Parathyroid hormone-related protein antibodies and related methods

Abstract

This disclosure relates to antibodies that specifically bind to parathyroid hormone-related protein (PTHrP). Immunoconjugates, nucleic acids, vectors, cells, and compositions comprising said antibodies are also provided. The antibodies, immunoconjugates, nucleic acids, vectors, cells, and compositions can be used for the diagnosis and treatment of diseases, particularly cancer and / or hypercalcemia.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 542,496, filed on October 4, 2023, the entire contents of which are incorporated herein by reference.

[0003] Merging of sequence lists

[0004] The computer-readable form of the sequence list “31165-P72536PC00_SequenceListing.xml” (99,090 bytes), created on October 4, 2024, is incorporated herein by reference. Technical Field

[0005] This invention relates to antibodies against parathyroid hormone-related protein (PTHrP), and more specifically, to antibodies specifically targeting PTHrP subtypes 1-173, as well as methods for diagnosing and treating diseases (particularly cancer). Background Technology

[0006] PTHrP is associated with hypercalcemia in various cancers, including breast cancer, colon cancer, skin cancer, kidney cancer, and lung cancer, as well as hematologic malignancies such as lymphoma, leukemia, and multiple myeloma. In addition, PTHrP is involved in the growth, invasion, and migration of tumor cells in breast cancer, prostate cancer, pancreatic cancer, and melanoma.

[0007] Antibodies against PTHrP and its specific subtypes are described in U.S. Patent Nos. 7,897,139, 8,501,929 and 10,463,726.

[0008] It can selectively bind to PTHrP and can be used for therapeutic inhibition of PTHrP. Figure 2P shows HC104-41-VLB (SEQ ID NO: 57) and human IGKV4-1. The 01 (SEQ ID NO: 76) sequence exhibits residue differences in the frame region and complementarity-determining region (CDR).

[0009] The inventors have identified antibodies capable of specifically binding to the PTHrP isoform PTHrP1-173, and these antibodies exhibit desirable properties. Other aspects described herein include: immunoconjugates containing said antibodies, nucleic acids encoding said antibodies, vectors containing said nucleic acids, cells expressing said antibodies, and compositions containing said antibodies. Methods of using these compositions are also described.

[0010] Therefore, in one aspect, this document provides an antibody or fragment thereof capable of specifically binding to PTHrP, comprising a heavy chain variable region and a light chain variable region; said heavy chain variable region comprising complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3; said light chain variable region comprising complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, wherein the amino acid sequence of said CDR comprises the following sequence:

[0011] In one embodiment, the amino acid sequence of the CDR of the antibody or fragment includes the following sequence:

[0012] or

[0013] In one embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 20; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 20, wherein the CDR sequence is as described in SEQ ID NO: 1, 3, and 7; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 1, 3, and 7; and the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 26; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 26, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 8-10.

[0014] In one embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 21; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 21, wherein the CDR sequence is as described in SEQ ID NO: 2, 4, and 7; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 2, 4, and 7; and the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 26; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 26, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 8-10.

[0015] In one embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 22; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 22, wherein the CDR sequence is as described in SEQ ID NO: 1, 3, and 7; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 1, 3, and 7; and the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 26; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 26, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 8-10.

[0016] In one embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 23; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 23, wherein the CDR sequence is as described in SEQ ID NO: 1, 5, and 7; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 1, 5, and 7; and the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 26; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 26, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 8-10.

[0017] In one embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 24; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 24, wherein the CDR sequence is as described in SEQ ID NO: 1, 3, and 7; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 1, 3, and 7; and the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 26; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 26, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 8-10.

[0018] In one embodiment, the heavy chain variable region comprises: i) the amino acid sequence as described in SEQ ID NO: 25; ii) an amino acid sequence having 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 25, wherein the CDR sequence is as described in SEQ ID NO: 1, 6, and 7; or iii) a sequence derived from a conservative substitution of the amino acid sequence described in i), wherein the CDR sequence is as described in SEQ ID NO: 1, 6, and 7; and the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 26; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 26, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a sequence derived from a conservative substitution of the amino acid sequence described in i), wherein the CDR sequence is as described in SEQ ID NO: 1, 6, and 7.

[0019] In one embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 20; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 20, wherein the CDR sequence is as described in SEQ ID NO: 1, 3, and 7; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 1, 3, and 7; and the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 27; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 27, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 8-10.

[0020] In one embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 21; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 21, wherein the CDR sequence is as described in SEQ ID NO: 2, 4, and 7; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 2, 4, and 7; and the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 27; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 27, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 8-10.

[0021] In one embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 23; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 23, wherein the CDR sequence is as described in SEQ ID NO: 1, 5, and 7; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 1, 5, and 7; and the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 27; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 27, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 8-10.

[0022] In one embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 24; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 24, wherein the CDR sequence is as described in SEQ ID NO: 1, 3, and 7; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 1, 3, and 7; and the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 27; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 27, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 8-10.

[0023] In one embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 25; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 25, wherein the CDR sequence is as described in SEQ ID NO: 1, 6, and 7; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 1, 6, and 7; and the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 27; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 27, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 8-10.

[0024] In one embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 20; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 20, wherein the CDR sequence is as described in SEQ ID NO: 1, 3, and 7; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 1, 3, and 7; and the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 28; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 28, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 8-10.

[0025] In one embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 21; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 21, wherein the CDR sequence is as described in SEQ ID NO: 2, 4, and 7; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 2, 4, and 7; and the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 28; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 28, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 8-10.

[0026] In one embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 23; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 23, wherein the CDR sequence is as described in SEQ ID NO: 1, 5, and 7; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 1, 5, and 7; and the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 28; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 28, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 8-10.

[0027] In one embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 24; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 24, wherein the CDR sequence is as described in SEQ ID NO: 1, 3, and 7; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 1, 3, and 7; and the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 28; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 28, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 8-10.

[0028] In one embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 25; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 25, wherein the CDR sequence is as described in SEQ ID NO: 1, 6, and 7; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 1, 6, and 7; and the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 28; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 28, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 8-10.

[0029] In one embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 73; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 73, wherein the CDR sequence is as described in SEQ ID NO: 2, 4, and 7; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 2, 4, and 7; and the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 26, 27, or 28; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 26, 27, or 28, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as shown in SEQ ID NO: 8-10.

[0030] In one embodiment, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 20, 21, 23, 24 or 25, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 26.

[0031] In one embodiment, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 20, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 26.

[0032] In one embodiment, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 21, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 26.

[0033] In one embodiment, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 23, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 26.

[0034] In one embodiment, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 24, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 26.

[0035] In one embodiment, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 25, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 26.

[0036] In one embodiment, the heavy chain variable region is contained in the heavy chain, and / or the light chain variable region is contained in the light chain, wherein the heavy chain may further contain the amino acid sequence of SEQ ID NO: 62 and / or 63, and / or the light chain may further contain the amino acid sequence of SEQ ID NO: 64 and / or 65.

[0037] In one respect, immunoconjugates comprise antibodies or fragments as described herein, as well as detectable markers.

[0038] In one aspect, a nucleic acid encodes a heavy chain variable domain or a light chain variable domain of an antibody or a fragment thereof as described herein.

[0039] In one embodiment, the nucleic acid i) encodes a heavy chain variable region, the nucleic acid encoding the heavy chain variable region comprising any one of SEQ ID NO: 11, 12, 14, 15, or 16, or comprising a sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any one of SEQ ID NO: 11, 12, 14, 15, or 16; ii) encodes a light chain variable region, the nucleic acid encoding the light chain variable region comprising any one of SEQ ID NO: 17, 18, or 19, or comprising at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any one of SEQ ID NO: 17, 18, or 19; or iii) encodes both the heavy chain variable region and the light chain variable region, the nucleic acids encoding the heavy chain variable region and the light chain variable region comprising the following sequences respectively: SEQ ID NO: 11 and 17; SEQ ID NO: 12 and 17; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17 ... SEQ ID NO: 14 and 17; SEQ ID NO: 15 and 17, SEQ ID NO: 16 and 17, SEQ ID NO: 12 and 18, SEQ ID NO: 16 and 18, SEQ ID NO: 11 and 19, SEQ ID NO: 12 and 19, SEQ ID NO: 14 and 19, SEQ ID NO: 15 and 19, or SEQ ID NO: 16 and 19, or sequences having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 11 and 17; SEQ ID NO: 12 and 17; SEQ ID NO: 14 and 17; SEQ ID NO: 11 and 17; SEQ ID NO: 15 and 17, SEQ ID NO: 16 and 17, SEQ ID NO: 12 and 18, SEQ ID NO: 16 and 18, SEQ ID NO: 11 and 17. SEQ ID NO: 12 and 19, SEQ ID NO: 14 and 19, SEQ ID NO: 15 and 19, or SEQ ID NO: 16 and 19, optionally, wherein the nucleic acid further comprises one or more nucleotide sequences encoding one or more amino acid sequences of SEQ ID NO: 62-65.

[0040] In one respect, the vector contains nucleic acids as described herein.

[0041] In one embodiment, the vector is a viral vector, optionally an adenovirus vector, an adenovirus-associated vector, or a retroviral vector, optionally a lentiviral vector.

[0042] In one respect, the cell expresses antibodies or fragments or nucleic acids as described herein, or contains vectors as described herein.

[0043] In one embodiment, the cells are selected from mammalian cells, optionally CHO cells or HEK-293 cells or insect cells, optionally Sf9 cells, Sf21 cells, TNI cells or S2 cells.

[0044] In one aspect, the composition comprises an antibody or fragment, an immunoconjugate, a nucleic acid, a vector, or cells as described herein, and optionally also comprises a diluent.

[0045] In one aspect, antibodies or fragments, immunoconjugates, nucleic acids, or vectors, as described herein, are used to manufacture drugs for treating hypercalcemia or cancer in subjects in need.

[0046] In one respect, antibodies or fragments, immunoconjugates, nucleic acids, vectors, cells or compositions as described herein are included in the kit.

[0047] On the one hand, the method for reducing or inhibiting human PTHrP activity described herein includes: contacting cells or tissues expressing human PTHrP with an antibody or fragment thereof that specifically binds to human PTHrP, wherein the antibody or fragment thereof includes a light chain variable region and a heavy chain variable region, the heavy chain variable region includes complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, the light chain variable region includes complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, wherein the amino acid sequence of the CDR includes the following sequence:

[0048] Optionally, the antibody or fragment thereof is the antibody or fragment thereof described herein.

[0049] In one aspect, there is a use of an antibody or fragment thereof capable of specifically binding to human PTHrP to reduce or inhibit human PTHrP activity, as described herein, the use comprising contacting cells or tissues expressing said human PTHrP with said antibody or fragment thereof, said antibody or fragment thereof comprising a light chain variable region and a heavy chain variable region, said heavy chain variable region comprising complementation-determining regions CDR-H1, CDR-H2, and CDR-H3, said light chain variable region comprising complementation-determining regions CDR-L1, CDR-L2, and CDR-L3, wherein the amino acid sequence of said CDR comprises the following sequence:

[0050] Optionally, the antibody or fragment thereof is the antibody or fragment thereof described herein.

[0051] In one implementation, the cells or tissue are located in vivo within the subject, and / or the contact includes administering an antibody or a fragment thereof to the subject in need, or using the antibody or a fragment thereof in the subject.

[0052] In one aspect, a method of treating a subject in need is provided, comprising administering to the subject an effective amount of an antibody or a fragment thereof, or a composition comprising said antibody or a fragment thereof, or an immunoconjugate, nucleic acid, or a carrier as described herein.

[0053] In one aspect, the use of the antibody or a fragment thereof, or a composition comprising the antibody or a fragment thereof, or an immunoconjugate, nucleic acid, or carrier as described herein, in treating a subject in need.

[0054] In one embodiment, the subject has been diagnosed with cancer, optionally breast cancer, lung cancer, colon cancer, pancreatic cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, or melanoma.

[0055] In one embodiment, the cancer is in the pre-metastatic stage.

[0056] In one embodiment, the method is used to treat hypercalcemia.

[0057] In one embodiment, the subject has been diagnosed with hypercalcemia, optionally hypercalcemia associated with malignancy.

[0058] Other features and advantages of this document will become apparent from the following detailed description. However, it should be understood that while the detailed description and specific embodiments illustrate the implementation described herein, these are for illustrative purposes only. Those skilled in the art, upon reading this detailed description, can make various changes and modifications within the spirit and scope of this document. Attached Figure Description

[0059] An implementation example from this paper will now be described with reference to the accompanying drawings, in which: Figure 1A The amino acid sequence of the variable region (VH) of the heavy chain of mouse HC104 hybridoma is shown (SEQ ID NO: 31).

[0060] Figure 1B The amino acid sequence of an antibody (HC104-146-VHA; SEQ ID NO: 32) disclosed in this invention is shown.

[0061] Figure 1C HC104-146-VHA (SEQ ID NO: 32) and IGHV1-46 are shown. 01 Differences in amino acid residues in the frame region and complementarity-determining region (CDR) of the human germline VH (SEQ ID NO: 70) sequence.

[0062] Figure 1D The amino acid sequences of the two antibodies (SEQ ID NO: 33 and 34) disclosed in this invention are shown.

[0063] Figure 1E shows the amino acid sequence of the antibody (HC104-146-VHB; SEQ ID NO: 35) in this paper.

[0064] Figure 1F shows HC104-146-VHB (SEQ ID NO: 35) and IGHV1-46. 01 Differential residues in the frame region and complementarity-determining region (CDR) of the human germline (SEQ ID NO: 70) sequence.

[0065] Figure 1G shows the amino acid sequence of the antibody (HC104-1692-VHA; SEQ ID NO: 36) described in this paper.

[0066] Figure 1H shows HC104-1692-VHA (SEQ ID NO: 36) and IGHV1-69-2. 01 Differential residues in the frame region and complementarity-determining region (CDR) of the human germline (SEQ ID NO: 71) sequence.

[0067] Figure 1I shows the amino acid sequences of the antibodies in this paper (SEQ ID NO: 37 and 38).

[0068] Figure 1J shows the amino acid sequence of one of the antibodies described in this paper (HC104-1692-VHB; SEQ ID NO: 39).

[0069] Figure 1K shows HC104-1692-VHB (SEQ ID NO: 39) and human germline IGHV1-69-2. The amino acid residue differences present in the frame region and complementarity-determining region (CDR) of the 01 (SEQ ID NO: 71) sequence.

[0070] Figure 1L shows the amino acid sequence of one of the antibodies described in this paper (HC104-551-VHA; SEQ ID NO: 40).

