Recombinant antibodies, pharmaceutical compositions containing the same, and their use in treating cancer

Abstract

The present invention discloses recombinant antibodies or fragments thereof. According to some embodiments of the present disclosure, each recombinant antibody exhibits binding affinity and neutralizing effect against parathyroid hormone-related protein. The present invention further discloses pharmaceutical compositions comprising said antibodies or antibody fragments, and their use in the treatment of cancer.

Description

[Technical field]

[0001] The present disclosure relates to the field of disease treatment. More specifically, the present invention relates to recombinant antibodies and their use in the treatment of cancer. [Background technology]

[0002] Cancer is a disease characterized by the development of abnormal cells that divide uncontrollably in a subject and may destroy normal tissues and / or organs. It is one of the leading causes of death worldwide, causing approximately 10 million deaths in 2020.

[0003] The most common cancers are breast, lung, colorectal, and prostate cancer. According to the World Health Organization (WHO), approximately one-third of cancer deaths are attributable to tobacco, high body weight, alcohol consumption, low fruit and vegetable intake, and / or lack of physical activity. Cancer-causing infectious diseases such as human papillomavirus (HPV) and hepatitis are responsible for approximately 30% of cancer cases in low-income and low-middle-income countries.

[0004] Cancer treatment varies depending on the type and progression of the cancer. Traditional cancer treatment methods include surgery, radiation therapy, and chemotherapy.

[0005] However, these treatments usually cause various complications and side effects, such as infections, blood clots, bleeding, nausea and vomiting, diarrhea, neurological and muscular injuries, incontinence, and sexual and fertility problems. Immunotherapy offers an alternative line of cancer treatment that aims to specifically stimulate immune responses against targeted cancer cells through inhibition of immune checkpoints or to enhance the ability of immune cells (e.g., T cells and B cells) to target and destroy cancer cells. However, cancer patients have reported significant side effects due to drug overstimulation and nonspecific toxicity from immunotherapy, including neurotoxicity, cytokine release syndrome (CRS), allergies, organ inflammation, and autoimmune disorders.

[0006] Therefore, there is a need for the development of new methods and / or drugs for treating cancer. Summary of the Invention

[0007] The following presents a simplified summary of the disclosure in order to provide the reader with a basic understanding. This summary is not an extensive overview of the disclosure and does not identify key / critical elements of the invention or delineate the scope of the invention. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented later.

[0008] A first aspect of the present invention is a recombinant antibody or a fragment thereof (e.g., a single chain variable fragment, scFv). The recombinant antibody or antibody fragment structurally comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, the VH domain comprising a first heavy chain complementarity determining region (CDR-H1), a second heavy chain CDR (CDR-H2) and a third heavy chain CDR (CDR-H3), and the VL domain comprising a first light chain CDR (CDR-L1), a second light chain CDR (CDR-L2) and a third light chain CDR (CDR-L3).

[0009] According to some embodiments of the present disclosure, CDR-H1, CDR-H2 and CDR-H3 comprise the amino acid sequences of "GYXFTDY" (SEQ ID NO: 1), "DTSDSY" (SEQ ID NO: 2) and "GDY", respectively, and the "X" residue at position 3 of SEQ ID NO: 1 is isoleucine (I) or threonine (T). In this embodiment, CDR-L1, CDR-L2 and CDR-L3 comprise the amino acid sequences of "QSLLESDGKTY" (SEQ ID NO: 3), "LVS" and "CQGTHFPWT" (SEQ ID NO: 4), respectively.

[0010] According to one embodiment of the present disclosure, the recombinant antibody is named 16G2, and CDR-H1, CDR-H2 and CDR-H3 comprise the amino acid sequences of "GYIFTDY" (SEQ ID NO: 5), "DTSDSY" (SEQ ID NO: 2) and "GDY", respectively, and CDR-L1, CDR-L2 and CDR-L3 comprise the amino acid sequences of "QSLLESDGKTY" (SEQ ID NO: 3), "LVS" and "CQGTHFPWT" (SEQ ID NO: 4), respectively.

[0011] Depending on the desired purpose, the antibody 16G2 can be produced in the form of a murine antibody (i.e., both the variable and constant domains of the antibody are of mouse origin), a chimeric antibody (i.e., the variable and constant domains of the antibody are of mouse and human origin, respectively), or a humanized antibody (i.e., the framework sequences of the variable domains of the antibody are modified by antibody variants that naturally occur in humans).

[0012] According to one embodiment, the antibody 16G2 is produced in the form of a murine antibody, the VH and VL domains of which comprise amino acid sequences having 85% or more identity with SEQ ID NOs: 7 and 8, respectively. In one embodiment, the VH and VL domains of the murine antibody 16G2 comprise amino acid sequences having 100% identity with SEQ ID NOs: 7 and 8, respectively, i.e. the VH and VL domains of the murine antibody 16G2 comprise the amino acid sequences of SEQ ID NOs: 7 and 8, respectively.

[0013] According to another embodiment, the antibody 16G2 is produced in the form of a chimeric antibody, the VH and VL domains of which each comprise an amino acid sequence having 85% or more identity with SEQ ID NO: 7, preferably the VH and VL domains of the chimeric antibody 16G2 each comprise the amino acid sequences of SEQ ID NOs: 7 and 8. According to this embodiment, the chimeric antibody 16G2 further comprises a heavy chain constant (CH) domain and a light chain constant (CL) domain of human origin, the CH and CL domains each comprise an amino acid sequence having 85% or more identity with SEQ ID NOs: 18 and 19, respectively, preferably the CH and CL domains of the chimeric antibody 16G2 each comprise the amino acid sequences of SEQ ID NOs: 18 and 19, respectively.

[0014] According to yet another embodiment, antibody 16G2 is produced in the form of a humanized antibody, the VH domain of which comprises an amino acid sequence having 85% or more identity to SEQ ID NO: 9, 10, 11 or 12, and the VL domain of which comprises an amino acid sequence having 85% or more identity to SEQ ID NO: 13, 14 or 15. In one embodiment, the VH domain of humanized antibody 16G2 comprises the amino acid sequence of SEQ ID NO: 9, 10, 11 or 12, and the VL domain of humanized antibody 16G comprises the amino acid sequence of SEQ ID NO: 13, 14 or 15. According to a specific embodiment, the VH domain of human antibody 16G2 comprises the amino acid sequence of SEQ ID NO: 11, and the VL domain of human antibody 16G2 comprises the amino acid sequence of SEQ ID NO: 13.

[0015] According to some embodiments of the present disclosure, the recombinant antibody is named 5B3, and its CDR-H1, CDR-H2 and CDR-H3 comprise the amino acid sequences of "GYTFTDY" (SEQ ID NO: 6), "DTSDSY" (SEQ ID NO: 2) and "GDY", respectively, and its CDR-L1, CDR-L2 and CDR-L3 comprise the amino acid sequences of "QSLLESDGKTY" (SEQ ID NO: 3), "LVS" and "CQGTHFPWT" (SEQ ID NO: 4), respectively. As can be understood, the antibody 5B3 can be made as a murine antibody, a chimeric antibody or a humanized antibody depending on the purpose.

[0016] According to one embodiment, the antibody 5B3 is produced in the form of a murine antibody, and the VH and VL domains comprise amino acid sequences having 85% or more identity with SEQ ID NOs: 16 and 17, respectively. In one embodiment, the VH and VL domains of the murine antibody 5B3 comprise the amino acid sequences of SEQ ID NOs: 16 and 17, respectively.

[0017] According to another embodiment, the antibody 5B3 is produced in the form of a chimeric antibody, the VH and VL domains of which comprise amino acid sequences having 85% or more identity with SEQ ID NOs: 16 and 17, respectively, and preferably the VH and VL domains of the chimeric antibody 5B3 comprise the amino acid sequences of SEQ ID NOs: 16 and 17, respectively. In this embodiment, the chimeric antibody 5B3 further comprises CH and CL domains of human origin, the CH and CL domains of which comprise amino acid sequences having 85% or more identity with SEQ ID NOs: 18 and 19, respectively, and preferably the CH and CL domains of the chimeric antibody 5B3 comprise the amino acid sequences of SEQ ID NOs: 18 and 19, respectively.

[0018] Also disclosed is the use of an antibody of the present disclosure in the preparation of a medicament or pharmaceutical composition for treating cancer in a subject, said medicament or pharmaceutical composition comprising an antibody or antibody fragment of any embodiment or example of the present disclosure, and optionally a pharma- ceutically acceptable carrier.

[0019] Another aspect of the present disclosure relates to a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of an antibody, antibody fragment, medicament or pharmaceutical composition of the present disclosure.

[0020] In some embodiments, the cancer is a parathyroid hormone-related protein (PTHrP)-positive cancer (i.e., a cancer that expresses or secretes PTHrP), such as gastric cancer, lung cancer, bladder cancer, breast cancer, pancreatic cancer, renal cancer, colon cancer, cervical cancer, ovarian cancer, brain cancer, prostate cancer, hepatocellular carcinoma, melanoma, esophageal cancer, multiple myeloma, or head and neck squamous cell carcinoma.

[0021] Subjects that can be treated with the present recombinant antibodies, medicaments, pharmaceutical compositions and / or methods are mammals, preferably human.

