Human antibodies that bind RET and use methods thereof

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 the RET (rearranged during transfection) receptor tyrosine kinase, as well as compositions containing these antibodies, and methods of treatment using these antibodies.

[0002] Sequence Listing A formal copy of the sequence listing has been electronically filed via EFS-Web simultaneously with this specification as an ASCII format sequence listing with the file name 10582WO01_SeqList_ST25.TXT, creation date April 9, 2020, and size approximately 168 kilobytes. The sequence listing contained in this ASCII format document is part of this specification and is hereby incorporated by reference in its entirety.

Background Art

[0003] The RET (rearranged during transfection) receptor tyrosine kinase is expressed during development in diverse 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, (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). It 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). RET no The RET knockout mice show renal agenesis and lack enteric neurons in the gastrointestinal tract. Very similar phenotypes are observed in both GFRα1 and GDNF knockout mice, 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 line-derived neurotrophic factor (GDNF) family, including GDNF, artemin, neurturin, and persephin. GDNF family ligands interact with and activate RET only in the presence of one of 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 of the coreceptors GFRα1, GFRα2, GFRα3, and GFRα4 are GDNF, neurturin (NRTN), artemin (ARTN), and persephin (PSPN), respectively, although 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 and familial medullary thyroid cancer (Mulligan, LM, et al, (1994), Nat. Genet. 6:70-74) . Furthermore, a large percentage of sporadic medullary thyroid cancers contain somatic activating mutations in RET (Fusco, A. et al, (1987), Nature 328:170-172; Grieco, M. et al, (1990), Cell 60:557-563). These mutations can occur in the kinase domain or the extracellular domain, resulting in unpaired cysteines that are 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, the expression of RET and GFRα1 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 cancers. 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 functional cooperation between these two pathways. Thus, RET signaling appears to be an important 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] Activation of RET is initiated by the binding of GDNF to GFRα1. The GDNF / GFRα1 complex then binds to RET, resulting in receptor dimerization and activation. There are several small molecules that have the ability to inhibit RET, including drugs (vandetanib) that show 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 have been identified (Borrello, MG, et al, (2013), Expert Opin. Ther. Targets, 17(4):403-419). Unfortunately, due to a lack of specificity, some of these compounds showed adverse events in clinical trials and could not be further developed.

[0008] To date, there have been no reports of therapeutic anti-RET monoclonal antibodies for use in the clinical setting for treating tumors that express RET. The studies reported herein describe the generation of fully human monoclonal antibodies that bind to RET and prevent the interaction of RET with one or more GDNF family members that form a complex with its corresponding coreceptor.

[0009] The domain structure of the RET extracellular region is shown in Figure 1 and 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 has not been elucidated, the GDNF / GFRα1 complex appears to contact the RET extracellular domain at multiple sites that include the fourth cadherin-like domain and the cysteine-rich domain. For this reason, antibodies directed against multiple domains of RET may potentially be able to inhibit signaling. Antibodies against RET are described in US6861509 and US2009 / 0136502 and can be found. However, considering the role that RET plays in tumor cell growth and proliferation, and the fact that there are few approved agents for targeting this molecule, there remains a need for inhibitors of RET, such as human antibodies that specifically bind to RET that are very potent and do not produce adverse effects that would preclude their clinical use.

Prior Art Documents

Patent Documents

[0010]

Patent Document 1

Patent Document 2

Summary of the Invention

Means for Solving the Problems

[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 persephin) that have formed a complex with its corresponding coreceptor (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 related embodiments, the human anti-RET antibody described herein prevents the interaction of RET with the artemin / GFRα3 complex. In related embodiments, the human anti-RET antibody described herein prevents the interaction of RET with the neurturin / GFRα2 complex, or the persephin / 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] Considering the role of RET in the development of multiple endocrine neoplasia syndromes and other cancers, the antibodies of the present invention that antagonize / inhibit the signaling activity of RET can be used in the treatment of these tumor syndromes and cancers to inhibit the growth / proliferation of tumor cells. Examples of cancer conditions that can be treated using the RET antagonist antibodies of the present invention include, but are not limited to, thyroid tumors, lung tumors, pancreatic tumors, skin cancer, breast cancer, and leukemia. The thyroid tumors that can be treated using the antagonist anti-RET antibodies of the present invention can include papillary thyroid carcinoma (PTC) or medullary thyroid carcinoma (MTC). The medullary thyroid carcinomas that can be treated using the antagonist anti-RET antibodies of the present invention can include hereditary MTC selected from the group consisting of MEN2A, MEN2B, and familial medullary thyroid carcinoma (FMTC) syndromes, or the medullary thyroid carcinoma can be sporadic MTC. The antibodies of the present invention can also be used to treat the pain associated with these cancer conditions, as well as the pain associated with other diseases, disorders, or conditions in which RET activity or signaling can play a role.

[0014] The antibody can be used as an independent treatment or in combination with a second agent useful for treating a disease or disorder associated with RET expression. In certain embodiments, the antibody can be administered therapeutically in combination with a second agent to treat a disease or disorder or to ameliorate at least one symptom associated with the disease or disorder. If the antibody inhibits RET activity or signaling and is being considered for use, for example, in treating a cancer condition, the second agent can be a chemotherapeutic agent or a myeloid recovery agent, or can be radiation therapy for treating a tumor. If the antibody inhibits RET activity or signaling and is being considered for use in treating pain associated with a particular condition and the treatment allows for the use of a second analgesic, the second agent can be any agent useful for reducing the pain associated with that condition, such as aspirin or another NSAID, morphine, a steroid (e.g., prednisone), a nerve growth factor (NGF) inhibitor (e.g., a small molecule NGF antagonist or an anti-NGF antibody), an anti-Nav 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, IKK1 / 2, CTLA-4Ig, or an opioid.

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

[0016] Thus, in a first aspect, the invention is an isolated human monoclonal antibody or antigen-binding fragment thereof that specifically binds to the RET (rearranged during transfection) receptor tyrosine kinase, wherein the antibody has the following characteristics: (a) being a fully human antibody; (b) having a K -7 in the range of about 1.0×10 -12 M to about 1.0×10 D M as measured by surface plasmon resonance; (c) inhibiting or blocking the binding or interaction of one or more GDNF family member ligands (GDNF, neurturin, artemin, and persephin) with RET that form a complex with their corresponding coreceptors (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 persephin; (e) enhancing RET internalization / degradation after binding of an antibody to the RET receptor; (f) comprising 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) comprising 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 Provided is an isolated human monoclonal antibody or an antigen-binding fragment thereof having one or more of the following.

[0017] In one embodiment, the isolated human monoclonal antibody or an antigen-binding fragment thereof that specifically binds to RET blocks the binding of human RET to the GDNF:GFRα1 co-complex at an IC 50 value in the range of about 100 pM to about 7.0 nM.

[0018] In a related embodiment, the isolated human monoclonal antibody or an antigen-binding fragment thereof that specifically binds to RET blocks the binding of human RET to the GDNF:GFRα1 co-complex at an IC 50 value in the range of about 250 pM to about 5.2 nM.

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

[0020] In related embodiments, an isolated human monoclonal antibody or an antigen-binding fragment thereof that specifically binds to RET blocks the binding of human RET to the GDNF:GFRα1 co-complex by about 57% to about 97%.

[0021] In one embodiment, an isolated human monoclonal antibody or an antigen-binding fragment thereof that specifically binds to RET inhibits GDNF-mediated RET signaling at an IC 50 value in the range of greater than about 50 pM to 100 nM.

[0022] In related embodiments, an isolated human monoclonal antibody or an antigen-binding fragment thereof that specifically binds to RET inhibits GDNF-mediated RET signaling at an IC 50 value in the range of greater than about 143 pM to 100 nM.

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

[0024] In related embodiments, an isolated human monoclonal antibody or an antigen-binding fragment thereof that specifically binds to RET inhibits GDNF-mediated RET signaling by about 60% to about 100%.

[0025] In one embodiment, an isolated human monoclonal antibody or an antigen-binding fragment thereof that specifically binds to RET inhibits artemin-mediated RET signaling at an IC 50 value in the range of about 100 pM to about 500 nM.

[0026] In related embodiments, an isolated human monoclonal antibody or antigen-binding fragment thereof that specifically binds to RET inhibits artemin-mediated RET signaling at an IC 50 value in the range of about 250 pM to about 341 nM.

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

[0028] In one embodiment, an isolated human monoclonal antibody or antigen-binding fragment thereof that specifically binds to RET comprises 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 antigen-binding fragment thereof that specifically binds to RET comprises 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 antigen-binding fragment thereof that specifically binds to RET comprises 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, the isolated human monoclonal antibody or antigen-binding fragment thereof 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, the isolated human monoclonal antibody or antigen-binding fragment thereof that specifically binds to RET comprises 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 the CDRs within the specified HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify the boundaries of CDRs include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Generally, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia 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 are also available for identifying CDR sequences within antibodies. sequences.

[0034] In one embodiment, an isolated human monoclonal antibody or antigen-binding fragment thereof that specifically binds to RET is: (a) an 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) an 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) An HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 232, 248, 264, 280, and 296; (d) An LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268, 284, and 300; (e) An LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 222, 238, 254, 270, 286, and 302; and (f) An LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 224, 240, 256, 272, 288, and 304 is included.

[0035] In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof comprising a heavy and light chain sequence pair selected from the group consisting of SEQ ID NO: 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 thereof that specifically binds to RET and competes with respect to specific binding to RET.

[0036] In one embodiment, the present invention provides an isolated antibody or an antigen-binding fragment thereof that specifically binds to RET and binds to the same epitope on RET recognized by an antibody comprising a heavy 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 is a fully human monoclonal antibody or an antigen-binding fragment thereof that specifically binds to RET and has the following characteristics: (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 thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (ii) comprising an 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, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (iii) an 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, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and an 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, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (iv) comprising an 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, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity;(v) an 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, or a substantially similar sequence thereof 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 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 thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (viii) about 1 x 10; -7 M ~ approx. 1×10 -12 K in the M range D (ix) demonstrating binding of human RET to the GDNF:GFRα1 co-complex with an IC of less than about 5.2 nM; 50 or (x) capable of blocking ligand-dependent RET signaling with an IC in the range of about 143 pM to greater than 100 nM. 50 The present invention provides fully human monoclonal antibodies or antigen-binding fragments thereof that demonstrate one or more of the following: an ability to inhibit approximately 60 to 100% of the IL-1 receptor agonist activity;

[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. A recombinant expression vector having the nucleic acid of the present invention, and a host cell into which such a vector has been introduced are also encompassed by the present invention in the same manner as a method for producing an antibody by culturing the host cell under conditions that allow the production of the antibody and recovering the produced antibody.

