EA045797B1 - RADIOACTIVE LABELING OF POLYPEPTIDES - Google Patents

Description

Link to sequence listing submitted electronically

This application contains a sequence listing provided electronically via EFS-Web as a sequence listing in ASCII format with the file name Sequence_Listing, created on December 7, 2018 and having a size of 13.2 kb. The sequence listing provided by EFS-Web is part of the specification and is incorporated herein by reference in its entirety.

Cross reference to related applications

This application pursuant to 35 USC §119(e) claims priority to US Provisional Patent Application No. 62/599,830, filed December 18, 2017, the disclosure of which is incorporated herein by reference in its entirety.

Scope of the invention

The invention relates to methods for radioactive labeling of polypeptides, such as antibodies. In particular, the invention relates to methods of using click chemistry to label a polypeptide with a radioactive metal ion. The invention also relates to pharmaceutical compositions and uses of radiolabeled polypeptides.

Prerequisites for creating the invention

Alpha-particle-emitting radionuclides hold great promise for the treatment of malignancy due to their combination of high energy with short-range action, allowing for significant killing that is primarily localized to tumor cells (Kim, Y.S. and M.W. Brechbiel, An overview of targeted alpha therapy. Tumour Biol, 2012. 33(3): p. 573-90). Targeted delivery of alpha emitters using an antibody, scaffold protein, small molecule ligand, aptamer, or other binding moiety specific for a cancer antigen provides a method for selectively delivering a radionuclide to tumors to improve their effectiveness and reduce side effects. In conventional practice, the binding moiety is attached to a chelator, which binds to an alpha-emitting radioactive metal to form a radioactive complex. Many such examples use a monoclonal antibody (mAb) as the targeting ligand to produce what is known as a radioactive immunoconjugate.

Actinium-225 ( ²²⁵ Ac) is an alpha-emitting radioisotope that is 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). The 10-day half-life of ²²⁵ Ac is long enough to facilitate production of the radioactive conjugate, but short enough to match the circulating pharmacokinetics of delivery vehicles such as antibodies. Therefore, radioactive ^225Ac immunoconjugates are of particular interest. Additionally, ²²⁵ Ac decays through a series of stages such that it eventually emits 4 alpha particles before reaching the stable isotope ²⁰⁹ Bi, thereby increasing efficiency. Another radioisotope of interest for medical applications is lutetium-177 ( ¹⁷⁷ Lu), which emits both gamma radiation, suitable for imaging, and medium-energy beta radiation, suitable for radiation therapy. It has been shown that Lu-labeled peptides exhibit reduced damage to normal tissue, and that ¹⁷⁷ Lu-labeling allows the use of a single radiolabeled agent 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 that are used for therapeutic purposes include, for example, beta or alpha emitters such as, for example, thorium, radium, ³² P, ⁴⁷ Sc, ⁶⁷ Cu, ⁷⁷ As, ⁸⁹ Sr, ⁹⁰ Y, ⁹⁹ Tc, ¹⁰⁵ Rh, ¹⁰⁹ Pd, ¹ⁿ Ag, ¹³¹ I, ¹⁵³ Sm, ¹⁵⁹ Gd, ¹⁶⁵ Dy, ¹⁶⁶ 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. Other radioisotopes that are used for imaging include gamma-emitting radioisotopes such as, for example, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁶ Y, ⁸⁹ Zr, and ^m In.

Previous clinical and preclinical research programs have typically used 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) to chelate actinium. However, it is known that chelation of actinium using DOTA can be challenging (Deal, K.A., et al., Improved in vivo stability of actinium-225 macrocyclic complexes. J Med Chem, 1999. 42(15): p. 2988-92 ), and each antibody often requires stringent conditions or high levels of DOTA. As a result, two different approaches were used, known as 1-stage and 2-stage radiolabeling methods, each of which has its own disadvantages.

The first to develop was a 2-stage method, which includes 2 chemical stages involving actinium (McDevitt, MR, et al., Tumor therapy with targeted atomic nanogenerators. Science, 2001. 294(5546): p. 1537-40). ^225Ac was chelated with the bifunctional chelator (BFC) DOTA isothiocyanate (DOTA-SCN) with high radiochemical yield (~95%) at pH 4.5-5 using 2 M acetate buffer at 55-60°C for 30 min. Ac-DOTA-SCN was then brought into contact with a targeting antibody to form a radioactive immunoconjugate. The main disadvantage of the 2-step method is that ~90% of SCN does not withstand labeling conditions, therefore ~90% of the introduced Ac is conjugated to non-reactive forms of DOTA, which cannot be conjugated to the antibody. This results not only in low yields (typically only about 10%) and higher costs, but also in reduced specific activity, which can limit the effectiveness of the final conjugate.

More recently, a 1-step method was developed for actinium (Maguire, WF, et al., Efficient 1step 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 has only 1 stage of a chemical reaction involving actinium. First, DOTA-SCN was conjugated to the antibody. ²²⁵ Ac was then chelated with DOTA mAb under mild conditions (37°C, pH 7.5), achieving up to 80% radiochemical yield. However, to achieve high yields, high levels of DOTA (∼10 or more per antibody) had to be conjugated. At high chelator:antibody ratio (CAR), in this case high DOTA:Ab ratio (DAR), species are more prone to impaired immunoreactivity; furthermore, although the average DAR may be 10, it is likely that ²²⁵ Ac is chelated in the population at ratios even higher than the average. Thus, this method reduces the risk of ²²⁵ Ac binding to the least active fraction of the antibody chelator. Additionally, the antibody and DOTA-mAb conjugate must be processed under metal-free conditions to avoid chelation of common metals such as iron, zinc, and copper, which pose significant challenges during the manufacturing process.

Click chemistry is a chemical approach introduced by Sharpless in 2001 that describes chemical reactions specifically tailored to produce substances quickly and reliably by combining small 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 may be classified as click reactions) include, but are not limited to, the formation of esters, thioesters, amides (eg, such as peptide coupling) from activated acids or acyl halides; nucleophilic substitution reactions (eg, such as nucleophilic halide substitution or ring opening of strained ring systems); Huisgen azide-alkyne cycloaddition (eg, 1,3-dipolar cycloaddition between an azide and an alkyne to form a 1,2,3-triazole linker); thiol-ene addition; imine formation; Diels-Alder reactions between tetrazines and trans-cyclooctene (TCO); and Michael additions (eg maleimide addition).

Click reactions between alkynes and azides typically require the addition of a copper catalyst to accelerate the 1,3-cycloaddition reaction and are known as copper-catalyzed azidalkyne cycloaddition (CuAAC) reactions. However, click reactions between cyclooctyne or cyclooctyne derivatives and azides generally do not require the addition of a copper catalyst, but instead proceed via voltage-promoted azide-alkyne cycloaddition (SPAAC) (Debets, M.F., et al., Bioconjugation with strained alkenes and alkynes. Ace Chem Res, 2011. 44(9): p. 805-15).

Site-specificity has become the most important focus in the development of antibody-drug conjugates (ADCs) (Agarwal, P. and C.R. Bertozzi, Site-specific antibody-drug conjugates: the nexus of bioorthogonal chemistry, protein engineering, and drug development. Bioconjug Chem, 2015. 26(2): pp. 176-92), as it has been demonstrated that both the efficacy and safety of ADCs can be improved using site-specific conjugation methods compared to random conjugation. It is believed that similar benefits in terms of safety and efficacy can be achieved for radioactive immunoconjugates.

As stated above, there remains a current need for efficient methods for producing stable radioactive immunoconjugates with high specific activity and high yield.

Summary of the invention

The invention addresses this need by providing methods for applying click reactions to radiolabeled polypeptides such as antibodies. In the method according to the invention, an azide-modified antibody and a radioactive complex containing a radioactive metal ion associated with a chelating moiety containing an alkyne group are used in click chemistry to obtain stable radioactive immunoconjugates having a low chelator:antibody ratio (CAR) and high radiochemical yields, in this case, a reduced use of radioactive metal is necessary, and metal-free conditions are required only at the stage of obtaining the initial radioactive complex. The methods of the invention simplify previous methods for preparing radioactive immunoconjugates with increased safety, efficiency and uniformity.

In one general aspect, the invention relates to a method of labeling a polypeptide with a radioactive metal ion, comprising:

a. providing a modified polypeptide comprising a polypeptide covalently linked to a first click reaction partner;

b. providing a radioactive complex containing a radioactive metal ion associated with a chelating moiety, wherein the chelating moiety comprises a chelating agent covalently linked to a second click reaction partner; And

c. bringing the modified polypeptide into contact with a radioactive complex under conditions

- 2 045797 x allowing a first click partner to react with a second click partner to thereby label the polypeptide with a radioactive metal ion.

In another general aspect, the invention relates to a pharmaceutical composition comprising a radiolabeled polypeptide obtained using the method of the invention and a pharmaceutically acceptable carrier.

In another general aspect, the invention relates to a method of treating a neoplastic disease or disorder in a subject in need thereof, comprising administering to the subject a pharmaceutical composition according to the invention.

In another general aspect, the invention relates to a combination or set comprising:

a. a modified polypeptide comprising a polypeptide covalently linked to a first click reaction partner; And

b. a radioactive complex containing a radioactive metal ion associated with a chelating moiety, wherein the chelating moiety comprises a chelating agent covalently linked to a second click reaction partner;

wherein the combination or set must be used to label the polypeptide with a radioactive metal ion.

In other general aspects, the invention relates to a therapeutic or diagnostic agent (theranostic agent) containing a radiolabeled polypeptide obtained using the method of the invention.

Brief description of graphic materials

The foregoing summary of the invention, as well as the following detailed description of the invention, will be better understood when taken in conjunction with the accompanying drawings. It should be understood that the invention is not limited to the exact embodiments shown in the drawings.

The graphic materials show the following:

In fig. 1 shows a diagram of radioactive labeling of an antibody in accordance with the method of the present invention; the figure shows random conjugation, whereby when azides are site-specifically conjugated to a monoclonal antibody (mAb), a similar radiolabeling scheme is used;

in fig. 2 shows a synthesis scheme for an improved two-step production of ⁸⁹ Zr-DOTA mAb using click reactions in accordance with an embodiment of the application;

in fig. Figure 3 shows cellular binding of In-111 radioimmunoconjugates: bound radioactivity increases with increasing number of cells; in particular:

in fig. 3A shows binding to the prostate cancer cell line C4-2B (PSMA+, transferrin receptor+) using a radioactive PSMA-binding antibody (PSMB127)-In-111 immunoconjugate, and a radioactive human transferrin-In-111 conjugate, in accordance with embodiments of the application. ; and in fig. 3B shows binding to the human epidermoid carcinoma cell line A431 (EGFR+) by radioactive immunoconjugates of the EGFR-binding antibodies cetuximab and panitumumab with In-111 in accordance with embodiments of the application, and the absence of binding of these conjugates to a control (EGFR−) human AML cell line MOLM-13;

in fig. 4 shows the kinetics of cellular internalization of In-111 in the C4-2B prostate cancer cell line treated with a radioactive anti-PSMA mAb immunoconjugate to In-111 in accordance with an embodiment of the application; surface-bound In-111 quickly disappeared from the cell surface and was redistributed intracellularly; and in fig. Figure 5 shows the results of a tumor xenotransplantation study in mice; mice were implanted with LNCaP human prostate cancer cells; When tumors reached 100 mm ³ , mice were treated with a single dose of click-radiolabeled anti-PSMA mAb (PSMB127) actinium conjugate according to one of the activity range applications or isotype control, a human IgG4 antibody that binds to a viral target not present in this radioactive conjugate system; in particular:

in fig. Figure 5A shows the tumor volume for each group; sizes were measured until less than half the group remained;

in fig. 5B shows survival curves for control mAb groups; and in fig. Figure 5C shows the survival curves for the anti-PSMA mAb groups.

