Radiolabeled biomolecules and use thereof

Abstract

To provide radiolabeled biomolecules and methods for radiolabeling biomolecules with radioactive halogen atoms, where the methods minimize loss of the radioactive halogen due to dehalogenation in vivo, preserve the biological activity of the biomolecules, maximize retention of radioactivity in cancer cells, and minimize retention of radioactivity in normal tissues after in vivo administration.SOLUTION: Some of such radiolabeled biomolecules comprise a radioactive metal atom in place of or in addition to the radioactive halogen. The biomolecules have affinity for particular types of cells and may specifically bind to certain cells, such as cancer cells. Relevant biomolecules include antibodies, monoclonal antibodies, antibody fragments, peptides, other proteins, nanoparticles and aptamers.SELECTED DRAWING: None

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to compounds and precursors thereof useful for radiolabeling biomolecules, and to radiolabeled biomolecules that can effectively retain radioactivity from biomolecules internalized within cells, making such compounds useful in the diagnosis and treatment of disease, particularly cancer. [Background technology]

[0002] background Radioiodination is one of the most convenient methods to radiolabel biological molecules. Several radioisotopes of iodine are available for cancer imaging and targeted radiotherapy. Radioisotopes of iodine are supplied as alkaline solutions, and iodine is converted to -1(I - These exist in the +1 oxidation state (iodide). Standard biomolecule radioiodination methods require oxidation of iodine to the +1 oxidation state for electrophilic substitution to tyrosine amino acids present in biomolecules, e.g., antibodies, other proteins and peptides. Challenges with monoclonal antibodies (mAbs) and peptides radioiodized in this way include in vivo instability to proteolysis within cells after internalization, deiodination, and loss of radioactivity from tumor cells as a result of both processes. It is widely recognized that after internalization (which can occur as a result of binding to receptors and specific antigens), within the cell, radioiodized antibodies and peptides are proteolytically degraded to radioactive iodotyrosine, which is efficiently transported out of the cell by membrane amino acid transporters. The released radioactive iodotyrosine is deiodized by deiodinase enzymes found in tissues, and the free radioactive iodine is redistributed and accumulates in organs with sodium iodide symporter expression, particularly the thyroid, stomach and salivary glands. Thus, the amount of radiolabel retained in the tumor is reduced, with a concomitant increase in uptake of radioactivity in normal tissues.

[0003] One of the drawbacks of antibodies is their long blood half-life, which leads to high background levels after systemic administration and consequently low tumor-to-background ratios. Also, traditional antibodies diffuse rather slowly into solid tumors, which prevents them from uniformly reaching and binding to receptors / antigens throughout the tumor mass.

[0004] Although several compounds have been identified in the art, they are unstable and difficult to produce in commercial quantities. Thus, there is a need for improved prosthetic compounds that can be used to radiolabel biomolecules for targeted radiotherapy and imaging applications.

[0005] Additionally, the size of the antibody is particularly problematic for tumors in the brain due to delivery limitations imposed by the blood-brain barrier, such that uptake of the antibody into tumor cells, particularly brain metastases, is low. The present invention addresses problems associated with treating cancer, including cancers that have metastasized to the brain, with a composition that can be taken up and retained by tumor cells, while reducing the amount of radiolabel taken up by normal tissues, particularly the kidneys. Summary of the Invention [Means for solving the problem]

[0006] Summary of the Invention The present invention relates to methods, compounds, and compositions for radiolabeling biomolecules (also referred to as macromolecules) with radioactive halogen atoms in a manner that minimizes the loss of radioactive halogens due to dehalogenation in vivo, preserves the biological activity of the biomolecule, maximizes retention in diseased cells such as cancer cells, and minimizes retention of radioactivity in normal tissues after in vivo administration. Biomolecules have an affinity for a particular cell type. That is, biomolecules can specifically bind to particular cells, such as cancer cells. The compositions of the present invention include radiolabeled biomolecules. Such biomolecules include antibodies, monoclonal antibodies, antibody fragments, peptides, other proteins, nanoparticles, and aptamers. Such examples of biomolecules for the purposes of the present invention include diabodies, scFv fragments, DARPins, fibronectin type III-based scaffolds, affibodies, VHH molecules (also known as single domain antibody fragments (sdAbs) and nanobodies), nucleic acid or protein aptamers, and nanoparticles. In addition, larger molecules such as proteins over 50 kDa, including antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, and F(ab')2 fragments, may be used in the practice of the present invention.In addition, nanoparticles having a size of less than 50 nm may be used in the practice of the present invention.

[0007] The methods of the present invention utilize prosthetic compounds that are effective for radiolabeling. As such, the present disclosure provides such radiolabeled compounds (referred to herein as "prosthetic compounds"), and precursors for obtaining such prosthetic compounds (referred to herein as "radioactive halogen precursors"). The present disclosure further provides radiolabeled macromolecules (e.g., biomolecules) comprising such prosthetic compounds / radicals and one or more macromolecules. In some such embodiments, these radiolabeled macromolecules are targeted radiotherapeutic agents. The prosthetic compounds and radiolabeled compounds of the present invention are useful, for example, in disease diagnosis and targeted radiotherapy.

[0008] In one embodiment of the present disclosure, a compound in the form of a prosthetic compound or a radioactive halogen precursor represented by formula I: [ka] (In the formula, X is CH or N; L1 and L3 are independently selected from a bond, a substituted or unsubstituted alkyl chain, a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain, and a polyethylene glycol (PEG) chain; MMCM is a polymeric conjugate moiety; L2 is a substituted or unsubstituted alkyl chain, a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain, or a polyethylene glycol (PEG) chain containing at least three oxygen atoms, where L2 optionally contains a brush border enzyme cleavable peptide; CG is selected from guanidine; PO3H; SO3H; one or more charged D- or L-amino acids, such as arginine, phosphono / sulfophenylalanine, glutamate, aspartate, and lysine; a hydrophilic carbohydrate moiety; a polyethylene glycol (PEG) chain; and Z-guanidine (also referred to herein as "guanidino-Z"); Z is (CH2) n and; n is greater than 1; m is 0 to 4 (when X=CH) or 0 to 3 (when X=N); and Y is an alkylmetal moiety (in the radioactive halogen precursor) or a radioactive halogen (in the prosthetic compound), where the radioactive halogen is 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I, 131 I, 211 At, or a pharma- ceutically acceptable salt or solvate thereof.

[0009] In certain preferred embodiments, m=1.

[0010] In some embodiments, Y is an alkylmetal moiety (when the compound is a radioactive halogen precursor) selected from the group consisting of trimethylstannyl (SnMe3), tri-n-butylstannyl (SnBu3), and trimethylsilyl (SiMe3). 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I and 211 At (when the compound is a prosthetic compound).

[0011] In some embodiments, the MMCM is an active ester or (Gly) m (m is 1 or greater). In some embodiments, the MMCM is selected from the group consisting of an N-hydroxysuccinimide (NHS) ester, a tetrafluorophenol (TFP) ester, an isothiocyanate group, or a maleimide group. One exemplary MMCM is Gly-Gly-Gly.

[0012] In some embodiments, L2 is (CH2) p (p=1-6 or p=2-6). In some embodiments, the optional brush border enzyme-cleavable peptide, when present in L2, is selected from the group consisting of Gly-Lys, Gly-Tyr, and Gly-Phe-Lys.

[0013] In certain embodiments, the compound (prosthetic compound or radioactive halogen precursor) has the following structure of formula 1a: [ka] It is expressed by:

[0014] In certain embodiments, the compound is N-succinimidyl 3-guanidinomethyl-5-[ 131 Iodobenzoate (iso-[ 131 I]SGMIB) or N-succinimidyl 3-[ 211At] Astato-5-guanidinomethylbenzoate (iso-[ 211 At]SAGMB).

[0015] In another aspect of the present invention, the present disclosure provides a compound in the form of a prosthetic compound or a radioactive halogen precursor represented by formula 2: [ka] (In the formula, MC is a multidentate metal chelating moiety; Cm is a thiourea, amide, or thioether; L4 is selected from a bond, a substituted or unsubstituted alkyl chain, a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain optionally having NH, CO or S at one or both termini, and a polyethylene glycol (PEG) chain; and T is provided as a compound (prosthetic compound or radioactive halogen precursor) disclosed herein (e.g., of Formula 1, e.g., Formula 1A), or a pharma- ceutically acceptable salt or solvate thereof.

[0016] In some embodiments, MC is a macrocyclic structure. In some exemplary prosthetic compounds, MC is selected from DOTA, TETA, NOTP and NOTA. In some embodiments, MC is an acyclic polydentate ligand. In some exemplary prosthetic compounds, MC is selected from EDTA, EDPTP and DTPA.

[0017] In certain embodiments, Y is an alkylmetal moiety (when the compound is a radioactive halogen precursor). The alkylmetal moiety in the radioactive halogen precursor is, for example, selected from the group consisting of trimethylstannyl (SnMe3), tri-n-butylstannyl (SnBu3) and trimethylsilyl (SiMe3). Such precursors may be useful in the production of prosthetic compounds and radiolabeled biomolecules disclosed herein, as described herein. In other embodiments, Y is a radioactive halogen (when the compound is a prosthetic compound), for example, 75Br, 76 Br, 77 Br, 123 I, 124 I, 125 I, 131 I or 211 At.

[0018] The present disclosure further provides radiolabeled biomolecules comprising a prosthetic compound disclosed herein attached to a biomolecule, and also provides intermediates comprising a radioactive halogen precursor disclosed herein attached to a biomolecule, which can react to form a radiolabeled biomolecule.

[0019] The biomolecule can be various. In certain embodiments, the biomolecule is selected from the group consisting of an antibody, an antibody fragment, a VHH molecule, an aptamer, or a variant thereof. In certain embodiments, the biomolecule is a VHH. In certain embodiments, the VHH targets HER2. In some embodiments, the VHH comprises an amino acid sequence selected from the sequences set forth in SEQ ID NOs: 1-5.

[0020] The present disclosure further provides a pharmaceutical composition comprising a radiolabeled biomolecule disclosed herein in association with a pharma- ceutically acceptable adjuvant, diluent or carrier.In a further aspect of the present disclosure, a method for treating cancer is provided, comprising administering to an individual in need thereof an effective amount of a radiolabeled biomolecule disclosed herein and / or an effective amount of a pharmaceutical composition disclosed herein.

[0021] To provide an understanding of embodiments of the present disclosure, reference is made to the accompanying drawings, which are not necessarily to scale. The drawings are illustrative only and are not to be construed as limiting the present disclosure. [Brief description of the drawings]

[0022] [Figure 1]Figure 1 provides non-reducing SDS-PAGE / fluorescence imaging profiles of (A) [211At]SAGMB-5F7 VHH, (B) [131I]SGMIB-5F7 VHH, (C) iso-[211At]SAGMB-5F7 VHH, and (D) iso-[131I]SGMIB-5F7 VHH, with molecular weight standards in the left lane for comparison;

[0023] [Diagram 2] Figure 2 provides the results of saturation binding assays performed on HER2-expressing BT474M1 breast carcinoma cells with 5F7 VHHs labeled with (A) [131I]SGMIB, (B) iso-[131I]SGMIB, (C) [211At]SAGMB, and (D) iso-[211At]SAGMB;

[0024] [Diagram 3] Figure 3 provides plots of internalization of [211At]SAGMIB-5F7 VHH and iso-[211At]SAGMB-5F7 VHH into BT474M1 cells in vitro, with Figure 3A showing total cell-bound (internalized + surface-bound) radioactivity and Figure 3B showing internalized radioactivity;

[0025] [Figure 4] Figure 4 provides plots of internalization of [131I]SGMIB-5F7 VHH and iso-[131I]SGMIB-5F7 VHH into BT474M1 cells in vitro, with Figure 4A showing total cell-bound (internalized + surface-bound) radioactivity and Figure 4B showing internalized radioactivity;

[0026] [Diagram 5]Figure 5 shows the biodistribution of [211At]SAGMB-5F7 VHH and iso-[211At]SAGMB-5F7 VHH in SCID mice bearing BT474M1 xenografts compared with uptake in tumors, data were obtained from paired labeling studies after administration of [131I]SGMIB-5F7 / [211At]SAGMB-5F7 VHH and iso-[131I]SGMIB-57 / iso-[211At]SAGMB-5F7 VHH tandems;

[0027] [Figure 6] Figure 6 shows the biodistribution of [131I]SGMIB-5F7 VHH and iso-[131I]SGMIB-5F7 VHH in SCID mice bearing BT474M1 xenografts compared with uptake in tumors, data obtained from paired labeling studies after administration of [131I]SGMIB-5F7 / [211At]SAGMB-5F7 VHH and iso-[131I]SGMIB-57 / iso-[211At]SAGMB-5F7 VHH tandems;

[0028] [Figure 7] Figure 7 shows the biodistribution of [211At]SAGMB-5F7 and iso-[211At]SAGMB-5F7 VHH in SCID mice bearing BT474M1 xenografts compared to uptake in the kidney, data were obtained from paired labeling studies after administration of [131I]SGMIB-5F7 / [211At]SAGMB-5F7 VHH and iso-[131I]SGMIB-57 / iso-[211At]SAGMB-5F7 VHH tandems;

[0029] [Figure 8]Figure 8 shows the biodistribution of [131I]SGMIB-5F7 VHH and iso-[131I]SGMIB-5F7 VHH in SCID mice bearing BT474M1 xenografts compared to uptake in the kidney, data obtained from paired labeling studies after administration of [131I]SGMIB-5F7 / [211At]SAGMB-5F7 VHH and iso-[131I]SGMIB-57 / iso-[211At]SAGMB-5F7 VHH tandems;

[0030] [Figure 9] Figure 9 provides data on the uptake of [211At]SAGMB-5F7 VHH and iso-[211At]SAGMB-5F7 VHH in the thyroid (Figure 9A) and stomach (Figure 9B) of SCID mice bearing BT474M1 xenografts, data were obtained from paired labeling studies after administration of [131I]SGMIB-5F7 / [211At]SAGMB-5F7 VHH and iso-[131I]SGMIB-57 / iso-[211At]SAGMB-5F7 VHH tandems;

[0031] [Figure 10] Figure 10 provides data on the uptake of [131I]SGMIB-5F7 and iso-[131I]SGMIB-5F7 in the thyroid (Figure 10A) and stomach (Figure 10B) of SCID mice bearing BT474M1 xenografts, data obtained from paired labeling studies after administration of [131I]SGMIB-5F7 / [211At]SAGMB-5F7 VHH and iso-[131I]SGMIB-57 / iso-[211At]SAGMB-5F7 VHH tandems;

[0032] [Figure 11]FIG. 11 shows the tumor-to-tissue ratios obtained from the biodistribution of [211At]SAGMB-5F7 VHH and iso-[211At]SAGMB-5F7 VHH in SCID mice bearing BT474M1 xenografts, data obtained from paired labeling studies after administration of [131I]SGMIB-5F7 / [211At]SAGMB-5F7 VHH and iso-[131I]SGMIB-5F7 / iso-[211At]SAGMB-5F7 VHH tandems; and

[0033] [Figure 12] FIG. 12 shows tumor-to-tissue ratios obtained from the biodistribution of [131I]SGMIB-5F7 VHH and iso-[131I]SGMIB-5F7 in SCID mice bearing BT474M1 xenografts, data obtained from paired labeling studies after administration of [131I]SGMIB-5F7 / [211At]SAGMB-5F7 VHH and iso-[131I]SGMIB-5F7 / iso-[211At]SAGMB-5F7 VHH tandems; and

[0034] [Figure 13] FIG. 13 is a table providing paired label biodistribution of [211At]SAGMB-5F7 VHH and [131I]SGMIB-5F7 VHH in SCID mice bearing subcutaneous B474M1 human breast carcinoma xenografts; and

[0035] [Figure 14] FIG. 14 is a table providing paired label biodistribution of iso-[211At]SAGMB-5F7 VHH and iso-[131I]SGMIB-5F7 VHH in SCID mice bearing subcutaneous B474M1 human breast carcinoma xenografts. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] Detailed Description of the Invention The present disclosure will now be described in more detail below with reference to exemplary embodiments. These exemplary embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art. Indeed, the present disclosure may be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0037] Compounds, compositions and methods are provided for diagnosing and treating diseases, including cancer. In general, the compounds of the present disclosure include a radiolabeled prosthetic compound / radical or radiolabeled prosthetic group attached to a macromolecule (e.g., a biomolecule) that functions as a targeting moiety (providing a targeted radiotherapeutic agent). As such, the present disclosure encompasses the radiolabeled prosthetic compounds and radicals themselves, as well as macromolecules having such radiolabeled prosthetic compounds / radicals attached thereto (referred to in some embodiments herein as "radiolabeled biomolecules" or "targeted radiotherapeutic agents").

[0038] The present disclosure also encompasses such compounds and radicals (alone and / or in combination with biomolecules) that contain alkylmetal moieties (referred to herein as "radioactive halogen precursors") from which prosthetic groups and / or targeted radiotherapeutic agents can be produced. Advantageously, in some embodiments, the preparation of such precursors involves the addition of larger radioactive halogens (such as, but not limited to, 18 Larger than F 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I, 131 I and 211 This enables the production of prosthetic compounds and targeted radiotherapeutics that contain ribozymes (including At).

[0039] The labeled prosthetic compound / radical or radioactive halogen precursor (alone or attached to a polymer) generally comprises, in addition to the radioactive halogen or its precursor, a charged group (CG) and a polymeric conjugate moiety (MMCM). As described in more detail below, each of these components may be attached to one or more cleavable (or non-cleavable) linkers. In some embodiments, the targeted radiotherapeutic agent comprises a biomolecule (targeting moiety), a radiolabeled prosthetic group or template, and optionally a chelator (either macrocyclic or acyclic).

[0040] The radiolabeled compounds, particularly radiolabeled biomolecules and methods of use described herein result in greater uptake of radioactivity in target cells, higher retention of radioactivity in target cells after internalization, and less uptake of radioactivity in normal cells; for example, less thyroid and kidney uptake of radioactivity. The targeted radiotherapy of the present invention can selectively deliver radionuclides to malignant cell populations. The advantage of targeted radiotherapy is that it can select radionuclides with properties that best match the constraints of the intended clinical application. As an example, in the case of central nervous system (CNS) tumors, radiation will be advantageously selected with tissue coverage that minimizes irradiation of normal CNS tissue.