[0071] Figure 1M shows HC104-551-VHA (SEQ ID NO: 40) and human germline IGHV5-51. The 01 (SEQ ID NO: 72) sequence contains differential residues in the frame region and complementarity-determining region (CDR).

[0072] Figure 1N shows the amino acid sequences of the antibodies in this paper (SEQ ID NO: 41, 42 and 73).

[0073] Figure 10 shows the amino acid sequence of the antibody (HC104-551-VHB; SEQ ID NO: 43) in this paper.

[0074] Figure 1P shows HC104-551-VHB (SEQ ID NO: 43) and human germline IGHV5-51. The 01 (SEQ ID NO: 72) sequence contains differential residues in the frame region and complementarity-determining region (CDR).

[0075] Figure 1Q shows the amino acid sequence of one of the antibodies described in this paper (HC104-551-VHC; SEQ ID NO: 44).

[0076] Figure 1R shows HC104-551-VHC (SEQ ID NO: 44) and human germline IGHV5-51. The amino acid residue differences present in the frame region and complementarity-determining region (CDR) of the 01 (SEQ ID NO: 72) sequence.

[0077] Figure 2A shows the amino acid sequence of the variable region (VL) of the light chain in mouse HC104 hybridoma (SEQ ID NO: 45).

[0078] Figure 2B shows the amino acid sequence of one of the antibodies described in this paper (HC104-320-VLA; SEQ ID NO: 46).

[0079] Figure 2C shows HC104-320-VLA (SEQ ID NO: 46) and human germline IGKV2-30. The 02 (SEQ ID NO: 74) sequence contains differential residues in the frame region and complementarity-determining region (CDR).

[0080] Figure 2D shows the amino acid sequence of the antibody in this paper (SEQ ID NO: 47 and 48).

[0081] Figure 2E shows the amino acid sequence of the antibody in this paper (HC104-320-VLB; SEQ ID NO: 49).

[0082] Figure 2F shows HC104-320-VLB (SEQ ID NO: 49) and human germline IGKV2-30. The 02 (SEQ ID NO: 74) sequence contains differential residues in the frame region and complementarity-determining region (CDR).

[0083] Figure 2G shows the amino acid sequence of the antibody (HC104-218-VLA; SEQ ID NO: 50) in this paper.

[0084] Figure 2H shows HC104-218-VLA (SEQ ID NO: 50) and human germline IGKV2-18. The amino acid residue differences present in the frame region and complementarity-determining region (CDR) of the 01 (SEQ ID NO: 75) sequence.

[0085] Figure 2I shows the amino acid sequences of the antibodies (SEQ ID NO: 51 and 52) in this paper.

[0086] Figure 2J shows the amino acid sequence of the antibody (HC104-218-VLB; SEQ ID NO: 53) in this paper.

[0087] Figure 2K shows HC104-218-VLB (sequence ID: 53) and human germline IGKV2-18. The 01 (Sequence ID: 75) sequence contains differential residues in the frame region and complementarity-determining region (CDR).

[0088] Figure 2L shows the amino acid sequence of one of the antibodies (HC104-41-VLA; sequence ID: 54) used in this paper.

[0089] Figure 2M shows HC104-41-VLA (SEQ ID NO: 54) and human germline IGKV4-1. The 01 (SEQ ID NO: 76) sequence contains differential residues in the framework and complementarity-determining regions (CDRs).

[0090] Figure 2N shows the amino acid sequences of the two antibodies (sequence IDs: 55 and 56) in this paper.

[0091] Figure 2O shows the amino acid sequence of the antibody (HC104-41-VLB; SEQ ID NO: 57) in this paper.

[0092] Figure 2P shows HC104-41-VLB (SEQ ID NO: 57) and the human germline IGKV4-1. The 01 (SEQ ID NO: 76) sequence contains differential residues in the frame region and complementarity-determining region (CDR).

[0093] Figures 3A and 3B show the final sample quality control results using reducing and non-reducing PAGE analysis and Coomassie brilliant blue staining. MW represents the molecular weight standard. The sample loading amount per lane was 2 µg.

[0094] Figures 4A to 4T show the SEC-HPLC spectra of HC104-551-VHA-230-VLA (A), HC104-551-VHB-230-VLA (B), HC104-146-VHA-230-VLA (C), and HC104-146-VHB-230-VLA (D), and HC104-1692-VHA-230-VLA (E), HC104-1692-VHB-230-VLA (F), HC104-551-VHA-41-VLA (G), HC104-551-VHB-41-VLA (H), HC104-146-VHB-41-VLA (I), HC104-1692-VHA-41-VLA (J), and HC104-1692-VHB-41-VLA. (K), HC104-551-VHA-218-VLA (L), HC104-551-VHB-218-VLA (M), HC104-146-VHB-218-VLA (N), HC104-1692-VHA-218-VLA (O), HC104-1692-VHB-218-VLA (P), HC104_hIgG1 (Q), HC104_hIgG1 (R), blank control (S), and blank control (T). Approximate molecular weights are as follows: 6.115 minutes: 670 kDa; 8.213 minutes: 150 kDa; 9.691 minutes: 45 kDa; 10.624 minutes: 17 kDa; 12.514 minutes: 1 kDa.

[0095] Figure 5 shows the thermostability analysis of the five humanized variants and chimeric antibodies. The graphs represent the fluorescence ratio (F350 / F330) and its corresponding first derivative.

[0096] Figures 6A to 6G show the sensorgram curves for the following antibodies: H104-551-VHA-230-VLA (A), H104-551-VHB-230-VLA (B), HC104-146-VHB-230-VLA (C), HC104-1692-VHA-230-VLA (D), HC104-1692-VHB-230-VLA (E), HC104-chimeric antibody (F), and mouse 104 from 20846 (G). Detailed Implementation

[0097] The inventors have identified an antibody that can specifically bind to the PTHrP isoform PTHrP1-173.

[0098] I. Definition

[0099] As used herein, the term “amino acid” includes all naturally occurring amino acids as well as modified L-amino acids. For example, the atoms of an amino acid can contain different isotopes. For instance, an amino acid can have hydrogen replaced by deuterium, nitrogen-14 replaced by nitrogen-15, carbon-12 replaced by carbon-13, and other similar changes.

[0100] As used herein, "conservative amino acid substitution" refers to replacing one amino acid residue with another without altering the intended properties of the protein. Suitable conservative amino acid substitutions can be achieved by substituting amino acids with similar hydrophobicity, polarity, and R-group size. Examples of conservative amino acid substitutions include:

[0101] As used herein, the term "sequence identity" refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences. To determine the percentage of sequence identity between two amino acid sequences or two nucleic acid sequences, the sequences need to be aligned for optimal alignment (e.g., vacancies can be introduced into the first amino acid sequence or nucleic acid sequence to achieve optimal alignment with the second amino acid sequence or nucleic acid sequence). Then, amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are compared. When a position in the first sequence is identical to the corresponding position in the second sequence, the two molecules are identical at that position. The percentage of sequence identity between two sequences is a function of the number of identical positions in the sequences (i.e., percentage of sequence identity = number of identical overlapping positions / total number of positions × 100%). In one embodiment, the two sequences are of the same length. Mathematical algorithms can also be used to determine the percentage of sequence identity between two sequences. A non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm proposed by Karlin and Altschul (1990, Proc. Natl. Acad. Sci. USA 87:2264-2268), which was modified by Karlin and Altschul (1993, Proc. Natl. Acad. Sci. USA 90:5873-5877). This algorithm has been integrated into the NBLAST and XBLAST programs developed by Altschul et al. (1990, J. Mol. Biol. 215:403). BLAST nucleotide searches can be performed using NBLAST nucleotide program parameters, for example, setting the score = 100 and the word length = 12, to obtain nucleotide sequences homologous to the nucleic acid molecule described herein. BLAST protein searches can be performed using XBLAST program parameters, for example, setting the score to 50 and the word length to 3, to obtain amino acid sequences homologous to the protein molecule described herein. To obtain vacancy-bearing alignment results for comparison, the Gapped BLAST method described by Altschul et al. (1997) in Nucleic Acids Res. 25:3389-3402 can be used. Alternatively, PSI-BLAST can be used for iterative searching to detect distant relationships between molecules (ibid.). When using the BLAST, Gapped BLAST, and PSI-BLAST procedures, the default parameters of the respective procedures (e.g., XBLAST and NBLAST) can be used (see the NCBI website).Another example of a mathematical algorithm used for sequence comparison is the one proposed by Myers and Miller (1988) in CABIOS 4:11-17. This algorithm has been integrated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When comparing amino acid sequences using the ALIGN program, the PAM120 residue weight table can be used, with a vacancy length deduction of 12 and a vacancy deduction of 4. The percentage of sequence identity between two sequences can be determined using a method similar to that described above, regardless of whether vacancy is allowed. When calculating the percentage of sequence identity, only sequences that are complete matches are typically counted.

[0102] Sequence consistency with the reference sequence can be any percentage between 70% and 99%, or any value falling within the range of 70% to 99%, such as 81%, 82%, 83%, 84%, and 85%, or 81.1%, 81.2%, 81.3%, 81.4%, and 81.5%, etc.

[0103] For antibodies, the sequence identity percentage can be determined by maximally aligning the antibody sequence using IgBlast, other tools, or other methods (such as IMGT, Kabat numbering rules). After alignment, if the target antibody region (e.g., the entire mature variable region of the heavy or light chain) is compared with the same region of the reference antibody, the sequence identity percentage between the target antibody region and the reference antibody region is calculated by dividing the number of identical amino acid positions in the target antibody region and the reference antibody region by the total number of aligned positions in both regions (excluding vacancies), and then multiplying by 100.

[0104] As used herein, the term "antibody" includes monoclonal antibodies, polyclonal antibodies, single-chain antibodies, humanized antibodies, and other chimeric or fully human antibodies, as well as their binding fragments. It also includes vectored antibodies or intracellular antibodies. Antibodies can be derived from recombinant sources and / or produced in transgenic animals. It also includes human antibodies that can be produced using biochemical techniques or isolated from antibody libraries. Humanized or chimeric antibodies may contain sequences from one or more homotypes or classes.

[0105] As used herein, a “fragment” refers to a portion of an antibody or antibody chain that has fewer amino acid residues than the intact antibody or antibody chain and is capable of binding to an antigen or competing with the intact antibody. Exemplary fragments include, but are not limited to, Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, miniantibodies, biantibodies, and their multimers. Fragments can be obtained by chemical or enzymatic treatment of the intact antibody or antibody chain. Fragments can also be obtained by recombinant methods (see below). For example, the F(ab')2 fragment can be generated by pepsin treatment. The resulting F(ab')2 fragment can be further processed to reduce disulfide bonds, thereby generating the Fab' fragment. Papain digestion can generate the Fab fragment. Fab, Fab', and F(ab')2, scFv, dsFv, ds-scFv, dimers, miniantibodies, biantibodies, bispecific antibody fragments, and other fragments can also be constructed using recombinant expression techniques.

[0106] As used herein, the term "complementarity-determining region" or "CDR" refers to a specific hypervariable region in an antibody that is generally considered to facilitate epitope binding. Computational methods for identifying CDR sequences include Kabat, Chothia, IgBlast, and IMGT. The CDRs listed in this disclosure are defined using the Kabat nomenclature. Those skilled in the art can also identify CDR sequences based on Kabat nomenclature by referring to the sequences contained herein.

[0107] The term "PTHrP" refers to parathyroid hormone-related protein, such as PTHrP provided in Uniprot accession number P12272, and includes all naturally occurring variants, including isoforms 1-173, 1-139, and 1-141. PTHrP is also commonly referred to as parathyroid hormone-related protein, parathyroid hormone-like (PTHLH), or parathyroid hormone-like protein (PLP). PTHrP, PTHLH, and PLP are generally considered to be interchangeable terms.

[0108] As used herein, the term "detectable marker" refers to a portion, such as a peptide sequence or fluorescent protein, that can be attached to or incorporated into the peptides, antibodies, or other compounds described herein, and that can directly or indirectly generate a detectable signal. For example, the marker may be a radioactive, opaque, positron-emitting radionuclide (e.g., for PET imaging) or radioisotope, such as 3H, 13N, 14C, 18F, 32P, 35S, 123I, 125I, or 131I; a fluorescent compound (e.g., an AlexaFluor™ fluorophore) or a chemiluminescent compound (e.g., fluorescein isothiocyanate, rhodamine, or luciferin); an enzyme, such as alkaline phosphatase, β-galactosidase, or horseradish peroxidase; an imaging agent; or a metal ion. Detectable markers can also be detected indirectly, for example, using a secondary antibody.

[0109] As used herein, the term "nucleic acid sequence" refers to a sequence of nucleoside or nucleotide monomers composed of naturally occurring bases, sugars, and interglycosylation (backbone) links. The term also includes modified or substituted sequences composed of non-naturally occurring monomers or portions thereof. The nucleic acid sequence of this application may be a deoxyribonucleic acid (DNA) sequence or a ribonucleic acid (RNA) sequence and may contain naturally occurring bases such as adenine, guanine, cytosine, thymidine, and uracil. These sequences may also contain modified bases. Examples of such modified bases include aza- and deadenine adenine, guanine, cytosine, thymidine, and uracil, as well as xanthine and hypoxanthine. Nucleic acids may be double-stranded or single-stranded and represent a sense strand or antisense strand. Furthermore, the term "nucleic acid" includes complementary nucleic acid sequences as well as codon-optimized or synonymous codon equivalents. Nucleic acids may be mRNA.

[0110] As used herein, the term "vector" includes any intermediate carrier of a nucleic acid molecule that enables the nucleic acid molecule to be introduced, for example, into prokaryotic and / or eukaryotic cells and / or integrated into the genome, including plasmids, bacteriophages, or viral vectors such as retroviral vectors, adenovirus-associated vectors, etc. As used herein, the term "plasmid" generally refers to a construct of extrachromosomal genetic material, typically a circular double-stranded DNA that can replicate independently of chromosomal DNA.

[0111] As used herein, "specific binding" means that an antibody recognizes its target antigen and binds to it with a higher affinity than it has with structurally different antigens and / or sequence-modified or mutated antigens. For example, the dissociation constant (KD) of a multivalent antibody to its target antigen is at least 1e-6, 1e-7, 1e-8, 1e-9, or 1e-10. Preferably, the affinity is greater than 1e-8. An antigen-binding fragment containing a variable region (e.g., a Fab fragment) may have an affinity for its target antigen that is 10 to 100 times lower than that for multivalent interactions with unfragmented antibodies.