Detailed Description of the Invention

In the Background to the Invention section and in the text of this application, citations or descriptions of various publications, articles and patents are provided; each of these references being incorporated herein by reference in their entirety. The discussion of documents, acts, materials, devices, products, and the like that have been included in the present description is provided as context for the present invention. Such discussion is not an admission that any or all such sources are part of the prior state of knowledge with respect to any inventions described or claimed.

All technical and scientific terms used herein, unless otherwise noted, have the same meaning as would be commonly understood by one skilled in the art to which this invention relates. Otherwise, certain terms used herein have the meanings set forth herein. All patents, published patent applications and publications cited herein are incorporated by reference as if set forth in their entirety herein.

It should be noted that in this document and in the attached claims, the singular form includes objects in the plural unless the context clearly indicates otherwise.

Throughout this specification and the following claims, unless the context otherwise requires, the word contain and its variations such as contains and containing are to be understood to mean the inclusion of said integer or stage, or group of integers or stages, but not the exclusion of any another integer or stage or group of integers or stages. When used herein, the term containing may be replaced by the term including, or sometimes when used herein may be replaced by the term having.

As used herein, the term consisting of excludes any element, step or ingredient not mentioned in the claim element. As used herein, the term essentially consisting of does not exclude materials or steps that do not materially affect the essential and novel features of the claims. Any of the above terms containing, including, including and having, when used herein in the context of an aspect or embodiment of the description, may be replaced by the term consisting of or consisting essentially of to vary the scope of the description.

In the context of this document, the connecting term and/or between a plurality of recited elements should be understood to include both individual and combined options. For example, if two elements are connected and/or, the first option refers to the possibility of using the first element without the second. The second option refers to the possibility of using a second element without the first. The third option refers to the possibility of using the first and second elements together. Either of these options is intended to be consistent with the meaning and therefore satisfy the requirement of the term and/or within the context of this document. It is also intended that the simultaneous application of more than one of these options corresponds to the meaning and, accordingly, satisfies the requirement of the term and/or.

In an attempt to assist the reader of the application, the description has been divided into various paragraphs or sections or directed to various embodiments of the application. These divisions should not be construed as separating the essence of a paragraph or section or embodiments from the essence of another paragraph or section or embodiments. On the contrary, one skilled in the art will appreciate that the description is broad in scope and covers all combinations of the various sections, paragraphs and sentences that may be contemplated. The discussion of any embodiment is intended to be exemplary only and is not intended to imply that the scope of the description, including the claims, is limited to those examples.

Radioactive labeling of polypeptides using the click reaction

In contrast to known procedures, the methods of the present invention provide an improved method for producing radioactive immunoconjugates that are suitable, for example, for medical use in subjects, eg humans, in need thereof. In particular, the methods described herein address the major limitations of existing methods by providing processes for both high yield metal ion chelation, including, but not limited to, ²²⁵ Ac, ¹ⁿ In and ⁸⁹ Zr, and low DAR . The invention allows for the production of a single batch of azide-labeled polypeptides, such as an azide-mAb conjugate, which can then be used to produce a radiolabeled agent for diagnostic (for example, when labeled with ⁸⁹ Zr or ¹ⁿ In) or therapeutic (for example, when labeled with ²²⁵ Ac) purposes , in which a radiolabel is attached at the same site(s) within a batch of azide-labeled polypeptides, which can be produced either by site-specific modification or by random azide conjugation. For example, in random azide conjugation, samples of a batch of azide-labeled polypeptides containing one distribution of azide-modified sites can be radiolabeled for various purposes using click reactions according to the invention.

The method of the invention, which is based on click reactions and is called click reaction radiolabeling, involves (1) producing a modified polypeptide, such as an antibody, that contains a click reaction partner, for example, an azide moiety; (2) producing a radioactive complex containing a radioactive metal ion, for example, ²²⁵ Ac, ⁿ¹ In or ⁸⁹ Zr, associated with a chelating moiety, where the chelating moiety contains a chelating agent covalently linked to a second click reaction partner, for example an alkyne group, such as

- 4 045797

DOTA-dibenzocyclooctine (DOTA-DBCO) or deferoxamine-DBCO (DFO-DBCO); and (3) performing a reaction between click reaction partners of the modified peptide and the radioactive complex, such as a voltage-promoted azide-alkyne cycloaddition (SPAAC) between an azide moiety and an alkyne group.

The method according to the invention allows the radioactive metal to be chelated under conditions of low or high pH and/or high temperature to achieve maximum efficiency, which can be achieved without the risk of inactivation of the alkyne reaction partner. Efficient chelation and efficient SPAAC reaction between the azide-mAb and the radioactive complex allows the preparation of radioimmunoconjugates with high radiochemical yield even at low azide:mAb ratios. In the method according to the invention, the only step in which trace metals must be excluded is the chelation of radioactive metal ions into the chelating moiety; antibody production, purification and conjugation steps should not be carried out under metal-free conditions.

As used herein, click chemistry refers to a chemical concept introduced by Sharpless that describes chemical reactions specifically designed to rapidly and reliably form covalent bonds by combining small units containing reactive groups (see Kolb, et al., supra). Click chemistry does not refer to a specific reaction, but rather a concept that includes, but is not limited to, reactions that mimic reactions found in nature. In some embodiments, click reactions are modular, have a wide range of applications, produce high reaction products, produce safe byproducts, are stereospecific, exhibit a large thermodynamic driving force that favors reaction with a single reaction product, and/or can be carried out under physiological conditions . In some embodiments, the click reaction exhibits high atom economy, can be carried out under simple reaction conditions, uses readily available starting materials and reagents, does not use toxic solvents, or uses a solvent that is mild or easily removable such as water, and/or allows easy isolation of the product by non-chromatographic methods such as crystallization or distillation. In certain embodiments, the click reaction is a Huisgen cycloaddition or a 1,3-dipolar cycloaddition between an azide (-N3) and an alkyne or alkyne moiety to form a 1,2,4triazole linker.

In a General aspect, the invention relates to a method of labeling a polypeptide, aptamer or low molecular weight ligand with a radioactive metal ion, comprising:

a. providing a modified polypeptide comprising a polypeptide covalently linked to a first click reaction partner;

b. providing a radioactive complex containing a radioactive metal ion associated with a chelating moiety, wherein the chelating moiety comprises a chelating agent covalently linked to a second click reaction partner; And

c. contacting the modified polypeptide with a radioactive complex under conditions allowing a first click reaction partner to react with a second click reaction partner to thereby label the polypeptide with a radioactive metal ion.

As used herein, the term polypeptide refers to a polymer consisting of amino acid residues, their associated natural structural variants, and their synthetic non-natural analogues linked by 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. The polypeptide may be natural, recombinant or synthetic, or any combination thereof. Synthetic polypeptides can be synthesized, for example, using an automatic polypeptide synthesizer. In preferred embodiments, the polypeptide is an antibody, preferably a monoclonal antibody, or a fragment thereof, such as an antigen binding fragment thereof. In accordance with preferred embodiments, the antibody or fragment thereof is specific for a cancer antigen. In other embodiments, the polypeptide is an engineered domain or scaffold protein.

As used herein, the term antibody or immunoglobulin is used in a broad sense and refers to immunoglobulin or antibody molecules, including polyclonal antibodies, monoclonal antibodies including murine/rat, human, human-adapted, humanized and chimeric monoclonal antibodies and antigen binding fragments thereof.

In a general sense, antibodies are proteins or peptide chains that specifically bind to a specific antigen. The structures of antibodies are well known. Immunoglobulins can be classified into 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. Accordingly, the antibodies of the present invention may be any of five major classes or corresponding subclasses. Preferably, the antibodies of this invention are IgG1, IgG2, IgG3 or IgG4. Lungs

- 5 045797 antibody chains of any vertebrate species can be classified, depending on the amino acid sequences of their constant domains, into one of two clearly distinct types, namely kappa and lambda. Accordingly, the antibodies of the invention may contain a kappa or lambda light chain constant domain. In certain embodiments, the antibodies of the invention comprise heavy and/or light chain constant regions, murine antibodies, or human antibodies. Each of the four IgG subclasses has different biological functions, known as effector functions. These effector functions are typically mediated through interaction with the Fc receptor (FcyR) or through C1q binding and complement fixation. Binding to FcyR can lead to antibody-dependent cell-mediated cytolysis, while binding to complement factors can lead to complement-mediated cell lysis. An antibody useful in the invention may have no or minimal effector function but retain its ability to bind FcRn.

As used herein, the term antigen binding fragment refers to an antibody fragment such as, for example, a diabody, Fab, Fab', F(ab')2, Fv fragment, disulfide bond stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (ds-diabody), single-chain antibody molecule (scFv), single-domain antibody (sdAb), scFv dimer (divalent antibody), multispecific antibody formed from an antibody moiety containing one or more CDR, camel single domain antibody, nanoantibody, domain antibody, divalent domain antibody, or any other antibody fragment that binds to an antigen but does not contain the structure of a full antibody. The antigen-binding fragment has the ability to bind to the same antigen to which the parent antibody or parent antibody fragment binds. As used herein, the term single chain antibody refers to an industry standard single chain antibody that comprises a heavy chain variable region and a light chain variable region linked by a short peptide of about 15 to about 20 amino acids in size. As used herein, the term single-domain antibody refers to a field-standard single-domain antibody that contains a heavy chain variable region and a heavy chain constant region, or that contains only a heavy chain variable region.

As used herein, the term scaffold or scaffold protein refers to any protein that has a target binding domain and that can bind to a target. The framework contains a framework region, which is typically structural, and a binding domain, which contacts the target and mediates specific binding. The scaffold binding domain need not be defined by a single contiguous scaffold sequence. In some cases, the scaffold may be part of a larger binding protein, which itself may be part of a multimeric binding protein that contains multiple scaffolds. Some binding proteins may be bi- or multispecific in the sense that they can bind to two or more different epitopes. The scaffold may be derived from a single chain antibody, or the scaffold may be derived from a non-antibody.

The polypeptides of the invention may be covalently linked to a first click reaction partner using any method of chemical or enzymatic modification of the polypeptide known to those skilled in the art in light of this disclosure. Methods for randomly modifying polypeptides can use amine-reactive groups that react with primary amines that are present at the N-terminus of each polypeptide chain and on the side chain of lysine residues. Examples of amine-reactive groups suitable for use in the invention include, but are not limited to, NHS-substituted N-hydroxysuccinimide (NHS), such as sulfo-NHS, isothiocyanate, and tetra- and per-fluorophenyl ether. Methods for randomly modifying polypeptides can use thiol-reactive groups that react with thiols or sulfhydryls that exist on the side chain of cysteine residues. Examples of thiol-reactive groups suitable for use in the invention include, but are not limited to, maleimide, haloacetyl and phenyloxadiazole sulfone. In preferred embodiments, the modified polypeptide is prepared by contacting a side chain, preferably the amino side chain of a lysine, with an electrophile covalently linked to a first click reaction partner (eg, NHS azide).

The method according to the invention further allows for the production of site-specific radiolabeled polypeptides. The click reaction radiolabeling method of the invention facilitates the site-specific preparation of radioactive immunoconjugates by taking advantage of known 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 azidealkyne cycloadditions. Angew Chem Int Ed Engl, 2014. 53(28): pp. 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 attaching molecules to proteins or antibodies in a site-specific manner are known in the art, and any method for site-specific labeling of antibodies is known in the art.

