RU2821548C2 - Human ret-binding antibodies and methods of use thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed are fully human antibodies which bind to receptor tyrosine kinase RET, nucleic acids coding said antibodies, expression vectors containing said nucleic acids. Also disclosed is the use of antibodies to RET for treating diseases, disorders or pathological conditions associated with expression, activation or transmission of a signal of the receptor tyrosine kinase RET gene or its rearranged form. Also disclosed is the use of the antibodies according to the invention for treating cancer and pain associated with cancer, or for relieving pain associated with other pathological conditions associated at least in part with expression, activation or transmission of RET signals.

EFFECT: disclosed antibodies specifically binding RET can be useful for slowing growth or proliferation of tumour cells, as well as for relieving pain associated with cancer and other pathological conditions.

32 cl, 1 dwg, 6 tbl, 6 ex

Description

Field of technology to which the present invention relates

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

List of sequences

[0002] An official copy of the sequence listing has been filed concurrently with the description electronically via EFS-Web as a sequence listing in ASCII format with file name 10582WO01_SeqList_ST25.TXT, creation date April 09, 2020, and approximately 168 kilobytes in size. The sequence listings contained herein in ASCII format are part of this specification and are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

[0003] Receptor tyrosine kinase RET (REarranged during Transfection) is expressed during development in various tissues, including the peripheral and central nervous system and the kidney (Arighi, E. et al, (2005), Cytokine Growth Factor Rev 16:441-467; Borrello, MG, et al, (2013), Expert Opin. Targets, 17(4):403-419; Golden, JP, et al. Neurol 398:139-150; Golden, JP, et al. (1999), Exp. It is also expressed in neural crest 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. No RET knockout mice exhibit renal agenesis and lack enteric neurons in the digestive tract. A very similar phenotype is observed in both GFRα1 and GDNF knockout mice, supporting a major role for GDNF/GFRα1 in the activation of RET signaling during development.

[0004] RET is a signaling receptor for ligands of the glial-derived neurotrophic factor (GDNF) family, which includes GDNF, artemin, neurturin and persefin. GDNF family ligands interact and activate RET only in the presence of one of four GPI-linked co-receptors known as GDNF family α-receptors, GFRcc(1-4) (Baloh, RH, et al. (2000), Curr Opin Neurobiol 10:103 -110;Borrello, MG, et al (2013), Expert Opin. Targets, 17(4):403-419). The primary ligands for the coreceptors GFRα1, GFRα2, GFRα3 and GFRα4 are GDNF, neurturin (NRTN), artemin (ARTN) and persephine (PSPN), respectively, although cross-reactions between ligands and coreceptors have been observed in vitro.

[0005] The role of RET as a factor in tumorigenesis has been established through activating mutations, which are often observed in the multiple endocrine neoplasia syndromes MEN2A and MEN2B and in hereditary medullary thyroid cancer (Mulligan, LM, et al, (1994), Nat. Genet 6:70-74). In addition, somatic activating mutations in RET are present in a large proportion of sporadic medullary thyroid cancers (Fusco, A. et al, (1987), Nature 328:170-172; Grieco, M. et al, (1990), Cell 60 :557-563). Such mutations may occur in the kinase domain or in the extracellular domain, where they lead to the formation of unpaired cysteines, which are thought to promote ligand-independent dimerization and activation of RET. Thus, the carcinogenic potential of RET in humans has been clearly established through genetic studies.

[0006] In addition to its role in endocrine cancers, recent work has 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 observation, RET and GFRα1 are predominantly expressed in a subset of estrogen receptor-positive breast cancers. In addition, GDNF-induced RET signaling promotes anchorage-independent growth of estrogen receptor-positive breast cancer cells and enhances the effects of estrogen on the growth and survival of these cells, suggesting a functional interaction between these two pathways. Thus, RET signaling appears to be an important factor in influencing the development of the oncogenic phenotype in breast cancer cells (Wang, S. et al (2012), Breast Cancer Res Treat 133(2):487-500; Stine, ZE, et al, (2011), Human Molecular Genetics 20(19):3746-3756).

[0007] RET activation is initiated by the binding of GDNF to GFRα1. The GDNF/GFRα1 complex then binds to RET, resulting in dimerization and activation of the receptor. There are several small molecules that have the ability to inhibit RET, including an agent (vandetanib) that is active in patients with medullary thyroid cancer (Wells, SA et al, (2012), J Clin Oncol 30:134 -141; Leboulleux, S. et al, (2012), Lancet Oncol 13:897-905). Other small molecules have been identified that bind to and inhibit RET signaling (Borrello, MG, et al, (2013), Expert Opin. Ther. Targets, 17(4):403-419). Unfortunately, due to a lack of specificity, some of these compounds experienced side effects in clinical trials that prevented further development.

[0008] To date, there have been no reports of therapeutic anti-RET monoclonal antibodies for use in the clinical setting to treat RET-expressing tumors. The studies presented herein describe the generation of fully human monoclonal antibodies that bind to RET and prevent RET from interacting with one or more members of the GDNF family in complex with their respective co-receptors.

[0009] The domain structure of the extracellular region of RET is shown in FIG. 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 yet been determined, it appears that the GDNF/GFRα1 complex makes contacts with the extracellular domain of RET at several sites, including the fourth cadherin-like domain and the cysteine-rich domain. Thus, antibodies to certain RET domains can effectively inhibit signaling. Antibodies to RET have been described and can be found in US6861509 and US2009/0136502.

[0010] However, given the role that RET plays in the growth and proliferation of tumor cells, and given the fact that there are several agents approved to target this molecule, there is still a need for RET inhibitors, such as human antibodies , which specifically bind to RET, are highly effective and do not cause side effects that would prevent approval for clinical use.

Brief Disclosure of the Present Invention

[0011] The present invention provides fully human monoclonal antibodies (mAbs) or antigen binding fragments thereof that specifically bind to RET and inhibit the binding or interaction of RET with one or more GDNF family member ligands (GDNF, neurturin, artemin and persefin) in a complex with their respective co-receptors (GFRα1, GFRα2, GFRα3 and GFRα4, respectively). In accordance with one embodiment, human anti-RET antibodies described herein prevent the interaction of RET with the GDNF/GFRα1 complex. In accordance with a related embodiment, human anti-RET antibodies described herein prevent the interaction of RET with the artemin/GFRα3 complex. According to a related embodiment, human anti-RET antibodies described herein prevent RET from interacting with the neurturin/GFRα2 complex or the persefin/GFRα4 complex.

[0012] From the research described herein, it appears that such antibodies are capable of modulating ligand-dependent RET signaling. In accordance with certain embodiments, antibodies have been identified that antagonize ligand-dependent RET signaling.

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

[0014] Antibodies can be used as a separate therapy or can be used in combination with a second agent useful for treating a disease or disorder associated with RET expression. In accordance with certain embodiments, antibodies can be administered therapeutically in combination with a second agent to treat a disease or disorder or to alleviate at least one symptom associated with a disease or disorder. If the antibody inhibits RET activity or signaling and is being considered for use in treating, for example, a cancerous condition, the second agent may be a chemotherapeutic agent or a bone marrow repair agent, or may be radiation therapy to treat the tumor. If the antibody inhibits RET activity or signaling and is being considered for the treatment of pain associated with a particular pathological condition, and if the treatment requires the use of a second analgesic agent, the second agent may be any agent that is also useful in relieving pain associated with such pathological condition, such as aspirin or other NS AID, morphine, steroids (eg, prednisone), nerve growth factor (NGF) inhibitor (eg, small molecule NGF antagonist or anti-NGF antibody), anti-Na _v 1.7 antibody or small molecule Na _v 1.7 inhibitor, Na _v antagonist 1.8 (e.g., anti-Na _v 1.8 antibody or small molecule Na _v 1.8 inhibitor), Na _v 1.9 antagonist (e.g., anti-Na _v 1.9 antibody or small molecule Na _v 1.9 inhibitor), cytokine inhibitor (e.g., interleukin-1 (IL-1) inhibitor 1) (such as rilonacept (“IL-1 decoy”) or anakinra (KINERET®), a small molecule IL-1 antagonist or anti-IL-1 antibody; an IL-18 inhibitor (such as a small molecule IL-18 antagonist or anti-IL antibody -18); an IL-6 or IL-6R inhibitor (such as a small molecule IL-6 antagonist, an anti-IL-6 antibody, or an anti-IL-6 receptor antibody), caspase-1, p38, IKK1/2, CTLA-4Ig inhibitors, or an opioid.

[0015] Antibodies of the present invention may be full length (eg, an IgG1 or IgG4 antibody) or may only be an antigen binding portion (eg, a Fab, F(ab') ₂ or scFv fragment) and may be modified to alter functionality , for example, to eliminate residual effector functions (Reddy et al, (2000), J. Immunol. 164:1925-1933).

[0016] Accordingly, according to a first aspect, the present invention provides an isolated human monoclonal antibody or antigen binding fragment thereof that specifically binds to a receptor tyrosine kinase RET (rearranged during transfection), the antibody having one or more of the following characteristics:

(a) is a fully human antibody;

(b) characterized by a K _D ranging from about 1.0×10 ^-7 M to about 1.0×10 ^-12 M as measured by surface plasmon resonance;

(c) inhibits or blocks the binding or interaction of RET with one or more member ligands of the GDNF family (GDNF, neurturin, artemin and persefin) in complex with their respective co-receptors (GFRα1, GFRα2, GFRα3 and GFRα4, respectively);

(d) inhibits RET signaling mediated by one or more GDNF family member ligands selected from GDNF, neurturin, artemin and persefin;

(e) enhances RET internalization/degradation following antibody binding to the RET receptor;

(f) contains a heavy chain variable region (HCVR) with an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194 , 210, 226, 242, 258, 274 and 290; or

(g) contains a light chain variable region (LCVR) with an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202 , 218, 234, 250, 266, 282 and 298.

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

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

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

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

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

[0022] In a related embodiment, an isolated human monoclonal antibody or antigen binding fragment thereof that specifically binds RET inhibits GDNF-mediated RET signaling with an IC ₅₀ value ranging from about 143 pM to greater than 100 nM.

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

[0024] According to a related embodiment, an isolated human monoclonal antibody or antigen binding fragment thereof that specifically binds RET inhibits GDNF-mediated RET signaling by an amount ranging from about 60% to about 100%.

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

[0026] In a related embodiment, an isolated human monoclonal antibody or antigen binding fragment thereof that specifically binds RET inhibits artemin-mediated RET signaling with an IC ₅₀ value ranging from about 250 pM to about 341 nM.

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

[0028] In accordance with one embodiment, an isolated human monoclonal antibody or antigen binding fragment thereof that specifically binds to RET comprises a heavy chain variable region (HCVR) with an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 18 , 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274 and 290.

[0029] In accordance with one embodiment, an isolated human monoclonal antibody or antigen binding fragment thereof that specifically binds to RET comprises a light chain variable region (LCVR) with an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 26 , 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282 and 298.

[0030] In accordance with one embodiment, an isolated human monoclonal antibody or antigen binding fragment thereof that specifically binds to RET comprises a heavy chain variable region (HCVR) with an amino acid sequence selected from the group consisting of SEQ ID NO: 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 NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282 and 298.

[0031] In accordance with one embodiment, an 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 NO: 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.

[0032] In accordance with one embodiment, an isolated human monoclonal antibody or antigen binding fragment thereof that specifically binds to RET 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 NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274 and 290; and LCVR comprising three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within the amino acid sequence of LCVR selected from the group consisting of SEQ ID NO: 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 in HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs in said HCVR and/or LCVR amino acid sequences disclosed herein. Exemplary conventional methods that can be used to identify CDR boundaries include, for example, the Kabat determination, the Chothia determination, and the AbM determination. In general terms, 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 275:927–948; and Martin et al, (1989), Proc. Natl Acad. Sci. USA 86:9268-9272. Public databases are also available to identify CDR sequences in antibodies.

[0034] In accordance with one embodiment, an isolated human monoclonal antibody or antigen binding fragment thereof that specifically binds to RET comprises:

(a) an HCDR1 domain with an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 228, 244 , 260, 276 and 292;

(b) an HCDR2 domain with an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 230, 246 , 262, 278 and 294;

(c) an HCDR3 domain with 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 with 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 with 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 with 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.

[0035] In accordance with one embodiment, the present invention provides an isolated antibody or antigen binding fragment thereof that specifically binds to RET, which competes for specific binding to RET with an antibody or antigen binding fragment comprising pairs of heavy and light chain sequences selected from 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.

[0036] In accordance with one embodiment, the present invention provides an isolated antibody or antigen binding fragment thereof that specifically binds to RET that binds the same epitope on RET that is recognized by the antibody comprising pairs of heavy and light chain sequences 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.

[0037] In accordance with one embodiment, the present invention provides a fully human monoclonal antibody or antigen binding fragment thereof that specifically binds to RET, wherein the antibody or fragment thereof has one or more of the following characteristics: (i) contains an HCVR with the 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 that is at least 90%, at least 95%, at least 98%, or at least 99% identical to such sequence; (ii) contain an LCVR with an amino acid sequence selected from the group consisting of SEQ ID NO: 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 thereto that is at least 90%, at least 95%, at least 98%, or at least 99% identical to such sequence; (iii) contain an HCDR3 domain with 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, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to such sequence; and an LCDR3 domain with 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, or a substantially similar sequence thereto that is at least 90%, at least 95%, at least 98%, or at least 99% identical to such sequence; (iv) contain an HCDR1 domain with an amino acid sequence selected from the group consisting of SEQ ID NO: 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 that is at least 90%, at least 95%, at least 98%, or at least 99% identical to such sequence; (v) an HCDR2 domain with an amino acid sequence selected from the group consisting of SEQ ID NO: 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 thereto that is at least 90%, at least 95%, at least 98%, or at least 99% identical to such sequence; (vi) an LCDR1 domain with 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, or a substantially similar sequence thereto that is at least 90%, at least 95%, at least 98%, or at least 99% identical to such sequence; (vii) and an LCDR2 domain with 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, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to such sequence; (viii) have a K _D ranging from about 1×10 ^-7 M to about 1×10 ^-12 M; (ix) are capable of blocking the binding of human RET to the GDNF:GFRα1 co-complex with an IC ₅₀ value of less than about 5.2 nM; or (x) have the ability to inhibit ligand-dependent RET signaling by approximately 60-100% with an IC ₅₀ value ranging from approximately 143 pM to greater than 100 nM.

[0038] According to a second aspect, the present invention relates to nucleic acid molecules encoding antibodies or fragments thereof that specifically bind to RET. Recombinant expression vectors carrying the nucleic acids of the present invention and the host cells into which such vectors have been introduced are also covered by the present invention, as are methods for producing antibodies by culturing the host cells under conditions allowing antibody production and by isolating the produced antibodies.

[0039] In accordance with one embodiment, the present invention provides an antibody or fragment thereof comprising an HCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 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 that is at least 90%, at least 95%, at least 98 % or at least 99% homologous with it.

[0040] In accordance with one embodiment, the antibody or fragment thereof further comprises an LCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 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 that is at least 90%, at least 95%, at least 98%, or at least at least 99% homologous with it.

[0041] In accordance with one embodiment, the present invention also provides an antibody or antigen binding fragment of an antibody further comprising an HCDR3 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 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 that is at least 90%, at least 95%, is at least 98% or at least 99% identical to such sequence; and an LCDR3 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 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 that is at least 90%, at least 95%, at least 98%, or at least 99% identical to such sequence.