[0022] The many features and advantages of the present disclosure will be better understood from the following detailed description when considered in conjunction with the accompanying drawings. [Brief description of the drawings]

[0023] The invention will be better understood from the following detailed description, taken in conjunction with the accompanying drawings, which are briefly described below.

[0024] [Figure 1] 1 shows the effect of BGM-2121 on inhibiting the proliferation of BxPC-3 cancer cells according to Example 1.3 of the present disclosure.

[0025] [Diagram 2] FIG. 1 shows the effect of BGM-2121 on inhibiting migration of HARA-B cancer cells according to Example 1.3 of the present disclosure. IgG: isotype antibody that serves as a negative control in the study. *p<0.05; **p<0.01.

[0026] [Diagram 3] FIG. 2 shows the therapeutic effect of mouse antibody 16G2 against non-small cell lung cancer (NSCLC) according to Example 2.1 of the present disclosure. (A) shows the tumor volume of mice treated with the given treatment. (B) shows the serum calcium level of mice treated with the given treatment. IgG represents the isotype antibody that serves as a negative control in this study. *p<0.05; **p<0.01; ***p<0.001; ns (not significant)≧0.05.

[0027] [Figure 4] 1 shows the therapeutic effect of mouse antibody 16G2 against pancreatic cancer according to Example 2.1 of the present disclosure. (A) shows the tumor volume of mice treated with the given treatment. (B) shows the serum calcium level of mice treated with the given treatment. IgG indicates the isotype antibody that serves as a negative control in this study. *p<0.05; **p<0.01.

[0028] [Diagram 5]1 shows the therapeutic effect of humanized antibody BGM-2121 according to Example 2.2 of the present disclosure on BxPC3 subcutaneous tumor model. (A) shows the tumor volume of mice administered with hIgG or BGM-2121 twice a week, once a week, or once every two weeks. (B) shows the tumor volume of mice administered with hIgG or BGM-2121 once every three weeks. (C) shows the survival probability of mice administered with hIgG or BGM-2121 once every three weeks. hIgG refers to the human IgG isotype antibody, which serves as the negative control in this study. *p<0.05; ***p<0.001.

[0029] [Figure 6] FIG. 2 shows the therapeutic effect of humanized antibody BGM-2121 on the orthotopic tumor model of BxPC3 according to Example 2.2 of the present disclosure. (A) shows the tumor weight of mice treated with the indicated treatments. (B) shows the serum creatine phosphokinase (CPK) level of mice treated with the indicated treatments. hIgG indicates the human IgG isotype antibody, which serves as a negative control in this study. *p<0.05; ns≧0.05.

[0030] [Figure 7] Figure 2 shows the therapeutic effect of humanized antibody BGM-2121 according to Example 2.2 of the present disclosure on CFPAC-1 subcutaneous tumor model. (A) shows the tumor volume of mice treated with the indicated treatments. (B) shows the tumor weight of mice treated with the indicated treatments. Arrows indicate the injection date of antibody (AB). hIgG indicates the human IgG isotype antibody that serves as the negative control in this study. *p<0.05. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] A preferred embodiment of the present disclosure will be described in detail below with reference to the accompanying drawings, but the present disclosure is not limited to such an embodiment. The description shows the functions of the example and a sequence of steps for constructing and operating the example. However, the same or equivalent functions and sequences may be achieved by different examples.

[0032] I. Definition

[0033] For convenience, the terms used in the present specification, examples, and claims are summarized below. Scientific and technical terms used in the present specification are to be generally understood by those skilled in the art unless otherwise defined. It is also understood that singular terms include the plural of the same term, and plural terms include the singular, unless expressly stated otherwise. Specifically, in the present specification and claims, the singular forms "a" and "an" also include plural references, unless the context clearly indicates otherwise. Also, in the present specification and claims, "at least one" and "one or more" have the same meaning, including one, two, or more than two.

[0034] Although the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Also, the term "about" as used herein generally means within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the term "about" means within an acceptable standard error of the mean as considered by one of ordinary skill in the art. Other than in the operating or working examples, or unless expressly specified, all numerical ranges, amounts, values, and percentages, such as amounts of materials, lengths of time, temperatures, operating conditions, ratios of amounts, etc. disclosed herein should be understood in all instances to be modified by the term "about". Thus, unless indicated to the contrary, the numerical parameters set forth in the present disclosure and the appended claims are approximations that can be varied as necessary. At the very least, each numerical parameter should be interpreted in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0035] The term "antibody" (Ab) is used in the broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific or multivalent antibodies (e.g., bispecific antibodies), chimeric antibodies, humanized antibodies, and antibody fragments, so long as they exhibit the desired biological activity. An "antibody fragment" or "fragment of an antibody" refers to a portion of a full-length antibody, typically the antigen-binding or variable domains (VH and VL domains) of a full-length antibody. Antibody fragments include, for example, fragment antigen-binding (Fab), Fab', F(ab')2, single-chain variable fragments (scFv), diabodies, linear antibodies, single-chain antibody molecules, multispecific antibodies consisting of antibody fragments, and the like.

[0036] "Complementarity determining region" (CDR) refers to the hypervariable region of an antibody molecule that forms a surface complementary to the three-dimensional surface of a bound antigen. From the N-terminus to the C-terminus, the heavy and light chains of an antibody each contain three CDRs (CDR-1, CDR-2, CDR-3). Thus, the antigen-binding site contains six CDRs in total: three CDRs (CDR-H1, CDR-H2, CDR-H3) in the variable domain of the heavy chain and three CDRs (CDR-L1, CDR-L2, CDR-L3) in the variable domain of the light chain.

[0037] The "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of an antibody. These domains are usually the most variable parts of an antibody and contain the antigen-binding site. "Variable" refers to the fact that certain parts of the variable domain differ widely in sequence among antibodies and are used for the binding specificity of each particular antibody to its particular antigen. However, the variability is not evenly distributed throughout the variable domain of an antibody. In both the light and heavy chain variable domains, it is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions. The more highly conserved parts of the variable domains are called frameworks (FRs). The variable domains of native heavy and light chains each consist of four FR regions, which adopt a mainly beta-sheet configuration and are bound by three CDRs. The CDRs form loops that connect the beta-sheet structures and sometimes form part of the beta-sheet structures. The CDRs of each chain are held in close proximity by the FR regions and, with the CDRs of the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).

[0038] As discussed herein, minor changes in the amino acid sequence of the antibody are considered to be included in the present disclosure, provided that the changes in the amino acid sequence maintain at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity. The antibodies of the present disclosure may be specifically modified to modify characteristics of the antibody that are not related to bioactivity. For example, amino acid residues in the framework (FR) region of the antibody may be altered and / or deleted without affecting the bioactivity (therapeutic ability against cancer) of the antibody in the present study. In particular, conservative amino acid substitutions are contemplated. Conservative substitutions are those that occur in groups of amino acid residues whose side chains are related. Genetically encoded amino acid residues are generally classified into the following families: (1) acidic = aspartic acid, glutamic acid, (2) basic = lysine, arginine, histidine, (3) non-polar = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, (4) uncharged polar = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. More preferred families are serine and threonine in the aliphatic-hydroxy family, asparagine and glutamine in the amide-containing family, alanine, valine, leucine and isoleucine in the aliphatic family, and phenylalanine, tryptophan and tyrosine in the aromatic family. For example, substitutions of amino acids with structurally related amino acids, such as leucine to isoleucine or valine, aspartic acid to glutamic acid, and threonine to serine, are not expected to have a significant effect on the binding or properties of the molecule, especially if the substitution does not involve an amino acid residue in the CDR, which is the antigen-binding site. Whether an amino acid change results in a functional peptide can be easily determined by measuring the specific activity of the peptide derivative. Fragments or analogs of proteins / peptides can be easily prepared by those skilled in the art. Preferred amino and carboxy termini of fragments or analogs occur near the boundaries of functional domains.

[0039] "Percentage of identity (%)" is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular amino acid sequence. The maximum percent sequence identity is obtained by aligning the sequences and introducing gaps as necessary. It is noted that conservative substitutions are not considered as part of sequence identity. Alignment for determining percentage sequence identity can be achieved by various methods within the art, for example, by publicly available computer software, such as BLAST, BLAST-2, ALIGN or Garin (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment. The parameters include any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. For the purposes of this specification, sequence comparison between two amino acid sequences is performed by the computer program Blastp (protein-protein BLAST), which is provided online by the National Center for Biotechnology Information (NCBI). The percentage amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (also referred to herein as a given amino acid sequence A having a particular % amino acid sequence identity with a given amino acid sequence B) is calculated by the following formula: Here, X is the number of amino acid residues obtained as identical matches by aligning sequences A and B using the sequence alignment program BLAST, and Y is the total number of amino acid residues in the shorter of sequences A and B.

[0040] As used herein, "treatment", "treatment" and "therapeutic treatment" are interchangeable and include prevention, improvement, alleviation and / or management of some or all of the symptoms, secondary disorders or conditions associated with cancer. As used herein, "treatment" refers to the application or administration of an antibody of the present disclosure to a subject having a symptom, secondary disorder or condition associated with cancer for the purpose of alleviating, improving, alleviating, inhibiting delayed onset, inhibiting progression, reducing severity and / or reducing incidence of one or more symptoms, secondary disorders or characteristics associated with cancer. Examples of symptoms, secondary disorders and / or conditions associated with cancer include, but are not limited to, hypercalcemia (calcium levels in the blood above normal), nausea, vomiting, loss of appetite, constipation, fatigue, muscle weakness, exacerbation, bone pain or fracture, swelling or lumps, bleeding, cough, fever, night sweats, coma, pain, etc. Subjects having only early signs of such symptoms, diseases and / or conditions may be treated with the purpose of reducing the risk of developing symptoms, secondary disorders and / or conditions associated with cancer. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced, as defined herein. Alternatively, treatment is "effective" if the progression of a symptom, disease or condition is reduced or halted.