[0112] As used herein, “specific binding” means that an antibody or its fragment binds to an epitope in the PTHrP subtype 1-173 (“PTHrP1-173”) sequence, or binds to any fragment containing the C-terminal portion of PTHrP1-173, such as amino acids 140-173.

[0113] As used in this article, “selectivity” or “preferential” means that the antibody selectively / preferentially binds to a certain PTHrP form and its affinity is increased by at least 3, 5, 10, 20, 100, 250 or 500 times.

[0114] As used herein, the terms “animal” or “subject” include all members of the animal kingdom, including mammals, and may or may not include humans.

[0115] As used herein, the term "treatment" or "therapy" is widely understood in the art to refer to a method of achieving a beneficial or anticipated outcome, including clinical outcomes. Beneficial or anticipated clinical outcomes may include, but are not limited to: relief or improvement of one or more symptoms or diseases, reduction of disease severity, stabilization (i.e., non-exacerbation) of a disease state, prevention of disease spread, delay or slowing of disease progression, improvement or relief of a disease state, reduction of disease relapse, and remission (whether partial or complete), regardless of whether the remission is detectable. "Treatment" or "therapy" may also refer to extended survival compared to expected survival without treatment. Subjects may be treated with antibodies or fragments thereof, immunoconjugates, or compositions described herein to prevent disease progression.

[0116] For the purpose of understanding the scope of this disclosure, the term “comprising” and its derivatives are intended as closing terms to expressly indicate the presence of listed features, elements, components, groups, integers and / or steps and exclude the presence of other unlisted features, elements, components, groups, integers and / or steps.

[0117] The numerical ranges indicated by endpoints in this document encompass all numerical values ​​and their fractions within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It should also be understood that all numerical values ​​and their fractions are to be considered as being modified by the term “about.” Furthermore, it should be understood that references without a specific quantity are plural unless explicitly defined as singular in the context. The term “about” refers to approximately 0.1% to 50%, 5%–50%, or 10%–40% of the referenced numerical value, optionally 10%–20%, and further optionally 10% or 15%.

[0118] Furthermore, the definitions and embodiments described in certain sections are intended to be applicable to other applicable embodiments described herein as understood by those skilled in the art. For example, different aspects of the invention will be defined in more detail in the following paragraphs. Unless otherwise expressly stated, each aspect so defined may be combined with any other aspect or pluralities. In particular, any feature designated as optional or advantageous may be combined with any other feature designated as optional or advantageous or pluralities.

[0119] II. Antibodies, Immunoconjugates, Nucleic Acids, Vectors, and Cells

[0120] This article contains antibodies or fragments thereof that specifically bind to the C-terminus of PTHrP and specifically bind to the PTHrP1-173 subtype. As shown in this article, the humanized antibody possesses desirable properties.

[0121] Therefore, in one aspect, this document provides an antibody or fragment thereof that specifically binds to PTHrP, the antibody or fragment thereof comprising a light chain variable region and a heavy chain variable region. The heavy chain variable region comprises complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprises complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3. In one embodiment, the amino acid sequence of CDR comprises:

[0122] In one embodiment, the amino acid sequence of the CDR comprises one or more of the following sequence combinations:

[0123] or

[0124] In one embodiment, the amino acid sequence of the CDR comprises one or more sequences shown in Table 3 or Table 4 below.

[0125] In one embodiment, the sequences of the heavy chain and light chain variable regions comprise one or more sequences shown in Table 7 or Table 8 below.

[0126] In one embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 20; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 20, wherein the CDR sequence is as described in SEQ ID NO: 1, 3, and 7; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 1, 3, and 7; and the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 26; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 26, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 8-10.

[0127] In one embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 21; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 21, wherein the CDR sequence is as described in SEQ ID NO: 2, 4, and 7; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 2, 4, and 7; and the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 26; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 26, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 8-10.

[0128] In one embodiment, the antibody or fragment includes a heavy chain variable region comprising: i) an amino acid sequence as described in SEQ ID NO: 73; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 73, wherein the CDR sequence is as described in SEQ ID NO: 2, 4, and 7; or iii) a conserved substituted amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 2, 4, and 7; and the antibody includes a light chain variable region comprising: i) an amino acid sequence as described in SEQ ID NO: 26; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 26, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substituted amino acid sequence of i), wherein the CDR sequence is as shown in SEQ ID NO: 8-10.

[0129] In one embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 22; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 22, wherein the CDR sequence is as described in SEQ ID NO: 1, 3, and 7; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 1, 3, and 7; and the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 26; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 26, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 8-10.

[0130] In one embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 23; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 23, wherein the CDR sequence is as described in SEQ ID NO: 1, 5, and 7; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 1, 5, and 7; and the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 26; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 26, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 8-10.

[0131] In one embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 24; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 24, wherein the CDR sequence is as described in SEQ ID NO: 1, 3, and 7; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 1, 3, and 7; and the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 26; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 26, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 8-10.

[0132] In one embodiment, the heavy chain variable region comprises: i) the amino acid sequence as described in SEQ ID NO: 25; ii) an amino acid sequence having 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 25, wherein the CDR sequence is as described in SEQ ID NO: 1, 6, and 7; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 1, 6, and 7; and the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 26; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 26, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 8-10.

[0133] In one embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 20; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 20, wherein the CDR sequence is as described in SEQ ID NO: 1, 3, and 7; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 1, 3, and 7; and the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 27; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 27, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 8-10.

[0134] In one embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 21; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 21, wherein the CDR sequence is as described in SEQ ID NO: 2, 4, and 7; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 2, 4, and 7; and the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 27; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 27, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 8-10.

[0135] In one embodiment, the antibody or fragment includes a heavy chain variable region comprising: i) an amino acid sequence as described in SEQ ID NO: 73; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 73, wherein the CDR sequence is as described in SEQ ID NO: 2, 4, and 7; or iii) a conserved substituted amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 2, 4, and 7; and the antibody includes a light chain variable region comprising: i) an amino acid sequence as described in SEQ ID NO: 27; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 27, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substituted amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 8-10.

[0136] In one embodiment, the antibody or fragment includes a heavy chain variable region comprising: i) an amino acid sequence as described in SEQ ID NO: 22; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 22, wherein the CDR sequence is as described in SEQ ID NO: 1, 3, and 7; or iii) a conserved substituted amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 1, 3, and 7; and the antibody includes a light chain variable region comprising: i) an amino acid sequence as described in SEQ ID NO: 27; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 27, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substituted amino acid sequence of i), wherein the CDR sequence is as shown in SEQ ID NO: 8-10.

[0137] In one embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 23; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 23, wherein the CDR sequence is as described in SEQ ID NO: 1, 5, and 7; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 1, 5, and 7; and the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 27; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 27, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 8-10.

[0138] In one embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 24; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 24, wherein the CDR sequence is as described in SEQ ID NO: 1, 3, and 7; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 1, 3, and 7; and the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 27; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 27, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 8-10.

[0139] In one embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 25; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 25, wherein the CDR sequence is as described in SEQ ID NO: 1, 6, and 7; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 1, 6, and 7; and the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 27; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 27, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 8-10.

[0140] In one embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 20; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 20, wherein the CDR sequence is as described in SEQ ID NO: 1, 3, and 7; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 1, 3, and 7; and the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 28; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 28, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 8-10.

[0141] In one embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 21; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 21, wherein the CDR sequence is as described in SEQ ID NO: 2, 4, and 7; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 2, 4, and 7; and the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 28; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 28, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 8-10.

[0142] In one embodiment, the antibody or fragment includes a heavy chain variable region comprising: i) an amino acid sequence as described in SEQ ID NO: 73; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 73, wherein the CDR sequence is as described in SEQ ID NO: 2, 4, and 7; or iii) a conserved substituted amino acid sequence of i), wherein the CDR sequence is as described in 2, 4, and 7; and the antibody includes a light chain variable region comprising: i) an amino acid sequence as described in SEQ ID NO: 28; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 28, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substituted amino acid sequence of i), wherein the CDR sequence is as shown in SEQ ID NO: 8-10.

[0143] In one embodiment, the antibody or fragment includes a heavy chain variable region comprising: i) an amino acid sequence as described in SEQ ID NO: 22; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 22, wherein the CDR sequence is as described in SEQ ID NO: 1, 3, and 7; or iii) a conserved substituted amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 1, 3, and 7; and the antibody includes a light chain variable region comprising: i) an amino acid sequence as described in SEQ ID NO: 28; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 28, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substituted amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 8-10.

[0144] In one embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 23; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 23, wherein the CDR sequence is as described in SEQ ID NO: 1, 5, and 7; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 1, 5, and 7; and the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 28; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 28, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 8-10.

[0145] In one embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 24; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 24, wherein the CDR sequence is as described in SEQ ID NO: 1, 3, and 7; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 1, 3, and 7; and the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 28; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 28, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 8-10.

[0146] In one embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 25; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 25, wherein the CDR sequence is as described in SEQ ID NO: 1, 6, and 7; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 1, 6, and 7; and the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 28; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 28, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 8-10.

[0147] In one embodiment, the antibody or fragment includes a heavy chain variable region comprising: i) the amino acid sequence of SEQ ID NO: 68; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 68, wherein the CDR sequence is as described in SEQ ID NO: 1, 3, and 7; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as described in SEQ ID NO: 1, 3, and 7; and the antibody includes a light chain variable region comprising: i) the amino acid sequence of SEQ ID NO: 69; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 69, wherein the CDR sequence is as described in SEQ ID NO: 8-10; or iii) a conserved substitution amino acid sequence of i), wherein the CDR sequence is as shown in SEQ ID NO: 8-10.

[0148] In one embodiment, the antibody or fragment thereof includes a heavy chain variable region comprising SEQ ID NO: 20, 21, 23, 24 or 25 and a light chain variable region comprising SEQ ID NO: 26.

[0149] In one embodiment, the antibody or a fragment thereof includes a heavy chain variable region comprising the amino acid sequence SEQ ID NO: 20 and a light chain variable region comprising the amino acid sequence SEQ ID NO: 26.

[0150] In one embodiment, the antibody or a fragment thereof includes a heavy chain variable region comprising the amino acid sequence SEQ ID NO: 21 and a light chain variable region comprising the amino acid sequence SEQ ID NO: 26.

[0151] In one embodiment, the antibody or a fragment thereof includes a heavy chain variable region comprising the amino acid sequence SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence SEQ ID NO: 26.

[0152] In one embodiment, the antibody or a fragment thereof includes a heavy chain variable region comprising the amino acid sequence SEQ ID NO: 24 and a light chain variable region comprising the amino acid sequence SEQ ID NO: 26.

[0153] In one embodiment, the antibody or a fragment thereof includes a heavy chain variable region comprising the amino acid sequence SEQ ID NO: 25 and a light chain variable region comprising the amino acid sequence SEQ ID NO: 26.

[0154] In one embodiment, the antibody or its fragment has higher yield, better stability, and / or lower aggregation compared to a reference antibody.

[0155] In one embodiment, the antibody or a fragment thereof has a higher binding affinity compared to a reference antibody.

[0156] In some embodiments, the reference antibody is one or more humanized antibodies, chimeric antibodies as described herein, and / or mouse 104 monoclonal antibodies having heavy chain variable regions and light chain variable regions, the structures of which are shown in SEQ ID NO: 81 and 82, respectively.

[0157] In one embodiment, the antibody or a fragment thereof has higher stability and / or lower aggregation than a reference mouse 104 monoclonal antibody.

[0158] In one embodiment, the antibody or a fragment thereof has a higher binding affinity than a reference mouse 104 monoclonal antibody.

[0159] In one embodiment, the conserved substitution amino acid sequence includes the CDR sequences listed in Table 3 or Table 4 below.

[0160] In one embodiment, the heavy chain variable region is contained in the heavy chain, and / or the light chain variable region is contained in the light chain.

[0161] In one embodiment, the heavy chain further comprises the amino acid sequence of SEQ ID NO: 62 and / or 63.

[0162] In one embodiment, the light chain further comprises the amino acid sequence of SEQ ID NO: 64 and / or 65.

[0163] In one embodiment, the heavy chain further comprises the amino acid sequence of SEQ ID NO: 62, and / or the light chain further comprises the amino acid sequence of SEQ ID NO: 64.

[0164] In one embodiment, the heavy chain comprises the amino acid sequence of SEQ ID NO: 63, and / or the light chain comprises the amino acid sequence of SEQ ID NO: 65.

[0165] In one embodiment, the amino acid sequence identity of the antibody is 83.7%, 80.6%, 84.7%, 90.0%, 86.0%, 80.2%, 88.8%, and 86.7%. The homology with the reference amino acid sequence is 92.9%, 91.0%, 89.0%, 83.2%, or 95.9%, respectively.

[0166] In one embodiment, the amino acid sequences of the heavy and light chain variable regions may contain any number of consecutive amino acids, for example, in the range of 25-50, 50-75, 75-100, 100-125, etc., as described in SEQ ID 20-28, and contain at least one sequence mutation as described herein.

[0167] A CDR may contain one or two amino acid substitutions, optionally including conserved amino acid substitutions. As described in the examples and shown in the tables and figures, several CDRs disclosed herein have high sequence homology. For example, CDR-H2 of variant 4 differs from CDR-H2 of variant 12 by one amino acid. Therefore, in one embodiment, the CDRs described herein contain one or two amino acid substitutions, optionally, which may be conserved amino acid substitutions. In one embodiment, the substitution occurs at residues that differ between the CDRs described herein. For example, the amino acid substitution occurs at residues in a CDR mentioned herein (e.g., CDR-H2) that differ from another corresponding CDR mentioned herein (e.g., another CDR-H2), and may include any amino acid at the position corresponding to the different amino acid, provided that the amino acid can maintain specific binding to PTHrP, or optionally, a conserved amino acid substitution.

[0168] In other embodiments, the variable chain may contain conserved amino acid substitutions, and the CDR sequence is as shown herein, for example, as shown in Table 3 or Table 4.