[0042] In accordance with one embodiment, the present invention provides an antibody or antigen binding 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 that is at least 90%, at least 95%, is at least 98% or at least 99% identical to such sequence; HCDR2 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 5, 21, 37, 53,69, 85, 101, 117, 133, 149, 165, 181, 197,213,229, 245, 261, 277 and 293 , or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to such sequence; 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 thereto that is at least 90%, at least 95%, at least 98%, or at least 99% identical to such sequence; 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 that is at least 90%, at least 95%, at least 98%, or at least 99% identical to such sequence.

[0043] According to a third aspect, the present invention provides a human antibody or antigen binding fragment specific for RET comprising HCVR encoded by nucleotide sequence segments derived from germline sequences _VH , _DH and _JH , and LCVR encoded by nucleotide sequence segments , derived from the germline sequence V _K and J _K .

[0044] The present invention relates to antibodies with a modified glycosylation pattern. In some applications, modification may be useful, for example, removing undesired glycosylation sites or, for example, removing the fucose moiety to enhance antibody-dependent cell-mediated cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, modification of galactosylation can be performed to modify complement dependent cytotoxicity (CDC).

[0045] According to 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 RET, and a pharmaceutically acceptable carrier or diluent. In accordance with one embodiment, the present invention provides a pharmaceutical composition comprising two fully human monoclonal antibodies or antigen binding fragments thereof that either bind to the same epitope or bind to two different epitopes on RET, and a pharmaceutically acceptable carrier or diluent . It should be understood that any combination of the antibodies described herein can be used in the pharmaceutical composition to achieve the desired results in the patient population in need of such therapy. For example, two antibodies that recognize and/or bind RET can be used in the composition.

[0046] In accordance with one embodiment, the composition contains an antibody that binds to RET and has the amino acid sequence pair HCVR/LCVR 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.

[0047] In accordance with one embodiment, the pharmaceutical composition contains at least one antibody that binds RET, wherein said antibody contains three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) contained in any of the amino acid sequences of the heavy chain variable region chain (HCVR) selected from the group consisting of SEQ ID NO: 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 in any 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 accordance with one embodiment, antibodies of the present invention or compositions containing one or more antibodies of the present invention can be used to inhibit at least one RET-related activity or function expressed on a cell. In accordance with one embodiment, the cell may be a tumor cell. In accordance with one embodiment, the activity may involve transducing cellular signals.

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

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

[0051] The second therapeutic agent may be any agent that is advantageously combined with an antibody or fragment thereof of the present invention, for example, if the anti-RET antibody is an RET inhibitor used to treat a cancer condition, the second agent may be selected from a chemotherapeutic agent , a radionuclide, an RET-specific siRNA, a second RET-specific antibody, a small molecule RET inhibitor, and a bone marrow reparative agent such as G-CSF, GM-CSF or M-CSF, or a biological agent having colony-stimulating or bone marrow reparative properties activity. In accordance with certain embodiments, the second therapeutic agent may be an agent that helps neutralize or reduce any one or more possible side effects associated with an antibody or antigen binding fragment of an antibody of the present invention when such one or more side effects occur. In accordance with certain embodiments, the second therapeutic agent may be an agent useful for reducing pain associated with certain pathological conditions characterized by pain and/or inflammation. This second agent may include a nerve growth factor (NGF) inhibitor (eg, a small molecule NGF antagonist or anti-NGF antibody), aspirin or another NSAID, morphine, steroids (eg, prednisone), an anti-Na _v 1.7 antibody, or a small molecule Na _v 1.7 inhibitor. , Na _v 1.8 antagonist (eg, Na _v 1.8 antibody or Na _v 1.8 small molecule inhibitor), Na _v 1.9 antagonist (eg, Na _v 1.9 antibody or Na _v 1.9 small molecule inhibitor), cytokine inhibitor (eg, interleukin-1 inhibitor (IL-1) (such as rilonacept (IL-1 decoy); Regeneron) or anakinra (KINERET®, Amgen), a small molecule IL-1 antagonist or anti-IL-18 inhibitor (such as a small molecule antagonist; IL-18 or anti-IL-18 antibody); IL-6 or IL-6R inhibitor (such as small molecule IL-6 antagonist, anti-IL-6 antibody or anti-IL-6 receptor antibody), caspase-1, p38, IKK1 inhibitors /2, CTLA-4Ig or opioid.

[0052] It will also be understood that the antibodies and pharmaceutically acceptable compositions of the present invention can be used in combination therapy, that is, the antibodies and pharmaceutically acceptable compositions can be administered simultaneously with, before, or after one or more other necessary therapeutic agents or medical procedures. The specific combination of forms of therapy (therapeutic agents or procedures) to be used in a combination regimen will take into account the compatibility of the necessary therapeutic agents and/or procedures and the desired therapeutic effect to be achieved. It will also be understood that the forms of therapy used may achieve the desired effect for the same disorder (eg, the antibody may be administered concomitantly with another agent used to treat the same disorder), or they may achieve different effects (eg , monitoring for any side effects). As used herein, additional therapeutic agents that are typically administered to treat or prevent a particular disease or condition are suitable for the disease or condition being treated.

[0053] If a small molecule RET inhibitor is to be combined with an antibody of the present 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, AEE73288, 51, CHIR258, SER-7055, OSI-930, AVT-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 administered simultaneously, dosages may be adjusted accordingly, as is known in the art.

[0055] A fifth aspect of the present invention relates to a method of 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 of provide the patient with an antibody or antigen binding fragment of any of the anti-RET antibodies described herein together with a pharmaceutically acceptable carrier or diluent.

[0056] In accordance with 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 cancer. In accordance with one embodiment, the disorder or pathological condition associated with the expression, activation or signaling of the receptor tyrosine kinase gene RET or a rearranged form thereof is selected from the group consisting of acute pain, chronic pain, neuropathic pain, inflammatory pain, arthritis, osteoarthritis , migraine, cluster headaches, trigeminal neuralgia, herpetic neuralgia, common forms of neuralgia, neurodegenerative disorders, neuroendocrine disorders, visceral pain, acute gout, postherpetic neuralgia, diabetic neuropathy, lumbosacral radiculitis, spinal pain, pain in the head or neck, severe or unrelieved pain, breakthrough pain, post-operative pain, toothache, rhinitis, cancer pain, or bladder-related disorders.

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

[0058] In accordance with one embodiment, the tumor is a solid tumor or a blood cell tumor.

[0059] In accordance with one embodiment, 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.

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

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

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

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

[0064] In accordance with one embodiment, the skin cancer is melanoma.

[0065] In accordance with one embodiment, the blood cell tumor is a leukemia.

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

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

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

[0069] In accordance with one embodiment, an anti-RET antibody of the present invention can interfere with or prevent the interaction of RET with one or more GDNF family member ligands (GDNF, neurturin, artemin and persefin) in complex with their respective co-receptors (GFRα1, GFRα2 , GFRα3 and GFRα4, respectively). In accordance with one embodiment, human anti-RET antibodies described herein can interfere with or prevent the interaction of RET with the GDNF/GFRα1 complex. According to a related embodiment, human anti-RET antibodies described herein can interfere with or prevent the interaction of RET with the artemin/GFRα3 complex. In other related embodiments, human anti-RET antibodies described herein can interfere with or prevent the interaction of RET with neurturin/GFRα2 or persefin/GFRα4 complexes.

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

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

[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 to be treated with the anti-RET antibody is a cancer condition, the second therapeutic agent may 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 inhibitor RET, an antitumor agent, siRNA specific for RET, and a second antibody specific for RET. If an anti-RET antibody is provided for the treatment of pain associated with a cancerous condition, or for the treatment of pain associated with other pathological conditions that may be attributable, at least in part, to RET activation or signaling, the second therapeutic agent may be selected from any one or more of the following: a nerve growth factor (NGF) inhibitor (eg, small molecule NGF antagonist or anti-NGF antibody), aspirin or other NSAID, morphine, steroids (eg, prednisone), anti-Na _v 1.7 antibody, or small molecule Na _v 1.7 inhibitor , Na _v 1.8 antagonist (e.g. anti-Na _v 1.8 antibody or small molecule Na _v 1.8 inhibitor), Na _v 1.9 antagonist (e.g. anti-Na _v 1.9 antibody or small molecule Na _v 1.9 inhibitor), cytokine inhibitor (e.g. interleukin-1 inhibitor (IL-1) (such as rilonacept (“IL-1 decoy”); Regeneron) or anakinra (KINERET®, Amgen), a small molecule IL-1 antagonist or anti-IL-18 antibody (such as a small molecule antagonist; IL-18 or anti-IL-18 antibody); an IL-6 or IL-6R inhibitor (such as a small molecule IL-6 antagonist, anti-IL-6 antibody, or anti-IL-6 receptor antibody), caspase-1, p38, IKK1/2, CTLA-4Ig inhibitors, or an opioid.

[0073] Other embodiments will become apparent upon consideration of the following detailed description.

Brief description of drawings

[0074] FIG. 1. Schematic diagram of the human RET receptor.

Detailed Disclosure of the Present Invention

[0075] Before disclosing the present methods, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, since such methods and conditions may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

[0076] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention relates. As used herein, the term “about” when used in relation to a specific specified numerical value means that the value may differ from the specified value by no more than 1%. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (eg, 99.1, 99.2, 99.3, 99.4, etc.).

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

Definitions

[0078] "Rearranged during transfection", also called "RET", is a receptor tyrosine kinase that is expressed during development in various tissues, including the peripheral and central nervous system and the kidney. The LET oncogene was identified in 1985 by Takahashi et al., who reported a novel gene rearrangement with transforming activity in NIH/3T3 cells that were transfected with human lymphoma DNA (see Takahashi, M., et al., (1985) Cell , 42:581-588). RET was later confirmed to be an oncogene that rearranges somatically in the DNA of patients with papillary thyroid carcinoma (PTC) and was later named RET/PTC. (Fusco, A. et al., (1987), Nature, 328:170-172; Grieco, M. et al., (1990), Cell, 60:557-63). The RET protein consists of three domains: an extracellular ligand-binding domain, a hydrophobic transmembrane domain, and a cytoplasmic portion with a tyrosine kinase domain separated by a 27-amino acid insert (see Fig. 1). There are two main isoforms of RET resulting from alternative splicing. The short and long isoforms of RET, which differ by 9 and 51 unrelated C-terminal amino acids, 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 have transforming activity as measured by plaque formation assays (Rossel, M. et al. (1997), Oncogene, 14:265-75).

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

[0080] The cDNA sequence and amino acid sequence of RET isoform C (also known as RET9) are provided in GenBank under accession numbers NM_020630.4 and NP 065681.1, respectively, and are provided herein under SEQ ID NOs: 311 and 312, respectively. RET or immunogenic fragments thereof can be used in the production of human monoclonal antibodies specific for RET. The RET protein or fragments thereof can be produced recombinantly using standard methods known in the art. Exemplary fusion proteins containing the RET ectodomain are shown at SEQ ID NOs: 305, 306, 307 and 309. Such fusion proteins can be used as immunogens or can be used to target therapeutics to cells or tissues expressing RET.

[0081] RET is a signaling receptor for ligands of the “glial-derived neurotrophic factor (GDNF) family,” which includes GDNF (see GenBank accession number NP 000505.1), artemin (see GenBank accession number Q5T4W7), neurturin (see GenBank accession number Q5T4W7), neurturin (see GenBank accession number NP 000505.1 GenBank accession NM_004558) and persephine (see GenBank accession AF040962). GDNF family ligands interact with RET and activate it only in the presence or complex with one of four GPI-linked “coreceptors” known as GDNF family □ receptors: GFRα1 (see GenBank accession number NP 005255.1), GFRα2 (see GenBank accession number NP 005255.1), GFRα2 (see GenBank accession number NM_001495.4), GFRα3 (see GenBank accession number NP 001487.2) and GFRα4 (see GenBank accession number NM_022139 for GFRα4a and NM 145762.2 for GFRα4b) (Baloh, RH, et al. (2000) Curr Opin Neurobiol 10:103–110; Borrello, MG, et al (2013), Expert Opin Targets, 17(4):403–419). The primary ligands of the coreceptors GFRα1, GFRα2, GFRα3 and GFRα4 are GDNF, neurturin (NRTN), artemin (ARTN) and persephine (PSPN), respectively.

[0082] The term “IC ₅₀ ” refers to the “half-maximal inhibitory concentration”, the value of which measures the effectiveness of inhibition by a compound (eg, an anti-RET antibody) in relation to biological or biochemical utility. Using this quantitative indicator, the amount required for a particular inhibitor is indicated to half inhibit a given biological process.

[0083] As used herein, the terms “treat,” “treatment,” and “treatment process” refer to either reducing the progression of a disease, disorder, or pathological condition that is attributable in part to or associated with RET expression in a cell or tissue in a subject, e.g. , slowing the rate of tumor cell proliferation in patients with a tumor expressing RET, upon administration of an antagonistic/inhibitory antibody of the present invention, or reducing pain associated with a cancerous condition or pain associated with any other disease or condition that is at least at least partly due to the expression of RET.

[0084] As used herein, the terms “prevent,” “prevention process,” and “prevention” refer to inhibiting the development or onset of a disease, disorder, or pathological condition attributable in part to or associated with RET expression in a cell or tissue in a subject, e.g. , certain forms of cancer, or to inhibit tissue damage that occurs in a patient following injury, or to inhibit or alleviate pain associated with a disease or condition attributable in part to RET expression.

[0085] The term "antibody" as used herein is intended to mean immunoglobulin molecules consisting of four polypeptide chains: two heavy (H) chains and two light (L) chains linked together by disulfide bonds (i.e., "full-length molecules antibodies"), as well as their multimers (for example, IgM) or their antigen-binding fragments. Each heavy chain consists of a heavy chain variable region (“HCVR” or “ _VH ”) and a heavy chain constant region (which includes the _CH 1, _CH 2 and _CH 3 domains). Each light chain consists of a light chain variable region ("LCVR" or " _VL ") and a light chain constant region ( _CL ). The V _H and V _L regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), alternating with regions that are more conserved, called framework regions (FR). Each V _H and V _L includes three CDRs and four FRs, located from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In accordance with certain embodiments of the present invention, the FR antibody (or antigen binding fragment thereof) may be identical to human germline sequences or may be naturally or artificially modified. The consensus amino acid sequence can be determined based on analysis based on direct comparison of two or more CDRs.

[0086] It is also possible to replace one or more CDR residues or eliminate one or more CDRs. The scientific literature describes antibodies for which one or two CDRs may not be important for binding function. Padlan et al. (1995 FASEB J. 9:133-139) analyzed the contact sites between antibodies and their antigens 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 did not have amino acids coming into contact with the antigen (see also Vajdos et al. 2002 J Mol Biol 320:415-428).

