Antibodies for parathyroid hormone related protein and methods related thereto

Abstract

The disclosure pertains to antibodies that specifically bind to Parathyroid Hormone Related Protein (PTHrP). Also provided are immunoconjugates, nucleic acids, vectors, cells and compositions comprising the antibodies. The antibodies, immunoconjugates, nucleic acids, vectors, cells and compositions can be used in the diagnosis and treatment of disease, particularly cancer and / or hypercalcemia.

Description

Antibodies for parathyroid hormone related protein and methods related theretoRelated application

[0001] This application claims benefit of United States Provisional Patent Application serial no. 63 / 542,496 filed October 4, 2023, incorporated herein by reference in its entirety.Incorporation of sequence listing

[0002] A computer readable form of the Sequence Listing “31165- P72536PC00_SequenceListing.xml” (99,090 bytes), created on October 4, 2024, is herein incorporated by reference.Field

[0003] The present disclosure relates to antibodies to Parathyroid Hormone Related Protein (PTHrP) and more particularly to antibodies that are specific for PTHrP isoform 1-173, as well as methods of diagnosis and treatment of disease, particularly cancer.Background

[0004] PTHrP has been implicated in hypercalcemia associated with cancers including breast, colon, skin, renal and lung cancers and hematological malignancies such as lymphomas, leukemias and multiple myelomas, as well as in growth, invasion and migration in breast, prostate, pancreatic and melanoma tumour cells.

[0005] Antibodies to PTHrP and to specific PTHrP isoforms are described in U.S. Patent Nos. 7,897,139, 8,501 ,929 and 10,463,726.

[0006] Antibodies that selectively bind PTHrP and that can be used to therapeutically inhibit PTHrP and to specific PTHrP isoforms are desirable.Summary

[0007] The inventors have identified antibodies that specifically bind the PTHrP isoform PTHrPI- 173 and which exhibit desirable properties. Other aspects described herein include immunoconjugates comprising the antibodies, nucleic acids encoding the antibodies, vectors comprising the nucleic acids, cells expressing the antibodies and compositions comprising the antibodies. Methods of use are also herein described.

[0008] Accordingly, in an aspect, the present disclosure provides an antibody or fragment thereof that specifically binds PTHrP, comprising a heavy chain variable region and a light 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, wherein amino acid sequences of said CDRs comprise the sequences:CDR-H1 : SYWXiQ, wherein: SEQ ID NO: 29,Xi = I or MCDR-H2: wherein:AIYPGDGDTSYX2X3X4FX5G, X2= A, S or T SEQ ID NO: 30,X3 = E, P or QX4= K or SX5= K or QCDR-H3: GDYDYDERAMDY, SEQ ID NO: 7,CDR-L1: RSSQSIGHSNGNTYLE, SEQ ID NO: 8,CDR-L2: KVSNRFS, SEQ ID NO: 9, andCDR-L3: FQGSHDPFT, SEQ ID NO: 10.

[0009] In an embodiment, the amino acid sequences of the CDRs of the antibody or fragment comprise the sequences:CDR-H1: SYWMQ, SEQ ID NO: 1 ,CDR-H2: AIYPGDGDTSYTQKFKG, SEQ ID NO: 3,CDR-H3: GDYDYDERAMDY, SEQ ID NO: 7,CDR-L1 : RSSQSIGHSNGNTYLE, SEQ ID NO: 8,CDR-L2: KVSNRFS, SEQ ID NO: 9, andCDR-L3: FQGSHDPFT, SEQ ID NO: 10CDR-H1: SYWIQ, SEQ ID NO: 2,CDR-H2: AIYPGDGDTSYSPSFQG, SEQ ID NO: 4,CDR-H3: GDYDYDERAMDY, SEQ ID NO: 7,CDR-L1 : RSSQSIGHSNGNTYLE, SEQ ID NO: 8,CDR-L2: KVSNRFS, SEQ ID NO: 9, andCDR-L3: FQGSHDPFT, SEQ ID NO: 10CDR-H1: SYWMQ, SEQ ID NO: 1 ,CDR-H2: AIYPGDGDTSYAQKFQG, SEQ ID NO: 5,CDR-H3: GDYDYDERAMDY, SEQ ID NO: 7,CDR-L1 : RSSQSIGHSNGNTYLE, SEQ ID NO: 8,CDR-L2: KVSNRFS, SEQ ID NO: 9, andCDR-L3: FQGSHDPFT, SEQ ID NO: 10 ; orCDR-H1: SYWMQ, SEQ ID NO: 1 ,CDR-H2: AIYPGDGDTSYAEKFQG, SEQ ID NO: 6,CDR-H3: GDYDYDERAMDY, SEQ ID NO: 7,CDR-L1 : RSSQSIGHSNGNTYLE, SEQ ID NO: 8,CDR-L2: KVSNRFS, SEQ ID NO: 9, andCDR-L3: FQGSHDPFT, SEQ ID NO: 10

[0010] In an embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 20, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 20, wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 3 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 3 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 26, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 26, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0011] In an embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 21 , ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 21 , wherein the CDR sequences are as set forth in SEQ ID NOs: 2, 4 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 2, 4 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 26, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 26, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) a conservatively substituted amino acid sequence of i), wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0012] In an embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 22, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 22, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 3 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 3 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 26, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 26, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0013] In an embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 23, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 23, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 5 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forthin SEQ ID NOs: 1 , 5 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 26, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 26, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0014] In an embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 24, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 24, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 3 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 3 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 26, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 26, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0015] In an embodiment, the heavy chain variable region comprises: i) an amino acid sequence as set forth in SEQ ID NO: 25, ii) the amino acid sequence of 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 25, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 6 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 6 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 26, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 26, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0016] In an embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 20, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 20, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 3 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 3 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 27, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 27, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0017] In an embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 21 , ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 21 , wherein the CDR sequences are as set forth in SEQ ID NOs: 2, 4 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 2, 4 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 27, ii) an amino acid sequence with at least 80%, at least 90%, or at least95% sequence identity to SEQ ID NO: 27, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or Hi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0018] In an embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 23, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 23, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 5 and 7, or Hi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 5 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 27, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 27, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or Hi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0019] In an embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 24, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 24, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 3 and 7, or Hi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 3 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 27, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 27, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or Hi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0020] In an embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 25, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 25, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 6 and 7, or Hi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 6 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 27, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 27, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or Hi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0021] In an embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 20, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 20, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 3 and 7, or Hi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 3 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 28, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 28, wherein the CDR sequences are as set forth in SEQ ID NOs:8-10, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0022] In an embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 21 , ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 21 , wherein the CDR sequences are as set forth in SEQ ID NOs: 2, 4 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 2, 4 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 28, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 28, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0023] In an embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 23, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 23, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 5 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 5 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 28, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 28, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0024] In an embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 24, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 24, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 3 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 3 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 28, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 28, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0025] In an embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 25, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 25, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 6 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 6 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 28, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 28, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0026] In an embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 73, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 73, wherein the CDR sequences are as set forth in SEQ ID NOs: 2, 4 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 2, 4 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 26, 27 or 28, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 26, 27 or 28, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0027] In an 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.

[0028] In an embodiment, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 20 and the light chain variable region comprise the amino acid sequence of SEQ ID NO: 26.

[0029] In an embodiment, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 21 and the light chain variable region comprise the amino acid sequence of SEQ ID NO: 26.

[0030] In an embodiment, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 23 and the light chain variable region comprise the amino acid sequence of SEQ ID NO: 26.

[0031] In an embodiment, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 24 and the light chain variable region comprise the amino acid sequence of SEQ ID NO: 26.

[0032] In an embodiment, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 25 and the light chain variable region comprise the amino acid sequence of SEQ ID NO: 26.

[0033] In an embodiment, the heavy chain variable region is comprised in a heavy chain and / or the light chain variable region is comprised in a light chain, optionally wherein the heavy chain further comprises the amino acid sequence of SEQ ID NO: 62 and / or 63 and / or wherein the light chain further comprises the amino acid sequence of SEQ ID NO: 64 and / or 65.

[0034] In an aspect, an immunoconjugate comprises an antibody or fragment as herein described and a detectable label.

[0035] In an aspect, a nucleic acid encodes an antibody heavy chain variable domain, or antibody light chain variable domain of an antibody or fragment as herein described.

[0036] In an embodiment, the nucleic acid i) encodes a heavy chain variable domain, the nucleic acid encoding heavy chain variable domain comprising the sequence of any one of SEQ ID NOs: 11 , 12, 14, 15, or 16, or a sequence with at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to any one of SEQ ID NOs: 11, 12, 14, 15, or 16; ii) encodes a light chain variable domain, the nucleic acid encoding light chain variable domain comprising the sequence of any one of SEQ ID NOs: 17, 18 or 19, or a sequence with at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to any one of SEQ ID NOs: 17, 18 or 19; or iii) encodes a heavy chain variable domain and a light chain variable domain, the nucleic acid encoding the heavy chain variable domain and light chain variable domain, respectively, comprising the sequences: SEQ ID NOs: 11 and 17; SEQ ID NOs: 12 and 17; SEQ ID NOs: 14 and 17; SEQ ID NOs: 15 and 17, SEQ ID NOs: 16 and 17, SEQ ID NOs: 12 and 18, SEQ ID NOs: 16 and 18, SEQ ID NOs: 11 and 19, SEQ ID NOs: 12 and 19, SEQ ID NOs: 14 and 19, SEQ ID NOs: 15 and 19, or SEQ ID NOs: 16 and 19, or sequences with at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NOs: 11 and 17; SEQ ID NOs: 12 and 17; SEQ ID NOs:14 and 17; SEQ ID NOs: 15 and 17, SEQ ID NOs: 16 and 17, SEQ ID NOs: 12 and 18, SEQ ID NOs:16 and 18, SEQ ID NOs: 11 and 19, SEQ ID NOs: 12 and 19, SEQ ID NOs: 14 and 19, SEQ ID NOs:15 and 19, or SEQ ID NOs: 16 and 19, optionally, wherein the nucleic acid further comprises one or more nucleotide sequences encoding one or more of the amino acid sequences of SEQ ID NOs: 62-65.

[0037] In an aspect, a vector comprises a nucleic acid as herein described.

[0038] In an embodiment, the vector is a viral vector, optionally, an adenoviral vector, an adenoviral-associated vector, or a retroviral vector, optionally a lentiviral vector.

[0039] In an aspect, a cell expresses an antibody or fragment, or a nucleic acid as herein described, or comprises a vector as herein described.

[0040] In an embodiment, the cell is selected from a mammalian cell, optionally a CHO cell or a HEK-293 cell, or an insect cell, optionally a Sf9 cell, Sf21 cell, Tni cell, or S2 cell.

[0041] In an aspect, a composition comprises an antibody or fragment, immunoconjugate, nucleic acid, vector, or a cell as herein described, optionally further comprising a diluent.

[0042] In an aspect, an antibody or fragment, an immunoconjugate, a nucleic acid, or a vector as herein described is for manufacture of a medicament for treating hypercalcemia or cancer in a subject in need thereof.

[0043] In an aspect, an antibody or fragment, an immunoconjugate, a nucleic acid, a vector, a cell or a composition as herein described is comprised in a kit.

[0044] In an aspect, a method of reducing or inhibiting activity of a human PTHrP as herein described comprises contacting a cell or tissue expressing the human PTHrP with an antibody or fragment thereof that specifically binds the human PTHrP, wherein the antibody or fragment thereof comprises a light chain variable region and a heavy chain variable region, the heavy chain variableregion 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, wherein amino acid sequences of said CDRs comprise the sequences:CDR-H1 : SYWXiQ, wherein: SEQ ID NO: 29,Xi = I or MCDR-H2: wherein:AIYPGDGDTSYX2X3X4FX5G, X2= A, S or T SEQ ID NO: 30,X3= E, P or QX4= K or SX5= K or QCDR-H3: GDYDYDERAMDY, SEQ ID NO: 7,CDR-L1: RSSQSIGHSNGNTYLE, SEQ ID NO: 8,CDR-L2: KVSNRFS, SEQ ID NO: 9, andCDR-L3: FQGSHDPFT, SEQ ID NO: 10, optionally wherein the antibody or fragment thereof is an antibody or fragment as herein described.

[0045] In an aspect, a use of an antibody or fragment thereof that specifically binds the human PTHrP, for reducing or inhibiting activity of a human PTHrP, as herein described comprises contacting a cell or tissue expressing the human PTHrP wherein the antibody or fragment thereof comprises 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, wherein amino acid sequences of said CDRs comprise the sequences:CDR-H1 : SYWX1Q, wherein: SEQ ID NO: 29,Xi = I or MCDR-H2: wherein:AIYPGDGDTSYX2X3X4FX5G, Xv2 > - A A, s or T l SEQ ID NO: 30,Xs = E, P or QX4= K or SX5= K or QCDR-H3: GDYDYDERAMDY, SEQ ID NO: 7,CDR-L1: RSSQSIGHSNGNTYLE, SEQ ID NO: 8,CDR-L2: KVSNRFS, SEQ ID NO: 9, andCDR-L3: FQGSHDPFT, SEQ ID NO: 10, optionally wherein the antibody or fragment thereof is an antibody or fragment as herein described.