[0169] The antibodies or fragments disclosed herein can be defined as containing both heavy chain and light chain variable regions. The heavy chain variable region contains 121 residues, numbered H1 to H113 according to Kabat nomenclature (including H1-H52, H52a, H53-H82, H82a-c, H83-H100, H100a-d, and H101-H113); the light chain variable region contains 112 residues, numbered L1 to L107 according to Kabat nomenclature (including L1-L27, L27a-e, and L28-L107). For example, please refer to the Kabat nomenclature of the variable heavy chain and variable light chain regions in the antibody amino acid (aa) sequences provided in Tables 1 and 2 below: Table 1: Examples of Kabat numbering for variable heavy chain region sequences

[0170] Table 2: Examples of Kabat numbering for variable light chain region sequences

[0171] In one embodiment, as shown in the example above, making the following residue substitutions at the positions defined by the Kabat number will not eliminate the desired properties of the protein:

[0172] In one embodiment, the antibody is a monoclonal antibody. In one embodiment, the antibody is a chimeric antibody, such as a humanized antibody. In one embodiment, the antibody is a single-chain antibody.

[0173] In one embodiment, the antibody is affinity purified.

[0174] In one embodiment, the antibody undergoes affinity maturation.

[0175] The binding ability of the antibody can be assessed by, for example, ELISA or FACS (e.g., EC50); or optionally by Kd value (e.g., by Biacore or Octet).

[0176] The bioactivity of the antibody can be assessed by relevant in vitro cell assays and by comparison with a reference antibody.

[0177] The cross-reactivity of the antibody can be compared with that of a relevant homologous species (e.g., by in vitro binding activity).

[0178] The biophysical properties of antibodies can be determined by methods such as SEC-HPLC to determine the content of high molecular weight soluble aggregates, SDS-PAGE electrophoresis under non-reducing and reducing conditions, and / or differential scanning calorimetry (DSC) analysis using a Microcal™ VP capillary DSC system to determine the Tm values ​​of Fab, CH2 and CH3.

[0179] In one embodiment, an antibody or a fragment thereof is conjugated to a detectable marker. In one embodiment, the detectable marker is a positron-emitting radionuclide. Positron-emitting radionuclides can be used, for example, in PET imaging.

[0180] Therefore, on the other hand, immunoconjugates comprise antibodies or fragments thereof as described herein, as well as detectable markers.

[0181] In one embodiment, the nucleic acid encodes an antibody, an antibody heavy chain, an antibody light chain, a variable region of the antibody heavy chain, or a variable region of the antibody light chain, or a fragment thereof, as described herein. For example, the nucleic acid may encode a heavy chain comprising the CDR-H1, CDR-H2, and / or CDR-H3 regions described herein and more specifically as shown in Table 3 below, or a light chain comprising the CDR-L1, CDR-L2, and / or CDR-L3 regions described herein and more specifically as shown in Table 4 below.

[0182] In one embodiment, the nucleic acid comprises any one of SEQ ID NO: 11-19, and may optionally further comprise one or more nucleotide sequences encoding the amino acid sequences of SEQ ID NO: 58-61 or SEQ ID NO: 62-65.

[0183] In one embodiment, the nucleic acid i) encodes a heavy chain variable region, wherein the nucleic acid encoding the heavy chain variable region comprises any sequence or any portion thereof of SEQ ID NO: 11, 12, 14, 15, or 16, or has at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any sequence thereof of SEQ ID NO: 11, 12, 14, 15, or 16; ii) encodes a light chain variable region, wherein the nucleic acid encoding the light chain variable region comprises any sequence or any portion thereof of SEQ ID NO: 17, 18, or 19, or has at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any sequence thereof of SEQ ID NO: 17, 18, or 19; or iii) encodes both the heavy chain variable region and the light chain variable region, wherein the nucleic acids encoding the heavy chain variable region and the light chain variable region respectively comprise the following sequences or any portions thereof: SEQ ID NO: 11 and 17; SEQ ID NO: 12 SEQ ID NO: 14 and 17; SEQ ID NO: 15 and 17, SEQ ID NO: 16 and 17, SEQ ID NO: 12 and 18, SEQ ID NO: 16 and 18, SEQ ID NO: 11 and 19, SEQ ID NO: 12 and 19, SEQ ID NO: 14 and 19, SEQ ID NO: 15 and 19, or SEQ ID NO: 16 and 19, or sequences having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 11 and 17; SEQ ID NO: 12 and 17; SEQ ID NO: 14 and 17; SEQ ID NO: 15 and 17, SEQ ID NO: 16 and 17, SEQ ID NO: 12 and 18, SEQ ID NO: 16 and 18, SEQ ID NO: 15 and 17; SEQ ID NO: 16 and 17, SEQ ID NO: 17 and 18, SEQ ID NO: 16 and 18 ... 11 and 19, SEQ ID NO: 12 and 19, SEQ ID NO: 14 and 19, SEQ ID NO: 15 and 19, or SEQ ID NO: 16 and 19.

[0184] In one embodiment, the identity of the nucleic acid sequence can be any percentage ranging from 70% to 99.5% with respect to the reference sequence.

[0185] In one respect, the expression cassette or vector contains the nucleic acid disclosed herein.

[0186] In one embodiment, the vector is a separate vector. The vector can be any vector, including those suitable for producing antibodies and / or their binding fragments or expressing the peptide sequences described herein. Suitable examples of vectors include adenoviral vectors, adenovirus-associated vectors, or retroviral vectors, and optionally lentiviral vectors.

[0187] Nucleic acid molecules can be integrated into suitable expression vectors in a known manner to ensure protein expression. Possible expression vectors include, but are not limited to, entrapment, plasmid, or modified viruses (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses). The vector should be compatible with the host cells used. The expression vector is suitable for transformation of the host cells, meaning that the expression vector contains a nucleic acid molecule encoding the antibody described herein.

[0188] In one respect, the cell expresses the antibody described herein or a fragment thereof or nucleic acid, or contains the vector described herein.

[0189] In one embodiment, the cells are isolated and / or recombinant cells expressing the antibodies or fragments thereof described herein, or containing the vectors disclosed herein. In one embodiment, the cells are fusion cells, such as hybridoma cells. Recombinant cells can be generated using any cell line suitable for producing peptides, such as cells suitable for producing antibodies and / or their binding fragments. For example, to introduce nucleic acids (e.g., vectors) into cells, the cells can be transfected, transformed, or infected, depending on the vector used.

[0190] Suitable host cells include a variety of prokaryotic and eukaryotic host cells. For example, the proteins described herein can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculoviruses), yeast cells, or mammalian cells.

[0191] In one embodiment, the cell is a eukaryotic cell selected from yeast, plant, nematode, insect, bird, fish, reptile, and mammalian cells.

[0192] In one embodiment, the insect cell is an Sf9 cell, an Sf21 cell, a Tni cell, or an S2 cell.

[0193] In another embodiment, the mammalian cell is a myeloma cell, a spleen cell, or a hybridoma cell.

[0194] Suitable yeast and fungal host cells for expressing antibodies or peptides include, but are not limited to, various species of *Saccharomyces cerevisiae*, *Schizosaccharomyces pombe*, *Pichia*, *Kluyveromyces*, and *Aspergillus*. Examples of vectors used for expression in *Saccharomyces cerevisiae* include pYepSec1, pMFa, pJRY88, and pYES2 (Invitrogen, San Diego, CA). Transformation protocols for yeast and fungi are well known to those skilled in the art.

[0195] Potentially applicable mammalian cell types include, but are not limited to, HEK-293, COS, BHK, CHO, HeLa, and NS-1 cells, with HEK-293 or CHO cells being optional. Suitable expression vectors for guiding mammalian cell expression typically include promoters (e.g., viral materials such as polyomavirus, adenovirus 2, cytomegalovirus, and simian virus 40) and other transcriptional and translational control sequences.

[0196] III. Composition

[0197] In another aspect, a composition is provided comprising an antibody or a fragment thereof, an immunoconjugate, a nucleic acid, a vector, or cells, as described herein.

[0198] In one embodiment, the composition is a pharmaceutical composition.

[0199] In one embodiment, the composition comprises a pharmaceutically acceptable carrier, diluent, and / or excipient.

[0200] In one embodiment, the composition comprises a diluent. Diluents suitable for nucleic acids and vectors include, but are not limited to, water, saline solutions, and ethanol. Diluents suitable for peptides (including antibodies or fragments thereof) and / or cells include, but are not limited to, saline solutions, pH buffer solutions, and glycerol solutions, or other solutions suitable for freezing peptides and / or cells.

[0201] In one embodiment, the composition comprises an antibody or a fragment thereof or a portion thereof as described herein. In another embodiment, the composition comprises an antibody or a portion thereof as described herein, and a diluent.

[0202] In one embodiment, the composition is a sterile composition.

[0203] In one embodiment, the composition is a pharmaceutical composition. The pharmaceutical composition can be used, for example, as described herein, to treat a subject who requires the composition. For example, a subject suffering from hypercalcemia (such as malignancy-associated hypercalcemia) or cancer (such as breast cancer, lung cancer, colon cancer, pancreatic cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, or melanoma).

[0204] In one embodiment, the composition comprises one or more antibodies or fragments described herein.

[0205] In one embodiment, the composition is used in the method described herein.

[0206] IV. Reagent Kit and Packaging

[0207] This invention relates to a kit or package comprising: i) an antibody or fragment thereof; ii) an immunoconjugate; iii) isolated nucleic acid; iv) a vector; v) cells; or vi) a composition described herein, wherein the kit or package is contained in a vial (e.g., a sterile vial or other container) and optionally includes a reference reagent and / or instructions for use.

[0208] In one embodiment, the kit is an ELISA.

[0209] In one embodiment, the kit is a multiplex detection or planar array kit.

[0210] In one embodiment, the kit contains the antibody or fragment thereof described herein, which is contained in a container (e.g., a sterile vial).

[0211] In one embodiment, the kit includes instructions for use with the ELISA or method described herein.

[0212] In one embodiment, this document is used for the method described in the kit.

[0213] VI. Methods

[0214] This invention relates to a method for reducing or inhibiting the activity of human PTHrP isoenzyme, comprising contacting cells or tissues expressing human PTHrP isoenzyme with an antibody or fragment thereof that specifically binds to PTHrP, as described herein. The antibody or its binding fragment includes a light chain variable region and a heavy chain variable region, the heavy chain variable region including complementation-determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region including complementation-determining regions CDR-L1, CDR-L2, and CDR-L3, wherein the amino acid sequence of the CDRs includes:

[0215] Any antibody described herein may be used. In one embodiment, a method for reducing or inhibiting the activity of a human PTHrP isoform comprises: contacting cells or tissues expressing a human PTHrP isoform with an antibody or fragment thereof that specifically binds to PTHrP, said antibody or fragment thereof comprising a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, the light chain variable region comprises complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequence of said CDR comprises one of the following sequence combinations:

[0216] For example, in one embodiment, a method for reducing or inhibiting human PTHrP subtype activity includes contacting cells or tissues expressing human PTHrP subtypes with an antibody or fragment thereof that specifically binds to PTHrP, as described herein, wherein the amino acid sequence of the CDR comprises one or more sequences shown in Tables 3 and 4 below.

[0217] In one embodiment, contacting cells or tissues expressing the human PTHrP subtype with an antibody or a fragment thereof includes expressing an immunoconjugate, nucleic acid, or vector in the cells or tissues, as described herein.

[0218] In one embodiment, the cells or tissues expressing the human PTHrP subtype are located within the subject.

[0219] On the other hand, a method for treating a subject in need of treatment includes administering to the subject an effective amount of an antibody or fragment thereof that specifically binds to PTHrP, the antibody or fragment thereof comprising a light chain variable region and a heavy chain variable region, the heavy chain variable region comprising complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, the light chain variable region comprising complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequence of the CDR comprising the following sequence:

[0220] Any antibody described herein may be used. In one embodiment, a method for reducing or inhibiting the activity of human PTHrP isoforms comprises administering to a subject in need an effective amount of an antibody or fragment thereof that specifically binds to PTHrP, the antibody or fragment thereof comprising a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, the light chain variable region comprises complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequence of the CDR comprises one of the following sequence combinations:

[0221] For example, a method of treating a subject in need of treatment includes administering to the subject in need of treatment an effective amount of an antibody or fragment thereof that specifically binds to PTHrP, the antibody or fragment thereof comprising a light chain variable region and a heavy chain variable region, the heavy chain variable region comprising complementarity-determining regions CDR-H1, CDR-H2 and CDR-H3, the light chain variable region comprising complementarity-determining regions CDR-L1, CDR-L2 and CDR-L3, and the amino acid sequences of the CDRs are shown in Tables 3 and 4 below.

[0222] In one embodiment, the method is a method of treating cancer in a subject requiring treatment, comprising: administering to the subject an effective amount of an antibody or a fragment thereof, a composition comprising said antibody or a fragment thereof, an immunoconjugate, a nucleic acid, or a vector, as described herein.

[0223] In one embodiment, the cancer is in the pre-metastatic stage.

[0224] In one embodiment, the method is a method for treating hypercalcemia (e.g., malignant tumor-associated hypercalcemia) for a subject in need, including administering to the subject an effective amount of an antibody or a fragment thereof, a composition containing an antibody or a fragment thereof, an immunoconjugate, a nucleic acid, or a vector, as described herein.

[0225] In other respects, uses of the antibodies, immunoconjugates, nucleic acids, and vectors described herein are also provided. For example, in one embodiment, use of an antibody, immunoconjugate, nucleic acid, or vector is provided to reduce or inhibit human PTHrP isotype activity in a subject of need. Another embodiment provides the use of an antibody, immunoconjugate, nucleic acid, or vector to prepare a medicament for reducing or inhibiting human PTHrP isotype activity and / or treating cancer or hypercalcemia in a subject of need. In one embodiment, an antibody, immunoconjugate, nucleic acid, or vector is provided for reducing or inhibiting human PTHrP isotype activity in a subject of need.

[0226] In one embodiment, the subject has (e.g., has been diagnosed with) any cancer that expresses PTHrP, such as breast cancer, lung cancer, colon cancer, pancreatic cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, or melanoma. In another embodiment, the subject has breast cancer. In yet another embodiment, the subject has pancreatic cancer.

[0227] In one embodiment, the subject has (e.g., has been diagnosed with) malignant tumor-related hypercalcemia.

[0228] In one embodiment, an effective amount of the antibody described herein or a fragment thereof or an immunoconjugate, or a composition comprising the antibody or a fragment thereof or an immunoconjugate, is selectively used in combination with another treatment for hypercalcemia or cancer.

[0229] The antibody or its fragment or immunoconjugate may be included in the compositions described herein, for example, in combination with a pharmaceutically acceptable carrier, diluent, and / or excipient, and formulated in a vesicular manner to improve delivery. Combinations of antibodies (e.g., two or more antibodies or their fragments) and / or immunoconjugates may also be used.

