Anti-RET antibodies as cancer therapeutics and diagnostic tools

Abstract

The invention described herein provides antibodies or antigen-binding fragments thereof, or polypeptides comprising the same (such as CAR in CAR-T cells), which can be useful for, for example, in treating certain cancer that expresses / over-expresses RET proto-oncogene, for treating certain cancer that expresses / over-expresses RET proto-oncogene, such as NB, AML, small cell lung cancer, Ewing sarcoma, breast cancer, and other cancer types with RET expression. The antibodies or antigen-binding fragments thereof of the invention are also useful for detection of RET expression / overexpression, and related diagnostic use.

Description

[0001] ANTI-RET ANTIBODIES AS CANCER THERAPEUTICS AND DIAGNOSTIC TOOLS

[0002] REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 556934, filed on February 23, 2024, the entire content of which is incorporated herein by reference.

[0004] BACKGROUND OF THE INVENTION

[0005] The RET proto-oncogene is a receptor tyrosine kinase (RTK) which functions as a transmembrane glycoprotein. The RET gene contains three functional domains: an extracellular ligand-binding domain, a transmembrane domain, and an intracellular tyrosine kinase domain.

[0006] RET receptor tyrosine kinase signaling in development is linked to cancers. The RET (Ret Proto-Oncogene) proto-oncogene encodes a single-pass transmembrane receptor tyrosine kinase. During development, RET promotes proliferation and migration of neural crest-derived cells which is essential for the formation of the enteric nervous system and kidney.

[0007] Physiologically, the activation of RET is triggered by Glial Cell-line Derived Neurotrophic Factor (GDNF) ligands (GFLs) that bind to their co-receptors, the GDNF Family Receptors a (GFRal-4). The GFL and GFRa association leads to RET dimerization to form a GFL(2)-GFRa(2)-RET(2) heterohexamer complex which induces the tyrosine phosphorylation of intracellular kinase domain, followed by recruitment of adaptor proteins to activate multiple downstream signaling pathways, including RAF / MEK / ERK and PI3K / AKT / mT0R.

[0008] However, the developmental function of RET in cell proliferation and migration can be “hijacked” by cancer cells. Usually, the oncogenic mechanisms of RET in tumors include RET point mutations, gene rearrangements or overexpression. These result in constitutive activation of RET tyrosine kinase domain and changes in expression of transcription factors, adhesion proteins and matrix remodeling proteins which promote survival, proliferation, and metastasis of cancer cells.

[0009] The role of RET as an oncogenic driver in multiple types of cancers has been well recognized. For example, RET rearrangement has been reported in 20%-40% papillary thyroid cancer (PTC) and l%-2% non-small cell lung cancer (NSCLC). RET point mutation has been identified in 100% multiple endocrine neoplasia types 2A and 2B (MEN2A and MEN2B) and familial medullary thyroid carcinoma (FMTC). In addition to oncogenic mutations, the activation of wild-type RET has been linked to tumorigenesis and invasion in a much broader group of cancers including neuroblastoma (NB), acute myeloid leukemia (AML), small cell lung cancer, Ewing sarcoma, and breast cancer, etc. For example, AML cell lines exhibited the second highest RET expression among 1036 human cell lines from 36 cancer entities in the Cancer Cell Line Encyclopedia (CCLE). The whole cancer genome sequencing data indicates that AML patients lack acquired point mutations, DNA copy number amplifications, or gene rearrangements of RET gene. However, it has been reported that elevated RET mRNA expression can be detected in 13.5% AML patient samples and RET protein is highly expressed in 83 of the 119 patients (69.7%). Wild-type RET overexpression has been shown to promote NB and AML tumor cell proliferation.

[0010] Considering RET’s important role in diverse cancers, selective RET inhibitors (selpercatinib and pralsetinib) have been developed and showed efficacy in targeting RET- driven malignancies, which prompted an FDA approval for treating cancers with RET gene alteration.

[0011] SUMMARY OF THE INVENTION

[0012] The instant disclosure provides antibody and antigen-binding fragment thereof that specifically binds an extracellular domain of wild-type RET protein (sometimes referred to collectively as “anti-RET antibodies” herein below), which is targeted as a novel druggable target for treating cancer (e.g., cancer characterized by expression / over-expression of RET). A novel anti-RET antibody that binds to the novel druggable target, i.e., extracellular domain of RET, was generated as a novel therapeutic for treating cancers. The anti-RET antibody was also used as a diagnostic tool to diagnose and / or monitor the progression of cancers. Anti-RET Antibodies were generated as off-the-shelf multi-indication cancer therapeutics and diagnostic tools.

[0013] In one aspect, the disclosure provides an antibody or antigen-binding fragment thereof specific for an extracellular domain of the RET (Ret Proto-Oncogene) proto-oncogene, comprising: (1) a heavy chain variable region (VH) comprising a heavy chain CDR1 sequence of SEQ ID NO: 1, a heavy chain CDR2 sequence of SEQ ID NO: 2, and a heavy chain CDR3 sequence of SEQ ID NO: 3; and, (2) a light chain variable region (VL) comprising a light chain CDR1 sequence of SEQ ID NO: 4, a light chain CDR2 sequence of SEQ ID NO: 5, and a light chain CDR3 sequence of SEQ ID NO: 6.

[0014] In some embodiments, the antibody or antigen-binding fragment thereof comprises the VH sequence of SEQ ID NO: 7, and the VL sequence of SEQ ID NO: 8. In some embodiments, the antibody or antigen-binding fragment thereof comprises the heavy chain sequence of SEQ ID NO: 9, and the light chain sequence of SEQ ID NO: 10.

[0015] In some embodiments, the antibody or antigen-binding fragment thereof inhibits RET signaling in a cancer cell characterized by expression of a mutant or wild-type RET.

[0016] In some embodiments, the antibody or antigen-binding fragment thereof is a humanmouse chimeric antibody, a humanized antibody, a human antibody, a CDR-grafted antibody, or a resurfaced antibody.

[0017] In some embodiments, the antibody or antigen-binding fragment thereof is a Fab, Fab’, F(ab’)2, Fd, single chain Fv or scFv, disulfide linked Fv, V-NAR domain, IgNar, intrabody, IgGACH2, minibody, F(ab’)3, tetrabody, triabody, diabody, single-domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.

[0018] In one aspect, the present disclosure provides a polypeptide comprising the VH and / or VE sequence of the antibody or antigen-binding fragment thereof described herein.

[0019] In certain aspects, the present disclosure provides a polynucleotide or set of polynucleotides encoding the VH and / or VE sequence of the antibody or antigen-binding fragment thereof described herein.

[0020] In another aspect, the present disclosure provides a vector comprising the polynucleotide or set of polynucleotides described herein.

[0021] In some embodiments, the vector is an expression vector (e.g., a mammalian expression vector, a yeast expression vector, an insect expression vector, or a bacterial expression vector).

[0022] In one aspect, the present disclosure provides a cell comprising the antibody or antigen-binding fragment thereof, the polypeptide, the polynucleotide, or the vector as described herein.

[0023] In another aspect, the present disclosure provides a method of producing the antibody or antigen-binding fragment thereof or the polypeptide described herein, wherein the method comprises: (a) culturing the cell described herein; and (b) isolating said antibody, antigenbinding fragment thereof, or polypeptide from said cultured cell.

[0024] In another aspect, the present disclosure provides a polypeptide comprising the VH CDR1-3 and / or the VL CDR1-3 of the antibody or antigen-binding fragment thereof described herein. In some embodiments, the polypeptide comprises the VH CDR1-3 and the VL CDR1-3 of the antibody or antigen-binding fragment thereof described herein.

[0025] In some embodiments, the polypeptide is a chimeric antigen receptor (CAR).

[0026] In some embodiments, the CAR comprises: (a) an antigen-recognition domain or an scFv comprising the VH CDR1-3 and the VL CDR1-3 of the antibody or antigen-binding fragment of any one of claims 1-4; and, (I)(b 1) a hinge region (e.g., a membrane-proximal region from an immune molecule such as IgG, CD8, and CD28) and a transmembrane domain (e.g., a membrane-proximal component of an endodomain, such as CD28 transmembrane domain) and (cl) an intracellular T cell signaling domain comprising an immunoreceptor tyrosine -based activation motif (ITAM) (such as the ITAM in the cytoplasmic domain of CD3-zeta), optionally further comprising one or more chimeric domain(s) from a co- stimulatory protein (such as CD28, CD27, CD134 (0X40), and CD137 (4-1BB)), or (II)(b2) an intracellular T cell signaling domain comprising the cytoplasmic domain of CD3s, or (III) (b3) a TRA CC domain linked to the VH of the antigen-recognition domain, and a TRB CC domain linked to the VL of the antigen-recognition domain.

[0027] The present disclosure also provides a T cell comprising the CAR described herein.

[0028] In some embodiments, the T cell is an autologous T cell isolated from a patient to whom the T cell is to be administered, or an allogeneic T cell isolated from a healthy donor.

[0029] In certain aspect, the disclosure provides an immunoconjugate (or antibody-drug conjugate or ADC) having the following formula Ab-[-L-D]n, wherein Ab is an antibody or antigen-binding fragment thereof or the polypeptide described herein, that is covalently linked to one or more units of linker-drug moieties -[-L-D], wherein L is a linker and D is a cytotoxic drug; and, n is an integer from 1 to 20 (e.g., from 1-12); and wherein each linkerdrug moiety may have the same or different linker L or cytotoxic drug D.

[0030] In some embodiments, each linker-drug moiety -[-L-D] is covalently linked to Ab via a side chain amino group of Lys. In some embodiments, each linker-drug moiety -[-L-D] is covalently linked to Ab via a sidechain thiol group of Cys.

[0031] In some embodiments, each linker-drug moiety -[-L-D] is covalently linked to Ab via a site-specifically incorporated non-natural amino acid.

[0032] In some embodiments, each linker L comprises a peptide unit. In some embodiments, the peptide unit comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-10, or 2-5 amino acid residues.

[0033] In some embodiments, the linker L is non-cleavable by protease (e.g., cathepsin). In some other embodiments, the linker L is a cleavable linker cleavable by protease (e.g., cathepsin), acidic environment, or redox state change.

[0034] In some embodiments, the cytotoxic drug is a DNA intercalating agent, a microtubule binder, a topoisomerase I inhibitor, or a DNA minor groove binder.

[0035] In some aspects, the present disclosure provides, a method of treating cancer in a patient in need thereof, wherein the cancer is characterized / caused by activation of RET proto-oncogene (e.g., a mutant RET proto-oncogene) or associated with RET overexpression, the method comprising administering to the patient a therapeutically effective amount of an antibody or antigen binding fragment thereof specific for an extracellular domain of a RET proto-oncogene, or a CAR-T cell comprising a CAR comprising the antibody or antigen binding fragment thereof, or an immunoconjugate comprising the antibody or antigen binding fragment thereof.

[0036] In some embodiments, the antibody is a mouse antibody, a chimeric antibody, a humanized antibody, or a human antibody.

[0037] In some embodiments, the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment as described herein; the CAR is a CAR as described herein; the CAR-T cell is the T cell as described herein; or the immunoconjugate is an immunoconjugate as described herein.

[0038] In some embodiments, the cancer is neuroblastoma (NB), acute myeloid leukemia (AML), small cell lung cancer (SCLC), Ewing sarcoma, breast cancer, and other cancer types with RET expression.

[0039] In some embodiments, the cancer is AML or NB.

[0040] In some embodiments, the AML is characterized by elevated RET mRNA expression.

[0041] In some embodiments, the method further comprises administering to the patient a second therapeutic intervention (e.g., radiotherapy, chemotherapy, and / or immune therapy).

[0042] In some embodiments, the cancer is NB, and the second therapeutic intervention is a standard-of-care or a conventional chemotherapeutic agent (such as an ALK inhibitor).

[0043] In some embodiments, the patient is resistant to chemotherapy, or has relapsed from chemotherapy.

[0044] In one aspect, also provided is a method of detecting the presence of RET (Ret ProtoOncogene) proto-oncogene product (e.g., wild-type or mutant RET protein) in a sample, the method comprises contacting the sample with the antibody or antigen-binding fragment thereof as described herein.

[0045] In some embodiments, the sample is a tissue sample (e.g., a tissue section such as a paraffin-embedded or frozen tumor tissue section, or a cultured cell) from a subject having a cancer, or at high risk of having the cancer, optionally, the cancer is characterized / caused by activation of RET proto-oncogene.

[0046] In some embodiments, the antibody or antigen-binding fragment thereof is detected by immunohistochemistry (IHC) using a labeled secondary antibody specific for the constant region of the antibody or antigen-binding fragment thereof.

[0047] In certain aspect, the disclosure provides a method of identifying a subject who has a cancer characterized / caused by activation of RET proto-oncogene or associated with RET overexpression, comprising determining the level of RET expression in a sample according to the method of detecting the presence of RET product as described herein.

[0048] In another aspect, the disclosure provides a method of treating a subject who has a cancer characterized / caused by activation of RET proto-oncogene or associated with RET overexpression, comprising administering to said subject a therapeutically effective amount of a therapeutically effective amount of an antibody or antigen binding fragment thereof specific for an extracellular domain of a RET proto-oncogene, or a CAR-T cell comprising a CAR comprising the antibody or antigen binding fragment thereof, or an immunoconjugate comprising the antibody or antigen binding fragment thereof, wherein the level of expression of RET is determined according to the method of detecting the presence of RET product as described herein.

[0049] BRIEF DESCRIPTION OF THE DRAWINGS

[0050] FIGs. 1A-1B show that anti-RET antibodies specifically bind to RET's extracellular domain in NIH-3T3 cells with stable human RET expression. In FIGs. 1A and IB, immunofluorescence assay was performed in NIH-3T3 with human RET ectopic expression (NIH3T3-RET) (FIG. 1A) and parental cells (FIG. IB). Mouse IgG was used as a negative control.

[0051] FIGs. 2A-2G show that anti-RET antibodies specifically bind to RET-expressed NB cell lines and inhibit RET signaling and NB cell proliferation. Immunofluorescence assay was performed in NGP (FIG. 2A), CHLA255 (FIG. 2B), and NB975 (FIG. 2C) cells. Mouse IgG was used as control. NB cell lines, NGP (FIG. 2D), CHLA255 (FIG. 2E), and NB975 (FIG. 2F) were treated with the mouse control or anti-RET#l antibody for indicated concentrations. CCK-8 cell proliferation assay was performed in NGP and CHLA255 cells treated with control or anti-RET#l antibody for 12 days and in NB975 cells treated with antibodies for 6 days. FIG. 2G: NGP cells were starved with serum-free medium for 12h and then treated with control or ant-RET#l antibodies (2 pg / ml) for 1 hour before GDNF stimulation.

[0052] FIGs 3A-3D show that anti-RET antibodies specifically bind to RET's extracellular domain. Flow cytometry analysis was performed in A549 cells stably expressed human RET (A549-hRET) or mouse Ret (A549-mRet) (FIG. 3A), as well as the human NB cell line SK- N-AS and the murine NB cell line NB975 (FIG. 3B). Immunofluorescence (IF) (FIG. 3C) and immunohistochemistry (IHC) (FIG. 3D) staining of xenograft human NB tumor tissue using anti-RET antibody.

[0053] FIG. 4 shows that anti-RET antibody can effectively induce ADCC against murine NB cells. In this assay, mouse spleen cells isolated as effect cells were co-cultured with murine NB cell line NB975. Anti-RET antibody or control antibody was added for 24 hours. Then cell viability was measured.

[0054] FIGs. 5A-5C show that anti-RET antibody treatment leads to a complete remission in an immunocompetent NB mouse model. FIG. 5A shows a schematic diagram summarizing the experimental plan for the murine NB cell line NB975 model using C57BL / 6 recipients. FIG. 5B shows representative serial BLI images depicting NB burden of NB975-Fluc engrafted mice treated with control, anti-RET antibody generated from parental or FUT8 / GMDS double knockout 293F cells. FIG. 5C shows Kaplan-Meier survival plot for the groups of mice shown in FIG. 5B.

[0055] FIGs. 6A-6C show that anti-RET antibody binds to RET positive AML cells and inhibits cell proliferation. Flow cytometry analysis was performed in A549 cells stably expressed human RET (A549-hRET) or mouse Ret (A549-mRet) (FIG. 6A), and human AML cell lines THP-1, MOLM-13, Kasumi-1, KG-1, and human Peripheral Blood Mononuclear Cells (PBMC) (FIG. 6B). FIG. 6C shows RET antibody inhibited cell proliferation in RET positive AML cells. KG-1 cell line was used as a control.

[0056] FIGs. 7A-7E show that humanized RET antibody binds to RET-positive cancer cells, inhibits cell proliferation, and trigs ADCC against NB cells. FIG. 7A shows flow cytometry analysis showing specific binding of the humanized RET antibody to the A549 cell line expressing human RET, but not to the parental cells. Human NB cell lines CHLA136 and NGP were analyzed by flow cytometry (FIG. 7B) and cell proliferation assays (FIG. 7C). FIG. 7D shows that the humanized anti-RET antibody (l|jg / mL) triggered ADCC against both CHLA136 and NGP cell lines (5,000 cells) after 24 hours of co-culture with NK cells (25,000 cells) in 96-well plate. FIG. 7E shows co-culturing NK cells with NB cells in the presence of the humanized anti-RET antibody led to increased IFNy secretion from NK cells.

[0057] FIGs. 8A-8B show that humanized RET antibody treatment inhibits tumor growth in an NB xenograft mouse model. FIG. 8A is a schematic diagram summarizing the experimental plan for the human NB cell line NGP model using NOD.Cg-Prkdcscid I12rgtmlWjl / SzJ (NSG) recipients. FIG. 8B shows representative serial BLI images depicting NB burden of NGP-Fluc engrafted mice treated with control or humanized RET antibody.

[0058] FIGs. 9A-9C show that humanized anti- RET antibody binds to RET positive cancer cell lines and inhibits their proliferation. FIG. 9A shows flow cytometry analysis performed in RET-positive cancer cell lines. FIG. 9B shows that humanized RET antibody inhibited cell proliferation in RET positive cancer cell lines. FIG. 9C shows that humanized anti-RET antibody mediated ADCC against cancer cells.

[0059] FIGs. 10A-10B show that RET antibody has a synergistic anti-tumor effect with standard chemotherapy and the ALK inhibitor in NB cells. FIG. 10A shows the synergistic anti-tumor effect of anti-RET antibody combined with doxorubicin or CPT in NB cells. CI<1 indicates synergy. ED, median effective doses. FIG. 10B shows synergistic anti-tumor effect of anti-RET antibody combined with the ALK inhibitor alectinib.

[0060] FIGs. 11A-11E show determination of the efficacy of RET-CAR T cells to lyse NB cell lines. FIG. 11A shows representation of human RET-CAR construct. FIGs. 11B-11C show co-culture of Jurkat cells expressing RET#1 CAR with NB cell lines NGP (FIG. 11B) and CHLA255 (FIG. 11C). NB cell proliferation was determined by a luciferase activity assay. FIGs. 11D-11E show the percentage of specific lysis of RET-CAR T cells against NGP (FIG. HD) and CHLA255 (FIG. HE) cells. P <0.01 (**), or P <0.001 (***).

