Bispecific binding agents and uses thereof

Abstract

To provide compositions, methods, and uses for treating cancer.SOLUTION: There are provided herein, compositions, methods, and uses involving (i) bispecific binding agents that specifically bind to a cancer antigen, (ii) clearing agents, and (iii) radiotherapeutic agents for treating cancer. Also there are provided uses and methods for treating HER2-related cancers.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 641,645, filed March 12, 2018, and U.S. Provisional Patent Application No. 62 / 813,592, filed March 4, 2019, each of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application incorporates by reference the Sequence Listing submitted herewith as a text file entitled "Sequence_Listing_13542-061-228.txt", created on March 7, 2019, and having a size of 144,888 bytes.

[0003] 1. Field Provided herein is a composition, method and use involving a bispecific binding agent that specifically binds to i) a first target that is a cancer antigen expressed by cancer, and ii) a second target that is not a cancer antigen.The bispecific binding agent described herein is useful in a method for treating cancer.Also provided herein is a method and use involving (i) a bispecific binding agent that specifically binds to a cancer antigen, (ii) ablation agent, and (iii) radiotherapy agent for treating cancer. [Background technology]

[0004] 2.Background The pharmacokinetics of full-size IgG monoclonal antibodies as carriers of therapeutic radioisotopes (i.e., radioimmunotherapy) typically exhibits an unfavorable therapeutic index (e.g., the ratio of radiation absorbed dose to tumor divided by the dose to radiosensitive tissues such as blood (see, e.g., Larson et al., 2015, "Radioimmunotherapy of human tumours." Nature Reviews Cancer; 15: 347-60)) for radioimmunotherapy, along with dose-limiting hematologic toxicity. Alternatively, a pretargeted radioimmunotherapy ("PRIT") strategy can be utilized that separates the antibody-mediated targeting step from the step of administering a cytotoxic ligand to shorten the residence time of the ligand in circulation (see, e.g., Kraeber-Bodere et al., 2015, "A pretargeting system for tumor PET imaging and radioimmunotherapy." Front Pharmacol. 6:54). A typical PRIT strategy involves three steps: (i) a tumor targeting step; (ii) a removal step; and (iii) a radiotherapy step. First, a bispecific tumor targeting agent (e.g., a bispecific antibody) having one specificity for a tumor antigen is administered to a subject, so that the bispecific tumor targeting agent can be pre-localized to a tumor. Second, a removal agent is administered to a subject to remove circulating bispecific tumor targeting agents (e.g., unbound bispecific tumor targeting agents in blood) from the blood. Third, a radiolabeled small molecule hapten or peptide is administered to a subject, which binds to tumor-bound bispecific tumor targeting agents and kills tumor cells. The removal step allows the amount of bispecific tumor targeting agents in circulation to be reduced, and allows the interaction between the bispecific tumor targeting agent and the radiolabeled small molecule hapten or peptide in blood to be reduced.Indeed, the ablation step improves the therapeutic index of the PRIT method by reducing radiation exposure to non-targeted tissues, particularly blood, thereby allowing the administration of high doses of radiolabeled small molecule haptens or peptides without dose-limiting toxicity.

[0005] However, major drawbacks of current PRIT methods include the inability to target antigens that are rapidly internalized (see, e.g., Walter et al., 2010, Cancer Biotherapy and Radiopharmaceuticals, 25(2):125-142). Unlike antibody-drug conjugates, which rely on binding to cell surface receptors and internalization upon binding to the cell for delivery of their payload, non-internalizing antibodies / cell surface targets are believed to be optimal for PRIT (e.g., Boerman et al., 2003, Pretargeted Radioimmunotherapy of Cancer: Progress Step by Step*. J. Nucl. Med. 44(3):400-411; Casalini et al., 1997, Tumor Pretargeting: Role of Avidin / Streptavidin on Monoclonal Antibody Internalization. J. Nucl. Med.; 38(9):1378-1381; Walter et al., 2010, Pretargeted Radioimmunotherapy for Hematologic and Other Malignancies, Cancer Biother Radiopharm.; 25(2):125-142; Cheal et al., 2014, “Preclinical evaluation of multistep targeting of diasialoganglioside GD2 using an IgG-scFv bispecific antibody with high affinity for GD2 and DOTA metal complex.” Molecular Cancer Therapeutics; 13:1802-12; Cheal et al., 2016, “Theranostic pretargeted radioimmunotherapy of colorectal cancer xenografts in mice using picomolar affinity Y-86- or Lu-177-DOTA-Bn binding scFv C825 / GPA33 IgG bispecificimmunogonjugates.” European Journal of Nuclear Medicine and Molecular Imaging; 43:925-37; Green et al., 2016, See “Comparative Analysis of Bispecific Antibody and Streptavidin-Targeted Radioimmunotherapy for B-cell Cancers.” Cancer Research; 76(22):6669-6679). Antigens that are rapidly internalized cannot be targeted by PRIT (see Walter et al., 2010, Pretargeted Radioimmunotherapy for Hematologic and Other Malignancies, Cancer Biother Radiopharm.; 25(2):125-142; see also Boerman et al., 2003, Pretargeted Radioimmunotherapy of Cancer: Progress Step by Step*. J. Nucl. Med. 44(3):400-411). However, many cancers are associated with antigens that are internalized into the cancer cell; for example, the cancer antigen human epidermal growth factor receptor 2 ("HER2") is susceptible to endocytosis into the cell (see, e.g., Austin et al.(See, e.g., Wang, et al., 2004, Molecular Biology of the Cell, 15:5268-5282). Thus, there is an unmet need for methods of treating cancers associated with antigens that are internalized into cells (e.g., antigens that are internalized into cells). Furthermore, due to the long time between the tumor targeting and ablative steps (typically 24-120 hours (e.g., Knox et al., 2000, Clinical Cancer Research, 6:406-414; Bodet-Milin et al. J Nucl Med 2016, vol 57, no 10, 1505-1511; Bodet-Milin et al. Front Med (Lausanne) 2015 Nov 27, 2:84; Schoffelen, R., Woliner-van der Weg, W., Visser, EP et al. Eur J Nucl Med Mol Imaging (2014) 41: 1593; Forero, et al. Blood 2004 104(1) 227-36; Weiden, et al. Cancer Biother Radiopharm 2000 15(1): 15-29), current PRIT procedures often require multiple visits or an overnight stay in the hospital, which can be costly and labor intensive for patients. Thus, improved, more efficient PRIT procedures are also needed. Summary of the Invention [Problem to be solved by the invention]

[0006] Provided herein are methods of treating cancer in a subject. [Means for solving the problem]

[0007] 3. Overview Provided herein is a method of treating cancer in a subject in need thereof, comprising the steps of: (a) administering to the subject a therapeutically effective amount of a bispecific binding agent, wherein the bispecific binding agent comprises a first molecule covalently attached (optionally via a linker) to a second molecule, said first molecule comprising a first binding site, said first binding site specifically binding to a first target, said first target being a cancer antigen expressed by said cancer, and said second molecule comprising a second binding site, said second binding site specifically binding to a second target, said second target being not a cancer antigen; (b) administering to the subject a therapeutically effective amount of the bispecific binding agent. and (c) after step (b) of administering a therapeutically effective amount of a clearing agent to the subject, the clearing agent binds to the second binding site and functions to reduce bispecific binding agent circulating in the blood of the subject; and (c) after step (b) of administering a therapeutically effective amount of a clearing agent to the subject, the radiotherapeutic agent (i) comprises a second target bound to a metal radionuclide, the second target being a metal chelator; or (ii) comprises a second target bound to a metal chelator, the metal chelator being bound to a metal radionuclide. In specific embodiments, the therapeutically effective amount of the bispecific binding agent is between 100 mg and 700 mg, 200 mg and 600 mg, 200 mg and 500 mg, 300 mg and 400 mg, about 300 mg, about 450 mg, about 500 mg, about 600 mg, or about 625 mg, and the subject is a human. In another specific embodiment, the therapeutically effective amount of the bispecific binding agent is 250 mg-700 mg, 300 mg-600 mg, or 400 mg-500 mg, and the subject is a human.In specific embodiments, the cancer antigen is HER2, CA6, CD138, CD19, CD22, CD27L, CD30, CD33, CD37, CD56, CD66e, CD70, CD74, CD79b, EGFR, EGFRvIII, FRα, GCC, GPNMB, mesothelin, MUC16, NaPi2b, Nectin 4, PSMA, STEAP1, Trop-2, 5T4, AGS-16, alpha v beta 6, CA19.9, CAIX, CD174, CD180, CD227, CD326, CD79a, CEACAM5, CRIPTO, DLL3, DS6, endothelin B receptor, FAP, GD2, mesothelin, PMEL 17, SLC44A4, TENB2, TIM-1, CD98, endosialin / CD248 / TEM1, fibronectin extracellular domain B, LIV-1, mucin 1, p-cadherin, peritosin, Fyn, SLTRK6, tenascin c, VEGFR2, PRLR, CD20, CD72, fibronectin, GPA33, splice isoform of tenascin C, TAG-72, B7-H3, L1CAM, Lewis Y, and polysialic acid. In a preferred embodiment, the cancer antigen is HER2. In a specific embodiment, the cancer antigen is an antigen that is internalized into cancer cells. In specific embodiments, the cancer antigens internalized into the cancer cells are HER2, CA6, CD138, CD19, CD22, CD27L, CD30, CD33, CD37, CD56, CD66e, CD70, CD74, CD79b, EGFR, EGFRvIII, FRα, GCC, GPNMB, mesothelin, MUC16, NaPi2b, nectin4, PSMA, STEAP1, Trop-2, 5T4, AGS-16, alpha v beta 6, CA19.9, CAIX, CD174, CD180, CD227, CD326, CD79a, CEACAM5, CRIPTO, DLL3, DS6, endothelin B receptor, FAP, GD2, mesothelin, PMEL 17, SLC44A4, TENB2, TIM-1, CD98, endosialin / CD248 / TEM1, fibronectin extracellular domain B, LIV-1, mucin 1, p-cadherin, peritosin, Fyn, SLTRK6, tenascin c, VEGFR2, and PRLR.In a preferred embodiment, the cancer antigen that is internalized into cancer cells is HER2. In another embodiment, the cancer antigen is an antigen that is not internalized into cancer cells. In a specific embodiment, the cancer antigen that is not internalized into cancer cells is selected from the group consisting of CD20, CD72, fibronectin, GPA33, splice isoform of tenascin-C, and TAG-72. In a preferred embodiment, the cancer antigen is HER2, and the metal chelator is DOTA or its derivative.

[0008] Also provided herein is a method of treating cancer in a subject in need thereof, comprising the steps of: (a) administering to the subject a first therapeutically effective amount of a bispecific binding agent, wherein the first therapeutically effective amount of the bispecific binding agent is between 100 mg and 700 mg, between 200 mg and 600 mg, between 200 mg and 500 mg, between 300 mg and 400 mg, about 300 mg, about 450 mg, about 500 mg, about 600 mg, or about 625 mg, and the bispecific binding agent comprises a first molecule covalently linked (optionally via a linker) to a second molecule, wherein the cancer expresses HER2, and the first molecule comprises an antibody or antigen-binding fragment thereof, or a single chain variable fragment (scFv), wherein the antibody or antigen-binding fragment thereof, or scFv (i) binds to HER2 on the cancer, and (ii) binds to all three of the heavy chain complementarity determining regions (CDRs) of SEQ ID NO:20 and all three of the light chain CDRs of SEQ ID NO:19. wherein the second molecule comprises a second binding site, the second binding site specifically binds to a second target, and the second target is not a cancer antigen; (b) after step (a) of administering a therapeutically effective amount of a bispecific binding agent to the subject, administering a therapeutically effective amount of a clearing agent to the subject, the clearing agent binds to the second binding site and functions to reduce bispecific binding agent circulating in the blood of the subject; and (c) after step (b) of administering a therapeutically effective amount of a clearing agent to the subject, the radiotherapy agent (i) comprises a second target bound to a metal radionuclide, and the second target is a metal chelator; or (ii) comprises a second target bound to a metal chelator, and the metal chelator is bound to a metal radionuclide, and the subject is a human. In a specific embodiment, the first therapeutically effective amount of the bispecific binding agent is about 450 mg.

[0009] In specific embodiments of the bispecific binding agent, the first molecule of the bispecific binding agent comprises an antibody or antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof comprising the first binding site, hi specific embodiments, the antibody is an immunoglobulin.

[0010] In a specific embodiment for a bispecific binding agent, where the first molecule comprises an immunoglobulin that comprises a first binding site that specifically binds to HER2, the heavy chain within the immunoglobulin comprises all three heavy chain CDRs of SEQ ID NO: 20 and the light chain within the immunoglobulin comprises all three light chain CDRs of SEQ ID NO: 19. In a specific embodiment, the heavy chain variable (V H ) domain comprises SEQ ID NO: 20. In a specific embodiment, the sequence of the V H The sequence of the domain comprises a humanized form of SEQ ID NO: 20. In a specific embodiment, the light chain variable (V L ) domain comprises SEQ ID NO: 19. In a specific embodiment, the sequence of the V L In a specific embodiment, the sequence of the domain comprises a humanized form of SEQ ID NO: 19, the sequence of the immunoglobulin heavy chain comprises any of SEQ ID NOs: 14-17. In a preferred embodiment, the sequence of the immunoglobulin heavy chain comprises SEQ ID NO: 15. In a more preferred embodiment, the sequence of the immunoglobulin heavy chain comprises SEQ ID NO: 16. In a specific embodiment, the sequence of the immunoglobulin light chain comprises SEQ ID NO: 11.

[0011] For bispecific binding agents, in specific embodiments, the second molecule is an scFv comprising a second binding site. In specific embodiments where the second molecule is an scFv, the second target is DOTA or a derivative thereof. In specific embodiments where the second molecule is an scFv and the second target is DOTA or a derivative thereof, the V H The sequence of the domain comprises all three of the CDRs of SEQ ID NO: 21 and the V L The sequence of the domain comprises all three of the CDRs of SEQ ID NO: 22. In a specific embodiment, the V H The sequence of the domain is SEQ ID NO: 21. In a specific embodiment, the V H The sequence of the domain comprises a humanized form of SEQ ID NO: 21. In a specific embodiment, the V HThe sequence of the domain is a humanized form of SEQ ID NO: 21. In a specific embodiment, the humanized form of SEQ ID NO: 21 is SEQ ID NO: 37. In a specific embodiment, the V L The sequence of the domain is SEQ ID NO: 22. In a specific embodiment, the V L The sequence of the domain comprises a humanized form of SEQ ID NO: 22. In a specific embodiment, the V L The sequence of the domain is a humanized form of SEQ ID NO: 22. In a specific embodiment, the humanized form of SEQ ID NO: 22 is SEQ ID NO: 38. In a specific embodiment, the sequence of the scFv comprises any of SEQ ID NOs: 31-36. In a specific embodiment, the sequence of the scFv is any of SEQ ID NOs: 31-36. In a specific embodiment, the sequence of the scFv comprises any of SEQ ID NOs: 39-44. In a specific embodiment, the sequence of the scFv is any of SEQ ID NOs: 39-44. In a preferred embodiment, the sequence of the scFv comprises SEQ ID NO: 33 (e.g., the sequence of the scFv is SEQ ID NO: 33). In a more preferred embodiment, the sequence of the scFv comprises SEQ ID NO: 44 (e.g., the sequence of the scFv is SEQ ID NO: 44).

[0012] In a specific embodiment where the first molecule is an immunoglobulin, the immunoglobulin comprises two identical heavy chains and two identical light chains, a first light chain and a second light chain, said first light chain being fused (optionally via a first peptide linker) to a second molecule, a first scFv comprising a second binding site, to create a first light chain fusion polypeptide, and said second light chain being fused (optionally via a second peptide linker) to a second scFv to create a second light chain fusion polypeptide, and said first light chain fusion polypeptide and said second light chain fusion polypeptide are identical. In specific embodiments, the first light chain fusion polypeptide comprises a first peptide linker and the second light chain fusion polypeptide comprises a second peptide linker, and the sequences of the first and second peptide linkers are 5-30, 5-25, 5-15, 10-30, 10-20, 10-15, 15-30, or 15-25 amino acids in length. In specific embodiments, the peptide linker is 7-32, 7-27, 7-17, 12-32, 12-22, 12-17, 17-32, or 17-27 amino acid residues in length. In specific embodiments, the first light chain fusion polypeptide comprises a first peptide linker and the second light chain fusion polypeptide comprises a second peptide linker, and the sequences of the first and second peptide linkers are any of SEQ ID NOs: 23 and 25-30. In specific embodiments, the V H Domain and V L The sequence of the intrascFv peptide linker between the domains is 5 to 30, 5 to 25, 5 to 15, 10 to 30, 10 to 20, 10 to 15, 15 to 30, or 15 to 25 amino acids in length. H Domain and V L The sequence of the intrascFv peptide linker between the domains is any one of SEQ ID NOs: 23 and 25 to 30. In a preferred embodiment, the V H Domain and V L The sequence of the intra-scFv peptide linker between the domains is SEQ ID NO: 27. In a more preferred embodiment, the V H Domain and VL The sequence of the intra-scFv peptide linker between the domains is SEQ ID NO: 30. In a specific embodiment, the first target is HER2. In a specific embodiment, the second target is DOTA or a derivative thereof.

[0013] In a specific embodiment of the bispecific binding agent, where the first molecule is an immunoglobulin, the immunoglobulin comprises two identical heavy chains and two identical light chains, a first light chain and a second light chain, said first light chain being fused (optionally via a first peptide linker) to a second molecule, a first scFv comprising a second binding site, to create a first light chain fusion polypeptide, and said second light chain being fused (optionally via a second peptide linker) to a second scFv to create a second light chain fusion polypeptide, where said first light chain fusion polypeptide and said second light chain fusion polypeptide are identical, where the first target is HER2 and the second target is DOTA or a derivative thereof. In a specific embodiment, the heavy chain in the immunoglobulin comprises all three heavy chain CDRs of SEQ ID NO:20 and the light chain in the immunoglobulin comprises all three light chain CDRs of SEQ ID NO:19. In a specific embodiment, the V in the heavy chain in the immunoglobulin comprises all three light chain CDRs of SEQ ID NO:19. H The sequence of the domain comprises SEQ ID NO: 20. In a specific embodiment, the V H The sequence of the domain comprises a humanized form of SEQ ID NO: 20. In a specific embodiment, in an immunoglobulin, in a light chain, the V L The sequence of the domain comprises SEQ ID NO: 19. In a specific embodiment, the V L The sequence of the domain comprises a humanized form of SEQ ID NO: 19. In a specific embodiment, the sequence of the heavy chain in the immunoglobulin comprises any of SEQ ID NOs: 14-17. In a preferred embodiment, the sequence of the heavy chain in the immunoglobulin comprises SEQ ID NO: 15. In a more preferred embodiment, the sequence of the heavy chain in the immunoglobulin comprises SEQ ID NO: 16. In a specific embodiment, the sequence of the light chain in the immunoglobulin comprises SEQ ID NO: 11. In a specific embodiment, the V in the first scFv comprises a humanized form of SEQ ID NO: 19. In a specific embodiment, the sequence of the heavy chain in the immunoglobulin comprises any of SEQ ID NOs: 14-17. In a preferred embodiment, the sequence of the heavy chain in the immunoglobulin comprises SEQ ID NO: 15. In a more preferred embodiment, the sequence of the heavy chain in the immunoglobulin comprises SEQ ID NO: 16. In a specific embodiment, the sequence of the light chain in the immunoglobulin comprises SEQ ID NO: 11. H The sequence of the domain comprises all three of the CDRs of SEQ ID NO: 21 and V LThe sequence of the domain comprises all three of the CDRs of SEQ ID NO: 22. In a specific embodiment, the V H In a specific embodiment, the sequence of the V domain in the first scFv is SEQ ID NO: 21. H The sequence of the domain comprises a humanized form of SEQ ID NO: 21. In a specific embodiment, the V H The sequence of the domain is a humanized form of SEQ ID NO: 21. In a specific embodiment, the humanized form of SEQ ID NO: 21 is SEQ ID NO: 37. In a specific embodiment, the V L The sequence of the domain is SEQ ID NO: 22. In a specific embodiment, the V L The sequence of the domain comprises a humanized form of SEQ ID NO: 22. In a specific embodiment, the V L The sequence of the domain is a humanized form of SEQ ID NO: 22. In a specific embodiment, the humanized form of SEQ ID NO: 22 is SEQ ID NO: 38. In a specific embodiment, the sequence of the first scFv comprises any of SEQ ID NOs: 31-36. In a specific embodiment, the sequence of the first scFv is any of SEQ ID NOs: 31-36. In a specific embodiment, the sequence of the first scFv comprises any of SEQ ID NOs: 39-44. In a specific embodiment, the sequence of the scFv is any of SEQ ID NOs: 39-44. In a preferred embodiment, the sequence of the first scFv comprises SEQ ID NO: 33 (e.g., the sequence of the first scFv is SEQ ID NO: 33). In a more preferred embodiment, the sequence of the scFv comprises SEQ ID NO: 44 (e.g., the sequence of the scFv is SEQ ID NO: 44). In a specific embodiment, the sequence of the first light chain fusion polypeptide is any of SEQ ID NOs: 5-10. In a preferred embodiment, the sequence of the first light chain fusion polypeptide is SEQ ID NO: 7. In a specific embodiment, the sequence of the first light chain fusion polypeptide is any of SEQ ID NOs: 5 to 10 and the sequence of the heavy chain is any of SEQ ID NOs: 14 to 17. In a preferred embodiment, the sequence of the first light chain fusion polypeptide is SEQ ID NO: 7 and the sequence of the heavy chain is SEQ ID NO: 15. In a preferred embodiment, the sequence of the first light chain fusion polypeptide is SEQ ID NO: 50 and the sequence of the heavy chain is SEQ ID NO: 16.

[0014] In specific embodiments of the bispecific binding agent, the bispecific binding agent comprises an Fc domain, hi specific embodiments, the bispecific binding agent is at least 100 kDa, at least 150 kDa, at least 200 kDa, at least 250 kDa, between 100 and 300 kDa, between 150 and 300 kDa, or between 200 and 250 kDa. In a specific embodiment for a bispecific binding agent where the bispecific binding agent comprises an immunoglobulin, the heavy chain within the immunoglobulin is mutated to destroy an N-linked glycosylation site, hi a specific embodiment, the heavy chain has an amino acid substitution that replaces an asparagine, which is an N-linked glycosylation site, with an amino acid that does not function as a glycosylation site. In a specific embodiment of a bispecific binding agent comprising an immunoglobulin, the heavy chain within the immunoglobulin is mutated to disrupt the C1q binding site. In a specific embodiment for bispecific binding agents, the bispecific binding agent does not activate complement.

[0015] In a specific embodiment, the bispecific binding agent does not bind to Fc receptors in its soluble or cell-bound form. In a specific embodiment where the bispecific binding agent comprises an scFv, the scFv is a disulfide-stabilized scFv.

[0016] In specific embodiments of the methods of treating cancer provided herein, the bispecific binding agent is administered to the subject intravenously, subcutaneously, intramuscularly, parenterally, transdermally, transmucosally, intraperitoneally, intrathoracically, or to any other body compartment, such as intrathecally, intraventricularly, or intraparenchymally. In a preferred embodiment, the bispecific binding agent is administered to the subject intravenously.

[0017] Also provided herein is a clearing agent for use in the method of treating cancer provided herein. In a specific embodiment, the clearing agent comprises a second target (i.e., the second target of the bispecific binding agent used in the method of treating cancer) that is bound to a molecule that is cleared from the circulation primarily by the liver, the fixed phagocyte system, the spleen, or the bone marrow. In a specific embodiment, the clearing agent comprises a 500 kDa aminodextran conjugated to the second target. In a specific embodiment, the clearing agent comprises about 100-150 second target molecules per 500 kDa aminodextran.

[0018] In specific embodiments of the methods of treating cancer provided herein, step (b) of administering a therapeutically effective amount of a clearing agent to the subject occurs within 10 hours, within 8 hours, within 6 hours, within 4 hours, within 2 hours, within 1-12 hours, 2-12 hours, 1-2 hours, 1-3 hours, 1-4 hours, 2-6 hours, 2-8 hours, 2-10 hours, 4-6 hours, 4-8 hours, 4-10 hours, 2 hours, or 4 hours after step (a) of administering a therapeutically effective amount of a bispecific binding agent to the subject. In another specific embodiment, step (b) of administering a therapeutically effective amount of a clearing agent to the subject occurs about 2 hours, about 4 hours, about 6 hours, about 8 hours, or about 10 hours after step (a) of administering a therapeutically effective amount of a bispecific binding agent to the subject. In another specific embodiment, the bispecific binding agent is at least 100 kDa and step (b) of administering a therapeutically effective amount of a clearing agent to the subject occurs within 4 hours of step (a) of administering a therapeutically effective amount of the bispecific binding agent to the subject.

[0019] In a specific embodiment of the method for treating cancer provided herein, the removing agent is administered intravenously to the subject. In a specific embodiment of the method for treating cancer provided herein, the therapeutically effective amount of the removing agent is an amount that results in a molar ratio of the therapeutically effective amount of the bispecific binding agent administered to the subject to the therapeutically effective amount of the removing agent administered to the subject of 10:1, and the subject is a human. In a specific embodiment of the method for treating cancer provided herein, the therapeutically effective amount of the removing agent is an amount that results in a reduction of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% in the serum concentration of the bispecific binding agent 1 hour, 2 hours, 3 hours, or 4 hours after step (b) of administering a therapeutically effective amount of the removing agent to the subject.

[0020] Also provided herein is a radiotherapeutic agent for use in the method of treating cancer provided herein.In a specific embodiment, the radiotherapeutic agent comprises (i) a second target that is bound to a metal radionuclide (i.e., the second target of the bispecific binding agent used in the method of treating cancer), the second target being a metal chelator.In another specific embodiment, the radiotherapeutic agent comprises (ii) a second target that is bound to a metal chelator (i.e., the second target of the bispecific binding agent used in the method of treating cancer), the second target being a metal chelator that is bound to a metal radionuclide. In a specific embodiment for a radiotherapeutic agent, the metal chelator is selected from the group consisting of 1,4,7,10-traazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or a derivative thereof, DOTA-Bn or a derivative thereof, p-aminobenzyl-DOTA or a derivative thereof, diethylenetriaminepentaacetic acid (DTPA) or a derivative thereof, and DOTA-deferoxamine. In a specific embodiment, the metal chelator is DOTA or a derivative thereof. In another specific embodiment, the metal chelator is DOTA-Bn or a derivative thereof. In specific embodiments for radiotherapeutic agents, the metal of said metallic radionuclide is selected from the group consisting of lutetium (Lu), actinium (Ac), astatine (At), bismuth (Bi), cerium (Ce), copper (Cu), dysprosium (Dy), erbium (Er), europium (Eu), gadolinium (Gd), gallium (Ga), holmium (Ho), iodine (I), indium (In), lanthanum (La), lead (Pb), neodymium (Nd), praseodymium (Pr), promethium (Pm), rhenium (Re), samarium (Sm), scandium (Sc), terbium (Tb), thulium (Tm), ytterbium (Yb), yttrium (Y), and zirconium (Zr).In specific embodiments for radiotherapeutic agents, the metal of said metallic radionuclide is selected from the group consisting of lutetium (Lu), actinium (Ac), astatine (At), bismuth (Bi), cerium (Ce), copper (Cu), dysprosium (Dy), erbium (Er), europium (Eu), gadolinium (Gd), gallium (Ga), holmium (Ho), iodine (I), indium (In), lanthanum (La), lead (Pb), neodymium (Nd), praseodymium (Pr), promethium (Pm), radium (Ra), rhenium (Re), samarium (Sm), scandium (Sc), terbium (Tb), thorium (Th), thulium (Tm), ytterbium (Yb), yttrium (Y), and zirconium (Zr). In a specific embodiment for the radiotherapeutic agent, the metal radionuclide is: 211 At, 225 Ac, 227 Ac, 212 Bi, 213 Bi, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 157 Gd, 166 Ho, 124 I, 125 I, 131 I, 111 In, 177 Lu, 212 Pb, 186 Re, 188 Re, 47 Sc, 153 Sm, 166 Tb, 89 Zr, 86 Y, 88 Y, and 90 In a specific embodiment for a radiotherapeutic agent, the metal radionuclide is selected from the group consisting of: 211 At, 225 Ac, 227 Ac, 212 Bi, 213 Bi, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 157 Gd, 166 Ho,124 I, 125 I, 131 I, 111 In, 177 Lu, 212 Pb, 223 Ra, 186 Re, 188 Re, 47 Sc, 153 Sm, 166 Tb, 227 Th, 89 Zr, 86 Y, 88 Y, 90 In a specific embodiment for a radiotherapeutic agent, the metal radionuclide is selected from the group consisting of: 177 Lu, 227 In a preferred embodiment, the metal radionuclide is 177 Lu. In a specific embodiment where the radiotherapeutic agent comprises (ii) a second target bound to a metal chelator, said metal chelator being bound to a metal radionuclide, the second molecule comprises streptavidin and the second target comprises biotin. In another specific embodiment where the radiotherapeutic agent comprises (ii) a second target bound to a metal chelator, said metal chelator being bound to a metal radionuclide, the second target comprises histamine succinylglycine.

[0021] In specific embodiments of the methods of treating cancer provided herein, step (c) of administering a therapeutically effective amount of a radiotherapeutic agent to the subject occurs 1-2 hours, 1-3 hours, 1-4 hours, 2-6 hours, 2-8 hours, 2-10 hours, 4-6 hours, 4-8 hours, 4-10 hours, within 1 hour, within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours after step (b) of administering a therapeutically effective amount of a clearing agent to the subject. In another specific embodiment, step (c) of administering a therapeutically effective amount of a radiotherapeutic agent to the subject occurs about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, or about 6 hours after step (b) of administering a therapeutically effective amount of a clearing agent to the subject. In another specific embodiment, step (c) of administering a therapeutically effective amount of a radiotherapeutic agent to the subject occurs about 1 hour after step (b) of administering a therapeutically effective amount of a clearing agent to the subject. In another specific embodiment, step (c) of administering to the subject a therapeutically effective amount of a radiotherapeutic agent occurs within 16 hours of step (a) of administering to the subject a therapeutically effective amount of a bispecific binding agent.

[0022] In specific embodiments of the methods of treating cancer provided herein, the radiotherapeutic agent is administered to the subject intravenously, subcutaneously, intramuscularly, parenterally, transdermally, transmucosally, intraperitoneally, intrathoracically, or to any other body compartment, such as intrathecally, intraventricularly, or intraparenchymally. In a preferred embodiment, the radiotherapeutic agent is administered to the subject intravenously. In specific embodiments, the therapeutically effective amount of the radiotherapeutic agent is between 25 mCi and 250 mCi, between 50 mCi and 200 mCi, between 75 mCi and 175 mCi, or between 100 mCi and 150 mCi, and the subject is a human. The radiotherapeutic agent is an alpha-emitting radioisotope, e.g., 225 In a specific embodiment, Ac, the therapeutically effective amount of the radiotherapeutic agent is between 0.108 mCi and 0.351 mCi, and the subject is a human.

[0023] The method of treating cancer provided herein may be repeated for a subject two, three or more times. In a specific embodiment of the method of treating cancer, the method further comprises: (d) administering a second therapeutically effective amount of a bispecific binding agent to the subject within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week of step (c) of administering a therapeutically effective amount of a radiotherapy agent to the subject; (e) administering a second therapeutically effective amount of a clearing agent to the subject after step (d) of administering a second therapeutically effective amount of a bispecific binding agent; and (f) administering a second therapeutically effective amount of a radiotherapy agent to the subject after step (e) of administering a second therapeutically effective amount of a clearing agent to the subject. In a specific embodiment, step (e) of administering a therapeutically effective amount of a clearing agent to the subject is performed within 12 hours of step (d) of administering a second therapeutically effective amount of a bispecific binding agent to the subject. In specific embodiments, the second therapeutically effective amount of the bispecific binding agent is 100 mg to 700 mg, 200 mg to 600 mg, 200 mg to 500 mg, 300 mg to 400 mg, about 300 mg, about 450 mg, about 500 mg, about 600 mg, or about 625 mg. In specific embodiments, the second therapeutically effective amount of the clearing agent is an amount that results in a 10:1 molar ratio of the therapeutically effective amount of the bispecific binding agent administered to the subject to the therapeutically effective amount of the clearing agent administered to the subject. In specific embodiments, the therapeutically effective amount of the clearing agent is an amount that results in at least a 90%, at least a 91%, at least a 92%, at least a 93%, at least a 94%, at least a 95%, at least a 96%, at least a 97%, at least a 98%, or at least a 99% reduction in the serum concentration of the bispecific binding agent 1 hour, 2 hours, 3 hours, or 4 hours after step (b) of administering the therapeutically effective amount of the clearing agent to the subject. In specific embodiments, the second therapeutically effective amount of the radiotherapeutic agent is between 25 mCi and 250 mCi, between 50 mCi and 200 mCi, between 75 mCi and 175 mCi, or between 100 mCi and 150 mCi.In a specific embodiment, the second therapeutically effective amount of the bispecific binding agent is administered to the subject intravenously, subcutaneously, intramuscularly, parenterally, transdermally, transmucosally, intraperitoneally, intrathoracically, or to any other body compartment, such as intrathecally, intraventricularly, or intraparenchymally. In a preferred embodiment, the second therapeutically effective amount of the bispecific binding agent is administered to the subject intravenously. In a specific embodiment, the second therapeutically effective amount of the clearing agent is administered to the subject intravenously. In a specific embodiment, the second therapeutically effective amount of the radiotherapy agent is administered to the subject intravenously, subcutaneously, intramuscularly, parenterally, transdermally, transmucosally, intraperitoneally, intrapleurally, or to any other body compartment, such as intrathecally, intraventricularly, or intraparenchymally. In a preferred embodiment, the second therapeutically effective amount of the radiotherapy agent is administered to the subject intravenously.

[0024] In another specific embodiment of the method of treating cancer, the method further comprises the steps of: (g) administering to the subject a third therapeutically effective amount of a bispecific binding agent within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week of step (f) administering to the subject a second therapeutically effective amount of radiation therapy; (h) administering to the subject a third therapeutically effective amount of a clearing agent after step (g) of administering to the subject the third therapeutically effective amount of the bispecific binding agent; and (i) administering to the subject a third therapeutically effective amount of a radiation therapy agent after step (h) of administering to the subject the third therapeutically effective amount of the clearing agent. In a specific embodiment, step (g) of administering to the subject a therapeutically effective amount of a clearing agent occurs within 12 hours of step (g) of administering to the subject the second therapeutically effective amount of the bispecific binding agent. In specific embodiments, the third therapeutically effective amount of the bispecific binding agent is 100 mg to 700 mg, 200 mg to 600 mg, 200 mg to 500 mg, 300 mg to 400 mg, about 300 mg, about 450 mg, about 500 mg, about 600 mg, or about 625 mg. In specific embodiments, the third therapeutically effective amount of the clearing agent is an amount that results in a 10:1 molar ratio of the therapeutically effective amount of the bispecific binding agent administered to the subject to the therapeutically effective amount of the clearing agent administered to the subject. In specific embodiments, the therapeutically effective amount of the clearing agent is an amount that results in at least a 90%, at least a 91%, at least a 92%, at least a 93%, at least a 94%, at least a 95%, at least a 96%, at least a 97%, at least a 98%, or at least a 99% reduction in the serum concentration of the bispecific binding agent 1 hour, 2 hours, 3 hours, or 4 hours after step (b) of administering a therapeutically effective amount of the clearing agent to the subject. In specific embodiments, the third therapeutically effective amount of the radiotherapeutic agent is between 25 mCi and 250 mCi, between 50 mCi and 200 mCi, between 75 mCi and 175 mCi, or between 100 mCi and 150 mCi. In specific embodiments, the third therapeutically effective amount of the bispecific binding agent is administered to the subject intravenously, subcutaneously, intramuscularly, parenterally, transdermally, transmucosally, intraperitoneally, intrapleurally, or into any other body compartment, such as intrathecally, intraventricularly, or intraparenchymally.In a preferred embodiment, the third therapeutically effective amount of the bispecific binding agent is administered intravenously to the subject. In a specific embodiment, the third therapeutically effective amount of the clearing agent is administered intravenously to the subject. In a specific embodiment, the third therapeutically effective amount of the radiotherapy agent is administered intravenously, subcutaneously, intramuscularly, parenterally, transdermally, transmucosally, intraperitoneally, intrathoracically, or to any other body compartment, such as intrathecally, intraventricularly, or intraparenchymally. In a preferred embodiment, the third therapeutically effective amount of the radiotherapy agent is administered intravenously to the subject. In a specific embodiment, the bispecific binding agents of the methods of treating cancer presented herein are contained in a pharmaceutical composition that further comprises a pharma- ceutically acceptable carrier.

