Human antibody that conjugates RET and method of use

Abstract

To provide human antibodies that bind RET and use methods thereof.SOLUTION: The invention provides a fully human antibody that binds to RET receptor tyrosine kinase, a composition comprising the antibody and use methods thereof. The antibody of the invention is useful to treat diseases, disorders or conditions associated with expression, activation or signaling of a RET receptor tyrosine kinase gene or a rearranged form thereof including cancerous conditions and pain associated with the cancer, or for alleviating the pain associated with other conditions caused by at least in part, by the RET expression, activation or signaling. The RET specific antibody may be also useful for retarding tumor cell growth or tumor cell proliferation and alleviating pain associated with cancer or other conditions. The antibody may be also useful for diagnosing a disease, a disorder or a condition associated with RET activation or signaling.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to human antibodies and antigen-binding fragments of human antibodies that specifically bind to RET (rearranged during transfection) receptor tyrosine kinase, as well as compositions containing these antibodies, and therapeutic methods using these antibodies.

[0002] Sequence List An official copy of the sequence listing is submitted electronically via EFS-Web at the same time as this specification, as an ASCII format sequence listing with the filename 10582WO01_SeqList_ST25.TXT, created on April 9, 2020, and approximately 168 kilobytes in size. The sequence listing contained in this ASCII format document is part of this specification and is incorporated herein by reference in its entirety. [Background technology]

[0003] RET (transfection rearrangement) receptor tyrosine kinase is expressed during development in a variety of tissues, including the peripheral and central nervous systems and the kidney (Arighi, E. et al, (2005), Cytokine Growth Factor Rev 16:441-467; Borrello, MG, et al. al, (2013), Expert Opin. Ther. Targets, 17(4):403-419; Golden, JP, et al. (1998), J. Comp. Neurol. 398:139-150; Golden, JP, et al. (1999), (Exp. Neurol. 158:504-528). This is also expressed in neural crest-derived cells and regulates cell proliferation, migration, and survival (Coulpier, M. et al, (2002), J Biol Chem, 277:1991-1999; Golden, JP, et al. (1998), J. Comp. Neurol. 398:139-150; Golden, JP, et al. (1999), Exp. Neurol. 158:504-528). RETno Knockout mice exhibit renal aplasia and lack intestinal neurons in the gastrointestinal tract. Both GFRα1 and GDNF knockout mice show very similar phenotypes, confirming the major role of GDNF / GFRα1 in the activation of RET signaling during development.

[0004] RET is a signaling receptor for ligands of the glial cell-derived neurotrophic factor (GDNF) family, including GDNF, artemin, neurturin, and parcefin. GDNF family ligands interact with and activate RET only in the presence of one of the four GPI-linked coreceptors known as GDNF family α receptors GFRα(1-4) (Baloh, RH, et al. (2000), Curr Opin Neurobiol 10:103-110; Borrello, MG, et al (2013), Expert Opin. Ther. Targets, 17(4):403-419). The main ligands for the coreceptors GFRα1, GFRα2, GFRα3, and GFRα4 are GDNF, neurturin (NRTN), artemin (ARTN), and parcefin (PSPN), respectively, but crosstalk between ligands and coreceptors has been observed in vitro.

[0005] The role of RET as a driver of tumorigenesis has been established by activating mutations frequently observed in multiple endocrine neoplasia syndromes MEN2A and MEN2B, as well as in familial medullary thyroid carcinoma (Mulligan, LM, et al, (1994), Nat. Genet. 6:70-74). Furthermore, a large percentage of sporadic medullary thyroid carcinomas contain somatic activation mutations in RET (Fusco, A. et al, (1987), Nature 328:170-172; Grieco, M. et al, (1990), Cell 60:557-563). These mutations affect the kinase domain or This can occur in the extracellular domain, leading to unpaired cysteine, which is thought to promote ligand-independent RET dimerization and activation. Thus, the tumorigenic potential of RET in humans has been clearly established through genetic studies.

[0006] In addition to its role in endocrine cancers, recent studies have identified RET as a potential therapeutic target in breast cancer. RET and GFRα1 are expressed in breast cancer cell lines and primary human breast cancer samples. Interestingly, RET and GFRα1 expression can be induced by estrogen in vitro. Consistent with this finding, RET and GFRα1 are preferentially expressed in the estrogen receptor-positive subset of breast cancer. Furthermore, GDNF-induced RET signaling promotes anchorage-independent growth of estrogen receptor-positive breast cancer cells, enhances the effect of estrogen on the growth and survival of these cells, and demonstrates a functional coordination between these two pathways. Thus, RET signaling appears to be a key driver of the oncogenic phenotype in breast cancer cells (Wang, C. et al (2012), Breast Cancer Res Treat 133(2):487-500; Stine, ZE, et al, (2011), Human Molecular Genetics 20(19):3746-3756) .

[0007] RET activation is initiated by the binding of GDNF to GFRα1. The GDNF / GFRα1 complex then binds to RET, leading to receptor dimerization and activation. Several small molecules exist that have the ability to inhibit RET, including a drug (vandetanib) that shows activity in patients with medullary thyroid cancer (see Wells, SA et al, (2012), J Clin Oncol 30:134-141; Leboulleux, S. et al, (2012), Lancet Oncol 13:897-905). Other small molecules that bind to RET and inhibit RET signaling also exist. This has been established (Borrello, MG, et al, (2013), Expert Opin. Ther. Targets, 17(4):403-419). Unfortunately, due to a lack of specificity, some of these compounds are Adverse events occurred during clinical trials, preventing further development.

[0008] To date, there have been no reports of therapeutic anti-RET monoclonal antibodies for use in clinical settings to treat tumors expressing RET. The studies reported herein describe the production of a fully human monoclonal antibody that binds to RET and prevents interaction between RET and one or more GDNF family members that form a complex with its corresponding co-receptor.

[0009] Figure 1 shows the domain structure of the extracellular region of RET, which consists of four cadherin-like domains followed by a cysteine-rich domain (see Borrello, MG, et al (2013), Expert Opin. Ther. Targets, 17(4):403-419). Although the structure of the active RET signaling complex is not elucidated, the GDNF / GFRα1 complex appears to contact the RET extracellular domain at multiple sites, including the fourth cadherin-like domain and the cysteine-rich domain. Therefore, antibodies targeting multiple domains of RET may potentially inhibit signaling. Antibodies against RET are described in US6861509 and US2009 / 0136502 and can be found. However, given the role that RET plays in tumor cell growth and proliferation, and the fact that there are few approved drugs to target this molecule, there is still a need for RET inhibitors, such as highly potent human antibodies that specifically bind to RET without producing adverse effects that would prevent approval for clinical use. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] U.S. Patent No. 6861509 [Patent Document 2] U.S. Patent Application Publication No. 2009 / 0136502 [Overview of the Initiative] [Means for solving the problem]

[0011] The present invention provides a fully human monoclonal antibody (mAb) or an antigen-binding fragment thereof that specifically binds to RET and inhibits the binding or interaction of RET with one or more GDNF family member ligands (GDNF, neurturin, artemin, and parcefin) that form complexes with their corresponding co-receptors (GFRα1, GFRα2, GFRα3, and GFRα4, respectively). In one embodiment, the human anti-RET antibody described herein prevents the interaction of RET with the GDNF / GFRα1 complex. In a related embodiment, the human anti-RET antibody described herein prevents the interaction of RET with the artemin / GFRα3 complex. In a related embodiment, the human anti-RET antibody described herein prevents the interaction of RET with the neurturin / GFRα2 complex or the parcefin / GFRα4 complex.

[0012] The studies described herein demonstrate that these antibodies are capable of modulating ligand-dependent RET signaling. In certain embodiments, antibodies that antagonize ligand-dependent RET signaling have been identified.

[0013] Given the role that RET plays in the development of multiple endocrine neoplasia syndromes and other cancers, the antibodies of the present invention, which antagonize / inhibit RET signaling activity, can be used in the treatment of these tumor syndromes and cancers to inhibit the growth / proliferation of tumor cells. Examples of cancerous conditions that can be treated with the RET antagonist antibodies of the present invention include, but are not limited to, thyroid tumors, lung tumors, pancreatic tumors, skin cancers, breast cancers, and leukemias. Thyroid tumors that can be treated with the antagonist anti-RET antibodies of the present invention may include papillary thyroid carcinoma (PTC) or medullary thyroid carcinoma (MTC). Medullary thyroid carcinoma that can be treated with the antagonist anti-RET antibodies of the present invention may include hereditary MTC selected from the group consisting of MEN2A, MEN2B, and familial medullary thyroid carcinoma (FMTC) syndromes, or medullary thyroid carcinoma may be sporadic MTC. The antibodies of the present invention can also be used to treat pain associated with these cancerous conditions, as well as pain associated with other diseases, disorders, or conditions in which RET activity or signaling may play a role.

[0014] Antibodies may be used as a standalone treatment or in combination with a second agent useful for treating a disease or disorder associated with RET expression. In certain embodiments, antibodies may be administered therapeutically in combination with a second agent to treat a disease or disorder, or to improve at least one symptom associated with the disease or disorder. If an antibody inhibits RET activity or signaling and its use is being investigated for treating, for example, a cancerous condition, the second agent may be a chemotherapeutic agent or a bone marrow resuscitation agent, or radiotherapy for treating a tumor. If an antibody inhibits RET activity or signaling and its use is being investigated for treating pain associated with a particular condition, and the treatment allows for the use of a second analgesic, the second agent may be any agent that is also useful for reducing pain associated with that condition, such as aspirin or another NSAID, morphine, steroids (e.g., prednisone), nerve growth factor (NGF) inhibitors (e.g., small NGF antagonists or anti-NGF antibodies), or anti-Na.v 1.7 antibody or Na v 1.7 small molecule inhibitor, Na v 1.8 antagonist (e.g., anti-Na v 1.8 antibody or Na v 1.8 small molecule inhibitor), Na v 1.9 antagonist (e.g., anti-Na v 1.9 antibody or Na v 1.9 small molecule inhibitor), cytokine inhibitor (e.g., interleukin-1 (IL-1) inhibitor (e.g., rilonacept ("IL-1 trap") or anakinra (KINERET (registered trademark)), small molecule IL-1 antagonist or anti-IL-1 antibody; IL-18 inhibitor (e.g., small molecule IL-18 antagonist or anti-IL-18 antibody); IL-6 or IL-6R inhibitor (e.g., small molecule IL-6 antagonist, anti-IL-6 antibody, or anti-IL-6 receptor antibody), caspase-1 inhibitor, p38, IKKI / 2, CTLA-4Ig, or an opioid.

[0015] The antibodies of the present invention can be full-length (e.g., IgG1 or IgG4 antibodies) or may contain only the antigen-binding portion (e.g., Fab, F(ab')2, or scFv fragments) and may be modified to affect functionality, e.g., to eliminate residual effector function (Reddy et al., (2000), J. Immunol. 164:1925-1933).

[0016] Thus, in a first aspect, the present invention is an isolated human monoclonal antibody or an antigen-binding fragment thereof that specifically binds to the RET (Rearranged during Transfection) receptor tyrosine kinase, the antibody having the following characteristics: (a) being a fully human antibody; (b) having a K -7 in the range of about 1.0×10 [[ID=;30]] -12 M to about 1.0×10 D M as measured by surface plasmon resonance; (c) Inhibiting or blocking the binding or interaction of RET with one or more GDNF family member ligands (GDNF, neurturin, artemin, and parcefin) that form a complex with its corresponding co-receptor (GFRα1, GFRα2, GFRα3, and GFRα4, respectively); (d) Inhibiting RET signaling mediated by one or more GDNF family member ligands selected from GDNF, neurturin, artemin, and parcephin; (e) To enhance the internal translocation / degradation of RET receptors after antibody binding to them; (f) Containing a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, and 290; or (g) Containing a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, and 298. The present invention provides an isolated human monoclonal antibody or its antigen-binding fragment having one or more of the following:

[0017] In one embodiment, an isolated human monoclonal antibody or its antigen-binding fragment that specifically binds to RET binds human RET to the GDNF:GFRα1 cocomplex in an IC50 range of approximately 100 pM to approximately 7.0 nM. 50 Block by value.

[0018] In related embodiments, an isolated human monoclonal antibody or its antigen-binding fragment that specifically binds to RET binds human RET to the GDNF:GFRα1 cocomplex in an IC50 range of approximately 250 pM to approximately 5.2 nM. 50 Block by value.

[0019] In one embodiment, an isolated human monoclonal antibody or its antigen-binding fragment that specifically binds to RET blocks the binding of human RET to the GDNF:GFRα1 cocomplex by approximately 40% to 100%.

[0020] In related embodiments, isolated human monoclonal antibodies or their antigen-binding fragments that specifically bind to RET block the binding of human RET to the GDNF:GFRα1 cocomplex by approximately 57% to approximately 97%.

[0021] In one embodiment, an isolated human monoclonal antibody or its antigen-binding fragment that specifically binds to RET inhibits GDNF-mediated RET signaling in the IC range of approximately 50 pM to 100 nM. 50 Inhibit it with a value.

[0022] In a related embodiment, an isolated human monoclonal antibody or its antigen-binding fragment that specifically binds to RET inhibits GDNF-mediated RET signaling in the range of IC values ​​greater than approximately 143 pM to 100 nM. 50 Inhibit it with a value.

[0023] In one embodiment, an isolated human monoclonal antibody or its antigen-binding fragment that specifically binds to RET inhibits GDNF-mediated RET signaling by approximately 40% to 100%.

[0024] In related embodiments, isolated human monoclonal antibodies or their antigen-binding fragments that specifically bind to RET inhibit GDNF-mediated RET signaling by approximately 60% to 100%.

[0025] In one embodiment, an isolated human monoclonal antibody or its antigen-binding fragment that specifically binds to RET transmits artemin-mediated RET signaling in an IC range of about 100 pM to about 500 nM. 50 Inhibit it with a value.

[0026] In a related embodiment, an isolated human monoclonal antibody or its antigen-binding fragment that specifically binds to RET is used to mediated artemin-mediated RET signaling in an IC range of approximately 250 pM to approximately 341 nM. 50 Inhibit it with a value.

[0027] In one embodiment, an isolated human monoclonal antibody or its antigen-binding fragment that specifically binds to RET inhibits artemin-mediated RET signaling by approximately 57% to 100%.

[0028] In one embodiment, an isolated human monoclonal antibody or its antigen-binding fragment that specifically binds to RET includes a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, and 290.

[0029] In one embodiment, an isolated human monoclonal antibody or its antigen-binding fragment that specifically binds to RET includes a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, and 298.

[0030] In one embodiment, an isolated human monoclonal antibody or its antigen-binding fragment that specifically binds to RET includes a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, and 290; and a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, and 298.

[0031] In one embodiment, an isolated human monoclonal antibody or its antigen-binding fragment that specifically binds to RET comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 122, 130 / 138, 146 / 154, 162 / 170, 178 / 186, 194 / 202, 210 / 218, 226 / 234, 242 / 250, 258 / 266, 274 / 282, and 290 / 298.

[0032] In one embodiment, an isolated human monoclonal antibody or its antigen-binding fragment that specifically binds to RET includes an HCVR containing three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within an HCVR amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, and 290; and an LCVR containing three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within an LCVR amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, and 298.

[0033] Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify CDR boundaries include, for example, Kabat's definition, Chothia's definition, and AbM's definition. Generally, Kabat's definition is based on sequence variability, Chothia's definition is based on the location of structural loop regions, and AbM's definition is a compromise between Kabat's and Chothia's approaches. See, for example, Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., (1997), J. Mol. Biol. 273:927-948; and Martin et al., (1989), Proc. Natl. Acad. Sci. USA 86:9268-9272. Public databases also include CDR distribution within antibodies. It can be used to identify columns.

[0034] In one embodiment, an isolated human monoclonal antibody or its antigen-binding fragment that specifically binds to RET is: (a) HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 228, 244, 260, 276, and 292; (b) HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 230, 246, 262, 278, and 294; (c) HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 232, 248, 264, 280, and 296; (d) LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268, 284, and 300; (e) LCDR2 domains having amino acid sequences selected from the group consisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 222, 238, 254, 270, 286, and 302; and (f) LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 224, 240, 256, 272, 288, and 304 Includes.

[0035] In one embodiment, the present invention provides an antibody or antigen-binding fragment comprising a heavy chain and light chain sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 122, 130 / 138, 146 / 154, 162 / 170, 178 / 186, 194 / 202, 210 / 218, 226 / 234, 242 / 250, 258 / 266, 274 / 282, and 290 / 298, and an isolated antibody or antigen-binding fragment that specifically binds to RET, and competes for specific binding to RET.

[0036] In one embodiment, the present invention provides an isolated antibody or its antigen-binding fragment that specifically binds to RET, which binds to the same epitope on RET recognized by an antibody comprising a heavy-chain and light-chain sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 122, 130 / 138, 146 / 154, 162 / 170, 178 / 186, 194 / 202, 210 / 218, 226 / 234, 242 / 250, 258 / 266, 274 / 282, and 290 / 298.

