Reagents for site-specific labeling of proteins with radioactive halogens and methods for making and using same - Patents.com

Abstract

The present disclosure provides, in part, methods for site-specific labeling of proteins / peptides with radioactive halogens and compositions resulting from said methods.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to the benefit of U.S. Provisional Patent Application No. 63 / 239,463, filed September 1, 2021, and U.S. Provisional Patent Application No. 63 / 279,355, filed November 15, 2021. The entire contents of the foregoing applications are incorporated herein in their entireties.

[0002] FIELD OF THEINVENTION The subject matter disclosed herein relates generally to site-selectively radiolabeled biomolecules that are stable in vivo. [Background technology]

[0003] 2. Background of the Invention The ability of many biomolecules to selectively and specifically target cellular biomarkers has long been exploited in both molecular imaging (e.g., positron emission tomography, "PET") and targeted radiotherapy, in which biomolecules are labeled with radioactive atoms for selective delivery to cancer cells with the goal of killing them. This exploitation employs ligation of a radioactive payload to the biomolecule. Some of the best radionuclides for this purpose are the radioactive halogens. For example, 18 F is used for PET imaging, 131 I is used in beta particle therapy, 211 At is used in alpha particle therapy.

[0004] For peptides and proteins, radiolabeled molecules are usually prepared by ligating reactive bifunctional probes to amino acids in the biomolecule, most often lysines. This control of the conjugation site is fairly easy when using small peptides that barely have more than one or two copies of each amino acid, but it becomes a much bigger problem with larger biomolecules. For example, most antibodies contain dozens of lysines distributed throughout their macromolecular structure. Indiscriminate attachment of radioactive molecules or precursor molecules that will be labeled in a subsequent step to these lysines can lead to the formation of thousands of different protein conjugates that differ in the position of the modified lysine and / or the number of different lysines in the individual proteins that are modified. Naturally, this so-called "random" bioconjugation approach is poorly reproducible and produces a very heterogeneous population of conjugates. In addition, this strategy can lead to the payload being grafted incorrectly to the bioactive site of the biomolecule, rendering some of the conjugates non-functional.

[0005] To circumvent these problems, researchers have turned to strategies that allow better control over the ligation reaction site, known as "site-specific" bioconjugation. Conjugating maleimide-containing probes to cysteine, a thiol-containing amino acid present in low abundance in proteins, has become a staple of the field and has been widely used over the past 30 years. The popularity of this strategy is primarily based on its simplicity and efficiency. Many proteins contain disulfide bridges, which can be easily reduced to form reactive thiols, or free cysteines can be added to proteins using standard recombinant techniques. Michael addition between maleimides and sulfhydryl groups can be performed at physiological pH and room temperature and reliably results in the formation of succinimidyl thioether bonds within an hour (see Figure 1).

[0006] Although ligation between thiols and maleimides represents an undeniable improvement over random conjugation, this reaction also has drawbacks. Indeed, maleimide-based conjugates show limited stability in physiological media, since the conjugation can undergo a retro-Michael reaction that results in the release of the payload or its transfer to other endogenous molecules containing free thiols (most often serum albumin, cysteine, and glutathione) by transthiolation. In the context of nuclear imaging and radiotherapy, this retro-Michael reaction can release the radioactive payload through a thiol exchange reaction, resulting in in vivo radiolabeling of endogenous biomolecules (see Figure 1). This "leakage" causes high uptake in non-target tissues and concomitant low uptake in target tissues, ultimately resulting in increased radiation dose to healthy tissues, reduced imaging contrast, and low target-to-normal tissue radiation dose ratios, resulting in reduced therapeutic efficacy.

[0007] It would therefore be desirable to generate a method for radiolabeling proteins at specific sites on the protein using a linking strategy that is selective and stable under the physiological conditions experienced at the desired target site.

[0008] The discussion above is merely provided for general background information and is not intended to be used as an aid in determining the scope of the claimed subject matter. Summary of the Invention [Problem to be solved by the invention]

[0009] [Means for solving the problem]

[0010] The present disclosure provides novel precursors of radiohalogenation, processes for preparing the radiohalogenated prosthetic molecules, and processes for preparing radiolabeled proteins or peptides using the radiohalogenated prosthetic molecules. Effect of the Invention

[0011] In embodiments, the radiohalogenated prosthetic molecule or radiohalogenated support precursor has the structure (I): [ka] (In the formula, a is 1 to 6; b is 1 to 6; c is 1 to 6; L 1 is -C(O)- or [ka] and; L 2 is a bond or -(CH 2 ) n where n is 1 to 6; G is guanidine, guanidine with one or more carbamate protecting groups, PO 3 H, SO 3 H, P.O. 2 -OtBu, SO 2 -OtBu, arginine, phosphonophenylalanine, sulfophenylalanine, glutamic acid, aspartic acid, lysine, a hydrophilic carbohydrate moiety, or a polyethylene glycol (PEG) chain; Y is CH or N; X is SnR 2 3 , B(OH) 2 , Bpin, or a radioactive halogen; R 1 is C 1-6 is alkyl; and Each R 2 is independently C 1-6 (It is alkyl) has.

[0012] In embodiments, the radiohalogenated prosthetic molecule or radiohalogenated support precursor has the structure (II): [ka] (In the formula, a is 1 to 6; b is 1 to 6; c is 1 to 6; L is -C(O)- or [ka] and; X is SnR 2 3 , B(OH) 2 , Bpin, or a radioactive halogen; R 1 is C 1-6 is alkyl; Each R 2 is independently C 1-6 is alkyl; and Each R 3 are independently a carbamate protecting group or H. has.

[0013] In embodiments, the radiohalogenated prosthetic molecule or radiohalogenated support precursor has the structure (III): [ka] has.

[0014] In embodiments, the radiohalogenated prosthetic molecule or radiohalogenated support precursor has the structure (IV): [ka] has.

[0015] In an embodiment of the present disclosure, R in any of compounds (I) to (IV) 1 is methyl, ethyl, or propyl. In an embodiment of the present disclosure, in any of compounds (I) to (IV), a is 1 or 2, b is 2 or 3, and c is 1 or 2. In an embodiment of the present disclosure, X in any of the radioactive halogenated support precursors of compounds (I) to (IV) is SnR 2 3 , B(OH) 2 , or Bpin, R 2is methyl, ethyl, or n-butyl. In an embodiment of the present disclosure, X in any of the radiohalogenated prosthetic molecules of compounds (I)-(IV) is 18 F, 122 I, 123 I, 124 I, 125 I, 131 I, 75 Br, 76 Br, 77 Br, 80m Br, and 211 At.

[0016] In embodiments, the radiolabeled protein or peptide comprises one or more radioactive halogenated prosthetic molecules attached to the protein or peptide via a thioether bond, and the one or more radioactive halogenated prosthetic molecule-protein / peptide conjugates have the structure (V): [ka] (In the formula, a is 1 to 6; b is 1 to 6; c is 1 to 6; L 1 is -C(O)- or [ka] and; L 2 is a bond or -(CH 2 ) n where n is 1 to 6; G is guanidine, guanidine with one or more carbamate protecting groups, PO 3 H, SO 3 H, P.O. 2 -OtBu, SO 2 -OtBu, arginine, phosphonophenylalanine, sulfophenylalanine, glutamic acid, aspartic acid, lysine, a hydrophilic carbohydrate moiety, or a polyethylene glycol (PEG) chain; Y is CH or N; X is a radioactive halogen; R 1 is C1-6 is alkyl; and Pep is a protein or peptide or one or more of the radioactive halogenated prosthetic molecule-protein / peptide conjugates is a pharma- ceutically acceptable salt of structure (V).

[0017] In an embodiment, the radiolabeled protein / peptide has the structure (VI): [ka] (In the formula, a is 1 to 6; b is 1 to 6; c is 1 to 6; L is -C(O)- or [ka] and; X is a radioactive halogen; R 1 is C 1-6 is alkyl; Each R 3 is independently a carbamate protecting group or H; and Pep is a protein or peptide has.

[0018] In an embodiment, the radioactive protein / peptide has the structure (VII): [ka] (In the formula, Pep is a protein / peptide) has.

[0019] In an embodiment, the radiolabeled protein / peptide has the structure (VIII): [ka] (In the formula, Pep is a protein or peptide has.

[0020] In an embodiment of the present disclosure, X in any of compounds (V) to (VIII) is 18 F, 122 I, 123 I, 124 I, 125 I, 131 I, 75 Br, 76 Br, 77 Br, 80m Br, and 211 At. In an embodiment of the present disclosure, the protein / peptide in any of compounds (V) to (VIII) comprises at least one cysteine ​​residue, and one or more radioactive labels are attached to the protein / peptide via the cysteine ​​residue. In an embodiment of the present disclosure, the protein / peptide comprises a C-terminal glycine-cysteine ​​tail, and the radioactive label is attached to the protein / peptide via the C-terminal glycine-cysteine ​​tail. In another embodiment, the protein / peptide comprises an N-terminal glycine-cysteine ​​tail, and the radioactive label is attached to the protein / peptide via the N-terminal glycine-cysteine ​​tail. The protein / peptide may be an antibody, for example, a single domain antibody fragment. The protein / peptide is a tumor-targeting protein / peptide.

[0021] In an embodiment, a method for performing molecular imaging in a subject comprises administering to the subject an effective amount of a radiolabeled protein / peptide of any of compounds (V) to (VIII).

[0022] In an embodiment, the method of treating cancer in a subject comprises administering to the subject an effective amount of a radiolabeled protein / peptide of any of Compounds (V)-(VIII).

[0023] In an embodiment, the method of labeling a protein or peptide with a radioactive halogen comprises: (i) Structure (II): [ka] (In the formula, a is 1 to 6; b is 1 to 6; c is 1 to 6; X is a radioactive halogen; R 1 is C 1-6 is alkyl; Each R 3 is independently a carbamate protecting group or H; and L is C(O) or [ka] is) to obtain a compound having the formula: (ii)R 3 is H, reacting compound (II) with a protein or peptide, or 3 is a carbamate protecting group, reacting compound (II) with a peptide; Includes.

[0024] In an embodiment, the method of labeling a protein / peptide with a radioactive halogen further comprises removing a carbamate protecting group (e.g., a Boc group) from the compound prior to reacting the compound with the protein. In an embodiment, the protein or peptide is an antibody fragment, e.g., a single domain antibody fragment. In an embodiment, the protein / peptide is a tumor targeting protein / peptide.

[0025] In an embodiment of the disclosure, the method further comprises modifying the protein / peptide by adding a C-terminal glycine-cysteine ​​tail to the protein / peptide. An exemplary glycine-cysteine ​​tail has the structure G n C, where n is an integer from 2 to 10. The radioactive halogen or radioactive halogen support precursor-prosthetic molecule reacts with a cysteine ​​residue of the glycine-cysteine ​​tail to form a thioether bond between the prosthetic molecule and the protein / peptide. In another embodiment, the protein / peptide comprises an N-terminal glycine-cysteine ​​tail and the radioactive label is attached to the protein / peptide via the N-terminal glycine-cysteine ​​tail.

[0026] In embodiments, the radiolabeled protein / peptide produced by this method has the structure (VII): [ka] (In the formula, Pep is a protein or peptide has.

[0027] In embodiments, the radiolabeled protein / peptide produced by this method has the structure (VIII): [ka] (In the formula, Pep is a protein or peptide has.

[0028] In order that the features of the present disclosure may be understood, the present invention may be described in detail by reference to certain embodiments, some of which are illustrated in the accompanying drawings. It should be noted, however, that the drawings are merely illustrative of certain embodiments of the present invention and therefore should not be considered as limiting the scope of the present invention, which encompasses other equally effective embodiments. The drawings are not necessarily to scale. Generally, emphasis is placed on illustrating the features of certain embodiments of the present invention. Therefore, for a better understanding of the present invention, reference should be made to the following detailed description, which may be read in conjunction with the following drawings. [Brief description of the drawings]

[0029] [Figure 1] FIG. 1 shows the conjugation of a thiol group in a protein or peptide with a maleimide derivative containing a radiolabeled moiety, and the exchange reaction of the product thiol with an endogenous thiol. [Diagram 2] FIG. 2 shows the conjugation of 5F7GGC to iso-[ 131 I]GMIB-PODS and iso-[ 211 At]AGMB-PODS. [Diagram 3]FIG. 3 shows the synthesis scheme of [177Lu]Lu-DOTA-PODS and [177Lu]Lu-DOTA-PODS-5F7GGC. [Figure 4] FIG. 4 shows data from a surface plasmon resonance (SPR) assay used to determine the binding affinity (Kd) of the 5F7GGC conjugate and the 5F7GGC conjugate for the HER2 extracellular domain. [Figure 5A] FIG. 5A shows data from a saturation binding assay using HER2-expressing BT474 human breast cancer cells to determine the binding affinity of iso-[ 131 I]GMIB-PODS-5F7GGC. [Figure 5B] FIG. 5B shows data from a saturation binding assay of iso-[ 131 I]GMIB-PODS-5F7GGC measured in SKOV-3 cells. [Figure 5C] FIG. 5C shows data from a saturation binding assay of iso-[211At]AGMB-PODS-5F7GGC measured in BT474 cells. [Figure 6A-B] Figures 6A and 6B show the results of an in vitro paired-label internalization assay in HER2-positive BT474 breast cancer cells co-incubated with [I]MEGMIB-5F7GGC and iso-[I]GMIB-PODS-5F7GGC. Data shown are the surface-bound (A) and internalized (B) fractions of radioactivity initially bound to the cells after 1 hour incubation at 4°C. [Figure 7A-B] 7A and 7B show the results of an in vitro paired-label internalization assay in HER2-positive BT474 breast cancer cells co-incubated with iso-[ 131 I]GMIB-PODS-5F7GGC and iso-[ 211 At]AGMB-PODS-5F7GGC. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] Detailed Description of the Invention The present disclosure provides novel precursors for radiohalogenation, processes for preparing radiohalogenated prosthetic molecules, and processes for preparing radiolabeled proteins or peptides using radiohalogenated prosthetic molecules. Although it is known that radiohalogenated proteins and peptides can be used for molecular imaging and targeted radiotherapy, the common techniques used to label proteins and peptides have a lack of selectivity and / or a lack of stability when administered to a subject. The present disclosure overcomes these problems by using radiohalogenated prosthetic molecules that can be conjugated to proteins / peptides to selectively form thioether bonds with cysteine ​​residues on the protein. Radiolabeled protein conjugates made with the described radiolabeled prosthetic molecules show high stability in vitro, high internalization and retention in tumor cells in vitro, and excellent tumor uptake in vivo.