[0025] In a specific embodiment, the cancer treated according to the methods presented herein expresses HER2, the cancer is breast cancer, gastric cancer, osteosarcoma, desmoplastic small round cell carcinoma, ovarian cancer, prostate cancer, pancreatic cancer, glioblastoma multiforme, gastric junction adenocarcinoma, gastroesophageal junction adenocarcinoma, cervical cancer, salivary gland cancer, soft tissue sarcoma, leukemia, melanoma, Ewing's sarcoma, rhabdomyosarcoma, head and neck cancer, or neuroblastoma. In a specific embodiment, the cancer is a metastatic tumor. In a specific embodiment, the metastatic tumor is a peritoneal metastasis. In a specific embodiment, the cancer treated according to the methods presented herein expresses HER2, the method of treating cancer further comprises administering to the subject an agent that increases HER2 expression by cells. In specific embodiments, agents that increase HER2 expression by cells, for example by transient depletion of caveolin 1 (CAV1), prolonging the half-life of HER2 and increasing its availability at the cell membrane, may be used; an example of such an agent is lovastatin. In specific embodiments where the cancer treated according to the methods presented herein expresses HER2, the cancer is resistant to treatment with trastuzumab, cetuximab, lapatinib, erlotinib, or any other small molecule or antibody that targets the HER family of receptors. [Brief description of the drawings]

[0026] [Figure 1A] Figure 1 shows in vitro characterization of anti-HER2-C825 BsAb. Figure 1A: Biochemical purity of HER2-C825 by SE-HPLC chromatogram (UV: 280 nm). The main peak (after 15.933 min) is the fully assembled BsAb peak (>96% integrated area under curve) with an approximate molecular weight of 210 kDa. After 25 min is the salt buffer peak. [Figure 1B] Figure 1B shows in vitro characterization of anti-HER2-C825 BsAb.B: Biacore sensorgram of BsAb binding to BSA-(Y)-DOTA-Bn. [Figure 1C] Figure 1C: In vitro characterization of anti-HER2-C825 BsAb. FACS histograms of antibody binding to the HER2(+) breast cancer cell line AU565. The top left histogram records the antibody concentration (μg per 106 cells); Rituxan was used as a negative control (MFI of 5). [Diagram 2] Figure 1 shows that anti-HER2-C825 BsAb bound to the surface of HER2(+) tumors is rapidly internalized. Anti-HER2-C825 was radioiodinated and in vitro radiotracer binding studies were performed with HER2(+) BT-474 cells to determine the internalization and intracellular processing of 131I-anti-HER2-C825 at 37°C. Data are presented as mean ± standard deviation (SD); n=3. [Diagram 3]Figure 1 shows an ex vivo biodistribution study of 177Lu radioactivity in various tissues of groups of nude mice (n=4 per group) bearing subcutaneous (sc) BT-474 tumors to optimize BsAb to anti-HER2 DOTA-PRIT (5.5-5.6MBq; approx. 30pmol) with 177Lu-DOTA-Bn. No clearing agent step was performed. The uptake of 177Lu radioactivity (percent injected radioactivity (IA / g%); mean ± SD) was determined 24 hours after injection in tumors following anti-HER2-DOTA-PRIT with various doses of BsAb (0.25, 0.50, or 0.75mg BsAb per mouse; 1.19-3.57nmol per mouse). No significant differences in tumor uptake of 177Lu radioactivity were observed between groups receiving 0.25 mg BsAb per mouse or 0.50 mg BsAb per mouse (ns) (P>0.05), suggesting that 0.25 mg BsAb per mouse was optimal. [Figure 4] Figure 1 shows highly tumor targeting of 177Lu radioactivity with minimal uptake in normal tissues including blood and kidney by optimized anti-HER2-DOTA-PRIT as early as 1 hour post-injection. Sequential biodistribution data from 1 to 336 hours post-injection of 5.5-6.1MBq (approximately 30pmol) pretargeted 177Lu-DOTA-Bn show tissue uptake (uptake as IA / g%; note: y-axis is in logarithmic scale) in subcutaneous BT-474 tumors and selected normal tissues. Data over the 24 hour post-injection period, when tumor uptake was maximal, is also presented in Table 10 (see Section 6 below). Data for all time points studied are presented in tabular form in Table 12 (see Section 6 below). [Figure 5A]Representative histology, immunohistochemistry (IHC), and autoradiography (Autorad.) assaying for BsAb and pretargeted 177Lu radioactivity distribution within BT-474 tumors. Figure 5A: IHC of anti-HER2-C825 BsAb (0.25 mg, 1.19 nmol) 24 hours after injection. Scale bar is 1000 μm. Using image-based densitometry, the % positive areas for BsAb-IHC and HER2-IHC were 51 and 61%, respectively, resulting in a (BsAb-IHC) / (HER2-IHC) ratio of 0.84. [Figure 5B] Representative histology, immunohistochemistry (IHC), and autoradiography (Autorad.) assaying for BsAb and pretargeted 177Lu radioactivity distribution within BT-474 tumors. Figure 5B: H&E and autoradiography (Autorad.) for pretargeted 177Lu-DOTA-Bn (55.5MBq, 300pmol) 24 hours after injection. Scale bar is 2000 μm. [Figure 6A] FIG. 6A: One cycle of anti-DOTA-PRIT with 55.5MBq IA results in complete response (CR) in mice with small BT-474 tumors, but is generally ineffective in mice bearing medium BT-474 tumors. Tumor volumes are presented as mean ± standard error of the mean (SEM). Black arrows indicate the day of injection of 177Lu-DOTA-Bn. FIG. 6A: Treatment of mice bearing small tumors with one cycle of anti-HER2-DOTA-PRIT + 55.5MBq 177Lu-DOTA-Bn versus control group. [Figure 6B]FIG. 6B: One cycle of anti-DOTA-PRIT with 55.5MBq IA results in complete response (CR) in mice with small BT-474 tumors, but is generally ineffective in mice bearing medium BT-474 tumors. Tumor volumes are presented as mean ± standard error of the mean (SEM). Black arrows indicate the day of injection of 177Lu-DOTA-Bn. FIG. 6B: Treatment of mice bearing medium tumors with one cycle of anti-HER2-DOTA-PRIT + 11.1, 33.3, or 55.5MBq 177Lu-DOTA-Bn versus control group. [Figure 7] Anti-HER2 DOTA-PRIT+177Lu-DOTA-Bn for theranostics. Planar scintigraphy of groups of mice bearing subcutaneous BT-474 xenografts (red arrows; palpable: 30 mm3) treated with anti-HER2 DOTA-PRIT+177Lu-DOTA-Bn (left image) or non-targeted 177Lu-DOTA-Bn (right image). While uptake of 177Lu radioactivity by pretargeted specific tumors was evident, mice administered 55.5 MBq 177Lu-DOTA-Bn showed mainly uptake in the kidney, consistent with renal clearance of 177Lu-DOTA-Bn. All images are presented on the same scale. [Figure 8] Figure 1 shows that split anti-DOTA-PRIT with IA of 167MBq resulted in 100% CR in mice with medium-sized xenografts and no recurrence after 85 days. Tumor volumes are presented as mean ± SEM. Black arrows indicate the day of injection of 177Lu-DOTA-Bn. [Figure 9A]Monitoring of fractionated anti-HER2-DOTA-PRIT treatment by SPECT / CT. The imaging field was limited to the caudal half of the animal (midline to the tail) to focus on the tumor (white arrow). Yellow arrows point to the bladder when appropriate. Figure 9A: Representative SPECT / CT images (left to right: coronal and transverse sections, maximum intensity projection (MIP) through the center of the tumor) of a BT-474 tumor-bearing animal 24 hours after injection of control IgG-DOTA-PRIT or cycle 1 177Lu-DOTA-Bn (55.5MBq, 300pmol) pretargeted with anti-HER2-DOTA-PRIT. [Figure 9B] Monitoring of fractionated anti-HER2-DOTA-PRIT treatment by SPECT / CT. The imaging field was limited to the caudal half of the animal (midline to the tail) to focus on the tumor (white arrow). Yellow arrows point to the bladder when appropriate. FIG. 9B: Representative sequential SPECT / CT MIP images of a BT-474 tumor-bearing animal receiving fractionated anti-HER2-DOTA-PRIT (left) taken 24 hours after each cycle of radioactive injection. Image-derived region of interest (ROI) values ​​for tumor uptake (mouse 1 (M1), M2, and M3) 24 hours after injection of 177Lu-DOTA-Bn (55.5 MBq, 300 pmol) in cycles 1, 2, or 3 are presented as MBq / g (mean ± SD). [Figure 10] Figure 1 shows fractionated anti-HER2-DOTA-PRIT+177Lu-DOTA-Bn for theranostics. SPECT / CT images of 3 out of 8 animals (subcutaneous BT-474 tumors; randomly selected animals, mice 1 (M1), M2, and M3) undergoing fractionated 3-cycle treatment with anti-HER2-DOTA-PRIT+55.5MBq 177Lu-DOTA-Bn. White arrows point to tumors in the lower flank. [Figure 11]Figure 1 shows the body weight of animals from before treatment (baseline, day 0) with 1 cycle anti-HER2 DOTA-PRIT + 11.1-55.5 MBq of 177Lu-DOTA-Bn from approximately 85-200 days after treatment. Data are presented as mean ± SD. [Figure 12A] Figure 12A, B, and C show animal weights from pre-treatment (baseline, day 0) to 80 days after treatment with treated controls. Black arrows indicate the day of injection of 177Lu-DOTA-Bn. Asterisks indicate the date of euthanasia or the date of death due to excessive weight loss (i.e., when body weight drops to 80% of baseline). [Figure 12B] Figure 12A, B, and C show animal weights from pre-treatment (baseline, day 0) to 80 days after treatment with treated controls. Black arrows indicate the day of injection of 177Lu-DOTA-Bn. Asterisks indicate the date of euthanasia or the date death was found due to excessive weight loss (i.e., when body weight drops to 80% of baseline). [Figure 12C] Figure 12A, B, and C show animal weights from pre-treatment (baseline, day 0) to 80 days after treatment with treated controls. Black arrows indicate the day of injection of 177Lu-DOTA-Bn. Asterisks indicate the day of euthanasia or death was found due to excessive weight loss (i.e., when body weight drops to 80% of baseline). [Figure 12D] Figure 12D shows the weight of animals before treatment (baseline, day 0) with split anti-HER2-DOTA-PRIT (Figure 12D) and after 80 days of treatment. Black arrows indicate the day of injection of 177Lu-DOTA-Bn. Asterisks indicate the day of euthanasia or the day of death due to excessive weight loss (i.e., when the body weight drops to 80% of baseline). [Figure 13]Morphological analysis via H&E staining of BT-474 tumor inoculation sites after 85 days shows that treatment with anti-HER2-DOTA-PRIT resulted in cures (5 of 8 cases) or microscopic residual disease (3 of 8 cases), while controls (10 of 10 cases) showed the presence of bulk tumors (see Table 24 for detailed description). [Figure 14] FIG. 1 is a diagram showing the DOTA-PRIT method. [Figure 15] FIG. 1 shows sequential PET imaging of 124I-anti-HER2-C825 in nude mice bearing subcutaneous BT-474 tumors. [Figure 16] Figure 1 shows BT-474 xenografts of mass 100-200 mg. Uptake of 177Lu-DOTA-Bn injected 28 hours after anti-HER2-C825 BsAb antibody injection. No clearing agent was used, and total antigen doses varied from 0.025 mg to 0.75 mg in individual experiments. Tumors were harvested 24 hours after radioactive injection ("pi") or 52 hours after initial antibody injection. Uptake, dependent on binding of radioactive hapten to high affinity Fv fragments conjugated to antibodies, was shown to be a function of dose, increasing to a plateau at approximately 125-250 micrograms. [Figure 17] Figure 14 shows the pooling of 177Lu-DOTA-B at the tumor site and the antibody concentration in the blood, calculated as a function of the administered dose. This assumes that the uptake curves in Figure 15 apply. Note that the uptake curve follows a regular uptake binding curve. Total binding increases with saturation kinetics until it plateaus. [Figure 18]Representative SPECT / CT images of subcutaneous BT-474 tumor-bearing mice (216 mm3 by external caliper measurement) 24 hours after injection of 177Lu-DOTA-Bn (55.5 MBq, approx. 300 pmol) pretargeted with anti-HER2-DOTA-PRIT. The imaging field was limited to the caudal half of the animal (midline to the tail) to focus on the tumor (white arrow). Mice were euthanized immediately after imaging and the radioactivity concentration in the tumor was determined by ex vivo biodistribution (attenuation-corrected percent injected radioactivity per gram of tissue (IA / g%)) to be 6.06 (5.53±0.27IA / g% for n=3). MIP=maximum intensity projection. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] 5. Detailed Description Provided herein is a method of treating cancer in a subject in need thereof, comprising: (a) administering to the subject a therapeutically effective amount of a bispecific binding agent; (b) administering to the subject a therapeutically effective amount of a clearing agent within 12 hours of administering to the subject a therapeutically effective amount of the bispecific binding agent (a), the clearing agent binds to the second binding site and functions to reduce the bispecific binding agent circulating in the blood of the subject; and after administering to the subject a therapeutically effective amount of the clearing agent (b), administering to the subject a therapeutically effective amount of a radiotherapy agent. The bispecific binding agent for use in the method of treating cancer provided herein is capable of specifically binding to (i) a cancer antigen expressed by the cancer to be treated by the method; (ii) the clearing agent; and (iii) a radiotherapy agent. In certain embodiments, the bispecific binding agent for use in the method of treating cancer provided herein specifically binds simultaneously to (i) a cancer antigen expressed by the cancer to be treated; and (ii) a radiotherapy agent. Without being bound to a particular theory, the bispecific binding agent forms a crosslink between the cancer cell and the radiotherapeutic agent, allowing the radiotherapeutic agent to kill the cancer cell bound to the bispecific binding agent. Surprisingly, the method of treating cancer provided herein is still effective even when targeting a cancer antigen that is internalized into the cancer cell (see, e.g., section 6). Furthermore, the method of treating cancer provided herein is advantageous because the step of administering the clearing agent can be performed as early as 1 hour after administration of the bispecific binding agent (compared to the standard waiting time of 24-120 hours between administration of the tumor targeting agent and administration of the clearing agent), e.g., in less than 16 hours.

[0028] Also provided herein are bispecific binding agents (see, e.g., Section 5.2), clearing agents (see, e.g., Section 5.3), and radiotherapeutic agents (see, e.g., Section 5.4) for use in the methods described herein. Also provided herein are compositions (e.g., pharmaceutical compositions) and kits (see, e.g., Section 5.5) comprising the bispecific binding agents, clearing agents, and / or radiotherapeutic agents.

[0029] 5.1 Methods of Treating Cancer Provided herein, in specific embodiments, is a method for treating cancer in a subject in need thereof, comprising the steps of: (a) administering to the subject a therapeutically effective amount of a bispecific binding agent, wherein the bispecific binding agent comprises a first molecule covalently linked (optionally via a linker) to a second molecule, the first molecule comprising a first binding site, the first binding site specifically binding to a first target, the first target being a cancer antigen expressed by the cancer, and the second molecule comprising a second binding site, the second binding site specifically binding to a second target, the second target being not a cancer antigen; (b) administering to the subject a therapeutically effective amount of the bispecific binding agent. and after step (b) of administering a therapeutically effective amount of a clearing agent to the subject, the second target being bound to a metal radionuclide, the second target being a metal chelator; or (ii) a second target bound, preferably covalently bound, to a metal chelator, the metal chelator being bound to a metal radionuclide.

[0030] Also provided herein is a method of treating cancer in a subject in need thereof, comprising the steps of: (a) administering to the subject a first therapeutically effective amount of a bispecific binding agent, wherein the first therapeutically effective amount of the bispecific binding agent is between 100 mg and 700 mg, between 200 mg and 600 mg, between 200 mg and 500 mg, between 300 mg and 400 mg, about 300 mg, about 450 mg, about 500 mg, about 600 mg, or about 625 mg, and the bispecific binding agent comprises a first molecule covalently linked (optionally via a linker) to a second molecule, and the cancer expresses HER2, and the first molecule comprises an antibody or antigen-binding fragment thereof, or an scFv, wherein the antibody or antigen-binding fragment thereof, or scFv (i) binds to HER2 on the cancer, and (ii) comprises all three of the heavy chain CDRs of SEQ ID NO:20 and all three of the light chain CDRs of SEQ ID NO:19, and the second molecule comprises a second binding Also provided is a method in which the subject is a human, the method comprising the steps of: (a) administering a therapeutically effective amount of a bispecific binding agent to the subject, the second binding site specifically binding to a second target, the second target not being a cancer antigen; (b) after step (a) of administering a therapeutically effective amount of a clearing agent to the subject, the clearing agent binding to the second binding site and functioning to reduce the amount of bispecific binding agent circulating in the blood of the subject; and (c) after step (b) of administering a therapeutically effective amount of a clearing agent to the subject, the radiotherapy agent (i) comprising a second target bound to a metal radionuclide, the second target being a metal chelator; or (ii) comprising a second target bound, preferably covalently bound, to a metal chelator, the metal chelator being bound to a metal radionuclide, wherein the subject is a human. In a specific embodiment, the therapeutically effective amount of the bispecific binding agent is about 450 mg.

[0031] Also provided herein is a method of treating cancer in a subject in need thereof, comprising the steps of: (a) administering to the subject a first therapeutically effective amount of a bispecific binding agent, wherein the first therapeutically effective amount of the bispecific binding agent is between 100 mg and 700 mg, between 200 mg and 600 mg, between 200 mg and 500 mg, between 300 mg and 400 mg, about 300 mg, about 450 mg, or about 500 mg; the bispecific binding agent comprises a first molecule covalently linked (optionally via a linker) to a second molecule; the cancer expresses HER2; and the first molecule comprises an antibody or antigen-binding fragment thereof, or an scFv, wherein the antibody or antigen-binding fragment thereof, or scFv (i) binds to HER2 on the cancer; and (ii) comprises all three of the heavy chain CDRs of SEQ ID NO:20 and all three of the light chain CDRs of SEQ ID NO:19; and the second molecule is linked to a second binding molecule. and (b) after step (a) of administering a therapeutically effective amount of the bispecific binding agent to the subject, administering a therapeutically effective amount of a clearing agent to the subject, wherein the clearing agent binds to the second binding site and functions to reduce bispecific binding agent circulating in the blood of the subject; and (c) after step (b) of administering a therapeutically effective amount of a clearing agent to the subject, wherein the radiotherapeutic agent (i) comprises a second target bound to a metal radionuclide, and the second target is a metal chelator; or (ii) comprises a second target bound to a metal chelator, and the metal chelator is bound to a metal radionuclide, wherein the subject is a human.

[0032] In a specific embodiment, the bispecific binding agent is a bispecific binding agent described in Section 5.2. In a preferred embodiment, the first target of the bispecific binding agent is HER2 and the second target of the bispecific binding agent is DOTA. In a specific embodiment, the therapeutically effective amount of the bispecific binding agent is as described in Section 5.7. In a specific embodiment, the bispecific binding agent is administered to a subject via a route of administration described in Section 5.7.

[0033] In specific embodiments, the clearing agent is a clearing agent described in Section 5.3 or Section 6. In specific embodiments, a therapeutically effective amount of the clearing agent is as described in Section 5.7. In specific embodiments, the clearing agent is administered to the subject via a route of administration described in Section 5.7. In specific embodiments, step (b) of administering a therapeutically effective amount of the clearing agent to the subject occurs within 10 hours, within 8 hours, within 6 hours, within 4 hours, within 2 hours, within 1-12 hours, within 2-12 hours, within 1-2 hours, within 1-3 hours, within 1-4 hours, within 2-6 hours, within 2-8 hours, within 2-10 hours, within 4-6 hours, within 4-8 hours, within 4-10 hours, within 2 hours, or within 4 hours after step (a) of administering a therapeutically effective amount of a bispecific binding agent to the subject. In another specific embodiment, step (b) of administering a therapeutically effective amount of the clearing agent to the subject occurs about 2 hours, about 4 hours, about 6 hours, about 8 hours, or about 10 hours after step (a) of administering a therapeutically effective amount of a bispecific binding agent to the subject. In specific embodiments, the bispecific binding agent is at least 100 kDa and step (b) of administering a therapeutically effective amount of a clearing agent to the subject occurs within 4 hours of step (a) of administering a therapeutically effective amount of the bispecific binding agent to the subject. In specific embodiments, step (b) of administering a therapeutically effective amount of a clearing agent to the subject occurs at a time point that is up to 10% longer or up to 10% shorter than the times described herein after step (a) of administering a therapeutically effective amount of a bispecific binding agent to the subject.

[0034] In specific embodiments, the radiotherapeutic agent is a radiotherapeutic agent described in Section 5.4 or Section 6. In a preferred embodiment, the radiotherapeutic agent comprises DOTA or a derivative thereof bound to a metallic radionuclide. In a preferred embodiment, the radiotherapeutic agent comprises DOTA or a derivative thereof bound to a metallic radionuclide, the metallic radionuclide is 177Lu. In specific embodiments, the therapeutically effective amount of the radiotherapeutic agent is as described in Section 5.7. In specific embodiments, the radiotherapeutic agent is administered to the subject via a route of administration described in Section 5.7. In specific embodiments, step (c) of administering a therapeutically effective amount of a radiotherapeutic agent to the subject occurs 1-2 hours, 1-3 hours, 1-4 hours, 2-6 hours, 2-8 hours, 2-10 hours, 4-6 hours, 4-8 hours, 4-10 hours, within 1 hour, within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours after step (b) of administering a therapeutically effective amount of a clearing agent to the subject. In specific embodiments, step (c) of administering a therapeutically effective amount of a radiotherapeutic agent to the subject occurs about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, or about 6 hours after step (b) of administering a therapeutically effective amount of a clearing agent to the subject. In a specific embodiment, step (c) of administering a therapeutically effective amount of a radiotherapeutic agent to the subject occurs about 1 hour after step (b) of administering a therapeutically effective amount of a clearing agent to the subject. In a specific embodiment, step (c) of administering a therapeutically effective amount of a radiotherapeutic agent to the subject occurs within 16 hours after step (a) of administering a therapeutically effective amount of a bispecific binding agent to the subject. In a specific embodiment, step (c) of administering a therapeutically effective amount of a radiotherapeutic agent to the subject occurs at a time point that is up to 10% longer or up to 10% shorter than the time points described herein after step (b) of administering a therapeutically effective amount of a clearing agent to the subject. In a specific embodiment, step (c) of administering a therapeutically effective amount of a radiotherapeutic agent to the subject occurs at a time point that is up to 10% longer or up to 10% shorter than the time points described herein after step (a) of administering a therapeutically effective amount of a bispecific binding agent to the subject.

[0035] The cancer treated according to the methods described herein can be any cancer known to those skilled in the art. In a specific embodiment, the cancer is a cancer described in Section 5.6 or Section 6. In a specific embodiment, the cancer is a cancer described in Table 1 below. Those skilled in the art will understand that the cancer treated according to the methods described herein will determine the identity of the first target of the bispecific binding agent (see, e.g., Sections 5.2 and 6) utilized in the methods presented herein. For example, in the methods of treating cancer presented herein, the cancer treated to use a bispecific binding agent with HER2 as the first target is a cancer that expresses HER2 (e.g., breast cancer). In a specific embodiment, the cancer is a HER2 expressing cancer, including but not limited to breast cancer, gastric cancer, osteosarcoma, desmoplastic small round cell carcinoma, squamous cell carcinoma of the head and neck, ovarian cancer, prostate cancer, pancreatic cancer, glioblastoma multiforme, gastric junction adenocarcinoma, gastroesophageal junction adenocarcinoma, cervical cancer, salivary gland cancer, soft tissue sarcoma, leukemia, melanoma, Ewing's sarcoma, rhabdomyosarcoma, neuroblastoma, or any other neoplastic tissue that expresses the HER2 receptor. In a specific embodiment, the HER2 expressing cancer is resistant to treatment with trastuzumab, cetuximab, lapatinib, erlotinib, or any other small molecule or antibody that targets the HER family of receptors. In a specific embodiment, tumors that are resistant to treatment with trastuzumab, cetuximab, lapatinib, erlotinib, or any other small molecule or antibody targeting the HER family of receptors are responsive to treatment with the bispecific binding agents of the invention. In a specific embodiment, HER2-expressing cancers are resistant to treatment with necitumumab, panitumumab, pertuzumab, or ado-trastuzumab emtansine. In a specific embodiment, HER2-expressing cancers that are resistant to treatment with necitumumab, panitumumab, pertuzumab, or ado-trastuzumab emtansine are responsive to treatment with the bispecific binding agents of the invention.In specific embodiments, a cancer is considered to be resistant to a treatment (e.g., trastuzumab, cetuximab, necitumumab, panitumumab, pertuzumab, ado-trastuzumab emtansine, lapatinib, erlotinib, or any small molecule that targets the HER family of receptors) if it fails to respond or responds incompletely (response less than complete remission) after treatment, or progresses or recurs.

[0036] In a specific embodiment, the methods of treating cancer presented herein are carried out as part of a multi-cycle regimen, as described in Section 5.7. In specific embodiments, the subject is a subject described in Section 5.6. In specific embodiments, treatment can be treatment to achieve a beneficial or desired clinical outcome, including, but not limited to, alleviation of symptoms, reduction in spread of disease, stabilization of the disease state (i.e., non-progression), delay or slowing of disease progression, improvement or inhibition of the disease state, and remission (whether partial or complete). In specific embodiments, "treatment" can also be treatment to prolong survival as compared to expected survival if not receiving treatment.

[0037] 5.2 Bispecific binding factors Provided herein are bispecific binding agents for use in the methods of treating cancer provided herein (see, for example, Sections 5.1 and 6). The bispecific binding agents described herein comprise a first molecule covalently bound (possibly via a linker) to a second molecule, the first molecule comprising a first binding site, the first binding site specifically binding to a first target, the first target being a cancer antigen expressed by the cancer, and the second molecule comprising a second binding site, the second binding site specifically binding to a second target, the second target being not a cancer antigen. In a specific embodiment, the bispecific binding agent is a bispecific binding agent described in Section 6. The first molecule of the bispecific binding agent mediates the binding of the bispecific binding agent to a cancer cell. In particular, the first molecule of the bispecific binding agent comprises a first binding site that specifically binds to a first target, said first target being a cancer antigen expressed by the cancer to be treated with the bispecific binding agent according to the methods provided herein (see, e.g., Sections 5.1 and 6).

[0038] In a specific embodiment, the first molecule comprises an antibody or an antigen-binding fragment thereof, said antibody or an antigen-binding fragment thereof comprising a first binding site. In a specific embodiment, the antibody of the first molecule of the bispecific binding agent is an immunoglobulin. The antibody in the bispecific binding agent of the present invention can be, by way of non-limiting example, a monoclonal antibody, a naked antibody, a chimeric antibody, a humanized antibody, or a human antibody. As used herein, the term "immunoglobulin" is used in accordance with its well-known meaning in the art and comprises two heavy chains and two light chains. Methods for making antibodies are described herein below.

[0039] In a specific embodiment, where the first molecule comprises an antibody or an antigen-binding fragment thereof, and the antibody or an antigen-binding fragment thereof comprises a first binding site, the antibody is a human antibody. Those skilled in the art know how to make human antibodies, for example, by phage display using antibody libraries derived from human immunoglobulin sequences, by using transgenic mice, by immunizing mice transplanted with human peripheral blood leukocytes, spleen cells, or bone marrow (e.g., the Trioma method with XTL), by using in vitro activated B cells, or by using a technique called "guided selection". See, e.g., U.S. Pat. Nos. 4,444,887, 4,716,111, 5,567,610, and 5,229,275; and PCT Publication Nos. 98 / 46645, 98 / 60433, 98 / 24893, 98 / 16664, 96 / 34096, 96 / 33735, 91 / 10741, Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Riss, (1985); and Boerner et al., J. Immunol., 147(1):86-95, (1991).

[0040] A chimeric antibody is a recombinant protein that contains the variable domains, including the complementarity determining regions (CDRs), of an antibody from one species, preferably a rodent antibody, while the constant domains of the antibody molecule are derived from the constant domains of a different species, e.g., from the constant domains of a human antibody when human applications are envisaged. In veterinary applications, the constant domains of the chimeric antibody may be derived from the constant domains of other species, e.g., horse, monkey, cow, pig, cat or dog. For example, for use of a bispecific binding agent in a method for treating cancer in dogs, the constant domains of the chimeric antibody that form part of the bispecific binding agent may be derived from the constant domains of a canine antibody.

[0041] A humanized antibody is an antibody produced by recombinant DNA technology, in which some or all of the amino acids of the light or heavy chain of a human immunoglobulin that are not required for antigen specificity (e.g., constant and framework regions of the variable domain) are used to replace the corresponding amino acids from the light or heavy chain of the cognate non-human antibody. By way of example, a humanized form of a non-human (e.g., murine) antibody against a given antigen has, in both its heavy and light chains, (1) the constant regions of a human antibody; (2) framework regions derived from the variable domain of a human antibody; and (3) CDRs derived from a non-human antibody. If necessary, one or more residues in the human framework regions can be changed to the residues at the corresponding positions in the murine antibody so as to preserve or improve the binding affinity of the humanized antibody to the antigen. This change is often referred to as a "back mutation." Similarly, forward mutations can be made to revert to the murine sequence for desired reasons, e.g., for stability or affinity to the antigen. Without being bound by any theory, humanized antibodies contain significantly fewer non-human components and therefore are generally less likely to induce an immune response in humans compared to chimeric human antibodies. Those skilled in the art know how to make humanized antibodies. For example, see EP 0239400; Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239: 1534-1536 (1988); Queen et al., Proc. Nat. Acad. ScL USA 86:10029 (1989); U.S. Patent No. 6,180,370; and Orlandi et al., Proc. Natl. Acad. Sd. USA 86:3833 (1989).

[0042] The antigen-binding fragment may be a Fab fragment, a F(ab')2 fragment of an antibody described herein, or a portion that contains amino acid residues that confer to the antibody its specificity for an antigen (e.g., complementarity determining region (CDR)). The antibody may be from any animal species, including rodents (e.g., mice, rats, or hamsters) and humans. Methods for making antigen-binding fragments of antibodies are known in the art. For example, antigen-binding fragments may be made by enzymatic cleavage, synthetically, or recombinantly, as known in the art and / or described herein.

[0043] As used herein, the term "variable region" or "variable domain" of an antibody is used interchangeably and is generally known in the art. In general, the spatial orientation of CDRs and FRs in the variable domain is as follows, from N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Without wishing to be bound by a particular mechanism or theory, it is believed that the CDRs of the light chain and the heavy chain are primarily responsible for the interaction and specificity of the antibody with the antigen. In certain embodiments, the variable region is a rodent (e.g., mouse or rat) variable region. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent (e.g., mouse or rat) CDRs and human FRs. In certain embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or mouse CDRs and primate (e.g., non-human primate) FRs.

[0044] In the art, CDRs are defined in various ways, including the Kabat, Chothia, and IMGT definitions, as well as exemplary definitions. The Kabat definition is based on sequence diversity (Kabat, Elvin A. et al., Sequences of Proteins of Immunological Interest. Bethesda: National Institutes of Health, 1983). In terms of the Kabat numbering system, (i) V H CDR1 is typically located at amino acid positions 31 to 35 of the heavy chain, which may include one or two additional amino acids following amino acid position 35 (referred to as 35A and 35B in the Kabat numbering scheme); (ii) V H CDR2 is typically located at amino acid positions 50 to 65 of the heavy chain; H CDR2 is typically located at amino acid positions 95 to 102 of the heavy chain (Kabat, Elvin A. et al., Sequences of Proteins of Immunological Interest. Bethesda: National Institutes of Health, 1983). In accordance with the Kabat numbering system, (i) V L CDR1 is typically located at amino acid positions 24 to 34 of the light chain; L CDR2 is typically located at amino acid positions 50 to 56 of the light chain; LCDR3 is typically located at amino acid positions 89-97 of the light chain (Kabat, Elvin A. et al., Sequences of Proteins of Immunological Interest. Bethesda: National Institutes of Health, 1983). As is well known to those skilled in the art, when using the Kabat numbering system, the actual linear amino acid sequence of an antibody variable domain may contain fewer or more amino acids due to shortening or elongation of FRs and / or CDRs, and thus the Kabat amino acid numbers are not necessarily the same as the linear amino acid numbers.

[0045] The Chothia definition is based on the location of structural loop regions (Chothia et al., (1987) J Mol Biol 196: 901-917; and U.S. Patent No. 7,709,226). The term "CDR according to Chothia" and similar terms are recognized in the art and refer to the CDR sequences of an antibody determined according to the method of Chothia and Lesk, 1987, J. Mol. Biol., 196:901-917, also referred to herein as "CDR according to Chothia" (see also e.g. U.S. Patent No. 7,709,226; and Martin, A., "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001)). In light of the Chothian numbering system, the numbering system of Kabat is used to H The amino acid residues in the region are numbered as follows: (i) V H CDR1 is typically located at amino acid positions 26 to 32 of the heavy chain; HCDR2 is typically located at amino acid positions 53 to 55 of the heavy chain; H CDR3 is typically located at amino acid positions 96-101 of the heavy chain. In a specific embodiment, the CDR3 is located at amino acid positions 96-101 of the heavy chain using the Kabat numbering system in accordance with the Chothia numbering system. H The amino acid residues in the region are numbered as follows: (i) V H CDR1 is typically located at amino acid positions 26 to 32 or 34 of the heavy chain; H CDR2 is typically located at amino acid positions 52 to 56 of the heavy chain (in one embodiment, CDR2 is at positions 52A to 56 [position 52A follows position 52 in the numbering]); (iii) V H CDR3 is typically located at amino acid positions 95-102 of the heavy chain (in one embodiment, there are no amino acids at positions numbered 96-100). In accordance with the Chothia numbering system, the Kabat numbering system is used to identify the V L The amino acid residues in the region are numbered as follows: (i) V L CDR1 is typically located at amino acid positions 26 to 33 of the light chain; L CDR2 is typically located at amino acid positions 50 to 52 of the light chain; L CDR3 is typically located at amino acid positions 91-96 of the light chain. In a specific embodiment, the CDR3 is located at amino acid positions 91-96 of the light chain using the Kabat numbering system in accordance with the Chothia numbering system. L The amino acid residues in the region are numbered as follows: (i) V L CDR1 is typically located at amino acid positions 24 to 34 of the light chain; L CDR2 is typically located at amino acid positions 50 to 56 of the light chain; L CDR3 is typically located at amino acid positions 89-97 of the light chain (in one embodiment, there are no amino acids at positions numbered 96-100). These CDR positions according to Chothia may vary depending on the antibody and can be determined according to methods known in the art.

[0046] The IMGT definition is according to IMGT (the international ImMunoGeneTics information system®, whose founder and operator is Marie-Paule Lefranc, Montpellier, France; the IMGT® website is imgt.org; see, e.g., Lefranc, M.-P., 1999, The Immunologist, 7:132-136 and Lefranc, M.-P. et al., 1999, Nucleic Acids Res., 27:209-212, both of which are incorporated herein by reference in their entireties). In terms of the IMGT numbering system, (i) V H CDR1 is typically located at amino acid positions 25 to 35 of the heavy chain; H CDR2 is typically located at amino acid positions 51 to 57 of the heavy chain; H CDR2 is typically located at amino acid positions 93 to 102 of the heavy chain. In accordance with the IMGT numbering system, (i) V L CDR1 is typically located at amino acid positions 27 to 32 of the light chain; L CDR2 is typically located at amino acid positions 50 to 52 of the light chain; L CDR3 is typically located at amino acid positions 89-97 of the light chain.

[0047] The cancer antigen that is the first target of the bispecific binding agent described herein can be any cancer antigen known in the art. Non-limiting examples of cancer antigens and non-limiting examples of cancers that express said antigens are provided in Table 1 below. In a preferred embodiment, the cancer antigen is HER2.

[0048] Table 1. [Table 1] TIFF2025039567000002.tif231155 TIFF2025039567000003.tif94155

[0049] Traditionally, pretargeted radioimmunotherapy ("PRIT") strategies have been implemented against antigens that are expressed on the cell surface and are not susceptible to endocytosis (see, e.g., Casalini et al., Journal of Nuclear Medicine, 1997; 38:1378-1381.; Liu, et al., Cancer Biother Radiopharm, 2007; 22(1):33-39.; Knight, et al., Molecular Pharmaceutics, 2017; 14(7): 2307-2313.; edited by Baum, Richard P. Therapeutic Nuclear Medicine 2014, Springer-Verlag Berlin Heidelberg, pg 612; edited by Oldham, Robert K. and Dillman, Robert O. Principles of Cancer Biotherapy 2009, Springer, ph 486).Without being bound to a particular theory, it has been found that internalization of binding agents (e.g., antibodies) bound to cancer antigens does not result in optimal presentation of tumor targeting agents (e.g., bispecific antibodies) on the surface of cancer cells for interaction with radiotherapy agents (see, e.g., Boerman et al., 2003, Pretargeted Radioimmunotherapy of Cancer: Progress Step by Step*. J. Nucl. Med. 44(3):400-411; Casalini et al., 1997, Tumor Pretargeting: Role of Avidin / Streptavidin on Monoclonal Antibody Internalization. J. Nucl. Med.; 38(9):1378-1381; Walter et al., 2010, Pretargeted Radioimmunotherapy for Hematologic and Other Malignancies, Cancer Biother Radiopharm.; 25(2):125-142). However, the Examples described herein (see Section 6) reveal that when used in accordance with the methods of treating cancer described herein (see, e.g., Sections 5.1 and 6), a high therapeutic index can be achieved for the bispecific binding agents described herein that target HER2, which are surprisingly susceptible to endocytosis (see, e.g., Austin et al., 2004, Molecular Biology of the Cell, 15:5268-5282). Thus, in a specific embodiment, the cancer antigen is an antigen that is internalized into the cancer cell.Non-limiting examples of cancer antigens that are internalized into cancer cells include HER2, CA6, CD138, CD19, CD22, CD27L, CD30, CD33, CD37, CD56, CD66e, CD70, CD74, CD79b, EGFR, EGFRvIII, FRα, GCC, GPNMB, mesothelin, MUC16, NaPi2b, nectin 4, PSMA, STEAP1, Trop-2, 5T4, AGS-16, alpha v beta 6, CA19.9, CAIX, CD174, CD180, CD227, CD326, CD79a, CEACAM5, CRIPTO, DLL3, DS6, endothelin B receptor, FAP, GD2, mesothelin, PMEL 17, SLC44A4, TENB2, TIM-1, CD98, endosialin / CD248 / TEM1, fibronectin extracellular domain B LIV-1, mucin 1, p-cadherin, peritosin, Fyn, SLTRK6, tenascin c, VEGFR2, and PRLR. See, e.g., Table 1 for a list of exemplary cancers that express the above cancer antigens.

[0050] In another specific embodiment, the cancer antigen is an antigen that is not internalized into cancer cells. Non-limiting examples of cancer antigens that are not internalized into cancer cells include CD20, CD72, fibronectin, GPA33, splice isoforms of tenascin-C, and TAG-72. For example, see Table 1 for a list of exemplary cancers that express the above cancer antigens. In a specific embodiment, where the cancer antigen is an ovarian cancer antigen, the first molecule is antibody MX35. In a specific embodiment, where the cancer antigen is Fyn3, the first molecule is antibody SC-16. In a specific embodiment, where the cancer antigen is B7-H3, the first molecule is antibody 8H9.