[0037] In one embodiment, the present invention relates to a fully human monoclonal antibody or an antigen-binding fragment thereof that specifically binds to RET, characterized by: (i) comprising an HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, and 290, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (i i) Including an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, and 298, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (iii) SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 232, 2 An amino acid sequence selected from the group consisting of 48, 264, 280, and 296, or an HCDR3 domain having a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 224, 240, 256, 272, 288, and 304, or at least 90%, at least 95%, (iv) It includes an LCDR3 domain having a substantially similar sequence with 98% or at least 99% sequence identity; (iv) It includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 228, 244, 260, 276, and 292, or an HCDR1 domain having a substantially similar sequence with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity;(v) an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 230, 246, 262, 278, and 294, or an HCDR2 domain having a substantially similar sequence with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (vi) an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268, 284, and 300 (vii) an LCDR1 domain having a column or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 222, 238, 254, 270, 286, and 302, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (viii) approximately 1 × 10; -7 M ~ approx. 1×10 -12 K in the range of M D (ix) The binding of human RET to the GDNF:GFRα1 cocomplex is less than approximately 5.2 nM IC 50 The ability to block at a certain value; or (x) ligand-dependent RET signaling in the range of IC greater than approximately 143 pM to 100 nM 50 The present invention provides a fully human monoclonal antibody or its antigen-binding fragment that demonstrates one or more of the ability to inhibit by approximately 60-100% in terms of value.

[0038] In a second aspect, the present invention provides a nucleic acid molecule encoding an antibody or a fragment thereof that specifically binds to RET. Recombinant expression vectors having the nucleic acid of the present invention, and host cells into which such vectors have been introduced, are also included by the present invention, as are methods for producing antibodies by culturing host cells under conditions that enable antibody production and recovering the produced antibodies.

[0039] In one embodiment, the present invention provides an antibody or fragment thereof containing an HCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 17, 33, 49, 65, 81, 97, 113, 129, 145, 161, 177, 193, 209, 225, 241, 257, 273, and 289, or substantially identical thereto having at least 90%, at least 95%, at least 98%, or at least 99% homology.

[0040] In one embodiment, the antibody or fragment thereof further comprises an LCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 9, 25, 41, 57, 73, 89, 105, 121, 137, 153, 169, 185, 201, 217, 233, 249, 265, 281, and 297, or substantially identical thereto having at least 90%, at least 95%, at least 98%, or at least 99% homology.

[0041] In one embodiment, the present invention also relates to an HCDR3 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7, 23, 39, 55, 71, 87, 103, 119, 135, 151, 167, 183, 199, 215, 231, 247, 263, 279, and 295, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and sequence We also provide antibodies or antigen-binding fragments of antibodies containing an LCDR3 domain encoded by a nucleotide sequence selected from the group consisting of numbers 15, 31, 47, 63, 79, 95, 111, 127, 143, 159, 175, 191, 207, 223, 239, 255, 271, 287, and 303, or by a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0042] In one embodiment, the present invention relates to an HCDR1 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 19, 35, 51, 67, 83, 99, 115, 131, 147, 163, 179, 195, 211, 227, 243, 259, 275, and 291, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; an HCDR2 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 5, 21, 37, 53, 69, 85, 101, 117, 133, 149, 165, 181, 197, 213, 229, 245, 261, 277, and 293, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; SEQ ID NOs: 11, The present invention provides an antibody or fragment thereof comprising an LCDR1 domain encoded by a nucleotide sequence selected from the group consisting of 27, 43, 59, 75, 91, 107, 123, 139, 155, 171, 187, 203, 219, 235, 251, 267, 283, and 299, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and an LCDR2 domain further comprising an LCDR2 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 13, 29, 45, 61, 77, 93, 109, 125, 141, 157, 173, 189, 205, 221, 237, 253, 269, 285, and 301, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0043] In a third aspect, the present invention relates to V H , D H , and J H HCVR, and V, are encoded by nucleotide sequence segments derived from germline sequences. K and J K It features a RET-specific human antibody or antigen-binding fragment containing an LCVR encoded by a nucleotide sequence segment derived from a germline sequence.

[0044] The present invention encompasses antibodies having modified glycosylation patterns. In some applications, modifications to remove undesirable glycosylation sites, or removal of fucose moieties to enhance antibody-dependent cell-mediated cytotoxicity (ADCC) function, may be useful (see Shield et al. (2002) JBC 277:26733). In other applications, complement-dependent cell-mediated cytotoxicity ( Galactosylation can be performed to modify the CDC.

[0045] In a fourth aspect, the present invention provides a pharmaceutical composition comprising at least one isolated fully human monoclonal antibody or its antigen-binding fragment that binds to RET, and a pharmaceutically acceptable carrier or diluent. In one embodiment, the present invention provides a pharmaceutical composition comprising two fully human monoclonal antibodies or their antigen-binding fragments that bind to the same epitope on RET or to two different epitopes, and a pharmaceutically acceptable carrier or diluent. It should be understood that any combination of antibodies described herein may be used in a pharmaceutical composition to achieve desired results in a patient population requiring such treatment. For example, two antibodies that recognize and / or bind to RET may be used in a composition.

[0046] In one embodiment, the composition includes an antibody conjugated to RET and having an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 122, 130 / 138, 146 / 154, 162 / 170, 178 / 186, 194 / 202, 210 / 218, 226 / 234, 242 / 250, 258 / 266, 274 / 282, and 290 / 298.

[0047] In one embodiment, the pharmaceutical composition comprises at least one antibody conjugating RET, wherein the antibody contains three heavy chain complementarity determining regions (HCDR1, It includes HCDR2 and HCDR3, as well as three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within any one of the light chain variable region (LCVR) amino acid sequences selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, and 298.

[0048] In one embodiment, at least one activity or function related to RET expressed on a cell can be inhibited using the antibody of the present invention or a composition containing one or more antibodies of the present invention. In one embodiment, the cell may be a tumor cell. In one embodiment, the activity may be cellular signaling.

[0049] In one embodiment, the present invention features a composition comprising an antibody or antigen-binding fragment of an antibody of the present invention and a second therapeutic agent.

[0050] The second therapeutic agent may be a small molecule drug, a protein / polypeptide, an antibody, a nucleic acid molecule, such as an antisense molecule, or siRNA. The second therapeutic agent may be synthetic or naturally derived.

[0051] The second therapeutic agent may be any agent that can be advantageously combined with the antibody or fragment of the present invention. For example, if the anti-RET antibody is an inhibitor of RET used to treat a cancerous condition, the second agent may be selected from chemotherapeutic agents, radionuclides, RET-specific siRNAs, RET-specific second antibodies, small-molecule RET inhibitors, and bone marrow resuscitation agents, such as G-CSF, GM-CSF, or M-CSF, or biological agents having colony-stimulating or bone marrow resuscitation activity. In certain embodiments, the second therapeutic agent may be an agent that helps to counteract or reduce any possible side effects that may occur, if side effects related to the antibody or antigen-binding fragment of the antibody of the present invention may occur. In certain embodiments, the second therapeutic agent may be an agent that is useful for reducing pain associated with a particular condition characterized by pain and / or inflammation. Such a second agent may be a nerve growth factor (NGF) inhibitor (e.g., a small-molecule NGF antagonist or anti-NGF antibody), aspirin or another NSAID, morphine, a steroid (e.g., prednisone), or an anti-Na. v 1.7 antibody or Na v 1.7 Small molecule inhibitors, Na v 1.8 Antagonists (e.g., anti-Na) v 1.8 antibody or Na v 1.8 small molecule inhibitors), Na v 1.9 Antagonists (e.g., anti-Na) v 1.9 antibody or Na v This may include small molecule inhibitors (1.9), cytokine inhibitors (e.g., interleukin-1 (IL-1) inhibitors (e.g., lilonacept ("IL-1 trap"); Regeneron), or anakinra (KINERET®, Amgen), small molecule IL-1 antagonists or anti-IL-1 antibodies; IL-18 inhibitors (e.g., small molecule IL-18 antagonists or anti-IL-18 antibodies); IL-6 or IL-6R inhibitors (e.g., small molecule IL-6 antagonists, anti-IL-6 antibodies, or anti-IL-6 receptor antibodies), caspase-1 inhibitors, p38, IKK1 / 2, CTLA-4Ig, or opioids).

[0052] Similarly, the antibodies and pharmaceutically acceptable compositions of the present invention are recognized as being usable in combination therapy, i.e., the antibodies and pharmaceutically acceptable compositions can be administered simultaneously with, before, or after one or more other desired therapeutic agents or medical procedures. Specific combinations of treatments (therapeutic agents or procedures) for use in a combination regimen take into account the suitability of the desired therapeutic agents and / or procedures, as well as the desired therapeutic effect to be achieved. Similarly, the treatments used are recognized as being able to achieve the desired effect with respect to the same disorder (e.g., an antibody can be administered simultaneously with another agent used to treat the same disorder) or as being able to achieve different effects (e.g., control of any adverse effects). When used herein, additional therapeutic agents that are normally administered to treat or prevent a particular disease or condition are appropriate for the disease or condition being treated.

[0053] When a small molecule RET inhibitor is intended to be combined with the antibody of the present invention, the small molecule RET inhibitor may be vandetanib, sorafenib, sunitinib, cabozantinib, motesanib, RPI-1, PP-1 and NVP-AST478, cediranib, (AZD2171), gefitinib, erlotinib, SU14813, batalanib, (BAY43-9006), XL-647, XL-999, or AG-013736. The following can be selected from the group consisting of BIBF1120, TSU68, GW786034, AEE788, CP-547632, KRN951, CHIR258, CEP-7055, OSI-930, ABT-869, E7080, ZK-304709, BAY57-9352, L-21649, BMS582664, XL-880, XL-184, XL-820, RPI-1, PP-1, and NVP-AST478.

[0054] When multiple therapeutic drugs are administered simultaneously, the dosage may be adjusted accordingly, as is recognized in the relevant technical field.

[0055] A fifth aspect of the present invention provides a method for treating a disorder or condition related to the expression, activation or signaling of the RET receptor tyrosine kinase gene or a rearranged form thereof, or pain associated with such a disorder or condition, comprising administering to a patient in need an antibody of one of the anti-RET antibodies described herein or an antigen-binding fragment thereof, together with a pharmaceutically acceptable carrier or diluent.

[0056] In one embodiment, the disorder or condition is a cancer selected from the group consisting of thyroid cancer, lung cancer, pancreatic cancer, skin cancer, breast cancer, and blood-derived cancers. In one embodiment, the disorder or condition related to the expression, activation, or signaling of the RET receptor tyrosine kinase gene or a rearranged form thereof is acute pain, chronic pain, neuropathic pain, inflammatory pain, arthritis, osteoarthritis, migraine, cluster headache, trigeminal neuralgia, herpetic neuralgia, systemic neuralgia, neurodegenerative disorders, neuroendocrine disorders, visceral pain, acute gout The following conditions are selected from the group consisting of postherpetic neuralgia, diabetic neuropathy, sciatica, back pain, head and neck pain, severe or intractable pain, breakthrough pain, postoperative pain, toothache, rhinitis, cancer pain, or bladder dysfunction.

[0057] In a related aspect, the present invention provides a method for inhibiting tumor growth or proliferation of tumor cells, wherein the tumor or tumor cells express RET or a rearranged form thereof, and the method comprises administering an antibody of the present invention or an antigen-binding fragment thereof to a patient in need thereof.

[0058] In one embodiment, the tumor is a solid tumor or a tumor of blood origin.

[0059] In one embodiment, the solid tumor is selected from the group consisting of thyroid tumors, lung tumors, pancreatic tumors, skin tumors, and breast tumors.

[0060] In one embodiment, the thyroid tumor is either papillary thyroid carcinoma (PTC) or medullary thyroid carcinoma (MTC).

[0061] In one embodiment, the medullary thyroid carcinoma is a hereditary MTC selected from the group consisting of MEN2A, MEN2B, and familial medullary thyroid carcinoma (FMTC) syndrome, or the medullary thyroid carcinoma is a sporadic MTC.

[0062] In one embodiment, the lung tumor is lung adenocarcinoma.

[0063] In one embodiment, the lung tumor is non-small cell lung cancer (NSCLC).

[0064] In one embodiment, the skin tumor is a melanoma.

[0065] In one embodiment, the tumor of blood origin is leukemia.

[0066] In one embodiment, the leukemia is chronic myelomonocytic leukemia.

[0067] In related embodiments, the present invention provides a method for downregulating RET expression and / or function, comprising administering an antibody of the present invention or an antigen-binding fragment thereof.

[0068] In one embodiment, downregulation of RET expression and / or function results in downregulation of a downstream signaling pathway selected from the group consisting of the RAS / RAF / ERK and PI3K pathways. In a particular embodiment, downregulation of RET expression and / or function results in downregulation of a signaling pathway selected from the group consisting of the PKC, SRC, and STAT3 pathways.

[0069] In one embodiment, the anti-RET antibody of the present invention may interfere with or prevent the interaction between RET and one or more GDNF family member ligands (GDNF, neurturin, artemin, and parcefin) that form complexes with their corresponding co-receptors (GFRα1, GFRα2, GFRα3, and GFRα4, respectively). In one embodiment, the human anti-RET antibody described herein may interfere with or prevent the interaction of RET with the GDNF / GFRα1 complex. In a related embodiment, the human anti-RET antibody described herein may interfere with or prevent the interaction of RET with the artemin / GFRα3 complex. In another related embodiment, the human anti-RET antibody described herein may interfere with or prevent the interaction of RET with the neurturin / GFRα2 or parcefin / GFRα4 complex.

[0070] Once activated, RET recruits a diverse range of signaling molecules that mediate biological responses. RET can activate various signaling pathways, such as RAS / RAF / ERK (extracellular signal-regulated kinases), phosphatidylinositol 3-kinase (PI3K) / AKT, PKC, and SRC. These signaling pathways are activated via the binding of adapter proteins to intracellular tyrosine residues of RET phosphorylated by their own kinase activity.

[0071] Therefore, in certain embodiments of the present invention, the anti-RET antibody of the present invention may block biological responses at least partially attributable to the activation of other signaling pathways by RET. In certain embodiments, the anti-RET antibody of the present invention may interfere with signal transduction through pathways involving RET and RAS. In certain embodiments, the anti-RET antibody may interfere with cell proliferation, migration, or invasion, or with the phosphorylation of ERK1 / 2 (extracellular signal-regulated kinase 1 / 2). In some embodiments, the anti-RET antibody may interfere with signal transduction through pathways involving RET and PI3K (phosphatidylinositol-3-kinase). In certain embodiments, the anti-RET antibody may interfere with cell proliferation, migration, or invasion, or with the phosphorylation of Akt (protein kinase B).

[0072] The antibody or antigen-binding fragment may be administered to the patient in combination with a second therapeutic agent suitable for treating the disease, disorder, or condition. If the disease or condition treated by the anti-RET antibody is a cancerous condition, the second therapeutic agent may be selected from the group consisting of chemotherapeutic agents, radionuclides (alone or as part of a drug-targeted regimen), antibody-drug conjugates, small molecule RET inhibitors, antitumor agents, RET-specific siRNAs, and RET-specific second antibodies. If the anti-RET antibody is intended to treat pain associated with a cancerous condition, or pain associated with other conditions that may be at least partially attributable to RET activation or signaling, the second therapeutic agent may be: nerve growth factor (NGF) inhibitors (e.g., small molecule NGF antagonists or anti-NGF antibodies), aspirin or another NSAID, morphine, steroids (e.g., prednisone), anti-Na. v 1.7 antibody or Na v 1.7 Small molecule inhibitors, Na v 1.8 Antagonists (e.g., anti-Na) v 1.8 antibody or Na v 1.8 small molecule inhibitors), Na v 1.9 Antagonists (e.g., anti-Na) v 1.9 antibody or Na vOne or more of the following may be selected: 1.9 small molecule inhibitors), cytokine inhibitors (e.g., interleukin-1 (IL-1) inhibitors (e.g., lilonacept ("IL-1 trap"); Regeneron), or anakinra (KINERET®, Amgen), small molecule IL-1 antagonists or anti-IL-1 antibodies; IL-18 inhibitors (e.g., small molecule IL-18 antagonists or anti-IL-18 antibodies); IL-6 or IL-6R inhibitors (e.g., small molecule IL-6 antagonists, anti-IL-6 antibodies, or anti-IL-6 receptor antibodies), caspase-1 inhibitors, p38, IKK1 / 2, CTLA-4Ig, or opioids).

[0073] Other embodiments will become apparent from the summary of the detailed description below. [Brief explanation of the drawing]

[0074] [Figure 1] Figure 1 is a schematic diagram of the human RET receptor. [Modes for carrying out the invention]

[0075] Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, as the methods and conditions may vary. Similarly, the technical terms used herein are for the purpose of describing only specific embodiments and are not intended to limit the scope of the present invention, as it is limited only by the appended claims.