[0061] FIGs. 12A-12D shows evaluation of the efficacy of RET CAR T cells to lyse the THP- 1 cell line in vitro. FIG. 12A shows representation of human RET-CAR constructs. FIGs. 12B- 12C show that RET-CAR T cells release cytokines, IL-2 (FIG. 12B) and IFNy (FIG. 12C) by co-culture with THP-1 cells. FIG. 12D shows the percentage of specific lysis of RET-CAR T cells against THP-1 cells.

[0062] FIGs. 13A-13B show that RET-CAR T cells demonstrate anti-leukemic effects in THP- 1 AML xenogeneic mouse model. FIG. 13A shows achematic diagram summarizing the experimental plan for the THP-l-FLuc AML xenograft model using NSG mouse recipients. FIG. 13B shows representative serial BLI images depicting leukemia burden of THP-1 -Flue engrafted mice treated with non-transduced (NTD), RET-CAR-28Z, or RET-CAR-BBZ T cells. Data are represented colorimetrically (photons s-1 cm-1) with the scale bars indicating upper (max) and lower (min) BLI thresholds.

[0063] DETAIL DESCRIPTION OF THE INVENTION

[0064] 1. Overview

[0065] The invention described herein is partly based on the finding that certain anti-RET (Ret Proto-Oncogene) antibodies, such as the ones described herein, are effective to treat diseases such as cancer.

[0066] Wild-type RET gene is overexpressed in many tumors, including NB and AML, and RET signaling promotes tumor growth. The present application provides antibodies and antigen -binding fragments thereof specific for an extracellular (ligand-binding) domain of RET protein, which antibodies and antigen-binding fragments thereof may be used to treat cancers in which RET activity is enhanced or increased (e.g., RET gain-of-function mutation, such as constitutively active RET mutation), or RET-dependent cancers, such as cancer having a RET overexpression, or a RET point mutation.

[0067] An anti-RET antibody of the invention binds to the druggable target, e.g., an epitope in the extracellular domain of RET, and serves as a therapeutic for treating cancers with activating RET mutations yet retains the ability to be bound by the anti-RET antibody of the invention.

[0068] In certain embodiments, the anti-RET antibody binds to the epitope in the extracellular domain of RET to block RET signaling.

[0069] In certain embodiments, the anti-RET antibody inhibits tumor growth by blocking RET signaling and inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) to kill tumor cells.

[0070] Thus one aspect of the invention provides an isolated monoclonal antibody, or an antigen-binding fragment thereof, specific for an epitope in the extracellular domain of RET (“a RET epitope”). The antibody or antigen-binding fragment of the invention can be obtained against the isolated RET epitope, or raised against a fusion protein or chemical conjugate thereof comprising said isolated RET epitope and a carrier protein (such as albumin, preferably BSA or ovalbumin, or keyhole limpet hemocyanin (KLH)), according to methods known in the art (see below).

[0071] In certain embodiments, the anti-RET monoclonal antibody comprises: (1) a heavy chain variable region (VH) comprising a heavy chain CDR1 sequence of SEQ ID NO: 1, a heavy chain CDR2 sequence of SEQ ID NO: 2, and a heavy chain CDR3 sequence of SEQ ID NO: 3; and (2) a light chain variable region (VL) comprising a light chain CDR1 sequence of SEQ ID NO: 4, a light chain CDR2 sequence of SEQ ID NO: 5, and a light chain CDR3 sequence of SEQ ID NO:4.

[0072] In certain embodiments, the antibody or antigen-binding fragment comprises the VH sequence of SEQ ID NO: 7, and the VL sequence of SEQ ID NO: 8.

[0073] In certain embodiments, the antibody comprises the heavy chain sequence of SEQ ID NO: 9, and the light chain sequence of SEQ ID NO: 10.

[0074] In certain embodiments, the isolated anti-RET monoclonal antibody or antigen-binding fragment thereof binds to said RET epitope, or a cell having said RET epitope, with a KD of about 10 nM, about 5 nM, or about 2 nM or less.

[0075] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is a mouse antibody, a human-mouse chimeric antibody, a humanized antibody, a human antibody, a CDR-grafted antibody, or a resurfaced antibody.

[0076] In certain embodiments, the antigen-binding fragment thereof is an Fab, Fab’, F(ab’)2, Fd, single chain Fv or scFv, disulfide linked Fv, V-NAR domain, IgNar, intrabody, IgGACH2, minibody, F(ab’)3, tetrabody, triabody, diabody, single-domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.

[0077] In a related aspect, the invention provides an isolated monoclonal antibody, or an antigen-binding fragment thereof, wherein said isolated monoclonal antibody or antigenbinding fragment thereof binds to the same epitope of RET that is bound by a reference monoclonal antibody, or competes with said reference monoclonal antibody for binding to the same epitope of RET, wherein said reference monoclonal antibody comprises: (1) a heavy chain variable region (VH) comprising a heavy chain CDR1 sequence of SEQ ID NO: 1, a heavy chain CDR2 sequence of SEQ ID NO: 2, and a heavy chain CDR3 sequence of SEQ ID NO: 3; and (2) a light chain variable region (VL) comprising a light chain CDR1 sequence of SEQ ID NO: 4, a light chain CDR2 sequence of SEQ ID NO: 5, and a light chain CDR3 sequence of SEQ ID NO:4.

[0078] Another aspect of the invention provides a polypeptide comprising the HCVR and / or the LCVR of any one of the subject anti-RET antibodies or antigen binding fragments thereof.

[0079] Another aspect of the invention provides a polynucleotide encoding any of the subject polypeptides.

[0080] Another aspect of the invention provides a vector comprising any of the subject polynucleotides. In certain embodiments, the vector is an expression vector (e.g., a mammalian expression vector, a yeast expression vector, an insect expression vector, or a bacterial expression vector).

[0081] Another aspect of the invention provides a cell comprising any of the subject anti-RET antibody or antigen-binding fragment thereof, any of the subject polypeptide, any of the subject polynucleotide, or any of the subject vector.

[0082] In certain embodiments, the cell expresses any of the subject antibody or antigenbinding fragment thereof, or any of the subject polypeptide.

[0083] In certain embodiments, the cell is an HEK293F cell, a BHK cell, a CHO cell, or a COS cell.

[0084] In certain embodiments, the cell comprises any of the subject anti-RET antibody or antigen-binding fragment thereof, or any of the subject polypeptide, on the surface of the cell.

[0085] In certain embodiments, the polypeptide is a fusion protein, such as a chimeric antigen T cell receptor (or CAR).

[0086] In certain embodiments, the cell is a T-cell bearing a chimeric antigen receptor (CAR- T cell) comprising any of the subject antibody or antigen-binding fragment thereof, or any of the subject polypeptide.

[0087] Chimeric antigen receptor (CAR) and CAR T cells

[0088] Chimeric antigen T cell receptor (CAR-T) is also known as chimeric antigen receptor (CAR), chimeric immunoreceptor, chimeric T cell receptor, or artificial T cell receptor. It is an engineered receptor that grafts an arbitrary specificity onto an immune effector T cell. Typically, these receptors are used to graft the specificity of a monoclonal antibody onto a T cell, with transfer of their coding sequence facilitated by retroviral or lentiviral vectors. The receptors are called chimeric because they are composed of parts from different sources. CAR- T may be used in treating cancer using adoptive cell transfer in which T cells are removed from a patient and modified so that they express receptors specific to the patient’s particular cancer, such as RET expressed on cancer cells. The T cells, which can then recognize and kill the cancer cells, are reintroduced into the patient. Modification of T-cells sourced from donors other than the patient may also be used similarly.

[0089] In certain embodiments, the CAR of the subject invention comprises an extracellular antigen binding domain that binds to RET, a transmembrane (TM) region, one or more costimulatory domain, and an intracellular signal transduction domain. In certain embodiments, the CAR further comprises a hinge / spacer domain between the antigen-binding domain and the TM domain. The hinge and TM domains may originate from the same protein, or from diff erent proteins.

[0090] In certain embodiments, the extracellular antigen binding region may be an sc-Fv, Fab, scFab or sdgG fragment thereof.

[0091] In certain embodiments, the CAR-T of the subject invention is a fusion of a subject single-chain variable fragments (scFv) derived from any of the subject monoclonal anti-RET antibody, fused to a transmembrane domain (such as the CD8 transmembrane domain) and an endodomain (such as the CD28 / 4-lBB-CD3-zeta endodomain).

[0092] In certain embodiments, the scFv is preceded by a signal peptide to direct the nascent protein to the endoplasmic reticulum and subsequent surface expression. Any eukaryotic signal peptide sequence may be used. In certain embodiments, the signal peptide natively attached to the amino-terminal is used (e.g., in a scFv with orientation light chain - linker - heavy chain, the native signal of the light-chain is used).

[0093] In certain embodiments, a flexible spacer is added to allow the scFv to orient in different directions to enable optimal antigen binding. The spacer is preferably flexible enough to allow the antigen binding domain to orient in different directions to facilitate antigen recognition. In certain embodiments, the hinge region from IgGl is used as the spacer. In certain embodiments, the CH2CH3 region of immunoglobulin and portions of CD3 is used as the spacer. For most scFv based constructs, the IgGl hinge usually suffices.

[0094] In certain embodiments, the construct comprises a transmembrane domain that is a typical hydrophobic alpha helix derived from the original molecule of the signaling endodomain that protrudes into the cell and transmits the desired signal. In certain embodiments, the transmembrane domain from the most membrane proximal component of the endodomain, such as the CD3-zeta transmembrane domain, is used.

[0095] In certain embodiments, the transmembrane domain comprises the transmembrane region of CD3^, CD4, CD8, CD28, 0X40 or CD137.

[0096] In some embodiments, the transmembrane region comprises the transmembrane region of a CD8 transmembrane domain, such as CD8a transmembrane domain.

[0097] In some embodiments, the CAR further comprises a hinge region between the extracellular antigen binding domain and the transmembrane domain. In certain embodiments, the hinge region is from a CD8 hinge region, such as the CD8a hinge.

[0098] In certain embodiments, the hinge region and the TM region can be from the same protein, e.g., both from the CD8 protein. In certain embodiments, the hinge region and the TM region can be from different proteins, e.g., the hinge region may be from the CD8a protein, while the TM region can be from the TM region of CD3 or CD28, etc. In certain embodiments, the CAR comprises one or more signal transduction domain(s) capable of activating the immune cell in which the CAR is expressed.

[0099] In certain embodiments, the CAR comprises one or more (e.g., two) signal transduction domain(s) capable of stimulating T-cell activation. The signal transduction domain may also be referred to as a an “endodomain”

[0100] In certain embodiments, the one or more signal transduction domain(s) can include, without limitation, one or more ol'TCR^, FcRy, FcRp, FcRs, CD3y, CD35, CD3s, CD3(^, signal transduction domain of CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments, the CAR comprises a CD3^ endodomain.

[0101] In certain embodiments, the endodomain is the CD3-zeta endodomain containing 3 ITAMs, which transmits an activation signal to the T cell after the antigen is bound by the antigen binding fragment of the invention.

[0102] In certain embodiments, the endodomain further comprises intracellular signaling domains from a costimulatory protein receptor (e.g., that of CD28, 41BB, ICOS) fused to the cytoplasmic tail (N- or C-terminal to the CD3-zeta domain) of the construct to provide additional signals to the T cell.

[0103] In some embodiments, the CAR further comprises one or more co- stimulatory domain from one or more of: CD2, CD3, CD4, CD5, CD7, CD27, CD28, CD30, CD40, CD83, CD86, CD127, CD134, CD137 / 4-1BB, 4-1BBL, OX-40, PD-1, LFA-1, Lek, DAP10, LIGHT, NKG2C, B7-H3, CD3^, or ICOS. In certain embodiments, the one or more co-stimulatory domain comprises an intracellular signal transduction region from CD3^, FcsRIy, PKC9, or ZAP70. In some embodiments, the CAR comprises a CD28 co-stimulatory domain. In certain embodiments, the CAR comprises an ITAM from 4-1BB (CD137), which acts as the costimulatory signaling domain of the CAR, and serves to enhance antigen activation and increase potency. In certain other embodiments, the CAR comprises an ITAM from the costimulatory domain of CD28, which also increases CAR-mediated T cell activation.

[0104] In certain embodiments, the endodomain combines multiple signaling domains, such as CD3z-CD28-41BB or CD3z-CD28-OX40, to augment potency, or to transmit a proliferative / survival signal.

[0105] In certain embodiments, the chimeric antigen receptor of the invention further comprises a Strep-tag II sequence (an eight-residue minimal peptide sequence (Trp-Ser-His- Pro-Gln-Phe-Glu-Lys) that exhibits intrinsic affinity toward streptavidin), to provides engineered T cells with an identification marker for rapid purification.

[0106] Another aspect of the invention provides a polynucleotide encoding the CAR of the invention described herein.

[0107] In some embodiments, the nucleic acid is a synthetic nucleic acid. In some embodiments, the nucleic acid is a DNA molecule. In some embodiments, the nucleic acid is an RNA molecule (e.g., an mRNA molecule encoding the CAR). In some embodiments, the mRNA is capped, polyadenylated, substituted with 5-methyl cytidine, substituted with pseudouridine, or a combination thereof.

[0108] In some embodiments, the nucleic acid (e.g., DNA) is operably linked to a regulatory element (e.g., a promoter) in order to control the expression of the nucleic acid. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is an organism-specific promoter.

[0109] Suitable promoters are known in the art and include, for example, a pol I promoter, a pol II promoter, a pol III promoter, a T7 promoter, a U6 promoter, a Hl promoter, retroviral Rous sarcoma virus LTR promoter, a cytomegalovirus (CMV) promoter, a SV40 promoter, a dihydrofolate reductase promoter, and a P-actin promoter.

[0110] In certain embodiments, the nucleic acid molecule encoding the CAR proteins, derivatives or functional fragments thereof are codon-optimized for expression in a host cell or organism. The host cell may include established cell lines (such as T cells) or isolated primary cells. The nucleic acid can be codon optimized for use in any organism of interest, in particular human immune cells. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.orjp / codon / , and these tables can be adapted in a number of ways. See Nakamura et al., Nucl. Acids Res. 28:292, 2000 (incorporated herein by reference). Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.).

[0111] An example of a codon optimized sequence is a CAR coding sequence optimized for expression in a eukaryote, e.g., humans (i.e. being optimized for expression in humans), or for another eukaryote, animal or mammal as herein discussed). Whilst this is preferred, it will be appreciated that other examples are possible and codon optimization for a host species other than human, or for codon optimization for specific organs is known. In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g. about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at http: / / www.kazusa.orjp / codon / and these tables can be adapted in a number of ways. See Nakamura, Y., et al. “Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28:292 (2000). Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, PA), are also available. In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a CAR corresponds to the most frequently used codon for a particular amino acid.

[0112] In some embodiments, the polynucleotide(s) or nucleic acid(s) of the invention are present in a vector (e.g., a viral vector).

[0113] The term “vector” as used herein generally refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors include, but are not limited to, nucleic acid molecules that are single- stranded, double-stranded, or partially doublestranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g., circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art.

[0114] In certain embodiments, the vector can be a cloning vector, or an expression vector. The vectors can be plasmids, phagemids, Cosmids, etc. The vectors may include one or more regulatory elements that allow for the propagation of the vector in a cell of interest (e.g., a mammalian cell such as a human immune cell like T / NK cell).

[0115] In certain embodiments, the vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.

[0116] In certain embodiments, the vector is a viral vector, wherein virally-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g., retroviruses, lentiviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, HSV, and adeno-associated viruses (AAV)). Viral vectors also include polynucleotides carried by a virus for transfection into a host cell.

[0117] In certain embodiments, viral vectors such as retrovirus, lentivirus or transposon may be used to integrate the transgene bearing the subject CAR-T construct into the host cell genome.

[0118] In certain embodiments, the vector is a lentiviral vector. In certain embodiments, the lentiviral vector is a self-inactivating lentiviral vector. See, for example, Zufferey et al., “SelfInactivating Lentivirus Vector for Safe and Efficient In vivo Gene Delivery.” J Virol. 72(12): 9873-9880, 1998 (incorporated herein by reference).

[0119] In certain embodiments, the vector is based on the Sleeping Beauty (SB) transposon, which has been used as a non- viral vector for introducing genes into genomes of vertebrate animals and for gene therapy. Because the SB system is composed solely of DNA, the costs of production and delivery are considerably reduced compared to viral vectors. SB transposons have been used to genetically modify T cell in human clinical trials.

[0120] In certain embodiments, the vector is capable of autonomous replication in a host cell into which they are introduced. In certain embodiments, the vector (e.g., non-episomal mammalian vectors) is integrated into the genome of a host cell upon introduction into the host cell and thereby are replicated along with the host genome. In certain embodiments, the vector, referred to herein as “expression vector,” is capable of directing the expression of genes to which they are operatively linked. Vectors for and that result in expression in a eukaryotic cell are “eukaryotic expression vectors.”

[0121] In certain embodiments, non-integrating vectors or episomal DNA / RNA constructs, such as plasmids or mRNA, can be used instead.

[0122] In certain embodiments, a vector that is stably maintained in the T cell without being integrated into the genome is used to enable long-term transgene expression without the risk of insertional mutagenesis or genotoxicity.

[0123] In certain embodiments, the vector is a recombinant expression vector that comprises a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell. The recombinant expression vector may include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively linked to the nucleic acid sequence to be expressed. Here, “operably linked” means that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell).

[0124] The term “regulatory element” includes promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). Such regulatory elements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissuespecific regulatory sequences). A tissue-specific promoter may direct expression primarily in a desired tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e.g., liver, pancreas), or particular cell types (e.g., lymphocytes such as T cells, or NK cells). Regulatory elements may also direct expression in a temporal-dependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific.

[0125] In some embodiments, a vector comprises one or more pol III promoter (e.g., 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof. Examples of pol III promoters include, but are not limited to, U6 and Hl promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al, Cell, 41 :521- 530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the P-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter.

[0126] Also encompassed by the term “regulatory element” are enhancer elements, such as WPRE; CMV enhancers; the R-U5’ segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit b- globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981).

[0127] It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, etc. A vector can be introduced into host cells to thereby produce transcripts, proteins, or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein.

[0128] In certain embodiments, the vector is a lentiviral or AAV vector, which can be selected for targeting particular types of cells (e.g., with tissue and / or cell type-specific tropism).

[0129] The vectors of the invention can be introduced into a target cell, such as a primary T / NK cell, or an “off-the-shelf’ allogeneic T / NK cell, using any of many art-recognized methods, such as transfection, lipid vectors, infection, electroporation, microinjection, parenteral injections, aerosol, gene guns, or use of ballistic particles, etc.

[0130] In certain embodiments, transfection includes chemical transfection that introduces the vector by, e.g., calcium phosphate, lipid, or protein complexes. Calcium phosphate, DEAE- dextran, liposomes, and lipoplexes (for oral delivery of gene) surfactants and perfluro chemical liquids for aerosol delivery of gene.

[0131] In certain embodiments, lipid vectors are generated by a combination of plasmid DNA and a lipid solution that result in the formation of a liposome, which can be fused with the cell membranes of a variety of cell types, thus introducing the vector DNA into the cytoplasm and nucleus, where the encoded gene is expressed. In certain embodiments, folate is linked to DNA or DNA-lipid complexes to more efficiently introduce vectors into cells expressing high levels of folate receptor. Other targeting moieties can be similarly used to target the delivery of the vectors to specific cell types targeted by the targeting moieties.

[0132] In certain embodiments, the vector DNA is internalized via receptor- mediated endocytosis.