- 6 045797 known to those skilled in the art, can be used in the invention taking into account this description. Examples of methods for site-specific modification of antibodies suitable for use in the invention include, but are not limited to, the inclusion of engineered cysteine residues (e.g., THIOMAB™), the use of unnatural amino acids or glycans (e.g., selenocysteine, p-AcPhe, formylglycine-forming enzyme (FGE, SMARTag™, etc.), and enzymatic methods (for example, the use of glycotransferase, endoglycosidase, microbial or bacterial transglutaminase (MTG or BTG), sortase A, etc.). In preferred embodiments, the modified polypeptide is an antibody or antigen-binding fragment thereof that is produced by tailoring the antibody or antigen-binding fragment thereof with a bacterial endoglycosidase specific for the β-1,4 linkage between core GlcNac residues at the Fc glycosylation site of the antibody, e.g. , GlycINATOR (Genovis), which leaves the internal GlcNAc intact on the Fc, allowing for site-specific incorporation of azidosugars at that site. The match or an antigen-binding fragment thereof can then react with an azide-labeled sugar, such as UDP-N-azidoacetylgalactosamine (UDP-GalNaz) or UDP-6azido-6-deoxy-GalNAc, in the presence of a glycosyltransferase, such as GalT galactosyltransferase or GalNAc transferase, to thus obtaining a modified antibody or antigen-binding fragment thereof. In other preferred embodiments, the modified polypeptide is an antibody or antigen binding fragment thereof that is produced by deglycosylation of the antibody or antigen binding fragment thereof with an amidase. The resulting deglycosylated antibody or antigen-binding fragment thereof can then be reacted with an azidoamine, preferably 3-azidopropylamine, 6-azidohexylamine or any azido-linker amine or any azido-alkylamine such as an azido-polyethylene glycol (PEG)-amine, e.g. -(2-aminoethyl)-O'-(2-azidoethyl)tetraethylene glycol, O-(2-aminoethyl)-O'-(2azidoethyl)pentaethylene glycol, O-(2-aminoethyl)-O'-(2-azidoethyl)triethylene glycol etc. or in the presence of microbial transglutaminase, thereby obtaining a modified antibody or antigen-binding fragment thereof.

As used herein, the term aptamer refers to a single-stranded oligonucleotide (single-stranded DNA or RNA molecule) that can specifically bind to its target with high affinity. The aptamer can be used as a molecule targeting various organic and inorganic substances.

As used herein, the term low molecular weight ligand refers to a low molecular weight organic compound. As used herein, small molecule ligands can refer to compounds that are less than about 1000 daltons in size and can be synthesized in the laboratory or found in nature.

As used herein, the term click reaction partner or click reaction handle refers to a reagent or reactive group that can participate in a click reaction. The click reaction partner may be a moiety that is rare in natural biomolecules and is chemically inert to the biomolecule, but, for example, when reacted with an azide-reactive or alkyne-reactive group, the reaction can occur efficiently under biologically relevant conditions, e.g. cell culture conditions, such as the absence of excess heat or aggressive reagents. Typically, click reactions require at least two molecules containing click reaction partners that can interact with each other. Such click-reaction partners that react with each other are sometimes referred to herein as click-reaction handle pairs or click-reaction pairs. In some embodiments, the click reaction partners are an azide and a strained alkyne, such as cyclooctyne or any other alkyne. In other embodiments, the click reaction partners are reactive dienes and suitable tetrazine dienophiles. For example, transcyclooctene, norbornene or biscyclononene can be coupled with a suitable tetrazine dienophile as a click reaction pair. In yet other embodiments, tetrazoles can act as latent sources of nitrilimines, which can combine with non-activated alkenes in the presence of ultraviolet light to form a click reaction pair, called a photo click reaction pair. In other embodiments, the click reaction partners are cysteine and maleimide. For example, a cysteine from a peptide (eg, GGGC) can interact with a maleimide that is bound to a chelating agent (eg, NOTA). Other suitable click reaction staples 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 other embodiments, the click reaction partners are Staudinger ligation moieties 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-response partners are described in US20130266512 and in WO2015073746, the corresponding click-response partner descriptions in both cases are included herein

- 7 045797 via link. In preferred embodiments, one of the first and second click partners contains an alkyne group and the other click partner contains an azide. In other preferred embodiments, one of the first and second click partners contains an alkene group and the other click partner contains a diene.

As used herein, the term alkyne, alkyne group, or alkyne moiety refers to a functional group containing a carbon-carbon triple bond. Alkyne moieties include terminal alkynes and cycloalkynes, preferably terminal alkynes and cycloalkynes that react with azide groups. A terminal alkyne has at least one hydrogen atom bonded to a carbon atom with a triple bond. A cycloalkyne is a cycloalkyl ring containing one or more triple bonds. Examples of cycloalkynes include, but are not limited to, cyclooctyne and cyclooctyne derivatives such as bicyclononine (BCN), cyclooctyne difluoride (DIFO), dibenzocyclooctine (DIBO), keto-DIBO, biarylazacyclooctynone (BARAC), dibenzoazacyclooctine (DIBAC), dimethoxyazacyclooctine (DIMAC) , dibenzocyclooctine (DBCO), difluorobenzocyclooctine (DIFBO), monobenzocyclooctine (MOBO) and tetramethoxy-DIBO (TMDIBO). In preferred embodiments, one of the first and second click partners comprises a cycloalkyne, preferably DBCO. In preferred embodiments, the other click reaction partner comprises an azide, preferably an NHS azide.

As used herein, the term diene refers to a compound having two carbon-carbon double bonds, the double bonds being conjugated at the 1,3 position. Diene double bonds can be cis or trans. Examples of dienes include, but are not limited to, a tetrazine or tetrazole group.

As used herein, the term alkene, alkene group, or alkene moiety refers to an unsaturated hydrocarbon molecule that contains a carbon-carbon double bond. In certain embodiments, an alkene may contain from 2 to 100 carbon atoms. Examples of alkenes include, but are not limited to, norbornene and transcyclooctene (TCO). In other preferred embodiments, one of the first and second click partners contains an alkene group, preferably norbornene or TCO. In preferred embodiments, the other click reaction partner contains a diene, preferably a tetrazine or tetrazole group.

As used herein, the term covalently linked means that the polypeptide is attached to a first click reaction partner by at least one covalent bond, and that the chelatin is attached to a second click reaction partner by at least one covalent bond. The connection can be direct, that is, without a linker, or indirect, that is, through a linker.

As used herein, the term linker refers to a chemical moiety that attaches a polypeptide or chelating agent to a click reaction partner. Any suitable linker known to those skilled in the art in light of this disclosure may be used in the invention. Linkers 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-based linker, or a cleavable linker such as a disulfide bond or a protease cleavage site such as valine-citrulline-RAB.

As used herein, the term radioactive metal ion or radiometal ion refers to one or more isotopes of elements that emit particles and/or photons. The invention may use any radioactive metal known to those skilled in the art given this description. Examples of radioactive metals suitable for use in the 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, ¹ⁿ Ag, ¹ⁿ In, ¹¹⁷ Sn, 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, ²²⁷ Th, and ²⁵⁵ Fm. As used herein, the term diagnostic emitter refers to a radioactive metal ion that is useful in a diagnostic or imaging application. Examples of diagnostic emitters include, but are not limited to, gamma emitters such as ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁶ Y, ⁸⁹ Zr, and ⁿ¹ In. As used herein, the term therapeutic emitter refers to a radioactive metal ion that is useful in use for therapeutic purposes. Examples of therapeutic emitters include, but are not limited to, 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, 111Ag, 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. In preferred embodiments, the radioactive metal ion is Ac. In accordance with preferred embodiments, the polypeptide may be labeled with non-metallic radioactive labels for use in pre-targeting or theranostic applications. Examples of non-metallic radioactive tracers suitable for use in the invention include, but are not limited to, ¹²⁵ I and ¹⁸ F.

- 8 045797

The radioactive complexes described herein contain a radioactive metal ion associated with a chelating moiety. In accordance with embodiments of the invention, the chelating moiety comprises a chelating agent covalently linked to a click reaction partner, sometimes referred to herein as a bifunctional chelator.

As used herein, the term chelating agent or chelator refers to a chemical compound that can chelate a radioactive metal, such as Ac, or a metal through coordination bonding. Any chelating agent known to those skilled in the art in light of this description may be used in the invention. In one embodiment, the chelating agent contains a macrocycle. Examples of macrocycle chelating agents suitable for use in the invention include, but are not limited to, deferoxamine (DFO), ethylenediaminetetraacetic acid (EDTA), and diethylenetriaminepentaacetic acid (DTPA). In another embodiment, the chelating agent contains an open chain ligand. Examples of open chain ligand chelating agents suitable for use in the invention include, but are not limited to, 1,4,7,10-tetraazacyclododecaneN,N',N,N'-tetraacetic acid (DOTA), 1,4 ,7,10,13,16-hexaazacyclohexadecane-N, N',N,N',N, N'-hexacetic acid (NEHA), 1,4,7,10,13-pentaazacyclopentanadecane-N, N',N ,N',N-penta acetic acid (PEPA), 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), 1,4,7,10tetraazacyclododecane-1,4,7,10-tetrapropionic acid (DOTPA), 1,4,8,11-tetraazacyclotetradecane1,4,8,11-tetrapropionic acid (TETRA) and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetramethylene phosphonic acid (DOTMP ). According to preferred embodiments, the chelating agent contains the structure of formula (I):

formula (I) wherein R1, _R2 , _R3 and R are each independently CHQCO ₂ X, wherein Q is independently hydrogen, C1-C4 alkyl or (C1-C2 alkyl)phenyl, and X is independently hydrogen , benzyl, C1-C4 alkyl; and Z is (CH ₂ ) _n Y, where n is 1-10, and

Y is an electrophilic or nucleophilic moiety covalently linked to a second click reaction partner;

alternatively, Z is hydrogen; and each of R1, _R2 , _R3 and R4 independently represents CHQCO2X, where

Q is independently hydrogen, C1-C4 alkyl, or (C1-C2 alkyl)phenyl, and X is independently hydrogen, benzyl, C1-C4 alkyl, or an electrophilic or nucleophilic moiety covalently linked to a second click reaction partner.

According to preferred embodiments, the chelating moiety comprises the structure of formula (II):

O'OH formula (II).

According to preferred embodiments, the chelating moiety contains the structure of formula (III):

- 9 045797

formula (III).

In an embodiment, the invention relates to a method for labeling a polypeptide with two or more radioactive metal ions using the method of the invention. For example, a method of double labeling a polypeptide with ions of two radioactive metals includes:

a. providing a modified polypeptide comprising a polypeptide covalently linked to a first click reaction partner and a second click reaction partner;

b. providing a first radioactive complex comprising a first radioactive metal ion linked to a chelating moiety, wherein the chelating moiety comprises a chelating agent covalently linked to a third click reaction partner; And

c. providing a second radioactive complex comprising a second radioactive metal ion linked to a chelating moiety, wherein the chelating moiety comprises a chelating agent covalently linked to a fourth click reaction partner; And

d. contacting the modified polypeptide with the first and second radioactive complexes under conditions allowing the first click partner to react with the third click partner, and the second click partner to react with the fourth click partner, thereby marking polypeptide with ions of the first and second radioactive metals.

