant" refers to statistical significance, and typically refers to a difference of two standard deviations (2SD) or greater.

[0037] The term "subject" as used herein refers to a mammal, including but not limited to dogs, cats, horses, cattle, pigs, sheep, goats, chickens, rodents or primates. The subject can be a domestic pet (e.g., dog, cat), agricultural livestock (e.g., cattle, horses, pigs, chickens, etc.), laboratory animals (e.g., mice, rats, rabbits, etc.), but is not limited thereto. The subject includes a human subject. A human subject can be a child, an adult or an elderly subject. A human subject can be of any gender.

[0038] As used herein, the terms "effective amount" and "therapeutically effective amount" include an amount sufficient to prevent or improve the manifestation of a disease or medical condition (such as a malignancy). It should be understood that there are many methods known in the art to determine the effective amount for a given application. For example, pharmacological methods for determining dosage can be used in a therapeutic setting. In therapeutic or preventive applications, the amount of the composition administered to the subject will depend on the type and severity of the disease and the characteristics of the individual, such as general health, age, sex, body weight, and resistance to drugs. It will also depend on the extent, severity, and type of the disease. A skilled person can determine the appropriate dosage based on these and other factors. The composition can also be administered in combination with one or more additional therapeutic compounds.

[0039] The term "treatment" or "improvement" as used herein, when used to refer to a disease, disorder or medical condition, refers to a therapeutic treatment for a condition, wherein the purpose is to reverse, alleviate, improve, inhibit, slow down or stop the progression or severity of a symptom or condition. The term "treatment" includes reducing or alleviating at least one adverse effect or symptom of a condition. If one or more symptoms or clinical parameters are improved, the treatment is usually "effective". Alternatively, if the progression of the condition is reduced or stopped, the treatment is "effective". "Treatment" includes not only the improvement of symptoms or parameters, but also includes stopping or at least slowing down the progression or deterioration of symptoms expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, the alleviation of one or more symptoms, the reduction of the degree of defects, the stabilization (i.e., non-deterioration) of tumors or malignant tumor states, tumor growth and / or metastasis, and the delay or slowing down of increased life expectancy compared to what would be expected in the absence of treatment.

[0040] As used herein, the term "long-term" administration means that the therapeutic agent or drug is administered for a period of at least 12 weeks. This includes administering a therapeutic agent or drug so that it is effective over a period of at least 12 weeks or it is effective for at least 12 weeks, and does not necessarily mean that the administration itself occurs for 12 weeks, such as if a sustained-release composition or a long-acting therapeutic agent or drug is used. Therefore, the subject receives treatment for a period of at least 12 weeks. Typically, long-term administration lasts for at least 4, 5, 6, 7, 8, 9 months or more, or lasts for at least 1, 2, 3, 5, 7 or 10 years or more.

[0041] The administration of the compositions contemplated herein can be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, infusion, implantation or transplantation. In a preferred embodiment, the composition is administered parenterally. As used herein, the phrases "administered parenterally" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, and include but are not limited to intravascular, intralymphatic, intralymphatic, intravenous, intraportal, intrahepatic artery, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, intranasal, intratracheal, intrathecal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. In one embodiment, the compositions contemplated herein are administered to a subject by direct injection into a tumor, lymph node or site of infection.

[0042] The terms "reduce", "reduce", "reduction" or "inhibit" are all used herein to indicate a reduction in a statistically significant amount. In some embodiments, "reduce" or "reduce" or "inhibit" generally means a reduction of at least 10% compared to a reference level (e.g., in the absence of a given treatment or agent), and may include, for example, a reduction of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more. "Reduce" or "inhibit" as used herein does not encompass complete inhibition or reduction compared to a reference level. "Complete inhibition" is 100% inhibition compared to a reference level. The reduction may preferably be reduced to a level that is within the normal range and accepted for individuals without a given disease.

[0043] The terms "increased", "increase", "enhancement" or "activation" are all used herein to indicate an increase in a statistically significant amount. In some embodiments, the terms "increased", "increase", "enhancement" or "activation" may represent an increase of at least 10% compared to a reference level, such as an increase of at least about 20% or at least about 30% or at least about 40% or at least about 50% or at least about 60% or at least about 70% or at least about 80% or at least about 90% or up to and including an increase of 100% or any increase between 10-100% compared to a reference level, or an increase of at least about 2 times or at least about 3 times or at least about 4 times or at least about 5 times or at least about 10 times, or any increase between 2 times to 10 times or greater compared to a reference level. In markers or symptoms, "increase" is a statistically significant increase in such levels.

[0044] Cancer-related definitions :

[0045] As used herein, the term "cancer" generally refers to a class of diseases or conditions in which abnormal cells divide uncontrollably and can migrate and invade nearby tissues. Cancer cells can also spread to other parts of the body through the blood and lymphatic system. There are several major types of cancer. Carcinomas are cancers that begin in the skin or tissues lining or covering internal organs. Sarcomas are cancers that begin in bones, cartilage, fat, muscles, blood vessels, or other connective or supporting tissues. Leukemias are cancers that begin in blood-forming tissues (such as bone marrow) and cause large numbers of abnormal blood cells to be produced and enter the blood. Lymphomas and multiple myeloma are cancers that begin in immune system cells. Central nervous system cancers are cancers that begin in brain and spinal cord tissues.

[0046] In some embodiments, cancer is a primary cancer. In some embodiments, cancer is a malignant cancer, which is also referred to as a malignant tumor. As used herein, the term "malignant" refers to a cancer in which a tumor cell group shows uncontrolled growth (i.e., division beyond normal limits), invasion (i.e., invasion and destruction of adjacent tissues) and metastasis (i.e., spread to other locations of the body via lymph or blood). As used herein, the term "metastasis" refers to cancer spreading from one part of the body to another. The tumor formed by the cells that spread is called a "metastatic tumor" or "metastasis". Metastatic tumors contain cells similar to those in the original (primary) tumor.

[0047] As used herein, the term "benign" or "non-malignant" refers to a tumor that may grow larger but does not spread to other parts of the body. Benign tumors are self-limited and rarely invade or metastasize.

[0048] "Cancer cell" or "tumor cell" refers to a single cell of a cancerous growth or tissue. A tumor generally refers to a mass or lesion formed by an abnormal growth of cells, which may be benign, premalignant, or malignant. Most cancer cells form solid tumors, but some, such as leukemias, do not necessarily form solid tumors. For those cancer cells that form solid tumors, the terms cancer (cell) and tumor (cell) are used interchangeably.

[0049] A subject with cancer or a tumor is one in which objectively measurable cancer cells are present in the subject's body. Included in this definition are malignant, actively proliferating cancers and potentially dormant tumors or micrometastases. Cancers that migrate from their original location, invade, and colonize other vital organs can ultimately lead to the death of the subject through deterioration of the function of the affected organs. Hematopoietic cancers, such as leukemias, can overwhelm the normal hematopoietic compartments in the subject's body, leading to hematopoietic failure in the form of anemia, thrombocytopenia, and neutropenia, ultimately leading to death.

[0050] Examples of cancer include, but are not limited to, carcinoma, adenocarcinoma, intraepithelial neoplasm; blastomas such as neuroblastoma; brain and CNS cancers such as glioblastoma multiforme (GBM); head and neck cancer; eye cancers such as retinoblastoma; oral cancer (e.g., lip, tongue, mouth, throat, and pharynx); salivary gland cancer; thyroid cancer; thymic cancer; respiratory cancers such as lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); peritoneal cancer; digestive system cancer; esophageal cancer; stomach cancer (including gastrointestinal cancer); hepatocellular carcinoma; hepatoma; biliary tract cancer; pancreatic cancer; colon and rectal cancer; urinary system cancers such as bladder cancer and kidney cancer; prostate cancer; testicular cancer; breast cancer; ovarian cancer; cervical cancer; uterine cancer or endometrial cancer; vulvar cancer; skin cancer; basal cell carcinoma, squamous cell carcinoma; melanoma; sarcomas such as rhabdomyosarcoma, osteosarcoma, and chondrosarcoma; connective tissue cancer; leukemia , bone cancer; lymphomas, including Hodgkin and non-Hodgkin lymphomas; multiple myeloma; other carcinomas and sarcomas; and B-cell lymphomas (including low-grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small noncleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom’s macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD) and abnormal blood vessel proliferation associated with keloids, edema (such as that associated with brain tumors), and Meigs syndrome.

[0051] "Cancer cells" are cancerous, precancerous, malignant or transformed cells in vivo, in vitro or in tissue culture, with spontaneous or induced phenotypic changes that do not necessarily involve the uptake of new genetic material. Although transformation can be caused by infection with a transforming virus and the incorporation of new genomic nucleic acids or the uptake of exogenous nucleic acids, it can also occur spontaneously or after exposure to a carcinogen, thereby mutating endogenous genes. Transformation / cancer is associated with, for example, morphological changes, cell immortalization, abnormal growth control, focus formation, anchoring independence, contact inhibition or loss of growth density restrictions, growth factor or serum independence, up-regulated / highly expressed tumor-specific markers, invasiveness or metastasis, and growth of tumors in suitable animal hosts (such as immunocompromised, immunodeficient or even immunocompetent (for cancers of mouse origin) mice).

[0052] The subject may be a subject who has been diagnosed or determined to have or has a condition (e.g., cancer) that requires treatment, or one or more complications associated with such a condition, and who may, but need not, have received treatment for the condition or one or more complications associated with the condition. The subject may also be a subject who has not been previously diagnosed as having a condition that requires treatment, or one or more complications associated with such a condition. For example, the subject may exhibit one or more risk factors for the condition, or one or more complications associated with the disease, or a subject who does not exhibit risk factors. A "subject in need of treatment for a particular condition" may be a subject who has the condition, is diagnosed with the condition, or is at risk of developing the condition.

