Radioactive labeling of polypeptides

Abstract

Improved methods for radiolabeling antibodies using click chemistry are described, as are pharmaceutical compositions and uses related to the radiolabeled antibodies produced by the methods.

Description

[Technical Field]

[0001] (Reference to electronically submitted sequence listings) This application includes a sequence listing in ASCII format filed on December 7, 2018, with the filename "Sequence Listing," and having a size of approximately 13.2 kB. The sequence listing submitted via EFS-Web is part of this specification and is incorporated herein by reference in its entirety.

[0002] (Cross-reference of related applications) This application is granted priority to U.S. Provisional Patent Application No. 62 / 599,830, filed December 18, 2017, pursuant to Section 119(e) of the U.S. Patent Act, the entirety of which disclosure is incorporated herein by reference.

[0003] (Field of Invention) The present invention relates to a method for radiolabeling polypeptides such as antibodies. More specifically, the present invention relates to a method for labeling polypeptides with radioactive metal ions using click chemistry. The present invention also relates to pharmaceutical compositions of radiolabeled polypeptides and their uses. [Background technology]

[0004] Alpha-emitting radionuclides are highly promising for cancer therapy due to their combination of high energy and short-range action, offering the potential to be highly localized and potently kill tumor cells (Kim, Y.Sand MWBrechbiel, An overview of targeted alpha therapy. Tumor Biol, 2012. 33(3):p.573-90). Targeted delivery of alpha-emitters using antibodies, scaffold proteins, small molecule ligands, aptamers, or other binding sites specific to cancer antigens provides a method for selective delivery of radionuclides to tumors, thereby enhancing their potency and mitigating off-target effects. In common implementations, the binding site is attached to a chelator that binds to an alpha-emitting metal to generate a radioactive complex. Many such examples use monoclonal antibodies (mAbs) as targeted ligands to generate what are known as radioimmune complexes.

[0005] Actinium-225 ( 225 Ac) is an alpha-emitting isotope of particular interest for medical applications (Miederer et al., Realizing the potential of the Actinium-225 radionuclide generator in targeted alpha particle therapy applications. Adv Drug Deliv Rev, 2008. 60(12):71-82). 225 The 10-day half-life of Ac is long enough to promote the formation of radioactive complexes, but short enough to match the circulating pharmacokinetics of delivery vehicles such as antibodies. Therefore, 225 Ac's radioactive immune complexes are of particular interest. In addition, 225 Ac is a stable isotope. 209 Its potency is increased by decaying through a series of processes that ultimately emit four alpha particles before reaching Bi. Another radioactive isotope for medical use is lutetium-177, which emits both gamma rays suitable for imaging and medium-energy beta rays suitable for radiotherapy. 177 It is Lu. 177The Lu-labeled peptide has demonstrated a reduction in normal tissue damage, 177 Lu-labeling has been shown to enable the use of a single radiopharmaceutical for both therapy and imaging (Kwekkeboom DJ, et al. [177Lu-DOTAOTyr3]octreotate: comparison with [111In-DTPAo]octreotide in patients. Eur J Nucl Med. 2001;28:p.1319-1325). Other radioisotopes used for therapeutic applications include, for example, beta emitters or alpha emitters such as thorium, radium, 32 P, 47 Sc, 67 Cu, 77 As, 89 Sr, 90 Y, 99 Tc, 105 Rh, 109 Pd, 111 Ag, 131 I, 153 Sm, 159 Gd, 165 Dy, 166 [[ID=3۲]]Ho, 169 Er, 186 Re, 188 Re, 194 Ir, 198 Au, 199 Au, 211 At, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 255 Fm, and 227 Th, etc. Other radioisotopes used for imaging applications include, for example, 62 Cu, 64 Cu, 67 Ga, 68 Ga, 86 Y, 89 Zr, and 111 gamma-emitting radioisotopes such as In, etc.

[0006] Previous clinical and preclinical programs have used 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for actinium chelation. However, DOTA chelation of actinium is known to be difficult (Deal, KA, et al., Improved in vivo stability of actinium-225 macrocyclic complexes. J Med Chem, 1999. 42(15): p.2988-92), often requiring harsh conditions or high levels of DOTA per antibody. As a result, two different approaches known as "one-step" and "two-step" radiolabeling methods have been employed, each with its own drawbacks.

[0007] The first method developed was a "two-step" approach involving two chemical steps containing actinium (McDevitt, MR, et al., Tumor therapy with targeted atomic nanogenerators. Science, 2001. 294(5546): p.1537-40). 225 Ac was chelated with a bifunctional chelator (BFC) DOTA-isothiocyanate (DOTA-SCN) in 2M acetate buffer at 55°C to 60°C for 30 minutes at pH 4.5 to 5 with a high radiochemical yield (approximately 95%). Subsequently, 225 Ac DOTA-SCN was reacted with a targeted antibody to generate a radioactive immune complex. The main drawback of the two-step method is that approximately 90% of the SCN cannot withstand the labeling conditions, so the input... 225 Approximately 90% of Ac is conjugated into DOTA, a non-reactive form that cannot be conjugated into the antibody. This results in low yields (typically only about 10%), high costs, and reduced specific activity, which can limit the effectiveness of the final conjugate.

[0008] The "one-step" method was recently developed for actinium (Maguire, WF, et al., Efficient 1-step radiolabeling of monoclonal antibodies to high specific activity with 225Ac for alpha-particle radioimmunotherapy of cancer. J Nucl Med, 2014. 55(9): p.1492-8). This method involves only one chemical reaction step involving actinium. DOTA-SCN was first conjugated to the antibody. Then, 225 Ac was chelated to DOTA-mAbs under mild conditions (37°C, pH 7.5), yielding a maximum radiochemical yield of 80%. However, achieving high yields required the conjugation of high levels of DOTA (approximately 10 or more per antibody). Species with a high chelator-to-antibody ratio (CAR), in this case a high DOTA-to-Ab ratio (DAR), are more likely to have reduced immunoreactivity, and furthermore, the average DAR may be 10. 225 Ac may be chelating a higher proportion of the population than average. Therefore, this method is likely to be effective. 225 There is a risk of ligating Ac to at least the active fraction of the antibody-chelator complex. Furthermore, to avoid chelation of common metals such as iron, zinc, and copper, the antibody and DOTA-mAb complex must be handled under metal-free conditions, which presents a significant challenge to the production process.

[0009] Click chemistry is a chemical approach introduced by Sharpless in 2001, a chemical method tuned to rapidly and reliably produce substances by combining small amounts of units. See, for example, Kolb, Finn and Sharpless, Angewandte Chemie International Edition (2001) 40:2004-2021; Evans, Australian Journal of Chemistry (2007) 60:384-395. Coupling reactions (some of which can be classified as "click chemistry") include, but are not limited to, the formation of esters, thioesters, and amides from activated acids or acyl halides (e.g., peptide coupling); nucleophilic substitution reactions (e.g., nucleophilic substitution of halides or ring opening of strained ring systems); azide-alkyne and hysgen cycloaddition reactions (e.g., 1,3-dipole cycloaddition reactions between azide and alkyne to form 1,2,3-triazole linkers); thioline addition reactions; imine formation; Diels-Alder reactions between tetrazine and trans-cyclooctene (TCO); and Michael addition reactions (e.g., maleimide addition reactions).

[0010] Click chemistry reactions between alkynes and azides typically require the addition of a copper catalyst to facilitate the 1,3-cycloaddition reaction and are known as copper-catalyzed azide-alkyne cycloaddition (CuAAC). However, click chemistry reactions between cyclooctin or cyclooctin derivatives and azides typically do not require the addition of a copper catalyst and instead proceed via strain-promoted azide-alkyne cycloaddition (SPAAC) (Debets, MF, et al., Bioconjugation with strained alkenes and alkynes. Acc Chem Res, 2011. 44(9): p.805-15).

[0011] Site specificity has become a major area of ​​focus in the field of antibody-drug conjugates (ADCs) because it has been demonstrated that site-specific methods can enhance both the efficacy and safety of ADCs compared to random conjugation (Agarwal, P. and CRBertozzi, Site-specific antibody-drug conjugates: the nexus of bioorthogonal chemistry, protein engineering, and drug development. Bioconjug Chem, 2015.26(2):p.176-92). Similar safety and efficacy benefits are thought to be achievable with respect to radioimmune conjugates. [Overview of the project] [Problems that the invention aims to solve]

[0012] As described above, there is still a need in the technological field for efficient methods to produce stable radioimmune complexes with high specific activity and high yield. [Means for solving the problem]

[0013] The present invention satisfies this need by providing a method for using click chemistry to radiolabel polypeptides such as antibodies. The method of the present invention requires a reduction in the use of radioactive metals and requires only metal-free conditions in the steps used to generate the initial radioactive complex, while using an azide-modified antibody and a radioactive complex containing a radioactive metal ion associated with a chelated moiety containing an alkyne group in a click chemistry reaction to produce a stable radioimmune complex with a low chelator-to-antibody ratio (CAR) and high radiation chemistry yield. The method of the present invention simplifies previous methods for producing radioimmune complexes while increasing safety, efficacy, and uniformity.

[0014] In a general embodiment, the present invention relates to a method for labeling polypeptides with radioactive metal ions, wherein the method is: a. To provide a modified polypeptide comprising a polypeptide covalently bonded to a first click reaction partner, b. To provide a radioactive complex containing a radioactive metal ion associated with the chelate moiety, wherein the chelate moiety contains a chelating agent covalently bonded to a second click reaction partner. c. The modified polypeptide is brought into contact with a radioactive complex under conditions that allow the polypeptide to be labeled with a radioactive metal ion by the reaction of a first click reaction partner with a second click reaction partner.

[0015] In another general embodiment, the present invention relates to a pharmaceutical composition comprising a radiolabeled polypeptide prepared by the method of the present invention and a pharmaceutically acceptable carrier.

[0016] In another general embodiment, the present invention relates to a method for treating a neoplastic disease or disorder in a subject requiring treatment of a neoplastic disease or disorder, comprising administering the subject a pharmaceutical composition of the present invention.

[0017] In another general embodiment, the present invention is a. A modified polypeptide comprising a polypeptide covalently bonded to a first click reaction partner, b. A combination or kit comprising a radioactive complex containing a radioactive metal ion associated with a chelate moiety, wherein the chelate moiety contains a chelating agent covalently bonded to a second click reaction partner, Here, the combination or kit is used to label polypeptides with radioactive metal ions.

[0018] In other general embodiments, the present invention relates to therapeutic or diagnostic agents ("theranostic agents") comprising a radiolabeled polypeptide prepared by the method of the present invention. [Brief explanation of the drawing]

[0019] The above-mentioned "Means for Solving the Problem" and the following "Modes for Carrying Out the Invention" will be better understood when read in conjunction with the attached drawings. It should be understood that the present invention is not limited to the embodiments shown in the drawings.

[0020] The drawings are as follows: [Figure 1] A schematic diagram of the radiolabeling of antibodies according to the method of the present invention is shown. Random conjugation is shown in the figure, and a similar radiolabeling scheme is used when the azide is site-specifically conjugated to a monoclonal antibody (mAb). [Figure 2] This invention presents an improved two-step synthesis scheme for the preparation of 89Zr-DOTA-mAb via click chemistry, according to one embodiment of this application. [Figure 3] The cell binding of the In-111 radioimmune complex is shown, and the bound radioactivity increases with increasing cell number. In particular, A shows the binding of the PSMA-binding antibody ("PSMB127") In-111 radioimmune complex and the human transferrin In-111 radioimmune complex to the human prostate cancer cell line C4-2B (PSMA+, transferrin receptor+) according to embodiments of this application, and B shows the binding of the EGFR-binding antibody, cetuximab and panitumumab In-111 radioimmune complex to the human epidermal carcinoma cell line A431 (EGFR+) according to embodiments of this application, and the lack of binding of these complexes to the control (EGFR-) human AML cell line MOLM-13. [Figure 4] One embodiment of this application demonstrates the dynamics of intracellular migration of In-111 in human prostate cancer cell line C4-2B treated with the anti-PSMA mAb In-111 radioactive immune complex, where surface-bound In-111 rapidly disappeared from the cell surface and was redistributed within the cell. [Figure 5A]The results of a mouse tumor xenograft study are presented, in which mice were transplanted with human prostate cancer LNCaP cells, and when the tumor reached 100 mm3, the mice were treated with a single dose of a click radiolabeled anti-PSMA mAb ("PSMB127") actinium radioconjugate according to one embodiment of this application, either within the radioactivity range or isotype control (a human IgG4 antibody that binds to a viral target not present in this system radioconjugate). In detail, the tumor volume for each group is shown, and the size is plotted until less than half of the group survived. [Figure 5B] The results of a mouse tumor xenograft study are presented, in which human prostate cancer LNCaP cells were transplanted into mice, and when the tumor reached 100 mm3, the mice were treated with a single dose of a click radiolabeled anti-PSMA mAb ("PSMB127") actinium radiocomplex according to one embodiment of this application, either within the radioactivity range or isotype control (a human IgG4 antibody that binds to a viral target not present in this system radiocomplex). In detail, the survival curve of the control mAb group is shown. [Figure 5C] The results of a mouse tumor xenograft study are presented, in which human prostate cancer LNCaP cells were transplanted into mice, and when the tumor reached 100 mm3, the mice were treated with a single dose of a click radiolabeled anti-PSMA mAb ("PSMB127") actinium radioconjugate according to one embodiment of this application, either within the radioactivity range or isotype control (a human IgG4 antibody that binds to a viral target not present in this system radioconjugate). In detail, the survival curves of the anti-PSMA mAb group are shown. [Modes for carrying out the invention]

[0021] In the background of the invention, and throughout this specification, various publications, articles, and patents are cited or referenced. Each of these references is incorporated herein by reference in its entirety. The considerations of documents, operations, materials, devices, articles, etc., included herein are for the purpose of providing context for the invention. Such considerations do not constitute an endorsement that any or all of these things constitute prior art with respect to any invention disclosed or claimed.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in which the present invention pertains. Otherwise, any particular term referenced herein has the meaning set forth herein. All patents, published patent applications and publications referenced herein are incorporated by reference as if they were included in their entirety herein.

[0023] It should be noted that the singular forms “a,” “an,” and “the” used herein and in the appended claims refer to multiple objects unless otherwise clearly indicated by the context.

[0024] Throughout this specification and the following claims, unless contextually required, the terms “comprise” and variations such as “comprises” and “comprising” will be understood to mean that they include a specified integer or step or group of integers or steps, but not any other integer or step or group of integers or steps. When used herein, the term “comprise” may be replaced with the terms “contain” or “including,” or sometimes with the term “have.”

[0025] As used herein, “consisting of” excludes any element, step, or component not specified in the elements of the claims. As used herein, “essentially consisting of” does not exclude materials or steps that do not substantially affect the basic and novel features of the claims. As used herein in relation to aspects or embodiments of the present invention, any of the above terms “comprising,” “containing,” “including,” and “having” may be replaced with the terms “consisting of” or “essentially consisting of.”

[0026] When used herein, the connecting term "and / or" between multiple enumerated elements is understood to encompass both individual and combined options. For example, when two elements are connected by "and / or", the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of the first and second elements together. It is understood that any one of these options satisfies the requirements of the term "and / or" as used herein. The simultaneous applicability of two or more of the options is also understood to be included in the meaning and thus satisfies the requirements of the term "and / or".

[0027] To assist readers of this application, the description of this specification is divided into various paragraphs or sections, or directed toward various embodiments of this application. These separations should not be considered to decouple the substance of one paragraph, section, or embodiment from the substance of another paragraph, section, or embodiment. On the contrary, those skilled in the art will understand that the description of this specification has broad applicability and encompasses all possible combinations of paragraphs, sentences, and texts. Any consideration of any embodiment is meant to be illustrative and is not intended to suggest that the scope of this disclosure, including the claims, is limited to these embodiments.

[0028] Click radiolabeling of polypeptides In contrast to known procedures, the method of the present invention provides an improved method for generating radioimmune complexes, which are suitable, for example, for medical applications in subjects requiring it, such as humans. In particular, the methods described herein are not limited to, 225 Ac, 111 In and 89 This invention addresses the major limitations of current methods by providing a process for both high-yield chelation and low DAR of metal ions containing Zr. The present invention addresses the radiolabeled ions used for diagnostic purposes (e.g., 89 Zr or 111(When labeled with In) or for therapeutic purposes (e.g., 225 This invention enables the production of a single batch of azide-labeled polypeptides, such as azide-mAb complexes, that can be used for production (when labeled with Ac), and the radiolabeling can be attached to the same site(s) within the batch of azide-labeled polypeptides and obtained by either site-specific modification or random azide complexation. For example, in the case of random azide complexation, a sample of a batch of azide-labeled polypeptides containing a single distribution of azide modification sites can be radiolabeled for different purposes using the click chemistry of the present invention.