[0031] I. Peptides / Proteins As used herein, the term "peptide" refers to a chain of amino acids linked together by amide bonds. Examples of peptides include oligopeptides and polypeptides. As used herein, the term "oligopeptide" refers to a peptide that is composed of less than 15 amino acids. As used herein, the term "polypeptide" refers to a peptide that is composed of 15 or more amino acids. As used herein, the term "protein" refers to a polypeptide that is composed of 50 or more amino acids.

[0032] In a preferred embodiment, the protein / peptide used to form the radiolabeled protein / peptide conjugate is a tumor-targeting protein / peptide. As used herein, a "tumor-targeting protein / peptide" is a protein / peptide that binds to a target molecule on a tumor cell, including a target molecule that is overexpressed in tumor cells (e.g., expressed at a measurably increased level in tumor cells compared to normal cells) and / or a target molecule that is specifically expressed in tumor cells (e.g., not substantially expressed in normal cells). Thus, a tumor-targeting protein / peptide may bind to a tumor-associated antigen or receptor and / or a tumor-specific antigen or receptor. A "tumor-associated antigen or receptor" is an antigen or receptor that is found at high levels in tumor cells, but may also be expressed at lower levels in non-tumor cells. A "tumor-specific antigen or receptor" is an antigen or receptor that is found exclusively or predominantly in cancer cells. Numerous tumor-targeting proteins / peptides, tumor-associated antigens and receptors, and tumor-specific antigens and receptors are known in the art and are routinely used.

[0033] Tumor targeting proteins / peptides that "specifically bind" or "preferentially bind" to tumor or cancer cells are terms well known in the art, and methods for determining such specific or preferential binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates with a particular cell or substance more frequently, faster, for a longer period, and / or with a higher affinity. A tumor targeting protein / peptide "specifically binds" or "preferentially binds" to a target or antigen if it binds with higher affinity, avidity, more readily, and / or for a longer period than it binds to other molecules. It should also be understood that a tumor targeting protein / peptide that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (but can include) exclusive binding.

[0034] Preferably, the tumor targeting agent (TTA) used in the present invention has a high binding affinity, e.g., about 10 -9 Dissociation constant K less than M D (k off / k on ).

[0035] Representative tumor targeting proteins include antibodies. An "antibody" is an immunoglobulin molecule that recognizes and binds to a specific target or antigen, such as a carbohydrate, polynucleotide, lipid, or polypeptide, through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the terms "antibody" and "antibodies" encompass any type of antibody, including, but not limited to, standard antibodies, monoclonal antibodies, polyclonal antibodies, bispecific antibodies, multispecific antibodies, heteroconjugate antibodies, recombinantly produced antibodies, humanized antibodies, chimeric antibodies, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, antibody fragments (described further below), and fusion proteins comprising an antibody or an antigen-binding fragment thereof, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, and any other modified configuration of an immunoglobulin molecule, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.

[0036] A standard antibody comprises two heavy (H) and light (L) polypeptide chains linked by covalent disulfide bonds and non-covalent interactions. Each light chain is composed of one variable domain (VL) and one constant domain (CL). The light chains of antibodies from vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains. Each heavy chain contains one variable domain (VH) and a constant region, which in the case of IgG, IgA, and IgD antibodies, contains three domains called CH1, CH2, and CH3 (IgM and IgE have a fourth domain, CH4).

[0037] An "antibody fragment" comprises at least a portion of an intact antibody sufficient to function as a targeting agent as described herein. Antibody fragments include "antigen-binding fragments," which generally refer to polypeptide fragments of immunoglobulins or antibodies that specifically bind or react with a selected antigen, target, or immunogenic determinant thereof, or compete for specific antigen binding with the intact antibody from which the fragment is derived. Antibody fragments include Fab fragments, Fab' fragments, F(ab') fragments, and F(ab') fragments. 2 These include, but are not limited to, fragments, Fd fragments, Fv fragments, Fc fragments, scFv fragments, Fc fusions including nanobody-Fc fusions, dual variable domain (DVD) Fabs, single chain antibodies, single domain antibodies (also known as sdAbs, nanobodies and VHH antibodies, e.g. VNAR antibodies). In a preferred embodiment of the invention, the tumor targeting protein is a single domain antibody fragment (sdAb), also known as a VHH molecule or a nanobody.

[0038] In one embodiment, the antibody is a "multispecific antibody" that binds to two or more antigens or epitopes. In some cases, the multispecific antibody binds to two, three, four, five or more target antigens or epitopes. The target antigens or epitopes can be present on the same cell or on separate cells. The target antigens can be two or more separate unique antigens or different epitopes of the same antigen. A "bispecific" antibody or antibody fragment binds to two target antigens or epitopes.

[0039] In certain embodiments, the antibodies useful in the present invention are fully human, humanized, or chimeric antibodies.

[0040] In other aspects, antibodies useful in the present invention are obtained from Camelidae species such as dromedary, Bactrian camel, wild Bactrian camel, llama, alpaca, vicuña, and guanaco. In particular, antibodies generated in Camelidae species have only three hypervariable regions (CDRs) compared to traditional IgG-derived antibody formats, and therefore these binders preferably recognize and bind conformational epitopes such as those formed by the enzymatic pocket of the regulatory domain.

[0041] As used herein, the term "antibody" or "antibodies" further includes antibody mimetics, i.e., synthetic proteins that exhibit high affinity specific binding similar to antibodies, such as DARPins, affibodies, knottins, affilins, affimers, affitins, alphabodies, anticalins, avimers, finomers, kunitx domain peptides, monobodies, nanoCLAMPs, and the like.

[0042] Representative tumor antigens / receptors to which tumor targeting proteins / peptides useful in the present invention bind include, for example, A33, αvβ3, AFP, AKAP-4, ALK, AR, B7-DC (PD-L2), B7H3, B7-H3, BCMA, BCR-ABL, BRCA mutations, BORIS, C1orf186, CA9, CA-125, CA19-9, CA6, CAIX, CAMPATH-1, CEA, CD19, CD20, CD25, CD30, CD33, CD37, CD45, CD5, CLDN16, CLDN6, CLDN18.2, CMET, CS-1, CCNB1, CXCR2, CYP1B1, DLL3, EGF, EGFR, EGFRvIII, (de2-7 EGFR), EMR2, ENG, EPCAM, EPHA2, ERG, ETV6-AML, EWSR1, FAP, FBP, folate receptor, FOSL1, FRA, FucGM1, G250, GAGE, G D2, GD3, GloboH, GLP-3, GM2, gp100, GPC3, GRP94 (endoplasmin), HER2, Her-2 / Neu, HER3, HLA-DR, HMWMAA, HPV E6, HPV E7, hTERT, IL-2 receptor, LCK, LGMN, LewisY, LIV1, LMP2, LRRC15, LY6E, MAD-CT-1, MAD-CT-2, MAGE A1, MAGE A4, MAGE C2, MAGE-A3, MelanA / MART1, MSLN, mesothelin, ML-IAP, MMP, MUC1, MUC15, MUC16, MYCN, NA17, NAPI2B, NY-BR-1, NY-ESO-1, OY-TES1, p53 mutant, p53 non-mutant, PAGE4, ​​PAP, PARP (e.g., PARP-1), PAX3, PAX5, PDGFR-B, PD-L, PD-L1, PLAV1, polySia, PR1, PSA, PSCA, PSMA, PTK7, Ras mutant, RGS5, RhoC, RON, ROR1, ROR2, SART3, sLe (animal), somatostatin receptor, SP17, SSX2, STn, STRA6, tenascin, TEM1, Tie 3, TIM-3, TMEM238, TMPRSS3, TMPRSS4, RAIL1, TROP2, TRP-2, UPK1B, VEGFR1, VEGFR2, VISTA, VTCN1 (B7-H4), WT1, XAGE 1, and TAG-72.

[0043] Examples of proteins / peptides that can bind to radioactive halogenated prosthetic molecules include, but are not limited to, antibodies that bind to molecules expressed by cancer cells. In embodiments, the antibodies are selective for HER2-expressing cancers. In one embodiment, the protein / peptide targeting agent is an anti-HER2 5F7 sdAb as described in Pruszynski et al., "Targeting Breast Carcinoma with Radioiodinated anti-HER2 Nanobody," Nuclear Medicine and Biology 40 (2013) 52-59. The anti-HER2 5F7 sdAb can be modified to couple to a radioactive halogenated prosthetic molecule by modifying the anti-HER2 5F7 sdAb to have a C-terminal glycine-cysteine ​​tail. Additional anti-HER2 single domain antibodies are described in U.S. Pat. No. 10,174,117 to Baty et al. and PCT Publication WO 2022 / 152862 to Perez et al., both of which are incorporated herein by reference. Anti-HER2 nanobodies are described in U.S. Patent Application Publication No. 2020 / 0306392 to Ting et al., which is incorporated herein by reference. Modified anti-HER2 sdAbs can also be used. For example, VHH_1028 was described in Feng et al., "A Modified Anti-HER2 sdAb for Radiopharmaceutical Therapy of HER2-Expressing Cancers," which is incorporated herein by reference. 131 Evaluation of an I-labeled HER2-specific single domain antibody fragment 131 It is a modified anti-HER2 sdAb that has been modified to ensure the absence of lysine residues in the CDR loops, as described in "I-labeled HER2-specific single domain antibody fragment for the radiopharmaceutical therapy of HER2-expressing cancers" Sci Rep 12, 3020 (2022).

[0044] In some embodiments of the invention, the tumor targeting protein is "internalized", i.e., upon binding to the target antigen or receptor, is taken up by the cell along with the radiolabel attached to the protein or a fragment thereof. As is understood in the art, antibodies can be engineered to be internalizing or otherwise selected for this property. See, e.g., Zhou et al., Arch Biochem Biophys, 2012, 15;526(2):107-13.

[0045] If the tumor targeting protein / peptide is not naturally internalized, one or more cell penetrating agents may be coupled to the tumor targeting protein / peptide to facilitate intracellular delivery of the radiolabeled protein / peptide. The cell penetrating agent may protect the radiolabeled protein / peptide from endosomal trapping and / or lysosomal degradation.

[0046] Representative cell-penetrating agents include cell-penetrating peptides (CPPs), which are typically peptides 10-30 amino acids (aa) in length and are either arginine-rich and amphipathic or lysine-rich and hydrophobic. CPPs can be, for example, cationic, amphipathic or hydrophobic peptides, dendrimeric, constrained or cross-linked peptides, e.g., composed primarily of Tyr, Trp and Phe. See, e.g., Herce et al., Nat Chem, 2017, 9:762-771.

[0047] In some embodiments of the present invention, nuclear localization peptides (NLPs) can be used to promote nuclear localization of radiolabeled proteins / peptides.For example, a nucleophilic peptide consisting of at least four arginines (R) and lysines (K) in a hexapeptide flanked by proline and glycine helix breakers can be used as the NLP of radiolabeled peptides.See, for example, Chen et al. J Nucl Med, 2006, 47:827-836.

[0048] II. Radioactive halogenated prosthetic molecules One aspect of the present disclosure is a compound having structure (I): [ka] (In the formula, a is 1 to 6; b is 1 to 6; c is 1 to 6; L 1 is -C(O)- or [ka] and; L 2 is a bond or -(CH 2 ) n where n is 1 to 6; G is guanidine, guanidine with one or more carbamate protecting groups, PO 3 H, SO 3 H, P.O. 2 -OtBu, SO 2 -OtBu, arginine, phosphonophenylalanine, sulfophenylalanine, glutamic acid, aspartic acid, lysine, a hydrophilic carbohydrate moiety, or a polyethylene glycol (PEG) chain; Y is CH or N; X is a radioactive halogen; and R 1 is C 1-6 (It is alkyl) The present invention provides a radioactive halogenated prosthetic molecule having the formula:

[0049] The terminal guanidine group can be unprotected or can be protected with one or more carbamate protecting groups. Exemplary carbamate protecting groups include, but are not limited to, tert-butyloxycarbonyl ("Boc"), allyloxycarbonyl ("Alloc"), fluorenylmethyloxycarbonyl ("Fmoc"), 2-(trimethylsilyl)ethoxycarbonyl ("TeoC"), and carboxybenzyl ("CBz"). The guanidine moiety can be protected with one, two, three, or four carbamate protecting groups. In a specific embodiment, the guanidine is a Boc-protected guanidine, such as a mono-Boc-protected guanidine, a di-Boc-protected guanidine, a tri-Boc-protected guanidine, or a tetra-Boc-protected guanidine.