[0041] Here, an "effective amount" refers to an amount of a component sufficient to obtain a desired response. For therapeutic purposes, an effective amount is also an amount in which the therapeutically beneficial effects outweigh the toxic or adverse effects of the component. An effective amount of a drug is not necessary for the treatment of a disease state, but provides a treatment that delays, inhibits, or prevents the onset of a disease state or improves the symptoms of a disease state. The effective amount may be divided into one or more suitable dosage forms and administered one or more times over a specified period of time. A particular effective or sufficient amount will vary depending on the particular condition being treated, the physical condition of the patient (e.g., the patient's weight, age, sex, etc.), the type of mammal or animal being treated, the duration of treatment, the nature of concurrent treatment (if any), and the particular formulation used, as well as the structure of the compound or its derivatives. An effective amount may be, for example, grams, milligrams, micrograms, or milligrams per kg of body weight (mg / kg). Alternatively, an effective amount may be expressed in terms of the concentration of the active component (e.g., an antibody of the present disclosure), such as molar concentration, mass concentration, volume concentration, molar concentration, molar fraction, mass fraction, or mixture ratio. One of skill in the art can calculate the human equivalent dose (HED) of a drug (such as the antibody) based on the dose determined from the animal model. For example, the maximum safe dose for use in human subjects may be estimated according to the U.S. Food and Drug Administration (FDA) industry guide entitled "Estimation of the Maximum Safe Starting Dose for Early Clinical Trials of Therapeutic Agents in Healthy Adult Volunteers."

[0042] "Subject" refers to an animal, including the human species, that is treatable by the antibodies, medicaments, pharmaceutical compositions and / or methods of the disclosure. "Subject" refers to both males and females, unless specifically indicating one gender.

[0043] II. Description of the Invention

[0044] (i) Antibodies—Mouse 16G2 and Mouse mAb 5B3

[0045] The present invention aims to treat diseases, particularly PTHrP-related diseases (eg, cancer), in a subject, as well as to provide pharmaceutical formulations for such purposes.

[0046] Accordingly, a first aspect of the present disclosure is directed to two monoclonal antibodies (mAbs), designated "16G2" and "5B3," respectively. According to embodiments of the present disclosure, the mAbs of the present disclosure exhibit binding affinity and neutralizing activity with parathyroid hormone-related protein (PTHrP), a hormone that acts as an endocrine, autocrine, paracrine and endocrine regulator, mediates cancer growth, progression and metastasis, and can treat cancer and PTHrP-induced hypercalcemia.

[0047] According to some embodiments of the present disclosure, the mAbs of the present invention are produced by immunization techniques (ie, immunizing animals with specific peptides).

[0048] In general, the polypeptide (PTHrP polypeptide) can be synthesized by conventional methods such as t-BOC or FMOC protection of α-amino group. In either method, the peptide is synthesized stepwise, one amino acid at a time, from the C-terminus. The polypeptide of the present disclosure can also be synthesized by known solid-phase peptide synthesis methods.

[0049] Next, the synthetic polypeptide can be used to immunize a host animal such as a mouse, rat, or rabbit to produce antibodies. The immunization can be performed according to a commonly used procedure. The immunization interval is not particularly limited. Immunization can be performed 2-10 times over several days to several weeks, preferably every week, until a desired antibody titer is achieved. For example, the host animal can be vaccinated by subcutaneously injecting the synthetic polypeptide every week for eight consecutive weeks.

[0050] After the final immunization, spleen cells and local lymph nodes are removed. After immunization, blood samples are periodically taken and centrifuged to separate serum. The obtained serum is subjected to measurement of antibody titer by any suitable method, such as enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), etc. In a preferred embodiment, the antibody titer is measured by ELISA. Next, animals that show high antibody titers against the synthetic polypeptide are given the final immunization. Antibody-producing cells are prepared from spleen cells and local lymph nodes of the immunized animals. In preparing the antibody-producing cells, it is preferable to remove tissue debris and red blood cells as much as possible. For this purpose, a commercially available red blood cell remover can be used. Alternatively, ammonium chloride and Tris buffer can be prepared and used. The antibody-producing cells thus prepared are immediately fused with immortal cells such as myeloma cells to prepare hybridoma cells, which continue to grow semi-permanently while producing antibodies. Generally available cell lines derived from animals such as mice can be used. The preferred cell lines used in the present invention are those that do not survive in HAT selection medium containing hypoxanthine, thymidine, and aminopterin, but survive only when fused with antibody-producing cells. Examples of myeloma cells include, but are not limited to, mouse myeloma cell lines (such as myeloma FO cells) and human myeloma cell lines (such as Karpas 707H). Cell fusion is usually performed by mixing spleen cells or lymph node cells with commercially available myeloma cells in the presence of a cell fusion promoter such as polyethylene glycol (PEG) with an average molecular weight of about 200 to 20,000 daltons. Alternatively, cell fusion can be performed using a commercially available cell fusion device that utilizes electrical stimulation such as electroporation. After fusion, the resulting cells are diluted and cultured in HAT medium.

[0051] The desired hybridoma is selected from the fused cells. The fused cells are cultured in HAT medium to form colonies. Next, the supernatant of each culture well is collected and the presence or absence of antibody titer against the polypeptide is examined. Confirmation methods can include ELISA, EIA, and RIA. Once antibody-positive wells are identified, they are cultured in HT medium that does not contain aminopterin and contains only hypoxanthine and thymidine. After culturing for a while, the antibody titer in the culture supernatant is confirmed again. Finally, the selected cells are cloned to obtain single cells. Clones with high specificity against the polypeptide are selected and grown to a certain extent to establish hybridomas.

[0052] The mAb produced by the hybridoma can be isolated or prepared by a known method. For example, the antibody may be prepared from the culture supernatant of the hybridoma cultured in a medium with a low serum concentration. Alternatively, the hybridoma may be injected into the abdominal cavity of an animal, and the resulting ascites droplets may be collected to prepare the antibody. Purification or isolation of the antibody can be performed by a method such as affinity column, gel filtration chromatography, or ion exchange chromatography. These known methods can be appropriately selected and used.

[0053] The mAbs 16G2 and 5B3 thus produced contain three CDRs in the VH domain (CDR-H1, CDR-H2, CDR-H3) and three CDRs in the VL region (CDR-L1, CDR-L2, CDR-L3).

[0054] According to some embodiments of the present disclosure, CDR-H1, CDR-H2, and CDR-H3 of MAB 16G2 comprise the amino acid sequences of "GYIFTDY" (SEQ ID NO: 5), "DTSDSY" (SEQ ID NO: 2), and "GDY," respectively, and CDR-L1, CDR-L2, and CDR-L3 of MAB 16G2 comprise the amino acid sequences of "QSLLESDGKTY" (SEQ ID NO: 3), "LVS," and "CQGTHFPWT" (SEQ ID NO: 4), respectively.

[0055] As an example, the amino acid sequences of the VH domain and VL domain of mAb 16G2 are provided below as SEQ ID NOs: 7 and 8, respectively, with the CDRs (CDR-H1, CDR-H2, CDR-H3 of the VH domain and CDR-L1, CDR-L2, CDR-L3 of the VL domain) (underlined) marked in bold.

[0056] SEQ ID NO:7 (VH domain of mAb 16G2) QVQLQQPGAELGMPGASVKMSCKAS GYIFTDY WMHWVKQRPGQGLEWIGAI DTSDSYS SYNQKFQGKATLTVDESSSTAYMQLSSLTSEDSAVYYCTL GDY WGQGTTLTVSS

[0057] SEQ ID NO:8 (VL domain of mAb 16G2) DVVMTQTPLTLSVTIGQPASMSCKSS QSLLESDGKTY LNWLLQRPGQSPKRLIY LVS KLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC CQGTHFPWT FGGGTKLEIK

[0058] Since the binding affinity and specificity of an antibody are mainly determined by its CDR sequences, it is understood that the framework (FR) sequences of the VH and VL domains can be altered (e.g., substituted with conserved or non-conserved amino acid residues) without affecting the binding affinity and / or specificity of the antibody. The FR sequences are preferably conservatively substituted with one or more suitable amino acids having similar properties, such as substituting leucine (a non-polar amino acid residue) with isoleucine, alanine, valine, proline, phenylalanine, or tryptophan (another non-polar amino acid residue); substituting aspartic acid (an acidic amino acid residue) with glutamic acid (another acidic amino acid residue); or substituting lysine (a basic amino acid residue) with arginine or histidine (another basic amino acid residue).