[0039] In one embodiment, the present invention provides an antibody or a fragment thereof comprising 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 a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereto.

[0040] In one embodiment, the antibody or the 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 a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereto.

[0041] In one embodiment, the present invention also provides an antibody or an antigen-binding fragment of an antibody comprising 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 thereto; and an LCDR3 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 15, 31, 47, 63, 79, 95, 111, 127, 143, 159, 175, 191, 207, 223, 239, 255, 271, 287, and 303, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0042] In one embodiment, the present invention provides an antibody or a fragment thereof further comprising an HCDR1 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 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 thereto; an HCDR2 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 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 thereto; an LCDR1 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 11, 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 thereto; and an LCDR2 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 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 thereto.

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

[0044] The present invention encompasses antibodies having an altered glycosylation pattern. In some applications, modifications that remove undesirable glycosylation sites, or the removal of fucose moieties to increase, for example, antibody-dependent cell cytotoxicity (ADCC) function, may be useful (see Shield et al. (2002) JBC 277:26733). In other applications, modifications of galactosylation can be made to modify complement-dependent cytotoxicity ( CDC).

[0045] In a fourth aspect, the present invention provides a pharmaceutical composition comprising at least one isolated fully human monoclonal antibody or antigen-binding fragment thereof 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 antigen-binding fragments thereof 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 the antibodies described herein may be used in a pharmaceutical composition to achieve the desired results in a population of patients in need of such treatment. For example, two antibodies that recognize and / or bind RET may be used in the composition.

[0046] In one embodiment, the composition comprises an antibody that binds RET and has 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 that binds to RET, and the antibody comprises three heavy-chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within any one of the heavy-chain variable region (HCVR) amino acid sequences 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 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, the antibody of the present invention or a composition containing one or more antibodies of the present invention can be used to inhibit at least one activity or function associated with RET expressed on the cell surface. In one embodiment, the cell can be a tumor cell. In one embodiment, the activity can be cell signaling.

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

[0050] The second therapeutic agent can 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 can be synthetic or of natural origin.

[0051] The second therapeutic agent can be any agent that is advantageously combined with the antibody or fragment thereof of the present invention. For example, when an anti-RET antibody is an inhibitor of RET used to treat a cancerous condition, the second agent can be selected from chemotherapeutic agents, radionuclides, siRNA specific for RET, a second antibody specific for RET, small molecule RET inhibitors, and myeloid recovery agents such as G-CSF, GM-CSF, or M-CSF, or biological agents having colony-stimulating or myeloid recovery activity. In certain embodiments, the second therapeutic agent can be an agent that serves to counteract or reduce any possible side effects that can occur when side effects related to the antibody or antigen-binding fragment of the antibody of the present invention occur. In certain embodiments, the second therapeutic agent can be an agent useful for reducing pain associated with certain conditions characterized by pain and / or inflammation. Such second agents can include 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 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 inhibitors (e.g., interleukin-1 (IL-1) inhibitors (e.g., rilonacept (“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, it is recognized that the antibodies and pharmaceutically acceptable compositions of the present invention can be used 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. The specific combination of therapies (therapeutics or procedures) to be used in a combination regimen takes into account the compatibility of the desired therapeutic agents and / or procedures, as well as the desired therapeutic effect to be achieved. Similarly, it is recognized that the therapies used can achieve the desired effect for the same disorder (e.g., the antibody can be administered simultaneously with another agent used to treat the same disorder), or they can achieve different effects (e.g., control of any adverse effects). As 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 contemplated to be combined with an antibody of the invention, the small molecule RET inhibitor may be selected from the group consisting of vandetanib, sorafenib, sunitinib, cabozantinib, motesanib, RPI-1, PP-1 and NVP-AST478, cediranib, (AZD2171), gefitinib, erlotinib, SU14813, vatalanib, (BAY43-9006), XL-647, XL-999, AG-013736 , 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 agents are co-administered, dosages may be adjusted accordingly, as recognized in the pertinent art.

[0055] A fifth aspect of the present invention is a method for treating a disorder or condition associated with the expression, activation, or signaling of the RET receptor tyrosine kinase gene or a rearranged form thereof, or pain associated with the disorder or condition, the method comprising administering to a patient in need thereof an antibody of any 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 cancer. In one embodiment, the disorder or condition associated with 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, generalized neuralgia, neurodegenerative disorder, neuroendocrine disorder, visceral pain, acute gout , postherpetic neuralgia, diabetic neuropathy, sciatica, back pain, head and neck pain, severe or refractory pain, breakthrough pain, postoperative pain, toothache, rhinitis, cancer pain, or bladder disorder.

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

[0058] In one embodiment, the tumor is a solid tumor or a blood-derived tumor.

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

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

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

[0065] In one embodiment, the blood-derived tumor is leukemia.

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

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

[0068] In one embodiment, downregulating 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 certain embodiments, downregulating 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 can interfere with or prevent the interaction between RET and one or more GDNF family member ligands (GDNF, neurturin, artemin, and persephin) that form a complex with its corresponding co-receptor (GFRα1, GFRα2, GFRα3, and GFRα4, respectively). In one embodiment, the human anti-RET antibody described herein can interfere with or prevent the interaction of RET with the GDNF / GFRα1 complex. In related embodiments, the human anti-RET antibody described herein can interfere with or prevent the interaction of RET with the artemin / GFRα3 complex. In other related embodiments, the human anti-RET antibody described herein can interfere with or prevent the interaction of RET with the neurturin / GFRα2 or persephin / GFRα4 complex.

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

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

[0072] The antibody or antigen-binding fragment can be administered to a patient in combination with a second therapeutic agent suitable for treating a disease, disorder, or condition. If the disease or condition being treated by the anti-RET antibody is a cancerous condition, the second therapeutic agent can be selected from the group consisting of a chemotherapeutic agent, a radionuclide (alone or as part of a drug targeting regimen), an antibody-drug conjugate, a small molecule RET inhibitor, an anti-tumor agent, an siRNA specific for RET, and a second antibody specific for RET. If the anti-RET antibody is envisioned for treating pain associated with a cancerous condition or other conditions that may be at least partially attributable to RET activation or signaling, the second therapeutic agent can be the following: a nerve growth factor (NGF) inhibitor (e.g., a small molecule NGF antagonist or an anti-NGF antibody), aspirin or another NSAID, morphine, a steroid (e.g., prednisone), an anti-Na v 1.7 antibody or Na v 1.7 small molecule inhibitor, Na v 1.8 antagonist (e.g., an anti-Na v 1.8 antibody or Na v 1.8 small molecule inhibitor), Na v 1.9 antagonist (e.g., an anti-Na v 1.9 antibody or Na vIt may be selected from any one or more of a small molecule inhibitor of 1.9, a cytokine inhibitor (for example, an interleukin-1 (IL-1) inhibitor (for example, rilonacept (“IL-1 trap”); Regeneron), or anakinra (KINERET (registered trademark), Amgen), a small molecule IL-1 antagonist or an anti-IL-1 antibody; an IL-18 inhibitor (for example, a small molecule IL-18 antagonist or an anti-IL-18 antibody); an IL-6 or IL-6R inhibitor (for example, a small molecule IL-6 antagonist, an anti-IL-6 antibody, or an anti-IL-6 receptor antibody), an inhibitor of caspase-1, p38, IKK1 / 2, CTLA-4Ig, or an opioid.

[0073] Other embodiments will become apparent from the summary of the following detailed description.

Brief Description of the Drawings

[0074]

Figure 1

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 because the methods and conditions can vary. Similarly, the technical terms used herein are for the purpose of describing only specific embodiments, and it should be understood that they are not intended to be limiting since the scope of the present invention is limited only by the appended claims.

[0076] Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which the present invention belongs. As used herein, the term “about” when used in relation to a specific recited numerical value means that the value can vary by up to 1% from the recited value. For example, as 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 can be used in the practice or testing of the present invention, but the preferred methods and materials are described below. All publications mentioned herein are hereby incorporated by reference in their entirety.

[0078] Definitions "Rearrangement during transfection", also called "RET", is a receptor tyrosine kinase that is expressed during development in a variety of tissues including the peripheral and central nervous systems and the kidney. The RET cancer gene was identified in 1985 by Takahashi et al., who reported a novel gene rearrangement with transforming 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 a cancer gene It is somatically rearranged in the DNA of patients with papillary thyroid carcinoma (PTC) 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 is composed of three domains: an extracellular ligand-binding domain, a hydrophobic transmembrane domain, and a cytoplasmic portion in which the tyrosine kinase domain is split by a 27-amino acid insertion (see Figure 1). There are two major isoforms of RET generated by alternative splicing. The short and long RET isoforms differ 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). Both isoforms show transforming activity 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 provided in GenBank as accession numbers NM_020975.4 and NP_066124.1, respectively, and are provided herein as SEQ ID NO: 309 and SEQ ID NO: 310, respectively.

[0080] The cDNA and amino acid sequences of the 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 fragment can be used to prepare human monoclonal antibodies specific for RET. The RET protein or its fragment can be recombinantly produced using standard methods known in the art. Exemplary fusion proteins containing the extracellular domain of RET are shown as SEQ ID NOs: 305, 306, 307, and 309. These fusion proteins can 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 line-derived neurotrophic factor (GDNF) family", including GDNF (see GenBank accession number NP_000505.1), artemin (see GenBank accession number Q5T4W7), neurturin (see GenBank accession number NM_004558), and persephin (see GenBank accession number AF040962). GDNF family ligands are in the presence or complex with one of four GPI-linked "coreceptors" known as GDNF family alpha receptors 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 numbers 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). Interacts with RET and activates it only when a body is formed. The main ligands of co-receptors GFRα1, GFRα2, GFRα3, and GFRα4 are GDNF, neurturin (NRTN), artemin (ARTN), and persephin (PSPN), respectively.

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

[0083] As used herein, the terms "treat", "treatment", and "treating" refer to a reduction in 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, e.g., slowing the growth rate of tumor cells in a patient having a tumor that expresses RET when an antagonist / inhibitory antibody of the present invention is administered, or reducing pain associated with a cancerous condition, or pain associated with any other disease or condition that is at least partially caused by RET expression.