[0230] The compositions, antibodies, immunogens, and immunoconjugates described herein can be administered via, for example, parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraventricular, intrathecal, intrasheath, intraorbital, intraocular, intraspinal, intracisional, intraperitoneal, intranasal, aerosol, or oral routes.

[0231] In one embodiment, the composition is administered systemically.

[0232] Furthermore, a method for detecting human PTHrP isoforms or fragments thereof in a subject includes: i) contacting a test sample (e.g., a biological sample) from the subject with the antibody or fragment thereof described herein, under conditions that allow for the generation of antibody-antigen complexes; ii) measuring the amount of antibody-antigen complexes in the test sample; and iii) comparing the amount of antibody-antigen complexes in the test sample with a control, wherein the detection of antibody-antigen complexes in the test sample, compared with the control, indicates that the sample contains human PTHrP isoforms and / or tumor cells expressing human PTHrP isoforms, and / or indicates that the subject has hypercalcemia (e.g., malignant tumor-associated hypercalcemia) or cancer (e.g., breast cancer or pancreatic cancer).

[0233] On the other hand, a method for detecting human PTHrP isoforms or fragments thereof in a subject includes: i) contacting a first test sample (e.g., a biological sample) with a sample obtained from the subject prior to treatment with the antibody or fragment thereof described herein to generate an antibody-antigen complex; ii) measuring the amount of antibody-antigen complex in the first test sample; iii) obtaining a second test sample (e.g., a biological sample) from the subject after treatment; iv) measuring the amount of antibody-antigen complex in the second test sample; and v) comparing the amount of antibody-antigen complex in the first test sample with the amount of antibody-antigen complex in the second test sample, wherein a relatively lower amount of antibody-antigen complex in the second test sample compared to the first test sample indicates a relatively lower level of human PTHrP isoforms or fragments thereof and / or tumor cells expressing human PTHrP isoforms or fragments thereof in the subject's body, and / or indicates a response to treatment in the subject. In another embodiment, one or more additional test samples may be obtained from the subject, wherein the amount of antibody-antigen complexes in the additional test samples is compared with the amount of antibody-antigen complexes in a previous test sample (e.g., a first or second test sample or a control sample) collected from the same patient to monitor the subject's response to treatment or disease progression (e.g., metastasis) or relapse. In some embodiments, the antibodies used in vivo are humanized. In one embodiment, the measurement method may be, for example, immunofluorescence.

[0234] In one embodiment, the sample is a biological sample. In other embodiments, the sample comprises blood, serum, plasma, tissue, cells, tumor, or extracts thereof. The sample may also be a component. In one embodiment, the sample is taken from a human subject. In one embodiment, the sample is tumor biopsy tissue.

[0235] On the other hand, in one embodiment of a method for detecting human PTHrP isoforms or fragments thereof in subjects, the sample is taken from a subject who has or is suspected of having hypercalcemia (e.g., malignancy-related hypercalcemia) or cancer (e.g., breast cancer, lung cancer, colon cancer, pancreatic cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, or melanoma). In other embodiments, the sample is taken from a subject who has (e.g., has been diagnosed with) breast cancer or pancreatic cancer.

[0236] Various methods can be used to determine the presence of human PTHrP isotypes or fragments thereof in a sample using the antibodies or fragments described herein, including immunoassays such as flow cytometry, dot or groove blots, Western blotting, ELISA, and immunoprecipitation combined with SDS-PAGE immunocytochemistry. Other available immunological methods include singlet and multiplex immunoassay platforms. Any non-humanized or humanized antibodies described herein can be used in these examples.

[0237] In one embodiment, the label or labeled antibody described herein is used for diagnostic assays or tests.

[0238] A platform based on single magnetic beads can be used, in which individual immune complexes are isolated on paramagnetic magnetic beads and detected.

[0239] Multiplex immunoassay platforms suitable for these purposes include magnetic bead or particle-based platforms as well as planar array platforms.

[0240] A bead- or particle-based platform can be used, in which the capture antibody is immobilized on particles such as fluorescently stained microbeads or paramagnetic microbeads, and a second detection antibody is used to detect the analyte.

[0241] As described herein, antibodies can also be labeled or modified and used to detect the location of human PTHrP subtypes or fragments thereof. This can be combined with diagnostic imaging to detect and / or locate the presence or location of labeled or modified antibodies bound in vivo. Any humanized antibody or fragment thereof, such as Fab, Fab', and F(ab')2 described herein, can be used in these embodiments. For example, Fab can be used to detect the presence of human PTHrP subtypes or fragments thereof, and / or tumor cells expressing human PTHrP subtypes or fragments thereof.

[0242] The foregoing is a general description of this application. A more comprehensive understanding of this application can be achieved by referring to the following specific examples. These examples are for illustrative purposes only and are not intended to limit the scope of this application. Depending on the specific circumstances, changes to the form or substitutions for equivalents may be considered. Although certain terminology is used herein, it is for descriptive purposes only and not for limitation.

[0243] The following non-limiting examples illustrate the content disclosed herein: Example Example 1 Humanization of HC104 mouse monoclonal antibodies by CDR grafting The mouse HC104 monoclonal antibody was humanized using CDR grafting technology. The humanization process included constructing a molecular model to aid in the selection of residues to be retained or replaced.

[0244] The CDR transplantation protocol used was a modernized version of the method developed by Greg Winter and colleagues at the Cambridge Medical Research Council. The definition of CDR was based on the Kabat nomenclature. The IMGT mouse and human V gene databases were searched using IgBLAST (http: / / www.ncbi.nlm.nih.gov / igblast / ) developed by NCBI, with the variable region sequence of HC104 mice as input, to select the human frame recipient region for transplantation of the CDR region from HC104 mice.

[0245] Humanization of the HC104 mouse monoclonal antibody (mAb) was achieved by transplanting the complementarity-determining region (CDR) into a human germline antibody sequence. HC104 was obtained by immunizing mice with a 33-amino acid-long peptide that corresponds to the C-terminal region (140-173) of the parathyroid hormone-like hormone or PTHrP (Uniprot P12272) isoform.

[0246] Humanization was achieved by transplanting three complementarity-determining regions (CDRs) (as defined by Kabat nomenclature) from the light chain variable region (VL) of the HC104 mouse antibody into a human VL. Similarly, three CDRs from the heavy chain variable region (VH) were transplanted into the human VH. Several amino acid residues in the selected human VH framework region were replaced with amino acid residues present in the mouse VH (i.e., so-called reversion mutations). Using the molecular model of the HC104 mouse monoclonal antibody Fv, the framework region residues were retained in humanized version A, while in subsequent humanized versions, these residues were replaced with their corresponding human VH residues. Guided by the molecular model, CDR residues were also replaced according to Kabat nomenclature in subsequent version B to further enhance humanization.

[0247] For the VH version of HC104 mice transplanted with CDR, three human strains were selected: IGHV1-46 01. IGHV1-69-2 01 and IGHV5-51 01. Mouse HC104 VH and human IGHV1-46 01. The sequence identity across the entire V gene (including CDR1 and CDR2, but excluding CDR3, which is not part of the V gene) is 71.4%; it is similar to the human germline IGHV1-69-2. The sequence identity of 01 was 68.4%; it was similar to IGHV5-51. The sequence identity of 01 was 63.3% (including CDR1 and CDR2, but excluding CDR3, which does not belong to the V gene) (see Table 3).

[0248] Table 3: Percentage of sequence identity between mouse V gene or its humanized version and different human strains (V gene)

[0249] To construct a CDR transplantation version of the HC104 mouse VL, we selected three human lines: IGKV2-30 02, IGKV2-18 01 and IGKV4-1 01. IGKV2-30 01 and IGKV2-18 The sequence identity of 01 with mouse HC104 VL across the entire V gene (including two-thirds of CDR1, CDR2, and CDR3) was 81% and 77%, respectively. IGKV4-1 The sequence identity between 01 and HC104 VL is 64.35%.

[0250] We selected combinations of heavy and light chains from humanized VH and humanized VL versions. For VH, humanized versions, including IGHV5-51, were constructed through computer simulation based on three different human lineages. There are two versions (A and B) of 01 and one version C, for a total of 7 different humanized versions of VH. For VL, humanized versions were constructed through computer simulation based on three different human lineages, with two versions (A and B) for both VH and VL, for a total of 6 different humanized versions of VL. In total, 6(VH) × 6(VL) = 42 combinations of humanized variants were obtained.

[0251] It expresses 18 VH / VL combinations: humanized versions A and B of VH are combined with version A of VL, that is, 6(VH) × 3(VL) = 18 humanized variants are selected.

[0252] The table below lists details of 18 humanized combinations of VH and VL.

[0253] Table 4: 18 combinations of humanized VH and VL

[0254] Table 1 lists the percentage of sequence identity (i.e., human percentage) across the entire V gene between the HC104 mouse parental monoclonal antibody and different CDR transplanted humanized versions.

[0255] For more details, please refer to Examples 2 and 3.

[0256] Example 2

[0257] Heavy chain

[0258] Figure 1A The amino acid sequence of mouse HC104 hybridoma VH is shown (CDR regions are highlighted according to the Kabat numbering scheme).

[0259] Select human lineage IGHV1-69-2 01. IGHV1-69-2 01 and IGHV5-51 01 serves as the recipient sequence for CDR transplantation. Using IgBLAST (https: / / www.ncbi.nlm.nih.gov / igblast / igblast.cgi) with the mouse HC104 VH amino acid sequence as input, a search was conducted in the IMGT human germline VH gene database to select the human framework receptor VH region for transplantation into the mouse HC104 CDR region. The closest matching entry was determined based on sequence alignment between the parental antibody and the human germline.

[0260] A large number of human phylogenetic sequences were analyzed, and three optimal candidate sequences were selected: two of them belong to human family 1 (IGHV1-69-2). 01 and IGHV1-69-2 01), a family belonging to human family 5 (IGHV5-51) 01).

[0261] In frame 4, the gene segment corresponding to the J gene in mouse HC104 VH was identified as being related to the mouse germline IGHJ4. 01. Highest gene homology. The mouse IGHJ4... 01. Comparison of the gene fragment 01 with the human J gene fragment in the CDR3 and FR4 regions revealed similarities to the human J4 fragment. 01 The fragment has the highest homology.

[0262] IGHV1-46 01 Human lineage as a frame receptor region

[0263] Humanized Version A

[0264] Mouse CDRs defined according to the Kabat nomenclature (highlighted in gray in Figure 1A) were transplanted into IGHV1-46. In step 01, the sequences shown in Figures 1B and 1C are obtained.

[0265] Humanized version A (HC104-146-VHA) and human germline IGHV1-46 The sequence identity of the V gene in 01 is 83.7% (82 out of 98 amino acid residues in the V gene are identical). As shown in Figure 1C, in the frame region and CDR region, the humanized version A is similar to the human germline IGHV1-46. The difference residues between 0 and 1 are highlighted in gray. The full-length amino acid sequence of version A is shown in Figure 1D.

[0266] Humanized Version B

[0267] Compared to version A, version B has 5 amino acid residues replaced with germline residues (i.e., mouse residues are replaced with the corresponding IGHV1-46 residues). 01. Humanized Residues). In frame 2, Kabat Ile (I) H48 is replaced with Met (M). In CDR2, Kabat Thr (T) H60 and Kabat Lys (K) H64 are replaced with Ala (A) and Gln (Q), respectively. In frame 3, Kabat Ser (S) H75 and Kabat Arg (R) H76 are replaced with Thr (T) and Ser (S), respectively. As shown in Figure 1D, the amino acid residues that differ between humanized versions A and B are highlighted in gray.

[0268] Humanized version B (HC104-146-VHB) and IGHV1-46 The V gene sequence identity of the 01 human germline viral strains is 88.8% (87 out of the 98 amino acid residues in the V gene are identical). For example... Figure 1F As shown, the differing residues between humanized versions A and B are highlighted in gray. The full-length amino acid sequence of version B is as follows. Figure 1D As shown.

[0269] IGHV1-69-2 01 Human lineage As a framework receptor region

[0270] Humanized Version A

[0271] Mouse CDRs defined according to the Kabat nomenclature (highlighted in gray in Figure 1A) were transplanted into IGHV1-69-2. In 01, to obtain Figure 1G and 1H sequence shown.

[0272] Humanized version A (HC104-1692-VHA) and human germline IGHV1-69-2 The V gene sequence identity of 01 is 80.61% (79 of the 98 amino acid residues in the V gene are identical). In Figure 1H, HC104-1692-VHA is similar to the human germline IGHV1-69-2. 01. Difference residues in the framework region and complementarity-determining region (CDR) are highlighted in gray. The full-length amino acid sequence of version A is shown in Figure 1I.

[0273] D-3 Humanized Version B

[0274] Compared to version A, version B has 6 amino acid residues replaced with germline residues (i.e., mouse residues are replaced with the corresponding IGHV1-46). 01 Human germline residues). In frame 2, Kabat Ile (I) H48 is replaced by Met (M). In CDR2, Kabat Thr (T) H60, Kabat Gln (Q) H61, and Kabat Lys (K) H64 are replaced by Ala (A), Glu (E), and Gln (Q), respectively. In frame 3, Kabat Ser (S) H75 and KabatArg (R) H76 are replaced by Thr (T) and Asp (D), respectively.

[0275] As shown in Figure 1J, the amino acid residues that differ between humanized versions A and B are highlighted in gray.

[0276] Humanized version B (HC104-1692-VHB) and human germline IGHV1-69-2 The sequence identity of the V gene in 01 is 86.7% (85 out of 98 amino acid residues in the V gene are identical). As shown in Figure 1K, HC104-1692-VHB is similar to the human germline IGHV1-69-2. 01. Amino acid residues that differ in the framework region and complementarity-determining region (CDR) are highlighted in gray. The full-length amino acid sequence of version B is shown in Figure 1I.

[0277] IGHV5-51 01 Human germline as a framework receptor region

[0278] Humanized Version A

[0279] The mouse CDR defined according to the Kabat nomenclature (highlighted in gray in Figure 1A) was transplanted into IGHV5-51. In 01, the detailed sequences shown in Figures 1L and 1M are obtained.

[0280] Humanized Version A (HC104-551-VHA) and IGHV5-51 01. The human lineage shares 84.7% sequence identity (83 out of 98 amino acid residues in the V gene are identical). As shown in Figure 1M, HC104-551-VHA and the human lineage IGHV5-51... 01. Differences in the frame region and CDR are highlighted in gray. The full-length amino acid sequence of version A is shown in Figure 1N.