[0076] Unless otherwise defined, all scientific and technical terms used herein have the same meanings as commonly understood by those skilled in the art in the field to which this invention pertains. Where used herein, the term “about” in relation to a particular enumerated number means that the value may vary by no more than 1% from the enumerated value. For example, where used herein, the expression “about 100” includes 99 and 101, as well as all values ​​in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0077] Any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, but preferred methods and materials are described below. All publications referenced herein are incorporated herein by reference in their entirety.

[0078] definition "Transfection-induced rearrangement," also known as "RET," is a receptor tyrosine kinase expressed during development in a variety of tissues, including the peripheral and central nervous systems, as well as the kidneys. The RET oncogene was identified in 1985 by Takahashi et al., who reported a novel gene rearrangement exhibiting transform activity in NIH / 3T3 cells transfected with human lymphoma DNA (Takahashi, M., et al., (1985)). See Cell, 42:581-588). RET was later confirmed to be an oncogene. This protein is rearranged somatically in the DNA of papillary thyroid carcinoma (PTC) patients and was later called RET / PTC (Fusco, A. et al., (1987), Nature, 328:170-172; Grieco, M. et al., (1990), Cell, 60:557-63). The RET protein consists of a cytoplasmic portion divided by a 27-amino acid insertion into three domains: an extracellular ligand-binding domain, a hydrophobic transmembrane domain, and a tyrosine kinase domain (see Figure 1). There are two main isoforms of RET produced by alternative splicing. The short and long RET isoforms are distinguished by 9 and 51 unrelated C-terminal amino acids, and are called RET9 and RET51, respectively. They are highly conserved across a wide range of species (Carter, MT, et al. (2001), Cytogenet Cell Genet, 95:169-76). All isoforms exhibit transform activity as determined by a focus formation assay (Rossel, M. et al. (1997), Oncogene, 14:265-75).

[0079] The cDNA sequence and amino acid sequence of isoform A of RET (also known as RET51) are available in GenBank under accession numbers NM_020975.4 and NP_066124.1, respectively, and are provided herein as Sequence IDs 309 and 310, respectively.

[0080] The cDNA sequence and amino acid sequence of RET isoform C (also known as RET9) are provided in GenBank under accession numbers NM_020630.4 and NP_065681.1, respectively, and are provided herein as SEQ ID NOs. 311 and 312, respectively. RET or its immunogenic fragments may be used to prepare human monoclonal antibodies specific to RET. RET proteins or fragments may be produced by recombination using standard methods known in the art. Exemplary fusion proteins containing the ectodomain of RET are shown as SEQ ID NOs. 305, 306, 307, and 309. These fusion proteins may be used as immunogens or to target therapeutic agents to cells or tissues expressing RET.

[0081] RET is a signaling receptor for ligands of the "glial cell-derived neurotrophic factor (GDNF) family," which includes GDNF (see GenBank accession number NP_000505.1), artemin (see GenBank accession number Q5T4W7), neurturin (see GenBank accession number NM_004558), and parcefin (see GenBank accession number AF040962). GDNF family ligands are known as GDNF family α receptors, specifically GFRα1 (see GenBank accession number NP_005255.1), GFRα2 (see GenBank accession number NM_001495.4), GFRα3 (see GenBank accession number NP_001487.2), and GFRα4 (see GenBank accession number NM_022139 for GFRα4a and NM_145762.2 for GFRα4b) (Baloh, RH, et al. (2000), Curr Opin Neurobiol 10:103-110; Borrello, MG, et al (2013), Expert Opin. Ther. Targets, 17(4):403-419). They are present in the presence of or complex with one of these four GPI-linked "coreceptors." It interacts with and activates RET only when it has formed a body. The main ligands for the coreceptors GFRα1, GFRα2, GFRα3, and GFRα4 are GDNF, neurturin (NRTN), artemin (ARTN), and parcefin (PSPN), respectively.

[0082] The term “IC 50 "Half-maximal inhibitory concentration" refers to the value that measures the effectiveness of a compound's (e.g., anti-RET antibody) inhibition for biological or biochemical utility. This quantitative measure indicates the amount of a particular inhibitor required to inhibit a given biological process by half.

[0083] As used herein, the terms “to treat,” “treatment,” and “to treat” mean reducing the progression of a disease, disorder, or condition that is partially caused by or related to RET expression in cells or tissues in a subject, for example, slowing the growth rate of tumor cells in a patient with a tumor that expresses RET upon administration of the antagonist / inhibitory antibody of the present invention, or reducing pain associated with a cancerous condition, or any other disease or condition that is at least partially caused by RET expression.

[0084] As used herein, the terms “prevent,” “prevent,” and “prevent” mean a disease, disorder, or condition partially caused by or related to RET expression in the cells or tissues of interest, such as the inhibition of the development or progression of a particular cancer, or the inhibition of tissue damage occurring in a patient after an injury, or the inhibition or improvement of pain associated with a disease or condition partially caused by RET expression.

[0085] The term “antibody,” as used herein, is intended to refer to an immunoglobulin molecule consisting of four polypeptide chains, i.e., two heavy (H) chains and two light (L) chains interconnected by disulfide bonds (i.e., a “complete antibody molecule”), as well as its polymer (e.g., IgM) or its antigen-binding fragment. Each heavy chain has a heavy chain variable region ("HCVR" or "V"). H) and heavy chain constant region (domain C H 1, C H 2, and C H It consists of 3). Each light chain is composed of a light chain variable region ("LCVR" or "V"). L ) and light chain steady region (C L ) is composed of V H and V L The region can be further subdivided into highly variable regions called complementary determination regions (CDRs), which are dotted with more conserved regions called framework regions (FRs). H and V L It consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the present invention, the FRs of the antibody (or its antigen-binding fragment) may be identical to the human germline sequence or may be modified naturally or synthetically. The amino acid consensus sequence may be defined based on a parallel analysis of two or more CDRs.

[0086] Substitution of one or more CDR residues or omission of one or more CDRs is also possible. Antibodies that can bind without one or two CDRs have been described in scientific papers. Padlan et al. (1995 FASEB J. 9:133-139) analyzed the contact region between an antibody and its antigen based on published crystal structures and concluded that only about one-fifth to one-third of the CDR residues actually contact the antigen. Padlan also found many antibodies in which one or two CDRs do not have amino acids that contact the antigen (see also Vajdos et al. 2002 J Mol Biol 320:415-428).

[0087] CDR residues not in contact with the antigen can be identified by molecular modeling and / or empirically based on previous studies from the region of the Kabat CDR located outside the Chothia CDR (e.g., residues H60-H65 in CDRH2 are often unnecessary). If a CDR or its residues are omitted, they are usually replaced by an amino acid occupying the corresponding position in another human antibody sequence or a consensus of such a sequence. The position of the substitution within the CDR and the amino acid to be substituted can also be selected empirically. Empirical substitutions can be conserved or non-conserved.

[0088] The fully human monoclonal antibodies disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR region of the heavy and light chain variable domains compared to the corresponding germline sequence. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from public antibody sequence databases. The present invention includes antibodies derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more frameworks and / or CDR regions mutate to a corresponding residue in the germline sequence from which the antibody originates, or to a corresponding residue in another human germline sequence, or to a conserved amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as “germline mutations”), and antigen-binding fragments thereof. Those skilled in the art can readily produce a number of antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof, starting from the heavy and light chain variable region sequences disclosed herein. In certain embodiments, V H and / or V LIn other embodiments, all of the framework and / or CDR residues within the domain undergo a reversion to residues found in the original germline sequence from which the antibody originates. In other embodiments, only certain residues, for example, only mutant residues found within the first eight amino acids of FR1, or within the last eight amino acids of FR4, or only mutant residues found within CDR1, CDR2, or CDR3 undergo a reversion to the original germline sequence. In other embodiments, one or more of the framework and / or CDR residues mutate to corresponding residues in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody originally originates). Furthermore, the antibody of the present invention may contain any combination of two or more germline mutations within the framework and / or CDR region, for example, certain individual residues mutate to corresponding residues in a particular germline sequence, while certain other residues different from the original germline sequence are maintained or mutate to corresponding residues in a different germline sequence. After obtaining them, antibodies and antigen-binding fragments containing one or more germline mutations can be easily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (if applicable), or reduced immunogenicity. Antibodies and antigen-binding fragments obtained by this general method are included in the present invention.

[0089] The present invention also includes complete monoclonal antibodies comprising a variant of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present invention includes antibodies having HCVR, LCVR, and / or CDR amino acid sequences having, for example, fewer than 10, fewer than 8, fewer than 6, or fewer than 4 conservative amino acid substitutions compared to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0090] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human mAbs of the present invention may include, for example, amino acid residues not encoded by human germline immunoglobulin sequences (mutations introduced, for example, by random or site-directed mutagenesis in vitro or by somatic mutation in vivo) in the CDR, particularly CDR3. However, as used herein, the term "human antibody" is not intended to include mAbs in which a CDR sequence derived from the germline of another mammalian species (e.g., mouse) is grafted onto a human FR sequence.

[0091] The terms "specifically bind" or "specifically bind to ~" mean that an antibody or its antigen-binding fragment forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is at least approximately 1 × 10⁻⁶. -6 Equilibrium dissociation constants M or less (e.g., smaller K) D Antibodies can be characterized by exhibiting stronger binding. Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis and surface plasmon resonance. As described herein, antibodies that specifically bind to RET have been identified by surface plasmon resonance, e.g., BIACORE®. Furthermore, polyspecific antibodies that bind to the RET protein and one or more additional antigens, or bispecific antibodies that bind to two different regions of RET, are nevertheless considered to be “specifically binding” antibodies as used herein.

[0092] The term "high affinity" refers to an antibody that, when measured by surface plasmon resonance, e.g., BIACORE® or solution affinity ELISA, has at least 10% affinity. -7 M, at least 10 -8 M; preferably 10 -9 M;comfort10 -10 M, more comfortable 10 -11 M, more comfortable 10 -12 M's K DThis refers to mAbs that have binding affinity to RET, which is expressed as such.

[0093] The term "slow offrate," "Koff," or "kd" refers to a surface plasmon resonance, such as 1 × 10⁻¹⁶, as determined by BIACORE®. -3 s -1 Or less, preferably 1 × 10 -4 s -1 This refers to an antibody that dissociates from RET with a rate constant of less than or equal to that of RET.

[0094] The terms “antigen-binding moiety” and “antigen-binding fragment” of an antibody, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically modified polypeptide or glycoprotein that specifically binds to an antigen to form a complex. The terms “antigen-binding moiety” or “antibody fragment” of an antibody, as used herein, refer to one or more fragments of an antibody that retain the ability to bind to RET.

[0095] In specific embodiments, the antibody or antibody fragment of the present invention may be conjugated to a therapeutic portion, such as a small molecule RET inhibitor, an antitumor agent, a radionuclide, a growth factor, a bone marrow resuscitation agent, or a colony-stimulating factor, or any other therapeutic portion useful for treating diseases, disorders, or conditions related to RET expression, or damaged tissue, such as cancer ("immunoconjugate" or "antibody-drug conjugate").

[0096] As used herein, “isolated antibody” is intended to mean an antibody that substantially does not contain other antibodies (Abs) having different antigen specificities (for example, an isolated antibody that specifically binds RET or a fragment thereof substantially does not contain any Abs that specifically bind antigens other than RET).

[0097] When used herein, “blocking antibody” or “neutralizing antibody” (or “antibody that neutralizes RET activity”) is intended to refer to an antibody whose binding to RET results in the inhibition of at least one biological activity of RET, such as cellular signaling. For example, the antibodies of the present invention may help block the binding of RET to its ligand or one of its GFRα coreceptors, or may prevent or treat diseases associated with RET expression. Alternatively, the antibodies of the present invention may demonstrate the ability to improve at least one symptom of a disease or condition associated with RET expression. This inhibition of the biological activity of RET can be evaluated by measuring one or more indicators of RET biological activity by one or more of several standard in vitro assays (e.g., any of the assays described herein) or by one or more in vivo assays known in the art (e.g., animal models that examine the inhibition of tumor cell growth in vivo) after administration of one or more of the antibodies described herein.

[0098] As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that enables real-time analysis of biomolecular interactions by detecting changes in protein concentration within a biosensor matrix, for example, using BIACORE® systems (Pharmacia Biosensor AB, Uppsala, Sweden, and Piscataway, NJ).

[0099] The term “K D When used herein, is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

[0100] The term “epitope” refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and have different biological effects. The term “epitope” also refers to the site on an antigen to which B and / or T cells respond. This also refers to the region of the antigen to which the antibody binds. Epitopes can be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and have residues that directly contribute to the affinity of the interaction. Epitopes can also be conformational, consisting of nonlinear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, may have specific three-dimensional structural features and / or specific charge features.

[0101] The term “substantial identity,” or “substantially identical,” when referring to a nucleic acid or fragment thereof, means that, when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions, nucleotide sequence identity exists in at least about 90%, more preferably at least about 95%, 96%, 97%, 98%, or 99% of the nucleotide bases, as measured by any well-known algorithm for sequence identity, such as FASTA, BLAST, or GAP, as considered below. A nucleic acid molecule having substantial identity with a reference nucleic acid molecule may, in certain cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0102] When applied to polypeptides, the term “substantial similarity” or “substantially identical” means that two peptide sequences share at least 90% sequence identity, and more preferably at least 95%, 98%, or 99% sequence identity, when optimally aligned using default gap weighting, such as by program GAP or BESTFIT. Preferably, non-identical residue positions differ by conserved amino acid substitutions. A “conservative amino acid substitution” is a substitution in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of the protein. If two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity may be adjusted upward to correct the conservative nature of the substitutions. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331, incorporated herein by reference. Examples of amino acids having side chains with specific chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conserved amino acid substituents are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative permutation is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443 45, which is incorporated herein by reference. A “moderately conservative” permutation is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0103] Polypeptide sequence similarity is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications, including conserved amino acid substitutions. For example, GCG software includes programs such as GAP and BESTFIT, which, along with default parameters, can be used to determine sequence homology or sequence identity between closely related polypeptides, e.g., homologous polypeptides from different species, or between a wild-type protein and its mutant protein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, along with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides best-in-class alignment of overlapping regions and percent sequence identity between the query sequence and the search sequence (Pearson (2000) above). The sequences of the present invention can be compared to a database containing numerous sequences from different organisms. Another preferred algorithm in such cases is the computer program BLAST, particularly BLASTP or TBLASTN, which use default parameters. For example, Altschul et al. (1990) J. Mol. Biol., each of which is incorporated herein by reference. See 215: 403-410 and (1997) Nucleic Acids Res. 25:3389-3402.

[0104] In specific embodiments, the antibody or antibody fragment for use in the method of the present invention may be monospecific, bispecific, or polyspecific. A polyspecific antibody may be specific to a different epitope of one target polypeptide, or may contain antigen-binding domains specific to epitopes of one or more target polypeptides. An exemplary bispecific antibody format that can be used in the context of the present invention is a first immunoglobulin (Ig) C H 3 domains and 2nd Ig C HIt requires the use of 3 domains, and the first and second Ig C H The three domains differ from each other by at least one amino acid, and the difference of at least one amino acid reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody lacking the amino acid difference. In one embodiment, the first Ig C H The 3 domains bind to protein A, and the second Ig C H The 3 domains contain mutations that reduce or eliminate protein A binding, such as the H95R modification (according to IMGT exon numbering; H435R if using EU numbering). Second C H 3 may further include the Y96F modification (by IMGT; Y436F if by EU). Second C H Further modifications that may be found within 3 include: for IgG1 mAb, D16E, L18M, N44S, K52N, V57M, and V82I (according to IMGT; according to EU, D356E, L358M, N384S, K392N, V397M, and V422I); for IgG2 mAb, N44S, K52N, and V82I (according to IMGT; according to EU, N384S, K392N, and V422I); and for IgG4 mAb, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (according to IMGT; according to EU, Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I). The above-mentioned changes in the bispecific antibody format are intended to be within the scope of the present invention.