[0087] CDR residues that do not come into contact with the antigen can be identified based on the results of previous studies (for example, residues H60-H65 in CDRH2 are often unnecessary) among the Kabat CDR regions located outside the Chothia CDR by molecular modeling and/or experimentally. Deletion of a CDR or residue(s) thereof typically results in a replacement with an amino acid occupying a corresponding position in the sequence of another human antibody or the consensus sequence of such sequences. The positions for substitution in the CDRs and amino acids for substitution can also be selected experimentally. Empirical substitutions may 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 regions and/or CDR regions of the heavy and light chain variable domains relative to the corresponding germline sequences. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from publicly available antibody sequence databases. The present invention relates to antibodies and antigen binding fragments thereof that are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids in one or more framework regions and/or CDR regions are mutated at the corresponding germline sequence residue(s) , from which the antibody was derived, or at the corresponding residue(s) of another human germline sequence, or at a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as “germline mutations”). One of ordinary skill in the art, from the heavy and light chain variable region sequences disclosed herein, will readily be able to produce numerous antibodies and antigen binding fragments that contain one or more individual germline mutations or combinations thereof. In certain embodiments, all framework residues and/or CDR residues in the V _H and/or V _L domains are mutated back to residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, for example, only the mutated residues occurring in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or the mutated residues occurring only in CDR1, CDR2, or CDR3 . In other embodiments, one or more framework residues and/or CDR residues are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that is different from the germline sequence of which the antibody was originally obtained). However, the antibodies of the present invention may contain any combination of two or more germline mutations in framework regions and/or CDR regions, in which, for example, some individual residues are mutated to the corresponding residue of a particular germline sequence, while how certain other residues that differ from the original germline sequence are retained or are mutated into the corresponding residue of a different germline sequence. Once produced, antibodies and antigen binding fragments that contain one or more germline mutations can be readily tested for one or more desirable properties, such as improved binding specificity, increased binding affinity, improved or increased antagonistic or agonistic biological properties (under appropriate circumstances) , reduced immunogenicity, etc. The present invention relates to antibodies and antigen-binding fragments, which are generally obtained by this method.

[0089] The present invention also provides fully human monoclonal antibodies containing variants of any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein with one or more conservative substitutions. For example, the present invention relates to antibodies with amino acid sequences HCVR, LCVR and/or CDR, for example, 10 or less, 8 or less, 6 or less, 4 or less, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein.

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

[0091] The term “specifically binds” or “specifically binds to” or the like means that the antibody or 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 (eg, a lower KD value 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, and the like. The antibodies described herein, which specifically bind to RET, were identified using surface plasmon resonance, for example, on the BIACORE™ system. Moreover, as used herein, multispecific antibodies that bind to the RET protein and one or more additional antigens, or a bispecific antibody that binds to two different regions of RET, are still considered to be antibodies that “bind specifically.”

[0092] The term “high affinity” antibody refers to an mAb having a binding affinity for RET, expressed as K _D , that is at least 10 ^-7 M, at least 10 ^-8 M, preferably 10 ^-9 M, more preferably 10 ^-10 M, more preferably 10 ^-11 M, more preferably 10 ^-12 M, as measured by surface plasmon resonance, for example, on a BIACORE™ system, or by a solution affinity ELISA.

[0093] By "slow dissociation rate", "Koff" or "kd" is meant an antibody that dissociates from RET with a reaction rate constant that is 1×10 ^-3 s ^-1 or less, preferably 1×10 ^-4 s ^-1 or less, as determined by surface plasmon resonance, such as the BIACORE™ system.

[0094] The terms "antigen binding portion" of an antibody, "antigen binding fragment" of an antibody, and the like. as used herein, include any naturally occurring, enzymatically produced, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds 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 antibody fragments that retain the ability to bind RET.

[0095] In accordance with specific embodiments, the antibody or antibody fragments of the present invention can be conjugated to a therapeutic moiety ("immunoconjugate" or "antibody-drug conjugate"), such as a small molecule RET inhibitor, an anticancer agent, a radionuclide, a growth factor , a bone marrow regenerative agent or colony stimulating factor, or any other therapeutic moiety useful for treating a disease, disorder or pathological condition associated with RET expression, such as cancer or damaged tissue.

[0096] The term "isolated antibody" as used herein is intended to mean an antibody that is substantially free of other antibodies (Abs) having different antigen specificities (e.g., an isolated antibody that specifically binds to RET, or a fragment thereof that is substantially free contains Abs that specifically bind antigens other than RET).

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

[0098] The term “surface plasmon resonance” as used herein refers to an optical phenomenon that enables real-time analysis of biomolecular interactions by detecting changes in protein concentrations in a biosensor array, for example, using the BIAcore™ system (Biacore Life Sciences, a division of GE Healthcare, Piscataway, NJ).

[0099] As used herein, the term “K _D ” is intended to denote the equilibrium dissociation constant of a particular antibody-antigen interaction.

[00100] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in a variable region of an antibody molecule known as a paratope. One antigen can have more than one epitope. Thus, different antibodies can bind to different regions on the antigen and can have different biological effects. The term "epitope" also refers to a site on an antigen to which B and/or T cells respond. It also refers to the site of an antigen to which an antibody binds. Epitopes can be defined as structural or functional. Functional epitopes are usually a subset of structural epitopes and contain residues that directly contribute to the affinity of the interaction. Epitopes can also be conformational, i.e. consist of nonlinear amino acids. In certain embodiments, epitopes may include determinants that are reactive surface moieties of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific characteristics of three-dimensional structures and/or specific charge characteristics.

[00101] The term "substantially identical" or "substantially identical" with respect to a nucleic acid or fragment thereof indicates that when the corresponding nucleic acid sequence having insertions or deletions is optimally aligned with another nucleic acid sequence (or its complementary strand) the nucleotide sequence identity is at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% nucleotide bases as measured by a well-known sequence identity algorithm such as FASTA, BLAST or GAP, which are discussed below. A nucleic acid molecule having substantial identity with a reference nucleic acid molecule may, in certain cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[00102] In the context of polypeptides, the term “substantially similar” or “substantially similar” means that two peptide sequences, when optimally aligned, such as through the GAP or BESTFIT programs, using default gap opening penalties, are at least 90% identical , even more preferably at least 95%, 98% or 99% identical. Preferably, the positions of the residues that are not identical differ in 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 (eg, charge or hydrophobicity). In general, a conservative amino acid substitution will not significantly change the functional properties of the protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity can be adjusted upward to correct for the conservative nature of the substitution. Methods for making such adjustments are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331, which is incorporated herein by reference. Examples of groups of amino acids that contain side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic side chains with hydroxyl groups: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) main side chains: lysine, arginine and histidine; 6) acidic side chains: aspartate and glutamate; and 7) sulfur-containing side chains: cysteine and methionine. Preferred groups of conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate and asparagine-glutamine. Alternatively, a conservative replacement is any change with a positive value in the PAM250 log-likelihood function matrix disclosed in Gonnet et al. (1992) Science 256: 1443 45, which is incorporated herein by reference. A “moderately conservative” replacement is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[00103] Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences by measuring the similarity assigned to various substitutions, deletions and other modifications, including conserved amino acid substitutions. For example, GCG software contains programs such as GAP and BESTFIT that can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA with default or recommended parameters, programs in GCG version 6.1. FASTA (eg, FASTA2 and FASTA3) outputs alignment results and percent sequence identity for the regions with the best match between the query and search sequences (Pearson (2000), earlier). Another preferred algorithm when comparing a sequence of the present invention to a database containing a large number of sequences from various organisms is the BLAST computer program, especially 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.

[00104] In accordance with specific embodiments, the antibody or antibody fragment for use in the method of the present invention may be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific for different epitopes of a single target polypeptide or may 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 involves the use of a first immunoglobulin (Ig) C _H 3 domain and a second Ig C _H 3 domain, wherein the first and second Ig C _H 3 domains differ from each other in at least one way amino acid, and wherein the at least one amino acid difference reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody without the amino acid difference. In accordance with one embodiment, the first C _H 3 Ig domain binds to protein A, and the second C _H 3 Ig domain contains a mutation that reduces or eliminates protein A binding, such as modification H95R (according to IMGT exon numbering; H435R according to EU numbering ). The second C _H 3 may additionally have the modification Y96F (according to IMGT; Y436F according to EU). Additional modifications that can be found in the second C _H 3 include the following: D16E, L18M, N44S, K52N, V57M and V82I (according to IMGT; D356E, L358M, N384S, K392N, V397M and V422I according to EU) in the case of mAb IgG1; N44S, K52N and V82I (IMGT; EU N384S, K392N and V422I) for IgG2 mAb; and Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (according to IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V422I according to EU) for IgG4 mAbs. The variants of the bispecific antibody format described above are within the scope of the present invention.

[00105] By the phrase “therapeutically effective amount” is meant an amount that produces the desired effect in the person to whom it is administered. The exact amount will depend on the purpose of treatment and can be determined by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

General Disclosure of the Present Invention

[00106] "Rearranged during transfection", also called "RET", is a receptor tyrosine kinase that is expressed during development in various tissues, including the peripheral and central nervous system and the kidney (Arighi, E. et al. (2005), Cytokine Growth Factor Rev 16:441-67; Borrello, MG, et al, (2013), Expert Opin. Targets, 17(4): 403-419). The RET oncogene was identified in 1985 by Takahashi et al., who reported a novel gene rearrangement with transforming activity in NIH/3T3 cells that were transfected with human lymphoma DNA (see Takahashi, M., et al., (1985) Cell, 42:581-588). RET was later confirmed to be an oncogene that rearranges somatically in the DNA of patients with papillary thyroid carcinoma (PTC) and was later named RET/PTC. (Fusco, A. et al, (1987), Nature, 328:170-172; Grieco, M. et al, (1990), Cell, 60:557-63). The RET protein consists of three domains: an extracellular ligand-binding domain, a hydrophobic transmembrane domain, and a cytoplasmic portion with a tyrosine kinase domain separated by an insert of 27 amino acids (see Fig. 1). There are two main isoforms of RET resulting from alternative splicing. The short and long isoforms of RET, which differ by 9 and 51 unrelated C-terminal amino acids, 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 have transforming activity as measured by plaque formation assays (Rossel, M. et al. (1997), Oncogene, 14:265-75).

[00107] Genetic alterations of RET have been shown to be involved in the etiology of various forms of thyroid cancer, and more recent evidence suggests that RET is also involved in the development of various forms of lung adenocarcinoma (Viglietto, G. et al. (1995), Oncogene , 11:1207-10; Fischer, A.H., et al., (1998), Am J Pathol. 153:1443-50). Other studies suggest that RET may be involved in the development of other tumors, including breast, pancreatic, various forms of 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).

[00108] Vandetanib (ZD6474, CAPRELSA®, Astra Zeneca) is an orally available aminoquinazoline compound that was originally developed as a VEGFR2 inhibitor but was later found to have activity against RET, VEGFR3, EGFR, and PDGFR. Vandetanib is currently approved by the FDA and EMA for the treatment of advanced and metastatic medullary thyroid carcinoma (MTC) (Wells, SA, et al, (2012), J. Clin. Oncol. 30:134-41).

[00109] Sorafenib (BAY43-9006, NEXAVAR®, Bayer Pharmaceuticals) is a bisarylurea compound originally developed to target the serine-threonine kinase BRAF, but was subsequently found to be effective 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 the treatment of advanced liver and kidney cancers.

[00110] Sunitinib (SU11248, SUTENT®, Pfizer) is an indolinone compound that primarily targets the kinases VEGFR2, PDGFR, c-kit, FLT3 and RET ((Chow, LQ, et al, (2007), J. Clin. Oncol. 25:884-96). Sunitinib has been approved by the FDA for patients with imatinib-resistant GIST and for the treatment of advanced pancreatic neuroendocrine tumors and renal cell carcinoma.

[00111] Cabozantinib (Cometriq, formerly known as XL-184, Exelixis) is a small molecule multikinase inhibitor targeting MET, VEGFR2 and RET. It is currently in clinical trials in a large number of tumor types, including medullary thyroid cancer, prostate cancer, ovarian cancer, non-small cell lung cancer (NSCLC), hepatocellular, renal cell and breast cancer, as well as melanoma and glioblastoma ((Zhang, Y. et al, (2010), IDrugs 13:112-21).

[00112] Another RET-targeting agent in clinical development is motesanib (AMG-706, Amgen), which is a multikinase inhibitor that targets VEGFR1-3, Flt3, Kit, PDGFR and RET.

[00113] Other RET inhibitors in preclinical development include RPI-1, which is an indoline compound; PP-1, which is a pyrazolopyrimidine compound active against RET and Src; and NVP-AST478, which is a biphenylurea compound with effective RET kinase activity in vitro and in vivo (Cuccuru, G. et al. (2004), J Natl Cancer Inst 96:1006-14).

[00114] However, one problematic aspect of the above-mentioned RET inhibitors is that they are not specific to RET, that is, they appear to act through multiple mechanisms and, as such, may potentially have other adverse effects in vivo. For example, some of the above inhibitors cause side effects such as hypertension and QTc prolongation. Thus, the non-selective profile of such agents may limit the spectrum of therapeutic action. It would be useful to find agents such as an anti-RET antibody that selectively binds to RET, which could result in superior clinical efficacy with a more favorable safety profile.

[00115] Accordingly, there is still a need for effective therapies against RET-induced tumors, and further, there is a need to identify an RET-specific agent for the prevention and treatment of other diseases, disorders or pathological conditions associated with RET expression , without the adverse side effects associated with the agents described above. Such specificity and efficacy can be achieved through the use of anti-RET antibodies, such as those described herein.

[00116] In certain embodiments, antibodies of the present invention are obtained from mice immunized with a primary immunogen, such as whole human RET protein, or a recombinant form of the protein, or a fragment thereof, or a fusion protein that contains 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 recombinantly produced RET fusion protein (see SEQ ID NO: 305 , 306, 307 and 313), followed by immunization with a secondary immunogen (purified human RET protein) or an immunogenically active fragment of the RET protein, such as the ectodomain from RET.

[00117] The immunogen may be DNA encoding a 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 its active fragment.

[00118] The immunogen may be derived from the extracellular domain of the RET protein, which spans amino acid residues 1-635 of any of SEQ ID NOs: 305, 307, 310, 312 and 313 (including the signal sequence); from amino acid residues 1-636 of the sequence under SEQ ID NO: 306 (including the signal sequence). The immunogen may be derived from a fragment of any of the above-mentioned regions of the RET protein.

[00119] The full-length amino acid sequence of RET51 is provided under SEQ ID NO: 310 and is also provided under GenBank accession number NP_066124.1. The full-length amino acid sequence of RET9 is provided under SEQ ID NO: 312 and is also provided under GenBank accession number NP_065681.1. Exemplary immunogens include the recombinant constructs shown under SEQ ID NO: 307 or 313.

[00120] In certain embodiments, antibodies that specifically bind to RET can be made using fragments of the above regions or peptides that extend beyond the designated regions by about 5 to about 20 amino acid residues from either one or both N- or the C-termini of the regions described herein. In certain embodiments, any combination of the above regions or fragments thereof can be used in the production of RET-specific antibodies. In certain embodiments, any one or more of the above RET regions or fragments thereof can be used to produce monospecific, bispecific, or multispecific antibodies.