[0050] In one embodiment, the radiohalogenated prosthetic molecule has the structure (II): [ka] (In the formula, a is 1 to 6; b is 1 to 6; c is 1 to 6; L is -C(O)- or [ka] and; X is a radioactive halogen; R 1 is C 1-6 is alkyl; Each R 2 is independently C 1-6 is alkyl; and Each R 3 are independently a carbamate protecting group or H. has.

[0051] In some specific embodiments, the radiohalogenated prosthetic molecule has the structure (II), where R 1 is methyl, ethyl, or propyl, a is 1 or 2, b is 2 or 3, and c is 1 or 2.

[0052] The radioactive halogens which may be used are: 18 F, 122 I, 123 I, 124 I, 125 I, 131 I, 75 Br, 76 Br, 77 Br, 80m Br, and 211 Including, but not limited to, At.

[0053] An example of a radioactive halogenated prosthetic molecule is the compound of structure (III): [ka] (In the formula, a is 1 to 6; b is 1 to 6; c is 1 to 6; X is a radioactive halogen; R 1 is C 1-6 is alkyl; Each R 2 is independently C 1-6 is alkyl; Each R 3 are independently a carbamate protecting group or H. It is a compound having the formula:

[0054] Another specific example of a radioactive halogenated prosthetic molecule has the structure (IV): [ka] (In the formula, a is 1 to 6; b is 1 to 6; c is 1 to 6; X is a radioactive halogen; R 1 is C 1-6 is alkyl; Each R 2 is independently C 1-6 is alkyl; Each R 3 are independently a carbamate protecting group or H. It is a compound having the formula:

[0055] III. Protein / peptide-radiohalogenated prosthetic molecule conjugates In embodiments, the radioactively halogenated prosthetic molecules described herein can be covalently attached to a protein / peptide to form a radioactively labeled protein / peptide. In one embodiment, the radioactively labeled protein / peptide comprises one or more radioactively halogenated prosthetic molecules attached to the protein / peptide via a thioether bond, wherein the radioactively labeled protein / peptide has the structure (V): [ka] (In the formula, a is 1 to 6; b is 1 to 6; c is 1 to 6; L is C(O) or [ka] and; L 2 is a bond or -(CH 2 ) n where n is 1 to 6; G is guanidine, guanidine with one or more carbamate protecting groups, PO 3 H, SO 3 H, P.O. 2 -OtBu, SO 2 -OtBu, arginine, phosphonophenylalanine, sulfophenylalanine, glutamic acid, aspartic acid, lysine, a hydrophilic carbohydrate moiety, or a polyethylene glycol (PEG) chain; Y is CH or N; X is a radioactive halogen; R 1 is C 1-6 is alkyl; and Pep is a protein / peptide) or the radiolabeled protein / peptide is a pharma- ceutically acceptable salt of structure (V).

[0056] In a specific embodiment, the radiolabeled protein / peptide has the structure (VI): [ka] (In the formula, a is 1 to 6; b is 1 to 6; c is 1 to 6; X is a radioactive halogen; L is C(O) or [ka] and Pep stands for protein / peptide) or the radiolabeled protein / peptide is a pharma- ceutically acceptable salt of structure (VI).

[0057] In an embodiment, the radioactive halogen is 18 F, 122 I, 123 I, 124 I, 125 I, 131 I, 75 Br, 76 Br, 77 Br, 80m Br, and 211 At.

[0058] In an embodiment, the protein / peptide contains at least one cysteine ​​residue, wherein one or more radioactive halogenated prosthetic molecules are attached to the cysteine ​​residue of the protein / peptide.

[0059] In a specific embodiment, the protein / peptide can be an antibody. For example, the protein / peptide can be a single domain antibody fragment. In a specific embodiment, the protein / peptide is a tumor targeting protein.

[0060] In some embodiments, the protein / peptide may be modified by incorporating one or more cysteine ​​amino acids into the protein / peptide prior to coupling with the radiolabel. In one embodiment, at least one cysteine ​​is incorporated into the C-terminus of the protein / peptide. For example, a glycine-cysteine ​​tail may be added to the C-terminus of the protein / peptide. Methods for incorporating C-terminal cysteine ​​groups on proteins are discussed in Pruszynski et al., "Targeting Breast Carcinoma with Radioiodinated anti-HER2 Nanobody," Nuclear Medicine and Biology 40 (2013) 52-59, which is incorporated herein by reference.

[0061] In embodiments, a specific example of a radiohalogenated protein / peptide has the structure (VII): [ka] where Pep is a protein / peptide. It is a compound having the formula:

[0062] In another embodiment, a specific example of a radioactive halogenated protein / peptide has the structure (VIII): [ka] where Pep is a protein / peptide. It is a compound having the formula:

[0063] IV. Synthesis of Radioactive Halogenated Prosthetic Molecules The radiohalogenated prosthetic molecules described herein can be synthesized by coupling a phenyloxadiazolylmethylsulfone (PODS)-containing linker to a radiolabeled support compound. The PODS linker contains a phenyloxadiazolylmethylsulfone that is used to selectively form a thioether bond with a cysteine ​​in a protein / peptide. The phenyloxadiazolylmethylsulfone is located at one end of the molecule. The remainder of the PODS linker contains a linker group that is used to couple the phenyloxadiazolylmethylsulfone to the radiolabeled support. The PODS linker has the general structure (IX): [ka] (In the formula, a is 1 to 6; b is 1 to 6; c is 1 to 6; R 1 is C 1-6 (It is alkyl) has.

[0064] In a preferred embodiment, the PODS linker has the structure (IX), where a is 1 or 2, b is 2 or 3, c is 1 or 2, and R 1 is methyl, ethyl, or propyl. An exemplary PODS linker (hereinafter known as "PODS") is shown below at (X). [ka]

[0065] Methods for preparing PODS linkers are taught in US Pat. No. 11,000,604, which is incorporated herein by reference.

[0066] The terminal amine of the PODS linker (-NH 2 ) is coupled to a radiolabeled support compound. In one embodiment, the radiolabeled support compound has the structure (XI): [ka] (In the formula, L 1 teeth, [ka] or [ka] and; L 2 is a bond or -(CH 2 ) n where n is 1 to 6; G is guanidine, guanidine with one or more carbamate protecting groups, PO 3 H, SO 3 H, P.O. 2 -OtBu, SO 2 -OtBu, arginine, phosphonophenylalanine, sulfophenylalanine, glutamic acid, aspartic acid, lysine, a hydrophilic carbohydrate moiety, or a polyethylene glycol (PEG) chain; Y is CH or N; X is SnR 2 3 , B(OH) 2 , Bpin, or a radioactive halogen; Each R 2 is independently C 1-6 (It is alkyl) has.

[0067] In a specific embodiment, the radiolabeled support compound has the structure (XII): [ka] (In the formula, L 1 teeth, [ka] or [ka] and; X is SnR 2 3 , B(OH) 2 , Bpin, or a radioactive halogen; Each R 2 is independently C 1-6 (It is alkyl) has.

[0068] Specific radiolabeled support compounds are N-succinimidyl 3-guanidinomethyl-5-(trimethylstannyl)benzoate (iso-SGMTB, XIII); N-succinimidyl 3-guanidinomethyl-5-[ * I]Iodobenzoate (iso-[ * I]SGMIB, XIV);N-Succinimidyl 3-[ 211 At] Astato 5-guanidinomethylbenzoate salt (iso[ 211 At]SAGMB, XV]; N-Maleimidoethyl 3-guanidinomethyl-5-(trimethylstannyl)benzamide (MEGMTB, XVI); N-Maleimidoethyl 3-[ 211 At] astat-5-guanidinomethylbenzamide (MEAGMB, XVII) and N-maleimidoethyl 3-guanidine SAGMB iodomethyl-5-iodobenzamide (MEGMIB, XVIII). [ka] Where: * I is an isotope of iodine, including, but not limited to, stable (127) or radioactive (131, 123, 125, and other) isotopes of iodine.

[0069] Methods for preparing radiolabeled support compounds are taught in U.S. Patent Application Publication No. 2020 / 0188541, which is incorporated herein by reference.

[0070] The PODS linker is coupled to a radiohalogenated support precursor to form a radiohalogenated support precursor-PODS conjugate. When the support has a maleimide group, the general reaction for coupling is shown in Scheme (1). [ka]

[0071] Coupling of the terminal amine of the PODS linker with the maleimide group of the radioactive halogen support precursor is carried out under basic conditions. Typical basic conditions include reaction in the presence of a tertiary amine, such as trimethylamine or diisopropylethylamine. In another embodiment, a buffered aqueous solution (pH>7) can be used as the reaction medium.

[0072] A general reaction scheme for coupling using the active ester N-hydroxysuccinimide is shown in Scheme (2). [ka]

[0073] Conjugation of the terminal amine of the PODS linker with the N-hydroxysuccinimide active ester group of the radioactive halogen support precursor is carried out under basic conditions similar to reaction scheme (1). Typical basic conditions include reaction in the presence of a tertiary amine such as trimethylamine or diisopropylethylamine. In other embodiments, a buffered aqueous solution (pH>7) can be used as the reaction medium.

[0074] Details regarding N-hydroxysuccinimide active esters and similar coupling reactions of maleimides with amines can be found in U.S. Patent Application Publication No. 2020 / 0188541, which is incorporated herein by reference.

[0075] In an embodiment of the present disclosure, the final step in preparing a radioactive halogenated prosthetic molecule is the conversion of a trialkyltin or trialkylboronic acid group to a radioactive halogen. In general, a trialkyltin-substituted aromatic compound (e.g., present in a precursor of a radioactive halogen support) is reacted with a radioactive halogen in the presence of an oxidizing agent. The oxidizing agent oxidizes the radioactive halogen, allowing the substitution of the trialkyltin moiety by the radioactive halogen to occur. This process is carried out in a suitable solvent or solvent system for dissolving the reactants. Scheme (3) illustrates this process for an amide-bearing PODS radioactive halogen support. A similar process is used to convert a trialkyltin-substituted maleimide-coupled PODS precursor to a radioactive halogenated support. In a preferred embodiment, the oxidizing agent is N-chlorosuccinimide. Methods for the formation of radioactive halogenated compounds are discussed in the article "Recent Advances in Synthetic Methods for Radioiodination" by Dubost et al., J. Org. Chem. 2020, 85, 8300-8310, which is incorporated herein by reference. The conversion of boronic acid and ester precursors to radioactive halogenated derivatives is typically carried out using nucleophilic radiohalides, and this conversion does not require an oxidizing agent (Kondo et al., Journal of Labelled Compounds and radiopharmaceuticals (2021), 64(8), 336-345; Reilly et al., Organic Letters (2018), 20(7), 1752-1755). [ka]

[0076] V. Synthesis of Radiolabeled Proteins and Peptides The present disclosure further describes a method for radiolabeling a protein / peptide, such as a method for radiolabeling a tumor-targeting protein, which is generally shown in the following reaction scheme (4). Protein / peptide + radiolabel → protein / peptide - (radiolabel) x Scheme (4) where x is at least 1.

[0077] x relates to the number of groups present on the protein / peptide that can react with a radioactive halogenated prosthetic molecule.

[0078] In one embodiment, radiolabels can be coupled to proteins / peptides using an oxadiazolylsulfone-based coupling method. This coupling technique is shown in Scheme (5). In this reaction (Reaction Scheme (5)), oxadiazolylsulfone reagents react selectively with thiols to form stable protein / peptide radiolabeled conjugates. [ka]

[0079] The reaction is typically carried out by mixing the protein / peptide with the radioactive halogenated prosthetic molecule. In some embodiments, the peptide is reacted with a reducing agent to cleave disulfide bonds prior to reaction with the radioactive halogenated prosthetic molecule. In embodiments, the reducing agent is tris(2-carboxyethyl)phosphine ("TCEP") or dithiothreitol (DTT). Scheme (6) shows an example of the conjugation of a radiolabel of the present disclosure to a protein / peptide. In the reaction shown, a tail group containing a pendant cysteine ​​group is added to the protein / peptide of interest. However, it should be understood that the same reaction can be carried out on a cysteine ​​amino acid present in an unmodified protein / peptide. [ka]

[0080] In another embodiment of the present disclosure, the protein / peptide is coupled to a radiohalogenated support precursor, followed by radiolabeling of the protein / peptide radiohalogenated support precursor. Upon coupling of the protein to the radiohalogenated support, the guanidine protecting group is preferably removed. This method is generally shown in the following reaction scheme (7): [ka]

[0081] The reactions used in Scheme (7) are similar, if not identical, to those used in the corresponding transformations in the previous reaction schemes.

[0082] VI.Applications The radiolabeled conjugates prepared as described herein have versatile uses in therapy and imaging, i.e., any application that would benefit from targeted delivery of a radionuclide, such as a radioactive halogen.

[0083] A. Treatment with Radiolabeled Conjugates It will be understood that the radiolabeled protein / peptide conjugates of the present disclosure, whether administered alone or in combination with additional anti-cancer agents or radiation therapy, can be used to treat cancer or other neoplastic disorders. The radiohalogenated protein / peptide conjugates disclosed herein can be used to treat any of a variety of cancers and other neoplastic conditions recognized in the art, for example solid tumors such as ovarian, breast, adrenal, liver, kidney, bladder, gastrointestinal, cervical, uterine, prostate, pancreatic, lung, thyroid, and brain tumors. The radiohalogenated protein / peptide conjugates disclosed herein can also be used to treat hematological malignancies.