[0046] In an embodiment, the cell or tissue is in vivo in a subject and / or the contacting comprises administering or use of the antibody or fragment thereof to a subject in need thereof.

[0047] In an aspect, a method of treating a subject in need thereof comprises administering to the subject an effective amount of the antibody or fragment or a composition comprising said antibody or fragment, or an immunoconjugate, a nucleic acid, or a vector as herein described.

[0048] In an aspect, a use of an effective amount of the antibody or fragment or a composition comprising said antibody or fragment, or an immunoconjugate, a nucleic acid, or a vector as herein described for treating a subject in need thereof.

[0049] In an embodiment, the subject has been diagnosed with cancer, optionally breast cancer, lung cancer, colon cancer, pancreatic cancer, ovarian cancer, prostate cancer, squamous cell cancer, or melanoma.

[0050] In an embodiment, the cancer is pre-metastatic.

[0051] In an embodiment, the method is for treating hypercalcemia.

[0052] In an embodiment, the subject has been diagnosed with hypercalcemia, optionally malignancy-associated hypercalcemia.

[0053] Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating embodiments of the disclosure are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.Brief description of the drawings

[0054] An embodiment of the present disclosure will now be described in relation to the drawings in which:

[0055] Figure 1A shows the amino acid sequence of the mouse HC104 hybridoma heavychain variable region (VH) (SEQ ID NO: 31 ).

[0056] Figure 1 B shows the amino acid sequence of an antibody of the disclosure (HC104- 146-VHA; SEQ ID NO: 32).

[0057] Figure 1C shows residues that differ between HC104-146-VHA (SEQ ID NO: 32) and IGHV1-46*01 human germline VH (SEQ ID NO: 70) sequences in the frameworks and CDRs.

[0058] Figure 1 D shows amino acid sequences of antibodies of the disclosure (SEQ ID Nos: 33 and 34).

[0059] Figure 1 E shows the amino acid sequence of an antibody of the disclosure (HC104- 146-VHB; SEQ ID NO: 35).

[0060] Figure 1F shows residues that differ between HC104-146-VHB (SEQ ID NO: 35) and IGHV1-46*01 human germline (SEQ ID NO: 70) sequences in the frameworks and CDRs.

[0061] Figure 1G shows the amino acid sequence of an antibody of the disclosure (HC104- 1692-VHA; SEQ ID NO: 36).

[0062] Figure 1 H shows residues that differ between HC104-1692-VHA (SEQ ID NO: 36) and IGHV1-69-2*01 human germline (SEQ ID NO: 71 ) sequences in the frameworks and CDRs.

[0063] Figure 11 shows amino acid sequences of antibodies of the disclosure (SEQ ID NOs: 37 and 38).

[0064] Figure 1J shows the amino acid sequence of an antibody of the disclosure (HC104- 1692-VHB; SEQ ID NO: 39).

[0065] Figure 1 K shows residues that differ between HC104-1692-VHB (SEQ ID NO: 39) and human germline IGHV1-69-2*01 (SEQ ID NO: 71 ) sequences in the frameworks and CDRs.

[0066] Figure 1 L shows the amino acid sequence of an antibody of the disclosure (HC104- 551-VHA; SEQ ID NO: 40).

[0067] Figure 1 M shows residues that differ between HC104-551-VHA (SEQ ID NO: 40) and human germline IGHV5-51*01 (SEQ ID NO: 72) sequences in the frameworks and CDRs.

[0068] Figure 1 N shows amino acid sequences of antibodies of the disclosure (SEQ ID NOs: 41 , 42 and 73).

[0069] Figure 10 shows the amino acid sequence of an antibody of the disclosure (HC104- 551-VHB; SEQ ID NO: 43).

[0070] Figure 1P shows residues that differ between HC104-551-VHB (SEQ ID NO: 43) and human germline IGHV5-51*01 (SEQ ID NO: 72) sequences in the frameworks and CDRs.

[0071] Figure 1Q shows the amino acid sequence of an antibody of the disclosure (HC104- 551-VHC; SEQ ID NO: 44).

[0072] Figure 1 R shows residues that differ between HC104-551-VHC (SEQ ID NO: 44) and human germline IGHV5-51*01 (SEQ ID NO: 72) sequences in the frameworks and CDRs.

[0073] Figure 2A shows the amino acid sequence of the mouse HC104 hybridoma light-chain variable region (VL) (SEQ ID NO: 45).

[0074] Figure 2B shows the amino acid sequence of an antibody of the disclosure (HC104- 320-VLA; SEQ ID NO: 46).

[0075] Figure 20 shows residues that differ between HC104-320-VLA (SEQ ID NO: 46) and human germline IGKV2-30*02 (SEQ ID NO: 74) sequences in the frameworks and CDRs.

[0076] Figure 2D shows amino acid sequences of antibodies of the disclosure (SEQ ID NOs: 47 and 48).

[0077] Figure 2E shows the amino acid sequence of an antibody of the disclosure (HC104- 320-VLB; SEQ ID NO: 49).

[0078] Figure 2F shows residues that differ between HC104-320-VLB (SEQ ID NO: 49) and human germline IGKV2-30*02 (SEQ ID NO: 74) sequences in the frameworks and CDRs.

[0079] Figure 2G shows the amino acid sequence of an antibody of the disclosure (HC104- 218-VLA; SEQ ID NO: 50).

[0080] Figure 2H shows residues that differ between HC104-218-VLA (SEQ ID NO: 50) and human germline IGKV2-18*01 (SEQ ID NO: 75) sequences in the frameworks and CDRs.

[0081] Figure 2I shows amino acid sequences of antibodies of the disclosure (SEQ ID NOs: 51 and 52).

[0082] Figure 2J shows the amino acid sequence of an antibody of the disclosure (HC104- 218-VLB; SEQ ID NO: 53).

[0083] Figure 2K shows residues that differ between HC104-218-VLB (SEQ ID NO: 53) and human germline IGKV2-18*01 (SEQ ID NO: 75) sequences in the frameworks and CDRs.

[0084] Figure 2L shows the amino acid sequence of an antibody of the disclosure (HC104-41- VLA; SEQ ID NOs: 54).

[0085] Figure 2M shows residues that differ between HC104-41-VLA (SEQ ID NO: 54) and human germline IGKV4-1*01 (SEQ ID NO: 76) sequences in the frameworks and CDRs.

[0086] Figure 2N shows amino acid sequences of antibodies of the disclosure (SEQ ID NOs: 55 and 56).

[0087] Figure 20 shows the amino acid sequence of an antibody of the disclosure (HC104- 41-VLB; SEQ ID NO: 57).

[0088] Figure 2P shows residues that differ between HC104-41-VLB (SEQ ID NO: 57) and human germline IGKV4-1*01 (SEQ ID NO: 76) sequences in the frameworks and CDRs.

[0089] Figure 3A and 3B show final sample quality control using Reduced and Non-reduced PAGE analysis and Coomassie blue staining. MW = Molecular weight marker. 2 g samples were loaded per lane.

[0090] Figures 4A to 4T show SEC-HPLC profiles for HC104-551 -VHA-230-VLA (A), HC104- 551-VHB-230-VLA (B), HC 104-146- VHA-230-VLA (C), HC104-146-VHB-230-VLA (D), 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), 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_hlgG1 (Q), HC104_hlgG1 (R), blank control (S), and blank control (T). Approximate MW is as follows: 6.115 min: 670 kDa; 8.213 min: 150 kDa; 9.691 min: 45k Da; 10.624 min: 17 kDa; 12.514 min: 1 kDa.

[0091] Figure 5 shows the thermal stability analysis of five humanize variants and the chimeric antibody. Plots depict the fluorescence ratio (F350 / F330) and the corresponding first derivative.

[0092] Figures 6Ato G show sensorgram profiles for H104-551-VHA-230-VLA (A), H104-551- VHB-230-VLA (B), HC104-146-VHB-230-VLA (C), HC 104-1692- VHA-230-VLA (D), HC104-1692- VHB- 230-VLA (E), HC104-chimeric (F) and mouse104 from 20846 (G).Detailed Description of the Disclosure

[0093] The inventors have identified antibodies that specifically bind the PTHrP isoform PTHrPI- 173.I. Definitions

[0094] As used herein, the term “amino acid” includes all of the naturally occurring amino acids as well as modified L-amino acids. The atoms of the amino acid can, for example, include different isotopes. For example, the amino acids can comprise deuterium substituted for hydrogen, nitrogen-15 substituted for nitrogen-14, and carbon-13 substituted for carbon-12 and other similar changes.

[0095] As used herein, “conservative amino acid substitution” means one in which one amino acid residue is replaced with another amino acid residue without abolishing the protein's desired properties. Suitable conservative amino acid substitutions can be made by substituting amino acids with similar hydrophobicity, polarity, and R-group size for one another. Examples of conservative amino acid substitution include:

[0096] 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 percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical overlapping positions / total number of positions times'! 00%). In one embodiment, the two sequences are the same length. The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. A, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264- 2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, word length=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the present application. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score-50, word length=3 to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., the NCBI website). Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0097] The sequence identity to a reference sequence can be any % between 70-99%, or any number falling within the range of 70-99%, for example, 81%, 82%, 83%, 84% and 85%, or 81.1%, 81.2%, 81.3%, 81.4% and 81.5%, etc.

[0098] For antibodies, percentage sequence identities can be determined when antibody sequences maximally aligned by IgBlast, tool or other (e.g. IMGT, Kabat numbering convention). After alignment, if a subject antibody region (e.g., the entire mature variable region of a heavy or light chain) is being compared with the same region of a reference antibody, the percentage sequence identity between the subject and reference antibody regions is the number of positions occupied by the same amino acid in both the subject and reference antibody region divided by the total number of aligned positions of the two regions, with gaps not counted, multiplied by 100 to convert to percentage.

[0099] As used herein, the term “antibody” includes monoclonal antibodies, polyclonal antibodies, single chain, humanized and other chimeric antibodies, or fully human antibodies, as well as binding fragments thereof. Also included are vectorized antibodies or intrabodies. The antibody may be from recombinant sources and / or produced in transgenic animals. Also included are human antibodies that can be produced through using biochemical techniques or isolated from a library. Humanized or chimeric antibody may include sequences from one or more than one isotype or class.

[0100] As used herein, the term “fragment” refers to a part or portion of an antibody or antibody chain comprising fewer amino acid residues than an intact or complete antibody or antibody chain and which binds the antigen or competes with intact antibody. Exemplary fragments include without limitations Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, and multimers thereof. Fragments can be obtained via chemical or enzymatic treatment of an intact or complete antibody or antibody chain. Fragments can also be obtained by recombinant means (vide infra). For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be constructed by recombinant expression techniques.

[0101] The term “complementarity determining region” or “CDR” as used herein refers to particular hypervariable regions of antibodies that are commonly presumed to contribute to epitope binding. Computational methods for identifying CDR sequences include Kabat, Chothia, IgBlast and IMGT. The CDRs listed in the present disclosure are defined using the Kabat nomenclature. A person skilled in the art having regard to the sequences comprised herein would also be able to identify CDR sequences based on Kabat.

[0102] The term “PTHrP” refers to Parathyroid Hormone Related Protein, for example, human PTHrP as provided in Uniprot accession number P12272, and includes all naturally occurring variants thereof, including isoforms 1-173, 1-139 and 1-141. PTHrP is also commonly referred to as ParathyroidHormone Like Hormone (PTHLH) or Parathyroid Hormone Like Protein (PLP). PTHrP, PTHLH and PLP would commonly be understood to be interchangeable terms.

[0103] The term “detectable label” as used herein refers to moieties such as peptide sequences, fluorescent proteins that can be appended or introduced into a peptide, antibody or other compound described herein and which is capable of producing, either directly or indirectly, a detectable signal. For example, the label may be radio-opaque, positron-emitting radionuclide (for example for use in PET imaging), or a radioisotope, such as3H,13N,14C,18F,32P,35S,123l,125l,1311; a fluorescent (fluorophore e.g. Alexa Fluor™) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine or luciferin; an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase; an imaging agent; or a metal ion. The detectable label may be also detectable indirectly for example using secondary antibody.

[0104] As used herein, the term “nucleic acid sequence” refers to a sequence of nucleoside or nucleotide monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present application may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The sequences may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil, and xanthine and hypoxanthine. The nucleic acid can be either double stranded or single stranded and represents the sense or antisense strand. Further, the term "nucleic acid" includes the complementary nucleic acid sequences as well as codon optimized or synonymous codon equivalents. The nucleic acid may be a mRNA.