[0053] Biopharmaceutical Definition :

[0054] As used herein, the terms "protein" and "polypeptide" are used interchangeably herein to refer to a series of amino acid residues interconnected by peptide bonds between the α-amino and carboxyl groups of adjacent residues. The terms "protein" and "polypeptide" refer to polymers of amino acids, including modified amino acids (e.g., phosphorylated, glycosylated, SUMOylated, farnesylated, etc.) and amino acid analogs, regardless of their size or function. "Protein" and "polypeptide" are generally used to refer to relatively large polypeptides, but the term "peptide" is generally used to refer to small polypeptides, but the usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to gene products and fragments thereof. Therefore, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments and analogs of the aforementioned.

[0055] In the embodiments described herein, variants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants, and / or conservatively substituted variants of any of the specific polypeptides described are further contemplated. With respect to amino acid sequences, the skilled artisan will recognize that a single substitution, deletion, insertion, or suppressor mutation of a nucleic acid, peptide, polypeptide, or protein sequence that changes a single amino acid or a small portion of amino acids in the coding sequence is a "conservatively modified variant," wherein the change results in the replacement of an amino acid with a chemically similar amino acid, and the desired activity of the polypeptide is retained. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles consistent with the present disclosure.

[0056] In some embodiments, the polypeptides described herein (or nucleic acids encoding such polypeptides) may be functional fragments of one of the amino acid sequences described herein. As used herein, a "functional fragment" is a peptide fragment or segment that retains at least 50% of the activity of a wild-type reference polypeptide as determined as described below. A functional fragment may comprise conservative substitutions of the sequences disclosed herein.

[0057] In some embodiments, the polypeptides described herein may be variants of the sequences described herein. In some embodiments, the variants are conservatively modified variants. For example, conservative substitution variants may be obtained by mutation of a native nucleotide sequence. As referred to herein, a "variant" is a polypeptide substantially homologous to a native or reference polypeptide, but due to one or more deletions, insertions, substitutions or suppression mutations, it has an amino acid sequence different from the amino acid sequence of a native or reference polypeptide. The DNA sequence encoding the variant polypeptide encompasses sequences that contain one or more nucleotide insertions, deletions or substitutions compared to a native or reference DNA sequence, but encodes a variant protein or fragment thereof that retains activity or function. A variety of PCR-based site-directed mutagenesis methods are known in the art, and those of ordinary skill may apply these methods.

[0058] As used herein, the term "nucleic acid" or "nucleic acid sequence" refers to any molecule, preferably a polymer molecule, of a unit or any analog thereof that is incorporated into ribonucleic acid, deoxyribonucleic acid or an artificial nucleic acid analog (e.g., peptide nucleic acid, morpholino nucleic acid and locked nucleic acid, glycol nucleic acid, threose nucleic acid and hexitol nucleic acid). Nucleic acid can be single-stranded or double-stranded. A single-stranded nucleic acid can be a nucleic acid chain of a denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid that is not derived from any double-stranded DNA. On the one hand, nucleic acid can be DNA. On the other hand, nucleic acid can be RNA. Suitable DNA can include, for example, genomic DNA or cDNA. Suitable RNA can include, for example, mRNA.

[0059] In some embodiments, polypeptides, nucleic acids or cells as described herein may be engineered. "Engineering" as used herein refers to aspects that have been artificially manipulated. For example, when at least one aspect of a polypeptide (e.g., its sequence) has been artificially manipulated to be different from the aspects that exist in nature, the polypeptide is considered to be "engineered". As common practice and as understood by those skilled in the art, the offspring of engineered cells are usually still referred to as "engineered" even if the actual manipulation is performed on a previous entity.

[0060] In some embodiments, a nucleic acid encoding a polypeptide as described herein (e.g., an antibody or an antibody reagent) is contained in a vector. In some aspects described herein, a nucleic acid sequence encoding a given polypeptide as described herein or any module thereof is operably connected to a vector. The vector may include, but is not limited to, a cloning vector, an expression vector, a plasmid, a phage, a transposon, a cosmid, a chromosome, a virus, an oncolytic virus-like vesicle (VLV), a virion, an extracellular vesicle, etc.

[0061] As used herein, the term "expression vector" refers to a vector for the expression of a sequence that guides RNA or a polypeptide from a vector connected to a transcriptional regulatory sequence. The expressed sequence is usually, but not necessarily, heterologous to the cell. An expression vector may contain more elements, for example, an expression vector may have two replication systems, thereby allowing it to be maintained in two organisms, such as in human cells for expression, and in a prokaryotic host for cloning and amplification. The term "expression" refers to a cellular process involving the production of RNA, peptides and proteins, and secretion of peptides or proteins under appropriate circumstances, including, but not limited to, transcription, transcript processing, translation and protein folding, modification, degradation, shuttling, reorganization and processing when applicable. "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translating mRNA transcribed from a gene. The term "gene" means a nucleic acid sequence that is transcribed (DNA) into RNA in vitro, in vitro or in vivo when operably linked to an appropriate regulatory sequence. A gene may include regions preceding and following the coding region, such as a 5' untranslated sequence (5'UTR) or "leader" sequence and a 3'UTR, a "trailer" sequence or "leader", and intervening sequences (introns) or spacers between individual coding segments (exons).

[0062] As used herein, the term "isolated" or "partially purified" refers to a nucleic acid or polypeptide separated from at least one other component (e.g., nucleic acid or polypeptide) in the case of a nucleic acid or polypeptide, wherein the other components are present together with the nucleic acid or polypeptide as found in the natural source of the nucleic acid or polypeptide, and / or when expressed by a cell or secreted in the case of a secreted polypeptide, the nucleic acid or polypeptide is present together. Chemically synthesized nucleic acids or polypeptides or nucleic acids or polypeptides synthesized using in vitro transcription / translation are considered to be "isolated". The term "purified" or "substantially purified" refers to an isolated nucleic acid or polypeptide, which is at least 95% of the subject nucleic acid or polypeptide by weight, including at least 96%, at least 97%, at least 98%, at least 99% or more. In some embodiments, the antibodies described herein, their antigen-binding portions (e.g., scFv or nanobodies), bispecific or trispecific T cell adapters, bispecific or trispecific NK cell adapters, immune cell recruiters (recruiters) or chimeric antigen receptors (CARs) are separated. In some embodiments, the antibodies, antibody reagents, antigen binding portions thereof, adapters, recruiters, or CARs described herein are purified.

[0063] As used herein, "engineering" refers to aspects that have been manipulated manually. For example, when antibodies, antibody reagents, antigen binding portions thereof (e.g., scFv or nano antibodies) or trispecific T cell adapters, bispecific or trispecific NK cell adapters, immune cell recruiters (such as bifunctional checkpoint inhibitory T cell adapters (CiTE), simultaneous multiple interaction T cell adapters (SMITE), immune mobilization monoclonal TCRs (ImmTACs) for cancer), CAR sequences are manipulated manually to be different from the antibody sequences present in nature, these antibodies, antibody reagents, antigen binding portions thereof, adapters, recruiters or CARs are considered to be "engineered". As common practice and those skilled in the art understand, even if the actual manipulation is performed on a previous entity, the progeny and copies of engineered polynucleotides and / or polypeptides are still generally referred to as "engineered".

[0064] Antibody related definitions :

[0065] As used herein, "epitope" can be formed on a polypeptide by continuous amino acids and non-continuous amino acids juxtaposed by the tertiary folding of the protein. Epitopes formed by continuous amino acids are usually retained when exposed to denaturing solvents, but epitopes formed by tertiary folding are usually lost when treated with denaturing solvents. Epitopes usually include at least 3 amino acids in a unique spatial conformation, and more commonly at least 5, about 9 or about 8-10 amino acids. "Epitope" includes structural units that are usually bound by immunoglobulin VH / VL pairs. The epitope defines the minimum binding site of an antibody and represents a target specific to the antibody. In the case of a single domain antibody, an epitope represents a structural unit bound by a separate variable domain. The terms "antigenic determinant" and "epitope" are also used interchangeably herein. In certain embodiments, an epitope determinant includes a chemically active surface grouping of a molecule, such as an amino acid, a sugar side chain, a phosphoryl group or a sulfonyl group, and in certain embodiments, may have specific three-dimensional structural features and / or specific charge features.

[0066] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules containing an antigen binding site that immunospecifically binds to an antigen. The term also refers to antibodies comprising two immunoglobulin heavy chains and two immunoglobulin light chains, as well as various forms including full-length antibodies and antigen-binding portions thereof; including immunoglobulin molecules, monoclonal antibodies, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, Fab, Fab', F(ab')2, Fv, disulfide-linked Fv, scFv, single domain antibodies (dAb), diabodies, nanobodies, multispecific (e.g., trispecific) antibodies, dual-specific antibodies, bispecific antibodies, anti-idiotypic antibodies, functionally active epitope binding portions thereof, and / or bifunctional hybrid antibodies.

[0067] Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region (CL). The light chain constant region consists of the CL domain. The VH and VL regions can be further divided into hypervariable regions called complementarity determining regions (CDRs) and interlaced with conserved regions called framework regions (FRs). Thus, each VH and VL region consists of three CDRs and four FRs, which are arranged from N-terminus to C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. This structure is well known to those skilled in the art.

[0068] The term "CDR" as used herein refers to the complementary determining region within the variable sequence of an antibody. There are three CDRs in each of the variable regions of the heavy and light chains, which are designated as CDR1, CDR2 and CDR3 for each variable region. The exact boundaries of these CDRs are defined differently according to different systems. The system described by Kabat et al. (1987 and 1991) not only provides a clear residue numbering system applicable to any variable region of an antibody, but also provides the precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Padlan (1995), MacCallum (1996), Chothia and Lesk (1987) and Chothia et al. (1989) describe other boundaries of the defined CDRs that overlap with the Kabat CDRs. There are also other CDR boundary definitions that may not strictly follow one of the above systems, but will still overlap with the Kabat CDRs, although they may be shortened or extended, but considering predictions or experimental results, these residues or residue groups or even the entire CDR will not significantly affect antigen binding. The methods used herein may use CDRs defined according to any of these systems, although preferred embodiments use the Kabat defined CDRs.