[0041] The compounds provided herein (e.g., radioactive halogen precursors, prosthetic compounds, intermediates and targeted radiotherapeutics) are prepared by methods that enhance retention of radionuclides, particularly (in certain embodiments) radioactive halogens, in target disease cells, e.g., cancer cells, using labeling techniques that generate exocytosis-resistant charged catabolic products that cannot cross lysosomal or cellular membranes after intracellular proteolysis. Compounds of the invention include charged catabolic products where a portion of the molecule bearing the label is inert to lysosomal degradation and is trapped intracellularly after proteolysis.

[0042] Certain prosthetic compounds and precursors thereof (i.e., radioactive halogen precursors) encompassed by the present disclosure are of formula 1 [ka] Formula 1: General structure of class I compounds and derivatives and variants thereof.

[0043] The present invention includes prosthetic compounds / radicals and their precursors (referred to as "Class I type compounds") of the general structure of formula 1, which comprises a homo (X=CH) or hetero (X=N) aromatic ring attached thereto; a polymeric conjugate moiety (MMCM) for coupling the prosthetic compound / radical or precursor to a polymer, a radioactive halogen or a radioactive halogen precursor (Y); and one or more charged substituents (CG). Each of these components can be attached to the aromatic ring via a linker (L1, L2, L3) or can be directly bonded to the aromatic ring (i.e., when L1 and / or L2 and / or L3 are bonds). Each of these components shown in formula 1 is described in more detail below.

[0044] In some embodiments, Y is a radioactive halogen (when Formula 1 represents a radiolabeled prosthetic compound / radical). Such a radioactive halogen is 18 F, 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I, 131 I and 211 Advantageously, in some embodiments, the radioactive halogen may be selected from 18 is greater than F. In certain embodiments, the radioactive halogen Y is 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I, 131 I and 211 In certain embodiments, the radioactive halogen Y is selected from 75 Br, 76 Br, 77 Br and 211 In one particular embodiment, the radioactive halogen Y is selected from211 At.

[0045] In other embodiments, Y is an alkylmetal moiety (when Formula 1 represents a radioactive halogen precursor / radical). Exemplary alkylmetal moieties include, but are not limited to, trialkylmetal precursors, including trimethylstannyl (SnMe3), tri-n-butylstannyl (SnBu3), and trimethylsilyl (SiMe3).

[0046] Y can be directly attached to the aromatic ring (L3=direct bond) or can be attached to the aromatic ring via a linker (L3), which can be, for example, a spacer, such as a substituted or unsubstituted alkyl chain, a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain or a short polyethylene glycol (PEG) chain (1-10 ethylene glycol units).

[0047] Charged groups (CG) are typically present in the prosthetic groups disclosed herein (i.e., m is 1 or greater). Typically, m is 1; however, more than one CG can be attached to the ring, such as m=2, m=3, and (when X=CH) m can be 4. When more than one CG is attached to the ring, each such CG (and corresponding L2) can be the same or different. In certain embodiments, as noted below (as shown in Formula 2), when another moiety can be attached to the ring of Formula 1, and such additional moiety is charged, m can be 0 (i.e., in some embodiments, the additional moiety can effectively function as a "charged group").

[0048] Charged groups are typically groups that are charged under physiological conditions of the internal cellular environment. In some embodiments, the charged group (CG) comprises a guanidine, a PO3H group, or a SO3H group. In some embodiments, the CG is a guanidinoalkyl group containing more than one carbon. In some embodiments, the CG is a guanidino hydrophilic group (e.g., an amino or hydroxyl-containing group) and / or an alkyloxycarbonylguanidine group. In other embodiments, the CG comprises one or more charged D-amino acids, such as arginine, glutamate, aspartate, lysine, and / or phosphono / sulfophenylalanine. In yet further embodiments, the CG comprises a hydrophilic carbohydrate moiety. In some embodiments, the compound may comprise one, two, or three CG moieties (and optionally the corresponding linker group L2) to increase intracellular capture in cancer cells.

[0049] CG can be directly bonded to the aromatic ring (L2=direct bond) or can be bonded to the aromatic ring via a linker (L2). L2 can be, for example, any of the above containing a spacer, such as a substituted or unsubstituted alkyl chain (e.g., a simple substituted or unsubstituted alkyl chain, e.g., methylene), a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain, a PEG chain of at least three oxygens, or a brush border enzyme cleavable peptide, such as Gly-Lys, Gly-Tyr, or Gly-Phe-Lys. Note that in certain embodiments, when CG is guanidine and L2 is an unsubstituted alkyl chain, the unsubstituted alkyl chain contains 2 or more carbon atoms.

[0050] In some embodiments of the present invention, a metabolizable spacer or cleavable linker L2 (e.g., brush border enzyme cleavable linker) is located between CG and aromatic ring. These formulations can avoid increased uptake and retention of radioactivity in the kidney, since the CG moiety is cleaved in the kidney, eliminating the charge and releasing the (now neutral or less charged) radioactive species from the renal tubule cells of the kidney, which are then rapidly excreted in urine. Brush border enzyme cleavable linkers have been used previously with radioactivity, but are not used in this method to create a "charge switch", and the labeling reagent is retained in the tumor because it is charged, but is removed in the kidney because it loses charge.

[0051] Such linkers include linker sequences targeting meprin β, a metalloprotease expressed in renal brush border membranes (Jodal et al., (2015) PLoS One Apr 9;10(4):e0123443); C-terminal lysine linked to an antibody fragment via the epsilon amino group of lysine or C-terminal (N(epsilon)-amino-1,6-hexane-bis-vinylsulfone) lysine, which exhibits reduced renal uptake by exploiting the lysine-specific carboxypeptidase activity of a renal brush border enzyme that cleaves the radiolabeled peptide linker prior to uptake by proximal tubule cells (Li et al., (2002) Bioconjug Chem 13(5):985-995); L-tyrosine, O-tyrosine, L-asparagine, L-glutamine, N-Boc-L-lysine (Akizawa et al., (2013) Bioconjugate Chem 24:291-299); glycyl-lysine (Arano et al. (1999) Cancer Research 59:128-134), all of which are incorporated herein by reference.

[0052] In some embodiments, the MMCM is an active ester. In this specification, an active ester is defined as an ester that can be conjugated with an amine group present on a macromolecule / biomolecule (e.g., a peptide or protein) under mild conditions, i.e., conditions that do not result in loss of biological function of the macromolecule / biomolecule. Exemplary such MMCM groups include, but are not limited to, N-hydroxysuccinimide (NHS) or tetrafluorophenol (TFP) esters, isothiocyanate groups, or maleimide groups. Such MMCMs generally result in random (non-site-specific) labeling of amine groups on proteins or peptides. In other embodiments, the MMCM provides for site-specific conjugation to be performed using the enzyme sortase, which results in conjugation to only one site (either the N-terminus or C-terminus of a protein). In this case, the MMCM is, for example, the tripeptide GlyGlyGly.

[0053] The MMCM can be directly attached to the aromatic ring (L1=direct bond) or can be attached to the aromatic ring via a linker (L1), where L1 can be, for example, a spacer, such as a substituted or unsubstituted alkyl chain, a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain, or a short polyethylene glycol (PEG) chain (1-10 ethylene glycol units).

[0054] The positions of these three moieties (-L1-MMCM, -L2-CG and -L3-Y) on the aromatic ring can vary. When X is CH, these three moieties can be located at any of the positions of the aromatic ring. In some such embodiments, the -L2-CG and -L3-Y moieties are located at the 3rd and 4th positions (or the 4th and 3rd positions, respectively) relative to the (1st) -L1-MMCM moiety. In some such embodiments, the -L2-CG and -L3-Y moieties are located at the 3rd and 5th positions relative to the -L1-MMCM moiety, such that the aromatic ring contains the reference moieties at the 1st, 3rd and 5th positions. When X is N, these three moieties can be located at any of the remaining five positions of the ring (including, but not limited to, the 2nd, 4th and 6th positions of the ring).

[0055] Specific prosthetic compound within the scope of formula 1 for labeling the targeting molecules of the present invention, and radioactive halogen precursors, include compounds of formula 1A and derivatives and variants thereof, as shown below: As shown, in formula 1A, X is CH (i.e., the aromatic ring is a benzene ring), L2 is a methylene group, and three moieties (-L1-MMCM, -L3-Y, and -CH2-CG) are present at the 1-, 3-, and 5-positions of the aromatic ring. [ka] Formula 1A: General structure of subclass IA type compounds

[0056] The present invention also includes compounds thereof having the general structure of Formula 2, shown below (referred to as "Class II compounds"). MC-Cm-L4-Cm-T Formula 2: General structure of class II compounds

[0057] Such compounds include a polydentate metal chelating moiety (MC), a linker (L4) with conjugate moieties (Cm) on both ends of L4, and a radiohalogenated template or radiohalogen precursor template (T). T can be, for example, a compound of formula 1 or a compound of formula 1A (a compound containing MMCM), as shown above. In some embodiments, T is a prosthetic compound / radical, and in some embodiments, T is a radiohalogen precursor compound / radical. In some such embodiments, as mentioned above, m=0, and the "MC-Cm-L4-Cm" portion of formula 2 provides the desired functionality of the L2-CG portion in formula 1 above (i.e., the MC-Cm-L4-Cm substituent is a sufficiently "charged group"). In other such embodiments, m=1, 2 or 3, and the aromatic ring of "T" has at least four substituents (i.e., L1-MMCM, L3-Y, L2-CG and Cm-L4-Cm-MC), and may optionally include one or more additional L2-CG substituents.

[0058] L4 can be as defined above for L1 and L3. As such, L4 can be a direct bond or can be, for example, a spacer, such as a substituted or unsubstituted alkyl chain, a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain, or a short polyethylene glycol (PEG) chain (1-10 ethylene glycol units). L4 is also as defined above, but with NH, CO (carbonyl) or S (thioether) at one or both termini.

[0059] Cm can be, for example, a thiourea, an amide, or a thioether. For example, in some embodiments, Cm is a thiourea (e.g., when the chelating moiety and the conjugate functional group of T are isothiocyanates), an amide (when the chelating moiety and the conjugate functional group of T are NHS or TFP active esters or acyl halides), or a thioether (when the chelating moiety and the conjugate functional group of T are maleimides).

[0060] Generally, T is a radiolabeled moiety containing MMCM (through which a macromolecule may be coupled to the compound) or a radioactive halogen precursor. As mentioned above, in some embodiments, T can be a compound / radical of formula 1 or a compound / radical of formula 1A. In other embodiments, T can be a compound / radical of formula 1A or a compound / radical of formula 1B, as described in, but not limited to, Choi et al. (2014) Nucl. Other radioactive halogen templates (T) may be used, including iso-SGMIB, disclosed in Med Biol 41(10):802-812, which is incorporated herein by reference; SIPC, disclosed in Reist et al., (1997) Nucl Med Biol 24(7):639-648, which is incorporated herein by reference; or SDMB, disclosed in U.S. Pat. No. 5,302,700, which is incorporated herein by reference.

[0061] The MC can be any polydentate moiety and can be cyclic or acyclic. The composition of the MC can vary. The MC can be uncomplexed (lacking metal) or can contain a stable (non-radioactive) or radioactive form of a metal, preferably a trivalent metal (M +3 ), for example, lutetium, yttrium, indium, actinium or gallium, and MC is connected to the linker using one of the free COOH groups present on MC or through any other position on MC, including one of the MC backbone carbons. Certain specific radiometals that can be complexed to MC include, but are not limited to: 177 Lu, 64 Cu, 111 In, 90 Y, 225 Ac, 213 Bi, 212 Pb, 212 Bi, 67 Ga, 68 Ga, 89 Zr and 227and Th. This list is not exhaustive and these exemplary radiometals are trivalent, however it is noted that certain MCs that may be used in accordance with the present invention may bind to metals of other valencies and such MCs and radiometals are also encompassed herein.

[0062] In some embodiments, the inclusion of a radioactive metal bound to the MC may eliminate the need for a radioactive atom elsewhere on the molecule (e.g., as "Y" when T=formula 1 / 1a moiety in formula 2). As such, in a compound of formula 2, "T" may or may not include a radioactive atom (e.g., a halogen). In some embodiments, T includes a moiety shown in formula 1 / 1a above, and the "Y" group is a non-radioactive halogen (e.g., non-radioactive bromine or iodine). In other embodiments, compounds of formula 2 are provided that include both a radioactive halogen (e.g., as "Y" when T=formula 1 / 1a moiety in formula 2) and a radioactive metal (bound to the MC, such as the radioactive metals mentioned above). In certain embodiments, such a strategy would allow, for example, the use of the same prosthetic agent for multiple isotopes. In certain examples, compounds of formula 2 may be used in combination with low energy beta emitters (e.g., 131 I) + high energy beta emitters (e.g., 90 Y); or alpha emitters (e.g., 225 Ac) metals and beta-emitter halogens (e.g., 131 I); or alpha-emitter halogens (e.g., 211 At) and beta-emitter radioactive metals (e.g., 177 Provided at:

[0063] In some embodiments, MC is a macrocyclic ligand consisting of a ring containing 8 or more atoms, and has at least three negatively charged substituents, such as carboxyl or phosphonate groups.Exemplary macrocyclic ligands suitable as MC groups include 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) and 1,4,7-triazacyclononane-1,4,7-tri(methylenephosphonic acid) (NOTP).In other embodiments, MC is MeO-DOTA, as disclosed in Gali et al., Anticancer Research (2001), 21(4A), 2785-2792, which is incorporated herein by reference.

[0064] An example of a Class II compound is shown in Formula 2A below, where MC is a macrocyclic ligand that includes DOTA, and the radiohalogenated template T is a moiety that corresponds to Formula 1. [ka] Exemplary Class II Compounds Having Formula 2A: DOTA MC

[0065] The parentheses on the left side of formula 2A are intended to convey that the specific site on MC (DOTA) to which the Cm group is attached is not limited (i.e., Cm can be attached to DOTA at various sites thereon). Similarly, the parentheses on the right side of formula 2A are intended to convey that the specific site on the ring of "T" to which the Cm group is attached is not limited (i.e., Cm can be attached to T at various sites on the ring). Again, as mentioned above, CG-L2 may or may not be present. In some embodiments, the benzene ring of T of formula 2A includes four substituents (including linkages MC, L2-MMCM, L3-Y, and L2-CG). In other embodiments, the benzene ring of T of formula 2A includes three substituents (including linkages MC, L2-MMCM, and L3-Y). The latter embodiment is particularly relevant when linkage MC is charged (i.e., it can replace the provision of the desired functionality of the "L2-CG" substituent).

[0066] In some embodiments, MC is an acyclic ligand consisting of a chain containing six or more atoms, and has at least three negatively charged substituents, such as carboxyl or phosphonate groups. Exemplary acyclic ligands suitable as MC groups include diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetramethylenephosphonic acid (EDTMP) and ethylenediaminetetraacetic acid (EDTA). An example of a class II compound is shown in Formula 2B below, where MC is an acyclic ligand that includes DTPA, and the radioactive halogenated template T is a moiety corresponding to Formula 1. [ka] Exemplary Class II Compounds with Formula 2B: DTPA(Acyclic)MC

[0067] As mentioned above with respect to formula 2A, the left bracket of formula 2B is intended to convey that the specific site on MC (DTPA) to which the Cm group is attached is not limited (i.e., Cm can be attached to DTPA at various sites thereon). Similarly, the right bracket of formula 2B is intended to convey that the specific site on the ring of "T" to which the Cm group is attached is not limited (i.e., Cm can be attached to T at various sites on the ring). Again, as mentioned above, CG-L2 may or may not be present. In some embodiments, the benzene ring of T of formula 2B includes four substituents (including linkages MC, L2-MMCM, L3-Y, and L2-CG). In other embodiments, the benzene ring of T of formula 2A includes three substituents (including linkages MC, L2-MMCM, and L3-Y). The latter embodiment is particularly relevant when linkage MC is charged (i.e., it can replace the provision of the desired function of the "L2-CG" substituent).

[0068] In some particular embodiments, MC=DOTA, L4=-NH(CH2)6NH-, T=3-iodo-5-succinimidyloxycarbonyl-benzoyl, Cm=amide, and MMCM=N-hydroxysuccinimide ester, maleimide-containing moiety, or (Gly) for site-specific conjugation using sortase. n There is provided a compound of formula 2, wherein:

[0069] The above formulas containing MMCM can be further functionalized with an attachment macromolecule (e.g., a biomolecule), and as such, in some embodiments, encompasses compounds of any of the formulas provided herein above that further include a macromolecule (e.g., a biomolecule) coordinated thereto via an MMCM. Thus, the present disclosure encompasses intermediates (including radioligand precursors and biomolecules) and radiolabeled biomolecules (including prosthetic groups and biomolecules), both of which may or may not include a metal chelating moiety.

[0070] The present disclosure further provides methods of synthesizing the prosthetic compound and radiolabeled biomolecules described herein. In some embodiments, the methods generally include preparing a compound of formula 1, where Y=alkyl metal radioactive halogen precursor. In certain embodiments, the methods generally include preparing a compound of formula 2, where Y=alkyl metal radioactive halogen precursor. In some embodiments, the use of such precursors allows for the preparation of larger radioactive "Y" groups, such as, but not limited to, 18 Larger than F 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I, 131 I and 211 This allows for the preparation of prosthetic compounds and radiolabeled biomolecules that contain At. In some embodiments, a macromolecule can be coordinated to MMCM, where Y is in the form of an alkylmetal radioactive halogen precursor; subsequent reactions then provide products where Y is in the form of the desired radioactive halogen atom. Definition:

[0071] "C m -C n Alkyl" by itself or C m -C n Haloalkyl, C m -C n Alkyl carbonyl, C m -C nCompound expressions such as alkylamines represent straight or branched aliphatic hydrocarbon radicals having the specified number of carbon atoms, for example, C1-C4 alkyl means an alkyl radical having 1 to 4 carbon atoms. C1-C6 alkyl has the corresponding meaning, including all straight and branched isomers of pentyl and hexyl. Preferred alkyl radicals for use in the present invention are C1-C6 alkyl, including methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl and n-hexyl, in particular C1-C4 alkyl, such as methyl, ethyl, n-propyl, isopropyl, t-butyl, n-butyl and isobutyl. Typically, methyl and isopropyl are preferred. Alkyl groups can be unsubstituted or substituted with one or more substituents, which can be the same or different, each of which is independently selected from halo, alkenyl, alkynyl, aryl, cycloalkyl, cyano, hydroxy, -O-alkyl, -O-aryl, -alkylene-O-alkyl, alkylthio, -NH, -NH(alkyl), -N(alkyl), -NH(cycloalkyl), -OC(=O)-alkyl, -OC(=O)-aryl, -OC(=O)-cycloalkyl, -C(=O)OH, and -C(=O)O-alkyl. Unless otherwise indicated, it is generally preferred that alkyl groups are unsubstituted.