[0084] Further neoplastic conditions amenable to treatment according to the invention are adrenal gland tumors, AIDS-related cancers, alveolar soft part sarcoma, astrocytic tumors, bladder cancer (squamous cell carcinoma and transitional cell carcinoma), bone cancer (adamantinoma, aneurysmal bone cyst, osteochondroma, osteosarcoma), brain and spinal cord cancer, metastatic brain tumors, breast cancer, carotid body tumors, cervical cancer, chondrosarcoma, chordoma, chromophobe renal cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, cutaneous benign fibrous histiocytoma, desmoplastic small round cell tumor, ependymoma, Ewing's tumor, extraskeletal myxoid chondrosarcoma, fibroplasia imperfecta, fibrous dysplasia of bone, cancer of the gallbladder and bile duct, gestational trophoblastic disease, germ cell tumors, head and neck cancer, pancreatic islet cell tumors, Kaposi's sarcoma, kidney cancer (nephroblastoma, papillary renal cell carcinoma), leukemia, lipoma / benign lipoma. , liposarcoma / malignant lipoma, liver cancer (hepatoblastoma, hepatocellular carcinoma), lymphoma, lung cancer (small cell carcinoma, adenocarcinoma, squamous cell carcinoma, large cell carcinoma, etc.), medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid carcinoma, parathyroid tumor, childhood cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, rare blood disorder, renal metastatic cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, thyroid metastatic cancer, and uterine cancer (cervical carcinoma, endometrial carcinoma, and leiomyoma).

[0085] In certain embodiments, the radiohalogenated protein / peptide conjugates are used to treat breast cancer, including invasive or infiltrating breast cancer, recurrent breast cancer, and / or refractory breast cancer. In some particular embodiments, the breast cancer is a HER2+ cancer.

[0086] The radiolabeled conjugates disclosed herein can be used as breakthrough targeted therapy for the treatment of cancer, particularly cancers characterized by solid tumors. An important advantage of the disclosed conjugates is the optimization of therapeutic index by reducing or eliminating side effects resulting from non-specific targeting of therapeutic radionuclides. Thus, the disclosed conjugates can be used at reduced doses and / or less aggressive dosing regimens as a result of improved therapeutic index. In particular, the conjugates disclosed herein can show improved therapeutic outcomes, including, but not limited to, reduced tumor size, delayed tumor growth, reduced metastasis, and / or extended life span. Such improvement is at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 99%, when compared to any therapeutic effect observed in a subject administered the radioactive halogen without protein / peptide targeting. In other embodiments, such improvement is at least about 2-fold, or at least about 5-fold, or at least about 10-fold, or at least about 20-fold, or at least about 50-fold, or at least about 100-fold, or more, when compared to any therapeutic effect observed in a subject administered the radioactive halogen alone, i.e., without targeting as in the radiolabeled protein / peptide conjugates described herein.

[0087] B. Imaging with Radiolabeled Conjugates A subject may also be administered the protein / peptide conjugates described in the present disclosure for imaging of tumors or other biological features. Radiolabeled protein / peptide conjugates may be prepared, for example, with proteins / peptides that target specific types of tumor cells. Administering the protein / peptide conjugate to a subject may localize the radioactive halogen at the site of interest. Standard imaging techniques of radioisotopes may then be used to determine the presence and / or extent of a particular tumor type, including detection and monitoring of any of the tumor types described above, including monitoring tumor progression and / or response to treatment. In certain embodiments, the radiolabeled protein / peptide conjugates may be used for imaging of breast cancer, more specifically HER2+ breast cancer, including any of the specific types of breast cancer described herein above.

[0088] C. Formulation, Administration, and Dosage The disclosed conjugates of the present invention may be formulated as desired using art-recognized techniques. In some embodiments, the therapeutic compositions of the present invention may be administered as is or with minimal additional components, while other therapeutic compositions may be formulated to contain suitable pharma- ceutically acceptable carriers (e.g., vehicles, adjuvants, and diluents) including excipients and auxiliary agents well known in the art, including, for example, pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents, radioprotectants, and the like. Specific non-limiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. Specific non-limiting exemplary radioprotectants include ascorbic acid, gentisic acid, ethanol, and combinations thereof.

[0089] In general, the compounds and compositions of the present invention can be administered to a subject by a variety of routes, including, but not limited to, oral, intravenous, intraarterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, buccal, intrarectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal, or otherwise by implantation or inhalation. The compositions can be formulated into preparations in solid, semi-solid, liquid, or gas form suitable for the particular mode of administration, including, for example, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and aerosols.

[0090] The particular dosing regimen for administering the conjugates of the invention, i.e., dose, timing and repetition, will depend on the particular subject and the subject's medical history, as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance rate, etc.). The frequency of administration can be determined and adjusted over the course of therapy. A therapeutically effective dose is a dose sufficient to provide a clinical benefit to a subject, including, for example, a dose sufficient to reduce tumor size, maintain a reduction in tumor size, reduce or slow tumor growth, slow the onset of metastasis, improve longevity, etc. The administered dose may be adjusted or attenuated to manage potential side effects and / or toxicity.

[0091] As used herein, the terms "subject" and "patient" are used interchangeably and refer to both human and non-human animals. The term "non-human animal" in the present disclosure includes all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. The methods and compositions disclosed herein can be used on samples in vitro (e.g., isolated cells or tissues) or in vivo on a subject (i.e., a living organism, such as a patient).

[0092] The compositions provided herein can be used in a number of in vitro and in vivo applications, including treatment of disease, imaging of tissues, etc. For example, in one embodiment, the compositions can be used for imaging of tumors or other tissues, comprising administering to a subject an effective amount of a composition provided herein.

[0093] As used herein, the term "administering" an agent, such as a therapeutic entity, to an animal or cell is intended to refer to distributing, delivering or applying the substance to an intended target. With respect to a therapeutic agent, the term "administering" is intended to refer to contacting or distributing, delivering or applying the therapeutic to a subject by any suitable route for delivery of the therapeutic to a desired location within an animal, including delivery by either parenteral or oral routes, intramuscular injection, subcutaneous / intradermal injection, intravenous injection, intrathecal administration, intrabuccal administration, transdermal delivery, topical administration, and administration by intranasal or airway routes.

[0094] In other embodiments, the compositions provided herein are used for the prevention and / or treatment of disease in a subject. As used herein, "treatment", "therapy" and / or "therapeutic regimen" refer to a clinical intervention made in response to a disease, disorder or physiological condition exhibited by or to which a patient may be susceptible. The purpose of treatment includes alleviating or preventing symptoms, slowing or halting the progression or worsening of a disease, disorder or condition, and / or ameliorating a disease, disorder or condition. As used herein, the terms "prevent", "preventing", "prevention", "prophylactic treatment" and the like refer to reducing the likelihood of developing a disease, disorder or condition in a subject who does not have the disease, disorder or condition but is at risk or susceptible to developing the disease, disorder or condition.

[0095] A therapeutically effective amount is an amount of the disclosed radiolabeled protein / peptide that results in a desired therapeutic effect, e.g., increased longevity and / or quality of life. In the context of cancer, desired therapeutic effects include reduction in tumor size, slowing of tumor growth, reduction or slowing of metastasis, reduction in tumorigenicity, reduction or amelioration of symptoms or indicators / biomarkers associated with cancer, etc. A therapeutically effective amount can be administered in a single dose or multiple doses.

[0096] For imaging applications, an effective amount is an amount of the radiolabeled protein / peptide of the present disclosure that, after administration to a patient, results in measurable in vivo or ex vivo detection with sufficient sensitivity to assess the biodistribution of the radiolabeled protein / peptide above any background or non-specific detection. Any detection technique known in the art may be used, such as SPECT / CT imaging, PET, PET / CT, and PET / MRI. For therapeutic applications, an effective amount for imaging applications may be administered in a single dose or multiple doses.

[0097] Working Example The following examples are included to demonstrate certain embodiments of the invention.

[0098] Materials and Methods [ in 0.1M NaOH 125 Sodium iodide (629 GBq / mg) was obtained from PerkinElmer (Boston, Mass.). 131 [I]Sodium iodide (936 GBq / mg, 236.097 GBq / mL) was obtained from International Isotopes Inc. (Idaho Falls, ID). All reagents and solvents were purchased from Fisher Scientific unless otherwise noted. 2 -iso-SGMTB, Boc 2 -iso-SGMTB, Boc 2-MEGMTB, PODS, and PODS-DOTA were synthesized according to reported methods (Feng, Y. et al., "Site-specific radioiodination of an anti-HER2 single domain antibody fragment with a residualizing prosthetic agent" Nuclear Medicine and Biology, 2021.92:p.171-183; Adumeau, P., M.Davydova, and B. M.Zeglis, "Thiol-reactive bifunctional chelators for the creation of site-selectively modified radioimmunoconjugates with improved stability" Bioconjugate Chemistry, 2018.29(4):p.1364-1372; and Davydova, M. et al., "Synthesis and bioconjugation of thiol-reactive reagents for the creation of site-selectively modified immunoconjugates," Journal of Visualized Experiments: JoVE, 2019(145), each of which is incorporated herein by reference.Details of the production, purification and characterization of anti-HER2 sdAb-5F7GGC have been reported (Feng, Y. et al., "Site-specific radioiodination of an anti-HER2 single domain antibody fragment with a residualizing prosthetic agent," Nuclear Medicine and Biology, 2021.92:p.171-183 and Pruszynski, M. et al., "Targeting breast carcinoma with radioiodinated anti-HER2 Nanobody," Nuclear Medicine and Biology, 2013.40(1):p.52-59, both of which are incorporated herein by reference). 125 / 131 [I]MEGMIB-5F7GGC was synthesized according to a reported method (Feng, Y. et al., "Site-specific radioiodination of an anti-HER2 single domain antibody fragment with a residualizing prosthetic agent" Nuclear Medicine and Biology, 2021.92:p.171-183). 211At]MEAGMB-5F7GGC was synthesized using essentially the same procedure. Anti-HER2 mAb, trastuzumab (Roche / Genentech), was obtained from Duke University Medical Center Pharmacy. All equipment was calibrated and maintained according to standard quality control practices and procedures. Reverse-phase HPLC (RP-HPLC) purification was performed on a Shimadzu HPLC system (Shimadzu Scientific Instruments, Kyoto, Japan). Analytical HPLC columns (250 × 2 mm, 5 μm, 300 Å) and preparative HPLC columns (250 × 10 mm, 5 μm, 300 Å) were purchased from Phenomenex (Jupiter Proteo HPLC columns, Phenomenex, Torrance, CA, USA). HPLC analysis was performed using LabSolutions LC / GC software (Shimadzu Scientific Instruments, Kyoto, Japan). Evaporation of the solvent was performed either with a vacuum evaporator (Biotage V-10 Touch, V10-2XX, Biotage, Uppsala, Sweden) or with a rotary evaporator (Hei-VAP Core, Heidolph, Schwabach, Germany). Gel permeation (GP) HPLC was utilized for the purification and identification of the 5F7GGC prosthetic molecule conjugate, using an Agilent PL Multisolvent 20 column and eluting with pure Milli-Q® water as the mobile phase. Purification of the radiolabeled compound was performed on an Agilent 1260 Infinity system using a reversed-phase HPLC column (Agilent Poroshell 120 C18, 2.7 μm, 4.6 × 50 mm) eluted with a gradient consisting of water (solvent A) and acetonitrile (solvent B), both containing 0.1% TFA, at a flow rate of 2 mL / min. The proportion of B was increased linearly from 40% to 80% over 8 min. The system was equipped with a 1260 Infinity Multiple wavelength detector (Santa Clara, CA).For radioactivity monitoring, one system was connected to a Dual Scan-RAM flow activity detector / TLC scanner and the other to a Flow-RAM detector (Lablogic, Tampa, FL). Both the HPLC and gamma detectors were controlled by LabLogic Laura software. A CRC-7 dose calibrator (Capintec, Pittsburgh, PA) was used to measure higher levels of radioactivity, and either an LKB 1282 (Wallac, Finland) or a Perkin Elmer Wizard II (Shelton, CT) automated gamma counter was used to assess lower levels of radioactivity.