Antigen-binding antibody fragments

[00121] Unless specifically stated otherwise, as used herein, the term “antibody” is to be understood to include antibody molecules containing two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., “full antibody molecules”), as well as their antigen-binding fragments. The terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, etc. as used herein, include any naturally occurring, enzymatically produced, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds 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 antibody fragments that retain the ability to specifically bind RET. An antibody fragment can include a Fab fragment, an E(ab') ₂ fragment, an Fv fragment, a dAb fragment, a fragment containing a CDR, or an isolated CDR. Antigen-binding antibody fragments can be produced, for example, from complete antibody molecules using any suitable standard techniques, such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding the variable and (optionally) constant domains of the antibody. 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 chemically manipulated or manipulated using molecular biology techniques, for example, to organize one or more variable and/or constant domains into a suitable configuration or to introduce codons, insert cysteine residues, modify, add or remove amino acids, and etc.

[00122] 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 molecules (scFv); (vi) dAb fragments and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody (eg, a dedicated complementarity determining region (CDR) such as the CDR3 peptide), or a restricted FR3-CDR3-FR4 peptide conformational freedom. Also used herein, the expression "antigen binding fragment" includes other engineered molecules such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, tri-bodies, tetrabodies, minibodies, nanobodies (eg monovalent antibodies, bivalent antibodies, etc.), small modular protein (SMIP) immunopharmaceuticals and shark IgNAR variable domains.

[00123] An antigen binding fragment of an antibody will typically contain at least one variable domain. The variable domain can be of any size or amino acid composition and will typically contain at least one CDR that is in frame with or adjacent to one or more framework sequences. In antigen binding fragments having a V _H domain linked to a V _L domain, the V _H and V _L domains may be arranged relative to each other in any suitable order. For example, the variable region may be dimeric and contain dimers V _H -V _H , V _H -V _L or V _L -V _L . Alternatively, the antigen binding fragment of the antibody may comprise a monomeric V _H or V _L domain.

[00124] In accordance with certain embodiments, an antigen binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable or constant domains that may be present in an antigen binding fragment of an antibody 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 _L . In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains can either be directly connected to each other or can be connected by a full or partial hinge or linker region. The hinge region may consist of at least 2 (eg, 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. In addition, the antigen binding fragment of an antibody of the present invention may comprise a homodimer or heterodimer (or other multimer) in any of the above configurations of variable and constant domains in non-covalent association with each other and/or with one or more monomeric _VH or _VL domains ( for example, through one or more disulfide bonds).

[00125] As with full antibody molecules, antigen binding fragments can be monospecific or multispecific (eg, bispecific). A multispecific antigen-binding antibody fragment will typically contain at least two different variable domains, with each variable domain capable of specifically binding to a distinct antigen or to a distinct epitope on a single antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of an antigen binding fragment of an antibody of the present invention using standard techniques available in the art.

Obtaining human antibodies

[00126] Methods for generating human antibodies in transgenic mice are known in the art. Any such known methods can be used in the context of the present invention to obtain human antibodies that specifically bind to RET.

[00127] Using VELOCIMMUNE® technology (see, for example, US Pat. No. 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method for generating monoclonal antibodies, high-affinity chimeric anti-RET antibodies with a human variable region and a murine constant region are initially isolated. VELOCIMMUNE® technology involves the creation of a transgenic mouse having a genome containing human heavy and light chain variable region loci operably linked to endogenous mouse constant region loci such that the mouse responds to antigen stimulation by producing an antibody containing the human variable region and the mouse constant region . DNA encoding the variable regions of the heavy and light chains of the antibody is isolated and operably linked to DNA encoding the human constant regions of the heavy and light chains. The DNA is then expressed in a cell capable of expressing a fully human antibody.

[00128] Typically, VELOCIMMUNE® mice are challenged with an antigen of interest and lymphatic cells (such as B cells) are isolated from the mice that express the antibodies. Lymphatic cells can be fused with a myeloma cell line to produce immortalized hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. The DNA encoding the heavy chain and light chain variable regions can be isolated and combined with the required isotype heavy chain and light chain constant regions. Such an antibody protein may be produced in a cell, such as a CHO cell. Alternatively, DNA encoding antigen-specific chimeric antibodies or light and heavy chain variable domains can be isolated directly from antigen-specific lymphocytes.

[00129] Initially, high-affinity chimeric antibodies with a human variable region and a mouse constant region are isolated. Just as in the experimental section below, antibodies are characterized and selected for the required characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with the necessary human constant regions to create a fully human antibody of the present invention, for example, wild type or modified IgG1 or IgG4. Although the selected constant region may vary according to the specific application, the variable region must have the characteristics of high affinity antigen binding and target specificity.

[00130] In accordance with certain embodiments, the antibodies of the present invention have affinities (K _D ) ranging from about 1.0 x 10 ^-7 M to about 1.0 x 10 ^-12 M, as measured by antigen binding, immobilized either in the solid phase or in the liquid phase. To create fully human antibodies, the mouse constant regions are replaced with the required human constant regions. Although the selected constant region may vary according to the specific application, the variable region must have the characteristics of high affinity antigen binding and target specificity.

Bioequivalents

[00131] Antibodies to RET and antibody fragments of the present invention cover proteins with amino acid sequences that differ from the sequences of the described antibodies, but which retain the ability to bind RET. Such variant antibodies and antibody fragments are characterized by the presence of one or more of the additions, deletions or substitutions of amino acids when compared to the original sequence, but exhibit biological activity that is substantially equivalent to that of the described antibodies. Likewise, the antibody-encoding DNA sequences of the present invention include sequences that are characterized by the presence of one or more additions, deletions or substitutions of nucleotides relative to the disclosed sequence, but which encode an antibody or antibody fragment that is substantially bioequivalent to the antibody or antibody fragment of the present invention.

[00132] Two antigen-binding proteins or antibodies are considered biological equivalents if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives, the rate and/or extent of absorption of which does not differ significantly from those when administered the same molar dose under similar experimental conditions, or in single doses. doses, or in multiple doses. Some antibodies are considered equivalents or pharmaceutical alternatives if they are equivalent in their extent of absorption, but not in their rate of absorption, and may still be considered bioequivalent because such differences in absorption rate are intended and taken into account when labeling is introduced, and are not essential to achieve effective concentrations of the drug in the body, for example, with long-term use, and are considered medically insignificant for the specific pharmaceutical product being studied.

[00133] In accordance with one embodiment, two antigen binding proteins are bioequivalent if they do not differ clinically significant in their safety, purity, or activity.

[00134] According to one embodiment, two antigen binding proteins are bioequivalent if the reference product can be substituted one or more times in a patient for the reference product with the biological product, and vice versa, without a perceived increased risk of adverse effects, including a clinically significant change in immunogenicity or reduced efficacy, compared to with long-term therapy without such replacement.

[00135] In one embodiment, two antigen binding proteins are bioequivalent if they both act by a common mechanism or mechanisms of action with respect to the condition or conditions of use, provided such mechanisms are known.

[00136] Bioequivalence can be demonstrated through in vivo and/or in vitro methods. Biological equivalence measurements include, for example, (a) in vivo testing in humans or other mammals, in which the concentration of an antibody or its metabolites is measured in blood, plasma, serum, or other body fluids over time; (b) in vitro testing, the results of which correlate with and can be reasonably predicted from in vivo bioavailability testing in humans; (c) in vivo testing in humans and other mammals, in which the relevant early pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) a well-controlled clinical trial that establishes the safety, efficacy, or bioavailability or bioequivalence of the antibody.

[00137] Bioequivalent variants of the antibodies of the present invention can be constructed, for example, by making various substitutions of residues or sequences or removing terminal or internal residues or sequences that are not essential for biological activity. For example, cysteine residues that are not significant for biological activity can be removed or replaced with other amino acids to prevent the formation of unwanted or inappropriate intramolecular disulfide bridges upon renaturation. In other contexts, bioequivalent antibodies may include antibody variants containing amino acid changes that modify the glycosylation characteristics of the antibodies, such as mutations that eliminate or remove glycosylation sites.

Antibodies to RET containing Fc variants

[00138] In accordance with certain embodiments of the present invention, there are provided anti-RET antibodies comprising an Fc domain containing one or more mutations that enhance or weaken the binding of the antibody to the FcRn receptor, for example, at an acidic pH compared to a neutral pH. pH. For example, the present invention relates to anti-RET antibodies containing a mutation in the C _H 2 or C _H 3 region of the Fc domain, wherein the mutation(s) increases the affinity of the Fc domain for FcRn in an acidic environment (for example, in the endosome, where the pH varies by range from about 5.5 to about 6.0). The result of such mutations may be an increase in the half-life of the antibody in the blood serum when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modification at position 250 (eg, E or Q); 250 and 428 (for example, L or F); 252 (for example, L/Y/F/W or T), 254 (for example, S or T) and 256 (for example, S/R/Q/E/D or T), or modification at position 428 and/or 433 (for example, H/L/R/S/P/Q or K), and/or 434 (for example, H/F or Y), or a modification at position 250 and/or 428, or a modification at position 307 or 308 ( eg 308F, V308F) and 434. According to one embodiment, this modification includes modification 428L (eg M428L) and 434S (eg N434S); modification 428L, 2591 (for example, V259I) and 308F (for example, V308F); modification 433K (for example, H433K) and 434 (for example, 434Y); modification 252, 254 and 256 (for example, 252Y, 254T and 256E); modification 250Q and 428L (for example, T250Q and M428L); and modification 307 and/or 308 (for example, 308F or 308P).

[00139] For example, the present invention relates to anti-RET antibodies containing an Fc domain containing one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (eg, T250Q and M248L); 252Y, 254T and 256E (for example, M252Y, S254T and T256E); 428L and 434S (for example, M428L and N434S); and 433K and 434F (for example, H433K and N434F). All possible combinations of the above mutations in the Fc domains and other mutations in the variable domains of antibodies disclosed herein are considered to be within the scope of the present invention.

Biological characteristics of antibodies

[00140] Generally, the antibodies of the present invention may function by binding to RET and thereby block or prevent RET activation and/or signaling. Antibodies of the present invention may also function by binding to RET and thereby interfere with or prevent the interaction or binding of RET with one or more members of the GDNF family in complex with their respective co-receptors, for example: GDNF/GFRα1, neurturin/GFRα2, artemin/GFRα3 or persefin /GFRα4. Based on the fact that the carcinogenic potential of RET has been established in humans, an antagonistic antibody that specifically binds to RET may have a beneficial effect in inhibiting the growth of tumor cells in patients suffering from a cancerous condition.

[00141] In certain embodiments, antibodies of the present invention may function by blocking or inhibiting the activity of RET upon binding to any region or fragment of a full-length protein having the amino acid sequence set forth in SEQ ID NO: 310 (RET51), also set forth in SEQ ID NO: 310 (RET51). GenBank accession NP_066124.1 and SEQ ID NO: 312 (RET9), also provided under GenBank accession NP_065681.1. Antibodies can also bind to any region that is found in the sequence of SEQ ID NO: 310 or 312, or to a fragment that is found in the sequence of SEQ ID NO: 310 or 312.

[00142] In accordance with one embodiment, the present invention provides a fully human monoclonal antibody or antigen binding fragment thereof that binds to the RET protein, wherein the antibody or fragment thereof has one or more of the following characteristics:

(a) is a fully human antibody;

(b) characterized by a K _D ranging from about 1.0×10 ^-7 M to about 1.0×10 ^-12 M as measured by surface plasmon resonance;

(c) inhibits or blocks the binding or interaction of RET with one or more member ligands of the GDNF family (GDNF, neurturin, artemin and persefin) in complex with their respective co-receptors (GFRα1, GFRα2, GFRα3 and GFRα4, respectively);

(d) inhibits RET signaling mediated by one or more GDNF family member ligands selected from GDNF, neurturin, artemin and persefin;

(e) enhances RET internalization/degradation following antibody binding to the RET receptor;

(f) contains a heavy chain variable region (HCVR) with an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194 , 210, 226, 242, 258, 274 and 290; or

(g) contains a light chain variable region (LCVR) with an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202 , 218, 234, 250, 266, 282 and 298.

[0142] Certain anti-RET antibodies of the present invention are capable of binding to the RET protein and inhibiting RET-related activation and/or signaling. In this case, antibodies can perform their function by inhibiting tumor growth, which depends on the activation of RET signaling for growth. Such RET antagonist antibodies may be used alone to treat a cancerous condition or may be used as an adjuvant therapy with any other anticancer agent, such as a small molecule chemotherapeutic or radiation therapy, or a bone marrow regenerative agent.

[0143] In certain embodiments, anti-RET antibodies may be capable of inhibiting multiple signaling pathways, including the RAS/RAF pathway, resulting in activation of the mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 (Trapp, M. et al., (1999), J Biol. Chem. 274:20885-94; Santoro, M. et al., (1994), Mol. Cell Biol. 14:663-75; :2207-14; Worby, CA, et al., (1996), J Biol Chem, 271:23619-22), phosphatidylinositol 3-kinase (PI3K), resulting in activation of the serine-threonine kinase Akt (Trupp). , M. et al., (1999), J Biol. Chem. 274:20885-94; van Weering, DHJ, (1997), J Biol Chem 272:249-54; 2000), 275:3568-76; Maeda, K. et al, (2004), 323: 345-54).

[0144] Non-limiting exemplary in vitro assays measuring the ability of anti-RET antibodies of the present invention to block RET binding to the GFRal/GDNF complex and in vitro assays measuring the effect of antibodies on RET signaling, activation, or internalization are illustrated in Examples 4 and 5, respectively. B In Example 3, binding affinities and kinetic constants of human anti-RET antibodies were determined using surface plasmon resonance and measurements were performed on a Biacore 4000 or T200 instrument. In Example 4, the ability of antibodies to block RET binding to the GFRα1/GDNF co-complex was tested using a competitive sandwich ELISA assay. Example 5 demonstrates the ability of the antibodies of the present invention to inhibit ligand-dependent RET signaling in a serum response factor (SRE) luciferase reporter assay. More specifically, the data presented in Example 5 show that the anti-RET antibodies of the present invention exhibit a distinct spectrum of inhibitory activity against RET signaling in the presence of glial family ligands, i.e. GDNF and artemina.

Epitope mapping and related techniques

[0145] To determine whether an antibody “interacts with one or more amino acids” in a polypeptide or protein, various techniques known to those of ordinary skill in the art can be used. Examples of techniques that can be used include, for example, standard epitope cross-competition assays such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY). Other methods include alanine scanning mutagenesis analysis, peptide blot analysis (Reineke (2004) Methods Mol Biol 248: 443-63), peptide digestion assay, crystallographic studies and NMR analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be used (Tomer (2000) Protein Science 9: 487-496). Another method that can be used to identify amino acids in a polypeptide with which an antibody reacts is hydrogen/deuterium exchange, determined by mass spectrometry. In general terms, the hydrogen/deuterium exchange method involves labeling a protein of interest with deuterium, followed by binding of the antibody to the deuterium-labeled protein. The protein/antibody complex is then transferred to water, and the exchangeable protons in the amino acids that are protected by the antibody complex undergo back-deuterium-to-hydrogen exchange at a slower rate than the exchangeable protons in amino acids that are not part of the interface . As a result, amino acids that form part of the protein/antibody interface can retain deuterium and thus have a relatively higher mass compared to amino acids not included in the interface. After antibody dissociation, the target protein is subjected to protease digestion and mass spectrometric analysis, thereby identifying deuterium-labeled residues that correspond 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. 75:256A-265A.