[0072] "C2-C n"Alkenyl" refers to a straight-chain or branched aliphatic hydrocarbon radical containing at least one carbon-carbon double bond and having the specified number of carbon atoms, e.g., C2-C4 alkenyl means an alkenyl radical having from 2 to 4 carbon atoms; C2-C6 alkenyl means an alkenyl radical having from 2 to 6 carbon atoms. Non-limiting alkenyl groups include ethenyl, propenyl, n-butenyl, 3-methylbut-2-enyl, n-pentenyl and hexenyl. An alkenyl group can be unsubstituted or substituted with one or more substituents, which can be the same or different, each of which is independently selected from halo, alkenyl, alkynyl, aryl, cycloalkyl, cyano, hydroxy, -O-alkyl, -O-aryl, -alkylene-O-alkyl, alkylthio, -NH, -NH(alkyl), -N(alkyl), -NH(cycloalkyl), -OC(=O)-alkyl, -OC(=O)-aryl, -OC(=O)-cycloalkyl, -C(=O)OH, and -C(=O)O-alkyl. Unless otherwise indicated, it is generally preferred that an alkenyl group is unsubstituted.

[0073] "C2-C n"Alkynyl" refers to a straight-chain or branched aliphatic hydrocarbon radical containing at least one carbon-carbon triple bond and having the specified number of carbon atoms, e.g., C2-C4 alkynyl means an alkynyl radical having from 2 to 4 carbon atoms; C2-C6 alkynyl means an alkynyl radical having from 2 to 6 carbon atoms. Non-limiting alkenyl groups include ethynyl, propynyl, 2-butynyl and 3-methylbutynyl, pentynyl and hexynyl. Alkynyl groups can be unsubstituted or substituted with one or more substituents, which can be the same or different, each substituent being independently selected from halo, alkenyl, alkynyl, aryl, cycloalkyl, cyano, hydroxy, -O-alkyl, -O-aryl, -alkylene-O-alkyl, alkylthio, -NH, -NH(alkyl), -N(alkyl), -NH(cycloalkyl), -OC(O)-alkyl, -OC(O)-aryl, -OC(O)-cycloalkyl, -C(O)OH, and -C(O)O-alkyl. Unless otherwise indicated, it is generally preferred that alkynyl groups are unsubstituted.

[0074] As used herein, "C m -C n The term "haloalkyl" refers to an alkyl group in which at least one C atom is a halogen (e.g., C m -C n Haloalkyl groups may contain 1 to 3 halogen atoms, preferably C substituted with iodine, bromine or fluorine. m -C n Represents an alkyl group. Typical haloalkyl groups are C 1- C2 haloalkyl, where halo represents iodo. Exemplary haloalkyl groups include iodomethyl, diiodomethyl and triiodomethyl. As used herein, only one of the halogens may be radioactive.

[0075] As used herein, "C m -C n The term "hydroxyalkyl" refers to a C alkyl group in which at least one C atom is substituted with a hydroxy group.m -C n Represents an alkyl group. Typical C m -C n Hydroxyalkyl groups are C alkyl groups in which one C atom is replaced by one hydroxyl group. m -C n Exemplary hydroxyalkyl groups include hydroxymethyl and hydroxyethyl.

[0076] As used herein, "C m -C n The term "alkylene" refers to a straight-chain or branched divalent alkyl radical having the indicated number of carbon atoms. m -C n An alkylene radical is a C1-C3 alkylene. Non-limiting examples of alkylene groups include -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH(CH3)CH2CH2-, -CH(CH3)-, and -CH(CH(CH3)2)-.

[0077] "C m -C n "Alkoxy" is the radical C m -C n Alkyl-O-(C m -C n Alkyl is as defined above. Of particular interest is C1-C4 alkoxy, including methoxy, ethoxy, n-propoxy, isopropoxy, t-butoxy, n-butoxy, sec-butoxy and isobutoxy. Typically, methoxy and isopropoxy are preferred. C1-C6 alkoxy has the corresponding meaning and is extended to include all straight and branched isomers of pentoxy and hexoxy.

[0078] The term "Me" means methyl and "MeO" means methoxy. The term "amino" refers to the radical -NH2. The term "halo" refers to a halogen radical, such as fluoro, chloro, bromo, iodo or astat. Typically, halo groups are iodo, bromo or astat. The term "aryl" refers to an aromatic ring, such as a phenyl, biphenyl or naphthyl group.

[0079] The term "heterocycloalkyl" refers to a stable saturated monocyclic 3- to 12-membered ring containing 1-4 heteroatoms independently selected from O, S, and N. In one embodiment, the stable saturated monocyclic 3- to 12-membered ring contains 4 N heteroatoms. In a second embodiment, the stable saturated monocyclic 3- to 12-membered ring contains 2 heteroatoms independently selected from O, S, and N. In a third embodiment, the stable saturated monocyclic 3- to 12-membered ring contains 3 heteroatoms independently selected from O, S, and N. Heterocycloalkyl groups can be unsubstituted or substituted with one or more substituents, which can be the same or different, each of which is selected from the group consisting of halo, alkenyl, alkynyl, aryl, cycloalkyl, cyano, hydroxy, -O-alkyl, -O-aryl, -alkylene-O-alkyl, alkylthio, -NH, -NH(alkyl), -N(alkyl), -NH(cycloalkyl), -OC(O)-alkyl, -OC(O)-aryl, -OC(O)-cycloalkyl, -C(O)OH, and -C(O)O-alkyl. Unless otherwise specified, it is generally preferred that heterocycloalkyl groups are unsubstituted.

[0080] The term "heteroaryl" refers to a stable aromatic ring containing from 1 to 4 heteroatoms independently selected from O, S, and N. In a preferred embodiment, the heteroaryl moieties useful in this disclosure have 6 ring atoms. In one embodiment of the present invention, the stable aromatic ring system contains one heteroatom which is N.

[0081] "Amino C m -C nThe term "alkyl" refers to a C alkyl group, as defined above, substituted with an amino group (i.e., one hydrogen atom of the alkyl moiety is replaced with an NH group). m -C n Represents an alkyl group. Typically, "amino C m -C n "Alkyl" is amino C1-C6 alkyl.

[0082] "Amino C m -C n The term "alkylcarbonyl" refers to a C alkyl group as defined above in which one hydrogen atom of the alkyl moiety is replaced by an NH group. m -C n It represents an alkylcarbonyl radical. Typically, it is represented by "amino C m -C n "Alkylcarbonyl" is amino C1-C6 alkylcarbonyl. Amino C m -C n Examples of alkylcarbonyl include, but are not limited to, glycyl: C(=O)CH2NH2, alanyl: C(=O)CH(NH2)CH3, valinyl: C=OCH(NH2)CH(CH3)2, leucinyl: C(=O)CH(NH2)(CH2)3CH3, isoleucinyl: C(=O)CH(NH2)CH(CH3)(CH2CH3) and norleucinyl: C(=O)CH(NH2)(CH2)3CH3, etc. This definition is not limited to naturally occurring amino acids.

[0083] Relevant terms are to be interpreted in accordance with the definitions provided above and common usage in the art.

[0084] As used herein, the term "(=O)" forms a carbonyl moiety when attached to a carbon atom. It should be noted that an atom can only bear an oxo group if the atom's valence allows. The term "monophosphate, diphosphate and triphosphate esters" refers to the groups: [ka] Refers to...

[0085] The term "thiomonophosphate, thiodiphosphate and thiotriphosphate" refers to the groups: [ka] Refers to...

[0086] As used herein, the radical positions on any molecular moiety used in a definition may be anywhere on such moiety so long as it is chemically stable. When any variable occurs more than one time in any moiety, each definition is independent.

[0087] Whenever used herein, "compounds of formula 1", "compounds of formula 1A", "compounds of formula 2" or "compounds of the invention" or similar phrases are meant to include the compounds of formula 1 and the subgroup of compounds of formula 1, the compounds of formula 2 and the subgroup of compounds of formula 2 (including possible stereochemical isomers) and the pharma- ceutically acceptable salts and solvates thereof.

[0088] The term "solvate" covers any pharma- ceutically acceptable solvates that the compounds of formula 1 and 2 and their salts can form. Such solvates are, for example, hydrates, alcoholates, such as ethanolates, propanolates, etc., especially hydrates.

[0089] Generally, the compound names used in this application are generated using ChemDraw Professional 16.0. Additionally, if the stereochemistry of a structure or portion of a structure is not shown, for example, in bold or dashed lines, the structure or portion of the structure should be interpreted as encompassing all stereoisomers thereof.

[0090] Linkers may also be selected to facilitate attachment of each moiety to the core structure. For example, as discussed in more detail below with respect to the preferred synthetic routes of prosthetic compounds, representative linkers are bifunctional alkyl chains (e.g., -CH2-, -C2H4-, -C3H6-, etc.) having 1-6 carbon atoms (one of which may be replaced with a cyclic (hydrocarbon ring) or heterocyclic (heterocyclic) radical). Representative heterocyclic radicals have at least one nitrogen atom in the heterocyclic ring. Thus, specific examples of such heterocyclic radicals are diazinyl, diazolyl, triazinyl, triazolyl, tetrazinyl and tetrazolyl radicals. These and other heterocyclic or other cyclic radicals may be optionally fused to another cyclic or heterocyclic radical, or may be fused to another cyclic or heterocyclic radical that is itself part of a fused ring system (e.g., a triazolyl radical may be fused to an eight-membered ring or heterocyclic radical that is itself fused to two six-membered rings, such as when a triazolyl radical (or other nitrogen atom-substituted heterocyclic hydrocarbon radical) is fused to a dibenzoazocanyl radical). Thus, linkers containing three or more fused rings, such as hydrocarbon rings, heterocycles, and combinations of these rings, are possible. Exemplary charged group linkers L2 are divalent substituted or unsubstituted alkyl chains, substituted or unsubstituted alkenyl chains, or substituted or unsubstituted alkynyl chains having 1 to 6 carbon atoms. In general, L1, L2, L3 and / or L4 can be (can include) a substituted or unsubstituted divalent alkyl radical having 1 to 6 carbon atoms, where one or more carbon atoms can be substituted and / or replaced with a heteroatom, such as NH, O or S, or with another alkyl radical having 1 to 8 carbon atoms (e.g., resulting in the formation of a branched alkyl radical), which can be linear, branched or cyclic. For example, one carbon atom of an alkyl radical can be substituted to provide a carbonyl (C=O) group and the adjacent carbon atom can be replaced with NH, thereby resulting in a peptide / amide bond -(C=O)-NH-.Thus, representative linkers L1, L2, L3 and L4 may include divalent alkyl radicals having one or more of such peptide bonds, -NH- bonds, -(C=O)- bonds and / or cyclic -CH- bonds (including combinations of any two, three or four of such bonds) incorporated into the alkyl chain. In addition, in the case of divalent alkyl radicals of L1, L2 and / or L3, carbon-carbon double bonds and / or carbon-carbon triple bonds may be formed between one or more pairs of adjacent carbon atoms to provide divalent unsaturated (e.g., olefinic) alkyl radicals.

[0091] The selection of an appropriate labeling method for a biomolecule requires careful consideration of the molecular fate after interaction with the biological environment. In the case of radioactively iodized proteins and peptides, avoiding the action of deiodinase enzymes, such as those normally involved in thyroid hormone metabolism, is a major concern. N-Succinimidyl 3-[ 131 Reagents such as [I]iodobenzoate (SIB) result in proteins that do not undergo appreciable deiodination in vivo, based on the non-tyrosine analog structure of the site where the radiolabel resides. However, when the labeled protein or peptide undergoes cellular internalization after binding to a cell surface receptor or antigen, depending on its intracellular pathway, even with SIB labeling, there can be a significant loss of label from the target cell.

[0092] In some embodiments, the targeted radiation therapy methods of the present invention may utilize radioactive halogens that emit radiation at a tissue range of less than 15 mm. These include alpha emitters, e.g. 211 At, beta emitters, e.g. 131 I and Auger electron emitters, e.g. 77 Br, 123 I and 125 I. Radioactive halogens, e.g. 75 Br, 76 Br, 124 positron emission tomography (PET) using radioactive halogens, e.g. 123 I, 131 I and 77The diagnostic imaging methods of the present invention utilize radiation at tissue ranges greater than 5 mm such that radiation can be detected outside the body by single photon emission computed tomography (SPECT) using Br or the like; or intraoperative imaging, which may be performed using any of the above radioactive halogens. See U.S. Patent No. 5,302,700, which is incorporated herein by reference. In particular, 131 I emits low energy beta particles with a maximum tissue range of 2.3 mm. Stein et al. (2003) Cancer Res 63:111-118, which is incorporated herein by reference. Theranostic methods of the present invention can be used to 1) utilize the same radioactive halogen to perform targeted radiotherapy and diagnostic imaging (e.g., 131 I, 123 I, 77 Br), or 2) utilize different radioactive halogens of the same element to perform targeted radiotherapy and diagnostic imaging (e.g. 124 I and 131 I; 123 I and 131 I; 77 Br and 76 Br; 77 Br and 75 Br, etc. In some embodiments (eg, using compounds of formula 2), other radiometals that are attached to the metal chelating portion of the molecule may be used.

[0093] Exemplary biomolecules that can be coupled to the radiolabeled prosthetic compound include molecules that specifically bind to cell surface receptors, antigens, or transporters. Exemplary cell surface antigens or receptors include those that are internalized by cells. Biomolecules are internalized by cells over a period of seconds, minutes, hours, or days. Preferred biomolecules are rapidly internalized (i.e., most biomolecules are internalized after a few minutes to a few hours). Biomolecules are internalized within 10 -6 M or less, preferably 10 -8 M -1 or less than the affinity constant (K D ) is considered to be specifically binding.

[0094] The biomolecule may be an antibody, an antibody fragment or a synthetic peptide that specifically binds to a cell surface antigen, receptor or transporter. Antibodies include monoclonal antibodies (mAbs) and antibody fragments include VHH molecules (also known as single domain antibody fragments (sdAbs) or nanobodies). In a preferred embodiment, the biomolecule is an internalizing antibody or antibody fragment. Any antibody that specifically binds to a cell surface antigen and is internalized by the cell is an internalizing antibody. The antibody may be an immunoglobulin of any class (i.e., IgG, IgA, IgD, IgE or IgM) and may be obtained by immunization of a mammal, such as a mouse, rat, rabbit, goat, sheep, primate, human or other suitable species, including those of the Camelidae family. The antibody may be polyclonal (i.e., obtained from the serum of an animal immunized with a cell surface antigen or a fragment thereof). The antibody can also be monoclonal (i.e., formed by immunizing a mammal with a cell membrane or surface ligand or antigen or a fragment thereof, fusing lymphoid or splenic cells from the immunized mammal with a myeloma cell line, and isolating a specific hybridoma clone, as known in the art). The antibody can also be a recombinant antibody, e.g., a chimeric or interspecies antibody produced by recombinant DNA techniques. A preferred internalizing antibody is a humanized antibody that contains a human immunoglobulin constant region together with a mouse variable region that has specificity for binding to a cell surface antigen (see, e.g., Reist et al., 1997). When a fragment of an antibody is used, the fragment should be able to specifically bind to the cell surface antigen. The fragment can, for example, contain at least a portion of an immunoglobulin light chain variable region and at least a portion of an immunoglobulin heavy chain variable region. The biomolecule can also be a synthetic polypeptide that specifically binds to a cell surface antigen. For example, a biomolecule can be a synthetic polypeptide that includes at least a portion of an immunoglobulin light chain variable region and at least a portion of an immunoglobulin heavy chain variable region, as described in U.S. Pat. No. 5,260,203 or as otherwise known in the art.

[0095] Many of the known molecular targets of labeled mAbs are internalizing antigens and receptors. B cell lymphomas (Press et al., 1994; Hansen et al., 1996), T cell leukemias (Geissler et al., 1991) and neuroblastoma cells (Novak-Hofer et al., 1994) all have antigens that are rapidly internalized. Internalizing receptors have been used to target mAbs to tumors. These include wild-type epidermal growth factor receptor (EGFR; gliomas and squamous cell carcinomas; Brady et al., 1992; Baselga et al., 1994), the p185 c-erbB-2 oncogene product HER2 (breast and ovarian carcinomas; De These include the receptor for mAbs that bind to cell surface antigens (Santes et al., 1992; Xu et al., 1997) and transferrin receptors (gliomas and other tumors; Laske et al., 1997). Indeed, it has been suggested that internalization can occur with virtually any mAb that binds to a cell surface antigen (Mattes et al., 1994; Sharkey et al., 1997a).

[0096] The advantage of mAb internalization for radioimmunotherapy is that it has the potential to increase the radiation absorbed dose delivered to the cell nucleus, as long as the radioactivity is trapped on the target cell for an extended period of time. Radiation dosimetry calculations are performed using multicellular range beta-emitters. 131 Even I suggests that a shift in the site of disintegration from the cell membrane to cytoplasmic vesicles could increase the radiation dose received by the cell nucleus by two-fold (Daghighian et al., 1996), thereby potentially increasing treatment. On the other hand, the drawback of mAb internalization is that this event exposes the mAb to further catabolic processes, which may result in the release of radioactivity from the tumor cells, a reduction in the radiation dose to the cancer cells, and an increase in the radiation dose to normal tissues in the body.