[0099] Boc 2 Synthesis of -iso-GMTB-PODS (XIX) [ka] N,N-diisopropylethylamine (DIPEA, 8.0 μL, 45.8 μmol, 3.0 equiv.) was added to a solution of PODS (24.8 mg, 45.8 μmol, 3.0 equiv.) in 1.0 mL of anhydrous dimethylformamide (DMF). 2 A solution of 10-iso-SGMTB (10 mg, 15.3 μmol, 1.0 equiv.) was added to the above and the resulting solution was stirred overnight at room temperature protected from light. To determine the progress of the reaction, RP-HPLC was performed using an analytical column (1 mL / min over 35 min, 5–95% acetonitrile in water (0.1% TFA); t R : 25.4 min). When the reaction was judged to be complete, the DMF was completely evaporated in a vacuum evaporator. The resulting crude product was reconstituted in 2 mL of acetonitrile, and the solution was filtered through a syringe filter. The product was purified by RP-HPLC using a preparative column (6 mL / min in 35 min, 5–95% acetonitrile in water (0.1% TFA); t R The purity of the isolated samples was assessed using analytical RP-HPLC (1 mL / min over 35 min, 5–95% acetonitrile in water with 0.1% TFA; product: t R(: 25.4 min; purity >95%). The solvent of the pooled HPLC fractions containing the product was removed by lyophilization overnight to give a white powder (4.9 mg, 30.0% yield). 1 H NMR (500 MHz, CDCl 3 ) δ: 9.90 (s, 1H), 9.50 (s, 1H), 7.99 (d, 2H, J = 8.8 Hz), 7.74 (m, 3H), 7.58 (s, 1H), 7.45 (s, 1H), 7.07 (s, 1H), 6.87 (s, 1H), 5.17 (s, 2H), 3.47-3.64 (m, 18H), 3.36 (q, 2H, J = 5.9 Hz), 2.66 (m, 2H), 2.57 (m, 2H), 1.90 (quintet, 2H, J = 6.2 Hz), 1.74 (quintet, 2H, J = 6.0 Hz), 1.48 (s, 9H), 1.36 (s, 9H), 0.27 (s, 9H). 13 C NMR (500 MHz, DMSO-d 6 ) δ: 171.8, 171.5, 167.0, 166.2, 163.2, 162.2, 160.1, 154.5, 144.1, 142.5, 138.5, 137.5, 134.4, 132.9, 129.0, 126.7, C 45 H 69 N 8 O 13 SSn (M+H) + ESI-MS m / z calculated: 1080.85; found: 1081.0.

[0100] Boc 2 Synthesis of -iso-GMIB-PODS(XX) [ka] DIPEA (6.4 μL, 36.5 μmol, 3.0 equiv.) was added to a solution of PODS (19.7 mg, 36.5 μmol, 3.0 equiv.) in 1.0 mL of DMF. Then, Boc 2 A solution of 1.0-iso-SGMIB (7.5 mg, 12.2 μmol, 1.0 equiv.) was added to the PODS mixture. The reaction mixture was stirred at room temperature overnight. The reaction progress was followed using an analytical column (1 mL / min in 35 min, 5-95% acetonitrile in water with 0.1% TFA; t R The reaction was monitored by RP-HPLC (t: 23.5 min). When the reaction was judged to be complete, the DMF was completely evaporated in a vacuum evaporator. The resulting crude product was reconstituted in 2 mL of acetonitrile and the solution was filtered through a syringe filter. The product was purified by RP-HPLC using a preparative column (6 mL / min over 35 min, 5–95% acetonitrile in water with 0.1% TFA; t R The purity of the isolated sample was assessed using analytical RP-HPLC (1 mL / min, 5–95% acetonitrile in water with 0.1% TFA, 35 min; t R : 23.5 min; purity >95%. The pooled HPLC fractions containing the product were lyophilized overnight to give a white powder (8.5 mg, 67.0% yield): 1 H NMR (500 MHz, CDCl 3 ) δ: 9.75 (s, 1H), 9.5 (s, 1H), 8.00 (d, 2H, J = 8.8 Hz), 7.85 (s, 1H), 7.65 (d, 2H, J = 8.5 Hz), 7.60 (s, 1H), 7.56 (s, 1H), 7.30 (s, 1H), 6.87 (s, 1H), 5.13 (s, 2H), 3.52-3.69 (m, 15H), 3.39 (q, 2H, J = 6.0 Hz), 3.51 (s, 3H), 2.70 (m, 2H), 2.59 (m, 2H), 1.90 (quintet, 2H, J = 6.1 Hz), 1.74 (quintet, 2H, J = 5.9 Hz), 1.48 (s, 9H), 1.36 (s, 9H). 13C NMR (500 MHz, DMSO-d 6 ) δ: 171.8, 171.4, 166.2, 165.0, 163.1, 162.2, 159.9, 154.3, 144.2, 141.7, 138.7, 137.0, 134.2, 129.0, 126.0, 119.6, C 42 H 60 N 8 O 13 SI (M+H) + ESI-MS m / z calculated: 1043.94; found: 1044.0.

[0101] Synthesis of iso-GMIB-PODS (XXI) [ka] Boc in 2.0 mL of dichloromethane 2 To a solution of 1-iso-GMIB-PODS (5.0 mg, 4.8 μmol) was added trifluoroacetic acid (TFA; 0.7 mL) and the mixture was stirred at room temperature for 3 h. Volatiles were evaporated using a rotary evaporator, the crude product was reconstituted in 2 mL of acetonitrile, and the resulting solution was filtered through a syringe filter. The product was purified by RP-HPLC using a preparative column (6 mL / min over 35 min, 5–95% acetonitrile in water with 0.1% TFA; t R The purity of the isolated sample was assessed using analytical RP-HPLC (1 mL / min over 35 min, 5–95% acetonitrile in water with 0.1% TFA; t R : 15.4 min; purity >95%. The pooled HPLC fractions containing the product were lyophilized overnight to give a white powder (4.0 mg, 87.1% yield; trifluoroacetate salt): 1 H NMR (500 MHz, DMSO-d 6) δ: 10.42 (s, 1H), 8.57 (t, 1H, J = 5.3 Hz), 8.12 (s, 1H), 8.03 (m, 3H), 7.86 (m, 3H), 7.79 (d, 2H, J = 7.8 Hz), 7.28 (s, 3H), 4.38 (d, 2H, J = 6.0 Hz), 3.69 (s, 3H), 3.25 - 3.54 (m, 14H), 3.07 (q, 2H, J = 6.3 Hz), 2.61 (t, 2H, J = 6.9 Hz), 2.41 (t, 2H, J = 7.0 Hz), 1.74 (quintet, 2H, J = 6.6 Hz), 1.61 (quintet, 2H, J = 6.6 Hz). 13 C NMR (500 MHz, DMSO-d 6 ) δ: 171.8, 171.5, 166.2, 162.2, 144.1, 129.0, 119.6, 116.4, 70.2, 70.1, 70.0, 68.7, 68.5, 43.4, 40.9, 36.3, 32.2, 30.5, 29.8, 29.1. C 32 H 46 N 8 O 9 SI (M + H) + Calculated ESI-MS m / z for: 844.7; Observed: 843.6.

[0102] Iso-[ 131 Synthesis of I]GMIB-PODS-5F7GGC (XXII)

Chem.

[0103] Iso-[ 211 Synthesis of At]AGMB-PODS-5F7GGC(XXIII) [ka] Iso-[ 211 A previously reported method for the synthesis of At]SAGMB(6) was used to 211 At activity level iso-[ 211 The synthesis of [At]AGMB-PODS was applied. The general synthetic scheme for the synthesis is depicted in Figure 2. Astatine-211 (approximately 370 MBq; 200 μL) in NCS / methanol was reacted with Boc 2 The reaction was added to a vial containing -iso-GMTB-PODS (50 μg, 0.05 μmol), the vial was briefly vortexed, and the reaction was allowed to proceed for 15 min at 20° C. Volatiles were evaporated under a stream of argon, and the residual activity was reconstituted in 40% acetonitrile in water (100 μL). This solution was injected onto a reversed-phase analytical HPLC column and eluted with the same gradient as above. The product Boc 2 -Iso-[ 211 At]AGMB-PODS(t R The HPLC fractions containing acetonitrile (2.2 mL) were pooled and most of the acetonitrile was removed using a stream of argon. Ethyl acetate (1 mL) was added to obtain Boc 2 -Iso-[ 211At]AGMB-PODS was extracted, the mixture was vortexed for 10 seconds, and the ethyl acetate layer was transferred to a half-drum vial. The ethyl acetate was removed using an argon stream, and TFA was added and incubated at room temperature for 10 minutes. TFA was evaporated under an argon stream, and residual TFA was removed by coevaporation with ethyl acetate (100 μL x 3). A freshly reduced amount of 5F7GGC was added, and the mixture was incubated at 37°C for 45 minutes. Iso-[ 211 At]AGMB-PODS-5F7GGC was isolated using a PD-10 column as described above.

[0104] [ 211 Synthesis of At]MEAGMB-5F7GGC(XXIV) [ka] [ 211Synthesis of [At]MEAGMB and subsequent conjugation to 5F7GGC was performed by adapting previously reported methods (Choi et al., “Astatine-211 labeled anti-HER2 5F7 single domain antibody fragment conjugates: radiolabeling and preliminary evaluation,” Nucl. Med. Biol. 2018;56:10-20; Feng et al., “Site-specific radioiodination of an anti-HER2 single domain antibody fragment with a residualizing prosthetic agent,” Nucl. Med. Biol. 2021;92:171-83). Briefly, 5F7 single domain antibody fragment conjugates were prepared using NCS / methanol as previously described (Choi et al., “Astatine-211 labeled anti-HER2 5F7 single domain antibody fragment conjugates: radiolabeling and preliminary evaluation.” Nucl. Med. Biol. 2018;56:10-20). 211 At (approximately 370 MBq; 200 μL) was dissolved in Boc 2-MEGMTB (50 μg, 0.07 μmol) was added to a vial containing the eluted t-MEGMTB, followed by the addition of 2 μL of glacial acetic acid. The vial was vortexed briefly and the reaction was allowed to proceed for 20 min at 20° C. The volatiles were evaporated under a stream of argon and the residual activity was redissolved in 40% acetonitrile in water (100 μL). This solution was injected onto a reverse-phase HPLC column and eluted with a gradient consisting of 0.1% TFA in water (solvent A) and 0.1% TFA in acetonitrile (solvent B) at a flow rate of 2 mL / min. The percentage of B was increased linearly from 40% to 80% over 8 min. The HPLC fractions containing the product (t R = 4.0 min) were pooled and most of the acetonitrile was removed using a stream of argon. 2 -[ 211 The At]MEAGMB was extracted with 2 x 1 mL of ethyl acetate and transferred to a half-dram glass vial. The ethyl acetate was evaporated with argon, and TFA (100 μL) was added, and the Boc 2 -[ 211 Deprotection of At]MEAGMB was allowed to proceed for 10 min at 20° C. Then, TFA was removed under an argon stream, followed by coevaporation with ethyl acetate (100 μL×3). The monomeric sdAB 5F7GGC (approximately 100 μg, 60 μL) obtained as above was converted to [ 211 [At] was added to a vial containing MEAGMB and conjugation was carried out for 45 min at 37° C. The labeled sdAb was isolated by gel filtration on a PD-10 column using PBS as the mobile phase. 211 Fractions containing At]MEAGMB-5F7GGC were pooled for use in the biological experiments described below. [ka]

[0105] [ 177 Synthesis of [Lu]Lu-DOTA-PODS-5F7GGC [ 177A general synthetic scheme for the synthesis of [Lu]Lu-DOTA-PODS-5F7GGC is shown in Figure 3. Lutetium-177, produced at the University of Missouri Research Reactor, was purchased from the National Isotope Development Center at Oak Ridge National Laboratory. Upon receipt, 177 Lu (185 MBq, 10 µL) was diluted with 0.1 M HCl (50 µL) and added to a 1 mL Eppendorf tube, followed by addition of DOTA-PODS (150 µL, 1 mg / mL, 0.2 M NH 4 OAc, pH=6.3) was added. The reaction mixture was vortexed for 30 seconds and left at room temperature for 30 minutes. The reaction mixture was then diluted with 10 mL of water and passed through a preconditioned C18 Sep-Pak® cartridge (Waters; 60 mg). Product activity was eluted from the cartridge with ethanol (400 μL) into a half-dram glass vial, which was evaporated with a gentle argon stream. The monomeric 5F7GGC sdAb, freshly obtained as described above, was purified by HPLC using [ 177 The conjugation was carried out for 45 min at 30° C. The conjugate was then treated with 50 mM EDTA to remove any accidentally bound phosphate ions. 177 Lu was removed. The labeled sdAb was isolated by gel filtration on a PD-10 column using PBS as the mobile phase. 177 Fractions containing [Lu]Lu-DOTA-PODS-5F7GGC were pooled for use in the biological experiments described below.

[0106] Cell culture conditions Reagents for cell culture were obtained from Thermo Fisher Scientific (Waltham, MA) unless otherwise stated. Cells were cultured at 37 °C for 24 h at 5% CO 2Cultured in a humidified incubator at 37°C. HER2-positive BT474 human breast cancer cells were obtained from Duke University Cell Culture Facility and grown in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% FBS and supplemented with 10 mg / mL bovine insulin. SKOV-3 human ovarian cancer cells were obtained from Duke University Cell Culture Facility and grown in McCoy's 5A medium containing 10% fetal bovine serum and 1% penicillin-streptomycin.

[0107] Quality control of labeled sdAb conjugates The radiochemical purity of the labelled sdAb was assessed by SDS-PAGE / phosphor imaging. Immunoreactive fractions were determined by the Lindmo method (Lindmo, T. et al., "Determination of the immunoreactive function of radiolabeled monoclonal antibodies by linear extrapolation to binding at infinite antigen excess," Journal of Immunological Methods, 1984.72(1):77-89, incorporated herein by reference) according to reported procedures (Foulon, C. F. et al., "Radioiodination via D-amino acid peptide enhances cellular retention and tumor xenograft targeting of an internalizing anti-epidermal growth factor receptor variant III monoclonal antibody," Cancer Research, 2000.60(16):4453-4460, incorporated herein by reference). Iso-[ 131The HER2 binding affinity of I]GMIB-PODS-5F7GGC was measured using SKOV-3 and BT474 cells expressing HER2 by the saturation binding assay described above (Zhou, Z. et al., "Fluorine-18 labeling of an anti-HER2 VHH using a residualizing prosthetic group via a strain-promoted click reaction: Chemistry and preliminary evaluation," Bioorganic & Medicinal Chemistry, 2018.26(8):p.1939-1949 and Vaidyanathan, G. et al., "N-Succinimidyl 3-((4-(4-[ 18 F]fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(guanidinomethyl)benzoate ([ 18 F]SFBTMGMB): Internalization of biomolecules 18 F-labeled residual (N-Succinimidyl 3-((4-(4-[18F] fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(guanidinomethyl)benzoate([ 18 F]SFBTMGMB): a residualizing label for 18 "F-labeling of internalizing biomolecules" Organic & Biomolecular Chemistry, 2016.14(4):1261-1271, both of which are incorporated herein by reference. Nonspecific binding was determined in a parallel assay by co-incubating cells with a 100-fold molar excess of trastuzumab.