In preferred embodiments, one of the first and second click partners contains an alkyne group, and the other of the first and second click partners contains an azide, and wherein one of the third and fourth click partners contains an alkene group, and the other of the third and fourth click reaction partners contains a diene.

In accordance with preferred embodiments, the first or second radioactive metal ion is a diagnostic emitter and the other is a therapeutic emitter. In accordance with preferred embodiments, both the first and second radioactive metal ions are therapeutic emitters.

Conditions for carrying out click reactions are known in the art, and any conditions for carrying out click reactions known to those skilled in the art, taking into account this description, can be used in the invention. Examples of conditions include, but are not limited to, incubation of the modified polypeptide and 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.

The products of the click reaction radiolabeling methods of the invention can be analyzed using methods known to those skilled in the art in light of this disclosure. For example, LC/MS analysis can be used to determine the ratio of chelator to labeled polypeptide; analytical size exclusion chromatography can be used to determine the oligomeric state of polypeptides and polypeptide conjugates; radiochemical yield can be determined using flash thin layer chromatography (e.g. iTLC-SG), and radiochemical purity can be determined using size exclusion HPLC. Examples of methods are described herein, for example in the examples below.

Pharmaceutical compositions and methods of treatment

The click reaction radiolabeling method of the invention can be modified into a pre-targeting approach (Kraeber-Bodere, F., et al., A pretargeting system for tumor PET imaging and radioimmunotherapy. Front Pharmacol, 2015. 6: p. 54) . First, the azido-mAb is administered, it binds to the target cells, and is allowed to be cleared from the circulation over time or removed by a clearing agent. Subsequently, the radioactive complex is administered and undergoes a SPAAC reaction with the azido-mAb bound at the target site, while the remaining unbound radioactive complex is rapidly cleared from the circulation (Deal, K.A., et al., Improved in vivo stability of actinium-225 macrocyclic complexes J Med Chem, 1999, 42(15): pp. 2988-92). This pre-targeting technique provides a method of enhancing the localization of radioactive metal ions at a target site in a subject

Accordingly, in another general aspect, the invention relates to a pharmaceutical composition comprising a radiolabeled polypeptide obtained using the method of the invention and a pharmaceutically acceptable carrier.

- 10 045797

As used herein, the term carrier refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposomal encapsulation, or other material well known in the art for use in pharmaceutical formulations. It should be understood that the characteristics of the carrier, excipient or diluent will depend on 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 adversely affect the effectiveness of the composition of the present invention or the biological activity of the composition of the present invention. In certain embodiments, subject to this disclosure, any pharmaceutically acceptable carrier suitable for use in an antibody-based or radioactive complex-based pharmaceutical composition may be used in the invention.

In accordance with specific embodiments, the compositions described herein are formulated to be suitable for the intended route of administration to a patient. For example, the compositions described herein may be formulated to be suitable for intravenous, subcutaneous, intramuscular, or intratumoral administration.

In certain embodiments, the modified polypeptide and the radioactive complex may be administered in the same or different compositions.

In another general aspect, the invention relates to a method of treating a neoplastic disease or disorder in a subject in need thereof, comprising administering to the subject a pharmaceutical composition according to the invention.

In accordance with specific embodiments, the method of the invention comprises administering a therapeutically effective dose of a pharmaceutical composition of the invention, wherein the composition comprises a radiolabeled polypeptide for targeting cells associated with a neoplastic disease or disorder such that, upon targeting, ^225Ac alpha particles and its products breakdown agents are delivered to the target cells and cause a cytotoxic effect, thereby providing treatment for the neoplastic disease or disorder.

In accordance with specific embodiments, therapeutically effective amounts of the modified polypeptide and radioactive complex are administered in different compositions.

As used herein, the term therapeutically effective amount refers to an amount of an active ingredient or component that induces a desired biological or medical response in a patient. The therapeutically effective amount can be determined empirically and routinely depending on the intended purpose. For example, in vitro assays may not necessarily be used to determine the optimal dose range. The selection of a specific effective dose can be made (eg, through clinical trials) by one skilled in the art, taking into account several factors, including the disease being treated or prevented, symptoms, the patient's body weight, the state of the patient's immune system, and other factors known to one of skill in the art. The exact dose to be used in a formulation also depends on the route of administration and the severity of the disease and should be determined based on the judgment of the healthcare professional and the condition of each patient. Effective doses can be calculated from dose response curves obtained in in vitro test systems or animal models.

As used herein, the terms treat, treater, and treatment are intended to mean an improvement or change in at least one measurable physical parameter associated with a disease, disorder, or condition that would benefit from administration of a radioactive metal ion, such as a neoplastic disease or disorder. which are not necessarily clearly expressed in the subject, but may be clearly expressed in the subject. The terms treat, treater, and treatment can also mean inducing regression, preventing progression, or at least slowing the progression of a disease, disorder, or condition. In a specific embodiment, treat, treat, and treat refer to alleviating, preventing the development or manifestation of, or reducing the duration of one or more symptoms associated with a disease, disorder, or condition that would benefit from administration of a radioactive metal ion, such as a neoplastic disease or disorder. In a specific embodiment, the terms treat, treater, and treatment refer to the prevention of recurrence of a disease, disorder, or condition. In a specific embodiment, the terms treat, treater, and treatment refer to improving the survival of a patient having a disease, disorder, or condition. In a specific embodiment, the terms treat, treater, and treatment refer to treating a disease, disorder, or condition in a patient.

Examples of neoplastic diseases or disorders include, but are not limited to, disseminated malignancy, solid malignancy, hypertrophy, coronary artery disease or vascular occlusion, a disease or disorder associated with an infected cell, microorganism, or virus, or a disease or disorder associated

- 11 045797 noe with an inflammatory cell, such as rheumatoid arthritis (RA).

As used herein, the term subject refers to an animal, preferably a mammal. In certain embodiments, the patient is a mammal, including non-primate mammals (e.g., camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, rabbit, guinea pig, marmoset, or mouse ) or primates (such as monkey, chimpanzee or human). In specific embodiments, the patient is a human.

Any scheme for using a modified polypeptide and a radioactive complex can be used taking into account this description. Generally, when the modified polypeptide and the radioactive complex are administered in different compositions, the radioactive complex can be administered at any time after administration of the modified antibody.

In accordance with specific embodiments, compositions used to treat a neoplastic disease or disorder can be used in combination with other agents that are effective in treating the related neoplastic diseases or disorders.

As used herein, the term combination in the context of administering two or more drugs to a patient refers to the use of more than one therapy. Use of the term in combination does not limit the order in which the drugs are administered to a subject. For example, the first therapeutic agent (e.g., a composition described herein) may be administered 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), after (for example, after 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) of administration of the second therapeutic agent to the patient or simultaneously with such administration.

In another general aspect, the invention relates to a theranostic agent comprising a radiolabeled antibody produced by the method of the invention and a pharmaceutically acceptable carrier, wherein the immunological properties of the radiolabeled antibody are retained.

As used herein, the term theranostic refers to the ability to provide any of diagnostic and therapeutic functions. In one embodiment, the theranostic agent provides both diagnostic and therapeutic functions. In another embodiment, the theranostic agent is an active pharmaceutical agent without a diagnostic function. In yet another embodiment, the theranostic agent is an agent useful for diagnosis, but does not have a therapeutic function.

In accordance with preferred embodiments, the radioactive metal ion is a diagnostic emitter, preferably ⁸⁹ Zr. In other preferred embodiments, the radioactive metal ion is a therapeutic emitter, preferably ²²⁵ Ac. In other preferred embodiments, the theranostic agent is used to provide both diagnostic and therapeutic functions to a subject in need thereof.

Combinations and sets

This document proposes a combination containing:

a. a modified polypeptide comprising a polypeptide covalently linked to a first click reaction partner; And

b. a radioactive complex containing a radioactive metal ion associated with a chelating moiety, wherein the chelating moiety comprises a chelating agent covalently linked to a second click reaction partner;

wherein the combination must be used to label the polypeptide with a radioactive metal ion.

In certain embodiments, the combination of the invention is a reaction mixture used to label a polypeptide with a radioactive metal ion. In other embodiments, the combination is a container or kit used to produce a radiolabeled polypeptide in vitro or in vivo. Such combinations may also further bear labels or instructions in a form prescribed by a government agency regulating the manufacture, use or distribution of pharmaceutical or biological products, said labels reflecting such agency's approval for the manufacture, use or distribution of the drug for administration to humans. The combinations covered herein may be used in the above methods of labeling a polypeptide with a radioactive metal ion or treating a neoplastic disease or disorder in a subject in need thereof.

Embodiments

The present invention also provides the following non-limiting embodiments.

Embodiment 1 is a method of labeling a polypeptide with a radioactive ion

- 12 045797 metal, including:

A. providing a modified polypeptide comprising a polypeptide covalently linked to a first click reaction partner;

b. providing a radioactive complex containing a radioactive metal ion associated with a chelating moiety, wherein the chelating moiety comprises a chelating agent covalently linked to a second click reaction partner; And

With. contacting the modified polypeptide with a radioactive complex under conditions allowing a first click reaction partner to react with a second click reaction partner to thereby label the polypeptide with a radioactive metal ion.

Embodiment 1a is the method of Embodiment 1 wherein the chelate contains a macrocycle.

Embodiment 1b is the method of Embodiment 1, wherein the chelating agent contains an open chain ligand.

Embodiment 2 is the method of Embodiment 1, wherein one of the first and second click partners contains an alkyne group and the other click partner contains an azide.

Embodiment 3 is the method of Embodiment 2, wherein the first click partner contains an azide group and the second click partner contains an alkyne group.

Embodiment 3a is the method of Embodiment 2 or 3, wherein the alkyne group includes a terminal alkyne.

Embodiment 3b is the method of Embodiment 2 or 3, wherein the alkyne group includes cycloalkynes, preferably cyclooctyne or a cyclooctyne derivative.

Embodiment 3c is the method of Embodiment 3b, wherein the alkyne group includes bicyclononine (BCN).

Embodiment 3d is the method of Embodiment 3b, wherein the alkyne group includes cyclooctyne difluoride (DIFO).

Embodiment 3e is the method of Embodiment 3b, wherein the alkyne group includes dibenzocyclooctine (DIBO).

Embodiment 3f is the method of Embodiment 3b, wherein the alkyne group includes biarylazacyclooctynone (BARAC).

Embodiment 3g is the method of Embodiment 3b, wherein the alkyne group includes dibenzoazacyclooctine (DIBAC).

Embodiment 3h is the method of Embodiment 3b, wherein the alkyne group includes dimethoxyazacyclooctine (DIMAC).

Embodiment 3i is the method of Embodiment 3b, wherein the alkyne group includes dibenzocyclooctine (DBCO).

Embodiment 3j is the method of Embodiment 3b, wherein the alkyne group includes difluorobenzocyclooctine (DIFBO).

Embodiment 3k is the method of Embodiment 3b, wherein the alkyne group includes monobenzocyclooctine (MOBO).

Embodiment 3l is the method of Embodiment 3b, wherein the alkyne group includes tetramethoxy-DIBO (TMDIBO).

Embodiment 3m is the method of any of Embodiments 2-31, wherein the azide group includes an NHS azide.

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

Embodiment 4a is the method of Embodiment 4, wherein the diene includes a tetrazine or tetrazole group.

Embodiment 4b is the method of Embodiment 4 or 4a, wherein the alkene group includes norbornene.

Embodiment 4c is the method of Embodiment 4 or 4a, wherein the alkene group contains trans-cyclooctene (TCO).