[0105] As used herein, the term “vector” comprises any intermediary vehicle for a nucleic acid molecule which enables said nucleic acid molecule, for example, to be introduced into prokaryotic and / or eukaryotic cells and / or integrated into a genome, and include plasmids, phagemids, bacteriophages or viral vectors such as retroviral based vectors, Adeno Associated viral vectors and the like. The term “plasmid” as used herein generally refers to a construct of extrachromosomal genetic material, usually a circular DNA duplex, which can replicate independently of chromosomal DNA.

[0106] As used herein, “specifically binds” in reference to an antibody means that the antibody recognizes its target antigen and binds its target with greater affinity than it does to a structurally different antigen and / or to an antigen with modified or mutated sequence. For example, a multivalent antibody binds its target with KD of at least 1e-6, at least 1e-7, at least 1e-8, at least 1e-9 or at least 1e-10. Affinities greater than at least 1e-8 are preferred. An antigen binding fragment such as Fab fragment comprising one variable domain, may find its target with a 10 fold or 100 fold less affinity than a multivalent interaction with a non-fragmented antibody.

[0107] As used herein, “specifically binds” in the context of PTHrP means that the antibody or fragment thereof binds an epitope in the sequence of PTHrP isoform 1-173 (“PTHrP1-173”) or any fragment thereof comprising a C-terminus portion of PTHrP1-173, e.g., amino acids 140-173.

[0108] As used herein, the term “selective” or “preferential” with respect to an antibody refers to an antibody that selectively / preferentially binds a form of PTHrP with at least 3 fold, or at least 5 fold, at least 10 fold, at least 20 fold, at least 100 fold, at least 250 fold, or at least 500 fold or more greater affinity.

[0109] As used herein, the term “animal” or "subject" includes all members of the animal kingdom including mammals, optionally including or excluding humans.

[0110] As used herein, the term “treating" or “treatment” is well understood in the art and refers to an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable. “Treating” and “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Subjects can be treated with an antibody or fragment thereof, immunoconjugate, or composition described herein to prevent progression.

[0111] In understanding the scope of the present disclosure, the term “consisting” and its derivatives, as used herein, are intended to be close ended terms that specify the presence of stated features, elements, components, groups, integers, and / or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and / or steps.

[0112] The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1 , 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about." Further, it is to be understood that "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. The term “about” means plus or minus 0.1 to 50%, 5-50%, or 10-40%, optionally 10- 20%, further optionally 10% or 15%, of the number to which reference is being made.

[0113] Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. For example, in the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being optional or advantageous may be combined with any other feature or features indicated as being optional or advantageous.II. Antibodies, Immunoconjuqates, Nucleic Acids, Vectors and Cells

[0114] The present disclosure identifies humanized antibodies or fragments thereof specific for the C-terminal end of PTHrP and specifically for the PTHrP1-173 isoform. As demonstrated herein, the humanized antibodies exhibit desirable properties.

[0115] Accordingly, in an aspect, the present disclosure provides an antibody or fragment thereof that specifically binds PTHrP and comprises 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 an embodiment, the amino acid sequences of the CDRs comprise:CDR-H1 : SYWXiQ, wherein: SEQ ID NO: 29,Xi = I or MCDR-H2: wherein:AIYPGDGDTSYX2X3X4FX5G, X2= A, S or T SEQ ID NO: 30,Xs = E, P or QX4= K or SX5= K or QCDR-H3: GDYDYDERAMDY, SEQ ID NO: 7,CDR-L1: RSSQSIGHSNGNTYLE, SEQ ID NO: 8,CDR-L2: KVSNRFS, SEQ ID NO: 9, andCDR-L3: FQGSHDPFT, SEQ ID NO: 10.

[0116] In an embodiment, amino acid sequences of the CDRs comprise one or more of the following sequence combinations:CDR-H1: SYWMQ, SEQ ID NO: 1 ,CDR-H2: AIYPGDGDTSYTQKFKG, SEQ ID NO: 3,CDR-H3: GDYDYDERAMDY, SEQ ID NO: 7,CDR-L1 : RSSQSIGHSNGNTYLE, SEQ ID NO: 8,CDR-L2: KVSNRFS, SEQ ID NO: 9, andCDR-L3: FQGSHDPFT, SEQ ID NO: 10CDR-H1: SYWIQ, SEQ ID NO: 2,CDR-H2: AIYPGDGDTSYSPSFQG, SEQ ID NO: 4,CDR-H3: GDYDYDERAMDY, SEQ ID NO: 7,CDR-L1 : RSSQSIGHSNGNTYLE, SEQ ID NO: 8,CDR-L2: KVSNRFS, SEQ ID NO: 9, andCDR-L3: FQGSHDPFT, SEQ ID NO: 10CDR-H1: SYWMQ, SEQ ID NO: 1 ,CDR-H2: AIYPGDGDTSYAQKFQG, SEQ ID NO: 5,CDR-H3: GDYDYDERAMDY, SEQ ID NO: 7,CDR-L1 : RSSQSIGHSNGNTYLE, SEQ ID NO: 8,CDR-L2: KVSNRFS, SEQ ID NO: 9, andCDR-L3: FQGSHDPFT, SEQ ID NO: 10 orCDR-H1: SYWMQ, SEQ ID NO: 1 ,CDR-H2: AIYPGDGDTSYAEKFQG, SEQ ID NO: 6,CDR-H3: GDYDYDERAMDY, SEQ ID NO: 7,CDR-L1 : RSSQSIGHSNGNTYLE, SEQ ID NO: 8,CDR-L2: KVSNRFS, SEQ ID NO: 9, andCDR-L3: FQGSHDPFT, SEQ ID NO: 10

[0117] In an embodiment, the amino acid sequences of the CDRs comprise one or more of the sequences shown in Tables 3 or 4 below.

[0118] In an embodiment, the sequences of the heavy chain and light chain variable regions comprise one or more of the sequences shown in Tables 7 or 8 below.

[0119] In an embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 20, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 20, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 3 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 3 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 26, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 26, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0120] In an embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 21 , ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 21 , wherein the CDR sequences are as set forth in SEQ ID NOs: 2, 4 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 2, 4 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 26, ii) an amino acid sequence with at least 80%, at least 90%, or at least95% sequence identity to SEQ ID NO: 26, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) a conservatively substituted amino acid sequence of i), wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0121] In an embodiment, the antibody or fragment comprises a heavy chain variable region comprising: i) an amino acid sequence as set forth in SEQ ID NO: 73, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 73, wherein the CDR sequences are as set forth in SEQ ID NOs: 2, 4 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 2, 4 and 7, and wherein the antibody comprises a light chain variable region comprising i) an amino acid sequence as set forth in SEQ ID NO: 26, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 26, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0122] In an embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 22, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 22, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 3 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 3 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 26, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 26, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0123] In an embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 23, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 23, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 5 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 5 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 26, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 26, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0124] In an embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 24, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 24, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 3 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 3 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 26, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 26, wherein the CDR sequences are as set forth in SEQ ID NOs:8-10, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0125] In an embodiment, the heavy chain variable region comprises: i) an amino acid sequence as set forth in SEQ ID NO: 25, ii) the amino acid sequence of 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 25, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 6 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 6 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 26, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 26, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0126] In an embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 20, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 20, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 3 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 3 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 27, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 27, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0127] In an embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 21 , ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 21 , wherein the CDR sequences are as set forth in SEQ ID NOs: 2, 4 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 2, 4 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 27, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 27, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10).

[0128] In an embodiment, the antibody or fragment comprises a heavy chain variable region comprising: i) an amino acid sequence as set forth in SEQ ID NO: 73, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 73, wherein the CDR sequences are as set forth in SEQ ID NOs: 2, 4 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 2, 4 and 7,, and wherein the antibody comprises a light chain variable region comprising i) an amino acid sequence as set forth in SEQ ID NO: 27, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 27, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) aconservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0129] In an embodiment, the antibody or fragment comprises a heavy chain variable region comprising: i) an amino acid sequence as set forth in SEQ ID NO: 22, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 22, wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 3 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 3 and 7, and wherein the antibody comprises a light chain variable region comprising i) an amino acid sequence as set forth in SEQ ID NO: 27, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 27, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0130] In an embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 23, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 23, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 5 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 5 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 27, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 27, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0131] In an embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 24, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 24, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 3 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 3 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 27, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 27, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0132] In an embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 25, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 25, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 6 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 6 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 27, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 27, wherein the CDR sequences are as set forth in SEQ ID NOs:8-10, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0133] In an embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 20, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 20, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 3 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 3 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 28, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 28, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10).

[0134] In an embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 21 , ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 21 , wherein the CDR sequences are as set forth in SEQ ID NOs: 2, 4 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 2, 4 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 28, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 28, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0135] In an embodiment, the antibody or fragment comprises a heavy chain variable region comprising: i) an amino acid sequence as set forth in SEQ ID NO: 73, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 73, wherein the CDR sequences are as set forth in SEQ ID NOs: 2, 4 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 2, 4 and 7, and wherein the antibody comprises a light chain variable region comprising i) an amino acid sequence as set forth in SEQ ID NO: 28, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 28, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0136] In an embodiment, the antibody or fragment comprises a heavy chain variable region comprising: i) an amino acid sequence as set forth in SEQ ID NO: 22, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 22, wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 3 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 3 and 7,, and wherein the antibody comprises a light chain variable region comprising i) an amino acid sequence as set forth in SEQ ID NO: 28, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 28, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) aconservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0137] In an embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 23, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 23, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 5 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 5 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 28, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 28, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0138] In an embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 24, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 24, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 3 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 3 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 28, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 28, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0139] In an embodiment, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 25, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 25, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 6 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 6 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 28, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 28, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0140] In an embodiment, the antibody or fragment comprises a heavy chain variable region comprising: i) an amino acid sequence of SEQ ID NO: 68, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 68, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 3 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 3 and 7, and wherein the antibody comprises a light chain variable region comprising: i) the amino acid sequence of SEQ ID NO: 69, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 69, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

[0141] In an embodiment, the antibody or fragment thereof comprises 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.

[0142] In an embodiment, the antibody or fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 26.

[0143] In an embodiment, the antibody or fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 26.

[0144] In an embodiment, the antibody or fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 26.

[0145] In an embodiment, the antibody or fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 24 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 26.

[0146] In an embodiment, the antibody or fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 25 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 26.

[0147] In an embodiment, the antibody or fragment thereof has better production, better stability and / or lower aggregation than a reference antibody.

[0148] In an embodiment, the antibody or fragment thereof has increased binding affinity compared to a reference antibody.

[0149] In some embodiments the reference antibody is one or more humanized antibodies, the chimeric antibody described herein and / or the mouse 104 monoclonal antibody with a heavy chain variable region and a light chain variable region as shown in SEQ ID NOs: 81 and 82, respectively.

[0150] In an embodiment, the antibody or fragment thereof has greater stability and / or lower aggregation than the reference mouse 104 monoclonal antibody.

[0151] In an embodiment, the antibody or fragment thereof has greater binding affinity than the reference mouse 104 monoclonal antibody.

[0152] In an embodiment, the conservatively substituted amino acid sequence comprises the CDR sequences as set out in Table 3 or 4 below.

[0153] In an embodiment, the heavy chain variable region is comprised in a heavy chain and / or the light chain variable region is comprised in a light chain.

[0154] In an embodiment, the heavy chain further comprises the amino acid sequence of SEQ ID NO: 62 and / or 63.

[0155] In an embodiment, the light chain further comprises the amino acid sequence of SEQ ID NO: 64 and / or 65.

[0156] In an 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.

[0157] In an 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.

[0158] In an embodiment, the antibody has an amino acid sequence identity of 83.7%, 80.6%, 84.7%, 90.0%, 86.0%, 80.2%, 88.8%, 86.7%, 92.9%, 91.0%, 89.0%, 83.2% or 95.9% to a reference amino acid sequence.

[0159] In an embodiment, the heavy chain and light chain variable region amino acid sequences can comprise any sequential number of amino acids falling within the ranges of, for example, 25-50, 50-75, 75-100, 100-125, etc., amino acids as described in SEQ ID NOs: 20-28 which includes at least one sequence mutation as described herein.

[0160] The CDRs may comprise 1 or 2 amino acid substitutions, optionally conservative amino acid substitutions. As described in the Examples and illustrated in the Tables and Figures, several of the CDRs disclosed herein present high sequence homology. For example, CDR-H2 of variant 4 differs from CDR-H2 of variant 12 by one amino acid. Accordingly, in an embodiment, a CDR described herein comprises 1 or 2 amino acid substitutions, optionally conservative amino acid substitution(s). In an embodiment, the substitution is at a residue that varies between CDRs described herein. For example, the amino acid substitution is at a residue of a CDR referenced herein (e.g. CDR-H2) that differs from another corresponding CDR referenced herein (e.g. another CDR-H2) and may comprise any amino acid at the position corresponding to the differing amino acid(s) and which retains specific binding to PTHrP, optionally it may be replaced with conversative substitution(s).