[0069] The term "antigen binding portion" of an antibody refers to one or more parts of an antibody as described herein, which still have binding affinity and avidity as defined herein above. Portions of an intact antibody are already able to perform the antigen binding function of an antibody. Under the term "antigen binding portion" of an antibody, examples of binding portions include (i) a Fab portion, i.e., a monovalent portion consisting of VL, VH, CL and CH1 domains; (ii) a F(ab')2 portion, i.e., a bivalent portion comprising two Fab portions interconnected in the hinge region via a disulfide bridge; (iii) a Fd portion consisting of VH and CH1 domains; (iv) a Fv portion consisting of the VL and VH domains of a single arm of an antibody; and (v) a dAb portion consisting of a VH domain or consisting of VH, CH1, CH2, DH3 or VH, CH2, CH3 (dAbs or single domain antibodies comprising only a VL domain are also shown to specifically bind to a target epitope). Although the two domains of the Fv part, i.e., VL and VH, are encoded by different genes, they can be further connected to each other using a synthetic linker, such as a poly-G4S amino acid sequence (disclosed as "G4S" of SEQ ID NO: 29 in U.S. Patent No. 10,253,111) and a recombinant method, so that they can be prepared as a single protein chain, wherein the VL and VH regions are combined to form a monovalent molecule (referred to as a single-chain variable fragment (scFv)). The term "antigen-binding portion" of an antibody is also intended to include such single-chain antibodies. Other forms of single-chain antibodies, such as nanobodies, "diabodies" are also included herein. A truncated version (modified single variable domain) of a heavy chain antibody (HCAb, VHH) from Camelidae is also referred to as a nanobody, which contains only one-tenth of the mass of a conventional antibody, but retains a similar high binding affinity to the antigen. Diabodies are bivalent bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but a linker that is too short to allow the two domains to bind on the same chain is used, thereby forcing the domains to pair with complementary domains of different chains and form two antigen binding sites. An immunoglobulin constant domain refers to a heavy chain or light chain constant domain. The amino acid sequences of human IgG heavy and light chain constant domains are known in the art.

[0070] As used herein, the term "antibody reagent" refers to a polypeptide that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and specifically binds to a given antigen. Antibody reagents may include antibodies or polypeptides containing the antigen-binding domains of antibodies. In some embodiments, antibody reagents may include monoclonal antibodies or polypeptides containing the antigen-binding domains of monoclonal antibodies. For example, an antibody may include a heavy chain (H) variable region (abbreviated herein as VH) and a light chain (L) variable region (abbreviated herein as VL). In another example, an antibody includes two heavy chain (H) variable regions and two light chain (L) variable regions. The term "antibody reagent" encompasses antigen-binding fragments of antibodies (e.g., single-chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv and domain antibodies (dAb) fragments and complete antibodies.

[0071] Antibodies may have structural features of IgA, IgG, IgE, IgD, IgM (and their subtypes and combinations). Antibodies may be from any source, including mice, rabbits, pigs, rats, and primates (human and non-human primates), as well as primatized (e.g., macaque / human chimeric) antibodies. Antibodies also include minibodies (artificial short chains of amino acids copied from fully functional natural antibodies), midibodies, humanized antibodies, chimeric (e.g., mouse / human) antibodies.

[0072] Antibodies as described herein, their antigen binding portions, adapters, recruiters or CARs can be part of larger immune adhesion molecules, which are formed by covalent or non-covalent association of antibodies or antibody moieties with one or more other proteins or peptides. Related to such immune adhesion molecules is the use of streptavidin core regions to prepare tetrameric scFv molecules, and the use of cysteine ​​residues, marker peptides and C-terminal polyhistidine, such as hexa-histidine tags (disclosed as SEQ ID NO:30 "hexa-histidine tags" in U.S. Patent No. 10,253,111) to produce divalent and biotinylated scFv molecules.

[0073] In some embodiments, the antibodies, antibody reagents, antigen binding portions thereof, adapters, recruiters or CARs described herein can be immunoglobulin molecules, monoclonal antibodies, chimeric antibodies, CDR-grafted antibodies, primatized antibodies, humanized antibodies, fragment antibodies, Fab, Fab′, F(ab′)2, Fv, disulfide-linked Fv, scFv, single domain antibodies, multidomain antibodies, multispecific (e.g., trispecific) antibodies, dual-specific antibodies, bispecific antibodies, anti-idiotypic antibodies, diabodies, nanobodies, minibodies, midibodies, and functionally active epitope binding portions thereof.

[0074] In some embodiments, the antibody or its antigen binding portion is a fully human antibody. In some embodiments, the antibody, its antigen binding portion is a humanized antibody or an antibody reagent. In some embodiments, the antibody, its antigen binding portion is a fully humanized antibody or an antibody reagent. In some embodiments, the antibody or its antigen binding portion is a chimeric antibody, a primatized antibody or an antibody reagent. In some embodiments, the antibody, its antigen binding portion is a recombinant polypeptide. In some embodiments, CAR comprises an extracellular domain that binds to a full-length subtype of ASPH, wherein the extracellular domain comprises a humanized or chimeric antibody or its antigen binding portion.

[0075] The term "human antibody" refers to an antibody whose variable region and constant region correspond to or are derived from human germline immunoglobulin sequences, for example, as described by Kabat et al. (1991). However, human antibodies may contain amino acid residues that are not encoded by human germline immunoglobulin sequences, for example, in CDR, and particularly in CDR3 (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutations in vivo). Recombinant human antibodies as described herein have variable regions and may also contain constant regions derived from human germline immunoglobulin sequences. See Kabat et al. (1991). However, according to specific embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or in vivo somatic mutagenesis in the case of using transgenic animals due to human Ig sequences), so that the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that are not naturally present in the human antibody germline library in vivo, although they are related to or derived from the VH and VL sequences of the human germline. According to a specific embodiment, the recombinant antibody is produced by selective mutagenesis or back mutation (a change in the nucleotide pairs in the mutant gene, which restores the original sequence and thus the original phenotype, a process leading to recovery) or both. Preferably, the mutagenesis results in an affinity for the target greater than that of the parent antibody, and / or an affinity for non-target structures less than that of the parent antibody. One of ordinary skill in the art can generate humanized antibodies from the sequences and information provided herein without much experimentation. In one approach, there are four general steps for humanizing monoclonal antibodies, see, for example, U.S. Patent Nos. 5,585,089, 6,835,823, and 6,824,989. These steps are: (1) determining the nucleotides and predicted amino acid sequences of the starting antibody light chain and heavy chain variable domains; (2) designing the humanized antibody, i.e., deciding which antibody framework region to use during the humanization process; (3) actual humanization methods / techniques; and (4) transfection and expression of the humanized antibody.

[0076] In some embodiments, antibodies, antibody reagents, antigen-binding portions thereof, adapters, recruiters and / or CARs as described herein may be variants of sequences described herein, such as conservative substitution variants of antibody polypeptides. In some embodiments, variants are conservatively altered variants. Conservative substitution variants can be obtained, for example, by mutations of natural nucleotide sequences. As referred to herein, "variants" are polypeptides that are substantially homologous to natural or reference polypeptides, but have amino acid sequences that are different from the amino acid sequences of natural or reference polypeptides due to one or more deletions, insertions, substitutions or suppression mutations. The DNA sequence encoding the variant polypeptide encompasses sequences that contain one or more nucleotide additions, deletions, substitutions or suppression mutations compared to natural or reference DNA sequences, but encode variant proteins or portions thereof that retain activity (e.g., antigen-specific binding activity to related target polypeptides, such as full-length subtypes of ASPH). A variety of PCR-based site-specific mutagenesis methods are also known in the art, and ordinary technicians can apply these methods.

[0077] Typically, the CDR regions in humanized antibodies and human antibody variants are substantially identical, and more typically, identical to the corresponding CDR regions in the mouse or human antibody from which they are derived. In some embodiments, one or more conservative amino acid substitutions may be made to the CDR residues without significantly affecting the binding affinity or avidity of the resulting humanized immunoglobulin or human antibody variant. In some embodiments, substitutions in the CDR regions may enhance binding affinity and / or avidity.

[0078] The term "chimeric antibody" refers to an antibody containing sequences of variable regions of heavy and light chains from one species and constant region sequences from another species, such as antibodies having mouse heavy and light chain variable regions connected to human constant regions. Humanized antibodies have variable region framework residues substantially from human antibodies (referred to as acceptor antibodies) and complementary determining regions substantially from non-human antibodies, such as mouse antibodies (referred to as donor immunoglobulins). Constant regions (if present) are also substantially or completely from human immunoglobulins. Human variable domains are generally selected from human antibodies whose framework sequences show a high degree of sequence identity with (e.g., mouse) variable region domains derived from CDRs. Heavy and light chain variable region framework residues may be substantially similar to regions of identical or different human antibody sequences. Human antibody sequences may be sequences of naturally occurring human antibodies, or may be consensus sequences of several human antibodies.

[0079] In addition, the technology developed for producing "chimeric antibodies" by splicing genes from mice or other species (e.g., rats, rabbits) (antibody molecules with appropriate antigen specificity) with genes from human antibody molecules with appropriate biological activity can also be used. The variable segment of a chimeric antibody is usually connected to at least a portion of an immunoglobulin constant region (Fc) (usually a constant region of a human immunoglobulin). Well-known procedures can be used to separate human constant region DNA sequences from various human cells, such as immortalized B cells. The antibody may contain a light chain and a heavy chain constant region. The heavy chain constant region may include CH1, hinge, CH2, CH3, and sometimes CH4 regions. For therapeutic purposes, the CH2 domain may be deleted or omitted.

[0080] In addition, recombinant humanized antibodies can be further optimized to reduce potential immunogenicity while maintaining functional activity for human treatment. Functional activity refers to a polypeptide that can exhibit one or more known functional activities associated with a recombinant antibody, an antigen binding portion thereof, or a CAR as described herein. Such functional anti-cancer activities include recognition, binding, and direct or indirect killing of cancer cells. In addition, a functionally active polypeptide means that as measured in a specific assay (e.g., a bioassay), the polypeptide shows an activity similar to but not necessarily identical to that of a reference antibody, an antigen binding portion thereof, an adapter, a collector, or a CAR (including mature forms) as described herein, with or without dose dependence. When there is a dose dependency, it need not be the same as the dose dependency of the reference antibody, antigen binding portion thereof, adapter, recruiter or CAR, but rather it is substantially similar to the dose dependency of a given activity compared to the reference antibody, antigen binding portion thereof, adapter, recruiter or CAR as described herein (i.e., the candidate polypeptide will exhibit a higher activity, or not less than about 1 / 25, about 1 / 10, or about 1 / 3 the activity relative to the antibody, antigen binding portion, adapter, recruiter and / or CAR described herein).