[0105] The term "therapeutic dose" refers to the amount administered that produces the desired effect. The exact amount depends on the purpose of the treatment and can be determined by those skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0106] General explanation "Transfection rearrangement," also known as "RET," is a receptor tyrosine kinase expressed during development in various tissues, including the peripheral and central nervous systems and the kidneys (Arighi, E. et al. (2005), Cytokine Growth Factor Rev 16:441-67; Borrello, MG, et al., (2013), Expert Opin. Ther. Targets, 17(4): 403-419). The RET oncogene was identified in 1985 by Takahashi et al., who reported a novel gene rearrangement exhibiting transform activity in NIH / 3T3 cells transfected with human lymphoma DNA (Takahashi, M., et al., (1985)). See Cell, 42:581-588). RET was later confirmed to be an oncogene. This protein is rearranged somatically in the DNA of papillary thyroid carcinoma (PTC) patients and was later called RET / PTC (Fusco, A. et al., (1987), Nature, 328:170-172; Grieco, M. et al., (1990), Cell, 60:557-63). The RET protein consists of a cytoplasmic portion divided by a 27-amino acid insertion into three domains: an extracellular ligand-binding domain, a hydrophobic transmembrane domain, and a tyrosine kinase domain (see Figure 1). There are two main isoforms of RET produced by alternative splicing. The short and long RET isoforms are distinguished by 9 and 51 unrelated C-terminal amino acids, and are called RET9 and RET51, respectively. They are highly conserved across a wide range of species (Carter, MT, et al. (2001), Cytogenet Cell Genet, 95:169-76). All isoforms exhibit transform activity as determined by a focus formation assay (Rossel, M. et al. (1997), Oncogene, 14:265-75).

[0107] Genetic modification of RET has been shown to be involved in the pathogenesis of thyroid cancer, and more recent data suggest that RET is also involved in lung adenocarcinoma (Viglietto, G. et al. (1995), Oncogene, 11:1207-10; Fischer, AH, et al., (1998), Am J Pathol). (153:1443-50). Other studies have shown that RET is associated with more conditions including breast, pancreatic, leukemia, and melanoma. This suggests that it may be related to certain tumors (Ballerini, P. et al, (2012), Leukemia, 26:2384-9; Sawai, H. et al. (2005), 65(24):11536-44; Narita, N. et al. al., (2009), Oncogene, 28:3058-68).

[0108] Vandetanib (ZD6474, CAPRELSA®, Astra Zeneca) is an orally administered aminoquinazoline compound initially developed as a VEGFR2 inhibitor, but later found to be active against RET, VEGFR3, EGFR, and PDGFR. Vandetanib is currently approved by the FDA and EMA for the treatment of advanced and metastatic medullary thyroid carcinoma (MTC) (Wells, SA, et al., (2012), J. Clin. Oncol. 30:134-41).

[0109] Sorafenib (BAY43-9006, NEXAVAR®, Bayer Pharmaceuticals) is a bisarylurea compound initially developed to target the serine / threonine kinase BRAF, but has since been found to be potent against Flt-3, VEGFR1-3, PDGFR, c-kit, and RET (Wilhelm, S. et al., (2006), Nat Rev Drug Discov, 5:835-44). Sorafenib has been approved by the FDA for advanced liver and kidney cancer.

[0110] Sunitinib (SU11248, SUTENT®, Pfizer) is an indolinone compound that primarily targets VEGFR2, PDGFR, c-kit, FLT3, and RET kinase (Chow, LQ, et al., (2007), J. Clin. Oncol. 25:884-96). Sunitinib has been approved by the FDA for use in patients with imatinib-resistant GIST as well. It has been approved for the treatment of advanced pancreatic endocrine tumors and renal cell carcinoma.

[0111] Cabozantinib (Cometriq, formerly known as XL-184, Exelixis) is a small molecule multi-kinase inhibitor that targets MET, VEGFR2, and RET. It is currently in clinical trials in numerous tumor types, including medullary thyroid cancer, prostate cancer, ovarian cancer, non-small cell lung cancer (NSCLC), hepatocellular carcinoma, renal cell carcinoma, and breast cancer, as well as melanoma and glioblastoma ((Zhang, Y. et al., (2010), IDrugs 13:112-21).

[0112] Another RET-targeting agent currently in clinical development is motesanib (AMG-706, Amgen), which is a multi-kinase inhibitor that targets VEGFR1-3, Flt3, Kit, PDGFR, and RET.

[0113] Other RET inhibitors in preclinical development include the indoline compound RPI-1; PP-1, a pyrazolopyrimidine compound active against RET and Src; and NVP-AST478, a biphenyl-urea compound with potent anti-RET kinase activity in vitro and in vivo (Cuccuru, G. et al. (2004), J Natl). Cancer Inst 96:1006-14).

[0114] However, one problematic aspect of the above-mentioned RET inhibitors is that they are not specific to RET; that is, they appear to act through multiple mechanisms and therefore may potentially exert other adverse effects in vivo. For example, certain inhibitors mentioned above induce adverse events such as hypertension and QTc prolongation. Thus, the non-selective profile of these drugs can limit the treatment window. Identifying drugs such as anti-RET antibodies that selectively bind to RET would be useful and could result in superior clinical efficacy with a more favorable safety profile.

[0115] Therefore, there is still a need for effective treatment of RET-driven tumors, and furthermore, there is a need to identify RET-specific agents to prevent and treat other diseases, disorders, or conditions associated with RET expression without the adverse side effects associated with the aforementioned agents. Such specificity and efficacy can be achieved through the use of anti-RET antibodies, such as those described herein.

[0116] In certain embodiments, the antibodies of the present invention are obtained from mice immunized with a primary immunogen, such as the whole human RET protein, or a recombinant form or fragment thereof, or a fusion protein containing the extracellular / ectodomain of human RET (see GenBank accession number NP_066124.1 (SEQ ID NO: 310) or GenBank accession number NP_065681.1 (SEQ ID NO: 312)), or a recombinantly produced RET fusion protein (see SEQ ID NOs: 305, 306, 307, and 313), followed by a secondary immunogen (purified human RET protein) or an immunogenically active fragment of the RET protein, such as the ectodomain of RET.

[0117] The immunogen may be the human RET protein (see GenBank accession number NM_020975.4 and SEQ ID NO: 309 for isoform A; or GenBank accession number NM_020630.4 and SEQ ID NO: 311 for isoform C), or DNA encoding an active fragment thereof.

[0118] The immunogen may originate from the extracellular domain of the RET protein, specifically amino acid residues 1-635 of any of sequence numbers 305, 307, 310, 312, and 313 (including the signal sequence); or from amino acid residues 1-636 of sequence number 306 (including the signal sequence). The immunogen may also originate from any fragment of the aforementioned regions of the RET protein.

[0119] The full-length amino acid sequence of RET51 is shown as SEQ ID NO: 310, also known as GenBank accession number NP_066124.1. The full-length amino acid sequence of RET9 is shown as SEQ ID NO: 312, also known as GenBank accession number NP_065681.1. Exemplary immunogens may be the recombinant constructs shown in SEQ ID NO: 307 or 313.

[0120] In certain embodiments, antibodies that specifically bind to RET may be prepared using a fragment of the above-described region, or a peptide extending about 5 to about 20 amino acid residues beyond the specified region from either or both of the N- or C-terminal ends of the region described herein. In certain embodiments, any combination of the above-described region or fragments may be used in the preparation of a RET-specific antibody. In certain embodiments, one or more of the above-described regions or fragments of RET may be used to prepare monospecific, bispecific, or polyspecific antibodies.

[0121] Antigen-binding fragments of antibodies Unless otherwise specified, the term “antibody” as used herein is understood to encompass an antibody molecule (i.e., a “complete antibody molecule”) containing two immunoglobulin heavy chains and two immunoglobulin light chains, as well as its antigen-binding fragment. The terms “antigen-binding moiety” of an antibody, “antigen-binding fragment” of an antibody, etc., as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen and forms a complex. The terms “antigen-binding moiety” or “antibody fragment” as used herein refer to one or more fragments of an antibody that retain the ability to specifically bind to RET. Antibody fragments may include Fab fragments, F(ab')2 fragments, Fv fragments, dAb fragments, fragments containing CDRs, or isolated CDRs. Antigen-binding fragments of an antibody may be derived from a complete antibody molecule using, for example, any suitable standard technique, such as proteolytic digestion, or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and (if necessary) constant domains. Such DNA is known and / or readily available from, for example, commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or by using molecular biological techniques to, for example, arrange one or more variable and / or constant domains into a suitable configuration, introduce codons, create cysteine ​​residues, modify, add, or delete amino acids, etc.

[0122] Non-restrictive examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv(scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues mimicking the hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR), e.g., CDR3 peptide), or constrained FR3-CDR3-FR4 peptides. Other manipulated molecules, such as domain-specific antibodies, single-domain antibodies, domain deletion antibodies, chimeric antibodies, CDR graft antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also included within the expression "antigen-binding fragment" as used herein.

[0123] The antigen-binding fragment of an antibody typically contains at least one variable domain. The variable domain can be a domain of any size or amino acid composition and generally contains at least one CDR adjacent to or in-frame with one or more framework sequences. L V associated with the domain H In an antigen-binding fragment having a domain, V H and V L Domains can be positioned in any suitable arrangement relative to each other. For example, the variable region is of dimeric form, V H -V H , V H -V L , or V L -V L It may contain a dimer. Alternatively, the antigen-binding fragment of the antibody may contain a monomer V H or V L It may contain a domain.

[0124] In certain embodiments, an antigen-binding fragment of an antibody can contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that can be found within the antigen-binding fragments of the antibodies of the present invention include: (i) V H -C H 1; (ii) V H -C H 2; (iii) V H -C H 3; (iv) V H -C H 1-C H 2; (v) V H -C H 1-C H 2-C H 3; (vi) V H -C H 2-C H 3; (vii) V H -C L ; (viii) V L -C<​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Examples include: In any configuration of variable and constant domains including any of the exemplary configurations described above, the variable and constant domains may be directly linked to each other or linked by a complete or partial hinge or linker region. The hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids, thereby providing a flexible or semi-flexible link between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragment of the antibody of the present invention may be linked to each other and / or one or more monomers. H Or V L The domain may include homodimers or heterodimers (or other polymers) of either the variable or constant domain configuration described above, through non-covalent association with the domain (e.g., by disulfide bonds).

[0125] Like complete antibody molecules, antigen-binding fragments can be monospecific or polyspecific (e.g., bispecific). A polyspecific antigen-binding fragment of an antibody typically comprises at least two distinct variable domains, each capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any polyspecific antibody format, including the exemplary bispecific antibody formats disclosed herein, may be adapted for use in the context of the antibody antigen-binding fragments of the present invention using routine techniques available in the art.

[0126] Preparation of human antibodies Methods for generating human antibodies in transgenic mice are known in the art. Any such known method can be used in the context of the present invention to produce human antibodies that specifically bind to RET.

[0127] First, a high-affinity chimeric antibody against RET having human variable regions and mouse constant regions is isolated using VELOCIMMUNE® technology (see, for example, US6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method for generating monoclonal antibodies. VELOCIMMUNE® technology involves generating a transgenic mouse having a genome containing human heavy and light chain variable regions operably ligated to an endogenous mouse constant region locus so that the mouse produces an antibody containing human variable regions and mouse constant regions in response to antigen stimulation. The DNA encoding the heavy and light chain variable regions of the antibody is isolated and operably ligated to the DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in cells capable of expressing a fully human antibody.

[0128] Generally, VELOCIMMUNE® mice are challenged with the target antigen, and lymphocytes (e.g., B cells) are collected from mice that express antibodies. Lymphocytes may be fused to myeloma cell lines to prepare immortal hybridoma cell lines. Such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the target antigen. DNA encoding the variable regions of the heavy and light chains may be isolated and ligated to the desired isotype constant regions of the heavy and light chains. Such antibody proteins can be produced in cells such as CHO cells. Alternatively, antigen-specific chimeric antibodies or DNA encoding the variable domains of the light and heavy chains may be directly isolated from antigen-specific lymphocytes.

[0129] First, a high-affinity chimeric antibody having a human variable region and a mouse constant region is isolated. The antibody is characterized and selected for desirable features, including affinity, selectivity, and epitopes, as in the experimental section below. The mouse constant region is replaced with the desired human constant region to generate the fully human antibody of the present invention, e.g., wild-type or modified IgG1 or IgG4. The selected constant region may vary depending on the specific use, but the high-affinity antigen-binding and target-specific features reside in the variable region.

[0130] In a particular embodiment, the antibody of the present invention, when measured by binding to an antigen immobilized on a solid phase or in a solution phase, yields approximately 1.0 × 10⁻⁶ -7 M ~ approx. 1.0×10 -12 Affinity in the range of M (K D The present invention provides a fully human antibody by replacing the mouse constant region with a desired human constant region. The selected constant region can vary depending on the specific use, but the high affinity antigen binding and target specificity features reside in the variable region.

[0131] biological equivalent The anti-RET antibodies and antibody fragments of the present invention comprise proteins having amino acid sequences that differ from the sequences of the antibodies described but retain the ability to bind RET. Such variant antibodies and antibody fragments exhibit biological activity that is essentially equivalent to that of the antibodies described, but with one or more additions, deletions, or substitutions of amino acids compared to the parent sequence. Similarly, the antibody-coding DNA sequences of the present invention comprise sequences encoding antibodies or antibody fragments that are essentially biologically equivalent to the antibodies or antibody fragments of the present invention, but with one or more additions, deletions, or substitutions of nucleotides compared to the sequences of the present disclosure.

[0132] Two antigen-binding proteins or antibodies are considered bioequivalent or pharmaceutically equivalent if, for example, they are administered at the same molar dose under similar experimental conditions, whether in single or multiple doses, and their absorption rates and extents do not show significant differences. Some antibodies are considered equivalent or pharmaceutically equivalent if their absorption rates are similar but their extents are not. Such differences in absorption rates may be intentional, reflected in labeling, and not essential for achieving effective body drug concentrations for chronic use, and are not considered medically significant with respect to the specific drug product being studied, so they may still be considered bioequivalent.

[0133] In one embodiment, two antigen-binding proteins are bioequivalent if there is no clinically significant difference in their safety, purity, and potency.

[0134] In one embodiment, two antigen-binding proteins are bioequivalent to continuous treatment without such switching, if a patient can be switched once or multiple times between a reference product and a bioequivalent product without expecting an increased risk of adverse effects, including clinically significant changes in immunogenicity or decreased efficacy.

[0135] In one embodiment, two antigen-binding proteins are bioequivalent to the extent that such mechanisms are known, if they both act by a common mechanism or mechanism of action with respect to the conditions of use (one or more).

[0136] Bioequivalence can be demonstrated by in vivo and / or in vitro methods. Measurements of bioequivalence include, for example, (a) in vivo studies in humans or other mammals in which the concentration of the antibody or its metabolites is measured as a function of time in blood, plasma, serum, or other body fluids; (b) in vitro studies that correlate with and reasonably predict human in vivo bioavailability data; (c) in vivo studies in humans or other mammals in which the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) well-controlled clinical trials to establish the safety, efficacy, or bioavailability or bioequivalence of the antibody.

[0137] Bioequivalent variants of the antibody of the present invention can be constructed, for example, by creating various substitutions of residues or sequences, or by deleting terminal or internal residues or sequences that are not necessary for biological activity. For example, deleting or replacing a cysteine ​​residue that is not essential for biological activity with another amino acid can prevent the formation of unwanted or inaccurate intramolecular disulfide crosslinks during reconstruction. In other circumstances, bioequivalent antibodies may include antibody variants that include amino acid changes that alter the glycosylation characteristics of the antibody, such as mutations that eliminate or remove glycosylation.

[0138] Anti-RET antibody containing Fc variant According to a particular embodiment of the present invention, an anti-RET antibody is provided comprising an Fc domain containing one or more mutations that enhance or decrease antibody binding to the FcRn receptor at acidic pH compared to neutral pH. For example, the present invention provides an Fc domain with C H 2 or C HThe present invention relates to an anti-RET antibody containing mutations in three regions, wherein the mutations increase the affinity of the Fc domain to FcRn in an acidic environment (e.g., in endosomes where the pH is in the range of approximately 5.5 to 6.0). Such mutations may result in an increased serum half-life of the antibody when administered to animals. Unrestrictive examples of such Fc modifications include, for example, modifications at position 250 (e.g., E or Q); positions 250 and 428 (e.g., L or F); position 252 (e.g., L / Y / F / W or T), position 254 (e.g., S or T), and position 256 (e.g., S / R / Q / E / D or T); or modifications at position 428 and / or 433 (e.g., H / L / R / S / P / Q or K), and / or position 434 (e.g., H / F or Y); or modifications at position 250 and / or 428; or modifications at position 307 or 308 (e.g., 308F, V308F), and position 434. In one embodiment, modifications include the 428L (e.g., M428L) and 434S (e.g., N434S) modifications; the 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications; the 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; the 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications; the 250Q and 428L modifications (e.g., T250Q and M428L); and the 307 and / or 308 modifications (e.g., 308F or 308P).

[0139] For example, the present invention includes an anti-RET antibody comprising an Fc domain containing one or more pairs or groups of mutations selected from the group consisting of 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); and 433K and 434F (e.g., H433K and N434F). All possible combinations of the aforementioned Fc domain mutations and other mutations within the antibody variable domain disclosed herein are intended to be within the scope of the present invention.