[0097] The antigen or receptor that is internalized by cells can be eventually localized in endosomes or lysosomes.Targeting or internalization moiety is a moiety that binds to target diseased cells, such as cancer cells, and is internalized after binding to cell surface receptors, transporters, antigens found on cell surface, such as transmembrane receptors, extracellular growth factors.In this way, the compound of the present invention can target any population of diseased cells or tumor cells.Therefore, it can be widely used to target any cancer, tumor or malignant growth. The compounds of the present invention are capable of inhibiting the functions of human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), its tumor-specific mutant EGFRvIII, vascular endothelial growth factor (VEGF), VEGFA / B, EGFR (HER1 / ERBB1), HER2 (ERBB2 / neu), ALK, Ax1, CD20, CD30, CD38, CD47, CD52, CDK4, CDK6, PD-1, PD-L1, KIT, VEGFR1 / 2 / 3, BAFF, HDAC, proteasome, ABL, FLT3, KIT, MET, RET, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-36, IL-19, IL-19, IL-19, IL-25, IL-19, IL-26, IL-27, IL-37, IL-19, IL-19, IL-28, IL-29, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-45, IL-55, IL-56, IL-57, IL-58, IL-59, IL-60, IL-61, IL-72, IL-83, IL-84, IL-85, IL-86, IL- -6, IL-6R, IL-1β, EGFR (HER1 / ERBB1), MEK, ROS1, BRAF, ABL, RANKL, B4GALNT1 (GD2), SLAMF7, (CS1 / CD319 / CRACC), mTOR, BTK, P13Kδ, PDGFR, PDGFRα, PDGFRβ, CTLA4, PARP, HDAC, FGFR1-3, RAF, RET, JAK1 / 2, JAK3, Smoothened, MEK, BCL2, PTCH, PIGF, EMP2, CSF-1R, LYPD3, etc. See, e.g., Abramson, R. (2017) Overview for Targeted Therapies for Cancer, My Cancer Genome, available on the world wide web in the overview-of-targeted-therapies-for-cancer section of the "mycancergenome" website.

[0098] In some embodiments, the targeting moiety may be selected from anti-HER2 VHH sequences, such as those shown in SEQ ID NOs: 1-5, as well as fragments and variants thereof that retain the binding specificity of said sequences. That is, the present invention encompasses fragments, analogs, mutants, variants and derivatives of radiolabeled VHH domains. These oligoclonal VHHs may target a broad range of epitopes on the HER2 receptor. Some of the VHHs do not compete with trastuzumab for binding to HER2. In some embodiments, the fragments, analogs, mutants, variants and / or derivatives of the VHH sequences provided herein have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with at least one of SEQ ID NOs: 1-5. See Table 1.

[0099] In determining the degree of sequence identity between two amino acid sequences, one skilled in the art may consider "conservative" amino acid substitutions, which may be generally described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of similar chemical structure and has little or no effect on the function, activity or other biological properties of the polypeptide. An amino acid sequence and a nucleic acid sequence are exactly the same if they have 100% sequence identity over their entire length.

[0100] As used herein, when a sequence is defined as being "at least X% identical" to a reference sequence (e.g., "a polypeptide at least 95% identical to SEQ ID NO:2"), "X% identical" refers to the absolute percent identity unless otherwise indicated. The term "absolute percent identity" refers to the percentage of sequence identity determined by scoring identical amino acids or nucleic acids as 1 and scoring any substitutions as zero, regardless of the similarity of the mismatched amino acids or nucleic acids. In a typical sequence alignment, the "absolute percent identity" of two sequences is presented as a percentage of amino acid or nucleic acid "identity". When optimal alignment of two sequences requires the insertion of gaps in one or both of the sequences, the amino acid residues of one sequence that align with the gaps in the other sequence are counted as mismatches for purposes of determining the percent identity. Gaps can be internal or external (i.e., truncations). Absolute percent identity can be readily determined using, for example, the Clustal W program, version 1.8, June 1999 (Thompson et al. (1994) Nucleic Acids Res 22:4673-4680), using default parameters.

[0101] As shown, the radiolabeled biomolecules of the present invention can be targeted to any disease or malignant cell population. In some cases, it may be preferable to use small biomolecules. Brain metastases are cancer cells that have spread to the brain from a primary tumor in another organ in the body. Metastatic tumors are the most common mass lesions in the brain. An estimated 24-45% of all cancer patients have brain metastases. Lung, breast, melanoma, colon and kidney cancers commonly spread to the brain. Brain metastases are associated with poor survival and high morbidity. Improved treatment for metastatic brain tumors is an aspect of the present invention.

[0102] The calculated pore size of breast cancer brain metastases is less than 10 nm in diameter. (Mittapali et al., (2017) Cancer Res 77(2):238-246). Thus, small molecules are needed to effectively target and treat metastatic brain tumors. For use in the diagnosis and treatment of metastatic brain tumors, the target biomolecules of the present invention are small molecules, including but not limited to affibodies, designed ankyrin repeat proteins (DARPins), aptamers and VHH molecules (also known as single domain antibody fragments (sdAbs) or nanobodies), which are collectively referred to herein as small biomolecules. Other "small molecule" scaffolds are characterized by mass / size, e.g., less than 10 nm in size or less than 25 kDa. As shown, these small biomolecules are designed to bind to a portion of the cancer cell. For example, VHHs can be prepared to specifically bind to a receptor on the cancer cell, e.g., human epidermal growth factor receptor-2 (HER2) or any of the other receptors mentioned above. See, e.g., U.S. Patent No. 9,234,028; U.S. Patent No. 9,309,515; U.S. Patent No. 8,524,244; U.S. Patent No. 9,234,065; Liu et al., (2012) J Transl. Med. 10:148; Gijs et al., (2016) Pharmaceuticals (Basel) 9(2):29; Moosavian et al., (2015) Iran J. Basic Med. Sci. 18(6):576-586; Mahlknecht et al., (2012) Proc. Natl. Acad. Sci. 110:8170-8175.

[0103] Due to their small size, VHHs, aptamers and other small biomolecules can efficiently diffuse and distribute throughout solid tumors, and due to their high binding specificity and affinity to the target antigen, high tumor uptake of small biomolecules can be observed. Importantly, their blood half-life is significantly shorter than full-length antibodies or larger target proteins, thereby allowing rapid clearance of the unbound fraction of small biomolecules by the kidney, resulting in a higher tumor-to-normal tissue ratio immediately after their administration. VHHs are easily generated with nanomolar to picomolar affinities by cloning from immunized camels or llamas and selection by phage display panning. Also, VHHs or sdAbs are stable and easily produced in large quantities using industrial-grade methods and qualified bacteria, yeast or mammalian cells. Compared to other small protein-based targeting vectors, VHHs generally offer significant advantages in terms of stability, solubility, expression yield, assembly of multimers, and the ability to recognize hidden or uncommon epitopes. See U.S. Patent Nos. 6,248,516; 6,300,064; 6,846,634; 6,846,634; 6,696,245; 9,243,065; and 7,696,320, all of which are incorporated herein by reference.

[0104] Aptamers are oligonucleotide or peptide molecules that bind to specific targeting molecules. Aptamers can be nucleic acid molecules (DNA, RNA, XNA) or can be composed of short chains of oligonucleotides (peptide molecules that consist of one or more short variable peptide domains). Aptamers provide molecular recognition properties that are easily generated by chemical synthesis, have desirable storage properties, and induce little or no immunogenicity in therapeutic applications. See Keefe et al. (2010) Nature Reviews Drug Discovery 9:537-550; Ellington and Szostak (1990) Nature 346:818-822; Tuerk and Gold (1990) Science 249:505-510; Kulbachinskiy, AV (2007) Biochemistry 72:1505-1518 (all of which are incorporated herein by reference).

[0105] As used herein, the "(calculated average) effective dose" of radiation in a subject refers to the tissue-weighted sum of equivalent doses in all designated tissues and organs of the body. It takes into account the type of radiation and the properties of each organ or tissue irradiated. It is the central quantity of dose limitations for radiation protection in the international system of radiation protection devised by the International Commission on Radiological Protection (ICRP). The SI unit of effective dose is the Sievert (Sv), which is 1 Joule per kilogram (J / kg). Effective dose replaced the previous "effective dose equivalent" in the ICRP system of dose in 1991. In procedures using radiopharmaceuticals, the effective dose is typically expressed per unit of injected activity (i.e., mSv / MBq). Thus, the effective dose of an individual patient will depend on the injected activity of the radiopharmaceutical, expressed in MBq, and the calculated average effective dose, expressed in mSv / MBq.

[0106] The effective dose of radiopharmaceuticals is calculated using OLINDA / EXM® software, approved by the FDA in 2004. OLINDA / EXM® personal computer code performs dose calculations and kinetic modeling of radiopharmaceuticals (OLINDA / EXM stands for Organ Level Internal Dose Assessment / Exponential Modeling). OLINDA® calculates radiation doses to different organs of the body from systemically administered radiopharmaceuticals and performs regression analysis of user-provided biokinetic data to assist in such calculations for nuclear medicine drugs. These calculations are used to perform risk / benefit assessments of the use of such drugs in nuclear medicine diagnostic and therapeutic applications. The technique uses several standard body models for adults, children, pregnant women, etc. that are widely accepted and used in the field of internal dose. The calculations are useful to pharmaceutical industry developers, nuclear medicine professionals, educators, regulators, researchers, and others who study the acceptable radiation doses that should be delivered when radiopharmaceuticals are given to patients or research subjects.

[0107] The calculated effective dose depends on the standard body model selected and the voiding bladder model selected. The values ​​provided herein are calculated using a female adult model and a voiding bladder interval of 1 hour.

[0108] Thus, in certain embodiments, prevention and / or treatment of cancer is achieved by administering to a subject in need thereof a radiolabeled small biomolecule as disclosed herein, i.e., an aptamer, VHH or functional fragment thereof, characterized in that the small biomolecule has a calculated mean effective dose in the subject of 0.001-0.05 mSv / MBq, such as, but not limited to, 0.02-0.05 mSv / MBq, more preferably 0.02-0.04 mSv / MBq, and most preferably 0.03-0.05 mSv / MBq.

[0109] Thus, the dose of radioactivity delivered to the patient per administration must be high enough to be effective, but below the dose that would result in dose-limiting toxicity (DLT). 131 For pharmaceutical compositions containing iodine radiolabeled antibodies, a maximum tolerated dose (MTD) must be determined that should not be exceeded in the therapeutic setting.

[0110] The proteins and peptides (hereinafter collectively referred to as biomolecules) envisaged herein and / or compositions comprising them are administered according to a suitable treatment regimen to prevent and / or treat the disease or disorder to be prevented or treated.In general, the clinician will be able to determine the suitable treatment regimen.In general, the treatment regimen comprises administering one or more small biomolecules, such as VHH sequences or polypeptides, or one or more compositions comprising them, in one or more pharmacologic effective amounts or doses.

[0111] Conveniently, the desired dose may be presented in a single dose or as divided doses (which may also be further divided) administered at suitable intervals. The administration regimen may include chronic (i.e., at least 2 weeks, e.g., for several months or years) or daily treatment. In particular, the administration regimen may vary from once a day to once a year, e.g., once a day and once every 12 months, e.g., but not limited to, once a week. Thus, depending on the desired duration and effectiveness of the treatment, the pharmaceutical small biomolecule compositions disclosed herein may be administered once or several times, or even intermittently, e.g., daily for several days, weeks or months at different dosages. The application amount of the small biomolecule compositions disclosed herein depends on the nature of the cancer or other disease to be treated. Multiple administrations may be preferred to achieve effective radiation dose delivery to the cancer while avoiding DLT. However, the radiolabeled material is typically administered at intervals of 4 to 24 weeks, preferably at intervals of 8 to 20 weeks. Those skilled in the art will know how to divide the administration into two or more applications, which may be applied immediately following one another or at some other predetermined interval ranging, for example, from one day to one week.

[0112] In particular, the biomolecules disclosed herein may be used in combination with other medicamentously active compounds or substances that are or may be used in the prevention and / or treatment of the diseases and disorders cited herein, which may or may not result in synergistic effects. Examples of such compounds and substances, as well as routes, methods and pharmaceutical formulations or compositions for administering them, will be apparent to the clinician.

[0113] In the context of the present invention, "in combination with", "in combination therapy" or "in combination treatment" shall mean that the radiolabeled biomolecules disclosed herein, such as VHHs, aptamers, etc., are applied to the patient together with one or more other pharma- ceutical active compounds or substances, in a regimen in which the patient may benefit from the beneficial effects of such a combination. In particular, both treatments are applied to the patient in close temporal proximity. In a preferred embodiment, both treatments are applied to the patient within 4 weeks (28 days). More preferably, both treatments are applied within 2 weeks (14 days), more preferably within 1 week (7 days). In a preferred embodiment, the two treatments are applied within 2 or 3 days. In another preferred embodiment, the two treatments are applied on the same day, i.e. within 24 hours. In another embodiment, the two treatments are applied within 4 hours or within 2 hours or within 1 hour. In another embodiment, the two treatments are applied in parallel, i.e. simultaneously, or the two administrations overlap in time.

[0114] In certain non-limiting embodiments, the radiolabeled biomolecule of the present invention is applied together with one or more therapeutic antibodies or therapeutic antibody fragments. Thus, in these certain non-limiting embodiments, targeted radiotherapy using radiolabeled biomolecule is combined with conventional immunotherapy using one or more therapeutic antibodies or therapeutic antibody fragments. In further specific embodiments, the radiolabeled biomolecule is used in combination therapy or treatment methods using one or more therapeutic antibodies or therapeutic antibody fragments, such as, but not limited to, combination treatment with trastuzumab (Herceptin®) and / or pertuzumab (Perjeta®).

[0115] For example, the radiolabeled biomolecule and one or more therapeutic antibodies or therapeutic antibody fragments, such as but not limited to trastuzumab (Herceptin®) and / or pertuzumab (Perjeta®), may be infused simultaneously or the infusions may overlap in time. When the two drugs are administered simultaneously, they may be formulated together in one single pharmaceutical preparation, or they may be mixed together from two different pharmaceutical preparations immediately prior to administration, for example by dissolving or diluting in one single infusion. In another embodiment, the two drugs are administered separately, i.e., as two independent pharmaceutical compositions. In a preferred embodiment, the administration of the two treatments is such that tumor cells in the patient's body are exposed simultaneously to effective amounts of a cytotoxic drug and radiation. In another preferred embodiment, an effective amount of the radiolabeled biomolecule of the present invention and one or more therapeutic antibodies or therapeutic antibody fragments, such as but not limited to trastuzumab (Herceptin®) and / or pertuzumab (Perjeta®), are both present at the tumor site simultaneously. The present invention also encompasses the use of additional agents administered in addition to the defined combination. This may be, for example, one or more additional chemotherapeutic agents. It may also be one or more agents applied to prevent, inhibit or ameliorate undesirable side effects of any of the other drugs given. For example, cytokines that stimulate the proliferation of white blood cells may be applied to ameliorate the effects of leukopenia or neutropenia.

[0116] According to a further aspect, there is provided the use of a radiolabeled biomolecule as envisaged herein that specifically binds to a tumor-specific or cancer cell-specific target molecule of interest for the preparation of a medicament for the prevention and / or treatment of at least one cancer-related disease and / or disorder involving the tumor-specific or cancer cell-specific target molecule. Thus, the present application provides a biomolecule that specifically binds to a tumor-specific or cancer cell-specific target, such as those described above, for use in the prevention and / or treatment of at least one cancer-related disease and / or disorder involving the tumor-specific or cancer cell-specific target. In certain embodiments, there is provided a method for the prevention and / or treatment of at least one cancer-related disease and / or disorder, comprising administering to a subject in need thereof a pharmacoactive amount of one or more biomolecules as envisaged herein (including VHH sequences or functional fragments thereof, polypeptides, aptamers, etc.) and / or pharmaceutical compositions.

[0117] The subject or patient to be treated with the radiolabeled biomolecule described herein can be any warm-blooded animal, but is particularly a mammal, more particularly a human, suffering from or at risk of cancer-related disease and / or other disease disorder.The effectiveness of the biomolecule described herein (i.e., VHH sequence or its functional fragment, aptamer, polypeptide, etc.) and the composition comprising it can be tested using any suitable in vitro assay, cell-based assay, in vivo assay and / or animal model known per se, or any combination thereof, depending on the specific disease or disorder involved.The suitable assay and animal model will be clear to those skilled in the art.

[0118] Depending on the tumor-specific or cancer cell-specific target involved, one of skill in the art will generally be able to select appropriate in vitro assays, cellular assays or animal models to test the biomolecules described herein for binding to tumor-specific or cancer cell-specific molecules; and for therapeutic and / or prophylactic effects with respect to one or more cancer-related diseases and disorders.

[0119] Thus, there is provided a biomolecule comprising or consisting essentially of at least one radiolabeled biomolecule or a functional fragment thereof for use as a medicament, more particularly for use in a method for the treatment of a disease or disorder associated with cancer, including solid tumors.

[0120] In certain embodiments, the radiolabeled biomolecules contemplated herein are used to treat and / or prevent cancer and neoplastic conditions. Examples of cancer or neoplastic conditions include, but are not limited to, fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelioma, lymphangiosarcoma, lymphangioendothelioma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer, esophageal cancer, rectal cancer, pancreatic cancer, ovarian cancer, prostate cancer, uterine cancer, head and neck cancer, skin cancer, brain cancer, squamous cell carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cysts, and the like. These include adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular cancer, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, or Kaposi's sarcoma. The biomolecules contemplated herein may also be used to treat a variety of proliferative disorders.Examples of proliferative disorders include hematopoietic neoplastic disorders and cell proliferation and / or differentiation disorders, including, but not limited to, epithelial hyperplasia, sclerosing adenoma, and ductal papilloma; tumors, including stromal tumors, such as fibroadenoma, phyllodes tumor, and sarcoma, and epithelial tumors, such as ductal papilloma; carcinomas of the breast, including intraepithelial (non-invasive) carcinomas, such as ductal carcinoma in situ (including Paget's disease) and lobular carcinoma in situ, and invasive (invasive) carcinomas, including, but not limited to, invasive ductal carcinoma, invasive lobular carcinoma, medullary carcinoma, colloid (mucinous) carcinoma, tubular carcinoma, invasive papillary carcinoma, other malignant neoplasms, gynecomastia carcinoma, bronchial carcinomas, including paraneoplastic syndromes, bronchioloalveolar carcinoma, neuroendocrine tumors, such as bronchial carcinoids, other tumors, and metastatic tumors; Membrane pathologies such as inflammatory pleural effusion, noninflammatory pleural effusion, pneumothorax and pleural tumors such as solitary fibrous tumor (pleural fibroma), malignant mesothelioma, non-neoplastic polyp, adenoma, familial syndromes, colorectal carcinogenesis, colorectal carcinoma, carcinoid tumor, nodular hyperplasia, adenoma and malignant tumors such as primary and metastatic tumors of the liver, tumors of the coelomic epithelium, serous tumors, mucinous tumors, endometrioid tumors, clear cell adenocarcinoma, cystadenofibroma, Brenner tumor, surface epithelial tumors; germ cell tumors such as mature (benign) teratomas, monodermal teratomas, immature malignant teratomas, germinomas, endodermal sinus tumors, choriocarcinomas; sex cord stromal tumors such as granulosa theca cell tumor, theca cell fibroma, androblastoma, leech cell tumor and gonadoblastoma; and metastatic tumors such as Krukenberg tumor.