Embodiment 5 is the method of any of Embodiments 1-4c, wherein the polypeptide is an antibody or an antigen-binding fragment thereof.

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

Embodiment 6a is a method according to any of embodiments 1-6, wherein the modified polypeptide is produced by randomly conjugating one or more azide groups to the polypeptide.

- 13 045797

Embodiment 6b is the method of any of embodiments 1-6, wherein the modified polypeptide is a modified antibody or antigen binding fragment thereof obtained by site-specific incorporation of a first click reaction partner.

Embodiment 6c is the method of Embodiment 6b, wherein the modified antibody or antigen-binding fragment thereof is prepared by tailoring the antibody or antigen-binding fragment thereof with a bacterial endoglycosidase specific for the β-1,4 linkage between the core GlcNac residue(s) at the site (ah) glycosylating an Fc antibody to produce a tailored antibody or an antigen-binding fragment thereof, and contacting the tailored antibody or an antigen-binding fragment thereof with an azidosugar, preferably a UDP-GalNaz azidosugar substrate, in the presence of a glycotransferase, preferably galactosyltransferase GalT.

Embodiment 6d is the method of Embodiment 6b, wherein the modified antibody or antigen-binding fragment thereof is prepared by deglycosylating the antibody or antigen-binding fragment thereof with an amidase to produce a deglycosylated antibody or antigen-binding fragment thereof, and contacting the deglycosylated antibody or antigen-binding fragment thereof with an azidoamine, preferably 3 -azidopropylamine, in the presence of transglutaminase from microorganisms.

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

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

Embodiment 7 is the method according to any of Embodiments 1 to 6d, wherein ^the radioactive metal ion is ³² P, ⁴⁷ Sc, ⁶⁷ Cu, ⁷⁷ As, ⁸⁹ Sr, 90 Y, ⁹⁹ Tc, ¹⁰⁵ Rh, ¹⁰⁹ Pd, ^m 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 ^m In.

Embodiment 7a is the method according to any of Embodiments 1 to 6d, wherein the radioactive metal ion is ²²⁵ Ac.

Embodiment 7b is the method according to any of embodiments l-6d, wherein the radioactive metal ion is ¹¹¹ In.

Embodiment 7c is the method according to any of embodiments l-6d, wherein the radioactive metal ion is ⁸⁹ Zr.

Embodiment 8 is the method of any of Embodiments 1-7c, wherein the chelating moiety is covalently linked to the second click reaction partner via a linker.

Embodiment 9 is the method of any of Embodiments 1 to 8, further comprising contacting a side chain electrophile, preferably a lysine side chain amino group or introduced into the polypeptide, with a sulfhydryl group covalently linked to the first click partner to producing a modified polypeptide, preferably an NHS azide.

Embodiment 10 is a method according to any of embodiments 1-9, wherein the modified polypeptide comprises a polypeptide covalently linked, directly or via a linker, to an azide, tetrazine or tetrazole group.

Embodiment 11 is a method according to any of embodiments 1-10, wherein the chelating agent contains a macrocycle, preferably the structure of formula (I):

formula (I),

- 14 045797 where each of R1, R2, R ₃ and R4 independently represents CHQCO2X, where

Q is independently hydrogen, C1-C4 alkyl or (C1-C2 alkyl)phenyl, and

X independently represents hydrogen, benzyl, C1-C4 alkyl; And

Z is (CH2) _n Y, where n is 1-10, and

Y is an electrophilic or nucleophilic moiety covalently linked to a second click reaction partner;

alternatively, Z is hydrogen; and each of R ₁ , R ₂ , R ₃ and R ₄ independently represents CHQCO ₂ X, where

Q is independently hydrogen, C1-C4 alkyl or ( _C1 - _C2 alkyl)phenyl, and

X independently represents hydrogen, benzyl, C1-C4 alkyl, or an electrophilic or nucleophilic moiety covalently bonded to a second click reaction partner.

Embodiment 12 is the method according to any of embodiments 1-11, wherein the chelating moiety contains the structure of formula (II):

formula (II).

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

formula (III).

Embodiment 12b is the method of any of Embodiments 1 to 10, wherein the chelating moiety comprises 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'-hexacetic acid (NEHA), 1,4,7 ,10,13-pentaazacyklopentanadecane-N,N',N,N',N-pentaacetic acid (PEPA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA) macrops (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), 1,4,8,11tetraazacyclotetradecane-1,4,8,11-tetrapropionic acid (TETRA), and 1,4,7,10-tetraazacyclododecane1,4,7,10-tetramethylenephosphonic acid (DOTMP).

Embodiment 12c is the method of Embodiment 12b, wherein the chelating agent includes 1,4,7,10-tetraazacyclododecane-N,N',N,N'-tetraacetic acid (DOTA).

Embodiment 12d is the method of Embodiment 12b, wherein the chelating agent contains deferoxamine (DFO).

Embodiment 13 is a method of labeling a polypeptide, preferably an antibody or an antigen binding fragment thereof, with a radioactive metal ion, preferably ²²⁵ Ac, ^U1 In or ⁸⁹ Zr, comprising:

a. providing a modified polypeptide, preferably a modified antibody or antigen-binding fragment thereof, comprising a polypeptide, or an antibody or antigen-binding fragment thereof, covalently linked to an azide, tetrazine or tetrazole group;

b. providing a radioactive complex containing a radioactive metal ion, preferably ²²⁵ Ac, ^m In or ⁸⁹ Zr, associated with a chelating moiety, wherein the chelating moiety

- 15 045797 contains a chelating agent covalently bonded to an alkyne or alkene group; And

With. contacting the modified polypeptide or antibody or antigen-binding fragment thereof with a radioactive complex under conditions allowing an azide, tetrazine or tetrazole group to react with an alkyne or alkene group to thereby label the polypeptide or antibody or antigen-binding fragment thereof with a radioactive metal ion, preferably ²²⁵ Ac, ⁿ¹ In or ⁸⁹ Zr, where the chelating agent contains the structure of formula (I):

formula (I), wherein each of R1, _R2 , R3 and R4 independently represents CHQCO2X, where

Q is independently hydrogen, C1-C4 alkyl or ( _C1 - _C2 alkyl)phenyl, and

X independently represents hydrogen, benzyl, C1-C4 alkyl; And

Z represents (CH ₂ ) _n Y, where n is 1-10, and

Y is an electrophilic or nucleophilic moiety covalently linked to an alkyne group;

alternatively, Z is hydrogen; and each of R1, _R2 , R3 and R4 independently represents CHQCO2X, where

Q is independently hydrogen, C1-C4 alkyl or ( _C1 - _C2 alkyl)phenyl, and

X independently represents hydrogen, benzyl, C1-C4 alkyl, or an electrophilic or nucleophilic moiety covalently bonded to an alkyne group.

Embodiment 13a is the method of Embodiment 13, wherein the chelating agent includes 1,4,7,10-tetraazacyclododecane-N,N',N,N'-tetraacetic acid (DOTA).

Embodiment 13b is the method of Embodiment 13, wherein the chelating moiety contains the structure of formula (II):

formula (II).

Embodiment 13d is a method of labeling a polypeptide, preferably an antibody or an antigen binding fragment thereof, with a radioactive metal ion, preferably ²²⁵ Ac, ⁿ¹ In or ⁸⁹ Zr, comprising:

a. providing a modified polypeptide, preferably a modified antibody or antigen-binding fragment thereof, comprising a polypeptide, or an antibody or antigen-binding fragment thereof, covalently linked to an azide, tetrazine or tetrazole group;

b. providing a radioactive complex containing a radioactive metal ion, preferably ²²⁵ Ac, ¹¹¹ In or ⁸⁹ Zr, associated with a chelating moiety, wherein the chelating moiety comprises a chelating agent covalently linked to an alkyne or alkene group; And

c. contacting the modified polypeptide or antibody or antigen-binding fragment thereof with a radioactive complex under conditions that allow an azide, tetrazine or tetrazole group to react with an alkyne or alkene group to thereby label the polypeptide or antibody or antigen-binding fragment thereof with a radioactive metal ion, preferably ²²⁵ Ac, ¹ⁿ In or ⁸⁹ Zr, where the chelating agent contains an open chain ligand, preferably deferoxamine (DFO).

Embodiment 14 is the method of any of Embodiments 13-13c, wherein the chelating agent is covalently linked to the alkyne or alkene group via a linker.

- 16 045797

Embodiment 15 is the method of any of Embodiments 13-14, further comprising contacting a side chain electrophile, preferably a lysine side chain amino group, or incorporated into a polypeptide, preferably an antibody or antigen binding fragment thereof, with a sulfhydryl group covalently linked to an azide, preferably an NHS azide, to obtain a modified polypeptide or antibody or an antigen binding fragment thereof.

Embodiment 16 is the method of any of embodiments 13-15, wherein the polypeptide, preferably an antibody or antigen binding fragment thereof, is covalently linked to an azide via a linker.

Embodiment 17 is the method of Embodiment 13, wherein the polypeptide is an antibody or an antigen-binding fragment thereof, the radioactive metal ion is ²²⁵ Ac, ⁿ¹ In or ⁸⁹ Zr, and the chelating moiety contains the structure of formula (II):

formula (II).

Embodiment 17a is the method of Embodiment 13c, wherein the polypeptide is an antibody or an antigen binding fragment thereof, the radiometal ion is ²²⁵ Ac, of formula (III):

^m In or ⁸⁹ Zr, and the chelating moiety contains the structure

formula (III).

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

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

Embodiment 18 is a method of double labeling a polypeptide with two radioactive metal ions, comprising:

a. providing a modified polypeptide comprising a polypeptide covalently linked to a first click reaction partner and a second click reaction partner;

b. providing a first radioactive complex comprising a first radioactive metal ion linked to a chelating moiety, wherein the chelating moiety comprises a chelating agent covalently linked to a third click reaction partner; And

c. providing a second radioactive complex comprising a second radioactive metal ion linked to a chelating moiety, wherein the chelating moiety comprises a chelating agent covalently linked to a fourth click reaction partner; And

d. contacting the modified polypeptide with the first and second radioactive complexes under conditions allowing the first click partner to react with the third click partner, and the second click partner to react with the fourth click partner, thereby marking polypeptide with ions of the first and second radioactive metals.

- 17 045797

Embodiment 19 is the method of Embodiment 18, wherein one of the first and second click partners contains an alkyne group, and the other of the first and second click partners contains an azide, and wherein one of the third and fourth partners click reaction partner contains an alkene group, and the other of the third and fourth click reaction partners contains a diene.

Embodiment 20 is the method of Embodiment 18 or 19, wherein the first or second radioactive metal ion is a diagnostic emitter and the other is a therapeutic emitter.

Embodiment 21 is the method according to Embodiment 18 or 19, in which both the first and second radioactive metal ions are therapeutic emitters.

Embodiment 21a is the method according to Embodiment 20 or 21, in which the diagnostic emitter is ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁶ Y, ⁸⁹ Zr, or ⁿ¹ In.

Embodiment 21b is the method according to any of embodiments 20-21a, wherein ^the therapeutic emitter is ³² P, ⁴⁷ Sc, ⁶⁷ Cu, ⁷⁷ As, ⁸⁹ Sr, 90 Y, ⁹⁹ Tc, ¹⁰⁵ Rh, 1 ⁰ ^ ^m 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 or ²²⁷ Th.

Embodiment 22 is a pharmaceutical composition comprising a radiolabeled polypeptide prepared by the method of any one of embodiments 1-21b and a pharmaceutically acceptable carrier.