[0281] E-3 Humanized Version B

[0282] Compared to version A, in version B, eight amino acid residues were replaced with germline residues (i.e., mouse residues were replaced with the corresponding IGHV5-51 residues). 01 Human germline residues). In CDR2, Kabat Met (M) H34 is replaced with Ile (I). In frame 2, Kabat Ile (I) H48 is replaced with Met (M). In CDR2, Kabat Thr (T) H60, Kabat Gln (Q) H61, Kabat Lys (K) H62, and Kabat Lys (K) H64 are replaced with Ser (S), Pro (P), Ser (S), and Gln (Q), respectively. In frame 3, Kabat Ser (S) H75 and Kabat Arg (R) H76 are replaced with Ile (I) and Ser (S), respectively. As shown in Figure 10, the amino acid residues that differ between humanized versions A and B are highlighted in gray.

[0283] Humanized version B (HC104-551-VHB) and human germline IGHV5-51 The sequence identity of the V gene in HC104-551-VHB is 92.9% (91 out of 98 residues in the V gene are identical). As shown in Figure 1P, HC104-551-VHB is similar to the human germline IGHV5-51. 01 Residues showing differences in the framework region and complementarity-determining region (CDR) are highlighted in green and Kamen blue, respectively. The full-length amino acid sequence of version B is shown in Figure 1N.

[0284] E-4 Humanized Version C

[0285] Compared to version B, version C has three amino acid residues replaced with germline residues (i.e., mouse residues are replaced with the corresponding IGHV5-51 residues). 01 Human germline residues). In frame 1, Kabat Ala (A) H24 and Kabat Thr (T) H28 are replaced with Gly (G) and Ser (S), respectively. In frame 3, Kabat Leu (L) H69 is replaced with Ile (I).

[0286] As shown in Figure 1Q, the amino acid residues that differ between humanized versions B and C are highlighted in gray.

[0287] Humanized version C (HC104-551-VHC) and IGHV5-51 01. The sequence identity of the human lineage is 95.9% (94 out of 98 residues in the V gene are identical). As shown in Figure 1R, the differing residues between HC104-551-VHB and HC104-551-VHC are highlighted in gray. The complete amino acid sequence of version C is shown in Figure 1N.

[0288] The heavy chain amino acid CDR sequences of humanized variants defined according to the Kabat nomenclature are summarized in Table 5 below: Table 5: Amino acid sequences of heavy chain CDRs

[0289] Example 3

[0290] Light chain

[0291] See Figure 2A, which shows the amino acid sequence of the mouse mAb HC104 virus light chain (VL) (the CDR region defined according to Kabat nomenclature is highlighted).

[0292] Using IgBLAST with the mouse VL region amino acid sequence as input, the IMGT human VL gene database was searched to select the human framework receptor VL region for transplantation into the CDR region of HC104 mice. The closest matching entries were determined based on sequence alignment between the parental antibody and the human lineage.

[0293] A large number of human germline sequences were analyzed, and three human lineages were selected: IGKV2-30. 02, IGKV2-18 01 and IGKV-4-1 01, a humanized version used to build a CDR port. IGKV2-30 02 showed the highest sequence identity with HC104 VL, at 81% (81 out of 100 amino acid residues were identical), followed by IGKV2-18. 01, with an identity of 77% (77 out of 100 amino acid residues are identical), IGKV4-1 01 The identity is 64.35% (65 out of 101 amino acid residues are the same).

[0294] The gene segment corresponding to the J gene in mouse HC104 VL is related to the mouse-specific J gene IGKJ2. The sequence of 01(FTFGSGTKLEIK) is completely identical. The mouse IGKJ2... 01. Comparison of the CDR3 and FR4 regions of the human J gene fragment with those of the human J gene fragment revealed that the human J gene fragment IGLJ2... 01 has the highest overall homology.

[0295] GKV2-30 02 Human lineage as a framework receptor region

[0296] C-2 Humanized Version A

[0297] The mouse CDR defined according to the Kabat number (highlighted in gray in Figure 2A) was transplanted into IGKV2-30. In step 02, the detailed sequences shown in Figures 2B and 2C are obtained.

[0298] Humanized version A (HC104-320-VLA) and IGKV2-30 The sequence identity of 02 is 90.0% (90 out of 100 amino acid residues in the V gene are identical). The full-length amino acid sequence of version A is shown in Figure 2D.

[0299] C-3 Humanized Version B

[0300] Compared to version A, version B contains one germline amino acid residue (i.e., mouse residues are replaced by the corresponding IGKV2-30). 02 Human germline residue substitution). In CDR1, Kabat Ile (I) L27b is replaced by Leu (L).

[0301] As shown in Figure 2E, the amino acid residues that differ between humanized versions A and B are highlighted in gray.

[0302] Humanized version B (HC104-320-VLB) and IGKV2-30 The sequence identity of 02 is 91.0% (91 out of 100 residues in the V gene are identical). As shown in Figure 2F, the residues highlighted in gray are the positions in the CDR region where humanized versions A and B differ. The full-length amino acid sequence of version B is shown in Figure 2D.

[0303] IGKV2-18 01 Human lineage as a frame receptor region

[0304] D-2 Humanized Version A

[0305] The mouse CDR defined according to the Kabat number (highlighted in gray in Figure 2A) was transplanted into IGKV2-18. In 01, the detailed sequences shown in Figures 2G and 2H are obtained.

[0306] Humanized version A (HC104-218-VLA) and IGKV2-18 The sequence identity of 01 is 86.0% (86 out of 100 amino acid residues in the V gene are identical). As shown in Figure 2H, the gray highlighted areas are the humanized version A and the human germline IGKV2-18. The differential sites within the CDR region between 0 and 1. The full-length amino acid sequence of version A is shown in Figure 2I.

[0307] D-3 Humanized Version B

[0308] Compared to version A, version B contains three germline mutations in amino acid residues (i.e., mouse residues are replaced by the corresponding IGKV2-18). 01. Human germline residue substitutions. In frame region 1 (FR1), Kabat Val (V) L2 is substituted with Ile (I). In CDR1, Kabat Ile (I) L27b is substituted with Leu (L). In frame region 3 (FR3), KabatThr (T) L69 is substituted with Ser (S). As shown in Figure 2J, the amino acid residues that differ between humanized versions A and B are highlighted in gray.

[0309] Humanized version B (HC104-218-VLB) and IGKV2-18 The sequence identity of 01 is 89.0% (89 out of 100 residues in the V gene are identical). As shown in Figure 2K, the residues highlighted in gray represent the different positions within the CDR region of humanized versions A and B. The full-length amino acid sequence of version B is shown in Figure 2I.

[0310] IGKV4-1 01 Human lineage as a frame receptor region

[0311] E-2 Humanized Version A

[0312] The mouse CDR defined according to the Kabat number (highlighted in gray in Figure 2A) was transplanted into IGKV4-1. In 01, the detailed sequences shown in Figures 2L and 2M are obtained.

[0313] Humanized version A (HC104-41-VLA) and IGKV4-1 The sequence identity of 01 is 81.2% (81 out of 101 amino acid residues in the V gene are identical). The full-length amino acid sequence of version A is shown in Figure 2N.

[0314] E-3 Humanized Version B

[0315] Compared to version A, version B has three amino acid residues replaced with germline residues (i.e., mouse-derived residues are replaced with the corresponding IGKV4-1 residues). 01 Human germline residues). In frame region 1 (FR1), Kabat Val (V) L2 is substituted with Ile (I). In complementarity-determining region 1 (CDR1), Kabat (Arg) L24 is substituted with Lys (K), and KabatIle (I) L27b is substituted with Val (V). As shown in Figure 20, the amino acid residues that differ between humanized versions A and B are highlighted in gray.

[0316] Humanized version B (HC104-41-VLB) and IGKV4-1 The sequence identity of 01 is 84.1% (84 out of 101 residues in the V gene are identical). As shown in Figure 2P, the residues highlighted in gray are the positions in the CDR region where humanized versions A and B differ. The full-length amino acid sequence of version B is shown in Figure 2N.

[0317] The light chain amino acid CDR sequences of the humanized variants defined according to the Kabat nomenclature are summarized in Table 6 below: Table 6: Amino acid sequences of light chain CDRs

[0318] Example 4

[0319] Expression and purification of HC104 humanized recombinant antibody

[0320] Genes and expression vectors

[0321] Genes encoding recombinant antibodies (rAbs)

[0322] Using the heavy chain (HC) and light chain (LC) variable region sequences of 18 humanized variant antibodies obtained by computer simulation humanization as described in Examples 1-3 as starting materials, a full-length human IgG1κ antibody was synthesized.

[0323] cDNAs encoding the HC and LC variable regions were chemically synthesized, and their expression in CHO cells was optimized. These cDNAs were subcloned into mammalian cell expression vectors containing the IgG1 heavy chain constant region and κ light chain constant region backbone. Sequences encoding signal peptides were added to the 5' / N ends. These sequences are shown in Table 7 below.

[0324] In Table 7, sequences not underlined represent the heavy and light chain variable regions of humanized antibody variants 1-18. Examples of heavy and light chain secretion signal sequences used are shown in bold / underlined, while examples of human IgG1 heavy chain constant region and human κ light chain constant region backbones used are shown in non-bold / non-underlined.

[0325] Table 7: Nucleotide sequences of the variable regions of the heavy and light chains

[0326] expression carrier

[0327] The expected protein sequences are shown in Table 8 below.

[0328] In Table 8, the ununderlined sequences represent the heavy and light chain variable regions of humanized antibody variants 1-18. Examples of heavy and light chain secretion signal sequences used are shown in bold / underlined, while examples of the human IgG1 heavy chain constant region and human κ light chain constant region backbones used are shown in non-bold / non-underlined.

[0329] Table 8: Amino acid sequences of the variable regions of the heavy and light chains

[0330] Small-scale production and purification trials

[0331] As mentioned above, the construct uses an endotoxin-free DNA preparation.

[0332] Various antibodies were expressed by combining heavy chains (HC) and light chains (LC), as shown in Table 9 below: Table 9: Antibodies

[0333] The plasmid was transiently co-transfected into CHO cells. Cell culture medium samples were collected when cell viability dropped below 50% (14 days post-transfection), and the recombinant and chimeric antibodies were then purified on protein A resin using standard methods. Clarification via 0.22 µm membrane filtration Equilibrate, bind, and wash with PBS buffer at pH 7.5. Elution was performed by adjusting the pH with citric acid. Neutralize with 1 M Tris-HCl buffer (pH 9.0) Target components were analyzed and combined using PAGE electrophoresis, and Final quality control was performed by PAGE electrophoresis: qualitative analysis and quantitative analysis by SDS-PAGE electrophoresis.

[0334] The eluted fractions were combined and buffer-exchanged with PBS buffer at pH 7.5 by dialysis. The final sample was filtered through a 0.22 µm Millipore filter. Figure 3A (recombinant antibody) and Figure 3b (chimera) show the purification process and final quality control results, respectively.

[0335] Results of small-scale production and purification trials

[0336] Figures 3A and 3B show the final sample quality control results.

[0337] The yield and purity are summarized in Table 10 below.

[0338] Table 10. Yield and purity of recombinant antibody production After purification, it was used for 30ml culture testing.

[0339] The full-length antibody was observed based on the non-reducing PAGE analysis shown in Figure 2. Final buffer: PBS pH 7.5

[0340] Conclusion: Transient expression of humanized variants showed excellent yield and high purity, including variants 1, 2, 4-6, 8 and 12-18, as well as the HC104 chimeric antibody (number 19).

[0341] Example 5

[0342] SEC-HPLC Aggregation Level Analysis

[0343] The aggregation levels of the expressed antibodies (variants 1-8, 10-14, and 16-18, and the HC104 chimeric antibody referred to herein as variant 19) were assessed using SEC-HPLC analysis. The analyses were performed using a Waters 2695 high-performance liquid chromatograph equipped with a photodiode array detector (2996).

[0344] Buffer preparation

[0345] Mobile phase: 100 mM sodium sulfate, 100 mM phosphate, pH 6.7 buffer (Na₂SO₄, 14.2 g / L; Na₂HPO₄·12H₂O, 15.6 g / L; NaH₂PO₄, 6.8 g / L). All buffers were degassed.

[0346] Sample preparation

[0347] Protein preparation: Take an additional copy from each final sample, store at -80°C overnight, and dilute the protein sample to 1 mg / mL with double-distilled water. Centrifuge the sample at 12000 g for 10 minutes and collect the supernatant.

[0348] Analysis conditions

[0349] Blank control: Sample diluted 5-fold with double-distilled water. Column: G3000SWXL, TOSOH, 7.8 × 300 mm. HPLC system: Waters 2695. Elution gradient: Isocratic elution with mobile phase. Flow rate: 0.8 ml / min. Temperature: 25°C. Injection volume: 30 μl. Detection wavelength: 280 nm. Data acquisition time: 25 min. Equilibration with 100% mobile phase for 5 min. Injection sequence: First inject a blank control, then inject the sample. Data analysis is as follows.

[0350] SEC-HPLC analysis results

[0351] Figures 4A to 4T The SEC-HPLC chromatograms of variants 1-8, 10-14, and 16-18, the HC104 chimeric antibody (number 19), and the blank control are shown. The purity of the intact antibody monomolecules detected in the samples was analyzed, and the results are shown in Table 9 below. When multiple peaks were detected, the data corresponding to the peaks of intact antibody monomolecules are indicated in bold. If only a single peak was detected, an estimated value of the concentration (purity %) is given.

[0352] Table 11. SEC-HPLC Analysis Data

[0353] Conclusion: SEC-HPLC analysis showed that antibody samples 1, 2, 4, 5, 6, and the chimeric antibody were rationally selected heterologous quadruple chain antibodies with optimal concentrations, consisting of two heavy chains and two light chains, with an aggregation level <5%. Samples 3 and 11 contained a small number of aggregates (<20%), while samples 7, 8, 10, 12, 13, 14, 16, 17, 18, and the murine antibody contained a higher proportion of aggregates (>30%).

[0354] Example 6

[0355] ELISA analysis

[0356] BSA-PTHrP 140-173

[0357] EC50 values ​​were calculated using an ELISA method. Microplates coated with BSA-PTHrP 140-173 were blocked with 3% BSA. Antibody dilutions ranged from 10 μg / ml to 0.00064 μg / ml. PBST buffer was added at 100 μl / well, and the plates were incubated at 37°C for 1 hour. Goat anti-human HRP conjugate antibody was then added, and the plates were incubated for 30 minutes. Readings were taken from OD450 to 630.