[0140] Biological characteristics of antibodies Generally, the antibodies of the present invention may function by binding to RET and thereby acting to block or prevent RET activation and / or signaling. The antibodies of the present invention may also function by binding to RET and thereby interfering with or preventing the interaction or binding of RET to one or more GDNF family members, e.g., GDNF / GFRα1, neuturin / GFRα2, artemin / GFRα3, or parcefin / GFRα4, which form a complex with its corresponding coreceptor. Based on the fact that the oncogenic potential of RET is established in humans, antagonist antibodies that specifically bind to RET may prove to have beneficial effects in inhibiting tumor cell growth in patients suffering from cancerous conditions.

[0141] In certain embodiments, the antibody of the present invention may function by blocking or inhibiting RET activity by binding to any region or fragment of the full-length protein whose amino acid sequence is shown in SEQ ID NO: 310 (RET51), also known as GenBank accession number NP_066124.1, and SEQ ID NO: 312 (RET9), also known as GenBank accession number NP_065681.1. The antibody may also bind to any region found in SEQ ID NO: 310 or 312, or to any fragment found within SEQ ID NO: 310 or 312.

[0142] In one embodiment, the present invention relates to a fully human monoclonal antibody or its antigen-binding fragment that binds to a RET protein, characterized by the following: (a) It must be a fully human antibody; (b) When measured by surface plasmon resonance, approximately 1.0 × 10 -7 M ~ approx. 1.0×10 -12 K in the range of M D To demonstrate; (c) Inhibiting or blocking the binding or interaction of RET with one or more GDNF family member ligands (GDNF, neurturin, artemin, and parcefin) that form a complex with its corresponding co-receptor (GFRα1, GFRα2, GFRα3, and GFRα4, respectively); (d) Inhibiting RET signaling mediated by one or more GDNF family member ligands selected from GDNF, neurturin, artemin, and parcephin; (e) To enhance the internal translocation / degradation of RET receptors after antibody binding to them; (f) Containing a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, and 290; or (g) Containing a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, and 298. The present invention provides a fully human monoclonal antibody or an antigen-binding fragment thereof exhibiting one or more of the following characteristics.

[0143] Certain anti-RET antibodies of the present invention can bind to the RET protein and inhibit RET-related activation and / or signaling. In doing so, the antibody may function to inhibit tumor growth whose growth depends on the activation of RET signaling. Such antagonist anti-RET antibodies may be used alone to treat cancerous conditions, or as adjunct therapy in combination with any other anticancer agent, such as small molecule chemotherapy agents, or radiotherapy, or bone marrow regenerative agents.

[0144] In certain embodiments, anti-RET antibodies may be able to inhibit multiple signaling pathways, including the RAS / RAF pathway, thereby inhibiting mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 (Trupp, M. et al., (1999), J Biol.). Chem. 274:20885-94; Santoro, M. et al., (1994), Mol. Cell Biol. 14:663-75; van Weering, DHJ, et al. (1995), 11:2207-14; Worby, CA, et al., (1996), J Biol Chem, 271:23619-22), phosphatidylinositol 3-quinol Activation of ze (PI3K) occurs, which in turn leads to the activation of serine / threonine kinase Akt (Trupp, M. et al., (1999), J Biol. Chem. 274:20885-94; van Weering, DHJ, (1997), J Biol Chem 272:249-54; Segouffin-Cariou, C., et al. (2000), 275:3568-76; Maeda, K. et al, (2004), 323: 345-54).

[0145] Examples 4 and 5 illustrate non-restrictive, exemplary in vitro assays for measuring the ability of the anti-RET antibody of the present invention to block the binding of RET to the GFRα1 / GDNF cocomplex, and in vitro assays for measuring the effect of the antibody on RET signaling, activation, or internal translocation, respectively. In Example 3, the binding affinity and rate constant of the human anti-RET antibody were determined by surface plasmon resonance, and measurements were performed using a Biacore 4000 or T200 instrument. In Example 4, the ability of the antibody to block the binding of RET to the GFRα1 / GDNF cocomplex was tested using a competitive sandwich ELISA assay. Example 5 demonstrates the ability of the antibody of the present invention to inhibit ligand-dependent RET signaling in a serum response factor (SRE)-luciferase reporter assay. More specifically, the data presented in Example 5 show that the anti-RET antibody of the present invention exhibits broad inhibitory activity against RET signaling in the presence of the glial family ligands GDNF and artemin.

[0146] Epitope mapping and related technologies Using various techniques known to those skilled in the art, it is possible to determine whether an antibody "interacts with one or more amino acids" in a polypeptide or protein. Exemplary techniques include routine cross-blocking assays, such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY), which can be performed. Other methods include alanine scan mutation analysis, peptide blot analysis (Reineke (2004) Methods Mol Biol 248:443-63), and peptide cleavage. Examples include analytical crystallographic studies and NMR analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of the antigen can be used (Tomer (2000) Protein Science 9: 487-496). Another method that can be used to identify amino acids in polypeptides that antibodies interact with is hydrogen / deuterium exchange detected by mass spectrometry. Generally, hydrogen / deuterium exchange involves deuterium labeling of the protein of interest, followed by the binding of the antibody to the deuterium-labeled protein. When the protein / antibody complex is then transferred to water, the exchangeable protons in amino acids protected by the antibody complex undergo reverse exchange from deuterium to hydrogen at a slower rate than the exchangeable protons in amino acids that are not part of the interface. As a result, amino acids that form part of the protein / antibody interface retain deuterium and may therefore exhibit a relatively larger mass compared to amino acids not included in the interface. After antibody dissociation, the target protein is subjected to protease cleavage and analysis by mass spectrometry to reveal the deuterium-labeled residues corresponding to the specific amino acids that the antibody interacts with. For example, see Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0147] The term "epitope" refers to a site on an antigen to which B and / or T cells respond. B cell epitopes can be formed from both continuous amino acids or discontinuous amino acids juxtaposed by the three-dimensional folding of proteins. Epitopes formed from continuous amino acids are typically retained even when exposed to denaturing solvents, while epitopes formed by three-dimensional folding are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least 3, more commonly at least 5 or 8-10 amino acids in a unique spatial conformation.

[0148] Modification-assisted profiling (MAP), also known as antigen-based antibody profiling (ASAP), is a method for classifying a large number of monoclonal antibodies (mAbs) targeting the same antigen according to the similarity of their binding profiles to chemically or enzymatically modified antigen surfaces (the entirety of which is specifically incorporated herein by reference, US2004 / 0101920). Each category may reflect unique epitopes that are either distinctly different from or partially overlapping with epitopes represented by other categories. This technique enables rapid filtering of genetically identical antibodies so that the characterization can focus on genetically distinct antibodies. When applied to hybridoma screening, MAP can facilitate the identification of rare hybridoma clones that produce mAbs with desired characteristics. Using MAP, the antibodies of the present invention may be sorted into groups of antibodies that bind to different epitopes.

[0149] The present invention includes anti-RET antibodies that bind to the same epitope as any of the specific exemplary antibodies listed in Table 1 herein. Similarly, the present invention also includes anti-RET antibodies that compete with any of the specific exemplary antibodies listed in Table 1 herein for binding to RET or a fragment thereof.

[0150] By using routine methods known in the art, it is possible to easily determine whether an antibody binds to the same epitope as the reference anti-RET antibody, or whether it competes for binding to it. For example, to determine whether a test antibody binds to the same epitope as the reference RET antibody of the present invention, the reference antibody is bound to the RET protein or peptide under saturated conditions. The ability of the test antibody to bind to the RET molecule is then evaluated. If the test antibody can bind to RET after saturated binding by the reference anti-RET antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-RET antibody. On the other hand, if the test antibody cannot bind to the RET molecule after saturated binding by the reference anti-RET antibody, then the test antibody may bind to the same epitope to which the reference anti-RET antibody of the present invention binds.

[0151] To determine whether an antibody competes for binding with the reference anti-RET antibody, the above binding methodology is performed in two directions: In the first direction, the reference antibody is bound to the RET molecule under saturated conditions, and then the binding of the test antibody to the RET molecule is evaluated. In the second direction, the test antibody is bound to the RET molecule under saturated conditions, and then the binding of the reference antibody to the RET molecule is evaluated. If, in either direction, only the first (saturated) antibody is able to bind to the RET molecule, it is concluded that the test antibody and the reference antibody compete for binding to RET. As will be recognized by those skilled in the art, an antibody that competes for binding with a reference antibody does not necessarily have to bind to the same epitope as the reference antibody, but can sterically block the binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0152] Two antibodies bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other to an antigen. That is, as measured in a competitive binding assay, a 1x, 5x, 10x, 20x, or 100x excess of one antibody inhibits the binding of the other by at least 50%, but preferably 75%, 90%, or even 99% (see, for example, Junghans et al., Cancer Res. 1990 50:1495-1502). Alternatively, if essentially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other, the two antibodies have the same epitope. If some amino acid mutations that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other, the two antibodies have overlapping epitopes.

[0153] Subsequently, additional routine experiments (e.g., peptide mutation and binding analysis) can be performed to confirm whether the observed lack of binding of the test antibody is indeed due to binding to the same epitope as the reference antibody, or whether steric blockage (or another phenomenon) is the cause of the observed lack of binding. These types of experiments can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0154] Immunoconjugate The present invention encompasses human RET monoclonal antibodies ("immunoconjugates") conjugated to a therapeutic portion, such as an agent capable of inhibiting the proliferation of tumor cells or improving at least one symptom associated with a RET-related condition, such as a cancerous condition. Such agents may be a second, different antibody against RET or an antitumor chemotherapeutic agent, or a radionuclide that acts to kill tumor cells when targeted at tumor cells expressing RET. The type of therapeutic portion that can be conjugated to an anti-RET antibody takes into account the condition being treated and the desired therapeutic effect to be achieved. Alternatively, if the desired therapeutic effect is treating complications or symptoms associated with RET expression by a particular tissue, or any other condition resulting from RET expression, such as cancer, for example, it may be advantageous to conjugate a suitable agent to treat the complications or symptoms of the condition or to mitigate any side effects of the antibody of the present invention. Examples of agents suitable for forming immunoconjugates are known in the art; see, for example, WO05 / 103081.

[0155] multispecific antibody The antibodies of the present invention may be monospecific, bispecific, or polyspecific. Polyspecific antibodies may be specific to different epitopes of a single target polypeptide, or they may contain antigen-binding domains specific to one or more target polypeptides. For example, Tutt et al. See al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. The antibodies of the present invention can be ligated to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or a fragment thereof can be functionally ligated to one or more other molecular entities, such as another antibody or antibody fragment (e.g., by chemical coupling, gene fusion, non-covalent association, or other methods) to produce a bispecific or polyspecific antibody having a second binding specificity.

[0156] An exemplary bispecific antibody format that can be used in the context of the present invention is a first immunoglobulin (Ig) C H3 Domain and second Ig C H3 With the use of the domain, the first and second Ig C H3 The domains differ from each other by at least one amino acid, and the difference of at least one amino acid reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody lacking an amino acid difference. In one embodiment, a first Ig C H3 The domain binds to protein A and a second Ig C H3 The domain contains mutations that reduce or eliminate protein A binding, such as the H95R modification (according to IMGT exon numbering; H435R if using EU numbering). Second C H3 This may further include the Y96F modification (by IMGT; Y436F if by EU). Second C H3Further modifications that may be found within the formulation include: for IgG1 antibodies, D16E, L18M, N44S, K52N, V57M, and V82I (according to IMGT; according to EU, D356E, L358M, N384S, K392N, V397M, and V422I); for IgG2 antibodies, N44S, K52N, and V82I (according to IMGT; according to EU, N384S, K392N, and V422I); and for IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (according to IMGT; according to EU, Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I). The variations on the two-specific antibody format described above are intended to be within the scope of the present invention.

[0157] Therapeutic administration and formulations The present invention provides a therapeutic composition comprising the anti-RET antibody or its antigen-binding fragment. Administration of the therapeutic composition according to the present invention is performed together with a suitable carrier, excipient, and other agents that are incorporated into the formulation to improve transfer, delivery, and tolerability. Numerous suitable formulations can be found in prescription collections known to all pharmacists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations may be, for example, in powder or pass. Examples include ointments, gels, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (e.g., LIPOFECTIN®), DNA conjugates, anhydrous absorbent pastas, oil-in-water and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowaxes. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0158] The dose of each antibody of the present invention may vary depending on the age and size of the recipient, the target disease, condition, route of administration, etc. When the antibodies of the present invention are used to treat RET-related diseases or conditions in a patient, or to treat one or more symptoms associated with a condition dependent on RET activation or signaling in a patient, such as a particular tumor expressing RET, or to reduce the severity of a disease, it is usually advantageous to administer each antibody of the present invention intravenously or subcutaneously as a single dose of about 0.01 to about 30 mg / kg body weight, more preferably about 0.1 to about 20 mg / kg body weight, or about 0.1 to about 15 mg / kg body weight, or about 0.02 to about 7 mg / kg body weight, about 0.03 to about 5 mg / kg body weight, or about 0.05 to about 3 mg / kg body weight, or about 1 mg / kg body weight, or about 3.0 mg / kg body weight, or about 10 mg / kg body weight, or about 20 mg / kg body weight. Multiple doses may be administered as needed. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. In certain embodiments, the antibody or antigen-binding fragment of the present invention may be administered as an initial dose of at least about 0.1 mg to about 800 mg, about 1 to about 600 mg, about 5 to about 300 mg, or about 10 to about 150 mg, about 100 mg, or about 50 mg. In certain embodiments, after the initial dose, a second or more subsequent doses of the antibody or antigen-binding fragment may be administered in amounts that are approximately the same as or less than the initial dose, and these subsequent doses are spaced at intervals of at least 1 to 3 days; at least 1 week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.

[0159] Various delivery systems are known, such as liposome encapsulation, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis, and these can be used to administer the pharmaceutical composition of the present invention (see, for example, Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of delivery include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through the epithelium or mucosal lining (e.g., oral mucosa, nasal mucosa, rectal and intestinal mucosa), or in combination with other bioactive agents. Administration may be systemic or topical. It can be delivered as an aerosolized formulation (see US2011 / 0311515 and US2012 / 0128669). The delivery of drugs useful for treating respiratory diseases by inhalation is becoming more widely accepted (see AJ Bitonti and JA Dumont, (2006), Adv. Drug Deliv. Rev, 58:1106-1118). In addition to being effective in treating local lung diseases, it is also used for other purposes. This delivery mechanism may also be useful for systemic delivery of antibodies (see Maillet et al. (2008), Pharmaceutical Research, Vol. 25, No. 6, 2008).

[0160] Pharmaceutical compositions can also be delivered in vesicles, particularly liposomes (e.g., Langer (See Science 249:1527-1533, 1990).

[0161] In certain circumstances, pharmaceutical compositions can be delivered by a controlled-release system. In one embodiment, a pump may be used. In another embodiment, a polymer material may be used. In yet another embodiment, the controlled-release system may be positioned near the target of the composition, thus requiring only a small fraction of the systemic dose.

[0162] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injection, intravenous drip infusion, etc. These injectable preparations may be prepared by known methods. For example, injectable preparations may be prepared by dissolving, suspending, or emulsifying the above-mentioned antibody or a salt thereof in a sterile aqueous or oily medium commonly used for injection. As aqueous mediums for injection, there are isotonic solutions containing, for example, physiological saline, glucose, and other adjuvants, which may be used in combination with appropriate solvents such as, for example, alcohol (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. As oily mediums, for example, sesame oil, soybean oil, etc., may be used in combination with solvents such as, for example, benzyl benzoate, benzyl alcohol. The injectable preparations thus prepared are preferably filled into appropriate ampoules.

[0163] The pharmaceutical compositions of the present invention can be delivered subcutaneously or intravenously by standard needles and syringes. In addition, with respect to subcutaneous delivery, pen delivery devices offer convenient application in the delivery of the pharmaceutical compositions of the present invention. Such pen delivery devices may be reusable or disposable. Reusable pen delivery devices generally utilize replaceable cartridges containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. Disposable pen delivery devices do not have replaceable cartridges. Rather, disposable pen delivery devices are pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the pharmaceutical composition in the reservoir is empty, the entire device is discarded.

[0164] Numerous reusable pen and auto-injector delivery devices have applications in the subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples include, to name just a few, AUTOPEN® (Owen Mumford, Inc., Woodstock, UK), DISETRONIC® pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75 / 25® pen, HUMALOG® pen, HUMALIN 70 / 30® pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN® I, II, and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR® (Novo Nordisk, Copenhagen, Denmark), BD® pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN®, OPTIPEN PRO®, and OPTIPEN®. Examples of disposable pen delivery devices applicable to the subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, STARLET® and OPTICLIK® (sanofi-aventis, Frankfurt, Germany). Examples include, but are not limited to, SOLOSTAR® pens (sanofi-aventis), FLEXPEN® (Novo Nordisk), and KWIKPEN® (Eli Lilly), SURECLICK® auto-injectors (Amgen, Thousands Oaks, CA), PENLET® (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA® pens (Abbott Labs, Abbott Park, IL).