[0029] The present invention, which relies on click chemistry and is called "click radiolabeling," involves (1) obtaining a modified polypeptide, such as an antibody, containing a first click chemistry reaction partner, for example, an azide moiety, and (2) a radioactive metal ion associated with the chelated moiety, for example, 225 Ac, 111 In, or 89 (3) to obtain a radioactive complex containing Zr, wherein the chelate portion comprises a second click chemistry reaction partner, such as a chelating agent covalently bonded to an alkyne group, such as DOTA-dibenzocyclooctin (DOTA-DBCO) or deferoxamine-DBCO (DFO-DBCO), and (4) a reaction between the radioactive complex and a click chemistry reaction partner of a modified peptide, such as a strain-enhanced azide-alkyne cycloaddition reaction (SPAAC) between the azide portion and the alkyne group.

[0030] The method of the present invention can maximize efficiency by enabling chelation of radioactive metals under low pH or high pH and / or high temperature conditions, which can be achieved without the risk of inactivating alkyne reaction partners. Efficient chelation between azide-mAb and radioactive complex and efficient SPAAC reaction enable the production of radioactive immune complexes in high radiochemical yield even at low azide:mAb ratios. In the method of the present invention, the only step that must exclude trace metals is the chelation of radioactive metal ions to the chelation moiety, and the steps of antibody production, purification, and complexation do not need to be carried out under metal-free conditions.

[0031] As used herein, the term “click chemistry” refers to the chemical philosophy introduced by Sharpless, describing chemistry tuned to rapidly and reliably generate covalent bonds by joining together small units containing reactive groups (see Kolb, et al. above). Click chemistry is not a specific reaction but refers to a concept that includes, but is not limited to, reactions that mimic reactions found in nature. In some embodiments, click chemistry reactions are modular, broad-ranging, have high chemical yields, produce harmless byproducts, are stereospecific, exhibit a strong thermodynamic drive for selecting reactions with a single reaction product, and / or can be carried out under physiological conditions. In some embodiments, click chemistry reactions exhibit high atomic economy, can be carried out under simple reaction conditions, use readily available starting materials and reagents, do not use toxic solvents or use harmless or easily removable solvents such as water, and / or provide isolation of simple products by non-chromatographic methods such as crystallization or distillation. In certain embodiments, the click chemistry reaction is a Huygen ring addition reaction or a 1,3-dipolar cycloaddition reaction between the azide (-N3) and the alkyne or alkyne moiety, forming a 1,2,4-triazole linker.

[0032] In general embodiments, the present invention relates to a method for labeling polypeptides, aptamers, or small molecule ligands with radioactive metal ions, wherein the method is: a. To provide a modified polypeptide comprising a polypeptide covalently bonded to a first click reaction partner, b. To provide a radioactive complex containing a radioactive metal ion associated with the chelate moiety, wherein the chelate moiety contains a chelating agent covalently bonded to a second click reaction partner. c. The modified polypeptide is brought into contact with a radioactive complex under conditions that allow the polypeptide to be labeled with a radioactive metal ion by the reaction of a first click reaction partner with a second click reaction partner.

[0033] As used herein, the term “polypeptide” refers to a polymer composed of naturally occurring structural variants and their synthetic, non-naturally derived analogs linked via peptide bonds. The term “polypeptide” refers to a polypeptide of any size, structure, or function. Typically, a polypeptide is at least three amino acids long. Polypeptides can be naturally occurring, recombinant, synthetic, or any combination thereof. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. According to a preferred embodiment, the polypeptide is an antibody, preferably a monoclonal antibody, or a fragment thereof, an antigen-binding fragment thereof, etc. According to a preferred embodiment, the antibody or fragment thereof is specific to a cancer antigen. According to another embodiment, the polypeptide is a genetically engineered domain or scaffold protein.

[0034] As used herein, the terms “antibody” or “immunoglobulin” are used in a broad sense and include immunoglobulins or antibody molecules, including polyclonal antibodies, mouse, human, human-compatible, humanized, and chimeric monoclonal antibodies, and monoclonal antibodies, including their antigen-binding fragments.

[0035] Generally, an antibody is a protein or peptide chain that exhibits binding specificity to a particular antigen, and is referred to herein as a “target.” The structure of antibodies is well known. Immunoglobulins can be assigned to five main classes, namely IgA, IgD, IgE, IgG, and IgM, depending on the amino acid sequence of the heavy chain constant domain. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Therefore, the antibody of the present invention can be any of the five main classes or the corresponding subclasses. Preferably, the antibody of the present invention is IgG1, IgG2, IgG3, or IgG4. The antibody light chain of any vertebrate species can be assigned to one of two distinctly different types, namely κ and λ, based on the amino acid sequence of their constant domains. Therefore, the antibody of the present invention can contain a κ or λ light chain constant domain. According to a particular embodiment, the antibody of the present invention comprises the heavy chain and / or light chain constant region of a mouse antibody or a human antibody. Each of the four IgG subclasses has a different biological function known as an effector function. These effector functions are generally mediated by interaction with the Fc receptor (FcγR) or by binding to C1q and fixation of complement. Binding to FcγR can lead to antibody-dependent cell-mediated cytolysis, while binding to complement factors can lead to complement-mediated cytolysis. Antibodies useful in this invention may have no effector function or only minimal effector function, but retain their ability to bind to FcRn.

[0036] As used herein, the term “antigen-binding fragment” refers to antibody fragments such as diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (dsdiabodies), single-chain antibody molecules (scFv), single-domain antibodies (sdab), scFv dimers (bivalent diabodies), multispecific antibodies formed from a portion of an antibody containing one or more CDRs, camelized single-domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragments that bind to an antigen but do not contain a complete antibody structure. Antigen-binding fragments can bind to the same antigen to which a parent antibody or parent antibody fragment binds. As used herein, the term “single-chain antibody” refers to conventional single-chain antibodies in this art, including heavy-chain and light-chain variable regions linked by short peptides of about 15 to about 20 amino acids. As used herein, the term "single-domain antibody" refers to a conventional single-domain antibody in this art that includes a heavy chain variable region and a heavy chain constant region, or includes only the heavy chain variable region.

[0037] As used herein, the terms “scaffold” or “scaffold protein” refer to any protein having a target-binding domain and capable of binding to a target. A scaffold comprises a “framework” that is largely structural and a “binding domain” that contacts the target and provides specific binding. The binding domain of a scaffold does not have to be defined by a single continuous sequence of the scaffold. In certain cases, a scaffold may be part of a larger binding protein, or it itself may be part of a multimeric binding protein containing multiple scaffolds. A particular binding protein may be bispecific or multispecific in that it can bind to two or more different epitopes. A scaffold may be derived from a single-chain antibody, or it may not be antibody-derived.

[0038] By using any method for chemical or enzymatic modification of polypeptides known to those skilled in the art in consideration of this disclosure, the polypeptides of the present invention can be covalently bonded to a first click reaction partner. Amine-reactive groups that react with primary amines present at the N-terminus and the side chains of lysine residues of each polypeptide chain can be used in the random modification of polypeptides. Examples of amine-reactive groups suitable for use in the present invention include, but are not limited to, N-hydroxysuccinimide (NHS), substituted NHS, e.g., sulfo-NHS, isothiocyanates, and tetra and perfluorophenyl esters. Thiol-reactive groups that react with thiols or sulfhydryls present at the side chains of cysteine ​​residues can be used in the random modification of polypeptides. Examples of thiol-reactive groups suitable for use in the present invention include, but are not limited to, maleimide, haloacetyl, and phenyloxadiazole sulfone. According to a preferred embodiment, the modified polypeptide is obtained by reacting an electrophile covalently bonded to a first click reaction partner (e.g., NHS-azide) with a lysine side chain, preferably an amino side chain.

[0039] The method of the present invention further enables the production of site-specific radiolabeled polypeptides. The click radiolabeling method of the present invention facilitates site-specific generation of radioactive immune complexes by utilizing established methods for site-specific introduction of azide groups into antibodies (Li, X., et al. Preparation of well-defined antibody-drug conjugates through glycan remodeling and strain-promoted azide-alkyne cycloadditions. Angew Chem Int Ed Engl, 2014. 53(28): p.7179-82; Xiao, H., et al., Genetic incorporation of multiple unnatural amino acids into proteins in mammalian cells. Angew Chem Int Ed Engl, 2013. 52(52): p.14080-3). Methods for site-specific attachment of molecules to proteins or antibodies are known in the art, and any method for site-specific labeling of antibodies known to those skilled in the art can be used in the present invention in consideration of this disclosure. Examples of methods for site-specifically modifying antibodies suitable for use in the present invention include, but are not limited to, the incorporation of modified cysteine ​​residues (e.g., THIOMAB®), non-natural amino acids or glycans (e.g., selenocysteine, p-AcPhe, formylglycine-producing enzymes (FGE, SMARTag®), etc.), and the use of enzymatic methods (e.g., glycotransferase, endoglycosidase, microbial or bacterial transglutaminase (MTG or BTG), saltase A, etc.). In a preferred embodiment, the modified polypeptide is an antibody or its antigen-binding fragment obtained by trimming the antibody or its antigen-binding fragment with a bacterial endoglycosidase specific to the β-1,4 linkage between core GlcNac residues in the Fc-glycosylation site of an antibody, such as GlycINATOR(Genovis), which leaves the innermost GlcNAc intact in Fc, enabling site-specific incorporation of azido sugars at that site.Next, the trimmed antibody or its antigen-binding fragment can be reacted with an azide-labeled sugar such as UDP-N-azidoacetylgalactosamine (UDP-GalNaz) or UDP-6-azido6-deoxyGalNac in the presence of a glycosyltransferase such as GalT galactosyltransferase or GalNac transferase to obtain a modified antibody or its antigen-binding fragment. According to another preferred embodiment, the modified polypeptide is an antibody or its antigen-binding fragment obtained by glycosylation of the antibody or its antigen-binding fragment with amidase. Next, the deglycosylated antibody or its antigen-binding fragment can be reacted with an azidoamine, preferably 3-azidopropylamine, 6-azidohexylamine, or any azidlinkeramine or any azidoalkylamine, azido-polyethylene glycol (PEG)-amine, such as O-(2-aminoethyl)-O'-(2-azidoethyl)tetraethylene glycol, O-(2-aminoethyl)-O'-(2-azidoethyl)pentaethylene glycol, O-(2-aminoethyl)-O'-(2-azidoethyl)triethylene glycol, or reacted in the presence of a microbial transglutaminase to obtain a modified antibody or its antigen-binding fragment.

[0040] As used herein, the term "aptamer" refers to a single-stranded oligonucleotide (single-stranded DNA or RNA molecule) that can bind specifically to its target with high affinity. Aptamers can be used as molecules that target a variety of organic and inorganic substances.

[0041] As used herein, the term “small molecule ligand” refers to a low molecular weight organic compound. As used herein, a small molecule ligand may refer to a compound having a size of less than approximately 1000 daltons and may refer to a compound that can be synthesized in the laboratory or may be found in nature.

[0042] As used herein, the terms “click reaction partner” or “click chemistry handle” refer to a reactant or reactive group that can be added to a click chemistry reaction. Click reaction partners are rarely found in naturally occurring biomolecules and are chemically inert to biomolecules, but when reacted with, for example, an azide-reactive or alkyne-reactive group, the reaction can proceed efficiently under biologically relevant conditions, such as cell culture conditions, including the absence of excessive heat or harsh reactants. Generally, a click chemistry reaction requires at least two molecules, including click reaction partners that can react with each other. Such click reaction partners that are reactive with each other may be referred to herein as a click chemistry handle pair or click chemistry pair. In some embodiments, the click reaction partners are azides and strained alkynes, such as cyclooctyne, or any other alkyne. In other embodiments, the click reaction partners are reactive dienes and preferred tetrazinediene derivatives. For example, trans-cyclooctene, norbornene, or biscyclononene can be paired with tetrazinediene derivatives suitable as click reaction partners. In yet another embodiment, tetrazole can be paired with an unactivated alkene in the presence of ultraviolet light to form a click reaction partner called a “photoclick” reaction partner. In another embodiment, the click reaction partners are cysteine ​​and maleimide. For example, cysteine ​​derived from a peptide (e.g., GGGC) can be reacted with maleimide associated with a chelating agent (e.g., NOTA). Other suitable click chemistry handles are known to those skilled in the art (see, for example, Spicer et al., Selective chemical protein modification. Nature Communications. 2014;5:p.4740). In another embodiment, the click reaction partners are Staudinger ligation components such as phosphine and azide.In other embodiments, the click reaction partners are Diels-Alder reaction components such as dienes, such as tetrazine, and alkenes, such as trans-cyclooctene (TCO) or norbornene. Exemplary click reaction partners are described in U.S. Patent Application Publication No. 20130266512 and International Publication No. 2015073746, and relevant descriptions of both click reaction partners are incorporated herein by reference. According to a preferred embodiment, one of the first and second click reaction partners comprises an alkyne group, and the other click reaction partner comprises an azide. According to another preferred embodiment, one of the first and second click reaction partners comprises an alkene group, and the other click reaction partner comprises a diene.

[0043] As used herein, the terms “alkyne,” “alkyne group,” or “alkyne moiety” refer to a functional group containing a carbon-carbon triple bond. Examples of alkyne moieties include terminal alkynes and cyclic alkynes, preferably terminal alkynes and cyclic alkynes reactive with an azide group. Terminal alkynes have at least one hydrogen atom bonded to the triple-bonded carbon atom. Cyclic alkynes are cycloalkyl rings containing one or more triple bonds. Examples of cyclic alkynes include, but are not limited to, bicyclononine (BCN), difluorocyclooctin (DIFO), dibenzocyclooctin (DIBO), keto-DIBO, biarylazacyclooctinone (BARAC), dibenzoazacyclooctin (DIBAC), dimethoxyazacyclooctin (DIMAC), dibenzocyclooctin (DBCO), difluorobenzocyclooctin (DIFBO), monobenzocyclooctin (MOBO), and tetramethoxyDIBO (TMDIBO), as well as cyclooctin and cyclooctin derivatives. In a preferred embodiment, one of the first and second click reaction partners comprises a cyclic alkyne, preferably DBCO. In a preferred embodiment, the other click reaction partner comprises an azide, preferably NHS-azide.

[0044] As used herein, the term “diene” refers to a compound having two carbon-carbon double bonds, with these double bonds linked at positions 1 and 3. The double bonds in a diene can be either cis or trans. Examples of dienes include, but are not limited to, tetrazine or tetrazole groups.

[0045] As used herein, the terms “alkene,” “alkene group,” or “alkene moiety” refer to an unsaturated hydrocarbon molecule containing a carbon-carbon double bond. According to certain embodiments, an alkene may contain 2 to 100 carbon atoms. Examples of alkenes include, but are not limited to, norbornene and trans-cyclooctene (TCO). According to other preferred embodiments, one of the first and second click reaction partners comprises an alkene group, preferably norbornene or TCO. According to other preferred embodiments, the other click reaction partner comprises a diene, preferably a tetrazine or tetrazole group.

[0046] As used herein, the term “covalently bonded” means that a polypeptide is attached to a first click reaction partner via at least one covalent bond, and a chelating agent is attached to a second click reaction partner via at least one covalent bond. The bond may be direct, i.e., without a linker, or indirect, i.e., via a linker.

[0047] As used herein, the term “linker” refers to a chemical moiety that connects a polypeptide or chelating agent to a click reaction partner. Any suitable linker known to those skilled in the art in consideration of this disclosure can be used in the present invention. The linker may be, for example, a single covalent bond, a substituted or unsubstituted alkyl, a substituted or unsubstituted heteroalkyl moiety, a polyethylene glycol (PEG) linker, a peptide linker, a sugar linker, or a cleavable linker such as a disulfide bond or a protease cleavage site such as valine-citrulline-PAB.