[0084] As used herein, the terms "prevent", "preventing", and "prevention" refer to inhibiting a disease, disorder, or condition that is partially caused by or related to RET expression in cells or tissues of a subject, e.g., inhibiting the occurrence or development of a particular cancer, or inhibiting tissue damage that occurs in a patient after an injury, or inhibiting or ameliorating pain associated with a disease or condition that is partially caused by RET expression.

[0085] The term "antibody", as used herein, is intended to refer to an immunoglobulin molecule composed 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 multimers thereof (e.g., IgM) or antigen-binding fragments thereof. Each heavy chain contains a heavy chain variable region ("HCVR" or "V H”) and a heavy chain constant region (Domain C H 1, C H 2, and C H 3). Each light chain consists of a light chain variable region (“LCVR” or “V L ”) and a light chain constant region (C L ). V H and V L regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each V H and V L consists of three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the present invention, the FRs of the antibody (or antigen-binding fragment thereof) can be identical to human germline sequences or can be modified naturally or synthetically. Amino acid consensus sequences can be defined based on the alignment 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 function without one or two CDRs with respect to binding have been described in the scientific literature. Padlan et al. (1995 FASEB J. 9:133-139) analyzed the contact regions 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 have no amino acids that contact the antigen (see also Vajdos et al. 2002 J Mol Biol 320:415-428).

[0087] CDR residues that have not contacted the antigen can be identified, based on previous studies, by molecular modeling and / or empirically, from the regions of Kabat CDRs that lie outside the Chothia CDRs (e.g., residues H60 - H65 in CDRH2 are often not required). When a CDR or a residue thereof is omitted, this is usually replaced by the amino acid occupying the corresponding position in another human antibody sequence or the consensus of such sequences. The positions of substitutions within the CDRs and the amino acids to substitute can also be selected empirically. Empirical substitutions can be conservative or non-conservative substitutions.

[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 regions of the heavy and light chain variable domains as compared to the corresponding germline sequences. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein, for example, to germline sequences available from public antibody sequence databases. The present invention includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residues of the germline sequence from which the antibody is derived, or to the corresponding residues of another human germline sequence, or to conservative amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). One of ordinary skill in the art can readily generate 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 LAll of the framework and / or CDR residues within the domain revert to the residues found in the original germline sequence from which the antibody is derived. In other embodiments, only certain residues, e.g., only the mutated residues found within the first 8 amino acids of FR1, or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2, or CDR3, revert to the original germline sequence. In other embodiments, one or more of the framework and / or CDR residues mutate to the corresponding residues of a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody was originally derived). Further, the antibodies of the invention can contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., certain individual residues mutate to the corresponding residues of a particular germline sequence, while certain other residues different from the original germline sequence are maintained or mutate to the corresponding residues of a different germline sequence. After being obtained, the antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, such as improvement of binding specificity, increase in binding affinity, improvement or enhancement (where applicable) of antagonist or agonist biological properties, reduction of immunogenicity, etc. Antibodies and antigen-binding fragments obtained by this general method are encompassed by the present invention.

[0089] The invention also includes full monoclonal antibodies comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the invention includes antibodies having HCVR, LCVR, and / or CDR amino acid sequences having conservative amino acid substitutions of, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., 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 invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo), for example, in the CDRs, particularly CDR3. However, as used herein, the term "human antibody" is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) are grafted onto human FR sequences.

[0091] The terms "specifically binds" or "binds specifically to", etc. mean that an antibody or an antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1×10 -6 M or less (e.g., a smaller K D indicates stronger binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. As described herein, antibodies that specifically bind to RET have been identified by surface plasmon resonance, e.g., BIACORE™. Moreover, multispecific 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 "specifically binding" antibodies as used herein.

[0092] The term "high affinity" antibody, when measured by surface plasmon resonance, e.g., BIACORE™ or solution affinity ELISA, has a K -7 of at least 10 -8 M, at least 10 -9 M; preferably 10 -10 M; more preferably 10 -11 M, more preferably 10 -12 M, more preferably 10 DRefers to an mAb having binding affinity for RET as denoted.

[0093] The terms "slow off-rate", "Koff", or "kd" mean an antibody that dissociates from RET with a rate constant of 1 × 10 -3 s -1 or less, preferably 1 × 10 -4 s -1 or less, as determined by surface plasmon resonance, e.g., BIACORE™.

[0094] The terms "antigen-binding portion 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 to form a complex. The terms "antigen-binding portion of an antibody" or "antibody fragment", as used herein, refer to one or more fragments of an antibody that retain the ability to bind to RET.

[0095] In a specific embodiment, the antibody or antibody fragment of the invention can be conjugated to a therapeutic moiety, e.g., a small molecule RET inhibitor, an anti-tumor agent, a radionuclide, a growth factor, a myeloid recovery agent, or a colony stimulating factor, or any other therapeutic moiety useful for treating a disease, disorder, or condition associated with RET expression, such as cancer, or damaged tissue ("immunoconjugate" or "antibody-drug conjugate").

[0096] "Isolated antibody", as used herein, is intended to refer to an antibody that substantially does not contain other antibodies (Abs) having different antigen specificities (e.g., an isolated antibody that specifically binds RET or a fragment thereof substantially does not contain Abs that specifically bind antigens other than RET).

[0097] "Blocking antibody" or "neutralizing antibody" (or "antibody that neutralizes RET activity"), as used herein, is intended to refer to an antibody whose binding to RET results in inhibition of at least one biological activity of RET, such as cell signaling. For example, the antibodies of the invention may help block binding of RET to one of its ligands or GFRα coreceptors, or may prevent or treat a disease associated with RET expression. Alternatively, the antibodies of the invention may demonstrate the ability to ameliorate at least one symptom of a disease or condition associated with RET expression. This inhibition of RET biological activity 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 in vivo assays known in the art after administration of one or more of the antibodies described herein (e.g., an animal model for examining inhibition of tumor cell growth in vivo).

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

[0099] The term "K D ", as 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 the specific antigen-binding site in the variable region of an antibody molecule known as a paratope. A single antigen can have more than one epitope. Thus, different antibodies can bind to different areas on the antigen and can have different biological effects. The term "epitope" also refers to the site on an antigen to which B and / or T cells respond. It also refers to the region of an antigen to which an antibody binds. Epitopes can be defined structurally or functionally. Functional epitopes are generally a subset of structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes can also be conformational, composed of non-linear amino acids. In certain embodiments, an epitope can include a determinant that is a chemically active surface group of a molecule such as an amino acid, sugar side chain, phosphoryl group, or sulfonyl group, and in certain embodiments, can 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 a fragment thereof, indicates that when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions, there is nucleotide sequence identity of 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 discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule can, in certain instances, 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 terms "substantial similarity" or "substantially similar" mean that two peptide sequences share at least 90% sequence identity, more preferably at least 95%, 98%, or 99% sequence identity when optimally aligned using default gap weights, such as by the programs GAP or BESTFIT. Preferably, non-identical residue positions differ by conservative 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). In general, conservative amino acid substitutions do not substantially change 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 for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331, which is incorporated herein by reference. Similar chem Examples of groups of amino acids having side chains with learning characteristics 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 conservative amino acid substituents are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, conservative substitutions are any change having 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" substitution is any change having a non-negative value in the PAM250 log-likelihood matrix.

[0103] The sequence similarity of polypeptides 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 conservative amino acid substitutions. For example, GCG software contains programs such as GAP and BESTFIT, which can be used with default parameters to determine sequence homology or sequence identity between related polypeptides, such as 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 with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides the alignment of the best overlapping regions and percent sequence identity between a query sequence and a search sequence (Pearson (2000) supra). When comparing the sequences of the present invention to a database containing a large number of sequences from different organisms Another preferred algorithm when comparing is the computer program BLAST, particularly BLASTP or TBLASTN using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and (1997) Nucleic Acids Res. 25:3389-3402, each of which is incorporated herein by reference.

[0104] In a specific embodiment, the antibodies or antibody fragments for use in the methods of the present invention can be monospecific, bispecific, or multispecific. Multispecific antibodies can be specific for different epitopes of one target polypeptide or can contain antigen-binding domains specific for epitopes of more than one target polypeptide. An exemplary bispecific antibody format that can be used in the context of the present invention is a first immunoglobulin (Ig) C H 3 domain and a second Ig C HRequires the use of three domains and the first and second Ig C H The three domains differ from each other by at least one amino acid, and the at least one amino acid difference 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 three domains bind protein A, and the second Ig C H The three domains contain mutations that reduce or abolish protein A binding, such as the H95R modification (according to IMGT exon numbering; H435R according to EU numbering). The second C H The three may further include the Y96F modification (according to IMGT; Y436F according to EU). The second C H Additional modifications that may be found within the three: for IgG1 mAb, D16E, L18M, N44S, K52N, V57M, and V82I (according to IMGT; D356E, L358M, N384S, K392N, V397M, and V422I according to EU); for IgG2 mAb, N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I according to EU); and for IgG4 mAb, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (according to IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I according to EU). Changes to the bispecific antibody format described above are contemplated to be within the scope of the present invention.

[0105] The phrase "therapeutically effective amount" means an amount that produces the desired effect when administered. The exact amount depends on the purpose of the treatment and can be determined by one of ordinary skill in the art using known techniques (see, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0106] General description "Rearrangement during transfection", also known as "RET", is a receptor tyrosine kinase that 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 16:441-67; Borrello, MG, et al., (2013), Expert Opin. Ther. Targets, 17(4): 403-419). The RET cancer gene was identified in 1985 by Takahashi et al., who reported a novel gene rearrangement with transforming 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 a cancer gene and was found to be somatically rearranged in the DNA of patients with papillary thyroid carcinoma (PTC), which 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 is composed of three domains: an extracellular ligand-binding domain, a hydrophobic transmembrane domain, and a cytoplasmic portion in which the tyrosine kinase domain is split by a 27-amino acid insertion (see Figure 1). There are two major isoforms of RET generated by alternative splicing. The short and long RET isoforms differ 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). Both isoforms exhibit transforming activity in a focus formation assay (Rossel, M. et al. (1997), Oncogene, 14:265-75).

[0107] Genetic modification of RET has been shown to be associated with the etiology of thyroid cancer, and more recent data suggest that RET is also associated with 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 suggested that RET may be associated with additional tumors including breast, pancreas, leukemia, and melanoma (Ballerini, P. et al, (2012), Leukemia, 26:2384-9; Sawai, H. et al. (2005), 65(24):11536-44; Narita, N. et al., (2009), Oncogene, 28:3058-68).