[0108] Determining internalization in vitro Iso-[ 131The internalization and cellular retention of [I]GMIB-PODS-5F7GGC was assessed using procedures reported for similar molecules (Choi, J. et al., "Astatine-211 labeled anti-HER2 5F7 single domain antibody fragment conjugates: Radiolabeling and preliminary evaluation." Nuclear Medicine and Biology, 2018.56:10-20 and Zhou, Z. et al., "Fluorine-18 labeling of the HER2-targeting single-domain antibody 2Rs15d using a residualizing label and preclinical evaluation. Molecular Imaging and Biology, 2017.19(6):867-877, both of which are incorporated herein by reference. Briefly, cells were incubated with radiolabeled sdAb and the internalized fraction was measured at different time points. 125 I]MEGMIB-5F7GGC and tandem iso-[ 131Paired labeling assays of [I]GMIB-PODS-5F7GGC were performed in BT474 cells according to reported procedures (Feng, Y. et al., "Site-specific radioiodination of an anti-HER2 single domain antibody fragment with a residualizing prosthetic agent," Nuclear Medicine and Biology, 2021.92:p.171-183 and Choi, J. et al., "Astatine-211 labeled anti-HER2 5F7 single domain antibody fragment conjugates: Radiolabeling and preliminary evaluation," Nuclear Medicine and Biology, 2018;56:p.10-20). Briefly, cells were incubated with radiolabeled sdAb for 30 min at 4°C, then washed with PBS, replaced with fresh medium, and the internalized fraction was measured at different time points. In these assays, nonspecific uptake was assessed in parallel experiments by co-incubating cells with a 100-fold molar excess of trastuzumab.

[0109] [ 211 At]MEAGMB-5F7GGC and iso-[ 211 In vitro stability of At]AGMB-PODS-5F7GGC 211 The in vitro stability of the At-labeled conjugates was evaluated in PBS, 50 mM cysteine ​​solution and human serum albumin (50% in PBS). 211At-labeled sdAb conjugate (3.7MBq, approximately 12μg of sdAb) was added to 1mL of each solution in a 10mL centrifuge tube. The tubes containing the conjugates were vortexed for 10 seconds and left at room temperature. The radiochemical purity of each conjugate was determined by SDS-PAGE and phosphor imaging of aliquots after 3 and 21 hours of incubation.

[0110] Biodistribution All experiments using animals were performed under protocols approved by the Duke University IACUC. After implanting an estrogen pellet (17β-estradiol) subcutaneously in the back of the neck, 5-week-old female athymic mice (~25 g) were injected with 20 × 10 IgG in 50% protein / peptide containing a C-terminal GGC tail in the above medium (100 μL). 6 Subcutaneous BT474 breast cancer xenografts were established by inoculating BT474 cells, in which the radiolabel was coupled to the protein / peptide via C-terminal GGC-tailed Matrigel (Corning Inc., NY). Four biodistribution studies were performed: 125 I]MEGMIB-5F7GGC and tandem iso-[ 131 I]GMIB-PODS-5F7GGC;BT474 xenograft-bearing athymic mice were treated with iso-[ 211 At]AGMB-PODS-5F7GGC and tandem iso-[ 131 I]GMIB-PODS-5F7GGC;BT474 xenograft-bearing athymic mice in a paired labeling format 125 I]MEGMIB-5F7GGC and tandem iso-[ 131 A 1:1 study was performed using [I]GMIB-PODS-5F7GGC. In these studies, each mouse was administered 0.11-0.22MBq (1-2μg of sdAb) of each labeled conjugate via the tail vein. At each time point after tracer administration, blood and urine were collected and the mice were sacrificed by an overdose of isofluorane. Tumors and other tissues were harvested, blotted dry, weighed, and 125 I,131 I and 211 At activity was counted using an automated gamma counter along with relevant injected standards, and from these counts, the percentage of injected dose (%ID) per gram of tissue (%ID / g) per organ, and tumor to normal tissue ratios were calculated.

[0111] Chemical and radiochemical results Boc 2 -iso-SGMTB, Boc 2The synthesis of -iso-SGMTB and PODS has been reported previously (Feng, Y. et al., "Site-specific radioiodination of an anti-HER2 single domain antibody fragment with a residualizing prosthetic agent" Nuclear Medicine and Biology, 2021.92:p.171-183; Adumeau, P., M. Davydova, and B. M. Zeglis, "Thiol-reactive bifunctional chelators for the creation of site-selectively modified radioimmunoconjugates with improved stability" Bioconjugate Chemistry, 2018.29(4):p.1364-1372; and Davydova, M. et al., "Synthesis and bioconjugation of thiol-reactive reagents for the creation of site-selectively modified immunoconjugates" Journal of Visualized Experiments: JoVE, 2019(145)). Boc 2 -iso-SGMTB and Boc 2 The conjugation of Boc-iso-SGMTB to PODS was carried out using the general protocol under basic conditions, respectively. 2 -iso-GMIB-PODS and Boc 2-iso-GMTB-PODS was isolated in reasonable yield using preparative reversed-phase HPLC. iso-GMIB-PODS-5F7GGC, DOTA-PODS-5F7GGC, and Lu-DOTA-PODS-5F7GGC were synthesized with similar yields (approximately 76%). The purity of these immunoconjugates was measured using GP-HPLC and was all greater than 95%. Their molecular weights were determined by LC-MS to be 13795.4 (calculated 13795.2), 14046.0 (calculated 14046.2), and 14216.7 (calculated 14217.2), respectively. Non-radioactive iso-SGMTB-PODS was synthesized using its radiolabeled analog iso-[ 131 I]GMIB-PODS was used as a standard for identification. 2 -Iso-[ 131 The radiochemical yield (RCY) and radiochemical purity (RCP) of I]GMIB-PODS were 70±8% (n=11) and more than 99% (RP-HPLC), respectively. 131 Conjugation of iso-[I]GMIB-PODS was achieved with an RCY of 58±9% (n=11). 131 The RCP of I]GMIB-PODS-5F7GGC was over 99%. 211 The synthesis of iso-[At]AGMB-PODS was performed with RCY of 66±5% (n=6) and RCP of >99% for each synthesis. 211 The RCY for the conjugation of At]AGMB-PODS with 5F7GGC was 64±7% (n=6), and the RCP of the radiolabeled sdAb determined by SDS-PAGE and GP-HPLC was >99%. 177 Lu]Lu-DOTA-PODS was synthesized in nearly quantitative yield, and 177 Lu]Lu-DOTA-PODS-5F7GGC was synthesized with 35±15% RCY (n=2) and RCP of >99% as determined by SDS-PAGE and GP-HPLC.

[0112] Characterization of sdAb conjugates The binding affinity (Kd) of 5F7GGC and 5F7GGC conjugates to the HER2 extracellular domain was measured by surface plasmon resonance (SPR). The Kd of 5F7GGC was determined by multi-cycle kinetic titration, and the Kd of 5F7GGC conjugates was measured by single-cycle kinetic titration. The results are shown in Figure 4 and Table 1. SPR revealed binding constants (Kd) of 0.10, 0.19, and 0.09 nM for iso-GMIB-PODS-5F7GGC, DOTA-PODS-5F7GGC, and Lu-DOTA-PODS-5F7GGC with recombinant HER2-Fc protein, respectively. d ) was revealed. The immunoreactive fraction (IRF) determined by the Lindmo assay (Lindmo, "Determination of the immunoreactive function of radiolabeled monoclonal antibodies by linear extrapolation to binding at infinite antigen excess" J Immunol.Methods.1984.72(1):p.77-89) was determined to be 100% iso-[ 131 I]GMIB-PODS-5F7GGC was 84.0 ± 1.8% (n = 2), and iso-[ 211 At]AGMB-PODS-5F7GGC: 85.8 ± 2.1% (n = 2); 211 At]MEAGMB-5F7GGC was 69.0% (n = 1), and 177 For [Lu]Lu-DOTA-PODS-5F7GGC, the rate was 71.2% (n=1).

[0113] FIG. 5A shows the iso-[ 131 FIG. 5B shows the binding affinity of iso-[I]GMIB-PODS-5F7GGC measured in SKOV-3 cells. 131 FIG. 5C shows the binding affinity of iso-[I]GMIB-PODS-5F7GGC measured in BT474 cells. 211As shown in the figure, the binding affinity of iso-[At]AGMB-PODS-5F7GGC was confirmed by saturation binding assay using HER2-expressing SKOV-3 and BT474 cell lines. 131 K of 3.3 ± 0.5 nM and 5.9 ± 0.8 nM for [I]GMIB-PODS-5F7GGC, respectively. d Using the BT474 cell line, iso-[ 211 At]AGMB-PODS-5F7GGC, 211 At]MEAGMB-5F7GGC, and [ 177 K of 4.7 ± 0.8, 3.4 ± 0.6, and 5.6 ± 0.9 nM for [Lu]Lu-DOTA-PODS-5F7GGC, respectively. d values ​​were obtained.

[0114] Table 1 [Table 1]

[0115] Cell Retention and Internalization Figure 6 shows the iso-[ 125 I]MEGMIB-5F7GGC and iso-[ 131 Figure 6 shows an in vitro paired-label internalization assay in HER2-positive BT474 breast cancer cells co-incubated with [I]GMIB-PODS-5F7GGC. Results are presented as surface-bound (Figure 6A) and internalized (Figure 6B) fractions of radioactivity initially bound to the cells after 1 hour incubation at 4°C. 131 I]GMIB-PODS-5F7GGC and [ 125 In paired-label internalization assays comparing iso-[I]MEGMIB-5F7GGC, cells after the first hour of incubation at 4°C showed significantly higher levels of iso-[ 131 I]GMIB-PODS-5F7GGC was 6.2 ± 0.3%, whereas [ 125 The percentage of this initially bound iso-[I]MEGMIB-5F7GGC that was internalized in cells after subsequent incubation at 37°C was 5.8 ± 0.4% (the difference was not statistically significant: P > 0.05).131 The fraction of I]GMIB-PODS-5F7GGC was 28.4±3.3%, 33.5±1.8%, and 29.0±0.8% at 1, 2, and 4 hours, respectively. 125 For [I]MEGMIB-5F7GGC, these values ​​were 25.0±3.4%, 30.5±2.2%, and 26.7±0.5%. A significantly higher internalization fraction was observed at the 4-h time point, 125 I]MEGMIB-5F7GGC compared to iso-[ 131 I]GMIB-PODS-5F7GGC was observed only in the iso-[ 131 I]GMIB-PODS-5F7GGC and [ 125 The surface-bound fraction for [I]MEGMIB-5F7GGC was not significantly different at any time point.

[0116] Figure 7 shows the iso-[ 131 I]GMIB-PODS-5F7GGC and iso-[ 211 Figure 7 shows an in vitro paired-label internalization assay in HER2-positive BT474 breast cancer cells co-incubated with iso-[At]AGMB-PODS-5F7GGC. Results are presented as surface-bound (Figure 7A) and internalized (Figure 7B) fractions of radioactivity initially bound to the cells after 1 hour incubation at 4°C. 131 The cellular uptake and internalization of I]GMIB-PODS-5F7GGC was evaluated using iso-[ 211 No significant difference in uptake was observed after 1 h incubation at 4°C, and the input activity bound to BT474 cells was determined to be higher than that of iso-[At]AGMB-PODS-5F7GGC. 131 I]GMIB-PODS-5F7GGC and [ 211 At]AGMB-PODS-5F7GGC were 7.4±0.8% and 7.5±1.0%, respectively. 131 The surface-bound fraction of [I]GMIB-PODS-5F7GGC ranged from 17.7% to 28.5% of the initially bound activity, and the internalized fraction ranged from 34.4% to 48.8%. 211For [At]AGMB-PODS-5F7GGC, the surface-bound fraction ranged from 15.4% to 29.0%, and the internalized fraction ranged from 34.1% to 41.8%. 131 I-labeled sdAb 211 The differences between the At-tagged sdAbs were not statistically significant at any time point.

[0117] [ 211 At]MEAGMB-5F7GGC and iso-[ 211 In vitro stability of At]AGMB-PODS-5F7GGC Iso-[ 211 At]AGMB-PODS-5F7GGC showed excellent stability in both PBS and cysteine ​​solutions, with an RCP of >95% after 21 hours. Decreased stability was observed in HSA, with residual sdAb-associated activity at 3 and 21 hours. 211 At was 86% and 76%, respectively. 211 At]AGMB-PODS-5F7GGC, [ 211 The in vitro stability of At]MEAGMB-5F7GGC was rather low, with two 211 No significant differences were observed between the At-labeled sdAbs. 211 For At]MEAGMB-5F7GGC, no substantial radioactivity was associated with intact sdAb in both cysteine ​​solution and HSA at 3 and 21 hours.