[0048] As used herein, the term "radioactive metal ion" or "radioactive metallic ion" refers to one or more isotopes of an element that emits particles and / or photons. Any radioactive metal known to one of ordinary skill in the art in view of the present disclosure can be used in the present invention. Examples of radioactive metals suitable for use in the present invention include, but are not limited to, 32 P, 47 Sc, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 77 As, 86 Y, 89 Zr, 89 Sr, 90 Y, 99 Tc, 105 Rh, 109 Pd, [[ID=3)1]] 111 Ag, 111 In, 117 Sn, 131 I, 153 Sm, 159 Gd, 165 Dy, / [[ID=)45]] 166 Ho, 169 Er, 177 Lu, 186 Re, 188 [[ID=SS4]]Re, / 194 Ir, 198 )]]Au, 199 Au, 211 At, 212 Pb, 212 Bi, 213 Bi, 223 / Ra, 225 Ac, 227 / [[ID=)74]]Th, and 255 Fm. As used herein, the term "diagnostic emitter" refers to a radioactive metal ion useful in diagnostic or imaging applications. Examples of diagnostic emitters include, but are not limited to, 62 [[ID=7B]]Cu, 64 Cu, 67 Ga, 68 Ga, 86 Y, 89 Zr, and 111 It should be noted that there seem to be some formatting or encoding issues in the original text, especially in the lines like "<...>" where it's not clear if they are part of a proper chemical symbol or something else. The translation is done as accurately as possible based on the presented text.Examples include gamma emitters such as In. As used herein, the term “therapeutic emitter” refers to a radioactive metal ion useful in therapeutic applications. Examples of therapeutic emitters, but not limited to, include beta or alpha emitters, such as thorium, radium, 32 P, 47 Sc, 67 Cu, 77 As, 89 Sr, 90 Y, 99 Tc, 105 Rh, 109 Pd, 111 Ag, 131 I, 153 Sm, 159 Gd, 165 Dy, 166 Ho, 169 Er, 177 Lu, 186 Re, 188 Re, 194 Ir, 198 Au, 199 Au, 211 At, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 225 Ac, 255 Fm and 227 Th is one example. According to a preferred embodiment, radioactive metal ions are 225 It is Ac. According to other embodiments, polypeptides can be labeled with nonmetallic radioactive labels for use in pre-targeting or theranostic applications. Examples of nonmetallic radioactive labels suitable for use in the present invention include, but are not limited to, 125 I and 18 F is one example.

[0049] The radioactive complexes described herein contain a radioactive metal ion associated with the chelated moiety. According to embodiments of the present invention, the chelated moiety contains a chelating agent covalently bonded to a click reaction partner and is sometimes referred to herein as a "bifunctional chelator".

[0050] As used herein, the terms "chelating agent" or "chelator" are used in the following context:225 This refers to radioactive metals such as Ac, or chemical compounds in which metals can be chelated via coordinate bonds. Any chelating agent known to those skilled in the art in consideration of this disclosure can be used in the present invention. In one embodiment, the chelating agent comprises a macrocyclic molecule. Examples of macrocyclic chelating agents suitable for use in the present invention include, but are not limited to, deferoxamine (DFO), ethylenediaminetetraacetic acid (EDTA), and diethylenetriaminepentaacetic acid (DTPA). In another embodiment, the chelating agent comprises an open-chain ligand. Examples of chelating agents containing open-chain ligands suitable for use in the present invention include, but are not limited to, 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), 1,4,7,10,13,16-hexaazacyclohexadecane-N,N',N'',N''',N'''',N'''''-hexaacetic acid (HEHA), 1,4,7,10,13-pentazacyclopentanadecane-N,N',N'',N''',N''''-pentaacetic acid (PEPA), and Macropa (Thiele et al., An Eighteen-Membered Macrocyclic Ligand for Actinium-225 Targeted Alpha Therapy. Angew Chem Int Ed Engl. 2017 Nov). Examples include 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrapropionic acid (DOTPA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetrapropionic acid (TETPA), and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetramethylenephosphonic acid (DOTMP). According to a preferred embodiment, the chelating agent is of formula (I):

[0051] [ka] Structure (In the formula, R1, R2, R3, and R4 are each independently CHQCO2X, Q is independently hydrogen, C1-C4 alkyl, or (C1-C2 alkyl)phenyl. X is independently hydrogen, benzyl, or a C1-C4 alkyl group. Z is (CH2) n Y is, n is between 1 and 10. Y is an electrophilic or nucleophilic moiety covalently bonded to the second click reaction partner. Alternatively, Z is hydrogen; and R1, R2, R3, and R4 are each independently CHQCO2X. Q is independently hydrogen, C1-C4 alkyl, or (C1-C2 alkyl)phenyl. X is independently an electrophilic or nucleophilic moiety covalently bonded to hydrogen, benzyl, a C1-C4 alkyl group, or a second click reaction partner. Includes.

[0052] According to a preferred embodiment, the chelated portion is formula (II):

[0053] [ka] It includes the structure.

[0054] According to a preferred embodiment, the chelated portion is formula (III):

[0055] [ka] Includes.

[0056] In one embodiment, the present invention relates to a method for labeling polypeptides with two or more radioactive metal ions using the method of the present invention. For example, a method for labeling polypeptides with two radioactive metal ions is: a. To provide a modified polypeptide comprising a polypeptide covalently bonded to a first click reaction partner and a second click reaction partner, b. To provide a first radioactive complex comprising a first radioactive metal ion associated with the chelate moiety, wherein the chelate moiety comprises a chelating agent covalently bonded to a third click reaction partner. c. To provide a second radioactive complex containing a second radioactive metal ion associated with the chelate moiety, wherein the chelate moiety contains a chelating agent covalently bonded to a fourth click reaction partner. d. The modified polypeptide is brought into contact with first and second radioactive complexes under conditions that enable the polypeptide to be labeled with first and second radioactive metal ions by the reaction of a first click reaction partner with a third click reaction partner and a second click reaction partner with a fourth click reaction partner.

[0057] In a preferred embodiment, one of the first and second click reaction partners comprises an alkyne group, the other of the first and second click reaction partners comprises an azide, one of the third and fourth click reaction partners comprises an alkene group, and the other of the third and fourth click reaction partners comprises a diene.

[0058] In a preferred embodiment, one or the second radioactive metal ion is a diagnostic emitter, and the other is a therapeutic emitter. In a preferred embodiment, both the first and second radioactive metal ions are therapeutic emitters.

[0059] Conditions for carrying out click chemistry reactions are known in the art, and any conditions for carrying out click chemistry reactions known to those skilled in the art in consideration of this disclosure can be used in the present invention. Examples of conditions, but not limited to, include incubating the modified polypeptide and the radioactive complex in a ratio of 1:1 to 1000:1 at a pH of 4 to 10 and a temperature of 20°C to 70°C.

[0060] The products of the click radiolabeling method of the present invention can be analyzed using methods known to those skilled in the art in consideration of this disclosure. For example, the ratio of chelator to labeled polypeptide can be determined using LC / MS analysis, the oligomeric state of polypeptides and polypeptide complexes can be determined using analytical size exclusion chromatography, the radiochemical yield can be determined by simple thin-layer chromatography (e.g., iTLC-SG), and the radiochemical purity can be measured by size exclusion HPLC. Exemplary methods are described herein, for example, in the following examples.

[0061] Pharmaceutical composition and therapeutic method The click radiolabeling method of the present invention may be modified into a pretargeting approach (Kraeber-Bodere, F., et al., A pretargeting system for tumor PET imaging and radioimmunotherapy. Front Pharmacol, 2015.6: p.54). First, an azide mAb is administered to bind to target cells and is removed from circulation over time or removed with a scavenging agent. Subsequently, a radioactive complex is administered and undergoes a SPAAC reaction with the azide mAbs bound to the target site, after which the remaining unbound radioactive complex is rapidly removed from circulation (Deal, KA, et al., Improved in vivo stability of actinium-225 macrocyclic complexes. J Med Chem, 1999.42(15): p.2988-92). This pretargeting technique provides a method for enhancing the localization of radioactive metal ions at the target site.

[0062] Therefore, in another general embodiment, the present invention relates to a pharmaceutical composition comprising a radiolabeled polypeptide prepared by the method of the present invention and a pharmaceutically acceptable carrier.

[0063] As used herein, the term “carrier” refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposome encapsulation, or other material known in the art for use in pharmaceutical formulations. It will be understood that the properties of a carrier, excipient, or diluent are determined by the route of administration for a particular application. As used herein, the term “pharmaceutically acceptable carrier” refers to a non-toxic material that does not interfere with the effect of the composition according to the present invention or the biological activity of the composition according to the present invention. Depending on the particular embodiment, any pharmaceutically acceptable carrier suitable for use in antibody-based or radiocomplex-based pharmaceutical compositions may be used in the present invention in consideration of this disclosure.

[0064] In specific embodiments, the compositions described herein are formulated to suit the intended route of administration to the subject. For example, the compositions described herein can be formulated to suit intravenous, subcutaneous, intramuscular, or intratumor administration.

[0065] According to certain embodiments, the modified polypeptide and the radioactive complex can be administered in the same or different compositions.

[0066] In another general embodiment, the present invention relates to a method for treating a neoplastic disease or disorder in a subject requiring treatment of a neoplastic disease or disorder, the method comprising administering the subject a pharmaceutical composition of the present invention.

[0067] According to a particular embodiment, the method of the present invention comprises administering a therapeutically effective dose of the pharmaceutical composition of the present invention, wherein the composition comprises a radiolabeled polypeptide for targeting cells associated with a neoplastic disease or disorder, and upon targeting, 225 Alpha particles from Ac and its daughter nuclides are delivered to target cells, causing cytotoxic effects on the target cells, thereby treating neoplastic diseases or disorders.

[0068] According to certain embodiments, therapeutically effective amounts of modified polypeptides and radioactive complexes are administered in different compositions.

[0069] As used herein, the term “therapeutic effective dose” refers to the amount of the active ingredient or component that elicits the desired biological or pharmacological response in a subject. The therapeutic effective dose can be determined by empirical and general methods for the purpose described. For example, in vitro assays may be used, if desired, to help identify the optimal dose range. The selection of a specific effective dose can be determined by a person skilled in the art (e.g., by clinical trials) based on consideration of several factors, including the disease being treated or prevented, associated symptoms, the patient’s weight, the patient’s immune status, and other factors known to a person skilled in the art. Furthermore, the exact dose used in a formulation should be determined according to the route of administration and the severity of the disease, and should be determined according to the physician’s judgment and the circumstances of each patient. The effective dose can be estimated from dose-response curves derived from in vitro or animal model test systems.

[0070] As used herein, the terms “treat,” “treating,” and “treatment” are intended to mean the improvement or restoration of at least one measurable physical parameter associated with a disease, disorder, or condition in which the administration of radioactive metal ions may be beneficial, such as a neoplastic disease or disorder, which may not necessarily be recognizable in the subject, but may be recognizable in the subject. The terms “treat,” “treating,” and “treatment” may also mean causing regression of a disease, disorder, or condition, preventing its progression, or at least slowing its progression. In certain embodiments, “treat,” “treating,” and “treatment” mean the alleviation, prevention of progression or onset, or reduction of the duration of one or more symptoms associated with a disease, disorder, or condition in which the administration of radioactive metal ions may be beneficial, such as a neoplastic disease or disorder. In specific embodiments, "to treat," "to treat," and "treatment" refer to preventing the recurrence of a disease, disability, or condition. In specific embodiments, "to treat," "to treat," and "treatment" refer to improving the survival rate of a subject with a disease, disability, or condition. In specific embodiments, "to treat," "to treat," and "treatment" refer to the disappearance of a disease, disability, or condition in a subject.

[0071] Examples of neoplastic diseases or disorders include, but are not limited to, disseminated carcinoma, solid tumor carcinoma, hypertrophy, coronary artery disease or vascular occlusive disease, diseases or disorders associated with infected cells, microorganisms or viruses, or diseases or disorders associated with inflammatory cells such as rheumatoid arthritis (RA).

[0072] As used herein, the term “subject” refers to an animal, preferably a mammal. In specific embodiments, the subject is a mammal, including non-primates (e.g., camels, donkeys, zebras, cattle, pigs, horses, goats, sheep, cats, dogs, rats, rabbits, guinea pigs, marmosets, or mice) or primates (e.g., monkeys, chimpanzees, or humans). In specific embodiments, the subject is a human.

[0073] Any dosing schedule for the modified polypeptide and the radiocomplex can be used in consideration of this disclosure. Generally, if the modified polypeptide and the radiocomplex are administered in different compositions, the radiocomplex can be administered at any time after the modified antibody has been administered.

[0074] According to certain embodiments, compositions used in the treatment of neoplastic diseases or disorders can be used in combination with other active ingredients effective in treating the related neoplastic diseases or disorders.

[0075] As used herein, the term “combined use” refers to the use of multiple therapeutic agents in relation to the administration of two or more therapeutic agents to a subject. The use of the term “combined use” does not limit the order in which the treatments are administered to the subject. For example, a first therapeutic agent (e.g., a composition described herein) may be administered to a subject before (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks prior), simultaneously with, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks prior) the administration of a second therapeutic agent to the subject.

[0076] In another general embodiment, the present invention relates to a theranostic agent comprising a radiolabeled antibody prepared by the method of the present invention and a pharmaceutically acceptable carrier, wherein the immunological properties of the radiolabeled antibody are preserved.

[0077] As used herein, the term "theranostic" refers to the ability to provide either a diagnostic or therapeutic function. In one embodiment, a theranostic agent provides both diagnostic and therapeutic functions. In another embodiment, a theranostic agent is an active agent without a diagnostic function. In yet another embodiment, a theranostic agent is an agent useful for diagnosis but without a therapeutic function.

[0078] According to a preferred embodiment, the radioactive metal ion is used in a diagnostic emitter, preferably 89 It is Zr. According to another preferred embodiment, the radioactive metal ion is a therapeutic emitter, preferably 225 Ac is used. According to a preferred embodiment, the theranostic agent is used to provide both diagnostic and therapeutic functions to a target that requires it.

[0079] Combinations and kits This specification provides combinations including the following: a. A modified polypeptide comprising a polypeptide covalently bonded to a first click reaction partner, b. A combination or kit comprising a radioactive complex containing a radioactive metal ion associated with a chelate moiety, wherein the chelate moiety contains a chelating agent covalently bonded to a second click reaction partner, Here, the combination is used to label the polypeptide with a radioactive metal ion.

[0080] According to certain embodiments, the combination of the present invention is a reaction mixture used to label polypeptides with radioactive metal ions. According to other embodiments, the combination is a pack or kit used to produce radiolabeled polypeptides in vitro or in vivo. Optionally, the combination may be accompanied by a cautionary note or instructions in a format specified by a government agency regulating the manufacture, use, or sale of a drug or biological product, which reflects the agency's authorization for manufacture, use, or sale for human administration. The combinations incorporated herein can be used in methods for labeling the above-mentioned polypeptides with radioactive metal ions, or in methods for treating neoplastic diseases or disorders in subjects requiring treatment of neoplastic diseases or disorders.

[0081] Embodiment The present invention also provides the following non-limiting embodiments.

[0082] Embodiment 1 is a method for labeling polypeptides with radioactive metal ions, a. To provide a modified polypeptide comprising a polypeptide covalently bonded to a first click reaction partner, b. To provide a radioactive complex containing a radioactive metal ion associated with the chelate moiety, wherein the chelate moiety contains a chelating agent covalently bonded to a second click reaction partner. c. The modified polypeptide is brought into contact with a radioactive complex under conditions that allow the polypeptide to be labeled with a radioactive metal ion by the reaction of a first click reaction partner with a second click reaction partner.

[0083] Embodiment 1a is the method according to Embodiment 1, wherein the chelating agent contains a macrocyclic molecule.

[0084] Embodiment 1b is the method according to Embodiment 1, wherein the chelating agent comprises an open-chain ligand.

[0085] Embodiment 2 is the method of Embodiment 1, wherein one of the first and second click reaction partners comprises an alkyne group and the other click reaction partner comprises an azide.

[0086] Embodiment 3 is the method of Embodiment 2, wherein the first click reaction partner comprises an azide group and the second click reaction partner comprises an alkyne group.

[0087] Embodiment 3a is the method according to Embodiment 2 or 3, wherein the alkyne group includes a terminal alkyne.

[0088] Embodiment 3b is the method according to Embodiment 2 or 3, wherein the alkyne group comprises a cyclic alkyne, preferably cyclooctin or a cyclooctin derivative.

[0089] Embodiment 3c is the method according to Embodiment 3b, wherein the alkyne group comprises bicyclononyne (BCN).

[0090] Embodiment 3d is the method according to Embodiment 3b, wherein the alkyne group comprises difluorinated cyclooctyne (DIFO).