[0059] Based on conservative substitutions, a skilled artisan can substitute amino acid residues in the FR sequences of the VH and VL domains of mAb 16G2 without affecting the activity and / or efficacy of mAb 16G2 (i.e., neutralizing PTHrP and treating cancer). Thus, antibodies containing substituted amino acids in the FR sequences of the VH and VL domains are intended to be within the scope of the present disclosure. According to certain embodiments, the VH domain of mAb 16G2 comprises an amino acid sequence having 85% or more (i.e., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to SEQ ID NO:7, and the VL domain of mAb 16G2 comprises an amino acid sequence having 85% or more (i.e., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to SEQ ID NO:8. According to some preferred embodiments, the VH domain and VL domain of mAb 16G2 comprise amino acid sequences having at least 90% identity to SEQ ID NOs:7 and 8, respectively. More preferably, the VH and VL domains of mAb 16G2 comprise amino acid sequences having 95% or greater identity to SEQ ID NOs: 7 and 8, respectively.

[0060] According to some embodiments of the present disclosure, CDR-H1, CDR-H2, and CDR-H3 of mAb 5B3 comprise the amino acid sequences of "GYTFTDY" (SEQ ID NO: 6), "DTSDSY" (SEQ ID NO: 2), and "GDY," respectively, and CDR-L1, CDR-L2, and CDR-L3 of mAb 5B3 comprise the amino acid sequences of "QSLLESDGKTY" (SEQ ID NO: 3), "LVS," and "CQGTHFPWT" (SEQ ID NO: 4), respectively.

[0061] As an example, the amino acid sequences of the VH domain and VL domain of mAb 5B3 are provided below as SEQ ID NOs: 16 and 17, respectively, with the CDRs (CDR-H1, CDR-H2, CDR-H3 of the VH domain, and CDR-L1, CDR-L2, CDR-L3 of the VL domain) (underlined) marked in bold.

[0062] SEQ ID NO: 16 (VH domain of mAb 5B3) QVQLQQPGAELVMPGASVKMSCKAS GYTFTDY WMHWVKQRPGQGLEWIGAL DTSDSY ASYNQRFKGKATLTVDASSSTAYMQLSSLTSEDSAVYYCTL GDY WGQGTTLTVSS

[0063] SEQ ID NO: 17 (VL domain of mAb5B3) DVVMTQTPLTLSVTFGQPASISCKSS QSLLESDGKTY LNWLLQRPGQSPKRLIY LVS KLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC CQGTHFPWT FGGGTKLEIK

[0064] As described above, the FR sequences of the VH and VL domains may be altered (e.g., substituted with conserved or non-conserved amino acid residues) without affecting the binding affinity and / or specificity of the antibody, and one of skill in the art may substitute amino acid residues in the FR sequences of the VH and VL domains of mAb 5B3 without affecting the activity and / or efficacy of mAb 5B3 (i.e., neutralization of PTHrP and treatment of cancer). Thus, antibodies containing substituted amino acids in the FR sequences of the VH and VL domains are intended to be within the scope of the present disclosure. According to certain embodiments, the VH domain of mAb 5B3 comprises an amino acid sequence having 85% or greater (i.e., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to SEQ ID NO: 16, and the VL domain of mAb 5B3 comprises an amino acid sequence having 85% or greater (i.e., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to SEQ ID NO: 17. Preferably, the VH and VL domains of mAb 5B3 comprise amino acid sequences having 90% or greater identity to SEQ ID NOs: 16 and 17, respectively. More preferably, the VH and VL domains of mAb 5B3 comprise amino acid sequences having 95% or greater identity to SEQ ID NOs: 16 and 17, respectively.

[0065] As can be understood, the mAb of the present disclosure (i.e., mAb 16G2 or 5B3) can be prepared by DNA cloning based on the above amino acid sequence. Specifically, a person skilled in the art can prepare a DNA construct containing a base sequence encoding the VH and VL sequences of the mAb of the present disclosure, and then introduce the DNA construct into a suitable host cell that does not produce immunoglobulin protein (e.g., E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, myeloma cells) to synthesize the desired mAb in the recombinant host cell.

[0066] Depending on the purpose, the mAb of the present invention can be produced in the form of Immunoglobulin G (IgG), Immunoglobulin A (IgA), Immunoglobulin M (IgM), Immunoglobulin D (IgD), or Immunoglobulin E (IgE).

[0067] (ii) Antibodies - Chimeric mAb 16G2 and Chimeric mAb 5B3

[0068] Each of the mAbs described in section (i) of this disclosure (including mAbs 16G2 and 5B3) is useful for generating chimeric antibodies, i.e., antibodies having variable domains from an original species (e.g., mouse) and constant domains from another species (e.g., human), to reduce the immunogenicity of the antibody in a subject.

[0069] Thus, the present disclosure also provides two chimeric antibodies derived from the murine mAbs 16G2 and 5B3, respectively, in which the constant domains of the murine mAbs have been replaced with the constant domains of a human antibody.

[0070] Thus, the VH domain of chimeric mAb 16G2 comprises the amino acid sequence of SEQ ID NO:7, and the VL domain of chimeric mAb 16G2 comprises the amino acid sequence of SEQ ID NO:8, as described in section (i) of this disclosure.

[0071] According to some embodiments, the VH domain of chimeric mAb 5B3 comprises the amino acid sequence of SEQ ID NO: 16, and the VL domain of chimeric mAb 5B3 comprises the amino acid sequence of SEQ ID NO: 17, as described in section (i) of this disclosure.

[0072] According to these embodiments, chimeric mAb 16G2 and chimeric mAb 5B3 each comprise a CH domain of SEQ ID NO: 18 linked to its VH domain and a CL domain of SEQ ID NO: 19 linked to its VL domain. The amino acid sequences of CH and CL are shown below.

[0073] SEQ ID NO: 18 (CH domain of chimeric mAb) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0074] SEQ ID NO: 19 (CL domain of chimeric mAb) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0075] As described above, the constant domains and FR sequences of the VH and VL domains can be altered (e.g., substituted with conserved or non-conserved amino acid residues) without affecting the binding affinity and / or specificity of the antibody, and one of skill in the art can substitute amino acid residues of the constant domains and / or FR sequences of the VH and VL domains of the chimeric mAb without affecting its activity and / or effect (e.g., neutralization of PTHrP and treatment of cancer). Thus, antibodies comprising substituted amino acids in their constant regions and / or FR sequences of the VH and VL domains are intended to be within the scope of the present disclosure.

[0076] Depending on the purpose, chimeric mAbs can be made in the form of IgG, IgA, IgM, IgD or IgE.

[0077] (iii) Antibody - humanized mAb 16G2

[0078] Alternatively, each of the mAbs described in section (i) of this disclosure (including mAbs 16G2 and 5B3) are useful for the production of humanized antibodies, i.e., the amino acid sequence of a non-human antibody (e.g., a murine antibody) is altered to become more similar to antibody variants naturally produced in humans, thereby minimizing the immunogenicity of the antibody in human subjects.

[0079] Thus, the present disclosure further provides different humanized VH, VL sequences, including humanized VH1 (SEQ ID NO: 9), humanized VH2 (SEQ ID NO: 10), humanized VH3 (SEQ ID NO: 11), humanized VH4 (SEQ ID NO: 12), humanized VL1 (SEQ ID NO: 13), humanized VL2 (SEQ ID NO: 14), and humanized VL3 (SEQ ID NO: 15). The amino acid sequences of the human VH and VL domains are provided below, where the CDRs (i.e., CDR-H1, CDR-H2, CDR-H3 of the VH domain, and CDR-L1, CDR-L2, CDR-L3 of the VL domain; underlined) are marked in bold in order.

[0080] SEQ ID NO:9 (humanized VH1 domain of mAb 16G2) QVQLVQSGAEVKKPGASVKVSCKAS GYIFTDY WMHWVRQAPGQGLEWMGAI DTSDSY SSYNQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTL GDY WGQGTTVTVSS

[0081] SEQ ID NO: 10 (humanized VH2 domain of mAb 16G2) QVQLVQSGAEVKKPGASVKVSCKAS GYIFTDY WMHWVRQAPGQGLEWIGAI DTSDSY SSYNQKFQGRATMTVDTSTSTVYMELSSLRSEDTAVYYCTL GDY WGQGTTVTVSS

[0082] SEQ ID NO:11 (humanized VH3 domain of mAb 16G2) QVQLVQSGAEVKKPGASVKVSCKAS GYIFTDYWMHWVKQAPGQGLEWIGAI DTSDSY SSYNQKFQGRATMTVDTSTSTVYMELSSLRSEDTAVYYCTL GDY WGQGTTVTVSS

[0083] SEQ ID NO: 12 (humanized VH4 domain of mAb 16G2) QVQLVQSGAEVKKPGASVKVSCKAS GYIFTDY WMHWVKQAPGQGLEWIGAI DTSDSY SSYNQKFQGRATMTVDESTSTVYMELSSLRSEDTAVYYCTL GDY WGQGTTVTVSS

[0084] SEQ ID NO: 13 (humanized VL1 domain of mAb 16G2) DVVMTQSPLSLPVTLGQPASISC KSSQSLLESDGKTY LNWFQQRPGQSPRRLIY LVS KLDSGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC CQGTHFPWT FGGGTKLEIK

[0085] SEQ ID NO: 14 (humanized VL2 domain of mAb 16G2) DVVMTQSPLSLPVTLGQPASISC KSSQSLLESDGKTY LNWLLQRPGQSPRRLIY LVS KLDSGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC CQGTHFPW TFGGGTKLEIK

[0086] SEQ ID NO: 15 (humanized VL3 domain of mAb 16G2) DVVMTQSPLSLPVTLGQPASMSC KSSQSLLESDGKTY LNWLLQRPGQSPKRLIY LVS KLDSGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC CQGTHFPWT FGGGTKLEIK

[0087] As described above, the FR sequences of the VH and VL domains can be altered (e.g., substituted with conserved or non-conserved amino acid residues) without affecting the binding affinity and / or specificity of the antibody, and a person skilled in the art can substitute amino acid residues in the FR sequences of the VH and VL domains of the humanized mAb without affecting its activity and / or effect (e.g., neutralization of PTHrP and treatment of cancer). Thus, antibodies containing substituted amino acids in the FR sequences of the humanized VH and VL domains are intended to be within the scope of the present disclosure.