[0161] In other embodiments, the variable chains can comprise conservative amino acid substitutions and the CDR sequences are as indicated herein, for example, a CDR sequence as set out in Table 3 or 4.

[0162] The antibodies or fragments thereof as herein disclosed can be defined as comprising a heavy chain variable region comprising 121 residues numbered H1 to H113 (including H1 -H52, H52a, H53-H82, H82a-c, H83-H100, H100a-d, and H101-H113) according to the Kabat nomenclature and a light chain variable region comprising 112 residues numbered L1 to L107 (including L1 -L27, L27a-e, and L28-L107) according to the Kabat nomenclature. See, for example, the Kabat numbering provided for the variable heavy chain region and variable light chain region of an antibody amino acid (aa) sequence in Tables 1 and 2 below:Table 1 : Example of Kabat numbering for a variable heavy chain region sequenceTable 2: Example of Kabat numbering for a variable light chain region sequence

[0163] In an embodiment, the following residue substitutions at positions defined by Kabat numbering, as exemplified above, can be made without abolishing the protein's desired properties:

[0164] In an embodiment, the antibody is a monoclonal antibody. In an embodiment, the antibody is a chimeric antibody such as a humanized antibody. In an embodiment, the antibody is a single chain antibody.

[0165] In an embodiment, the antibody is affinity purified.

[0166] In an embodiment, the antibody is affinity matured.

[0167] The binding capacity of the antibody can be assessed, for example, by ELISA or FACS (e.g., EC50); or optionally as Kd (e.g., by Biacore or Octet).

[0168] The biological activity of the antibody can be assessed in a relevant in vitro cellular assay compared with a reference antibody.

[0169] The cross-reactivity of the antibody can be compared with a relevant orthologue species (e.g., as in vitro binding activity).

[0170] A determination of the biophysical properties of the antibody can be carried out using, for example, SEC-HPLC profile to determine the level of high molecular weight soluble aggregates, SDS- PAGE under non-reducing and reducing conditions, and / or analysis by differential scanning calorimetry (DSC) using MicrocalTM VP-capillary DSC system to determine the Tm of Fab, CH2 and CH3.

[0171] In an embodiment, the antibody or fragment thereof is conjugated to a detectable label. In an embodiment, the detectable label is a positron-emitting radionuclide. A positron-emitting radionuclide can be used for example in PET imaging.

[0172] Accordingly, in another aspect, an immunoconjugate comprises an antibody or fragment thereof as herein described and a detectable label.

[0173] In an embodiment, a nucleic acid encodes an antibody, antibody heavy chain, antibody light chain, antibody heavy chain variable domain, or antibody light chain variable domain, or fragment thereof, as herein described. The nucleic acid may, for example, encode a heavy chain comprising CDR-H1 , CDR-H2 and / or CDR-H3 regions described herein and more particularly in Table 3 below or encoding a light chain comprising CDR-L1 , CDR-L2 and / or CDR-L3 regions described herein and more particularly in Table 4 below.

[0174] In an embodiment, the nucleic acid comprises any one of SEQ ID NOs: 11-19, and optionally further comprises SEQ ID NOs: 58-61 or one or more nucleotide sequences encoding an amino acid sequence of SEQ ID NOs: 62-65.

[0175] In an embodiment, the nucleic acid i) encodes a heavy chain variable domain, the nucleic acid encoding heavy chain variable domain comprising the sequence, or any part thereof, of any one of SEQ ID NOs: 11 , 12, 14, 15, or 16, or a sequence with at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to any one of SEQ ID NOs: 11, 12, 14, 15, or 16; ii) encodes a light chain variable domain, the nucleic acid encoding light chain variable domain comprising the sequence, or any part thereof, of any one of SEQ ID NOs: 17, 18 or 19, or a sequence with at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to any one of SEQ ID NOs: 17, 18 or 19; or iii) encodes a heavy chain variable domain and a light chain variable domain, the nucleic acid encoding the heavy chain variable domain and light chain variable domain, respectively, comprising the sequences, or any parts thereof: SEQ ID NOs: 11 and 17; SEQ ID NOs: 12 and 17; SEQ ID NOs: 14 and 17; SEQ ID NOs: 15 and 17, SEQ ID NOs: 16 and 17, SEQ ID NOs: 12 and 18, SEQ ID NOs: 16 and 18, SEQ ID NOs: 11 and 19, SEQ ID NOs: 12 and 19, SEQ ID NOs: 14 and 19, SEQ ID NOs: 15 and 19, or SEQ ID NOs:16 and 19, or sequences with at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NOs: 11 and 17; SEQ ID NOs: 12 and 17; SEQ ID NOs: 14 and 17; SEQ ID NOs: 15 and 17,SEQ ID NOs: 16 and 17, SEQ ID NOs: 12 and 18, SEQ ID NOs: 16 and 18, SEQ ID NOs: 11 and 19,SEQ ID NOs: 12 and 19, SEQ ID NOs: 14 and 19, SEQ ID NOs: 15 and 19, or SEQ ID NOs: 16 and 19.

[0176] In an embodiment, the nucleic acid sequence identity can be any % falling within the range of 70-99.5% identity with a reference sequence.

[0177] In an aspect, an expression cassette or vector comprises a nucleic acid herein disclosed.

[0178] In an embodiment, the vector is an isolated vector. The vector can be any vector, including vectors suitable for producing an antibody and / or binding fragment thereof or expressing a peptide sequence described herein. Examples of suitable vectors include adenoviral vectors, adenoviral-associated vectors, or retroviral vectors, optionally lentiviral vectors.

[0179] The nucleic acid molecules may be incorporated in a known manner into an appropriate expression vector which ensures expression of the protein. Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses (e.g. replication defective retroviruses, adenoviruses and adeno-associated viruses). The vector should be compatible with the host cell used. The expression vectors are suitable for transformation of a host cell, which means that the expression vectors contain a nucleic acid molecule encoding an antibody described herein.

[0180] In an aspect, a cell expresses an antibody or fragment thereof or nucleic acid herein described, or comprises a vector as herein described.

[0181] In an embodiment, the cell is an isolated and / or recombinant cell, expressing an antibody or fragment thereof described herein or comprising a vector herein disclosed. In an embodiment, the cell is a fused cell such as a hybridoma. The recombinant cell can be generated using any cell suitable for producing a polypeptide, for example suitable for producing an antibody and / orbinding fragment thereof. For example, to introduce a nucleic acid (e.g. a vector) into a cell, the cell may be transfected, transformed or infected, depending upon the vector employed.

[0182] Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. For example, the proteins described herein may be expressed in bacterial cells such as E. coli, insect cells (using baculovirus), yeast cells or mammalian cells.

[0183] In an embodiment, the cell is a eukaryotic cell selected from a yeast, plant, worm, insect, avian, fish, reptile and mammalian cell.

[0184] In an embodiment, the insect cell is a Sf9 cell, Sf21 cell, Tni cell, or S2 cell.

[0185] In another embodiment, the mammalian cell is a myeloma cell, a spleen cell, or a hybridoma cell.

[0186] Yeast and fungi host cells suitable for expressing an antibody or peptide include, but are not limited to Saccharomyces cerevisiae, Schizosaccharomyces pombe, the genera Pichia or Kluyveromyces and various species of the genus Aspergillus. Examples of vectors for expression in yeast S. cerivisiae include pYepSed, pMFa, pJRY88, and pYES2 (Invitrogen Corporation, San Diego, CA). Protocols for the transformation of yeast and fungi are well known to those of ordinary skill in the art.

[0187] Mammalian cells that may be suitable include, among others: HEK-293, COS, BHK, CHO, HeLa, and NS-1 cells, optionally HEK-293 cells or further optionally CHO cells. Suitable expression vectors for directing expression in mammalian cells generally include a promoter (e.g., derived from viral material such as polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40), as well as other transcriptional and translational control sequences.III. Compositions

[0188] In a further aspect, is provided a composition comprising an antibody or fragment thereof, an immunoconjugate, a nucleic acid, a vector or a cell as herein described.

[0189] In an embodiment, the composition is a pharmaceutical composition.

[0190] In an embodiment, the composition comprises a pharmaceutically acceptable carrier, diluent, and / or excipient.

[0191] In an embodiment, the composition comprises a diluent. Suitable diluents for nucleic acids and vectors include but are not limited to water, saline solutions and ethanol. Suitable diluents for polypeptides, including antibodies or fragments thereof and / or cells include but are not limited to saline solutions, pH buffered solutions and glycerol solutions or other solutions suitable for freezing polypeptides and / or cells.

[0192] In an embodiment, the composition comprises an antibody or fragment thereof or part thereof described herein. In another embodiment, the composition comprises an antibody or part thereof described herein and a diluent.

[0193] In an embodiment, the composition is a sterile composition.

[0194] In an embodiment, the composition is a pharmaceutical composition. The pharmaceutical composition can, for example be used in a method described herein such as for treating a subject in need thereof, e.g. a subject with hypercalcemia, for example, malignancy-associated hypercalcemia, or cancer, for example, breast cancer, lung cancer, colon cancer, pancreatic cancer, ovarian cancer, prostate cancer, squamous cell cancer or melanoma.

[0195] In an embodiment, the composition comprises one or more antibodies or fragments as described herein.

[0196] In an embodiment, the composition is for use in a method as herein described.IV. Kits and Packages

[0197] A further aspect relates to a kit or package comprising i) an antibody or fragment thereof, ii) an immunoconjugate iii) an isolated nucleic acid, iv) a vector, v) a cell, or vi) a composition as described herein comprised in a vial such as a sterile vial or other housing and optionally a reference agent and / or instructions for use thereof.

[0198] In an embodiment, the kit is an ELISA.

[0199] In an embodiment, the kit is a multiplex assay or a planar array kit.

[0200] In an embodiment, the kit comprises an antibody or fragment thereof described herein contained in a container such as a sterile vial.

[0201] In an embodiment, the kit comprises instructions for use for an ELISA or a method described herein.

[0202] In an embodiment, the kit is for use in a method as described herein.VI. Methods

[0203] A further aspect relates to methods of reducing or inhibiting activity of a human PTHrP isoform comprising contacting a cell or tissue expressing a human PTHrP isoform with an antibody or fragment thereof that specifically binds PTHrP as described herein. The antibody or binding fragment thereof comprises 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 with the amino acid sequences of said CDRs comprising:CDR-H1 : SYWXiQ, wherein: SEQ ID NO: 29,Xi = I or MCDR-H2: wherein:AIYPGDGDTSYX2X3X4FX5G, SEQ ID NO: 30,X3= E, P or QX4= K or SX5= K or QCDR-H3: GDYDYDERAMDY, SEQ ID NO: 7,CDR-L1: RSSQSIGHSNGNTYLE, SEQ ID NO: 8,CDR-L2: KVSNRFS, SEQ ID NO: 9, andCDR-L3: FQGSHDPFT, SEQ ID NO: 10.

[0204] Any of the antibodies described herein can be used. In an embodiment, the method of reducing or inhibiting activity of a human PTHrP isoform comprises contacting a cell or tissue expressing a human PTHrP isoform with an antibody or fragment thereof that specifically binds PTHrP and comprises 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 and the light chain variable region comprises complementarity determining regions CDR-L1, CDR-L2 and CDR-L3 and the amino acid sequences of the CDRs comprise one of the following sequence combinations:

[0205] For example, in an embodiment, the method of reducing or inhibiting activity of a human PTHrP isoform comprises contacting a cell or tissue expressing a human PTHrP isoform with an antibody or fragment thereof that specifically binds PTHrP as herein described, wherein the amino acid sequences of the CDRs comprise one or more of the sequences shown in Tables 3 and 4 below.

[0206] In an embodiment, contacting a cell or tissue expressing a human PTHrP isoform with an antibody or fragment thereof comprises expressing an immunoconjugate, a nucleic acid or a vector as herein described in the cell or tissue.

[0207] In an embodiment, the cell or tissue expressing a human PTHrP isoform is in vivo in a subject.

[0208] A further aspect relates to a method of treating a subject in need thereof, comprising administering to the subject an effective amount of an antibody or fragment thereof that specifically binds PTHrP, comprising a light chain variable region and a heavy chain variable region, the heavychain 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 with the amino acid sequences of said CDRs comprising the sequences:CDR-H1 : SYWXiQ, wherein: SEQ ID NO: 29,Xi = I or MCDR-H2: wherein:AIYPGDGDTSYX2X3X4FX5G, Xv2 > - A A, o or T l SEQ ID NO: 30,X3= E, P or QX4= K or SX5= K or QCDR-H3: GDYDYDERAMDY, SEQ ID NO: 7,CDR-L1: RSSQSIGHSNGNTYLE, SEQ ID NO: 8,CDR-L2: KVSNRFS, SEQ ID NO: 9, andCDR-L3: FQGSHDPFT, SEQ ID NO: 10.