[0091] Embodiment 3e is the method according to Embodiment 3b, wherein the alkyne group comprises dibenzocyclooctin (DIBO).

[0092] Embodiment 3f is the method according to Embodiment 3b, wherein the alkyne group comprises biarylazacyclooctyne (BARAC).

[0093] Embodiment 3g is the method according to Embodiment 3b, wherein the alkyne group comprises dibenzoazacyclooctin (DIBAC).

[0094] Embodiment 3h is the method according to Embodiment 3b, wherein the alkyne group comprises dimethoxyazacyclooctyne (DIMAC).

[0095] Embodiment 3i is the method according to Embodiment 3b, wherein the alkyne group comprises dibenzocyclooctyne (DBCO).

[0096] Embodiment 3j is the method according to Embodiment 3b, wherein the alkyne group comprises difluorobenzocyclooctin (DIFBO).

[0097] Embodiment 3k is the method according to Embodiment 3b, wherein the alkyne group comprises monobenzocyclooctin (MOBO).

[0098] Embodiment 3l is the method according to Embodiment 3b, wherein the alkyne group comprises tetramethoxyDIBO (TMDIBO).

[0099] Embodiment 3m is a method according to any one of Embodiments 2 to 3l, wherein the azide group comprises an NHS-azide.

[0100] Embodiment 4 is the method according to Embodiment 1, wherein one of the first and second click reaction partners contains an alkene group and the other click reaction partner contains a diene.

[0101] Embodiment 4a is the method according to Embodiment 4, wherein the diene contains a tetrazine or tetrazole group.

[0102] Embodiment 4b is the method according to Embodiment 4 or 4a, wherein the alkene group comprises norbornene.

[0103] Embodiment 4c is the method according to Embodiment 4 or 4a, wherein the alkene group comprises trans-cyclooctene (TCO).

[0104] Embodiment 5 is the method according to any one of Embodiments 1 to 4c, wherein the polypeptide is an antibody or an antigen-binding fragment thereof.

[0105] Embodiment 6 is the method of Embodiment 5, wherein the antibody is a monoclonal antibody or an antigen-binding fragment thereof.

[0106] Embodiment 6a is a method according to any one of Embodiments 1 to 6, wherein the modified polypeptide is obtained by randomly compounding one or more azide groups into the polypeptide.

[0107] Embodiment 6b is the method according to any one of Embodiments 1 to 6, wherein the modified polypeptide is a modified antibody or its antigen-binding fragment obtained by site-specific incorporation of a first click reaction partner.

[0108] Embodiment 6c is the method of Embodiment 6b, wherein a modified antibody or its antigen-binding fragment is trimmed with a bacterial endoglycosidase specific to the β-1,4 linkage between core GlcNac residues (which may be multiple) of the Fc-glycosylation site of the antibody to obtain a trimmed antibody or its antigen-binding fragment, and the trimmed antibody or its antigen-binding fragment is reacted with an azide sugar, preferably a UDP-GalNaz azide sugar substrate, in the presence of a glycosyltransferase, preferably a GalT galactosyltransferase.

[0109] Embodiment 6d is the method of Embodiment 6b, wherein a modified antibody or its antigen-binding fragment is deglycosylated with amidase to obtain a deglycosylated antibody or its antigen-binding fragment, and the deglycosylated antibody or its antigen-binding fragment is reacted with an azidoamine, preferably 3-azidopropylamine, in the presence of microbial transglutaminase.

[0110] Embodiment 6e is an antibody that binds to human prostate-specific membrane antigen (PSMA) or its antigen-binding fragment, and preferably the antibody comprises the HC CDR1 sequence of SEQ ID NO: 3, the HC CDR2 sequence of SEQ ID NO: 4, the HC CDR3 sequence of SEQ ID NO: 5, the light chain (LC) CDR1 sequence of SEQ ID NO: 6, the LC CDR2 sequence of SEQ ID NO: 7, and the LC CDR3 sequence of SEQ ID NO: 8, according to any one of Embodiments 6 to 6d.

[0111] Embodiment 6f is the method of Embodiment 6e, wherein the antibody comprises the HC sequence of SEQ ID NO: 9 and the LC sequence of SEQ ID NO: 10.

[0112] Embodiment 7 describes a scenario in which radioactive metal ions are present. 32 P, 47 Sc, 67 Cu, 77 As, 89 Sr, 90 Y, 99 Tc, 105 Rh, 109 Pd, 111 Ag, 131 I, 153 Sm, 159 Gd, 165 Dy, 166 Ho, 169 Er, 177 Lu, 186 Re, 188 Re, 194 Ir, 198 Au, 199 Au, 211 At, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 225 Ac, 255 Fm, 227 Th, 62 Cu, 64 Cu, 67 Ga, 68 Ga, 86 Y, 89 Zr, or 111 The method is described in any one of embodiments 1 to 6d.

[0113] Embodiment 7a is a radioactive metal ion 225 This is a method according to any one of embodiments 1 to 6d, wherein the material is Ac.

[0114] Embodiment 7b is a radioactive metal ion 111 The method is described in any one of embodiments 1 to 6d.

[0115] Embodiment 7c is a radioactive metal ion 89 This is a method according to any one of embodiments 1 to 6d, wherein the value is Zr.

[0116] Embodiment 8 is a method according to any one of Embodiments 1 to 7c, wherein the chelated portion is covalently bonded to a second click reaction partner via a linker.

[0117] Embodiment 9 is the method according to any one of Embodiments 1 to 8, further comprising reacting a sulfhydryl group covalently bonded to a first click reaction partner with an electrophile on the side chain, preferably an amino side chain of lysine on or introduced into the polypeptide, to obtain a modified polypeptide, preferably an NHS-azide.

[0118] Embodiment 10 is a method according to any one of Embodiments 1 to 9, wherein the modified polypeptide comprises a polypeptide in which an azide, tetrazine, or tetrazole group is covalently bonded directly or via a linker.

[0119] Embodiment 11 is a chelating agent that is a macrocyclic molecule, preferably of formula (I):

[0120] [ka] Structure (In the formula, R1, R2, R3, and R4 are each independently CHQCO2X, Q is independently hydrogen, C1-C4 alkyl, or (C1-C2 alkyl)phenyl. X is independently hydrogen, benzyl, or a C1-C4 alkyl group. Z is (CH2) n Y, n is from 1 to 10, Y is an electrophilic or nucleophilic moiety covalently attached to a second click reaction partner, alternatively, Z is hydrogen; and R1, R2, R3 and R4 are each independently CHQCO2X, Q is independently hydrogen, C1-C4 alkyl or (C1-C2 alkyl)phenyl, X is independently hydrogen, benzyl, C1-C4 alkyl, or an electrophilic or nucleophilic moiety covalently attached to a second click reaction partner) is a method according to any one of embodiments 1 to 10.

[0121] Embodiment 12 is a method according to any one of embodiments 1 to 11, wherein the chelating moiety has the formula (II):

[0122]

Chemical formula

[0123] Embodiment 12a is a method according to any one of embodiments 1 to 11, wherein the chelating moiety comprises a chelating agent having an open-chain ligand, preferably, the chelating moiety has the formula (III):

[0124]

Chemical formula

[0125] Embodiment 12b is the method according to any one of Embodiments 1 to 10, comprising a chelating agent selected from the group consisting of 1,4,7,10-tetraazacyclododecane-N,N’,N’’,N’’’-tetraacetic acid (DOTA), deferoxamine (DFO), 1,4,7,10,13,16-hexaazacyclohexadecane-N,N’,N’’,N’’’,N’’’’,N’’’’’-hexaacetic acid (HEHA), 1,4,7,10,13-pentaazacyclopentadecane-N,N’,N’’,N’’’,N’’’’-pentaacetic acid (PEPA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), Macropa (Thiele et al., An Eighteen-Membered Macrocyclic Ligand for Actinium-225 Targeted Alpha Therapy. Angew Chem Int Ed Engl. 2017 Nov 13;56(46):p.14712-14717), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrapropionic acid (DOTPA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetrapropionic acid (TETPA), and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetramethylenephosphonic acid (DOTMP).

[0126] Embodiment 12c is the method according to Embodiment 12b, wherein the chelating agent comprises 1,4,7,10-tetraazacyclododecane-N,N’,N’’,N’’’-tetraacetic acid (DOTA).

[0127] Embodiment 12d is the method according to Embodiment 12b, wherein the chelating agent comprises deferoxamine (DFO).

[0128] Embodiment 13 is a radioactive metal ion, preferably 225 Ac,[[ID=##]] 111 In or 89A method for labeling a polypeptide, preferably an antibody or its antigen-binding fragment, using Zr, a. To provide a modified polypeptide, preferably a modified antibody or its antigen-binding fragment, comprising a polypeptide covalently bonded to an azide, tetrazine, or tetrazole group, or an antibody or its antigen-binding fragment. b. Radioactive metal ions, preferably associated with the chelated moiety. 225 Ac, 111 In or 89 To provide a radioactive complex containing Zr, wherein the chelating portion comprises a chelating agent covalently bonded to an alkyne or alkene group, and c. The azide, tetrazine, or tetrazole group reacts with an alkyne or alkene group to bind a polypeptide, antibody, or its antigen-binding fragment to a radioactive metal ion, preferably. 225 Ac, 111 In, or 89 This includes contacting a modified polypeptide or antibody or its antigen-binding fragment with a radioactive complex under conditions that enable labeling with Zr, The chelating agent is given by formula (I):

[0129] [ka] (In the formula, R1, R2, R3, and R4 are each independently CHQCO2X, Q is independently hydrogen, C1-C4 alkyl, or (C1-C2 alkyl)phenyl. X is independently hydrogen, benzyl, or a C1-C4 alkyl group. Z is (CH2) n Y is, n is between 1 and 10. Y is an electrophilic or nucleophilic moiety covalently bonded to the alkyne group. Alternatively, Z is hydrogen; and R1, R2, R3, and R4 are each independently CHQCO2X. Q is independently hydrogen, C1-C4 alkyl, or (C1-C2 alkyl)phenyl. X is independently an electrophilic or nucleophilic moiety covalently bonded to hydrogen, benzyl, C1-C4 alkyl, or alkyne group. This method includes the structure of [the object].

[0130] Embodiment 13a is the method according to Embodiment 13, wherein the chelating agent comprises 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA).

[0131] Embodiment 13b has a chelate portion of formula (II):

[0132] [ka] This is the method according to Embodiment 13, which includes the structure of [the specified element].

[0133] Embodiment 13d contains radioactive metal ions, preferably 225 Ac, 111 In or 89 A method for labeling a polypeptide, preferably an antibody or its antigen-binding fragment, using Zr, a. To provide a modified polypeptide, preferably a modified antibody or its antigen-binding fragment, comprising a polypeptide covalently bonded to an azide, tetrazine, or tetrazole group, or an antibody or its antigen-binding fragment. b. Radioactive metal ions, preferably associated with the chelated moiety. 225 Ac, 111 In or 89 To provide a radioactive complex containing Zr, wherein the chelating portion comprises a chelating agent covalently bonded to an alkyne or alkene group, and c. The azide, tetrazine, or tetrazole group reacts with an alkyne or alkene group to bind a polypeptide, antibody, or its antigen-binding fragment to a radioactive metal ion, preferably. 225 Ac, 111 In, or 89Contacting a modified polypeptide or antibody or an antigen-binding fragment thereof with a radioactive complex under conditions that allow labeling with Zr, wherein the chelating agent comprises an open-chain ligand, preferably desferrioxamine (DFO), is a method.

[0134] Embodiment 14 is the method according to any one of Embodiments 13 to 13C, wherein the chelating agent is covalently bonded to an alkyne or alkene group via a linker.

[0135] Embodiment 15 further comprises reacting a sulfhydryl group covalently bonded to an azide, preferably NHS-azide, with an electrophile on the side chain, preferably the amino side chain of lysine introduced onto a polypeptide or onto a polypeptide, preferably an antibody or an antigen-binding fragment thereof, to obtain a modified polypeptide, or an antibody or an antigen-binding fragment thereof, the method according to any one of Embodiments 13 to 14.

[0136] Embodiment 16 is the method according to any one of Embodiments 13 to 15, wherein the polypeptide, preferably an antibody or an antigen-binding fragment thereof, is covalently bonded to an azide via a linker.

[0137] Embodiment 17 is that the polypeptide is an antibody or an antigen-binding fragment thereof, and the radioactive metal ion is 225 Ac, 111 In or 89 Zr, and the chelating moiety has the formula (II):

[0138]

Chemical formula

[0139] Embodiment 17a is that the polypeptide is an antibody or an antigen-binding fragment thereof, and the radioactive metal ion is 225 Ac, 111 In or 89 Zr, and the chelating moiety has the formula (III):

[0140] [ka] This is the method according to Embodiment 13c, which includes the structure of [the specified element].

[0141] Embodiment 17b is the method according to any one of Embodiments 13 to 17a, wherein the polypeptide is an antibody that binds to human prostate-specific membrane antigen (PSMA) or its antigen-binding fragment, and preferably the antibody comprises the HC CDR1 sequence of SEQ ID NO: 3, the HC CDR2 sequence of SEQ ID NO: 4, the HC CDR3 sequence of SEQ ID NO: 5, the light chain (LC) CDR1 sequence of SEQ ID NO: 6, the LC CDR2 sequence of SEQ ID NO: 7, and the LC CDR3 sequence of SEQ ID NO: 8.

[0142] Embodiment 17c is the method according to Embodiment 17b, wherein the antibody comprises the HC sequence of SEQ ID NO: 9 and the LC sequence of SEQ ID NO: 10.

[0143] Embodiment 18 is a method for doubly labeling a polypeptide with two radioactive metal ions, a. To provide a modified polypeptide comprising a polypeptide covalently bonded to a first click reaction partner and a second click reaction partner, b. To provide a first radioactive complex comprising a first radioactive metal ion associated with the chelate moiety, wherein the chelate moiety comprises a chelating agent covalently bonded to a third click reaction partner. c. To provide a second radioactive complex containing a second radioactive metal ion associated with the chelate moiety, wherein the chelate moiety contains a chelating agent covalently bonded to a fourth click reaction partner. d. The modified polypeptide is brought into contact with first and second radioactive complexes under conditions that enable the polypeptide to be labeled with first and second radioactive metal ions by the reaction of a first click reaction partner with a third click reaction partner and a second click reaction partner with a fourth click reaction partner.

[0144] Embodiment 19 is the method according to Embodiment 18, wherein one of the first and second click reaction partners comprises an alkyne group, the other of the first and second click reaction partners comprises an azide, one of the third and fourth click reaction partners comprises an alkene group, and the other of the third and fourth click reaction partners comprises a diene.

[0145] Embodiment 20 is the method according to Embodiment 18 or 19, wherein the first or second radioactive metal ion is a diagnostic emitter and the other is a therapeutic emitter.

[0146] Embodiment 21 is the method according to Embodiment 18 or 19, wherein both the first and second radioactive metal ions are therapeutic emitters.

[0147] Embodiment 21a is a diagnostic emitter, 62 Cu, 64 Cu, 67 Ga, 68 Ga, 86 Y, 89 Zr, or 111 This is the method according to embodiment 20 or 21.

[0148] Embodiment 21b is a therapeutic emitter, 32 P, 47 Sc, 67 Cu, 77 As, 89 Sr, 90 Y, 99 Tc, 105 Rh, 109 Pd, 111 Ag, 131 I, 153 Sm, 159 Gd, 165 Dy, 166 Ho, 169 Er, 177 Lu, 186 Re, 188 Re, 194 Ir, 198 Au, 199 Au, 211 At, 212 Pb, 212Bi, 213 Bi, 223 Ra, 225 Ac, 255 Fm, or 227 This is the method according to any one of embodiments 20 to 21a, wherein Th is the method.

[0149] Embodiment 22 is a pharmaceutical composition comprising a radiolabeled polypeptide prepared by any one of Embodiments 1 to 21b and a pharmaceutically acceptable carrier.

[0150] Embodiment 23 is a method for treating or diagnosing a disease or disorder, particularly a neoplastic disease or disorder, in a subject requiring treatment or diagnosis of such a disease or disorder, comprising administering the composition of Embodiment 22 to the subject.

[0151] Embodiment 24 is the method according to Embodiment 23, wherein the pharmaceutical composition comprises two compositions administered sequentially, the first composition comprising a modified polypeptide and the second comprising a radioactive complex (which may be more than one).

[0152] Embodiment 25 is a theranostic agent comprising a radiolabeled antibody prepared by the method of any one of Embodiments 1 to 21b and a pharmaceutically acceptable carrier, wherein the immunological properties of the radiolabeled antibody are preserved.