[0051] In a preferred embodiment, the cancer antigen is HER2. HER2 is a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases. In a specific embodiment, HER2 is human HER2. GenBank™ Accession No. NM_004448.3 (SEQ ID NO: 1) provides an exemplary human HER2 nucleic acid sequence. GenBank™ Accession No. NP_004439.2 (SEQ ID NO: 2) provides an exemplary human HER2 amino acid sequence. In another specific embodiment, HER2 is canine HER2. GenBank™ Accession No. NM_001003217.1 (SEQ ID NO: 3) provides an exemplary canine HER2 nucleic acid sequence. GenBank™ Accession No. NP_001003217.1 (SEQ ID NO: 4) provides an exemplary canine HER2 amino acid sequence.

[0052] In a specific embodiment of the bispecific binding agents of the invention, the first molecule is an antibody or antigen-binding fragment thereof that specifically binds to HER2. In a preferred embodiment, the antibody is an immunoglobulin that specifically binds to HER2. In specific embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of trastuzumab (see, e.g., Baselga et al. 1998, Cancer Res 58(13): 2825-2831), M-111 (see, e.g., Higgins et al., 2011, J Clin Oncol, 29(Suppl): Abstract TPS119), pertuzumab (see, e.g., Franklin et al., 2004, Cancer Cell, 5: 317-328), ertumaxomab (see, e.g., Kiewe and Thiel, 2008, Expert Opin Investig Drugs, 17(10): 1553-1558), MDXH210 (see, e.g., Schwaab et al., 2001, Journal of Immunotherapy ... 24(1): 79-87), 2B1 (see, e.g., Borghaei et al., 2007, J Immunother, 30: 455-467), and MM-302 (see, e.g., Wickham and Futch, 2012, Cancer Research, 72(24): Supplement 3).

[0053] In a specific embodiment, where the first molecule is an antibody or antigen-binding fragment thereof that binds to HER2, the heavy chain in the antibody or antigen-binding fragment thereof that specifically binds to HER2 is the variable heavy chain (V H The light chain in an antibody or antigen-binding fragment thereof that specifically binds to HER2 contains all three heavy chain complementarity determining regions (CDRs) of the light chain variable (V) domain of trastuzumab. LIn a specific embodiment, the heavy chain in an antibody or antigen-binding fragment thereof that specifically binds to HER2 comprises all three heavy chain CDRs of SEQ ID NO: 14, and the light chain in an antibody or antigen-binding fragment thereof that specifically binds to HER2 comprises all three light chain CDRs of SEQ ID NO: 11. In a specific embodiment, the heavy chain in an antibody or antigen-binding fragment thereof that specifically binds to HER2 comprises all three heavy chain CDRs of SEQ ID NO: 15, and the light chain in an antibody or antigen-binding fragment thereof that specifically binds to HER2 comprises all three light chain CDRs of SEQ ID NO: 16. H The domain is V of trastuzumab. H In a specific embodiment, the antibody or antigen-binding fragment thereof that specifically binds to HER2 comprises a V H The sequence of the domain comprises SEQ ID NO: 20 (see Table 4). In a specific embodiment, the antibody or antigen-binding fragment thereof that specifically binds to HER2 comprises the V domain of trastuzumab. H V of trastuzumab, which has five or fewer amino acid mutations compared to the native sequence of the domain H In a specific embodiment, the light chain V domain in the light chain of an antibody or antigen-binding fragment thereof that specifically binds to HER2 is L The domain is V of trastuzumab. L In a specific embodiment, the V domain in the light chain of an antibody or antigen-binding fragment thereof that specifically binds to HER2. L The sequence of the domain comprises SEQ ID NO: 19 (see Table 4). In a specific embodiment, the antibody or antigen-binding fragment thereof that specifically binds to HER2 comprises the V domain of trastuzumab. L V of trastuzumab, which has five or fewer amino acid mutations compared to the native sequence of the domain L In a specific embodiment, the V domain of trastuzumab is H Domain and / or V L V of an antibody or antigen-binding fragment thereof compared to the respective native sequences of the domains H Domain and / or V L One or more of the amino acid mutations in the V domain of trastuzumab H Domain and / or V LThese are conservative amino acid substitutions with respect to the respective native sequences of the domains.In a preferred embodiment, the antibody that specifically binds to HER2 is an immunoglobulin.

[0054] Conservative amino acid substitutions are those that occur within a family of amino acids whose side chains are related.Generally, the amino acids encoded by genes are divided into (1) acidic family, including aspartic acid and glutamic acid; (2) basic family, including arginine, lysine, and histidine; (3) non-polar family, including isoleucine, alanine, valine, proline, methionine, leucine, phenylalanine, tryptophan; and (4) uncharged polar family, including cysteine, threonine, glutamine, glycine, asparagine, serine, and tyrosine; in addition, the aliphatic hydroxy family includes serine and threonine. In addition, the amide-containing family includes asparagine and glutamine. In addition, the aliphatic family includes alanine, valine, leucine, and isoleucine. In addition, the aromatic family includes phenylalanine, tryptophan, and tyrosine.Finally, the sulfur-containing side chain family includes cysteine ​​and methionine. By way of example, one of skill in the art would reasonably expect that the replacement of a single leucine with an isoleucine or valine, a single aspartic acid with a glutamic acid, a single threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid, particularly if the replacement does not involve an amino acid within a framework region, would not have a significant effect on the binding or properties of the resulting molecule. Preferred conservative amino acid substitution groups include lysine-arginine, alanine-valine, phenylalanine-tyrosine, glutamic acid-aspartic acid, valine-leucine-isoleucine, cysteine-methionine, and asparagine-glutamine.

[0055] In a specific embodiment, the first molecule is an antibody or antigen-binding fragment thereof that binds to HER2, the antibody or antigen-binding fragment thereof comprises a heavy chain of trastuzumab. In a specific embodiment, the sequence of the heavy chain comprises the sequence of any one of SEQ ID NOs: 14-17 (see Table 2). In a specific embodiment, the antibody or antigen-binding fragment thereof comprises a variant of the heavy chain of trastuzumab (see, e.g., SEQ ID NOs: 14-17 (see Table 2)). In a preferred embodiment, the sequence of the heavy chain in the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 15. In a more preferred embodiment, the sequence of the heavy chain in the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 16. In a specific embodiment, the antibody or antigen-binding fragment thereof comprises a variant of the heavy chain of trastuzumab having no more than five amino acid mutations compared to the native sequence of the heavy chain of trastuzumab. In a specific embodiment, the antibody or antigen-binding fragment thereof comprises a light chain of trastuzumab. In a specific embodiment, the sequence of the light chain in the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 11. In a specific embodiment, the antibody or antigen-binding fragment thereof comprises a variant of the light chain of trastuzumab. In a specific embodiment, the antibody or antigen-binding fragment thereof comprises a variant of the light chain of trastuzumab, which has 5 or less amino acid mutations compared to the native sequence of the light chain of trastuzumab. In a specific embodiment, one or more of the amino acid mutations of the heavy and / or light chain of the antibody or antigen-binding fragment thereof compared to the native sequence of the heavy and / or light chain of trastuzumab, respectively, are conservative amino acid substitutions in the context of the native sequence of the heavy and / or light chain of trastuzumab, respectively.

[0056] [Table A] TIFF2025039567000005.tif30170

[0057] [Table B]

[0058] Table 4: V of trastuzumabL Domains and V H An array of domains. [Table 4]

[0059] In a specific embodiment where the first molecule is an antibody or antigen-binding fragment thereof that binds to HER2, the antibody or antigen-binding fragment thereof binds to the same epitope as a HER2-specific antibody known in the art. In a specific embodiment, the antibody or antigen-binding fragment thereof binds to the same epitope as trastuzumab. Binding to the same epitope may be determined by assays known to those of skill in the art, such as, for example, mutational analysis or crystallography studies. In a specific embodiment, the antibody or antigen-binding fragment thereof competes with an antibody known in the art for binding to HER2. In a specific embodiment, the antibody or antigen-binding fragment thereof competes with trastuzumab for binding to HER2. Competition for binding to HER2 may be determined by assays known to those of skill in the art, such as, for example, flow cytometry. In a specific embodiment, the antibody or antigen-binding fragment thereof binds to the V epitope of a HER2-specific antibody known in the art. H V with at least 85%, 90%, 95%, 98%, or at least 99% similarity to the domain H In a specific embodiment, the antibody or antigen-binding fragment thereof comprises the V domain of a HER2-specific antibody known in the art. H The V domain of a HER2-specific antibody known in the art contains between one and five conservative amino acid substitutions. H In a specific embodiment, the antibody or antigen-binding fragment thereof comprises the V domain of a HER2-specific antibody known in the art. L V with at least 85%, 90%, 95%, 98%, or at least 99% similarity to the domain L In a specific embodiment, the antibody or antigen-binding fragment thereof comprises the V domain of a HER2-specific antibody known in the art. LThe V domain of a HER2-specific antibody known in the art contains between one and five conservative amino acid substitutions. L In a specific embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain V domain as set forth in Table 2 above. H Domain (e.g., any one of SEQ ID NOs: 14 to 17) H In a specific embodiment, the antibody or antigen-binding fragment thereof comprises a light chain V domain as set forth in Table 3 above. L Domain (i.e., V L domain).

[0060] The sequences of the variable regions of the anti-HER2 antibodies described herein may be modified by insertions, substitutions, and deletions, provided that the resulting antibody retains the ability to specifically bind to HER2, as determined, for example, by ELISA, flow cytometry, and BiaCore™. One skilled in the art may confirm this retention of activity by performing functional assays described herein below, such as, for example, binding and cytotoxicity assays. In a specific embodiment in which the first molecule is an immunoglobulin that binds to HER2, the immunoglobulin is an IgG1 immunoglobulin.

[0061] In specific embodiments, the first molecule of the bispecific binding agent is covalently linked to the second molecule of the bispecific binding agent via a linker. In specific embodiments, the linker covalently linking the first molecule to the second molecule is a peptide linker. In specific embodiments, the peptide linker is between 5-30, 5-25, 5-15, 10-30, 10-20, 10-15, 15-30, or 15-25 amino acid residues in length. In specific embodiments, the peptide linker is between 7-32, 7-27, 7-17, 12-32, 12-22, 12-17, 17-32, or 17-27 amino acid residues in length. In specific embodiments, the peptide linker exhibits one or more characteristics suitable for a peptide linker known to those of skill in the art. In specific embodiments, the peptide linker includes amino acids that allow for solubility of the peptide linker, such as, for example, serine and threonine. In a specific embodiment, the peptide linker comprises an amino acid that allows flexibility of the peptide linker, such as, for example, glycine. In a specific embodiment, the peptide linker connects the N-terminus of the first molecule to the C-terminus of the second molecule. In a preferred embodiment, the peptide linker connects the C-terminus of the first molecule to the N-terminus of the second molecule. In a specific embodiment, the peptide linker is a linker described in Table 5 below (e.g., any one of SEQ ID NOs: 23 and 25-30). In another specific embodiment, the peptide linker is a linker described in Table 5 below (e.g., any one of SEQ ID NOs: 51-56). In a specific embodiment, the peptide linker is SEQ ID NO: 23. In a preferred embodiment, the peptide linker is SEQ ID NO: 53.

[0062] Table 5: Peptide linker sequences [Table 5]

[0063] In another specific embodiment, the first molecule of the bispecific binding agent is directly covalently bound to the second molecule of the bispecific binding agent (i.e., there is no linker between the first and second molecules of the bispecific binding agent).

[0064] The second molecule of the bispecific binding agent mediates the interaction of the bispecific binding agent with the radiotherapeutic agent, where the radiotherapeutic agent comprises (i) a second target (second target of the bispecific binding agent) that is bound to a metal radionuclide, the second target being a metal chelator; or (ii) a second target (second target of the bispecific binding agent) that is bound, preferably covalently bound, to a metal chelator, the second target being bound to a metal radionuclide. In particular, the second molecule of the bispecific binding agent comprises a second binding site that specifically binds to the second target. In a specific embodiment, the second target is a metal chelator that forms part of the radiotherapeutic agent. In another specific embodiment, the second target is a molecule that is bound, preferably covalently bound, to a metal chelator that forms part of the radiotherapeutic agent.

[0065] In a specific embodiment, the second molecule comprises an antibody or an antigen-binding fragment thereof, said antibody or an antigen-binding fragment thereof comprising a second binding site. In a preferred embodiment, the second molecule comprises a single chain variable fragment (scFv), said scFv comprising a second binding site. scFv is an art-recognized term. scFv refers to the V of an immunoglobulin. H Domain and V L domain, the fusion protein retains the same antigen specificity as the whole immunoglobulin. H The domains are connected via peptide linkers (such peptide linkers are sometimes referred to herein as "inter-scFv peptide linkers"), L are merged into a domain.

[0066] In specific embodiments of the invention where the second molecule is an scFv, the scFv has an intra-scFv peptide linker that is between 5-30, 5-25, 5-15, 10-30, 10-20, 10-15, 15-30, or 15-25 amino acid residues in length. In specific embodiments, the intra-scFv peptide linker exhibits one or more characteristics suitable for a peptide linker known to those of skill in the art. In specific embodiments, the intra-scFv peptide linker comprises amino acids that allow for solubility of the intra-scFv peptide linker, such as, for example, serine and threonine. In specific embodiments, the intra-scFv peptide linker comprises amino acids that allow for flexibility of the intra-scFv peptide linker, such as, for example, glycine. In specific embodiments, the intra-scFv peptide linker comprises a V H The N-terminus of the domain is V L In a specific embodiment, the intra-scFv peptide linker is H The C-terminus of the domain is V L The inter-scFv peptide linker is connected to the N-terminus of the domain. In a specific embodiment, the intra-scFv peptide linker is a linker described in Table 5 above (e.g., any one of SEQ ID NOs: 23 and 25-30). In a specific embodiment, the intra-scFv peptide linker is SEQ ID NO: 27. In a specific embodiment, the intra-scFv peptide linker is SEQ ID NO: 30.

[0067] In a specific embodiment, the second target of the bispecific binding agent is a metal chelator.In such an embodiment, the second target of the bispecific binding agent is a metal chelator of a radiotherapeutic agent (see, for example, Section 5.4) that is used in combination with the bispecific binding agent in the method of treating cancer presented herein (see, for example, Sections 5.1 and 6).The metal chelator can be any metal chelator known in the art. Non-limiting examples of metal chelating agents include 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and its metal chelating derivatives (e.g., p-aminobenzyl-DOTA (benzyl-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid with an amino group at the para ("p") position of the benzene ring), DOTA-Bn (benzyl-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid), and DOTA-deferoxamine), and diethylenetriaminepentaacetic acid (DTPA) and its metal chelating derivatives. In the specific embodiment where the second molecule is a metal chelator, the metal chelator is DOTA or its metal chelator derivative (e.g., DOTA-Bn or DOTA-deferoxamine), or DTPA or its metal chelator derivative.Non-limiting examples of DOTA derivatives are described in Leon-Rodriquez & Kovacs, 2008, The Synthesis and Chelation Chemistry of DOTA-Peptide Conjugates, Bioconjugate Chemistry; 19(2):391-402, which is incorporated herein by reference in its entirety.In a preferred embodiment, the second target is DOTA-Bn.

[0068] In specific embodiments, where the second molecule is an antibody or an antigen-binding fragment thereof or an scFv that binds to a metal chelator, the binding of the bispecific binding agent to the metal chelator (through the second molecule) does not significantly impair the chelating ability of the metal chelator. For example, in specific embodiments, the binding of the bispecific binding agent to the metal chelator (through the second molecule) does not reduce the chelating ability of the metal chelator by more than 3%, 5%, 10%, 15%, 20%, 30%, or 40% compared to the chelating ability of the metal chelator before interaction with the second molecule. Methods for determining the chelating ability of a metal chelator in the presence and absence of the bispecific binding agent described herein are known in the art, such as, for example, Antczak, et al., Bioconjugate Chem. 2006, 17, 1551-1560 for metal chelatation methods.

[0069] In a specific embodiment in which the second molecule is an antibody or antigen-binding fragment thereof or an scFv that binds to a metal chelator, the second molecule specifically binds to DOTA or a metal chelate derivative thereof (e.g., DOTA-Bn). In a preferred embodiment, the second molecule is an scFv that specifically binds to DOTA or a metal chelate derivative thereof (e.g., DOTA-Bn). In another preferred embodiment, the second molecule is an scFv that specifically binds to DOTA-Bn. In specific embodiments, the second molecule is, for example, 2D12.5 (see, e.g., Orcutt et al., "Engineering an antibody with picomolar affinity to DOTA chelates of multiple radionuclides for pretargeted radioimmunotherapy and imaging." Nucl Med Biol 2011;38:223-33), a murine scFv with high affinity for benzyl-1,4,7,10-tetraazocyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA-Bn), C825 (see, e.g., Orcutt et al., 2011, Nucl. Med. Biol. 38:223-233; and U.S. Pat. No. 8,648,176), each of which is incorporated herein by reference in its entirety. Imaging Biol. 2011, 13(2) 215-21, any of the anti-DOTA (or metal chelate derivative thereof) antibodies or antigen-binding fragments thereof or scFv V known in the art. H Domains and V L Includes the domain.

[0070] In a specific embodiment, where the second molecule is an antibody or antigen-binding fragment thereof or scFv that binds to DOTA or its metal chelate derivatives, the second molecule binds to the same epitope as an antibody or antigen-binding fragment thereof or scFv known in the art that specifically binds to DOTA or its metal chelate derivatives (e.g., DOTA-Bn). In a specific embodiment, the second molecule binds to the same epitope as C825. Binding to the same epitope can be determined by assays known to those of skill in the art, such as, for example, mutational analysis or crystallography studies. In a specific embodiment, the second molecule competes for binding to DOTA or its metal chelate derivatives (e.g., DOTA-Bn) with an antibody or antigen-binding fragment thereof or scFv known in the art that specifically binds to DOTA or its metal chelate derivatives (e.g., DOTA-Bn). In a specific embodiment, the second molecule that specifically binds to DOTA or its metal chelate derivatives (e.g., DOTA-Bn) competes with C825 for binding to DOTA-Bn. Competition for binding to DOTA or a metal chelate derivative thereof (e.g., DOTA-Bn) can be determined by assays known to those of skill in the art, such as, for example, flow cytometry. In a specific embodiment, the second molecule is a VFv of an antibody or antigen-binding fragment thereof or scFv that specifically binds to DOTA or a metal chelate derivative thereof (e.g., DOTA-Bn) known in the art. H V with at least 85%, 90%, 95%, 98%, or at least 99% similarity to the domain H In a specific embodiment, the second molecule comprises a V domain of an antibody or antigen-binding fragment thereof, or scFv, that specifically binds DOTA or a metal chelate derivative thereof (e.g., DOTA-Bn), known in the art, that contains between one and five conservative amino acid substitutions compared to an antibody or antigen-binding fragment thereof, or scFv, that specifically binds DOTA or a derivative thereof (e.g., DOTA-Bn). HIn a specific embodiment, the second molecule comprises a V domain of an antibody or antigen-binding fragment thereof, or an scFv, known in the art, that specifically binds to DOTA or a metal chelate derivative thereof (e.g., DOTA-Bn). L V with at least 85%, 90%, 95%, 98%, or at least 99% similarity to the domain L In a specific embodiment, the second molecule comprises a V domain of an antibody or antigen-binding fragment thereof, or scFv that specifically binds DOTA or a metal chelate derivative thereof (e.g., DOTA-Bn) known in the art, which contains between one and five conservative amino acid substitutions compared to an antibody or antigen-binding fragment thereof, or scFv that specifically binds DOTA or a metal chelate derivative thereof (e.g., DOTA-Bn). L Includes the domain.

[0071] In a specific embodiment, in which the second molecule is an antibody or an antigen-binding fragment thereof or an scFv that specifically binds to DOTA-Bn, the V H The domain is C825V H A second intramolecular V L The domain is C825V L In a specific embodiment, the V H The domain comprises all three CDRs of SEQ ID NO:21 and V L The domain comprises all three CDRs of SEQ ID NO:22.

[0072] In a preferred embodiment, the second molecule is an antibody or antigen-binding fragment thereof or scFv that specifically binds DOTA or a metal chelate derivative thereof (e.g., DOTA-Bn), the second molecule is derived from mouse C825 and thus the V of mouse C825. H Domains and V LIn a specific embodiment, the second molecule is an scFv. In a specific embodiment, the scFv is derived from mouse C825 and contains the V domain of native mouse C825. H Domain and / or V L In a specific embodiment, the scFv V domain has no more than five amino acid mutations compared to the sequence of the V domain. H The sequence of the domain is SEQ ID NO: 21. In a specific embodiment, the V L The sequence of the domain is SEQ ID NO: 22. In a specific embodiment, the sequence of the scFv comprises any one of the mouse sequences set forth in Table 7 below (e.g., any one of SEQ ID NOs: 31-36). In a preferred embodiment, the sequence of the scFv comprises SEQ ID NO: 33. In a specific embodiment, the scFv comprises the V domain of mouse C825. H V of mouse C825, with 5 or fewer amino acid mutations compared to the native sequence of the domain H In a specific embodiment, the scFv comprises a variant of the V domain of mouse C825. L V of mouse C825, with 5 or fewer amino acid mutations compared to the native sequence of the domain L In a specific embodiment, the scFv comprises a variant of the V domain of mouse C825. H V of mouse C825, with 5 or fewer amino acid mutations compared to the native sequence of the domain H A variant of the domain, V H In a specific embodiment, the scFv comprises the V domain of mouse C825. L V of mouse C825, with 5 or fewer amino acid mutations compared to the native sequence of the domain L A variant of the domain, V L In a specific embodiment, the scFv comprises the V domain of mouse C825. H V of mouse C825 with five or fewer amino acid mutations compared to the native sequence of the domain H Domain variant V H The scFv contains the V domain of mouse C825. LV of mouse C825 with five or fewer amino acid mutations compared to the native sequence of the domain L A variant of the domain, V L Includes the domain.

[0073] Table 6: V of mouse C825 and humanized C825 H Domain sequence and V L Domain Array [Table 6]

[0074] [Table C] TIFF2025039567000011.tif250156 TIFF2025039567000012.tif100155

[0075] In a specific embodiment, the second molecule is an antibody or antigen-binding fragment thereof or scFv that specifically binds to DOTA or a metal chelate derivative thereof, the V H The sequence of the domain comprises a humanized form of SEQ ID NO: 21. In a specific embodiment, where the second molecule is an antibody or antigen-binding fragment thereof or scFv that specifically binds to DOTA or a metal chelate derivative thereof, the V H The sequence of the domain is a humanized form of SEQ ID NO: 21. In a preferred embodiment, the humanized form of SEQ ID NO: 21 is SEQ ID NO: 37. In a specific embodiment, the V L The sequence of the domain comprises a humanized form of SEQ ID NO: 22. In a specific embodiment, the V LThe sequence of the domain is a humanized form of SEQ ID NO: 22. In a preferred embodiment, the humanized form of SEQ ID NO: 22 is SEQ ID NO: 38. In a preferred embodiment, the second molecule is an scFv. In a specific embodiment, the sequence of the scFv comprises any one of the humanized sequences set forth in Table 7 above (e.g., any one of SEQ ID NOs: 39-44). In a specific embodiment, the sequence of the scFv is any one of the humanized sequences set forth in Table 7 above (e.g., any one of SEQ ID NOs: 39-44). In a preferred embodiment, the sequence of the scFv comprises SEQ ID NO: 44 (e.g., the sequence of the scFv is SEQ ID NO: 44). In a specific embodiment, the scFv comprises a humanized form of V H A humanized form of C825 having five or fewer amino acid changes compared to the sequence of domain V H Domain variant V H In a specific embodiment, the scFv comprises a humanized form of the V domain. L A humanized form of C825 having five or fewer amino acid changes compared to the sequence of domain V L Domain variant V L Methods for making humanized antibodies are known to those skilled in the art and are described above.

[0076] The sequence of the variable region of the second molecule that specifically binds DOTA or its metal chelate derivative (e.g., DOTA-Bn) can be modified by insertion, substitution, and deletion, provided that the resulting scFv retains the ability to bind DOTA or its metal chelate derivative, as determined, for example, by ELISA, flow cytometry, and BiaCore™. One skilled in the art can confirm this retention of activity by performing functional assays described herein below, such as, for example, binding and cytotoxicity assays.

[0077] In a preferred embodiment of the bispecific binding agent of the present invention, the first molecule is an immunoglobulin and the second molecule is an scFv. In a specific embodiment, the immunoglobulin of the first molecule comprises two identical heavy chains and two identical light chains, a first light chain and a second light chain, said first light chain being fused (possibly via a first peptide linker) to a second molecule, a first scFv comprising a second binding site, to create a first light chain fusion polypeptide, and said second light chain being fused (possibly via a second peptide linker) to a second scFv to create a second light chain fusion polypeptide, and said first light chain fusion polypeptide and said second light chain fusion polypeptide are identical. Since said first light chain fusion polypeptide and said second light chain fusion polypeptide are identical, the first peptide linker and second peptide linker of the bispecific binding agent are identical, and the first scFv and second scFv of the bispecific binding agent are identical. In specific embodiments, the first light chain fusion polypeptide comprises a first peptide linker and the second light chain fusion polypeptide comprises a second peptide linker, and the sequences of the first and second peptide linkers are 5-30, 5-25, 5-15, 10-30, 10-20, 10-15, 15-30, or 15-25 amino acids in length. In specific embodiments, the first light chain fusion polypeptide comprises a first peptide linker and the second light chain fusion polypeptide comprises a second peptide linker, and the sequences of the first and second peptide linkers are 7-32, 7-27, 7-17, 12-32, 12-22, 12-17, 17-32, or 17-27 amino acids in length. In a specific embodiment, the first light chain fusion polypeptide comprises a first peptide linker and the second light chain fusion polypeptide comprises a second peptide linker, and the sequences of the first peptide linker and the second peptide linker are selected from the group consisting of SEQ ID NO: 23 and 25-30. In a specific embodiment, the sequence of the first peptide linker and the second peptide linker is SEQ ID NO: 23. In a specific embodiment, the first scFv comprises a V H Domain and V LThe scFv comprises an intra-scFv peptide linker between the domains. In a specific embodiment, the sequence of the intra-scFv peptide linker is 5 to 30, 5 to 25, 5 to 15, 10 to 30, 10 to 20, 10 to 15, 15 to 30, or 15 to 25 amino acids in length. In a specific embodiment, the sequence of the intra-scFv peptide linker is selected from the group consisting of any one of SEQ ID NOs: 23 and 25 to 30. In a specific embodiment, the sequence of the intra-scFv peptide linker is SEQ ID NO: 27. In a specific embodiment, the sequence of the intra-scFv peptide linker is SEQ ID NO: 30.

[0078] In a specific embodiment of the bispecific binding agent of the invention, the first molecule is an immunoglobulin that specifically binds HER2 and the second molecule is an scFv that specifically binds DOTA-Bn. In a specific embodiment, the immunoglobulin of the first molecule comprises two identical heavy chains and two identical light chains, a first light chain and a second light chain, said first light chain being fused (optionally via a first peptide linker) to a second molecule, a first scFv comprising a second binding site, to create a first light chain fusion polypeptide, and said second light chain being fused (optionally via a second peptide linker) to a second scFv to create a second light chain fusion polypeptide, and said first light chain fusion polypeptide and said second light chain fusion polypeptide are identical. In specific embodiments, the first light chain fusion polypeptide comprises a first peptide linker and the second light chain fusion polypeptide comprises a second peptide linker, and the sequences of said first and second peptide linkers are 5-30, 5-25, 5-15, 10-30, 10-20, 10-15, 15-30, or 15-25 amino acids in length. In specific embodiments, the first light chain fusion polypeptide comprises a first peptide linker and the second light chain fusion polypeptide comprises a second peptide linker, and the sequences of said first and second peptide linkers are 7-32, 7-27, 7-17, 12-32, 12-22, 12-17, 17-32, or 17-27 amino acids in length. In a specific embodiment, the first light chain fusion polypeptide comprises a first peptide linker and the second light chain fusion polypeptide comprises a second peptide linker, and the sequences of the first peptide linker and the second peptide linker are selected from the group consisting of SEQ ID NOs: 23 and 25-30 (see Table 8). In a specific embodiment, the first light chain fusion polypeptide comprises a first peptide linker and the second light chain fusion polypeptide comprises a second peptide linker, and the sequences of the first peptide linker and the second peptide linker are selected from the group consisting of SEQ ID NOs: 51-56 (see Table 8). In a specific embodiment, the sequence of the first peptide linker and the second peptide linker is SEQ ID NO: 23.In a specific embodiment, the sequence of the first peptide linker and the second peptide linker is SEQ ID NO: 53. In a specific embodiment, the heavy chain in the immunoglobulin is a heavy chain described herein. In a specific embodiment, the light chain in the immunoglobulin is a light chain described herein. In a specific embodiment, the heavy chain in the immunoglobulin comprises all three heavy chain CDRs of SEQ ID NO: 20, and the light chain in the immunoglobulin comprises all three light chain CDRs of SEQ ID NO: 19. In a specific embodiment, the V in the heavy chain in the immunoglobulin. H The sequence of the domain comprises SEQ ID NO: 20. In a preferred embodiment, the V L The sequence of the domain comprises SEQ ID NO: 19. In a specific embodiment, the sequence of the heavy chain of the immunoglobulin comprises any of SEQ ID NOs: 14-17. In a preferred embodiment, the sequence of the heavy chain of the immunoglobulin comprises SEQ ID NO: 15. In another preferred embodiment, the sequence of the heavy chain of the immunoglobulin comprises SEQ ID NO: 16. In a preferred embodiment, the sequence of the light chain of the immunoglobulin comprises SEQ ID NO: 11. In a specific embodiment, the V in the heavy chain of the immunoglobulin comprises any of SEQ ID NOs: 14-17. In a preferred embodiment, the sequence of the heavy chain of the immunoglobulin comprises SEQ ID NO: 15. In another preferred embodiment, the sequence of the heavy chain of the immunoglobulin comprises SEQ ID NO: 16. In a preferred embodiment, the sequence of the light chain of the immunoglobulin comprises SEQ ID NO: 11. H The sequence of the domain comprises a humanized form of SEQ ID NO: 20. In a specific embodiment, the V L The sequence of the domain comprises a humanized form of SEQ ID NO: 19. In a specific embodiment, the first scFv comprises the V H Domain and V L In a specific embodiment, the sequence of the intra-scFv peptide linker is 5 to 30, 5 to 25, 5 to 15, 10 to 30, 10 to 20, 10 to 15, 15 to 30, or 15 to 25 amino acids in length. In a specific embodiment, the sequence of the intra-scFv peptide linker is selected from the group consisting of any one of SEQ ID NOs: 23 and 25 to 30. In a preferred embodiment, the sequence of the intra-scFv peptide linker is SEQ ID NO: 27. In a preferred embodiment, the sequence of the intra-scFv peptide linker is SEQ ID NO: 30. In a specific embodiment, the V H The sequence of the domain comprises all three of the CDRs of SEQ ID NO: 21 and VL The sequence of the domain comprises all three of the CDRs of SEQ ID NO: 22. In a specific embodiment, the V H The sequence of the domain is SEQ ID NO: 21. In a specific embodiment, the V L The sequence of the domain is SEQ ID NO: 22. In a specific embodiment, the V H The sequence of the domain comprises a humanized form of SEQ ID NO: 21. In a specific embodiment, the humanized form of SEQ ID NO: 21 is SEQ ID NO: 37. In a specific embodiment, the V L The sequence of the domain comprises a humanized form of SEQ ID NO: 22. In a specific embodiment, the humanized form of SEQ ID NO: 22 is SEQ ID NO: 38. In a specific embodiment, the first scFv is an scFv described herein. In a specific embodiment, the first scFv comprises a sequence of any of SEQ ID NOs: 31-36. In a preferred embodiment, the scFv comprises a sequence of SEQ ID NO: 33. In a specific embodiment, the first scFv comprises a sequence of any of SEQ ID NOs: 39-44. In a preferred embodiment, the scFv comprises a sequence of SEQ ID NO: 44. In a specific embodiment, the sequence of the first light chain fusion polypeptide is any of SEQ ID NOs: 5-10. In a preferred embodiment, the sequence of the first light chain fusion polypeptide is SEQ ID NO: 7. In a specific embodiment, the sequence of the first light chain fusion polypeptide is any of SEQ ID NOs: 45-50. In a preferred embodiment, the sequence of the first light chain fusion polypeptide is SEQ ID NO: 50. In a specific embodiment, the sequence of the first light chain fusion polypeptide is any of SEQ ID NOs: 5 to 10, and the sequence of the heavy chain is any of SEQ ID NOs: 14 to 17. In a preferred embodiment, the sequence of the first light chain fusion polypeptide is any of SEQ ID NOs: 7, and the sequence of the heavy chain is any of SEQ ID NOs: 15. In a specific embodiment, the sequence of the first light chain fusion polypeptide is any of SEQ ID NOs: 45 to 50, and the sequence of the heavy chain is any of SEQ ID NOs: 14 to 17. In a preferred embodiment, the sequence of the first light chain fusion polypeptide is any of SEQ ID NOs: 50, and the sequence of the heavy chain is any of SEQ ID NO: 16.

[0079] [Table D] TIFF2025039567000014.tif245170 TIFF2025039567000015.tif242170 TIFF2025039567000016.tif141170

[0080] In a preferred embodiment of the bispecific binding agents of the invention, the bispecific binding agent comprises a first molecule covalently bound via a linker to a second molecule, said first molecule comprising a first binding site, said first binding site specifically binding to a first target, said first target being a cancer antigen expressed by said cancer, said cancer antigen being HER2, said second molecule comprising a second binding site, said second binding site specifically binding to a second target, said second target being DOTA-Bn, wherein the first molecule comprises an immunoglobulin comprising the first binding site, said immunoglobulin being HER2. The fusion polypeptide comprises two identical heavy chains and two identical light chains, a first light chain and a second light chain, wherein the first light chain is fused (optionally via a first peptide linker) to a second molecule, a first scFv comprising a second binding site, to create a first light chain fusion polypeptide, and the second light chain is fused (optionally via a second peptide linker) to a second scFv to create a second light chain fusion polypeptide, wherein the first light chain fusion polypeptide and the second light chain fusion polypeptide are identical, wherein the sequence of the first light chain fusion polypeptide is SEQ ID NO:7 and the sequence of the heavy chain is SEQ ID NO:15.

[0081] In a more preferred embodiment of the bispecific binding agents of the invention, the bispecific binding agent comprises a first molecule covalently bound via a linker to a second molecule, said first molecule comprising a first binding site, said first binding site specifically binding to a first target, said first target being a cancer antigen expressed by said cancer, said cancer antigen being HER2, said second molecule comprising a second binding site, said second binding site specifically binding to a second target, said second target being DOTA-Bn, wherein said first molecule comprises an immunoglobulin comprising the first binding site, said immunoglobulin comprising the first binding site. The antibody comprises two identical heavy chains and two identical light chains, a first light chain and a second light chain, wherein the first light chain is fused (optionally via a first peptide linker) to a second molecule, a first scFv comprising a second binding site, to create a first light chain fusion polypeptide, and the second light chain is fused (optionally via a second peptide linker) to a second scFv to create a second light chain fusion polypeptide, wherein the first light chain fusion polypeptide and the second light chain fusion polypeptide are identical, and wherein the sequence of the first light chain fusion polypeptide is SEQ ID NO:50 and the sequence of the heavy chain is SEQ ID NO:16.

[0082] In another specific embodiment, the second target is a molecule that is bound to the metal chelator of the radiotherapeutic agent described herein. In such an embodiment, the second molecule and the second target (to which the second molecule binds) can be members of any known binding pair (e.g., ligand-receptor), but must be selected so that the interaction between the second molecule of the bispecific binding agent and the second target that is bound to the metal chelator of the radiotherapeutic agent does not significantly impair the chelation of the metal radionuclide of the radiotherapeutic agent. In a specific embodiment, the second target comprises biotin, and the second molecule comprises streptavidin or avidin. In a specific embodiment, the second target comprises histamine succinylglycine, and the second molecule comprises an antibody or an antigen-binding fragment thereof or an scFv that binds to histamine succinylglycine.

[0083] In order to use the bispecific binding agent described herein in the method of treating cancer in a subject of a particular species, the bispecific binding agent is used to bind to the first target of this particular species. For example, to treat humans, the first target of the bispecific binding agent binds to the human homolog of the first target. For example, if the first target of the bispecific binding agent is HER2 and a cancer expressing HER2 is treated in humans, the bispecific binding agent comprises a first binding site that specifically binds to human HER2. In another example, to treat dogs, the first target of the bispecific binding agent binds to the canine homolog of the first target. For example, if the first target of the bispecific binding agent is HER2 and a cancer expressing HER2 is treated in dogs, the bispecific binding agent comprises a first binding site that specifically binds to canine HER2. Bispecific binding agents that are cross-reactive with the first targets of various species can be used to treat subjects in these species. For example, the anti-HER2 antibody trastuzumab is predicted to bind to both human HER2 and canine HER2 due to the relative conservation of the epitope within HER2 recognized by trastuzumab. See also, e.g., Singer et al., 2012, Mol Immunol, 50: 200-209.

[0084] In addition, to use the bispecific binding agent described herein in the methods of treating cancer presented herein in a subject of a particular species, the bispecific binding agent, preferably the constant region of the immunoglobulin portion of the bispecific binding agent, is derived from this particular species. For example, to treat a human, the bispecific binding agent may comprise an antibody that is an immunoglobulin that comprises a human constant region. In another example, to treat a dog, the bispecific binding agent may comprise an antibody that is an immunoglobulin that comprises a dog constant region. In a specific embodiment, when treating a human, the immunoglobulin is a humanized immunoglobulin. In another specific embodiment, when treating a human, the immunoglobulin is a human immunoglobulin.