[0121] Imaging of radioactivity after administration of a biomolecule labeled with the claimed prosthetic compound can be performed by standard radiological methods, including, for example, scanning the body with a gamma camera (radioscintigraphy), single photon emission computed tomography (SPECT) and positron emission tomography (PET) (see, for example, Bradwell et al., Immunology Today 6:163-170, 1985). For in vivo use, the labeled prosthetic compound coupled to the biomolecule should be given in a diagnostically or therapeutically acceptable amount. A therapeutically acceptable amount is an amount that, when administered in one or more doses, produces the desired therapeutic effect, e.g., tumor shrinkage, with a toxicity level acceptable for clinical treatment. Such a dose will cause sufficient absorption of radiation in tumor cells to damage these cells, e.g., by breaking DNA. Such a dose should preferably cause minimal damage to adjacent and distant healthy cells.

[0122] Both the dose of a particular composition and the means of administration of the composition can be determined based on the particular quality of the composition, the symptoms, age and weight of the patient, the progression of the particular disease being treated, and other relevant factors. When the composition contains an antibody, the effective dosage of the composition is within the range of about 5 μg to about 50 μg / kg of patient body weight, about 50 μg to about 5 mg / kg, about 100 μg to about 500 μg / kg of patient body weight, and about 200 to about 250 μg / kg. A diagnostically acceptable amount of radioactivity is an amount that allows detection of radioactivity from a labeled biomolecule required for diagnosis at a toxicity level acceptable for diagnosis.

[0123] Various embodiments are provided herein below.

[0124] Embodiment 1: A compound represented by formula I (including a prosthetic compound or a radioactive halogen precursor): [ka] (In the formula, X is CH or N; L1 and L3 are independently selected from a bond, a substituted or unsubstituted alkyl chain, a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain, and a polyethylene glycol (PEG) chain; MMCM is a polymeric conjugate moiety; L2 is a substituted or unsubstituted alkyl chain, a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain, or a polyethylene glycol (PEG) chain containing at least three oxygen atoms, where L2 optionally contains a brush border enzyme cleavable peptide; CG is selected from guanidine; PO3H; SO3H; one or more charged D-amino acids, arginine or phosphono / sulfophenylalanine, glutamate, aspartate, lysine; a hydrophilic carbohydrate moiety; a polyethylene glycol (PEG) chain; and guanidino-Z; Z is (CH2) n and; n is greater than 1; and Y is an alkylmetal radioactive halogen precursor; or 18 F, 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I, 131 I and 211 At) or a pharma- ceutically acceptable salt or solvate thereof.

[0125] Embodiment 2: The compound of embodiment 1, wherein Y is an alkylmetal radioactive halogen precursor selected from the group consisting of trimethylstannyl (SnMe3), tri-n-butylstannyl (SnBu3), and trimethylsilyl (SiMe3).

[0126] Embodiment 3: Y is 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I, 131I and 211 The compound of embodiment 1, wherein R is a radioactive halogen selected from the group consisting of At.

[0127] Embodiment 4: The MMCM is an active ester or (Gly) m The compound of any of embodiments 1-3, wherein m is 1 or more.

[0128] Embodiment 5: The compound of any one of embodiments 1 to 3, wherein the MMCM is selected from the group consisting of N-hydroxysuccinimide (NHS), a tetrafluorophenol (TFP) ester, an isothiocyanate group, or a maleimide group.

[0129] Embodiment 6: The compound of any one of embodiments 1-3, wherein MMCM is Gly-Gly-Gly.

[0130] Embodiment 7: L2 is (CH2) p The compound according to any one of embodiments 1 to 6, wherein (p=1 to 6).

[0131] Embodiment 8: The compound of any one of embodiments 1-7, wherein the optional brush border enzyme-cleavable peptide is selected from the group consisting of Gly-Lys, Gly-Tyr, and Gly-Phe-Lys.

[0132] Embodiment 9: A compound having the following structure: [ka] The compound according to any one of embodiments 1 to 8, wherein the compound is represented by:

[0133] Embodiment 10: N-Succinimidyl 3-guanidinomethyl-5-[ 131 I]iodobenzoate or N-succinimidyl 3-[ 211 At]astat-5-guanidinomethylbenzoate.

[0134] Embodiment 11: A radiolabeled biomolecule or intermediate comprising a compound according to any one of embodiments 1 to 10 attached to a biomolecule.

[0135] Embodiment 12: A radiolabeled biomolecule or intermediate according to embodiment 11, wherein said biomolecule is selected from the group consisting of an antibody, an antibody fragment, a VHH molecule, an aptamer or a variant thereof.

[0136] Embodiment 13: A radiolabeled biomolecule or intermediate according to embodiment 11 or 12, wherein the labeled biomolecule is a VHH.

[0137] Embodiment 14: The radiolabeled biomolecule or intermediate of embodiment 13, wherein said VHH targets HER2.

[0138] Embodiment 15: A radiolabeled biomolecule or intermediate according to embodiment 14, wherein said VHH comprises an amino acid sequence selected from the sequences set forth in SEQ ID NOs: 1 to 5.

[0139] Embodiment 16: A pharmaceutical composition comprising a radiolabeled biomolecule according to any of embodiments 11 to 15, wherein said compound is in the form of a prosthetic compound, in association with a pharma- ceutically acceptable adjuvant, diluent or carrier.

[0140] Embodiment 17: A compound represented by formula 2 (including a prosthetic compound or a radioactive halogen precursor): [ka] (In the formula, MC is a multidentate metal chelating moiety; Cm is a thiourea, amide, or thioether; L4 is selected from a bond, a substituted or unsubstituted alkyl chain, a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain optionally having NH, CO or S at one or both termini, and a polyethylene glycol (PEG) chain; and T is a compound according to any one of embodiments 1-10), or a pharma- ceutically acceptable salt or solvate thereof.

[0141] Embodiment 18: The compound of embodiment 17, wherein MC is a macrocyclic structure.

[0142] Embodiment 19: The compound of embodiment 17, wherein MC is selected from DOTA, TETA, NOTP and NOTA.

[0143] Embodiment 20: The compound of embodiment 17, wherein MC is an acyclic polydentate ligand.

[0144] Embodiment 21: The compound of embodiment 17, wherein MC is selected from EDTA, EDTMP and DTPA.

[0145] Embodiment 22: The compound of any one of embodiments 17-21, further comprising a metal bound to said MC.

[0146] Embodiment 23: The metal is: 177 Lu, 64 Cu, 111 In, 90 Y, 225 Ac, 213 Bi, 212 Pb, 212 Bi, 67 Ga, 68 Ga, 89 Zr and 227 The compound of embodiment 21, selected from the group consisting of Th.

[0147] Embodiment 24: The compound of any one of embodiments 17-23, wherein Y is an alkylmetal moiety (and said compound is a radioactive halogen precursor).

[0148] Embodiment 25: The compound of embodiment 24, wherein the alkylmetal moiety is selected from the group consisting of trimethylstannyl (SnMe3), tri-n-butylstannyl (SnBu3), and trimethylsilyl (SiMe3).

[0149] Embodiment 26: Y is a radioactive halogen, e.g. 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I, 131 I or 211 The compound according to any one of embodiments 17 to 23, wherein At is At (and said compound is a prosthetic compound).

[0150] Embodiment 27: A radiolabeled biomolecule or intermediate comprising a compound according to any one of embodiments 17 to 26 attached to a biomolecule.

[0151] Embodiment 28: A radiolabeled biomolecule or intermediate according to embodiment 27, wherein said biomolecule is selected from the group consisting of an antibody, an antibody fragment, a VHH molecule and an aptamer.

[0152] Embodiment 29: The radiolabeled biomolecule or intermediate of embodiment 27, wherein the labeled biomolecule is a VHH.

[0153] Embodiment 30: The radiolabeled biomolecule or intermediate of embodiment 29, wherein said VHH targets HER2.

[0154] Embodiment 31: A radiolabeled biomolecule or intermediate according to embodiment 30, wherein said VHH comprises an amino acid sequence selected from the sequences set forth in SEQ ID NOs: 1 to 5.

[0155] Embodiment 32: A pharmaceutical composition comprising a radiolabeled biomolecule according to any of embodiments 27 to 31, wherein said compound is a prosthetic compound, in association with a pharma- ceutically acceptable adjuvant, diluent or carrier.

[0156] Embodiment 33: A method for treating cancer, comprising administering to an individual in need thereof an effective amount of a radiolabeled biomolecule according to any one of embodiments 11-15 or 27-31, or an effective amount of the pharmaceutical composition according to embodiment 16 or 32.

[0157] The present disclosure includes not only any combination of two, three, four or more of the above embodiments, but also combinations of any two, three, four or more features or elements described in this disclosure, regardless of whether such features or elements are explicitly combined in a particular embodiment herein.

[0158] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

[0159] experiment Example 1: SIB-Arg [ka] Scheme 1: Synthetic approach to the SIB-Arg standard [ka] Scheme 2: Synthetic approach to the tin precursor of SIB-Arg [ka] Scheme 3: Synthetic approach to radiohalogenated SIB-Arg

[0160] A solution of D-arginine (174.2 mg; 1 mmol in 3.5 ml) in 0.1 M sodium carbonate buffer (pH 8.5) is slowly added to a solution of bis(2,5-dioxopyrrolidin-1-yl) 5-iodoisophthalate (486.2 mg; 1 mmol) in tetrahydrofuran (THF; 5.0 ml). The mixture is stirred at room temperature and the progress of the reaction is followed by thin layer chromatography (TLC). After evaporation of the solvent, the crude material is subjected to reversed-phase semi-preparative high performance liquid chromatography (HPLC). Following the same procedure, 1 mmol (524 mg) of bis(2,5-dioxopyrrolidin-1-yl) 5-(trimethylstannyl)isophthalate is conjugated with 1 mmol of D-arginine. The tin precursor is radiohalogenated, purified and then conjugated to the polymer using standard conditions. Example 2: Arg-Gly-Tyr-PEG-SIB

[0161] A molecule containing the guanidine-bearing amino acid arginine, a brush border enzyme-cleavable linker dipeptide GlyTyr, connected to a SIB moiety via a PEG linker (Arg-Gly-Tyr-PEG-SIB) is shown in Schemes 4-6. Using standard iododestannylation reactions, radiolabeled versions of this molecule, such as Arg-Gly-Tyr-PEG-[ 131 I]SIB is obtained from the corresponding tin precursor. [ka] Scheme 4: Synthesis of Arg-Gly-Tyr-PEG-SIB standard [ka] Scheme 5: Synthesis of the tin precursor of Arg-Gly-Tyr-PEG-SIB [ka] Scheme 6: Synthesis of radiohalogenated Arg-Gly-Tyr-PEG-SIB

[0162] N-acetylargininyl-glycyl-tyrosine is synthesized by solid-phase peptide synthesis and coupled to PEG diamine (n=2-4). Alternatively, PEG diamine can be anchored to trityl chloride resin and three amino acids can be attached consecutively. The resulting peptide derivative (1 mmol) is reacted with a mixture of THF and 0.1 M sodium carbonate buffer (pH 8.5) containing bis(2,5-dioxopyrrolidin-1-yl) 5-iodoisophthalate (486.2 mg; 1 mmol). The progress of the reaction is followed by reversed-phase HPLC and, once complete, the product is isolated by reversed-phase semi-preparative HPLC. The tin precursor is synthesized in a similar manner by replacing bis(2,5-dioxopyrrolidin-1-yl) 5-iodoisophthalate with bis(2,5-dioxopyrrolidin-1-yl) 5-(trimethylstannyl)isophthalate. The tin precursor is radiohalogenated and purified for conjugation to macromolecules using standard conditions. Example 3: DOTA-PEG-SIB

[0163] The synthesis scheme of DOTA-PEG-SIB is shown in Scheme 7. The same approach can be used to synthesize the tin precursor. Standard conditions can be used to label the tin precursor with radioactive iodine; the DOTA moiety present in both the iodo and tin derivatives can be complexed with non-radioactive lutetium. Unlike SIB-DOTA (Vaidyanathan et al., (2012) Bioorg. Med. Chem. 20(24):6929-6939), all four COOH groups of the DOTA macrocycle are available for complexation with metal ions, and a PEG linker replaces the hydrophobic six-carbon alkyl chain. Also, importantly, the linker can contain a brush border cleavable amino acid sequence. [ka] Scheme 7: Synthesis of DOTA-PEG-SIB

[0164] A mixture of 5-(tert-butoxy)-5-oxo-4-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanoic acid (DOTAGA tetra-t-Bu ester; 30 mg, 43 μmol), N-hydroxysuccinimide (13.8 mg, 120 μmol), N-Boc-2-{2-[2-(2-amino-ethoxy)-ethoxy]-ethoxy}-ethylamine (35 mg, 120 μmol) and EDC (18.6 mg, 120 μmol) in DMF (0.5 mL) is stirred at 20 °C overnight. It is then purified by semi-preparative reverse phase HPLC to give tri-tert-butyl 2,2',2''-(10-(2,2,24,24-tetramethyl-4,18,22-trioxo-3,8,11,14,23-pentaoxa-5,17-diazapentacosan-21-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate as an oil (16 mg, 16 μmol, 39% yield). LRMS (LCMS-ESI) m / z: 975.7 (M+H) + Trifluoroacetic acid (300 μl) is added to the above product (16 mg, 16 μmol) and the resulting solution is stirred at 20° C. overnight. The TFA is evaporated to give 2,2',2''-(10-(1-amino-16-carboxy-13-oxo-3,6,9-trioxa-12-azahexadecan-16-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid as an oil (10 mg, 15.4 μmol, 96% yield). LRMS (LCMS-ESI) m / z: 651.3 (M+H) + The above product is coupled to bis(2,5-dioxopyrrolidin-1-yl) 5-iodoisophthalate by reacting 1 equivalent of each reagent and 1 equivalent of N,N-diisopropylethylamine in DMF. The product is purified by reverse phase HPLC. 177 Conjugated to macromolecules for subsequent labeling with radiometals such as Lu. Example 4: Preconjugation-Concept

[0165] The preceding examples show an approach that consists of first synthesizing a radioactive halogenated molecule (from tin or other alkyl metal precursors) and then coupling the radiolabeled molecule to a macromolecule. An alternative approach is to first react a precursor of the radioactive halogen with the macromolecule and then radiolabel this protein-precursor conjugate. This second approach is called preconjugation and has several potential advantages, including reduced synthesis time (important for radioactivity) and increased overall yield. In the preconjugation alternative, which is a common approach for radiometal labeling rather than radiohalogen labeling, due to the difference in their chemical nature, a tin-containing precursor molecule is first conjugated to the macromolecule. Such a derivatized macromolecule can then be radioactively halogenated, but preferably this procedure is carried out at a pH below 6.5. This approach is shown in Scheme 8, using the agent shown in Scheme 7 (complexation with non-radioactive lutetium). [ka] Scheme 8: Preconjugation of proteins with the Lu-DOTA-PEG-SIB tin precursor and radiohalogenation of the resulting conjugate.

[0166] or, 177 For labeling with radiometals such as Lu, iodo derivatives with uncomplexed DOTA moieties can be used. For example, 177 Lu, 177 For LuCl (2 Ci / ml), dilute 10 µl of 0.05 M HCl into 0.15 M ammonium acetate buffer and react with 100-1000 µg of DOTA-PEG-SIB. Once the reaction is complete, purify by standard size-exclusion chromatography methods.

[0167] Additionally, tin derivatives bearing uncomplexed DOTA moieties can be conjugated to polymers and then labeled with both radioactive metals and radioactive halogens. Example 5: Preconjugation - Experimental Approach

[0168] Following the same procedure as described above for the iodo derivative, the DOTAGA derivative 2,2',2''-(10-(1-amino-16-carboxy-13-oxo-3,6,9-trioxa-12-azahexadecan-16-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid is coupled to bis(2,5-dioxopyrrolidin-1-yl)5-(trimethylstannyl)isophthalate. It is then complexed with non-radioactive lutetium. For this, 50 μmol of the tin derivative are treated with 10 ml of 0.4 M acetate buffer (pH 5.2) containing 5 equivalents of LuCl3. The progress of the reaction is followed by reversed-phase HPLC and the lutetium complex is purified by semi-preparative reversed-phase HPLC. The complex is then conjugated to a macromolecule. For this, a solution of the polymer in 0.2 M sodium carbonate buffer (pH 8.5) (10 nmol / ml) is added to a solution of the prosthetic agent in DMSO (25 mM; 5 μl, 125 nmol) and the mixture is incubated for 1 h at 20° C. The resulting polymer-prosthetic group conjugate is isolated and simultaneously buffer exchanged into 0.2 M acetate (pH 5.5) by filtration through a VivaSpin ultrafiltration unit (GE Healthcare) with an appropriate molecular weight cut-off (e.g., 10 kDa for VHHs). The modified polymer is then radiohalogenated at pH 5. Example 6: Pre-iodination of polymers with carrier iodine prior to radioiodination

[0169] In the above strategy (pre-conjugating an alkyl metal prosthetic agent followed by radiohalogenation), one drawback of radioiodination in particular is that in addition to the desired radiolabeling site (i.e., the moiety bearing the alkyl metal group), constituent tyrosine residues present in the macromolecule may also be radioiodized. The problem with placing radioiodine on tyrosine is that the radioactivity is released once in the body and does not localize to the macromolecule in the cancer cell due to the action of endogenous deiodinases. This can be minimized by performing the radioiodination at a lower pH (4-5), but it cannot be avoided entirely. One approach to circumvent this potential problem is to first introduce non-radioactive iodine to these tyrosine residues and then subject the macromolecule to radioiodination. When mediated by these same endogenous deiodinases, the non-radioactive iodine on the constituent tyrosine residues is removed, thereby restoring the original tyrosine structure and likely maintaining the affinity of the macromolecule for its intended target. Non-radioactive iodination of proteins may be achieved simply by treating the protein with an excess of sodium iodide in the presence of an oxidizing agent such as chloramine-T.