[0153] Embodiment 26 is a diagnostic emitter in which radioactive metal ions are preferably 89 The theranostic agent described in Embodiment 25 is Zr.

[0154] Embodiment 27 is a radioactive metal ion that is used in a therapeutic emitter, preferably 225 Ac is the theranostic agent described in Embodiment 25.

[0155] Embodiment 27a is a radioactive metal ion 111 In is the theranostic agent described in Embodiment 25.

[0156] Embodiment 27b is a theranostic agent according to any one of Embodiments 25 to 27a, wherein the polypeptide is an antibody that binds to human prostate-specific membrane antigen (PSMA) or its antigen-binding fragment, and preferably the antibody comprises the HC CDR1 sequence of SEQ ID NO: 3, the HC CDR2 sequence of SEQ ID NO: 4, the HC CDR3 sequence of SEQ ID NO: 5, the light chain (LC) CDR1 sequence of SEQ ID NO: 6, the LC CDR2 sequence of SEQ ID NO: 7, and the LC CDR3 sequence of SEQ ID NO: 8.

[0157] Embodiment 27c is the method according to Embodiment 27b, wherein the theranostic agent comprises the HC sequence of SEQ ID NO: 9 and the LC sequence of SEQ ID NO: 10.

[0158] Embodiment 27d is a radiolabeled antibody of formula (IV):

[0159] [ka] It has the formula (IV) (DOTA-Ac-DBCO-protein), or, 225 Ac is 32 P, 47 Sc, 67 Cu, 77 As, 89 Sr, 90 Y, 99 Tc, 105 Rh, 109 Pd, 111 Ag, 131 I, 153 Sm, 159 Gd, 165 Dy, 166 Ho, 169 Er, 177 Lu, 186 Re, 188 Re, 194 Ir, 198 Au, 199 Au, 211 At, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 255 Fm, 227 Th,62 Cu, 64 Cu, 67 Ga, 68 Ga, 86 Y, 89 Zr, or 111 The theranostic agent according to any one of embodiments 25 to 27c, which is substituted with another radioactive metal ion such as In.

[0160] Embodiment 27e is a radiolabeled antibody of formula (V):

[0161] [ka] Formula (V) (DOTA-In-DBCO protein) The theranostic agent according to any one of embodiments 25 to 27c, having the formula.

[0162] Embodiment 27f is a radiolabeled antibody of formula (VI):

[0163] [ka] It has the formula (VI) (DFO-Zr DBCO-protein), or, 89 Zr, 32 P, 47 Sc, 67 Cu, 77 As, 89 Sr, 90 Y, 99 Tc, 105 Rh, 109 Pd, 111 Ag, 131 I, 153 Sm, 159 Gd, 165 Dy, 166 Ho, 169 Er, 177 Lu, 186 Re, 188 Re, 194 Ir, 198 Au, 199 Au, 211 At, 212 Pb, 212Bi, 213 Bi, 223 Ra, 225 Ac, 255 Fm, 227 Th, 62 Cu, 64 Cu, 67 Ga, 68 Ga, 86 Y, or 111 The theranostic agent according to any one of embodiments 25 to 27c, which is substituted with another radioactive metal ion such as In.

[0164] Embodiment 27g is a radiolabeled antibody of formula (VII):

[0165] [ka] Formula (VII) (DOTA-Zr-DBCO-protein) The theranostic agent according to any one of embodiments 25 to 27c, having the formula.

[0166] Embodiment 27h is a theranostic agent according to any one of Embodiments 25 to 27g, wherein the radiolabeled antibody has a chelator:antibody ratio (CAR) of less than 3.

[0167] Embodiment 27i is a theranostic agent according to any one of Embodiments 25 to 27h, wherein the radiolabeled antibody has a chelator:antibody ratio (CAR) of 2.

[0168] Embodiment 28 is, a. A modified polypeptide comprising a polypeptide covalently bonded to a first click reaction partner, b. A combination or preferably a kit comprising a radioactive complex containing a radioactive metal ion associated with a chelate moiety, wherein the chelate moiety contains a chelating agent covalently bonded to a second click reaction partner, Here, the combination is used to label the polypeptide with a radioactive metal ion.

[0169] Embodiment 28a is a combination or kit of Embodiment 28, wherein the chelating agent includes a macrocyclic molecule.

[0170] Embodiment 28b is the combination described in Embodiment 28, wherein the chelating agent includes an open-chain ligand.

[0171] Embodiment 29 is the combination or kit described in Embodiment 28, used for labeling polypeptides with radioactive metal ions via a reaction between first and second click reaction partners in vitro.

[0172] Embodiment 30 is the combination or kit described in Embodiment 28, used for labeling polypeptides with radioactive metal ions in vivo via a reaction between first and second click reaction partners.

[0173] Embodiment 31 is a composition comprising a modified polypeptide including a polypeptide covalently bonded to a first click reaction partner.

[0174] Embodiment 32 is a composition comprising a radioactive complex containing a radioactive metal ion associated with a chelated moiety, wherein the chelated moiety comprises a chelating agent covalently bonded to a second click reaction partner.

[0175] Embodiment 32a is the composition according to Embodiment 32, wherein the chelating agent comprises a macrocyclic molecule.

[0176] Embodiment 32b is the composition according to Embodiment 32, wherein the chelating agent comprises an open-chain ligand.

[0177] Embodiment 33 is a combination or kit according to any one of Embodiments 28 to 30, or a composition according to Embodiment 31 or 32, wherein the polypeptide is an antibody or its antigen-binding fragment.

[0178] Embodiment 33a is the method according to Embodiment 33, wherein the antibody can bind to human prostate-specific membrane antigen (PSMA) or its antigen-binding fragment, and preferably the antibody comprises the HC CDR1 sequence of SEQ ID NO: 3, the HC CDR2 sequence of SEQ ID NO: 4, the HC CDR3 sequence of SEQ ID NO: 5, the light chain (LC) CDR1 sequence of SEQ ID NO: 6, the LC CDR2 sequence of SEQ ID NO: 7, and the LC CDR3 sequence of SEQ ID NO: 8.

[0179] Embodiment 33b is the combination, kit, or composition described in Embodiment 33a, wherein the antibody comprises the HC sequence of SEQ ID NO: 9 and the LC sequence of SEQ ID NO: 10.

[0180] Embodiment 33c is a combination, kit, or composition according to Embodiment 33a or 33b, wherein the antibody or its antigen-binding fragment is covalently bound to an azide group.

[0181] Embodiment 33d is a combination, kit, or composition of Embodiment 33c in which an antibody or its antigen-binding fragment is randomly covalently bound to an azide group.

[0182] Embodiment 33e is a combination, kit, or composition of Embodiment 33c in which an antibody or its antigen-binding fragment is site-specifically covalently bound to an azide group.

[0183] Embodiment 33f is a combination, kit, or composition according to Embodiment 33e, wherein the antibody or its antigen-binding fragment is covalently bound to an azide group via a method comprising: trimming the antibody or its antigen-binding fragment with a bacterial endoglycosidase specific to the β-1,4 linkage between core GlcNac residues (which may be more than one) of the Fc-glycosylation site of the antibody to obtain a trimmed antibody or its antigen-binding fragment; and reacting the trimmed antibody or its antigen-binding fragment with an azide sugar, preferably a UDP-GalNaz azide sugar substrate, in the presence of a glycosyltransferase, preferably a GalT galactosyltransferase.

[0184] Embodiment 33g is a combination, kit, or composition according to Embodiment 33e, obtained by a method comprising deglycosylating a modified antibody or its antigen-binding fragment with amidase to obtain a deglycosylated antibody or its antigen-binding fragment, and reacting the deglycosylated antibody or its antigen-binding fragment with an azidoamine, preferably 3-azidopropylamine, in the presence of a microbial transglutaminase.

[0185] Embodiment 34 is a radioactive metal ion, 225 Ac, 111 In or 89 A combination, kit, or composition containing Zr, as described in any one of embodiments 33 to 33g.

[0186] Embodiment 35 is a chelating agent that is a macrocyclic molecule, preferably of formula (I):

[0187] [ka] Structure (In the formula, R1, R2, R3, and R4 are each independently CHQCO2X, Q is independently hydrogen, C1-C4 alkyl, or (C1-C2 alkyl)phenyl. X is independently hydrogen, benzyl, or a C1-C4 alkyl group. Z is (CH2) n Y is, n is between 1 and 10. Y is an electrophilic or nucleophilic moiety covalently bonded to the alkyne group. Alternatively, Z is hydrogen; and R1, R2, R3, and R4 are each independently CHQCO2X. Q is independently hydrogen, C1-C4 alkyl, or (C1-C2 alkyl)phenyl. X is independently an electrophilic or nucleophilic moiety covalently bonded to hydrogen, benzyl, C1-C4 alkyl, or alkyne group. This is a combination, kit, or composition as described in Embodiment 34, including the above.

[0188] Embodiment 36 is the combination, kit, or composition described in Embodiment 35, wherein the electrophilic or nucleophilic moiety is covalently bonded to the alkyne group via a linker.

[0189] Embodiment 37 has a chelate portion of formula (II):

[0190] [ka] The combination, kit, or composition described in Embodiment 35 or 36 includes the structure of [the specified element].

[0191] Embodiment 37a comprises a chelating agent having an open-chain ligand, preferably the chelating portion being of formula (III):

[0192] [ka] The combination, kit, or composition described in Embodiment 34 includes the structure of [the specified element].

[0193] Embodiment 38 is a combination, kit, or composition according to any one of Embodiments 34 to 37, wherein the polypeptide is covalently bonded to the azide via a linker.

[0194] Embodiment 39 is a method for treating or diagnosing a disease or disorder, particularly a neoplastic disease or disorder, comprising administering a theranostic agent described in any one of Embodiments 25 to 27d, or a combination described in any one of Embodiments 28 and 33 to 38, to the target.

[0195] Embodiment 40 is a method for treating or diagnosing a disease or disorder, particularly a neoplastic disease or disorder, in a subject requiring treatment or diagnosis of the disease or disorder, particularly a neoplastic disease or disorder, comprising administering the composition of Embodiment 31 and the composition of Embodiment 32 to the subject, preferably wherein the polypeptide is an antibody. [Examples]

[0196] The following embodiments of the present invention are intended to further illustrate the essence of the invention. It should be understood that the following embodiments are not intended to limit the invention, and the scope of the invention is defined by the appended claims.

[0197] Example 1: Random conjugation of azide / handle to antibody The monoclonal antibody (mAb), denoted as "PSMB127" and referred to herein as "anti-PSMA mAb," is a human IgG4 antibody that binds to human prostate-specific membrane antigen (PSMA). It contains the heavy chain (HC) CDR1 sequence of SEQ ID NO: 3, the HC CDR2 sequence of SEQ ID NO: 4, the HC CDR3 sequence of SEQ ID NO: 5, the light chain (LC) CDR1 sequence of SEQ ID NO: 6, the LC CDR2 sequence of SEQ ID NO: 7, and the LC CDR3 sequence of SEQ ID NO: 8, as well as the HC sequence of SEQ ID NO: 9 and the LC sequence of SEQ ID NO: 10. The anti-PSMA mAb was expressed and purified using standard chromatography.

[0198] The human IgG4 S228P / F234A / L235A (IgG4-PAA) antibody isotype control for the anti-PMSA mAb, referred to herein as "control mAb," has the HC sequence of SEQ ID NO: 1 and the LC sequence of SEQ ID NO: 2. Commercially available antibodies trastuzumab (Herceptin), cetuximab (Erbitux), pertuzumab (Perjeta), and panitumumab (Vectibix) were purchased from Roche, Lilly, Roche, and Amgen, respectively. Mouse anti-human Her2 mAbs were obtained from BioXCell (catalog number BE0277). Trastuzumab, pertuzumab, and anti-human Her2 mAbs bind to human Her2. Cetuximab and panitumumab bind to human EGFR.

[0199] Complexation: The antibody stock solution (1-10 mg / mL) in 10 mM sodium acetate pH 5.2, phosphate-buffered saline pH 7, or other compatible buffer was mixed with 20% (wt / wt) 1 M sodium carbonate buffer pH 9 to a final pH of approximately 9. NHS-PEG4-azide (Thermo catalog number 26130) was dissolved in DMSO to a final concentration of 100 mM, and 0.2% (wt / wt) of the stock solution was added to create a molar excess of approximately 3-10 relative to the mAb. After incubating the reaction mixture at 22°C for 10 minutes, the mixture was quenched by adding 1 M Tris (pH 7.5) to a final concentration of 50 mM Tris.

[0200] Purification: The azide-mAb complex was purified using methods such as Zeba desalting column with a 7K MW cutoff (Thermo), dialysis, standard protein A affinity chromatography, or another suitable method, and then replaced with compatible buffer (PBS; 20 mM HEPES 150 mM, NaCl pH 7.5; or 10 mM sodium acetate pH 5.2). After purification, the complex was concentrated to 10-20 mg / mL using an Amicon concentrator with a 50K MW cutoff (Millipore).

[0201] LC / MS analysis: The chelator-to-antibody ratio (CAR) was determined by LC / MS analysis using an Agilent G6224 MS-TOF instrument equipped with an Agilent PLRP-S column (300-Angstrom, 2.1 x 150 mm, catalog number PL1912-3301) (Table 1). Mass spectra were deconvoluted using the maximum entropy algorithm over the m / z range of 2000–3200 for masses of 140–170 kDa.

[0202] Analytical size exclusion chromatography (SEC): Analytical SEC was performed to determine the oligomeric state of the antibody and antibody complex, confirming that the complexation process did not result in aggregation. An Agilent 1200 series HPLC with a Tosoh TSKgel G3000SWxl (Tosoh Bioscience #08541) 7.8 mm × 30 cm column was used. Mobile phase: 1 × PBS, flow rate: 0.8 ml / ml, injection volume: 15 μL, protein concentration: 0.1–2 mg / mL.

[0203] [Table 1]

[0204] Example 2: Random conjugation of azide / handle to non-antibody polypeptide Non-antibody polypeptides, specifically transferrin and human holo-transferrin, were purchased from R&D Systems (catalog number 2914-HT) and dissolved in water to a concentration of 10 mg / mL. Human epidermal growth factor (EGF) was purchased from Sino Biological (catalog number 10605-HNAE).

[0205] Complexation: A stock solution of polypeptide (1-10 mg / mL) in 10 mM sodium acetate pH 5.2, phosphate-buffered saline pH 7, or another compatible buffer was mixed with 20% (wt / wt) 1 M sodium carbonate buffer pH 9 to a final pH of approximately 9. NHS-PEG4-azide (Thermo catalog number 26130) was dissolved in DMSO to a final concentration of 100 mM, and 0.2% (wt / wt) of the stock solution was added to create a molar excess of approximately 3-10 relative to the protein. The reaction mixture was incubated at 22°C for 10 minutes, and then quenched with 1 M Tris pH 7.5 to a final concentration of 50 mM. The complexation efficiency is shown in Table 2.

[0206] [Table 2]

[0207] Example 3: Site-specific incorporation of azido sugar into antibody glycan Antibody glycans were trimmed with GlycINATOR (Genovis), a bacterial endoglycosidase specific to β-1,4 linkages between core GlcNac residues within Fc glycosylation sites (which may include multiple sites), leaving the innermost GlcNac intact on the Fc for later site-specific incorporation of azido sugars. More specifically, GlycINATOR immobilized on agarose beads packed into a column (Genovis) was equilibrated in Tris-buffered saline pH 7.4 (TBS). 1 mL of 5-10 mg / mL mAb was added to the resin and incubated on a rocker at room temperature for 1 hour, then eluted by rotating at 100Xg for 1 minute. The column was eluted three times with 0.5 mL of TBS. The eluate containing the trimmed mAbs was pooled, and the supplied buffer additive (Genovis) was added along with the UDP-GALNaz azido sugar substrate and the GalT galactosyltransferase enzyme. The reaction mixture was stirred overnight at 30°C. The final azide mAb was purified using an mAb-selective column (GE) with an AKTA Avant instrument. Azide modification was confirmed by LC-MS, and the CAR was determined to be exactly 2.