[0085] In a specific embodiment, the bispecific binding agent comprises a variant Fc region, which comprises at least one amino acid modification compared to a wild-type Fc region such that the molecule does not bind or has reduced binding to Fc receptors (FcRs) in soluble or cell-bound form (including, for example, on immune effector cells such as NK cells, monocytes, and neutrophils). These FcRs include, but are not limited to, FcR1 (CD64), FcRII (CD32), and FcRIII (CD16). Affinity for neonatal Fc receptors, FcR(n), is unaffected and thus maintained in the bispecific binding agent. For example, if the immunoglobulin is an IgG, preferably the IgG has reduced or no affinity for Fc gamma receptors. In specific embodiments, mutations are introduced at one or more positions in the Fc region that directly contact the Fc gamma receptor, such as, for example, amino acids 234-239 (hinge region), amino acids 265-269 (B / C loop), amino acids 297-299 (C' / E loop), and amino acids 327-332 (F / G loop), so that the bispecific binding agent has reduced or no affinity for the Fc gamma receptor. See, for example, Sondermann et al., 2000, Nature, 406: 267-273, which are incorporated herein by reference in their entirety. Preferably, an IgG is made with the mutation N297A to disrupt binding to the Fc receptor. In specific embodiments, the affinity of the bispecific binding agent or fragment thereof for an Fc gamma receptor is determined, e.g., by a BiaCore™ assay, e.g., as described in Okazaki et al., 2004. J Mol Biol, 336(5):1239-49. In specific embodiments, bispecific binding agents comprising such variant Fc regions bind to Fc receptors on FcR-bearing immune effector cells with less than 25%, 20%, 15%, 10%, or 5% binding compared to a reference Fc region.Without being bound to a particular theory, bispecific binding agents comprising such variant Fc regions may have a reduced ability to induce a cytokine storm, hi preferred embodiments, bispecific binding agents comprising such variant Fc regions do not bind to Fc receptors in soluble or cell-bound form.

[0086] In specific embodiments, the bispecific binding agent comprises a variant Fc region, such as an Fc region with additions, deletions, and / or substitutions of one or more amino acids in the Fc region of an antibody presented herein, for example, to alter effector function or to enhance or decrease affinity of the antibody to FcR. In a preferred embodiment, the affinity of the antibody to FcR is decreased. In certain cases, such as, for example, in the case of an antibody whose mechanism of action involves blocking or antagonism, but not killing, of cells bearing a target antigen, reduced or eliminated effector function is desired. In specific embodiments, the Fc variants presented herein may be combined with other Fc modifications, including, but not limited to, modifications that alter effector function. In specific embodiments, such modifications provide additive, synergistic, or novel properties in antibodies or Fc fusions. Preferably, the Fc variants presented herein enhance the phenotype of the modification with which they may be combined.

[0087] In a specific embodiment, the bispecific binding agent of the present invention is not glycosylated or has a reduced glycosylation content compared to the wild-type immunoglobulin. In another specific embodiment, the bispecific binding agent comprises an immunoglobulin, the heavy chain in the immunoglobulin is not glycosylated or has a reduced glycosylation content compared to the wild-type heavy chain. Preferably, this is achieved by mutating the antibody or antigen-binding fragment of the first molecular portion of the bispecific binding agent in its Fc receptor to destroy one or more glycosylation sites (e.g., N-linked glycosylation sites). In another specific embodiment, the antibody or antigen-binding fragment of the bispecific binding agent is mutated to destroy one or more N-linked glycosylation sites. In certain preferred embodiments, the antibody or antigen-binding fragment of the bispecific binding agent is mutated to destroy N-linked glycosylation sites. In a specific embodiment, the heavy chain of the antibody or antigen-binding fragment thereof in the bispecific binding agent comprises an amino acid substitution replacing an asparagine, which is an N-linked glycosylation site, with an amino acid that does not function as a glycosylation site. In a preferred embodiment, reduced glycosylation content of the bispecific binding agent is achieved by deleting the glycosylation site in the Fc region of the bispecific binding agent by altering position 297 from asparagine to alanine (N297A). For example, in a specific embodiment, the bispecific binding agent comprises a heavy chain with the sequence of SEQ ID NO: 15 or 16. As used herein, a "glycosylation site" includes any specific amino acid sequence within an antibody to which an oligosaccharide (i.e., a carbohydrate containing two or more monosaccharides linked together) is specifically and covalently attached. The oligosaccharide side chains are typically linked to the backbone of the antibody via N- or O-linkages. N-linked glycosylation refers to the attachment of an oligosaccharide moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the conjugation of an oligosaccharide moiety to a hydroxyamino acid, eg, serine, threonine.Methods for modifying the glycosylation content of antibodies are well known in the art, see, for example, U.S. Pat. No. 6,218,149; European Patent No. 0359096; U.S. Patent Application Publication No. 2002 / 0028486; WO 03 / 035835; U.S. Patent Application Publication No. 2003 / 0115614; U.S. Pat. No. 6,218,149; U.S. Pat. No. 6,472,511; all of which are incorporated herein by reference in their entireties. In another embodiment, aglycosylation of bispecific binding agents of the invention can be achieved by recombinantly producing the bispecific binding agents in cells or expression systems incapable of glycosylation, such as, for example, bacteria. In another embodiment, aglycosylation or reduced glycosylation content of bispecific binding agents of the invention can be achieved by enzymatic removal of carbohydrate moieties at glycosylation sites.

[0088] In specific embodiments, the bispecific binding agent of the present invention does not bind or has reduced binding affinity to the complement component C1q (compared to a reference immunoglobulin or wild-type immunoglobulin). Preferably, this is achieved by introducing a mutation into the antibody or antigen-binding fragment thereof of the bispecific binding agent to disrupt the C1q binding site. In certain preferred embodiments, the method includes deleting the C1q binding site of the Fc region of the bispecific binding agent by modifying position 322 from lysine to alanine (K322A) (see, e.g., Idusogie et al., 2000. J Immunol. 164(8):4178-84 for a description of the K322A modification). For example, in specific embodiments, the bispecific binding agent comprises a heavy chain with the sequence of SEQ ID NO: 16 or 17. In specific embodiments, the affinity of the bispecific binding agent or fragment thereof for the complement component C1q is determined, e.g., by a BiaCore™ assay, e.g., as described in Okazaki et al., 2004. J Mol Biol, 336(5):1239-49. In specific embodiments, the bispecific binding agent comprising a disrupted C1q binding site binds to the complement component C1q with less than 25%, 20%, 15%, 10%, or 5% binding compared to a reference immunoglobulin or a wild-type immunoglobulin. In specific embodiments, the bispecific binding agent does not activate complement.

[0089] In a specific embodiment, the bispecific binding agent of the present invention comprises an immunoglobulin that (i) comprises at least one amino acid modification compared to a wild-type Fc region such that the molecule does not bind or has reduced binding to Fc receptors in soluble or cell-bound form; (ii) comprises one or more mutations in the Fc region to destroy an N-linked glycosylation site; and (iii) comprises no or reduced binding to the complement component C1q. For example, in a specific embodiment, the bispecific binding agent comprises an IgG that (i) eliminates or reduces binding to Fc receptors in soluble or cell-bound form; (ii) comprises a first mutation, N297A, in the Fc region to destroy an N-linked glycosylation site in the Fc region; and (iii) comprises a second mutation, K322A, in the Fc region to eliminate or reduce binding to the complement component C1q. See, for example, SEQ ID NO: 16.

[0090] In a specific embodiment, the bispecific binding agent comprises an Fc domain. In a preferred embodiment, the first molecule of the bispecific binding agent comprises an Fc domain. In specific embodiments, the bispecific binding agent is at least 100 kDa, at least 150 kDa, at least 200 kDa, at least 250 kDa, between 100 and 300 kDa, between 150 and 300 kDa, or between 200 and 250 kDa. In specific embodiments, the bispecific binding agent is at least 100 kDa.

[0091] The bispecific binding agents presented herein can bind to a first target and a second target with a wide range of affinities. The affinity or avidity of an antibody to an antigen can be determined experimentally using any suitable method. See, for example, Berzofsky, et al., "Antibody-Antigen Interactions," In Fundamental Immunology, Paul, WE, Ed., Raven Press: New York, NY (1984); Kuby, Janis Immunology, WH Freeman and Company: New York, NY (1992); and the methods described herein. The affinity measured for a particular antibody-antigen interaction may vary when measured under different conditions (e.g., salt concentration, pH). Therefore, the measurement of affinity and other antigen binding parameters is preferably done with standardized solutions of antibody and antigen and standardized buffer. The affinity, K D What is k off / k on Generally, K in the micromolar range is D are considered to have low affinity. Generally, K in the pmol range D is considered to be of high affinity.

[0092] In specific embodiments in which the first target of the bispecific binding agent is HER2, the bispecific binding agent preferably is selected from the group consisting of MDA-MB-361, MDA-MB-468, AU565, SKBR3, HTB27, HTB26, HCC1954, MCF7, OVCAR3, SKOV3, NCI-N87, KATO, HER2, and HER2-associated antibodies, as determined by assays known to those of skill in the art, such as, for example, ELISA, BiaCore™, flow cytometry, and cell-based assays. III, AGS, SNU-16, HT144, SKMEL28, M14, HTB63, RG160, RG164, CRL1427, U2OS, SKEAW, SKES-1, HTB82, NMB7, SKNBE(2)C, IMR32, SKNBE(2)S, SKNBE(1)N, NB5, 15B, 93-VU-147T, PCI-30, UD-SCC2, PCI-15B, SCC90, UMSCC47, NCI-H524, NCI-H69, NCI-H345, etc. In a specific embodiment, the bispecific binding agent binds to a HER2 positive cancer cell line with an EC50 in the nmol range. In specific embodiments, the use of bispecific binding agents in the methods described herein (see, e.g., Sections 5.1 and 6) reduces tumor progression, metastasis, and / or tumor size. See, e.g., Section 6.

[0093] 5.2.1 Generation of bispecific binding factors Also provided herein are methods for making the bispecific binding agents described in Sections 5.1 and 6. In specific embodiments provided herein are methods for making bispecific binding agents comprising a first molecule covalently linked (optionally via a linker) to a second molecule, wherein the first molecule comprises a first binding site, the first binding site specifically binds to a first target, the first target being a cancer antigen expressed by the cancer, and the second molecule comprises a second binding site, the second binding site specifically binds to a second target, the second target being not a cancer antigen. In specific embodiments herein, a method is provided for making a bispecific binding agent comprising a first molecule covalently linked (optionally via a linker) to a second molecule, where the cancer expresses HER2, and the first molecule comprises an antibody or antigen-binding fragment thereof, or an scFv, where the antibody or antigen-binding fragment thereof or scFv (i) binds to HER2 on the cancer and (ii) comprises all three of the heavy chain CDRs of SEQ ID NO: 20 and all three of the light chain CDRs of SEQ ID NO: 19, and the second molecule comprises a second binding site, where the second binding site specifically binds to a second target, and where the second target is not a cancer antigen.

[0094] The methods that can be used to produce the bispecific binding agents described herein are known to those skilled in the art, for example, by chemical synthesis, by purification from biological sources, or by recombinant expression, including, for example, production from mammalian cells or transgenic preparations.The methods described herein utilize conventional techniques of molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, synthesis and modification of oligonucleotides, nucleic acid hybridization, and related fields that are within the skill of the art, unless otherwise indicated.These techniques are described, for example, in the references cited herein and are fully explained in the literature. For example, Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annual updates); Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates) Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, See IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren et al. (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

[0095] In the art, there are various methods that can be used to produce bispecific binding agents. For example, bispecific binding agents can be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. One or more DNAs encoding the bispecific binding agents presented herein can be easily isolated and sequenced using conventional procedures, such as by using oligonucleotide probes that can specifically bind to the heavy and light chains of mouse antibodies, or genes encoding such chains from human, humanized, or other sources. Once isolated, the DNA is transferred into an expression vector and then transformed into host cells, such as NS0 cells, monkey COS cells, Chinese hamster ovary (CHO) cells, yeast cells, algae cells, chicken eggs, or myeloma cells that do not produce immunoglobulin proteins unless transformed, so that synthesis of the bispecific binding agent occurs in the recombinant host cells. The DNA may also be modified, for example, by substituting the constant domains of the desired species for the homologous human sequences in the coding sequences for the human heavy and light chains (U.S. Pat. No. 4,816,567; Morrison et al., supra), or by covalently joining to the immunoglobulin coding sequences all or part of the coding sequence for a non-immunoglobulin polypeptide. Such non-immunoglobulin polypeptides can be substituted for the constant domains of the bispecific binding agents provided herein. In specific embodiments, the DNA is as described in Section 5.2.1.1.

[0096] The bispecific binding agent presented herein can also be prepared using transgenic animals, such as mammals, such as goats, cows, horses, sheep, etc., that contain and express a transgene encoding at least one bispecific binding agent, such as an antibody protein, for example, to produce such an antibody in their milk. Such animals can be produced using known methods. For example, see references such as, but not limited to, U.S. Patent Nos. 5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; and 5,304,489, each of which is incorporated herein by reference in its entirety.

[0097] Bispecific binding agents provided herein can also be prepared, for example, using transgenic plants and cultured plant cells (e.g., but not limited to, tobacco and corn) that contain and express a transgene encoding at least one bispecific binding agent to produce such bispecific binding agents in a plant part or in cells cultured therefrom. Bispecific binding factors provided herein can also be prepared using bacteria transformed to contain and express at least one bispecific binding factor-encoding plasmid, e.g., bacteria transformed to produce such bispecific binding factors within the bacteria.

[0098] In specific embodiments, bispecific binding agents can be recovered and purified from recombinant cell culture by well-known methods, including, but not limited to, Protein A purification, Protein G purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography, and high performance liquid chromatography. See, e.g., Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001), e.g., chapters 1, 4, 6, 8, 9, and 10.

[0099] In specific embodiments, the bispecific binding agents provided herein include, for example, the products of chemical synthesis procedures and products made by recombinant techniques in eukaryotic hosts, including, for example, yeast cells, higher plant cells, insect cells, and mammalian cells. In specific embodiments, the bispecific binding agents are produced in a host such that they are not glycosylated. In specific embodiments, the bispecific binding agents are produced in a bacterial cell such that they are not glycosylated. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Sections 17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20, Colligan, Protein Science, supra, Chapters 12-14. Purified antibodies may be characterized by, for example, ELISA, ELISPOT, flow cytometry, immunocytology, Biacore™ analysis, Sapidyne KinExA™ kinetic exclusion assay, SDS-PAGE, and Western blot, or by HPLC analysis. See also Section 6 for detailed examples for the design and production of the bispecific binding agents described herein.

[0100] 5.2.1.1 Polynucleotides Provided herein, in specific embodiments, is a polynucleotide comprising a nucleotide sequence encoding a bispecific binding agent or fragment thereof (e.g., heavy and / or light chain fusion polypeptide) as described herein that specifically binds to a first target (e.g., HER2) and a second target (e.g., DOTA or a derivative thereof) as described in Sections 5.2 and 6. Also provided herein is a vector comprising such a polynucleotide. The polynucleotides and vectors can be used for recombinant production of the bispecific binding agent or fragment thereof.

[0101] The term "purified" includes preparations of polynucleotides or nucleic acid molecules having less than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (particularly less than about 10%) of other materials, such as cellular materials, culture medium, other nucleic acid molecules, chemical precursors, and / or other chemicals. In specific embodiments, nucleic acid molecules encoding bispecific binding agents described herein are isolated or purified. The nucleic acid molecule may be in the form of RNA, such as mRNA, hnRNA, whether obtained by cloning or produced synthetically, or any combination thereof, or in the form of DNA, including but not limited to cDNA and genomic DNA.

[0102] In specific embodiments, the polynucleotides used for recombinant production comprise a nucleotide sequence encoding a bispecific binding agent or fragment thereof described in Sections 5.2 and 6 (e.g., a heavy chain fusion polypeptide or a light chain fusion polypeptide), where the bispecific binding agent comprises a first molecule covalently linked (optionally via a linker) to a second molecule, where the first molecule comprises a first binding site, where the first binding site specifically binds to a first target, where the first target is a cancer antigen expressed by the cancer, and where the second molecule comprises a second binding site, where the second binding site specifically binds to a second target, where the second target is not a cancer antigen. In specific embodiments where the polynucleotides comprise a nucleotide sequence encoding a fragment of a bispecific binding agent, the polynucleotides may be combined to generate the bispecific binding agent, for example, ex vivo. For example, the translation product of a polynucleotide comprising a nucleotide sequence encoding a heavy chain of a bispecific binding agent and the translation product of a polynucleotide comprising a nucleotide sequence encoding a light chain fusion polypeptide of a bispecific binding agent can be combined, for example, to produce a bispecific binding agent ex vivo.

[0103] In another specific embodiment, the polynucleotide used for recombinant production comprises a nucleotide sequence encoding a bispecific binding agent, or a fragment thereof, as described in Sections 5.2 and 6, wherein the bispecific binding agent comprises a first molecule covalently linked (optionally via a linker) to a second molecule, wherein said cancer expresses HER2, said first molecule comprises an antibody or antigen-binding fragment thereof, or an scFv, said antibody or antigen-binding fragment thereof, or scFv that (i) binds to HER2 on said cancer and (ii) comprises all three of the heavy chain CDRs of SEQ ID NO:20 and all three of the light chain CDRs of SEQ ID NO:19, said second molecule comprises a second binding site, said second binding site specifically binds to a second target, and said second target is not a cancer antigen. In certain embodiments, the polynucleotide used for recombinant production comprises a nucleotide sequence encoding a bispecific binding agent, or a fragment thereof, that (i) specifically binds to HER2 and DOTA, or a derivative thereof, and (ii) comprises an amino acid sequence described herein.

[0104] In a specific embodiment, one or more portions of the bispecific binding factors described herein are produced by expression from a nucleotide sequence set forth in Table 9. In a preferred embodiment for producing a bispecific binding factor, the sequence of the light chain is SEQ ID NO: 11, and the nucleotide sequence encoding the light chain expressed to produce the light chain is SEQ ID NO: 13. In a preferred embodiment for producing a bispecific binding factor, the sequence of the scFv is SEQ ID NO: 33, and the nucleotide sequence encoding the scFv expressed to produce the scFv is SEQ ID NO: 38. In a preferred embodiment for producing a bispecific binding factor, the sequence of the light chain is SEQ ID NO: 11, the sequence of the scFv is SEQ ID NO: 33, the nucleotide sequence encoding the light chain expressed to produce the light chain is SEQ ID NO: 13, and the nucleotide sequence encoding the scFv expressed to produce the scFv is SEQ ID NO: 38. In a preferred embodiment for producing a bispecific binding factor, the sequence of the light chain is SEQ ID NO: 7, and the nucleotide sequence encoding the light chain fusion polypeptide expressed to produce the light chain fusion polypeptide is SEQ ID NO: 18. In a preferred embodiment for making a bispecific binding agent, the sequence of the heavy chain is SEQ ID NO:15 and the nucleotide sequence encoding the heavy chain expressed to make the heavy chain is SEQ ID NO:12.

[0105] Table 9: Exemplary nucleic acid sequences. [Table 9] TIFF2025039567000018.tif123168 TIFF2025039567000019.tif117168

[0106] Polynucleotides for use as provided herein may be obtained by any method known in the art. For example, if the nucleotide sequence encoding the bispecific binding agent or fragment thereof described herein is known, the polynucleotide encoding the bispecific binding agent or fragment thereof may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, in brief, involves the synthesis of overlapping oligonucleotides containing portions of the antibody-encoding sequence, annealing and ligating these oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

[0107] Alternatively, polynucleotides encoding a bispecific binding agent or fragment thereof for use as provided herein can be generated from nucleic acid derived from a suitable source. In cases where a clone containing a nucleic acid encoding a particular bispecific binding agent or fragment thereof is not available, but the sequence of the bispecific binding agent or fragment thereof is known, the nucleic acid encoding the bispecific binding agent or fragment thereof can be chemically synthesized or obtained from a suitable source (e.g., a cDNA library of an antibody, or a cDNA library generated from any tissue or cell expressing an antibody, such as a hybridoma cell selected to express an antibody provided herein, or a nucleic acid isolated therefrom, preferably polyA+RNA) by PCR amplification using synthetic primers hybridizing to the 3' and 5' ends of the sequence, or by cloning using an oligonucleotide probe specific for a particular gene sequence to identify a cDNA clone from a cDNA library encoding an antibody, for example. The amplified nucleic acid generated by PCR can then be cloned into a replicable cloning vector using any method known in the art. See, for example, Section 5.2.1.2.

[0108] In specific embodiments, the amino acid sequence of the antibody of the bispecific binding agent is an amino acid sequence known in the art. In such embodiments, the polynucleotide encoding such an antibody can be manipulated to create, for example, amino acid substitutions, deletions, and / or insertions, using methods known in the art for the manipulation of nucleotide sequences, such as recombinant DNA methods, site-directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, all of which are incorporated herein by reference in their entirety), to generate bispecific binding agents with different amino acid sequences. For example, such manipulations can be performed to deglycosylate the encoded amino acids or to destroy the ability of the antibody to bind to C1q, Fc receptors, or to activate the complement system.

[0109] The isolated nucleic acid molecules presented herein may include, but are not limited to, nucleic acid molecules comprising an open reading frame (ORF) optionally with one or more introns, e.g., at least one specified portion of at least one CDR, such as CDR1, CDR2, and / or CDR3 of at least one heavy or light chain; nucleic acid molecules comprising a coding sequence for an anti-HER2 antibody or variable region, an anti-DOTA (or derivative thereof) scFv, or a single chain fusion polypeptide; and nucleic acid molecules comprising a nucleotide sequence that differs substantially from the nucleotide sequences set forth above, but that, due to the degeneracy of the genetic code, still encodes at least one bispecific binding agent described herein and / or at least one bispecific binding agent known in the art.

[0110] The nucleic acid for use presented herein may advantageously contain multiple sequences in addition to the polynucleotide presented herein. For example, a multiple cloning site containing one or more endonuclease restriction sites may be inserted into the nucleic acid to aid in the isolation of the polynucleotide. In addition, a translatable sequence may also be inserted to aid in the isolation of the translated polynucleotide presented herein. For example, a hexahistidine marker sequence provides a convenient means for purifying the polypeptide presented herein. The nucleic acid presented herein (excluding coding sequences) may be a vector, adapter, or linker for cloning and / or expressing the polynucleotide presented herein.

[0111] Additional sequences may also be added to such cloning and / or expression sequences to optimize their function in cloning and / or expression, aid in the isolation of the polynucleotide, or improve the introduction of the polynucleotide into cells. The use of cloning vectors, expression vectors, adapters, and linkers is well known in the art (see, e.g., Ausubel, supra; or Sambrook, supra). In a specific embodiment, for antibody humanization, one or more of the CDRs of the antibody described herein may be inserted into the framework region using defined recombinant DNA methods. The framework region may be a naturally occurring framework region or a consensus framework region, but preferably is a human framework region (see, for example, Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for a list of human framework regions). Preferably, the polynucleotide generated by the combination of the framework region and the CDR encodes an antibody that specifically binds to HER2. One or more amino acid substitutions may be made within the framework region, and preferably, the amino acid substitutions improve the binding of the antibody to its antigen. In addition, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine ​​residues that participate in intrachain disulfide bonds to generate an antibody molecule that lacks one or more intrachain disulfide bonds. Other modifications to the polynucleotide are presented herein and are within the skill of the art.

[0112] In specific embodiments, the isolated or purified nucleic acid molecule, or a fragment thereof, can encode a fusion protein when linked to another nucleic acid molecule. The creation of fusion proteins is within the skill of the art and can involve the use of restriction enzymes or recombinant cloning methods (see, e.g., Gateway™ (Invitrogen)). See also U.S. Patent No. 5,314,995. In specific embodiments, the polynucleotides presented herein are in the form of a vector (eg, an expression vector), as described in Section 5.2.1.2.

[0113] 5.2.1.2 Cells and vectors Provided herein, in specific embodiments, are cells (e.g., ex vivo cells) that express (e.g., recombinantly express) the bispecific binding factors described herein for use in making the bispecific binding factors described herein. Also provided herein are vectors (e.g., expression vectors) that contain nucleotide sequences (see, e.g., Section 5.2.1.1) encoding the bispecific binding factors described herein or fragments thereof for recombinant expression in a host cell, preferably a mammalian cell, for use in making the bispecific binding factors described herein. Also provided herein are cells (e.g., ex vivo cells) that contain such vectors or nucleotide sequences for recombinantly expressing the bispecific binding factors described herein. Also provided herein are methods for making the bispecific binding factors described herein, comprising expressing such bispecific binding factors from cells (e.g., ex vivo cells). In a preferred embodiment, the cells are ex vivo cells.

[0114] In specific embodiments herein, provided are vectors comprising one or more polynucleotides described in Section 5.2.1.1 for use in making the bispecific binding agents described herein. In specific embodiments, the polynucleotides described in Section 5.2.1.1 may be cloned into a suitable vector and used to transform or transfect any suitable host suitable for the recombinant production of bispecific binding agents using methods well known in the art. In specific embodiments, the vector is a mammalian vector used for recombinant expression of the bispecific binding agent in a mammalian host or host cell. Non-limiting examples of mammalian expression vectors include vectors such as pIRESlneo, pRetro-Off, pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto, Calif.), pcDNA3.1(+ / -), pcDNA / Zeo(+ / -), or pcDNA3.1 / Hygro(+ / -) (Invitrogen), PSVL and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC37152), pSV2dhfr (ATCC37146), and pBC12MI (ATCC67109). Non-limiting examples of mammalian host cells that may be used in combination with such mammalian vectors include human Hela 293 cells, human H9 cells, and human Jurkat cells, mouse NIH3T3 cells and mouse C127 cells, Cos1 cells, Cos7 cells, and CV1 cells, quail QC1-3 cells, mouse L cells, and Chinese hamster ovary (CHO) cells.

[0115] In a specific embodiment, the vector is a viral vector, for example a retroviral vector, a parvovirus-based vector, for example an adeno-associated virus (AAV)-based vector, an AAV-adenovirus chimeric vector, and an adenovirus-based vector, and a lentivirus vector, for example a herpes simplex virus (HSV)-based vector.In a specific embodiment, the viral vector is engineered to make the virus replication-defective.In a specific embodiment, the viral vector is engineered to eliminate toxicity to the host.

[0116] In specific embodiments, the vectors or polynucleotides described herein may be transferred by conventional techniques into cells (e.g., cells ex vivo) and the resulting cells may be cultured by conventional techniques to produce the bispecific binding agents described herein. In a preferred embodiment, the cells are CHO cells. In a particularly preferred embodiment, the cells are CHO-S cells. In specific embodiments, the polynucleotides described herein may be expressed in stable cell lines that contain the polynucleotide integrated into a chromosome by introducing the polynucleotide into a cell.

[0117] 5.3 Remover Provided herein is a clearing agent for use in the methods of treating cancer provided herein (see, e.g., Section 5.1). As described above, when used in the methods of treating cancer provided herein, the clearing agent is administered to the subject after (e.g., within 12 hours) step (a) of administering a therapeutically effective amount of a bispecific binding agent to the subject. The clearing agent described herein functions to reduce the amount of bispecific binding agent circulating in the blood of the subject before administering a therapeutically effective amount of a radiotherapy agent to the subject. In a specific embodiment, the clearing agent comprises a molecule that is primarily cleared from the circulation by the liver, fixed phagocyte system, spleen, or bone marrow. Without being bound by any particular theory, administration of a clearing agent to a subject (i) after administration of a bispecific binding agent to the subject, but (ii) prior to administration of a radiotherapeutic agent to the subject, clears or reduces the bispecific binding agent circulating in the subject's blood, resulting in reduced exposure of non-targeted normal tissues (e.g., tissues that do not express a cancer antigen) in the subject to a subsequent administration of the radiotherapeutic agent. Thus, without being bound by any theory, administration of the clearing agent allows for an improved therapeutic index by limiting radiation from the radiotherapeutic agent in non-targeted normal tissues (i.e., tissues that do not express a cancer antigen), thereby allowing for higher doses of the radiotherapeutic agent to be administered to the subject without resulting in dose-limiting toxicity of the radiation.

[0118] For use in the methods of treating cancer presented herein, the clearing agent binds to the bispecific binding factor used in the methods of treating cancer. Therefore, for use in the methods of treating cancer presented herein, the clearing agent should be selected to bind to the bispecific binding factor used in the methods. Thus, those skilled in the art will understand that the clearing agent is selected based on the structure and specificity of the bispecific binding factor used in the methods. In a specific embodiment, the clearing agent comprises a second target (the second target of the bispecific binding factor) that is bound to a molecule that is cleared from the circulation, or a derivative of the second target that retains the ability to bind to the second molecule (preferably at the second binding site). The derivative of the second target described herein retains the ability to bind to the second molecule (preferably at the second binding site). In a specific embodiment, the clearing agent comprises a second target (the second target of the bispecific binding factor) or a derivative thereof that is bound to a molecule that is cleared from the circulation mainly by the liver, the fixed phagocyte system, the spleen, or the bone marrow. For example, if the second target of the bispecific binding agent is DOTA, the clearing agent for use in combination with the bispecific binding agent may comprise DOTA bound to a molecule that is primarily cleared from the circulation by the liver, the fixed phagocyte system, the spleen, or the bone marrow. In another example, if the second target of the bispecific binding agent is DOTA, the clearing agent for use in combination with the bispecific binding agent may comprise a derivative of DOTA (e.g., benzyl isothiocyanate-DOTA) bound to a molecule that is primarily cleared from the circulation by the liver, the fixed phagocyte system, the spleen, or the bone marrow. For use of a derivative of a second target (second target of the bispecific binding agent) in a clearing agent, the derivative of the second target must retain its ability to bind to the bispecific binding agent (specifically bind to the second binding site of the second molecule of the bispecific binding agent).

[0119] Those skilled in the art are aware of molecules that are mainly removed from the circulation by the liver, fixed phagocyte system, spleen or bone marrow. Non-limiting examples of molecules that are mainly removed from the circulation by the liver, fixed phagocyte system, spleen or bone marrow include aminodextran, galactosylated albumin, galactose, galactosamine, mannose, lactose, muramyl tripeptide, RGD peptide and glycyrrhizin (see, for example, Mishra et al., 2013, Efficient Hepatic Delivery of Drugs: Novel Strategies and Their Significance, BioMed Research International, vol. 2013, Article ID 32184, 20 pages). Those skilled in the art will understand that molecules that are mainly removed from the circulation by the liver, fixed phagocyte system, spleen or bone marrow include, for example, molecules that bind to surface receptor proteins on liver cells, spleen cells or bone marrow cells and are internalized into cells. For example, the clearing agent is intended to include a molecule that interacts with liver cells (e.g., hepatocytes) so that the clearing agent is primarily cleared from the circulation by the liver. For example, for clearance by the liver, the clearing agent may include a molecule that interacts with a receptor on the hepatocyte, such as an asialoglycoprotein receptor. In this embodiment, the clearing agent may include a galactosylated albumin that is bound to a second target (a second target of the bispecific binding agent) such that the second target in the clearing agent binds to the bispecific binding agent in the circulation, and the galactosylated albumin interacts with the asialoglycoprotein receptor on the hepatocyte (see, e.g., Stockert, Physiol Rev. 1995; 75:591-609), such that the bispecific binding agent bound to the clearing agent is internalized by the hepatocyte and cleared from the subject by the liver.

[0120] In a specific embodiment, the clearing agent comprises a 500 kDa aminodextran conjugated to a second target. In a specific embodiment, the second target is DOTA. In a specific embodiment, the clearing agent comprises a 500 kDa aminodextran conjugated to DOTA. In a specific embodiment, the clearing agent comprises a 500 kDa aminodextran conjugated to a derivative of the second target. In a specific embodiment, the second target is DOTA. In a specific embodiment where the second target is DOTA, the derivative of the second target is benzyl isothiocyanate-DOTA. In a specific embodiment, the clearing agent comprises a 500 kDa aminodextran conjugated to benzyl isothiocyanate-DOTA.

[0121] In a specific embodiment, the remover comprises about 100-150 second target molecules per 500 kDa of aminodextran. In a specific embodiment, the second target is DOTA. In a specific embodiment, the remover comprises about 100-150 DOTA molecules per 500 kDa of aminodextran. In a specific embodiment, the remover comprises about 100-150 derivative molecules of the second target per 500 kDa of aminodextran. In a specific embodiment, the second target is DOTA. In a specific embodiment where the second target is DOTA, the derivative of the second target is benzyl isothiocyanate-DOTA. In a specific embodiment, the remover comprises about 100-150 benzyl isothiocyanate-DOTA molecules per 500 kDa of aminodextran. In a specific embodiment where the second target is DOTA, the remover further comprises a non-radioactive lutetium or yttrium molecule. Those skilled in the art will appreciate that clearing agents suitable for use in the methods of treating cancer provided herein are preferably clearing agents that are easily manufactured, easily characterized, and have a consistent composition. For example, suitable clearing agents include agents that have a single chemical composition, such as, for example, fully synthetic dendrimer-conjugated agents.

[0122] The art is known to provide scavenger and methods for making scavenger (see, for example, Orcutt et al. Mol Cancer Ther 2012, 11(6) 1365-72, U.S. Pat. No. 6,075,010, U.S. Pat. No. 6,416,738, and International Patent Application Publication No. 2012 / 085789). For example, to make a scavenger containing 100-150 benzyl isothiocyanate-DOTA molecules per 500 kDa of aminodextran, the aminodextran is reacted in a large excess of benzyl isothiocyanate-DOTA to achieve a quantitative reaction. For a description of how to generate the clearing agents described herein, see, e.g., Orcutt et al., 2012, Effect of small-molecule-binding affinity on tumor uptake in vivo: a systematic study using a pretargeted bispecific antibody. Mol Cancer Ther; 11: 1365-72.

[0123] In a specific embodiment in which the clearing agent comprises a second target that is a metal chelator, the clearing agent further comprises a non-radioactive metal capable of interacting with the metal chelator. For example, when the clearing agent comprises DOTA or a derivative thereof, the non-radioactive metal used to generate the non-radioactive clearing agent is: 175 Lu or 89 Y. In a specific embodiment, the clearing agent comprises 100-150 benzyl isothiocyanate-DOTA molecules per 500 kDa of aminodextran, where benzyl isothiocyanate-DOTA is 175 It is complexed with Lu.

[0124] For use in the methods of treating cancer provided herein, it is preferred to utilize a clearing agent that removes unbound bispecific binding agents from the circulation within a few hours (sometimes referred to herein as "clearance"). In specific embodiments, the clearing agent removes unbound bispecific binding agents from the circulation in less than 24 hours, less than 23 hours, less than 22 hours, less than 21 hours, less than 20 hours, less than 19 hours, less than 18 hours, less than 17 hours, less than 16 hours, less than 15 hours, less than 14 hours, less than 13 hours, less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, less than 2 hours, or less than 1 hour. In specific embodiments, the clearing agent clears unbound bispecific binding agent from the circulation within 1-2 hours, 1-3 hours, 1-4 hours, 2-6 hours, 2-8 hours, 2-10 hours, 4-6 hours, 4-8 hours, 4-10 hours, within 1 hour, within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours.

[0125] In specific embodiments, a bispecific binding agent is considered to be cleared from the circulation if at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the bispecific binding agent is cleared from the circulation within 1 hour, 2 hours, 3 hours, or 4 hours after administration of the clearing agent to a subject. Methods for determining the percentage of a bispecific binding agent cleared from the circulation are known to those of skill in the art, see, for example, the assay described in Breitz, et al., J Nucl Med 2000 41(1) 131-40 and section 6.

[0126] 5.4 Radiotherapy Agents Also provided herein is a radiotherapeutic agent for use in the methods of treating cancer provided herein (see, e.g., Sections 5.1 and 6). As described above, when used in the methods of treating cancer provided herein, the radiotherapeutic agent is administered to the subject after step (b) of administering a therapeutically effective amount of a clearing agent to the subject. Without being bound by a particular theory, for use in the methods of treating cancer provided herein, the radiotherapeutic agent binds to the bispecific binding agent and mediates the killing of the cancer cells to which the bispecific binding agent binds, along with other cells, by cross-fire effect, radiation-induced bystander effect, and unirradiated site effect. A first molecule of the bispecific binding agent described herein (see, e.g., Sections 5.2 and 6) specifically binds to a cancer antigen (i.e., the first target of the bispecific binding agent) on cancer cells in the subject, and a second molecule of the bispecific binding agent specifically binds to a second target that forms part of the radiotherapeutic agent. Thus, without being bound by any theory, the bispecific binding agent forms a crosslink between the cancer cell and the radiotherapeutic agent, allowing the radiotherapeutic agent to kill the cancer cell bound to the bispecific binding agent. Thus, for use of the radiotherapeutic agent in combination with the bispecific binding agent in the method of treating cancer presented herein, a radiotherapeutic agent should be selected that includes a second target of the bispecific binding agent. The radiotherapeutic agent includes (i) a second target bound to a metal radionuclide, the second target being a metal chelator; or (ii) a second target bound to a metal chelator, the second target being bound to a metal radionuclide. Thus, one skilled in the art will understand that the radiotherapeutic agent for use in the method of treating cancer presented herein will be selected based on the structure and specificity of the bispecific binding agent used in the method. In a preferred embodiment, the radiotherapeutic agent includes DOTA or a derivative thereof bound to a metal radionuclide. In a preferred embodiment, the radiotherapeutic agent includes DOTA or a derivative thereof bound to a metal radionuclide, the metal radionuclide is 177 This is Lu.

[0127] In specific embodiments, the radiotherapeutic agent comprises (i) a second target (second target of the bispecific binding agent) that is bound to a metal radionuclide, where the second target is a metal chelator. For example, if the first molecule of the bispecific binding agent is an immunoglobulin that binds (through its first binding site) to HER2, a cancer antigen on cancer cells (i.e., the first target), and the second molecule of the bispecific binding agent is a single chain variable fragment (scFv) that binds (through its second binding site) to DOTA or a derivative thereof, which is a metal chelator (i.e., the second target), the radiotherapeutic agent can comprise DOTA or a derivative thereof, which is a metal chelator, bound to a metal radionuclide. In specific embodiments where the radiotherapeutic agent comprises DOTA or a derivative thereof, the metal radionuclide is 177 This is Lu.

[0128] In another specific embodiment, the radiotherapeutic agent comprises (ii) a second target (second target of the bispecific binding agent used in the method of treating cancer) that is bound, preferably covalently bound, to a metal chelator, said second target being bound to a metal radionuclide. For example, if the first molecule of the bispecific binding agent is an immunoglobulin that binds (through its first binding site) to HER2 (i.e., the first target), a cancer antigen on cancer cells, and the second molecule of the bispecific binding agent is streptavidin that binds (through its second binding site) to biotin (i.e., the second target), the radiotherapeutic agent may comprise biotin bound to a metal chelator that is bound to a metal radionuclide. In a specific embodiment, the second target is covalently bound to the metal chelator.