[0170] As an example of this approach, 0.5 M sodium phosphate buffer (pH 7.4) containing VHH protein is reacted with 15 equivalents each of sodium iodide and chloramine-T at room temperature for 5-10 min. The reaction is stopped by the addition of sodium bisulfite (2 molar equivalents of chloramine-T). The iodized protein is purified by gel filtration or ultrafiltration. Example 7:

[0171] Targeted radiotherapy of CNS diseases. An attractive strategy for treating cancers of the central nervous system (CNS) is targeted radiotherapy, using vectors such as the small biomolecules of the present invention to selectively deliver radionuclides to malignant cell populations. The advantage of targeted radiotherapy is that it is possible to select a radionuclide with properties that best match the constraints of the intended clinical application, which in the case of CNS tumors means selecting a tissue-wide radiation that minimizes irradiation of normal CNS tissues. For example, neoplastic meningitis (NM) appears as floating cancer cells in the CSF and as sheet-like deposits on the compartment walls. Radiation dosimetry calculations have shown that radionuclides that emit short-range radiation are optimal for the treatment of NM by maximizing radiation dose deposition to tumor cells while minimizing dose to the spinal cord.

[0172] VHH molecules. VHH molecules, also known as single domain antibody fragments (sdAbs) or nanobodies, originate from the Camelidae and are the smallest antigen-binding fragments of natural antibodies with a molecular weight (~15 kDa) one order of magnitude smaller than intact mAbs. Unlike artificial affibody scaffolds, VHHs are easily generated with nanomolar to picomolar affinities by cloning from immunized camels or llamas and selection by phage display panning. Compared to other small protein-based targeting vectors, VHHs generally offer significant advantages in terms of thermal and chemical stability, low immunogenicity, solubility, expression yield, assembly of multimers, and the ability to recognize hidden or uncommon epitopes. Currently, VHHs in both monomeric and multimeric formats are undergoing clinical evaluation as therapeutics for many diseases, including inflammation. A panel of anti-HER2 VHHs was developed to 99m Tc, 68 Ga, 18 F, 131 I and 177The VHHs were labeled with a variety of radionuclides, including Lu. These radiolabeled VHHs showed peak tumor uptake in the range of 3-6% ID / g and rapid clearance from all normal tissues except the kidney. The present invention shows significantly higher tumor uptake, lower accumulation in normal tissues including the kidney, improved radiolabeling efficiency, and provides more potent radiolabeled biomolecules for use in targeting internalizing receptors such as HER2 and HER1.

[0173] Alpha particle emitters: Rationale for CNS tumor targeted radiation therapy. Like external beam radiation currently used in CNS tumor treatment, 131 Beta emitters, such as I, are radiation with low energy transfer. Alpha particles, on the other hand, are high linear energy transfer (LET) radiation, so that their ability to kill cancer cells is not compromised by hypoxia, dose rate effects or cell cycle position, making them attractive for targeted radiotherapy of CNS tumors. Unlike the case of low LET radiation, resistance mechanisms do not limit the effectiveness of alpha particles, since cells have only a limited ability to repair DNA double-strand breaks induced by alpha particles, which have been shown to kill tumor cells by apoptotic mechanisms. The range of alpha particles in tissue is only 50-80 μm, corresponding to only a few cell diameters, which should be ideally suited to destroy free-floating tumor cells in the CSF, thin plate-like tumors on the spinal cord and intracranial metastases, while minimizing irradiation of tumor-adjacent normal CNS tissue. Thus, both beta and alpha emitters are encompassed by the present invention. Example 8: Radiolabeled iso-SAGMB and iso-SGMIB as prosthetic agents for targeted radiotherapy of HER-2 expressing cancers 1. Introduction

[0174] Human epidermal growth factor receptor 2 (HER2) is overexpressed in a proportion of patients with multiple types of cancer, including breast, non-small cell lung, gastric, colon, and ovarian. Up to 20-30% of breast cancers overexpress HER2, and HER2 has been shown to confer a more invasive phenotype, including a greater propensity for metastasis to the central nervous system (CNS). Also, a higher incidence of brain metastases and leptomeningeal carcinomatosis has been reported in patients treated with the anti-HER2 monoclonal antibody (mAb) trastuzumab. In many cases, trastuzumab extends survival by controlling systemic disease in many patients; however, due to poor delivery due to the blood-brain barrier impermeability of this large protein, this increases the chances of CNS lesions where trastuzumab is not effective.

[0175] Patients with HER2-positive CNS disease have a grim prognosis; therefore, there is an urgent need for treatments that may be more effective without impairment of neurological function, which can be an unfortunate side effect of nonspecific treatments, including conventional radiation therapy. An attractive approach to increase the specificity of cancer treatment is targeted radiation therapy, using mAbs or other vectors to selectively deliver cytotoxic radionuclides to cancer cells. In the context of disease within the CNS, alpha particles (radiation with a tissue range of only a small cell diameter (50-80 μm)) may be advantageous because they may minimize cross-fire irradiation of normal tissues. Also, alpha particles have a high relative biological efficacy, with only a few traverses per cell required to achieve their destruction.

[0176] As an initial investigation of the therapeutic potential of alpha particles for the treatment of HER2-positive cancers, 211 We labeled trastuzumab with At and assessed its cytotoxicity against three HER2-expressing human breast carcinoma lines in vitro. 211 The relative biological efficacy of At-labeled trastuzumab was approximately 10-fold higher than conventional external beam therapy, with a significant reduction in survival achieved, and a 100% reduction in survival per bound cell. 211 Subsequent studies were conducted in a HER2-positive breast cancer meningitis model, 211The therapeutic efficacy of a single intrathecal injection of At-labeled trastuzumab was evaluated. In some long-term survivors, a significant increase in median survival was observed; however, histopathological analysis revealed that in some animals, the neuraxis region was spared from treatment, even with direct injection into the intrathecal compartment. Intact mAbs are not recommended for use in the treatment of rheumatoid arthritis, as their large size precludes uniform delivery and, in the case of intravenous application, results in slow normal tissue clearance. 211 It is not ideal for use in combination with short-lived alpha emitters such as At.

[0177] To overcome these limitations, various smaller HER2-targeting proteins have been developed, including recombinant fragments, e.g., diabodies and minibodies, as well as smaller scaffolds, e.g., affibodies. Another attractive targeted radiotherapy platform, known as variable domain of heavy chain-only antibodies (VHHs) or nanobodies, which are single domain antibody fragments (sdAbs), derived from Camelidae heavy chain-only antibodies, has a molecular weight of 12-15 kDa. These VHHs can be produced relatively inexpensively with nM to pM affinities, high thermal and chemical stability, and low immunogenicity. Their small size also allows them to be rapidly cleared from blood and normal tissues and to efficiently penetrate tumors, which is a major limitation of the current approach. 211 This is a particularly advantageous property for use with short-lived α-emitters such as At. Finally, several VHHs with high HER2 affinity have been generated and reported to target HER2-positive cancers in animal models and recent clinical imaging studies.

[0178] 211 Reagent for labeling 5F7 VHH with At, N-succinimidyl 3-[ 211 At] astat-4-guanidinomethylbenzoate ([ 211 At]SAGMB) and the new residual agent N-succinimidyl 3-[ 211 At] Astato-5-guanidinomethylbenzoate (iso-[ 211 In parallel, we evaluated the potential utility of beta particle emitters such as β-particle neutron emitters (SAGMB). 131I-labeled analogue (N-succinimidyl 4-guanidinomethyl-3-[ 131 Iodobenzoate ([ 131 I]SGMIB) and N-succinimidyl 3-guanidinomethyl-5-[ 131 Iodobenzoate (iso-[ 131 We evaluated the potential utility of [I]SGMIB) in HER2-expressing breast carcinoma cells and xenografts. We assessed the tumor-targeting properties of the four residual agents in HER2-expressing breast carcinoma cells and xenografts. 2. Materials and Methods 2.1. Overview

[0179] Except where noted, all reagents were purchased from Sigma-Aldrich. Sodium [ 131 [I] iodide (44.4 TBq / mmol) was obtained from Perkin-Elmer Life and Analytical Sciences (Boston, MA, USA). 209 Bi(α,2n) 211Astatine-211 was produced by bombarding a natural bismuth metal target with 28 MeV α particles via the At reaction. Astatine-211 was isolated from the target by dry distillation, trapped in PEEK or PTFE tubing, and finally extracted with a solution (0.2 mg / mL) of methanol containing N-chlorosuccinimide (NCS) as previously described. Succinimidyl 4 / 5-((1,2-bis(tert-butoxycarbonyl)guanidino)methyl)-3-iodobenzoate (Boc2-SGMIB / iso-SGMIB) and their corresponding tin precursors (Boc2-SGMTB / iso-SGMTB) were synthesized as previously reported. High-performance liquid chromatography (HPLC) was performed using a Beckman Gold HPLC system equipped with a Model 126 programmable solvent module, a Model 166 NM variable wavelength detector, and a ScanRam RadioTLC scanner / HPLC detector combination (LabLogic; Brandon, FL, USA). HPLC data were acquired and processed using Laura software (LabLogic). Normal-phase HPLC was performed using a 4.6 × 250 mm Partisil silica column (10 μm; Alltech, Deerfield, IL, USA) and eluted in isocratic mode with a mixture of 75:25 hexane:ethyl acetate (v / v) containing 0.2% acetic acid at a flow rate of 1 mL / min. Disposable PD10 desalting columns for gel filtration were purchased from GE Healthcare (Piscataway, NJ, USA). Instant thin-layer chromatography (ITLC) was performed using silica gel impregnated glass fiber sheets (Pall Corporation, East Hills, NY, USA) with PBS (pH 7.4) as the mobile phase. The spread sheets were analyzed for radioactivity using a TLC scanner as described above or by cutting the sheets into small strips and counting in an automated gamma counter. Radioactivity levels in the various samples were assessed using either an LKB 1282 (Wallac, Finland) or a Perkin Elmer Wizard II (Shelton, CT, USA) automated gamma counter. 2.2. Anti-HER2 5F7 VHH molecule

[0180] The anti-HER2 5F7 VHH molecule was obtained as a gift from Ablynx NV (Ghent, Belgium) and selected from a phage library derived from a llama immunized with SKBR3 human breast carcinoma cells. Its production, purification and characterization were as previously described (Pruszynski M, Koumarianou E, Vaidyanathan G, Revets H, Devoogdt N, Lahoutte T, et al. Targeting breast carcinoma. with radioiodinated anti-HER2 Nanobody. Nucl Med Biol 2013;40:52-9, which is incorporated herein by reference. 2.3. Cells and cell culture conditions

[0181] Cell culture reagents were purchased from Invitrogen (Grand Island, NY, USA). BT474M1 human breast carcinoma cells were grown in DMEM / F12 medium containing 10% fetal calf serum (FCS), streptomycin (100 μg / mL) and penicillin (100 IU / mL) (Sigma-Aldrich, MO, USA). Cells were cultured at 37°C in a 5% CO2 humidified incubator. 2.4. 131 I]SGMIB and iso-[ 131 Synthesis of [I]SGMIB

[0182] In most experiments, radioiododestannylation of the corresponding tin precursors using tert-butyl hydroperoxide as the oxidizing agent and chloroform as the solvent, as previously reported, afforded [ 131 I]SGMIB and iso-[ 131[I]SGMIB was synthesized. See Vaidyanathan G, Zalutsky MR. Synthesis of N-succinimidyl 4-guanidinomethyl-3-[*I]iodobenzoate: a radio-iodination agent for labeling internalizing proteins and peptides. Nature Prot 2007;2:282-6 and Choi J, Vaidyanathan G, Koumarianou E, McDougald D, Pruszynski M, Osada T, et al., N-Succinimidyl guanidinomethyl iodobenzoate protein radiohalogenation agents: influence of isomeric substitution on radiolabeling and target cell residualization. Nucl Med Biol 2014;41:802-12, which are incorporated herein by reference. In a more recent run, NCS was used as the oxidizing agent and the reaction was carried out in methanol. For this, a solution of NCS in methanol (0.2 mg / mL; 100 μL), acetic acid (1 μL) and [ 131 [I] iodide (1–2 μL; 37–74 MBq), in that order, was added to a half-drum glass vial containing 50 μg of the required tin precursor, and the reaction was allowed to proceed for 15 min at 20 °C with occasional vial rotation. Most of the solvent was evaporated with a stream of argon, and the residue was partitioned between 200 μL each of ethyl acetate and water. The ethyl acetate layer was separated, dried over anhydrous sodium sulfate, and the ethyl acetate was evaporated. The residual radioactivity was reconstituted in HPLC mobile phase (200 μL) and injected onto a normal-phase column. The isolation and deprotection procedure was as follows: 211 At]SAGMB and iso-[ 211 At]SAGMB was as described below. 2.5. 211 At]SAGMB and iso-[ 211 Synthesis of [At]SAGMB

[0183] Astatine-211 in NCS / methanol (30-56 MBq) was added to a vial containing 200 μg of the required tin precursor, followed by 10 μL of acetic acid. The reaction mixture was incubated at 20 °C for 30 min and the methanol was evaporated with a gentle argon stream. The remaining mixture was redissolved in 20 μL of (75:25) hexane / ethyl acetate and injected onto a normal-phase HPLC column. Boc2-iso-[ 211 At]SAGMB or Boc2-[ 211 At]SAGMB(t R The HPLC fraction containing the 5'-aminopropyl ether (pH 7.0) was isolated and the solvent was evaporated under a stream of argon for 20 min. The Boc protecting group was removed by treatment with 100 μL of trifluoroacetic acid (TFA) at 20° C. for 10 min. The process of ethyl acetate addition (50 μL) and evaporation was performed three times to ensure complete removal of TFA. The residual radioactivity was then used directly for 5F7 VHH labeling. 2.6.5 Radiolabeling of F7 VHH

[0184] As previously reported, 131 I]SGMIB or iso-[ 131 Iodine-131 labeling of 5F7 VHH with [I]SGMIB was performed. See Choi J, Vaidyanathan G, Koumarianou E, McDougald D, Pruszynski M, Osada T, et al., N-Succinimidyl guanidinomethyl iodobenzoate protein radiohalogenation agents: influence of isomeric substitution on radiolabeling and target cell residualization. Nucl Med Biol 2014;41:802-12, which is incorporated herein by reference. 211 For labeling, a solution of 5F7 VHH (50 μL, 2 mg / mL) in 0.1 M borate buffer (pH 8.5) was added to [ 211 At]SAGMB or iso-[ 211[At]SAGMB radioactivity was added to the vial and the mixture was incubated for 20 min at 20° C. The labeled 5F7 VHH was purified by gel filtration through a PD-10 column eluted with phosphate buffered saline (PBS). Prior to use, the PD-10 column was pretreated with human serum albumin to minimize non-specific binding. 2.7. Quality Control Procedures

[0185] ITLC and SDS-PAGE 131 I- and 211 At-labeled 5F7 preparations were evaluated to determine protein-associated radioactivity and the presence of aggregates and multimeric species, respectively. For ITLC, PBS (pH 7.4) was used as the mobile phase; in this system, the intact protein remained at the starting point (R f = 0), whereas lower molecular weight radioactive species have an R of 0.7–0.8. f The immunoreactive fraction of the labeled 5F7 VHH conjugate was determined by the Lindmo method using magnetic beads coated with the HER2 extracellular domain or bovine serum albumin (BSA) as a negative control. Briefly, an aliquot (~5 ng) of labeled 5F7 was incubated with 2x concentrations of both HER2 and BSA-coated beads and the immunoreactive fraction was calculated as the specific binding estimated for infinite HER2 excess. 2.8. Binding Affinity of Radiolabeled 5F7 Conjugates

[0186] BT474M1 breast carcinoma cells 8 × 10 4The cells were plated in 24-well plates at a density of 1000 cells / well and incubated at 37 °C for 24 h. The cells were then allowed to acclimate at 4 °C for 30 min before the addition of increasing concentrations of radiolabeled 5F7 conjugate (0.1-100 nM). The cells were then incubated at 4 °C for 2 h, the medium containing unbound radioactivity was removed, and the cells were washed twice with cold PBS. Finally, the cells were solubilized by treatment with 1 N NaOH (0.5 mL) for 10 min at 37 °C. Cell-bound radioactivity was counted using an automated gamma counter. To determine nonspecific binding, parallel assays were performed as above, except that a 100-fold excess of trastuzumab was also added to the incubation medium. The data were fitted using GraphPad Prism software to obtain the K d value was determined. 2.9. Internalization Assay

[0187] Internalization and cell processing assays were performed in a paired labeling format using BT474M1 breast carcinoma cells. 8 × 10 cells 5 Three milliliters of medium containing cells at a density of 100 cells / well was plated in a 6-well plate and incubated at 37°C overnight, then at 4°C for 30 minutes. The medium was removed and 5 nmol of each [ 211 At]SAGMB-5F7+[ 131 I]SGMIB-5F7 or iso-[ 211 At]SAGMB-5F7+iso-[ 131 The wells were supplemented with fresh medium containing either [I]SGMIB-5F7 or [I-SGMIB-5F7] and the cells were incubated for an additional hour at 4°C. The cell culture supernatant containing unbound radioactivity was removed and fresh medium at 37°C was added. After 1, 2, 4, 6 and 24 hours of incubation at 37°C, the percentage of initial cell-associated radioactivity that was internalized, was on the cell membrane, or was released into the cell culture supernatant was determined as previously described. To determine nonspecific uptake, parallel experiments were performed as above except that a 100-fold molar excess of trastuzumab was also added to the wells. 2.10. Paired label biodistribution experiments

[0188] Animal experiments were performed in accordance with the guidelines established by the Duke University Institutional Animal Care and Use Committee. Subcutaneous BT474M1 tumor xenografts were established in SCID mice as previously described (Pruszynski M, Koumarianou et al., 2013). See E, Vaidyanathan G, Revets H, Devoogdt N, Lahoutte T, et al., Improved tumor targeting of anti-HER2 nanobody through N-succinimidyl 4-guanidinomethyl-3-iodobenzoate radiolabeling. J Nucl Med 2014;55:650-6, which is incorporated herein by reference. 3 Once a volume of 185 kBq had been reached, two paired label biodistribution studies were performed. Groups of five mice received tail vein injections of approximately 185 kBq of each labeled molecule. In the first experiment, 211 At]SAGMB-5F7 (178 MBq / mg) and [ 131 In the second, iso-[ 211 At]SAGMB-5F7 (85MBq / mg) and iso-[ 131 I]SGMIB-5F7 (89 MBq / mg) was injected into the tumor. Thus, the efficacy of each of the two isomeric configurations for tumor targeting and in vivo stability was evaluated. 211 At-for- 131 The effects of I replacement could be directly compared. Biodistribution was assessed at 1, 2, 4 and 21 hours post-injection; a second study included an additional time point of 14 hours. Blood and urine were collected and mice were sacrificed by isoflurane overdose. Tumor and normal tissues were isolated, blotted dry and weighed along with blood and urine. All tissue samples were counted along with 5% injection standard using an automated gamma counter. 131 I and 211 At radioactivity was counted and percent injected dose (%ID) / g of organ / tissue was calculated. 2.11.Statistical analysis

[0189] Data are presented as mean ± standard deviation. Differences in the behavior of labeled conjugates that were co-incubated (in vitro) or co-administered (in vivo) were analyzed for statistical significance using a paired two-tailed Student's t-test using Microsoft Office excel program, and differences in the behavior of labeled conjugates that were not co-incubated or co-administered were tested using an unpaired Student's t-test. Differences with a P value < 0.05 were considered statistically significant. 3.Results 3.1. Radiolabeling

[0190] The synthetic schemes for the four radiohalogenated 5F7 VHH conjugates are provided in Scheme 9. [ka] Scheme 9: 211 At]SAGMB / iso-[ 211 At]SAGMB was used 211 Synthesis of 5F7 VHH molecules with radioactive iodine using At or [*I]SGMIB / iso-[*I]SGMIB

[0191] Under the same conditions, iso-[ 211 The radiochemical yield of the synthesis of SAGMB-Boc2 was [ 211 At]SAGMB-Boc2 yields were 66.8 ± 2.4% (n = 7) compared with 62.6 ± 2.3% (n = 6). Although the difference between the two yields was small, it was statistically significant (P < 0.05). 211 The radiochemical yield for the synthesis of [At]SAGMB-Boc2 was similar to that previously reported when using TBHP as the oxidizing agent and chloroform as the solvent. Most of the experiments reported herein used TBHP as the oxidizing agent and [ 131 I]SGMIB and iso-[ 131 However, some studies have used NCS as the oxidant and methanol as the solvent to synthesize [I]SGMIB.131 I]SGMIB and iso-[ 131 I]SGMIB was synthesized in radiochemical yields of 69.2 ± 4.2% ( n = 4) and 84.0 ± 4.5% ( n = 2), which were significantly higher than those obtained using TBHP and chloroform, respectively.