[0209] Any of the antibodies described herein can be used. In an embodiment, the method of reducing or inhibiting activity of a human PTHrP isoform, comprises administering to a subject in need thereof an effective amount of an antibody or fragment thereof that specifically binds PTHrP and comprises 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 and the light chain variable region comprises complementarity determining regions CDR-L1, CDR-L2 and CDR-L3 and the amino acid sequences of the CDRs comprise one of the following sequence combinations:

[0210] For example, the method of method of treating a subject in need thereof comprises administering to a subject in need thereof an effective amount of an antibody or fragment thereof that specifically binds PTHrP, 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 with the amino acid sequences of said CDRs comprising the sequences shown in Tables 3 and 4 below.

[0211] In an embodiment, the method is a method of treating cancer in a subject in need thereof and comprises administering to the subject an effective amount of an antibody or fragment thereof, a composition comprising an antibody or fragment thereof, an immunoconjugate, a nucleic acid, or a vector as herein described.

[0212] In an embodiment, the cancer is pre-metastatic.

[0213] In an embodiment, the method is a method of treating hypercalcemia, for example malignancy-associated hypercalcemia, in a subject in need thereof and comprises administering to the subject an effective amount of an antibody or fragment thereof, a composition comprising an antibody or fragment thereof, an immunoconjugate, a nucleic acid, or a vector as herein described.

[0214] Also provided are in other aspects are uses of the antibodies, immunoconjugates, nucleic acids and vectors as described herein. For example, one embodiment provides use of an antibody, immunoconjugate, nucleic acid or vector for reducing or inhibiting activity of a human PTHrP isoform in a subject in need thereof. Another embodiment provides use of an antibody, immunoconjugate, nucleic acid or vector for the manufacture of a medicament for reducing or inhibiting activity of a human PTHrP isoform and / or treating cancer or hypercalcemia in a subject in need thereof. Another embodiment provides an antibody, immunoconjugate, nucleic acid or vector for use for reducing or inhibiting activity of a human PTHrP isoform in a subject in need thereof

[0215] In an embodiment, the subject has (e.g., has been diagnosed with) any cancer that expresses PTHrP, for example, breast cancer, lung cancer, colon cancer, pancreatic cancer, ovarian cancer, prostate cancer, squamous cell cancer, or melanoma. In a further embodiment, the subject has breast cancer. In yet a further embodiment, the subject has pancreatic cancer.

[0216] In an embodiment, the subject has (e.g., has been diagnosed with) malignancy-associated hypercalcemia.

[0217] In an embodiment, an effective amount of an antibody or fragment thereof or immunoconjugate of the disclosure described herein, or a composition comprising said antibody or fragment thereof or immunoconjugate, is administered optionally in combination with another hypercalcemia or cancer treatment.

[0218] The antibody or fragment thereof or immunoconjugate can, for example, be comprised in a composition as described herein for example in combination with a pharmaceutically acceptable carrier, diluent and / or excipient and formulated for example in vesicles for improving delivery. Combinations of antibodies (e.g. 2 or more antibodies or fragments thereof) and / or immunoconjugates can also be used.

[0219] The compositions, antibodies, immunogens, and immunoconjugates described herein can be administered for example, by parenteral, intravenous, subcutaneous, intramuscular, intracranial,intraventricular, intrathecal, intraorbital, ophthalmic, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol or oral administration.

[0220] In an embodiment, the composition is administered systemically.

[0221] In a further aspect, a method of detecting a human PTHrP isoform in a subject comprises i) contacting a test sample, for example a biological sample, from said subject with an antibody or fragment thereof as herein described under conditions permissive to produce an antibody-antigen complex, ii) measuring the amount of the antibody-antigen complex in the test sample, and Hi) comparing the amount of antibody-antigen complex in the test sample to a control, wherein detecting antibody-antigen complex in the test sample as compared to the control indicates that the sample comprises a human PTHrP isoform and / or tumour cells expressing a human PTHrP isoform, and / or indicates that the subject has hypercalcemia, for example, malignancy-associated hypercalcemia, or cancer, for example breast cancer or pancreatic cancer.

[0222] In a further aspect, a method of detecting a human PTHrP isoform or fragment thereof in a subject comprises i) contacting a first test sample, for example a biological sample, obtained from said subject prior to receipt of a treatment with an antibody or fragment thereof as herein described under conditions permissive to produce an antibody-antigen complex, ii) measuring the amount of the antibody-antigen complex in the first test sample, Hi) obtaining a second test sample, for example a biological sample, from said subject following receipt of the treatment, iv) measuring the amount of the antibody-antigen complex in the second test sample, and v) comparing the amount of antibody-antigen complex in the first test sample to the amount of antibody-antigen complex in the second test sample, wherein comparatively lower levels of antibody-antigen complex in the second test sample as compared to the first test sample indicates a presence of comparatively lower levels of a human PTHrP isoform or fragment thereof and / or tumour cells expressing a human PTHrP isoform or fragment thereof in the subject and / or indicates a response to the treatment by the subject. In a further embodiment, one or more further test samples can be obtained from the subject, wherein an amount of antibody-antigen complex in the further test sample is compared to an amount of antibody-antigen complex in a previous test sample (e.g. , the first or second test sample or a control sample) taken from the same patient to monitor response to treatment or progression (e.g., metastasis) or recurrence of disease in the subject. In some embodiments, the antibody for use in vivo is humanized. In an embodiment, the measuring may, for example, be by immunofluorescence.

[0223] In an embodiment, the sample is a biological sample. In further embodiments, the sample comprises blood, serum, plasma, tissue, cells, tumour, or an extract thereof. The sample can also be a fraction. In an embodiment, the sample is obtained from a human subject. In an embodiment, the sample is a tumour biopsy.

[0224] In an embodiment, the sample is from a subject having or suspected of having hypercalcemia, for example malignancy-associated hypercalcemia, or a cancer, for example, breastcancer, lung cancer, colon cancer, pancreatic cancer, ovarian cancer, prostate cancer, squamous cell cancer, or melanoma. In other embodiments, the sample is obtained from a subject that has (e.g. , has been diagnosed with) breast cancer or pancreatic cancer.

[0225] A number of methods can be used to determine if a human PTHrP isoform or fragment thereof is present in a sample using the antibodies or fragments thereof described herein, including immunoassays such as flow cytometry, dot or slot blots, Western blots, ELISA, and immunoprecipitation followed by SDS-PAGE immunocytochemistry. Other immune based methods that can be used include singleplex and multiplex immunoassay platforms. Any non-humanized or humanized antibody as herein described can be used in these embodiments.

[0226] In an embodiment, a labelled or tagged antibody as herein described is used in a diagnostic assay or test.

[0227] Singleplex bead-based platforms can be used in which individual immunocomplexes are isolated on paramagnetic beads and detected.

[0228] Multiplex immunoassay platforms suitable for these purposes include bead- or particlebased platforms, and planar array platforms.

[0229] Bead- or particle-based platforms can be used in which the capture antibody is immobilized on a particle such as a fluorescently dyed bead or paramagnetic bead, and analyte is detected with a second detection antibody.

[0230] An antibody as described herein can also be labelled or tagged and administered to a subject to detect the location of a human PTHrP isoform or fragment thereof, which can be used in conjunction with, for example, diagnostic imaging for detecting and / or locating a presence or a location of bound labelled or tagged antibody in vivo. Any humanized antibody or fragment thereof, for example a Fab, Fab', F(ab')2, as herein described can be used in these embodiments. For example, a Fab can be used to detect a presence of human PTHrP isoform or its fragments and / or tumour cells expressing a human PTHrP isoform or fragment thereof.

[0231] The above disclosure generally describes the present application. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for the purpose of illustration and are not intended to limit the scope of the application. Changes in form and substitution of equivalents are contemplated as circumstances might suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

[0232] The following non-limiting examples are illustrative of the present disclosure:ExamplesExample 1Humanization by CDR-grafting of HC104 mouse monoclonal antibody

[0233] Humanization of mouse HC104 monoclonal antibody was performed using CDR- grafting technology. The humanization process involved building a molecular model to aid in selecting residues to conserve or substitute.

[0234] The CDR-grafting protocol used is a modernized version of the approach of Greg Winter and colleagues at the Medical Research Council, Cambridge, UK. Definition of the CDRs is based on the Kabat nomenclature. Selection of human framework acceptor regions into which HC104 mouse CDR regions were grafted was accomplished by searching the IMGT mouse and human V gene databases using IgBLAST, developed at NCBI to facilitate analysis of immunoglobulin V region sequences (http: / / www.ncbi.nlm.nih.gov / igblast / ), using HC104 mouse variable region sequences as input.

[0235] HC104 mouse monoclonal antibody (mAb) was humanized by grafting the complementarity determining regions (CDRs) into human germline antibody sequences. HC104 was obtained by immunizing mice with a 33 amino acid long peptide corresponding to the C-terminal region (140 -173) of the isoform parathyroid hormone-like hormone or PTHrP (Uniprot P12272).

[0236] HC104 mouse antibody was humanized by grafting the three CDRs, as defined by the Kabat nomenclature, from the light chain variable region (VL) into a human germline VL. Similarly, the three CDRs from the heavy chain variable region (VH) were grafted into a human germline VH. Several amino acid residues in the framework regions of the selected human germline variable regions were changed to the amino acid residues that were present in the mouse variable regions (so called back- mutations). Using the molecular model of the Fv of the HC104 mouse monoclonal antibody, residues in the framework regions were retained in the humanized version A or substituted with their human germline counterparts, in the subsequent humanized versions. Under guidance of the molecular model, in subsequent version B, the CDR residues, as defined by Kabat, were also substituted to increase the degree of humanness.

[0237] For the CDR-grafted versions of the HC104 mouse VH, three human germlines, IGHV1-46*01, IGHV1-69-2*01 and IGHV5-51*01 were selected. Mouse HC104 VH has a sequence identity with human germline IGHV1 -46*01 of 71.4% across the whole V gene (including CDR1 and CDR2 but excluding CDR3 which is not part of the V gene human germline IGHV1 -69-2*01 with a sequence identity of 68.4% and IGHV5-51*01 with a sequence identify of 63.3% across the whole V gene (including CDR1 and CDR2 but excluding CDR3 which is not part of the V gene) (see Table 3).Table 3: Percentage identity between mouse V genes or their humanized versions with variable human germlines (V genes)

[0238] To make CDR-grafted versions of the HC104 mouse VL, three human germlines, IGKV2-30*02, IGKV2-18*01 and IGKV4-1*01, were selected. IGKV2-30*01 and IGKV2-18*01 have sequence identity with mouse HC104 VL across the whole V-gene (including CDR1 , CDR2 and two third of CDR3) of 81% and 77%, respectively. IGKV4-1*01 has sequence identity with HC104 VL of 64.35%.

[0239] A heavy and light chain combination between the humanized VH and humanized VL versions was selected. For the VH, humanized versions based on three different human germlines were made in silico, including 2 versions (A and B) and 1 version C for IGHV5-51*01 , for a total of 7 different humanized VH. For the VL humanized versions based on three human germlines with 2 versions (A and B) for both were made in silico, for a total of 6 different humanized VL, and a total of 6 (VH) x 6 (VL) = 42 humanized variant combinations.

[0240] 18 VH / VL combinations were expressed: humanized versions A and B of the VH together with version A only of the VL, or 6 (VH) x 3 (VL) = 18 humanized variants were selected.

[0241] See details of the 18 humanized VH and VL combinations in the table below.Table 4: 18 humanized VH and VL combinations

[0242] The percentage of sequence identity (i.e. percentage of humanness) across the whole V genes between the HC104 mouse parental monoclonal antibody and the different CDR-grafted humanized versions is presented in Table 1 above.

[0243] Further details are provided in Examples 2 and 3.Example 2Heavy chain

[0244] See Fig. 1 A for the amino acid sequence of the VH of the mouse HC104 hybridoma (CDR regions according to the Kabat numbering scheme are highlighted).

[0245] Human germlines IGHV1 -69-2*01 , IGHV1-69-2*01 and IGHV5-51*01 were selected as acceptor sequences for CDR-grafting. The selection of human framework acceptor VH regions into which the HC104 mouse CDR regions were grafted was accomplished by searching the IMGT human VH gene database using IgBLAST (https: / / www.ncbi.nlm.nih.gov / igblast / igblast.cgi) with the mouse HC104 VH amino acid sequence as input. Based on the sequence alignment of the Parental antibody to the human germlines, the closest matching entries were identified.

[0246] . A large number of human germline sequences were analyzed, and three human germlines were selected as best candidates; two belonging to the human family 1 ; IGH V1 -69-2*01 and IGHV1-69-2*01 and one belonging to the human family 5; IGHV5-51*01.