[0129] Metal chelators that can form part of the radiotherapeutic agent described herein are known in the art. Non-limiting examples of metal chelators include DOTA or its derivatives (e.g., DOTA-Bn and DOTA-deferoxamine), and DTPA or its derivatives. In a specific embodiment, the metal chelator is DOTA or its derivatives. In a specific embodiment, the metal chelator is DOTA-Bn.

[0130] Metals that may form part of the radiotherapeutic agents described herein are known in the art. Non-limiting examples of metals include lutetium (Lu), actinium (Ac), astatine (At), bismuth (Bi), cerium (Ce), copper (Cu), dysprosium (Dy), erbium (Er), europium (Eu), gadolinium (Gd), gallium (Ga), holmium (Ho), iodine (I), indium (In), lanthanum (La), lead (Pb), neodymium (Nd), praseodymium (Pr), promethium (Pm), rhenium (Re), samarium (Sm), scandium (Sc), terbium (Tb), thulium (Tm), ytterbium (Yb), yttrium (Y), and zirconium (Zr). In a specific embodiment, the metal is yttrium (Y). In a preferred embodiment, the metal is lutetium (Lu). Non-limiting examples of metal radionuclides include: 211 At, 225 Ac, 227 Ac, 212 Bi, 213 Bi, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 157 Gd, 166 Ho, 124 I, 125 I, 131 I, 111 In, 177 Lu, 212 Pb, 186 Re, 188 Re, 47 Sc, 153 Sm, 166 Tb, 89 Zr, 86 Y,88 Y, and 90 The metal radionuclide of the radiotherapeutic agent includes a metal chelator, such as Lu or Y. One skilled in the art will appreciate that the metal radionuclide of the radiotherapeutic agent is selected based on its ability to bind to the metal chelator of the radiotherapeutic agent. For example, if the metal chelator of the radiotherapeutic agent is DOTA, a metal radionuclide capable of binding to DOTA, such as Lu or Y, is used. In specific embodiments, the metal radionuclide has pmol affinity for the metal chelator. In addition, the metal radionuclide of the radiotherapeutic agent must be selected such that the radiotherapeutic agent, including the metal chelator bound to the radionuclide, retains the ability to be bound by the bispecific binding agent (i.e., via the second binding site of the bispecific binding agent). In specific embodiments, where the metal chelator of the radionuclide is DOTA or a derivative thereof, the metal radionuclide is 86 Y, 90 Y, 88 Y, or 177 In a preferred embodiment where the metal chelator of the radionuclide is DOTA or a derivative thereof, the metal radionuclide is 177 This is Lu. In another specific embodiment, the metal chelator of the radiotherapeutic agent described herein has Formula I:

[0131] [ka] (I) [In the formula, M 1 teeth, 175 Lu 3+ , 45 Sc 3+ , 69 Ga 3+ , 71 Ga 3+ , 89 Y 3+ , 113 In 3+ , 115 In 3+ , 139 La 3+ , 136 Ce 3+ , 138 Ce3+ , 140 Ce 3+ , 142 Ce 3+ , 151 EU 3+ , 153 EU 3+ , 159 Tb 3+ , 154 Gd 3+ , 155 Gd 3+ , 156 Gd 3+ , 157 Gd 3+ , 158 Gd 3+ ,or 160 Gd 3+ ;X 1 , X 2 , X 3 , and X 4 are each independently a lone pair (i.e., resulting in an oxygen anion) or H; X 5 , X 6 , and X 7 are each independently a lone pair of electrons (i.e., resulting in an oxygen anion) or H; and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22, or a pharma- ceutically acceptable salt thereof. In certain embodiments, n is 3.

[0132] In another specific embodiment of the compounds of formula I, X 1 , X 2 , X 3 , and X 4 At least two of X are each independently a lone pair. In specific embodiments of compounds of formula I, 1 , X 2 , X 3 , and X 4 Three of the electrons are independently lone pairs, and the remaining X 1 , X 2 , X 3 , or X 4 is H.

[0133] In specific embodiments, where the metal chelator of the radiotherapeutic agent described herein comprises a compound of Formula I, the radiotherapeutic agent further comprises a cation of a radionuclide. In specific embodiments, the compound of Formula I has a K of about 1 pM to 1 nM (e.g., about 1 to 10 pM; 1 to 100 pM; 5 to 50 pM; 100 to 500 pM; or 500 pM to 1 nM). d and can bind to the cation of the radionuclide. d is in the range of about 1 nM to about 1 pM, for example, about 1 nM, 950 pM, 900 pM, 850 pM, 800 pM, 750 pM, 700 pM, 650 pM, 600 pM, 550 pM, 500 pM, 450 pM, 400 pM, 350 pM, 300 pM, 250 pM, 200 pM, 150 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 9 pM, 8 pM, 7 pM, 6 pM, 5 pM, 4 pM, 3 pM, 2.5 pM, 2 pM, or 1 pM or less. In specific embodiments where the metal chelator of the radiotherapeutic agents described herein comprises a compound of Formula I, the metal chelator has the formula II:

[0134] [ka] (II) [In the formula, M 1 teeth, 175 Lu 3+ , 45 Sc 3+ , 69 Ga 3+ , 71 Ga 3+ , 89 Y 3+ , 113 In 3+ , 115 In 3+ , 139 La 3+ , 136 Ce 3+ , 138 Ce 3+ , 140 Ce 3+ , 142 Ce 3+ ,151 EU 3+ , 153 EU 3+ , 159 Tb 3+ , 154 Gd 3+ , 155 Gd 3+ , 156 Gd 3+ , 157 Gd 3+ , 158 Gd 3+ ,or 160 Gd 3+ And;M 2 is the cation of the radionuclide; X 1 , X 2 , X 3 , and X 4 are each independently a lone pair (i.e., resulting in an oxygen anion) or H; X 5 , X 6 , and X 7 are each independently a lone pair of electrons (i.e., resulting in an oxygen anion) or H; and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22, or a pharma- ceutically acceptable salt thereof. In certain embodiments, n is 3.

[0135] In specific embodiments of the metal chelators of formula I or II, X 5 , X 6 , and X 7 At least two of are each independently a lone pair. Additionally or alternatively, in some embodiments for bis-chelates, the cation of the radionuclide is a divalent cation or a trivalent cation. The cation of the radionuclide can be an alpha particle emitting isotope, a beta particle emitting isotope, an Auger emitter, or a combination of any two or more of these cations. Examples of alpha particle emitting isotopes are: 213 Bi, 211 At, 225 Ac, 152 Dy, 212 Bi, 223 Ra,219 Rn, 215 Po, 211 Bi, 221 Fr, 217 At, and 255 Examples of beta particle emitting isotopes include, but are not limited to, Fm. 86 Y, 90 Y, 89 Sr, 165 Dy, 186 Re, 188 Re, 177 Lu, and 67 Examples of Auger emitters include, but are not limited to, Cu. 111 In, 67 Ga, 51 Cr, 58 Co, 99m Tc, 103m Rh, 195m Pt, 119 Sb, 161 Ho, 189m Os, 192 Ir, 201 Tl, and 203 In some embodiments of the metal chelator of formula I or II, the cation of the radionuclide is 68 Ga, 227 Th, or 64 Cu.

[0136] For metal chelators of formula I or II, in some embodiments, the cation of the radionuclide has a decay energy in the range of 20-6,000 keV. Decay energies can be in the range of 60-200 keV for Auger emitters, 100-2,500 keV for beta emitters, and 4,000-6,000 keV for alpha emitters. Maximum decay energies of useful beta particle emitting nuclides can be in the range of 20-5,000 keV, 100-4,000 keV, or 500-2,500 keV. Decay energies of useful Auger emitters can be <1,000 keV, <100 keV, or <70 keV. Decay energies of useful alpha particle emitting radionuclides can be in the range of 2,000-10,000 keV, 3,000-8,000 keV, or 4,000-7,000 keV.

[0137] In a specific embodiment, the metal radionuclide of the radiotherapeutic agent is a theranostic isotope. Theranostic isotopes as used herein are metal radionuclides that can be used for both therapeutic (e.g., cancer treatment) and imaging (e.g., in vivo imaging) purposes. Thus, a radiotherapeutic agent that includes a theranostic isotope allows the radiotherapeutic agent to (i) kill targeted cancer cells and (ii) be imaged in vivo, for example, to monitor the presence, location, and amount of the radiotherapeutic agent in a subject, thereby allowing monitoring of cancer treatment. In a specific embodiment, the theranostic isotope is both a beta particle and gamma radiation emitter. Without being bound to a particular theory, the beta particle emission serves a therapeutic purpose (i.e., to kill cancer cells), and the gamma radiation emission allows gamma scintigraphy for imaging purposes. Additionally, gamma emission allows imaging by high-resolution single-photon emission computed tomography / computed tomography (SPECT / CT), e.g., for pre-treatment dosimetry and treatment monitoring of bispecific binding agents (e.g., Ljungberg et al., 2016, MIRD Pamphlet No. 26: Joint EANM / MIRD Guidelines for Quantitative 177 (See, for example, Delker et al., 2016, Dosimetry for (177)Lu-DKFZ-PSMA-617: a new radiopharmaceutical for the treatment of metastatic prostate cancer. European Journal of Nuclear Medicine and Molecular Imagine, 43:42-51). Non-limiting examples of isotopes for theranostics include: 177 Lu,155 Tb, 90 Y, 131 I, 166 Ho, 152 Sm, and 111 Non-limiting examples of isotope pairs that can be used for imaging and therapy include: 111 In / 90 Y, 111 In / 225 Ac, 124 I / 131 I, 68 Ga / 177 Lu, 68 Ga / 90 Y, 86 Y / 90 Y, 64 Cu / 67 Cu. Such isotope pairs are selected such that the therapeutic and diagnostic isotopes have similar binding properties to the metal chelator. Thus, also provided herein is a method of diagnosing or prognosing cancer, comprising carrying out the method of treating cancer of the present invention using a radiotherapeutic agent containing a theranostic isotope and detecting an image in a subject using the theranostic radionuclide.

[0138] As will be apparent to one of skill in the art, the metal radionuclide of the radiotherapeutic agent, when bound to a metal chelator, is preferably bound non-covalently (ie, by chelation) to the metal chelator.

[0139] For methods of producing radiotherapeutic agents, see, e.g., Cheal et al., 2014, Preclinical evaluation of multistep targeting of diasialoganglioside GD2 using an IgG-scFv bispecific antibody with high affinity for GD2 and DOTA metal complex, Molecular Cancer Therapeutics; 13:1803-12.

[0140] 5.5 Pharmaceutical Compositions and Kits Specific embodiments provided herein are compositions (e.g., pharmaceutical compositions) comprising a therapeutically effective amount of a bispecific binding agent described herein (see, e.g., Sections 5.2 or 6). Specific embodiments provided herein are compositions (e.g., pharmaceutical compositions) comprising a therapeutically effective amount of a clearing agent described herein (see, e.g., Sections 5.3 and 6). Specific embodiments provided herein are compositions (e.g., pharmaceutical compositions) comprising a therapeutically effective amount of a radiotherapeutic agent described herein (see, e.g., Sections 5.4 and 6). Also provided herein are kits comprising one or more compositions (e.g., pharmaceutical compositions) comprising a therapeutically effective amount of a bispecific binding agent described herein (see, e.g., Sections 5.2 or 6), one or more compositions (e.g., pharmaceutical compositions) comprising a therapeutically effective amount of a clearing agent described herein (see, e.g., Sections 5.3 and 6), and / or one or more compositions (e.g., pharmaceutical compositions) comprising a therapeutically effective amount of a radiotherapeutic agent described herein (see, e.g., Sections 5.4 and 6). The compositions may be used in the preparation of individual single unit dosage forms. The compositions comprising the bispecific binding agent provided herein or the radiotherapeutic agent provided herein may be formulated for intravenous, subcutaneous, intramuscular, parenteral, transdermal, transmucosal, intraperitoneal, or intrathoracic administration, or for administration to other body compartments, such as intrathecal, intraventricular, or intraparenchymal administration. The compositions comprising the clearing agents provided herein may be formulated for intravenous administration. In a specific embodiment of a composition comprising a bispecific binding agent, the composition is formulated for intraperitoneal administration to treat peritoneal metastasis. In a specific embodiment of a composition comprising a bispecific binding agent, the composition is formulated for intrathecal administration. In a specific embodiment of a composition comprising a bispecific binding agent, the composition is formulated for intrathecal administration to treat brain metastasis. See, e.g., Kramer et al., 2010, 97: 409-418. In a specific embodiment of a composition comprising a bispecific binding agent, the composition is formulated for intraventricular administration in the brain.In a specific embodiment of the composition comprising the bispecific binding agent, the composition is formulated for intraventricular administration to treat brain metastasis. See, for example, Kramer et al., 2010, 97: 409-418. In a specific embodiment of the composition comprising the bispecific binding agent, the composition is formulated for intraparenchymal administration in the brain. In a specific embodiment of the composition comprising the bispecific binding agent, the composition is formulated for intraparenchymal administration to treat brain tumor or brain tumor metastasis. See, for example, Luther et al., 2014, Neuro Oncol, 16: 800-806; and Clinical Trial No.: NCT01502917. In a preferred embodiment of the composition comprising the bispecific binding agent, the composition is formulated for intravenous administration. In a preferred embodiment of the composition comprising the clearing agent, the composition is formulated for intravenous administration. In a preferred embodiment of the composition comprising the radiotherapeutic agent, the composition is formulated for intravenous administration.

[0141] In specific embodiments, the compositions provided herein include at least one of any suitable auxiliary agent, such as, but not limited to, a diluent, binder, stabilizer, buffer, salt, lipophilic solvent, preservative (e.g., ascorbic acid), adjuvant, surfactant, or other incipient to stabilize and prevent aggregation. In specific embodiments, pharma- ceutically acceptable auxiliary agents are preferred. Non-limiting examples of such sterile solutions, and methods for preparing them, are well known in the art, such as, but not limited to, Gennaro, Ed., Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (Easton, Pa.) 1990. Pharmaceutically acceptable carriers suitable for the mode of administration, solubility, and / or stability of the bispecific binding agent, removal agent, or radiotherapy agent described herein can be routinely selected. In specific embodiments, the pharmaceutical compositions described herein are intended to be used in accordance with the methods provided herein (see, e.g., Sections 5.1 and 6).

[0142] 5.5.1 Kits Provided herein is a kit comprising one or more bispecific binding agents, clearing agents, and / or radiotherapeutic agents described herein, or one or more compositions described herein. In a specific embodiment, the kit comprises (i) a packaging material, and (ii) at least one vial comprising a bispecific binding agent or composition thereof described herein, at least one vial comprising a clearing agent or composition thereof described herein, and / or at least one vial comprising a radiotherapeutic agent or composition thereof described herein. In a specific embodiment, the vial comprises a solution of at least one bispecific binding agent, clearing agent, or radiotherapeutic agent, or composition thereof described herein, with a formulated buffer and / or preservative, but may be in an aqueous diluent. In specific embodiments, the compositions provided herein may be provided to a subject as a solution or as a duplicate vial containing a vial of lyophilized bispecific binding agent, clearing agent, or radiotherapeutic agent, or a composition thereof, to be reconstituted with a second vial containing water, preservatives, and / or excipients, preferably phosphate buffer and / or saline, and a selected salt in an aqueous diluent. A single solution vial, or a duplicate vial requiring reconstitution, may be reused multiple times and may be sufficient to treat a subject for a single or multiple cycles, thus resulting in a more convenient treatment regimen than currently available treatment regimens.

[0143] In specific embodiments, the kits containing the bispecific binding agents, clearing agents, and / or radiotherapeutic agents described herein, or compositions thereof, are useful for administration over a period of time ranging from immediate to 24 hours or more. In specific embodiments, the kits containing the bispecific binding agents, clearing agents, and / or radiotherapeutic agents described herein, or compositions thereof, may be safely stored, optionally at temperatures between about 2° C. and about 40° C., and retain the biological activity of the agent for extended periods of time, allowing the package labeling to indicate that the solution may be stored and / or used for periods of 6, 12, 18, 24, 36, 48, 72, or 96 hours or more. If a preserved diluent is used, such labeling may include use after 1 to 12 months, 6 months, 18 months, and / or up to 2 years.

[0144] Kits can be provided indirectly to subjects, such as those described in Section 5.6, by providing a pharmacy, clinic, or other such institution or facility with multiple vials containing a lyophilized bispecific binding agent, remover, or radiotherapy agent, or a vial of these compositions, to be reconstituted with a second vial containing a solution or aqueous diluent. The solution in this case can be up to a liter or even larger in size, resulting in a larger reservoir from which at least one small portion of the bispecific binding agent, remover, or radiotherapy agent solution is removed, either once or multiple times, for transfer into smaller vials and provided by the pharmacy or clinic to their customers and / or patients.

[0145] Recognized devices that include these single vial systems include pen injector devices for delivering solutions, such as BD Pens, BD Autojector®, Humaject®, manufactured or developed by, for example, Becton Dickensen (Franklin Lakes, NJ,), Disetronic (Burgdorf, Switzerland); Bioject (Portland, Oreg.); National Medical Products, Weston Medical (Peterborough, UK), Medi-Ject Corp (Minneapolis, Minn.). Recognized devices that include dual vial systems include pen injector systems for reconstituting lyophilized drugs in a cartridge to deliver a reconstituted solution, such as HumatroPen®.

[0146] In a specific embodiment, the kit includes packaging materials. In a specific embodiment, the packaging materials provide the conditions under which the product may be used, in addition to information required by regulatory agencies. In a specific embodiment, the packaging materials provide, for a multi-vial wet / dry product, instructions to the subject to reconstitute at least one bispecific binding agent, removal agent, and / or radiotherapy agent in an aqueous diluent to form a solution and use the solution for a period of 2-24 hours or more. For a single vial solution product, the label indicates that such a solution may be used for a period of 2-24 hours or more. In a preferred embodiment, the kit is useful for use as a human medicine. In a specific embodiment, the kit is useful for use as a veterinary medicine. In a preferred embodiment, the kit is useful for use as a canine medicine. In a preferred embodiment, the kit is useful for intravenous administration. In another preferred embodiment, the kit is useful for subcutaneous, intramuscular, parenteral, transdermal, transmucosal, intraperitoneal, intrathoracic, intrathecal, intraventricular, or intraparenchymal administration.

[0147] 5.6 Patient Population The subject treated according to the methods provided herein can be any mammal, such as a rodent, cat, dog, horse, cow, pig, monkey, primate, or human. In a specific embodiment, the subject is a dog. In a preferred embodiment, the subject is a human.

[0148] In a specific embodiment, the subject treated according to the method presented herein has been diagnosed with cancer.Non-limiting examples of cancer include bladder cancer, brain cancer, breast cancer (e.g., triple-negative breast cancer), cervical cancer, renal clear cell carcinoma, colon cancer, colorectal cancer, desmoplastic small round cell carcinoma, endometrial cancer, epithelial tumors (e.g., breast tumors, gastrointestinal tumors), esophageal cancer, Ewing's sarcoma, gastric cancer, gastroesophageal junction adenocarcinoma, gastroesophageal junction adenocarcinoma, glioblastoma (e.g., glioblastoma multiforme), glioma, gynecological malignancies, head and neck cancer, hepatocellular carcinoma, leukemia, lung cancer, lymphoma, melanoma, mesothelioma, myeloma, neuroblastoma, neuroendocrine tumor, The cancers include non-small cell lung cancer, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, small cell lung cancer, soft tissue sarcoma, head and neck squamous cell carcinoma, stromal cancer of most neoplasms (e.g., colorectal neoplasm, pancreatic neoplasm, among others), tumors associated with vasculature, tumors associated with papillomavirus, urothelial carcinoma, various cancers as markers of tumor-associated angiogenesis, Wilms' tumor, other cancer stem cells, and invasive epithelial tumors, and cancers associated with vasculature (see, for example, Table 1). In a specific embodiment, the cancer is a metastatic cancer. In a specific embodiment, the metastatic cancer includes peritoneal metastatic cancer.

[0149] Those skilled in the art will understand that the cancer to be treated by the bispecific binding agent described herein according to the method presented herein indicates the first target of the bispecific binding agent used in the method of treating cancer.For example, if the cancer to be treated is a cancer that expresses HER2, the bispecific binding agent used in the method of treating HER2-expressing cancer comprises a first binding site that specifically binds to HER2 (i.e., the first target of the bispecific binding agent).In other words, the cancer to be treated according to the method presented herein expresses a cancer antigen that is the first target of the bispecific binding agent.

[0150] In a preferred embodiment, the first target of the bispecific binding agent is HER2, and the subject treated according to the methods described herein has been diagnosed with a cancer that expresses HER2 (e.g., breast cancer, gastric cancer, osteosarcoma, desmoplastic small round cell carcinoma, squamous cell carcinoma of the head and neck, ovarian cancer, prostate cancer, pancreatic cancer, glioblastoma multiforme, gastric junction adenocarcinoma, gastroesophageal junction adenocarcinoma, cervical cancer, salivary gland carcinoma, soft tissue sarcoma, leukemia, melanoma, Ewing's sarcoma, rhabdomyosarcoma, neuroblastoma, or any other neoplastic tissue that expresses the HER2 receptor). In a specific embodiment for the treatment of a HER2-expressing cancer, the subject is resistant to treatment with trastuzumab, cetuximab, lapatinib, erlotinib, or any other small molecule or antibody that targets the HER family of receptors. In specific embodiments, cancers that are resistant to treatment with trastuzumab, cetuximab, lapatinib, erlotinib, or any other small molecule or antibody that targets the HER family of receptors are responsive to the methods of treating cancer of the invention (see, e.g., Sections 5.1 and 6).

[0151] In a specific embodiment, the subject treated according to the methods presented herein has previously been administered one or more chemotherapy regimens for metastatic disease, such as one or more chemotherapy regimens for brain metastases or peritoneal metastases. In a specific embodiment, the subject has not yet been administered treatment for metastatic disease.

[0152] 5.7 Dosage, Route of Administration, and Regimen In specific embodiments, the therapeutically effective amount of a bispecific binding agent administered to a subject according to the methods provided herein (see, e.g., Section 5.1) is a dose determined by the needs of the subject. In specific embodiments, the dose is determined by a physician according to the needs of the subject.

[0153] In specific embodiments, a therapeutically effective amount of a bispecific binding agent administered to a subject according to the methods of treating cancer provided herein is determined based on the concentration of a cancer antigen (i.e., the cancer antigen that is the primary target of the bispecific binding agent) on the subject's cancer cells and / or the extent of uptake of the bispecific binding agent by said cancer cells. In specific embodiments, the extent of uptake will be confirmed by theranostic methods and confirmed by biopsy or ex vivo tissue counting, both in laboratory and clinical settings. In specific embodiments, a therapeutically effective amount of a bispecific binding agent is determined based on the law of mass action (e.g., O'Donoghue et al., 2011, 124I-huA33 antibody uptake is driven by A33 antigen concentration in tissues from colorectal cancer patients imaged by immune-PET. J. Nucl. Med.; 52(12):1878-85; and see Section 6.3) and is determined based on results in animal model studies described in Section 6.3. Without being bound to a particular theory, a therapeutically effective amount of a bispecific agent is preferably an amount that provides sufficient bispecific binding agent to approach saturation of binding capacity (e.g., in the range of 50-90%) of the target cancer antigen on the cancer cells (i.e., the first target of the bispecific binding agent), since subsaturation of the cancer antigen allows a maximum amount of radiotherapeutic agent to bind to the bispecific binding agent bound to the cancer cells in the subject, thus providing therapeutic efficacy and / or enabling imaging results in vivo. In specific embodiments, a therapeutically effective amount of a bispecific binding agent is an amount that is estimated to achieve at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% saturation of the cancer antigen by the bispecific binding agent on the cancer cells according to the laws of mass action. In specific embodiments, a therapeutically effective amount of a bispecific binding agent is an amount that is estimated to achieve between 60%-100%, between 70%-99%, between 70%-95%, between 70%-90%, between 75%-85%, between 80%-90% saturation of the cancer antigen by the bispecific binding agent on the cancer cells according to the laws of mass action. In specific embodiments, a therapeutically effective amount of a bispecific binding agent is an amount that is estimated to achieve about 80% saturation of the cancer antigen by the bispecific binding agent on the cancer cells according to the laws of mass action.

[0154] In specific embodiments where the first target of the bispecific binding agent is HER2, the dose of the bispecific binding agent is less than the U.S. Food and Drug Administration ("FDA") approved dose of trastuzumab for the subject's cancer. See, e.g., Trastuzumab [Highlights of Prescribing Information], South San Francisco, CA: Genentech, Inc.; 2014. In specific embodiments, the therapeutically effective amount of the bispecific binding agent is about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% less than the FDA approved dose of trastuzumab. In specific embodiments, the therapeutically effective amount of the bispecific binding agent is 100 mg to 700 mg, 200 mg to 600 mg, 200 mg to 500 mg, 300 mg to 400 mg, about 300 mg, about 450 mg, about 500 mg, about 600 mg, or about 625 mg, and the subject is a human. "About" when used in the context of a therapeutically effective amount refers to an amount within 1%, 3%, 5%, or 10% of the recited amount. In specific embodiments, the therapeutically effective amount of the bispecific binding agent is between 250 mg and 700 mg, between 300 mg and 600 mg, or between 400 mg and 500 mg, and the subject is a human. In specific embodiments, the therapeutically effective amount of the bispecific binding agent is between 1.0 mg / kg and 8.0 mg / kg, between 2.0 mg / kg and 7.0 mg / kg, between 3.0 mg / kg and 6.5 mg / kg, between 4.0 mg / kg and 6.5 mg / kg, or between 5.0 mg / kg and 6.5 mg / kg.

[0155] In a specific embodiment, the therapeutically effective amount of the bispecific binding agent is administered via intravenous infusion over 30 minutes. In a specific embodiment, the therapeutically effective amount of the bispecific binding agent is administered via intravenous infusion over 30-90 minutes. In a specific embodiment, the bispecific binding agent is administered to the subject intravenously, subcutaneously, intramuscularly, parenterally, transdermally, transmucosally, intraperitoneally, intrathoracically, or to some other body compartment, such as intrathecally, intraventricularly, or intraparenchymally. In a preferred embodiment, the binding agent is administered to the subject intravenously.

[0156] In a specific embodiment, the therapeutically effective amount of the clearing agent administered to a subject according to the methods provided herein is an amount determined by the needs of the subject. One of skill in the art will understand that the therapeutically effective amount of the clearing agent depends on the structure of the clearing agent, the structure of the bispecific binding agent, and / or the therapeutically effective amount of the bispecific binding agent administered to the subject. In a specific embodiment, the therapeutically effective amount of the clearing agent is proportional to the therapeutically effective amount of the bispecific binding agent administered to the subject. In a specific embodiment, where the clearing agent comprises about 100-150 (Y or Lu)DOTA-Bn molecules per 500 kDa of aminodextran, the therapeutically effective amount of the clearing agent is an amount that provides a 10:1 molar ratio of the therapeutically effective amount of the bispecific binding agent administered to the subject to the therapeutically effective amount of the clearing agent administered to the subject. In other words, in a specific embodiment, the therapeutically effective amount of the bispecific binding agent administered to the subject is an amount that is a 10-fold molar excess of the therapeutically effective amount of the clearing agent administered to the subject. For example, for every 100 mg of bispecific binding factor having a molecular weight of 210 kDa, which is 0.476 micromoles, 25 mg of clearing agent having a molecular weight of 500 kDa, which is 0.05 micromoles, is administered to the subject. In a specific embodiment where the bispecific binding factor comprises a heavy chain and a light chain fusion polypeptide of SEQ ID NO: 15 and SEQ ID NO: 7, and the clearing agent comprises about 100-150 (Y or Lu)DOTA-Bn molecules per 500 kDa aminodextran, between 15 mg and 35 mg, between 20 mg and 35 mg, or between 20 mg and 30 mg of clearing agent is administered to the subject for every 100 mg of bispecific binding factor administered to the subject.

[0157] In specific embodiments, a therapeutically effective amount of a clearing agent is an amount that results in at least a 90%, at least a 91%, at least a 92%, at least a 93%, at least a 94%, at least a 95%, at least a 96%, at least a 97%, at least a 98%, or at least a 99% reduction in the serum concentration of the bispecific binding agent 1 hour, 2 hours, 3 hours, or 4 hours after step (b) of administering a therapeutically effective amount of the clearing agent to the subject. Methods known to those of skill in the art for determining the percent reduction in serum concentration of the bispecific binding agent are methods known in the art, such as ELISA on serum samples before and after administration of the clearing agent. In a preferred embodiment, the clearing agent is administered intravenously to the subject.

[0158] In specific embodiments, the therapeutically effective amount of a radiotherapeutic agent administered to a subject according to the methods provided herein is an amount determined by the needs of the subject. One of skill in the art will appreciate that the therapeutically effective amount of a radiotherapeutic agent will depend on the identity of the metal radionuclide of the radiotherapeutic agent. For example, if the metal radionuclide of the radiotherapeutic agent is: 177 In specific embodiments, the therapeutically effective amount of the radiotherapeutic agent is between 25 mCi and 250 mCi, between 50 mCi and 200 mCi, between 75 mCi and 175 mCi, or between 100 mCi and 150 mCi. 177 In another specific embodiment, the therapeutically effective amount of the radiotherapeutic agent is Lu or an equivalent beta-emitter, is between 50 mCi and 200 mCi.

[0159] In specific embodiments, a therapeutically effective amount of a radiotherapeutic agent is administered to a subject intravenously, subcutaneously, intramuscularly, parenterally, transdermally, transmucosally, intraperitoneally, intrathoracically, or to other body compartments, such as intrathecally, intraventricularly, or intraparenchymally. In a preferred embodiment, the radiotherapeutic agent is administered to a subject intravenously.

[0160] The method of treating cancer provided herein may also form part of a multi-cycle treatment regimen. For example, the method of treating cancer provided herein may be repeated two, three or more times on the same subject. In a specific embodiment, the method of treating cancer provided herein is repeated two times on the same subject. For example, in a specific embodiment, the method of treating cancer described in section 5.1 further comprises: (d) administering to the subject a second therapeutically effective amount of a bispecific binding agent within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week of administering to the subject a therapeutically effective amount of a radiotherapy agent; (e) administering to the subject a second therapeutically effective amount of a clearing agent after administering to the subject a second therapeutically effective amount of a bispecific binding agent after administering to the subject a second therapeutically effective amount of a clearing agent; and (f) administering to the subject a second therapeutically effective amount of a radiotherapy agent after administering to the subject a second therapeutically effective amount of a clearing agent after administering to the subject a second therapeutically effective amount of a clearing agent after administering to the subject a second therapeutically effective amount of a radiotherapy agent. In a specific embodiment, step (e) of administering to the subject a therapeutically effective amount of a clearing agent occurs within 12 hours of step (d) of administering to the subject a first therapeutically effective amount of a bispecific binding agent. In a specific embodiment, the methods of treating cancer provided herein are repeated on the same subject three times.For example, in specific embodiments, the methods of treating cancer described in Section 5.1 include the steps of: (d) administering to the subject a second therapeutically effective amount of a bispecific binding agent within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week of step (c) of administering to the subject a therapeutically effective amount of a radiation therapy agent; (e) after step (d) of administering to the subject the second therapeutically effective amount of the bispecific binding agent, administering to the subject a second therapeutically effective amount of a clearing agent; (f) after step (e) of administering to the subject a second therapeutically effective amount of a radiation therapy agent; (g) administering to the subject a third therapeutically effective amount of a bispecific binding agent within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week of administering to the subject a second therapeutically effective amount of radiation therapy; (h) administering to the subject a third therapeutically effective amount of a clearing agent after step (g) of administering to the subject a third therapeutically effective amount of a bispecific binding agent; and (i) administering to the subject a third therapeutically effective amount of a radiation therapy agent after step (h) of administering to the subject a third therapeutically effective amount of a clearing agent. In a specific embodiment, the step (e) of administering to the subject a therapeutically effective amount of a clearing agent is performed within 12 hours of the step (d) of administering to the subject a second therapeutically effective amount of a bispecific binding agent. In a specific embodiment, the step (g) of administering to the subject a therapeutically effective amount of a clearing agent is performed within 12 hours of the step (g) of administering to the subject a second therapeutically effective amount of a bispecific binding agent.

[0161] The second and / or third therapeutically effective amount of the bispecific binding factor in the multi-cycle method of treating cancer presented herein may be the same therapeutically effective amount or may be a different therapeutically effective amount compared to the therapeutically effective amount of the bispecific binding factor administered to the subject in step (a). In a specific embodiment, the second therapeutically effective amount of the bispecific binding factor is the same as the therapeutically effective amount of the bispecific binding factor administered to the subject in step (a). In a specific embodiment, the second therapeutically effective amount of the bispecific binding factor is less than the therapeutically effective amount of the bispecific binding factor administered to the subject in step (a). In a specific embodiment, the second therapeutically effective amount of the bispecific binding factor exceeds the therapeutically effective amount of the bispecific binding factor administered to the subject in step (a). In a specific embodiment, the third therapeutically effective amount of the bispecific binding factor is the same as the therapeutically effective amount of the bispecific binding factor administered to the subject in step (a). In a specific embodiment, the third therapeutically effective amount of the bispecific binding factor is less than the therapeutically effective amount of the bispecific binding factor administered to the subject in step (a). In specific embodiments, the third therapeutically effective amount of the bispecific binding agent exceeds the therapeutically effective amount of the bispecific binding agent administered to the subject in step (a). In specific embodiments, the second therapeutically effective amount of the bispecific binding agent is 100 mg to 700 mg, 200 mg to 600 mg, 200 mg to 500 mg, 300 mg to 400 mg, about 300 mg, about 450 mg, about 500 mg, about 600 mg, or about 625 mg. In specific embodiments, the third therapeutically effective amount of the bispecific binding agent is 100 mg to 700 mg, 200 mg to 600 mg, 200 mg to 500 mg, 300 mg to 400 mg, about 300 mg, about 450 mg, about 500 mg, about 600 mg, or about 625 mg. In specific embodiments, the second therapeutically effective amount of the bispecific binding agent is administered to the subject intravenously, subcutaneously, intramuscularly, parenterally, transdermally, transmucosally, intraperitoneally, intrathoracically, or to any other body compartment, such as intrathecally, intraventricularly, or intraparenchymally. In a preferred embodiment, the second therapeutically effective amount of the bispecific binding agent is administered to the subject intravenously.In specific embodiments, the third therapeutically effective amount of the bispecific binding agent is administered to the subject intravenously, subcutaneously, intramuscularly, parenterally, transdermally, transmucosally, intraperitoneally, intrathoracically, or to any other body compartment, such as intrathecally, intraventricularly, or intraparenchymally. In a preferred embodiment, the third therapeutically effective amount of the bispecific binding agent is administered to the subject intravenously.

[0162] In the multi-cycle method of treating cancer provided herein, the second therapeutically effective amount and / or the third therapeutically effective amount of the clearing agent may be the same therapeutically effective amount or may be a different therapeutically effective amount compared to the therapeutically effective amount of the clearing agent administered to the subject in step (b). In a specific embodiment, the second therapeutically effective amount of the clearing agent is the same as the therapeutically effective amount of the clearing agent administered to the subject in step (b). In a specific embodiment, the second therapeutically effective amount of the clearing agent is less than the therapeutically effective amount of the clearing agent administered to the subject in step (b). In a specific embodiment, the second therapeutically effective amount of the clearing agent exceeds the therapeutically effective amount of the clearing agent administered to the subject in step (b). In a specific embodiment, the third therapeutically effective amount of the clearing agent is the same as the therapeutically effective amount of the clearing agent administered to the subject in step (b). In a specific embodiment, the third therapeutically effective amount of the clearing agent is less than the therapeutically effective amount of the clearing agent administered to the subject in step (b). In a specific embodiment, the third therapeutically effective amount of the clearing agent exceeds the therapeutically effective amount of the clearing agent administered to the subject in step (b). In specific embodiments, the second therapeutically effective amount of the clearing agent is an amount that results in at least a 90%, at least a 91%, at least a 92%, at least a 93%, at least a 94%, at least a 95%, at least a 96%, at least a 97%, at least a 98%, or at least a 99% reduction in the serum concentration of the bispecific binding agent 1 hour, 2 hours, 3 hours, or 4 hours after step (b) of administering the therapeutically effective amount of the clearing agent to the subject. In specific embodiments, where the clearing agent comprises about 100-150 (Y)DOTA-Bn molecules per 500 kDa of aminodextran, the second therapeutically effective amount of the clearing agent is an amount that results in a 10:1 molar ratio of the therapeutically effective amount of the bispecific binding agent administered to the subject to the therapeutically effective amount of the clearing agent administered to the subject.In a specific embodiment, the third therapeutically effective amount of the clearing agent is an amount that results in at least a 90%, at least a 91%, at least a 92%, at least a 93%, at least a 94%, at least a 95%, at least a 96%, at least a 97%, at least a 98%, or at least a 99% reduction in the serum concentration of the bispecific binding agent 1 hour, 2 hours, 3 hours, or 4 hours after step (b) of administering the therapeutically effective amount of the clearing agent to the subject. In a specific embodiment, where the clearing agent comprises about 100-150 (Y)DOTA-Bn molecules per 500 kDa of aminodextran, the third therapeutically effective amount of the clearing agent is an amount that results in a 10:1 molar ratio of the therapeutically effective amount of the bispecific binding agent administered to the subject to the therapeutically effective amount of the clearing agent administered to the subject. In a preferred embodiment, the second therapeutically effective amount of the clearing agent is administered intravenously to the subject. In a preferred embodiment, the third therapeutically effective amount of the clearing agent is administered intravenously to the subject.