[0165] Advantageously, the above-mentioned pharmaceutical compositions for oral or parenteral use are prepared into dosage forms in unit doses suitable for the dosage of the active ingredient. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the above-mentioned antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose, and preferably about 5 to about 100 mg of the above-mentioned antibody in the form of an injection, and about 10 to about 250 mg in other dosage forms.

[0166] Administration regimen According to certain embodiments of the present invention, multiple doses of an antibody against RET may be administered to a subject over a predetermined period of time. A method according to this aspect of the present invention involves sequentially administering multiple doses of an antibody against RET to a subject. As used herein, “sequentially administering” means that each dose of the antibody against RET is administered to the subject at different times, for example, on different days separated by a predetermined interval (e.g., several hours, several days, several weeks, or several months). The present invention includes a method comprising sequentially administering to a patient one or more secondary doses of the antibody against RET and, optionally, one or more tertiary doses of the antibody against RET, after a single initial dose of the antibody against RET.

[0167] The terms “initial dose,” “secondary dose,” and “tertiary dose” refer to the chronological order of administration of antibodies against RET. Therefore, the “initial dose” is the dose administered at the beginning of the treatment regimen (also called the “baseline dose”); the “secondary dose” is the dose administered after the initial dose; and the “tertiary dose” is the dose administered after the secondary dose. The initial, secondary, and tertiary doses may all contain the same amount of antibody against RET, but generally they may differ in terms of administration frequency. However, in certain embodiments, the amounts of antibody against RET contained in the initial, secondary, and / or tertiary doses may differ from each other throughout the course of treatment (e.g., adjusted upward or downward as needed). In certain embodiments, two or more doses (e.g., 2, 3, 4, or 5) may be administered as a “loading dose” at the beginning of the treatment regimen, followed by subsequent doses administered at a less frequent rate (e.g., “maintenance doses”).

[0168] In one exemplary embodiment of the present invention, each secondary and / or tertiary dose is 1 to 26 weeks after the previous dose (e.g., 1, 1 1 / 2 , 2, 2 1 / 2 ,3,3 1 / 2 ,4,4 1 / 2 , 5, 5 1 / 2 , 6, 6 1 / 2 , 7, 7 1 / 2 , 8, 8 1 / 2 ,9,9 1 / 2 , 10, 10 1 / 2 , 11, 11 1 / 2 , 12, 12 1 / 2 , 13, 13 1 / 2 , 14, 14 1 / 2 , 15, 15 1 / 2 , 16, 16 1 / 2 , 17, 17 1 / 2 , 18, 18 1 / 2 , 19, 19 1 / 2 , 20, 20 1 / 2 , 21, 21 1 / 2 , 22, 22 1 / 2 , 23, 23 1 / 2 , 24, 24 1 / 2 , 25, 25 1 / 2 , 26, 26 1 / 2It is administered after (or a longer period of time). The phrase “immediately preceding dose,” as used herein, means the dose of antibody against RET administered to the patient immediately before the next dose in a sequence of multiple doses, without dose intervening.

[0169] A method according to this aspect of the present invention may involve administering to a patient any number of secondary and / or tertiary doses of an antibody against RET. For example, in one particular embodiment, only a single secondary dose is administered to the patient. In another embodiment, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Similarly, in one particular embodiment, only a single tertiary dose is administered to the patient. In another embodiment, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.

[0170] In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2 weeks after the previous dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2 to 4 weeks after the previous dose. Alternatively, the frequency at which secondary and / or tertiary doses are administered to the patient may vary throughout the course of the treatment regimen. The administration frequency may also be adjusted by the physician during the course of treatment as needed for individual patients after clinical examinations.

[0171] Therapeutic use of antibodies By binding to / interacting with RET proteins expressed in certain cells and tissues, this antibody is useful for preventing the interaction of RET proteins with one or more ligand / co-receptor complexes, such as GDNF / GFRα1, artemin / GFRα3, neurturin / GFRα2, or parcefin / GFRα4. Given the ability of the anti-RET antibody of the present invention to prevent or inhibit this interaction, the antagonist antibody of the present invention may prove useful for inhibiting tumor cell growth when tumor cell growth depends on RET signaling, or may prove useful for inhibiting pain associated with cancerous conditions and pain associated with other diseases or disorders in which RET activation or signaling plays a role. The antibody of the present invention may be used to slow tumor growth and / or metastasis in subjects with RET-expressing tumors, or to treat pain associated with cancerous conditions, when administered alone, in combination with another antitumor agent or treatment regimen, or with one or more agents used to further improve condition-related pain. Alternatively, the antibody of the present invention may be useful for improving at least one symptom associated with a cancerous condition.

[0172] The antibodies of the present invention are intended to be used alone or in combination with a second or third agent to treat RET-related diseases or conditions, or to alleviate at least one symptom or complication associated with RET-related diseases or conditions. “RET-related diseases or conditions” is any disease or condition in which RET is known to be expressed in diseased or condition-affected cells or tissues, and which preferably responds to treatment with small molecule therapeutic agents known to inhibit RET activation and / or signaling, or to treatment with the anti-RET antibodies of the present invention. The second or third agents may be delivered simultaneously with the antibodies of the present invention, or they may be administered separately before or after the antibodies of the present invention. The second and third agents may be organic small molecules or biological agents, such as proteins or polypeptides. The second or third agents may be synthetic or naturally derived. The second or third agent could be an antitumor agent, such as a chemotherapy agent or radiotherapy agent, or a bone marrow resuscitation agent, or another agent that reduces fever or pain, another second but different antibody that specifically binds to RET, an agent (e.g., an antibody) that binds to a RET ligand, such as GDNF, neuturin, artemin, or parcefin, or a coreceptor of RET, such as GFRα1, GFRα2, GFRα3, or GFRα4, or an siRNA specific to the RET molecule.

[0173] In further embodiments of the present invention, the antibody is used for the preparation of pharmaceutical compositions for treating patients suffering from RET-related diseases or conditions. In yet another embodiment of the present invention, the antibody is used for the preparation of pharmaceutical compositions for reducing tumor cell proliferation or reducing tumor burden in patients with tumors whose growth depends on RET signaling. In further embodiments of the present invention, the antibody is used as an adjunct therapy with any other agent useful for treating RET-related diseases or conditions, including chemotherapeutic agents, radiotherapy, bone marrow resuscitation agents, a second RET antibody, or any other antibody specific to the RET antigen, or an antibody specific to GDNF or GFRα1, or any other palliative therapy known to those skilled in the art.

[0174] The antibodies of the present invention are useful for treating, preventing, and / or improving any disease, disorder, or condition associated with RET activity, or for improving at least one symptom associated with a disease, disorder, or condition, or for reducing pain associated with such disease, disorder, or condition. Examples of conditions, diseases, and / or disorders that can be treated with the anti-RET antibodies of the present invention, and / or pain associated with such conditions, diseases, or disorders, include, but are not limited to, acute or chronic pain including neuropathic pain, inflammatory pain, arthritis, migraine, cluster headache, trigeminal neuralgia, herpetic neuralgia, systemic neuralgia, irritable bowel syndrome, inflammatory bowel syndrome, visceral pain including abdominal pain, osteoarthritis pain, gout, postherpetic neuralgia, diabetic neuropathy, radiculopathy, sciatica, back pain, head and neck pain, breakthrough pain, postoperative pain, bone pain, and cancer pain. Other conditions treatable by the antibodies and therapeutic methods of the present invention include thyroid cancer, familial medullary thyroid carcinoma (FMTC) syndrome, sporadic medullary carcinoma (MTC), multiple endocrine neoplasia syndromes MEN2A and MEN2B, prostate cancer, breast cancer, cervical cancer, colon cancer, or bladder cancer, as well as pain associated with these conditions. Cancers treatable by the antibodies of the present invention may be solid tumors, or they may be blood-derived tumors such as leukemia.The antibody or its antigen-binding fragment of the present invention is also effective in treating the following conditions: non-malignant acute, chronic, or fracture-related bone pain; rheumatoid arthritis, spinal stenosis; neuropathic low back pain; myofascial pain syndrome; fibromyalgia; temporomandibular joint pain; pancreatic pain; chronic headache; tension headache; HIV-related neuropathy; Charcot-Marie-tooth neuropathy; hereditary sensory neuropathy; peripheral neuropathy; painful neuroma; ectopic proximal and distal discharge; radiculopathy; chemotherapy-induced neuropathic pain; radiotherapy-induced neuropathic pain; post-mastectomy pain; central pain; spinal cord injury pain; post-stroke pain; thalamic pain; complex regional pain syndrome; phantom limb pain; intractable pain; musculoskeletal pain It can also be used to treat pain; joint pain; acute gout pain; mechanical lower back pain; neck pain; tendinitis; injury / exercise pain; pyelonephritis; appendicitis; cholecystitis; intestinal obstruction; hernia; chest pain including cardiac pain; pelvic pain, renal colic, acute labor pain including contractions (obstetric pain); cesarean section pain; burn and trauma pain; endometriosis; herpes zoster pain; sickle cell anemia; acute pancreatitis; sinusitis pain, orofacial pain including toothache; multiple sclerosis pain; leprosy pain; Behçet's disease pain; painful steatosis; phlebitis pain; Guillain-Barré pain; painful leg and mobile toes; Haglund syndrome; Fabry disease pain; bladder and genitourinary tract disorders; overactive bladder; painful bladder syndrome; interstitial cystitis; or prostatitis.

[0175] Combination therapy As described above, the method of the present invention, according to certain embodiments, involves administering one or more additional therapeutic agents to a target in combination with an antibody against RET. As used herein, the expression “in combination with” means that the additional therapeutic agent is administered before, after, or concurrently with the pharmaceutical composition containing the anti-RET antibody. The term “in combination with” also includes sequential or concurrent administration of the anti-RET antibody and the second therapeutic agent.

[0176] For example, when administered "before" the pharmaceutical composition containing the anti-RET antibody, the additional therapeutic agent may be administered approximately 72 hours, 60 hours, 48 ​​hours, 36 hours, 24 hours, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, or 10 minutes before the administration of the pharmaceutical composition containing the anti-RET antibody. When administered "after" the pharmaceutical composition containing the anti-RET antibody, the additional therapeutic agent may be administered approximately 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, or 72 hours after the administration of the pharmaceutical composition containing the anti-RET antibody. Administration "contemporaneously" or in conjunction with a pharmaceutical composition containing an anti-RET antibody means that the additional therapeutic agent is administered to the subject in a separate dosage form less than 5 minutes (before, after, or concurrently) with the administration of the pharmaceutical composition containing the anti-RET antibody, or that it is administered to the subject as a single combination drug formulation containing both the additional therapeutic agent and the anti-RET antibody.

[0177] Combination therapy may include any additional therapeutic agents that can be advantageously combined with the anti-RET antibody of the present invention and the antibody or bioactive fragment of the antibody of the present invention. For example, a second or third therapeutic agent, such as a chemotherapeutic agent or radiotherapy useful for inhibiting the proliferation of tumor cells in a target, may be used to help reduce the tumor burden in the patient. Alternatively, the antibody may be used as adjuvant therapy after surgical removal of the tumor, either alone or in combination with a chemotherapeutic agent, radiotherapy, or bone marrow regeneration agent. The antibody may also be used in combination with other therapies including a second antibody specific to RET or an antibody specific to a RET ligand as described above, or with an antibody or fusion molecule that conjugates GFRα1 (see SEQ ID NO: 308).

[0178] Diagnostic use of antibodies Furthermore, the anti-RET antibody of the present invention may be used to detect and / or measure RET in a sample, for example, for diagnostic purposes. Confirmation of a disease or condition thought to be associated with RET is assumed to be performed, for example, by measuring the presence of RET in a biopsy sample from a tumor (i.e., tumor cells) that depends on growth via RET signaling. An exemplary RET diagnostic assay may include, for example, contacting a sample obtained from a patient with the anti-RET antibody of the present invention, the anti-RET antibody being labeled with a detectable label or reporter molecule, or being used as a capture ligand to selectively isolate cells expressing the RET protein from a patient sample. Alternatively, an unlabeled anti-RET antibody can be used for diagnostic applications in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule is, 3 H, 14 C, 32 P, 35 S, or 125 This may include radioactive isotopes such as I; fluorescent or chemiluminescent moieties such as fluorescein isothiocyanate or rhodamine; or enzymes such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure RET containing F protein in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0179] The samples that can be used in the RET diagnostic assay according to the present invention include any tissue or body fluid samples that can be obtained from a patient containing a detectable amount of RET protein or fragments thereof under normal or pathological conditions. Generally, a baseline or standard level of RET protein is first established by measuring the level of RET in a specific sample obtained from a healthy patient (e.g., a patient without a disease or condition associated with the presence of RET). This baseline level of RET can then be compared to the level of RET measured in a sample obtained from an individual suspected of having a disease or condition associated with RET, or symptoms associated with such a condition. [Examples]

[0180] The following examples are provided to those skilled in the art to provide a complete disclosure and explanation of how the methods and compositions of the present invention are prepared and used, and are not intended to limit the scope that the inventors consider to be the present invention. While efforts have been made to ensure accuracy with respect to the numerical values ​​used (e.g., quantity, temperature, etc.), some experimental errors and deviations should be taken into account. Unless otherwise specified, parts are parts by weight, molecular weight is the average molecular weight, temperature is the temperature in Celsius, and atmospheric pressure is atmospheric pressure or approximately atmospheric pressure.

[0181] (Example 1: Generation of human antibodies against RET protein) Antibodies against RET can be generated using an immunogen comprising any one of the following. In certain embodiments, the antibodies of the present invention are obtained from mice immunized with a primary immunogen such as full-length RET protein (see, for example, SEQ ID NO: 310, also found in ATCC accession number NP_066124.1, for the human RET51 isoform, both having signal sequences from residues 1-28; and SEQ ID NO: 312, also found in ATCC accession number NP_065681.1, for the human RET9 isoform). Mice may be given one or more additional immunizations containing the same molecule, or they may be given additional immunizations with an immunogenic fragment thereof, for example, the human RET extracellular domain in the range of amino acids 1-635 of SEQ ID NO: 313, also found in ATCC accession number NP_066124.1, having a signal sequence in the range of amino acid residues 1-28. In a particular embodiment, mice are injected with full-length RET protein and then immunized with additional molecules prepared by one or a recombinant of the constructs shown as SEQ ID NOs. 305, 306, 307, and 313.

[0182] In certain embodiments, the antibodies of the present invention are obtained from mice immunized with a primary immunogen such as a bioactive RET molecule or immunogenic fragment of a RET protein, or a full-length protein or DNA encoding an active fragment thereof. The immunogen may be delivered to the animal via any route including, but not limited to, intramuscular, subcutaneous, intravenous, or nasal passages.

[0183] In certain embodiments, the full-length RET protein or a fragment thereof may be used to prepare monospecific, bispecific, or polyspecific antibodies.

[0184] As described above, full-length proteins or fragments used as immunogens, along with adjuvants to stimulate the immune response, were directly administered to VELOCIMMUNE® mice containing DNA encoding the variable regions of human immunoglobulin heavy chains and kappa light chains. The antibody immune response was monitored by RET immunoassay. Once the desired immune response was achieved, splenocytes were harvested and fused with mouse myeloma cells to maintain their viability and form hybridoma cell lines. Hybridoma cell lines were screened and selected to identify cell lines that produced RET-specific antibodies. Using this technique and the various immunogens described above, several chimeric antibodies (i.e., antibodies possessing both human variable domains and mouse constant domains) were obtained; a particular exemplary antibody thus produced was named, for example, H2M7086N.

[0185] Anti-RET antibodies were also isolated directly from antigen-positive B cells without fusing to myeloma cells, as described in US2007 / 0280945A1, which is incorporated specifically herein by reference in its entirety. Using this method, several fully human anti-RET antibodies (i.e., antibodies possessing both a human variable domain and a human constant domain) were obtained; the thus produced variant antibodies were named as follows: H4H8044P, H4H8045P, H4H8046P, H4H8048P, H4H8056P, H4H8058P, H4H8060P, H4H8062P, H4H8066P, H4H8067P, H4H8071P, H4H8076P, H4H8079P, H4H8080P, H4H8083P, H4H8084P, H4H8085P, and H4H8087P.

[0186] The biological properties of exemplary antibodies produced according to the method of this embodiment will be described in detail in the following examples.