[0088] According to one embodiment, the humanized mAb designated "humanized mAb 16G2-1" comprises a VH1 domain (SEQ ID NO: 9) and a VL1 domain (SEQ ID NO: 13). According to another embodiment, the humanized mAb designated humanized mAb 16G2-2 comprises a VH1 domain (SEQ ID NO: 9) and a VL2 domain (SEQ ID NO: 14). According to another embodiment, the humanized mAb designated humanized mAb 16G2-3 comprises a VH1 domain (SEQ ID NO: 9) and a VL3 domain (SEQ ID NO: 15).

[0089] According to one embodiment, the humanized mAb designated "humanized mAb 16G2-4" comprises a VH2 domain (SEQ ID NO: 10) and a VL1 domain (SEQ ID NO: 13). According to another embodiment, the humanized mAb designated "humanized mAb 16G2-5" comprises a VH2 domain (SEQ ID NO: 10) and a VL2 domain (SEQ ID NO: 14). According to another embodiment, the humanized mAb designated "humanized mAb 16G2-6" comprises a VH2 domain (SEQ ID NO: 10) and a VL3 domain (SEQ ID NO: 15).

[0090] According to one embodiment, the humanized mAb designated "humanized mAb 16G2-7" comprises a VH3 domain (SEQ ID NO:11) and a VL1 domain (SEQ ID NO:13). According to another embodiment, the humanized mAb designated "humanized mAb 16G2-8" comprises a VH3 domain (SEQ ID NO:11) and a VL2 domain (SEQ ID NO:14). According to another embodiment, the humanized mAb designated "humanized mAb 16G2-9" comprises a VH3 domain (SEQ ID NO:11) and a VL3 domain (SEQ ID NO:15).

[0091] According to one embodiment, the humanized mAb designated "humanized mAb 16g-10" comprises a VH4 domain (SEQ ID NO: 12) and a VL1 domain (SEQ ID NO: 13). According to another embodiment, the humanized mAb designated "humanized mAb 16g-11" comprises a VH4 domain (SEQ ID NO: 12) and a VL2 domain (SEQ ID NO: 14). According to another embodiment, the humanized mAb designated "humanized mAb 16g-12" comprises a VH4 domain (SEQ ID NO: 12) and a VL3 domain (SEQ ID NO: 15).

[0092] Depending on the purpose, humanized mAbs can be produced in the form of IgG, IgA, IgM, IgD, or IgE.

[0093] (iv) A pharmaceutical agent or composition comprising the mAb of the present disclosure.

[0094] According to some aspects of the present disclosure, the mAbs of the present disclosure (including the murine mAbs, chimeric mAbs and humanized mAbs described in each of sections (i)-(iii) of the present disclosure) exhibit binding affinity and neutralizing activity for PTHrP. Accordingly, another aspect of the present disclosure relates to a medicament or pharmaceutical composition for treating cancer, particularly cancer caused and / or associated with PTHrP. The medicament or pharmaceutical composition comprises a mAb of the present disclosure or a fragment thereof (e.g., scFv), and optionally a pharmaceutically acceptable carrier.

[0095] The mAb or antibody fragment of the present disclosure is typically present at a level of about 0.1%-99% by weight based on the total weight of the medicament or pharmaceutical composition. In some embodiments, the mAb or antibody fragment of the present disclosure is preferably present at 1% by weight or more based on the total weight of the medicament or pharmaceutical composition. In some embodiments, the mAb or antibody fragment is present at 5% by weight or more based on the total weight of the medicament or pharmaceutical composition. In yet other embodiments, the mAb or antibody fragment is present at a level of 10% by weight or more based on the total weight of the medicament or pharmaceutical composition. In yet other embodiments, the mAb or antibody fragment is present at a level of 25% by weight or more based on the total weight of the medicament or pharmaceutical composition.

[0096] A pharma- ceutically acceptable carrier includes any pharma- ceutically acceptable material or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulant, useful for carrying or delivering an active agent (e.g., the mAb or antibody fragment of the present invention) from one organ or part of the body to another. The carrier must be "acceptable" in terms of compatibility with other ingredients in the formulation, and is selected to minimize degradation of the active agent and to minimize adverse effects in the subject. Depending on the desired purpose, the pharmaceutical or pharmaceutical composition of the present disclosure may further include one or more pharma- ceutical acceptable additives, such as binders, flavoring agents, buffering agents, thickening agents, coloring agents, antioxidants, diluents, stabilizers, buffers, emulsifiers, dispersing agents, suspending agents, preservatives, and the like.

[0097] The choice of pharma- ceutically acceptable carrier to be used in combination with the mAb / antibody fragment of the present invention is essentially determined by the method of administration of the medicament or pharmaceutical composition. The medicament or pharmaceutical composition of the present disclosure can be administered to a subject via subcutaneous, intravenous or intramuscular injection.

[0098] The pharmaceutical or pharmaceutical composition for injection can be prepared in a sterile or non-aqueous solution, suspension, or emulsion. Non-aqueous solutions include, for example, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. The aqueous solutions include, for example, water, alcohol-based solutions, emulsions, suspensions such as saline, and buffered media. Common parenteral vehicles include aqueous sodium chloride solutions, Ringer's dextrose, dextrose, sodium chloride, lactid Ringer, fixed oils, and the like, and intravenous vehicles include fluids, nutrient replenishers, electrolyte replenishers (e.g., Ringer's dextrose-based), and the like.

[0099] (v) Uses of the Antibodies, Pharmaceutical Compositions, and Medicaments of the Invention

[0100] Another aspect of the present disclosure relates to a method of treating cancer in a subject, comprising administering to the subject an effective amount of a mAb, antibody fragment, pharmaceutical composition or medicament according to any aspect, embodiment or example of the present disclosure.

[0101] According to some embodiments of the present disclosure, the cancer is a PTHrP-positive cancer, which is a cancer that expresses or secretes PTHrP, and the cancers treatable by the mAb, antibody fragment, pharmaceutical composition, medicament and / or method of the present disclosure include, but are not limited to, gastric cancer, lung cancer, bladder cancer, breast cancer, pancreatic cancer, renal cancer, colorectal cancer, cervical cancer, ovarian cancer, brain cancer, prostate cancer, hepatocellular carcinoma, melanoma, esophageal cancer, multiple myeloma, or head and neck squamous cell carcinoma.In one embodiment, the cancer is lung cancer.In another exemplary embodiment, the cancer is pancreatic cancer.

[0102] According to some embodiments, the subject is a mouse. , 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 mg / kg) of the mAb or antibody fragment of the invention is administered to the subject. Preferably, about 1 mg / kg to 50 mg / kg of the mAb or antibody fragment of the invention is administered to the subject. More preferably, about 5 mg / kg to 20 mg / kg of the mAb or antibody fragment of the invention is administered to the subject. According to one example, about 10 mg / kg of the mAb or antibody fragment of the invention is sufficient to produce a therapeutic effect in a subject (i.e., inhibition of tumor growth, alleviation or amelioration of cancer symptoms such as hypercalcemia).

[0103] One of skill in the art can easily determine the human equivalent dose (HED) of the mAb or antibody fragment of the invention based on the dosages determined from the animal studies provided in the Examples of the invention. Effective amounts of the mAb / antibody fragment of the invention suitable for use in human subjects range from 10 μg to 10 mg per kg body weight for humans (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 0, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990 The HED may be at a dose of about 100 μg / kg or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg / kg. Preferably, the HED is about 100 μg to 5 mg / kg of body weight. The dosage may be administered in a single aliquot or in multiple aliquots. The skilled artisan or clinician may adjust the dosage or administration schedule depending on the physical condition of the patient or the severity of the disease.

[0104] The mAb, antibody fragment, pharmaceutical composition and / or medicament of the invention may be administered to a subject once or twice a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks or more, depending on the desired purpose. The progress of this treatment is easily monitored by conventional techniques and assays. As can be appreciated, the dosing regimen can vary over time. According to one embodiment, the mAb of the invention is administered to a subject twice a week. According to another embodiment, the mAb of the invention is administered to a subject once a week. According to yet another embodiment, the mAb of the invention is administered to a subject once every two weeks. According to a further embodiment, the mAb of the invention is administered to a subject once every three weeks.

[0105] The mAbs, antibody fragments, pharmaceutical compositions and / or medicaments of the invention may be administered to a subject by a route selected from the group consisting of nasal, topical, mucosal, and parenteral administration, which may be either subcutaneous, intramuscular, intravenous, or intraperitoneal injection.