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

[0109] Sorafenib (BAY43-9006, NEXAVAR®, Bayer Pharmaceuticals) is a bisarylurea compound that was initially developed to target the serine / threonine kinase BRAF but was later found to be a potent agent 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 cancer and kidney cancer.

[0110] Sunitinib (SU11248, SUTENT®, Pfizer) is an indolinone compound that targets primarily VEGFR2, PDGFR, c-kit, FLT3, and RET kinases ((Chow, LQ, et al., (2007), J. Clin. Oncol. 25:884-96). Sunitinib has been approved by the FDA for patients with imatinib-resistant GIST as well as 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 a number of tumor types including medullary thyroid cancer, prostate cancer, ovarian cancer, non-small cell lung cancer (NSCLC), hepatocellular carcinoma, renal cell and breast cancer, as well as melanoma and glioblastoma ((Zhang, Y. et al., (2010), IDrugs 13:112-21).

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

[0113] Another RET inhibitor in preclinical development is the indolinone compound RPI-1; PP-1 is a pyrazolopyrimidine compound that is active against RET and Src, and NVP-AST478 is a biphenyl-urea compound that has 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 RET inhibitors is that they are not specific to RET, i.e., they appear to act through multiple mechanisms and thus may potentially exhibit other adverse effects in vivo. For example, certain of the above inhibitors induce adverse events such as hypertension and QTc prolongation. Thus, the non-selective profile of these agents may limit the therapeutic window. Identifying agents such as anti-RET antibodies that selectively bind to RET can be useful and can result in excellent clinical efficacy with a more favorable safety profile.

[0115] Therefore, there remains a need for effective treatments for RET-driven tumors and for identifying RET-specific agents to prevent and treat other diseases, disorders, or conditions related to RET expression without having the adverse side effects associated with the above 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 invention are obtained from mice immunized with a primary immunogen, such as 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), and then immunized with a secondary immunogen (purified human RET protein) or an immunologically active fragment of the RET protein, such as the ectodomain of RET.

[0117] The immunogen can be DNA encoding 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 an active fragment thereof.

[0118] The immunogen may cover the extracellular domain of the RET protein spanning amino acid residues 1 to 635 of any of SEQ ID NOs: 305, 307, 310, 312, and 313 (including the signal sequence); and may be derived from amino acid residues 1 to 636 of SEQ ID NO: 306 (including the signal sequence). The immunogen may be derived from a fragment of any of the above regions of the RET protein.

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

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

[0121] Antigen-binding fragment of an antibody Unless otherwise indicated, as used herein, the term "antibody" is understood to include antibody molecules (i.e., "complete antibody molecules") comprising two immunoglobulin heavy chains and two immunoglobulin light chains, as well as antigen-binding fragments thereof. The terms "antigen-binding portion 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 to form a complex. The term "antigen-binding portion of an antibody" or "antibody fragment", as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to RET. Antibody fragments can include Fab fragments, F(ab')2 fragments, Fv fragments, dAb fragments, fragments containing CDRs, or isolated CDRs. Antigen-binding fragments of an antibody can be derived from complete antibody molecules using, for example, any suitable standard techniques, such as proteolytic digestion, or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding antibody variable and (optionally) constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated, for example, by chemical or molecular biology techniques, to arrange one or more variable and / or constant domains in a suitable configuration, or to introduce codons, create cysteine residues, modify, add, or delete amino acids, etc.

[0122] Non-limiting 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 that mimic the hypervariable regions of an antibody (e.g., isolated complementarity-determining regions (CDRs), such as CDR3 peptides), or constrained FR3-CDR3-FR4 peptides. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetra-bodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains are also encompassed within the expression “antigen-binding fragment” as used herein.

[0123] Antigen-binding fragments of an antibody typically contain 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. The V L domain associated with the V H domain in an antigen-binding fragment having a V H and V L domain can be positioned in any suitable arrangement relative to each other. For example, the variable region can be dimeric and contain a V H -V H 、V H -V L 、or V L -V L dimer. Alternatively, the antigen-binding fragment of an antibody can contain a monomeric V H or V L 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 H 1; (ix) V L -C H 2; (x) V L -C H 3; (xi) V L -C H 1-C H 2; (xii) V L -C H 1-C H 2-C H 3; (xiii) V L -C H 2-C H 3; and (xiv) V L -C Linclude. In any configuration of variable and constant domains including any of the above exemplary configurations, the variable and constant domains may be directly linked to each other, or may be 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 linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Moreover, the antigen-binding fragments of the antibodies of the present invention may associate by non-covalent binding to each other and / or to one or more monomeric V H or V L domains (e.g., by disulfide bonds) to include homodimers or heterodimers (or other multimers) of any of the above variable and constant domain configurations.

[0125] Similar to a complete antibody molecule, the antigen-binding fragment can be monospecific or multispecific (e.g., bispecific). The multispecific antigen-binding fragment of an antibody typically includes at least two different variable domains, each of which is capable of specifically binding to a different antigen or to different epitopes on the same antigen. Any multispecific antibody format including the exemplary bispecific antibody formats disclosed herein may be adapted for use in the context of the antigen-binding fragments of the antibodies 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] Using the VELOCIMMUNE® technology (see, e.g., US 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®), or any other known method for generating monoclonal antibodies, a high-affinity chimeric antibody against RET having human variable regions and mouse constant regions is first isolated. The VELOCIMMUNE® technology involves generating transgenic mice having a genome that includes human heavy and light chain variable regions operably linked to the endogenous mouse constant region locus such that the mouse produces antibodies containing human variable regions and mouse constant regions in response to antigen stimulation. DNA encoding the variable regions of the heavy and light chains of the antibody is isolated and operably linked to DNA encoding 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 antigen of interest, and lymphocytes (e.g., B cells) are recovered from the mice expressing the antibody. The lymphocytes may be fused to a myeloma cell line to prepare immortalized hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific for the antigen of interest. DNA encoding the variable regions of the heavy and light chains may be isolated and linked to the desired isotype constant regions of the heavy and light chains. Such antibody proteins may 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 isolated directly from antigen-specific lymphocytes.

[0129] First, isolate a high-affinity chimeric antibody having a human variable region and a mouse constant region. Characterize the antibody as in the following experimental section and select for desirable characteristics including affinity, selectivity, epitope, etc. Replace the mouse constant region with a desired human constant region to generate a fully human antibody of the invention, such as wild-type or modified IgG1 or IgG4. The selected constant region can vary depending on the specific use, but the high-affinity antigen-binding and target specificity characteristics reside in the variable region.

[0130] In certain embodiments, the antibodies of the invention have an affinity (K -7 M to about 1.0×10 -12 ) in the range of about 1.0×10 D M as measured by binding to an antigen either immobilized on a solid phase or in solution phase. Replace the mouse constant region with a desired human constant region to generate a fully human antibody of the invention. The selected constant region can vary depending on the specific use, but the high-affinity antigen-binding and target specificity characteristics reside in the variable region.

[0131] Biological equivalents The anti-RET antibodies and antibody fragments of the invention include proteins having an amino acid sequence that differs from the sequences of the recited antibodies but retains the ability to bind RET. Such variant antibodies and antibody fragments include one or more additions, deletions, or substitutions of amino acids when compared to the parent sequence, but exhibit a biological activity that is essentially equivalent to that of the recited antibodies. Similarly, the antibody-encoding DNA sequences of the invention include sequences that include one or more additions, deletions, or substitutions of nucleotides when compared to the disclosed sequences, but encode an antibody or antibody fragment that is essentially biologically equivalent to an antibody or antibody fragment of the invention.

[0132] Two antigen-binding proteins or antibodies are considered to be biologically equivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives that do not show a significant difference in the rate and extent of absorption when administered at the same molar dose under similar experimental conditions, either as a single dose or multiple doses. Some antibodies are considered to be equivalents or pharmaceutical alternatives when their extent of absorption is equivalent but their absorption rate is not, and such differences in absorption rate are intentional, reflected in the labeling, and considered not to be medically significant for the particular drug product being studied, for example, not essential for achieving a therapeutically effective drug concentration for chronic use, and still considered to be biologically equivalent.

[0133] In one embodiment, two antigen-binding proteins are biologically equivalent if there are no clinically meaningful differences in their safety, purity, and strength.

[0134] In one embodiment, two antigen-binding proteins are biologically equivalent if patients can be switched one or more times between the reference product and the biological product without an increased risk of adverse effects, including a clinically significant change in immunogenicity or a decrease in efficacy, and compared to continued treatment without such switching.

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

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

[0137] Biologically equivalent variants of the antibodies of the invention can be constructed, for example, by making various substitutions of residues or sequences, or deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine residues that are not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of unwanted or inaccurate intramolecular disulfide bridges upon refolding. In other situations, biologically equivalent antibodies can include antibody variants that contain amino acid changes that modify the glycosylation characteristics of the antibody, such as mutations that eliminate or remove glycosylation.

[0138] Anti-RET antibodies comprising Fc variants According to certain embodiments of the invention, there are provided anti-RET antibodies comprising an Fc domain that contains one or more mutations that enhance or reduce the binding of the antibody to the FcRn receptor at acidic pH as compared to neutral pH. For example, the invention pertains to the C H 2 or C HAn anti-RET antibody comprising a mutation in three regions, wherein the mutation increases the affinity of the Fc domain for FcRn in an acidic environment (e.g., in an endosome where the pH ranges from about 5.5 to about 6.0). Such a mutation can result in an increase in the serum half-life of the antibody when administered to an animal. Non-limiting 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); positions 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T); or modifications at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K), and / or 434 (e.g., H / F or Y); or modifications at positions 250 and / or 428; or modifications at positions 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); and modifications at 307 and / or 308 (e.g., 308F or 308P).

[0139] For example, the present invention includes an anti-RET antibody comprising an Fc domain comprising 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 domains disclosed herein are contemplated to be within the scope of the present invention.

[0140] Biological characteristics of the antibody Generally, the antibodies of the present invention can function by binding to RET and acting to block or prevent RET activation and / or signal transduction thereby. The antibodies of the present invention can also function by binding to RET and interfering with or preventing the interaction or binding of RET with one or more GDNF family members, such as GDNF / GFRα1, neurturin / GFRα2, artemin / GFRα3, or persephin / GFRα4, which form a complex with its corresponding co-receptor. Based on the fact that the tumorigenic ability of RET has been established in humans, antagonist antibodies that specifically bind to RET can be shown to have a beneficial effect in inhibiting tumor cell growth in patients suffering from a cancerous condition.