[0187] (Example 2: Amino acid sequences of the heavy and light chain variable regions) Table 1 lists heavy and light chain variable region amino acid sequence pairs and their corresponding antibody identifiers for selected antibodies specific to the RET protein. Antibodies are typically referred to herein according to the following nomenclature: an Fc prefix (e.g., "H4H", "H1M", "H2M") followed by a numerical identifier (e.g., "7086" as shown in Table 1), followed by a "P" or "N" suffix. Thus, according to this nomenclature, an antibody may be called, for example, "H2M7086N". The H4H, H1M, and H2M prefixes in antibody names used herein indicate a specific Fc region of the antibody. For example, an "H2M" antibody has a mouse IgG2 Fc, while an "H4H" antibody has a human IgG4 Fc. As will be recognized by those skilled in the art, an H1M or H2M antibody can be converted to an H4H antibody, and vice versa, but in either case the variable domain (including the CDR) indicated by the numerical identifier shown in Table 1 remains the same. Antibodies with the same numerical name but different suffixes (N, B, or P) refer to antibodies that have the same CDR sequence but possess heavy and light chain variations in regions outside the CDR sequence (i.e., the framework region). Thus, N, B, and P variants of a particular antibody have the same CDR sequence within their heavy and light chain variable regions, but their framework regions differ from one another. [Table 1-1] [Table 1-2]

[0188] (Example 3: Binding affinity and rate constant derived from surface plasmon resonance of a human monoclonal anti-RET antibody) The binding affinity and rate constant of human anti-RET antibodies are determined by surface plasmon resonance (Biacore). Determined at 25 °C and 37 °C by a T200 (Tables 2-3). Antibodies expressed as human IgG4 Fc (i.e., the "H4H" designation) were captured on an anti-human Fc sensor surface (mAb-capture format), and soluble monomeric (hRET.mmh; SEQ ID NO: 305, macaca fascicularis (mf) RET.mmh; SEQ ID NO: 306) or dimeric (hRET.mFc; SEQ ID NO: 307) RET proteins were injected onto the sensor surface. The binding equilibrium dissociation constant (K D ), and the dissociation half-life (t 1 / 2 ) were calculated from the kinetic rate constants: K D [M] = k d / k a ; and t 1 / 2 (min) = (ln 2 / (60 * k<s d ). The calculations were performed using Biacore T200 evaluation software v1.0.

[0189] Some of the antibodies of the present invention showed affinities below nanomolar concentrations for human and monkey RET proteins (Tables 2-3).

Table 2-1

Table 2-2

Table 2-3

Table 3-1

Table 3-2

[0190] (Example 4 Anti-RET Antibodies Potently Block the Binding of Human RET to the GFRα1 / GDNF Co-Complex) The ability of anti-RET antibodies to block the binding of human RET to plate-bound GDNF:GFRα1, which had previously formed a complex with RET, was evaluated using competitive sandwich ELISA. Most RET antibodies strongly blocked the binding of RET to the plate-bound GDNF / GFRα1 cocomplex (Table 4). 50 The values ​​ranged from 5.2 nM to below the theoretical minimum value of the assay (250 pM), and the maximum blockade ranged from 72% to 96%.

[0191] Detailed method Recombinant human dimer GDNF (R&D systems) and human GFRα1.mFc (SEQ ID NO: 308) were mixed in PBS in a 1:1 molar ratio to obtain a final co-complex concentration of approximately 2.0 μg / ml. The GDNF-GFRα1 co-complex was incubated at room temperature (RT) for 1 hour, and then coated onto 96-well microtiter plates overnight at 4°C. Nonspecific binding sites were blocked with BSA.

[0192] Separately, 1 nM biotinylated monomer RET protein (biot-hRET.mmh; SEQ ID NO: 305) was titrated with serially diluted antibodies in varying amounts ranging from 0 to 120 nM. The antibody-RET mixture was incubated at RT for 1 hour and then transferred to a microtiter plate pre-coated with hGDNF / hGFRα1 coconucle. After allowing binding to proceed at RT for 1 hour, the plate was thoroughly washed. Plate-bound biot-hRET.mmh was detected with HRP-conjugated streptavidin and developed with TMB. The plate was read at 450 nm, and data analysis was performed using a sigmoid dose-response model in Prism® software.

[0193] The IC was calculated as the antibody concentration required to block 50% of the binding of hRET to hGDNF / hGFRα1. 50The value was used as an indicator of blocking efficacy. The maximum blocking value represents the ability of the anti-RET antibody to block hRET binding compared to baseline. Baseline values ​​were calculated as absorbance measured with a constant amount of hRET in the dose curve (0% blocking) and absorbance measured without the addition of hRET (100% blocking). The percentage of blocking at the highest concentration of the tested antibody was determined using the absorbance value of the well containing the highest concentration of each antibody. IC 50 A summary of the maximum blocking percentages is shown in Table 4. [Table 4]

[0194] (Example 5: The anti-RET antibody inhibits ligand-dependent RET signaling in the SRE-luciferase reporter assay and exhibits strong internal translocation.) In this example, the effects of anti-RET antibodies on RET signaling and internal translocation were investigated using MCF7 and hRET-manipulated reporter cell lines.

[0195] The glial family ligands GDNF and artemin induce RET activation through the formation of high-affinity co-complexes with GFRα1 or GFRα3, respectively, bringing together two RET molecules and initiating phosphorylation of specific tyrosine residues. Transphosphorylation of RET activates several downstream intracellular cascades, and upregulation of RET signaling is associated with the pathology of several diseases, including cancer (Borrello, MG, et al., (2013), Expert. Opin. Ther. Targets 17(4): 403-419).

[0196] To test the ability of RET antibodies to block GDNF-mediated signaling, the MCF7 / SRE-Luc strain was created by transducing a serum response factor (SRE)-regulated luciferase reporter gene into the human mammary cancer cell line MCF7, which expresses RET and GFRα1. The antibody of the present invention showed potent inhibition of GDNF-stimulated RET signaling and IC 50The values ​​ranged from 143 pM to >100 nM (Table 5). The percentage of inhibition ranged from 60 to 100%. Several non-blocking antibodies were also identified; H4H8085P stimulated luciferase activity to 50% of the level observed by GDNF, while H4H8044P, H4H8076P, and H4H8046P were weaker activators of the luciferase response (2-5% activation).

[0197] To determine whether antibodies blocking GDNF-mediated RET signaling are also effective against artemin-induced activity, we constructed an engineered HEK293 / hGFRa3 / hRET SRELuc cell line. Most GDNF-dependent blockers of RET signaling were also blockers of artemin-dependent signaling activity in this cell line (Table 5; columns 5-6). Interestingly, H4H8048P was identified as a more potent blocker of artemin-dependent signaling compared to GDNF-dependent signaling, likely reflecting different epitopes to which the GDNF-GFRα1 and artemin-GFRα3 cocomplexes bind on the RET receptor.

[0198] Finally, to understand whether the observed blocking activity was obtained by the degradation of the RET receptor upon antibody binding, several antibodies were tested in an internal distribution assay (Table 6). Of the seven antibodies tested, H4H8087P was identified as the most strongly internally distributed, and H4H8079P and H4H7086P also showed potent internal distribution.

[0199] In conclusion, this example illustrates that the anti-RET antibody of the present invention exhibits broad-spectrum activating and inhibitory activity against RET signaling in the presence of the glial family ligands GDNF and artemin. [Table 5] [Table 6]

[0200] Detailed method Generation of MCF7 / SRE-luciferase stable cell line MCF7 cells naturally express RET and GFRα1. The production of MCF7 / SRELuc cells utilized stably integrated SRE-luciferase generated via transduction of MCF7 with the Cignal Lenti SRE reporter kit (SABiosciences) and selection with puromycin for two weeks. The lentivirus expresses the firefly luciferase gene under the control of a minimal CMV promoter and tandem repeats of the serum response element (SRE).

[0201] Generation of HEk293 / hGFRa1(or 3) / hRET / SRE-luciferase stable cell line Human GFRα (1 or 3) and hRET were stably introduced into HEK293 cells via sequential rounds of lipofectamine 2000-mediated transfection and selected in 500 μg / ml G418 (hGFRa1 or 3) and 100 μg / ml hygromycin B (hRET) for at least two weeks. Then, HEK293 double stable strains expressing hGFRa1 / hRET or hGFRa3 / hRET were transduced with the Cignal Lenti SRE reporter kit as described above to generate HEK293 / hGFRa1 / hRET / SRE-Luc cell line and artemin-responsive HEK293 / GFRa3 / hRET / SRE-Luc cell line.

[0202] Inhibition of GDNF-stimulated luciferase activity in MCF7 / SRE-luciferase engineered cell line 20,000 MCF7-SRE-luc cells were seeded in a PDL-coated 96-well plate in Optimem + 0.5% FBS and grown overnight at 37 °C, 5% CO2. For the inhibition curve, cells were incubated for 1 hour with serially diluted anti-hRET mAb in the range of 1.6 pM to 1 μM. Then, a fixed dose of human GDNF (4 - 10 pM) was added and the cells were incubated for an additional 6 hours.

[0203] To evaluate the activation properties of anti-RET mAbs, MCF7-SRE-Luc cells were incubated with serially diluted anti-hRET mAbs ranging from 1.6 pM to 1 μM for 6 hours in the absence of ligands.

[0204] The dose-response curves of GDNF were measured using serially diluted GDNF in the range of 0.05 pM to 10 nM. The GDNF was added to the wells without antibody and incubated at 37°C for 6 hours. Luciferase activity was measured using ONE GLO® reagent (Promega), and relative light units (RLU) were measured using a Victor luminometer (Perkin Elmer).

[0205] Inhibition of artemin-stimulated luciferase activity in HEK293 / hGFRa3 / hRET-manipulated cell lines Inhibition of artemin-stimulated luciferase activity in the HEK293 / hGFRa3 / hRET / SRE-Luc cell line was evaluated using an anti-RET antibody via the method described for MCF7 / SRE-Luc cells. To generate inhibition curves, cells were incubated with serially diluted anti-hRET antibodies ranging from 1.6 pM to 1 μM for 1 hour. Subsequently, cells were stimulated with a constant dose of h-artemin (100 pM) for 6 hours. Dose-response curves for artemin were generated by adding serially diluted h-artemin (0.17 pM to 10 nM) to cells at 37°C for 6 hours without antibody addition. Luciferase activity measurements and curve fitting were performed as described for GDNF-stimulated luciferase activity.

[0206] EC 50 / I C 50 Value calculation EC 50 / I C 50 The values ​​were determined using a 4-parameter logistic equation for a 12-point response curve with GraphPad Prism. The blockade percentage is reported for the highest antibody dose, and the data is reported as mean ± standard deviation (SD).

[0207] Quantitative analysis of the internal distribution characteristics of anti-RET antibodies To test the internal migration of anti-hRET mAbs, HEK293 / hGFRa1 / hRET / SRE-Luc cells were incubated with antibody (10 μg / ml) on ice for 30 minutes and then washed once. The cells were then incubated with alexa488 conjugate anti-hFc Fab secondary antibody for 30 minutes and washed a second time. The antibody was allowed to migrate internally at 37°C for 4 hours, or maintained at 4°C to prevent migration. Cells were fixed in 4% formaldehyde, and alexa488 on the cell surface was quenched by incubation at 4°C for 1 hour with anti-alexa488 quench antibody. Nuclei were stained with Hoechst dye, and images were acquired using ImageXpress micro XL (Molecular Devices).

[0208] The total Alexa488 intensity in intracellular vesicles of quenched samples at 37°C was quantified using Columbus image analysis software (Perkin Elmer). The total internal migration mAb intensity is expressed as the percentage of the mAb that most strongly migrated internally.

[0209] (Example 6: Production of bispecific antibodies) Various bispecific antibodies are generated for use in the practical application of the methods of the present invention. For example, RET-specific antibodies are generated in a bispecific format ("bi-specific antibody") in which variable regions that bind to distinct domains of the RET protein are linked together to confer dual domain specificity within a single binding molecule. A well-designed bispecific antibody can enhance the overall RET neutralizing efficacy by increasing both specificity and binding avidity. Variable regions specific to individual domains are paired on a structural scaffold, thereby allowing each region to bind simultaneously to a distinct epitope or to different regions within a single domain. In an example of a bispecific antibody, a heavy chain variable region (V) from a binder specific to one domain is used. H ) is converted from a series of binders specific to the second domain into a light chain variable region (VL ) and rearrange it, and that V H Without impairing the original singularity for the original V H Non-cognate V that can form a pair with L Identify the partner. In this way, a single V L segment (for example, V L 1) Two different V H Domain (for example, V H 1 and V H 2) Combined with the two connecting "arms" (V H 1-V L 1, and V H 2-V L It is possible to generate a bispecific antibody composed of 1). L The use of segments reduces system complexity, thereby simplifying and increasing the efficiency of the cloning, expression, and purification processes used to generate bispecific antibodies (see, for example, USSN 13 / 022759 and US2010 / 0331527).

[0210] Alternatively, antibodies conjugating RET and a second target, such as, but not limited to, a tumor antigen, may be prepared in a bispecific format using the techniques described herein or other techniques known to those skilled in the art. A variable region of an antibody that binds to a distinct region may be ligated with a variable region that binds to a relevant site on a different antigen, for example, to confer dual antigen specificity within a single binding molecule. A well-designed bispecific antibody of this property performs a dual function. For example, a bispecific antibody conjugating RET and one of its ligands may better inhibit tumor cell growth without the need to administer a composition containing two distinct antibodies. Each variable region can be conjugated to a distinct antigen by combining a variable region specific to RET with a variable region specific to one of its ligands, and forming a pair on a structural scaffold.