[0146] The term “epitope” refers to a site on an antigen to which B and/or T cells respond. B-cell epitopes can be formed from both nonessential amino acids and essential amino acids that come into contact as a result of the formation of the tertiary structure of the protein. Epitopes formed from non-essential amino acids are usually retained by denaturing solvents, while epitopes formed by tertiary structure formation are usually lost when treated with denaturing solvents. An epitope typically comprises at least 3 or, more typically, at least 5 or 8-10 amino acids in a unique spatial conformation.

[0147] Modification-based antibody analysis (MAP), also known as antigen structure-based antibody analysis (ASAP), is a method by which large quantities of monoclonal antibodies (mAbs) to the same antigen are classified according to the similarity of the binding profile of each antibodies with chemically or enzymatically modified antigen surfaces (US 2004/0101920, incorporated herein by reference in its entirety). Each category may reflect a unique epitope, either clearly different from or substantially identical to an epitope present in another category. This technique allows genetically identical antibodies to be quickly filtered out so that characterization efforts can focus on genetically distinct antibodies. When applied to hybridoma screening, MAP can facilitate the identification of rare hybridoma clones that produce mAbs with desired characteristics. MAP can be used to separate the antibodies of the present invention into groups of antibodies that bind different epitopes.

[0148] The present invention provides anti-RET antibodies that bind to the same epitope as any of the specific exemplary antibodies described herein in Table 1. Likewise, the present invention also provides anti-RET antibodies that compete for binding with RET or a fragment thereof with any of the specific exemplary antibodies described herein in Table 1.

[0149] Using standard methods known in the art, it is easy to determine whether an antibody binds to the same epitope as or competes for binding to a reference anti-RET antibody. For example, to determine whether a test antibody binds to the same epitope as a reference anti-RET antibody of the present invention, the reference antibody is allowed to bind to the RET protein or peptide under saturating conditions. The ability of the test antibody to bind to the RET molecule is then assessed. If the test antibody is able to bind to RET after saturating binding to the reference anti-RET antibody, then it can be concluded that the test antibody binds to a different epitope than the reference anti-RET antibody. On the other hand, if the test antibody is unable to bind to the RET molecule after saturating binding to the reference anti-RET antibody, then the test antibody may bind to the same epitope as the epitope bound by the reference anti-RET antibody of the present invention.

[0150] To determine whether an antibody competes for binding with a reference anti-RET antibody, the binding methodology described above is performed in two adaptations. In the first adaptation, the reference antibody is allowed to bind to the RET molecule under saturating conditions, followed by an assessment of the binding of the test antibody to the RET molecule. In a second adaptation, the test antibody is allowed to bind to the RET molecule under saturating conditions, followed by an assessment of the binding of the reference antibody to the RET molecule. If in both adaptations only the first (saturating) antibody is able to bind to the RET molecule, then it is concluded that the test antibody and the reference antibody are competing for RET binding. One of ordinary skill in the art will appreciate that an antibody that competes for binding to a reference antibody may not necessarily bind to an identical epitope as the reference antibody, but may sterically block binding of the reference antibody by binding to a matching or adjacent epitope.

[0151] Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) the other's binding to the antigen. Namely, a 1-, 5-, 10-, 20-, or 100-fold excess of one antibody inhibits the binding of the other by at least 50%, but preferably by 75%, 90%, or even 99%, as measured in a competition assay binding (see, for example, Junghans et at, Cancer Res. 1990 50:1495-1502). Alternatively, two antibodies have the same epitopes if virtually all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies are characterized by overlapping epitopes if multiple amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other.

[0152] Additional routine experimentation (e.g., mutation and peptide binding assays) can then be performed 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 is responsible for the lack of observed binding steric blocking (or other phenomenon). Experiments of this nature can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay that is available in the art.

Immunoconjugates

[0153] The present invention provides a human anti-RET monoclonal antibody conjugated to a therapeutic moiety ("immunoconjugate"), such as an agent that is capable of inhibiting the proliferation of tumor cells or attenuating at least one symptom associated with a RET-associated pathological condition, such like a cancerous condition. Such an agent may be a second non-RET antibody or an antitumor chemotherapeutic agent, or may be a radionuclide that, when targeted to a tumor cell expressing RET, acts to kill the tumor cell. For the type of therapeutic component that can be conjugated to an anti-RET antibody, the pathological condition to be treated and the desired therapeutic effect to be achieved will be taken into account. Alternatively, if the desired therapeutic effect is to treat the effects or symptoms associated with RET expression by certain tissues, or any other pathological condition resulting from RET expression, such as, but not limited to, cancer, it may be advantageous to conjugate an agent that is suitable for the treatment consequences or symptoms of a pathological condition or to alleviate any side effects of the antibodies of the present invention. Examples of suitable means for forming immunoconjugates are known in the art, see, for example, WO 05/103081.

Multispecific antibodies

[0154] Antibodies of the present invention can be monospecific, bispecific or multispecific. Multispecific antibodies may be specific for different epitopes of a single target polypeptide or may contain antigen binding domains specific for multiple target polypeptides. See, for example, Tutt et at, 1991, J. Immunol. 147:60-69; Kufer et at, 2004, Trends Biotechnol. 22:238-244. Antibodies of the present invention may be associated with or co-expressed with another functional molecule, for example, with another peptide or protein. For example, an antibody or fragment thereof may be operably linked (e.g., by chemical binding, genetic fusion, non-covalent binding, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to form a bispecific or multispecific antibody with the second binding specificity.

[0155] An exemplary bispecific antibody format that can be used in the context of the present invention involves 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 in at least one way amino acid, and wherein the at least one amino acid difference reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody without the amino acid difference. In accordance with one embodiment, the first C domain of _{the H3} Ig binds to protein A, and the second domain C of _{the H3} Ig contains a mutation that reduces or eliminates protein A binding, such as the H95R modification (according to IMGT exon numbering; H435R according to EU numbering). The second C _H3 may additionally have the modification Y96F (according to IMGT; Y436F according to EU). Additional modifications that can be found in the second _CH3 include the following: D16E, L18M, N44S, K52N, V57M and V82I (according to IMGT; D356E, L358M, N384S, K392N, V397M and V422I according to EU) in the case of IgG1 antibodies; N44S, K52N and V82I (IMGT; EU N384S, K392N and V422I) for IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (according to IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V422I according to EU) for IgG4 antibodies. The variants of the bispecific antibody format described above are within the scope of the present invention.

Therapeutic Administration and Formulations

[0156] The present invention relates to therapeutic compositions containing antibodies to RET or antigen binding fragments thereof according to the present invention. Administration of therapeutic compositions in accordance with the present invention will be accomplished with suitable carriers, excipients, and other agents that are included in the formulations to provide improved tolerance, delivery, durability, and the like. Many suitable formulations can be found in a reference book well known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. Such formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil emulsions in water" and "water in oil", carbowax emulsions (polyethylene glycols with different molecular weights), semi-liquid gels and semi-liquid mixtures containing carbowax. See. also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0157] The dosage of each of the antibodies of the present invention may vary depending on the age and size of the subject to be administered, the target disease, pathological conditions, route of administration, and the like. When using the antibodies of the present invention to treat a RET-related disease or condition in a patient, or to treat one or more symptoms associated with a condition that is dependent on RET activation or signaling, such as certain RET-expressing tumors in a patient , or to reduce the severity of the disease, it will be advantageous to administer each of the antibodies of the present invention intravenously or subcutaneously, typically in a single dose of about 0.01 to about 30 mg/kg of the subject's body weight, more preferably from about 0.1 to about 20 mg/kg subject's body weight, or about 0.1 to about 15 mg/kg subject's body weight, or about 0.02 to about 7 mg/kg subject's body weight, about 0.03 to about 5 mg/ kg of the subject's body weight, or about 0.05 to about 3 mg/kg of the subject's body weight, or about 1 mg/kg of the subject's body weight, or about 3.0 mg/kg of the subject's body weight, or about 10 mg/kg of the subject's body weight of the subject's body, or approximately 20 mg/kg of the subject's body weight. Multiple doses may be administered if necessary. Depending on the severity of the condition, the frequency of administration and duration of treatment can be adjusted. In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention can be administered at an initial dosage of at least about 0.1 mg to about 800 mg, about 1 to about 600 mg, about 5 to about 300 mg or from about 10 to about 150 mg, up to about 100 mg or up to about 50 mg. In certain embodiments, the initial dose may be followed by a second or more subsequent doses of antibodies or antigen-binding fragments thereof in an amount that may be about the same or less than the initial dose, with subsequent doses separated by at least 1-3 days, at least one 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.

[0158] Various delivery systems are known and can be used to administer the pharmaceutical composition of the present invention, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, for example, Wu et al . (1987) J. Biol. Chem. 262:4429-4432). Routes of administration include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered in any convenient way, for example, by infusion or bolus injection, absorption through epithelial or mucocutaneous integuments (for example, oral mucosa, nasal mucosa, rectal and intestinal mucosa, etc.), and it can be administered together with other biologically active means. Administration may be systemic or local. Delivery can be made as an aerosol formulation (see US2011/0311515 and US2012/0128669). The delivery of agents useful for the treatment of respiratory diseases by inhalation is becoming increasingly common (see A. J. Bitonti and J. A. Dumont, (2006), Adv. Drug Deliv. Rev, 58:1106-1118). In addition to being effective in the treatment of local pulmonary disease, such a delivery mechanism may also be suitable for systemic delivery of antibodies (see Maillet et al. (2008), Pharmaceutical Research, Vol. 25, No. 6, 2008).

[0159] The pharmaceutical composition can also be delivered in a vesicle, particularly a liposome (see, for example, Langer (1990) Science 249:1527-1533).

[0160] In certain situations, the pharmaceutical composition can be delivered in a controlled release system. According to one embodiment, a pump may be used. According to another embodiment, polymeric materials can be used. According to yet another embodiment, the controlled release system can be placed close to the target of the composition, thereby making only a portion of the systemic dose necessary.

[0161] Injectable drugs include dosage forms for intravenous, subcutaneous, intracutaneous, intradermal and intramuscular injections, intravenous drips, etc. Such injectable preparations can be prepared by generally known methods. For example, injectable preparations can be prepared, for example, by dissolving, suspending or emulsifying the above-described antibody or a salt thereof in a sterile aqueous or oily medium commonly used for injection. The aqueous injection medium is, for example, saline solution, isotonic solution containing glucose and other auxiliary agents, etc., which can be used in combination with an appropriate solubilizing agent such as alcohol (for example, ethanol), polyhydric alcohol ( e.g. propylene glycol, polyethylene glycol), nonionic surfactant [e.g. polysorbate 80, HCO-50 (adduct of polyoxyethylene (50 mol) and hydrogenated castor oil)], etc. As the oil medium, for example, sesame oil, soybean oil, etc. are used, which can be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injectable preparation thus obtained is preferably filled into a suitable ampoule.

[0162] The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, with regard to subcutaneous delivery, a pen-type delivery device can be easily used to deliver the pharmaceutical composition of the present invention. This pen-type delivery device can be reusable or disposable. A reusable pen-type delivery device typically uses a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition from the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen-type delivery device can then be reused. In the case of a disposable pen-type delivery device, there is no replaceable cartridge. More specifically, the disposable pen-type delivery device is filled with a pharmaceutical composition held in a reservoir of the device. Once the pharmaceutical composition reservoir is empty, the entire device is discarded.

[0163] For subcutaneous delivery of the pharmaceutical composition of the present invention, a variety of reusable pens and autoinjector delivery devices are used. Examples include, but are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Burgdorf, 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), to name a few. Examples of disposable pen-type delivery devices used in subcutaneous delivery of the pharmaceutical composition of the present invention include, but are not limited to, the SOLOSTAR™ pen (sanofi-aventis), FLEXPEN™ (Novo Nordisk) and KWIKPEN™ (Eli Lilly), auto-injector SURECLICK™ (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.) and HUMIRA™ pen (Abbott Labs, Abbott Park, IL), to name a few .

[0164] Advantageously, the pharmaceutical compositions described above for oral or parenteral administration are prepared in dosage forms in a unit dose adjusted in accordance with the dose of the active ingredients. Such dosage forms in a standard dose include, for example, tablets, pills, capsules, injectables (ampoules), suppositories, etc. The amount of the above-mentioned antibody contained is typically from about 5 to about 500 mg per unit dose dosage form; especially in the form of an injectable preparation, it is preferable that the above-mentioned antibody be contained in an amount of from about 5 to about 100 mg and from about 10 to about 250 mg in the case of other dosage forms.

Administration regimens

[0165] In accordance with certain embodiments of the present invention, multiple doses of an anti-RET antibody may be administered to a subject over a period of time. Methods in accordance with this aspect of the present invention involve sequentially administering multiple doses of an anti-RET antibody to a subject. As used herein, the term “sequential administration” means that each dose of anti-RET antibody is administered to a subject at a separate point in time, for example, on different days separated by a predetermined interval (for example, hours, days, weeks or months). The present invention provides methods that involve sequentially administering to a patient one initial dose of an anti-RET antibody, then one or more secondary doses of an anti-RET antibody, and then optionally one or more tertiary doses of an anti-RET antibody.

[0166] The terms "initial dose", "secondary doses" and "tertiary doses" refer to the time sequence of administration of the anti-RET antibody. Thus, the "starting dose" is the dose that is administered at the beginning of the treatment regimen (also referred to as the "starting dose"); “secondary doses” are doses that are administered after the initial dose; and “tertiary doses” are doses that are administered after secondary doses. Both the initial and secondary and tertiary doses may contain the same amount of anti-RET antibody, but usually may differ from each other regarding the frequency of administration. However, in certain embodiments, the amounts of anti-RET antibody contained in the initial, secondary, and/or tertiary doses are different from each other (eg, adjusted upward or downward as necessary) throughout the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of a treatment regimen as “boost doses,” followed by subsequent doses that are administered less frequently (e.g., “maintenance doses”). doses").

[0167] In accordance with one illustrative embodiment of the present invention, each secondary and/or tertiary dose is administered over 1-26 weeks (e.g., 1. eleven, 21, 21 ^½ , 22, or more) after the immediately preceding dose. As used herein, the phrase “immediately preceding dose” in the case of a multiple administration sequence means the dose of anti-RET antibody that is administered to a patient before the next dose in the sequence without any intervening doses.

[0168] Methods in accordance with this aspect of the present invention may involve administering to a patient any number of secondary or tertiary doses of the anti-RET antibody. For example, in accordance with certain embodiments, only one secondary dose is administered to the patient. In other embodiments, the patient is administered two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) secondary doses. Likewise, in certain embodiments, only one tertiary dose is administered to the patient. In other embodiments, the patient is administered two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) tertiary doses.

[0169] In embodiments that provide multiple secondary doses, each secondary dose may be administered at the same frequency as the remaining secondary doses. For example, each secondary dose can be administered to the patient 1-2 weeks after the immediately preceding dose. Likewise, in embodiments providing multiple tertiary doses, each tertiary dose may be administered at the same frequency as the remaining tertiary doses. For example, each tertiary dose may be administered to the patient 2-4 weeks after the immediately preceding dose. Alternatively, the frequency with which secondary and/or tertiary doses are administered to the patient may vary over the course of the treatment regimen. The physician may adjust the frequency of administration during the course of treatment depending on the needs of the individual patient after clinical evaluation.