[0081] In some embodiments, antibody reagents described herein (e.g., antibodies, adapters, recruiters, or CARs) are not naturally occurring biomolecules. For example, in the absence of human intervention and manipulation (e.g., manufacturing steps performed by humans), mouse antibodies produced for antigens of human origin do not occur in nature. Chimeric antibodies are also not naturally occurring biomolecules, for example, because they contain sequences obtained from multiple species and assembled into recombinant molecules. In certain specific embodiments, human antibody reagents described herein are not naturally occurring biomolecules, such as fully human antibodies for human antigens that are subjected to negative selection in nature and are not naturally found in the human body.

[0082] In some embodiments, antibodies, antibody reagents, antigen binding portions thereof, adapters, collectors and / or CARs are isolated polypeptides. In some embodiments, antibodies, antibody reagents, antigen binding portions thereof, adapters, collectors and / or CARs are purified polypeptides. In some embodiments, antibodies, antibody reagents, antigen binding portions thereof, adapters, collectors and / or CARs are engineered polypeptides.

[0083] "Avidity" is a measure of the strength of binding between an antigen binding molecule (such as an antibody or antigen binding portion thereof described herein) and the relevant antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the antigen binding molecule and the number of relevant binding sites present on the antigen binding molecule. Typically, an antigen binding protein (such as an antibody or antibody portion described herein) will be expressed as 10 -5 Up to 10 -12 mol / L or lower, such as 10 -7 Up to 10 -12 mol / L or less, or 10 -8 Up to 10 -12 The dissociation constant (K) in mol / L D )(i.e. 10 5 Up to 10 12 L / mol or higher, such as 10 7 Up to 10 12 Liter / mole or 10 8 Up to 10 12 The association constant (K A )) binds to its cognate or specific antigen. Any -4 mol / L K D Value (or any value below 10 4 M -1 K A The K values ​​for biological interactions that are considered meaningful (e.g., specific) are generally considered to represent nonspecific binding. D Usually in 10 -10 M(0.1nM) to 10 -5 M (10000nM). The stronger the interaction, the higher the K DThe lower. For example, the binding site on the antibody described herein or part thereof will bind to the desired antigen with an affinity of less than 500nM, such as less than 200nM or less than 10nM, such as less than 500pM. The specific binding of the antigen-binding protein to the antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassay (RIA), sandwich competition assay, enzyme-linked immunosorbent assay (ELISA), antigen drop assay, enzyme immunoassay (EIA) and different variants thereof known per se in the art; and other techniques as mentioned herein.

[0084] As used herein, "selective binding" or "specific binding" refers to the binding of a peptide (e.g., an antibody, an adapter, a recruiter, a CAR, or a portion thereof) described herein to a D 10 -5 M (10000nM) or lower, for example 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or less and the ability to bind to a target (such as an antigen present on the cell surface of a cancer cell). Specific binding may be affected by, for example, the affinity and avidity of a polypeptide agent and the concentration of a polypeptide agent. One of ordinary skill in the art may use any suitable method to determine the appropriate conditions for the selective binding of a polypeptide agent to a target as described herein, such as titrating a polypeptide agent in a suitable cell binding assay. A polypeptide that specifically binds to an antigen on a target will not be replaced by a non-similar competitor. In certain embodiments, when an antibody, an antigen-binding portion thereof, an adapter, a recruiter, or a CAR preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules, it is said to specifically bind to an antigen.

[0085] In some embodiments, an antibody, antigen binding portion thereof, adapter, recruiter, or CAR as described herein is present in an amount of 10 -5 M (10000nM) or lower, for example 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or lower dissociation constant (K D ) binds to the full-length isoform of ASPH. In some embodiments, an antibody, antigen-binding portion thereof, adapter, recruiter, or CAR as described herein is present in an amount of about 10 -5 M to 10-6 The dissociation constant (K D ) binds to the full-length isoform of ASPH. In some embodiments, an antibody, antigen-binding portion thereof, adapter, recruiter, or CAR as described herein is present in an amount of about 10 -6 M to 10 -7 The dissociation constant (K D ) binds to the full-length isoform of ASPH. In some embodiments, an antibody, antigen-binding portion thereof, adapter, recruiter, or CAR as described herein is present in an amount of about 10 -7 M to 10 -8 The dissociation constant (K D ) binds to the full-length isoform of ASPH. In some embodiments, an antibody, antigen-binding portion thereof, adapter, recruiter, or CAR as described herein is present in an amount of about 10 -8 M to 10 -9 The dissociation constant (K D ) binds to the full-length isoform of ASPH. In some embodiments, an antibody, antigen-binding portion thereof, adapter, recruiter, or CAR as described herein is present in an amount of about 10 -9 M to 10 -10 The dissociation constant (K D ) binds to the full-length isoform of ASPH. In some embodiments, an antibody, antigen-binding portion thereof, adapter, recruiter, or CAR as described herein is present in an amount of about 10 -10 M to 10 -11 The dissociation constant (K D ) binds to the full-length isoform of ASPH. In some embodiments, an antibody, antigen-binding portion thereof, adapter, recruiter, or CAR as described herein is present in an amount of about 10 -11 M to 10 -12 The dissociation constant (K D ) binds to the full-length isoform of ASPH. In some embodiments, the antibodies, antigen-binding portions thereof, adapters, recruiters, or CARs described herein are in the form of less than 10 -12 The dissociation constant (K D ) binds to the full-length isoform of ASPH.

[0086] Pharmaceutical composition

[0087] The compositions and methods of the present invention can be used to treat individuals in need. In certain embodiments, the individual is a mammal, such as a human, or a non-human mammal. When applied to an animal, such as a human, the composition or compound is preferably administered as a pharmaceutical composition, which comprises, for example, a compound of the present invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art, and include, for example, aqueous solutions, such as water or physiologically buffered saline or other solvents or vehicles, such as glycols, glycerol, oils, such as olive oil, corn oil, sunflower oil, grape seed oil, vegetable oil, fish oil or injectable organic esters. In a preferred embodiment, when such pharmaceutical compositions are used for human administration, particularly for invasive administration routes (i.e., bypassing the transport or diffusion route through the epithelial barrier, such as injection, pump infusion, transplantation or implantation), the aqueous solution is free of pyrogens, or is substantially free of pyrogens. For example, excipients may be selected to achieve delayed release of a medicament or selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage unit form, such as tablets, pills, capsules (including sprinkle capsules and gelatin capsules), granules, lyophilized agents for reconstitution, powders, solutions, syrups, suppositories, gels, sprays, aerosols (e.g., pressurized intraperitoneal aerosols), radioisotopes, interventions (e.g., transarterial embolization, radiofrequency ablation), injections, infusions, etc. The composition can also be present in a transdermal delivery system, such as a skin patch. The composition can also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.

[0088] Pharmaceutically acceptable carriers may contain physiologically acceptable agents, which act, for example, to stabilize, reduce degradation, exclude or remove, increase solubility, increase immunogenicity, or increase the absorption of compounds (such as compounds of the present invention). Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose, or dextran; antioxidants, such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins; adjuvants (such as alum, MF59, AS01 / 03 / 04, CpG1018) or other stabilizers or excipients. The selection of pharmaceutically acceptable carriers (including physiologically acceptable agents) depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition may be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) may also be a liposome or other polymer matrix, in which, for example, a compound of the present invention may be incorporated. For example, liposomes containing phospholipids or other lipids are non-toxic, physiologically acceptable and metabolizable carriers, which are relatively simple to manufacture and administer.

[0089] As used herein, the phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions, metals, radioisotopes / radiating particles, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, rejection, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0090] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, vegetable oil, cottonseed oil, safflower oil, sesame oil, olive oil, sunflower oil, grapeseed oil, fish oil, corn oil and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerol, sorbitol, , mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar, matrigel or hydrogels; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffered saline; (21) nanoparticles, such as liposomes, polymers, micelles, metal nanoparticles, carbon nanotubes, solid lipid nanoparticles, noisomes and dendrimers; (22) extracellular vesicles; and (23) other non-toxic compatible substances for pharmaceutical formulations.

[0091] The pharmaceutical composition (preparation) can be administered to a subject by any of several routes of administration including: oral administration (e.g., oral administration with aqueous or non-aqueous solutions or suspensions, tablets, pills, suppositories, patches, pastes, infusion pumps, capsules (including dispersible capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral, nasal, urethral, ​​rectal or vaginal mucosa (e.g., sublingual, aerosol); subcutaneous; intradermal, transdermal (e.g., as a patch applied to the skin); intramuscular; intravenous; and topical (e.g., as a cream, lotion, ointment or spray applied to the skin). The compound can also be formulated for inhalation. In certain embodiments, the compound can simply be dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable therefor can be found, for example, in U.S. Pat. Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970, and 4,172,896 and patents cited therein.

[0092] The preparation can be conveniently present in unit dosage form and can be prepared by any method well-known in the pharmaceutical field. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary according to the host being treated, the specific mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount in which the compound produces a therapeutic effect. Usually, in one hundred percent, the range of this amount is about 1% to about 99% of the active ingredient, preferably about 5% to about 70%, and most preferably about 10% to about 30%.

[0093] The method for preparing these preparations or compositions comprises the step of mixing active compound (such as compound of the present invention) with carrier and optional one or more auxiliary ingredients.In general, preparation is by uniformly and intimately mixing compound of the present invention with liquid carrier or finely divided solid carrier or both, and then if necessary, the product is shaped and prepared.