[0163] The second and / or third therapeutically effective amount of the radiotherapy agent in the multi-cycle method of treating cancer presented herein may be the same therapeutically effective amount or a different therapeutically effective amount compared to the therapeutically effective amount of the radiotherapy agent administered to the subject in step (c). In a specific embodiment, the second therapeutically effective amount of the radiotherapy agent is the same as the therapeutically effective amount of the radiotherapy agent administered to the subject in step (c). In a specific embodiment, the second therapeutically effective amount of the radiotherapy agent is less than the therapeutically effective amount of the radiotherapy agent administered to the subject in step (c). In a specific embodiment, the second therapeutically effective amount of the radiotherapy agent is greater than the therapeutically effective amount of the radiotherapy agent administered to the subject in step (c). In a specific embodiment, the third therapeutically effective amount of the radiotherapy agent is the same as the therapeutically effective amount of the radiotherapy agent administered to the subject in step (c). In a specific embodiment, the third therapeutically effective amount of the radiotherapy agent is less than the therapeutically effective amount of the radiotherapy agent administered to the subject in step (c). In specific embodiments, the third therapeutically effective amount of the radiotherapeutic agent exceeds the therapeutically effective amount of the radiotherapeutic agent administered to the subject in step (c). In specific embodiments, the second therapeutically effective amount of the radiotherapeutic agent is between 25 mCi and 250 mCi, between 50 mCi and 200 mCi, between 75 mCi and 175 mCi, or between 100 mCi and 150 mCi. In specific embodiments, the third therapeutically effective amount of the radiotherapeutic agent is between 25 mCi and 250 mCi, between 50 mCi and 200 mCi, between 75 mCi and 175 mCi, or between 100 mCi and 150 mCi. In specific embodiments, the second therapeutically effective amount of the radiotherapy agent is administered to the subject intravenously, subcutaneously, intramuscularly, parenterally, transdermally, transmucosally, intraperitoneally, intrathoracically, or to any other body compartment, such as intrathecally, intraventricularly, or intraparenchymally. In preferred embodiments, the second therapeutically effective amount of the radiotherapy agent is administered to the subject intravenously. In specific embodiments, the third therapeutically effective amount of the radiotherapy agent is administered to the subject intravenously, subcutaneously, intramuscularly, parenterally, transdermally, transmucosally, intraperitoneally, intrathoracically, or to any other body compartment, such as intrathecally, intraventricularly, or intraparenchymally.In a preferred embodiment, the third therapeutically effective amount of a radiotherapeutic agent is administered intravenously to the subject.

[0164] 5.8 Combination Therapy In specific embodiments, the bispecific binding agent provided herein may be administered in combination with one or more additional pharmacologic active agents, such as cancer chemotherapeutic agents. In specific embodiments, such combination therapy may be achieved by simultaneous, sequential, or separate administration of the individual components of the treatment. In specific embodiments, the bispecific binding agent and the one or more additional pharmacologic active agents may be synergistic, such that the dose of one or both of the components may be reduced compared to the dose of either component administered as a monotherapy. Alternatively, in specific embodiments, the bispecific binding agent and the one or more additional pharmacologic active agents may be additive, such that the dose of the bispecific binding agent and the dose of the one or more additional pharmacologic active agents are equivalent or similar to the dose of either component administered as a monotherapy.

[0165] In a specific embodiment, the bispecific binding agent provided herein is administered on the same day as one or more additional pharma- ceutically active agents. In a specific embodiment, the bispecific binding agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours before the one or more additional pharma- ceutically active agents. In a specific embodiment, the bispecific binding agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours after the one or more additional pharma- ceutically active agents. In a specific embodiment, the bispecific binding agent is administered 1, 2, 3, or more days before the one or more additional pharma- ceutically active agents. In a specific embodiment, the bispecific binding agent is administered 1, 2, 3, or more days after the one or more additional pharma- ceutically active agents. In a specific embodiment, the bispecific binding agent is administered 1, 2, 3, 4, 5, or 6 weeks before the one or more additional pharma- ceutically active agents. In specific embodiments, the bispecific binding agent is administered 1, 2, 3, 4, 5, or 6 weeks after the one or more additional pharma- ceutical active agents.

[0166] In a specific embodiment, where the cancer is breast cancer, the additional pharmacologic active agent is doxorubicin. In a specific embodiment, where the cancer is breast cancer, the additional pharmacologic active agent is cyclophosphamide. In a specific embodiment, where the cancer is breast cancer, the additional pharmacologic active agent is paclitaxel. In a specific embodiment, where the cancer is breast cancer, the additional pharmacologic active agent is docetaxel. In a specific embodiment, where the cancer is breast cancer, the one or more additional pharmacologic active agents are carboplatin.

[0167] In a specific embodiment, the additional pharma- ceutical active agent is an agent that increases cell death, apoptosis, autophagy, or necrosis of tumor cells. In a specific embodiment, the bispecific binding agent provided herein is administered in combination with two additional pharmacologic active agents, e.g., in combination with trastuzumab for HER2-expressing cancer (see Trastuzumab [Highlights of Prescribing Information]. South San Francisco, CA: Genentech, Inc.; 2014). In a specific embodiment where the cancer is a HER2-expressing cancer, the two additional pharmacologic active agents are doxorubicin and paclitaxel. In a specific embodiment where the cancer is a HER2-expressing cancer, the two additional pharmacologic active agents are doxorubicin and docetaxel. In a specific embodiment where the cancer is a HER2-expressing cancer, the two additional pharmacologic active agents are cyclophosphamide and paclitaxel. In a specific embodiment, the cancer is a HER2-expressing cancer, the two additional pharmacologic active agents are cyclophosphamide and docetaxel.In a specific embodiment, the cancer is a HER2-expressing cancer, the two additional pharmacologic active agents are docetaxel and carboplatin.In a specific embodiment, the cancer is a HER2-expressing cancer, the two additional pharmacologic active agents are cisplatin and capecitabine.In a specific embodiment, the cancer is a HER2-expressing cancer, the two additional pharmacologic active agents are cisplatin and 5-fluorouracil.

[0168] In a specific embodiment, the bispecific binding agents presented herein are administered following multi-modality, anthracycline-based therapy. In a specific embodiment, the bispecific binding agent provided herein is administered after one or more chemotherapy regimens for metastatic disease, for example, one or more chemotherapy regimens for brain metastasis or peritoneal metastasis.In a specific embodiment, the bispecific binding agent provided herein is administered in combination with tumor-reducing chemotherapy.In a specific embodiment, administration is performed after the subject is treated with tumor-reducing chemotherapy.

[0169] In a specific embodiment, the cancer is a cancer that expresses HER2, the additional pharmacologic agent is an agent that increases HER2 expression by cells, such as external radiation or radioimmunotherapy.See, for example, Wattenberg et al., 2014, British Journal of Cancer, 110: 1472.In a specific embodiment, the additional pharmacologic agent is an agent that directly controls HER2 signaling pathway, such as lapatinib.See, for example, Scaltiri et al., 2012, 28(6): 803-814. EXAMPLES

[0170] 6. Example Example 1 6.1 Preclinical model of theranostic anti-DOTA hapten bispecific pretargeted radioimmunotherapy against internalized solid tumor antigens: Curative treatment for HER2-positive breast cancer References cited in this Example are identified by numbers in parentheses. Corresponding citations are provided in Section 6.1.5.

[0171] 6.1.1 Introduction The pharmacokinetics of full-sized IgG monoclonal antibodies as carriers of therapeutic radioisotopes (i.e., radioimmunotherapy; RIT) typically exhibits an unfavorable therapeutic index ("TI"; defined herein as the ratio of radiation absorbed dose to tumor divided by dose to radiosensitive tissues such as blood [1]) for radioimmunotherapy ("RIT"). Alternatively, a pretargeting RIT ("PRIT") strategy can be utilized that separates the antibody-mediated targeting step from the step of administering a cytotoxic ligand to shorten the residence time of the ligand in circulation [2].

[0172] Cure in preclinical animal models with human carcinoma xenografts for carbohydrate targets (diasialoganglioside GD2[3] on human neuroblastoma xenografts) or glycoprotein targets (GPA33[4] on human colon cancer xenografts) with total injected activity ("IA") of approximately 30-111 MBq per mouse. 177 High TI targeting using the pretargeted "DOTA-PRIT" platform has been supported by cure with negligible toxicity following treatment with Lu-DOTA-Bn. DOTA-PRIT has also recently demonstrated a total IA of 26-37MBq in preclinical CD20(+) human lymphoma xenografts. 90 High-TI targeting with healing has been demonstrated using Y-DOTA-biotin C825-hapten [5].

[0173] DOTA-PRIT uses a single specificity for a tumor antigen and a radioactive metal complex of the low molecular weight chelator S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecanetetraacetic acid (e.g., a β-emitter, 177A non-radioactive bispecific binding agent (e.g., a bispecific antibody ("BsAb")) with a second specificity for a hapten such as "[M]-DOTA-Bn" as Lu-DOTA-Bn [6]) may be used. After sufficient uptake by the tumor and clearance of unbound BsAb (accelerated by a clearing agent ("CA")), 177 Lu-DOTA-Bn is administered, which is captured by tumor-localized BsAb or is otherwise rapidly cleared from the body by the kidney. In this example, variations on the DOTA-PRIT platform were investigated using HER2, an antigen that is expressed in a very wide spectrum of human cancers but is susceptible to endocytosis and has unique properties that distinguish it from other commonly studied PRIT targets. Unlike antibody-drug conjugates that rely on binding to cell surface receptors and internalization upon binding to cells for delivery of their payload, a non-internalizing antibody / cell surface target is believed to be optimal for PRIT. Specifically, in DOTA-PRIT, non-radioactive BsAb administered intravenously ("iv") accumulates in the tumor and serves as a receptor for the subsequently administered radiolabeled hapten (e.g., radiolabeled DOTA-Bn). Without being bound to a particular theory, it was previously hypothesized that if extensive internalization of BsAbs occurs at the tumor surface, this may have a profound effect on the efficiency of the hapten targeting step.

[0174] Anti-HER2 monoclonal antibodies are an example of internalizing antibodies [7]. HER2 (transmembrane tyrosine kinase receptor HER2 / neu or c-erbB-2; molecular weight (MW): 185 kD) is a member of the HER / erbB family of cell surface receptors, and high HER2 expression is prognostic for survival in several cancer types, including breast cancer [8], gastric cancer [9], and gynecological malignancies

[10] . Trastuzumab can prolong survival in breast or ovarian cancer patients with HER2-positive ("HER2(+)") disease [11, 12]. Unfortunately, despite initial responses to trastuzumab, resistance is common

[13] . Antibody-based delivery of cytotoxins

[14] or therapeutic radionuclides

[15] has been attempted to enhance the efficacy of trastuzumab. Several preclinical RIT studies with trastuzumab-radioisotope conjugates have also been described (e.g., studies with α-emitters [16–23] or β-emitters [24–28]) and, without being bound to any particular theory, it is hypothesized that the PRIT approach may potentially improve TI.

[0175] In this example, a theranostic isotope was incorporated for anti-HER2-DOTA-PRIT, allowing for simultaneous therapy and imaging. 177 Lu has a physical half-life of 6.73 days ("d") and is both a beta particle and gamma radiation emitter (beta maximum energy: 0.5 MeV; beta particle range in tissues Rmax: 2 mm; gamma: 208 keV, 11% abundance), allowing for therapy and gamma scintigraphy, respectively. Pretargeted against GD2 and GPA33 in tumors 177 The biological retention kinetics of Lu-DOTA-Bn are as follows: 177 The relatively short beta particle range of Lu, which is well-suited to the long physical half-life of Lu, is theoretically well suited to treating small tumor volumes (optimal tumor diameter for cure probability 0.9 = 2.0 mm

[29] ) while minimizing concomitant radiation damage to normal tissues. 177Lu was the focus of this study's work on DOTA-PRIT treatment of solid tumors. 177 The gamma radiation of Lu is 177 High-resolution single-photon emission computed tomography / computed tomography (SPECT / CT) imaging for pretreatment dosimetry and treatment monitoring is emerging as a quantitative clinical imaging modality for targeted therapy with 111L radiopharmaceuticals [30, 31].

[0176] The objectives of this example were: (1) to generate an anti-HER2-C825 BsAb that would enable proof-of-concept studies with anti-HER2-DOTA-PRIT; (2) to characterize the internalization kinetics of the anti-HER2-C825 BsAb / HER2 antigen complex on the surface of HER2(+) tumor cells; and (3) to demonstrate the internalization kinetics of the anti-HER2-DOTA-PRIT with 177 The objectives of this study were to confirm the highly specific tumor targeting of Lu-DOTA-Bn, and (4) to investigate whether TI was sufficient for safe and effective theranostic application of anti-HER2-DOTA-PRIT in mice bearing established subcutaneous ("sc") human HER2(+) breast cancer xenografts.

[0177] 6.1.2 Materials and Methods 6.1.2.1 Cloning and expression of anti-HER2-C825 BsAb The bispecific binding agent "HER2-C825 BsAb" was prepared in an IgG-scFv

[36] format using the sequences of trastuzumab

[37] and murine C825

[38] . The heavy chain of HER2-C825 BsAb (also sometimes referred to herein as "anti-HER2-C825") comprises the amino acid sequence of SEQ ID NO: 15, and the light chain fusion polypeptide of HER2-C825 BsAb comprises the amino acid sequence of SEQ ID NO: 7. The BsAb (molecular weight: approximately 210 kDa) was produced in CHO cells and purified by protein A affinity chromatography as previously described [3]. The control BsAb, huA33-C825, was made using the same platform as previously described [4]. Biochemical purity analysis for BsAbs was performed using SE-HPLC (column: TSKgel G3000SWxl; running buffer: 400 mM sodium perchlorate pH 6.0; flow rate: 0.5 mL / min) with detection of eluted BsAbs by UV absorbance at 280 nm.

[0178] 6.1.2.2 Surface plasmon resonance studies The Biacore T100 Biosensor, CM5 sensor chip, and related reagents were purchased from GE Healthcare. BSA-(Y)-DOTA-Bn conjugates were prepared as previously described [3]. Antigens were immobilized using an amine coupling kit (GE Healthcare). Purified BsAbs were analyzed and data were fitted to the Bivalent Analyte model using the Biacore T100 Evaluation software as previously described [3].

[0179] 6.1.2.3 Cell lines BT-474 is a ductal carcinoma with epithelial morphology, luminal B subtype, estrogen receptor alpha positive ("ER(+)"), progesterone positive / negative ("PR(+ / -)"), HER2(+), whereas MDA-MB-231 is an adenocarcinoma cell line with epithelial morphology, claudin-low subtype, and a triple-negative immune profile (ER(-), PR(-), and HER2(-))

[39] . Cell lines were obtained from the American Type Culture Collection (Manassas, VA) and routinely tested for mycoplasma negativity using a commercially available kit (Lonza). All cell lines were cultured at 37°C in 5% CO 2 The cells were maintained in a humidified incubator containing 0.1 mM NaCl for less than 10 passages and cultured in Dulbecco's modified Eagle's high glucose / F-12 (DME-HG / F-12) medium supplemented with non-essential amino acids (0.1 mM), 10% heat-inactivated fetal calf serum (FCS), 100 units / mL penicillin, and 100 μg / mL streptomycin.

[0180] 6.1.2.4 Internalization and Intracellular Processing of Anti-HER2-C825 Trastuzumab is known to be internalized upon binding to the surface HER2 antigen [7]. Based on previously described assays [7, 40], the radioactive tracer, 131 I-anti-HER2-C825 was used to evaluate the internalization kinetics of anti-HER2-C825 after binding to BT-474 cell surface HER2 antigen at 37° C. Briefly, cells were cultured at 5.0×10 cells / mL. 5 Cells were seeded at a density of 10x100 in 12-well plates and allowed to adhere overnight. 131Cells were preincubated with I-anti-HER2-C825 (prepared to a specific activity of 132 MBq / mg using the IODOGEN method

[41] , purified to >98% radiochemical purity using SEC, and diluted to 160 ng / mL, 0.8 nM in complete medium) for 1 h. Cells were then washed extensively with complete medium, chilled on ice, and the plates were placed in a 37°C incubator. Radioactivity distribution in the external medium, cell surface, as well as the fraction of radioactivity internalized by cells were determined by quantification in a gamma counter at various time points up to 24 h (n=3–6 per time point). Cell surface antibodies were stripped using an acid wash protocol consisting of treating cells with 2 M urea, 50 mM glycine, 150 mM NaCl, pH 2.4, three times for 5 min each on ice and pooling of the supernatants. 131 To determine whether I radioactivity was bound to the antibody (suggesting passive dissociation or "shedding"

[40] ) or was in the form of low molecular weight metabolites suggesting intracellular metabolism following exocytosis, the external medium was further assayed using trichloroacetic acid (TCA) precipitation (approximately 1 mL of collected medium was mixed with 0.9 mL of 20% w / v TCA). Controls included incubation at 4°C or diluted into medium. 131 Control wells consisted of wells consisting of I-anti-HER2-C825 alone. These control wells were also subjected to TCA precipitation to inhibit internalization and determine the basal catabolic (via degradation) rate of the tracer, respectively. For kinetic analysis, the data were fitted to a curve with a monophasic association using a nonlinear model using the Prism software package, Graphpad Software Inc., San Diego, CA.

[0181] 6.1.2.5 Xenograft models All animal experiments were approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center (New York, NY) and followed regulatory guidelines for proper and humane use of animals in research studies. Female athymic nude mice (6-8 weeks old) were obtained from Harlan / Envigo. Mice were acclimated for a minimum of 1 week. For the BT-474 tumor model, mice were implanted with estrogen (17β-estradiol; 60-day release, 0.72 mg per pellet; Innovative Research of America) by trocar injection 3 days (d) prior to cell inoculation. The MDA-MB-231 xenograft model did not require estrogen supplementation. For the establishment of all tumor xenografts, mice were injected subcutaneously into the lower flank with 5.0 × 10 cells in 200 μL of cell suspension in a 1:1 mixture of medium with reconstituted basement membrane (BD Matrigel™, Collaborative Biomedical Products Inc., Bedford, MA). 6 were inoculated and used within 3-4 weeks. Tumor volumes were estimated using the formula for the volume (V) of an ellipsoid: V = 4 / 3π (length / 2 × width / 2 × height / 2) with dimensions in millimeters (mm).

[0182] 6.1.2.6 Anti-HER2 DOTA-PRIT Reagents and Administration Protocols The three anti-HER2 DOTA-PRIT agents are an anti-HER2-C825 BsAb, an ablative agent, and a radiotherapeutic agent. 177 All reagents were administered intravenously ("iv") via the lateral tail vein. 177 In relation to the injection of Lu-DOTA-Bn, the following time points were observed: for anti-HER2-C825, at t=-28 hours (h), followed by the clearing agent at t=-4 hours, and at t=0 hours. 177The clearing agent (500 kDa dextran-(Y)-DOTA-Bn conjugate; 61 moles of (Y)-DOTA-Bn per mole of dextran) was prepared as previously described

[42] and formulated in saline for injection. 177 Lu-DOTA-Bn was also prepared according to a previously described method [4]. Radioactivity in the samples was measured using a CRC-15R dose calibrator (Capintec, Ramsey, NJ) using the appropriate settings for each isotope.

[0183] 6.1.2.7 Biodistribution Studies to Optimize Anti-HER2 DOTA-PRIT in Vivo To optimize the dose of DOTA-PRIT reagent for efficient tumor targeting in vivo, we used 5.6 MBq (approximately 30 pmol) per mouse. 177 In groups of BT-474 tumor-bearing mice administered Lu-DOTA-Bn radioactivity with the tracer, prior to therapeutic studies, dose titration experiments were performed for the BsAb and the clearing agent. To this end, the aim was to maximize the uptake of radioactivity in the tumor while 177 Specific TI criteria were established that were at least 20:1 for blood and 10:1 for kidney 24 hours after injection of Lu-DOTA-Bn. 177 For biodistribution assays for Lu radioactivity, 177 Groups were sacrificed 24 hours after injection of Lu-DOTA-Bn ("pi"). For biodistribution analysis, mice were 2 After euthanasia by (gas) asphyxiation, tumors and selected organs were harvested, rinsed with water, air-dried, weighed, and subjected to radioassay by gamma scintillation counting (Perkin Elmer Wallac Wizard 3 inch). Count rates were background- and decay-corrected using isotope-specific system calibration factors, converted to activity, normalized to administered activity, and expressed as IA / g% (mean ± SEM).

[0184] 6.1.2.8 Dosimetry calculations For dose calculation, use the optimized DOTA-PRIT protocol + 5.5-6.1 MBq (approximately 30 pmol) 177 Sequential biodistribution studies were performed with Lu-DOTA-Bn in BT-474 tumor-bearing mice (n=24). Groups of HER2(+) BT-474 tumor-bearing mice (n=4-5) were administered PRIT+5.5-6.1MBq (approximately 30pmol) of 177 Lu-DOTA-Bn was administered and the results were analyzed in tumor and selected normal tissues at 1.0 (n=5), 2.5 (n=5), 24 (n=5), 96 (n=5), and 336 (n=4) hours after injection. 177 All tissues were sacrificed for biodistribution analysis for Lu radioactivity (Table 15). For each tissue, the time-activity concentration data, without decay correction, were fitted to one, two or more complex exponential functions as appropriate using Excel and analytically integrated to obtain the cumulative activity concentration per unit irradiated activity (MBq per MBq x time / g). 177 For non-penetrating irradiation, the self-absorbed doses between tumors and selected organs are estimated assuming complete local absorption of Lu beta rays only and ignoring gamma and non-self dose contributions. 177 The Lu equilibrium dose constant (8.49 g × cGy / MBq × time) was used.

[0185] 6.1.2.9 Immunohistochemistry ("IHC") and Autoradiography Experiments For IHC on HER2-expressing tumors, groups of BT-474 tumor-bearing nude mice were intravenously administered 0.25 mg (1.19 nmol) of anti-HER2-C825. 24 hours after injection, animals were sacrificed and tumors were frozen in OCT. Detection of HER2 by IHC was performed at the Molecular Cytology Core Facility of Memorial Sloan Kettering Cancer Center using a Discovery XT processor (Ventana Medical Systems, Roche, Tucson-AZ). Tissue sections were blocked for 30 minutes (min) in 10% normal goat serum, 2% BSA in PBS. Sections were then incubated with rabbit polyclonal HER2 antibody (Enzo; Catalog No: alx-810-227) at a concentration of 5.0ug / ml for 5 hours, followed by incubation with biotinylated goat anti-rabbit IgG (Vector labs; Catalog No: PK6101) at 5.75ug / mL for 1 hour. Detection was performed with Blocker D, Streptavidin- HRP and DAB detection kit (Ventana Medical Systems). All reagents were used according to the manufacturer's instructions. For IHC to determine the distribution of anti-HER2-C825 antibodies, the same procedure was followed, except that the primary antibody step was omitted and biotinylated goat anti-human IgG (Vector; Catalog No: BA3000) antibody was used.

[0186] For ex vivo autoradiography, 177 Animals were sacrificed 24 hours after injection of Lu-DOTA-Bn, and tumors were excised, flash frozen, and embedded in OCT. Serial cryosections 10 μm thick were quickly cut and 177For the determination of the distribution of Lu radioactivity, the sections were exposed to a Phosphor plate overnight at -20°C. Digital autoradiography images with a pixel size of 25 μm were obtained as follows: The tumor sections were exposed to a Phosphor imaging plate (Fujifilm BAS-MS2325; Fujifilm Corporation) overnight at -20°C. Once the exposure was complete, the imaging plate was removed from the cassette and mounted on a Typhoon FLA 7000 (GE Healthcare, USA) to read the images. The image reader creates digital images with a 16-bit grayscale with a pixel size of 25 μm. These images are then converted into files in TIFF image format for subsequent analysis. Finally, H&E staining was performed to visualize the shape of the tumor in the serial sections, and images were collected in a similar manner. Images were manually registered using Photoshop CS6 software (Adobe Systems).

[0187] 6.1.2.10 Theranostic Anti-HER2-DOTA-PRIT Treatment To assess the toxicity and efficacy of anti-HER2-DOTA-PRIT therapy in an animal model for human breast cancer, treatment studies were performed in BT-474 tumor-bearing mice with subcutaneous xenografts of "small" or "medium" size (palpable ~30 mm). 3 Tumors with volumes in the range of 100–400 mm were classified as “small size”; 3 Tumor volumes in the range were classified as "medium size." Treatment groups were monitored for 85-200 days and survivors were subjected to histopathology studies (see below).

[0188] First, anti-HER2 DOTA-PRIT + 55.5MBq 177 Single cycle treatment with Lu-DOTA-Bn (300 pmol) was assessed in groups of mice bearing small subcutaneous xenografts and compared with treated controls (estimated dose to tumor: 22 Gy). The groups were monitored for 85 days after treatment. During this study, planar scintigraphy (using a method previously described [3]) was performed.177 used up to 70 h after injection of Lu-DOTA-Bn. 177 The targeting of Lu radioactivity to the tumor was verified. Groups of mice bearing medium-sized subcutaneous xenografts were then treated with 11.1, 33.3, or 55.5 MBq (60–300 pmol) of HER2 PRIT per mouse in a single-cycle dose-escalation study. 177 The groups were treated with Lu-DOTA-Bn and compared with a control group (estimated tumor absorbed radiation dose: 4.4-22 Gy). These groups were monitored for approximately 200 days after treatment to study tumor recurrence and chronic toxicity of anti-HER2-DOTA-PRIT.

[0189] In the third treatment study, 55.5MBq per mouse per cycle was administered. 177 A three-cycle fractionated DOTA-PRIT regimen with Lu-DOTA-Bn (300 pmol) was assessed in a group of mice bearing medium-sized subcutaneous xenografts (estimated absorbed radiation dose to tumors: 70 Gy). To achieve this, a total IA of 167 MBq per mouse was administered. 177 Lu-DOTA-Bn was administered in equal weekly doses for three cycles, with each of the three DOTA-PRIT reagents administered in each cycle (designated "anti-HER2-DOTA-PRIT"). To verify that efficacy was dependent on the tumor-specific targeting of anti-HER2-C825, a control treatment arm was included in which anti-HER2-C825 was replaced with anti-GPA33-targeting BsAb huA33-C825 [4] (designated "control IgG-DOTA-PRIT"). These groups were monitored for approximately 85 days after treatment. Three mice receiving treatment with anti-HER2-DOTA-PRIT, as well as one mouse receiving control IgG-DOTA-PRIT, were assayed for tumor targeting, 177 Patients were randomly selected for SPECT / CT imaging to quantify tumor uptake of Lu radioactivity.

[0190] 6.1.2.11 Treatment response and toxicity assessment Mice were monitored daily and weighed at least twice weekly for evidence of treatment-induced toxicity. 3 Animals were observed until sacrifice due to excessive tumor burden of 0.01 mm or less if the tumors raised motility concerns. Animals exhibiting weight loss of more than 15% from their initial (pretreatment) weight or weight loss of 20% or more from their pretreatment weight in 1 or 2 days were removed from the group at that time and sacrificed. To further assess for toxicity, randomly selected treated animals were subjected to histopathology evaluation of xenografts, kidneys, bone marrow (sternum, vertebrae, femur, and tibia), liver, and spleen (unless otherwise noted) by a board-certified veterinary pathologist at the Memorial Sloan Kettering Cancer Center Laboratory of Comparative Pathology. Hematology and clinical chemistry panels were also collected. CR was assessed for tumors that were not measurable (<10 mm 3 ) regression. Cure is defined as the absence of histopathological evidence of neoplasia at the tumor inoculation site at necropsy. Breast cancer xenografts developed distant metastases in 33.3% (2 of 6) of treated survivors after 200 days, but none were seen in animals assessed after 85 days (see Tables 21, 24, and 27).

[0191] 6.1.2.12 Statistics All statistics were determined using the Prism software package, Graphpad Software Inc., San Diego, Calif. Statistical comparisons between two separate groups were analyzed by Student's unpaired t-test when appropriate, and the statistical significance level was set at P<0.05.

[0192] 6.1.2.13 Abbreviations BsAb: bispecific antibody; TI: therapeutic index; CR: complete response; d: days; RIT: radioimmunotherapy; PRIT: pretargeted radioimmunotherapy; IA: injected radioactivity; [M]-DOTA-Bn: radiometal complex of the chelating agent S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecanetetraacetic acid; CA: clearing agent; MW: molecular weight; SPECT / CT: single photon emission computed tomography; sc: subcutaneous; h: time; pi: post injection; IA / g%: percent injected radioactivity per gram; SEM: standard error of the mean; IHC: immunohistochemistry; ROI: region of interest; RBC: red blood cells; HGB: hemoglobin; PLT: platelets; iv: intravenous; V: volume; min: minutes. 6.1.3 Results

[0193] 6.1.3.1 In vitro characterization of anti-HER2-C825 BsAb Biochemical purity analysis for anti-HER2-C825 by size-exclusion high performance liquid chromatography ("SE-HPLC") is shown in Figure 1A. SE-HPLC showed a main peak with an approximate molecular weight ("MW") of 210 kilodaltons ("kD") (96.5% by ultraviolet ("UV") analysis), as well as some minor peaks hypothesized to be aggregates that could be removed by gel filtration. The BsAb remained stable by SE-HPLC after multiple freeze / thaw cycles (data not shown). The binding affinity for the antigen, bovine serum albumin (BSA)-(Y)-DOTA-Bn, was measured by Biacore T100. Anti-HER2-C825 was 2.10 × 10 4 M -1 seconds -1 k on , 1.25×10 -4 seconds -1 k off , and a total K of 6.0 nM D (The control BsAb, huA33-C825 (1.90 × 10 4 M -1 seconds -1 k on , 2.20×10 -4 seconds-1 k off , and a total K of 11.6 nM D ; Fig. 1B). Binding to tumor targets was measured by flow cytometry. Anti-HER2-C825 was as efficient as parental trastuzumab in binding to the HER2(+) breast cancer cell line AU565 (Fig. 1C). In summary, anti-HER2-C825 retained high binding capacity to both HER2 and DOTA targets.

[0194] 6.1.3.2 Internalization kinetics and intracellular processing of anti-HER2-C825 To characterize the internalization kinetics and intracellular processing of anti-HER2-C825 by HER2-expressing (HER2(+)) cells, anti-HER2-C825 was immunofluorescently stained with iodine-131 ( 131 The antibody was radioactively iodinated with I) and subjected to in vitro cell binding studies with HER2(+) BT-474 cells at 37°C for up to 24 hours ("h"). 131 I-anti-HER2-C825 was rapidly internalized by BT-474 cells after incubation at 37°C, with 25.6 ± 1.16% of the added radioactivity showing peak internalization at 2 h (Figure 2).

[0195] In addition, when assayed after 4 h, the amount of internalized radioactivity at 4°C was, on average, approximately 15-fold lower than at 37°C (n=6; data not shown). After 2-24 h at 37°C, the internalized radioactivity was observed to decrease to 5.6±0.21%, suggesting that exocytosis had occurred. This was also evident by the rate at which the percentage of catabolized radioactivity (i.e., accumulation of low molecular weight catabolic products) was observed in the extracellular medium, with a K=0.054 and a half-life (R) of 12.82 h. 2 For the surface-bound radioactivity fraction, approximately 50% of the radioactivity was lost during the first 2 h of incubation at 37°C, whereas the remaining radioactivity decayed with a half-life of 8.5 h (R 2After 24 h of incubation (the time interval between injection of BsAb and clearing agent in DOTA-PRIT), 11.1 ± 0.48% of the initially bound anti-HER2-C825 remained on the cell surface.

[0196] In the medium after 0 to 24 hours at 37°C 131 Control experiments to assay the in vitro stability of I-anti-HER2-C825 showed that the percent change in antibody-associated radioactivity between these two time points was -2.1%, indicating that the catabolic radioactivity observed in the extracellular medium fraction was primarily 131 It is suggested that this is due to internalization and exocytosis of I-anti-HER2-C825.

[0197] 6.1.3.3 In vivo optimization of anti-HER2 DOTA-PRIT Optimized doses of BsAb and clearing agent for anti-HER2 DOTA-PRIT in vivo were determined to be 0.25 mg (1.19 nmol) and 62.5 μg (0.125 nmol dextran; 7.63 nmol (Y)-DOTA-Bn), respectively, using groups of BT-474 tumor-bearing mice. A summary of selected biodistribution data from these optimization efforts is presented in Table 10, while the remaining data are presented in FIG. 3 and Table 11. This targeting regimen resulted in tumor uptake of 7.58±0.78 (as percent injected radioactivity per gram IA / g%; mean±standard error of the mean (SEM)) at 24 hours post injection ("pi"), while uptake by the kidney, the tissue showing the greatest uptake in normal, was 0.73±0.05. Of note, in the absence of administration of a clearing agent step (i.e., anti-HER2-C825 administered at [time ("t") = -28 hours later], 177 Lu-DOTA-Bn [t=0 hours]), tumor-induced cytotoxicity 24 hours after injection 177 Uptake and blood distribution of Lu radioactivity 177Both Lu radioactivity uptakes were much higher, 19.94±3.54 IA / g% and 4.95±1.17 IA / g%, respectively, resulting in an unfavorable tumor-to-blood ratio (4.0±1.2) (Table 10). Optimization of the clearing agent dose reduced the mean tumor and blood uptakes by about 60% (from about 20 to 7.6 IA / g%) and about 95% (from about 5 to 0.3 IA / g%), respectively, compared to the control without clearing agent, significantly improving the tumor-to-blood ratio (26.7±9.0), but at the expense of reduced tumor uptake.

[0198] Table 10: Results from separate experiments designed to identify optimal anti-HER2-DOTA-PRIT protocols. 177 Selected biodistribution data 24 hours after injection of Lu-DOTA-Bn (approximately 5.6 MBq, 30 pmol unless otherwise noted) confirming HER2(+) tumor-specific targeting in mice bearing subcutaneous HER2(+) (BT-474) or subcutaneous HER2 negative ("HER2(-)") (MDA-MB-468) tumors. All tumors were in the 100-200 mg range ex vivo. nd = not determined. Int. = intestine. Data are presented as IA / g% (mean ± SEM). [Table 10] a 0.25 mg of anti-HER2-C825, 62.5 μg (25% (w / w)) of depletion agent, and approximately 5.6 MBq 177 Lu-DOTA-Bn b 0.25 mg of anti-HER2-C825, 62.5 μg (25% (w / w)) of clearing agent, and 55.5 MBq of 177Lu-DOTA-Bn c Without injection of BsAb or removal agent, approximately 16.8 MBq 177 Lu-DOTA-Bn (90 pmol) only

[0199] Table 11: In nude mice bearing subcutaneous BT-474 tumors 177 A multi-tissue approach for optimizing removal of anti-HER2 DOTA-PRIT with Lu-DOTA-Bn 177 Ex vivo biodistribution studies of Lu radioactivity. Groups of mice bearing HER2(+) tumors (n=4 per group) were injected with 0.25 mg (1.19 nmol) of anti-HER2-C825 [t=-28 h later] followed by 0.25 mg (1.19 nmol) of anti-HER2-C825 per mouse (mass / mass) administered as 0-70 μg per mouse; 0-0.14 nmol of dextran; 0-8.5 nmol of (Y)-DOTA-Bn per mouse (mass / mass) [t=-4 h later] and 5.5-5.6 MBq (approximately 30 pmol) of 177 Lu-DOTA-Bn [t=0 hours] and sacrificed 24 hours after injection for biodistribution in tumors and normal tissues. 177 Lu radioactivity concentration data are presented as IA / g% (mean ± standard deviation ("SD")). Tumor size is presented as grams (g) (mean ± SD). [Table 11]

[0200] To confirm HER2-specific targeting, a biodistribution study was performed in mice bearing subcutaneous HER2(-) MDA-MB-468. Pretargeted HER2(-) tumors were detected 24 hours after injection. 177 Tumor uptake of Lu radioactivity was approximately 7-fold lower (2.75±0.17 IA / g%) than in HER2(+)BT-474 tumors (19.94±1.7 IA / g%) (Table 10). In addition, 177Injection of Lu-DOTA-Bn alone (i.e., control without PRIT) showed negligible uptake in the tumor (0.07±0.01 IAG / g%) and minimal uptake in the blood (0.002±0.00 IAG / g%) and kidney (0.38±0.01 IAG / g%) 24 hours after injection. 177 It is evident that Lu-DOTA-Bn exhibits minimal systemic retention due to rapid renal clearance (Table 10).

[0201] To determine the time point of peak tumor uptake and perform dosimetric calculations for subsequent therapeutic studies, sequential biodistribution studies were performed using the optimized anti-HER2 DOTA-PRIT regimen at various time points from 1 to 336 hours after injection into BT-474 tumor-bearing mice. As shown in FIG. 4 (see also Tables 10 and 12), the pretargeted 177 Peak tumor uptake of 111Lu radioactivity (approximately 5.6 MBq / approximately 30 pmol) was observed to be 7.58±0.78 at 24 hours after injection, whereas the corresponding radioactivity in blood was 0.28±0.09 (tumor-to-blood ratio: 26.7±9.0) and in kidney was 0.73±0.05 (tumor-to-kidney ratio: 10.4±1.3). 177 Lu radioactivity remained relatively constant, ranging from approximately 5 to 8 IA / g%, from 1 to 24 hours after injection, suggesting that tumor targeting was quite rapid and that clearance of radioactivity from the tumor by the body was relatively slow. Intratumoral radioactivity dropped to 2.29 ± 0.41 IA / g% and 0.32 ± 0.06 IA / g% at 96 and 336 hours after injection, respectively, resulting in an approximate tumor clearance half-life of 38.6 hours (R 2 =0.894).

[0202] Table 12: Biodistribution determined using ex vivo data from nude mice bearing subcutaneous BT-474 tumors at the indicated time points (1-336 hours) post-injection. 177Lu activity data are presented as IA / g% (mean ± standard error of the mean ("SEM")). These data are also shown in Figure 4 and were used in the dosimetry calculations (Table 13). [Table 12]

[0203] To guide treatment studies and predict dose-limiting tissues, absorbed dose estimates were obtained for tumors and tissues assayed by biodistribution. As shown in Table 13, the absorbed dose estimates for blood, tumor, liver, spleen, and kidneys were: 177 The estimated absorbed doses (as cGy / MBq) of Lu-DOTA-Bn were 1.4, 39.9, 3.3, 0.3, and 5.6, respectively. The estimated dose to the kidney was the highest among normal tissues, resulting in a TI of 7. Based on the estimated maximum tolerated doses of 250-2000 cGy for blood (bone marrow) and kidney, respectively

[32] , the estimated maximum tolerated activity is approximately 180 MBq, with blood (bone marrow) as the dose-limiting tissue (TI for blood: 28).