[0133] The CAR of the invention can be introduced into various kinds of immune cells for CAR-mediated therapy. The immune cells into which the CAR of the invention can be introduced include T cells.

[0134] Thus in one aspect, the invention also provides a cell comprising any of the CAR of the invention, polynucleotide encoding the CAR protein, or vector of the invention comprising the polynucleotide of the invention.

[0135] In certain embodiments, the cell is a eukaryote. In certain embodiments, the cell is a human cell. In certain embodiments, the cell is an immune cell. In certain embodiments, the cell is a T cell, such as CD4+ or CD8+ T cell. In certain embodiments, the cell is a primary cell isolated from a patient into which cell a CAR-expressing vector is to be introduced to express the CAR before the cell is reintroduced to the patient. In certain embodiments, the cell is from a healthy donor into which cell a CAR-expressing vector is to be introduced to express the CAR before the cell is reintroduced to a patient different from the healthy donor. Optionally, the HLA-type of the healthy donor matches that of the patient.

[0136] In certain embodiments, the T cells and / or NK cells and / or monocytes and / or macrophages of the present invention can be obtained from a number of non-limiting source by various non-limiting method, comprising PBMCs, bone marrow, lymph node tissue, cord blood, thymus tissue, ascites, pleural effusion, spleen tissue, and tumors.

[0137] In some embodiments, the immune cells are isolated from patients in need of CAR- based therapy, e.g., from patients diagnosed with cancer or inflammatory disease. In this embodiment, the T cells are autologous.

[0138] As used herein, “autologous” refers to cell treatment subject, the cell line or cell population derived from the object.

[0139] In some embodiments, the immune cells are isolated from healthy donors that are not the patient in need of treatment. In this embodiment, the immune cells are derived from a heterologous host, preferably from a host that is human leukocyte antigen (HLA)-compatible.

[0140] In some embodiments, the T-cells comprise CD4+ T cells. In some embodiments, the T-cells comprise CD8+ T cells.

[0141] The subject CAR T cells can be prepared by any means known in the art. For example, expression constructs such as viral-based vectors (e.g., lentiviral vectors) comprising and capable of expressing the CAR polynucleotides of the invention can be used to transduce the isolated immune cells to obtain the subject CAR-T cells. One of the skills in the art can easily construct expression constructs such as viral vectors suitable for protein expression.

[0142] In certain embodiments, the cell (e.g., immune cell) further expresses a cytokine, such as IL-2, IL-7, IL-12, IL-15, or IL-21, or combination thereof. In certain embodiments, expression of the one or more cytokine is activated upon binding of the CAR to its target antigen. In certain embodiments, expression of the cytokine is under the control of a promoter that is activated by activation of the immune cell.

[0143] In certain embodiments, the cell further comprises a safety switch for down-regulating the activity of the immune cell.

[0144] In certain embodiments, the safety switch comprises a coding sequence for an iCaspase9 (inducible caspase-9) monomer that can be activated by dimerization with, e.g., FKBP, to trigger apoptosis of the immune cell.

[0145] Another aspect of the invention provides a fusion protein or chemical conjugate comprising the isolated RET epitope of the invention, and a carrier protein (such as albumin, preferably BSA or ovalbumin, or keyhole limpet hemocyanin (KLH)).

[0146] As is known in the art, a carrier protein is any protein used for coupling with peptides or other haptens that are not sufficiently large or complex on their own to induce an immune response and produce antibodies. The carrier protein, because it is large and complex, confers immunogenicity to the conjugated hapten, resulting in antibodies being produced against epitopes on the hapten and carrier.

[0147] Many proteins can be used as carriers and are chosen based on immunogenicity, solubility, and availability of useful functional groups through which conjugation with the hapten can be achieved. In certain embodiments, the carrier protein used in the instant invention is keyhole limpet hemocyanin (KLH) or an albumin, such as bovine serum albumin (BSA) or ovalbumin.

[0148] Many such carrier proteins that can be used in the instant invention are commercially available, such as the Thermo Scientific Imject Mariculture Keyhole Limpet Hemocyanin (me KLH); the Blue Carrier* Protein (a purified preparation of Concholepas concholepas hemocyanin (CCH) which exhibits most of the same immunogenic properties as the KLH); the Thermo Scientific Imject BSA (a highly purified (i.e., Fraction V) bovine serum albumin); cationized bovine serum albumin (cBSA) (prepared by modifying native BSA with excess ethylenediamine, essentially capping all negatively-charged carboxyl groups with positively- charged primary amines, resulting in a highly positively-charged protein (pl > 11) that has significantly increased immunogenicity compared to native BSA); and ovalbumin.

[0149] The RET epitopes of the invention can be fused to the carrier protein, or chemically conjugated to the carrier protein through, for example, any one or more of the surface primary amine groups of the carrier protein.

[0150] Different approaches are available for conjugating haptens / peptide epitopes to carrier proteins, depending on the functional groups available on the hapten / epitope, the required hapten / epitope orientation and distance from the carrier, and the possible effect of conjugation on biological and antigenic properties. For example, epitopes having primary amines (the N- terminus and the side chain of lysine residues), carboxylic groups (C-terminus or the side chain of aspartic acid and glutamic acid), and sulfhydryls (side chain of cysteine residues) can be targeted for conjugation using such groups. Generally, it is the many primary amines in a carrier protein that are used to couple haptens via a crosslinking reagent.

[0151] In certain embodiments, the protein-carrier and peptide-carrier conjugation is carried out using the carbodiimide crosslinker EDC (i.e., EDC conjugation via carboxyl and amine crosslinking).

[0152] In certain embodiments, the protein-carrier and peptide-carrier conjugation is carried out using maleimide conjugation (sulfhydryl crosslinking).

[0153] In certain embodiments, the protein-carrier and peptide-carrier conjugation is carried out using glutaraldehyde conjugation (amine-to-amine crosslinking).

[0154] Another aspect of the invention provides a method of producing any of the subject anti- RET antibody or antigen-binding fragment thereof, or any of the subject polypeptide, comprising: (a) culturing any of the subject cell; and, (b) isolating said antibody, antigenbinding fragment thereof, or polypeptide from said cultured cell. In certain embodiments, the cell is a eukaryotic cell.

[0155] Another aspect of the invention provides an immunoconjugate (or antibody-drug conjugate or ADC) having the following formula: Ab-[-L-D]n, wherein: Ab is any of the subject anti-RET antibody or antigen-binding fragment thereof, or any of the subject polypeptide thereof, that is covalently linked to one or more units of linker-drug moieties -[-L- D], wherein L is a linker and D is a cytotoxic drug; and, n is an integer from 1 to 20 (e.g., from 1-12); and wherein each linker-drug moiety may have the same or different linker L or cytotoxic drug D.

[0156] In certain embodiments, each linker-drug moiety -[-L-D] is covalently linked to Ab via a sidechain amino group of Lys.

[0157] In certain embodiments, each linker-drug moiety -[-L-D] is covalently linked to Ab via a sidechain thiol group of Cys.

[0158] In certain embodiments, each linker-drug moiety -[-L-D] is covalently linked to Ab via a site-specifically incorporated non-natural amino acid.

[0159] In certain embodiments, each linker L comprises a peptide unit.

[0160] In certain embodiments, the peptide unit comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-10, or 2- 5 amino acid residues.

[0161] In certain embodiments, the linker L is non-cleavable by protease (e.g., cathepsin).

[0162] In certain embodiments, the linker L is a cleavable linker cleavable by protease (e.g., cathepsin), acidic environment, or redox state change.

[0163] In certain embodiments, the cytotoxic drug is a DNA intercalating agent, a microtubule binder, a topoisomerase I inhibitor, or a DNA minor groove binder.

[0164] In certain embodiments, the cytotoxic drug is auristatin class such as monomethyl auristatin E (MMAE) and MM AL, maytansine class such as DM-1, DM-3, DM-4, calicheamicin such as ozogamicin, SN-38, or PBD (pyrrolobenzodiazepin).

[0165] In a related aspect, the D moiety is not a drug molecule per se, but an adaptor molecule (such as EITC) that can be tightly bound by a universal CAR-T specific for the adaptor molecule. According to this aspect of the invention, a single universal CAR-T cell, which binds with extraordinarily high affinity to an adaptor molecule such as EITC, are used to treat various cancer types when co-administered with bispecific SMDC (small molecule drug conjugate) adaptor molecules. These unique bispecific adaptors are constructed with an adaptor, such as FITC molecule, and a tumor-homing molecule, such as the antigen-binding fragment of the subject anti-RET antibody, to precisely bridge the universal CAR-T cell with the cancer cells, which causes localized T cell activation. Anti-tumor activity is induced only when both the universal CAR-T cells and the correct antigen- specific adaptor molecules are present. Anti-tumor activity and toxicity can be controlled further by adjusting the administered adaptor molecule dosing. Treatment of antigenically heterogeneous tumors can be achieved by administration of a mixture of the desired antigen- specific adaptors.

[0166] Another aspect of the invention provides a pharmaceutical composition comprising any of the subject anti-RET antibody or antigen-binding fragment thereof, or the polypeptide thereof, or the immunoconjugate thereof, and a pharmaceutically acceptable carrier or excipient.

[0167] Another aspect of the invention provides a method for inhibiting the growth of a cell expressing RET (e.g., RET expressed from a mutated RET gene), the method comprising contacting the cell with any of the subject anti-RET antibody or antigen-binding fragment thereof, or the subject polypeptide thereof, or the subject immunoconjugate thereof, or the subject pharmaceutical composition thereof.

[0168] A related aspect of the invention provides a method for treating a cancer / tumor characterized / caused by the cell expressing RET (e.g., RET expressed from a mutated RET gene), the method comprising contacting the cell with any of the subject anti-RET antibody or antigen-binding fragment thereof, or the subject polypeptide thereof, or the subject immunoconjugate thereof, or the subject pharmaceutical composition thereof.

[0169] In certain embodiments, the cell is a tumor cell.

[0170] In certain embodiments, the tumor cell comprises a mutated RET gene.

[0171] In certain embodiments, the mutated RET gene is capable of oncogenic RET ligandindependent phosphorylation, and / or leading to constitutive activation of a downstream signal transduction cascade (such as MAPK pathway, PI3K / AKT pathway, PLCy pahway, and SRC pathway).

[0172] In certain embodiments, the mutated RET gene is a RET point mutation. In certain embodiments, the mutated RET gene comprises a germline or sporadic mutation activating RET’s kinase domain. In certain embodiments, said mutation activating RET’s kinase domain is capable of triggering monomeric activation of RET. In certain embodiments, the mutation activating RET’s kinase domain comprises a RET M918T mutation, a C634R mutation, and / or a V804 gatekeeper mutation (such as V804M).

[0173] In certain embodiments, the mutated RET gene comprises an E511K mutation, an R114H mutation, an M I I09I / T mutation, an R525Q mutation, an R600Q mutation, a V706M mutation, an A756V mutation, an M255I mutation, an R163Q mutation, and / or a T636M mutation. In certain embodiments, the tumor cell is from a lung cancer (such as NSCLC or

[0174] SCLC).

[0175] In certain embodiments, the tumor cell is from a thyroid cancer. In certain embodiments, the thyroid cancer is a papillary thyroid cancer (PTC). In certain embodiments, the thyroid cancer is a medullary thyroid cancer (MTC, such as familial medullary thyroid cancer (FMTC), multiple endocrine neoplasia type 2A (MEN 2A) syndrome, or multiple endocrine neoplasia type 2B (MEN 2B) syndrome). In certain embodiments, the thyroid cancer is a poorly differentiated thyroid carcinoma. In certain embodiments, the thyroid cancer is an anaplastic thyroid cancer.

[0176] In certain embodiments, the tumor cell is from breast carcinoma, pheochromocytoma, neuroendocrine malignancy (including paraganglioma, pheochromocytoma, pulmonary or extra-pulmonary carcinoid tumor), parathyroid hyperplasia and adenoma, intestinal ganglioneuromas, mucosal neuromas, gastrointestinal malignancy (such as colorectal adenocarcinoma, gastrointestinal stromal tumor, or hepatocellular carcinoma), or pancreatic acinar cell carcinoma (PACC).

[0177] In certain embodiments, the tumor cell is from breast cancer (such as HER2-negative breast cancer), oesophagus cancer, stomach cancer, pancreas cancer, colon / rectum cancer, prostate cancer, and soft tissue cancer, a cancer of the thyroid gland (e.g., anaplastic or poorly differentiated), a cancer of the lung (e.g., pleomorphic, adenocarcinoma, large cell neuroendocrine), papillary thyroid cancer, esophageal cancer, salivary gland carcinoma (such as salivary intraductal carcinomas, salivary gland mammary analogue secretory carcinomas (MASCs)), spitzoid neoplasm (such as spitzoid melanomas and atypical spitzoid tumor), pediatric spindle-cell mesenchymal neoplasm, histiocytic neoplasm, cutaneous xanthogranuloma, pancreatic ductal adenocarcinoma, stomach adenocarcinoma, esophageal cancer, cholangiocarcinoma, bladder carcinoma, head and neck cancer, mesothelioma, low- grade glioma, atypical lung carcinoid tumor, chronic myeloproliferative neoplasm, hepatobiliary cancer, glioblastoma, and bladder urothelial carcinoma.

[0178] Another aspect of the invention provides a method of determining presence and / or abundance of RET in a sample from a subject, the method comprising contacting the sample with any of the subject anti-RET antibody or antigen-binding fragment thereof.

[0179] Another aspect of the invention provides a method of diagnosing a subject having cancer, wherein cells of the cancer expresses RET, the method comprising: (1) using the subject method to determine the presence and / or abundance of RET in a cancer sample from the subject in order to identify subject expressing (e.g., overexpressing) RET (e.g., any of the mutant RET described herein) in the cancer sample; (2) optionally selecting / referring / recommending the subject to treatment when the subject is identified as expressing (e.g., overexpressing) RET (e.g., any of the mutant RET described herein).

[0180] In certain embodiments, the method further comprising (3) administering to said subject a therapeutically effective amount of any of the subject anti-RET antibody or antigen-binding fragment thereof, or the polypeptide thereof, or the immunoconjugate thereof, or the pharmaceutical composition thereof; thereby diagnosing and treating the subject having cancer.

[0181] With the invention generally described above, certain specific aspects or embodiments of the invention are described further in the sections below.

[0182] 2. Definitions

[0183] The terms “antibody,” “antibody molecule,” and “antibody protein” are used interchangeably herein and shall be considered equivalent. They include an immunoglobulin molecule that recognizes and specifically binds to a target molecule, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the light chain and / or heavy chain variable regions of the immunoglobulin molecule. As used herein, the term “antibody” encompasses intact polyclonal antibodies, intact monoclonal antibodies, and may as an abbreviation include antibody fragments (such as Fab, Fab’, F(ab’)2, and Fv fragments), single chain Fv (scFv) mutants, multispecific antibodies such as bispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determination portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the desired biological activity. An antibody can be of any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc.

[0184] In some embodiments, an antibody is a non-naturally occurring, recombinantly generated antibody. In some embodiments, an antibody is purified from natural components. In some embodiments, an antibody is recombinantly produced. In some embodiments, an antibody is produced by a hybridoma or generated in a library of antibodies. “Complementarity determining regions (CDRs) of a monoclonal antibody” are understood to be those amino acid sequences involved in specific antigen binding according to Kabat (Kabat E. A., Wu T. T., Perry H. M., Gottesman K. S. and Foeller C. (1991) Sequences of Proteins of Immunological Interest (5th Ed.). NIH Publication No. 91-3242. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, Md., incorporated herein by reference) in connection with Chothia and Lesk (Chothia and Lesk (1987) J. Mol. Biol. 196:901-917, incorporated herein by reference).

[0185] As used herein, the term “framework modifications” refers to the exchange, deletion or addition of single or multiple amino acids in the variable regions surrounding the individual complementarity determining regions. Framework modifications may have an impact on the immunogenicity, producibility or binding specificity of an antibody protein.

[0186] An “antigen-binding fragment,” “antigen-binding portion,” or “fragment” for short, as used herein, refers to a shorter version of the antibody molecule, i.e. any polypeptide subset, characterized in that it is encoded by a shorter nucleic acid molecule than the full length sequence, but still retains its antibody binding activity (e.g., substantially the same binding specificity, although can be slightly worse binding affinity as measured by Kd).

[0187] These terms refer to a portion of an intact antibody and refer to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, and Fv fragments, linear antibodies, single chain antibodies, and multi- specific antibodies formed from antibody fragments.

[0188] The term “antigen-binding fragment” of an antibody includes one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by certain fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding fragment” of an antibody include (without limitation): (i) an Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CHI domains (e.g., an antibody digested by papain yields three fragments: two antigen-binding Fab fragments, and one Fc fragment that does not bind antigen); (ii) a F(ab’)2 fragment, abivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region (e.g., an antibody digested by pepsin yields two fragments: a bivalent antigen -binding F(ab’)2 fragment, and a pFc’ fragment that does not bind antigen) and its related F(ab’) monovalent unit; (iii) a Fd fragment consisting of the VH and CHI domains (i.e., that portion of the heavy chain which is included in the Fab); (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, and the related disulfide linked Fv; (v) a dAb (domain antibody) or sdAb (single domain antibody) fragment (Ward et al., Nature 341:544-546, 1989), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR).

[0189] Various techniques are known for the production of antibody fragments. Traditionally, these fragments are derived via proteolytic digestion of intact antibodies (for example Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117, 1993; Brennan et al., Science 229:81, 1985). In certain embodiments, antibody fragments are produced recombinantly. Fab, Fv, and scFv antibody fragments can all be expressed in and secreted from E. coli or other host cells, thus allowing the production of large amounts of these fragments. Such antibody fragments can also be isolated from antibody phage libraries. The antibody fragment can also be linear antibodies as described in U.S. Patent 5,641,870, for example, and can be monospecific or bispecific. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner.

[0190] A “monoclonal antibody” refers to a homogeneous antibody population involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants. The term “monoclonal antibody” encompasses both intact and full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab’, F(ab’)2, Fv), single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. Furthermore, “monoclonal antibody” refers to such antibodies made in any number of manners including but not limited to by hybridoma, phage selection, recombinant expression, and transgenic animals.

[0191] Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein (1975) Nature 256:495. Using the hybridoma method, a mouse, hamster, or other appropriate host animal, is immunized to elicit the production by lymphocytes of antibodies that will specifically bind to an immunizing antigen. Lymphocytes can also be immunized in vitro. Following immunization, the lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol, to form hybridoma cells that can then be selected away from unfused lymphocytes and myeloma cells. Hybridomas that produce monoclonal antibodies directed specifically against a chosen antigen as determined by immunoprecipitation, immunoblotting, or by an in vitro binding assay (e.g., radioimmunoassay (RIA); enzyme-linked immunosorbent assay (ELISA)) can then be propagated either in vitro culture using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986) or in vivo as ascites tumors in an animal. The monoclonal antibodies can then be purified from the culture medium or ascites fluid as described for polyclonal antibodies.

[0192] Alternatively monoclonal antibodies can also be made using recombinant DNA methods as described in U.S. Patent 4,816,567. The polynucleotides encoding a monoclonal antibody are isolated from mature B-cells or hybridoma cells, such as by RT- PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequence is determined using conventional procedures. The isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors, which when transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, monoclonal antibodies are generated by the host cells. Also, recombinant monoclonal antibodies or fragments thereof of the desired species can be isolated from phage display libraries expressing CDRs of the desired species as described (McCafferty et al., Nature 348:552-554, 1990; Clackson et al., Nature, 352:624-628, 1991; and Marks et al., J. Mol. Biol. 222:581-597, 1991).