[0141] In certain embodiments, the antibodies of the present invention can function by binding to and 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 shown as GenBank accession number NP_066124.1, and in SEQ ID NO: 312 (RET9), also shown as GenBank accession number NP_065681.1. The antibody can also bind to any region found in SEQ ID NO: 310 or 312, or a fragment found within SEQ ID NO: 310 or 312.

[0142] In one embodiment, the present invention is a fully human monoclonal antibody or an antigen-binding fragment thereof that binds to the RET protein and has the following characteristics: (a) being a fully human antibody; (b) having a K -7 in the range of about 1.0×10 -12 M to about 1.0×10 D M as measured by surface plasmon resonance; (c) inhibiting or blocking the binding or interaction of one or more GDNF family member ligands (GDNF, neurturin, artemin, and persephin) of RET with its corresponding coreceptors (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 persephin; (e) enhancing the internalization / degradation of RET after the binding of an antibody to the RET receptor; (f) comprising 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) comprising 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 Provided is a fully human monoclonal antibody or an antigen-binding fragment thereof that exhibits one or more of the above.

[0143] Certain anti-RET antibodies of the present invention can bind to the RET protein and inhibit RET-related activation and / or signaling. By doing so, the antibody can function to inhibit the growth of tumors whose growth depends on the activation of RET signaling. Such antagonist anti-RET antibodies may be used alone to treat cancer conditions or in combination therapy with any other anti-cancer agent, such as a chemotherapeutic small molecule, or radiation therapy, or a bone marrow recovery agent.

[0144] In certain embodiments, the anti-RET antibody may be capable of inhibiting multiple signaling pathways including the RAS / RAF pathway, thereby resulting in the activation of mitogen-activated protein kinase (MAPK) 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), the activation of phosphatidylinositol 3-kinase (PI3K) occurs, and as a result, the activation of serine / threonine kinase Akt occurs (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] Non-limiting exemplary in vitro assays for measuring the ability of the anti-RET antibodies of the invention to block the binding of RET to the GFRα1 / GDNF co-complex, and in vitro assays for measuring the effect of the antibodies on RET signaling, activation, or internalization are illustrated in Examples 4 and 5, respectively. In Example 3, the binding affinity and rate constants of the human anti-RET antibodies were determined by surface plasmon resonance, and the measurements were performed on a Biacore 4000 or T200 instrument. In Example 4, the ability of the antibodies to block the binding of RET to the GFRα1 / GDNF co-complex was tested using a competitive sandwich ELISA assay. Example 5 demonstrates the ability of the antibodies of the 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 antibodies of the invention exhibit a broad range of inhibitory activity against RET signaling in the presence of the glial family ligands GDNF and artemin.

[0146] Epitope mapping and related techniques Using various techniques known to those of skill in the art, it is possible to determine whether an antibody "interacts with one or more amino acids" within 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 mutagenesis, peptide blot analysis (Reineke (2004) Methods Mol Biol 248:443-63), peptide cleavage Analytical crystallography studies and NMR analysis can be mentioned. 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 within a polypeptide with which an antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. Generally, the hydrogen / deuterium exchange method involves deuterium labeling of the protein of interest, followed by binding of the antibody to the deuterium-labeled protein. Next, when the protein / antibody complex is transferred to water, the exchangeable protons within the amino acids protected by the antibody complex undergo back-exchange from deuterium to hydrogen at a slower rate than the exchangeable protons within amino acids that are not part of the interface. As a result, the amino acids that form part of the protein / antibody interface retain deuterium and can thus exhibit a relatively large mass compared to amino acids not included in the interface. After dissociation of the antibody, the target protein is subjected to protease cleavage and analysis by mass spectrometry, thereby revealing the deuterium-labeled residues corresponding to the specific amino acids with which the antibody interacts. See, for example, Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

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

[0148] Modified assistance profiling (MAP), also known as antigen structure-based antibody profiling (ASAP), is a method of classifying multiple monoclonal antibodies (mAbs) targeting the same antigen according to the similarity of the binding profiles of each antibody to a chemically or enzymatically modified antigen surface (US2004 / 0101920, which is specifically incorporated herein by reference in its entirety). Each category may reflect a unique epitope that is clearly different from or partially overlapping with the epitope represented by another category. This technique enables rapid filtering of genetically identical antibodies so that characterization can focus on antibodies that are genetically different. When applied to hybridoma screening, MAP can facilitate the identification of rare hybridoma clones that produce mAbs with desired characteristics. The antibodies of the present invention may be sorted using MAP into groups of antibodies that bind different epitopes.

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

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

[0151] To determine whether the antibody competes with respect to binding to the reference anti-RET antibody, the above binding methodology is carried out in two directions; in the first direction, the reference antibody is bound to the RET molecule under saturation 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 saturation conditions, and then the binding of the reference antibody to the RET molecule is evaluated. If in either direction only the first (saturating) antibody is capable of binding to the RET molecule, the test antibody and the reference antibody are concluded to compete with respect to binding to RET. As will be recognized by those skilled in the art, an antibody that competes with respect to binding to a reference antibody does not necessarily bind to the same epitope as the reference antibody, but can sterically block the binding of the reference antibody by binding to overlapping or adjacent epitopes.

[0152] Two antibodies bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other to the antigen. That is, when measured in a competitive binding assay, a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold 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, two antibodies have the same epitope 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. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other.

[0153] Next, experiments of additional routines (such as peptide mutation and binding analysis) can be carried out to confirm whether the observed lack of binding of the test antibody is actually due to binding to the same epitope as the reference antibody or whether steric hindrance (or another phenomenon) is the cause of the observed lack of binding. This type of experiment can be carried out 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 moiety such as a drug capable of inhibiting the growth of tumor cells or improving at least one symptom associated with a RET-related condition such as a cancerous state. Such a drug can be a second different antibody or an anti-tumor chemotherapeutic agent against RET, or a radionuclide that acts to kill tumor cells when targeted to tumor cells expressing RET. The type of therapeutic moiety that can be conjugated to the 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 to treat a complication or symptom associated with the expression of RET by a particular tissue or any other condition resulting from RET expression, such as, but not limited to, treating cancer, it may be advantageous to conjugate an appropriate drug to treat the complication or symptom of the condition or to reduce any side effects of the antibodies of the present invention. Examples of drugs suitable for forming immunoconjugates are known in the art; see, for example, WO05 / 103081.

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

[0156] Exemplary bispecific antibody formats that can be used in the context of the present invention involve the use of a first immunoglobulin (Ig) C H3 domain and a second Ig C H3 domain, wherein the first and second Ig C H3 domains differ from each other by at least one amino acid, and the at least one amino acid difference reduces the binding of the bispecific antibody to protein A as compared to a bispecific antibody lacking the amino acid difference. In one embodiment, the first Ig C H3 domain binds protein A and the second Ig C H3 domain contains a mutation that reduces or abolishes protein A binding, such as the H95R modification (according to IMGT exon numbering; H435R according to EU numbering). The second C H3 may further include the Y96F modification (according to IMGT; Y436F according to EU). The second C H3Additional modifications that can be found therein include: for IgG1 antibodies, D16E, L18M, N44S, K52N, V57M, and V82I (according to IMGT; for EU numbering, D356E, L358M, N384S, K392N, V397M, and V422I); for IgG2 antibodies, N44S, K52N, and V82I (IMGT; for EU numbering, N384S, K392N, and V422I); and for IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (according to IMGT; for EU numbering, Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I). Variations on the bispecific antibody formats 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 an anti-RET antibody of the invention or an antigen-binding fragment thereof. Administration of a therapeutic composition according to the present invention is administered in conjunction with a suitable carrier, excipient, and other agents incorporated into the formulation to provide delivery, transfer, improved tolerability, etc. Numerous suitable formulations can be found in formularies known to all pharmacists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (e.g., LIPOFECTIN™), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycols 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. Tars, soft gels, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (e.g., LIPOFECTIN(™)), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowaxes are included.

[0158] The dosage of each of the antibodies of the present invention can vary depending on the age and size of the subject to be administered, the target disease, condition, route of administration, etc. 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 that depends on RET activation or signaling in a patient, such as a particular tumor expressing RET, or to reduce the severity of a disease. When used, each of the antibodies of the present invention is usually administered intravenously or subcutaneously in 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. The frequency and duration of treatment can be adjusted according to the severity of the condition. In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention can be administered as a first 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 first dose, a second or subsequent dose of the antibody or antigen-binding fragment thereof may be administered in an amount that can be approximately the same as or less than the amount of the first dose, and the subsequent doses are separated by at least 1 day 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] A variety of delivery systems are known, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis, and the pharmaceutical compositions of the present invention can be administered using these (see, for example, Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Examples of introduction methods 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, such as by injection or bolus injection, by absorption through the epithelial or mucosal inner layer (e.g., oral mucosa, nasal mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other bioactive agents. Administration can be systemic or local. This can be delivered as an aerosolized formulation (see US2011 / 0311515 and US2012 / 0128669). Delivery of drugs useful for treating respiratory diseases by inhalation is becoming more widely accepted (see A. J. Bitonti and J. A. Dumont, (2006), Adv. Drug Deliv. Rev, 58:1106-1118). In addition to being effective for treating local lung diseases, such a delivery mechanism may also be useful for the systemic delivery of antibodies (see Maillet et al. (2008), Pharmaceutical Research, Vol. 25, No. 6, 2008).

[0160] The pharmaceutical composition can also be delivered in vesicles, particularly liposomes (see, for example, Langer (1990) Science 249:1527-1533).

[0161] In certain situations, the pharmaceutical composition can be delivered with a controlled release system. In one embodiment, a pump may be used. In another embodiment, a polymeric material can be used. In yet another embodiment, the controlled release system can be placed proximal to the target of the composition, thus requiring only a very small fraction of the systemic dose.

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

[0163] The pharmaceutical composition 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 have convenient applications in the delivery of the pharmaceutical composition of the present invention. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices generally utilize replaceable cartridges containing the pharmaceutical composition. When 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. Then, the pen delivery device can be reused. In disposable pen delivery devices, there are no replaceable cartridges. Rather, disposable pen delivery devices are pre-filled with the pharmaceutical composition held in a reservoir within the device. When the pharmaceutical composition in the reservoir is empty, the entire device is discarded.