[0204] Table 13: 177 Optimized Lu-DOTA-Bn in nude mice bearing subcutaneous HER2(+)BT-474 tumors based on sequential biodistribution data 1.0–336 h after injection. 177 Estimated absorbed dose for anti-HER2-DOTA-PRIT with Lu-DOTA-Bn. [Table 13]

[0205] Prior to the treatment study, the therapeutic dose of IA 177 A pilot SPECT / CT imaging study was performed with a group of BT-474 tumor-bearing mice receiving the optimized anti-HER2 DOTA-PRIT along with Lu-DOTA-Bn (approximately 56 MBq, 300 pmol). 177Twenty-four hours after injection of Lu-DOTA-Bn into the lower flank, tumors were delineated (Figure 3), suggesting high absolute tumor uptake as well as high tumor-to-blood ratios. Biodistribution performed immediately after imaging showed tumor, blood, and kidney uptake of 5.53±0.27, 0.29±0.05, and 0.56±0.08, respectively (Table 10), suggesting high TIs were maintained in the most critical tissues during treatment.

[0206] Intratumoral BsAb targeting and its relationship to HER2 expression and pretargeted 177 Tumor microdistribution of Lu radioactivity was determined by BsAb or pretargeting 177 BT-474 tumors excised 24 hours after each injection of Lu-DOTA-Bn were probed using immunohistochemistry ("IHC") and autoradiography. These studies demonstrated homogenous uptake of BsAb and pretargeted BsAb in HER2-positive tumor regions. 177 Lu radioactivity revealed a highly uniform and homogenous distribution in the tumor (Fig. 5).

[0207] 6.1.3.4 Treatment research Treatment studies were performed to determine the effect of tumor estimated dose on response over a wide range of starting tumor sizes, and in particular to determine whether it is feasible to achieve a high probability of cure with a dosimetry-based treatment plan that results in an estimated tumor absorbed dose of approximately 70 Gy. A summary of the three anti-HER2 DOTA-PRIT treatment studies is provided in Table 14.

[0208] Table 14: Summary of Anti-HER2 DOTA-PRIT Efficacy / Toxicity Studies [Table 14]

[0209] As shown in Figure 6A, 55.5 MBq 177Single cycle anti-HER2-DOTA-PRIT with Lu-DOTA-Bn (estimated delivered absorbed dose in tumor: 22 Gy) was effective in treating small subcutaneous xenografts, resulting in a high frequency of complete responses ("CR") (5 of 5, 100%), and survivors (4 of 5, 80%) were subjected to necropsy with no recurrence observed in any of the animals after 85 days. Mice receiving anti-HER2-DOTA-PRIT or 55.5 MBq of 177 Planar scintigraphy for the group of mice treated with Lu-DOTA-Bn alone clearly showed pretargeting specific uptake by the tumor 20 hours after injection (persisting in the tumor at least 70 hours after injection) (Figure 7). With the exception of one mouse (1 of 5, 20%) in the BsAb-only group that showed tumor regression to CR approximately 40 days after treatment with no subsequent recurrence, the control group generally showed no tumor response and tumors progressed without CR. At 40 days after treatment, no statistical significance was found between tumor volumes compared to the control group (data not shown). In addition, after 85 days, tumor sizes in the control group progressed on average to 380-3130% of pretreatment volume, while animals treated with BsAb alone showed minimal mean progression.

[0210] 11.1 to 55.5MBq 177 Treatment of mice bearing medium-sized subcutaneous xenografts with one-cycle anti-HER2-DOTA-PRIT with Lu-DOTA-Bn generally did not result in significant tumor responses compared to controls, suggesting that the estimated absorbed dose of 4.4-22 Gy in the tumors was insufficient to result in a high probability of tumor CR (Figure 6B). No statistical significance was found between tumor volumes relative to control or treated groups at 40 days after treatment (data not shown). A small proportion of anti-HER2-DOTA-PRIT-treated animals (4 of 15, 26.7%) showed CR by approximately 75-100 days after treatment, regardless of IA (2 of 5: 11.1 MBq and 1 of 5: 33.3 MBq or 55.5 MBq groups), likely an effect of the trastuzumab-like action of the BsAb.

[0211] We demonstrated that single-cycle treatment is ineffective for treating medium-sized tumors, whereas fractionated delivery of large absorbed doses of radiation in the tumor is highly effective. 177 Treatment with Lu-DOTA-Bn (55.5 MBq per cycle, estimated delivered absorbed dose in tumor: 66 Gy) resulted in 100% CR (8 of 8 cases), whereas tumor progression without CR was observed in treated controls (Figure 8). After 85 days, tumor volumes for the control untreated, BsAb-treated, or control IgG-DOTA-PRIT groups were 134±89%, 396±252%, or 114±155% of pretreatment volumes, respectively.

[0212] Selected mice undergoing fractionated treatment were subjected to SPECT / CT to verify and quantify tumor uptake. As shown in Figure 9A, tumor uptake in cycle 1 of control IgG-DOTA-PRIT or anti-HER2-DOTA-PRIT pretargeted mice was significantly increased in the control IgG-DOTA-PRIT or anti-HER2-DOTA-PRIT pretargeted mice. 177 Imaging of randomly selected mice 24 hours after injection of Lu-DOTA-Bn demonstrated tumor-specific targeting of radioactivity by anti-HER2-C825, whereas control IgG-DOTA-PRIT showed negligible tumor uptake. Three randomly selected mice receiving split anti-HER2-DOTA-PRIT were also imaged sequentially by SPECT / CT imaging. 177 Representative images for one of the animals 24 hours after injection of Lu-DOTA-Bn, cycles 1, 2, and 3, are presented in Figure 9B, while data for two mice are presented in Figure 10. Region of interest (ROI) analysis of tumor areas from the images was also performed, revealing the difference in tumor area in the tumors at each cycle of treatment. 177A graph presenting Lu radioactivity (expressed as MBq per gram of tumor: MBq / g) as a function of time (h) after initiation of treatment in cycle 1 is also presented in FIG. 9B, which shows that the mean uptake by the tumor was 177 The range of approximately 4.3 to 6.1 MBq / g was shown 24 hours after injection of Lu-DOTA-Bn.

[0213] 6.1.3.5 Toxicity In summary, in each of the treatment experiments, 177 No significant mean weight loss was observed compared to controls, including those receiving Lu-DOTA-Bn (Figures 11 and 12). Notably, at 55.5 MBq per cycle, 177 No acute toxicity was observed for the anti-HER2-DOTA-PRIT regimen with Lu-DOTA-Bn (FIG. 12), suggesting that even more aggressive treatment regimens may be safely used. Tables 15, 16, and 17 summarize the criteria by which animals were excluded from each treatment experiment. These included (1) euthanasia required due to excessive weight loss, (2) animals found dead, and (3) euthanasia required due to excessive tumor burden. We observed that in this BT-474 animal model, a small number of animals, regardless of treatment regimen (including untreated controls), showed rapid deterioration (e.g., urinary retention

[33] and endometrial hyperplasia

[34] ), possibly analogous to known effects of treatment with implanted estrogen pellets. Therefore, in the split treatment study, three randomly selected animals (one from each treatment group: untreated, BsAb only, and control IgG-DOTA-PRIT) that exhibited rapid weight loss within 12-22 days of treatment initiation were subjected to complete necropsy to determine the cause of clinical morbidity. Morbidity in three of three clinical animals (100%) was determined to be clearly attributable to adverse effects of estrogen treatment (see Table 17).

[0214] Table 15: Single cycle anti-HER2-DOTA-PRIT + up to 55.5MBq for mice bearing medium-sized tumors 177 For Lu-DOTA-Bn, the number of animals removed from the study based on predefined criteria of weight loss and tumor growth. The study endpoint was approximately 200 days after treatment. Survivors at approximately day 85: 3 of 5 from no treatment, 3 of 5 from BsAb alone, 55.5 MBq 177 Five out of five cases from Lu-DOTA-Bn alone and anti-HER2-DOTA-PRIT+55.5MBq 177 5 of 5 cases from Lu-DOTA-Bn. [Table 15]

[0215] Table 16: Single cycle anti-HER2-DOTA-PRIT + 55.5MBq for mice bearing small tumors 177 For Lu-DOTA-Bn, the number of animals removed from the study based on predefined criteria of weight loss and tumor growth. The study endpoint was approximately 85 days after treatment. Survivors at approximately day 85: 3 of 5 from no treatment, 3 of 5 from BsAb alone, 55.5MBq 177 Five out of five cases from Lu-DOTA-Bn alone and anti-HER2-DOTA-PRIT+55.5MBq 177 5 of 5 cases from Lu-DOTA-Bn. [Table 16]

[0216] Table 17: Number of animals removed from the study based on predefined criteria of weight loss and tumor growth for split anti-HER2-DOTA-PRIT, end of treatment approximately 85 days after study initiation. Survivors at approximately day 85 were 4 of 6 from no treatment, 3 of 5 from BsAb alone, IgG-DOTA-PRIT+ 177 Three of six cases from Lu-DOTA-Bn and anti-HER2-DOTA-PRIT+177 Three animals from the control group, including 8 out of 8 from Lu-DOTA-Bn, showed rapid deterioration in health and significant weight loss within 12-22 days of treatment initiation, and were submitted to necropsy to determine the cause of toxicity. One untreated mouse showed rapid weight loss from pretreatment weight after 12 days, was moribund and submitted to necropsy, while another untreated control mouse was found dead after 18 days. The moribund animal had mild focal unilateral suppurative pyelonephritis with intralesional cocci. One mouse from the BsAb-only group showed rapid weight loss, was moribund and submitted to necropsy after 20 days. This mouse showed neutrophilic pyelitis (bilateral) and pyelonephritis (unilateral) with intralesional bacteria (large cocci). A second mouse from the BsAb-only group was found dead after 35 days. Treatment with control IgG-DOTA-PRIT resulted in three animals experiencing rapid weight loss from pretreatment weight after days 4, 11, and 21. One mouse from this group was moribund and submitted to necropsy after day 22. The mouse was diagnosed with severe hypoplastic (aplastic) anemia due to starvation and hypoplastic growth plates in long bones, agonal bacterial emboli, and agonal hemorrhage. [Table 17]

[0217] 6.1.3.6 Hematology, Clinical Chemistry, and Histopathology Single cycle anti-HER2-DOTA-PRIT + 55.5MBq in mice bearing small subcutaneous tumors 177Approximately 85 days after treatment with Lu-DOTA-Bn, of the five animals (one mouse from the BsAb-only treatment group and four from the anti-HER2-DOTA-PRIT treatment group) with CR that were necropsied, four were cured: one from the BsAb-only group (1 of 3, 33.3%) and three from the anti-HER2-DOTA-PRIT treatment group (3 of 4, 75%). No significant treatment-related morphological changes were observed (Table 18). Hematology and clinical chemistry values ​​(Tables 19 and 20) were generally within normal ranges, with the exception of white blood cells ("WBC"), platelets ("PLT"), and neutrophils ("NEUT"), which were significantly lower (P=0.0137, 0.0195, or 0.017, respectively) in the anti-HER2-DOTA-PRIT treated group (n=4; WBC range: 2.13-2.36 K / μL; PLT range: 229-670 K / μL; NEUT range: 0.47-1.42 K / μL) compared with the untreated group (n=3; WBC range: 3.15-6.01 K / μL; PLT range: 686-946 K / μL; NEUT range: 1.51-2.47 K / μL). In addition, BUN (blood urea nitrogen) was significantly higher (P = 0.0202) in the anti-HER2-DOTA-PRIT treatment group (n = 4; BUN range: 26.0-39.0 mg / dL) compared with the non-treatment group (n = 3; BUN range: 20.0-23.0 mg / dL).

[0218] Table 18: Single cycle anti-HER2-DOTA-PRIT+ 177 Histopathology findings from BT-474 tumor-bearing mice (small tumors) receiving Lu-DOTA-Bn approximately 85 days after treatment. A total of 15 animals were assessed by necropsy. [Table 18] TIFF2025039567000031.tif154168 TIFF2025039567000032.tif204168 TIFF2025039567000033.tif60169

[0219] Table 19: 1-cycle DOTA-PRIT+55.5MBq 177 Hematology values ​​from subcutaneous BT-474 tumor-bearing mice (small tumors) receiving Lu-DOTA-Bn at approximately 85 days. RBC: red blood cells, HGB: hemoglobin, PLT: platelets, WBC: white blood cells, NEUT: neutrophils, LYMPH: lymphocytes, MONO: monocytes. Note: 2 animals: 55.5MBq 177 Mice treated with Lu-DOTA-Bn alone (PLT:57) and mice treated with BsAb alone (PLT:0) showed low values ​​for platelets, but because platelet aggregation was observed, values ​​were excluded from the analysis and considered to be an artifact of blood sampling. [Table 19]

[0220] Table 20: 1 cycle anti-HER2-DOTA-PRIT + 55.5MBq 177 Clinical chemistry values ​​from subcutaneous BT-474 tumor-bearing mice (small tumors) receiving Lu-DOTA-Bn at approximately 85 days. BUN: blood urea nitrogen, CREA: creatinine, ALP: alanine phosphatase, ALT: alanine aminotransferase, and AST: aspartate aminotransferase. [Table 20]

[0221] Anti-HER2-DOTA-PRIT + 11.1 to 55.5 MBq in mice bearing medium-sized subcutaneous tumors 177Approximately 200 days after treatment with Lu-DOTA-Bn, a total of 6 patients were alive, including 2 CRs, one each in the 11.1MBq and 55.5MBq groups. All CRs were determined to be cured. No significant treatment-related histopathology was seen (Table 21). Hematology and clinical chemistry values ​​were within normal ranges (Tables 22 and 23). Due to the lack of survival of untreated controls and the small number of survival of anti-HER2-DOTA-PRIT-treated animals at approximately 200 days, no statistical comparisons were made for hematology and clinical chemistry parameters for this study.

[0222] Table 21: Single cycle anti-HER2-DOTA-PRIT+ 177 Histopathology findings from BT-474 tumor-bearing mice (medium sized tumors) receiving Lu-DOTA-Bn approximately 200 days after treatment. A total of 6 animals were assessed by necropsy. [Table 21] TIFF2025039567000037.tif219166

[0223] Table 22: 1-cycle DOTA-PRIT+ 177 Hematology values ​​from BT-474 tumor-bearing mice (medium-sized tumors) receiving Lu-DOTA-Bn at about 200 days. RBC: red blood cells, HGB: hemoglobin, PLT: platelets, WBC: white blood cells, NEUT: neutrophils, LYMPH: lymphocytes, MONO: monocytes. Normal animals: Harlan, athymic nude, Hsd: Athymic Nude-Foxn 1nu, about 3 months old, female, without estrogen or xenograft implantation. [Table 22]

[0224] Table 23: Clinical chemistry values ​​from BT-474 tumor-bearing mice (medium size tumors) receiving one cycle of Anti-HER2-DOTA-PRIT+177Lu-DOTA-Bn at about 200 days. BUN: Blood urea nitrogen, CREA: Creatinine, ALP: Alanine phosphatase, ALT: Alanine aminotransferase, and AST: Aspartate aminotransferase. Normal animals: Harlan, athymic nude Hsd: Athymic Nude-Foxn 1nu, approximately 3 months old females without estrogen or xenograft implantation. [Table 23]

[0225] In the split treatment study, anti-HER2-DOTA-PRIT resulted in a high frequency of cures (5 of 8, 62.5%) after 85 days of treatment. The other three treated animals (3 of 8, 37.5%) showed microscopic residual disease consisting mainly of soft tissue consolidation with a few scattered neoplastic cells (Table 21). Representative H&E staining of tissue sections taken from the tumor inoculation site for all treatment groups is shown in Figure 13 (see also Table 24).

[0226] Table 24: Split anti-HER2-DOTA-PRIT+ 177 Histopathology findings from BT-474 tumor-bearing mice (medium-sized tumors) receiving Lu-DOTA-Bn at approximately day 85. A total of 18 animals were assessed by necropsy. AC: undifferentiated carcinoma, L: lymphoplasmacytic, N: normal, EM: extramedullary hematopoiesis, HH: hematopoietic hyperplasia, LH: lymphoid hyperplasia, FE: focal diffuse, FL: fibro-osseous lesions, FM: focal myelofibrosis, MH: myeloid hyperplasia, MF: multifocal, MFR: multifocal random, 1: minimal, 2: mild, 3: moderate, 4: marked. [Table 24] TIFF2025039567000041.tif151168 TIFF2025039567000042.tif192168 TIFF2025039567000043.tif81168

[0227] At 85 days after treatment, two anti-HER2-DOTA-PRIT animals (2 of 8) also showed significant hematology and clinical chemistry values ​​(Tables 25 and 26). One mouse showed mild anemia (7.70 M / μL; control range: 8.38-8.88 M / μL) and decreased hemoglobin ("HGB"; 12.7 g / dL; control range: 14.3-14.7 g / dL), as well as increased aspartate aminotransferase (154 U / L; control range: 57-81 U / L) and alanine phosphatase (128 U / L; control range: 64-110 U / L). Another mouse showed mild anemia (7.89 M / µL), decreased HGB (13.1 g / dL), and thrombocytopenia (500 K / µL; control range: 781-953 K / µL), but normal clinical chemistry values. No changes were observed in 6 of 8 animals. In summary, a few hematological parameters showed significantly different ranges in the anti-HER2-DOTA-PRIT group compared to the untreated group, but no significant differences were found for clinical chemistry: RBC and PLT were significantly lower (P<0.05) in the anti-HER2-DOTA-PRIT treated group (n=8; RBC range: 7.70-8.63 M / μL; PLT range: 500-794 K / μL) compared to the untreated group (n=4; RBC range: 8.38-8.88 M / μL; PLT range: 781-953 K / μL). One anti-HER2-DOTA-PRIT treated mouse (1 of 8, 12.5%) had significant bone marrow histopathological changes, which had focal myelofibrosis (severity score: 1; Table 27).

[0228] Table 25: Split control IgG-DOTA-PRIT, or 177Hematology values ​​from BT-474 tumor-bearing mice (medium sized tumors) receiving anti-HER2-DOTA-PRIT with Lu-DOTA-Bn (total activity administered per mouse is 167MBq), approximately 85 days after treatment initiation. RBC: red blood cells, HGB: hemoglobin, PLT: platelets, WBC: white blood cells, NEUT: neutrophils, LYMPH: lymphocytes, MONO: monocytes. [Table 25]

[0229] Table 26: Split control IgG-DOTA-PRIT, or 177 Clinical chemistry values ​​from BT-474 tumor-bearing mice (medium sized tumors) receiving anti-HER2-DOTA-PRIT with Lu-DOTA-Bn (167MBq per mouse) approximately 85 days after treatment initiation. BUN: blood urea nitrogen, CREA: creatinine, ALP: alanine phosphatase, ALT: alanine aminotransferase, and AST: aspartate aminotransferase. [Table 26]

[0230] Table 27: Pathology severity rating for significant shape changes. Split DOTA-PRIT+ 177 Summary of the distribution and incidence of morphological changes in the different groups, approximately 85 days after the start of treatment, from the group receiving Lu-DOTA-Bn. Treatment groups were no treatment, BsAb only (BsAb), control IgG-DOTA-PRIT+ 177 Lu-DOTA-Bn (IgG treatment), and anti-HER2-DOTA-PRIT+ 177 Lu-DOTA-Bn (anti-HER2 treatment) was included. See Table 28 for ratings. [Table 27] TIFF2025039567000047.tif102169

[0231] Table 28: Calculation of organ severity scores listed in Table 27 [Table 28] Histopathological lesion severity grading system: formula for calculating total score: spread score + distribution score; total severity per organ: sum of partial scores for a single histopathological lesion

[0232] 6.1.4 Discussion Safe and curative treatment for advanced human solid tumors is a major unmet need in oncology. These tumors include lung cancer, prostate cancer, breast cancer, pancreatic cancer, glioma, gastrointestinal malignancies; in other words, virtually all major tumors. This fact remains true despite major breakthroughs in targeting drugs, immune checkpoint blockers and tyrosine kinase inhibitors. In particular, the heterogeneity of solid tumors in time and space, de novo or acquired heterogeneity, and heterogeneity at DNA, RNA, and protein levels prevents true cure of advanced solid tumors in clinics with the most advanced molecular targeting drugs.

[0233] Without being bound by any particular theory, it is hypothesized that the DOTA-PRIT platform in this embodiment (see FIG. 14) has good potential to improve the specificity and efficacy of liquid radiation and drugs / toxins in the treatment of solid tumors. The DOTA-PRIT method optimizes RIT to allow targeting of large amounts of radiation to tumors while avoiding normal tissue. DOTA-PRIT targeting human xenograft tumors expressing GD2 and GPA33 has been studied in laboratory animals, and an effective treatment regimen has been developed for DOTA-PRIT targeting GD2 and GPA33, which can achieve 100% CR and high rate of histological cure while limiting toxicity. For GD2-positive tumors, 84.9 cGy / MBq gave a TI of 142 for blood and 23 for kidney, and for GPA33-positive tumors, 65.8 cGy / MBq gave a TI of 73 for blood and 12 for kidney. These two targeting systems have clinical utility for a variety of human solid tumors, including colon cancer, pancreatic cancer, peritoneal pseudomucosoma, and a subset of pancreatic cancer for GPA33, and neuroblastoma, glioma, sarcoma, and small cell lung cancer for GD2.

[0234] HER2 antigen is widely expressed in major human tumors, especially breast, ovarian, and gastrointestinal junction tumors.For this reason, in this embodiment, a DOTA-PRIT variant is developed to target HER2 for radiotherapy.In contrast to GPA33 and GD2, HER2 system is much more unstable in membrane and is also believed to be rapidly internalized when bound to its cognate antibody.Without being bound to a particular theory, it is inferred that the residence time of BsAb bound to tumor surface is crucial for the success of pretargeting RIT, and in this case, an antibody-antigen complex that does not internalize has a clear advantage. Notwithstanding this, and without being bound by any particular theory, the present example states that the TI of anti-HER2-DOTA-PRIT is lower than that of anti-GPA33-DOTA-PRIT or anti-GD2-DOTA-PRIT, but these studies were intended to support the proof-of-concept of anti-HER2-DOTA-PRIT with the hope that they would be useful in defining the kinetic limits of endocytosed antigens relative to PRIT.

[0235] We successfully prepared an anti-HER2-C825 product with sufficient affinity, biochemical purity, and yield for in vivo studies. The human BT-474 breast cancer cell line, a HER2-expressing tumor, was chosen as an animal model system for comparison of DOTA-PRIT treatment response. Using the optimized anti-HER2-DOTA-PRIT, a lower TI was indeed observed than the other two antigen systems: a TI of 28 for blood and 7 for kidney with 39.9 cGy / MBq to tumor. Based on preliminary in vitro internalization experiments, it was expected that TI would be affected, but that at 24 hours, sufficient surface-bound BsAb (11%) would remain to improve TI to a level reasonable for curative RIT.

[0236] For comparison with the other two DOTA-PRIT solid tumor systems, the most straightforward treatment regimen was a three-cycle fractionated regimen for medium-sized tumors in the size range of 100-400 mg. The fractionated treatment method was sufficient to achieve a tumoricidal absorbed radiation dose of approximately 70 Gy to the tumor. 177 A three-cycle regimen, which was also used for targeting GPA33 and GD2, was chosen because it was reasoned to be ideal for safe administration of Lu-DOTA-Bn radioactivity [1]. Control of HER2(+)BT-474 tumor growth, including cure, was confirmed with optimized administration. Three-cycle fractionated anti-HER2-DOTA-PRIT (total IA per mouse: 167MBq) was found to be well tolerated and highly effective, and animals showed no acute toxicity. The total dose of radiation to the tumor was approximately 70Gy, resulting in a high frequency of CR (8 out of 8, 100%) and complete eradication (5 out of 8, 62.5%) to cure of the tumor, as well as 37.5% microscopic residual disease (3 out of 8) after 85 days. No CRs recurred within 85 days. Using sequential SPECT / CT imaging, it was verified that efficient tumor targeting was achieved at each treatment cycle (Figure 18). Surviving animals in the control group showed tumor progression to 207±201% of pre-treatment volume after approximately 85 days, with no CRs or cures.

[0237] Regarding tumor response, a size-dependent effect was observed in efficacy studies, with small tumor size requiring one cycle of anti-HER2-DOTA-PRIT + 55.5MBq to achieve a high incidence of CR (5 of 5, 100%) and cure (3 of 4, 75% at 85 days). 177 However, a single dose treatment for medium-sized tumors was administered. 177Regardless of the dose of Lu-DOTA-Bn (11.1-55.5MBq), low frequencies of CR (4 of 15, 26.7%) or cures (2 of 6 evaluable animals at 200 days) were observed. In addition, a single dose of medium-sized tumors was observed to result in a final recurrent CR (1 of 3 evaluable, 33.3%) within 100 days after treatment. As described above, 3 cycles of treatment with 55.5MBq were highly effective against medium-sized tumors, resulting in a high frequency of CRs and cures.

[0238] In summary, this example involves the development of a high-TI theranostic approach for PRIT against HER2(+) disease. Curative treatment for HER2-expressing tumors is a major unmet need. There are limited treatment options for patients with HER2-overexpressing cancers, especially those that are resistant to trastuzumab and kinase inhibitors

[35] . The success of the HER2 antibody-antigen system is a reference point for comparison of other internalized antigen targets, and this example is used as a guide for further adaptation of DOTA-PRIT. This example suggests that high-TI targeting is feasible, with the potential for cure, while sparing normal tissue.

[0239] 6.1.5 References TIFF2025039567000049.tif244160 TIFF2025039567000050.tif245146 TIFF2025039567000051.tif244147 TIFF2025039567000052.tif23165

[0240] Example 2 6.2 Alteration of Tumor Targeting Interval with Anti-HER2-C825 and Subsequent Pretargeted 177 Effect of Lu-DOTA-Bn hapten on intratumoral uptake Anti-HER2-C825 (BsAb; described in section 6.1 above) tumor targeting and pharmacokinetics in mice (n=4) bearing HER2(+)BT-474 tumors were assessed using sequential PET imaging (Figure 15). Tumor uptake was 8.87±2.50, 10.16±4.08, 7.96±2.37, and 5.62±1.44 (as mean±SD) at 4, 12, 24, and 48 hours post-injection (pi), respectively. The approximate half-life in the tumor after peak uptake 12 hours post-injection is 36 hours. Blood radioactivity was 11.3±0.79, 6.57±0.44, 3.98±0.25, and 2.26±0.23 (as mean±SD) at 4, 12, 24, and 48 hours post-injection, respectively. The serum half-life was determined to be 8.1 hours (nonlinear fitting, monophasic decay; R2=0.9837).

[0241] Table 29: Presents a statistical comparison of uptake values ​​in tumor and blood observed at 24 hours post-injection with other imaging time points (4, 12, and 48 hours post-injection). [Table 29] Initially, a 24 hour interval between the injection of BsAb and the injection of the clearing agent was utilized. 131 According to the in vitro binding assay with I-anti-HER2-C825 and BT-474 cells, 131 The internalized fraction of I-anti-HER2-C825 was broad, with 89%, 46%, 41%, 14%, and 11% of the surface remaining at t = 1 min, 2 h, 4 h, 19 h, and 24 h, respectively (Figure 2). Two separate in vivo anti-HER2-DOTA-PRIT experiments were performed to assess the time interval between BsAb, in tissues, 177 The effect on Lu-DOTA-Bn uptake (24 hours after injection) (mean ± SD) was studied, see Tables 30 and 31.

[0242] Table 30 [Table 30]

[0243] Comparison of Group 1 vs. Group 2 (Table 30): When comparing Group 1 with Group 2, there was no significant difference between uptake by blood, tumor, or kidney (P=0.13, 0.49, and 0.23, respectively), indicating that blood concentrations of BsAb were approximately three-fold higher (11.3±0.79 and 3.98±0.25; P=1.05×10 -6 ; for 4 and 24 hours, respectively), suggesting that the clearing agents have comparable efficacy in vivo. Tumor targeting also did not decrease with decreasing time interval.

[0244] Comparison of Group 1 vs. Group 3 (Table 30): Comparing Group 1 with Group 3, there was a significant difference between blood or tumor uptake (P=0.019 and 0.047, respectively), suggesting that increasing the dose of the clearing agent is effective at decreasing blood uptake (mean: 0.06-0.03) but at the expense of decreasing tumor uptake (mean: 11.62-4.14). Tumor:blood and tumor:kidney ratios were similar for Groups 1 and 3. Group 4 (Table 30) shows that ablative agents are clearly required to improve tumor:blood and tumor:kidney ratios. Dose escalation of ablative agents between 0 and 62.5 μg is suggested to optimize anti-HER2-DOTA-PRIT with a 4 hour BsAb targeting interval.

[0245] In the second set of experiments (Table 31), the time intervals for BsAb as well as the removal agent and 177 The time interval with Lu-DOTA-Bn was also varied. 177 All animals were sacrificed 24 hours after injection of Lu-DOTA-Bn.

[0246] Table 31 [Table 31]

[0247] Comparison of Group 1 vs. Group 2 (Table 31): No significant differences were found between Group 1 and Group 2 for blood, but significant differences were found for tumor (P=0.002). Comparison of Group 1 vs. Group 3 (Table 31): There was a significant difference between Group 1 and Group 3 for blood (P=0.01), but no significant difference for tumor (P=0.09). Comparison of Group 1 vs. Group 4 (Table 31): No significant differences were found between Group 1 and Group 4 for blood, but significant differences were found for tumor (P=0.005).

[0248] These data confirm that in vivo uptake by HER2(+) tumor cells did not vary significantly between 4 and 24 hours (see Table 30). Thus, these data essentially describe the balanced tumor-blood (plasma) kinetics of tumor-targeting BsAb and provide a rationale for same-day PRIT (i.e., administration of BsAb, clearing agent, and radiolabeled DOTA on the same day). Furthermore, when comparing pretargeting with a 24-hour interval with pretargeting with a 4-hour interval with the same dose of clearing agent (62.5 μg), 124 Although blood concentrations of I-bispecific antibody are estimated to be much higher at 4 hours than at 24 hours, tumor and blood uptake are comparable (compare Groups 1 and 2 in Table 30).

[0249] Moreover, even using a 2-hour time interval, effective pretargeting was still demonstrated (see group 4 in table 31). It is surprising that the ability to perform same-day pretargeting and still obtain in vivo pharmacokinetics with large tumor-to-tissue ratios (e.g., with targeting with relatively large bispecific antibodies that are expected to be slowly taken up by tumors, the reference ratios for internalized targets are >50:1 for tumor:blood and >10:1 for tumor:kidney).

[0250] Example 3 6.3 Extension to human procedures Previously, it was determined that the extent of uptake of a radioactive antibody targeting the A33 antigen (an antigen that is anchored in the membrane and does not internalize) in human colon cancer was proportional to the amount of A33 receptors on the tumor (see, e.g., O'Donoghue JA, et al., 124I-huA33 antibody uptake is driven by A33 antigen concentration in tissues from colorectal cancer patients imaged by immuno-PET. J Nucl Med. 2011 Dec;52(12):1878-85). This observation is consistent with the equilibrium kinetics of antibody uptake in vivo, and the law of mass action is: 124 This means that it can be used to quantitatively characterize antibody-antigen interactions (in this case, huA33 antibody-GPA33 antigen interaction) in tumors and normal tissues after administration of I-huA33 (see O'Donoghue JA, et al. 124I-huA33 antibody uptake is driven by A33 antigen concentration in tissues from colorectal cancer patients imaged by immuno-PET. J Nucl Med. 2011 Dec;52(12):1878-85). It is not obvious that this approach can be used for internalized antigen-antibody systems. Therefore, in the laboratory, we explored the application of the law of mass action to the case of HER2 antigen on BT474 xenografts using HER2-C825 BsAb.

[0251] From the law of balanced mass action, the antibody concentration [L] and tumor receptor concentration [R] are Formula 1:K a = [LR] / [L]×[R] and solving for the fraction of tumor receptors bound to the antibody, [LR] / [R], gives Formula 2:K a ×[L]=[LR] / [R] It is believed to be the case. [L] is the K for antibodies measured in plasma. a Since is known, the saturation [LR] / [R] can be calculated. At a final dose of 250 micrograms (μg) per mouse, 124 After injection of I-anti-HER2-C825 into mice, it was found that there was an initial uptake into the tumor and clearance from the blood (Figure 15). At a certain time point (T), the system reached equilibrium and the signals in the tumor and plasma decreased in parallel, and at this time point after injection, the plasma concentration was 5% of the injected dose per mL, which translates to 0.060 nM per mL or 60 nM per L. 10 9 K of L / M a Using x 60nM / L and formula 2, at a dose of 250μg / mL, [LR] / [R]=60. At a dose of 5% per mL of blood, 250μg is 1.2nM of anti-HER2-C825, which is about 60nM / L at equilibrium. To scale up to humans, an estimate of the total volume of blood of about 5000mL is used below.

[0252] Without being bound by any theory, it is desirable to have enough antibody to approach saturation (saturation) of the binding capacity of the HER2 receptor in humans, since near saturation of the receptor has the greatest amount of hapten binding capacity at the tumor site. Without being bound by any theory, this is because the maximum amount of hapten binding capacity within the tumor is reached. 177 The clearing agent will result in uptake of Lu-DOTA-Bn. The clearing agent is used to remove all extraneous antibodies from the blood and other tissues, thereby resulting in a high therapeutic index.

[0253] Scaled up to human blood volume (approximately 5000 mL) and considering that mouse blood volume is approximately 2 mL, an approximately 2500-fold increase in the total injected dose (5000 mL blood in human / 2 mL blood in mouse), i.e., 2500 x 250 micrograms, would result in an equivalent concentration at equilibrium in human plasma. This would result in approximately 625 mg of HER2-C825 being injected to achieve [RL] / [R]=60 (98.4%). If half this dose or 312.5 mg were injected, [RL] / [R]=30 (97%), and at a dose of 156.5 mg, [RL] / [R]=15 (94%). Without being bound to a particular theory, all of these doses would be in close proximity to the full or capable of binding to the receptor, so there would be little change in bifunctional antibody uptake at the tumor site. These are doses that are very close to those commonly administered, e.g., doses of Herceptin that have been empirically determined to be near optimal for treatment (see Herceptin package insert; e.g., for gastric cancer, e.g., 8 mg / kg intravenously over 90 minutes as an initial infusion, or 560 mg for a 70 kg adult).

[0254] This general finding was confirmed by the following experiment: Antibody was injected, then 24 hours later, 177 In mice injected with Lu-DOTA-Bn, tumors were most effectively targeted at doses >100 micrograms (Figures 16 and 17). [R] is not precisely known, but is approximately equal to 10 cells per gram. 8 Using this number to calculate approximately 210,770 HER2 antigen sites per cell (assuming that, on average, one site per antibody is labeled), the graph in FIG. 17 can provide a crude estimate of [R], the "bmax", which is approximately 35 pmol per gram.

[0255] Figure 2 shows the time course of bifunctional HER2-C825 antibody uptake into BT474 tumors. By 4 hours after injection, uptake is near maximal, suggesting that it may not be necessary to wait long to initiate the process of antibody clearance by the clearing agent and to inject Lu177-DOTA-Bn to target the bifunctional antibody to the tumor. Without being bound to a particular theory, this time may be optimal for clinical applications, since all of the reagents can be easily administered in a single day, and the TI for tumor:blood and tumor:liver, as well as the absolute uptake in the tumor, remain within 80% of the maximal levels observed in this experiment at 10 hours (see Table 32). This uptake level results in cure in mice, and the TI is protective for the two most important organs, blood and kidney. In this case, the HER2-binding protein captured by the bispecific binding agent bound to the membrane, 177 The Lu radiometal is taken up and antibody-antigen internalization is likely to be useful since the radiometal is trapped withi...

Claims

1. 1. A method of treating cancer in a subject in need thereof, comprising: (a) administering to a subject a therapeutically effective amount of a bispecific binding agent, the bispecific binding agent comprising a first molecule covalently linked (optionally via a linker) to a second molecule, the first molecule comprising a first binding site, the first binding site specifically binding to a first target, the first target being a cancer antigen expressed by the cancer, and the second molecule comprising a second binding site, the second binding site specifically binding to a second target, the second target being not a cancer antigen; (b) administering to the subject a therapeutically effective amount of a clearing agent within 12 hours of step (a) administering to the subject a therapeutically effective amount of a bispecific binding agent, wherein the clearing agent binds to the second binding site and functions to reduce bispecific binding agents circulating in the blood of the subject; and (c) after step (b) of administering a therapeutically effective amount of a clearing agent to the subject, administering a therapeutically effective amount of a radiotherapeutic agent to the subject, wherein the radiotherapeutic agent comprises: (i) a second target bound to a metal radionuclide, the second target being a metal chelator; or (ii) a second target bound to a metal chelator, the metal chelator being bound to a metal radionuclide.

2. 2. The method of claim 1, wherein step (b) of administering a therapeutically effective amount of a clearing agent to the subject occurs within 10 hours, within 8 hours, within 6 hours, within 4 hours, within 2 hours, 1-12 hours, 2-12 hours, 1-2 hours, 1-3 hours, 1-4 hours, 2-6 hours, 2-8 hours, 2-10 hours, 4-6 hours, 4-8 hours, 4-10 hours, 2 hours, or 4 hours after step (a) of administering a therapeutically effective amount of a bispecific binding agent to the subject.

3. 2. The method of claim 1, wherein step (b) of administering a therapeutically effective amount of a clearing agent to the subject occurs about 2 hours, about 4 hours, about 6 hours, about 8 hours, or about 10 hours after step (a) of administering a therapeutically effective amount of a bispecific binding agent to the subject.

4. 4. The method of any one of claims 1 to 3, wherein step (c) of administering a therapeutically effective amount of a radiotherapeutic agent to the subject occurs 1 to 2 hours, 1 to 3 hours, 1 to 4 hours, 2 to 6 hours, 2 to 8 hours, 2 to 10 hours, 4 to 6 hours, 4 to 8 hours, 4 to 10 hours, within 1 hour, within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours after step (b) of administering a therapeutically effective amount of a clearing agent to the subject.

5. 4. The method of any one of claims 1 to 3, wherein step (c) of administering to the subject a therapeutically effective amount of a radiotherapeutic agent occurs about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, or about 6 hours after step (b) of administering to the subject a therapeutically effective amount of a clearing agent.

6. 4. The method of any one of claims 1 to 3, wherein step (c) of administering to the subject a therapeutically effective amount of a radiotherapeutic agent occurs about 1 hour after step (b) of administering to the subject a therapeutically effective amount of a clearing agent.