[0193] The polynucleotide(s) encoding a monoclonal antibody can further be modified in a number of different manners using recombinant DNA technology to generate alternative antibodies. In some embodiments, the constant domains of the light and heavy chains of, for example, a mouse monoclonal antibody can be substituted 1) for those regions of, for example, a human antibody to generate a chimeric antibody, or, 2) for a non- immunoglobulin polypeptide to generate a fusion antibody. In some embodiments, the constant regions are truncated or removed to generate the desired antibody fragment of a monoclonal antibody. Site-directed or high-density mutagenesis of the variable region can be used to optimize specificity, affinity, etc. of a monoclonal antibody.

[0194] The term “humanized antibody” refers to forms of non-human (e.g., murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (e.g., murine) sequences. Typically, humanized antibodies are human immunoglobulins in which residues from the complementary determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g., mouse, rat, rabbit, hamster) that have the desired specificity, affinity, and capability (Jones et al., Nature 321:522- 525, 1986; Riechmann et al., Nature 332:323-327, 1988; Verhoeyen et al., Science 239:1534- 1536, 1988).

[0195] Methods for engineering, humanizing or resurfacing non-human or human antibodies can also be used and are well known in the art. A humanized, resurfaced or similarly engineered antibody can have one or more amino acid residues from a source that is nonhuman, e.g., but not limited to, mouse, rat, rabbit, non-human primate or other mammal. These non-human amino acid residues are replaced by residues that are often referred to as “import” residues, which are typically taken from an “import” variable, constant or other domain of a known human sequence.

[0196] Such imported sequences can be used to reduce immunogenicity or reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, or any other suitable characteristic, as known in the art. In general, the CDR residues are directly and most substantially involved in influencing RET binding. Accordingly, part or all of the non-human or human CDR sequences are maintained while the non-human sequences of the variable and constant regions can be replaced with human or other amino acids.

[0197] Antibodies can also optionally be humanized, resurfaced, engineered or human antibodies engineered with retention of high affinity for the antigen RET and other favorable biological properties. To achieve this goal, humanized (or human) or engineered anti-RET antibodies and resurfaced antibodies can be optionally prepared by a process of analysis of the parental sequences and various conceptual humanized and engineered products using three- dimensional models of the parental, engineered, and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen, such as RET. In this way, framework (FR) residues can be selected and combined from the consensus and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.

[0198] Humanization, resurfacing or engineering of antibodies of the present invention can be performed using any known method, such as but not limited to those described in, Winter (Jones et al., Nature 321:522, 1986; Riechmann et al., Nature 332:323, 1988; Verhoeyen et al., Science 239:1534, 1988, Sims et al., J. Immunol. 151:2296, 1993; Chothia and Lesk, J. Mol. Biol. 196:901, 1987, Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285, 1992; Presta et al., J. Immunol. 151:2623, 1993; Raguska et al., Proc. Natl. Acad. Sci. U.S.A. 91(3):969-973, 1994; U.S. Pat. Nos. 5,639,641, 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370; 5,693,762; 5,530,101; 5,585,089; 5,225,539; 4,816,567; PCT / : US98 / 16280; US96 / 18978; US91 / 09630; US91 / 05939; US94 / 01234; GB89 / 01334; GB91 / 01134; GB92 / 01755; WO90 / 14443; WO90 / 14424; W090 / 14430; EP 229246; 7,557,189; 7,538,195; and 7,342,110, each of which is entirely incorporated herein by reference, including the references cited therein.

[0199] In certain alternative embodiments, the antibody to RET is a human antibody. Human antibodies can be directly prepared using various techniques known in the art. Immortalized human B lymphocytes immunized in vitro or isolated from an immunized individual that produce an antibody directed against a target antigen can be generated (See, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., 1991, J. Immunol, 147 (l):86-95; and U.S. Patent 5,750,373). Also, the human antibody can be selected from a phage library, where that phage library expresses human antibodies, as described, for example, in Vaughan et al., Nat. Biotech. 14:309- 314, 1996, Sheets et al., Proc. Nat’l. Acad. Sci. 95:6157-6162, 1998, Hoogenboom and Winter, J. Mol. Biol. 227:381, 1991, and Marks et al., J. Mol. Biol. 222:581, 1991). Techniques for the generation and use of antibody phage libraries are also described in U.S. Patent Nos. 5,969,108, 6,172,197, 5,885,793, 6,521,404; 6,544,731; 6,555,313; 6,582,915; 6,593,081; 6,300,064; 6,653,068; 6,706,484; and 7,264,963; and Rothe et al., J. Mol. Bio. doi: 10.1016 / j.jmb.2007.12.018, 2007 (each of which is incorporated by reference in its entirety). Affinity maturation strategies and chain shuffling strategies (Marks et al., Bio / Technology 10:779-783, 1992, incorporated by reference in its entirety) are known in the art and can be employed to generate high affinity human antibodies.

[0200] Humanized antibodies can also be made in transgenic mice containing human immunoglobulin loci that are capable upon immunization of producing the full repertoire of human antibodies in the absence of endogenous immunoglobulin production. This approach is described in U.S. Patents 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.

[0201] In some instances, the Fv framework region (FR) residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species that has the desired specificity, affinity, and capability. The humanized antibody can be further modified by the substitution of additional residues either in the Fv framework region and / or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and / or capability. In general, the humanized antibody will comprise substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDR regions that correspond to the non-human immunoglobulin whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Pats. 5,225,539 and 5,639,641, Roguska et al., Proc. Natl. Acad. Sci. USA 91(3):969-973, 1994; and Roguska et al., Protein Eng. 9(10):895- 904, 1996 (all incorporated herein by reference). In some embodiments, a “humanized antibody” is a resurfaced antibody. In some embodiments, a “humanized antibody” is a CDR- grafted antibody.

[0202] A “variable region” of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The variable regions of the heavy and light chain each consist of four framework regions (FR) connected by three complementarity determining regions (CDRs) also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies. There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, 5th ed., 1991, National Institutes of Health, Bethesda Md.); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al., J. Molec. Biol. 273:927-948, 1997). In addition, combinations of these two approaches are sometimes used in the art to determine CDRs.

[0203] The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0204] The amino acid position numbering as in Kabat, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991) (incorporated herein by reference). Using this numbering system, the actual linear amino acid sequence can contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain can include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues can be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence. Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917,1987). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop. This is because the Kabat numbering scheme places the insertions at H35A and H35B - if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34. The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software.

[0205] Loop Kabat AbM Chiothia

[0206] LI L24-L34 L24-L34 L24-L34

[0207] L2 L50-L56 L50-L56 L50-L56

[0208] L3 L89-L97 L89-L97 L89-L97

[0209] Hl H31-H35B H26-H35B H26-H32..34

[0210] (Kabat Numbering)

[0211] (Chothia Numbering)

[0212] H2 H50-H65 H50-H58 H52-H56

[0213] H3 H9S-H102 H95-H102 H95-H102

[0214] The term “human antibody” means an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any technique known in the art. In certain embodiments, the human antibody does not have non-human sequence. This definition of a human antibody includes intact or full-length antibodies, or antigen-binding fragments thereof.

[0215] The term “chimeric antibodies” refers to antibodies wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable region of both light and heavy chains corresponds to the variable region of antibodies derived from one species of mammals (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capability while the constant regions are homologous to the sequences in antibodies derived from another (usually human) to avoid or reduce the chance of eliciting an immune response in that species (e.g., human). In certain embodiments, chimeric antibody may include an antibody or antigen-binding fragment thereof comprising at least one human heavy and / or light chain polypeptide, such as, for example, an antibody comprising murine light chain and human heavy chain polypeptides.

[0216] For the purposes of the present invention, it should be appreciated that modified antibodies can comprise any type of variable region that provides for the association of the antibody with the polypeptides of a human RET. In this regard, the variable region can comprise or be derived from any type of mammal that can be induced to mount a humoral response and generate immunoglobulins against the desired tumor associated antigen. As such, the variable region of the modified antibodies can be, for example, of human, murine, nonhuman primate (e.g., cynomolgus monkeys, macaques, etc.) or lupine origin. In some embodiments both the variable and constant regions of the modified immunoglobulins are human. In other embodiments the variable regions of compatible antibodies (usually derived from a non-human source) can be engineered or specifically tailored to improve the binding properties or reduce the immunogenicity of the molecule. In this respect, variable regions useful in the present invention can be humanized or otherwise altered through the inclusion of imported amino acid sequences.

[0217] In certain embodiments, the variable domains in both the heavy and light chains are altered by at least partial replacement of one or more CDRs and, if necessary, by partial framework region replacement and sequence changing. Although the CDRs can be derived from an antibody of the same class or even subclass as the antibody from which the framework regions are derived, it is envisaged that the CDRs will be derived from an antibody of different class and in certain embodiments from an antibody from a different species. It may not be necessary to replace all of the CDRs with the complete CDRs from the donor variable region to transfer the antigen-binding capacity of one variable domain to another. Rather, it may only be necessary to transfer those residues that are necessary to maintain the activity of the antigenbinding site. Given the explanations set forth in U.S. Pat. Nos. 5,585,089, 5,693,761 and 5,693,762, it will be well within the competence of those skilled in the art, either by carrying out routine experimentation or by trial and error testing to obtain a functional antibody with reduced immunogenicity.

[0218] Alterations to the variable region notwithstanding, those skilled in the art will appreciate that the modified antibodies of this invention will comprise antibodies (e.g., full- length antibodies or immunoreactive fragments thereof) in which at least a fraction of one or more of the constant region domains has been deleted or otherwise altered so as to provide desired biochemical characteristics such as increased tumor localization or reduced serum halflife when compared with an antibody of approximately the same immunogenicity comprising a native or unaltered constant region. In some embodiments, the constant region of the modified antibodies will comprise a human constant region. Modifications to the constant region compatible with this invention comprise additions, deletions or substitutions of one or more amino acids in one or more domains. That is, the modified antibodies disclosed herein can comprise alterations or modifications to one or more of the three heavy chain constant domains (CHI, CH2, or CH3) and / or to the light chain constant domain (CL). In some embodiments, modified constant regions wherein one or more domains are partially or entirely deleted are contemplated. In some embodiments, the modified antibodies will comprise domain deleted constructs or variants wherein the entire CH2 domain has been removed (ACH2 constructs). In some embodiments, the omitted constant region domain will be replaced by a short amino acid spacer (e.g., 10 residues) that provides some of the molecular flexibility typically imparted by the absent constant region.

[0219] It will be noted that in certain embodiments, the modified antibodies can be engineered to fuse the CH3 domain directly to the hinge region of the respective modified antibodies. In other constructs it may be desirable to provide a peptide spacer between the hinge region and the modified CH2 and / or CH3 domains. For example, compatible constructs could be expressed wherein the CH2 domain has been deleted and the remaining CH3 domain (modified or unmodified) is joined to the hinge region with a 5-20 amino acid spacer. Such a spacer can be added, for instance, to ensure that the regulatory elements of the constant domain remain free and accessible or that the hinge region remains flexible. However, it should be noted that amino acid spacers can, in some cases, prove to be immunogenic and elicit an unwanted immune response against the construct. Accordingly, in certain embodiments, any spacer added to the construct will be relatively non-immunogenic, or even omitted altogether, so as to maintain the desired biochemical qualities of the modified antibodies.

[0220] Besides the deletion of whole constant region domains, it will be appreciated that the antibodies of the present invention can be provided by the partial deletion or substitution of a few or even a single amino acid. For example, the mutation of a single amino acid in selected areas of the CH2 domain may be enough to substantially reduce Fc binding and thereby increase tumor localization. Similarly, it may be desirable to simply delete that part of one or more constant region domains that control the effector function (e.g., complement C1Q binding) to be modulated. Such partial deletions of the constant regions can improve selected characteristics of the antibody (serum half-life) while leaving other desirable functions associated with the subject constant region domain intact. Moreover, as alluded to above, the constant regions of the disclosed antibodies can be modified, e.g., through the mutation or substitution of one or more amino acids, which may enhance the profile of the resulting construct. In this respect it may be possible to disrupt the activity provided by a conserved binding site (e.g., Fc binding) while substantially maintaining the configuration and immunogenic profile of the modified antibody. Certain embodiments can comprise the addition of one or more amino acids to the constant region to enhance desirable characteristics such as decreasing or increasing effector function or provide for more cytotoxin or carbohydrate attachment. In such embodiments it can be desirable to insert or replicate specific sequences derived from selected constant region domains.

[0221] The present invention further embraces variants and equivalents which are substantially homologous to the chimeric, humanized and human antibodies, or antibody fragments thereof, set forth herein. These can contain, for example, conservative substitution mutations, i.e., the substitution of one or more amino acids by similar amino acids. For example, conservative substitution refers to the substitution of an amino acid with another within the same general class such as, for example, one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid or one neutral amino acid by another neutral amino acid. What is intended by a conservative amino acid substitution is well known in the art, such as those defined hereinabove.

[0222] The terms “epitope” or “antigenic determinant” are used interchangeably herein and refer to that portion of an antigen capable of being recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, epitopes can be formed both from contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding are typically lost upon protein denaturing. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.

[0223] “Binding affinity” generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd) or the half-maximal effective concentration (EC50). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention. Specific illustrative embodiments are described herein.

[0224] The phrase “substantially similar,” or “substantially the same,” as used herein, denotes a sufficiently high degree of similarity between two numeric values (generally one associated with an antibody of the invention and the other associated with a reference / comparator antibody) such that one of skill in the art would consider the difference between the two values to be of little or no biological and / or statistical significance within the context of the biological characteristics measured by said values (e.g., Kd values). The difference between said two values is less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% as a function of the value for the reference / comparator antibody.

[0225] A polypeptide, antibody, polynucleotide, vector, cell, or composition which is “isolated” is a polypeptide, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some embodiments, an antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure.

[0226] Methods known in the art for purifying antibodies and other proteins also include, for example, those described in U.S. Patent Publication Nos. 2008 / 0312425, 2008 / 0177048, and 2009 / 0187005, each of which is hereby incorporated by reference herein in its entirety.

[0227] As used herein, “substantially pure” refers to material which is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0228] “A functional variant” of the antibody molecule according to the invention is an antibody molecule which possesses a biological activity (either functional or structural) that is substantially similar to the antibody molecule according to the invention, i.e. a substantially similar substrate specificity or cleavage of the substrate.

[0229] The term “functional variant” also includes “a fragment”, “an allelic variant” “a functional variant”, “variant based on the degenerative nucleic acid code” or “chemical derivatives.” Such a “functional variant” may carry one or several point mutations, one or several nucleic acid exchanges in the coding sequence, deletions or insertions or one or several amino acid exchanges, deletions or insertions. Said functional variant is still retaining its biological activity such as antibody binding activity, at least in part or even going along with an improvement said biological activity.

[0230] A “functional variant” of the antibody molecule according to the invention may also include an antibody molecule which possesses a biological activity (either functional or structural) that is substantially similar to the antibody molecule according to the invention, i.e. a substantially similar target molecule binding activity. An “allelic variant” is a variant due to the allelic variation, e.g. differences in the two alleles in humans. Said variant is still retaining its biological activity such as antibody target binding activity, at least in part or even going along with an improvement said biological activity.

[0231] A “variant based on the degenerative of the genetic code” is a variant due to the fact that a certain amino acid may be encoded by several different nucleotide triplets. Said variant is still retaining its biological activity such as antibody binding activity, at least in part or even going along with an improvement said biological activity.

[0232] A “fusion molecule” may be the antibody molecule according to the invention fused to e.g. a reporter such as a radiolabel, a chemical molecule such as a toxin or a fluorescent label or any other molecule known in the art.

[0233] As used herein, a “chemical derivative” according to the invention is an antibody molecule according to the invention chemically modified or containing additional chemical moieties not normally being part of the molecule. Such moieties may improve the molecule’s activity such as target destruction (e.g. killing of tumor cells) or may improve its solubility, absorption, biological half-life etc.

[0234] A molecule is “substantially similar” to another molecule if both molecules have substantially similar structures or biological activity. Thus, provided that two molecules possess a similar activity, they are considered variants as that term is used herein even if the structure of one of the molecules is not found in the other, or if the sequence of amino acid residues is not identical.

[0235] A “sample” or “biological sample” of the present invention is of biological origin, in specific embodiments, such as from eukaryotic organisms. In some embodiments, the sample is a human sample, but animal samples may also be used. Non-limiting sources of a sample for use in the present invention include solid tissue, biopsy aspirates, ascites, fluidic extracts, blood, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, tumors, organs, cell cultures and / or cell culture constituents, for example.

[0236] A “cancerous / tumor sample” is a sample that contains a cancerous cell. The method can be used to examine an aspect of expression of RET or a state of a sample, including, but not limited to, comparing different types of cells or tissues, comparing different developmental stages, and detecting or determining the presence and / or type of disease or abnormality.

[0237] For many uses of the antibodies according to the invention it is desirable to have the smallest possible antigen-binding, i.e., RET-binding units. Therefore in another preferred embodiment an antibody protein according to the invention is a Fab fragment (Fragment antigen -binding=Fab). These RET-specific antibody proteins according to the invention consist of the variable regions of both chains which are held together by the adjacent constant region. These may be formed by protease digestion, e.g. with papain, from conventional antibodies, but similar Fab fragments may also be produced in the mean time by genetic engineering. In another preferred embodiment an antibody protein according to the invention is an F(ab’)2 fragment, which may be prepared by proteolytic cleaving with pepsin.

[0238] Using genetic engineering methods it is possible to produce shortened antibody fragments which consist only of the variable regions of the heavy (VH) and of the light chain (VL). These are referred to as Fv fragments (Fragment variable=fragment of the variable part). In another preferred embodiment a RET-specific antibody molecule according to the invention is such an Fv fragment. Since these Fv-fragments lack the covalent bonding of the two chains by the cysteines of the constant chains, the Fv fragments are often stabilized. It is advantageous to link the variable regions of the heavy and of the light chain by a short peptide fragment, e.g. of 10 to 30 amino acids, preferably 15 amino acids. In this way a single peptide strand is obtained consisting of VH and VL, linked by a peptide linker. An antibody protein of this kind is known as a single-chain-Fv (scFv). Examples of scFv-antibody proteins of this kind known from the prior art are described in Huston et al. (1988, PNAS 16: 5879-5883). Therefore, in another preferred embodiment an RET-specific antibody protein according to the invention is a single-chain-Fv protein (scFv).

[0239] In recent years, various strategies have been developed for preparing scFv as a multimeric derivative. This is intended to lead, in particular, to recombinant antibodies with improved pharmacokinetic and biodistribution properties as well as with increased binding avidity. In order to achieve multimerization of the scFv, scFv were prepared as fusion proteins with multimerization domains. The multimerization domains may be, e.g. the CH3 region of an IgG or coiled coil structure (helix structures) such as Leucin-zipper domains. However, there are also strategies in which the interaction between the VH / VL regions of the scFv are used for the multimerization (e.g. di-, tri- and pentabodies). Therefore in another embodiment an antibody protein according to the invention is a RET-specific diabody antibody fragment. By diabody the skilled person means a bivalent homodimeric scFv derivative (Hu et al., 1996, PNAS 16: 5879-5883). The shortening of the Linker in an scFv molecule to 5-10 amino acids leads to the formation of homodimers in which an inter-chain VH / NL-superimposition takes place. Diabodies may additionally be stabilized by the incorporation of disulfide bridges. Examples of diabody-antibody proteins from the prior art can be found in Perisic et al. (1994, Structure 2: 1217-1226).