[0094] Formulations of the invention suitable for oral administration may be in the form of capsules (including dispersible capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), lyophilizates, powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid or as an oil-in-water or water-in-oil liquid emulsion or as an elixir or syrup or as a lozenge (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and / or as a mouthwash, each containing a predetermined amount of a compound of the invention as the active ingredient. The composition or compound may also be administered as a bolus, elixir or paste.

[0095] To prepare solid dosage forms for oral administration, capsules, including dispersible capsules and gelatin capsules, tablets (coated and uncoated, bilayer, miniature), pills, dragées, powders, granules and the like, the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol and / or silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or acacia; (3) wetting agents, such as glycerol; (4) disintegrants, such as agar-agar, gelatin, hydrogels, matrigel, calcium carbonate, potato starch or tapioca starch, alginic acid, certain silicates and sodium carbonate; (5) solution retarders. The pharmaceutical composition may also include a buffering agent, such as a dispersible capsule and a gelatin capsule, a tablet or a pill. A similar type of solid composition using excipients such as lactose (lactose or milk sugar) and high molecular weight polyethylene glycols may also be used as fillers in soft-filled gelatin capsules and hard-filled gelatin capsules.

[0096] Tablets may be made by compression or molding, optionally with one or more auxiliary ingredients. Compressed tablets may be prepared using a binder (e.g., gelatin or hydroxypropylmethylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), a surfactant or a dispersant. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0097] Tablets and other solid dosage forms of pharmaceutical compositions, such as dragees, capsules (including dispersible capsules and gelatin capsules), pills and granules, may be optionally scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the field of pharmaceutical formulation. They may also be formulated to provide slow or controlled release of the active ingredient therein, for example using hydroxypropylmethylcellulose, other polymer matrices, nanoparticles (such as liposomes) and / or microspheres in different proportions to provide the desired release profile. They may be sterilized, for example, by filtering through a bacterial retention filter or by incorporating a sterilizing agent as a sterile solid composition, which may be dissolved in sterile water or some other sterile injectable medium just before use. These compositions may also optionally contain an opacifier, and may be compositions that release the active ingredient only or preferentially in a certain part of the respiratory tract, urogenital tract, gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that may be used include polymeric substances and waxes. The active ingredient may also be in microencapsulated form, in the form of extracellular vesicles, with one or more of the above-mentioned excipients where appropriate.

[0098] Liquid dosage forms that can be used for oral administration include pharmaceutically acceptable emulsions, lyophilized agents for reconstitution, microemulsions, microcapsules, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage form may also have an inert diluent commonly used in the art, such as water or other solvents, cyclodextrins and derivatives thereof, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (e.g., fish oil, cottonseed oil, peanut oil, corn oil, germ oil, vegetable oil, grape seed oil, sunflower oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan and mixtures thereof.

[0099] In addition to inert diluents, the oral compositions may also include adjuvants such as alum, MF59, AS01 / 03 / 04, CpG1018, wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, coloring agents, perfuming agents and preservatives.

[0100] Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0101] Dosage forms for topical or transdermal administration include powders, sprays, creams, lotions, ointments, gels, hydrogels, matrix gels, solutions, patches, pastes, microneedles (for transdermal, intraocular, vaginal, transungual, cardiac, vascular, gastrointestinal and intracochlear delivery) and inhalants. The active compound can be mixed under sterile conditions with a pharmaceutically acceptable carrier, adjuvant, and with any preservatives, buffers, or propellants that may be required.

[0102] Ointments, creams, lotions, patches, pastes and gels may contain, in addition to the active compounds, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

[0103] Powders and sprays may contain, in addition to the active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays may additionally contain typical propellants, such as chlorofluorocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0104] Transdermal patches, pastes and microneedles have the additional advantage of providing controlled delivery of the compounds of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the active compound in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound through the skin. The rate of the flux can be controlled by providing a rate-controlling membrane or dispersing the compound in a polymer matrix or gel.

[0105] As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intraocular (such as intravitreal), intramuscular, intraarterial, intraarticular, intralymph node, intralymphatic, intratumoral, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intracochlear, intraperitoneal, intravaginal, transdermal, transtracheal, transungual, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions or sterile powders which may be reconstituted immediately before use into sterile injectable solutions or dispersions which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[0106] The example of suitable aqueous and non-aqueous carrier that can be used in the pharmaceutical composition of the present invention comprises water, ethanol, polyol (such as glycerol, propylene glycol, polyethylene glycol etc.) and suitable mixture thereof, vegetable oil, such as olive oil and injectable organic ester, such as ethyl oleate. The example of suitable aqueous and non-aqueous carrier that can be used in the pharmaceutical composition of the present invention comprises water, ethanol, polyol (such as glycerol, propylene glycol, polyethylene glycol etc.) and suitable mixture thereof, vegetable oil, such as olive oil and injectable organic ester, such as ethyl oleate. For example, can be by using coating material (such as lecithin), in the case of dispersion, by maintaining required particle size and by using surfactant to maintain suitable fluidity (flow / adhesion ratio).

[0107] These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. Protection from the action of microorganisms may be ensured by including various antibacterial, antiviral and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, etc. in the composition. In addition, extended absorption of injectable drug forms may be caused by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0108] Sometimes, in order to prolong the effect of a drug, it is desirable to slow down the absorption of a drug from a subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of a poorly water-soluble crystalline or amorphous substance. The absorption rate of the drug then depends on its dissolution rate. The dissolution rate can depend on the crystal size and crystalline form. Delayed absorption of a parenterally administered drug form is achieved by dissolving or suspending the drug in an oily vehicle.

[0109] Injectable reservoir forms are prepared by forming a microencapsulated matrix of the subject compound in a biodegradable polymer (such as polylactic acid-polyglycolide). Depending on the ratio of the drug to the polymer and the properties of the specific polymer used, the drug release rate can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Reservoir injectable formulations are also prepared by encapsulating the drug in nanoparticles (such as liposomes, extracellular vesicles, microcapsules or microemulsions) that are compatible with body tissues.

[0110] For use in the methods of the invention, the active compound may be given per se, or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably 0.5 to 90%) of the active ingredient in combination with a pharmaceutically acceptable carrier.

[0111] The introduction method can also be provided by a rechargeable or biodegradable device. In recent years, various slow-release polymer devices have been developed and tested in vivo for controlled delivery of drugs, including protein biopharmaceuticals. Various biocompatible polymers (including hydrogels, matrix gels), including biodegradable and non-degradable polymers, can be used to form implants to continuously release compounds at specific target sites.

[0112] Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0113] The selected dosage level will depend upon a variety of factors, including the activity of the specific compound or combination of compounds employed, or their esters, salts or amides, the route of administration, the time of administration, the excretion or clearance rate of the specific compound employed, the duration or frequency of treatment, other drugs that may interact with or affect the metabolism or efficacy of the compounds of the invention, compounds and / or materials (e.g., vaccines, antibodies and derivatives) used in combination with the specific compound employed, other therapeutic approaches (e.g., surgery, cell-based therapies, chemotherapy, radiation therapy, interventional therapies), the age, sex, weight, condition or comorbidities, diet, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0114] A doctor or veterinarian with ordinary skills in the art can easily determine and prescribe a therapeutically effective amount of the desired pharmaceutical composition. For example, a doctor or veterinarian can start the dosage of the pharmaceutical composition or compound at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. "Therapeutically effective amount" refers to a concentration of the compound sufficient to cause the desired therapeutic effect. The effective amount of the compound varies according to the subject's weight, sex, age, comorbidities, and medical history. Other factors affecting the effective amount may include, but are not limited to, the severity of the patient's condition, the condition being treated, the method of treatment, the compound, and, if necessary, the stability of another type of therapeutic agent administered with the compound of the present invention. A larger total dose can be delivered by multiple administrations of the agent. Methods for determining efficacy and dosage are known to those skilled in the art. See, for example, Isselbacher et al. (1996).

[0115] A suitable daily dose of an active compound used in the compositions and methods of the invention may be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend on the factors described above.

[0116] If desired, the effective daily dose of the active compound can be administered at appropriate intervals throughout the day, optionally in unit dosage form as one, two, three, four, five, six or more sub-doses. In certain embodiments of the invention, the active compound is administered twice or three times a day. In other embodiments, the active compound is administered once a day.

[0117] Patients receiving such treatment are any animal in need thereof, including primates, particularly humans; other mammals, such as horses, cattle, pigs, sheep, cats, and dogs; poultry; and pets in general.

[0118] In certain embodiments, the compounds of the invention may be used alone, in combination with another type of therapeutic agent, or in combination with treatment methods such as surgery, chemotherapy, radiation therapy, and interventional therapy.

[0119] The present disclosure includes pharmaceutically acceptable salts of the compounds of the present invention used in the compositions and methods of the present invention. In certain embodiments, the salts contemplated by the present invention include, but are not limited to, alkyl, dialkyl, trialkyl, or tetraalkyl ammonium salts. In certain embodiments, the salts contemplated by the present invention include, but are not limited to, L-arginine, benthamine, benzathine, betaine, calcium hydroxide, choline, danol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucosamine, hydrabamine, 1H-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, the salts contemplated by the present invention include, but are not limited to, Na, Ca, K, Mg, Zn, or other metal salts. In certain embodiments, the salts contemplated by the present invention include, but are not limited to, 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, l-ascorbic acid, l-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)-camphor-10-methanesulfonic acid, capric acid, decanoic acid, caproic acid, hexanoic acid), caprylic acid, octanoic acid acid), carbonic acid, cinnamic acid, citric acid, cyclohexylaminosulfonic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, d-glucoheptonic acid, d-gluconic acid, d-glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, l-malic acid, malonic acid, mandelic acid, methanesulfonic acid, naphthalene-1,5-dimethylsulfonic acid, naphthalene-2-methanesulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, l-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, l-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenate.

[0120] Pharmaceutically acceptable acid addition salts may also exist as various solvates, such as solvates with water, methanol, ethanol, dimethylformamide, etc. Mixtures of such solvates may also be prepared. The source of such solvates may be derived from the crystallization solvent, inherent in the preparation or crystallization solvent, or incidentally present in such solvent.