[0247] In framework 4, the gene segment of the mouse HC104 VH corresponding to the J gene was identified as most homologous to the mouse germline IGHJ4*01 gene. The mouse IGH J4*01 - segment gene was compared to the human J-segment genes over CDR3 and FR4 and found to be most homologous to human J4*01 -segment.IGHV1-46*01 human germlines as framework acceptor regionsHumanized version A

[0248] Mouse CDRs (highlighted in grey in Fig. 1A) as defined by the Kabat nomenclature were grafted into IGH V1 -46*01 to obtain the sequence shown in Figs. 1 B and 1C.

[0249] 83.7% identity (82 identical residues out of a total of 98 residues in the V gene) was found for humanized version A (HC104-146-VHA) with IGHV1-46*01 human germline. In the framework and CDR regions, as shown in Fig. 1 C, residues that differ between humanized version A and human germline IGHV1-46*01 are highlighted in grey. The full-length amino acid sequence of version A is shown in Fig. 1 D.Humanized version B

[0250] In version B, as compared to version A, 5 amino acid residues were germ lined (i.e. the mouse residue was substituted by the corresponding IGHV1 -46*01 human germline residue). In framework 2 Kabat lie (I) H48 was substituted with Met (M). In CDR2 Kabat Thr (T) H60 and Kabat Lys (K) H64 were substituted with Ala (A) and Gin (Q), respectively. In framework 3 Kabat Ser (S) H75 and Kabat Arg (R) H76 were substituted with Thr (T) and Ser (S), respectively. As shown in Fig. 1 D, the amino acid residues that differ between humanized version A and B are highlighted in grey.

[0251] 88.8% identity (87 identical residues out of a total of 98 residues in the V gene) was found for humanized version B (HC104-146-VHB) with IGHV1-46*01 human germline. As shown in Fig. 1 F, residues that differ between humanized version A and B are highlighted in grey. The full-length amino acid sequence of version B is shown in Fig. 1 D.IGHV1-69-2*01 human Qermline as framework acceptor regionsHumanized version A

[0252] Mouse CDRs (highlighted in grey in Fig. 1A) as defined by the Kabat nomenclature were grafted into IGHV1 -69-2*01 to obtain the sequence shown in Figs. 1G and 1 H..

[0253] 80.61% identity (79 identical residues out of a total of 98 residues in the V gene) was found for humanized version A (HC104-1692-VHA) with IGHV1-69-2*01 human germline. In Fig. 1 H, residues that differ between HC104-1692-VHA and human germline IGHV1-69-2*01 in framework regions and in CDRs are highlighted in grey. The full-length amino acid sequence of version A is shown in Fig. 11.D-3 Humanized version B

[0254] In version B, as compared to version A, 6 amino acid residues were germ lined (i.e. the mouse residue was substituted by the corresponding IGHV1 -46*01 human germline residue). In framework 2 Kabat lie (I) H48 was substituted with Met (M). In CDR2 Kabat Thr (T) H60, Kabat Gin (Q) H61 and Kabat Lys (K) H64 were substituted with Ala (A), Glu (E) and Gin (Q), respectively. In framework 3 Kabat Ser (S) H75 and Kabat Arg (R) H76 were substituted with Thr (T) and Asp (D), respectively.

[0255] As shown in Fig. 1 J, the amino acid residues that differ between humanized version A and B are highlighted in grey.

[0256] 86.7% identity (85 identical residues out of a total of 98 residues in the V gene) was found for humanized version B (HC104-1692-VHB) with IGHV1-69-2*01 human germline. As shown in Fig. 1K, residues that differ between HC104-1692-VHB and human germline IGHV1-69-2*01 in framework regions and in CDRs are highlighted in grey. The full-length amino acid sequence of version B is shown in Fig. 11.IGHV5-51*01 human Qermline as framework acceptor regionsHumanized version A

[0257] Mouse CDRs (highlighted in grey in Fig. 1A) as defined by the Kabat nomenclature were grafted into IGHV5-51*01 to obtain the detailed sequence shown in Figs. 1 L and 1 M.

[0258] 84.7% identity (83 identical residues out of a total of 98 residues in the V gene) was found for humanized version A (HC104-551 -VHA) with IGHV5-51*01 human germline. As shown in Fig. 1 M, residues that differ between HC104-551-VHA and human germline IGHV5-51*01 in framework regions and in CDRs are highlighted in grey. The full-length amino acid sequence of version A is shown in Fig. 1 N.E-3 Humanized version B

[0259] In version B, as compared to version A, 8 amino acid residues were germ lined (i.e. the mouse residue was substituted by the corresponding IGHV5-51*01 human germline residue). In CDR2 Kabat Met (M) H34 was substituted with lie (I). In framework 2 Kabat lie (I) H48 was substituted with Met (M). In CDR2 Kabat Thr (T) H60, Kabat Gin (Q) H61 , Kabat Lys (K) H62 and Kabat Lys (K) H64 were substituted with Ser (S), Pro (P), Ser (S) and Gin (Q), respectively. In framework 3 Kabat Ser (S) H75 and Kabat Arg (R) H76 were substituted with lie (I) and Ser (S), respectively. As shown in Fig. 10, the amino acid residues that differ between humanized version A and B are highlighted in grey.

[0260] 92.9% identity (91 identical residues out of a total of 98 residues in the V gene) was found for humanized version B (HC104-551-VHB) with IGHV5-51*01 human germline. As shown in Fig. 1 P, residues that differ between HC104-551-VHB and human germline IGHV5-51*01 in framework regions and in CDRs are highlighted in green and cayman blue, respectively. The full-length amino acid sequence of version B is shown in Fig. 1 N.E-4 Humanized version C

[0261] In version C, as compared to version B, 3 amino acid residues were germ lined (i.e. the mouse residue was substituted by the corresponding IGHV5-51*01 human germline residue). In framework 1 Kabat Ala (A) H24 and Kabat Thr (T) H28 were substituted with Gly (G) and Ser (S), respectively. In framework 3 Kabat Leu (L) H69 was substituted with lie (I).

[0262] As shown in Fig. 1 Q, amino acid residues that differ between humanized version B and C are highlighted in grey.

[0263] 95.9% identity (94 identical residues out of a total of 98 residues in the V gene) was found for humanized version C (HC104-551-VHC) with IGHV5-51*01 human germline. As shown in Fig. 1 R, residues that differ between HC104-551-VHB and HC104-551-VHC are highlighted in grey. The full length amino acid sequence of version C is shown in Fig. 1 N.

[0264] The heavy chain amino acid CDR sequences as defined by the Kabat nomenclature of the humanized variants are summarized in Table 5 below:Table 5: Amino acid sequences of heavy chain CDRsExample 3Light chain

[0265] See Fig. 2A for the amino acid sequence of the VL of the mouse mAb HC104 (CDR regions as defined by the Kabat nomenclature are highlighted).

[0266] The selection of human framework acceptor VL regions into which the HC104 mouse CDR regions were grafted was accomplished by searching the IMGT human VL gene database using IgBLAST with the mouse VL region amino acid sequence as input. Based on the sequence alignment of the Parental antibody to the human germlines, the closest matching entries were identified.

[0267] A large number of human germline sequences were analyzed and three human germlines, IGKV2-30*02, IGKV2-18*01 and IGKV-4-1*01, were selected for the CDR-grafted humanized versions. IGKV2-30*02 has the highest sequence identity with HC104 VL of 81% (81 identical residues out of a total of 100) followed by IGKV2-18*01 with 77% (77 identical residues out of a total of 100) and IGKV4-1*01 with 64.35% (65 identical residues out of a total of 101 ).

[0268] The gene segment of the mouse HC104 VL corresponding to the J gene was 100% identical in sequence to the unique mouse J gene; IGKJ2*01 (FTFGSGTKLEIK). The mouse IGKJ2*01 gene segment was compared to the human J-segment genes over CDR3 and FR4, and the human J- segment IGLJ2*01 was found to have the highest overall homology.IGKV2-30*02 human germline as framework acceptor regionsC-2 Humanized version A

[0269] Mouse CDRs (highlighted in grey in Fig. 2A) as defined by the Kabat numbering were grafted into IGKV2-30*02 to obtain the detailed sequence shown in Figs. 2B and 20.

[0270] 90.0% identity (90 identical residues out of a total of 100 residues in the V gene) was found for humanized version A (HC104-320-VLA) with IGKV2-30*02. The full-length amino acid sequence of version A is shown in Fig. 2D.C-3 Humanized version B

[0271] In version B, as compared to version A, one amino acid residue was germ lined (i.e. the mouse residue was substituted by the corresponding IGKV2-30*02 human germline residue). In CDR1 Kabat lie (I) L27b was substituted with Leu (L).

[0272] As shown in Fig. 2E, amino acid residues that differ between humanized version A and B are highlighted in grey.

[0273] 91.0% identity (91 identical residues out of a total of 100 residues in the V gene) was found for humanized version B (HC104-320- VLB) with IGKV2-30*02. As shown in Fig. 2F, residues highlighted in grey are positions that differ within CDRs between humanized version A and B. The full- length amino acid sequence of version B is shown in Fig. 2D.IGKV2-18*01 human qermline as framework acceptor regionsD-2 Humanized version A

[0274] Mouse CDRs (highlighted in grey in Fig. 2A) as defined by the Kabat numbering were grafted into IGKV2-18*01 to obtain the detailed sequence shown in Figs. 2G and 2H.

[0275] 86.0% identity (86 identical residues out of a total of 100 residues in the V gene) was found for humanized version A (HC104-218-VLA) with IGKV2-18*01. As shown in Fig. 2H, residues highlighted in grey are positions that differ within CDRs between humanized version A and the human germline IGKV2-18*01. The full-length amino acid sequence of version A is shown in Fig. 2I.D-3 Humanized version B

[0276] In version B, as compared to version A, three amino acid residues were germ lined (i.e. the mouse residue was substituted by the corresponding IGKV2-18*01 human germline residue). In frameworkl (FR1 ) Kabat Vai (V) L2 was substituted with lie (I). In CDR1 Kabat lie (I) L27b was substituted with Leu (L). In framework 3 (FR3) Kabat Thr (T) L69 was substituted with Ser (S). As shown in Fig. 2J, amino acid residues that differ between humanized version A and B are highlighted in grey.

[0277] 89.0% identity (89 identical residues out of a total of 100 residues in the V gene) was found for humanized version B (HC104-218- VLB) with IGKV2-18*01. As shown in Fig. 2K, residueshighlighted in grey are positions that differ within CDRs between humanized version A and B. The full- length amino acid sequence of version B is shown in Fig. 21.IGKV4-1*01 human germline as framework acceptor regionsE-2 Humanized version A

[0278] Mouse CDRs (highlighted in grey in Fig. 2A) as defined by the Kabat numbering were grafted into IGKV4-1*01 to obtain the detailed sequence shown in Figs. 2L and 2M.

[0279] 81.2% identity (81 identical residues out of a total of 101 residues in the V gene) was found for humanized version A (HC104-41-VLA) with IGKV4-1*01. The full-length amino acid sequence of version A is shown in Fig. 2N.E-3 Humanized version B

[0280] In version B, as compared to version A, three amino acid residues were germ lined (i.e. the mouse residue was substituted by the corresponding IGKV4-1*01 human germline residue). In frameworkl (FR1 ) Kabat Vai (V) L2 was substituted with lie (I). In CDR1 Kabat (Arg) L24 was substituted with Lys (K) and Kabat lie (I) L27b was substituted with Vai (V). As shown in Fig. 20, amino acid residues that differ between humanized version A and B are highlighted in grey.

[0281] 84.1% identity (84 identical residues out of a total of 101 residues in the V gene) was found for humanized version B (HC104-41-VLB) with IGKV4-1*01. As shown in Fig. 2P, residues highlighted in grey are positions that differ within CDRs between humanized version A and B. The full- length amino acid sequence of version B is shown in Fig. 2N.

[0282] The light chain amino acid CDR sequences as defined by the Kabat nomenclature of the humanized variants are summarized in Table 6 below:Table 6: Amino acid sequences of light chain CDRsExample 4HC104 humanized recombinant antibody expression and purificationGenes and Expression VectorGenes coding for the recombinant antibodies (rAbs)

[0283] Starting from sequences of the heavy chain (HC) and light chain (LC) variable regions of the 18 humanized variants antibodies resulting from in-silico humanization as described in Examples 1-3, full-length human lgG1 Kappa antibodies were made.

[0284] The cDNA coding for the variable regions of the HC and LC were chemically synthesized with optimization for expression in CHO cells and subcloned into mammalian cell expression vectors containing backbones for human lgG1 heavy chain constant region and human kappa light chain constant region. Sequences coding for signal peptides were added in 57Nter position. The sequences are illustrated in Table 7 below.

[0285] In Table 7, non-underlined sequences indicate heavy and light chain variable domain regions of humanized antibody variants 1-18. Examples of heavy chain and light chain secretion signaling sequences used are shown in bold / underline, and examples of human lgG1 heavy chain constant region and human kappa light chain constant region backbones used are shown as non- bolded / underlined.Table 7: Nucleotide sequences of heavy chain and light chain variable domain regionsExpression vector

[0286] The sequences for the expected proteins produced are illustrated in Table 8 below.