Therapeutic uses of antibodies

[0170] Due to their binding/interaction with the RET protein expressed on certain cells and tissues, the present antibodies are useful for preventing the interaction of the RET protein with one or more ligand/coreceptor complexes, for example: GDNF/GFRα1, artemin/GFRα3, neurturin/GFRα2 or persephine/GFRα4. Given the ability of the anti-RET antibodies of the present invention to prevent or inhibit this interaction, the antagonistic antibodies of the present invention may be useful in inhibiting the growth of tumor cells when the tumor cell is dependent on RET signaling for growth, or they may be useful in suppressing pain associated with cancer. condition, as well as pain associated with other diseases or disorders in which RET activation or signaling plays a role. The antibodies of the present invention can be used to inhibit the growth and/or metastasis of tumors in a subject with a tumor expressing RET, or to treat pain associated with a cancerous condition, when administered alone or in combination with another antineoplastic agent or according to another therapeutic regimen or with one or several drugs used to further relieve pain associated with a given pathological condition. Alternatively, the antibodies of the present invention may be useful for alleviating at least one symptom associated with a cancerous condition.

[0171] It is contemplated that the antibodies of the present invention may be used alone or in combination with a second agent or a third agent to treat a disease or condition associated with RET, or to alleviate at least one symptom or complication associated with a disease or condition related to RET. The term “RET-associated disease or condition” means any disease or condition in which RET is known to be expressed in cells or tissues affected by the disease or condition and in which a favorable response to treatment or small molecule therapeutic agent is achieved. , which is known to inhibit RET activation and/or signaling, or which produces a favorable response to treatment with an anti-RET antibody of the present invention. The second or third agent may be delivered simultaneously with the antibodies of the present invention, or they may be administered separately, either before or after the antibodies of the present invention. The second or third agent may be a small organic molecule or biological agent, such as a protein or polypeptide. The second or third agent may be synthetic or natural. The second or third agent may be an antineoplastic agent, such as a chemotherapy drug or radiation therapy, or a bone marrow repair agent, or other agents to reduce fever or pain, another second but different antibody that specifically binds RET, an agent (eg, antibody) that binds to a RET ligand, such as GDNF, neurturin, artemin, or persefin, or that binds to a RET coreceptor, such as GFRα1, GFRα2, GFRα3, or GFRα4, or an siRNA specific for a RET molecule.

[0172] In accordance with another embodiment of the present invention, the present antibodies are used to obtain a pharmaceutical composition for the treatment of patients suffering from a disease or condition associated with RET. In accordance with yet another embodiment of the present invention, the present antibodies are used to prepare a pharmaceutical composition for reducing the proliferation of tumor cells or reducing tumor burden in a patient who has a tumor that is dependent on RET signaling for growth. In accordance with a further embodiment of the present invention, the present antibodies are used as an adjuvant therapy with any other agent useful for treating an RET-related disease or condition, including a chemotherapeutic agent, radiation therapy, a bone marrow regenerative agent, a second antibody to RET, or any other antibody specific for RET antigen, or antibody specific for GDNF or GFRα1, or any other palliative therapy known to those skilled in the art.

[0173] The antibodies of the present invention are useful for treating, preventing and/or alleviating any disease, disorder or condition associated with RET activity, or for alleviating at least one symptom associated with such a disease, disorder or condition, or for relieving pain associated with such disease, disorder or condition. Exemplary pathological conditions, diseases and/or disorders and/or pain that are associated with such pathological conditions, diseases or disorders that can be treated with the anti-RET antibodies of the present invention include acute or chronic pain, including, but not limited to, neuropathic pain pain, inflammatory pain, arthritis, migraine, cluster headaches, trigeminal neuralgia, herpetic neuralgia, common forms of neuralgia, irritable bowel syndrome, inflammatory bowel syndrome, visceral pain, including abdominal pain, osteoarthritis pain, gout, postherpetic neuralgia , diabetic neuropathy, sciatica, sciatica, spinal pain, head or neck pain, breakthrough pain, post-operative pain, bone pain, cancer pain. Other pathological conditions that are amenable to treatment with the antibodies and therapeutic methods of the present invention include forms of thyroid cancer, familial medullary thyroid carcinoma (FMTC) syndrome, sporadic medullary carcinoma (MTC), multiple endocrine neoplasia syndromes MEN2A and MEN2B, cancer prostate, breast cancer, cervical cancer, colon cancer or bladder cancer and pain associated with these pathological conditions. Forms of cancer that are treatable with the antibodies of the present invention may be solid tumors or they may be blood cell tumors such as leukemia. The antibodies of the present invention or antigen-binding fragments thereof can also be used to treat the following pathological conditions: acute, chronic pain due to non-malignant diseases or pain due to bone fractures; rheumatoid arthritis, spinal stenosis; neuropathic pain in the lower spine; myofascial pain syndrome; fibromyalgia; pain in the temporomandibular joint; pain in the pancreas; chronic headache; tension headache; HIV-related neuropathy; Charcot-Marie-Tooth neuropathy; hereditary forms of sensory neuropathy; peripheral nerve damage; various forms of painful neuroma; ectopic proximal and distal discharge; radiculopathy; neuropathic pain caused by chemotherapy; neuropathic pain caused by radiation therapy; pain after mastectomy; central pain; pain due to spinal cord injury; post-stroke pain; thalamic pain; reflex sympathetic dystrophy; phantom pain; unbearable pain; musculoskeletal pain; joint pain; acute padagric pain; mechanical pain in the lower spine; neck pain; tendinitis; pain during injury/physical activity; pyelonephritis; appendicitis; cholecystitis; intestinal obstruction; hernias; pain in the chest, including heart pain; pain in the pelvic area, pain due to renal colic, acute obstetric pain, including pain during childbirth; pain during caesarean section; burn and traumatic pain; endometriosis; pain with herpes zoster; sickle cell anemia; acute pancreatitis; pain in the mouth and face, including pain due to sinusitis, toothache; pain in multiple sclerosis; leprous pain; pain due to Behçet's disease; adiposalgia; phlebitic pain; pain in Guillain-Barre disease; painful legs and pain when moving your toes; Haglund syndrome; pain due to Fabry disease; diseases of the bladder and genitourinary system; overactive bladder; painful bladder syndrome; interstitial cystitis; or prostatitis.

Combination therapy agents

[0174] As noted above, the methods of the present invention, in accordance with certain embodiments, provide for the administration of one or more additional therapeutic agents in combination with an anti-RET antibody to a subject. As used herein, the expression “in combination with” means that additional therapeutic agents are administered before, after, or simultaneously with a pharmaceutical composition containing an anti-RET antibody. The term “in combination with” also includes sequential or parallel administration of an anti-RET antibody and a second therapeutic agent.

[0175] For example, when administered "before" a pharmaceutical composition containing an anti-RET antibody, the additional therapeutic agent may be administered about 72 hours before, about 60 hours before, about 48 hours before, about 36 hours before, about 24 hours before, about 12 hours, approximately 10 hours, approximately 8 hours, approximately 6 hours, approximately 4 hours, approximately 2 hours, approximately 1 hour, approximately 30 minutes, approximately 15 minutes, or approximately 10 minutes before administration of the pharmaceutical a composition containing an anti-RET antibody. When administered "after" a pharmaceutical composition containing an anti-RET antibody, the additional therapeutic agent can be administered after about 10 minutes, after about 15 minutes, after about 30 minutes, after about 1 hour, after about 2 hours, after about 4 hours, after about 6 hours, approximately 8 hours, approximately 10 hours, approximately 12 hours, approximately 24 hours, approximately 36 hours, approximately 48 hours, approximately 60 hours, or approximately 72 hours after administration of a pharmaceutical composition containing antibodies to RET. Administration “simultaneously with” or with a pharmaceutical composition containing an anti-RET antibody means that the additional therapeutic agent is administered to a subject in a separate dosage form within less than 5 minutes (before, after, or simultaneously) with the administration of a pharmaceutical composition containing an anti-RET antibody or is administered to a subject in a single combination dosage form containing both an additional therapeutic agent and an anti-RET antibody.

[0176] Combination therapies may include an anti-RET antibody of the present invention and any additional therapeutic agent that can be advantageously combined with an antibody of the present invention or a biologically active fragment of an antibody of the present invention.

For example, a second or third therapeutic agent may be used to help reduce tumor burden in a subject, such as a chemotherapeutic agent or radiation therapy agent effective in inhibiting the proliferation of tumor cells in the subject. Alternatively, the antibodies may be used as an adjuvant therapy following surgical removal of a tumor and may be used alone or in combination with a chemotherapeutic agent, radiation therapy, or a bone marrow reconstitution agent. The antibodies can also be used in combination with other therapies as described above, including a second antibody specific for RET, or an antibody specific for a RET ligand, or an antibody or fusion molecule that binds GFRα1 (see SEQ ID NO : 308).

Diagnostic applications of antibodies

[0177] The anti-RET antibodies of the present invention can also be used to detect and/or measure RET in a sample, for example, for diagnostic purposes. It is contemplated that confirmation of a disease or pathological condition believed to be associated with RET can be achieved by measuring the presence of RET, for example, in a biopsy sample from a tumor (ie, tumor cells), which is growth dependent on RET signaling. Exemplary RET diagnostic assays may involve, for example, contacting a sample obtained from a patient with an anti-RET antibody of the present invention, wherein the anti-RET antibody is labeled with a detectable label or reporter group, or is used as a capture ligand to selectively isolate a cell, expressing RET protein from patient samples. Alternatively, an untagged anti-RET antibody may be used for diagnostic purposes in combination with a secondary antibody that is itself labeled with a detectable label. The detectable label or reporter group may be a radioisotope such as ³ H, ¹⁴ C, ³² P, ³⁵ S or ¹²⁵ 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 F protein-containing RET in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0178] Samples that can be used in diagnostic assays for RET in accordance with the present invention include any tissue or fluid sample obtained from a patient that contains detectable amounts of either RET protein or fragments thereof under normal or pathological conditions. Typically, to initially determine a baseline or reference level of RET protein, RET levels will be measured in a specific sample obtained from a healthy patient (eg, a patient not affected by a disease or condition associated with the presence of RET). This baseline RET level can then be compared to RET levels measured in samples obtained from individuals suspected of having an RET-related disease or condition or symptoms associated with such conditions.

EXAMPLES

[0179] The following examples are provided to provide those skilled in the art with complete disclosure and description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of the present invention. Attempts have been made to ensure accuracy in terms of numbers used (e.g. amounts, temperatures, etc.), but some experimental errors and deviations must be taken into account. Unless otherwise noted, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

Example 1

Creation of human antibodies to the RET protein

[0180] An immunogen, including any of the following, can be used to generate antibodies to RET. In certain embodiments, the antibodies of the present invention are obtained from mice immunized with a primary immunogen, such as full-length RET protein (see, for example, SEQ ID NO: 310 for the human RET51 isoform, which can also be found under ATCC accession number NP_066124. 1; and SEQ ID NO: 312 in the case of the human isoform RET9, which can also be found under ATCC accession number NP_065681.1, both of which have signal sequences of residues 1-28). Mice can be given one or more booster injections containing the same molecule, or they can be boosted with other fragments, such as the extracellular domain of human RET, which consists of amino acids in the range 1-635 of SEQ ID NO: 313 , which can also be found under ATCC registration number NP_066124.1 with a signal sequence consisting of amino acid residues 1-28). In certain embodiments, mice are administered full-length RET protein followed by boosting with any of the constructs set forth in SEQ ID NOs: 305, 306, 307, and 313, or a recombinantly produced molecule.

[0181] In certain embodiments, the antibodies of the present invention are obtained from mice immunized with a primary immunogen, such as a biologically active RET molecule, or an immunogenic fragment of a RET protein, or DNA encoding a full-length protein or an active fragment thereof. The immunogen can be delivered to an animal by any route, including, but not limited to, intramuscular, subcutaneous, intravenous, or intranasal.

[0182] In certain embodiments, full-length RET protein or fragments thereof can be used to produce monospecific, bispecific, or multispecific antibodies.

[0183] The full-length protein or fragment thereof, which were used as immunogens, as mentioned above, was administered directly with an adjuvant to stimulate an immune response to VELOCIMMUNE ^® mice containing DNA encoding the variable regions of the heavy chain and kappa light chain of human immunoglobulin. The immune response with antibody production was monitored using a RET immunoassay. Once the required immune response was achieved, splenocytes were collected and hybridized with murine myeloma cells to maintain their viability and create hybridoma cell lines. Hybridoma cell lines were screened and selected to identify cell lines that produced RET-specific antibodies. Using this technique and the various immunogens described above, several chimeric antibodies (ie, antibodies having human variable domains and murine constant domains) were produced; certain illustrative antibodies thus obtained are designated, for example, H2M7086N.

[0184] Antibodies to RET were also isolated directly from antigen-positive B cells without hybridization with myeloma cells, as described in U.S. 2007/0280945A1, which is expressly incorporated herein by reference in its entirety. Using this method, several fully human anti-RET antibodies (ie, antibodies having human variable domains and human constant domains) were produced; Example antibodies thus obtained were designated as follows: H4H8044R, H4H8045R, H4H8046R, H4H8048R, H4H8056R, H4H8058R, H4H8060R, H4H8062R, H4H8066R, H4H8067R, H4H8071R, H4H8076R, H 4N8079R, N4N8080R, N4N8083R, N4N8084R, N4N8085R and N4N8087R.

[0185] The biological properties of illustrative antibodies produced in accordance with the methods of this example are described in detail in the examples below.

Example 2

Amino acid sequences of the variable regions of the heavy and light chain

[0186] Table 1 presents pairs of amino acid sequences of the heavy and light chain variable regions of selected RET protein-specific antibodies and their corresponding antibody identifiers. As used herein, antibodies are generally designated according to the following nomenclature: the Fc prefix (e.g., “H4H,” “H1M,” “H2M”), followed by a numeric identifier (e.g., “7086,” as presented in Table 1), with followed by the suffix "P" or "N". Thus, according to this nomenclature, the antibody can be called, for example, “H2M7086N”. The prefixes H4H, H1M and H2M in the antibody designations used herein indicate a specific Fc region of the antibody. For example, antibody "H2M" has a mouse Fc IgG2, while antibody "H4H" has a human Fc IgG4. As will be appreciated by one of ordinary skill in the art, an H1M or H2M antibody can be converted to an H4H antibody and vice versa, but in any case, the variable domains (including CDRs), which are designated by the numeric identifiers shown in Table 1, will remain the same. Antibodies that have the same antibody numeric designator but differ by the letter suffix N, B, or P are those that have heavy and light chains with identical CDR sequences, but with sequence variations in regions that are outside the CDR sequences (i.e. in frame areas). Thus, the N, B and P variants of a particular antibody have identical CDR sequences in the heavy and light chain variable regions, but differ from each other in their framework regions.

Example 3

Surface Plasma Resonance Binding Affinities and Kinetic Constants for Human Anti-RET Monoclonal Antibodies

[0187] Binding affinities and kinetic constants for human anti-RET antibodies were determined using surface plasmon resonance (Biacore T200) at 25°C and 37°C (Tables 2-3). Antibodies expressed as human Fc IgG4 (i.e., "H4H" designations) were immobilized onto the surface of the anti-human Fc sensor (mAb immobilized format) and a soluble monomer (hRET.mmh; SEQ ID NO) was introduced onto the sensor surface : 305, RET.mmh from Macaca fascicularis (mf); SEQ ID NO: 306) or dimeric (hRET.mFc; SEQ ID NO: 307) RET protein. Equilibrium binding dissociation constants (K _D ) and half-dissociation periods (t _1/2 ) were calculated from the reaction rate constants as follows: K _D [M]=k _d /k _a ; and t _1/2 (min.)=(ln2/(60*k _d ). Calculations were performed using a trial release of the BiacoreT200 v1.0 program.