[0106] As can be appreciated, the methods of the invention can be administered to a subject alone or in combination with an additional treatment that has some effect in the prevention or treatment of cancer. The methods of the invention can be administered before, during or after administration of the additional treatment, depending on the intended / therapeutic purpose.

[0107] According to certain aspects of the disclosure, administration of the mAb, antibody fragment, pharmaceutical composition, or medicament of the invention inhibits tumor growth. According to some embodiments of the disclosure, administration of the mAb, antibody fragment, pharmaceutical composition, or medicament of the invention reduces calcium levels in the blood (e.g., serum) of a subject.

[0108] Subjects treatable by the methods of the present invention are essentially mammals, such as humans, mice, rats, guinea pigs, hamsters, monkeys, pigs, dogs, cats, horses, sheep, goats, cows, rabbits, etc. Preferably, the subject is a human.

[0109] The following examples are provided to clarify certain aspects of the present disclosure and to allow those skilled in the art to practice the present disclosure.These examples are not intended to limit the scope of the present invention.It is believed that those skilled in the art can utilize the present disclosure to the fullest extent based on the description in this specification.All publications cited in this specification are incorporated herein by reference in their entirety. Working Example

[0110] Materials and Methods

[0111] cell culture

[0112] The rat osteosarcoma cell line UMR-106 was cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The human lung cancer cell line HARA-B and the human pancreatic cell line BxPC-3 were cultured in RPMI medium supplemented with 10% FBS. The human pancreatic cell line CFPAC-1 was cultured in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% FBS.

[0113] Preparation of mouse mAbs 16G2 and 5B3

[0114] Mouse monoclonal antibodies were generated by immunization of mice. Specifically, BALB / c mice were immunized with an Escherichia coli (E. coli)-expressed fusion protein containing a PTHrP peptide and a polyhistidine tag (His tag) fused to the C-terminus of the PTHrP peptide. After immunization, B cells were extracted from the spleens of the immunized mice and fused with myeloma cells. The titers of antibodies secreted by the hybridomas thus generated were measured by ELISA, and hybridoma clones (clones 16G2 and 5B3) expressing antibodies that exhibited binding affinity and specificity for PTHrP were identified. The selected clones 16G2 and 5B3 were cultured to promote antibody production. Antibodies isolated from the supernatants of the culture media of clones 16G2 and 5B3 were purified and then subjected to sequence analysis and activity assays.

[0115] The obtained antibodies were named "mouse mAb 16G2" and "mouse mAb 5B3," respectively, and the VH and VL sequences of the mouse antibodies are summarized in Table 1.

[0116] Table 1: VH and VL sequences of mouse mAbs 16G2 and 5B3 [Table 1] *CDR sequences (including CDR-H1, CDR-H2, and CDR-H3 of the VH domain, and CDR-L1, CDR-L2, and CDR-L3 of the VL domain) are marked in bold, in order.

[0117] Preparation of chimeric mAbs 16G2, 5B3

[0118] The nucleotide sequences encoding the VH and VL domains of the murine mAbs 16G2 and 5B3, respectively, were cloned into the expression vector pcDNA3.4, which contains the constant domain of human IgG. The vectors containing both the non-human variable domains and the human constant domains were transformed into the host cell EXPICHO-S for expression. TM The chimeric monoclonal antibodies were isolated from the culture medium supernatant and subsequently purified and sequenced.

[0119] The chimeric mAb thus obtained comprises the VH and VL domains of a mouse mAb (i.e., the VH and VL domains of mouse mAb 16G2 or mouse mAb 5B3) and the constant domains of a human antibody, wherein the CL domain comprises the amino acid sequence of SEQ ID NO: 18 and the CH domain comprises the amino acid sequence of SEQ ID NO: 19.

[0120] Preparation of humanized mouse mAb 16G2

[0121] The murine-derived framework regions within the VH and VL domains of the chimeric mAb were first identified and replaced with human framework regions while maintaining the critical binding regions. Structural and functional evaluation was performed to ensure successful humanization of these regions. The altered variable domains were then cultured in host cells EXPICHO-STM The humanized mAb was expressed in and optimized for maximum production of the humanized mAb. After purification, the humanized mAb was subjected to sequence analysis and activity assay.

[0122] In this study, four humanized VH domains (including VH1, VH2, VH3, and VH4 domains) and three humanized VL domains (including VL1, VL2, and VL3 domains) were produced. The amino acid sequences of the humanized VH and VL domains are summarized in Table 2.

[0123] Table 2: Amino acid sequences of humanized VH and VL domains [Table 2] *CDR sequences (including CDR-H1, CDR-H2, and CDR-H3 of the VH domain, and CDR-L1, CDR-L2, and CDR-L3 of the VL domain) are marked in bold, in order.

[0124] The humanized mAbs containing humanized VH and VL regions are designated "humanized mAb 16G2-1" (mAb containing humanized VH1 and VL1 sequences), "humanized mAb 16G2-2" (mAb containing humanized VH1 and VL2 sequences), "humanized mAb 16G2-3" (mAb containing humanized VH1 and VL3 sequences), "humanized mAb 16G2-4" (mAb containing humanized VH2 and VL1 sequences), "humanized mAb 16G2-5" (mAb containing humanized VH2 and VL2 sequences), "humanized mAb 16G2-6" (mAb containing humanized VH2 and VL3 sequences), "humanized mAb 16G2-7" (mAb containing humanized VH3 and VL1 sequences; also referred to as "BGM-2121" in this study), and "humanized mAb 16G2-8" (mAb containing humanized VH3 and VL1 sequences; also referred to as "BGM-2121" in this study), respectively. The mAbs were named "humanized mAb 16G2-8" (mAb comprising humanized VH3 and VL2 sequences), "humanized mAb 16G2-9" (mAb comprising humanized VH3 and VL3 sequences), "humanized mAb 16G2-10" (mAb comprising humanized VH4 and VL1 sequences), "humanized mAb 16G2-11" (mAb comprising humanized VH4 and VL2 sequences) and "humanized mAb 16G2-12" (mAb comprising humanized VH4 and VL3 sequences).

[0125] Surface Plasmon Resonance (SPR)

[0126] The affinity between antigen and antibody is determined by BIACORE TM Measurements were performed using an SPR system. Briefly, antibodies were injected onto the sensor chip and immobilized as capturers using a mouse antibody capture kit. Antigens were diluted to multiple concentrations (50, 25, 12.5, 6.25, 3.125, 1.5625 nM) and injected onto the surfaces of flow cells 1 and 2 as the binding phase, followed by HBS-EP+ running buffer (HEPES 10 mM, NaCl 150 mM, EDTA 3 mM, 0.005% TWEEN® 20) as the dissociation phase. All data were recorded at 25°C and processed using software.

[0127] Enzyme-linked immunosorbent assay (ELISA)

[0128] Biotinylated PTHrP fragment containing the N-terminal 34 residues of human PTHrP (hereinafter, "PTHrP(1-34) peptide") and biotinylated parathyroid hormone (PTH) fragment containing the N-terminal 34 residues of human PTH (hereinafter, "PTH(1-34) peptide") were each diluted to a final concentration of 0.2 μg / mL and then added to a 96-well plate, and the plate was incubated at room temperature for 2 hours. After washing and blocking procedures, the diluted antibody was added to the wells and incubated at room temperature for 2 hours. Secondary antibody was then added to the wells. After further washing, TMB (3,3',5,5'-tetramethylbenzidine) substrate was added to induce color development. The reaction was stopped with HCL, and absorbance was measured at 450 nm with 570 nm as the reference. The absorbance data obtained was analyzed by software using nonlinear regression to determine the half-maximal effective concentration (EC50).

[0129] Cell-based neutralization assay

[0130] For the cell-based cAMP neutralization assay, UMR-106 cells were plated in 96-well plates at 1.5 × 10 4The cells were seeded at a density of 1000 cells / well and incubated at 37 °C for 3 days. Afterwards, the medium was removed and washed with serum-free medium, and the cells were incubated in experimental medium (DMEM containing 10 mM HEPES and 0.5 mM isobutylmethylxanthine (IBMX)) for 30 min at 37 °C. The experimental medium was removed, and recombinant PTHrP (1-34) peptide (20 ng / mL) (4-fold dilution: from 2.5 μg / mL to 0.04 μg / mL) with or without anti-PTHrP antibody was added and incubated at 37 °C for 30 min. After incubation, the medium was aspirated and treated with 250 μl of 0.1 M HCL at room temperature for 20 min. The cells were harvested, transferred to a V-bottom 96-well dish, and centrifuged at 1,000 x g for 10 min at 4 °C. The supernatant was transferred to a clean test tube, and the absorbance was measured at wavelengths of 405–420 nm using an ELISA reader.

[0131] In vitro neutralization assay (competitive ELISA)

[0132] PTH1R protein (1 μg / mL) in coating buffer was added to the ELISA plate and incubated overnight at 4° C., followed by washing with PBS-T washing buffer (1× PBS containing 0.05% TWEEN® 20). Protein coating was blocked by incubation with blocking buffer (washing buffer containing 2% (w / v) bovine serum albumin (BSA)) for 1.5 h at 37° C. Biotinylated PTHrP was diluted to 0.5 μg / mL in dilution buffer (washing buffer containing 0.5% (w / v) BSA) and anti-PTHrP antibody was diluted to 40, 10, 2.5, and 0.625 μg / mL in dilution buffer. The mixture of biotinylated PTHrP and anti-PTHrP antibody was added to the plate and incubated at 37° C. for 1 h. Next, 100 μL of streptavidin-HRP (0.1 μg / mL) working solution was added to the plate and incubated at 37° C. for 1 h, after which 100 μL of TMB substrate working solution was added to each well. After 10 min, the reaction was stopped by adding 100 μL of 1N HCl. The absorbance was measured at a wavelength of 450 nm using an ELISA reader.