[0211] The bispecific binder is tested for binding and functional blockade of a target antigen, e.g., RET, in any of the above assays with respect to the antibody. For example, bispecific interactions are evaluated using standard methods for measuring soluble protein binding, e.g., Biacore, ELISA, size exclusion chromatography, multi-angle laser light scattering, direct scanning calorimetry, and other methods. Binding of a bispecific antibody to both RET and one of its ligands is determined through the use of an ELISA binding assay in which synthetic peptides representing different antigens are coated into wells of a microtiter plate, and binding of the bispecific antibody is determined through the use of a secondary detection antibody. Binding experiments can also be performed using surface plasmon resonance experiments, where the real-time binding interaction of the peptide to the antibody is measured by flowing the peptide or bispecific antibody across a sensor surface in which the bispecific antibody or peptide is captured, respectively. Functional in vitro blockade of both RET and one of its ligands by a bispecific antibody is determined using any bioassay, such as the assays described herein, or by in vivo protective studies in appropriate animal models, such as tumor-bearing animal models. The present invention provides, for example, the following items: (Item 1) An isolated human monoclonal antibody or its antigen-binding fragment that specifically binds to RET (transfection rearrangement) receptor tyrosine kinase, wherein the antibody has the following characteristics: (a) It must be a fully human antibody; (b) When measured by surface plasmon resonance, approximately 1.0 × 10 -7 M ~ approx. 1.0×10 -12 K in the range of M D To demonstrate; (c) Inhibiting or blocking the binding or interaction of RET with one or more GDNF family member ligands (GDNF, neurturin, artemin, and parcefin) that form a complex with its corresponding co-receptor (GFRα1, GFRα2, GFRα3, and GFRα4, respectively); (d) Inhibiting RET signaling mediated by one or more GDNF family member ligands selected from GDNF, neurturin, artemin, and parcephin; (e) To enhance the internal translocation / degradation of RET after the antibody has bound to the RET receptor; (f) Containing a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, and 290; or (g) Containing a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, and 298. An isolated human monoclonal antibody or its antigen-binding fragment having one or more of the following: (Item 2) The aforementioned antibody binds human RET to the GDNF:GFRα1 cocomplex in an IC50 range of approximately 100 pM to approximately 7.0 nM. 50 An isolated human monoclonal antibody or its antigen-binding fragment, as described in item 1, which blocks at a certain value. (Item 3) The antibody binds human RET to the GDNF:GFRα1 cocomplex in an IC50 range of approximately 250 pM to approximately 5.2 nM. 50 An isolated human monoclonal antibody or its antigen-binding fragment, as described in item 2, which blocks at a certain value. (Item 4) An isolated human monoclonal antibody or its antigen-binding fragment as described in item 3, wherein the percentage of human RET blocking the GDNF:GFRα1 cocomplex is in the range of approximately 40% to 100%. (Item 5) An isolated human monoclonal antibody or its antigen-binding fragment as described in item 4, wherein the blockade percentage of human RET against the GDNF:GFRα1 cocomplex is in the range of approximately 57% to approximately 97%. (Item 6) GDNF-mediated RET signaling is observed in IC values ​​in the range of approximately 50 pM to 100 nM. 50 An isolated human monoclonal antibody or its antigen-binding fragment, as described in item 1, that is inhibited by a certain value. (Item 7) GDNF-mediated RET signaling is observed in IC values ​​in the range of approximately 143 pM to greater than 100 nM. 50 An isolated human monoclonal antibody or its antigen-binding fragment, as described in item 6, that is inhibited by a certain value. (Item 8) An isolated human monoclonal antibody or its antigen-binding fragment as described in item 6, which inhibits GDNF-mediated RET signaling by approximately 40% to 100%. (Item 9) An isolated human monoclonal antibody or its antigen-binding fragment as described in item 7, which inhibits GDNF-mediated RET signaling by approximately 60% to 100%. (Item 10) Artemin-mediated RET signaling occurs in ICs in the range of approximately 100 pM to 500 nM. 50 An isolated human monoclonal antibody or its antigen-binding fragment, as described in item 1, that is inhibited by a certain value. (Item 11) Artemin-mediated RET signaling occurs in ICs in the range of approximately 250 pM to 341 nM. 50 An isolated human monoclonal antibody or its antigen-binding fragment, as described in item 10, that is inhibited by a certain value. (Item 12) An isolated human monoclonal antibody or its antigen-binding fragment as described in item 11, which inhibits artemin-mediated RET signaling by approximately 57% to approximately 100%. (Item 13) An isolated human monoclonal antibody or its antigen-binding fragment as described in item 1, comprising an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 122, 130 / 138, 146 / 154, 162 / 170, 178 / 186, 194 / 202, 210 / 218, 226 / 234, 242 / 250, 258 / 266, 274 / 282, and 290 / 298. (Item 14) An isolated human monoclonal antibody or its antigen-binding fragment that specifically binds to RET, wherein the antibody contains three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) within an HCVR amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, and 290. Isolated human monoclonal antibodies or antigen-binding fragments thereof, comprising HCVR and LCVR containing three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within an LCVR amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, and 298. (Item 15) (a) HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 228, 244, 260, 276, and 292; (b) HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 230, 246, 262, 278, and 294; (c) HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 232, 248, 264, 280, and 296; (d) LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268, 284, and 300; (e) LCDR2 domains having amino acid sequences selected from the group consisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 222, 238, 254, 270, 286, and 302; and (f) LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 224, 240, 256, 272, 288, and 304 The isolated human monoclonal antibody or antigen-binding fragment described in item 14, including the following: (Item 16) An isolated antibody or antigen-binding fragment that competes for specific binding to RET with an antibody or antigen-binding fragment containing heavy and light chain sequence pairs selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 122, 130 / 138, 146 / 154, 162 / 170, 178 / 186, 194 / 202, 210 / 218, 226 / 234, 242 / 250, 258 / 266, 274 / 282, and 290 / 298. (Item 17) An isolated antibody or its antigen-binding fragment that binds to the same epitope on a RET recognized by an antibody containing heavy and light chain sequence pairs selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 122, 130 / 138, 146 / 154, 162 / 170, 178 / 186, 194 / 202, 210 / 218, 226 / 234, 242 / 250, 258 / 266, 274 / 282, and 290 / 298. (Item 18) An isolated nucleic acid molecule encoding an antibody or antigen-binding fragment as described in any of items 1-17. (Item 19) An expression vector containing the nucleic acid molecule described in item 18. (Item 20) A pharmaceutical composition comprising one or more antibodies or antigen-binding fragments thereof that specifically bind to RET as described in any of items 1 to 17, and a pharmaceutically acceptable carrier or diluent. (Item 21) A method for treating a disorder or condition related to the expression, activation or signaling of the RET receptor tyrosine kinase gene or a rearranged form thereof, or pain associated with said disorder or condition, comprising administering to a patient in need of such treatment one or more antibodies or antigen-binding fragments thereof described in any of items 1 to 17, or a pharmaceutical composition comprising one or more antibodies described in any of items 1 to 17. (Item 22) The method according to item 21, wherein the disorder or condition relating to the expression, activation, or signaling of the RET receptor tyrosine kinase gene or a rearranged form thereof is cancer, and the cancer is selected from the group consisting of thyroid cancer, lung cancer, pancreatic cancer, skin cancer, breast cancer, and hematological cancers. (Item 23) The method according to item 21, wherein the disorder or condition related to the expression, activation, or signaling of the RET receptor tyrosine kinase gene or a rearranged form thereof is selected from the group consisting of acute pain, chronic pain, neuropathic pain, inflammatory pain, arthritis, osteoarthritis, migraine, cluster headache, trigeminal neuralgia, herpetic neuralgia, systemic neuralgia, neurodegenerative disorders, neuroendocrine disorders, visceral pain, acute gout, postherpetic neuralgia, diabetic neuropathy, sciatica, back pain, head and neck pain, severe or intractable pain, breakthrough pain, postoperative pain, toothache, rhinitis, cancer pain, or bladder disorder. (Item 24) A method for inhibiting tumor growth or proliferation of tumor cells, wherein the tumor or tumor cells express RET or a rearranged form thereof, and the method comprises administering to a patient in need one or more antibodies or antigen-binding fragments thereof described in any of items 1 to 17, or a pharmaceutical composition comprising one or more antibodies described in any of items 1 to 17. (Item 25) The method according to item 24, wherein the tumor is a solid tumor or a tumor of blood origin. (Item 26) The method according to item 25, wherein the solid tumor is selected from the group consisting of thyroid tumors, lung tumors, pancreatic tumors, skin tumors, and breast tumors. (Item 27) The method according to item 26, wherein the thyroid tumor is papillary thyroid carcinoma (PTC) or medullary thyroid carcinoma (MTC). (Item 28) The method according to item 27, wherein the medullary thyroid carcinoma is a hereditary MTC selected from the group consisting of multiple endocrine neoplasia type 2 or 3 (MEN2A, MEN2B) and familial medullary thyroid carcinoma (FMTC) syndrome, or the medullary thyroid carcinoma is a sporadic MTC. (Item 29) The method according to item 26, wherein the lung tumor is lung adenocarcinoma. (Item 30) The method according to item 26, wherein the lung tumor is non-small cell lung cancer (NSCLC). (Item 31) The method according to item 26, wherein the skin tumor is a melanoma. (Item 32) The method according to item 25, wherein the blood-derived tumor is leukemia. (Item 33) The method described in item 32, wherein the leukemia is chronic myelomonocytic leukemia. (Item 34) A method for downregulating RET expression and / or function, comprising administering to a patient in need one or more antibodies or antigen-binding fragments thereof described in any of items 1 to 17, or a pharmaceutical composition comprising one or more antibodies described in any of items 1 to 17. (Item 35) The method according to item 34, wherein downregulating the expression and / or function of RET results in downregulation of a downstream signaling pathway selected from the group consisting of the RAS / RAF pathway and the PI3K pathway. (Item 36) The method according to any one of items 21 to 35, wherein the antibody or antigen-binding fragment is administered to the patient in combination with a second therapeutic agent. (Item 37) The method according to item 36, wherein the second therapeutic agent is selected from the group consisting of a low molecular weight tyrosine kinase inhibitor, an antitumor agent, an siRNA specific to RET, a second antibody specific to RET, and an analgesic. (Item 38) The aforementioned small molecule tyrosine kinase inhibitors include vandetanib, cediranib (AZD2171), gefitinib, erlotinib, SU14813, batalanib, sorafenib, sorafenib (BAY43-9006), sunitinib, cabozantinib, motesanib, XL-647, XL-999, AG-013736, BIBF1120, TSU68, GW786034, and AEE. The method according to item 37, selected from the group consisting of 788, CP-547632, KRN951, CHIR258, CEP-7055, OSI-930, ABT-869, E7080, ZK-304709, BAY57-9352, L-21649, BMS582664, XL-880, XL-184, XL-820, RPI-1, PP-1, and NVP-AST478. (Item 39) The method according to item 37, wherein the antitumor agent is selected from the group consisting of chemotherapeutic agents, radionuclides, and antibody-drug conjugates. (Item 40) The aforementioned analgesic may be a nerve growth factor (NGF) inhibitor (e.g., a small molecule NGF antagonist or anti-NGF antibody), aspirin or another NSAID, morphine, a steroid (e.g., prednisone), or an anti-Na. v 1.7 antibody or Na v 1.7 Small molecule inhibitors, Na v 1.8 Antagonists (e.g., anti-Na) v 1.8 antibody or Na v 1.8 small molecule inhibitors), Na v 1.9 Antagonists (e.g., anti-Na) v 1.9 antibody or Na vThe method described in item 37, selected from the group consisting of 1.9 small molecule inhibitors, cytokine inhibitors (e.g., interleukin-1 (IL-1) inhibitors (e.g., lilonacept ("IL-1 trap"); Regeneron) or anakinra (KINERET®, Amgen), small molecule IL-1 antagonists or anti-IL-1 antibodies; IL-18 inhibitors (e.g., small molecule IL-18 antagonists or anti-IL-18 antibodies); IL-6 or IL-6R inhibitors (e.g., small molecule IL-6 antagonists, anti-IL-6 antibodies, or anti-IL-6 receptor antibodies), caspase-1 inhibitors, p38, IKK1 / 2, CTLA-4Ig, or opioids).

Claims

1. An isolated human monoclonal antibody or its antigen-binding fragment that specifically binds to RET (transfection rearrangement) receptor tyrosine kinase, The heavy chain variable region (HCVR) amino acid sequence shown in SEQ ID NO: 210 and the light chain variable region (LCVR) amino acid sequence shown in SEQ ID NO: 218; or The heavy chain variable region (HCVR) amino acid sequence shown in SEQ ID NO: 242 and the light chain variable region (LCVR) amino acid sequence shown in SEQ ID NO: 250 An isolated human monoclonal antibody or its antigen-binding fragment, containing [the specified substance].

2. The isolated human monoclonal antibody or its antigen-binding fragment according to claim 1, wherein the antibody is a fully human antibody.

3. An isolated human monoclonal antibody or its antigen-binding fragment that specifically binds to RET, wherein the antibody is Three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within the HCVR amino acid sequence shown in SEQ ID NO: 210 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the LCVR amino acid sequence shown in SEQ ID NO: 218; or Three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within the HCVR amino acid sequence shown in SEQ ID NO: 242 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the LCVR amino acid sequence shown in SEQ ID NO: 250 Includes, The CDR is identified according to the definition of Kabat, Chothia, or AbM. Isolated human monoclonal antibody or its antigen-binding fragment.

4. The isolated human monoclonal antibody or its antigen-binding fragment according to claim 3, wherein the antibody is a fully human antibody.

5. The isolated human monoclonal antibody or its antigen-binding fragment according to claim 3, comprising HCDR1 having the amino acid sequence of SEQ ID NO: 212, HCDR2 having the amino acid sequence of SEQ ID NO: 214, HCDR3 having the amino acid sequence of SEQ ID NO: 216, LCDR1 having the amino acid sequence of SEQ ID NO: 220, LCDR2 having the amino acid sequence of SEQ ID NO: 222, and LCDR3 having the amino acid sequence of SEQ ID NO:

224.

6. The isolated human monoclonal antibody or its antigen-binding fragment according to claim 3, comprising HCDR1 having the amino acid sequence of SEQ ID NO: 244, HCDR2 having the amino acid sequence of SEQ ID NO: 246, HCDR3 having the amino acid sequence of SEQ ID NO: 248, LCDR1 having the amino acid sequence of SEQ ID NO: 252, LCDR2 having the amino acid sequence of SEQ ID NO: 254, and LCDR3 having the amino acid sequence of SEQ ID NO:

256.

7. An isolated nucleic acid molecule encoding the HCVR or LCVR of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

8. An expression vector comprising the nucleic acid molecule described in claim 7.

9. A pharmaceutical composition comprising one or more antibodies or antigen-binding fragments thereof that specifically bind to RET, as described in any one of claims 1 to 6, and a pharmaceutically acceptable carrier or diluent.

10. The pharmaceutical composition according to claim 9 for treating in a patient a disorder or condition related to the expression, activation or signaling of the RET receptor tyrosine kinase gene or a rearranged form thereof, or pain associated with said disorder or condition.

11. The pharmaceutical composition according to claim 10, wherein the disorder or condition relating to the expression, activation, or signaling of the RET receptor tyrosine kinase gene or a rearranged form thereof is cancer, and the cancer is selected from the group consisting of thyroid cancer, lung cancer, pancreatic cancer, skin cancer, breast cancer, and blood-derived cancer.

12. The pharmaceutical composition according to claim 10, wherein the disorder or condition related to the expression, activation, or signaling of the RET receptor tyrosine kinase gene or a rearranged form thereof is selected from the group consisting of acute pain, chronic pain, neuropathic pain, inflammatory pain, arthritis, osteoarthritis, migraine, cluster headache, trigeminal neuralgia, herpetic neuralgia, systemic neuralgia, neurodegenerative disorders, neuroendocrine disorders, visceral pain, acute gout, postherpetic neuralgia, diabetic neuropathy, sciatica, back pain, head and neck pain, severe or intractable pain, breakthrough pain, postoperative pain, toothache, rhinitis, cancer pain, and bladder disorders.

13. A pharmaceutical composition according to claim 9 for inhibiting tumor growth or proliferation of tumor cells in a patient, wherein the tumor or tumor cells express RET or a rearranged form thereof.

14. The pharmaceutical composition according to claim 13, wherein the tumor is a solid tumor or a tumor of blood origin.

15. The pharmaceutical composition according to claim 14, wherein the solid tumor is selected from the group consisting of thyroid tumors, lung tumors, pancreatic tumors, skin tumors, and breast tumors.

16. The aforementioned lung tumor is lung adenocarcinoma; The thyroid tumor is either papillary thyroid carcinoma (PTC) or medullary thyroid carcinoma (MTC); The lung tumor in question is non-small cell lung cancer (NSCLC); The aforementioned skin tumor is melanoma; The aforementioned blood-derived tumor is leukemia; or The aforementioned leukemia is chronic myelomonocytic leukemia. The pharmaceutical composition according to claim 15.

17. The pharmaceutical composition according to claim 16, wherein the medullary thyroid carcinoma is a hereditary MTC selected from the group consisting of multiple endocrine neoplasia type 2 or 3 (MEN2A, MEN2B) and familial medullary thyroid carcinoma (FMTC) syndrome, or the medullary thyroid carcinoma is a sporadic MTC.

18. The pharmaceutical composition according to claim 9 for downmodulating RET signaling and / or function in a patient.

19. The pharmaceutical composition according to claim 18, wherein downmodulating the RET signaling and / or function results in downmodulation of a downstream signaling pathway selected from the group consisting of the RAS / RAF pathway and the PI3K pathway.

20. The pharmaceutical composition according to any one of claims 10 to 19, wherein the antibody or antigen-binding fragment is administered to the patient in combination with the second therapeutic agent.

21. The pharmaceutical composition according to claim 20, wherein the second therapeutic agent is selected from the group consisting of a low molecular weight tyrosine kinase inhibitor, an antitumor agent, a RET-specific siRNA, a RET-specific second antibody, and an analgesic.

22. The aforementioned small molecule tyrosine kinase inhibitors include vandetanib, cediranib (AZD2171), gefitinib, erlotinib, SU14813, batalanib, sorafenib, sorafenib (BAY43-9006), sunitinib, cabozantinib, motesanib, XL-647, XL-999, AG-013736, BIBF1120, TSU68, GW786034, and AEE788. A pharmaceutical composition according to claim 21, selected from the group consisting of CP-547632, KRN951, CHIR258, CEP-7055, OSI-930, ABT-869, E7080, ZK-304709, BAY57-9352, L-21649, BMS582664, XL-880, XL-184, XL-820, RPI-1, PP-1, and NVP-AST478.

23. The antitumor agent is selected from the group consisting of chemotherapeutic agents, radionuclides, and antibody-drug conjugates; The aforementioned analgesic is a nerve growth factor (NGF) inhibitor, aspirin or another NSAID, morphine, steroid, or anti-Na. ｖ 1.7 Antibody, Na ｖ 1.7 Small molecule inhibitors, Na ｖ 1.8 Antagonist, Na ｖ 1.9 Selected from the group consisting of antagonists, cytokine inhibitors, small molecule IL-1 antagonists, anti-IL-1 antibodies; IL-18 inhibitors; IL-6 or IL-6R inhibitors, caspase-1 inhibitors, p38, IKK1 / 2, CTLA-4Ig, and opioids; or The aforementioned analgesics include low molecular weight NGF antagonists, anti-NGF antibodies, prednisone, and anti-Na. ｖ 1.8 antibody, Na ｖ 1.8 Small molecule inhibitors, anti-Na ｖ 1.9 antibody, Na ｖ Selected from the group consisting of 1.9 small molecule inhibitors, interleukin-1 (IL-1) inhibitors, lilonacept, anakinra, small molecule IL-18 antagonists, anti-IL-18 antibodies, small molecule IL-6 antagonists, anti-IL-6 antibodies, and anti-IL-6 receptor antibodies. The pharmaceutical composition according to claim 21.

24. The analgesic agent is a low molecular weight NGF antagonist, an anti-NGF antibody, prednisone, an anti-Na ｖ 1.8 antibody, Na ｖ 1.8 low molecular weight inhibitor, anti-Na ｖ 1.9 antibody, Na ｖ selected from the group consisting of a 1.9 low molecular weight inhibitor, an interleukin-1 (IL-1) inhibitor, rilonacept, anakinra, a low molecular weight IL-18 antagonist, an anti-IL-18 antibody, a low molecular weight IL-6 antagonist, an anti-IL-6 antibody, and an anti-IL-6 receptor antibody, The pharmaceutical composition according to claim 21.

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