Embodiment 23 is a method of treating or diagnosing a disease or disorder, particularly a neoplastic disease or disorder, in a subject in need thereof, comprising administering to the subject a pharmaceutical composition according to Embodiment 22.

Embodiment 24 is the method of Embodiment 23, wherein the pharmaceutical composition comprises two sequentially administered compositions, the first of which contains the modified polypeptide and the second of which contains a radioactive complex or radioactive complexes.

Embodiment 25 is a theranostic agent comprising a radiolabeled antibody produced by the method of any one of embodiments 121b and a pharmaceutically acceptable carrier, wherein the immunological properties of the radiolabeled antibody are retained.

Embodiment 26 is a theranostic agent according to embodiment 25, wherein the radioactive metal ion is a diagnostic emitter, preferably ⁸⁹ Zr.

Embodiment 27 is the theranostic agent of Embodiment 25, wherein the radioactive metal ion is a therapeutic emitter, preferably ²²⁵ Ac.

Embodiment 27a is the theranostic agent according to Embodiment 25, wherein the radioactive metal ion is ⁿ¹ In.

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

Embodiment 27c is the theranostic agent of Embodiment 27b, wherein the antibody comprises the HC sequence of SEQ ID NO: 9 and the LC sequence of SEQ ID NO: 10.

Embodiment 27d is a theranostic agent according to any of embodiments 25-27c, wherein the radiolabeled antibody has formula (IV):

- 18 045797 hq ₂ c cq ₂ h

formula ( ^IV ) (DOTA-Ac-DBCO protein), alternatively, ²²⁵ Ac is replaced by another radioactive metal ion such as ³² P, ⁴⁷ Sc, ⁶⁷ Cu, ^{77 As, 89} Sr, ⁹⁰ Y, ⁹⁹ Tc, ¹⁰⁵ Rh, ¹⁰⁹ Pd, ^m 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 In.

Embodiment 27e is a theranostic agent according to any of embodiments 25-27c, wherein the radiolabeled antibody has formula (V):

formula (V) (DOTA-In-DBCO-protein).

Embodiment 27f is a theranostic agent according to any of embodiments 25-27c, wherein the radiolabeled antibody has formula (VI):

formula (VI) (DFO-Zr-DBCO protein), alternatively, ⁸⁹ Zr is replaced by another radioactive metal ion such as 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, or ^m In.

Embodiment 27g is a theranostic agent according to any of embodiments 25-27c, wherein the radiolabeled antibody has formula (VII):

- 19 045797 formula (VII) (DOTA-Zr-DBCO-protein).

Embodiment 27h is a theranostic agent according to any of embodiments 25-27g, wherein the radiolabeled antibody has a chelator ratio:

antibody (CAR) less than 3.

Embodiment 27i is a theranostic agent according to any of embodiments 25-27h, wherein the radiolabeled antibody has a chelator:antibody ratio (CAR) of 2.

Embodiment 28 is a combination, preferably a set, comprising:

a. a modified polypeptide comprising a polypeptide covalently linked to a first click reaction partner; And

b. a radioactive complex containing a radioactive metal ion associated with a chelating moiety, wherein the chelating moiety comprises a chelating agent covalently linked to a second click reaction partner;

wherein the combination must be used to label the polypeptide with a radioactive metal ion.

Embodiment 28a is a combination or set according to embodiment 28, wherein the chelating agent contains a macrocycle.

Embodiment 28b is the combination or set of Embodiment 28 wherein the chelating agent contains an open chain ligand.

Embodiment 29 is the combination or kit of Embodiment 28 to be used to label a polypeptide with a radioactive metal ion through a reaction between first and second click partners in vitro.

Embodiment 30 is the combination or kit of Embodiment 28 to be used to label a polypeptide with a radioactive metal ion through a reaction between first and second click partners in vivo.

Embodiment 31 is a composition comprising a modified polypeptide comprising a polypeptide covalently linked to a first click reaction partner.

Embodiment 32 is a composition comprising a radioactive complex comprising a radioactive metal ion bound to a chelating moiety, wherein the chelating moiety comprises a chelating agent covalently linked to a second click reaction partner.

Embodiment 32a is the composition of Embodiment 32 wherein the chelating agent contains a macrocycle.

Embodiment 32b is the composition of Embodiment 32 wherein the chelating agent contains an open chain ligand.

Embodiment 33 is a combination or kit according to any of embodiments 28-30 or a composition according to embodiment 31 or 32, wherein the polypeptide is an antibody or an antigen-binding fragment thereof.

Embodiment 33a is a combination or kit or composition according to embodiment 33, wherein the antibody is capable of binding to human prostate specific membrane antigen (PSMA) or an antigen binding fragment thereof, preferably the antibody comprises the HC CDR1 sequence SEQ ID NO: 3, the HC CDR2 sequence SEQ ID NO: 4, HC CDR3 sequence SEQ ID NO: 5, light chain (LC) CDR1 sequence SEQ ID NO: 6, LC CDR2 sequence SEQ ID NO: 7, and LC CDR3 sequence SEQ ID NO: 8.

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

Embodiment 33c is a combination or kit or composition according to embodiment 33a or 33b, wherein the antibody or antigen binding fragment thereof is covalently linked to an azide group.

Embodiment 33d is the combination or kit or composition of Embodiment 33c, wherein the antibody or antigen binding fragment thereof is covalently linked to an azide group in a random manner.

Embodiment 33e is a combination or kit or composition according to embodiment 33c, wherein the antibody or antigen binding fragment thereof is site-specifically covalently linked to an azide group.

Embodiment 33f is the combination or kit or composition of Embodiment 33e, wherein the antibody or antigen-binding fragment thereof is covalently linked to an azide group by a method comprising: tailoring the antibody or antigen-binding fragment thereof with a β-1-specific bacterial endoglycosidase, 4-linkage between core GlcNac residue(s) at the antibody Fc glycosylation site(s) to produce a tailored antibody or antigen-binding fragment thereof, and contacting the tailored antibody or antigen-binding fragment thereof with an azido-sugar, preferably a UDP azido-sugar substrate

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GalNaz, in the presence of a glycotransferase, preferably galactosyltransferase GalT.

Embodiment 33g is the combination or kit or composition of Embodiment 33e, wherein the modified antibody or antigen-binding fragment thereof is prepared by a method comprising deglycosylation of the antibody or antigen-binding fragment thereof with an amidase to produce a deglycosylated antibody or antigen-binding fragment thereof, and contacting the deglycosylated an antibody or an antigen-binding fragment thereof with an azidoamine, preferably 3-azidopropylamine, in the presence of microbial transglutaminase.

Embodiment 34 is a combination, set, or composition according to any of embodiments 33-33g, wherein the radioactive metal ion includes ²²⁵ Ac, ¹¹¹ In, or ⁸⁹ Zr.

Embodiment 35 is a combination, kit, or composition according to embodiment 34, wherein the chelating agent contains a macrocycle, preferably the structure of formula (I):

formula (I) wherein each of R1, R2, R3 and R4 independently represents CHQCO2X, where

Q is independently hydrogen, C1- _C4 alkyl or ( _C1 - _C2 alkyl)phenyl, and

X independently represents hydrogen, benzyl, C1-C4 alkyl; And

Z represents (CH2)nY, where n is 1-10, and

Y is an electrophilic or nucleophilic moiety covalently linked to an alkyne group;

alternatively, Z is hydrogen; and each of R ₁ , R ₂ , R ₃ and R ₄ independently represents CHQCO ₂ X, where

Q is independently hydrogen, C1-C4 alkyl or ( _C1 - _C2 alkyl)phenyl, and

X independently represents hydrogen, benzyl, C ₁ -C ₄ alkyl, or an electrophilic or nucleophilic moiety covalently bonded to an alkyne group.

Embodiment 36 is the combination, set, or composition of Embodiment 35, wherein the electrophilic or nucleophilic moiety is covalently linked to the alkyne group via a linker.

Embodiment 37 is a combination, kit or composition according to embodiment 35 or 36, wherein the chelating moiety contains the structure of formula (II):

formula (II)

Embodiment 37a is the combination, kit or composition of Embodiment 34, wherein the chelating moiety comprises a chelating agent having an open chain ligand, preferably the chelating moiety contains the structure of formula (III):

- 21 045797 formula (III).

Embodiment 38 is a combination, kit, or composition according to any of embodiments 34-37, wherein the polypeptide is covalently linked to an azide via a linker.

Embodiment 39 is a method of treating or diagnosing a disease or disorder, particularly a neoplastic disease or disorder, in a subject in need thereof, comprising administering to the subject a theranostic agent according to any one of embodiments 25-27d or a combination according to any one of embodiments 28 and 33 -38.

Embodiment 40 is a method of treating or diagnosing a disease or disorder, particularly a neoplastic disease or disorder, in a subject in need thereof, comprising administering to the subject a composition of embodiment 31 and a composition of embodiment 32, wherein the polypeptide is preferably an antibody.

Examples

To further illustrate the nature of the present invention, the following examples of the invention are provided. It should be understood that the following examples do not limit the invention, and that the scope of the invention is defined by the appended claims.

Example 1 Random Azide/Staple Conjugation to Antibody

Monoclonal Antibodies (mAbs): A human IgG4 antibody that binds to human prostate specific membrane antigen (PSMA), referred to herein as the anti-PSMA mAb designated PSMB127, has the heavy chain (HC) CDR1 sequence SEQ ID NO: 3, sequence HC CDR2 SEQ ID NO: 4, HC CDR3 sequence SEQ ID NO: 5, light chain (LC) CDR1 sequence SEQ ID NO: 6, LC CDR2 sequence SEQ ID NO: 7, and LC CDR3 sequence SEQ ID NO: 8, and has the HC sequence SEQ ID NO: 9 and the LC sequence SEQ ID NO: 10. The anti-PSMA mAb was expressed and purified using standard chromatographic techniques.

The human IgG4 isotype control antibody S228P/F234A/L235A (IgG4-PAA) anti-PMSA mAb, referred to herein as the control mAb, has the HC sequence SEQ ID NO: 1 and the LC sequence SEQ ID NO: 2. Commercial Antibody Trastuzumab (Herceptin) , cetuximab (Erbitux), pertuzumab (Pergeta), and panitumumab (Vectibix) were purchased from Roche, Lilly, Roche, and Amgen, respectively. Mouse anti-human Her2 mAb was obtained from BioXCell (catalog no. BE0277). Trastuzumab, pertuzumab, and anti-human Her2 mAbs bind to human Her2. Cetuximab and panitumumab bind to human EGFR.

Conjugation: A stock solution of antibody (1-10 mg/ml) in 10 mM sodium acetate, pH 5.2, phosphate buffered saline, pH 7, or other compatible buffer, mixed with 20% (v/v) 1 M sodium carbonate buffer, pH 9 to final pH ~9. NHS-PEG4 azide (Thermo, catalog no. 26130) was dissolved in DMSO to a final concentration of 100 mM, and 0.2% (v/v) starting material was added to obtain a molar excess of ~3-10 relative to the mAb. The reaction mixture was incubated at 22°C for 10 minutes, then quenched by adding 1 M Tris pH 7.5 to a final concentration of 50 mM Tris.

Purification: The azide-mAb conjugate was purified and buffer exchanged to compatible buffer (PBS; 20 mM HEPES 150 mM, NaCl pH 7.5; or 10 mM sodium acetate, pH 5.2), using a method such as Zeba desalting columns with molecular weight cut-off 7 kDa (Thermo), dialysis; standard affinity chromatography based on protein A binding; or other compatible method. After purification, the conjugate was concentrated to 10-20 mg/ml using Amicon concentrators with a molecular weight cut-off of 50 kDa (Millipore).