[0240] By minibody the skilled person means a bivalent, homodimeric scFv derivative. It consists of a fusion protein which contains the CH3 region of an immunoglobulin, preferably IgG, most preferably IgGl as the dimerization region which is connected to the scFv via a Hinge region (e.g. also from IgGl) and a Linker region. The disulfide bridges in the Hinge region are mostly formed in higher cells and not in prokaryotes. In another preferred embodiment an antibody protein according to the invention is a RET-specific minibody antibody fragment. Examples of minibody- antibody proteins from the prior art can be found in Hu et al. (1996, Cancer Res. 56: 3055-61).

[0241] By tribody the skilled person means a: trivalent homotrimeric scFv derivative (Kortt et al. 1997 Protein Engineering 10: 423-433). ScFv derivatives wherein VH-VL are fused directly without a linker sequence lead to the formation of trimers.

[0242] The skilled person will also be familiar with so-called miniantibodies which have a bi- , tri- or tetravalent structure and are derived from scFv. The multimerization is carried out by di-, tri- or tetrameric coiled coil structures (Pack et al., 1993 Biotechnology II:, 1271-1277; Lovejoy et al. 1993 Science 259: 1288-1293; Pack et al., 1995 J. Mol. Biol. 246: 28-34).

[0243] Therefore in one embodiment an antibody protein according to the invention is a RET- specific multimerized molecule based on the abovementioned antibody fragments and may be, for example, a tribody, a tetravalent miniantibody or a pentabody.

[0244] Humanized RET-specific antibody proteins can be generated by molecular biology methods known in the art.

[0245] The variable regions of the antibody proteins of the present invention are typically linked to at least a portion of the immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Human constant region DNA sequences can be isolated in accordance with well-known procedures from a variety of human cells, but preferably immortalized B cells (see Kabat et al., supra, and WO 87 / 02671). Hence the antibody proteins of the invention may contain all or only a portion of the constant region as long as they exhibit specific binding to the RET antigen. The choice of the type and extent of the constant region depends on whether effector functions like complement fixation or antibody dependent cellular toxicity are desired, and on the desired pharmacological properties of the antibody protein. The antibody protein of the invention will typically be a tetramer consisting of two light chain / heavy chain pairs, but may also be dimeric, i.e. consisting of a light chain / heavy chain pair, e.g. a Fab or Fv fragment.

[0246] Therefore, in a further embodiment the invention relates to antibody proteins according to the invention, characterized in that they have a variable light chain region and a variable heavy chain region, each joined to a human constant region. In particular, the variable region of the light chain was joined to a human kappa constant region and the variable region of the heavy chain was joined to a human gamma- 1 constant region. Other human constant regions for chimerizing light and heavy chains are also available.

[0247] Humanization of the variable region of a murine antibody may be achieved employing methods known in the art. EP 0239400 discloses grafting of the CDRs of a murine variable region into the framework of a human variable region. WO 90 / 07861 discloses methods of reshaping a CDR- grafted variable region by introducing additional framework modifications. WO 92 / 11018 discloses methods of producing humanized Ig combining donor CDRs with an acceptor framework that has a high homology to the donor framework. WO 92 / 05274 discloses the preparation of framework mutated antibodies starting from a murine antibody. Further prior art references related to humanization of murine monoclonal antibodies are EP 0368684; EP 0438310; WO 92 / 07075, or WO 92 / 22653. All are incorporated herein by reference.

[0248] In another embodiment, the invention relates to an antibody molecule according to the invention characterized that each of said variable region of the light chain and said variable region of the heavy chain region is separately joined to a human constant region.

[0249] In another embodiment, the invention relates to an antibody molecule according to the invention, wherein said human constant region of the light chain is a human kappa constant region.

[0250] In another embodiment, the invention relates to an antibody protein according to the invention, wherein said human constant region of the heavy chain is a human IgGl constant region.

[0251] The antibody proteins of the invention provide a highly specific tool for targeting therapeutic agents to the RET antigen. Therefore, in a further aspect, the invention relates to antibody proteins according to the invention, wherein said antibody protein is conjugated to a therapeutic agent, optionally via a linker, in an antibody-drug-conjugate (ADC). Of the many therapeutic agents known in the art, therapeutic agents selected from the group consisting of radioisotopes, toxins, toxoids, inflammatogenic agents, enzymes, antisense molecules, peptides, cytokines, and chemotherapeutic agents are preferred. Among the radioisotopes, gamma, beta and alpha-emitting radioisotopes may be used as a therapeutic agent. P-emitting radioisotopes are preferred as therapeutic radioisotopes.186Rhenium,188Rhenium,131Iodine and90Yttrium have been proven to be particularly useful P-emitting isotopes to achieve localized irradiation and destruction of malignant tumor cells. Therefore, radioisotopes selected from the group consisting of186Rhenium,188Rhenium,131Iodine and90Yttrium are particularly preferred as therapeutic agents conjugated to the antibody proteins of the invention. For example, for the radioiodination of an antibody of the invention, a method as disclosed in WO 93 / 05804 may be employed.

[0252] The term “immunoconjugate,” “conjugate,” or “ADC” as used herein refers to a compound or a derivative thereof that is linked to a cell binding agent (i.e., an anti-RET antibody or fragment thereof) and is defined by a generic formula: A-L-C, wherein C = cytotoxin, L = linker, and A = cell binding agent (CBA), such as anti-RET antibody or antibody fragment. Immunoconjugates can also be defined by the generic formula in reverse order: C- L-A.

[0253] A “linker” is any chemical moiety that is capable of linking a compound, usually a drug, such as a cytotoxic agent described herein, to a cell-binding agent such as an anti-RET antibody or a fragment thereof in a stable, covalent manner. Linkers can be susceptible to or be substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, at conditions under which the compound or the antibody remains active. Suitable linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups. Linkers also include charged linkers, and hydrophilic forms thereof as described herein and know in the art.

[0254] The terms “cancer cell,” “tumor cell,” and grammatical equivalents refer to the total population of cells derived from a tumor or a pre-cancerous lesion, including both non- tumorigenic cells, which comprise the bulk of the tumor cell population, and tumorigenic stem cells (cancer stem cells). As used herein, the term “tumor cell” will be modified by the term “non-tumorigenic” when referring solely to those tumor cells lacking the capacity to renew and differentiate to distinguish those tumor cells from cancer stem cells.

[0255] The term “subject” refers to any animal (e.g., a mammal), including, but not limited to humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.

[0256] Administration “in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[0257] The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. Such formulation can be sterile.

[0258] An “effective amount” of an antibody or immunoconjugate as disclosed herein is an amount sufficient to carry out a specifically stated purpose. An “effective amount” can be determined empirically and in a routine manner, in relation to the stated purpose.

[0259] The term “therapeutically effective amount” refers to an amount of an antibody or other drug effective to “treat” a disease or disorder in a subject or mammal. In the case of cancer, the therapeutically effective amount of the drug can reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and in a certain embodiment, stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and in a certain embodiment, stop) tumor metastasis; inhibit, to some extent, tumor growth; relieve to some extent one or more of the symptoms associated with the cancer; and / or result in a favorable response such as increased progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), complete response (CR), partial response (PR), or, in some cases, stable disease (SD), a decrease in progressive disease (PD), a reduced time to progression (TTP), or any combination thereof. See the definition herein of “treating.” To the extent the drug can prevent growth and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic.

[0260] A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0261] A “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer, regardless of mechanism of action. Terms such as “treating” or “treatment” or “to treat” or “alleviating” or “to alleviate” refer to therapeutic measures that cure, slow down, lessen symptoms of, and / or halt progression of a diagnosed pathologic condition or disorder. Thus, those in need of treatment include those already diagnosed with the disorder, and may also include those who have minimal residual disease, or resistant disease, or relapsed disease. In certain embodiments, a subject is successfully “treated” for cancer according to the methods of the present invention if the patient shows one or more of the following: a reduction in the number of or complete absence of cancer cells; a reduction in the tumor size; inhibition of or an absence of cancer cell infiltration into peripheral organs including, for example, the spread of cancer into soft tissue and bone; inhibition of or an absence of tumor metastasis; inhibition or an absence of tumor growth; relief of one or more symptoms associated with the specific cancer; reduced morbidity and mortality; improvement in quality of life; reduction in tumorigenicity, tumorigenic frequency, or tumorigenic capacity, of a tumor; reduction in the number or frequency of cancer stem cells in a tumor; differentiation of tumorigenic cells to a non-tumorigenic state; increased progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), complete response (CR), partial response (PR), stable disease (SD), a decrease in progressive disease (PD), a reduced time to progression (TTP), or any combination thereof.

[0262] “Polynucleotide” or “nucleic acid,” as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, “caps,” substitution of one or more of the naturally occurring nucleotides with an analog, intemucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, cabamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s). Further, any of the hydroxyl groups ordinarily present in the sugars can be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or can be conjugated to solid supports. The 5’ and 3’ terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls can also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2’-0-methyl-, 2’-0-allyl, 2’ -fluoro- or 2’- azido-ribose, carbocyclic sugar analogs, alpha-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages can be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S (“thioate”), P(S)S (“dithioate”), (0)NR2 (“amidate”), P(O)R, P(O)OR*, CO or CH2 (“formacetal”), in which each R or R is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[0263] The term “vector” means a construct, which is capable of delivering, and expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[0264] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that, because the polypeptides of this invention are based upon antibodies, in certain embodiments, the polypeptides can occur as single chains or associated chains. In some embodiments, a polypeptide, peptide, or protein is non-naturally occurring. In some embodiments, a polypeptide, peptide, or protein is purified from other naturally occurring components. In some embodiments, the polypeptide, peptide, or protein is recombinantly produced.

[0265] The terms “identical” or percent “identity” in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences. One such non- limiting example of a sequence alignment algorithm is the algorithm described in Karlin et al., Proc. Natl. Acad. Sci. 87:2264-2268, 1990, as modified in Karlin et al., Proc. Natl. Acad. Sci. 90:5873-5877, 1993, and incorporated into the NBLAST and XBLAST programs (Altschul et al., Nucleic Acids Res. 25:3389-3402, 1991). In certain embodiments, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997; BLAST-2, WU- BLAST-2 (Altschul et al., Methods in Enzymology 266:460-480, 1996), ALIGN, ALIGN-2 (Genentech, South San Francisco, California) or Megalign (DNASTAR) are additional publicly available software programs that can be used to align sequences. In certain embodiments, the percent identity between two nucleotide sequences is determined using the GAP program in GCG software (e.g., using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 90 and a length weight of 1, 2, 3, 4, 5, or 6). In certain alternative embodiments, the GAP program in the GCG software package, which incorporates the algorithm of Needleman and Wunsch (J. Mol. Biol. (48):444- 453, 1970) can be used to determine the percent identity between two amino acid sequences (e.g., using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5). Alternatively, in certain embodiments, the percent identity between nucleotide or amino acid sequences is determined using the algorithm of Myers and Miller (CAB IOS, 4:11 17, 1989). For example, the percent identity can be determined using the AEIGN program (version 2.0) and using a PAM120 with residue table, a gap length penalty of 12 and a gap penalty of 4. Appropriate parameters for maximal alignment by particular alignment software can be determined by one skilled in the art. In certain embodiments, the default parameters of the alignment software are used. In certain embodiments, the percentage identity “X” of a first amino acid sequence to a second sequence amino acid is calculated as 100 x (Y / Z), where Y is the number of amino acid residues scored as identical matches in the alignment of the first and second sequences (as aligned by visual inspection or a particular sequence alignment program) and Z is the total number of residues in the second sequence. If the length of a first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be longer than the percent identity of the second sequence to the first sequence.

[0266] As a non-limiting example, whether any particular polynucleotide has a certain percentage sequence identity (e.g., is at least 80% identical, at least 85% identical, at least 90% identical, and in some embodiments, at least 95%, 96%, 97%, 98%, or 99% identical) to a reference sequence can, in certain embodiments, be determined using the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489, 1981, to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.

[0267] In some embodiments, two nucleic acids or polypeptides of the invention are substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. In certain embodiments, identity exists over a region of the sequences that is at least about 10, about 20, about 40-60 residues in length or any integral value there between, or over a longer region than 60-80 residues, at least about 90-100 residues, or the sequences are substantially identical over the full length of the sequences being compared, such as the coding region of a nucleotide sequence for example.

[0268] A “conservative amino acid substitution” is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. In certain embodiments, conservative substitutions in the sequences of the polypeptides and antibodies of the invention do not abrogate the binding of the polypeptide or antibody containing the amino acid sequence, to the antigen(s), i.e., the CD123 / IL-3Ra to which the polypeptide or antibody binds. Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate antigen-binding are well-known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187, 1993; Kobayashi et al., Protein Eng. 12(10):879-884, 1999; and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417, 1997).

[0269] Another aspect of the present invention provides an antibody protein according to the invention linked to a therapeutic agent, wherein said therapeutic agent is a therapeutic agent selected from the group consisting of radioisotopes, toxins, toxoids, pro-drugs and chemotherapeutic agents.

[0270] In certain embodiments, the therapeutic agent is linked to the antibody protein via a linker selected from the group of MAG-3 (U.S. Pat. No. 5,082,930 A, EP 0247866 Bl (page 2 lines 55-56-page 3 lines 1-23)); MAG-2 GABA (U.S. Pat. No. 5,681,927 A, EP 0284071 Bl (page 6 lines 9-29)); and N2S2 ((=phenthioate) U.S. Pat. Nos. 4,897,255 A, 5,242,679 A, EP 0188256 Bl (page 2, lines 38-page 3, lines 18)), (Ac)Phe-Lys(Alloc)-PABC-PNP, 6- Maleimidohexanoic acid N-hydroxysuccinimide ester, 6-Quinoxalinecarboxylic acid, 2,3- bis(bromomethyl)-Fmoc-Val-Cit-PAB, Fmoc-Val-Cit-PAB-PNP, Mc-Val-Cit-PABC-PNP, Val-cit-PAB-OH, all herein incorporated by reference.

[0271] In certain embodiments, the radioisotope is selected from the group consisting of186Rhenium,188Rhenium,131Iodine and90Yttrium.

[0272] In certain embodiments, the antibody proteins according to the invention are labelled. Such RET-specific labelled antibody allows for the localization and / or detection of the RET antigen in vitro and / or in vivo.

[0273] A label is defined as a marker that may be directly or indirectly detectable. An indirect marker is defined as a marker that cannot be detected by itself but needs a further directly detectable marker specific for the indirect marker. Preferred labels for practicing the invention are detectable markers. From the large variety of detectable markers, a detectable marker may be selected from the group consisting of enzymes, dyes, radioisotopes, digoxygenin, and biotin.

[0274] In certain embodiments, the label is a detectable marker, such as one selected from the group consisting of enzymes, dyes, radioisotopes, digoxygenin, and biotin.

[0275] In certain embodiments, antibody proteins according to the invention are conjugated to an imageable agent. A large variety of imageable agents, especially radioisotopes, are available from the state of the art. In certain embodiments, the imageable agent is gamma-emitting isotopes, such as125Iodine. In certain embodiments, the antibody protein has specific activity of from about 0.5 to about 15 mCi / mg, or from about 0.5 to about 14 mCi / mg, or about 1 to about 10 mCi / mg, or about 1 to about 5 mCi / mg, and about 2 to 6 mCi / mg or 1 to 3 mCi / mg.

[0276] 3. Compositions and. Pharmaceutical Compositions

[0277] The present invention includes a composition (e.g., a pharmaceutical composition) comprising the subject antibodies or antigen-binding fragments thereof, immuno-conjugates thereof, or CAR T cells described herein, and a carrier (e.g., a pharmaceutically acceptable carrier). The present invention also includes a composition (e.g., a pharmaceutical composition) comprising the subject antibodies or antigen-binding fragments thereof, conjugate thereof, or CAR T cells described herein , and a carrier (a pharmaceutically acceptable carrier), and further comprising a second therapeutic agent. The present compositions are useful for inhibiting abnormal cell growth or treating a proliferative disorder in a mammal (e.g., human), including hematologic cancer, leukemia, or lymphoma.

[0278] In particular, the present invention provides pharmaceutical compositions comprising one or more of the RET-binding agents or immuno-conjugates thereof described herein. In certain embodiments, the pharmaceutical compositions further comprise a pharmaceutically acceptable vehicle. These pharmaceutical compositions find use in inhibiting tumor growth and treating cancer in human patients, including hematologic cancer, leukemia, or lymphoma.

[0279] In certain embodiments, formulations are prepared for storage and use by combining a purified antibody, or immuno-conjugate thereof of the present invention with a pharmaceutically acceptable vehicle (e.g. carrier, excipient) (Remington, The Science and Practice of Pharmacy 20th Edition Mack Publishing, 2000). Suitable pharmaceutically acceptable vehicles include, but are not limited to, nontoxic buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides (e.g., less than about 10 amino acid residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; carbohydrates such as monosaccharides, disaccharides, glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and non-ionic surfactants such as TWEEN or polyethylene glycol (PEG).

[0280] A pharmaceutically acceptable carrier can contain physiologically acceptable compounds that act, for example, to stabilize or to increase the absorption of an AMPA glutamate receptor agonist, antagonist or modulator. Such physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients (see also e.g. Remington’s Pharmaceutical Sciences (1990), 18th ed. Mack Publ., Easton). One skilled in the art would know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration of the composition.

[0281] Suitable pharmaceutically acceptable carriers, diluents, and excipients are generally well known and can be determined by those of ordinary skill in the art as the clinical situation warrants. Examples of suitable carriers, diluents and / or excipients include: (1) Dulbecco's phosphate buffered saline, pH about 7.4, containing or not containing about 1 mg / mL to 25 mg / mL human serum albumin, (2) 0.9% saline (0.9% w / v NaCl), and (3) 5% (w / v) dextrose; and may also contain an antioxidant such as tryptamine and a stabilizing agent such as Tween 20.

[0282] The pharmaceutical compositions described herein can be administered in any number of ways for either local or systemic treatment. Administration can be topical (such as to mucous membranes including vaginal and rectal delivery) such as transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders; pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal); oral; or parenteral including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial (e.g., intrathecal or intraventricular) administration. In some particular embodiments, the administration is intravenous. The pharmaceutical compositions described herein can also be used in vitro or in ex vivo.

[0283] In an animal or human body, it can prove advantageous to apply the pharmaceutical compositions as described above via an intravenous or other route, e.g. systemically, locally or topically to the tissue or organ of interest, depending on the type and origin of the disease or problem treated, e.g. a tumor. For example, a systemic mode of action is desired when different organs or organ systems are in need of treatment as in e.g. systemic autoimmune diseases, or allergies, or transplantations of foreign organs or tissues, or tumors that are diffuse or difficult to localise. A local mode of action would be considered when only local manifestations of neoplastic or immunologic action are expected, such as, for example local tumors.