[0287] In Table 8, non-underlined sequences indicate heavy and light chain variable domain regions of humanized antibody variants 1-18. Examples of heavy chain and light chain secretion signaling sequences used are shown in bold / underline, and examples of human lgG1 heavy chain constant region and human kappa light chain constant region backbones used are shown as non- bolded / underlined.Table 8: Amino acid sequences of heavy chain and light chain variable domain regionsSmall-scale production and purification tests

[0288] An endotoxin-free DNA preparation was used for the constructions obtained as described above.

[0289] Various antibodies were expressed by combining HC and LC as follows in T able 9:Table 9: Antibodies

[0290] The plasmids were transiently co-transfected in CHO cells. Cell culture medium samples were collected when viability dropped under 50% (14 days after transfection) and the rAbs and chimeric antibody were then purified on a protein A resin using a standard method:• Clarification by 0.22 pm filtration• Equilibration, binding, and wash with PBS pH 7.5• Elution by pH shift with citric acid• Neutralization with 1 M Tris-HCI pH 9.0• Analysis by PAGE and pool of the fractions of interest, and• Final QC by PAGE: qualitative and quantitative by SDS-PAGE.

[0291] Elution fractions were pooled and buffer exchanged vs PBS, pH 7.5 by dialysis method. Final samples were filtered by 0.22 pm Millipore filter. Purification profiles and final QC are illustrated in Fig. 3A (rAbs) and Fig. 3b (chimera).Results of the small-scale production and purification tests

[0292] Figure 3A, B shows final sample QC.

[0293] The yield and purity obtained are summarized in Table 10 shown below.Table 10. Yield and purity obtained for the recombinant antibody productions★Obtained after purification for the 30ml-culture test.**Based on full length antibody observed on non-reduced PAGE analyses in Figure 2. Final buffer: PBS pH 7.5

[0294] Conclusion: Transient expression of the humanized variants showed very good yield and high purity for various antibodies, including for variants 1 , 2, 4-6, 8 and 12-18 and HC104 chimeric antibody (identified as no. 19).Example 5SEC-HPLC aggregation level analysis

[0295] SEC-HPLC analysis was performed to evaluate the aggregation level of the antibodies that expressed as described above (variant nos. 1-8, 10-14 and 16-18 and HC104 chimeric antibodyherein referred to as variant number 19). The analyses were performed with a HPLC Waters 2695 device equipped with a Photodiode Array Detector (2996).Buffer preparation

[0296] Mobile phase: 100mM Sodium sulfate, 100mM Phosphate salt, pH 6.7 buffer (Na2SO4, 14.2g / L, Na2HPO4 12 H2O 15.6 g / L, NaH2P04 6.8 g / L). Air bubbles were removed from all buffers.Sample preparation

[0297] Protein preparation: starting from one extra aliquot of each final sample stored at -80°C for one night, protein samples were diluted to 1 mg / ml with double distilled (DD) water. Samples were centrifuged at 12000g for 10 minutes and supernatants were collected.Analysis conditions

[0298] Blank control: sample was diluted 5x with DD water. Column: G3000SWXL, TOSOH, 7.8x300mm. HPLC: Waters 2695. Elution gradient: mobile phase isocratic elution. Flow rate: 0.8 ml / min. Temperature: 25°C. Injection volume: 30 pl. Detection wavelength: 280nm. Collected date time: 25min. Equilibrated with mobile phase 100% for 5 minutes. Samples Ran: One blank control injected and then samples injected. Data was analyzed.Results of the SEC-HPLC analysis

[0299] SEC-HPLC profiles obtained for variant nos. 1-8, 10-14 and 16-18, HC104 chimeric (identified as no. 19) antibody and blank controls are shown in Figs. 4A to 4T. The purity of full antibody single molecules detected in the sample was analyzed and results are shown in Table 9 below. When several peaks were detected, data corresponding to the peak of full antibody single molecules is indicated in bold. Where a single peak was detected, an estimate of the concentration (purity %) is given.Table 11. SEC-HPLC dataConclusion: SEC-HPLC analyses show that antibody samples 1 , 2, 4, 5, 6 and chimeric exhibit the best concentration of ideal heterotetrametric antibodies composed of two heavy and two light chains and aggregation levels <5%. Samples 3 and 11 contain some aggregates (<20%), while 7, 8, 10, 12, 13, 14, 16, 17, 18 and mouse antibody contain a large percentage of aggregates (> 30%).Example 6ELISA analysisBSA-PTHrP 140-173

[0300] EC50 values were calculated by ELISA using BSA-PTHrP 140-173 coated plates, 3% BSA blocking, and antibody dilutions of from 10 ug / ml to 0.00064 ug / ml, PBST, 100 pl / well, 37°C, 1 h, followed by incubation with a goat anti human HRP conjugated antibody for 30 min. Readings were at OD450- 630.

[0301] EC50 values were determined for humanized variants 1 -8, 10-14 and 16-18 and HC104 chimeric antibody (identified as no. 19) and several exhibited an improved EC50 compared to HC104 chimeric antibody, including variants 1-8, 11-13, and 17.Example 7Temperature Stability

[0302] Differential Scanning Fluorimetry Analysis (DSF) was performed using a Nanotemper- nanoDSF system with a standard method. The mAbs (50 g in PBS. pH 7.5 buffer) were subject to a linear thermal ramp of 1°C / min. from 20° to 95°C. Tryptophan fluorescence at 350nm and 330nm was collected at a rate of 10 datapoints per minute. Unfolding transition midpoints were determined automatically from the 1st derivative of the fluorescence ratio (F350 / F330).Table 12: Results of the DSF analysis

[0303] Conclusion: The thermal stability curves of the humanised antibodies showed two unfolding transitions in the plot of the fluorescence ratio (F330 / F350) versus temperature (Fig. 5). The two distinct unfolding events were attributed to the different thermal stabilities of Fab- and Fc-domains of the antibodies. DSF analyses showed that variants 1, 2 and 3 had the highest thermal stability with a Tm1 around 72°C and T m2 >80°C.Example 8Determination of KD of antibody / antigen interaction

[0304] Five humanized antibody variants were analyzed via OpenSPR™ for determination of the KD of each antibody for their antigen. PThrP 1-173 (2.64 mg / ML) was used as a ligand. Each of the humanized antibodies were used at a concentration of 1.00 mg / mL and 95% purity. HC104-chimeric (3.80 mg / mL) and mouse 104 from 20846 (1.00 mg / mL) were used as controls.

[0305] Briefly, the PThrP 1-173 antigen was immobilized on COOH sensor chip. A solution containing antibody at various concentrations was flown over PTHrP 1 -173. Real time measurement of kinetics parameters: on / off rates (Ka and Kd) were collected and binding affinity constant (KD) was determined.

[0306] Experimental Design and parameters• Buffer: PBS-P+ (0.01 M phosphate buffer pH7.4, 2.7mM KCL, 0.137 mM NaCI, 0.05% Surfactant P20)• Regeneration buffer: 10 mM Gly-HCI (pH 2.0)• Analyte testing concentration: shown in results (below)• Sample rate: 30 pM• Regeneration buffer flow rate: 150 pL / min• Binding reaction temperature: 20 °C• Sensor: COOH• Instrument: OpenSPRMethod1. COOH chip was installed according to the standard operating procedure of the OpenSPR™ instrument.2. At the beginning, ran at the maximum flow rate (150 L / min), and the buffer was PBS-P+ (pH7.4).3. After reaching the signal baseline, 200 uL 80% IPA (isopropyl alcohol) was loaded, and ran for 10s to exhaust bubble. After the baseline was reached, the sample ring was rinsed with buffer and emptied with air.4. Buffer flow rate was adjusted to 20 uL / min after signal reached the baseline.5. Loaded 200 uL EDC (400 mM) / NHS (100 mM) (1 :1 ) solution (20 uL / min, 4min).6. Loaded 200uL of the diluted ligand TNFalpha diluted with activation buffer (PBS pH=7.5), ran for 4 min (20 uL / min). Flushed the sample ring with buffer solution and emptied it with air.7. Filled 200 uL Blocking solution (20 uL / min, 4 min), washed the sample ring with PBS and emptied it with air.8. Observed baseline for 5 minutes to ensure the stability.9. The analyte was diluted with PBS-P+, the concentration was shown in the experimental results, and the sample rate was 30pL / min. The binding time of the analyte and ligand was 180 s, and the dissociation time was 300s.10. The analysis software used for the experimental results was TraceDrawer (Ridgeview Instruments AB, Sweden), and the analysis method was One to One analysis model.Results are shown in T able 13, below. Note: KD is the equilibrium dissociation constant, a ratio of kd / ka between the antibody and its antigen. KD and affinity are inversely related: the lower the KD value the higher the affinity of the antibody.Table 13: Kinetics parameters and affinity of antibody / antiqen interactionDiscussion and Conclusion

[0307] Generally, weak binding is regarded as 10-3 mM, middle binding as 10-6 M and strong binding as 10-9 nM. Proteins and small molecules typically are typically in the range of 10-3-10-6 M, weak antigen & antibodies in the range of 10-6-10-7 M and strong antigen and antibody in the range of 10-8-10-10 M. Most antibodies have KD values in the low micromolar ( 10~6) to nanomolar ( 10~7-10~9) range. High affinity antibodies are generally considered to be in the low nanomolar range (10~9) with very high affinity antibodies being in the picomolar ( 10~12) range.

[0308] All 7 antibodies tested against PThrP 1-173 had a high affinity (all reached at least 10’7to 10-8KD) for example H104-551-VHA-230-VLA and H 104-551-VHB-230-VLA had KD in the 10-8 range, indicative of a strong interaction; favourable kinetics parameters, indicative of a faster association and slower dissociation; sensorgram shape demonstrating a clear concentration-response relationship at much lower concentrations (Figs. 6A-6G).Example 9Mammosphere Formation Assay

[0309] A mammosphere growth assay was performed on MDA-231 cells prior to and following treatment with anti-PTHrP monoclonal antibodies H104-551-VHA-230-VLA and H 104-551-VHB-230- VLA.

[0310] Briefly, 5x 103cells / well MDA-MB-231 human breast cancer cells were plated into UltraLow Adherent 24-well plates(Corning® Costar®) in 1 ml of complete MammoCult™ Medium((Stem Cell Technologies). Cells were incubated at 37°C and 5% CO2 for 7 days(baseline) and mammospheres were collected and trypsinized. Severaly anti-PTHrP humanized antibodies described herein (10 micrograms / ml) and the chimera were added to the culture medium (compared to a control with no antibody) and mammospheres allowed to grow for 7 days and the number of mammospheres counted using the Lionheart system (Agilent Bio Tek), a fully automated, high throughput microscope which uses Olympus objectives.

[0311] The number of untreated mammospheres (controls) doubled (compared to day 0) whereas treatment with the humanized antibodies tested strongly inhibited mammosphere growth as compared to baseline (at least 50% decrease compared to day 0). The humanized antibodies tested performed better than the chimera in inhibiting mammosphere growth.Example 10Uses of humanized PTHrP antibodies

[0312] The present disclosure relates to inhibition of PTHrP production and / or signaling, inhibition of PTHrP isoform production and / or signaling, and inhibition of receptors and / or signaling pathways activated by the PTHrP isoform. The antibodies, immunoconjugates, nucleic acids and vectors disclosed herein can be used for inhibiting PTHrP and / or PTHrP1-173 isoform as a treatmentfor disease, for example hypercalcemia (e.g., malignancy-associated hypercalcemia) or cancer (e.g., breast cancer, lung cancer, pancreatic cancer, prostate cancer, melanoma and squamous cancers of several types). T reatment with the herein disclosed antibodies or immunoconjugates, for example, may include treating cancer, inhibiting tumour growth and / or inhibiting metastasis.

[0313] The antibodies disclosed herein can further be used for inhibition of PTHrP and / or PTHrP1-173 isoform production and / or signaling, and inhibition of receptors and / or signaling pathways activated by the PTHrP isoform in models of human hypercalcemia, for example malignancy-associated hypercalcemia, or cancer, for example, breast cancer, lung cancer, prostate cancer, pancreatic cancer, melanoma and squamous cancers of several types. These uses may include, for example, use in mouse models of human hypercalcemia, mouse models of human melanoma (e.g., transplanting A375 cells into mouse cardiac ventricle), mouse models of human breast cancer (e.g., nude mice transplanted with the human breast cancer cell line MDA-MB-435 into mammary fat pad), mouse models of human pancreatic cancer, in vitro models of human prostate cancer, in vitro models for squamous skin cancer or squamous skin cell carcinoma, models of tumor progression in keratinocytes (e.g., keratinocytes immortalized with human papilloma virus 16 to give rise to the non-tumorigenic HPK1 A cell line, which is subsequently transformed into cancer cells by overexpression of an activated H-Ras oncogene to give rise to HPK1 Aras cells that develop into a classical squamous tumor).