LC/MS Analysis: The chelator:antibody ratio (CAR) was determined using LC/MS analysis on an Agilent G6224 MS-TOF instrument equipped with an Agilent PLRP-S column (300 Å, 2.1 x 150 mm; catalog no. PL1912-3301) (Table 1). The mass spectrum was deconvoluted using the maximum entropy algorithm in the m/z range 2000–3200 for masses 140–170 kDa.

Analytical size exclusion chromatography (SEC): Analytical SEC was performed to determine the oligomeric state of antibodies and antibody conjugates and ensure that the conjugation process does not result in aggregation. An Agilent 1200 series HPLC system was used with a Tosoh TSKgel G3000SWxl column (Tosoh Bioscience, no. 08541) 7.8 x 30 cm; mobile phase 1X PBS; flow rate 0.8 ml/min; injection volume 15 µl; protein concentration 0.1-2 mg/ml.

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Table 1

Conjugation efficiency

Antibody Isotype CAR anti-PSMA mAb Human IgG4 2.4 control mAb Human IgG4 2.0 Trastuzumab Humanized IgGl 2.5 Cetuximab Chimeric human/mouse IgGl 3.6 Pertuzumab Humanized IgGl 2.3 Panitumumab Human IgG2 3.0 Antibody against human Neg2 Mouse IgG2A 3.1

Example 2 Random Azide/Staple Conjugation to Non-Antibody Polypeptides

Non-antibody polypeptides: Transferrin, human holotransferrin, was purchased from R&D Systems (catalog no. 2914-NT) and dissolved in water to 10 mg/ml. EGF, human epidermal growth factor (EGF), was purchased from Sino Biological (catalog no. 10605HNAE).

Conjugation: A stock solution of polypeptides (1-10 mg/ml) in 10 mM sodium acetate, pH 5.2, phosphate-buffered saline, pH 7, or other compatible buffer, mixed with 20% (v/v) 1 M sodium carbonate buffer, pH 9 to final pH ~9. HH8-PEG4-azide (Thermo, catalog no. 26130) was dissolved in DMSO to a final concentration of 100 mM, and 0.2% (v/v) starting material was added to obtain a molar excess of ~3-10 relative to the protein. The reaction mixture was incubated at 22°C for 10 minutes, then quenched with 1 M Tris pH 7.5 to a final concentration of 50 mM. The efficiency of conjugation is given in table. 2.

table 2

E^ efficiency of conjugation Protein Chelator:protein ratio Transferrin 3.6 EGF 1.1

Example 3. Site-specific incorporation of azidosugars into antibody glycans

Antibody glycans were adjusted using GlycINATOR (Genovis), a bacterial endoglycosidase specific for the P-1,4 linkage between core GlcNac residues at the Fc glycosylation site(s), leaving the internal GlcNAc portion intact on the Fc, which can then be used for site-specific incorporation azidosugars. More specifically, GlycINATOR immobilized on agarose beads loaded onto a column (Genovis) was equilibrated in pH 7.4 Tris-buffered saline (TBS). 1 ml of mAb at a concentration of 5-10 mg/ml was added to the resin and incubated on a shaker for 1 hour at room temperature. The mAb was eluted by spinning at 100xg for 1 minute. The column was eluted 3 more times with 0.5 ml TBS. Eluates containing the customized mAb were pooled and buffer supplement (Genovis) was added along with the azidosugar substrate UDP-GalNaz and the galactosyltransferase enzyme GalT. The mixture was heated overnight at 30°C. The final azide mAb was purified using a mAb Select (GE) column on an ACT Avant instrument. Azide modification was confirmed by LC-MS with a CAR determination of exactly 2.

Example 4 Site-specific incorporation of 3-azidopropylamine with microbial transglutaminase (MTG)

Azido groups were site-specifically introduced into the antibody at positions Gln295 essentially 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 deglycosylated using Rapid PNGase F (New England Biolabs), an amidase that cleaves between the internal GlcNAc and asparagine residues of high-mannose, hybrid, and complex oligosaccharides and allows complete deglycosylation and release of all N-glycans, including N-glycans from both conserved (e.g. Asn297 Fc) and non-conserved (e.g. Fab N-glycans) glycosylation sites. 10 ml of antibody at a concentration of 1 mg/ml in sodium acetate buffer pH 5.2 was incubated with 5 μl of PNGase F overnight at 37°C, and deglycosylation was confirmed by LC-MS. PNGase F was removed using 4 cycles of concentration and dilution using an Amicon device (50 kDa cutoff). For conjugation of 3-azidopropylamine (3-APA; Click Chemistry Tools), deglyco

-23 045797 sylated mAb (0.5-1 mg/ml) was adjusted to pH 7-7.5 by adding 2% (v/v) 0.5 M HEPES pH 7.5. 100 equivalents of 3-APA were added along with 5-10% w/v. transglutaminase Activa TI (Ajinomoto), MTG. The reaction mixture was incubated at 37°C for 1-4 hours, followed by purification of the azide-modified mAb on an mAbSelect Sure column using standard chromatographic methods. The conjugate was characterized by LC-MS and the CAR was found to be 2.

Example 5. Chelation of a radioactive metal with a bifunctional chelator (BFC) 225 225

Synthesis of Ac-DOTA-GA-DBCO: Ac(NO ₃ ) ₃ was purchased from Oak Ridge National Laboratory. 1,4,7,10-tetraazacyclododececane,1-(glutaric acid)-4,7,10-triacetic acid-(3-aminopropanoic acid)dibenzocyclooctine (DOTA-GA-DBCO) was custom synthesized. The synthesis was performed according to Bernhard et al. Chem. pharm. J. 2012, 18, 7834-7841. DBCO-amine (3-amino-1-[(5-aza-3,4:7,8dibenzocyclooct-1-yne)-5-yl]-1-propanone, Sigma) was contacted with DOTA-GA anhydride and the product purified using reverse phase HPLC.

Quantification of actinium-225 was carried out using a Capintec CRC55TW dose calibrator, whereby ²²⁵ Ac(NO3)3 was dissolved in 0.1 N. HCl to obtain a solution of 10 mCi/ml. In a plastic test tube to a solution of tetramethylammonium acetate (1 M solution, 7.5 μl, 7.5 μmol), DOTAGA-DBCO (1 mg/ml in water, 2.5 μl, 3.4 nmol) and NaOH (0.1 n., 2.5 μl, 0.25 μmol) ²²⁵ Ac(NO3) ₃ (10 mCi/ml in 0.1 N HCl, 5 μl, 50 μCi, 0.0038 nmol) was added. The pH of the mixture was found to be ~6.5 using litmus pH paper. The tube was placed on a shaking block at 80°C and 290 rpm for 30 min, and the tube was allowed to cool to room temperature.

Synthesis of ¹¹¹ In-DOTA-GA-DBCO: ¹¹¹ InCl3 in 0.05 M HCl was purchased from GE Healthcare. In a plastic test tube to a solution of tetramethylammonium acetate (1 M solution, 7.5 μl, 7.5 μmol), DOTAGA-DBCO (1 mg/ml in water, 2.5 μl, 3.4 nmol) and HCl (0.1 n., 5 µl) ¹¹¹ InCl3 in 0.05 n was added. HCl (5 µL, 104.3 µCi measured in Capintec CRC-55TW dose calibrator, 0.0022 nmol). The pH of the mixture was found to be ~5.5 using litmus pH paper. The tube was placed on a shaking block at 60°C and 290 rpm for 30 min, and the tube was allowed to cool to room temperature.

Synthesis of ⁸⁹ Zr-DFO-DBCO: ⁸⁹ Zr-oxalate was purchased from 3D Imaging. DFO-DBCO was purchased from Macrocyclics (Plano, TX, catalog no. B-773), dissolved in DMSO to 0.5 mg/mL, and diluted in water to 25 μg/mL.

mCi of Zr-89 was transferred to a metal-free microcentrifuge tube and 1 M oxalic acid was added to achieve a total volume of 80 μL. 12 μL of 2 M potassium carbonate was added in 2 μL increments and the mixture was stirred with a pipette tip until no more bubbles formed. Then 120 μl of 1 M HEPES was added, followed by 300 μl of water. The pH of the solution was tested and more 2 M potassium carbonate was added to raise the pH to 6-6.5 if necessary. 136 μL of DFODBCO stock solution (3.4 μg) was added and the reaction mixture was incubated for 1 hour at room temperature. The chelate was analyzed by determining 0.5 μl of the reaction mixture on a TLC Green plate (Biodex) and eluting with 20% NaCl. After elution, the plate was scanned with a PerkinElmer Cyclone Plus phosphorescent system to ensure that most of the Zr-89 remained at the original level, indicating DFO-DBCO chelation.

Synthesis of ⁸⁹ Zr-DOTA-GA-DBCO: To a strong anion exchange cartridge Waters Sep-pak Light QMA (acrylic acid/acrylamide copolymer on silicon dioxide, surface functional groups: C(O)NH(CH2)3N(CH3)3 ⁺ Cl, pore size 300 A, particle size 37-55 μm, ion exchange capacity 230 μeq/g), MeCN (6 ml) was added, followed by 0.9% saline solution (10 ml), then water (10 ml). A solution of ⁸⁹ Zr(ox)2 in 1.0 M oxalic acid (2 µL, 290 µCi) was added to the pre-conditioned cartridge. The cartridge was in turn washed with deionized water (20 ml) to remove excess oxalic acid, followed by elution of ⁸⁹ ZrCl ₄ from the column with 1.0 M HCl (aq) (100 µl each) to obtain a total volume of 400 µl with a yield of 248 µCi (86%), with most of the activity occurring in the 3rd fraction. The combined aliquots were then evaporated until completely dry.

µl DOTA-GA-DBCO (1.0 mg/ml in metal-free water, 10 µg, 13.6 nmol) was added to ⁸⁹ ZrCl ₄ (268 µCi, 50 µl). The DOTA-GA-DBCO/ ⁸⁹ Zr solution was diluted in 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 min. The yield of the ⁸⁹ Zr-DOTA-GADBCO complex was determined to be 98% using an SPC25 column (Sigma Aldrich, cat. no. SPC25120-50G) eluted with 1% NH4OH. Unchelated ⁸⁹ Zr remains on the column, and the ⁸⁹ ZrDOTA-GA-DBCO complex elutes.

Example 6. Synthesis of click-labeled radioactive anti-PSMA mAb conjugates

Look at fig. 1 and 2 is a diagram of radioactive labeling of antibodies in accordance with the methods according to the invention.

Synthesis of anti-PSMA mAb-dibenzo-[1,2,3]-triazoloazocine-GA-DOTA-225Ac (site-specific,

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CAR=2): Random or site-specific azide modified antibody (site-specific, CAR=2 or random, average CAR between 1 and 4) in PBS or other compatible buffer (10-20 mg/ml) was added to the solution ²²⁵ Ac-DOTA -GA-DBCO prepared as described. The final pH of the mixture was ~6.5 using litmus pH paper. The reaction solution was stirred gently and left at room temperature for 3 hours before purification with a PD-10 column (GE Healthcare) preconditioned with 15 ml sodium acetate buffer, 10 mM, pH 6-6.5 or other compatible buffer. The reaction mixture was pipetted into the reservoir of a preconditioned PD-10 column, and the eluate was collected in plastic tubes. The reaction tube was washed with sodium acetate buffer (0.2 ml x 3), the washes were pipetted into the PD-10 column reservoir and the eluate was collected. Sodium acetate buffer was continuously added to the reservoir of the PD-10 column, and the eluate was collected in plastic tubes, with ~1 mL of eluate collected in each tube until a total of 10 mL of eluate was collected.