[0208] Example 4: Site-specific integration of 3-azidopropylamine by microbial transglutaminase (MTG) The azide group was site-specifically positioned relative to the antibody at position Gln295, as described (Dennler et al. Transglutaminase-based chemo-enzymatic conjugation approach yields homogeneous antibody-drug conjugates. Bioconj Chem 2014 Mar 19;25(3):p.569-78). The anti-PSMA mAb was cleaved between the innermost GlcNac residue and the asparagine residue of high-mannose, hybrid, and complex oligosaccharides, and deglycosylated with Rapid PNGase F (New England Biolabs), an amidase that enables complete deglycosylation and release of all N-glycans, including N-glycans derived from both conserved (e.g., Fc Asn297) and non-conserved (e.g., Fab N-glycan) glycosylation sites. 10 mL of 1 mg / mL antibody was incubated overnight at 37°C with 5 μL of PNGase F in sodium acetate buffer (pH 5.2), and deglycosylation was confirmed by LC-MS. PNGase F was removed by 4 cycles of concentration, and the mixture was diluted using an Amicon device (50 kDa cutoff). For the encombination with 3-azidopropylamine (3-APA; click chemistry tool), 2% (wt / wt) of 0.5 M HEPES pH 7.5 was added to adjust the pH of the deglycosylated mAb (0.5-1 mg / mL) to 7-7.5. 100 equivalents of 3-APA were added together with 5-10% wt / vol of the MTG, activin TI transglutaminase (Ajinomoto). After incubation of the reaction mixture at 37°C for 1-4 hours, the azido-modified mAbs were purified using standard chromatography on an mAbSelect Sure column. The composite was evaluated for its properties using LC-MS, and its CAR (Carbon Amount) was determined to be 2.

[0209] Example 5: Chelation of radioactive metals into a bifunctional chelator (BFC) 225 Synthesis of Ac-DOTA-Ga-DBCO: 225Ac(NO3)3 was purchased from Oak Ridge National Laboratory. 1,4,7,10-tetraazacyclododecane and 1-(glutaric acid)-4,7,10-triacetate-(3-amino-propanoic acid)dibenzocyclooctyne (DOTA-Ga-DBCO) were custom synthesized. The synthesis was based on Bernhard et al. Chem. Eur. J. 2012, 18, 7834-7841. DBCO-amine (3-amino-1-[(5-aza-3,4:7,8-dibenzocycloocta-1-in)-5-yl]-1-propanone (Sigma) was reacted with DOTA-GA anhydride, and the product was purified by reverse-phase HPLC.

[0210] The quantitative determination of actinium-225 was achieved using the Capintec CRC-55TW dose calibrator, thereby, 225 Ac(NO3)3 was dissolved in 0.1N HCl to prepare a 10 mCi / mL solution. In a plastic vial, tetramethylammonium acetate (1M solution, 7.5 μL, 7.5 μmol), DOTA-GA-DBCO (1 mg / mL aqueous solution, 2.5 μL, 3.4 nmol), and NaOH (0.1N, 2.5 μL, 0.25 μmol) were added. 225 Ac(NO3)3 (10 mCi / mL, 50 μCi, 0.0038 nmol in 5 μL of 0.1N HCl) was added. The pH of the mixture was observed to be approximately 6.5 using pH paper. The vial was placed on a shaking block at 80°C and 290 rpm for 30 minutes, and then cooled to room temperature.

[0211] 111 Synthesis of In-DOTA-GA-DBCO: in 0.05M HCl 111 I purchased InCl3 from GE Healthcare. The solution consists of tetramethylammonium acetate (1M solution, 7.5 μL, 7.5 μmol), DOTA-GA-DBCO (1 mg / mL aqueous solution, 2.5 μL, 3.4 nmol), and HCl (0.1N, 5 μL) in a plastic vial, with 0.05N HCl added. 111InCl3 (5 μL, 104.3 μCi, 0.0022 nmol as measured by a Capintec CRC-55TW dose calibrator) was added. The pH of the mixture was observed to be approximately 5.5 using pH paper. The vial was placed on a shaking block at 60°C and 290 rpm for 30 minutes, and then cooled to room temperature.

[0212] 89 Synthesis of Zr-DFO-DBCO: 89 Zr oxalate was purchased from 3D Imaging. DFO-DBCO was purchased from Macrocyclics (Plano, Texas, catalog number B-773), dissolved in DMSO to 0.5 mg / mL, and then diluted with water to 25 μg / mL.

[0213] 2 mCi of Zr-89 was transferred to a metal-free microcentrifuge tube, and 1 M oxalic acid was added to bring the total volume to 80 μL. 12 μL of 2 M potassium carbonate was added in 2 μL increments, and the mixture was agitated at the pipette tip until bubbling stopped. Next, 120 μL of 1 M HEPES was added, followed by 300 μL of water. The pH of the solution was tested, and additional 2 M potassium carbonate was added to raise the pH to 6–6.5 as needed. 136 μL of DFO-DBCO stock (3.4 μg) was added, and the reaction mixture was incubated at room temperature for 1 hour. 0.5 μL of the reaction mixture was spotted onto a TLC Green strip (Biodex) and analyzed by elution with 20% NaCl. After elution, the strip was scanned with a PerkinElmer Cyclone Plus phosphor imager to ensure that the majority of Zr-89 remained at baseline, demonstrating chelation by DFO-DBCO.

[0214] 89 Synthesis of Zr-DOTA-GA-DBCO: In water, add Sep-pak Light QMA strong anion exchange cartridge (acrylic acid / acrylamide copolymer on diol silica, surface sensitive: C(O)NH(CH2)3N(CH3)3 + Cl -(Pore size 300 Å, particle size 37-55 μm, ion exchange capacity 230 μeq / gram), MeCN (6 mL) was added, followed by 0.9% physiological saline (10 mL), then water (10 mL). In 1.0 M oxalic acid (2 μL, 290 μCi) 89 A solution of Zr(ox)2 was added to the pre-conditioned cartridge. The cartridge was then washed with deionized water (20 ml) to remove excess oxalic acid, and then treated with 1.0 M HCl (aqueous solution) (100 μL each). 89 ZrCl4 was eluted from the column, and a total volume of 400 μl was obtained with a recovery rate of 248 μCi (86%), with the majority of the radioactivity being in fraction 3. The combined organic extract was then evaporated and dried.

[0215] 10 μL of DOTA-GA-DBCO (1.0 mg / mL of metal-free water, 10 μg, 13.6 nmol) 89 It was added to ZrCl4 (268 μCi, 50 μL). DOTA-GA-DBCO / 89 The Zr solution was diluted with 150 μL of 1.0 M HEPES. The pH of the mixture was adjusted to pH 7.5 (Pandya et al., Zirconium tetraazamacrocycle complexes display extraordinary stability and provide a new strategy for zirconium-89-based radiopharmaceutical development. Chem Sci. 2017 Mar 1;8(3):p.2309-2314). The solution was then incubated at 90°C for 60 minutes. 89 The yield of the Zr-DOTA-GA-DBCO complex was determined to be 98% by elution with a 1% NH4OH solution using an SPC25 column (Sigma Aldrich part number SPC25120-50G). (Unchelated) 89 Zr remains on the column, 89 The Zr-DOTA-GA-DBCO complex is eluted.

[0216] Example 6: Synthesis of click-labeled radioactive composites of anti-PSMA mAbs Please refer to Figures 1 and 2 for a schematic diagram of the radiolabeled antibody produced by the method of the present invention.

[0217] Anti-PSMA mAb-dibenzo-[1,2,3]-triazoloazosin-Ga-DOTA-225Ac (site-specific, CAR=2): Random or site-specific azide-modified antibody (site-specific, CAR=2 or random, mean CAR of 1-4) was generated in PBS or other compatible buffer (10-20 mg / mL) as described. 225 The solution was added to Ac-DOTA-GA-DBCO. The final pH of the mixture was approximately 6.5 according to pH paper. The reaction solution was gently stirred and left at room temperature for 3 hours before purification on a PD-10 column (GE Healthcare) pre-conditioned with 15 mL of NaOAc buffer, 10 mM, pH 6-6.5, or another compatible buffer. The reaction mixture was pipetted into the reservoir of the pre-conditioned PD-10 column, and the eluate was collected in a plastic tube. The reaction vial was washed with NaOAc buffer (3 times with 0.2 mL each), the washing solution was pipetted into the reservoir of the PD-10 column, and the eluate was collected. NaOAc buffer was continuously applied to the reservoir of the PD-10 column, and the eluate was collected in a plastic tube, collecting approximately 1 mL of eluate in each tube until a total of 10 mL of eluate had been collected.

[0218] The purity of each recovered fraction was evaluated using iTLC-SG (Agilent) with a citrate-H2O-MeOH solution as the mobile phase. The pure fractions (or multiple fractions) were combined and the final product was obtained in 10 mM NaOAc buffer. The product solution was analyzed for chemical and radiochemical purity by HPLC. The antibody concentration in the product solution was measured by UV absorption using a standard curve. Subsequently, the radioactivity of the product solution was quantified using a Capintec CRC-55TW dose calibrator.

[0219] AC-225 Chelation Quality Control: A chelation challenge using diethylenetriaminepentaacetic acid (DTPA) was used for quality control of the purified product. A 10 mM Na5DTPA aqueous solution was used. 225 The DPTA was added to a sample solution containing Ac-labeled mAbs to adjust the ratio [DPTA] / [mAb] to 500-1,000. A 50 mM Na5DTPA aqueous solution was then added. 225 Alikates of the purified product were added to a solution containing Ac-labeled mAb to adjust the ratio of [DPTA] / [mAb] to 50,000–100,000. The two mixtures were placed on a shaking block at room temperature and 290 rpm for 30 minutes. The mixtures were spotted onto an iTLC-SG and developed using citrate-H2O-MeOH as the mobile phase. Under these conditions, the free mAb was obtained. 225 Ac moved to the solvent front and bound 225 Ac-mAb remains at baseline.

[0220] Anti-PSMA mAb-dibenzo-[1,2,3]-triazoloazosin-GA-DOTA- 111 Synthesis of In: Random or site-directed azide-modified anti-PSMA mAbs (site-directed, CAR=2 or random, mean CAR of 1-4) were generated in 10 mM NaOAc as described. 111 The reaction mixture was added to a solution of Ac-DOTA-GA-DBCO. The reaction solution was gently stirred and left at room temperature for 2 hours before passing it through the PD-10 column. The PD-10 column was pre-conditioned by passing 15 mL of NaOAc buffer, 10 mM, pH 6-6.5 through the column, and the washing solution was discarded. Next, the reaction mixture was pipetted into the reservoir of the pre-conditioned PD-10 column, and the eluate was collected in a plastic tube. The reaction vial was washed with NaOAc buffer (3 times with 0.2 mL each), and the washing solution was pipetted into the reservoir of the PD-10 column, and the eluate was collected. NaOAc buffer was continuously applied to the reservoir of the PD-10 column, and the eluate was collected in a plastic tube, collecting approximately 1 mL of eluate in each tube until a total of 10 mL of eluate had been collected.

[0221] The purity of each recovered fraction was evaluated by iTLC-SG using a 10 mM EDTA aqueous solution (pH=5-6) as the mobile phase. The pure fractions (multiple fractions may be present) were combined and the final product was obtained in 10 mM NaOAc buffer. The product solution was analyzed for chemical and radiochemical purity by HPLC. The antibody concentration in the product solution was measured by UV absorption using a standard curve. Subsequently, the radioactivity of the product solution was quantified using a Capintec CRC-55TW dose calibrator.

[0222] Quality control of In-111 chelation: A chelation challenge using DTPA was used for quality control of the purified product: A 10 mM Na5DTPA aqueous solution was used. 111 The mAb was added to a sample solution containing In-labeled mAbs to adjust the [DTPA] / [mAb] ratio to 1000-10,000. The mixture was placed on a shaking block at room temperature and 290 rpm for 30 minutes. The mixture was spotted onto an iTLC-SG and developed using 10 mM EDTA aqueous solution (pH=5-6) as the mobile phase. 111 In or loosely bonded 111 It moved to the solvent front and bonded tightly. 111 In-mAb remains at baseline.

[0223] Synthesis of anti-PSMA mAb-dibenzo-[1,2,3]-triazoloazosin-DFO-ZR-89 800 μg of randomly azide-modified anti-PSMA mAbs (average CARs of 1-4; a similar procedure can be performed with site-specific azide mAbs) in 10 mM HEPES, 50 mM NaCl, pH 7.5, or other compatible buffer was prepared as described. 89The reaction mixture was added to a Zr-DFO-DBCO solution, incubated at 37°C for 1.5 hours, and then passed through a PD-10 column. The PD-10 column was washed with 15 mL of isotonic saline, and the washing solution was discarded. The reaction mixture was then pipetted into a reservoir on a pre-conditioned PD-10 column, and the eluate was collected in a plastic tube. Saline solution was continuously applied to the reservoir on the PD-10 column, and the eluate was collected in a plastic tube, with approximately 0.5 mL of eluate collected in each tube until a total of 10 mL of eluate had been collected. The radioactivity of each fraction and the material remaining on the column was measured using a dose calibrator. The final product was obtained by pooling the product peaks, usually fractions 4-7. The product solution was analyzed for chemical and radiochemical purity by HPLC. The antibody concentration in the product solution was measured by UV absorption using a standard curve. The radioactivity of the product solution was quantified using a dose calibrator.

[0224] Anti-PMSA mAb-DOTA- 89 Zr synthesis Azide-modified anti-PSMA mAb complexes (CAR=1~4), modified by random azide complex formation in 20 mM HEPES 50 mM NaCl pH 7.5 (10.1 mg / mL, 200 μL, approximately 13.5 nmol), were generated as described above. 89 The compound was added to a solution of Zr-DOTA-GA-DBCO. The final pH of the mixture was adjusted to 7.0. The reaction solution was incubated at 37°C for 2 hours, followed by purification using a PD-10 column (GE Healthcare) with either 0.9% physiological saline, HEPES buffer, or PBS, and the product was obtained at 64%. 89 Zr-DOTA-mAb was obtained.

[0225] Quality control of Zr-89 chelation: For Zr-DFO-mAbs, purified products were analyzed by HPLC only. These complexes did not retain Zr-89 when challenged with DTPA or EDTA. Zr-DOTA-mAbs were challenged with 33 mM EDTA and incubated overnight at room temperature. Analysis of the challenged complexes by passing them through a PD-10 column revealed that they retained 80% of their radioactivity.

[0226] Example 7: Analytical characterization of click-labeled radioactive composites Determination of radiochemical transformation Radiochemical conversion (%RA conversion; see Tables 3-5) was determined by iTLC-SG (simplified thin-layer chromatography (iTLC) using binderless glass microfiber chromatography paper impregnated with silica gel (SG)). The %RA conversion value is calculated by dividing the integral of the radiation signals of the product peaks (with different retention times for the radioactive starting material and by-products) by the value obtained by integrating all radiation signal peaks present between the baseline and the solvent front. This percentage of the product is then expressed as the conversion rate.

[0227] For products incorporating Ac-225, the amount is approximately 0.1 to 1 μCi. 225 A sample solution containing Ac was spotted at the baseline of an iTLC-SG strip approximately 2 cm from the bottom. The iTLC-SG strip was developed using a citrate-water-methanol mobile phase (20 mL 0.4 M trisodium citrate / 3 mL 2 N HCl / 2.3 mL MeOH), dried at room temperature, and stored for a minimum of 6 hours before analysis (to reach secular equilibrium for 225Ac and all daughter nuclides). The iTLC-SG was scanned using a Bioscan AR2000 radioactive TLC imaging scanner with a 99 mTc setting.

[0228] For In-111 chelate, approximately 0.5 to 2.5 μCi was present at the baseline of the iTLC-SG strip, approximately 2 cm from the bottom edge. 111A sample solution containing In was spotted. The iTLC-SG strip was developed using 10 mM EDTA pH 5-6 as the mobile phase and then dried at room temperature. The dried iTLC-SG was scanned using a Bioscan AR2000 radioactive TLC imaging scanner with an In-111 setting.

[0229] In the case of Zr-89 chelate, the %RA conversion was determined by dividing the radioactivity relative to the product peak by the total radioactivity including the radioactivity in the PD-10 column, as determined by counting with a dose calibrator.

[0230] Determining the radiochemical purity of radiolabeled proteins. The radiochemical purity (%RA purity, see Tables 3-4) of Ac-225 and In-111 chelates was determined by SE-HPLC (size exclusion HPLC). For Ac-225, a Tosoh TSKgel column (G3000SW × 17.8 mm × 30 cm, 5 μm) was used, and the column was eluted with DPBS buffer (×1, calcium and magnesium-free). Flow rate: 0.7 mL / min, operation for 20 minutes, room temperature. After HPLC, the eluate was collected in pre-numbered vials, and the eluate fraction from 0.5 min or 1 min was collected in each vial. The vials containing the eluate were left at room temperature for more than 6 hours. 225 Ac allowed permanent equilibrium to be reached with its daughter nuclides. The radioactivity in each vial was then counted using a Capintec CRC-55TW well counter. Based on the radioactivity in the vials, a radiochromatogram was reconstructed.