[0314] The present disclosure further relates to antibodies specific for PTHrP and / or PTHrPI- 173 isoforms. The antibodies disclosed herein can further be used for disease diagnosis or detection of PTHrP and / or PTHrP1-173 isoforms as indicators of disease activity, for example, hypercalcemia (e.g., malignancy-associated hypercalcemia) or cancer (e.g., breast cancer, lung cancer, pancreatic cancer, prostate cancer, melanoma and squamous cancers of several types), or as indicators of metastatic tumour spread, optionally prior to development of hypercalcemia, or as prognostic indicators of possibly effective cancer treatments, for example, by tagging or labelling the herein disclosed antibodies with a diagnostic agent (e.g., a reporter molecule, a radioisotope, or a positron-emitting radionuclide) and analyzing binding in vivo or in vitro in tissue samples using imaging.

[0315] Humanized antibodies as disclosed herein can further be used in in vivo imaging and / or for in vivo therapeutic targeting of tumours and metastatic sites expressing PTHrP and / or its isoforms, which may include, for example, tagging or labelling the herein disclosed antibodies with a detectable label (e.g., a reporter molecule, a radioisotope, or a positron-emitting radionuclide) or therapeutic agents (e.g., cytotoxic agents, prodrugs or drugs). For example, a humanized Fab as herein disclosed can be labelled with a radionuclide and administered to a subject for detecting and / or locating human PTHrP isoform in vivo via PET imaging, which can be used to diagnose the cause of hypercalcemia or cancer in the subject.

[0316] The antibodies disclosed herein can further be used to measure or assess treatment effects on tumour growth (e.g., inhibition of tumour growth), metastatic spread and / or patient survival in vivo, and / or on cell growth and / or invasion in vitro, for example, by tagging or labelling the herein disclosed antibodies with a diagnostic agent (e.g., a reporter molecule, a radioisotope, or a positron-emitting radionuclide) and analyzing binding in vivo or in vitro in biological samples using imaging or diagnostic assays or tests. For example, an amount of antibody-antigen complex in a test sample obtained from a subject can be compared to an amount of antibody-antigen complex in a further test sample or a baseline or control sample to diagnose and / or monitor disease, for example, hypercalcemia or cancer, in the subject. A humanized antibody as herein disclosed could be used to treat the subject, and the same or a distinct humanized or non-humanized antibody or fragment thereof could be used to monitor response to the treatment or progression or recurrence of disease in the subject via analysis of binding in vitro, for example identifying cancer cells in biological samples in vitro using an antibody as herein described with immunohistochemistry or immunofluorescence.

Claims

Claims:

1. An antibody or fragment thereof that specifically binds PTHrP, comprising a heavy chain variable region and a light 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, wherein amino acid sequences of said CDRs comprise the sequences:CDR-H1 : SYWX1Q, wherein: SEQ ID NO: 29,Xi = I or MCDR-H2: wherein:AIYPGDGDTSYX2X3X4FX5G, X2= A, S or T SEQ ID NO: 30,Xs = E, P or QX4= K or SX5= K or QCDR-H3: GDYDYDERAMDY, SEQ ID NO: 7,CDR-L1: RSSQSIGHSNGNTYLE, SEQ ID NO: 8,CDR-L2: KVSNRFS, SEQ ID NO: 9, andCDR-L3: FQGSHDPFT, SEQ ID NO: 10.The antibody or fragment of claim 1 , wherein the amino acid sequences of the CDRs comprise the sequences:CDR-H1: SYWMQ, SEQ ID NO: 1,CDR-H2: AIYPGDGDTSYTQKFKG, SEQ ID NO: 3,CDR-H3: GDYDYDERAMDY, SEQ ID NO: 7,CDR-L1 : RSSQSIGHSNGNTYLE, SEQ ID NO: 8,CDR-L2: KVSNRFS, SEQ ID NO: 9, andCDR-L3: FQGSHDPFT, SEQ ID NO: 10CDR-H1: SYWIQ, SEQ ID NO: 2,CDR-H2: AIYPGDGDTSYSPSFQG, SEQ ID NO: 4,CDR-H3: GDYDYDERAMDY, SEQ ID NO: 7,CDR-L1 : RSSQSIGHSNGNTYLE, SEQ ID NO: 8,CDR-L2: KVSNRFS, SEQ ID NO: 9, andCDR-L3: FQGSHDPFT, SEQ ID NO: 10CDR-H1: SYWMQ, SEQ ID NO: 1,CDR-H2: AIYPGDGDTSYAQKFQG, SEQ ID NO: 5,CDR-H3: GDYDYDERAMDY, SEQ ID NO: 7,CDR-L1 : RSSQSIGHSNGNTYLE, SEQ ID NO: 8,CDR-L2: KVSNRFS, SEQ ID NO: 9, andCDR-L3: FQGSHDPFT, SEQ ID NO: 10 ; orCDR-H1: SYWMQ, SEQ ID NO: 1,CDR-H2: AIYPGDGDTSYAEKFQG, SEQ ID NO: 6,CDR-H3: GDYDYDERAMDY, SEQ ID NO: 7,CDR-L1 : RSSQSIGHSNGNTYLE, SEQ ID NO: 8,CDR-L2: KVSNRFS, SEQ ID NO: 9, andCDR-L3: FQGSHDPFT, SEQ ID NO:

103. The antibody or fragment of 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 with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 20, wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 3 and 7, or Hi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 3 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 26, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 26, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

4. The antibody or fragment of 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 with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 21 , wherein the CDR sequences are as set forth in SEQ ID NOs: 2, 4 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 2, 4 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 26, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 26, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) a conservatively substituted amino acid sequence of i), wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

5. The antibody or fragment of 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 with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 22, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 3 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 3 and 7, and wherein the light chain variable regioncomprises: i) the amino acid sequence of SEQ ID NO: 26, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 26, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or Hi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

6. The antibody or fragment of 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 with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 23, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 5 and 7, or Hi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 5 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 26, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 26, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or Hi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

7. The antibody or fragment of 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 with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 24, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 3 and 7, or Hi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 3 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 26, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 26, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or Hi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

8. The antibody or fragment of claim 1 or 2, wherein the heavy chain variable region comprises: i) an amino acid sequence as set forth in SEQ ID NO: 25, ii) the amino acid sequence of 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 25, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 6 and 7, or Hi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 6 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 26, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 26, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or Hi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

9. The antibody or fragment of 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 with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 20, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 3 and 7, or Hi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 3 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 27, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 27, wherein the CDR sequences are asset forth in SEQ ID NOs: 8-10, or Hi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

10. The antibody or fragment of 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 with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 21 , wherein the CDR sequences are as set forth in SEQ ID NOs: 2, 4 and 7, or Hi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 2, 4 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 27, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 27, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or Hi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

11. The antibody or fragment of 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 with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 23, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 5 and 7, or Hi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 5 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 27, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 27, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or Hi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

12. The antibody or fragment of 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 with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 24, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 3 and 7, or Hi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 3 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 27, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 27, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or Hi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

13. The antibody or fragment of claim 1 or 2, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 25, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 25, wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 6 and 7, or Hi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 6 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 27, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 27, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or Hi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

14. The antibody or fragment of 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 with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 20, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 3 and 7, or Hi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 3 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 28, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 28, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or Hi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

15. The antibody or fragment of claim 1 or 2, the heavy chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 21 , ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 21, wherein the CDR sequences are as set forth in SEQ ID NOs: 2, 4 and 7, or Hi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 2, 4 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 28, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 28, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or Hi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

16. The antibody or fragment of 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 with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 23, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 5 and 7, or Hi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 5 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 28, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 28, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or Hi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

17. The antibody or fragment of 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 with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 24, wherein the CDR sequences are as set forth in SEQ ID NOs: 1, 3 and 7, or Hi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 3 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 28, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 28, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or Hi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

18. The antibody or fragment of 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 with at least 80%, at least90%, or at least 95% sequence identity to SEQ ID NO: 25, wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 6 and 7, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 1 , 6 and 7, and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 28, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 28, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10.

19. The antibody or fragment of 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 with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 73, wherein the CDR sequences are as set forth in SEQ ID NOs: 2, 4 and 7, or iii) a conservatively substituted amino acid sequence of i), and wherein the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO: 26, 27 or 28, ii) an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 26, 27 or 28, wherein the CDR sequences are as set forth in SEQ ID NOs: 8-10, or iii) a conservatively substituted amino acid sequence of i).

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

22. An immunoconjugate comprising the antibody or fragment of any one of claims 1 to 21 and a detectable label.

23. A nucleic acid encoding the antibody or fragment, antibody heavy chain variable domain, or antibody light chain variable domain of any one of claims 1 to 22.

24. The nucleic acid of claim 23, wherein the nucleic acid i) encodes a heavy chain variable domain, the nucleic acid encoding heavy chain variable domain comprising the sequence of any one of SEQ ID NOs: 11 , 12, 14, 15, or 16, or a sequence with at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to any one of SEQ ID NOs: 11 , 12, 14, 15, or 16; ii) encodes a light chain variable domain, the nucleic acid encoding light chain variable domain comprising the sequence of any one of SEQ ID NOs: 17, 18 or 19, or a sequence with at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to any one of SEQ ID NOs: 17, 18 or 19; or iii) encodes a heavy chain variable domain and a light chain variable domain, the nucleic acid encoding the heavy chain variable domain and light chain variable domain, respectively, comprising the sequences: SEQ ID NOs: 11 and 17; SEQ ID NOs:12 and 17; SEQ ID NOs: 14 and 17; SEQ ID NOs: 15 and 17, SEQ ID NOs: 16 and 17, SEQ ID NOs:12 and 18, SEQ ID NOs: 16 and 18, SEQ ID NOs: 11 and 19, SEQ ID NOs: 12 and 19, SEQ ID NOs:14 and 19, SEQ ID NOs: 15 and 19, or SEQ ID NOs: 16 and 19, or sequences with at least 70%, 80%,85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NOs: 11 and 17; SEQ ID NOs: 12 and 17;SEQ ID NOs: 14 and 17; SEQ ID NOs: 15 and 17, SEQ ID NOs: 16 and 17, SEQ ID NOs: 12 and 18,SEQ ID NOs: 16 and 18, SEQ ID NOs: 11 and 19, SEQ ID NOs: 12 and 19, SEQ ID NOs: 14 and 19,SEQ ID NOs: 15 and 19, or SEQ ID NOs: 16 and 19, optionally, wherein the nucleic acid further comprises one or more nucleotide sequences encoding one or more of the amino acid sequences of SEQ ID NOs: 62-65.

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

26. The vector of claim 25, wherein the vector is a viral vector, optionally, an adenoviral vector, an adenoviral-associated vector, or a retroviral vector, optionally a lentiviral vector.

27. A cell expressing the antibody or fragment of any one of claims 1 to 21 , or the nucleic acid of claim 23 or 24, or comprising the vector of claim 25 or 26.

28. The cell of claim 27, wherein the cell is selected from a mammalian cell, optionally a CHO cell or a HEK-293 cell, or an insect cell, optionally a Sf9 cell, Sf21 cell, T ni cell, or S2 cell.

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

30. The antibody or fragment 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 manufacture of a medicament for treating hypercalcemia or cancer in a subject in need thereof.31 . A kit comprising the antibody or fragment of any one of claims 1 to 21 , the immunoconjugate of claim 22, the nucleic acid of claim 23 or 24, the vector of claim 25 or 26, the cell of claim 27 or 28 or the composition of claim 29.

32. A use of an antibody or fragment thereof that specifically binds the human PTHrP for reducing or inhibiting activity of a human PTHrP, comprising contacting a cell or tissue expressing the 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 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, wherein amino acid sequences of said CDRs comprise the sequences:CDR-H1 : SYWXiQ, wherein: SEQ ID NO: 29,Xi = I or MCDR-H2: wherein:AIYPGDGDTSYX2X3X4FX5G, SEQ ID NO: 30,X3= E, P or QX4= K or SX5= K or QCDR-H3: GDYDYDERAMDY, SEQ ID NO: 7,CDR-L1: RSSQSIGHSNGNTYLE, SEQ ID NO: 8,CDR-L2: KVSNRFS, SEQ ID NO: 9, andCDR-L3: FQGSHDPFT, SEQ ID NO: 10, optionally wherein the antibody or fragment thereof is the antibody or fragment thereof according to any one of claims 1 to 21.

33. The use of claim 32, wherein the cell or tissue is in vivo in a subject and / or wherein the contacting comprises use of the antibody or fragment thereof to a subject in need thereof.

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

35. The use of claim 34, wherein the subject has been diagnosed with cancer, optionally breast cancer, lung cancer, colon cancer, pancreatic cancer, ovarian cancer, prostate cancer, squamous cell cancer, or melanoma.

36. The use of claim 35, wherein the cancer is pre-metastatic.

37. The use of claim 34 wherein the use is for treating hypercalcemia.

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

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