[0164] A number of reusable pens and autoinjector delivery devices have application in the subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples include, but are by no means limited to, 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™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany). Examples of disposable pen delivery devices having application in the subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are by no means limited to, SOLOSTAR™ pen (sanofi-aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly), SURECLICK™ autoinjector (Amgen, Thousands Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.), and HUMIRA™ pen (Abbott Labs, Abbott Park, IL).

[0165] Advantageously, the pharmaceutical composition for oral or parenteral use as described above is prepared in 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, and the like. The amount of the antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose, and particularly in the form of an injection, the antibody is preferably contained in an amount of about 5 to about 100 mg, and in other dosage forms, in an amount of about 10 to about 250 mg.

[0166] Dosing regimen According to certain embodiments of the present invention, multiple doses of the antibody against RET may be administered to a subject over a determined period of time. The method according to this aspect of the present invention includes sequentially administering multiple doses of the antibody against RET to a subject. As used herein, "administering sequentially" means that each dose of the antibody against RET is administered to the subject at different times, for example, on different days at predetermined intervals (e.g., several hours, several days, several weeks, or several months). The present invention includes methods that include 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 "first dose", "second dose", and "third dose" refer to the chronological order of administration of the antibody against RET. Thus, the "first dose" is the dose administered at the beginning of the treatment regimen (also referred to as the "baseline dose"); the "second dose" is the dose administered after the first dose; and the "third dose" is the dose administered after the second dose. The first, second, and third doses may all contain the same amount of the antibody against RET, but generally may differ from each other with respect to the dosing frequency. However, in certain embodiments, the amounts of the antibody against RET contained in the first, second, and / or third doses may differ from each other during the course of the treatment (e.g., adjusted upward or downward as needed). In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as a "loading dose", followed by subsequent doses administered at a lower frequency (e.g., a "maintenance dose").

[0168] In one exemplary embodiment of the invention, each second and / or third dose is administered 1 to 26 weeks (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 / 2or after a longer period). As used herein, the phrase "immediately preceding dose" means, in a plurality of administrations in a sequence, the dose of the antibody to RET administered to a patient immediately prior to the administration of the very next dose in that sequence, without any intervening doses.

[0169] The method according to this aspect of the invention may comprise administering to a patient any number of secondary and / or tertiary doses of an antibody to RET. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Similarly, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, 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 immediately preceding 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 immediately preceding dose. Alternatively, the frequency at which secondary and / or tertiary doses are administered to the patient may vary over the course of the treatment regimen. The dosing frequency may also be adjusted by a physician during the course of treatment, as needed for an individual patient based on clinical tests.

[0171] Therapeutic use of the antibody By binding / interacting with the RET protein expressed in certain cells and tissues, this antibody is useful for preventing the interaction of the RET protein with one or more ligand / coreceptor complexes, such as GDNF / GFRα1, artemin / GFRα3, neurturin / GFRα2, or persephin / GFRα4. Considering 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 the growth of tumor cells depends on RET signaling, or for inhibiting pain associated with a cancerous condition and pain associated with other diseases or disorders in which RET activation or signaling plays a role. The antibody of the present invention, when administered alone or in combination with another anti-tumor agent or treatment regimen, or together with one or more agents used to further improve pain associated with the condition, may be used to delay the growth and / or metastasis of tumors in a subject having RET-expressing tumors, or to treat pain associated with a cancerous condition. 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 contemplated to be used alone or in combination with a second agent, or a third agent, to treat a RET-related disease or condition or to alleviate at least one symptom or complication associated with a RET-related disease or condition. A "RET-related disease or condition" is any disease or condition known to be expressed in cells or tissues in which RET is diseased or present and that preferably responds to treatment with a small molecule therapeutic agent known to inhibit RET activation and / or signaling, or preferably responds to treatment with an anti-RET antibody of the present invention. The second or third agent can be delivered concomitantly with the antibody of the present invention, or they can be administered separately, either before or after the antibody of the present invention. The second and third agents can be organic small molecules or biological agents, such as proteins or polypeptides. The second or third agent can be synthetic or of natural origin. The second or third agent can be an anti-tumor agent, such as a chemotherapeutic agent or radiation therapy, or a myeloid recovery agent, or another agent that reduces fever or pain, another second but different antibody that specifically binds RET, an agent (e.g., an antibody) that binds to a RET ligand, such as GDNF, neurturin, artemin, or persephin, or to a co-receptor of RET, such as GFRα1, GFRα2, GFRα3, or GFRα4, or an siRNA specific for the RET molecule.

[0173] In still further embodiments of the present invention, the antibody is used for the preparation of a pharmaceutical composition for treating a patient suffering from a RET-related disease or condition. In yet another embodiment of the present invention, the antibody is used for the preparation of a pharmaceutical composition for reducing the growth of tumor cells or reducing the tumor burden in a patient having a tumor whose growth is dependent on RET signaling. In a further embodiment of the present invention, the antibody is used as adjuvant therapy with any other agent useful for treating a RET-related disease or condition, including a chemotherapeutic agent, radiation therapy, a myeloid recovery agent, a second RET antibody, or any other antibody specific for a RET antigen, or an antibody specific for GDNF or GFRα1, or any other palliative treatment known to those of skill in the art.

[0174] The antibodies of the present invention are useful for the treatment, prevention, and / or amelioration of any disease, disorder, or condition associated with RET activity, or for ameliorating at least one symptom associated with a disease, disorder, or condition, or for reducing pain associated with such a disease, disorder, or condition. Exemplary 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 neuropathic pain, inflammatory pain, arthritis, migraine, cluster headache, trigeminal neuralgia, herpes neuralgia, systemic neuralgia, irritable bowel syndrome, inflammatory bowel syndrome, visceral pain including abdominal pain, osteoarthritis pain, gout, postherpetic neuralgia, diabetic neuropathy, radicular pain, sciatica, back pain, head and neck pain, protrusion pain, postoperative pain, bone pain, acute or chronic pain including cancer pain, but are not limited thereto. Other conditions that can be treated with the antibodies and treatment methods of the present invention include thyroid cancer, familial medullary thyroid cancer (FMTC) syndrome, sporadic medullary cancer (MTC), multiple endocrine neoplasia syndromes MEN2A and MEN2B, prostate cancer, breast cancer, cervical cancer, colon cancer, or bladder cancer, and pain associated with these conditions. The cancers that can be treated with the antibodies of the present invention can be solid tumors, or they can be blood-derived tumors such as leukemia.The antibody or antigen-binding fragment thereof of the present invention is also in the following conditions: non-malignant acute, chronic, or bone pain due to fracture; 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 nerve injury; painful neuroma; ectopic proximal and distal discharge; nerve root disorder; 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; joint pain; acute gout pain; mechanical low back pain; neck pain; tendinitis; injury / sports pain; pyelonephritis; appendicitis; cholecystitis; intestinal obstruction; hernia; chest pain including angina pectoris; pelvic pain, renal colic, obstetric pain including labor pain; cesarean section pain; burn and trauma pain; endometriosis; herpes zoster pain; sickle cell anemia; acute pancreatitis; orofacial pain including rhinosinusitis pain, toothache; multiple sclerosis pain; leprosy pain; Behçet's disease pain; painful lipodystrophy; phlebitis pain; Guillain-Barré pain; painful legs and moving toes; Haglund's syndrome; Fabry disease pain; bladder and urogenital diseases; overactive bladder; painful bladder syndrome; interstitial cystitis; or prostatitis and can also be used to treat them.

[0175] Combined therapy As described above, the method of the present invention, according to certain embodiments, comprises administering one or more additional therapeutic agents in combination with an antibody against RET to a subject. As used herein, the phrase "in combination with" means that the additional therapeutic agent is administered before, after, or simultaneously with the pharmaceutical composition comprising the anti-RET antibody. The term "in combination with" also includes sequential or simultaneous administration of the anti-RET antibody and a second therapeutic agent.

[0176] For example, when administered "before" a pharmaceutical composition comprising an anti-RET antibody, the additional therapeutic agent can be administered about 72 hours, about 60 hours, about 48 hours, about 36 hours, about 24 hours, about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, about 1 hour, about 30 minutes, about 15 minutes, or about 10 minutes before the administration of the pharmaceutical composition comprising the anti-RET antibody. When administered "after" a pharmaceutical composition comprising an anti-RET antibody, the additional therapeutic agent can be administered about 10 minutes, about 15 minutes, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours after the administration of the pharmaceutical composition comprising the anti-RET antibody. Administration "simultaneous with" or "concomitant with" a pharmaceutical composition comprising an anti-RET antibody means that the additional therapeutic agent is administered to the subject in a separate dosage form within less than 5 minutes (before, after, or simultaneously) of the administration of the pharmaceutical composition comprising the anti-RET antibody, or is administered to the subject as a single combined dosage formulation comprising both the additional therapeutic agent and the anti-RET antibody.

[0177] Combination therapy can include the anti-RET antibody of the invention and any additional therapeutic agent that can be advantageously combined with the antibody of the invention or a biologically active fragment of the antibody of the invention. For example, a second or third therapeutic agent, such as a chemotherapeutic agent or radiation therapy useful for inhibiting the growth of tumor cells in a subject, may be used to assist in reducing the tumor burden in a patient. Alternatively, the antibody may be used as adjuvant therapy after surgical removal of the tumor, alone or in combination with a chemotherapeutic agent, radiation therapy, or a bone marrow recovery agent. The antibody may also be used in combination with other therapies, such as a second antibody specific for RET or an antibody specific for a RET ligand as described above, or an antibody or fusion molecule that binds GFRα1 (see SEQ ID NO: 308).

[0178] Diagnostic uses of the antibody Also, 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 envisioned to be done, for example, by measuring the presence of RET in a biopsy sample (i.e., tumor cells) from a tumor that depends on growth through 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, where the anti-RET antibody is labeled with a detectable label or reporter molecule, or used as a capture ligand for selectively isolating cells that express the RET protein from the patient sample. Alternatively, an unlabeled anti-RET antibody can be used in diagnostic applications in combination with a secondary antibody that is labeled in a manner detectable by itself. The detectable label or reporter molecule can be 3 H, 14 C, 32 P, 35 S, or 125 a radioisotope such as I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure RET containing the F protein in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0179] Samples that can be used in the RET diagnostic assay according to the present invention include any tissue or body fluid sample that can be obtained from a patient containing a detectable amount of RET protein or a fragment thereof under normal or pathological conditions. Generally, the level of RET in a specific sample obtained from a healthy patient (e.g., a patient not suffering from a disease or condition associated with the presence of RET) is measured to first establish a baseline or standard level of the RET protein. 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.