[0118] Biodistribution Iso-[ 131 I]GMIB-PODS-5F7GGC and [ 125 The paired-labeled biodistribution of I]MEGMIB-5F7GGC was compared in athymic mice without BT474 subcutaneous xenografts (Table 2) and with BT474 subcutaneous xenografts (Table 3). In non-tumor bearing mice, iso-[ 131 Kidney uptake of radioactivity from [I]GMIB-PODS-5F7GGC (36.3 ± 5.2% ID / g) was 125I]MEGMIB-5F7GGC (15.7 ± 1.3% ID / g; P < 0.005). However, at 4 h p.i., there was no significant difference (P > 0.05) between the renal activity concentrations produced by both radioimmunoconjugates. At 1 h p.i., iso-[ 131 I]GMIB-PODS-5F7GGC was uptaken in the liver by 125 I]MEGMIB-5F7GGC was significantly higher than that from 1H-TETRAFLUOROACETATE (P<0.01), with the trend reversing at 4 hours pi (P<0.05). Minimal uptake and retention was observed in other organs. As shown in Table 3, iso-[ 131 Incorporation of [I]GMIB-PODS-5F7GGC 125 At 1 hour p.i., there was no significant difference between [I]MEGMIB-5F7GGC and [I]MEGMIB-5F7GGC (P>0.05 at all time points). The differences in tissue concentrations in normal organs were consistent with those observed in non-tumor-bearing mice. 125 The tumor-to-kidney activity concentration ratio of I]MEGMIB-5F7GGC (0.8 ± 0.1) was higher than that of iso-[ 131 I]GMIB-PODS-5F7GGC was significantly higher than that of GMIB-PODS-5F7GGC (0.3 ± 0.1; P < 0.0001). However, the tumor-to-kidney activity concentration ratios of the two conjugates were not significantly different at 4 hours p.i. 125 The tumor-to-liver activity concentration ratios of iso-[I]MEGMIB-5F7GGC were 9.2±1.5, 23.3±6.8, and 63.4±21.2 at 1, 4, and 24 hours p.i., respectively. 131 I] was significantly higher than that of GMIB-PODS-5F7GGC (P<0.0001).

[0119] Table 2 [Table 2] Table 3 [Table 3]

[0120] Iso-[ 131 I]GMIB-PODS-5F7GGC and its 211 At-labeled analogs are iso-[ 211 The biodistribution of At]AGMB-PODS-5F7GGC was directly compared in paired labeling experiments using athymic mice bearing BT474 xenografts (Table 4). High tumor uptake and retention was observed for both agents, with the differences between the agents not being statistically significant (P>0.05 at 1, 4, and 21 hours). Renal activity levels at 1 hour pi were also similar. However, there was a significant increase in iso-[ 211 The active concentration of At]AGMB-PODS-5F7GGC is 131 I]GMIB-PODS-5F7GGC was significantly higher than that of the iso-[ 131 The tumor-to-kidney activity concentration ratios of iso-[I]GMIB-PODS-5F7GGC were 2.8±0.5 and 8.0±1.2 at 4 and 21 hours p.i., respectively. 211 At]AGMB-PODS-5F7GGC: 1.5 ± 0.3 and 2.7 ± 0.4 (P < 0.01 at 4 h; P < 0.0001 at 21 h; no significant difference at 1 h). A similar trend was observed in the liver. At 1 h p.i., 211 At-labeled analogues (2.5±0.6) and 131 No significant difference was found between the tumor-to-liver activity concentration ratios of the I-labeled analogue (2.8 ± 0.8). However, 131 For I-labeled sdAb, significantly higher tumor-to-liver activity concentration ratios were observed: 131 12.2 ± 3.5 and 27.1 ± 8.9 for I at 4 and 21 h, respectively; 211 At 4 and 21 h, the values ​​were 7.7±1.8 and 11.6±3.4, respectively (P<0.05 at 4 h and P<0.01 at 21 h). 211 The active concentration of iso-[At]AGMB-PODS-5F7GGC in the stomach was significantly higher at 1 hour (P<0.05) and 4 hours. 131I]GMIB-PODS-5F7GGC. 211 The uptake of radioactivity in the thyroid gland from iso-[At]AGMB-PODS-5F7GGC at all time points was 131 I]GMIB-PODS-5F7GGC. Nevertheless, the absolute levels of activity in the stomach and thyroid of both radioimmunoconjugates were consistent with the low level of in vivo dehalogenation of both labeled sdAbs. 211 The At-labeled conjugates showed a higher degree of dehalogenation. 211 When [AT]AGMB-PODS-5F7GGC was evaluated as a single agent in athymic mice bearing BT474 xenografts, tissue activity levels were consistent with those seen in paired-labeling studies, with the exception of tumor uptake at 4 and 24 hours (Table 5). In single-labeling studies, tumor uptake was two-fold higher, 20.5±2.4%ID / g and 8.8±0.8%ID / g at 4 and 24 hours, respectively.

[0121] Table 4 [Table 4]

[0122] Table 5 [Table 5]

[0123] Iso-[ in athymic mice bearing BT474 xenografts 125 I]GMIB-PODS-5F7GGC and [ 177 To directly compare the in vivo performance of [Lu]Lu-DOTA-PODS-5F7GGC, a biodistribution study was performed. 125 The tumor uptake values ​​of I were 1 and 4 h, respectively. 177The radioactivity levels in the kidneys were similar at 1 hour (P>0.05), but were significantly higher than those in iso-[ 125 I]GMIB-PODS-5F7GGC (6.3±1.2%ID / g and 1.1±0.2%ID / g) 177 Lu]Lu-DOTA-PODS-5F7GGC (64.8 ± 13.7% ID / g and 40.8 ± 9.7% ID / g) (P < 0.0001 for both time points). As a result, the tumor-to-kidney activity concentration ratios were not significantly different between the two conjugates at 1 h ( 125 I is 0.3±0.1 and 177 Lu: 0.2±0.1; P>0.05). However, at 4 and 24 hours, iso-[ 125 I] The tumor-to-kidney activity concentration ratios of GMIB-PODS-5F7GGC were 2.9±0.7 and 5.8±2.0, and 0.2±0.0 and 0.2±0.1 (P<0.0001 at both time points). 177 Lu]Lu-DOTA-PODS-5F7GGC.

[0124] Table 6 [Table 6]

[0125] Consideration Using recombinant methods, we added a GGC sequence to the C-terminus of the anti-HER2 sdAb 5F7 and introduced a single cysteine ​​for site-specific labeling. 131 I] iodobenzoylmaleimide D-amino acid peptide-containing prosthetic molecule was utilized. Unfortunately, however, poor conjugation required modification of the sdAb with 2-iminothiolane to obtain sufficient product for biological evaluation. As a result, the bioconjugation reaction was no longer site-specific. Maleimidoethyl 3-(guanidinomethyl)-5-[ 131I]iodobenzoate (MEGMIB)-maleimide analogues of the residual prosthetic molecule iso-SGMTB were synthesized and evaluated for site-specific labeling of 5F7GGC. 131 I]SGMTB and [ 131 Both 5F7s randomly labeled on their lysines with MEGMIB-5F7GGC showed excellent tumor targeting in vivo. However, the maleimide version had high retention of activity in the liver, spleen, and kidneys at 24 hours post-injection, suggesting different metabolic patterns for the two radioimmunoconjugates. 211 The extension to At was performed using maleimidoethyl 3-[ 211 At] astat-5-(guanidinomethyl) iodobenzoate ([ 211 The synthesis of [At]MEAGMB) was briefly evaluated, but in vivo results were inconclusive. These results suggest that the activity of [At]MEAGMB in the presence of endogenously abundant thiol-containing species is 211 This can be explained by the rapid decomposition of [At]MEAGMB-5F7GGC.

[0126] Highly versatile array of iodine radionuclides and for targeted alpha particle therapy 211 We developed a site-specific labeling strategy for sdAbs with high in vivo stability that is applicable to both iso-[ 131 Randomly labeled sdAbs on lysines with reagents such as I]SGMTB can exhibit high affinity and stability, but this has often involved selecting sdAbs that do not contain lysines in the CDR regions or generating analogs in which the CDR lysines have been removed. Radiohalogens generated as described herein may exhibit features such as efficient and rapid conjugation at biologically relevant pH and / or improved stability in vitro and in vivo compared to corresponding conjugates labeled using maleimide chemistry. In particular, the PODS moiety can be attached to the iso-[I]SGMTB of the residual prosthetic molecule as previously validated. 131 I]SGMTB(29) and iso-[ 211 At]SAGMB(6) and iso-[ 131 I]GMIB-PODS and iso-[211 These new synthons were used to label 5F7-GGC with the maleimide-based prosthetic group [ 131 I]MEGMIB and [ 211 In addition, the conjugates synthesized using DOTA-PODS were compared directly with those synthesized using [At]MEAGMB. 177 Lu-labeled iso-[ 125 A direct comparison of [I]GMIB-PODS-5F7GGC and 5F7GGC, a paired labeling comparison of radiometal and radiohalogen labeled sdAbs, was also performed.

[0127] Tin precursor Boc 2 -iso-GMTB-PODS and the iodized standard iso-GMIB-PODS were synthesized in two steps in reasonable yields. 131 I]GMIB-PODS, iso-[ 211 At]AGMB-PODS and iso-[ 211 The synthesis of iso-[At]MEAGMB was carried out 211 At]SAGMB and [ 131 The conjugation of iso-GMIB-PODS to 5F7GGC gave higher yields than the conjugation of MEGMIB to 5F7GGC. Similarly, the conjugation of iso-[ 131 Conjugation of GMIB-PODS to 5F7GGC was performed using the 131 I]MEGMIB(16) conjugation had a higher RCY (P=0.0016). 131 I]GMIB-PODS and iso-[ 211 No significant difference was observed in the conjugation of At]AGMB-PODS to 5F7GGC in RCY. 177 The construction of [Lu]Lu-DOTA-PODS-5F7GGC was evaluated in two ways: (1) [ 177 Lu]LuCl 3 (1) synthesis and purification of DOTA-PODS-5F7GGC for direct labeling with 5F7GGC, and (2) [ 177Synthesis and purification of [Lu]Lu-DOTA-PODS. DOTA-PODS-5F7GGC was synthesized using [ 177 Lu]LuCl 3 When directly labeled with 177 Lu]Lu 3+ It was observed that DOTA-PODS-5F7GGC becomes loosely bound and dissociates upon injection into animals. A possible explanation is that 177 Lu]LuCl 3 forms colloids at neutral pH and co-elutes with the radioimmunoconjugate from the PD-10 column. 177 The non-radioactive versions of all of these sdAb immunoconjugates showed high binding affinity to the extracellular domain of HER2, demonstrating that conjugation with these prosthetic moieties does not reduce HER2 recognition. Furthermore, for all radioimmunoconjugates, the immunoreactivity and binding affinity to HER2-expressing cancer cells was high and consistent with that previously reported for other 5F7 radioconjugates.

[0128] All radioimmunoconjugates in this study showed high uptake and internalization into HER2-positive BT474 cells, although some significant differences in behavior were observed. 125 I]MEGMIB-5F7GGC, iso-[ 131 I]GMIB-PODS-5F7GGC showed higher intracellular retention after 4 h of incubation at 37°C, which is consistent with the 125 I]MEGMIB-5F7GGC compared to iso-[ 131 This likely reflects the higher in vitro stability of [I]GMIB-PODS-5F7GGC. 131 I]MEGMIB-5F7GGC was converted to iso-[ 125I]SGMTB-5F7 had lower in vitro stability. Similar differences in behavior were observed when comparing radioiodinated trastuzumab with D-amino acid residue peptide conjugates linked via both active ester and maleimide moieties. 211 The cellular uptake and retention of iso-[At]AGMB-PODS-5F7GGC was 131 I]GMIB-PODS-5F7GGC was not significantly different from that of GMIB-PODS-5F7GGC, consistent with the low extent of dehalogenation of both conjugates under in vitro conditions.

[0129] The contribution of the PODS moiety to the stability of the radioimmunoconjugate under in vitro conditions was investigated by comparing the iso-[ 211 At]AGMB-PODS-5F7GGC and iso-[ 211 The behavior of iso-[At]MEAGMB-5F7GGC, two of which contain thiol-containing substances that promote the retro-Michael-mediated degradation process, was investigated by comparing the behavior of iso-[ 211 At]AGMB-PODS-5F7GGC showed excellent stability. In contrast, iso-[ 211 At]MEAGMB-5F7GGC was rapidly degraded within 3 h by both cysteine ​​and HSA. 131 For iso-[I]MEGMIB-5F7GGC, significantly greater stability was seen earlier, with 90% of the radioimmunoconjugate remaining intact after 24 h of incubation in human serum (16). 131 I]MEGMIB-5F7GGC compared to iso-[ 211 The rather low stability of [At]MEAGMB-5F7GGC either before or after dissociation from the sdAb conjugate 211 This may be related to several factors such as higher dehalogenation of the At]MEAGMB moiety.