[0231] For In-111, a Tosoh TSKgel column (G3000SW × 17.8 mm × 30 cm, 5 μm) was used. The column was eluted with DPBS buffer (×1, calcium and magnesium-free). Flow rate: 0.7 mL / min, run for 20 minutes, room temperature. Radioactivity detection was performed using the above HPLC system and a Perkin Elmer Radiomatic 625TR radioactive flow detector, equipped with a 0.5 mL flow cell using the In-111 setting and an Ultima Flo™ M cocktail used at a flow rate of 1.4 mL / min.

[0232] For Zr-89 (see Table 5), a Tosoh TSKgel column (G3000SW × 17.8 mm × 30 cm, 5 μm) was used. The column was eluted with citrate-buffered saline. Flow rate: 1 mL / min, operation for 20 minutes, room temperature. Radioactivity was detected using the above HPLC system and a Beckman flow-through detector connected to a Bioscan Flow Count instrument.

[0233] [Table 3] * Step 1 is the chelation of actinium-225 to DOTA-GA-DBCO. ** Step 2 is a click reaction of a bifunctional chelate to the protein.

[0234] [Table 4] * Step 1 is the chelation of indium-111 to DOTA-GA-DBCO. ** Step 2 is a click reaction of a bifunctional chelate to the protein.

[0235] [Table 5]

[0236] Example 8: Click reaction between modified mAb and DOTA-GA-DBCO Random and site-specific azide mAbs (anti-PSMA mAbs, cetuximab, panitumumab, tratuzumab, pertuzumab) at concentrations of 1–10 mg / mL were mixed with 5–20-fold excess unchelated DOTA-GA-DBCO and incubated at room temperature or 37°C for 1–24 hours. The mAbs were desalted using a Zeba desalting column (Thermo), concentrated using an Amicon centrifuge (Millipore), re-diluted with buffer, and concentrated again to remove all remaining DBCO-DOTA. Complete click reactions of all free azides with DBCO-DOTA were confirmed by LC-MS.

[0237] Example 9: Click reaction between modified mAb and DFO-DBCO Random and site-specific anti-PSMA mAb azide-mAbs at concentrations of 1-10 mg / mL were mixed with 5-20 times excess unchelated DOTA-GA-DFO and incubated at room temperature or 37°C for 1-24 hours. The mAbs were desalted using a Zeba desalting column (Thermo), concentrated using an Amicon centrifuge (Millipore), re-diluted with buffer, and concentrated again to remove all remaining DBCO-DFO. Complete click reactions between all free azide and DBCO-DFO were confirmed by LC-MS.

[0238] Example 10: Cell Binding (FACS) The cell binding of azide-modified antibodies, DOTA-DBCO-azide-modified antibodies, and DFO-DBCO-azide-modified antibodies was compared with the parent mAbs of the conjugates listed in Table 1. Cell lines expressing mAb targets were treated with antibodies or conjugates within the concentration range, and binding was measured by flow cytometry. The binding of panitumumab and cetuximab conjugates to EGFR+A431 cells was evaluated. The binding of herceptin and pertuzumab to HER2+SK-BR-3 cells was evaluated. The binding of anti-PSMA mAbs to PSMA+C4-2b cells was evaluated.

[0239] Cell lines: C4-2B cells and human prostate cancer cell lines were obtained from Janssen Oncology (Springhouse, Pennsylvania). Human epidermal carcinoma cell line A431 and human breast cancer cell line SK-BR-3 were obtained from Janssen BioTherapeutics (Springhouse, Pennsylvania), which originally uses cells derived from ATCC (Manassas, Virginia). EGFR receptor-negative MOLM-13 human acute myeloid leukemia suspension cells were maintained in RPMI1640 + 25mM Hepes (Gibco) supplemented with 20% heat-inactivated fetal bovine serum (Gibco). Cells were grown using RPMI1640 + 25mM HEPES (Gibo, Waltham, Massachusetts) containing 10% FBS (Gibo, Waltham, Massachusetts).

[0240] Flow cytometry: Cells were detached from flasks using enzyme-free cell dissociation buffer (Gibco, Waltham, Massachusetts) and filtered through a 40 μm filter (Falcon). 5 x 10⁴ cells were collected per well in a 96-well U-bottom plate. 4Cells were seeded. The cells were incubated at 4°C for 1 hour with a compound antibody or parental antibody diluted in BSA staining buffer (BD Bioscience, San Jose, California). The cells were washed twice with staining buffer. Then, the cells were incubated in the dark at 4°C for 30 minutes with AlexaFluor647-tagged anti-human IgG secondary antibody (Jackson ImmunoResearch Laboratories). The secondary antibody was diluted 1:200 with staining buffer containing 3% donkey serum (Rockland Immunochemicals). During the last 10 minutes of incubation, SYTOX® Green Nucleic Acid Stain (ThermoFisher) was added to the cells at a final concentration of 30 nM. After washing the cells twice with staining buffer, they were resuspended in a final volume of 25 μL / well in staining buffer and read using an iQue Screener flow cytometer (Intellicyt). ForeCyt software was used to analyze the data. Live cells were determined by excluding events with high nucleic acid staining. Mean fluorescence intensity (MFI) was determined in living cells and graphed using GraphPad Prism 7 (GraphPad Software) as the logarithm of antibody concentration vs. MFI. Nonlinear regression curve fitting was added to the data, and EC 50 The value was calculated.

[0241] For all mAbs and complexes tested, the parent mAb and modified mAb showed similar cell binding (Table 6).

[0242] [Table 6]

[0243] Example 11: In-111 cell binding assay Cell binding was measured by radiometric assay using the In-111 radiolabeled protein listed in Table 4. Anti-PSMA mAbs and transferrin were tested in C4-2B cells (PSMA+ and transferrin+). Cetuximab and panitumumab were tested in A431 cells (EGFR+) and MOLM-13 cells (EGFR-).

[0244] Adherent cells were detached using enzyme-free cell dissociation buffer (Gibco). The detached adherent cells and recovered suspension cells were counted and washed with cold staining buffer (BD Biosciences). Various numbers of cells from 200 μL of staining buffer were added to microcentrifuge tubes and placed on ice. 0.5 μCi of In-111 labeled protein was added to each tube and incubated on ice for 1 hour. The cells were washed with cold PBS (Gibco) to remove unbound antibody and resuspended in 500 μL of cold PBS. The samples were transferred to counting vials and cell-associated radioactivity was measured by gamma counting (Hidex Automatic Gamma Counter).

[0245] Counts per minute (CPM) from the test samples were converted to In-111 μCi using the CPM value, which was calculated using a linear regression using a known amount of In-111 labeled protein. The μCi boundary value was converted to molar bounds using the following calculation: (μCi bounds / specific activity) / MW mAb or protein. Each data point is the mean ± SD of the triple reaction.

[0246] Click-labeled In-111 anti-PSMA mAbs and In-111 transferrin bound to C4-2B cells, and cell-associated radioactivity increased with increasing cell number (Figure 3A). Click-labeled In-111 anti-EGFR antibodies, panitumumab and cetuximab, bound to A431 cells, and cell-associated radioactivity increased with increasing cell number. Specific binding of click-labeled anti-EGFR antibodies was not detected in negative control MOLM-13 cells (Figure 3B).

[0247] Example 12: Indium Cell Uptake Assay The dynamics of In-111 click-labeled anti-PSMA mAb internalization in C4-2B cells were determined.

[0248] Cells are placed in 3 x 10 cells per 60 mm dish (Corning). 6 Cells were seeded and placed overnight in a humidified CO2 incubator at 37°C. The seeding medium was removed and replaced with 2 mL of cold staining buffer (BD Bioscience). The dishes were then placed on ice. 0.5 μCi of In-111-labeled antibody was added to each dish and incubated on ice for 1 hour. Cells were washed with cold PBS (Gibco) to remove unbound antibody from the cell surface. Cells were assayed for surface membrane-bound radioactivity and intracellular radioactivity at various time points, as described below.

[0249] Surface-bound radioactivity was stripped using an acid-wash stripping procedure: 1.5 mL of stripping buffer (containing 50 mL of glycine, 150 mL of NaCl pH 2.7, and pepsin (Amresco) added to 25 μg / mL) was added to the cells, and the dish was incubated on ice for 15 minutes. The stripping buffer was transferred to a counting vial. The cells were washed with cold PBS, and the wash solution was transferred to a counting vial. Radioactivity was assayed using a gamma counter (Hidex Automatic Gamma Counter). Surface-bound radioactivity was determined as the sum of the stripping buffer and PBS wash solution.

[0250] Intracellular radioactivity was assayed by preparing cell lysates: Surface-bound radioactivity was stripped, the cells were washed, and 1.5 mL of 1 M NaOH (Teknova) was added to the cells. The dish was incubated on ice for 5 minutes. The surface-strand cell lysates were transferred to a counting vial. The dish was washed with cold PBS, and the washing solution was transferred to the counting vial. Radioactivity was assayed by gamma counting (Hidex Automatic Gamma Counter). Intracellular radioactivity was determined as the sum of surface-strand cell lysates and PBS washing solution.

[0251] For the time=0 sample, cells were assayed for surface membrane-bound radioactivity and intracellular radioactivity immediately after initial antibody binding on ice. For the 10-minute, 30-minute, 1-hour, and 2-hour samples, 3 mL of cell culture medium was added to each dish after initial antibody binding, and the dishes were placed in a humidified CO2 incubator at 37°C. At each time point, the dishes were removed from the incubator and placed on ice. The cell culture medium was transferred to a counting vial, and the cells were washed with cold PBS. The PBS wash was collected in the counting vial. The cells were assayed for surface membrane-bound radioactivity and intracellular radioactivity as described. At each time point, unstripped samples were prepared by incubating the cells with PBS instead of stripping buffer before cell lysis. These samples were used to evaluate stripping efficiency, and the results were compared with the stripped samples.

[0252] The CPM from the test samples was converted to μCi In-111 using CPM values ​​from linear regression prepared using a known amount of In-111-labeled mAb. The localization rate (%) of In-111 mAb on the cell surface membrane (stripped samples) and inside cells (lysed samples) was expressed using the following formula: localization rate % = 100 * The values ​​were determined using (sample μCi / average total μCi), where total μCi refers to the sum of all recovered samples, including incubation medium, PBS wash, glycine rinse, and lysed cells. Each data point is the mean ± SD of the triple reaction.

[0253] Surface-bound In-111 rapidly disappeared from the cell surface and was redistributed within the cell. The stripping technique released approximately 80% of the cell surface-associated radioactivity at time 0, and by the end of incubation, only 20% had been released by stripping, with over 60% of the radioactivity remaining in the cell lysate (Figure 4).

[0254] Example 13: Efficacy in a mouse tumor xenograft model Dose determination study of anti-PSMA mAb-DOTA-AC-225: Male NSG mice (n=8 per group) were given 10 6 LNCap cells were subcutaneously transplanted to reduce the tumor size to 100-150 mm. 3 It was propagated in mice. Anti-PSMA mAb-azide-DOTA- with radioactivity ranges (10nCi, 25nCi, 70nCi, 200nCi) 225 Ac or isotype control, control mAb-azide-DOTA- 225 A single dose of Ac was administered intravenously. The amount injected per mouse was 10 μg of protein in total using cold antibody. Tumor measurements and body weight were recorded twice a week. Tumor size was 1,500 mm. 3 The animals were euthanized when they exceeded a certain threshold, or when their weight loss exceeded 20%.

[0255] The anti-PSMA mAb-DOTA-Ac-225 demonstrated tumor growth inhibition, particularly after a single dose at high radiation doses, and showed superior tumor growth inhibition compared to isotype controls at all doses (Figure 5A). All control mAb radiocomplexes at all doses exhibited survival curves similar to those of the vehicle control (Figure 5B; Table 7); the anti-PSMA mAb complex showed a clear dose-response, with survival rates increasing with increasing radiation dose (Figure 5C; Table 7). At 209 days, when the study was concluded, all three mice remained from the anti-PSMA mAb 200nCi group and showed no detectable tumors.

[0256] [Table 7]

[0257] The embodiments of the present invention are intended to be merely illustrative, and those skilled in the art will be able to recognize or confirm numerous equivalents to specific procedures of the present invention through routine experimentation alone. Any such equivalent will be considered to fall within the scope of the present invention and will be included in the following claims.

[0258] All references cited herein (including patent applications, patents, or documents) are incorporated herein for any purpose to the same extent as if each individual document or patent or patent application were specifically and individually indicated to be incorporated by reference in its entirety for any purpose. This may include the following aspects. . [1] A method for labeling polypeptides with radioactive metal ions, a. To provide a modified polypeptide comprising the polypeptide covalently bonded to a first click reaction partner, b. To provide a radioactive complex comprising the radioactive metal ion associated with the chelated portion, wherein the chelated portion comprises a chelating agent covalently bonded to a second click reaction partner. c. A method comprising contacting the modified polypeptide with the radioactive complex under conditions that enable the polypeptide to be labeled with the radioactive metal ion by the reaction of the first click reaction partner with the second click reaction partner. [2] The method according to [1], wherein one of the first and second click reaction partners comprises an alkyne group and the other click reaction partner comprises an azide, or where one of the first and second click reaction partners comprises an alkene group and the other click reaction partner comprises a diene. [3] The method according to [1] or [2] above, wherein the polypeptide is an antibody or an antigen-binding fragment thereof. [4] The method according to [3] above, wherein the antibody is an anti-PSMA monoclonal antibody. [5] The radioactive metal ions, 225 Ac, 111 In, or 89 The method described in any one of the above [1] to [4], wherein Zr is Zr. [6] The method according to any one of the above [1] to [4], further comprising reacting an electrophile on a side chain with a sulfhydryl group covalently bonded to the first click reaction partner to obtain the modified polypeptide. [7] The method according to any one of the above [1] to [5], wherein the modified polypeptide is a modified antibody or an antigen-binding fragment thereof obtained by site-specific incorporation of the first click reaction partner. [8] The method according to [7] above, wherein the modified antibody or its antigen-binding fragment is obtained by a method comprising: trimming the antibody or its antigen-binding fragment with a bacterial endoglycosidase specific to the β-1,4 linkage between core GlcNac residues of the Fc-glycosylation site of the antibody to obtain a trimmed antibody or its antigen-binding fragment; and reacting the trimmed antibody or its antigen-binding fragment with an azide-labeled sugar in the presence of a glycosyltransferase such as GalT galactosyltransferase or GalNac transferase to obtain the modified antibody or its antigen-binding fragment. [9] The method according to [8], wherein the azide-labeled sugar is UDP-N-azidoacetylgalactosamine (UDP-GalNaz) or UDP-6-azido6-deoxyGalNAc.

[10] The method according to [8] above, wherein the glycosyltransferase is GalT galactosyltransferase or GalNActransferase.

[11] The method according to [7] above, wherein the modified antibody or its antigen-binding fragment is obtained by a method comprising deglycosylating the antibody or its antigen-binding fragment with amidase to obtain a deglycosylated antibody or its antigen-binding fragment, and reacting the deglycosylated antibody or its antigen-binding fragment with an azidoamine in the presence of a microbial transglutaminase to obtain the modified polypeptide.

[12] The method according to

[11] , wherein the azidoamine is selected from 3-azidopropylamine, 6-azidohexylamine, O-(2-aminoethyl)-O'-(2-azidoethyl)tetraethylene glycol, O-(2-aminoethyl)-O'-(2-azidoethyl)pentaethylene glycol, and O-(2-aminoethyl)-O'-(2-azidoethyl)triethylene glycol.

[13] The method according to [1], wherein the modified polypeptide is covalently bonded to an azide, tetrazine, or tetrazole group, either directly or via a linker.