[0188] Some antibodies of the present invention had sub-nanomolar affinity values for human and simian RET protein (Tables 2-3).

Example 4

Antibodies to RET effectively block binding of human RET to the GFRα1/GDNF cooperative complex

[0189] The ability of anti-RET antibodies to block the binding of human RET to the preformed complex on the GDNF:GFRα1 plate was assessed using a competitive sandwich ELISA. Most anti-RET antibodies effectively blocked the binding of RET to the plate-bound GDNF/GFRα1 co-complex (Table 4). IC ₅₀ values ranged from 5.2 nM to levels that were below the theoretical low end of the assay (250 pM), with maximum blocking ranging from 72-96%.

Detailed description of methods

[0190] Recombinant human dimeric GDNF (R&D systems) and human GFRα1.mFc (SEQ ID: 308) were mixed in a 1:1 molar ratio in PBS to produce a final co-complex at a concentration of ~2.0 μg/mL. The GDNF-GFRα1 co-complex was incubated for 1 hour at room temperature (RT) before coating into 96-well microtiter plates overnight at 4°C. Nonspecific binding sites were blocked using BSA.

[0191] Separately, 1 nM biotinylated monomeric RET protein (biot-hRET.mmh; SEQ ID: 305) was titrated with various amounts of serially diluted antibodies in the range of 0-120 nM. Antibody-RET mixtures were incubated for 1 hour at RT and then transferred to microtiter plates precoated with hGDNF/hGFRα1 co-complex. Allow binding to occur for 1 hour at room temperature, followed by extensive washing. Plate-bound biot-hRET.mmh was detected using HRP-conjugated streptavidin and developed using TMB. The plates were read at 450 nm and a sigmoidal dose-response model in Prism™ software was used for data analysis.

[0192] As a measure of blocking efficiency, IC ₅₀ values were used, calculated as the concentration of antibody required to block 50% of hRET binding to hGDNF/hGFRα1. The maximum blocking values represent the ability of anti-RET antibodies to block hRET binding relative to baseline. Baseline values were calculated as uptake measured with a constant amount of hRET in the dose curve (0% blocking) and uptake measured without adding hRET (100% blocking). The absorbance values of the wells containing the highest concentration for each antibody were used to determine the percentage blocking at the highest antibody concentration tested. A summary of IC ₅₀ and maximum blocking percentage is shown in Table 4.

Example 5

Antibodies to RET inhibit ligand-dependent RET signaling in the SRE-luciferase assay and are highly internalized

[0193] In this example, the effects of anti-RET antibodies on RET signaling and internalization were studied using engineered MCF7 and hRET reporter cell lines.

[0194] The glial family ligands GDNF and artemin trigger RET activation by forming a high-affinity co-complex with GFRα1 or 3, respectively, bringing two RET molecules together and initiating phosphorylation of specific tyrosine residues. RET transphosphorylation activates several downstream intracellular cascades, and up-regulation of RET signaling has been implicated in the pathologies of several diseases, including cancer (Borrello, MG, et al., (2013), Expert. Opin. Ther. Targets 17(4): 403 -419).

[0195] To test the ability of anti-RET antibodies to block GDNF-mediated signaling, the human breast adenocarcinoma cell line MCF7 expressing RET and GFRα1 was modified by transduction with a serum response factor (SRE)-regulated luciferase reporter gene to generate the MCF7/SRE line -Luc. The antibodies of the present invention were effective in inhibiting GDNF-stimulated RET signaling with IC ₅₀ values ranging from 143 pM to >100 nM (Table 5). The percentage of inhibition varied in the range of 60-100%. Several non-blocking antibodies have also been identified; H4H8085P stimulated luciferase activity to 50% of the levels observed for GDNF, whereas H4H8044P, H4H8076P, and H4H8046P were weaker activators of the luciferase response (2-5% activation).

[0196] To determine whether antibodies that blocked GDNF-mediated RET signaling would also be effective against artemin-triggered activity, engineered HEK293/hGFRα3/hRET SRELuc cell lines were 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 found to be a more potent blocker of artemin-dependent signaling compared to GDNF-dependent signaling, possibly reflecting different epitopes bound by the GDNF-GFRα1 and artemin-GFRα3 cooperative complexes on the RET receptor .

[0197] Finally, to understand whether the observed blocking activity could be due to disruption of the RET receptor by antibody binding, several antibodies were tested in an internalization assay (Table 6). Among the seven antibodies tested, H4H8087P was identified as the most potent internalizer, with H4H8079P and H4H7086P also exhibiting strong internalization.

[0198] In conclusion, this example illustrates that the anti-RET antibodies of the present invention have a distinct spectrum of activating and inhibitory activities on RET signaling in the presence of glial family ligands, i.e. GDNF and artemina.

Detailed description of methods

Creation of stable cell lines with MCFJ/SRE-luciferase

[0199] MCF7 cells naturally express RET and GFRα1. To generate MCF7/SRELuc cells, a stably integrated SRE luciferase was used, obtained by transducing MCF7 using the Cignal Lenti SRE Reporter kit (SABiosciences) and 2 weeks of selection in puromycin. The lentivirus expresses the firefly luciferase gene under the control of the CMV minimal promoter and tandem repeats of serum response elements (SREs).

Generation of stable cell lines with Hek293/hGFRa1 (or 3)/hRET/SRE-luciferase

[0200] Human GFRα (1 or 3) and hRET were introduced on a stable basis into HEK293 cells through successive rounds of Lipofectamine 2000-mediated transfection and selected for at least two weeks in 500 μg/ml G418 (hGFRa1 or 3) and 100 μg /ml hygromycin B (hRET). Dual-factor modified stable HEK293 lines expressing hGFRa1/hRET or hGFRa3/hRET were then transduced using the Cignal Lenti SRE Reporter kit as described above to generate the HEK293/hGFRal/hRET/SRE-Luc cell line and the artemin-sensitive cell line HEK293/GFRa3/hRET/SRE-Luc.

Inhibition of GDNF-stimulated luciferase activity in MCF7/SRE-luciferase engineered cell lines

[0201] Twenty thousand MCF7-SRE-luc cells were seeded in 96-well PDL-coated plates in Optimem + 0.5% FBS and grown overnight at 37°C in an atmosphere of 5% CO ₂ . To construct inhibition curves, cells were incubated for 1 hour with serially diluted anti-hRET mAbs ranging in concentration from 1.6 pM to 1 μM. A constant dose of human GDNF (4-10 pM) was then added and the cells were incubated for an additional 6 hours.

[0202] To evaluate the activating properties of the anti-RET mAb, MCF7-SRE-Luc cells were incubated for 6 hours with serially diluted anti-hRET mAbs ranging from 1.6 pM to 1 µM under ligand-free conditions.

[0203] GDNF dose response curves were generated using serially diluted GDNF in the concentration range from 0.05 pM to 10 nM, added to wells without antibodies and incubated for 6 hours at 37°C. Luciferase activity was measured using ONE GLO™ reagent (Promega), and relative light units (RLU) were measured using a Victor Luminometer (Perkin Elmer).

Inhibition of artemin-stimulated luciferase activity in HEK293/hGFRa3/hRET engineered cell lines

[0204] Inhibition of artemin-stimulated luciferase activity in HEK293/hGFRa3/hRET/SRE-Luc cell lines was assessed using anti-RET antibodies using the method described for MCF7/SRE-Luc cells. To obtain inhibition curves, cells were incubated for 1 hour with serially diluted anti-hRET antibodies at concentrations ranging from 1.6 pM to 1 μM. Cells were then stimulated with a constant dose of human artemin (hArtemin) (100 μM) for 6 hours. Artemin dose response curves were generated by adding serially diluted hArtemin (0.17 pM to 10 nM) to the cells for 6 hours at 37°C without adding antibody. Luciferase activity measurements and curve fitting were performed as described for GDNF-stimulated luciferase activity.

Calculation of EC ₅₀ /IC ₅₀ values

[0205] EC ₅₀ /IC ₅₀ values were determined from a four-parameter logistic equation using a 12-point response curve using GraphPad Prism. Percent blocking is presented for the highest dose of antibody, and data are presented as mean ± standard deviation (SD).

Quantitative Analysis of Internalization Properties of Anti-RET Antibodies [0206] To test the internalization properties of hRET mAbs, HEK293/hGFRa1/hRET/SRE-Luc cells were incubated with antibodies (10 μg/ml) for 30 minutes on ice, followed by one wash. Cells were then incubated with alexa488 dye-conjugated secondary antibodies with anti-hFc Fab for 30 minutes, followed by a second wash. Antibodies were allowed to internalize for 4 hours at 37°C or left at 4°C to prevent internalization. Cells were fixed in 4% formaldehyde, and alexa488 dye on the cell surface was blocked by incubation with alexa488 blocking antibody for 1 hour at 4°C. Nuclei were stained with Hoechst dye and imaged on a knageXpress micro XL (Molecular Devices).

[0207] The total intensity of alexa488 dye in intracellular vesicles at 37°C in blocked samples was quantified using Columbus image analysis software (Perkin Elmer). The total intensity of internalized mAbs was expressed as a percentage of the most potent internalized mAb.

Example 6

Creation of a bispecific antibody

[0208] Various bispecific antibodies have been created for use in the methods of the present invention. For example, RET-specific antibodies have been produced in a bispecific format (“bispecific molecule”), in which the variable regions that bind to different domains of the RET protein are linked together to confer specificity for two domains in a single binding molecule. Appropriately designed bispecific molecules can enhance overall RET neutralizing efficacy by increasing both binding specificity and avidity. Variable regions with specificity for individual domains are paired on a structural framework, which allows each region to simultaneously bind to individual epitopes or to different regions in the same domain. In one example of a bispecific molecule, heavy chain variable regions ( _VH ) from a binding molecule with a single domain specificity were recombined with light chain variable regions ( _VL ) from a number of binding molecules with a second domain specificity to identify unrelated _VL partners that could form pairs with the original _VH without violating the original specificity of such _VH . Thus, a single V _L segment (for example, V _L 1) could be combined in combination with two different V _H domains (for example, V _H 1 and V _H 2) to produce a bispecific molecule consisting of two binding arms. (V _H 1-V _L 1 and V _H 2-V _L 1). The use of a separate V _L segment reduced the complexity of the system and thus simplified and increased the efficiency of the cloning, expression and purification methods used to obtain the bispecific molecule (see, for example, USSN13/022759 and US2010/0331527).

[0209] Alternatively, antibodies that bind to RET and a second target, such as, but not limited to, a tumor antigen, can be produced in a bispecific molecule format using the techniques described herein or other techniques known to those skilled in the art. Antibody variable regions that bind to different sites can be coupled to variable regions that bind to corresponding sites, for example, on another antigen, to provide specificity for two antigens in one binding molecule. Accordingly designed bispecific molecules of this nature performed a dual function. For example, in the case of a bispecific antibody that bound, for example, RET and one of its ligands, it was possible to better inhibit the growth of tumor cells without the need for a composition containing two separate antibodies. Variable regions with specificity for RET were combined with a variable region with specificity for one of its ligands and paired on a structural framework, allowing each variable region to bind to separate antigens.

[0210] Bispecific binding molecules are tested for binding and functional blocking of a target antigen, eg, RET, in any of the assays described above for antibodies. 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, have been used to evaluate bispecific interactions. The binding of bispecific antibodies to both RET and one of its ligands was determined using an ELISA binding assay in which synthetic peptides representing various antigens were coated into the wells of microtiter plates and binding of the bispecific molecule was determined using a secondary detection antibody. Binding experiments could also be performed using surface plasmon resonance experiments, in which the binding interaction of a peptide with an antibody is measured in real time by the passage of the peptide or bispecific molecule across a sensor surface on which the bispecific molecule or peptide, respectively, is immobilized. In vitro functional blocking of both RET and one of its ligands by a bispecific molecule was determined using any bioassay, such as the assays described herein, or through in vivo protection studies in appropriate animal models, such as animal models with tumors.

Claims (70)