[0133] cell viability

[0134] The number of viable cells following a particular treatment (i.e., 0, 25, 50, or 100 μg / mL of anti-PTHrP antibody) was measured by a luminescent cell viability assay, a quantitative method based on the amount of adenosine triphosphate (ATP) present within the cells. Briefly, 5×10 3 BxPC-3 cells were seeded into 96-well plates and incubated at 37°C for 24 hours. After removing the medium, 50 μl / well of RPMI supplemented with 5% FBS was added to the cells. The cells were incubated at 37°C for 2 hours (starvation period). Anti-PTHrP antibody or gemcitabine (as a positive control) was administered to the wells, and the cells were incubated at 37°C, 5% CO2 for 72, 96, or 108 hours. Pre-warmed luminescence reagent was added to the wells and incubated at room temperature for 10 minutes, after which luminescence was measured.

[0135] Wound healing assay

[0136] Human lung cancer cell line HARA-B cells (1 × 10 5 Cells) were seeded into cell culture insert systems and allowed to form confluent layers, then incubated at 37°C, 5% CO2 for 24 hours. After washing twice with 1X PBS, the cells were incubated in 70 μl of 2% FBS medium for 24 hours (starvation period). PTHrP(1-34) peptide (10 μg / mL) and antibodies (anti-PTHrP antibody or human IgG, 100 μg / mL) were precomplexed for 1 hour at room temperature. The cell culture insert systems were removed and the cells were washed twice with 1X PBS. The mixture of PTHrP peptide and antibody was added to each dish. Cell images were taken using a microscope after 0, 12, 24, 32, and 48 hours.

[0137] Animal models

[0138] (i) Treatment of non-small cell lung cancer with mouse mAb 16G2

[0139] Four- to six-week-old nude mice were used for xenograft tumor growth studies, and the animals were acclimated and housed in a pathogen-free environment.6 HARA-B cells were suspended in 200 μL of PBS and injected subcutaneously into the dorsal flank of mice. Tumor volume was determined by measuring the length (L) and width (W) of the tumor using a caliper and calculating with the formula (length × width × width / 2). After inoculation, mice were randomly divided into two groups and intraperitoneally (ip) administered anti-PTHrP antibody (10 mg / kg) or control IgG three times a week. Serum was collected and calcium was measured.

[0140] (ii) Treatment of pancreatic cancer with mouse mAb 16G2

[0141] Four to six week old nude mice were used for xenograft tumor growth studies, and the animals were acclimated and housed in a pathogen-free environment. For the xenograft tumor growth assay, 5 × 10 6 BxPC-3 cells were suspended in 200 μL PBS and injected subcutaneously into the dorsal flank of mice. Tumor volume was determined by measuring the length (L) and width (W) of the tumor using a caliper and calculating with the formula (L x W x W / 2). After inoculation, mice were randomly divided into two groups and administered anti-PTHrP antibody (10 mg / kg) or control IgG by intraperitoneal (ip) injection three times a week.

[0142] (iii) Treatment of pancreatic cancer with humanized mAb 16G2-7 (BGM-2121)

[0143] Four to six week old nude mice were used for xenograft tumor growth studies, and the animals were acclimated and housed in a pathogen-free environment. For the xenograft tumor growth assay, 5 × 10 6 BxPC-3 or CFPAC-1 cells were suspended in 200 μL PBS and injected subcutaneously into the dorsal flank of mice. Tumor volume was determined by measuring the length (L) and width (W) of the tumor using calipers and calculating with the formula (length × width × width / 2). After inoculation, mice were randomly divided into two groups and administered anti-PTHrP antibody (1, 3, or 10 mg / kg) or control hIgG by intravenous (iv) injection at the designated time points. Serum was collected and calcium was measured. For intrasplenic tumor injection, the left flank was incised under anesthesia. The spleen was gently pulled out and 2 × 10 6BxPC-3 cells were slowly injected into the spleen. After inoculation, mice were randomly divided into two groups and administered anti-PTHrP antibody (10 mg / kg) or control hIgG intravenously (iv) at the indicated time points. Serum was collected and calcium was measured.

[0144] statistical analysis

[0145] All experiments were repeated at least three times. Results were evaluated by Student's t-test. P values ​​less than 0.05 were considered significant. *p<0.05; **p<0.01; ***p<0.001; ns (not significant): ≥0.05.

[0146] Example 1: Characterization of the mAbs of the invention

[0147] 1.1 Binding affinity

[0148] In this example, the binding activity of the mAb of the present invention to PTHrP was evaluated. As described in the "Materials and Methods" section of this disclosure, the mAb of the present invention (i.e., mouse mAb, chimeric mAb, or humanized mAb) and PTHrP antigen were added to a sensor chip, respectively, and the binding activity between them was determined by SPR. The results are summarized in Tables 3 and 4.

[0149] The data in Table 3 showed that all mAbs tested (including mouse mAb 16G2, mouse mAb 5B3, chimeric mAb 16G2 and chimeric mAb 5B3) recognized and bound to PTHrP. Compared with mouse mAbs and reference Abs (Chugai antibody as a positive control), the chimeric antibodies showed higher binding affinity to PTHrP (KD values ​​of chimeric 16G2 mAb and chimeric 5B3 mAb were 1.15×10, respectively). -10 and 2.08 x 10 -10 (It was.)

[0150] Table 3: Antigen binding affinity of specific mAbs [Table 3] Ka: Binding rate constant Kd: dissociation rate constant KD: equilibrium dissociation constant; KD=kd / ka

[0151] The data in Table 4 show the binding affinity of the humanized mAbs (including humanized mAb 16G2-1 through humanized mAb 16G2-12) to PTHrP.

[0152] Table 4: Antigen-binding affinities of selected humanized mAbs [Table 4] Ka: Binding rate constant Kd: dissociation rate constant KD: equilibrium dissociation constant; KD=kd / ka Reference Ab: Chugai antibody; positive control Murine 16G2: Murine mAb 16G2 comprising the VH domain of SEQ ID NO:7 and the VL domain of SEQ ID NO:8.

[0153] The binding affinity of chimeric mAb 16G2 and mAb 16G2-7 (BGM-2121) to PTHrP(1-34) peptide or PTH(1-34) peptide was further confirmed by ELISA. According to the results in Table 5, chimeric mAb 16G2 and BGM-2121 recognized and bound to PTHrP(1-34) peptide with equilibrium dissociation constants (KD) of 2.44 nM and 1.79 nM, respectively. It was confirmed that chimeric mAb 16G2 and BGM-2121 did not show off-target binding to the PTH(1-34) fragment, which is a major regulator of calcium and phosphate metabolism in bone and kidney.

[0154] Table 5: Binding affinity of specific antibodies to PTHrP(1-34) or PTH(1-34) peptides [Table 5] ** Anti-hPTH: a commercially available antibody developed by Phoenix Pharmaceuticals that shows high binding affinity to human PTH(1-34) peptide. It was used as a control in this study.

[0155] 1.2 In vitro activity

[0156] In this example, the biological activity of the mAb of the present invention was analyzed by neutralization assay. The results are summarized in Tables 6 and 7.

[0157] Cell-based neutralization assays were performed using the rat osteosarcoma cell line UMR-106, which expresses endogenous PTH1 receptors. All mAbs tested, including mouse mAb 16G2, mouse mAb 5B3, chimeric mAb 16G2, and chimeric mAb 5B3, inhibited PTHrP-induced cAMP production in UMR-106 cells (data not shown). Results in Table 6 showed that chimeric mAb 16G2 (EC50 of 0.81 nM) and BGM-2121 (EC 50 Both chimeric mAbs 16G2 and BGM-2121 showed high potency in selectively inhibiting PTHrP(1-34) peptide-induced cAMP production (EC 50 >40nM).

[0158] Table 6: Neutralizing activity (EC50) of specific antibodies by cell-based neutralization assay [Table 6]

[0159] The data in Table 7 demonstrate that each of the humanized mAbs 16G2-1, 16G2-4, 16G2-7 (BGM-2121), and 16G2-10 of the present invention is useful for neutralizing the activity of the PTHrP antigen. The humanized 16G2-10 mAb had the highest competitive neutralizing activity (IC50 = 1.36 x 10 -8 ; Table 7), with humanized 16G2-7 mAb showing the highest cell-based neutralizing activity (IC50 = 8.926 × 10 -10 ;Table 7) are shown.

[0160] Table 7: Neutralizing activity of selected humanized mAbs [Table 7]

[0161] The data in Table 8 further confirmed that BGM-2121 bound to the biotin-labeled PTHrP (1-34) peptide and competed with PTH1R binding. The results suggested that BGM-2121 exerted neutralizing activity by inhibiting the interaction of the biotin-labeled PTHrP (1-34) peptide with the PTH1R receptor. According to the results in Table 8, both the chimeric mAb 16G2 antibody (EC50 of 22.5 nM) and BGM-2121 (EC50 of 15.4 nM) showed potent inhibition of human PTHrP (1-34) peptide.