Example

[0180] The following examples are set forth to provide a complete disclosure and description of how to make and use the methods and compositions of the present invention to those skilled in the art and are not intended to limit the scope that the inventors regard as the invention. Although efforts have been made to ensure accuracy with respect to the numerical values used (e.g., amounts, temperatures, etc.), some experimental error and deviation should be taken into account. Unless otherwise indicated, parts are by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near 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 invention are obtained from mice immunized with a primary immunogen such as the full-length RET protein (e.g., for the human RET51 isoform, which also has a signal sequence from residues number 1-28, see SEQ ID NO: 310 as also found in ATCC accession number NP_066124.1; and for the human RET9 isoform, see SEQ ID NO: 312 as also found in ATCC accession number NP_065681.1). The mice may be given one or more additional immunizing injections containing the same molecule, or may be boost-immunized with an immunogenic fragment thereof, e.g., the extracellular domain of human RET in the range of amino acids 1-635 of SEQ ID NO: 313, which has a signal sequence in the range of amino acids 1-28. In certain embodiments, after injecting the full-length RET protein into the mice, the mice are boost-immunized with any one or a molecule prepared recombinantly of the constructs shown as SEQ ID NOs: 305, 306, 307, and 313.

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

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

[0184] The full-length protein or fragment thereof used as an immunogen as described above was directly administered to VELOCIMMUNE® mice containing DNA encoding human immunoglobulin heavy and kappa light chain variable regions, together with an adjuvant for stimulating an immune response. The antibody immune response was monitored by a RET immunoassay. When the desired immune response was achieved, splenocytes were recovered, fused with mouse myeloma cells while maintaining their viability, and hybridoma cell lines were formed. The hybridoma cell lines were screened and selected to identify cell lines that produce RET-specific antibodies. Using this technique and the various immunogens described above, several chimeric antibodies (i.e., antibodies having human variable domains and mouse constant domains) were obtained; a particular exemplary antibody thus generated was named, for example, H2M7086N.

[0185] Anti-RET antibodies were also isolated directly from antigen-positive B cells without fusing with myeloma cells as described in US2007 / 0280945A1, which is hereby specifically incorporated by reference in its entirety. Using this method, several fully human anti-RET antibodies (i.e., antibodies having human variable domains and human constant domains) were obtained; exemplary antibodies thus generated 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 generated according to the method of this example are described in detail in the following examples.

[0187] (Example 2 Heavy and Light Chain Variable Region Amino Acid Sequences) Table 1 describes the heavy and light chain variable region amino acid sequence pairs of selected antibodies specific for the RET protein and their corresponding antibody identifiers. Antibodies are typically named herein according to the following nomenclature: a numerical identifier (e.g., "7086" as shown in Table 1) following an Fc prefix (e.g., "H4H", "H1M", "H2M"), followed by a "P" or "N" suffix. Thus, according to this nomenclature, an antibody can be called, for example, "H2M7086N". The H4H, H1M, and H2M prefixes in the antibody names used herein indicate specific Fc regions of the antibody. For example, the "H2M" antibody has a mouse IgG2 Fc, while the "H4H" antibody has a human IgG4 Fc. As will be recognized by those skilled in the art, the H1M or H2M antibody can be converted to an H4H antibody and vice versa, but in any case the variable domains (including the CDRs) indicated by the numerical identifiers shown in Table 1 remain the same. Antibodies having the same numerical antibody name but different suffix letters N, B, or P refer to antibodies having the same CDR sequences but having sequence variations in regions that do not enter the CDR sequences (i.e., the framework regions) in the heavy and light chains. Thus, the N, B, and P variants of a particular antibody have the same CDR sequences within their heavy and light chain variable regions, but differ from each other within their framework regions.

Table 1-1

Table 1-2

[0188] (Example 3 Binding Affinity and Rate Constants from Surface Plasmon Resonance of Human Monoclonal Anti-RET Antibodies) The binding affinity and rate constants of human anti-RET antibodies were determined by surface plasmon resonance (Biacore Determined at 25 °C and 37 °C by the T200 (Tables 2-3). Antibodies expressed as human IgG4 Fc (i.e., the "H4H" designation) were captured on the 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 (minutes) = (ln2 / (60 * k d ). The calculations were performed using Biacore T200 evaluation software v1.0.

[0189] Some antibodies of the present invention showed affinity below nanomolar concentration 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 preformed complex was evaluated using a competitive sandwich ELISA. Most of the RET antibodies strongly blocked the binding of RET to the plate-bound GDNF / GFRα1 co-complex (Table 4). The IC 50 values ranged from 5.2 nM to values below the theoretical minimum of the assay (250 pM), and the maximum blockade ranged from 72 - 96%.

[0191] Detailed method Recombinant human dimeric GDNF (R&D systems), and human GFRα1.mFc (SEQ ID NO: 308) were mixed in a 1:1 molar ratio in PBS 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 a 96-well microtiter plate was coated overnight at 4°C. Nonspecific binding sites were blocked with BSA.

[0192] Separately, 1 nM biotinylated monomeric RET protein (biot-hRET.mmh; SEQ ID NO: 305) was titrated with various amounts of serially diluted antibodies in the range between 0 - 120 nM. The antibody-RET mixture was incubated at RT for 1 hour and then transferred to the microtiter plate pre-coated with hGDNF / hGFRα1 co-complex. After allowing the binding to proceed for 1 hour at RT, it was washed thoroughly. 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 within Prism™ software.

[0193] The IC calculated as the concentration of antibody required to block 50% of the binding of hRET to hGDNF / hGFRα1 50Values were 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 the baseline. The baseline values were calculated as the absorbance measured with a fixed amount of hRET in the dose curve (0% block) and the absorbance measured without adding hRET (100% block). The blocking percentage 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 IC

Table 4

[0194] (Example 5 Anti-RET antibodies inhibit ligand-dependent RET signaling in the SRE-luciferase reporter assay and show strong internalization) In this example, the effects of anti-RET antibodies on RET signaling and internalization were investigated using MCF7 and hRET-engineered reporter cell lines.

[0195] The glial family ligands GDNF and artemin each induce RET activation through the formation of high-affinity co-complexes with GFRα1 or 3, bringing two RET molecules together and initiating phosphorylation of specific tyrosine residues. The transphosphorylation of RET activates several downstream intracellular cascades, and the upregulation of RET signaling is implicated in 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 human breast cancer cell line MCF7, which expresses RET and GFRα1, was transduced with a serum response factor (SRE)-regulated luciferase reporter gene to create the MCF7 / SRE-Luc strain. The antibodies of the present invention showed strong inhibition of GDNF-stimulated RET signaling, IC 50The values were in the range of 143 pM to >100 nM (Table 5). The percent inhibition was in the range of 60 - 100%. Several non-blocking antibodies were also identified; H4H8085P stimulated luciferase activity up to 50% of the levels observed with GDNF, while H4H8044P, H4H8076P, and H4H8046P were weaker activators of the luciferase response (2 - 5% activation).

[0197] To determine whether antibodies that block GDNF-mediated RET signaling are also effective against artemin-induced activity, an engineered HEK293 / hGFRa3 / hRET SRELuc cell line was constructed. 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 on the RET receptor where the GDNF-GFRα1 and artemin-GFRα3 co-complexes bind.

[0198] Finally, to understand whether the observed blocking activity is due to degradation of the RET receptor upon antibody binding, several antibodies were tested in an internalization assay (Table 6). Among the seven antibodies tested, H4H8087P was identified as the most strongly internalizing, and H4H8079P and H4H7086P also showed strong internalization.

[0199] In conclusion, this example illustrates that the anti-RET antibodies of the present invention exhibit a broad range of activation and inhibitory activities 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 2 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 2 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 - 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 characteristics of the anti-RET mAb, MCF7-SRE-Luc cells were incubated for 6 hours with serially diluted anti-hRET mAb in the range of 1.6 pM to 1 μM in the absence of ligand.

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

[0205] Inhibition of artemin-stimulated luciferase activity in HEK293 / hGFRa3 / hRET engineered cell line 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 the inhibition curve, cells were incubated for 1 hour with serially diluted anti-hRET antibody in the range of 1.6 pM to 1 μM. Then, the cells were stimulated for 6 hours with a fixed dose of h artemin (100 pM). The dose-response curve of artemin was generated by adding serially diluted h artemin (0.17 pM to 10 nM) to the cells at 37 °C for 6 hours without adding the antibody. Luciferase activity measurement and curve fitting were performed as described for GDNF-stimulated luciferase activity.

[0206] EC 50 / IC 50 value calculation EC 50 / IC 50 values were determined from a four-parameter logistic equation for a 12-point response curve using GraphPad Prism. The percent blockade was reported for the highest antibody dose, and the data were reported as mean ± standard deviation (SD).

[0207] Quantitative Analysis of the Internalization Characteristics of Anti-RET Antibodies To test the anti-hRET mAb for internalization, HEK293 / hGFRa1 / hRET / SRE-Luc cells were incubated on ice for 30 minutes with the antibody (10 μg / ml) and then washed once. The cells were then incubated for 30 minutes with an alexa488-conjugated anti-hFc Fab secondary antibody and then washed a second time. The antibody was allowed to internalize at 37°C for 4 hours or maintained at 4°C to prevent internalization. The cells were fixed in 4% formaldehyde and the alexa488 on the cell surface was quenched by incubation with an anti-alexa488 quenching antibody at 4°C for 1 hour. The nuclei were stained with a Hoechst dye and images were acquired in an ImageXpress micro XL (Molecular Devices).

[0208] The total alexa488 intensity in intracellular vesicles at 37°C in the quenched samples was quantified via Columbus image analysis software (Perkin Elmer). The total internalized mAb intensity was expressed as a percentage of the most strongly internalized mAb.

[0209] (Example 6 Generation of Bispecific Antibodies) Various bispecific antibodies are generated for use in the practice of the methods of the invention. For example, RET-specific antibodies are generated in a bispecific format (a "bi-specific antibody") in which variable regions that bind to separate domains of the RET protein are linked together such that a single binding molecule confers dual domain specificity. Appropriately designed bispecific antibodies can enhance overall RET neutralizing efficacy through increasing both specificity and binding avidity. Variable regions having specificity for individual domains are paired on a structural scaffold, thereby allowing each region to simultaneously bind to a distinct epitope or different regions within one domain. In one example of a bispecific antibody, the heavy chain variable region (V H ) from a binder having specificity for one domain is paired with the light chain variable region (VL ) and recombine it with the V H without interfering with the original specificity for, the original V H to identify non-cognate V that can pair with the L partner. In this way, a single V L segment (e.g., V L 1) can be combined with two different V H domains (e.g., V H 1 and V H 2) to generate a bispecific antibody composed of two binding "arms" (V H 1-V L 1, and V H 2-V L 1). The use of a single V L segment reduces the complexity of the system, thereby simplifying the cloning, expression, and purification processes used to generate bispecific antibodies and increasing their efficiency (see, e.g., USSN13 / 022759 and US2010 / 0331527).