[0284] The pharmaceutical compositions comprising antibody proteins of the present invention may be applied by different routes of application known to the expert, notably intravenous injection or direct injection into target tissues. For systemic application, the intravenous, intravascular, intramuscular, intraarterial, intraperitoneal, oral, or intrathecal routes are preferred. A more local application can be effected subcutaneously, intracutaneously, intracardially, intralobally, intramedullarly, intrapulmonarily or directly in or near the tissue to be treated (connective-, bone-, muscle-, nerve-, epithelial tissue). Depending on the desired duration and effectiveness of the treatment, pharmaceutical antibody compositions may be administered once or several times, also intermittently, for instance on a daily basis for several days, weeks or months and in different dosages.

[0285] For preparing suitable pharmaceutical compositions comprising antibody preparations for the applications described above, one may use known injectable, physiologically acceptable sterile solutions. For preparing a ready-to-use solution for parenteral injection or infusion, aqueous isotonic solutions, such as e.g. saline or corresponding plasma protein solutions are readily available. The pharmaceutical compositions may be present as lyophylisates or dry preparations, which can be reconstituted with a known injectable solution directly before use under sterile conditions, e.g. as a kit of parts. The final preparation of the antibody compositions of the present invention are prepared for injection, infusion or perfusion by mixing purified antibodies according to the invention with a sterile physiologically acceptable solution, that may be supplemented with known carrier substances or / and additives (e.g. serum albumin, dextrose, sodium bisulfite, EDTA).

[0286] The amount of the antibody applied depends on the nature of the disease. In cancer patients, the applied dose of a “naked” antibody which is comprised in the pharmaceutical composition according to the invention may be between 0.1 and 100 mg / m2, between 5 and 50 mg / m2 per application, 10 mg / m2to about 40 mg / m2, 10 mg / m2to about 30 mg / m2, also 20 mg / m2to about 30 mg / m2, and about 25 mg / m2body surface area. An antibody protein dose of about 50 mg / m2body surface area can also be used.

[0287] The dose of radioactivity applied to the patient per administration has to be high enough to be effective, but must be below the dose limiting toxicity (DLT). For pharmaceutical compositions comprising radiolabeled antibodies, e.g. with186Rhenium, the maximally tolerated dose (MTD) has to be determined which must not be exceeded in therapeutic settings. Application of radiolabeled antibody to cancer patients may then be carried out by repeated (monthly or weekly) intravenous infusion of a dose which is below the MTD (See e.g. Welt et al. (1994) J. Clin. Oncol. 12: 1193-1203). Multiple administrations are preferred, generally at weekly intervals; however, radiolabelled materials should be administered at longer intervals, i.e., 4-24 weeks apart, preferable 12-20 weeks apart. The artisan may choose, however, to divide the administration into two or more applications, which may be applied shortly after each other, or at some other predetermined interval ranging, e.g. from 1 day to 1 week.

[0288] Furthermore, the applied radioactivity dose will be in accordance with the guidelines outlined below. In general, the radioactivity dose per administration will be between 30 and 75 mCi / m2body surface area (BSA). Thus, the amount of radiolabelled antibody in the pharmaceutical composition according to the invention, labelled with186Rhenium,188Rhenium, ""'Technetium,133Iodine, or "Yttrium, preferably labelled with l86Rhenium, to be applied to a patient is 10, 20, 30, 40, 50 or 60 mCi / m2, preferably 50 mCi / m2. In one embodiment, the invention relates to a pharmaceutical composition, wherein the dose of said radiolabelled antibody according to the invention is MTD, 50 mCi / m2.

[0289] In certain embodiments, the pharmaceutical composition according to the invention further comprising one or more radioprotectants selected from the group of ascorbic acid, gentisic acid, reductic acid, erythrorbic acid, p-aninobenzoic acid, 4hydroxybenzoic acid, nicotinic acid, nicotinamide, 2-5-dihydroxy-l,4-benzenedisulfonic acid, povidone, inositol, and / or citrate. In certain embodiments, the radioprotectant is ascorbic acid.

[0290] An antibody or immunoconjugate of the invention can be combined in a pharmaceutical combination formulation, or dosing regimen as combination therapy, with a second compound, such as one that is known to be effective in treating a disease or disorder of interest. In some embodiments, the second compound is a anti-cancer agent. In some embodiments, the methods encompass administration of the second compound and an immunoconjugate of the invention that results in a better efficacy as compared to administration of the immunoconjugate alone. The second compound can be administered via any number of ways, including for example, topical, pulmonary, oral, parenteral, or intracranial administration. In some embodiments, the administration is oral. In some embodiments, the administration is intravenous. In some embodiments, the administration is both oral and intravenous.

[0291] An antibody or immunoconjugate can also be combined in a pharmaceutical combination formulation, or dosing regimen as combination therapy, with an analgesic, or other medications.

[0292] An antibody or immunoconjugate can be combined in a pharmaceutical combination formulation, or dosing regimen as combination therapy, with a second compound having anticancer properties. The second compound of the pharmaceutical combination formulation or dosing regimen can have complementary activities to the ADC of the combination such that they do not adversely affect each other. Pharmaceutical compositions comprising the RET- binding agent and the second anti-cancer agent are also provided.

[0293] In certain embodiments, the therapeutically effective amount of the subject antibodies or antigen-binding fragments thereof, or immuno-conjugates described herein, or a composition thereof, alone or in combination with a second therapeutic agent, preferentially inhibits the proliferation of leukemic stem cells (LSCs), leukemia progenitors (LPs), and / or leukemic blasts, over normal hematopoietic stem cells (HSCs). In certain embodiments, IC50 value or the half maximum concentration of the above subject agents to inhibit the proliferation of leukemic stem cells (LSCs), leukemia progenitors (LPs), and / or leukemic blasts, is at least 10-, 20-, 30-, 40-, 50-, 60-, 70-, 80-, 90-, 100-, 150-, 300-, 500-fold or more lower than that for the normal hematopoietic stem cells (HSCs).

[0294] Another aspect of the present invention provides a pharmaceutical composition for the treatment of a disease or condition, such as cancer, which comprises a CAR-T cell of the invention and a pharmaceutically acceptable carrier. In addition, the invention further claims CAR T cell of the invention in preparation for use of the medicine for treating the disease. The “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like physiologically compatible.

[0295] In certain embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).

[0296] 4. Method, of Treatment

[0297] The present invention includes a method of inhibiting abnormal cell growth or treating a proliferative disorder in a mammal (e.g., human) comprising administering to said mammal a therapeutically effective amount of the subject antibodies or antigen-binding fragments thereof, or immuno-conjugates described herein, or the subject CAR-T cell, or a composition thereof, alone or in combination with a second therapeutic agent.

[0298] A related aspect of the present invention provides the use of an antibody protein according to the invention in the manufacture of a medicament for treatment of cancer. Another aspect of the present invention relates to the use of antibody proteins according to the invention conjugated to a therapeutic agent as described above e.g., using an antibody-drug conjugate or ADC) for the treatment of cancer. Cancer includes any disease associated with malignant growth such as solid tumors, sarcomas and leukemias, as described herein below. A necessary precondition for such diseases is the expression of RET, e.g., a RET mutant as described herein or overexpression of RET.

[0299] The present invention also provides a method for inducing cell death in selected cell populations comprising contacting target cells or tissue containing target cells with an effective amount of the subject antibodies or antigen-binding fragments thereof, or immuno-conjugates, or CAR-T cells of the present invention. The target cells are cells to which the cell-binding agent of the conjugates or CARs can bind. The method of the invention for inducing cell death in selected cell populations, for inhibiting cell growth, and / or for treating cancer, can be practiced in vitro, in vivo, or ex vivo. For clinical in vivo use, the cytotoxic compounds or conjugates of the invention will be supplied as a solution or a lyophilized powder that are tested for sterility and for endotoxin levels. In certain embodiments, the abnormal cell growth or proliferative disorder in a mammal is a disease or condition associated with or characterized by the expression of RET, such as cancer.

[0300] Inhibitory mouse monoclonal anti-RET antibodies were generated and screened. Several anti-RET antibodies that can bind to the extracellular domain of the wild-type RET protein and inhibit RET signaling in cancer cells with inhibitory function were obtained.

[0301] To confirm these antibodies' binding specificity to RET, immunofluorescence staining with the purified antibodies was performed. Mouse NIH-3T3 cells were stably transfected with the human RET expression plasmid and empty vector control. In immunofluorescence staining assay, the representative anti-RET antibody could only stain NIH-3T3 cells with human RET expression but not NIH-3T3 cells with vector control.

[0302] It was further examined whether the anti-RET antibody could bind RET expressed in NB cells. A representative anti-RET antibody bound to human MYCN-amplified cell line NGP, non-amplified cell line CHLA255, and mouse NB cell line NB975.

[0303] Anti-RET antibodies also inhibited cell proliferation in NGP and NB975 cells. The effect of anti-RET antibodies on RET-mediated signaling in NB cells was also examined. It was found that GDNF-induced S6 phosphorylation in NB cells was blocked by anti-RET antibodies.

[0304] Together, these data demonstrated that this anti-RET antibody could bind to RET extracellular domain on the NB cell surface and block GDNF-induced RET signaling in NB cells. Both heavy and light chain cDNA were cloned into expression vectors that encode this antibody.

[0305] The specificity of the anti-RET antibody was further evaluated by performing flow cytometry analysis with the purified anti-RET antibodies. It was found that anti-RET antibody can detect RET which was stably expressed in A549 cells in flow cytometry assay. In addition, the anti-RET antibody is also bound to human NB cell line SK-N-AS, murine NB cell line NB975 and Human AML cell line THP1 in flow cytometry assay. Thus, the anti-RET antibody could be used in immunofluorescence staining of tissues, such as NB tissue sections.

[0306] Chemotherapy resistance contributes to treatment failure in NB and inevitably results in relapse. Therefore, the ability to enhance current chemotherapy efficacy and overcome established chemo-resistance is critical for treating high-risk NB. It was found that the RET inhibition by the inhibitory anti-RET antibodies had synergistic anti-tumor effects in combination with standard-of-care or conventional chemotherapeutic agents, such as in NB cells.

[0307] Furthermore, the anti-RET mouse monoclonal antibodies were humanized. Humanized anti-RET antibody mammalian expression vectors were generated by inserting the appropriate mouse complementarity-determining region (CDR) coding segments into a human antibody scaffold. Humanized anti-RET antibody was purified. It is bound to RET well compared to its original mouse version in flow cytometry analysis with NB cell lines. Additionally, the humanized anti-RET antibody also inhibited the cell proliferation in vitro and tumor growth in vivo of these cells. Furthermore, the humanized anti-RET antibody inhibits cell proliferation of cancer cells with wild-type RET expression including AML, small cell lung cancer, Ewing sarcoma, and breast cancer. Antibodies or CAR therapeutics based on the anti-RET antibody sequences can be used to treat NB, acute myeloid leukemia (AML), small cell lung cancer, Ewing sarcoma, and breast cancer that are addicted to high wild-type RET expression.

[0308] Thus in one aspect, the invention provides a method for inhibiting the growth of a cell expressing RET (e.g., RET expressed from a wild-type or mutated RET gene), the method comprising contacting the cell with any of the subject anti-RET antibody or antigen-binding fragment thereof, or the subject polypeptide thereof, or the subject immunoconjugate thereof, or the subject CAR-T cell, or the subject pharmaceutical composition thereof. In some embodiments, the method comprises administering a therapeutically effective amount of any of the subject anti-RET antibody or antigen-binding fragment thereof, or the subject polypeptide thereof, or the subject immunoconjugate thereof, or the subject CAR-T cell, or the subject pharmaceutical composition thereof to a subject in need thereof.

[0309] A related aspect of the invention provides a method for treating a cancer / tumor characterized / caused by the cell expression of RET (e.g., RET expressed from a wild-type or mutated RET gene), the method comprising contacting the cell with any of the subject anti- RET antibody or antigen-binding fragment thereof, or the subject polypeptide thereof, or the subject immunoconjugate thereof, or the subject CAR-T cell the subject pharmaceutical composition thereof. In some embodiments, the method comprises administering a therapeutically effective amount of any of the subject anti-RET antibody or antigen-binding fragment thereof, or the subject polypeptide thereof, or the subject immunoconjugate thereof, or the subject CAR-T cell, or the subject pharmaceutical composition thereof to a subject in need thereof.

[0310] Without be bound by theory, the treatment methods described herein are applicable to cancer or tumor expressing either wild-type RET or mutated RET. The antibody or antigenbinding fragment thereof targets the extracellular domain of RET. Thus, any of the antibody or antigen-binding fragment thereof, immunoconjugate, CAR-T cells or compositions thereof described herein can target a RET protein, even the mutated form, as long as the binding epitope on the extracellular domain of the RET protein is preserved (e.g., when the primary sequence of the epitope is not mutated, or when the conformation of the epitope remains substantially the same despite having a mutation in the primary sequence).

[0311] In certain embodiments, the cell is a tumor cell. In some embodiments, the tumor cell overexpresses RET (e.g., wild-type or mutated RET) compared to normal cells.

[0312] In certain embodiments, the tumor cell comprises wild-type RET gene.

[0313] In certain embodiments, the tumor cell comprises a mutated RET gene.

[0314] In certain embodiments, the mutated RET gene is capable of oncogenic RET ligandindependent phosphorylation, and / or leading to constitutive activation of a downstream signal transduction cascade (such as MAPK pathway, PI3K / AKT pathway, PLCy pahway, and SRC pathway).

[0315] In certain embodiments, the mutated RET gene is a RET point mutation. In certain embodiments, the mutated RET gene comprises a germline or sporadic mutation activating RET’s kinase domain. In certain embodiments, said mutation activating RET’s kinase domain is capable of triggering monomeric activation of RET. In certain embodiments, the mutation activating RET’s kinase domain comprises a RET M918T mutation, a C634R mutation, and / or a V804 gatekeeper mutation (such as V804M).

[0316] In certain embodiments, the mutated RET gene comprises an E511K mutation, an R114H mutation, an M I I09I / T mutation, an R525Q mutation, an R600Q mutation, a V706M mutation, an A756V mutation, an M255I mutation, an R163Q mutation, and / or a T636M mutation.

[0317] In certain embodiments, the tumor cell is from breast carcinoma, pheochromocytoma, neuroendocrine malignancy (including paraganglioma, pheochromocytoma, pulmonary or extra-pulmonary carcinoid tumor), parathyroid hyperplasia and adenoma, intestinal ganglioneuromas, mucosal neuromas, gastrointestinal malignancy (such as colorectal adenocarcinoma, gastrointestinal stromal tumor, or hepatocellular carcinoma), or pancreatic acinar cell carcinoma (PACC).

[0318] In certain embodiments, the tumor cell is from breast cancer (such as HER2-negative breast cancer), oesophagus cancer, stomach cancer, pancreas cancer, colon / rectum cancer, prostate cancer, and soft tissue cancer, a cancer of the thyroid gland (e.g., anaplastic or poorly differentiated), a cancer of the lung (e.g., pleomorphic, adenocarcinoma, large cell neuroendocrine), papillary thyroid cancer, esophageal cancer, salivary gland carcinoma (such as salivary intraductal carcinomas, salivary gland mammary analogue secretory carcinomas (MASCs)), spitzoid neoplasm (such as spitzoid melanomas and atypical spitzoid tumor), pediatric spindle-cell mesenchymal neoplasm, histiocytic neoplasm, cutaneous xanthogranuloma, pancreatic ductal adenocarcinoma, stomach adenocarcinoma, esophageal cancer, cholangiocarcinoma, bladder carcinoma, head and neck cancer, mesothelioma, low- grade glioma, atypical lung carcinoid tumor, chronic myeloproliferative neoplasm, hepatobiliary cancer, glioblastoma, and bladder urothelial carcinoma.

[0319] In certain embodiments, the tumor cell is from a lung cancer (such as NSCLC or SCLC).

[0320] In certain embodiments, the tumor cell is from a thyroid cancer. In certain embodiments, the thyroid cancer is a papillary thyroid cancer (PTC). In certain embodiments, the thyroid cancer is a medullary thyroid cancer (MTC, such as familial medullary thyroid cancer (FMTC), multiple endocrine neoplasia type 2A (MEN 2A) syndrome, or multiple endocrine neoplasia type 2B (MEN 2B) syndrome). In certain embodiments, the thyroid cancer is a poorly differentiated thyroid carcinoma. In certain embodiments, the thyroid cancer is an anaplastic thyroid cancer.

[0321] In certain embodiments, the tumor cell is from neuroblastoma (NB).

[0322] In certain embodiments, the tumor cell is from Ewing sarcoma.

[0323] In certain embodiments, the tumor cell is from breast cancer.

[0324] In some embodiments, the cancer is selected from the group consisting of neuroblastoma (NB), Ewing sarcoma, small cell lung cancer (SCLC), breast cancer, and any cancer in which the tumor cells express RET.

[0325] Neuroblastoma is a cancer that develops from immature nerve cells found in several areas of the body. Neuroblastoma most commonly affects children age 5 or younger, though it may rarely occur in older children. RET’s role in neuroblastoma has been established through numerous studies. Neuroblastoma tumor cells express significantly higher levels of RET when compared across many other cancer cell lines. Initial studies demonstrated that transgenic mice overexpressing RET develop neuroblastoma tumors. RET expression was also shown to be associated with increased neuroblastoma metastases in vivo, and RET expression is higher in neuroblastoma tumors from patients with stage 4 and high-risk disease. Neuroblastoma cell lines were also found to be the most sensitive of cancer cell lines in the CCLE to RET depletion with RNAi. RET inhibition has become an increasingly important therapeutic strategy for treatment of NB (see, Steen et al. “Targeting the RET tyrosine kinase in neuroblastoma: A review and application of a novel selective drug design strategy.” Biochem Pharmacol. 2023 Oct;216: 115751; incorporated herein by reference).

[0326] Ewing sarcoma is a type of cancer that begins as a growth of cells in the bones and the soft tissue around the bones. Ewing sarcoma mostly happens in children and young adults, although it can happen at any age.

[0327] Small-cell lung cancer (SCLC) is a highly malignant pulmonary neuroendocrine tumor representing 15% of all lung carcinomas and is strongly associated with cigarette smoking. No significant improvement in SCLC treatment has emerged over the past 30 years. Oncogenic somatic mutation of RET has been found in a subset of patients with SCLC (see Dabir et al. “RET Mutation and Expression in Small-Cell Lung Cancer” J Thoracic Onco. 2014, 9(9): 1316-1323; incorporated herein by reference). Tyrosine kinase inhibitors targeting RET have been explored for lung treatment, though acquired resistance to small molecule drugs have been observed.

[0328] Breast cancer is the second most common cancer diagnosed in women in the United States. The recent discovery of RET enrichment in breast cancer brain metastases suggests a role for RET inhibition specific to advanced disease. In breast cancer, RET has been primarily studied in the context of estrogen-receptor positive (ER+) disease. While RET expression and function is significantly associated with ER positivity, RET overexpression has also been identified in the ER negative (ER-), triple negative (TN) and HER2- amplified breast cancer sub-group. Gains in RET expression have been reported in multiple in vitro models of endocrine therapy-resistant breast cancer. Current understanding of RET supports a case for using RET-selective inhibitors for treatment of multiple breast cancer phenotype (see Pecar et al. “RET signaling in breast cancer therapeutic resistance and metastasis” Breast Cancer Res 25, 26 (2023); incorporated herein by reference).