[0358] EC50 values ​​were determined for humanized variants 1-8, 10-14, and 16-18, as well as the HC104 chimeric antibody (number 19). The results showed that the EC50 values ​​of several variants, including variants 1-8, 11-13, and 17, were improved compared to the HC104 chimeric antibody.

[0359] Example 7

[0360] Temperature stability

[0361] Differential scanning fluorescence (DSF) analysis was performed using a Nanotemper-nanoDSF system according to standard methods. mAb (50 µg, dissolved in pH 7.5 PBS buffer) was placed in a linear temperature program from 20°C to 95°C at a rate of 1°C / min. Tryptophan fluorescence at 350 nm and 330 nm was acquired at a rate of 10 data points per minute. The midpoint of the unfolding transition was automatically determined by the first derivative of the fluorescence ratio (F350 / F330).

[0362] Table 12: DSF Analysis Results

[0363] Conclusion: The thermostability curves of the humanized antibodies showed two development transitions in the fluorescence ratio (F330 / F350) versus temperature graph (Figure 5). These two different development events are attributed to the difference in thermostability between the antibody Fab and Fc domains. DSF analysis indicated that variants 1, 2, and 3 had the highest thermostability, with Tm1 approximately 72°C and Tm2 > 80°C.

[0364] Example 8

[0365] Determination of antibody / antigen interaction KD

[0366] Five humanized antibody variants were analyzed using OpenSPR™ to determine the KD value of each antibody relative to its antigen. PThrP 1-173 (2.64 mg / mL) was used as the ligand. Each humanized antibody was used at a concentration of 1.00 mg / mL with a purity of 95%. HC104 chimera (3.80 mg / mL) and mouse 104 from 20846 (1.00 mg / mL) were used as controls.

[0367] In short, the PThrP 1-173 antigen was immobilized on a COOH sensor chip. Solutions containing different concentrations of antibody were flowed through the PThrP 1-173. Kinetic parameters were measured in real time: binding / dissociation rates (Ka and Kd) were collected, and the binding affinity constant (KD) was determined.

[0368] Experimental design and parameters Buffer: PBS-P+ (0.01 M phosphate buffer, pH 7.4, 2.7 mM KCl, 0.137 mM NaCl, 0.05% surfactant P20) Regeneration buffer: 10 mM glycine-HCl (pH 2.0) Analyte test concentration: see results (below) Injection rate: 30 μL / min Regeneration buffer flow rate: 150 μL / min Combined reaction temperature: 20 ℃ Sensor: COOH Instrument: OpenSPR method 1. Install the COOH chip according to the standard operating procedure for OpenSPR™ instruments.

[0369] 2. In the initial stage, run at the maximum flow rate (150 μL / min) with PBS-P+ (pH 7.4) as the buffer.

[0370] 3. Once the signal reaches baseline, inject 200 μL of 80% isopropanol (IPA) and run for 10 seconds to expel air bubbles. After baseline recovery, rinse the sample loop with buffer and purge with air.

[0371] 4. Once the signal reaches the baseline, adjust the buffer flow rate to 20 μL / min.

[0372] 5. Inject 200 μL of EDC (400 mM) / NHS (100 mM) (1:1) solution (20 μL / min, 4 min).

[0373] 6. Load 200 μL of TNFα ligand diluted with activation buffer (PBS, pH=7.5) and run for 4 minutes (flow rate 20 μL / min). Rinse the sample loop with buffer and air dry.

[0374] 7. Add 200 μL of blocking solution (flow rate 20 μL / min, run for 4 minutes), rinse the sample loop with PBS, and air dry.

[0375] 8. Observe the baseline for 5 minutes to ensure stability.

[0376] 9. The analyte was diluted with PBS-P+ to the concentration shown in the experimental results. The injection flow rate was 30 μL / min. The binding time of the analyte to the ligand was 180 seconds, and the dissociation time was 300 seconds.

[0377] 10. The experimental results were analyzed using TraceDrawer software (Ridgeview Instruments AB, Sweden), and the analysis method was a one-to-one analysis mode.

[0378] The results are shown in Table 13 below. Note: KD is the equilibrium dissociation constant, which is the ratio of kd / ka between the antibody and its antigen. KD is inversely proportional to affinity: the lower the KD value, the higher the affinity of the antibody.

[0379] Table 13: Kinetic parameters and affinity of antibody / antigen interactions

[0380] Discussion and Conclusion

[0381] Typically, the KD value for weak binding is considered to be 10. - ³ M, the KD value for moderate binding is 10 -6 M, the KD value of strong binding is 10. -9 M. The KD value of proteins and small molecules is typically in the range of 10. - ³~10 -6 Within the M range, the KD values ​​of weak antigens and antibodies are in the range of 10. -6 ~10 -7 Within the M range, the KD values ​​of strong antigens and antibodies are in the range of 10. -8 ~10 - ¹ 0The KD value of most antibodies is in the low micromolar range (10). -6 ) to nanomoore (10 -7 ~10 -9 The KD value of high-affinity antibodies is typically in the low nanomolar range (10⁻⁶). -9 The KD value of antibodies with extremely high affinity is within the range of 10 picoseconds, while that of antibodies with very high affinity is within the range of 10 picoseconds. - Within the range of ¹²).

[0382] All seven antibodies tested against PThrP 1-173 showed high affinity (all reaching at least 10). -7 Up to 10 -8 KD), for example, the KD values ​​of H104-551-VHA-230-VLA and H104-551-VHB-230-VLA are in the range of 10. -8 Within the range, strong interactions are present; favorable kinetic parameters indicate faster binding and slower dissociation; sensing shape indicates a clear concentration-response relationship at much lower concentrations (Figures 6A-6G).

[0383] Example 9

[0384] Breast bulb formation test

[0385] Mammary globule growth assays were performed before and after treatment of MDA-231 cells with anti-PTHrP monoclonal antibodies H104-551-VHA-230-VLA and H104-551-VHB-230-VLA.

[0386] In summary, 5 × 10³ MDA-MB-231 human breast cancer cells / well were seeded in 1 ml of complete MammoCult™ medium (Stem Cell Technologies) in 24-well ultra-low adhesion cell culture plates (Corning® Costar®). Cells were cultured at 37°C and 5% CO2 for 7 days (baseline), and mammary globules were collected and digested with trypsin. Several anti-PTHrP humanized antibodies (10 μg / ml) and chimeras described herein were added to the medium (compared to the control group without antibodies), and mammary globules were allowed to grow for 7 days. The number of mammary globules was counted using the Lionheart system (Agilent BioTek). The Lionheart system is a fully automated high-throughput microscope using Olympus objectives.

[0387] Seven days after breast globule growth, the number of untreated breast globules was twice that of the group treated with the tested humanized antibody (compared to baseline on day 7), while the tested humanized antibody treatment significantly inhibited breast globule growth, reducing it by 50%. The tested humanized antibody was superior to the chimera in inhibiting breast globule growth.

[0388] Example 10

[0389] Uses of humanized PTHrP antibodies

[0390] This document discloses the inhibition of PTHrP production and / or signaling, the inhibition of PTHrP subtype production and / or signaling, and the inhibition of receptors and / or signaling pathways activated by PTHrP subtypes. The antibodies, immunoconjugates, nucleic acids, and vectors disclosed herein can be used to inhibit PTHrP and / or PTHrP1-173 subtypes to treat diseases such as hypercalcemia (e.g., malignancy-related hypercalcemia) or cancers (e.g., breast cancer, lung cancer, pancreatic cancer, prostate cancer, melanoma, and squamous cell carcinoma). For example, treatment using the antibodies or immunoconjugates disclosed herein can include treating cancer, inhibiting tumor growth, and / or inhibiting metastasis.

[0391] The antibodies disclosed in this article can also be used to inhibit the production and / or signaling of PTHrP and / or PTHrP1-173 isoforms, as well as to inhibit receptors and / or signaling pathways activated by PTHrP isoforms, for example, in human hypercalcemia models (such as malignant tumor-associated hypercalcemia) or cancer models (such as breast cancer, lung cancer, prostate cancer, pancreatic cancer, melanoma, and various types of squamous cell carcinoma). These uses may include, for example, constructing mouse models of human hypercalcemia, mouse models of human melanoma (e.g., transplanting A375 cells into the ventricles of mice), mouse models of human breast cancer (e.g., transplanting the human breast cancer cell line MDA-MB-435 into the mammary fat pads of nude mice), mouse models of human pancreatic cancer, in vitro models of human prostate cancer, in vitro models of squamous cell carcinoma or squamous skin cancer, and models of keratinocyte tumor progression (e.g., using human papillomavirus 16 immortalized keratinocytes to generate a non-tumorigenic HPK1A cell line, which is then converted into cancer cells by expressing the activated H-Ras oncogene, generating HPK1Aras cells, which eventually develop into classic squamous cell tumors).

[0392] This document also discloses specific antibodies against PTHrP and / or PTHrP1-173 subtypes. The antibodies disclosed herein can be further used for disease diagnosis or detection of PTHrP and / or PTHrP1-173 subtypes, as an indicator of disease activity, such as hypercalcemia (e.g., malignancy-associated hypercalcemia) or cancer (e.g., breast cancer, lung cancer, pancreatic cancer, prostate cancer, melanoma, and various types of squamous cell carcinoma), or as an indicator of metastatic tumor spread (optionally prior to the onset of hypercalcemia), or as a prognostic indicator for potentially effective cancer treatment, for example, by labeling or tagging the disclosed antibodies with diagnostic reagents (e.g., reporter molecules, radioisotopes, or positron-emitting radionuclides) and using imaging techniques to analyze in vivo or in vitro binding in tissue samples.

[0393] The humanized antibodies disclosed herein can be further used for in vivo imaging and / or in vivo treatment of tumors and metastatic sites targeting PTHrP and / or its subtypes. For example, the antibodies disclosed herein can be labeled with detectable markers (e.g., reporter molecules, radioisotopes, or positron-emitting radionuclides) or therapeutic agents (e.g., cytotoxic drugs, prodrugs, or drugs). For instance, the humanized Fab fragments disclosed herein can be labeled with radionuclides and administered to subjects to detect and / or locate human PTHrP subtypes in vivo via PET imaging, thereby diagnosing the cause of hypercalcemia or cancer in the subjects.

[0394] The antibodies disclosed herein can also be used to measure or assess the effects of treatment on tumor growth (e.g., inhibition of tumor growth), metastasis, and / or patient survival in vivo, and / or on cell growth and / or invasion in vitro. For example, the antibodies disclosed herein can be labeled with diagnostic reagents (e.g., reporter molecules, radioisotopes, or positron emission tomography radionuclides) and the binding of the antibody to the antigen in a biological sample can be analyzed using imaging or diagnostic analysis methods. For example, the amount of antibody-antigen complex in a test sample obtained from a subject can be compared with the amount of antibody-antigen complex in another test sample or a baseline or control sample to diagnose and / or monitor a subject's disease, such as hypercalcemia or cancer. The humanized antibodies disclosed herein can be used to treat subjects, and the same or different humanized or non-humanized antibodies or fragments thereof can be used to monitor a subject's response to treatment or disease progression or recurrence via in vitro binding analysis, for example, using the antibodies described herein to identify cancer cells in a biological sample in vitro via immunohistochemistry or immunofluorescence.

Claims

1. An antibody or fragment thereof that specifically binds to PTHrP, comprising a heavy chain variable region and a light chain variable region; wherein the heavy chain variable region comprises complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3; and the light chain variable region comprises complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3; wherein, The amino acid sequence of the CDR includes the following sequence: 。 2. The antibody or fragment thereof according to claim 1, wherein the amino acid sequence of the CDR comprises the following sequence: or 。 3. The antibody or fragment thereof according to claim 1 or 2, wherein the heavy chain variable region comprises: i) The amino acid sequence of SEQ ID NO: 20; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 20, wherein the CDR sequence is as shown in SEQ ID NO: 1, 3, and 7; or iii) The conserved substituted amino acid sequences of i), wherein the CDR sequences are as shown in SEQ ID NO: 1, 3 and 7; The light chain variable region includes: i) The amino acid sequence of SEQ ID NO: 26; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 26, wherein the CDR sequence is as shown in SEQ ID NO: 8-10; or iii) The conserved substituted amino acid sequence of i), wherein the CDR sequence is as shown in SEQ ID NO: 8-10.

4. The antibody or a fragment thereof according to claim 1 or 2, wherein the heavy chain variable region comprises: i) The amino acid sequence of SEQ ID NO: 21; ii) An amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 21, wherein the CDR sequence is as shown in 2, 4, and 7; or iii) The conserved substituted amino acid sequence of i), wherein the CDR sequence is shown in 2, 4 and 7; Furthermore, the light chain variable region includes: i) The amino acid sequence of SEQ ID NO: 26; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 26, wherein the CDR sequence is as shown in SEQ ID NO: 8-10; or iii) The conserved substituted amino acid sequence of i), wherein the CDR sequence is as shown in SEQ ID NO: 8-10.

5. The antibody or a fragment thereof according to claim 1 or 2, wherein the heavy chain variable region comprises: i) The amino acid sequence of SEQ ID NO: 22; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 22, wherein the CDR sequence is as shown in SEQ ID NO: 1, 3, and 7; or iii) The conserved substituted amino acid sequences of i), wherein the CDR sequences are as shown in SEQ ID NO: 1, 3 and 7; Furthermore, the light chain variable region includes: i) The amino acid sequence of SEQ ID NO: 26; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 26, wherein the CDR sequence is as shown in SEQ ID NO: 8-10; or iii) The conserved substituted amino acid sequence of i), wherein the CDR sequence is as shown in SEQ ID NO: 8-10.

6. The antibody or a fragment thereof according to claim 1 or 2, wherein the heavy chain variable region comprises: i) The amino acid sequence of SEQ ID NO: 23; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 23, wherein the CDR sequence is as shown in SEQ ID NO: 1, 5, and 7; or iii) The conserved substituted amino acid sequence of i), wherein the CDR sequence is as shown in SEQ ID NO: 1, 5 and 7; Furthermore, the light chain variable region includes: i) The amino acid sequence of SEQ ID NO: 26; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 26, wherein the CDR sequence is as shown in SEQ ID NO: 8-10; or iii) The conserved substituted amino acid sequence of i), wherein the CDR sequence is as shown in SEQ ID NO: 8-10.