[0162] Table 8: Neutralizing activity (EC50) of specific antibodies by competitive ELISA assay [Table 8]

[0163] These results demonstrated that each of the mAbs of the present invention (including mouse mAbs, chimeric mAbs, and humanized mAbs) exhibits PTHrP-binding affinity and can function as a neutralizing antibody to treat cancer (especially PTHrP-positive cancers) by inhibiting the activity of PTHrP.

[0164] 1.3 Inhibitory effect on cancer cells

[0165] As described in Materials and Methods, BxPC-3 cells were treated with an anti-PTHrP antibody of the invention (BGM-2121) or gemcitabine (positive control) for 72, 96, or 108 hours, after which cell viability was measured by a luminescent cell viability assay. The data in Figure 1 demonstrate that BGM-2121 had a dose-dependent effect on BxPC-3 cell viability.

[0166] Wound-healing assays further demonstrated the inhibitory effect of BGM-2121 on cell migration. Treatment with PTHrP(1-34) peptide significantly enhanced the migratory ability of HARA-B cells, whereas co-incubation with 100 μg / ml BGM-2121 inhibited the migration of PTHrP(1-34) peptide-stimulated cells (24 h after treatment; Figure 2), suggesting that BGM-2121 may inhibit the migratory ability of HARA-B cells.

[0167] Example 2: Antitumor effect of the mAb of the present invention

[0168] 2.1 Mouse mAb 16G2 treatment

[0169] In this example, two animal models were used to evaluate the therapeutic effect of mouse mAb 16G2 on cancer. As described in the "Materials and Methods" section of this disclosure, a lung cancer cell line (i.e., HARA-B) and a pancreatic cell line (i.e., BxPC-3) were injected into the dorsal flank of mice, respectively, followed by treatment with mouse mAb 16G2 or control antibody IgG. The results are shown in Figures 3-4, respectively.

[0170] Compared with the control antibody IgG, administration of mouse mAb 16G2 significantly inhibited tumor growth in the HARA-B model (NSCLC) (Figure 3A). Furthermore, serum calcium concentrations were significantly decreased in HARA-B tumor-bearing mice administered mouse 16G2 antibody compared with those administered control antibody IgG (Figure 3B).

[0171] The data in Figure 4 confirmed the antitumor effect of mouse mAb 16G2. Compared with the control antibody IgG, administration of mouse 16G2 significantly inhibited tumor growth in the BxPC-3 model (pancreatic cancer) (Figure 4A) and reduced serum calcium levels in BxPC-3 tumor-bearing mice (Figure 4B). There was no significant difference in the body weight of mice administered control antibody IgG and mouse mAb 16G2 (data not shown).

[0172] 2.2 BGM-2121 Processing

[0173] In this example, the antitumor effect of humanized mAb BGM-2121 was investigated. In the subcutaneous tumor model, nude mice were subcutaneously injected with BxPC3 cells in the flank, followed by intravenous administration of 10 mg / kg BGM-2121 or isotype hIgG antibody twice a week, once a week, once every two weeks, or once every three weeks. The data show that treatment with BGM-2121 once a week or once every two weeks significantly inhibits tumor growth compared to hIgG treatment (Figure 5A). The results showed that weekly administration of BGM-2121 (10 mg / kg) significantly reduced tumor volume by 30% compared to mice treated with hIgG (data not shown). Furthermore, administration of BGM-2121 (10 mg / kg) once every 3 weeks significantly suppressed tumor volume (approximately 65% ​​reduction) and significantly improved overall survival of tumor-bearing mice (approximately 30% increase) compared to the hIgG control group (Figures 5B and C).

[0174] In the orthotopic tumor model, BxPC3 cells were injected into the spleen of mice to establish a xenograft model simulating pancreatic liver metastasis. These mice were then intravenously administered 10 mg / kg BGM-2121 or isotype hIgG Ab on days 7, 14, 21, 28, and 35. Analysis of tumor weight revealed striking differences in situ, as mice treated with BGM-2121 showed a marked growth inhibition of pancreatic hyperplasia (approximately 58% inhibition) compared to the hIgG Ab treatment group (Figure 6A). Furthermore, serum CPK levels were reduced in BxPC-3-bearing mice treated with BGM-2121 compared to the hIgG control group (Figure 6B).

[0175] In addition to the BxPC3 tumor model, the antitumor effect of our BGM-2121 was further confirmed by the CFPAC-1 tumor model. CFPAC-1 cells were subcutaneously injected into the flank of nude mice, followed by intravenous administration of 10 mg / kg BGM-2121 or hIgG once a week for a total of 4 weeks. The data in Figure 7 show that administration of our BGM-2121 significantly inhibited tumor growth (about 40% inhibition, Figure 7A) and tumor weight (Figure 7B) compared to hIgG treatment.

[0176] In conclusion, the present invention provides two novel murine mAbs, 16G2 and 5B3, which are useful for producing chimeric or humanized antibodies. According to the embodiments of the present disclosure, the mAbs of the present invention exhibit binding affinity and neutralizing activity for PTHrP, and can function as therapeutic agents for treating cancer, especially PTHrP-positive cancer.

[0177] The above description of the embodiments is for illustrative purposes only, and it will be understood that various modifications may be made by those skilled in the art. The above specification, examples, and data provide a complete description of the structure and use of the exemplary embodiments of the invention. Although various embodiments of the invention have been described above with a certain degree of detail or with reference to one or more specific embodiments, those skilled in the art may make numerous modifications to the disclosed embodiments without departing from the spirit or scope of the invention.

[0178] To the extent that the above background, description, examples, and accompanying drawings disclose additional subject matter not included in the scope of the claims below, the invention is not publicly disclosed and the right to file one or more applications to claim such additional inventions is reserved.

Claims

1. comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, the VH domain comprises a first heavy chain complementarity determining region (CDR-H1), a second heavy chain CDR (CDR-H2) and a third heavy chain CDR (CDR-H3); the VL domain comprises a first light chain CDR (CDR-L1), a second light chain CDR (CDR-L2) and a third light chain CDR (CDR-L3); The CDR-H1, CDR-H2 and CDR-H3 each comprise the amino acid sequences of "GYXFTDY" (SEQ ID NO: 1), "DTSDSY" (SEQ ID NO: 2) and "GDY", respectively, where X in SEQ ID NO: 1 is isoleucine (I) or threonine (T); A recombinant antibody or a fragment thereof, wherein the CDR-L1, CDR-L2 and CDR-L3 comprise the amino acid sequences "QSLLESDGKTY" (SEQ ID NO: 3), "LVS" and "CQGTHFPWT" (SEQ ID NO: 4), respectively.

2. The CDR-H1, CDR-H2 and CDR-H3 each comprise the amino acid sequences "GYIFTDY" (SEQ ID NO: 5), "DTSDSY" (SEQ ID NO: 2) and "GDY", respectively; The recombinant antibody or fragment thereof of claim 1, wherein the CDR-L1, CDR-L2 and CDR-L3 respectively comprise the amino acid sequences "QSLLESDGKTY" (SEQ ID NO: 3), "LVS" and "CQGTHFPWT" (SEQ ID NO: 4).

3. The recombinant antibody or fragment thereof of claim 2, wherein the VH domain and the VL domain comprise the amino acid sequences of SEQ ID NOs: 7 and 8, respectively.

4. the VH domain comprises the amino acid sequence of SEQ ID NO: 9, 10, 11 or 12; The recombinant antibody or fragment thereof of claim 2, wherein the VL domain comprises the amino acid sequence of SEQ ID NO: 13, 14 or 15.

5. The recombinant antibody or fragment thereof of claim 4, wherein the VH and VL domains comprise the amino acid sequences of SEQ ID NOs: 11 and 13, respectively.

6. The CDR-H1, CDR-H2 and CDR-H3 each contain the amino acid sequences "GYTFTDY" (SEQ ID NO: 6), "DTSDSY" (SEQ ID NO: 2) and "GDY", respectively; The recombinant antibody or fragment thereof of claim 1, wherein the CDR-L1, CDR-L2 and CDR-L3 respectively comprise the amino acid sequences "QSLLESDGKTY" (SEQ ID NO: 3), "LVS" and "CQGTHFPWT" (SEQ ID NO: 4).

7. The recombinant antibody or fragment thereof of claim 6, wherein the VH and VL domains comprise the amino acid sequences of SEQ ID NOs: 16 and 17, respectively.

8. A pharmaceutical composition comprising the recombinant antibody or fragment thereof described in claim 1 and a pharma- ceutically acceptable excipient.

9. A method for treating cancer in a subject, comprising administering to the subject an effective amount of a recombinant antibody or fragment thereof described in claim 1, or a pharmaceutical composition described in claim 8.

10. 10. The method of claim 9, wherein the cancer is a parathyroid hormone-related protein (PTHrP)-expressing cancer.

11. 11. The method of claim 10, wherein the cancer is gastric cancer, lung cancer, bladder cancer, breast cancer, pancreatic cancer, renal cancer, colon cancer, cervical cancer, ovarian cancer, brain cancer, prostate cancer, hepatocellular carcinoma, melanoma, esophageal cancer, multiple myeloma, or head and neck squamous cell carcinoma.

12. The method of claim 9 , wherein the subject is a human.

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