1. An isolated human monoclonal antibody or antigen-binding fragment thereof that specifically binds to a receptor tyrosine kinase RET (REarranged during Transfection), wherein the antibody or antigen-binding fragment contains a heavy chain variable region (HCVR) containing three heavy chain CDRs (HCDR1 , HCDR2 and HCDR3), and a light chain variable region (LCVR) comprising three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained in the amino acid sequence pair HCVR/LCVR 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. 2. An isolated human monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is a full-length human antibody. 3. An isolated human monoclonal antibody or its antigen-binding fragment according to claim 1, containing: (a) HCDR1 with amino acid sequence SEQ ID NO:4, HCDR2 with amino acid sequence SEQ ID NO:6, HCDR3 with amino acid sequence SEQ ID NO:8, LCDR1 with amino acid sequence SEQ ID NO:12, LCDR2 with amino acid sequence SEQ ID NO :14, and LCDR3 with amino acid sequence SEQ ID NO:16; or (b) HCDR1 having the amino acid sequence SEQ ID NO:20, HCDR2 having the amino acid sequence SEQ ID NO:22, HCDR3 having the amino acid sequence SEQ ID NO:24, LCDR1 having the amino acid sequence SEQ ID NO:28, LCDR2 having the amino acid sequence SEQ ID NO: :30 and LCDR3 with amino acid sequence SEQ ID NO:32; or (c) HCDR1 having the amino acid sequence SEQ ID NO:36, HCDR2 having the amino acid sequence SEQ ID NO:38, HCDR3 having the amino acid sequence SEQ ID NO:40, LCDR1 having the amino acid sequence SEQ ID NO:44, LCDR2 having the amino acid sequence SEQ ID NO :46 and LCDR3 with amino acid sequence SEQ ID NO:48; or (d) HCDR1 having the amino acid sequence SEQ ID NO:52, HCDR2 having the amino acid sequence SEQ ID NO:54, HCDR3 having the amino acid sequence SEQ ID NO:56, LCDR1 having the amino acid sequence SEQ ID NO:60, LCDR2 having the amino acid sequence SEQ ID NO: :62 and LCDR3 with amino acid sequence SEQ ID NO:64; or (e) HCDR1 having the amino acid sequence SEQ ID NO:68, HCDR2 having the amino acid sequence SEQ ID NO:70, HCDR3 having the amino acid sequence SEQ ID NO:72, LCDR1 having the amino acid sequence SEQ ID NO:76, LCDR2 having the amino acid sequence SEQ ID NO :78 and LCDR3 with amino acid sequence SEQ ID NO:80; or (f) HCDR1 having the amino acid sequence SEQ ID NO:84, HCDR2 having the amino acid sequence SEQ ID NO:86, HCDR3 having the amino acid sequence SEQ ID NO:88, LCDR1 having the amino acid sequence SEQ ID NO:92, LCDR2 having the amino acid sequence SEQ ID NO :94 and LCDR3 with amino acid sequence SEQ ID NO:96; or (g) HCDR1 having the amino acid sequence SEQ ID NO:100, HCDR2 having the amino acid sequence SEQ ID NO:102, HCDR3 having the amino acid sequence SEQ ID NO:104, LCDR1 having the amino acid sequence SEQ ID NO:108, LCDR2 having the amino acid sequence SEQ ID NO: :110 and LCDR3 with amino acid sequence SEQ ID NO:112; or (h) HCDR1 having the amino acid sequence SEQ ID NO:116, HCDR2 having the amino acid sequence SEQ ID NO:118, HCDR3 having the amino acid sequence SEQ ID NO:120, LCDR1 having the amino acid sequence SEQ ID NO:124, LCDR2 having the amino acid sequence SEQ ID NO: :126 and LCDR3 with amino acid sequence SEQ ID NO:128; or (i) HCDR1 having the amino acid sequence SEQ ID NO:132, HCDR2 having the amino acid sequence SEQ ID NO:134, HCDR3 having the amino acid sequence SEQ ID NO:136, LCDR1 having the amino acid sequence SEQ ID NO:140, LCDR2 having the amino acid sequence SEQ ID NO: :142 and LCDR3 with amino acid sequence SEQ ID NO:144; or (j) HCDR1 having the amino acid sequence SEQ ID NO:148, HCDR2 having the amino acid sequence SEQ ID NO:150, HCDR3 having the amino acid sequence SEQ ID NO:152, LCDR1 having the amino acid sequence SEQ ID NO:156, LCDR2 having the amino acid sequence SEQ ID NO: :158 and LCDR3 with amino acid sequence SEQ ID NO:160; or (k) HCDR1 with the amino acid sequence SEQ ID NO:164, HCDR2 with the amino acid sequence SEQ ID NO:166, HCDR3 with the amino acid sequence SEQ ID NO:168, LCDR1 with the amino acid sequence SEQ ID NO:172, LCDR2 with the amino acid sequence SEQ ID NO :174 and LCDR3 with amino acid sequence SEQ ID NO:176; or (l) HCDR1 having the amino acid sequence SEQ ID NO:180, HCDR2 having the amino acid sequence SEQ ID NO:182, HCDR3 having the amino acid sequence SEQ ID NO:184, LCDR1 having the amino acid sequence SEQ ID NO:188, LCDR2 having the amino acid sequence SEQ ID NO: :190 and LCDR3 with the amino acid sequence SEQ ID NO:192; or (m) HCDR1 having the amino acid sequence SEQ ID NO:196, HCDR2 having the amino acid sequence SEQ ID NO:198, HCDR3 having the amino acid sequence SEQ ID NO:200, LCDR1 having the amino acid sequence SEQ ID NO:204, LCDR2 having the amino acid sequence SEQ ID NO :206 and LCDR3 with the amino acid sequence SEQ ID NO:208; or (n) HCDR1, with the amino acid sequence SEQ ID NO:212, HCDR2 with the amino acid sequence SEQ ID NO:214, HCDR3 with the amino acid sequence SEQ ID NO:216, LCDR1 with the amino acid sequence SEQ ID NO:220, LCDR2 with the amino acid sequence SEQ ID NO:222, and LCDR3 with amino acid sequence SEQ ID NO:224; or (o) HCDR1 with the amino acid sequence SEQ ID NO:228, HCDR2 with the amino acid sequence SEQ ID NO:230, HCDR3 with the amino acid sequence SEQ ID NO:232, LCDR1 with the amino acid sequence SEQ ID NO:236, LCDR2 with the amino acid sequence SEQ ID NO :238 and LCDR3 with amino acid sequence SEQ ID NO:240; or (p) HCDR1 with amino acid sequence SEQ ID NO:244, HCDR2 with amino acid sequence SEQ ID NO:246, HCDR3 with amino acid sequence SEQ ID NO:248, LCDR1 with amino acid sequence SEQ ID NO:252, LCDR2 with amino acid sequence SEQ ID NO :254, and LCDR3 with amino acid sequence SEQ ID NO:256; or (q) HCDR1 with amino acid sequence SEQ ID NO:260, HCDR2 with amino acid sequence SEQ ID NO:262, HCDR3 with amino acid sequence SEQ ID NO:264, LCDR1 with amino acid sequence SEQ ID NO:268, LCDR2 with amino acid sequence SEQ ID NO :270 and LCDR3 with amino acid sequence SEQ ID NO:272; or (r) HCDR1 with the amino acid sequence SEQ ID NO:276, HCDR2 with the amino acid sequence SEQ ID NO:278, HCDR3 with the amino acid sequence SEQ ID NO:280, LCDR1 with the amino acid sequence SEQ ID NO:284, LCDR2 with the amino acid sequence SEQ ID NO :286 and LCDR3 with amino acid sequence SEQ ID NO:288; or (s) HCDR1 having the amino acid sequence SEQ ID NO:292, HCDR2 having the amino acid sequence SEQ ID NO:294, HCDR3 having the amino acid sequence SEQ ID NO:296, LCDR1 having the amino acid sequence SEQ ID NO:300, LCDR2 having the amino acid sequence SEQ ID NO: :302, and LCDR3 with amino acid sequence SEQ ID NO:304. 4. The isolated human monoclonal antibody or antigen-binding fragment thereof according to claim 3, where HCDR1 contains the amino acid sequence of SEQ ID NO: 212, HCDR2 contains the amino acid sequence of SEQ ID NO: 214, HCDR3 contains the amino acid sequence of SEQ ID NO: 216, LCDR1 contains the amino acid sequence SEQ ID NO: 220, LCDR2 contains the amino acid sequence of SEQ ID NO: 222, and LCDR3 contains the amino acid sequence of SEQ ID NO: 224. 5. An isolated human monoclonal antibody or an antigen-binding fragment thereof according to claim 3, where HCDR1 contains the amino acid sequence of SEQ ID NO: 36, HCDR2 contains the amino acid sequence of SEQ ID NO: 38, HCDR3 contains the amino acid sequence of SEQ ID NO: 40, LCDR1 contains the amino acid sequence SEQ ID NO: 44, LCDR2 contains the amino acid sequence of SEQ ID NO: 46, and LCDR3 contains the amino acid sequence of SEQ ID NO: 48. 6. The isolated human monoclonal antibody or antigen-binding fragment thereof according to claim 3, where HCDR1 contains the amino acid sequence of SEQ ID NO: 148, HCDR2 contains the amino acid sequence of SEQ ID NO: 150, HCDR3 contains the amino acid sequence of SEQ ID NO: 152, LCDR1 contains the amino acid sequence SEQ ID NO: 156, LCDR2 contains the amino acid sequence of SEQ ID NO: 158, and LCDR3 contains the amino acid sequence of SEQ ID NO: 160. 7. An isolated human monoclonal antibody or an antigen-binding fragment thereof according to claim 3, containing: (a) HCVR having the amino acid sequence SEQ ID NO:2, and LCVR having the amino acid sequence SEQ ID NO:10; or (b) HCVR having the amino acid sequence of SEQ ID NO:18, and LCVR having the amino acid sequence of SEQ ID NO:26; or (c) HCVR having the amino acid sequence of SEQ ID NO:34, and LCVR having the amino acid sequence of SEQ ID NO:42; or (d) HCVR having the amino acid sequence of SEQ ID NO:50, and LCVR having the amino acid sequence of SEQ ID NO:58; or (e) HCVR having the amino acid sequence SEQ ID NO:66, and LCVR having the amino acid sequence SEQ ID NO:74; or (f) HCVR having the amino acid sequence SEQ ID NO:82, and LCVR having the amino acid sequence SEQ ID NO:90; or (g) HCVR having the amino acid sequence SEQ ID NO:98, and LCVR having the amino acid sequence SEQ ID NO:106; or (h) HCVR having the amino acid sequence of SEQ ID NO:114, and LCVR having the amino acid sequence of SEQ ID NO:122; or (i) HCVR having the amino acid sequence of SEQ ID NO:130, and LCVR having the amino acid sequence of SEQ ID NO:138; or (j) HCVR having the amino acid sequence of SEQ ID NO:146, and LCVR having the amino acid sequence of SEQ ID NO:154; or (k) HCVR having the amino acid sequence of SEQ ID NO:162, and LCVR having the amino acid sequence of SEQ ID NO:170; or (l) HCVR having the amino acid sequence of SEQ ID NO:178, and LCVR having the amino acid sequence of SEQ ID NO:186; or (m) HCVR having the amino acid sequence of SEQ ID NO:194, and LCVR having the amino acid sequence of SEQ ID NO:202; or (n) HCVR having the amino acid sequence of SEQ ID NO:210, and LCVR having the amino acid sequence of SEQ ID NO:218; or (o) HCVR having the amino acid sequence of SEQ ID NO:226, and LCVR having the amino acid sequence of SEQ ID NO:234; or (p) HCVR having the amino acid sequence of SEQ ID NO:242, and LCVR having the amino acid sequence of SEQ ID NO:250; or (q) HCVR having the amino acid sequence of SEQ ID NO:258, and LCVR having the amino acid sequence of SEQ ID NO:266; or (r) HCVR having the amino acid sequence of SEQ ID NO:274, and LCVR having the amino acid sequence of SEQ ID NO:282; or (s) HCVR having the amino acid sequence of SEQ ID NO:290, and LCVR having the amino acid sequence of SEQ ID NO:298. 8. The isolated human monoclonal antibody or antigen binding fragment thereof according to claim 7, wherein the antibody or antigen binding fragment thereof comprises an HCVR having the amino acid sequence of SEQ ID NO: 210, and an LCVR having the amino acid sequence of SEQ ID NO: 218. 9. The isolated human monoclonal antibody or antigen binding fragment thereof according to claim 7, wherein the antibody or antigen binding fragment thereof comprises an HCVR having the amino acid sequence of SEQ ID NO: 34, and an LCVR having the amino acid sequence of SEQ ID NO: 42. 10. The isolated human monoclonal antibody or antigen binding fragment thereof according to claim 7, wherein the antibody or antigen binding fragment thereof comprises an HCVR having the amino acid sequence of SEQ ID NO: 146, and an LCVR having the amino acid sequence of SEQ ID NO: 154. 11. An isolated nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof according to any one of claims. 1-10. 12. An expression vector containing a nucleic acid molecule according to claim 11. 13. The use of a pharmaceutical composition containing one or more antibodies or antigen-binding fragments thereof according to any one of paragraphs. 1-10 and a pharmaceutically acceptable carrier or diluent in the treatment of a disorder or condition associated with the expression, activation or signaling of the receptor tyrosine kinase gene RET or a rearranged form thereof, or pain associated with the disorder or condition, comprising administering the composition to a patient in need thereof . 14. Use according to claim 13, wherein the disorder or pathological condition associated with the expression, activation or signaling of the RET receptor tyrosine kinase gene or a rearranged form thereof is cancer, and wherein the cancer is selected from the group consisting of thyroid cancer, lung cancer , pancreatic cancer, skin cancer, breast cancer and blood cancer. 15. Use according to claim 13, wherein the disorder or pathological condition associated with the expression, activation or signaling of the RET receptor tyrosine kinase gene or a rearranged form thereof is selected from the group consisting of acute pain, chronic pain, neuropathic pain, inflammatory pain, arthritis , osteoarthritis, migraine, cluster headaches, trigeminal neuralgia, herpetic neuralgia, common forms of neuralgia, neurodegenerative disorders, neuroendocrine disorders, visceral pain, acute gout, postherpetic neuralgia, diabetic neuropathy, lumbosacral sciatica, spinal pain, headache or neck, severe or unrelieved pain, breakthrough pain, post-operative pain, toothache, rhinitis, cancer pain, or bladder-related problems. 16. The use of a pharmaceutical composition containing one or more antibodies or antigen-binding fragments thereof according to any one of paragraphs. 1-10 and a pharmaceutically acceptable carrier or diluent in inhibiting tumor growth or tumor cell proliferation, wherein the tumor or tumor cell expresses RET or a rearranged form thereof, wherein the composition is administered to a patient in need thereof. 17. Use according to claim 16, wherein the tumor is a solid tumor or a blood cell tumor. 18. Use according to claim 17, 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. 19. Use according to claim 18, wherein the thyroid tumor is papillary thyroid carcinoma (PTC) or medullary thyroid carcinoma (MTC). 20. The use of claim 19, wherein medullary thyroid carcinoma is a hereditary form of MTC selected from the group consisting of multiple endocrine neoplasia type 2 or 3 (MEN2A, MEN2B) and familial medullary thyroid carcinoma syndrome (FMTC) ), or wherein medullary thyroid carcinoma is a sporadic form of MTC. 21. Use according to claim 18, wherein the lung tumor is a lung adenocarcinoma. 22. Use according to claim 18, wherein the lung tumor is non-small cell lung cancer (NSCLC). 23. Use according to claim 18, wherein the skin tumor is melanoma. 24. Use according to claim 23, wherein the blood cell tumor is leukemia. 25. Use according to claim 24, wherein the leukemia is chronic myelomonocytic leukemia. 26. The use of a pharmaceutical composition containing one or more antibodies or antigen-binding fragments thereof according to any one of paragraphs. 1-10 and a pharmaceutically acceptable carrier or diluent in suppressing the expression and/or function of RET, providing for the administration of the composition to a patient in need thereof. 27. Use according to claim 26, wherein inhibition of RET expression and/or function results in dysregulation of a downstream signaling pathway selected from the group consisting of the RAS/RAF pathway and the PI3K pathway. 28. Application according to any one of paragraphs. 13-27, wherein the antibody or antigen binding fragment is administered to a patient in combination with a second therapeutic agent. 29. The use of claim 28, wherein the second therapeutic agent is selected from the group consisting of a small molecule tyrosine kinase inhibitor, an antitumor agent, an RET-specific siRNA, a second RET-specific antibody, and an anti-pain agent. 30. Use according to claim 29, wherein the small molecule tyrosine kinase inhibitor is selected from the group consisting of vandetanib, cediranib (AZD2171), gefitinib, erlotinib, SU14813, vatalanib, 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, BAY57-93 52, l- 21649, BMS582664, XL-880, XL-184, XL-820, RPI-1, PP-1 and NVP-AST478. 31. Use according to claim 29, wherein the antitumor agent is selected from the group consisting of a chemotherapeutic agent, a radionuclide and an antibody-drug conjugate. 32. Use according to claim 29, wherein the pain-relieving agent is selected from the group consisting of a nerve growth factor (NGF) inhibitor (e.g., a small molecule NGF antagonist or an anti-NGF antibody), aspirin or other NSAID, morphine, steroids (e.g., prednisone) , antibodies to Na _v 1.7 or a small molecule inhibitor of Na _v 1.7, an antagonist of Na _v 1.8 (for example, an antibody to Na _v 1.8 or a small molecule inhibitor of Na _v 1.8), an antagonist of Na _v 1.9 (for example, an antibody to Na _v 1.9 or a small molecule inhibitor of Na _v 1.9), a cytokine inhibitor (eg, an interleukin-1 (IL-1) inhibitor (such as rilonacept (IL-1 decoy); Regeneron) or anakinra (KINERET®, Amgen), a small molecule IL-1 antagonist, or an anti-IL antibody -1; an IL-18 inhibitor (such as a small molecule IL-18 antagonist or an anti-IL-18 antibody); an IL-6 or IL-6R inhibitor (such as a small molecule IL-6 antagonist, an anti-IL-6 antibody, or an anti-IL receptor antibody); -6), caspase-1, p38, IKK1/2, CTLA-4Ig or opioid inhibitors.