[0210] Alternatively, antibodies that bind 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 of skill in the art. Antibody variable regions that bind to separate regions may be linked together, for example, with variable regions that bind to related sites on different antigens, to confer dual antigen specificity within a single binding molecule. Appropriately designed bispecific antibodies of this nature perform a dual function. For example, in the case of a bispecific antibody that binds RET and one of its ligands, it may be possible to better inhibit tumor cell growth without the need to administer a composition containing two separate antibodies. By combining a variable region that is specific for RET with a variable region that is specific for one of its ligands and forming a pair on a structural scaffold, it is possible to bind each variable region to a separate antigen.

[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 an antibody. For example, standard methods for measuring soluble protein binding, such as Biacore, ELISA, size exclusion chromatography, multi-angle laser light scattering, direct scanning calorimetry, and other methods are used to evaluate the bispecific interaction. The binding of the 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 the different antigens are coated on the wells of a microtiter plate, and the binding of the bispecific antibody is determined through the use of a secondary detection antibody. The binding experiment can also be carried out using a surface plasmon resonance experiment, where the real-time binding interaction of the peptide to the antibody is measured by flowing the peptide or the bispecific antibody over the entire sensor surface to which the peptide or the bispecific antibody is respectively captured. The functional in vitro blockade of both RET and one of its ligands by the bispecific antibody is determined using any bioassay such as the assays described herein or by in vivo protection studies in a suitable animal model such as a tumor-bearing animal model. The present invention provides, for example, the following items. (Item 1) An isolated human monoclonal antibody or an antigen-binding fragment thereof that specifically binds to the RET (rearranged during transfection) receptor tyrosine kinase, wherein the antibody has the following characteristics: (a) being a fully human antibody; (b) having a K in the range of about 1.0×10 -7 M to about 1.0×10 -12 M as measured by surface plasmon resonance; D (c) inhibiting or blocking the binding or interaction of RET with one or more GDNF family member ligands (GDNF, neurturin, artemin, and persephin) that form a complex with its corresponding coreceptor (GFRα1, GFRα2, GFRα3, and GFRα4, respectively). (c) inhibiting or blocking the binding or interaction of RET with one or more GDNF family member ligands (GDNF, neurturin, artemin, and persephin) that form a complex with its corresponding coreceptor (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 persephin; (e) enhancing RET internalization / degradation after binding of the antibody to the RET receptor; (f) comprising 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) comprising 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 an antigen-binding fragment thereof having one or more of the above. (Item 2) The isolated human monoclonal antibody or an antigen-binding fragment thereof according to item 1, wherein the antibody blocks the binding of human RET to the GDNF:GFRα1 co-complex at an IC 50 value in the range of about 100 pM to about 7.0 nM. (Item 3) The isolated human monoclonal antibody or an antigen-binding fragment thereof according to item 2, wherein the antibody blocks the binding of human RET to the GDNF:GFRα1 co-complex at an IC 50 value in the range of about 250 pM to about 5.2 nM. (Item 4) The isolated human monoclonal antibody or an antigen-binding fragment thereof according to item 3, wherein the percentage of blockade of human RET to the GDNF:GFRα1 co-complex is in the range of about 40% to 100%. (Item 5) The isolated human monoclonal antibody or an antigen-binding fragment thereof according to item 4, wherein the percentage of blockade of human RET to the GDNF:GFRα1 co-complex is in the range of about 57% to about 97%. (Item 6) The isolated human monoclonal antibody or antigen-binding fragment thereof according to item 1, wherein GDNF-mediated RET signaling is inhibited at IC values in the range greater than about 50 pM to 100 nM. 50 (Item 7) The isolated human monoclonal antibody or antigen-binding fragment thereof according to item 6, wherein GDNF-mediated RET signaling is inhibited at IC values in the range greater than about 143 pM to 100 nM. 50 (Item 8) The isolated human monoclonal antibody or antigen-binding fragment thereof according to item 6, wherein GDNF-mediated RET signaling is inhibited by about 40% to about 100%. (Item 9) The isolated human monoclonal antibody or antigen-binding fragment thereof according to item 7, wherein GDNF-mediated RET signaling is inhibited by about 60% to about 100%. (Item 10) The isolated human monoclonal antibody or antigen-binding fragment thereof according to item 1, wherein artemin-mediated RET signaling is inhibited at IC values in the range of about 100 pM to about 500 nM. 50 (Item 11) The isolated human monoclonal antibody or antigen-binding fragment thereof according to item 10, wherein artemin-mediated RET signaling is inhibited at IC values in the range of about 250 pM to about 341 nM. 50 (Item 12) The isolated human monoclonal antibody or antigen-binding fragment thereof according to item 11, wherein artemin-mediated RET signaling is inhibited by about 57% to about 100%. (Item 13) ​​​​An isolated human monoclonal antibody or an antigen-binding fragment thereof according to item 1, comprising an HCVR / LCVR amino acid sequence pair selected from the group consisting of array numbers 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 an antigen-binding fragment thereof that specifically binds to RET, wherein the antibody comprises an HCVR comprising 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 comprising 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) An 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) An 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) An HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 232, 248, 264, 280, and 296; (d) An LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268, 284, and 300; (e) An LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 222, 238, 254, 270, 286, and 302; and (f) An LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 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 according to item 14, comprising: (Item 16) An isolated antibody or antigen-binding fragment thereof that competes with an antibody or antigen-binding fragment containing a heavy and light chain sequence pair selected from the group consisting of SEQ ID NO: 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 for specific binding to RET. (Item 17) An isolated antibody or antigen-binding fragment thereof that binds to the same epitope on RET recognized by an antibody comprising a heavy and light chain sequence pair selected from the group consisting of array numbers 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 thereof according to any one of Items 1 to 17. (Item 19) An expression vector comprising the nucleic acid molecule according to Item 18. (Item 20) A pharmaceutical composition comprising one or more of the antibodies or antigen-binding fragments thereof that specifically bind to RET according to any one 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 signal transduction of the RET receptor tyrosine kinase gene or a rearranged form thereof, or pain related to said disorder or condition, comprising administering to a patient in need thereof one or more of the antibodies or antigen-binding fragments thereof according to any one of Items 1 to 17, or a pharmaceutical composition comprising one or more of the antibodies according to any one of Items 1 to 17. (Item 22) The method according to Item 21, wherein the disorder or condition related to the expression, activation, or signal transduction 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. (Item 23) The method according to item 21, wherein the disorder or condition related to the expression, activation, or signal transduction of the RET receptor tyrosine kinase gene or its rearranged form is selected from the group consisting of acute pain, chronic pain, neuropathic pain, inflammatory pain, arthritis, osteoarthritis, migraine, cluster headache, trigeminal neuralgia, herpes neuralgia, systemic neuralgia, neurodegenerative disorder, neuroendocrine disorder, visceral pain, acute gout, postherpetic neuralgia, diabetic neuropathy, sciatica, back pain, head and neck pain, severe or refractory pain, breakthrough pain, postoperative pain, toothache, rhinitis, cancer pain, or bladder disorder. (Item 24) A method for inhibiting tumor growth or tumor cell proliferation, wherein the tumor or tumor cells express RET or its rearranged form, and the method comprises administering to a patient in need thereof one or more antibodies or antigen-binding fragments thereof according to any one of items 1 to 17, or a pharmaceutical composition comprising one or more antibodies according to any one of items 1 to 17. (Item 25) The method according to item 24, wherein the tumor is a solid tumor or a blood-derived tumor. (Item 26) The method according to item 25, wherein the solid tumor is selected from the group consisting of a thyroid tumor, a lung tumor, a pancreatic tumor, a skin tumor, and a breast tumor. (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 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 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 melanoma. (Item 32) The method according to item 25, wherein the blood-derived tumor is leukemia. (Item 33) The method according to 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 thereof one or more antibodies or antigen-binding fragments thereof according to any one of items 1 to 17, or a pharmaceutical composition comprising one or more antibodies according to any one of items 1 to 17. (Item 35) The method according to item 34, wherein the downregulation of RET expression and / or function results in the 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 small molecule tyrosine kinase inhibitor, an anti-tumor agent, an siRNA specific for RET, a second antibody specific for RET, and an analgesic. (Item 38) The method according to item 37, wherein the low molecular weight tyrosine kinase inhibitor is selected from the group consisting of vandetanib, cediranib, (AZD2171), gefitinib, erlotinib, SU14813, batatinib, sorafenib, sorafenib (BAY43-9006), sunitinib, cabozantinib, motesanib, XL-647, XL-999, AG-013736, 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. (Item 39) The method according to item 37, wherein the anti-tumor agent is selected from the group consisting of a chemotherapeutic agent, a radionuclide, and an antibody-drug conjugate. (Item 40) The analgesic is a nerve growth factor (NGF) inhibitor (e.g., a low molecular weight NGF antagonist or an anti-NGF antibody), aspirin or another NSAID, morphine, a steroid (e.g., prednisone), an anti-Na v 1.7 antibody or Na v 1.7 low molecular weight inhibitor, Na v 1.8 antagonist (e.g., an anti-Na v 1.8 antibody or Na v 1.8 low molecular weight inhibitor), Na v 1.9 antagonist (e.g., an anti-Na v 1.9 antibody or Na vThe method according to item 37, selected from the group consisting of a small molecule inhibitor of 1.9, a cytokine inhibitor (for example, an interleukin-1 (IL-1) inhibitor (for example, rilonacept ("IL-1 trap"); Regeneron) or anakinra (KINERET (registered trademark), Amgen), a small molecule IL-1 antagonist or an anti-IL-1 antibody; an IL-18 inhibitor (for example, a small molecule IL-18 antagonist or an anti-IL-18 antibody); an IL-6 or IL-6R inhibitor (for example, a small molecule IL-6 antagonist, an anti-IL-6 antibody, or an anti-IL-6 receptor antibody), an inhibitor of caspase-1, p38, IKK1 / 2, CTLA-4Ig, or an opioid).

Claims

【Claim 1】 The invention described in the specification.

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