7. The antibody or a fragment thereof according to claim 1 or 2, wherein the heavy chain variable region comprises: i) The amino acid sequence of SEQ ID NO: 24; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 24, wherein the CDR sequence is as shown in SEQ ID NO: 1, 3, and 7; or iii) The conserved substituted amino acid sequences of i), wherein the CDR sequences are as shown in SEQ ID NO: 1, 3 and 7; Furthermore, the light chain variable region includes: i) The amino acid sequence of SEQ ID NO: 26; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 26, wherein the CDR sequence is as shown in SEQ ID NO: 8-10; or iii) The conserved substituted amino acid sequence of i), wherein the CDR sequence is as shown in SEQ ID NO: 8-10.

8. The antibody or a fragment thereof according to claim 1 or 2, wherein the heavy chain variable region comprises: i) The amino acid sequence of SEQ ID NO: 25; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 25, wherein the CDR sequence is as shown in SEQ ID NO: 1, 6, and 7; or iii) The conserved substituted amino acid sequence of i), wherein the CDR sequence is as shown in SEQ ID NO: 1, 6 and 7; Furthermore, the light chain variable region includes: i) The amino acid sequence of SEQ ID NO: 26; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 26, wherein the CDR sequence is as shown in SEQ ID NO: 8-10; or iii) The conserved substituted amino acid sequence of i), wherein the CDR sequence is as shown in SEQ ID NO: 8-10.

9. The antibody or a fragment thereof according to claim 1 or 2, wherein the heavy chain variable region comprises: i) The amino acid sequence of SEQ ID NO: 20; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 20, wherein the CDR sequence is as shown in SEQ ID NO: 1, 3, and 7; or iii) The conserved substituted amino acid sequences of i), wherein the CDR sequences are as shown in SEQ ID NO: 1, 3 and 7; Furthermore, the light chain variable region includes: i) The amino acid sequence of SEQ ID NO: 27; ii) An amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 27, wherein the CDR sequence is as shown in SEQ ID NO: 8-10; or iii) The conserved substituted amino acid sequence of i), wherein the CDR sequence is as shown in SEQ ID NO: 8-10.

10. The antibody or a fragment thereof according to claim 1 or 2, wherein the heavy chain variable region comprises: i) The amino acid sequence of SEQ ID NO: 21; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 21, wherein the CDR sequence is as shown in SEQ ID NO: 2, 4, and 7; or iii) The conserved substituted amino acid sequences of i), wherein the CDR sequences are as shown in SEQ ID NO: 2, 4 and 7; Furthermore, the light chain variable region includes: i) The amino acid sequence of SEQ ID NO: 27; ii) An amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 27, wherein the CDR sequence is as shown in SEQ ID NO: 8-10; or iii) The conserved substituted amino acid sequence of i), wherein the CDR sequence is as shown in SEQ ID NO: 8-10.

11. The antibody or a fragment thereof according to claim 1 or 2, wherein the heavy chain variable region comprises: i) The amino acid sequence of SEQ ID NO: 23; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 23, wherein the CDR sequence is as shown in SEQ ID NO: 1, 5, and 7; or iii) The conserved substituted amino acid sequence of i), wherein the CDR sequence is as shown in SEQ ID NO: 1, 5 and 7; Furthermore, the light chain variable region includes: i) The amino acid sequence of SEQ ID NO: 27; ii) An amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 27, wherein the CDR sequence is as shown in SEQ ID NO: 8-10; or iii) The conserved substituted amino acid sequence of i), wherein the CDR sequence is as shown in SEQ ID NO: 8-10.

12. The antibody or a fragment thereof according to claim 1 or 2, wherein the heavy chain variable region comprises: i) The amino acid sequence of SEQ ID NO: 24; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 24, wherein the CDR sequence is as shown in SEQ ID NO: 1, 3, and 7; or iii) The conserved substituted amino acid sequences of i), wherein the CDR sequences are as shown in SEQ ID NO: 1, 3 and 7; Furthermore, the light chain variable region includes: i) The amino acid sequence of SEQ ID NO: 27; ii) An amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 27, wherein the CDR sequence is as shown in SEQ ID NO: 8-10; or iii) The conserved substituted amino acid sequence of i), wherein the CDR sequence is as shown in SEQ ID NO: 8-10.

13. The antibody or a fragment thereof according to claim 1 or 2, wherein the heavy chain variable region comprises: i) The amino acid sequence of SEQ ID NO: 25; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 25, wherein the CDR sequence is as shown in SEQ ID NO: 1, 6, and 7; or iii) The conserved substituted amino acid sequence of i), wherein the CDR sequence is as shown in SEQ ID NO: 1, 6 and 7; Furthermore, the light chain variable region includes: i) The amino acid sequence of SEQ ID NO: 27; ii) An amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 27, wherein the CDR sequence is as shown in SEQ ID NO: 8-10; or iii) The conserved substituted amino acid sequence of i), wherein the CDR sequence is as shown in SEQ ID NO: 8-10.

14. The antibody or a fragment thereof according to claim 1 or 2, wherein the heavy chain variable region comprises: i) The amino acid sequence of SEQ ID NO: 20; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 20, wherein the CDR sequence is as shown in SEQ ID NO: 1, 3, and 7; or iii) The conserved substituted amino acid sequences of i), wherein the CDR sequences are as shown in SEQ ID NO: 1, 3 and 7; Furthermore, the light chain variable region includes: i) The amino acid sequence of SEQ ID NO: 28; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 28, wherein the CDR sequence is as shown in SEQ ID NO: 8-10; or iii) The conserved substituted amino acid sequence of i), wherein the CDR sequence is as shown in SEQ ID NO: 8-10.

15. The antibody or fragment thereof according to claim 1 or 2, wherein, The heavy chain variable region includes: i) The amino acid sequence of SEQ ID NO: 21; ii) An amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 21, wherein the CDR sequence is as shown in SEQ ID NO: 2, 4, and 7; or iii) The conserved substituted amino acid sequences of i), wherein the CDR sequences are as shown in SEQ ID NO: 2, 4 and 7; Furthermore, the light chain variable region comprises: i) The amino acid sequence of SEQ ID NO: 28; ii) An amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 28, wherein the CDR sequence is as shown in SEQ ID NO: 8-10; or iii) The conserved substituted amino acid sequence of i), wherein the CDR sequence is as shown in SEQ ID NO: 8-10.

16. The antibody or a fragment thereof according to claim 1 or 2, wherein the heavy chain variable region comprises: i) The amino acid sequence of SEQ ID NO: 23; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 23, wherein the CDR sequence is as shown in SEQ ID NO: 1, 5, and 7; or iii) The conserved substituted amino acid sequence of i), wherein the CDR sequence is as shown in SEQ ID NO: 1, 5 and 7; Furthermore, the light chain variable region comprises: i) The amino acid sequence of SEQ ID NO: 28; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 28, wherein the CDR sequence is as shown in SEQ ID NO: 8-10; or iii) The conserved substituted amino acid sequence of i), wherein the CDR sequence is as shown in SEQ ID NO: 8-10.

17. The antibody or a fragment thereof according to claim 1 or 2, wherein the heavy chain variable region comprises: i) The amino acid sequence of SEQ ID NO: 24; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 24, wherein the CDR sequence is as shown in SEQ ID NO: 1, 3, and 7; or iii) The conserved substituted amino acid sequences of i), wherein the CDR sequences are as shown in SEQ ID NO: 1, 3 and 7; Furthermore, the light chain variable region includes: i) The amino acid sequence of SEQ ID NO: 28; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 28, wherein the CDR sequence is as shown in SEQ ID NO: 8-10; or iii) The conserved substituted amino acid sequence of i), wherein the CDR sequence is as shown in SEQ ID NO: 8-10.

18. The antibody or a fragment thereof according to claim 1 or 2, wherein the heavy chain variable region comprises: i) The amino acid sequence of SEQ ID NO: 25; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 25, wherein the CDR sequence is as shown in SEQ ID NO: 1, 6, and 7; or iii) The conserved substituted amino acid sequence of i), wherein the CDR sequence is as shown in SEQ ID NO: 1, 6 and 7; Furthermore, the light chain variable region includes: i) The amino acid sequence of SEQ ID NO: 28; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 28, wherein the CDR sequence is as shown in SEQ ID NO: 8-10; or iii) The conserved substituted amino acid sequence of i), wherein the CDR sequence is as shown in SEQ ID NO: 8-10.

19. The antibody or a fragment thereof according to claim 1 or 2, wherein the heavy chain variable region comprises: i) The amino acid sequence of SEQ ID NO: 73; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 73, wherein the CDR sequence is as shown in SEQ ID NO: 2, 4, and 7; or iii) The conserved substituted amino acid sequences of i), wherein the CDR sequences are as shown in SEQ ID NO: 2, 4 and 7; Furthermore, the light chain variable region includes: i) The amino acid sequence of SEQ ID NO: 26, 27 or 28; ii) an amino acid sequence having at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 26, 27, or 28, wherein the CDR sequence is as shown in SEQ ID NO: 8-10; or iii) The conserved substituted amino acid sequence of i), wherein the CDR sequence is as shown in SEQ ID NO: 8-10.

20. The antibody or fragment thereof according to claim 1 or 2, wherein the heavy chain variable region comprises SEQ ID NO: 20, 21, 23, 24 or 25, and the light chain variable region comprises SEQ ID NO:

26.

21. The antibody or fragment thereof according to any one of claims 1 to 20, wherein, The variable region of the heavy chain is contained in the heavy chain and / or the variable region of the light chain is contained in the light chain; optionally, the heavy chain further contains the amino acid sequence of SEQ ID NO: 62 and / or 63, and / or the light chain further contains the amino acid sequence of SEQ ID NO: 64 and / or 65.

22. An immunoconjugate comprising the antibody or a fragment thereof as described in any one of claims 1 to 21 and a detectable marker.

23. A nucleic acid encoding an antibody or a fragment thereof, an antibody heavy chain variable domain, or an antibody light chain variable domain as described in any one of claims 1 to 22.

24. The nucleic acid according to claim 23, wherein the nucleic acid: i) Encoding a heavy chain variable domain, wherein the nucleic acid encoding the heavy chain variable domain comprises any one of SEQ ID NO: 11, 12, 14, 15, 16, 66 or 67, or a sequence having at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with any one of SEQ ID NO: 11, 12, 14, 15, 16, 66 or 67; ii) Encoding a light chain variable domain, wherein the nucleic acid encoding the light chain variable domain comprises any one of the sequences in SEQ ID NO: 17, 18, or 19, or a sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any one of SEQ ID NO: 17, 18, or 19; or iii) Encoding a heavy chain variable domain and a light chain variable domain, wherein the nucleic acids encoding the heavy chain variable domain and the light chain variable domain respectively comprise the following sequences: SEQ ID NO: 11 and 17; SEQ ID NO: 12 and 17; SEQ ID NO: 14 and 17; SEQ ID NO: 15 and 17; SEQ ID NO: 16 and 17; SEQ ID NO: 12 and 18; SEQ ID NO: 16 and 18; SEQ ID NO: 11 and 19; SEQ ID NO: 12 and 19; SEQ ID NO: 14 and 19; SEQ ID NO: 15 and 19; or SEQ ID NO: 16 and 19; or with SEQ ID NO: 11 and 17; SEQ ID NO: 12 and 17; SEQ ID NO: 14 and 17; SEQ ID NO: 15 and 17; SEQ ID NO: 16 and 17; SEQ ID NO: 12 and 18; SEQ ID NO: 16 and 18; SEQ ID NO: 11 and 19; SEQ ID NO: 12 and 19; SEQ ID NO: 14 and 19; SEQ ID NO: 15 and 19; SEQ ID NO: 16 and 19; or SEQ ID NO: 66 and 67 sequences having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity; optionally, said nucleic acid further includes one or more nucleotide sequences encoding one or more amino acid sequences of SEQ ID NO: 62-65.

25. A vector comprising the nucleic acid of claim 23 or 24.

26. The vector according to claim 25, wherein the vector is a viral vector; optionally, it is an adenovirus vector, an adenovirus-associated vector, or a retroviral vector, preferably a lentiviral vector.

27. A cell expressing the antibody or a fragment thereof as described in any one of claims 1 to 21, or the nucleic acid as described in claim 23 or 24, or including the vector as described in claim 25 or 26.

28. The cell of claim 27, wherein the cell is selected from mammalian cells; optionally CHO cells or HEK-293 cells; or insect cells; optionally Sf9 cells, Sf21 cells, Tni cells or S2 cells.

29. A composition comprising an antibody or fragment thereof as described in any one of claims 1 to 21, an immunoconjugate as described in claim 22, a nucleic acid as described in claim 23 or 24, a vector as described in claim 25 or 26, or a cell as described in claim 27 or 28; optionally further comprising a diluent.

30. The antibody or fragment thereof of any one of claims 1 to 21, the immunoconjugate of claim 22, the nucleic acid of claim 23 or 24, or the vector of claim 25 or 26, for preparing a medicament for treating hypercalcemia or cancer in a subject with such need.

31. A kit comprising an antibody or fragment thereof as described in any one of claims 1 to 21, an immunoconjugate as described in claim 22, a nucleic acid as described in claim 23 or 24, a vector as described in claim 25 or 26, a cell as described in claim 27 or 28, or a composition as described in claim 29.

32. Use of an antibody or fragment thereof that specifically binds to human PTHrP for reducing or inhibiting the activity of human PTHrP, comprising contacting a cell or tissue expressing human PTHrP with the antibody or fragment thereof, wherein the antibody or fragment thereof comprises a light chain variable region and a heavy chain variable region, the heavy chain variable region comprising complementation-determining regions CDR-H1, CDR-H2, and CDR-H3, the light chain variable region comprising complementation-determining regions CDR-L1, CDR-L2, and CDR-L3, wherein the amino acid sequence of the CDR comprises the following sequence: Optionally, the antibody or fragment thereof is the antibody or fragment thereof according to any one of claims 1 to 21.

33. The use according to claim 32, wherein the cell or tissue is located in the subject's body, and / or wherein the contact comprises administering the antibody or a fragment thereof to a subject who requires it.

34. Use of an effective amount of the antibody or fragment thereof of any one of claims 1 to 21, or a composition comprising the antibody or fragment thereof, the immunoconjugate of claim 22, the nucleic acid of claim 23 or 24, or the vector of claim 25 or 26, for the treatment of a subject in need of such treatment.

35. The use according to claim 34, wherein the subject has been diagnosed with cancer, such as breast cancer, lung cancer, colon cancer, pancreatic cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, or melanoma.

36. The use according to claim 35, wherein the cancer is a pre-metastatic cancer.

37. The use according to claim 34, wherein the use is for treating hypercalcemia.

38. The use according to claim 37, wherein the subject has been diagnosed with hypercalcemia, optionally hypercalcemia associated with malignancy.

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