[14] The chelating agent is of formula (I): [ka] (In the formula, R 1 、R 2 、R 3 and R 4 CHQCO 2 X is, Q is independently hydrogen, C 1 ~C 4 Alkyl or (C 1 ~C 2 It is alkylphenyl, X is independently hydrogen, benzyl, and C 1 ~C 4 It is alkyl, Z is (CH2) n Y is, n is between 1 and 10. Y is an electrophilic or nucleophilic moiety covalently bonded to the second click reaction partner, Alternatively, Z is hydrogen; and R 1 、R 2 、R 3 and R 4 CHQCO 2 X is, Q is independently hydrogen, C 1 ~C 4 Alkyl or (C 1 ~C 2 It is alkylphenyl, X is independently hydrogen, benzyl, and C 1 ~C 4 The macrocycle comprises a structure having an alkyl group or an electrophilic or nucleophilic moiety covalently bonded to the second click reaction partner. Alternatively, the method according to [1] above, wherein the chelating agent comprises an open-chain ligand.

[15] The chelate portion is given by formula (II):

change

change

[16] 225 A C 、 111 In, or 89 A method for labeling an antibody or its antigen-binding fragment with Zr, a. To provide a modified antibody or antigen-binding fragment containing an antibody or antigen-binding fragment covalently bound to an azide, tetrazine, or tetrazole group, b. Associated with the chelated portion 225 A C 、 111 In or 89 To provide a radioactive complex containing Zr, wherein the chelating portion comprises a chelating agent covalently bonded to an alkyne or alkene group, and c. The azide, tetrazine, or tetrazole group reacts with the alkyne or alkene group to form the antibody or its antigen-binding fragment. 225 A C 、 111 In, or 89 This includes contacting the modified antibody or its antigen-binding fragment with a radioactive complex under conditions that enable labeling with Zr, The chelating agent is of formula (I):

change

[17] The method according to

[16] , further comprising reacting an electrophile on a side chain with a sulfhydryl group covalently bonded to the azide, tetrazine, or tetrazole group to obtain the modified antibody or its antigen-binding fragment.

[18] The method according to

[16] , wherein the modified antibody or its antigen-binding fragment is obtained by site-specific integration of the first click reaction partner.

[19] The method according to

[18] , wherein the modified antibody or its antigen-binding fragment is obtained by a method comprising: trimming the antibody or its antigen-binding fragment with a bacterial endoglycosidase specific to the β-1,4 bond between core GlcNac residues of the Fc-glycosylation site of the antibody to obtain a trimmed antibody or its antigen-binding fragment; and reacting the trimmed antibody or its antigen-binding fragment with an azide-labeled sugar in the presence of a glycosyltransferase to obtain a modified antibody or its antigen-binding fragment.

[20] The method according to

[19] , wherein the azide-labeled sugar is UDP-N-azidoacetylgalactosamine (UDP-GalNaz) or UDP-6-azido6-deoxyGalNAc.

[21] The method according to

[19] above, wherein the glycosyltransferase is selected from GalT galactosyltransferase or GalNActransferase.

[22] The method according to

[16] above, wherein the modified antibody or its antigen-binding fragment is obtained by deglycosylating the antibody or its antigen-binding fragment with amidase to obtain a deglycosylated antibody or its antigen-binding fragment, and by reacting the deglycosylated antibody or its antigen-binding fragment with an azidoamine in the presence of a microbial transglutaminase to obtain the modified polypeptide.

[23] The method according to

[22] , wherein the azidoamine is selected from 3-azidopropylamine, 6-azidohexylamine, O-(2-aminoethyl)-O'-(2-azidoethyl)tetraethylene glycol, O-(2-aminoethyl)-O'-(2-azidoethyl)pentaethylene glycol, and O-(2-aminoethyl)-O'-(2-azidoethyl)triethylene glycol.

[24] The chelate portion is given by formula (II):

change

change

[16] above, including the structure of .

[25] A method for doubly labeling a polypeptide with two radioactive metal ions, a. To provide a modified polypeptide comprising the polypeptide covalently bonded to a first click reaction partner and a second click reaction partner. b. To provide a first radioactive complex comprising the first radioactive metal ion associated with the chelated moiety, wherein the chelated moiety comprises a chelating agent covalently bonded to a third click reaction partner, c. To provide a second radioactive complex comprising the second radioactive metal ion associated with the chelated moiety, wherein the chelated moiety comprises a chelating agent covalently bonded to a fourth click reaction partner. d. A method comprising contacting the modified polypeptide with the radioactive complex under conditions that enable the polypeptide to be labeled with the first and second radioactive metal ions by the reaction of the first click reaction partner with the third click reaction partner and the reaction of the second click reaction partner with the fourth click reaction partner.

[26] The method according to

[25] , wherein one of the first and second click reaction partners comprises an alkyne group, the other of the first and second click reaction partners comprises an azide, one of the third and fourth click reaction partners comprises an alkene group, the other of the third and fourth click reaction partners comprises a diene, the first or second radioactive metal ion is a diagnostic emitter and the other is a therapeutic emitter, or both of the first and second radioactive metal ions are therapeutic emitters.

[27] A pharmaceutical composition comprising a radiolabeled polypeptide prepared by the method described in [1] or

[16] above, and a pharmaceutically acceptable carrier.

[28] A method for treating a neoplastic disease or disorder in a subject requiring treatment, comprising administering the pharmaceutical composition described in

[27] above to the subject.

[29] A theranostic agent comprising a radiolabeled antibody prepared by the method described in [1] or

[16] above, and a pharmaceutically acceptable carrier, wherein the immunological properties of the radiolabeled antibody are preserved.

[30] A theranostic agent prepared by the method described in [1] above, Formula (VIII):

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change

[31] The radioactive metal ions, 32 P、 47 Sc, 67 Cu, 77 As, 89 Sr、 90 Y、 99 Tc, 105 Rh, 109 Pd, 111 Ag, 131 I、 153 Sm, 159 Gd, 165 Dy, 166 Ho, 169 Er、 177 Lu, 186 Re,188 Re, 194 Ir、 198 Au, 199 Au, 211 At, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 225 Ac, 255 Fm, 227 Th, 62 Cu, 64 Cu, 67 Ga, 68 Ga, 86 Y、 89 Zr, or 111 A theranostic agent as described above

[29] , selected from In.

[32] combination, a. A modified polypeptide comprising a polypeptide covalently bonded to a first click reaction partner, b. A radioactive complex comprising a radioactive metal ion associated with a chelated moiety, wherein the chelated moiety comprises a chelating agent covalently bonded to a second click reaction partner, The combination is used to label the polypeptide with the radioactive metal ion.

Claims

1. A method for labeling polypeptides with radioactive metal ions, a. To provide a modified polypeptide comprising the polypeptide covalently bonded to a first click reaction partner, b. To provide a radioactive complex containing the radioactive metal ion associated with the chelated portion, wherein the chelated portion contains a chelating agent covalently bonded to a second click reaction partner. c. Contacting the modified polypeptide with the radioactive complex under conditions that enable the polypeptide to be labeled with the radioactive metal ion by the reaction of the first click reaction partner with the second click reaction partner, wherein the contact is performed without a copper catalyst. One of the first and second click reaction partners comprises an alkyne group and the other of the click reaction partners comprises an azide, or one of the first and second click reaction partners comprises an alkene group and the other of the click reaction partners comprises a diene. The chelating agent is defined by formula (II): 【Chemistry 1】 Structure, or formula (III): 【Chemistry 2】 A method including structure.

2. The method according to claim 1, wherein the polypeptide is an antibody or an antigen-binding fragment thereof.

3. The method according to claim 2, wherein the antibody is an anti-PSMA monoclonal antibody.

4. The aforementioned radioactive metal ions ２２５ Ac, １１１ In, or ８９ The method according to any one of claims 1 to 3, wherein the material is Zr.

5. The method according to any one of claims 1 to 3, further comprising reacting an electrophile on a side chain with a sulfhydryl group covalently bonded to the first click reaction partner to obtain the modified polypeptide.

6. The method according to any one of claims 1 to 3, wherein the modified polypeptide is a modified antibody or an antigen-binding fragment thereof obtained by site-specific integration of the first click reaction partner.

7. The method according to claim 6, wherein the modified antibody or its antigen-binding fragment is obtained by a method comprising: trimming the antibody or its antigen-binding fragment with a bacterial endoglycosidase specific to the β-1,4 linkage between core GlcNac residues in the Fc-glycosylation site of the antibody to obtain a trimmed antibody or its antigen-binding fragment; and reacting the trimmed antibody or its antigen-binding fragment with an azide-labeled sugar in the presence of a glycosyltransferase to obtain the modified antibody or its antigen-binding fragment.

8. The method according to claim 7, wherein the glycosyltransferase is GalT galactosyltransferase or GalNActransferase.

9. The method according to claim 7, wherein the azide-labeled sugar is UDP-N-azidoacetylgalactosamine (UDP-GalNaZ) or UDP-6-azido6-deoxyGalNAc.

10. The method according to claim 7, wherein the glycosyltransferase is GalT galactosyltransferase or GalNActransferase.

11. The method according to claim 6, wherein the modified antibody or its antigen-binding fragment is obtained by a method comprising: deglycosylating the antibody or its antigen-binding fragment with amidase to obtain a deglycosylated antibody or its antigen-binding fragment; and reacting the deglycosylated antibody or its antigen-binding fragment with an azidoamine in the presence of microbial transglutaminase to obtain the modified polypeptide.

12. The method according to claim 11, wherein the azidoamine is selected from 3-azidopropylamine, 6-azidohexylamine, O-(2-aminoethyl)-O'-(2-azidoethyl)tetraethylene glycol, O-(2-aminoethyl)-O'-(2-azidoethyl)pentaethylene glycol, and O-(2-aminoethyl)-O'-(2-azidoethyl)triethylene glycol.

13. The method according to claim 1, wherein the modified polypeptide comprises a polypeptide covalently bonded to an azide, tetrazine, or tetrazole group, either directly or via a linker.

14. ２２５ A Ｃ , １１１ In, or ８９ A method for labeling an antibody or its antigen-binding fragment with Zr, a. To provide a modified antibody or antigen-binding fragment containing an antibody or antigen-binding fragment covalently bound to an azide, tetrazine, or tetrazole group, b. Associated with the chelating moiety ２２５ A Ｃ , １１１ In or ８９ providing a radioactive complex containing Zr, wherein the chelating moiety comprises a chelating agent covalently bonded to an alkyne or alkene group, and c. The azide, tetrazine, or tetrazole group reacts with the alkyne or alkene group to form the antibody or its antigen-binding fragment. ２２５ A Ｃ , １１１ In, or ８９ The process involves contacting the modified antibody or its antigen-binding fragment with a radioactive complex under conditions that enable labeling with Zr, wherein the contact is performed without a copper catalyst. The chelating agent is defined by formula (II): 【Transformation 3】 Structure or formula (III): 【Chemistry 4】 A method including structure.

15. The method according to claim 14, further comprising reacting an electrophile on a side chain with a sulfhydryl group covalently bonded to the azide, tetrazine, or tetrazole group to obtain the modified antibody or its antigen-binding fragment.

16. The method according to claim 14, wherein the modified antibody or its antigen-binding fragment is obtained by site-specific incorporation of the azide, tetrazine, or tetrazole group.

17. The method according to claim 16, wherein the modified antibody or its antigen-binding fragment is obtained by a method comprising: trimming the antibody or its antigen-binding fragment with a bacterial endoglycosidase specific to the β-1,4 linkage between core GlcNac residues in the Fc-glycosylation site of the antibody to obtain a trimmed antibody or its antigen-binding fragment; and reacting the trimmed antibody or its antigen-binding fragment with an azide-labeled sugar in the presence of a glycosyltransferase to obtain a modified antibody or its antigen-binding fragment.

18. The method according to claim 17, wherein the azide-labeled sugar is UDP-N-azidoacetylgalactosamine (UDP-GalNaZ) or UDP-6-azido6-deoxyGalNAc.

19. The method according to claim 17, wherein the glycosyltransferase is selected from GalT galactosyltransferase or GalNActransferase.

20. The method according to claim 14, wherein the modified antibody or its antigen-binding fragment is obtained by a method comprising: deglycosylating the antibody or its antigen-binding fragment with amidase to obtain a deglycosylated antibody or its antigen-binding fragment; and reacting the deglycosylated antibody or its antigen-binding fragment with an azidoamine in the presence of microbial transglutaminase to obtain the modified antibody or its antigen-binding fragment.

21. The method according to claim 20, wherein the azidoamine is selected from 3-azidopropylamine, 6-azidohexylamine, O-(2-aminoethyl)-O'-(2-azidoethyl)tetraethylene glycol, O-(2-aminoethyl)-O'-(2-azidoethyl)pentaethylene glycol, and O-(2-aminoethyl)-O'-(2-azidoethyl)triethylene glycol.

22. A method for doubly labeling a polypeptide with two radioactive metal ions, a. To provide a modified polypeptide comprising the polypeptide covalently bonded to a first click reaction partner and a second click reaction partner. b. To provide a first radioactive complex comprising the first radioactive metal ion associated with the chelated portion, wherein the chelated portion comprises a chelating agent covalently bonded to a third click reaction partner. c. To provide a second radioactive complex comprising the second radioactive metal ion associated with the chelated portion, wherein the chelated portion comprises a chelating agent covalently bonded to a fourth click reaction partner. d. A method comprising contacting the modified polypeptide with the radioactive complex under conditions that enable the polypeptide to be labeled with the first and second radioactive metal ions by the reaction of the first click reaction partner with the third click reaction partner and the reaction of the second click reaction partner with the fourth click reaction partner, wherein the contact is performed without a copper catalyst.

23. The method according to claim 22, wherein one of the first and second click reaction partners comprises an alkyne group, the other of the first and second click reaction partners comprises an azide, one of the third and fourth click reaction partners comprises an alkene group, the other of the third and fourth click reaction partners comprises a diene, the first or second radioactive metal ion is a diagnostic emitter and the other is a therapeutic emitter, or both of the first and second radioactive metal ions are therapeutic emitters.

24. A pharmaceutical composition comprising a radiolabeled polypeptide prepared by the method of claim 1 or 14 and a pharmaceutically acceptable carrier.

25. A pharmaceutical composition according to claim 24, for use in a method of treating a neoplastic disease or disorder in a subject requiring treatment for a neoplastic disease or disorder, wherein the method comprises administering the pharmaceutical composition according to claim 24 to the subject.

26. A theranostic agent comprising a radiolabeled antibody prepared by the method of claim 1 or 14, and a pharmaceutically acceptable carrier, wherein the immunological properties of the radiolabeled antibody are preserved.

27. A theranostic agent prepared by the method described in claim 1, Formula (VIII): 【Transformation 5】 Or formula (IX): 【Transformation 6】 A theranostic agent having the following structure.

28. The aforementioned radioactive metal ions ３２ P, ４７ Sc, ６７ Cd, ７７ As, ８９ Sr, ９０ Y, ９９ Tc, １０５ Rh, １０９ Pd, １１１ Ag, １３１ I, １５３ Sm, １５９ Gd, １６５ Dy, １６６ Ho, １６９ Er, １７７ Lu, １８６ Re, １８８ Re, １９４ Ir, １９８ Au, １９９ Au, ２１１ At, ２１２ Pb, ２１２ Bi, ２１３ Bi, ２２３ Ra, ２２５ Ac, ２５５ Fm, ２２７ Th, ６２ Cd, ６４ Cd, ６７ Ga, ６８ Ga, ８６ Y, ８９ Zr, or １１１ A theranostic agent according to claim 26, selected from In.

29. It is a combination, a. A modified polypeptide containing a polypeptide covalently bonded to a first click reaction partner, b. A radioactive complex comprising a radioactive metal ion associated with a chelated portion, wherein the chelated portion comprises a chelating agent covalently bonded to a second click reaction partner, The above combination is used to label the polypeptide with the radioactive metal ion according to the method of claim 1, One of the first and second click reaction partners comprises an alkyne group and the other of the click reaction partners comprises an azide, or one of the first and second click reaction partners comprises an alkene group and the other of the click reaction partners comprises a diene. The chelating agent is of formula (I): 【Transformation 7】 (In the formula, Z is hydrogen, R １ , R ２ , R ３ and R ４ Each of them operates independently as CHQCO ２ X is, Q is independently hydrogen, C １ ~C ４ Alkyl or (C １ ~C ２ It is alkylphenyl, X is independently hydrogen, benzyl, and C １ ~C ４ The macrocycle comprises a structure having an alkyl group or a portion covalently bonded to the second click reaction partner. Alternatively, the chelating agent may be a combination comprising an open-chain ligand containing deferoxamine (DFO).

30. The method according to any one of claims 2, 3, or 14, wherein the radiolabeled antibody has a chelator-to-antibody ratio (CAR) of less than 3.

31. The method according to any one of claims 2, 3, or 14, wherein the radiolabeled antibody has a chelator:antibody ratio (CAR) of 2.

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