[0121] Wetting agents, emulsifiers, dispersants and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening agents, cooling or heating agents, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition.

[0122] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, polyunsaturated fatty acids (PUFA) such as ω-3 fatty acids, etc.; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, succimer, dimercaptonol, dimercaptopropanol (BAL), etc.

[0123] Unless otherwise defined herein, scientific and technical terms related to the application should have the meanings commonly understood by those of ordinary skill in the art to which the disclosure belongs. It should be understood that the present invention is not limited to the ad hoc methods described herein, schemes, animal models, (engineered or genetically modified) cells, organoids, constructs, carriers, carriers, adjuvants, compounds, drug delivery systems, antibodies and derivatives, vaccines and reagents, etc., and therefore can be changed. The terms used herein are only used to describe the purpose of specific embodiments and are not intended to limit the scope of the present invention, which is defined only by the claims. Definitions of commonly used terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, The Encyclopedia of Molecular Cell Biology and Molecular Medicine, Molecular Biology and Biotechnology: a Comprehensive Desk Reference, Immunology, Janeway's Immunobiology, Lewin's Genes XI, Molecular Cloning: A Laboratory Manual., Basic Methods in Molecular Biology, Laboratory Methods in Enzymology, Current Protocols in Molecular Biology (CPMB), Current Protocols in Protein Science (CPPS), and Current Protocols in Immunology (CPI).

[0124] In some embodiments of any aspect, the disclosure described herein does not relate to methods of cloning humans, methods of altering the germline genetic identity of humans, the use of human embryos for industrial or commercial purposes, or methods of altering the genetic identity of animals that may cause suffering to animals without providing substantial medical benefit to humans or animals, or animals produced by such methods.

[0125] Other terms are defined herein in the description of various aspects of the invention.

[0126] Aspartate beta-hydroxylase (ASPH)

[0127] Many pathways have been shown to contribute to the multi-step and multifaceted metastasis of malignancies. Aspartate β-hydroxylase (ASPH) is a key factor in the invasive transformation of malignancies by enhancing cell proliferation, migration, invasion, migration, angiogenesis, lymphogenesis, and dormancy-reactivation-exogenous at distant sites. ASPH also promotes tumor growth by inducing immunosuppression. These proto-oncogenic pathological properties are achieved in part via activation of Notch, TGFβ, HGF-cMET, HIF, and SRC signaling pathways. In different human tumors, growth factors and hypoxia transcriptionally upregulate ASPH expression, and specifically targeting this biomarker can have a wide range of clinical impacts on diagnosis, prognosis, and treatment.

[0128] ASPH is a naturally occurring enzyme that was originally identified by expression cloning of a cDNA library from a cancerous hepatobiliary cell line (Korioth et al. (1994); Lavaissiere et al. (1996)). There are 12 known ASPH splice isoforms. It has been determined that isoform 1 is expressed during development, minimally expressed in normal tissues, and then re-expressed at high levels in a variety of malignancies such as carcinomas (Jia et al. (1992); Patel et al. (2014)). Some isoforms lack the C-terminus, which contains the catalytic site for converting enzymatic activity. The ASPH truncated homologs Junctin and Humbug are expressed in a variety of normal tissues, primarily involved in calcium homeostasis. Isoform 1 is the only isoform that is overexpressed in tumors derived from the endoderm, mesoderm, and ectoderm.

[0129] Table 1 shows the amino acid sequence of ASPH (SEQ ID NO: 1; GENBANK Accession No. S83325; the His motif is underlined; the conserved sequence within the catalytic domain is in bold).

[0130]

[0131] The ASPH antibodies of the invention (described below) bind to a polypeptide within the amino acid sequence of the full-length ASPH isoform identified as NPVEDS (residues 286-291 of SEQ ID NO: 1, as indicated by the shaded residues in Table 1). NPVEDS is an LRC1 epitope because inhibition by peptides using this sequence abolished LRC1 binding to the full-length isoform of the ASPH protein, and there are no other sequences that bind to other regions of the molecule (data not shown).

[0132] Functionally, overexpression of ASPH induces tumor cell proliferation, migration, invasion, migration, angiogenesis, lymphopoiesis, cell stemness, colonization, and dormancy-reactivation-at distant sites by: (1) promoting activation of Notch, TGFβ, HGF-cMET, HIF, and SRC signaling cascades; (2) inhibiting apoptosis through caspase 3 cleavage; (3) enhancing cell proliferation via phosphorylation of RB1; (4) regulating cell senescence; (5) generating cancer stem-like progenitor cells; and (6) subverting the immune system and inducing immunosuppression (Dong et al. (2015); Huang et al. (2016); and Iwagami et al. (2016)). Transcriptional regulation of ASPH expression in human tumors is controlled by well-known growth factor and cytokine signaling cascades, such as insulin / IRS-1 / RAF / RAS / MAPK / ERK, insulin / IRS-1 / PI3K / AKT, and WNT / β-catenin signaling (Iwagami et al. (2016)).

[0133] In this context, ASPH is a key molecule that connects upstream growth factors, chemokines and cytokines to proto-oncogenic signaling pathways (such as Notch activation and subsequent downstream expression of Notch target genes). Notch signaling is a recognized pathological feature of cancer. Notch signaling promotes tumor growth and metastasis by transcriptionally upregulating the expression of cytokines and proteases (such as matrix metalloproteinases (MMPs)) and growth factors (such as VEGF) (Engin et al. (2009); Xu et al. (2016)). Both tissue invasion of tumor cells and tumor-induced angiogenesis require the participation of MMPs (Fang et al. (2000)). MMP expression may be an independent marker of poor prognosis (Berend et al. (1998)). In this case, subtype 1 is the only subtype with aspartyl / asparaginyl β-hydroxylase activity, which is essential for Notch activation.

[0134] ASPH is a member of the α-ketoglutarate-dependent dioxygenase family (Hou et al. (2011); Jawad et al. (2010)). ASPH catalyzes the hydroxylation of aspartyl and asparaginyl residues in epidermal growth factor (EGF)-like repeats of various proteins required for signal transduction, including Notch receptors and ligands. ASPH is conserved in mammalian evolution, and the human protein sequence is 85% identical to rat and mouse; the catalytic site in the C-terminal region is 100% conserved between the three species. As an oncofetal protein expressed during embryonic development and differentiation, ASPH is "turned off" at birth and during adulthood, only to reappear during tumorigenesis. Re-expression / reactivation of ASPH stimulates cell proliferation, migration, invasion, emigration, angiogenesis, lymphopoiesis, cell stemness, colonization, and dormancy-reactivation-at distant sites and is thought to generate and maintain the malignant phenotype in cancer (Dong et al. (2015); Huang et al. (2016); and Aihara et al. (2014)).

[0135] The known mechanisms underlying the transforming activity of ASPH are as follows: upregulation of ASPH occurs through well-known upstream growth factor signaling pathways such as Wnt / β-catenin, PI3K-AKT, and insulin-IGF-1 / IRS-1 / ERK-MAPK and hypoxia (Longato et al. (2009); Elvidge et al. (2006)). ASPH overexpression in malignancies activates Notch1 signaling through hydroxylation and direct binding to Notch1 receptors and ligands, followed by production of the Notch intracellular domain (NICD1), which translocates to the nucleus where it acts as part of a transcriptional complex that upregulates genes responsible for known hallmarks of cancer development and progression, such as c-Myc, MMPs, and VEGF. Other mechanisms have been reported to contribute to the oncogenic properties of ASPH, including inhibition of apoptosis, blocking of cellular senescence, enhancing cancer stem cell formation (Dong et al. (2015)), and dysregulation of the cell cycle controlled by phosphorylation of the retinoblastoma gene (RB) (Iwagami et al. (2016)).

[0136] Therefore, the ability to measure ASPH at an early stage of cancer is valuable and a potential game-changer. The present invention greatly fills the knowledge gap in early diagnosis, prognosis and treatment, with substantial patient care and commercial importance.

[0137] ASPH Antibodies

[0138] The present invention discloses the development and characterization of a unique monoclonal antibody (mAb) against a transmembrane protein, which can be used for early diagnosis, prognosis and treatment of a wide range of human malignancies. The present invention provides an antibody that binds to the full-length isoform of ASPH (hereinafter "LRC1"), the antibody comprising a light chain (SEQ ID NO: 11) and a heavy chain (SEQ ID NO: 10), wherein the light chain comprises an LCVR (SEQ ID NO: 9), and the heavy chain comprises an HCVR (SEQ ID NO: 8), wherein the LCVR comprises complementary determining regions LCDR1, LCDR2 and LCDR3, and the HCVR comprises complementary determining regions HCDR1, HCDR2 and HCDR3, wherein LCDR1 is SEQ ID NO: 5, LCDR2 is SEQ ID NO: 6, LCDR3 is SEQ ID NO: 7, HCDR1 is SEQ ID NO: 2, HCDR2 is SEQ ID NO: 3, and HCDR3 is SEQ ID NO: 4. The unique amino acid sequence of the cloned LRC1 monoclonal antibody has been determined and is provided in Table 2.

[0139]

[0140]

[0141] The unique nucleic acid sequences of the cloned LRC1 monoclonal antibodies have been determined and are provided in Table 3.