[0192] Boc2-[ 211 At]SAGMB and Boc2-iso-[ 211 At]SAGMB treatment 211 At]SAGMB and iso-[ 211 By reaction with At]SAGMB, 211 Labeling of 5F7 VHH with At was achieved. 211 At]SAGMB (39.5 ± 6.8%; n = 5) and [ 211 The conjugation efficiency of [At]SAGMB (38.4 ± 15.6%; n = 6) was similar (P > 0.05). 131 I]SGMIB and iso-[ 131 The conjugation efficiency of 5F7 labeling with [I]SGMIB was 28.9 ± 13.0% (n = 6) and 33.1 ± 7.1% (n = 6), respectively. The radiochemical purity obtained by ITLC analysis was 1.0 ± 1.0% for iso-[ 211 At]SAGMB-5F7,[ 211 At]SAGMB-5F7, iso-[ 131 I]SGMIB-5F7 and [ 131 I]SGMIB-5F7 were 98.9%, 97.8%, 98.6% and 98.4%. As shown in Figure 1, SDS-PAGE performed under non-reducing conditions demonstrated that more than 98% of the radioactivity of the four radiohalogenated 5F7 conjugates was present in a single band with a molecular weight of approximately 15 kDa, which corresponds to the molecular weight of the VHH monomer. 3.2. Immunoreactive fractions and binding affinities

[0193] To determine whether labeling of 5F7 VHH impairs HER2 binding, immunoreactive fractions were determined in a paired labeling format using the extracellular domain of HER2 as the molecular target. 211 At]SAGMB-5F7,[ 211 At]SAGMB-5F7, iso-[ 131 I]SGMIB-5F7 and [ 131 For [I]SGMIB-5F7, the immunoreactive fractions were determined to be 81.3±0.9%, 83.5±1.1%, 81.8±1.4% and 84.5±0.8%, suggesting that the 5F7 VHHs retain similar levels of immunoreactivity regardless of the prosthetic agent used. Dissociation constants (K d ) values ​​were less than 5 nM for the four labeled conjugates (Figure 2). 4 The data in Figure 2 were based on incubating 1000 cells / mL of the antibody with increasing amounts of labeled VHH conjugates and determining the specific cell-bound radioactivity as described herein. Plots were generated and Kd values ​​calculated using GraphPad Prism software. However, iso-[ 211 At]SAGMB-5F7 (3.0 ± 0.1 nM) 211 At]SAGMB-5F7 (4.5±0.4 nM) showed significantly higher affinity binding (P<0.05). 131 I]SGMIB-5F7 and [ 131 I]SGMIB-5F7K d The values ​​were 1.3±0.2 nM and 2.4±0.2 nM, respectively, indicating a higher affinity for the iso-conjugation conjugate. 131 I-labeled conjugates were 211 It had significantly higher binding affinity than its At-labeled 5F7 counterpart (P<0.05). 3.3. Internalization Assay

[0194] Paired-label internalization assays were performed using HER2-expressing BT474M1 cells to determine the 211 At]SAGMB-5F7 and iso-[ 211 At]SAGMB-5F7 (Figure 3) and [ 131 I]SGMIB-5F7 and iso-[ 131 The extent of intracellular capture of in vitro radioactivity by [I]SGMIB-5F7 (Figure 4) was determined. The data presented in Figure 3 were generated based on two versions of labeled 5F7 obtained from two different experiments. As shown in Figure 3, values ​​of 77.4 ± 0.8% and 67.2 ± 1.1%, respectively, were observed for cell-associated (membrane-bound + internalized) and [I]SGMIB-5F7 (Figure 4). 211 The percentage of initially bound radioactivity internalized by iso-[At]SAGMB-5F7 remained fairly constant over 24 hours. In general, changes in the nature of the prosthetic agent did not affect the persistence of radioactivity in HER2-positive cancer cells. For example, at 6 hours, iso-[ 211 At]SAGMB-5F7 and iso-[ 131 For [I]SGMIB-5F7, 69.5±1.2% and 73.2±1.7% of the initial bound radioactivity remained in the intracellular compartment. 131 I]SGMIB-5F7 and [ 211 At]SAGMB-5F7, the behavior of iso-[ 131 I]SGMIB-5F7 (49.0 ± 3.6%) and iso-[ 211 The intracellular radioactivity levels from At]SAGMB-5F7 (48.4 ± 5.5%) were significantly lower ( P < 0.05) than those observed from 1 to 6 h. 3.4. Biodistribution studies

[0195] Two paired-labeling experiments were performed in SCID mice bearing subcutaneous BT474M1 breast cancer xenografts to determine the 211 At]SAGMB-5F7 and iso-[ 211 The tissue distribution of At]SAGMB-5F7 was 131 Direct comparison was made with the I-labeled counterpart over a 21-hour period (which 211The results obtained for the 1000 Å decay (corresponding to about three half-lives of 1000 Å decay) are summarized in Tables 1 and 2, respectively. 211 Tumor uptake of [At]SAGMB-5F7 remained at 15-16%ID / g from 1 to 4 h after injection and then declined to 9.49 ± 1.22%ID / g at 21 h (Figure 5). Coadministration [At]SAGMB-5F7 was significantly higher than coadministration [At]SAGMB-5F7, except for the 21 h time point, where the value of the radioiodinated conjugate was approximately 20% higher (11.8 ± 1.5%ID / g; P < 0.05). 131 Similar tumor uptake values ​​were observed for iso-[I]SGMIB-5F7 (Figure 6). 211 Similar trends were observed for tumor uptake of iso-[At]SAGMB-5F7 compared to its radioiodinated counterpart. 211 Tumor accumulation of SAGMB-5F7 was 211 At] was nearly 50% higher than that of SAGMB-5F7 (Figure 5), reaching a peak of 23.4 ± 2.2% ID / g at 4 h (significant difference, P < 0.05, except at 21 h by unpaired t test). 131 I] SGMIB-5F7 tumor uptake was 131 I]SGMIB-5F7 (Figure 6). Except for the kidney, all four 5F7 radioconjugates, especially iso-[ 211 At]SAGMB-5F7 and iso-[ 131 Normal tissue uptake of [I]SGMIB-5F7 was low. In the kidney, the radioactivity levels of the iso-conjugate were significantly lower (P<0.05 by unpaired t-test) than those of the corresponding non-iso-conjugate (Figures 7 and 8). 211 For the At-labeled conjugates the differences were less pronounced. 211 For At-labeled compounds, the carbon-halogen bond strength is expected to be lower, so a comparison of the radioactivity levels in the thyroid and stomach (tissues known to sequester free astatides and iodide) can elucidate the relative in vivo stability of these conjugates.211 At and 131 The uptake of I activity is summarized in Figures 9 and 10, respectively. 211 Thyroid and stomach accumulation of At-labeled 5F7 conjugates was 131 1-labeled coadministered counterparts. However, thyroid and stomach radioactivity levels were significantly higher than those seen with the iso-[ 211 At]SAGMB-5F7, [ 211 At]SAGMB-5F7, which was approximately two-fold lower than that of iso-[ 211 This suggests that [At]SAGMB-5F7 is deastatinated to a lower extent in vivo.

[0196] As shown in Figure 11, in all tissues, iso-[ 211 At]SAGMB-5F7 tumor-to-normal tissue ratio is 211 At]SAGMB-5F7. For example, at 4 hours, the tumor-to-liver ratio, tumor-to-blood ratio, tumor-to-spleen ratio, and tumor-to-kidney ratio were significantly higher than those of [ 211 At] compared with 7.31 ± 1.26, 32 ± 4, 7.11 ± 1.47 and 0.67 ± 0.08 for SAGMB-5F7, iso-[ 211 In SAGMB-5F7, the values ​​were 18±4, 63±13, 21±3, and 1.50±0.25, respectively. Similarly, in all tissues, the iso-[ 131 I] The tumor-to-normal tissue ratio of SGMIB-5F7 is [ 131 I]SGMIB-5F7 (Figure 12). Finally, the tumor-to-normal tissue ratios of the radioiodized 5F7 VHH conjugates were significantly higher than those of the corresponding 211 was significantly higher than that of the At-labeled 5F7 VHH conjugate. 4. Discussion

[0197] In this study, we investigated two related prosthetic agents designed to trap radionuclides in HER2-expressing cancer cells after receptor-mediated internalization via the generation of positively charged labeled catabolites [ 211 At]SAGMB and iso-[ 211At]SAGMB is used to detect alpha particle-emitting radioactive halogens 211 We successfully labeled anti-HER2 5F7 VHH with At. 211 The high cytotoxicity of Atα particles is due to the following in vitro and in vivo compartmentalized conditions: 211 This has been demonstrated using At-labeled trastuzumab. 211 Although At has many potential advantages for targeted radiotherapy, the short tissue range of its α particles combined with a half-life of 7.2 hours necessitates the development of strategies to rapidly achieve uniform and long-term delivery to cancer cells with rapid clearance from normal tissues. Most approaches to achieve this goal utilize small molecules, such as mAb fragments; however, unlike in the case of whole mAbs, 211 The At-labeled mAb fragments showed high uptake in the thyroid and stomach, which is consistent with the in vivo free 211 Within the HER2 targeting space, this behavior is consistent with the release of N-succinimidyl 3-[ 211 At] astatobenzoate (SAB) (which is the corresponding 125 This was observed with affibodies (7 kDa) labeled with N-succinimidyl N-(4-[ 211 At] Astatophenethyl succinamate (SAPS) 211 We also labeled anti-HER2 diabodies with At and obtained some promising therapeutic responses, 211 No biodistribution results of the At-labeled diabody were reported.

[0198] Optimal 211 In attempting to develop At-labeled anti-HER2 constructs, it is important to not only consider the in vivo stability issues mentioned above, but also how to maximize the extent and duration of radioactivity inclusion in cancer cells following binding and internalization of the labeled molecule. In addition, a protein format must be selected that provides rapid tumor targeting at therapeutically relevant levels without prolonging residence time in normal tissues. The excellent results obtained with anti-HER2 VHHSGMIB conjugates have211 This study provided the motivation for evaluating the potential utility of guanidinomethyl-substituted prosthetic groups for labeling 5F7 VHH with At. 5F7 with (see Pruszynski M, Koumarianou E, Vaidyanathan G, Revets H, Devoogdt N, Lahoutte T, et al. Improved tumor targeting of anti-HER2 nanobody through N-succinimidyl 4-guanidinomethyl-3-iodobenzoate radiolabeling. J Nucl Med 2014;55:650-6, which is incorporated herein by reference) and without (see Vaidyanathan G, McDougald D, Choi J., Koumarianou E, Weitzel D, Osada T, et al. Preclinical evaluation of 18F-labeled anti-HER2 nanobody conjugates for imaging HER2 receptor expression by immuno-PET. J Nucl Med 2016;57:967-73) The VHH was evaluated after SGMIB in BT474M1 xenografts and SCID mice bearing both constructs, with peak tumor uptake occurring 2 hours post-injection, indicating that this VHH 211 These results suggest that At has localization kinetics compatible with the 7.2-h half-life of At. The version without the GGC tail exists as a pure monomer and as a mixture of monomer and 5F7-GGC dimers (Pruszynski M, Koumarianou E, Vaidyanathan G, Revets H, Devoogdt N, Lahoutte T, et al. Improved tumor targeting of anti-HER2 nanobody through The tailless 5F7 construct was chosen for use in these experiments because it showed significantly greater tumor localization compared to N-succinimidyl 4-guanidinomethyl-3-iodobenzoate radiolabeling. J Nucl Med 2014;55:650-6, which is incorporated herein by reference.

[0199] The size of the astatine atom is large compared to the iodine atom, 211 In labeling, steric hindrance may be a more important factor. 131 In SGMIB, 131 Based on the observed significantly higher radioiodination and protein conjugation yields compared to iso-[I]SGMIB, we chose iso-[ 211 At]SAGMB (1,3,5-isomer) and [ 211 Both iso-[ At]SAGMB (1,3,4-isomers) were evaluated. 211 Radiolabeling and VHH conjugation yields of SAGMB are 211 At] was higher than that of SAGMB, but these differences were not significant. Conjugation of these prosthetic groups and their radioactive iodinated counterparts resulted in monomeric products with excellent immunoreactivity and affinity (<5 nM) for binding to HER2-overexpressing BT474M1 breast carcinoma cells. 131 The results obtained for I]SGMIB-5F7 are 131I]SGMIB-5F7-GGC construct. See Pruszynski M, Koumarianou E, Vaidyanathan G, Revets H, Devoogdt N, Lahoutte T, et al., Improved tumor targeting of anti-HER2 nanobody through N-succinimidyl 4-guanidinomethyl-3-iodobenzoate radiolabeling. J Nucl Med 2014;55:650-6, which is incorporated herein by reference. For both isomers, 211 The affinity of the At-labeled 5F7 conjugate was determined by the corresponding 131 The β-amyloid binding was approximately half that of the I-labeled 5F7 VHH conjugate. Without being bound to any mechanism of action, the larger size of the astatine atom and 211 It is believed that the radiolytic effect of the Atα particle may reduce the binding affinity. 211 At]SAGMB-5F7 (3.0 ± 0.1 nM) and [ 211 The binding affinity of At]SAGMB-5F7 (4.5±0.4 nM) should be compatible with their use as targeted radiotherapy.

[0200] Maximizing radionuclide capture in cancer cells following binding and cellular processing of radiolabeled receptor targeting proteins should increase the efficacy of targeted radiotherapy. Internalization assays performed with both trastuzumab and 5F7 VHH demonstrated that labeling of these HER2 targeting proteins with either [*I]SGMIB or iso-[*I]SGMIB resulted in comparable intracellular capture of radioiodine for up to 6 hours; however, at the 24 hour time point, total cell binding and internalized radioactivity was significantly lower for iso-[*I]SGMIB. Choi J, Vaidyanathan G, Koumarianou E, McDougald D, Pruszynski M, Osada T, et al., N-Succinimidyl guanidinomethyl See iodobenzoate protein radiohalogenation agents: influence of isomeric substitution on radiolabeling and target cell residualization. Nucl Med Biol 2014;41:802-12, which is incorporated herein by reference. These results suggest that the residual capacity of iso-[*I]SGMIB is not as persistent as that of [*I]SGMIB, since its half-life is 7.2 hours. 211 Paired labeling experiments with BT474M1 breast carcinoma cells showed that iso-[ 211 At]SAGMB-5F7 and [ 211 Our results showed that for both the 1,3,4- and 1,3,5-isomers, the substitution of iodine for astatine did not affect retention capacity; however, for the latter, at the 24-h time point, the iso-[At]SAGMB-5F7 cell binding and intracellular radioactivity were directly compared to their radioactively iodinated counterparts. 211 At]SAGMB-5F7 and iso-[ 131 A significant decrease in intracellular capture was observed for both iso-[I]SGMIB-5F7 and 1,3,5-isomers. The mechanism responsible for this behavior is unclear, but it seems likely that a higher catabolic rate of the 1,3,5-isomer and / or release of labeled catabolic products may play a role. Nevertheless, iso-[I]SGMIB-5F7 and 1,3,5-isomers exhibited a significant decrease in intracellular capture. 211 At]SAGMB-5F7, 48.4±5.5% of the initial bound radioactivity was still internalized at 24 h. 211 This is encouraging, as over 90% of the At atoms will decay by this time.

[0201] The main focus of this study is 211 This is the evaluation of At-labeled 5F7 VHH conjugates, which, to the best of our knowledge, is the first attempt to evaluate this promising α-emitter for labeling VHH molecules. 211[At]SAGMB has been used successfully to label the intact internalizing mAb L8A4, which reacts with mutant forms of the epidermal growth factor receptor. However, extrapolating results from one type of protein construct to another must be done with caution. For example, N ε -(3-[ 131 Iodobenzoyl)-Lys 5 -N α -Maleimido-Gly 1 -GEEEK( 131 The I-IB-Mal-D-GEEEK) was shown to be an excellent reagent for labeling the intact mAb L8A4, but offered no advantage in terms of tumor uptake and a clear disadvantage in terms of kidney uptake for labeling the 5F7 VHH. Importantly, 211 At]SAGMB-5F7 and iso-[ 211 The high and long-term retention of radioactivity in HER2-expressing BT474M1 cancer cells observed in the internalization assay with [At]SAGMB-5F7 was reproduced in paired-label biodistribution studies performed in SCID mice bearing xenografts derived from the same BT474M1 cell line. These 211 The magnitude of tumor accumulation observed with the At-labeled 5F7 conjugate was 99m Tc, 177 Lu, 68 Ga and 18 Another HER2-targeting VHH (2Rs15d) labeled with F, and 211 These results were 2- to 3-fold higher than those reported for HER2-specific affibodies labeled with various radionuclides, including At.