7. 4. The method of any one of claims 1 to 3, wherein step (c) of administering to the subject a therapeutically effective amount of a radiotherapeutic agent occurs within 16 hours of step (a) of administering to the subject a therapeutically effective amount of a bispecific binding agent.

8. 8. The method of any one of claims 1 to 7, wherein the clearing agent comprises a second target that is bound to a molecule that is cleared from the circulation primarily by the liver, the fixed phagocyte system, the spleen, or the bone marrow.

9. The method of any one of claims 1 to 8, wherein the clearing agent comprises a 500 kDa aminodextran conjugated to a second target.

10. The method of any one of claims 1 to 9, wherein the clearing agent comprises about 100 to 150 second target molecules per 500 kDa of aminodextran.

11. 11. The method of any one of claims 1 to 10, wherein the metal chelator is selected from the group consisting of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or a derivative thereof, DOTA-Bn or a derivative thereof, p-aminobenzyl-DOTA or a derivative thereof, diethylenetriaminepentaacetic acid (DTPA) or a derivative thereof, and DOTA-deferoxamine.

12. The method according to any one of claims 1 to 10, wherein the metal chelating agent is DOTA or a derivative thereof.

13. The method according to any one of claims 1 to 10, wherein the metal chelator is DOTA-Bn or a derivative thereof.

14. 14. The method of any one of claims 1 to 13, wherein the metal of the metallic radionuclide is selected from the group consisting of lutetium (Lu), actinium (Ac), astatine (At), bismuth (Bi), cerium (Ce), copper (Cu), dysprosium (Dy), erbium (Er), europium (Eu), gadolinium (Gd), gallium (Ga), holmium (Ho), iodine (I), indium (In), lanthanum (La), lead (Pb), neodymium (Nd), praseodymium (Pr), promethium (Pm), rhenium (Re), samarium (Sm), scandium (Sc), terbium (Tb), thulium (Tm), ytterbium (Yb), yttrium (Y), and zirconium (Zr).

15. Metallic radionuclides, 211 At, 225 A c, 227 A c, 212 Bi, 213 Bi, 64 Cu, 67 Cu, 67 G.A. 68 G.A. 157 Gd, 166 Ho, 124 I, 125 I, 131 I, 111 In, 177 Lu, 212 Pb, 186 Re, 188 Re, 47 Sc, 153 Sm, 166 Tb, 89 Zr, 86 Y. 88 Y, and 90 The method of claim 14, wherein said compound is selected from the group consisting of:

16. Metallic radionuclides 177 The method of claim 14, wherein said compound is Lu.

17. The method of any one of claims 1 to 16, wherein the bispecific binding agent comprises an Fc domain.

18. 18. The method of any one of claims 1 to 17, wherein the bispecific binding agent is at least 100 kDa, at least 150 kDa, at least 200 kDa, at least 250 kDa, between 100 and 300 kDa, between 150 and 300 kDa, or between 200 and 250 kDa.

19. 19. The method of any one of claims 1 to 18, wherein the bispecific binding agent is at least 100 kDa and step (b) of administering to the subject a therapeutically effective amount of a clearing agent occurs within 4 hours of step (a) of administering to the subject a therapeutically effective amount of the bispecific binding agent.

20. The method of any one of claims 1 to 19, wherein the first molecule comprises an antibody or antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof comprising a first binding site.

21. 21. The method of claim 20, wherein the antibody is an immunoglobulin.

22. 22. The method of any one of claims 1 to 21, wherein the radiotherapeutic agent comprises (i) a second target bound to a metallic radionuclide, said second target being a metal chelator.

23. The method of any one of claims 1 to 22, wherein the second molecule comprises an antibody or antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof comprising a second binding site.

24. The method of any one of claims 1 to 22, wherein the second molecule comprises a single chain variable fragment (scFv), said scFv comprising a second binding site.

25. 22. The method of claim 21 , wherein the immunoglobulin comprises two identical heavy chains and two identical light chains, a first light chain and a second light chain, the first light chain being fused (optionally via a first peptide linker) to a second molecule, a first scFv comprising a second binding site, to create a first light chain fusion polypeptide, and the second light chain being fused (optionally via a second peptide linker) to a second scFv to create a second light chain fusion polypeptide, and the first light chain fusion polypeptide and the second light chain fusion polypeptide are identical.

26. 26. The method of claim 25, wherein a first light chain fusion polypeptide comprises a first peptide linker and said second light chain fusion polypeptide comprises a second peptide linker, and the sequences of said first peptide linker and said second peptide linker are 5-30, 5-25, 5-15, 10-30, 10-20, 10-15, 15-30, or 15-25 amino acids in length.

27. 26. The method of claim 25, wherein the first light chain fusion polypeptide comprises a first peptide linker and the second light chain fusion polypeptide comprises a second peptide linker, and the sequences of the first peptide linker and the second peptide linker are 7-32, 7-27, 7-17, 12-32, 12-22, 12-17, 17-32, or 17-27 amino acids in length.

28. 26. The method of claim 25, wherein the first light chain fusion polypeptide comprises a first peptide linker and the second light chain fusion polypeptide comprises a second peptide linker, and the sequences of the first peptide linker and the second peptide linker are any of SEQ ID NOs: 23 and 25-30.

29. 26. The method of claim 25, wherein the first light chain fusion polypeptide comprises a first peptide linker and the second light chain fusion polypeptide comprises a second peptide linker, and the sequences of the first peptide linker and the second peptide linker are any of SEQ ID NOs: 51-56.

30. In the first scFv, the heavy chain variable (V H ) domain and the light chain variable (V L 30. The method of any one of claims 25 to 29, wherein the sequence of the intra-scFv peptide linker between the domains is 5 to 30, 5 to 25, 5 to 15, 10 to 30, 10 to 20, 10 to 15, 15 to 30, or 15 to 25 amino acids in length.

31. Within the first scFv, V H Domain and V L The method according to any one of claims 25 to 30, wherein the sequence of the scFv intra-peptide linker between the domains is any one of SEQ ID NOs: 23 and 25 to 30.

32. Within the first scFv, V H Domain and V L The method according to any one of claims 25 to 30, wherein the sequence of the scFv intra-peptide linker between the domains is SEQ ID NO:

27.

33. Within the first scFv, V H Domain and V L The method according to any one of claims 25 to 30, wherein the sequence of the scFv intra-peptide linker between the domains is SEQ ID NO:

30.

34. V in the first scFv H The sequence of the domain comprises all three of the complementarity determining regions (CDRs) of SEQ ID NO: 21, and the V L The method of any one of claims 25 to 33, wherein the sequence of the domain comprises all three of the CDRs of SEQ ID NO:

22.

35. V in the first scFv H The method according to any one of claims 25 to 34, wherein the sequence of the domain is SEQ ID NO:

21.

36. V in the first scFv L The method according to any one of claims 25 to 35, wherein the sequence of the domain is SEQ ID NO:

22.

37. The method of any one of claims 25 to 34, wherein the sequence of the first scFv comprises any of SEQ ID NOs: 31 to 36.

38. The method of any one of claims 25 to 34, wherein the sequence of the first scFv comprises any of SEQ ID NOs: 39 to 44.

39. The method of any one of claims 25 to 34, wherein the sequence of the first scFv comprises SEQ ID NO:

33.

40. The method of any one of claims 25 to 34, wherein the sequence of the first scFv comprises SEQ ID NO:

44.

41. V in the first scFv H The method of any one of claims 25 to 34, wherein the sequence of the domain comprises a humanized form of SEQ ID NO:

21.

42. 42. The method of claim 41 , wherein the humanized form of SEQ ID NO:21 is SEQ ID NO:

37.

43. V in the first scFv L The method of any one of claims 25 to 34 and 41, wherein the sequence of the domain comprises a humanized form of SEQ ID NO:

22.

44. 44. The method of claim 43, wherein the humanized form of SEQ ID NO:22 is SEQ ID NO:

38.

45. 22. The method of any one of claims 1 to 21, wherein the radiotherapeutic agent comprises (ii) a second target bound to a metal chelator, said metal chelator being bound to a metallic radionuclide.

46. 46. ​​The method of claim 45, wherein the second molecule comprises streptavidin and the second target comprises biotin.

47. 46. ​​The method of claim 45, wherein the second target comprises histamine succinylglycine.

48. Cancer antigens include HER2, CA6, CD138, CD19, CD22, CD27L, CD30, CD33, CD37, CD56, CD66e, CD70, CD74, CD79b, EGFR, EGFRvIII, FRα, GCC, GPNMB, mesothelin, MUC16, NaPi2b, nectin4, PSMA, STEAP1, Trop-2, 5T4, AGS-16, alpha v beta 6, CA19.9, CAIX, CD174, CD180, CD227, CD326, CD79a, CEACAM5, CRIPTO, DLL3, DS6, endothelin B receptor, FAP, GD2, mesothelin, and PMEL. 17, SLC44A4, TENB2, TIM-1, CD98, endosialin / CD248 / TEM1, fibronectin extracellular domain B, LIV-1, mucin 1, p-cadherin, peritosin, Fyn, SLTRK6, tenascin-c, VEGFR2, PRLR, CD20, CD72, fibronectin, GPA33, splice isoform of tenascin-C, TAG-72, B7-H3, L1CAM, Lewis Y, and polysialic acid.

49. 49. The method of claim 48, wherein the cancer antigen is HER2.

50. The method according to any one of claims 1 to 47, wherein the cancer antigen is an antigen that is internalized into cancer cells.

51. Cancer antigens internalized into cancer cells include HER2, CA6, CD138, CD19, CD22, CD27L, CD30, CD33, CD37, CD56, CD66e, CD70, CD74, CD79b, EGFR, EGFRvIII, FRα, GCC, GPNMB, mesothelin, MUC16, NaPi2b, nectin4, PSMA, STEAP1, Trop-2, 5T4, AGS-16, alpha v beta 6, CA19.9, CAIX, CD174, CD180, CD227, CD326, CD79a, CEACAM5, CRIPTO, DLL3, DS6, endothelin B receptor, FAP, GD2, mesothelin, and PMEL. 17, SLC44A4, TENB2, TIM-1, CD98, endosialin / CD248 / TEM1, fibronectin extracellular domain B, LIV-1, mucin 1, p-cadherin, peritosin, Fyn, SLTRK6, tenascin c, VEGFR2, and PRLR.

52. 52. The method of claim 51, wherein the cancer antigen that is internalized into the cancer cell is HER2.

53. 45. The method of any one of claims 21 and 25-44, wherein the cancer antigen is HER2, the heavy chain in the immunoglobulin comprises all three heavy chain CDRs of SEQ ID NO: 20, and the light chain in the immunoglobulin comprises all three light chain CDRs of SEQ ID NO:

19.

54. The cancer antigen is HER2, and the V H The method of any one of claims 21, 25-44, and 53, comprising the sequence of the domain SEQ ID NO:

20.

55. The cancer antigen is HER2, and the V L The method of any one of claims 21, 25-44, 53, and 54, wherein the sequence of the domain comprises SEQ ID NO:

19.

56. 56. The method of any one of claims 21, 25-44, and 53-55, wherein the cancer antigen is HER2 and the sequence of the heavy chain in the immunoglobulin comprises any of SEQ ID NOs: 14-17.

57. 56. The method of any one of claims 21, 25-44, and 53-55, wherein the cancer antigen is HER2 and the sequence of the heavy chain in the immunoglobulin comprises SEQ ID NO:

15.

58. 56. The method of any one of claims 21, 25-44, and 53-55, wherein the cancer antigen is HER2 and the sequence of the heavy chain in the immunoglobulin comprises SEQ ID NO:

16.

59. 58. The method of any one of claims 21, 25-44, and 51-57, wherein the cancer antigen is HER2 and the sequence of the light chain in the immunoglobulin comprises SEQ ID NO:

11.

60. 40. The method of any one of claims 25-37 and 39, wherein the cancer antigen is HER2 and the sequence of the first light chain fusion polypeptide is any of SEQ ID NOs: 5-10.

61. 40. The method of any one of claims 25 to 37 and 39, wherein the cancer antigen is HER2 and the sequence of the first light chain fusion polypeptide is SEQ ID NO:

7.

62. 40. The method of any one of claims 25-37 and 39, wherein the cancer antigen is HER2, the sequence of the first light chain fusion polypeptide is any of SEQ ID NOs: 5-10, and the sequence of the heavy chain is any of SEQ ID NOs: 14-17.

63. 40. The method of any one of claims 25 to 37 and 39, wherein the cancer antigen is HER2, the sequence of the first light chain fusion polypeptide is SEQ ID NO:7 and the sequence of the heavy chain is SEQ ID NO:

15.

64. 26. The method of claim 25, wherein the cancer antigen is HER2 and the sequence of the first light chain fusion polypeptide is any of SEQ ID NOs: 45-50.

65. 65. The method of claim 64, wherein the cancer antigen is HER2 and the sequence of the first light chain fusion polypeptide is SEQ ID NO:

50.

66. 26. The method of claim 25, wherein the cancer antigen is HER2, the sequence of the first light chain fusion polypeptide is any of SEQ ID NOs: 45-50, and the sequence of the heavy chain is any of SEQ ID NOs: 14-17.

67. 67. The method of claim 66, wherein the cancer antigen is HER2, the sequence of the first light chain fusion polypeptide is SEQ ID NO:50, and the sequence of the heavy chain is SEQ ID NO:

16.

68. The method according to any one of claims 1 to 47, wherein the cancer antigen is an antigen that is not internalized into cancer cells.

69. 69. The method of claim 68, wherein the cancer antigen that is not internalized into cancer cells is selected from the group consisting of CD20, CD72, fibronectin, GPA33, a splice isoform of tenascin-C, and TAG-72.

70. 70. The method of any one of claims 1-69, wherein the therapeutically effective amount of the bispecific binding agent is between 100 mg and 700 mg, between 200 mg and 600 mg, between 200 mg and 500 mg, between 300 mg and 400 mg, about 300 mg, about 450 mg, about 500 mg, about 600 mg, or about 625 mg, and wherein the subject is a human.

71. 70. The method of any one of claims 1-69, wherein the therapeutically effective amount of the bispecific binding agent is 250 mg to 700 mg, 300 mg to 600 mg, or 400 mg to 500 mg, and the subject is a human.

72. 1. A method of treating cancer in a subject in need thereof, comprising: (a) administering to a subject a first therapeutically effective amount of a bispecific binding agent, wherein the first therapeutically effective amount of the bispecific binding agent is between 100 mg and 700 mg, between 200 mg and 600 mg, between 200 mg and 500 mg, between 300 mg and 400 mg, about 300 mg, about 450 mg, about 500 mg, about 600 mg, or about 625 mg, and wherein the bispecific binding agent comprises a first molecule covalently attached (optionally via a linker) to a second molecule, and wherein the cancer expresses HER2, the first molecule comprises an antibody or antigen-binding fragment thereof, or an scFv, the antibody or antigen-binding fragment thereof, or scFv (i) binds to HER2 on the cancer, and (ii) comprises all three of the heavy chain CDRs of SEQ ID NO:20 and all three of the light chain CDRs of SEQ ID NO:19, the second molecule comprises a second binding site, the second binding site specifically binds to a second target, and the second target is not a cancer antigen; (b) after step (a) of administering to the subject a therapeutically effective amount of a bispecific binding agent, administering to the subject a therapeutically effective amount of a clearing agent, said clearing agent binding to said second binding site and functioning to reduce bispecific binding agents circulating in the blood of the subject; and (c) after step (b) of administering to the subject a therapeutically effective amount of a clearing agent, administering to the subject a therapeutically effective amount of a radiotherapeutic agent, the radiotherapeutic agent (i) comprising a second target bound to a metallic radionuclide, the second target being a metal chelator; or (ii) comprising a second target bound to a metal chelator, the metal chelator being bound to a metallic radionuclide. Including, The subject is a human The method.

73. 73. The method of claim 72, wherein the first therapeutically effective amount of the bispecific binding agent is about 450 mg.

74. 74. The method of claim 72 or 73, wherein the clearing agent comprises a second target that is bound to a molecule that is cleared from the circulation primarily by the liver, the fixed phagocyte system, the spleen, or the bone marrow.

75. 75. The method of any of claims 72-74, wherein the clearing agent comprises a 500 kDa aminodextran conjugated to a second target.

76. 76. The method of any one of claims 72 to 75, wherein the clearing agent comprises about 100 to 150 second target molecules per 500 kDa of aminodextran.

77. 77. The method of any one of claims 72 to 76, wherein the metal chelator is selected from the group consisting of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or a derivative thereof, DOTA-Bn or a derivative thereof, p-aminobenzyl-DOTA or a derivative thereof, diethylenetriaminepentaacetic acid (DTPA) or a derivative thereof, and DOTA-deferoxamine.

78. 77. The method of any one of claims 72 to 76, wherein the metal chelator is DOTA or a derivative thereof.

79. 77. The method of any one of claims 72 to 76, wherein the metal chelator is DOTA-Bn or a derivative thereof.

80. 80. The method of any one of claims 72 to 79, wherein the metal of the metallic radionuclide is selected from the group consisting of lutetium (Lu), actinium (Ac), astatine (At), bismuth (Bi), cerium (Ce), copper (Cu), dysprosium (Dy), erbium (Er), europium (Eu), gadolinium (Gd), gallium (Ga), holmium (Ho), iodine (I), indium (In), lanthanum (La), lead (Pb), neodymium (Nd), praseodymium (Pr), promethium (Pm), rhenium (Re), samarium (Sm), scandium (Sc), terbium (Tb), thulium (Tm), ytterbium (Yb), yttrium (Y), and zirconium (Zr).

81. Metallic radionuclides, 211 At, 225 A c, 227 A c, 212 Bi, 213 Bi, 64 Cu, 67 Cu, 67 G.A. 68 G.A. 157 Gd, 166 Ho, 124 I, 125 I, 131 I, 111 In, 177 Lu, 212 Pb, 186 Re, 188 Re, 47 Sc, 153 Sm, 166 Tb, 89 Zr, 86 Y. 88 Y, and 90 81. The method of claim 80, wherein said compound is selected from the group consisting of: Y.

82. Metallic radionuclides 177 The method of claim 80, wherein said compound is Lu.

83. The method of any one of claims 72 to 82, wherein the bispecific binding agent comprises an Fc domain.

84. 83. The method of any one of claims 71-82, wherein the bispecific binding agent is at least 100 kDa, at least 150 kDa, at least 200 kDa, at least 250 kDa, between 100 and 300 kDa, between 150 and 300 kDa, or between 200 and 250 kDa.

85. V within an antibody or antigen-binding fragment thereof or within an scFv of the first molecule H The method of any one of claims 82 to 84, wherein the sequence of the domain comprises SEQ ID NO:

20.

86. V within an antibody or antigen-binding fragment thereof or within an scFv of the first molecule L The method of any one of claims 72 to 85, wherein the sequence of the domain comprises SEQ ID NO:

19.

87. 87. The method of any one of claims 72-86, wherein the first molecule comprises an antibody or antigen-binding fragment thereof, and the sequence of a heavy chain within the antibody or antigen-binding fragment thereof of the first molecule comprises any of SEQ ID NOs: 14-17.

88. 87. The method of any one of claims 72 to 86, wherein the first molecule comprises an antibody or antigen-binding fragment thereof, and the sequence of a heavy chain within the antibody or antigen-binding fragment thereof of the first molecule comprises SEQ ID NO:

15.

89. 87. The method of any one of claims 72 to 86, wherein the first molecule comprises an antibody or antigen-binding fragment thereof, and the sequence of a heavy chain within the antibody or antigen-binding fragment thereof of the first molecule comprises SEQ ID NO:

16.

90. 87. The method of any one of claims 72 to 86, wherein the first molecule comprises an antibody or antigen-binding fragment thereof, and the sequence of a light chain within the antibody or antigen-binding fragment thereof of the first molecule comprises SEQ ID NO:

11.

91. V within an antibody or antigen-binding fragment thereof or within an scFv of the first molecule H The method of any one of claims 72 to 84, wherein the sequence of the domain comprises a humanized form of SEQ ID NO:

20.

92. V within an antibody or antigen-binding fragment thereof or within an scFv of the first molecule L The method of any one of claims 72 to 84 and 91, wherein the sequence of the domain comprises a humanized form of SEQ ID NO:

19.

93. The method of any one of claims 72 to 92, wherein the first molecule comprises an antibody.

94. 94. The method of claim 93, wherein the antibody is an immunoglobulin.

95. 93. The method of any one of claims 72 to 92, wherein the first molecule comprises an antigen-binding fragment.

96. The method of any one of claims 72-86, 91, and 92, wherein the first molecule comprises an scFv.

97. The method of any one of claims 72 to 96, wherein the second molecule comprises a second antibody or a second antigen-binding fragment thereof.

98. The method of any one of claims 72 to 96, wherein the second molecule comprises a second scFv.

99. 85. The method of any one of claims 72-84, wherein a first molecule comprises an antibody, the antibody (i) binds to HER2 on the cancer, and (ii) comprises all three of the heavy chain CDRs of SEQ ID NO:20 and all three of the light chain CDRs of SEQ ID NO:19, the antibody is an immunoglobulin, the immunoglobulin comprises two identical heavy chains and two identical light chains, a first light chain and a second light chain, the first light chain is fused (optionally via a first peptide linker) to a second molecule, the second scFv comprising a second binding site, to create a first light chain fusion polypeptide, and the second light chain is fused (optionally via a second peptide linker) to a third scFv to create a second light chain fusion polypeptide, and the first light chain fusion polypeptide and the second light chain fusion polypeptide are identical.

100. 100. The method of claim 99, wherein the first light chain fusion polypeptide comprises a first peptide linker and the second light chain fusion polypeptide comprises a second peptide linker, and the sequences of the first and second peptide linkers are 5-30, 5-25, 5-15, 10-30, 10-20, 10-15, 15-30, or 15-25 amino acids in length.

101. 100. The method of claim 99, wherein the first light chain fusion polypeptide comprises a first peptide linker and the second light chain fusion polypeptide comprises a second peptide linker, and the sequences of the first peptide linker and the second peptide linker are any of SEQ ID NOs: 23 and 25-30.

102. 100. The method of claim 99, wherein the first light chain fusion polypeptide comprises a first peptide linker and the second light chain fusion polypeptide comprises a second peptide linker, and the sequence of the first peptide linker and the second peptide linker is SEQ ID NO:

23.

103. V in the second scFv H Domain and V L 102. The method of any one of claims 99 to 101, wherein the sequence of the intra-scFv peptide linker between the domains is 5 to 30, 5 to 25, 5 to 15, 10 to 30, 10 to 20, 10 to 15, 15 to 30, or 15 to 25 amino acids in length.

104. V in the second scFv H Domain and V L The method of any one of claims 99 to 101, wherein the sequence of the intra-scFv peptide linker between the domains is 7 to 32, 7 to 27, 7 to 17, 12 to 32, 12 to 22, 12 to 17, 17 to 32, or 17 to 27 amino acids in length.

105. V in the second scFv H Domain and V L The method of any one of claims 99 to 103, wherein the sequence of the scFv intra-peptide linker between the domains is any one of SEQ ID NOs: 23 and 25 to 30.

106. V in the second scFv H Domain and V L The method according to any one of claims 99 to 103, wherein the sequence of the scFv intra-peptide linker between the domains is any one of SEQ ID NOs: 51 to 56.

107. V in the second scFv H Domain and V L The method of any one of claims 99 to 103, wherein the sequence of the scFv intra-peptide linker between the domains is SEQ ID NO:

27.

108. V in the second scFv H Domain and V L The method of any one of claims 99 to 103, wherein the sequence of the scFv intra-peptide linker between the domains is SEQ ID NO:

30.

109. 109. The method of any one of claims 99 to 108, wherein the second binding site specifically binds DOTA.

110. V in the second scFv H The sequence of the domain comprises all three of the CDRs of SEQ ID NO: 21, and the V L The method of claim 109, wherein the sequence of the domain comprises all three of the CDRs of SEQ ID NO:

22.

111. V in the second scFv H The method of any one of claims 109 to 110, wherein the sequence of the domain is SEQ ID NO:

21.

112. V in the second scFv L The method of any one of claims 109 to 111, wherein the sequence of the domain is SEQ ID NO:

22.

113. The method of any one of claims 109 to 112, wherein the sequence of the first scFv comprises any of SEQ ID NOs: 31 to 36.

114. The method of any one of claims 105 or 108 to 110, wherein the sequence of the first scFv comprises any of SEQ ID NOs: 39 to 44.

115. The method of any one of claims 109 to 113, wherein the sequence of the first scFv comprises SEQ ID NO:

33.

116. V in the second scFv H The method of claim 109 or 110, wherein the sequence of the domain comprises a humanized form of SEQ ID NO:

21.

117. The method of claim 116, wherein the humanized form of SEQ ID NO:21 is SEQ ID NO:

37.

118. V in the second scFv L The method of any one of claims 109 to 110 and 116 to 117, wherein the sequence of the domain comprises a humanized form of SEQ ID NO:

22.

119. The method of claim 118, wherein the humanized form of SEQ ID NO:22 is SEQ ID NO:

38.

120. In the heavy chain, V H The method of any one of claims 99 to 119, wherein the sequence of the domain comprises SEQ ID NO:

20.

121. In the light chain, V L The method of any one of claims 99 to 120, wherein the sequence of the domain comprises SEQ ID NO:

19.

122. The method of any one of claims 99 to 121, wherein the sequence of the heavy chain comprises any of SEQ ID NOs: 14 to 17.

123. The method of any one of claims 99 to 121, wherein the sequence of the heavy chain comprises SEQ ID NO:

15.

124. The method of any one of claims 99 to 121, wherein the sequence of the heavy chain comprises SEQ ID NO:

16.

125. The method of any one of claims 99 to 123, wherein the sequence of the light chain comprises SEQ ID NO:

11.

126. The method of any one of claims 99-115 and 120-125, wherein the sequence of the light chain fusion polypeptide comprises any of SEQ ID NOs: 5-10.

127. The method of any one of claims 99-115 and 120-125, wherein the sequence of the light chain fusion polypeptide comprises SEQ ID NO:

7.

128. 100. The method of claim 99, wherein the sequence of the light chain fusion polypeptide comprises any of SEQ ID NOs: 5-10 and the sequence of the heavy chain comprises SEQ ID NOs: 14-17.

129. 100. The method of claim 99, wherein the sequence of the light chain fusion polypeptide comprises SEQ ID NO:7 and the sequence of the heavy chain comprises SEQ ID NO:

15.

130. 100. The method of claim 99, wherein the sequence of the light chain fusion polypeptide comprises any of SEQ ID NOs: 45-50.

131. 131. The method of claim 130, wherein the sequence of the light chain fusion polypeptide comprises SEQ ID NO:

50.

132. 100. The method of claim 99, wherein the sequence of the light chain fusion polypeptide comprises any of SEQ ID NOs: 45-50 and the sequence of the heavy chain comprises SEQ ID NOs: 14-17.

133. 133. The method of claim 132, wherein the sequence of the light chain fusion polypeptide comprises SEQ ID NO:50 and the sequence of the heavy chain comprises SEQ ID NO:

16.

134. 134. The method of any one of claims 72-133, wherein the radiotherapeutic agent comprises (i) a second target bound to a metallic radionuclide, said second target being a metal chelator.

135. 109. The method of any one of claims 72-108, wherein the radiotherapeutic agent comprises (ii) a second target bound to a metal chelator, said metal chelator being bound to a metallic radionuclide.

136. 136. The method of claim 135, wherein the second molecule comprises streptavidin and the second target comprises biotin.

137. 136. The method of claim 135, wherein the second target comprises histamine succinylglycine.

138. 138. The method of any one of claims 72-137, wherein step (c) of administering a therapeutically effective amount of a radiotherapeutic agent to the subject occurs 1-2 hours, 1-3 hours, 1-4 hours, 2-6 hours, 2-8 hours, 2-10 hours, 4-6 hours, 4-8 hours, 4-10 hours, within 1 hour, within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours after step (b) of administering a therapeutically effective amount of a clearing agent to the subject.

139. 138. The method of any one of claims 72-137, wherein step (c) of administering to the subject a therapeutically effective amount of a radiotherapeutic agent occurs about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, or about 6 hours after step (b) of administering to the subject a therapeutically effective amount of a clearing agent.

140. 138. The method of any one of claims 72-137, wherein step (c) of administering to the subject a therapeutically effective amount of a radiotherapeutic agent occurs about 1 hour after step (b) of administering to the subject a therapeutically effective amount of a clearing agent.

141. 138. The method of any one of claims 72-137, wherein step (c) of administering to the subject a therapeutically effective amount of a radiotherapeutic agent occurs within 16 hours of step (a) of administering to the subject a therapeutically effective amount of a bispecific binding agent.

142. 142. The method of any one of claims 53, 56-69, and 72-141, wherein the cancer is breast cancer, gastric cancer, osteosarcoma, desmoplastic small round cell carcinoma, ovarian cancer, prostate cancer, pancreatic cancer, glioblastoma multiforme, gastric junction adenocarcinoma, gastroesophageal junction adenocarcinoma, cervical cancer, salivary gland cancer, soft tissue sarcoma, leukemia, melanoma, Ewing's sarcoma, rhabdomyosarcoma, head and neck cancer, or neuroblastoma.

143. The method of any one of claims 53, 56-69, and 72-142, wherein the cancer is a metastatic tumor.

144. The method of claim 143, wherein the metastatic tumor is a peritoneal metastasis.

145. 143. The method of any one of claims 53, 56-69, and 72-142, further comprising the step of administering to the subject an agent that increases HER2 expression by the cells.

146. 143. The method of any one of claims 53, 56-69, and 72-142, wherein the cancer is resistant to treatment with trastuzumab, cetuximab, lapatinib, erlotinib, or any other small molecule or antibody that targets the HER family of receptors.

147. 15. The method of any one of claims 21, 25-44, 53-68, and 72-146, wherein the heavy chain in the immunoglobulin is mutated to disrupt an N-linked glycosylation site.

148. The method of claim 147, wherein the heavy chain comprises an amino acid substitution replacing an asparagine that is an N-linked glycosylation site with an amino acid that does not function as a glycosylation site.

149. 150. The method of any one of claims 21, 25-44, 53-68, and 72-148, wherein the heavy chain in the immunoglobulin is mutated to disrupt the C1q binding site.

150. 150. The method of any one of claims 1-149, wherein the bispecific binding agent does not activate complement.

151. The method of any one of claims 1 to 150, wherein the bispecific binding agent does not bind to an Fc receptor in a soluble or cell-bound form.

152. The method of any one of claims 24 to 44, 53 to 67, and 72 to 133, wherein the scFv is a disulfide-stabilized scFv.

153. 153. The method of any one of claims 1-152, wherein the bispecific binding agent is administered to the subject intravenously, subcutaneously, intramuscularly, parenterally, transdermally, transmucosally, intraperitoneally, intrathoracically, or to any other body compartment, such as intrathecally, intraventricularly, or intraparenchymally.

154. 153. The method of any one of claims 1-152, wherein the bispecific binding agent is administered intravenously to the subject.

155. The method of any one of claims 1 to 154, wherein the clearing agent is administered intravenously to the subject.

156. 155. The method of any one of claims 1-154, wherein the radiotherapeutic agent is administered to the subject intravenously, subcutaneously, intramuscularly, parenterally, transdermally, transmucosally, intraperitoneally, intrathoracically, or to any other body compartment, such as intrathecally, intraventricularly, or intraparenchymally.

157. The method of any one of claims 1 to 154, wherein the radiotherapeutic agent is administered intravenously to the subject.

158. The method of any one of claims 1 to 157, wherein the therapeutically effective amount of the clearing agent is an amount that results in a 10:1 molar ratio of the therapeutically effective amount of the bispecific binding agent administered to the subject to the therapeutically effective amount of the clearing agent administered to the subject, and the subject is a human.

159. The method of any one of claims 1-157, wherein the therapeutically effective amount of the clearing agent is an amount that results in a reduction of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% in the serum concentration of the bispecific binding agent 1 hour, 2 hours, 3 hours, or 4 hours after step (b) of administering a therapeutically effective amount of the clearing agent to the subject.

160. 160. The method of any one of claims 1-159, wherein the therapeutically effective amount of the radiotherapeutic agent is between 25 mCi and 250 mCi, between 50 mCi and 200 mCi, between 75 mCi and 175 mCi, or between 100 mCi and 150 mCi, and the subject is a human.

161. (d) administering to the subject a therapeutically effective amount of a second bispecific binding agent within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week of step (c) administering to the subject a therapeutically effective amount of a radiotherapeutic agent; (e) after step (d) of administering to the subject a second therapeutically effective amount of a clearing agent; and (f) after step (e) of administering to the subject a second therapeutically effective amount of a clearing agent, administering to the subject a second therapeutically effective amount of a radiotherapeutic agent. The method of any one of claims 1 to 160, comprising:

162. 162. The method of claim 161, wherein step (e) of administering to the subject a therapeutically effective amount of a clearing agent occurs within 12 hours of step (d) of administering to the subject a second therapeutically effective amount of a bispecific binding agent.

163. 163. The method of claim 161 or 162, wherein the second therapeutically effective amount of the bispecific binding agent is between 100 mg and 700 mg, between 200 mg and 600 mg, between 200 mg and 500 mg, between 300 mg and 400 mg, about 300 mg, about 450 mg, about 500 mg, about 600 mg, or about 625 mg.

164. 164. The method of any one of claims 161-163, wherein the second therapeutically effective amount of the clearing agent is an amount that results in a 10:1 molar ratio of the therapeutically effective amount of the bispecific binding agent administered to the subject to the therapeutically effective amount of the clearing agent administered to the subject.

165. The method of any one of claims 161-163, wherein the therapeutically effective amount of the clearing agent is an amount that results in a reduction of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% in the serum concentration of the bispecific binding agent 1 hour, 2 hours, 3 hours, or 4 hours after step (b) of administering a therapeutically effective amount of the clearing agent to the subject.

166. 166. The method of any one of claims 161-165, wherein the second therapeutically effective amount of the radiotherapeutic agent is between 25 mCi and 250 mCi, between 50 mCi and 200 mCi, between 75 mCi and 175 mCi, or between 100 mCi and 150 mCi.

167. 167. The method of any one of claims 161-166, wherein the second therapeutically effective amount of the bispecific binding agent is administered to the subject intravenously, subcutaneously, intramuscularly, parenterally, transdermally, transmucosally, intraperitoneally, intrathoracically, or to any other body compartment, such as intrathecally, intraventricularly, or intraparenchymally.

168. 167. The method of any one of claims 161-166, wherein the second therapeutically effective amount of the bispecific binding agent is administered intravenously to the subject.

169. 169. The method of any one of claims 161-168, wherein the second therapeutically effective amount of the clearing agent is administered intravenously to the subject.

170. 170. The method of any one of claims 161-169, wherein a second therapeutically effective amount of a radiotherapeutic agent is administered to the subject intravenously, subcutaneously, intramuscularly, parenterally, transdermally, transmucosally, intraperitoneally, intrathoracically, or to any other body compartment, such as intrathecally, intraventricularly, or intraparenchymally.

171. 170. The method of any one of claims 161-169, wherein the second therapeutically effective amount of a radiotherapeutic agent is administered intravenously to the subject.

172. (g) administering to the subject a third therapeutically effective amount of a bispecific binding agent within 1 day, within 2 days, within 3 days, within 4 days, within 5 days, within 6 days, or within 1 week of step (f) administering to the subject a second therapeutically effective amount of radiation therapy; (h) after step (g) of administering to the subject a third therapeutically effective amount of a clearing agent; and (i) after step (h) of administering to the subject a third therapeutically effective amount of a clearing agent, administering to the subject a third therapeutically effective amount of a radiotherapeutic agent. The method of any one of claims 161 to 171, comprising:

173. 173. The method of claim 172, wherein step (g) of administering a therapeutically effective amount of a clearing agent to the subject occurs within 12 hours of step (g) of administering a second therapeutically effective amount of a bispecific binding agent to the subject.

174. 174. The method of claim 172 or 173, wherein the third therapeutically effective amount of the bispecific binding agent is between 100 mg and 700 mg, between 200 mg and 600 mg, between 200 mg and 500 mg, between 300 mg and 400 mg, about 300 mg, about 450 mg, about 500 mg, about 600 mg, or about 625 mg.

175. 175. The method of any one of claims 172-174, wherein the third therapeutically effective amount of the clearing agent is an amount that results in a 10:1 molar ratio of the therapeutically effective amount of the bispecific binding agent administered to the subject to the therapeutically effective amount of the clearing agent administered to the subject.

176. The method of any one of claims 172-174, wherein the therapeutically effective amount of the clearing agent is an amount that results in a reduction of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% in the serum concentration of the bispecific binding agent 1 hour, 2 hours, 3 hours, or 4 hours after step (b) of administering a therapeutically effective amount of the clearing agent to the subject.

177. 177. The method of any one of claims 172-176, wherein the third therapeutically effective amount of the radiotherapeutic agent is between 25 mCi and 250 mCi, between 50 mCi and 200 mCi, between 75 mCi and 175 mCi, or between 100 mCi and 150 mCi.

178. 178. The method of any one of claims 172-177, wherein the third therapeutically effective amount of the bispecific binding agent is administered to the subject intravenously, subcutaneously, intramuscularly, parenterally, transdermally, transmucosally, intraperitoneally, intrathoracically, or to any other body compartment, such as intrathecally, intraventricularly, or intraparenchymally.

179. 178. The method of any one of claims 172-177, wherein the third therapeutically effective amount of the bispecific binding agent is administered intravenously to the subject.

180. 178. The method of any one of claims 172-177, wherein the third therapeutically effective amount of the clearing agent is administered intravenously to the subject.

181. 181. The method of any one of claims 172-180, wherein a third therapeutically effective amount of a radiotherapeutic agent is administered to the subject intravenously, subcutaneously, intramuscularly, parenterally, transdermally, transmucosally, intraperitoneally, intrathoracically, or to any other body compartment, such as intrathecally, intraventricularly, or intraparenchymally.

182. 181. The method of any one of claims 172-180, wherein the third therapeutically effective amount of a radiotherapeutic agent is administered intravenously to the subject.

183. The method of any one of claims 1 to 182, wherein the bispecific binding agent is contained in a pharmaceutical composition further comprising a pharma- ceutically acceptable carrier.

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