[0142]

[0143]

[0144]

[0145] Monoclonal antibodies against ASPH protein have been previously produced, but among hundreds of such antibodies, only the LRC1 antibody recognizes a unique 6 amino acid epitope and possesses these interesting, unexpected and highly desirable properties. The LRC1 antibody of the present invention binds to a polypeptide within the amino acid sequence of the full-length ASPH isoform identified as NPVEDS (residues 286-291 of SEQ ID NO: 1). The LRC1 antibody of the present invention provides improved sensitivity and specificity for assays designed to detect early tumor formation (including premalignant lesions and in situ malignant tumors) using liquid biopsies. This is due to the unique and unexpected properties of the LRC1 antibody, namely: (1) recognition of the full-length isoform of ASPH that has transforming activity when overexpressed in cells (data shown in Tables 4 and 5); (2) recognition for the first time of ASPH expression in tumors derived from the mesoderm, such as sarcomas (data shown in Figure 1-3); (3) detection of as little as 25 pg of full-length ASPH protein (antigen) bound to a solid support (data shown in Figure 4 ); (4) as a companion diagnostic test for malignant tumors, detecting the presence of ASPH in extracellular vesicles (ectosomes and exosomes) derived from cancer cells (data shown in Figure 5 ); (5) An immunoassay based on a single LRC1 mAb has been developed as a highly sensitive technique for early cancer diagnosis using 15-25 μL of blood from a finger prick (data shown in Figure 6-7 ), involving only the use of LRC1 mAb and lateral flow and microfluidic analysis using digital and optical control and automation; (6) the exact 6 amino acid epitope (i.e., NPVEDS) that LRC1 binds to on the full-length oncoprotein has been identified (shaded in Table 1), and this epitope is expressed on the cell surface of tumor cells and on extracellular vesicles derived from ASPH-positive cancer cells; and (7) the unique amino acid and nucleic acid sequences of the LRC1 mAb have been determined (provided in Tables 2 and 3, respectively). In summary, many embodiments of the present invention greatly improve the early detection and prognosis of cancer and can be used as targeting agents to deliver cytotoxic "payloads" or recruit cytotoxic immune cells to ASPH-expressing tumors. The use of a single antibody to have this degree of immunoreactivity against tumors derived from the endoderm, mesoderm, and ectoderm is unprecedented and relevant to human tumor formation.

[0146]

[0147]

[0148]

[0149] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the present disclosure to the precise form disclosed. Although the specific embodiments and examples of the present disclosure are described herein for illustrative purposes, various equivalent modifications can be made within the scope of the present disclosure as recognized by those skilled in the relevant art. Although the method steps or functions are presented in a given order, alternative embodiments can perform functions in different orders, or can perform functions substantially simultaneously. The teachings of the present disclosure provided herein can be applied to other procedures or methods where appropriate. The various embodiments described herein can be combined to provide further embodiments. If necessary, the aspects of the present disclosure can be modified to provide further embodiments of the present disclosure using the compositions, functions and concepts of the above-mentioned references and applications. Due to considerations of biological functional equivalence, some changes can be made in the protein amino acid sequence or structure without affecting the biological or chemical effects in terms of type or amount. In view of specific embodiments, these and other changes can be made to the present disclosure. All such changes are intended to be included within the scope of the appended claims.

[0150] The specific elements of any of the above embodiments may be combined with or replaced by the elements in other embodiments. Although the advantages associated with certain embodiments of the present disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments must exhibit such advantages to fall within the scope of the present disclosure.

[0151] The technology described herein is further illustrated by the following additional numbered embodiments, which should in no way be construed as further limiting.

[0152] 1. An antibody that binds to a full-length isoform of aspartate beta-hydroxylase (ASPH), the antibody comprising a light chain and a heavy chain, wherein the light chain comprises a light chain variable region (LCVR), and the heavy chain comprises a heavy chain variable region (HCVR), wherein the LCVR comprises complementarity determining regions LCDR1, LCDR2 and LCDR3, and the HCVR comprises complementarity determining regions HCDR1, HCDR2 and HCDR3, wherein LCDR1 consists of the amino acid sequence of SEQ ID NO:5, LCDR2 consists of the amino acid sequence of SEQ ID NO:6, LCDR3 consists of the amino acid sequence of SEQ ID NO:7, HCDR1 consists of the amino acid sequence of SEQ ID NO:2, HCDR2 consists of the amino acid sequence of SEQ ID NO:3, and HCDR3 consists of the amino acid sequence of SEQ ID NO:4.

[0153] 2. An antibody that binds to a full-length isoform of ASPH, the antibody comprising a light chain and a heavy chain, wherein the light chain comprises an LCVR, and the heavy chain comprises an HCVR, wherein the LCVR comprises the amino acid sequence of SEQ ID NO:9, and the HCVR comprises the amino acid sequence of SEQ ID NO:8.

[0154] 3. An antibody that binds to a full-length isoform of ASPH, the antibody comprising a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 11 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 10.

[0155] 4. An antibody that binds to a full-length isoform of ASPH, the antibody comprising two light chains and two heavy chains, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 11, and each heavy chain comprises SEQ ID NO: 10.

[0156] 5. A DNA molecule comprising a polynucleotide sequence encoding a light chain polypeptide having the amino acid sequence of SEQ ID NO: 11.

[0157] 6. A DNA molecule comprising a polynucleotide sequence encoding a heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 10.

[0158] 7. A recombinant host cell comprising the DNA molecule of embodiment 5 and the DNA molecule of embodiment 6, the cell being capable of expressing an antibody comprising a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is SEQ ID NO: 10, and the amino acid sequence of the light chain is SEQ ID NO: 11.

[0159] 8. A method for producing an antibody that binds to human ASPH, the antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 10, and the light chain comprises the amino acid sequence of SEQ ID NO: 11, the method comprising the following steps: (a) culturing the recombinant host cell of embodiment 7 under conditions such that the antibody is expressed; and (b) recovering the expressed antibody from the host cell.

[0160] 9. An antibody produced by the method of embodiment 8.

[0161] 10. A pharmaceutical composition comprising the antibody of any one of embodiments 1-4 and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0162] 11. An assay comprising:

[0163] (i) measuring the level of ASPH in a sample obtained from a subject, wherein the measuring comprises incubating the sample in a preparation having immobilized antibodies according to any one of embodiments 1 to 4;

[0164] (ii) comparing the ASPH level with a reference level; and

[0165] (iii) identifying the subject as:

[0166] (a) if the ASPH level is higher than the reference level, the patient has a malignant tumor; or

[0167] (b) If the ASPH level is equal to or lower than the reference level, there is no malignancy.

[0168] 12. The assay of embodiment 11, wherein the biological sample is serum and the detected ASPH level is compared to a reference level in the subject's serum.

[0169] 13. The assay according to any one of embodiments 11-12, wherein when the ASPH level is above the reference level, the assay further comprises providing a confirmed diagnosis and a treatment suitable for treating the malignancy.

[0170] 14. The assay of embodiment 13, wherein the treatment comprises administering to the subject a composition comprising a therapeutically effective amount of the antibody of any one of embodiments 1-4 linked to a cytotoxic agent.

[0171] 15. The assay of embodiment 13, wherein the confirmatory diagnosis is obtained using a diagnostic tool selected from the group consisting of: imaging, endoscopy, biopsy, histology, cytology, and pathology.

[0172] 16. The assay of any one of embodiments 11-15, wherein the malignancy is selected from the group consisting of head and neck cancer, thyroid cancer, thymic cancer, non-small cell lung cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, gallbladder cancer, bile duct cancer, pancreatic cancer, colon and rectal cancer, kidney cancer, prostate cancer, breast cancer, ovarian cancer, fallopian tube cancer and cervical cancer, soft tissue sarcoma, osteosarcoma, chondrosarcoma, malignant mesothelioma, myeloid and lymphoid leukemias, multiple myeloma, lymphoma, primary malignant CNS tumors of neuronal and glial cell origin and metastatic CNS tumors, skin cancer and melanoma.

[0173] 17. A method of monitoring the progress of treatment of a subject suffering from a malignant tumor, the method comprising:

[0174] (i) measuring a first ASPH level in a first sample obtained from a subject at a first time point, wherein the measuring comprises incubating the first sample in a preparation having an immobilized antibody according to any one of embodiments 1 to 4;

[0175] (ii) administering to the subject a therapeutic agent or a therapeutic method for treating a malignant tumor;

[0176] (iii) measuring a second ASPH level in a second sample obtained from the subject at a second time point, wherein the measuring comprises incubating the second sample in a preparation having immobilized antibodies according to any one of embodiments 1 to 4;

[0177] Wherein the second level measurement is significantly lower than the first level measurement indicates that the therapeutic agent or treatment method is effective.

[0178] 18. A method of treating a malignancy in a subject, the method comprising administering to the subject a therapeutically effective amount of the antibody and derivatives of any one of embodiments 1 to 4 linked to a cytotoxic agent.

[0179] 19. A method according to any one of embodiments 17-18, wherein the malignant tumor is selected from the following but not limited to: head and neck cancer, thyroid cancer, thymic cancer, non-small cell lung cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, gallbladder cancer, bile duct cancer, pancreatic cancer, colon and rectal cancer, kidney cancer, prostate cancer, breast cancer, ovarian cancer, fallopian tube cancer and cervical cancer, soft tissue sarcoma, osteosarcoma, chondrosarcoma, malignant mesothelioma, myeloid leukemia and lymphoid leukemia, multiple myeloma, lymphoma, primary malignant CNS tumors of neuronal and glial cell origin and metastatic CNS tumors, skin cancer and melanoma.

[0180] 20. A kit for detecting malignant tumor cells, comprising an antibody or an ASPH binding fragment and derivative thereof, wherein the antibody or ASPH binding fragment and derivative binds to residues 286-291 of SEQ ID NO: 1.

[0181] 21. The kit according to embodiment 20, further comprising means for detecting the binding of said antibodies and derivatives to said malignant tumor cells.

[0182] 22. A kit according to embodiment 22, wherein the means is a detectable marker.

[0183] 23. A kit according to embodiment 22, wherein the detectable marker is a radioactive compound.

[0184] 24. The kit according to embodiment 22, wherein the detectable marker is Gd +++ or Fe ++ .

[0185] 25. A kit according to any one of embodiments 20-24, wherein the malignant tumor cells are from a malignancy selected from the group consisting of head and neck cancer, thyroid cancer, thymic cancer, non-small cell lung cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, gallbladder cancer, bile duct cancer, pancreatic cancer, colon and rectal cancer, kidney cancer, prostate cancer, breast cancer, ovarian cancer, fallopian tube cancer and cervical cancer, soft tissue sarcoma, osteosarcoma, chondrosarcoma, malignant mesothelioma, myeloid and lymphoid leukemia, multiple myeloma, lymphoma, primary malignant CNS tumors of neuronal and glial cell origin and metastatic CNS tumors, skin cancer and melanoma.