[0130] Due to poor in vitro stability, iso-[ 211At]MEAGMB-5F7GGC was not further evaluated in biodistribution studies. 211 With the exception of the confirmatory single-labeling evaluation of iso-[At]AGMB-PODS-5F7GGC, biodistribution experiments were performed in a paired-labeling format, which facilitated direct comparison of the two radioimmunoconjugates and eliminated possible confounding variables (such as tumor size) that may have existed between groups of experimental animals. 131 I]GMIB-PODS-5F7GGC and iso-[ 125 When iso-[I]MEGMIB-5F7GGC was compared in tumor-bearing mice, a trend towards higher tumor uptake was observed for the PODS conjugate at 1 and 4 hours, but the difference was not significant. On the other hand, the maleimide-based conjugate showed more than two-fold lower renal activity levels at 1 hour, but not thereafter, in both tumor-bearing and non-tumor-bearing mice. By 24 hours, the opposite behavior was observed. 125 The low initial renal uptake of [I]MEGMIB-5F7GGC is consistent with previously published results and suggests that this behavior may reflect in vivo lability of the thiosuccinimide bond, generating labeled catabolic products that are rapidly excreted.

[0131] In a study of mice bearing HER2-expressing xenografts bearing both randomly labeled 5F7 and the closely related VHH_1028 sdAb, 131 Compared to I-labeled conjugates, 211 Considerable therapeutic efficacy was demonstrated for the At-labeled conjugate (VHH_1028 sdAb, see Feng et al., "Radiopharmaceuticals for Radiopharmaceutical Therapy of HER2-Expressing Cancers," incorporated herein by reference). 131 Evaluation of an I-labeled HER2-specific single domain antibody fragment 131I-labeled HER2-specific single domain antibody fragment for the radiopharmaceutical therapy of HER2-expressing cancers" Sci Rep 12, 3020 (2022). 211 We have developed a site-specific and biologically stable reagent for labeling sdAbs with At. To assess the effect of halogens on in vivo behavior, iso-[ 211 At]AGMB-PODS-5F7GGC and iso-[ 131 I]GMIB-PODS-5F7GGC were compared in a paired labeling format. 211 At 131 No significant difference was observed in tumor uptake of I, 211 At levels in the spleen, stomach, and thyroid 131 The results were significantly higher than the I level. 211 This is consistent with a higher degree of dehalogenation of the At-labeled sdAb. 211 At]SAGMB and iso-[ 131 A similar halogen-dependent trend in tumor and normal tissue uptake was reported for 5F7 randomly labeled with [I]SGMTB (Choi et al., “Astatine-211 labeled anti-HER2 5F7 single domain antibody fragment conjugates: radiolabeling and preliminary evaluation,” Nucl. Med. Biol. 2018;56:10-20). On the other hand, iso-[I]SGMTB, an sdAb that differs from 5F7 by a single amino acid and shows identical in vivo behavior, was reported. 211 The biodistribution of iso-[At]AGMB-VHH_1028 has been determined in athymic mice bearing BT474 subcutaneous xenografts in a single-label format. 211When At]AGMB-PODS-5F7GGC was evaluated in a single-labeled format, its tumor uptake was significantly higher than that of iso-[ 211 The uptake was approximately twice that reported for At]AGMB-VHH_1028. Furthermore, in the stomach, thyroid, and spleen, 211 At levels were approximately two-fold lower in the PODS-based radioimmunoconjugate, whereas iso-[ 211 These results suggest that the α-SAGMB conjugate is more stable in vivo than the α-SAGMB conjugate.

[0132] Finally, the use of PODS for site-specific labeling provides the opportunity to compare metal and halogen sdAb labeling strategies while eliminating heterogeneity as a confounding variable, since only a single species is generated. This is particularly important for sdAbs that bind internalizing targets such as HER2, since labeled sdAbs with metal-free chelates may have altered charge and potentially different internalization and / or capture in tumor cells. Iso-[ 125 I]GMIB-PODS-5F7GGC 177 A significantly higher tumor uptake was observed when compared to [Lu]Lu-DOTA-PODS-5F7GGC, whereas the opposite behavior was observed at 24 h. One possible limitation of the radioiodinated sdAb is that it showed a higher hepatobiliary organ uptake at earlier time points. 177 Retention of Lu was substantially higher than that of the radioiodinated agent, reaching a difference of approximately 40-fold at 24 h. The potential dose-limiting renal activity level was 177 This is consistent with levels previously reported for other sdAbs labeled with Lu. The results of this direct comparison suggest that for 5F7 (and possibly other sdAbs), radiohalogenation using a PODS-containing residual agent is preferred over the use of radiometals.

[0133] Further information is available in U.S. Provisional Application Nos. 62 / 507,477 (filed May 17, 2017) and 62 / 634,385 (filed February 23, 2018), as well as U.S. Pat. No. 11,000,604 (entitled "Reagent for Site-Selective Bioconjugates of Proteins or Antibodies," filed May 17, 2018, issued May 11, 2021), the contents of which are incorporated herein by reference.

[0134] This written description uses examples to disclose the disclosure, including the best mode, and to enable any person skilled in the art to practice the invention, including making and using any devices or systems, and performing any incorporated methods. The patentable scope of the disclosure is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements that have insignificant differences from the literal language of the claims.

Claims

1. 1. A radiohalogenated prosthetic group conjugate comprising one or more radioactive halogens attached to a protein or peptide via a thioether bond, said radiohalogenated prosthetic group conjugate having the structure (V): 【Chemical 1】 (In the formula: a is 1 to 6, b is 1 to 6, and c is 1 to 6; L １ is -C(O)- or 【Chemistry 2】 and L ２ is a bond or -(CH ２ ) ｎ - (where n is 1 to 6); G is guanidine, guanidine with one or more carbamate protecting groups, PO ３ H, SO ３ H, P.O. ２ -OtBu, SO ２ - OtBu, arginine, phosphonophenylalanine, sulfophenylalanine, glutamic acid, aspartic acid, lysine, a hydrophilic carbohydrate moiety, or a polyethylene glycol (PEG) chain; Y is CH or N; X is a radioactive halogen; R １ But C １－６ is alkyl; and Pep is a protein or peptide).

2. The radiohalogenated prosthetic group conjugate has the structure (VI): 【Chemistry 3】 (In the formula, R ３ are independently a carbamate protecting group or H. The radiohalogenated prosthetic group conjugate of claim 1.

3. The radiohalogenated prosthetic group conjugate has the structure (VII): 【Chemistry 4】 where Pep is a protein / peptide. The radiohalogenated prosthetic group conjugate of claim 2.

4. The radiohalogenated prosthetic group conjugate has the structure (VIII): 【Chemistry 5】 where Pep is a protein / peptide. The radiohalogenated prosthetic group conjugate of claim 2.

5. The radioactive halogen is １８ F. １２２ I, １２３ I, １２４ I, １２５ I, １３１ I, ７５ Br, ７６ Br, ７７ Br, ８０ｍ Br, or ２１１ The radiohalogenated prosthetic group conjugate of any one of claims 1 to 4, wherein the prosthetic group is selected from the group consisting of At.

6. The radioactive halogen is １２３ I, １２４ I, or １３１ 6. The radiohalogenated prosthetic group conjugate of claim 5, wherein the prosthetic group is selected from the group consisting of: I.

7. The radioactive halogen is ２１１ 6. The radiohalogenated prosthetic group conjugate of claim 5, wherein the prosthetic group is At.

8. The radiohalogenated prosthetic group conjugate of any one of claims 1 to 4, wherein the protein / peptide contains at least one cysteine ​​residue, and the protein / peptide is linked to the radiolabeled prosthetic group via the cysteine ​​residue.

9. 9. The radiohalogenated prosthetic group conjugate of claim 8, wherein the protein / peptide comprises a glycine-cysteine ​​tail of the structure GnC, where n is an integer from 2 to 10, and the protein / peptide is attached to the radiolabeled prosthetic group via the glycine-cysteine ​​tail.

10. 9. The radiohalogenated prosthetic group conjugate of claim 8, wherein the protein / peptide comprises a C-terminal GGC tail, and the protein / peptide is linked to the radiolabeled prosthetic group via the C-terminal GGC tail.

11. 9. The radiohalogenated prosthetic group conjugate of claim 8, wherein the protein / peptide is selected from the group consisting of an antibody, a single domain antibody fragment, or a tumor-targeting protein / peptide.

12. 12. The radiohalogenated prosthetic group conjugate of claim 11, wherein the protein / peptide binds to HER2.

13. The radioactive halogen is １２３ I, １２４ I, or １３１ I, or ２１１ At; the protein / peptide comprises a glycine-cysteine ​​tail of the structure GnC, where n is an integer between 2 and 10, and the protein / peptide is conjugated to the radiolabeled prosthetic group via the glycine-cysteine ​​tail; and the protein / peptide binds to HER2, The radiohalogenated prosthetic group conjugate of claim 1.

14. 2. The radiohalogenated prosthetic group conjugate of claim 1, wherein the radiohalogenated prosthetic group conjugate has the following structure: 【Chemistry 6】

15. 2. The radiohalogenated prosthetic group conjugate or a pharmaceutically acceptable salt thereof according to claim 1, which is used for performing molecular imaging.

16. The radioactive halogenated prosthetic group conjugate of claim 15, wherein the molecular imaging is performed to detect cancer cells.

17. 17. The radiohalogenated prosthetic group conjugate of claim 16, wherein the cancer cells are breast cancer cells.

18. 2. The radiohalogenated prosthetic group conjugate or a pharmaceutically acceptable salt thereof according to claim 1, for use in cancer treatment.

19. 19. The radiohalogenated prosthetic group conjugate of claim 18, wherein the cancer is selected from the group consisting of breast cancer, cervical cancer, ovarian cancer, or uterine cancer.

20. 20. The radiohalogenated prosthetic group conjugate of claim 19, wherein the cancer is HER2 breast cancer.

21. 1. A method for labeling a protein or peptide with a radioactive halogen, comprising: (i) obtaining a compound having structure (II), 【Chemistry 7】 During the ceremony: a is 1 to 6, b is 1 to 6, and c is 1 to 6; X is a radioactive halogen; R １ But C １－６ is alkyl; Each R ３ is independently a carbamate protecting group or H; and L １ is -C(O)- or 【Chemistry 8】 and (ii) R ３ is H, reacting a compound of structure (II) with said protein or peptide; or ３ is a carbamate protecting group, reacting a compound of structure (II) with said peptide A method comprising:

22. 22. The method of claim 21, further comprising the step of removing the carbamate protecting group from the compound prior to reacting the compound with the protein or peptide.

23. The radiohalogenated prosthetic group conjugate has the structure (VII): 【Chemistry 9】 where Pep is a protein / peptide.

22. The method of claim 21.

24. The radiohalogenated prosthetic group conjugate has the structure (VIII): 【Chemistry 10】 where Pep is a protein / peptide.

22. The method of claim 21.

25. The method of any one of claims 21 to 24, wherein said protein / peptide is selected from the group consisting of an antibody, a single domain antibody fragment, a tumor targeting protein / peptide and HER2.

26. further comprising the step of modifying said protein / peptide by adding a C-terminal GGC tail to said protein / peptide, The compound reacts with the cysteine ​​residue of the GGC tail to form a thioether bond between the compound and the protein / peptide. The method according to claims 21 to 24.

27. 1. A method for labeling a protein or peptide with a radioactive halogen, comprising the steps of: (i) obtaining a compound having structure (VI), 【Chemistry 11】 During the ceremony: a is 1 to 6, b is 1 to 6, and c is 1 to 6; X is SnR ２ ３ , B(OH)2 or Bpin; Each R ３ is independently a carbamate protecting group or H; and L １ is -C(O)- or 【Chemistry 12】 and Pep is a protein / peptide.

28. A compound having the structure (I): 【Chemistry 13】 During the ceremony: a is 1 to 6, b is 1 to 6, and c is 1 to 6; L １ is -C(O)- or 【Chemistry 14】 and L ２ is a bond or -(CH ２ ) ｎ - (where n is 1 to 6); G is guanidine, guanidine with one or more carbamate protecting groups, PO ３ H, SO ３ H, P.O. ２ -OtBu, SO ２ - OtBu, arginine, phosphonophenylalanine, sulfophenylalanine, glutamic acid, aspartic acid, lysine, a hydrophilic carbohydrate moiety, or a polyethylene glycol (PEG) chain; Y is CH or N; X is SnR ２ ３ , B(OH) ２ , Bpin, or a radioactive halogen; R １ But C １－６ is alkyl; and Each R ２ is independently C １－６ is alkyl compound.

29. A compound having the structure (II): 【Chemistry 15】 During the ceremony: a is 1 to 6, b is 1 to 6, and c is 1 to 6; L １ is -C(O)- or 【Chemistry 16】 and X is SnR ２ ３ , B(OH) ２ , Bpin, or a radioactive halogen; R １ But C １－６ is alkyl; Each R ２ is independently C １－６ is alkyl; Each R ３ are independently a carbamate protecting group or H 29. The compound of claim 28.

30. 30. The compound of claim 29 having the structure (III). 【Chemistry 17】

31. 30. The compound of claim 29 having the structure (IV). 【Chemistry 18】

32. R １ is methyl, ethyl or propyl; a is 1 or 2, b is 2 or 3, and c is 1 or 2; X is SnR ２ ３ , B(OH) ２ or Bpin; R ２ is methyl, ethyl, or n-butyl The compound according to any one of claims 28 to 31.

33. X is a radioactive halogen, said radioactive halogen being １８ F. １２２ I, １２３ I, １２４ I, １２５ I, １３１ I, ７５ Br, ７６ Br, ７７ Br, ８０ｍ Br, or ２１１ The compound according to any one of claims 28 to 31, selected from the group consisting of At.

34. The radioactive halogen is １２３ I, １２４ I, or １３１ 34. The compound of claim 33, wherein:

35. The radioactive halogen is ２１１ 34. The compound of claim 33, wherein At.

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