[0329] Another aspect of the invention relates to the use of an antibody protein according to the invention as defined supra in the manufacture of a medicament for treatment of cancer, wherein the amount of antibody protein per application is between 0.1 and 100 mg / m2, between 5 and 50 mg / m2, 10 mg / m2to about 40 mg / m2, 10 mg / m2to about 30 mg / m2, or 20 mg / m2to about 30 mg / m2, or about 25 mg / m2body surface area, or about 50 mg / m2body surface area.

[0330] In certain embodiments, an antibody protein conjugated to a radioisotope according to the invention as defined supra is used in the manufacture of a medicament for treatment of cancer, wherein the radioactivity dose per administration is between 30 and 75 mCi / m2body surface area (BSA). In certain embodiments, the antibody protein according to the invention is radiolabelled with186Rhenium,188Rhenium, ""'Technetium,131Iodine, or "Yttrium, such as186Rhenium. In yet another embodiment, the invention relates to the use of an antibody protein conjugated to a radioisotope according to the invention as defined supra in the manufacture of a medicament for treatment of cancer, wherein to antibody dose is 10, 20, 30, 40, 50 or 60 mCi / m2, or 50 mCi / m2.

[0331] In certain embodiments, an antibody protein conjugated to a radioisotope according to the invention as defined supra is used in the manufacture of a medicament for treatment of cancer, wherein the antibody protein has specific activity of from about 0.5 to about 15 mCi / mg, or from about 0.5 to about 14 mCi / mg, preferably about 1 to about 10 mCi / mg, preferably about 1 to about 5 mCi / mg, and most preferably 2 to 6 mCi / mg or 1 to 3 mCi / mg.

[0332] Preferred also is the use of an antibody protein conjugated to a radioisotope according to the invention as defined supra in the manufacture of a medicament for treatment of cancer, wherein said antibody or antibody derivative is in an aqueous solution at pH of from about 7 to about 8, and at a concentration of from about 0.5 to about 2.0 mg / ml.

[0333] The invention further relates to a method of cancer treatment, wherein an antibody protein according to the invention is administered once to several times to an individual in need thereof, said antibody protein selectively binds to RET, destroys tumor cells via the therapeutic agent linked to the antibody protein (such as an ADC) and the therapeutic success is monitored. Said antibody protein may be present as naked / unmodified antibody protein, modified antibody protein, such as fusion protein, or antibody protein conjugated to a therapeutic agent (such as ADC), which comprises contacting the tumor with an effective amount of said antibodies. The method of treating tumors as described above may be effective in vitro or in vivo. Cancer is any cancer as described above.

[0334] Cancer therapies and their dosages, routes of administration and recommended usage are known in the art and have been described in such literature as the Physician's Desk Reference (PDR). The PDR discloses dosages of the agents that have been used in treatment of various cancers. The dosing regimen and dosages of these aforementioned chemotherapeutic drugs that are therapeutically effective will depend on the particular cancer being treated, the extent of the disease and other factors familiar to the physician of skill in the art and can be determined by the physician. The contents of the PDR are expressly incorporated herein in its entirety by reference. One of skill in the art can review the PDR, using one or more of the following parameters, to determine dosing regimen and dosages of the chemotherapeutic agents and conjugates that can be used in accordance with the teachings of this invention. These parameters include: Comprehensive index; Manufacturer; Products (by company's or trademarked drug name); Category index; Generic / chemical index (non- trademark common drug names); Color images of medications; Product information, consistent with FDA labeling; Chemical information; Function / action; Indications & Contraindications; Trial research, side effects, warnings.

[0335] The amount of the antibody applied depends on the nature of the disease. In cancer patients, the applied dose of a “naked” antibody may be between 0.1 and 100 mg / m2, between 5 and 50 mg / m2per application, 10 mg / m2to about 40 mg / m2, 10 mg / m2to about 30 mg / m2, also 20 mg / m2to about 30 mg / m2, and about 25 mg / m2body surface area, or about 50 mg / m2 body surface area.

[0336] The dose of radioactivity applied to the patient per administration has be high enough to be effecfive, but must be below the dose limiting toxicity (DLT). For radiolabeled antibodies, e.g. with186Rhenium, the maximally tolerated dose (MTD) has to be determined which must not be exceeded in therapeutic settings. Application of radiolabeled antibody to cancer patients may then be carried out by repeated (monthly or weekly) intravenous infusion of a dose which is below the MTD (See e.g. Welt et al. (1994) J. Clin. Oncol. 12: 1193-1203). Multiple administrations are preferred, generally at weekly intervals; however, radiolabelled materials should be administered at longer intervals, i.e., 4-24 weeks apart, or 12-20 weeks apart. The artisan may choose, however, to divide the administration into two or more applications, which may be applied shortly after each other, or at some other predetermined interval ranging, e.g. from 1 day to 1 week.

[0337] Also provided a method of cancer treatment according to the invention (see above), wherein the antibody protein conjugated to a radioisotope according to the invention as defined supra has specific activity of from about 0.5 to about 15 mCi / mg, or from about 0.5 to about 14 mCi / mg, preferably about 1 to about 10 mCi / mg, preferably about 1 to about 5 mCi / mg, and most preferably 2 to 6 mCi / mg or 1 to 3 mCi / mg.

[0338] Also provided is a method of cancer treatment according to the invention (see above), wherein the antibody protein conjugated to a radioisotope according to the invention as defined supra is in an aqueous solution at pH of from about 7 to about 8, and at a concentration of from about 0.5 to about 2.0 mg / ml.

[0339] In a treatment using the CAR-T cell of the invention, the amount and the dosage level of the CAR-T cells in the pharmaceutical composition of the invention may be varied depending on specific patient need, the mode of administration, the type and / or degree of cancer in a subject, the desired therapeutic response, the tolerable toxicity to the patient, as well as other factors deemed relevant by an attending physician. That is, the selected dosage level may depend on a variety of pharmacokinetic factors including the particular composition used, the route of administration, the age of the patient, other pharmaceutical composition used in conjunction, duration and time of administration, rate of excretion or elimination, gender, weight, condition, general health condition and medical history, and like factors of the patient, as is generally known in the medical field. One of the ordinary skill in the art can empirically determine the effective amount of the invention without necessitating undue experimentation. Combined with the teachings provided herein, by choosing among the various active CAR-T cells and weighing factors such as potency, relative bioavailability, patient body weight, severity of adverse side-effects and preferred mode of administration, an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial toxicity in and of itself and yet is entirely effective to treat the particular subject.

[0340] In certain embodiments, the method further comprises administering an immune checkpoint inhibitor such as a PD-1 inhibitor (e.g. pembrolizumab, nivolumab, and cemiplimab), a PD-L1 inhibitor (e.g. atezolizumab, avelumab, and durvalumab), a CTLA-4 targeting agents (e.g. ipilimumab), or an immunomodulating agent (e.g. thalidomide and lenalidomide).

[0341] In some embodiments, the method further comprises administering to the subject radiotherapy and / or chemotherapy and / or surgery and / or other tumor-targeting drug (e.g., targeting monoclonal antibody of other antigen or small molecule compounds).

[0342] In certain embodiments, the chemotherapy includes one or more of all-trans retinoic acid, Actinomycin D, Adriamycin, anastrozole, Azacitidine, Azathioprine, Alkeran, Ara-C, Arsenic Trioxide (Trisenox), BiCNU Bleomycin, Busulfan, CCNU, Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide, Cytarabine, Cytoxan, DTIC, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, 5-flurouracil, Epirubicin, Epothilone, Etoposide, exemestane, Erlotinib, Fludarabine, Fluorouracil, Gemcitabine, Hydroxyurea, Herceptin, Hydrea, Ifosfamide, Irinotecan, Idarubicin, Imatinib, letrozole, Lapatinib, Leustatin, 6-MP, Mithramycin, Mitomycin, Mitoxantrone, Mechlorethamine, megestrol, Mercaptopurine, Methotrexate, Mitoxantrone, Navelbine, Nitrogen Mustard, Oxaliplatin, Paclitaxel, pamidronate disodium, Pemetrexed, Rituxan, 6-TG, Taxol, Topotecan, tamoxifen, taxotere, Teniposide, Tioguanine, toremifene, trimetrexate, trastuzumab, Valrubicin, Vinblastine, Vincristine, Vindesine, Vinorelbine, Velban, VP- 16, and / or Xeloda.

[0343] The method of the invention also provides in vitro method to kill cells, such as cancer cells. Examples of in vitro uses include treatments of autologous bone marrow prior to their transplant into the same patient in order to kill diseased or malignant cells: treatments of bone marrow prior to their transplantation in order to kill competent T cells and prevent graft-versus- host-disease (GVHD); treatments of cell cultures in order to kill all cells except for desired variants that do not express the target antigen; or to kill variants that express undesired antigen.

[0344] The conditions of non-clinical in vitro use are readily determined by one of ordinary skill in the art.

[0345] Examples of clinical ex vivo use are to remove tumor cells or lymphoid cells from bone marrow prior to autologous transplantation in cancer treatment or in treatment of autoimmune disease, or to remove T cells and other lymphoid cells from autologous or allogenic bone marrow or tissue prior to transplant in order to prevent GVHD. Treatment can be carried out as follows. Bone marrow is harvested from the patient or other individual and then incubated in medium containing serum to which is added the cytotoxic agent of the invention, concentrations range from about 10 pM to 1 pM, for about 30 minutes to about 48 hours at about 37°C. The exact conditions of concentration and time of incubation, i.e., the dose, are readily determined by one of ordinary skill in the art. After incubation the bone marrow cells are washed with medium containing serum and returned to the patient intravenously according to known methods. In circumstances where the patient receives other treatment such as a course of ablative chemotherapy or total-body irradiation between the time of harvest of the marrow and reinfusion of the treated cells, the treated marrow cells are stored frozen in liquid nitrogen using standard medical equipment.

[0346] 5. Nucleic Acid

[0347] A further aspect of the present invention is a nucleic acid, characterised in that it codes for an antibody or protein according to the invention. Said nucleic acid may be RNA or preferably DNA. Said DNA molecule may be chemically synthesized. First, suitable oligonucleotides can be synthesized with methods known in the art (e.g. Gait, M. J., 1984, Oligonucleotide Synthesis. A Practical Approach. IRL Press, Oxford, UK), which can be used to produce a synthetic gene. Methods to generate synthetic genes are known in the art (e.g. Stemmer et al. 1995, Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides, Gene 164(1): 49-53; Ye et al. 1992, Gene synthesis and expression in E. coli for pump, a human matrix metalloproteinase, Biochem Biophys Res Commun 186(1): 143-9; Hayden et Mandecki 1988, Gene synthesis by serial cloning of oligonucleotides, DNA 7(8): 571-7). These methods can be used to synthesize any DNA molecule disclosed in the present application.

[0348] The nucleic acid according to the invention may contain 5’ or 3’ or 5’ and 3’ untranslated regions. The nucleic acid according to the invention may contain other untranslated regions upstream and / or downstream. The untranslated region may contain a regulatory element, such as e.g. a transcription initiation unit (promoter) or enhancer. Said promoter may, for example, be a constitutive, inducible or development-controlled promoter. In certain embodiments, and without ruling out other known promoters, the constitutive promoters of the human Cytomegalovirus (CMV) and Rous sarcoma virus (RSV), as well as the Simian virus 40 (SV40) and Herpes simplex promoter. Inducible promoters according to the invention comprise antibiotic-resistance promoters, heat- shock promoters, hormone- inducible “Mammary tumour virus promoter” and the metallothioneine promoter. The nucleic acid according to the invention may code for a fragment of the antibody protein according to the invention. This refers to part of the polypeptide according to the invention.

[0349] 6. Vector

[0350] Another important aspect of the present invention is a recombinant DNA vector, characterized in that it contains a nucleic acid according to the invention. Examples are viral vectors such as e.g. Vaccinia, Semliki-Forest- Virus and Adenovirus. Vectors for use in 293T- cells have the SV40 origin of replication and make it possible to achieve high copy numbers of the plasmids. Vectors for use in insect cells are, for example, E. coli transfer vectors and contain e.g. the DNA coding for polyhedrin as promoter.

[0351] Another aspect of the present invention is a recombinant DNA vector according to the invention, characterized in that it is an expression vector.

[0352] Another aspect of the present invention is a recombinant DNA vector according to the invention, characterized in that it is vector pAD-CMV or a functional derivative thereof. Such derivatives are e.g. pAD-CMVl, pAD-CMV19 or pAD-CMV25.

[0353] The vector may be the ones disclosed in U.S. Pat. Nos. 5,648,267 A or 5,733,779 A comprising a nucleotide sequence according to the invention. Another aspect of the present invention is a recombinant DNA vector according to the invention, characterized in that it is vector N5KGlVal or a derivative thereof.

[0354] 7. Cell or Host Cell

[0355] Another aspect is a host, characterized in that it contains a vector according to the invention.

[0356] Another aspect is a host according to the invention, characterized that it is a eukaryotic host cell. The eukaryotic host cells according to the invention include fungi, such as e.g. Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, Trichoderma, insect cells (e.g. from Spodoptera frugiperda Sf-9, with a Baculovirus expression system), plant cells, e.g. from Nicotiana tabacum, mammalian cells, e.g. COS cells, BHK, CHO or myeloma cells.

[0357] In descendants of the cells of the immune system in which antibody proteins are also formed in our body, the antibody proteins according to the invention are particularly well folded and glycosylated. Mammalian host cells, preferably CHO or COS cells are preferred, e.g. a CHO DG44 (Urlaub and Chasin, Proc. Natl. Acad. Sci. U.S.A. 77(7): 4216-20 (1980)), or CHO-K1 (ATCC CCL-61) cells. Thus, another aspect is a host according to the invention according to the invention, characterized in that it is a BHK, CHO or COS cell, most preferred CHO DG44 or CHO-K1 (ATCC CCL-61) cells.

[0358] In certain embodiments, the host is a bacteriophage.

[0359] In certain embodiments, the host is a prokaryotic host cell. Examples of prokaryotic host cells are Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis.

[0360] The invention further relates to a process for preparing an antibody protein according to the invention, characterized in that it comprises the following steps: a host according to the invention is cultivated under conditions in which said antibody protein is expressed by said host cell and said antibody protein is isolated. The antibody according to the invention may be produced as follows. Nucleic acid molecules coding for the light chain and the heavy chain may be synthesized chemically and enzymatically by standard methods. First, suitable oligonucleotides can be synthesized with methods known in the art (details supra). Methods to generate synthetic genes from oligonucleotides are known in the art (details supra). These nucleic acid molecules encoding the antibody heavy and light chains may be cloned into an expression vector (either both chains in one vector molecule, or each chain into a separate vector molecule), which then is introduced into a host cell. The host cell may be a mammalian host cell (details supra), e.g. a COS, CHO (Chinese Hamster Ovary), or BHK cell. The host cell then is cultured in a suitable culture medium under conditions where the antibody is produced, and the antibody is then isolated from the culture according to standard procedures. Procedures for production of antibodies from recombinant DNA in host cells and respective expression vectors are well-known in the art (see e.g. WO 94 / 11523, WO 97 / 9351, EP 0481790).

[0361] The invention also relates to a process, wherein the host is a mammalian cell, preferably a CHO or COS cell.

[0362] In certain embodiments, the host cell is co-transfected with two plasmids which carry the expression units for the light or the heavy chain. EXAMPLES

[0363] The invention described herein is exemplified by the data presented in the series of slides incorporated herein below.

[0364] A representative humanized anti-RET antibody used in the example, including the heavy chain (human IgGl Fc) sequence, with the three CDR sequences doble-underlined, is provided below (SEQ ID NO: 9). The VH CDR1 (KASGYTFTTYGMS), CDR2 (WINTYS), and CDR3 (ARGSMDY) sequences are SEQ ID NOs: 1-3, respectively. The VH sequence is SEQ ID NO: 7.

[0365] QVQLVQS GSE LKKP GAP VKVS CKASGYTFTTYGMSWVROAP GQGLE WMGWINTYSGVP T YAQ GFTGRFVFSLDTSVSTAYLOISSLKAEDTAVYYCARGSMDYWGOGTLVTVSSASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSSGLYSLSSWTV PSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQD WLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK ( SEQ ID NO : 9 )

[0366] The humanized anti-RET antibody kappa light chain sequence, with the three CDR sequences double underlined, is provided below (SEQ ID NO: 10). The VL CDR1 (RSSQSLLDSDGKTYLN), CDR2 (YLVSELDS), and CDR3 (WQGTHFPL) sequences are SEQ ID NOs: 4-6, respectively. The VL sequence is SEQ ID NO: 8.

[0367] DWMTOSPLSLPVTLGOPASISCRSSQSLLDSDGKTYLNWFOORPGOSPRRLIYLVSELDSG VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPLTFGGGTKVEIKRTVAAPSVFIFP PSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC ( SEQ ID NO : 10 )

[0368] Many receptor tyrosine kinases (RTKs) have been shown to promote cancer development. Currently, several antibodies targeting RTKs such as EGFR and HER2 have been approved as therapeutics for cancer patients. As key regulators of the development and progression of many types of cancer, RTKs are ideal cancer targets for the development of antibody therapeutics.

[0369] Recent studies suggest that RET RTK promotes the development and progression of several types of cancers, including NB, breast cancer, Ewing sarcoma, acute myeloid leukemia (AML), and small cell lung cancer.

[0370] Physiologically, RET is activated by glial cell line-derived neurotrophic factor (GDNF) ligands that bind to coreceptor GDNF family receptor alphas (GFRas), leading to RET dimerization. GDNF-GFRal-RET signaling plays crucial roles in the development of the enteric nervous system, kidney, and lower urinary tract as well as in spermatogenesis.

[0371] It was hypothesized that RET is an ideal target for the development of antibody therapeutics. Here Applicant provides evidence that a humanized anti-RET antibody has been successfully developed, which antibody inhibited RET signaling and cell proliferation of NB, AML, small cell lung cancer, Ewing sarcoma, and breast cancer cell lines, and sensitized the cancer cells to chemotherapy, through binding to the extracellular domain of RET. Importantly, anti-RET antibody could inhibit NB growth in an immunocompetent mouse model. RET-CAR T cells significantly exerted effectiveness in killing AML cells in a xenogeneic mouse model.

[0372] In addition, this anti-RET antibody can also serve as a diagnostic tool for immunohistochemistry (IHC) staining of paraffin-embedded tumor tissue section and flow cytometry assay of tumor cells.

[0373] Example 1. Generation of inhibitory mouse monoclonal anti-RET antibodies.

[0374] To generate mouse anti-RET monoclonal (mAb) antibodies, mice were immunized with recombinant RET proteins, and splenocytes were isolated and fused with mouse myeloma SP2 / 0-AG14 cells (ATCC® CRL- 1581™) to form hybridomas. The antibody supernatant from individual clones was used to screen the RET extracellular domain's binders by ELISA assay. Then these binders were further examined for their effect on NB cell proliferation. Through this screening of more than 500 hybridoma clones, six anti-RET monoclonal antibodies were identified to be able to bind to the RET extracellular domain on the NB cell surface and inhibit NB cell proliferation. To confirm these antibodies' binding specificity to RET in NB cells, immunofluorescence staining with the purified antibodies was performed. First, mouse NIH- 3T3 cells were stably transfected with the human R£T-3Flag expression plasmid and empty vector control. In this immunofluorescence staining assay, the representative anti-RET antibody can only detect NIH-3T3 cells with human RET expression but not NIH-3T3 cells with vector control (FIGs. 1A and IB). In this assay, cells were fixed with paraformaldehyde without treatment with permeabilization buffer. It suggests that our anti-RET antibody can bind the extracellular domain of RET. Whether the anti-RET antibody could bind RET expressed in NB cells was further examined.