[0202] Regarding the possibility of isomeric substitution patterns influencing tumor radioactivity levels, at all time points, 131 I]SGMIB-5F7 and iso-[ 211 At]SAGMB-5F7 is 131 I]SGMIB-5F7 and [ 211At]SAGMB-5F7 showed a significant and unexpected 1.5-fold tumor delivery advantage compared to [At]SAGMB-5F7, however this does not appear to reflect differences in retention potency as similar intracellular entrapment was observed for both isomers up to the final time point in in vitro internalization assays. 211 At- and 131 Regarding the differences in the in vivo behavior of I-labeled VHH conjugates, [ 211 At]SAGMB-5F7 and iso-[ 211 At early time points, the localization of SAGMB-5F7 was significantly higher than that of their co-administration 131 The 1H-labeled analogues were comparable to those of the 1H-labeled analogues, but were approximately 20% lower at 21 h. This may reflect halogen-dependent differences in in vivo stability, most likely due to a higher rate of dehalogenation of astatine, consistent with its lower CX bond strength. For both isomers, the dehalogenation rates in the thyroid and stomach, tissues known to sequester free radioactive halide, were significantly higher. 211 At level is 131 This is supported by the observation that the β-amino acid salts are higher than those of I. However, 211 The radioactivity levels in the thyroid and stomach after injection of iso-[At]SAGMB-5F7 were 0.4–0.6% and 1.0–2.3%ID, respectively. 211 At]SAGMB-5F7 were 0.2-0.3% and 0.6-1.7%ID, respectively, which was due to the iso-[ 211 This suggests that the iso-[At]SAGMB conjugate is less deastatinated. 131 Gastric and thyroid radioactivity levels after injection of [I]SGMIB-5F7 were 131 I]SGMIB-5F7, suggesting an unexpected isomer-dependent difference in the in vivo stability of these radiohalogenated sdAb conjugates. 211 At]SAGMB-5F7 and iso-[ 211 At]SAGMB-5F7 in both the thyroid and stomach 211The extent of At incorporation was lower than that reported for a variety of low molecular weight proteins labeled using several different methods. 211 At]SAGMB-5F7 and iso-[ 211 At]SAGMB-5F7 211 Although the loss of At [astatide] was relatively low, it did not result in normal tissue toxicity (which is 211 This can increase the concentration of AT-labeled antibodies (which can be significantly reduced by the use of blocking agents, as was done in clinical studies with At-labeled antibodies).

[0203] Tumor to normal tissue ratios were generally higher for the radioiodinated conjugates compared to the astatinated versions, presumably reflecting the higher in vivo stability of the iodinated versions. Unexpectedly, when iso-prosthetic agents were used to label the 5F7 VHH, the tumor to normal tissue ratios were significantly higher for both radionuclides. As summarized in Tables 1 and 2, this may be due to several advantages in tumor uptake, but also in particular 131 For I-labeled conjugates, this also reflects the rather low radioactivity levels in normal tissues. A possible explanation for this behavior is the mass effect, where a certain mass of VHH molecule is required to block non-specific uptake of labeled VHH in normal organs such as the liver spleen and lungs. Xavier C, Vaneycken I, D'Huyvetter M, Heemskerk J, Keyaerts M, Vincke C, et al. Synthesis, preclinical validation, dosimetry, and toxicity of 68 See Ga-NOTA-anti-HER2 nanobodies for iPET imaging of HER2 receptor expression in cancer. J Nucl Med 2013;54:776-784, which is incorporated herein by reference. 211 At]SAGMB-5F7+[ 131 I]SGMIB-5F77 biodistribution experiments were performed at a total 5F7 VHH dose of 2.1 μg and iso-[211 At]SAGMB-5F7+iso-[ 131 In the I]SGMIB-5F7 study, a total 5F7 dose of 4.3 μg was administered, which may be relevant here. However, a total VHH dose of 2.1 μg was administered in this study [ 131 The biodistribution observed for I]SGMIB-5F7 was consistent with the 4.3 and 6.8 μg total 5F7 doses [ 131 This may not be a factor, as the results were very similar to those previously reported for I]SGMIB-5F7. Vaidyanathan G, McDougald D, Choi J., Koumarianou E, Weitzel D, Osada T, et al., Preclinical evaluation of 18 See F-labeled anti-HER2 nanobody conjugates for imaging HER2 receptor expression by immuno-PET. J Nucl Med 2016;57:967-73, which is incorporated herein by reference. Also, for anti-HER2 VHH 2Rs15d, significant differences in mass-dependent localization were observed at doses between 0.1 and 1 μg. 68 This was observed after labeling with Ga but not at doses of 1–10 μg, including the doses used in this study.

[0204] Notably, in the same animal model, iso-[ 125 I]SGMIB-trastuzumab and [ 131 Given the similarities in tissue distribution previously observed when comparing iso-[I]SGMIB-trastuzumab, the differences in biological behavior observed for the two isomeric versions with the same radiohalogen were unexpected. See Choi J et al., Nucl Med Biol 2014;41:802-12, which is incorporated herein by reference. However, VHH molecules are approximately 10 times smaller than intact mAbs, which may lead to more rapid degradation into species small enough to allow easy access to deiodinases and other enzymes that may lead to dehalogenation, such as cytochrome P450. iso-[I]SGMIB-trastuzumab125 I] The metabolic stability of SGMIB-5F7 is 131 The greater catabolic activity of [I]SGMIB-5F7 compared to [I]SGMIB-5F7 can be explained by the difference in catabolism of the two conjugates and the susceptibility of the labeled catabolites to in vivo deiodination. As summarized in a recent review, subtle differences in the design of the radioiodine compounds can lead to increased deiodination rates. Consistent with this, the deiodination of meta-iodobenzylguanidine (a structural element of iso-SGMIB) was less than that of ortho-iodobenzylguanidine (a structural element of SGMIB). Studies are planned to evaluate the chemical nature of the labeled catabolites generated from iso-SGMIB-VHH and SGMIB-VHH conjugates to better understand the mechanisms leading to the observed differences in their in vivo behavior.

[0205] A potential problem with using VHH molecules as a platform for targeted radiotherapy is the high accumulation and long-term retention of radioactivity in the kidney, which may result in dose-limiting nephrotoxicity. This behavior is similar to that of radiometals, e.g. 177 Lu and some residual radioactive halogenated agents, e.g. 131 It has been observed in I-IB-Mal-D-GEEEK. For example, 131 When I-IB-Mal-D-GEEEK was used to label 5F7-GGC, kidney levels were greater than 150% ID / g at 1-8 hours post-injection and approximately 100% ID / g at 24 hours. In contrast, all four radiohalogenated 5F7 conjugates evaluated in this study had high initial kidney radioactivity levels (60-100% ID / g) that declined rapidly, with renal clearance half-lives of approximately 1-2 hours. Remarkably, at all time points, 131 I- and 211 Kidney radioactivity levels for both At-labeled iso-conjugates were significantly lower than those observed for their corresponding 1,3,4 isomer conjugates, and the difference in kidney retention increased with time. 131 The kidney radioactivity levels observed 21 hours after injection of [I]SGMIB-5F7 were 131The renal radioactivity levels were more than four-fold lower than those of iso-[I]SGMIB-5F7. Radionuclide-dependent differences in renal radioactivity levels were also observed, but to a lesser extent than those between the two isomeric versions of a given radionuclide. Paradoxically, iso-[ 211 Kidney radioactivity levels after injection of co-administered iso-[At]SAGMB-5F7 were significantly higher than those after injection of co-administered iso-[ 131 I] higher than that of SGMIB-5F7, 211 Kidney radioactivity levels after injection of SAGMB-5F7 were significantly higher than those after coadministration of 131 [I]SGMIB-5F7 was lower than that of [I]SGMIB-5F7. At this time, we are unable to explain the differences in renal uptake and retention of the four 5F7 VHH radioconjugates, which were unexpected considering the similarity of the acylation agents in physical properties that may affect renal retention, e.g., polarity and hydrophilicity. Also, in previous studies, [ 131 I]SGMIB and [ 211 Intact mAb L8A4 labeled with [At]SAGMB and 131 I]SGMIB and iso-[ 125 No significant difference in renal uptake values ​​was noted between trastuzumab labeled with iso-[I]SGMIB and those labeled with trastuzumab. The mechanism responsible for their lower renal radioactivity levels is unclear, but iso-[ 211 At]SAGMB and iso-[ 131 [I]SGMIB conjugates are the reagent of choice for minimizing the absorbed radiation dose to the kidney from 5F7 and potentially other VHHs. If further reduction in kidney radiation dose is required, this should be achieved at least 177 It has been shown that this can be achieved by co-injection with the plasma expander gelofusine using Lu-labelled VHH conjugates.

[0206] In summary: 211 It was demonstrated that anti-HER2 5F7 VHH could be labeled with At in reasonable yields with excellent retention of affinity and immunoreactivity after labeling. 211 Studies in preclinical models with iso-[At]SAGMB-5F7 demonstrated high and sustained tumor targeting and rapid normal tissue clearance, and 211Even more favorable results were observed with iso-[At]SAGMB-5F7. 131 I]SGMIB-5F7 is 131 I]SGMIB-5F7 provided significantly improved tumor targeting. 211 At]SAGMB-5F7 and iso-[ 131 I]SGMIB-5F7 warrants further evaluation as an α- and β-particle-emitting targeted radiotherapy for the treatment of HER2-expressing malignancies.

[0207] VHH sequences that target HER2 and that can be used in carrying out the present invention include those set forth in SEQ ID NOs: 1-5. SEQ ID NO:1 Immunoglobulin heavy chain variable region, partial [Camelus dromedarius] DVQLVESGGGSVQGAAGGSLRLSCAASDITYSTDCMGWFRQAPGKEREGVATINNGRAITYYADSVKGRFTISQDNAKNTVYLQMNSLRPKDTAIYYCAARLRAGYCYPADYSMDYWGKGTQVTVSS SEQ ID NO:2 Immunoglobulin heavy chain variable region, partial [Camelus dromedarius] DVQLEESGGGSVQAGGSLRLSCAASGYIYSTYCMGWFRQAPGKEREGVAAINDVGGSVYYADSVKGRFTISQDIAQDTMYLQMNDLTPENTVTYTCAALRCLSDSDPDTRVHMYYDWGQGTQVTVSS SEQ ID NO:3 Immunoglobulin heavy chain variable region, partial [Camelus dromedarius] DVQLEESGGGSVQTGGSLRLSCAASGYTYSSACMGWFRQGPGKEREAVADVNTGGRRTYYADSVKGRFTISQDNTKDMRYLQMNNLKPEDTATYYCATGPRRRDYGLGPCDYNYWGQGTQVTVSS SEQ ID NO:4 Immunoglobulin heavy chain variable region, partial [Camelus dromedarius] EVQLEESGGGLVQPGGSLTLSCAASGYTFTNCAAGWYRQAPGKECELVASIFSGNRTNYADSVKGRFTISRDNTKDIVYLQMNSLKPEDTTVYYCDARTPCWGQGTQVTVSS SEQ ID NO:5 Immunoglobulin heavy chain variable region, partial [Camelus dromedarius] EVQLEESGGGSVQAGGSLRLSCAASGYTFLQLLHGWFRQAPGKEREVVARFNTDINKTFYLESVKGRFTLSQDNAKNTLYLQMNSLKPEDTAIYYCAASRPDSTCDYFAYRGQGTQVTVSS

[0208] All publications, patents, and patent applications mentioned in this specification are indicative of the level of those skilled in the art to which this invention pertains. All publications, patents, and patent applications are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, it will be apparent that certain changes and modifications can be practiced within the scope of the embodiments. The present invention provides, for example, the following items. (Item 1) A compound in the form of a prosthetic compound or a radioactive halogen precursor represented by formula I: [ka] (In the formula, X is CH or N; L1 and L3 are independently selected from a bond, a substituted or unsubstituted alkyl chain, a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain, and a polyethylene glycol (PEG) chain; MMCM is a polymeric conjugate moiety; L2 is a substituted or unsubstituted alkyl chain, a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain, or a polyethylene glycol (PEG) chain containing at least three oxygen atoms, where L2 optionally contains a brush border enzyme cleavable peptide; CG is selected from guanidine; PO3H; SO3H; one or more charged D- or L-amino acids selected from arginine, phosphono / sulfophenylalanine, glutamate, aspartate, and lysine; a hydrophilic carbohydrate moiety; a polyethylene glycol (PEG) chain; and Z-guanidine; Z is (CH2) n and; n is greater than 1; m is 0 to 3; and Y is an alkylmetal moiety; or 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I, 131 I and 211 At) or a pharma- ceutically acceptable salt or solvate thereof. (Item 2) 2. The compound according to claim 1, which is a radioactive halogen precursor, and Y is an alkyl metal moiety selected from the group consisting of trimethylstannyl (SnMe3), tri-n-butylstannyl (SnBu3) and trimethylsilyl (SiMe3). (Item 3) A prosthetic compound, wherein Y is 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I, 131 I and 211 The compound according to item 1, wherein the radioactive halogen is selected from the group consisting of At. (Item 4) MMCM is an active ester or (Gly) m(m is 1 or more). (Item 5) 2. The compound according to item 1, wherein the MMCM is selected from the group consisting of an N-hydroxysuccinimide (NHS) ester, a tetrafluorophenol (TFP) ester, an isothiocyanate group or a maleimide group. (Item 6) 2. The compound according to item 1, wherein MMCM is Gly-Gly-Gly. (Item 7) L2 is (CH2) p (p=1 to 6). (Item 8) 2. The compound according to claim 1, wherein the optional brush border enzyme-cleavable peptide is selected from the group consisting of Gly-Lys, Gly-Tyr and Gly-Phe-Lys. (Item 9) The following structure: [ka] The compound according to item 1, represented by: (Item 10) N-Succinimidyl 3-guanidinomethyl-5-[ 131 I]iodobenzoate or N-succinimidyl 3-[ 211 At] the compound according to item 9, comprising astato-5-guanidinomethylbenzoate. (Item 11) 13. A radiolabeled biomolecule or intermediate comprising a compound according to item 1 attached to a biomolecule. (Item 12) 12. The radiolabeled biomolecule or intermediate according to item 11, wherein the biomolecule is selected from the group consisting of an antibody, an antibody fragment, a VHH molecule, an aptamer or a variant thereof. (Item 13) Item 14. The radiolabeled biomolecule or intermediate according to Item 11, wherein the labeled biomolecule is a VHH. 14. The radiolabeled biomolecule or intermediate according to item 13, wherein said VHH targets HER2. (Item 15) 15. The radiolabeled biomolecule or intermediate according to item 14, wherein the VHH comprises an amino acid sequence selected from the sequences set forth in SEQ ID NOs: 1 to 5. (Item 16) 12. A pharmaceutical composition comprising the radiolabeled biomolecule according to item 11 in association with a pharma- ceutically acceptable adjuvant, diluent or carrier. (Item 17) A compound in the form of a prosthetic compound or a radioactive halogen precursor represented by formula 2: [ka] (In the formula, MC is a multidentate metal chelating moiety; Cm is a thiourea, amide, or thioether; L4 is selected from a bond, a substituted or unsubstituted alkyl chain, a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain optionally having NH, CO or S at one or both termini, and a polyethylene glycol (PEG) chain; T is a compound according to any one of items 1 to 10, or a pharma- ceutically acceptable salt or solvate thereof. (Item 18) 18. The compound according to item 17, wherein MC is a macrocyclic structure. (Item 19) 18. The compound according to item 17, wherein MC is selected from DOTA, TETA, NOTP and NOTA. (Item 20) 18. The compound according to item 17, wherein MC is an acyclic polydentate ligand. (Item 21) 18. The compound according to item 17, wherein MC is selected from EDTA, EDTMP and DTPA. (Item 22) 18. The compound according to item 17, which is a radioactive halogen precursor, and Y is an alkyl metal moiety selected from the group consisting of trimethylstannyl (SnMe3), tri-n-butylstannyl (SnBu3) and trimethylsilyl (SiMe3). (Item 23) A prosthetic compound, wherein Y is 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I, 131 I and 211 Item 18. The compound according to item 17, wherein the radioactive halogen is selected from At. (Item 24) 18. The compound according to item 17, further comprising a metal bound to said MC. (Item 25) The metal is 177 Lu, 64 Cu, 111 In, 90 Y, 225 Ac, 213 Bi, 212 Pb, 212 Bi, 67 Ga, 68 Ga, 89 Zr and 227 25. The compound according to item 24, wherein the radioactive metal is selected from the group consisting of Th. (Item 26) A radiolabeled biomolecule or intermediate comprising the compound according to claim 17 attached to the biomolecule. 27. The radiolabeled biomolecule or intermediate according to item 26, wherein the biomolecule is selected from the group consisting of an antibody, an antibody fragment, a VHH molecule and an aptamer. (Item 28) 27. The radiolabeled biomolecule or intermediate according to item 26, wherein the biomolecule is a VHH. (Item 29) 29. The radiolabeled biomolecule or intermediate according to item 28, wherein said VHH targets HER2. (Item 30) 30. The radiolabeled biomolecule or intermediate according to item 29, wherein the VHH comprises an amino acid sequence selected from the sequences set forth in SEQ ID NOs: 1 to 5. (Item 31) 27. A pharmaceutical composition comprising the radiolabeled biomolecule according to item 26 in association with a pharma- ceutically acceptable adjuvant, diluent or carrier. (Item 32) 12. A method for treating cancer, comprising administering to an individual in need thereof an effective amount of the radiolabeled biomolecule according to item 11. (Item 33) 27. A method for treating cancer, comprising administering to an individual in need thereof an effective amount of a radiolabeled biomolecule according to item 26.

Claims

[Claim 1] The invention described in this specification.

Images