[0186] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods, protocols, and materials are described below.

[0187] References

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[0194] Current Protocols in Immunology (CPI) (2003). John E. Coligan, ADAM Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (Eds.) John Wiley and Sons, Inc. (ISBN 0471142735, 9780471142737).

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[0201] Hou et al. (2011). “WISP-1 increases MMP-2 expression and cell motility in human chondrosarcoma cells.” Biochem. Pharmacol. 81:1286 - 1295. Huang et al. (2016). “Anti-Tumor Effects of Second Generation beta-Hydroxylase Inhibitors on Cholangiocarcinoma Development and Progression.” PLoS One 11:e0150336.

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[0207] Jia et al. (1992). “cDNA cloning and expression of bovine aspartyl(asparaginyl)beta-hydroxylase.” J. Biol. Chem. 267:14322-14327. Kabat et al. (1987) and (1991). Sequences Of Proteins of Immunological Interest. (National Institutes of Health, Bethesda, MD, USA)

[0208] Kabat et al. (1991). Sequences of Proteins of Immunological Interest. Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91 3242.

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[0218] Patel et al. (2014). “Mutations in ASPH cause facial dysmorphism, lens dislocation, anterior-segment abnormalities, and spontaneous filtering blebs, or Traboulsi syndrome.” Am. J. Hum. Genet. 94:755 - 759.

[0219] Soderstrom et al. (2001). “Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human chondrosarcomas.” APMIS 109:305 - 315.

[0220] Tang et al. (2011). “IL - 6 increases MMP - 13 expression and motility in human chondrosarcoma cells.” J. Biol. Chem. 286:11056 - 11066.

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[0223] Uria et al. (1998). “Collagenase - 3 (MMP - 13) expression in chondrosarcoma cells and its regulation by basic fibroblast growth factor.” Am. J. Pathol. 153:91 - 101.

[0224] Xu et al. (2016). “Metastasis - associated lung adenocarcinoma transcript 1 promotes the proliferation of chondrosarcoma cell via activating Notch - 1 signaling pathway.” Onco. Targets Ther. 9:2143 - 2151.

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[0226] All patents and other publications cited throughout this application, including literature citations, issued patents, published patent applications, and pending patent applications, are expressly incorporated herein by reference for the purpose of description and disclosure, e.g., the methods described in such publications can be used in conjunction with the technology described herein. These publications are provided only for their disclosures prior to the filing date of this application. In this regard, nothing should be construed as an admission that the inventors are not entitled to antedate such disclosures by prior invention or for any other reason. All statements regarding the dates of these documents or representations regarding their contents are based on the information available to the applicant and do not constitute any admission as to the correctness of the dates or contents of these documents.

[0227] It is believed that the foregoing written description is sufficient to enable those skilled in the art to practice these aspects and embodiments. The scope of these aspects and embodiments should not be limited by the examples provided, because these examples are intended to be a single illustration of one aspect, and other functionally equivalent embodiments are within the scope of the present disclosure. In addition to those shown and described herein, various modifications will be apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and purposes described herein are not necessarily covered by each embodiment. Those skilled in the art will recognize or be able to determine many equivalents of the specific embodiments described herein using only routine experiments. Such equivalents are intended to be covered by the appended claims.

Claims

1. An antibody that binds to the full-length isoform of aspartate beta-hydroxylase (ASPH), the antibody comprising a light chain and a heavy chain, wherein the light chain comprises a light chain variable region (LCVR), and the heavy chain comprises a heavy chain variable region (HCVR), in, The LCVR comprises complementarity determining regions LCDR1, LCDR2 and LCDR3, and the HCVR comprises complementarity determining regions HCDR1, HCDR2 and HCDR3, and Wherein LCDR1 consists of the amino acid sequence of SEQ ID NO:5, LCDR2 consists of the amino acid sequence of SEQ ID NO:6, LCDR3 consists of the amino acid sequence of SEQ ID NO:7, HCDR1 consists of the amino acid sequence of SEQ ID NO:2, HCDR2 consists of the amino acid sequence of SEQ ID NO:3, and HCDR3 consists of the amino acid sequence of SEQ ID NO:

4.

2. The antibody of claim 1, wherein the LCVR comprises the amino acid sequence of SEQ ID NO:9 and the HCVR comprises the amino acid sequence of SEQ ID NO:

8. 3 . The antibody of claim 1 , wherein the light chain comprises the amino acid sequence of SEQ ID NO: 11, and the heavy chain comprises the amino acid sequence of SEQ ID NO:

10. 4 . The antibody of claim 1 , comprising two light chains and two heavy chains, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 11, and each heavy chain comprises SEQ ID NO:

10.

5. A DNA molecule comprising a polynucleotide sequence encoding a light chain polypeptide having the amino acid sequence of SEQ ID NO:

11.

6. A DNA molecule comprising a polynucleotide sequence encoding a heavy chain polypeptide having the amino acid sequence of SEQ ID NO:

10.

7. A recombinant host cell comprising the DNA molecule of claim 5 and the DNA molecule of claim 6, wherein the cell is capable of expressing an antibody comprising a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is SEQ ID NO: 10, and the amino acid sequence of the light chain is SEQ ID NO:

11.

8. A method for producing an antibody that binds to human ASPH, the antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 10, and the light chain comprises the amino acid sequence of SEQ ID NO: 11, the method comprising the following steps: (a) culturing the recombinant host cell of claim 7 under conditions that allow expression of the antibody; and (b) recovering the expressed antibody from the host cell.

9. An antibody produced by the method of claim 8.

10. A pharmaceutical composition comprising the antibody of claims 1-4 and one or more pharmaceutically acceptable carriers, diluents or excipients.

11. An assay comprising: (i) measuring the level of ASPH in a sample obtained from a subject, wherein the measuring comprises incubating the sample in a preparation having an immobilized antibody according to any one of claims 1 to 4; (ii) comparing ASPH levels with reference levels; as well as (iii)(a) if the ASPH level is higher than the reference level, identifying the subject as having a malignant tumor; or (b) if the ASPH level is equal to or lower than the reference level, identifying the subject as being free of malignancy.

12. The assay of claim 11, wherein the biological sample is serum and the detected ASPH level is compared to a reference level in the subject's serum.

13. The assay according to any one of claims 11-12, wherein when the ASPH level is above the reference level, the assay further comprises providing a confirmed diagnosis and a treatment for treating the malignancy.

14. The assay of claim 13, wherein the treatment comprises administering to the subject a composition comprising a therapeutically effective amount of the antibody of any one of claims 1-4 linked to a cytotoxic agent.

15. The assay of claim 13, wherein the confirmatory diagnosis is obtained using a diagnostic tool selected from the group consisting of imaging, endoscopy, biopsy, histology, cytology, and pathology.

16. The assay according to any one of claims 11-15, wherein the malignancy is selected from the group consisting of: head and neck cancer, thyroid cancer, thymic cancer, non-small cell lung cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, gallbladder cancer, bile duct cancer, pancreatic cancer, colon and rectal cancer, kidney cancer, prostate cancer, breast cancer, ovarian cancer, fallopian tube cancer and cervical cancer, soft tissue sarcoma, osteosarcoma, chondrosarcoma, malignant mesothelioma, myeloid and lymphoid leukemias, multiple myeloma, lymphoma, primary malignant CNS tumors of neuronal and glial cell origin and metastatic CNS tumors, skin cancer and melanoma.

17. A method for monitoring the progress of treatment of a patient suffering from a malignant tumor, the method comprising: (i) measuring a first ASPH level in a first sample obtained from the subject at a first time point, wherein the measuring comprises incubating the first sample in a preparation having an immobilized antibody of any one of claims 1-4; (ii) administering to the subject a therapeutic agent or a therapeutic method for treating the malignant tumor; (iii) measuring a second ASPH level in a second sample obtained from the subject at a second time point, wherein the measuring comprises incubating the second sample in a preparation having an immobilized antibody of any one of claims 1-4; Wherein the second level measurement is significantly lower than the first level measurement indicates that the therapeutic agent or treatment method is effective.

18. A method of treating a malignancy in a subject, the method comprising administering to the subject a therapeutically effective amount of the antibody and derivatives of any one of claims 1-4 linked to a cytotoxic agent.

19. The method according to any one of claims 17-18, wherein the malignant tumor is selected from the group consisting of, but not limited to, head and neck cancer, thyroid cancer, thymic cancer, non-small cell lung cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, gallbladder cancer, bile duct cancer, pancreatic cancer, colon and rectal cancer, kidney cancer, prostate cancer, breast cancer, ovarian cancer, fallopian tube cancer and cervical cancer, soft tissue sarcoma, osteosarcoma, chondrosarcoma, malignant mesothelioma, myeloid and lymphoid leukemia, multiple myeloma, lymphoma, primary malignant CNS tumors of neuronal and glial cell origin and metastatic CNS tumors, skin cancer and melanoma.

20. A kit for detecting malignant tumor cells, comprising an antibody or an ASPH binding fragment and derivative thereof, wherein the antibody or ASPH binding fragment and derivative binds to residues 286-291 of SEQ ID NO:

1.

21. The kit of claim 20, further comprising means for detecting binding of said antibodies and derivatives to said malignant tumor cells.

22. The kit of claim 22, wherein the means is a detectable marker.

23. The kit of claim 22, wherein the detectable marker is a radioactive compound.

24. The kit of claim 22, wherein the detectable marker is Gd +++ or Fe ++ .

25. The kit according to any one of claims 20-24, wherein the malignant tumor cells are from a malignant tumor selected from the group consisting of head and neck cancer, thyroid cancer, thymic cancer, non-small cell lung cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, gallbladder cancer, bile duct cancer, pancreatic cancer, colon and rectal cancer, kidney cancer, prostate cancer, breast cancer, ovarian cancer, fallopian tube cancer and cervical cancer, soft tissue sarcoma, osteosarcoma, chondrosarcoma, malignant mesothelioma, myeloid and lymphoid leukemia, multiple myeloma, lymphoma, primary malignant CNS tumors of neuronal and glial cell origin and metastatic CNS tumors, skin cancer and melanoma.

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