[0375] As shown in FIGs 2A-2G, the representative anti-RET antibody could bind to human A / YCA-amplified cell line NGP, non-amplified cell line CHLA255, and murine NB cell line NB975 (FIGs. 2A-2C). Anti-RET antibodies also inhibited cell proliferation in NGP, CHLA255 and NB975 cells (FIGs. 2D-2F). The effect of anti-RET antibodies on RET- mediated signaling in NB cells was also examined. As shown in FIG. 2G, anti-RET antibodies blocked S6 phosphorylation.

[0376] To further confirm anti-RET antibodies' binding specificity to RET in NB cells, flow cytometry analysis was performed with the purified antibodies. A549 cells were stably transfected with the human / ?ET-3Flag and mouse Ret-3Fag expression plasmids. After validation by immunoblotting assay, all cells were stained with the anti-RET antibody (50 pg / ml) for 1 hour in 4 °C. The results show that the anti-RET antibody could specifically bind to A549-hRET and A549-mRet cells but could not bind to A549 cells (FIG. 3A). In addition, the anti-RET antibody could also bind to human NB cell line SK-N-AS and murine NB cell line NB975 (FIG. 3B). Additionally, the anti-RET antibody could detect the expression of RET in the xenograft human NB tumor tissue using immunofluorescence (IF) (FIG. 3C) and immunohistochemistry (IHC) staining (FIG. 3D). Additionally, a comprehensive tissue microarray screening of 31 different types of normal human tissues (T8234708, Biochain) showed no off-target binding by the anti-RET antibody (data not shown).

[0377] Example 2. RET antibody mediates Antibody-Dependent Cellular Cytotoxicity (ADCC) against NB cells and inhibits NB growth in an immunocompetent NB mouse model

[0378] The anti-RET antibodies work by targeting the RET protein, which is often overexpressed in NB cells. These antibodies mark the cancer cells for destruction by the immune system. A process called Antibody-Dependent Cellular Cytotoxicity (ADCC) then occurs, where immune cells- particularly Natural Killer (NK) cells-recognize the marked NB cells and release toxic substances to eliminate them. Mouse anti-RET antibody was found to effectively induce ADCC against murine NB cell line NB975 using mouse spleen cells as effector cells (FIG. 4).

[0379] To evaluate the efficacy of the anti-RET antibody against tumors in vivo, an immunocompetent mouse model of NB was employed. Firefly luciferase-expressing NB975 cells (NB975-Fluc) were injected intraperitoneally (i.p.) into C57BL / 6 mice (106cells per mouse). Mice then received either the anti-RET antibody or the anti-RET-dKO antibody (200 pg per mouse) three times a week for two weeks, delivered intraperitoneally (FIG. 5A). Tumor growth was monitored weekly using bioluminescent imaging. Compared to the control group, both antibody treatments (anti-RET and anti-RET-dKO) significantly inhibited NB tumor growth and extended the lifespan of the mice (FIGs. 5B-5C). Notably, in the group treated with the anti-RET-dKO antibody, no tumor recurrence was observed within 20 weeks after tumor implantation, suggesting that this treatment led to a complete remission without recurrence.

[0380] Example 3. RET antibody can bind to RET positive AML cells and inhibit cell proliferation.

[0381] Binding specificity of the RET antibody was validated, and whether it could inhibit AML proliferation was evaluated. A549 cells were stably transfected with the human RET- 3Flag and mouse Ret-3Fag expression plasmids. After validation by immunoblotting assay, all cells were stained with the RET antibody (50 pg / mL) for 1 hour in 4 °C. The results showed that the RET antibody could specifically bind to A549-hRET and A549-mRet cells but could not bind to A549 cells using flow cytometry (FIG. 6A). Moreover, our RET antibody could bind to the RET positive AML cell lines THP-1, MOLM-13, and Kasumi-1 but could not bind the RET-negative KG- la cell line or human PBMC (FIG. 6B). Furthermore, the RET antibody could inhibit THP-1, MOLM-13, and Kasumi-1 cell proliferation in vitro (FIG. 6C) but had no effect on KG-1 cell proliferation. These results suggest that our RET antibody can specifically bind to RET positive AML cells and inhibit their cell proliferation.

[0382] Example 4. The humanized anti-RET antibody can bind to RET, inhibit NB cell proliferation, and mediate ADCC

[0383] To enhance the therapeutic potential, humanized versions of the mouse anti-RET antibody were generated. This involved grafting the mouse antibody's antigen-binding regions (complementarity-determining regions or CDRs) onto a human antibody scaffold. Following purification, we evaluated the humanized antibody's functionality. As shown in FIGs. 7A-7E, the humanized antibody could effectively bind to RET-positive cancer cells, including human A549-RET cells (FIG. 7A), human NB cell lines CHLA136, and NGP (FIG. 7B). Furthermore, the humanized antibody inhibited the proliferation of these RET-positive NB cells (FIG. 7C) and triggered ADCC against them (FIG. 7D). Additionally, co-culturing NK cells with NB cells in the presence of the antibody stimulated IFNy secretion by NK cells (FIG. 7E), indicating that this antibody induced NK activation and killing of target NB cells.

[0384] The therapeutic potential of the humanized RET antibody in treating NB using a human NB xenograft model was further assessed. Firefly luciferase-expressing NGP cells (NGP-Fluc) were injected intraperitoneally (i.p.) into NSG mice (10A6 cells per mouse). The mice then received either a control treatment or the humanized RET antibody (200 pg per mouse) three times a week for three weeks, delivered intraperitoneally (FIG. 8A). Tumor growth was significantly inhibited in the humanized RET antibody treatment group compared to the control group (FIG. 8B).

[0385] Example 5. The humanized RET antibody can effectively bind to RET-positive cancer cell lines and inhibit their proliferation

[0386] The humanized RET antibody demonstrated a broad binding profile, recognizing RET expression across a diverse panel of cancer cell lines (FIG. 9A). This included binding to BC cell line MDA-MB-361, AML cell lines THP-1, SCLC cell line SHP-77, and Ewing's sarcoma cell line A-673. Importantly, the humanized RET mAb effectively inhibited the proliferation of these RET-positive cancer cell lines (FIG. 9B). Furthermore, the antibody induced ADCC against the targeted cancer cells (FIG. 9C).

[0387] Example 6. RET antibody has synergistic anti-tumor effect with conventional chemotherapeutic agents in NB cells

[0388] Chemotherapy resistance contributes to treatment failure in NB and inevitably results in relapse. Therefore, the ability to enhance current chemotherapy efficacy and overcome established chemo-resistance is critical for high-risk NB patients. It was found that the RET inhibition by the inhibitory anti-RET antibodies could have synergistic anti-tumor effects with standard chemotherapy in NB cells (FIG. 10A). Previous study shows that blocking ALK signaling by a small molecule alectinib, approved by the FDA for the first-line treatment of patients with ALK-positive metastatic non-small cell lung cancer in 2017, suppresses NB cell proliferation and tumor growth. Blocking both ALK and RET signaling was hypothesized to have synergistic anti-tumor effect. To test this hypothesis, the anti-RET antibody and the ALK inhibitor alectinib were used for synergy analysis in NB cells. As shown in FIG. 10B, significant inhibitory synergy between anti-RET antibodies and ALK inhibitor alectinib was detected in the treated NGP and SY5Y cells.

[0389] Example 7. RET-specific chimeric antigen receptor (CAR) T cells can be activated by NB and AML cells

[0390] A chimeric antigen receptor (CAR) construct was developed to engineer T cells with specificity via a monoclonal antibody sequence. In our design, single-chain Fv fragments or scFv were obtained by connecting the VH and VL domains with a single polypeptide linker. Then, the scFV was linked to a CD8 spacer hinge-transmembrane combined with a CD28 endodomain- CD3 (^-chain intracellular domain (Fig. 11A). To initially determine the specificity of RET- CARs to NB cells, we co-cultured Jurkat cells with RET-CAR expression with NB cells. Nontransduced Jurkat cells were used as the control. We found that Jurkat cells with RET-CAR expression inhibited NB cell proliferation compared to the non-transduced Jurkat cells (Fig. 11B, 11C). Since Jurkat cell line has no function to lyse the target cells even it is activated, therefore, these results suggest that RET-CAR could bind to NB target cells and inhibit NB cell proliferation by inhibiting RET signaling in NB cells. Furthermore, we confirmed the efficacy of RET-CAR T cells to lyse NB cells in culture. As expected, T cells expressing the RET-CAR could significantly lyse NB cells compared to the non-transduced T cells (Fig. 11D, HE).

[0391] The anti-tumor effect of RET-CAR T cell on AML cells was further determined. The structures of RET-CARs are shown in FIG. 12A. T cells expressing the RET-CARs were cocultured with THP-1 cells. Non-transduced T cells were used as the control. T cells expressing RET-CARs produced significant levels of IL2 (Fig. 12B, P<0.001) and IFNy (Fig. 12C, P<0.001) when stimulated with THP-1 cells compared to the control T cells. Finally, the efficacy of RET-CARs T cells was confirmed to lyse THP-1 cells in culture. As expected, T cells expressing the RET-CARs could significantly lyse THP-1 cells compared to the nontransduced T cells (Fig. 12D, P<0.001).

[0392] Example 8. RET-CAR T cells exhibit in vivo activity against RET positive AML cells.

[0393] The established highly aggressive xenogeneic THP-1 AML model was used to test the anti- AML in vivo activity of the RET-CAR T cells. This model allowed measurement of AML cell expansion using bio luminescence imaging (BLI) (FIG. 13A). NSG mice were transplanted with 1 x 106THP-1 -Flue cells intraperitoneally following sub-lethal irradiation with 3.5 Gy one day prior. Mice were subsequently left for 7 days for leukemia to develop. Mice were distributed randomly into each treatment group and intravenously injected with 5 x 106non-transduced (NTD) T cells, 5 x 106RET-CAR-28Z or RET-CAR-BBZ T cells in 100 pL PBS. BLI was performed at weekly intervals to track leukemia progression or regression.

[0394] The results showed that the THP-1 -Flue derived BLI signal was lower when the AML- bearing mice received the RET-CAR T cells compared to mice that received NTD T cells (FIG. 13B). This experiment suggests that CAR-T cells engineered to target the RET receptor tyrosine kinase are effective in killing RET positive AML cells in mice. The RET-CAR T cells treatment was well tolerated without noticeable adverse effects.

Claims

Claims:

1. An antibody or antigen-binding fragment thereof specific for an extracellular domain of the RET (Ret Proto-Oncogene) proto-oncogene, comprising:(1) a heavy chain variable region (VH) comprising a heavy chain CDR1 sequence of SEQ ID NO: 1, a heavy chain CDR2 sequence of SEQ ID NO: 2, and a heavy chain CDR3 sequence of SEQ ID NO: 3; and,(2) a light chain variable region (VL) comprising a light chain CDR1 sequence of SEQ ID NO: 4, a light chain CDR2 sequence of SEQ ID NO: 5, and a light chain CDR3 sequence of SEQ ID NO: 6.

2. The antibody or antigen-binding fragment of claim 1, comprising the VH sequence of SEQ ID NO: 7, and the VL sequence of SEQ ID NO: 8.

3. The antibody or antigen-binding fragment of claim 1 or 2, wherein the antibody comprises the heavy chain sequence of SEQ ID NO: 9, and the light chain sequence of SEQ ID NO: 10.

4. The antibody or antigen-binding fragment of any one of claims 1-3, which inhibits RET signaling in a cancer cell characterized by expression of a mutant or wild-type RET.

5. The antibody or antigen-binding fragment according to any one of claims 1-4, which is a human-mouse chimeric antibody, a humanized antibody, a human antibody, a CDR-grafted antibody, or a resurfaced antibody.

6. The antibody or antigen-binding fragment according to any one of claims 1-4, wherein said antigen-binding fragment thereof is an Fab, Fab’, F(ab’)2, Fd, single chain Fv or scFv, disulfide linked Fv, V-NAR domain, IgNar, intrabody, IgGACH2, minibody, F(ab’)3, tetrabody, triabody, diabody, single-domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.

7. A polypeptide comprising the VH and / or VL sequence of the antibody or antigenbinding fragment according to any one of claims 1-4.

8. A polynucleotide or set of polynucleotides encoding the VH and / or VL sequence of the antibody or antigen-binding fragment according to any one of claims 1-6 or the polypeptide of claim 7.

9. A vector comprising the polynucleotide or set of polynucleotides of claim 8.

10. The vector of claim 9, which is an expression vector (e.g., a mammalian expression vector, a yeast expression vector, an insect expression vector, or a bacterial expression vector).

11. A cell comprising the antibody or antigen -binding fragment of any one of claims 1-6, the polypeptide of claim 7, the polynucleotide of claim 8, or the vector of claim 9 or 10.

12. A method of producing the antibody or antigen-binding fragment of any one of claims 1-6, or the polypeptide of claim 7, comprising:(a) culturing the cell of claim 11 ; and(b) isolating said antibody, antigen-binding fragment thereof, or polypeptide from said cultured cell.

13. A polypeptide comprising the VH CDR1-3 and / or the VL CDR1-3 of the antibody or antigen -binding fragment of any one of claims 1-4.

14. The polypeptide of claim 13, comprising the VH CDR1-3 and the VL CDR1-3 of the antibody or antigen-binding fragment of any one of claims 1-4.

15. The polypeptide of claim 14, which is a chimeric antigen receptor (CAR).

16. The polypeptide of claim 15, wherein the CAR comprises:(a) an antigen-recognition domain or an scFv comprising the VH CDR1-3 and the VL CDR1-3 of the antibody or antigen-binding fragment of any one of claims 1-4; and,(I)(bl) a hinge region (e.g., a membrane-proximal region from an immune molecule such as IgG, CD8, and CD28) and a transmembrane domain (e.g., a membrane-proximal component of an endodomain, such as CD28 transmembrane domain); and(cl) an intracellular T cell signaling domain comprising an immunoreceptor tyrosine-based activation motif (IT AM) (such as the ITAM in the cytoplasmic domain of CD3-zeta), optionally further comprising one or more chimeric domain(s) from a co- stimulatory protein (such as CD28, CD27, CD134 (0X40), and CD137 (4-1BB)), or,(II)(b2) an intracellular T cell signaling domain comprising the cytoplasmic domain ofCD3s, or(III)(b3) a TRA CC domain linked to the VH of the antigen-recognition domain, and a TRB CC domain linked to the VL of the antigen-recognition domain.

17. A T cell comprising the CAR of claim 16.

18. The T cell of claim 17, which is an autologous T cell isolated from a patient to whom the T cell is to be administered, or an allogeneic T cell isolated from a healthy donor.

19. An immunoconjugate (or antibody-drug conjugate or ADC) having the following formula:Ab-[-L-D]n, wherein:Ab is an antibody or antigen -binding fragment thereof of any one of claims 1-6, or the polypeptide of claim 7, that is covalently linked to one or more units of linker-drug moieties -[-L-D], wherein L is a linker and D is a cytotoxic drug; and, n is an integer from 1 to 20 (e.g., from 1-12); and wherein each linker-drug moiety may have the same or different linker L or cytotoxic drug D.

20. The immunoconjugate of claim 19, wherein each linker-drug moiety -[-L-D] is covalently linked to Ab via a side chain amino group of Lys.

21. The immunoconjugate of claim 19, wherein each linker-drug moiety -[-L-D] is covalently linked to Ab via a sidechain thiol group of Cys.

22. The immunoconjugate of claim 19, wherein each linker-drug moiety -[-L-D] is covalently linked to Ab via a site-specifically incorporated non-natural amino acid.

23. The immunoconjugate of any one of claims 19-22, wherein each linker L comprises a peptide unit.

24. The immunoconjugate of claim 23, wherein the peptide unit comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-10, or 2-5 amino acid residues.

25. The immunoconjugate of any one of claims 19-24, wherein the linker L is non- cleavable by protease (e.g., cathepsin).

26. The immunoconjugate of any one of claims 19-24, wherein the linker L is a cleavable linker cleavable by protease (e.g., cathepsin), acidic environment, or redox statechange.

27. The immunoconjugate of any one of claims 19-26, wherein the cytotoxic drug is a DNA intercalating agent, a microtubule binder, a topoisomerase I inhibitor, or a DNA minor groove binder.

28. A method of treating cancer in a patient in need thereof, said cancer being characterized / caused by activation of RET proto-oncogene (e.g., a mutant RET proto-oncogene) or associated with RET overexpression, the method comprising administering to the patient a therapeutically effective amount of an antibody or antigen binding fragment thereof specific for an extracellular domain of a RET protooncogene, or a CAR-T cell comprising a CAR comprising the antibody or antigen binding fragment thereof, or an immunoconjugate comprising the antibody or antigen binding fragment thereof.

29. The method of claim 28, wherein the antibody is a mouse antibody, a chimeric antibody, a humanized antibody, or a human antibody.

30. The method of claim 29, wherein the antibody or antigen-binding fragment thereof is any one of claims 1-6; the CAR is the CAR of claim 16; the CAR-T cell is the T cell of claim 17 or 18; or the immunoconjugate is the immunoconjugate of any one of claims 19-27.

31. The method of any one of claims 28-30, wherein the cancer is neuroblastoma (NB), acute myeloid leukemia (AML), small cell lung cancer (SCLC), Ewing sarcoma, breast cancer, and other cancer types with RET expression.

32. The method of claim 31, wherein the cancer is AML or NB.

33. The method of claim 32, wherein the AML is characterized by elevated RET mRNA expression.

34. The method of any one of claims 28-33, further comprising administering to the patient a second therapeutic intervention (e.g., radiotherapy, chemotherapy, and / or immune therapy).

35. The method of claim 34, wherein the cancer is NB, and the second therapeutic intervention is a standard-of-care or a conventional chemotherapeutic agent (such as an ALK inhibitor).

36. The method of claim 35, wherein the patient is resistant to chemotherapy, or hasrelapsed from chemotherapy.

37. A method of detecting the presence of RET (Ret Proto-Oncogene) proto-oncogene product (e.g., wild-type or mutant RET protein) in a sample, the method comprises contacting the sample with the antibody or antigen-binding fragment thereof of any one of claims 1-6.

38. The method of claim 37, wherein the sample is a tissue sample (e.g., a tissue section such as a paraffin-embedded or frozen tumor tissue section, or a cultured cell) from a subject having a cancer, or at high risk of having the cancer, optionally, the cancer is characterized / caused by activation of RET proto-oncogene.

39. The method of claims 37 or 38, wherein the antibody or antigen-binding fragment thereof is detected by immunohistochemistry (IHC) using a labeled secondary antibody specific for the constant region of the antibody or antigen-binding fragment thereof.

40. A method of identifying a subject who has a cancer characterized / caused by activation of RET proto-oncogene or associated with RET overexpression, comprising determining the level of RET expression in a sample according to the method of any one of claims 37-39.

41. A method of treating a subject who has a cancer characterized / caused by activation of RET proto-oncogene or associated with RET overexpression, comprising administering to said subject a therapeutically effective amount of a therapeutically effective amount of an antibody or antigen binding fragment thereof specific for an extracellular domain of a RET proto-oncogene, or a CAR-T cell comprising a CAR comprising the antibody or antigen binding fragment thereof, or an immunoconjugate comprising the antibody or antigen binding fragment thereof, wherein the level of expression of RET is determined according to the method of any one of claims 37-39.

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