The purity of each collected fraction was assessed by iTLC-SG (Agilent) using citrate-H ₂ O-MeOH solution as the mobile phase. Pure fractions were pooled to obtain the final product in 10 mM sodium acetate buffer. The product solution was analyzed by HPLC for chemical and radiochemical purity. The concentration of antibodies in the product solution was determined by UV absorbance using a standard curve. The activity of the product solution was, in turn, quantified using a Capintec CRC-55TW dose calibrator.

Ac-225 Chelation Quality Control: Chelation testing was performed using diethylenetriaminepentaacetic acid (DTPA) as a quality control for the purified product. A 10 mM aqueous solution of Na ₅ DTPA was added to the sample solution containing mAb labeled with ²²⁵ Ac until the [DTPA]/[mkAt] ratio was 500-1000. A 50 mM aqueous solution of Na5DTPA was added to an aliquot of the purified product in a solution containing ²²⁵ Ac labeled mAb until the [DTPA]/[mkAt] ratio was 50,000-100,000. The two mixtures were placed on a shaking block at room temperature and 290 rpm for 30 minutes. The mixture was applied to iTLC-SG and citrate-H2O-MeOH solution was added as the mobile phase. Under these conditions, free Ac migrates to the solvent front, while bound ²²⁵ Ac-mAb remains at the baseline.

Synthesis of anti-PSMA mAb-dibenzo-[1,2,3]-triazoloazocine-CA-DOTA- ¹¹¹ In: Anti-PSMA mAb, randomly or site-specifically modified with azide, in 10 mM NaOAc (site-specific, CAR=2 or randomly, the average CAR between 1 and 4) was added to the ⁿ¹ InDOTA-GA-DBCO solution prepared as described. The reaction solution was stirred gently and left at room temperature for 2 hours before passing through a PD-10 column. The PD-10 column was preconditioned by passing 15 ml of sodium acetate buffer, 10 mM, pH 6-6.5, through the column and the washes were discarded. The reaction mixture was then pipetted into the reservoir of a preconditioned PD-10 column, and the eluate was collected in plastic tubes. The reaction tube was washed with sodium acetate buffer (0.2 ml x 3), the washes were pipetted into the PD-10 column reservoir and the eluate was collected. Sodium acetate buffer was continuously added to the reservoir of the PD-10 column, and the eluate was collected in plastic tubes, with ~1 mL of eluate collected in each tube until a total of 10 mL of eluate was collected.

The purity of each collected fraction was assessed by iTLC-SG using 10 mM EDTA aqueous solution (pH 5-6) as the mobile phase. Pure fractions were pooled to obtain the final product in 10 mM sodium acetate buffer. The product solution was analyzed by HPLC for chemical and radiochemical purity. The concentration of antibodies in the product solution was determined by UV absorbance using a standard curve. The activity of the product solution was, in turn, quantified using a Capintec CRC-55TW dose calibrator.

In-111 Chelation Quality Control: Chelation testing was performed using DTPA as a quality control for the purified product. A 10 mM aqueous solution of Na ₅ DTPA was added to the sample solution containing mAb labeled with ⁿ¹ In until [DTPA]/[mkAt] = 1000-10000. The mixture was placed on a shaking block at room temperature and 290 rpm for 30 minutes. The mixture was applied to iTLC-SG and 10 mM EDTA aqueous solution (pH 56) was added as the mobile phase. Free ¹ⁿ In or weakly bound ¹ⁿ In migrates to the solvent front, while strongly bound ^1G1 1n-mAb remains at the baseline.

Synthesis of anti-PSMA mAb-dibenzo-[1,2,3]-triazoloazocine-DFO-Zr-89

800 μg anti-PSMA mAb randomly modified with azide (average CAR between 1 and 4; same procedure can be done with site-specific azido mAb) in 10 mM HEPES, 50 mM NaCl pH 7.5 or other compatible buffer was added to the ⁸⁹ Zr-DFO-DBCO solution prepared as described and incubated at 37°C for 1.5 hours before passing through a PD-10 column. The PD-10 column was preconditioned by passing 15 mL of isotonic saline through the column and the wash was discarded. The reaction mixture was then pipetted into the reservoir of a preconditioned PD-10 column, and the eluate was collected in plastic tubes. Saline solution was continuously added to the reservoir of the PD-10 column, and the eluate was collected in plastic tubes, each

- 25 045797 0.5 ml of eluate was collected in a test tube until a total of 10 ml of eluate was collected. The activity of each fraction and the material remaining on the column was determined using a dose calibrator. The peak product, typically fractions 4-7, was combined to obtain the final product. The product solution was analyzed by HPLC for chemical and radiochemical purity. The concentration of antibodies in the product solution was determined by UV absorbance using a standard curve. The activity of the product solution was quantified using a dose calibrator.

Synthesis of anti-PMSA mAb-DOTA- ⁸⁹ Zr

An azide-modified anti-PSMA mAb (CAR=1-4) modified by random azide conjugation in 20 mM HEPES, 50 mM NaCl pH 7.5 (10.1 mg/ml, 200 μl, ~13.5 nmol) was added to the ⁸⁹ Zr-DOTA-GA-DBCO solution prepared as described above. 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 on a PD-10 column (GE Healthcare) or 0.9% saline, HEPES buffer, or PBS to obtain product ⁸⁹ Zr-DOTA-MKAT at 64%.

Quality control of Zr-89 chelation: For Zr-DFO-mAb, purified products were analyzed by HPLC only. These conjugates did not retain Zr-89 when tested with DTPA or EDTA. Zr-DOTA mAb was tested by adding EDTA to 33 mM and incubating overnight at room temperature. The conjugates were analyzed after testing by running through a PD-10 column and were found to retain 80% of the radioactivity.

Example 7. Analytical characterization of click-labeled radioactive conjugates

Determination of radiochemical conversion

Radiochemical conversion (% RA conversion; see Tables 3-5) was determined using iTLC-SG (instant thin layer chromatography (iTLC) using unbound glass fiber chromatography paper impregnated with silica gel (SG)). The % RA conversion value is calculated by dividing the integrated radio signal value of the product peak (various retention times of the radioactive starting material and by-products) by the value obtained by integrating all radio signal peaks present between the baseline and the solvent front. This product fraction is then expressed as percentage conversion.

For Ac-225-incorporated products, sample solutions containing ~0.1-1 μCi of ²²⁵ Ac were applied to the baseline of the iTLC-SG plate approximately 2 cm from the bottom edge. The iTLC-SG plate was prepared using a citrate-water-methanol mobile phase (20 mL 0.4 M trisodium citrate/3 mL 2 N HCl/2.3 mL MeOH), allowed to dry at room temperature, and stored for a minimum of 6 hours before analysis (until radioactive equilibrium of 225Ac and all daughter nuclides is achieved). iTLC-SG was scanned using a Bioscan AR2000 radio-TLC scanner set to 99mTc.

For In-111 chelates, sample solutions containing ~0.5-2.5 μCi ^{of 1n} In were applied to the baseline of the iTLC-SG plate, approximately 2 cm from the bottom edge. The iTLC-SG plate was prepared using 10 mM sodium EDTA, pH 5–6, as the mobile phase and then allowed to dry at room temperature. The dried iTLC-SG was scanned using a Bioscan AR2000 radioTLC scanner set to In-111.

For Zr-89% RA chelates, conversions were determined by dividing the activity at the peak of the product by the total activity, including the activity in the PD-10 column, determined by measurement using a dose calibrator.

Determination of radiochemical purity of radioactively labeled proteins

The radiochemical purity (% RA purity; see Tables 3-4) of the Ac-225 and In-111 chelates was determined using SEC-HPLC (size exclusion HPLC). In the case of Ac-225, a Tosoh TSKgel column (G3000SWxl 7.8 mm χ 30 cm, 5 µm) was used; the column was eluted with DPBS buffer (1x, without calcium and magnesium); flow rate: 0.7 ml/min; analysis time - 20 min; room temperature. After HPLC, the eluate was collected into pre-numbered tubes, with 0.5 min or 1 min of eluate fractions collected in each tube. The tubes containing the eluate were left at room temperature for >6 hours to allow ²²⁵ Ac to reach radioactive equilibrium with its daughter nuclides. The activity in each tube was then counted in the well of a Capintec CRC-55TW counter. The radiochromatogram was reconstructed based on the activity in the tubes.

In the case of In-111, a Tosoh TSKgel column (G3000SWxl 7.8 mm χ 30 cm, 5 µm) was used; the column was eluted with DPBS buffer (1x, without calcium and magnesium); flow rate: 0.7 ml/min; analysis time - 20 min; room temperature. Radioactivity detection was performed using the above HPLC system and a Perkin Elmer Radiomatic 625TR flow radio detector equipped with a 0.5 mL flow cell using the In-111 setting, and an Ultima FloTM M cocktail at a flow rate of 1.4 mL/min.

For Zr-89 (see Table 5), a Tosoh TSKgel column (G3000SWxl 7.8 mm χ 30 cm, 5 μm) was used. The column was eluted with citrate-buffered saline; flow rate: 1 ml/min; analysis time - 20 min; room temperature. Radioactivity detection was performed using the above system

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HPLC and Beckman flow detector coupled to a Bioscan Flow Count instrument.

Table 3

Radioactive labeling of proteins with actinium-225

mAb Stage 1* Stage 2** %RA conversions (iTLC) %RA conversions (iTLC) % RA purity (HPLC) Specialist. END (µCi/ml) Destiny, asset. (µCi/mg) General R.A. (µCi) CAR anti-PSMA mAb (azide, randomly) 88% 73% 80% 17.2 74.9 17.2 3-4 anti-PSMA mAb (glycan, site specific) 88% 77% 83% 7.2 102.8 7.2 2 anti-PSMA mAb (MTG, site-specific) 93% 70% 90% 31.3 74.2 31.3 2.0 control mAb (azide, randomly) 92% 68% 97% 28.1 56.1 28.1 2 Panitumumab (azide, random) 87% 65% 95% 24.9 66.6 24.9 3.0 Cetuximab (azide, random) 91% 67% 98% 33.7 76.4 33.7 3.6 hEGF (azide, randomly) 92% 48% 100% 19.63 1091 19.63 1.1

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Transferrin (azide, random) 94% 57% 78% 24.1 83.7 24.1 3.6 Trastuzumab (azide, random) 94% 60% 97% 9.88 62.9 9.88 2.5 Pertuzumab (azide, random) 94% 63% 92% 16.75 49.9 16.75 2.3 Antibody against human HER2 (azide, random) 85% 78% 94% 21.5 55.0 21.5 3.1

*Step 1 is the chelation of actinium-225 DOTA-GA-DBCO **Step 2 is the click reaction of the bifunctional chelate with the protein

Table 4

Radioactive labeling of proteins with indium-111

mAb Stage 1* Stage 2 ^* %RA conversions (iTLC) %RA conversions (iTLC) % RA purity (HPLC) Specialist. END (µCi/ml) Destiny, asset. (µCi/mg) Total RA (µCi) CAR anti-PSMA mAb (azide, randomly) 81% 79% 98% 57.6 139.5 72 2.4 Transferrin (azide, random) 79% 88% 90% 71.4 410.3 71.4 3.6 Panitumumab (azide, random) 46% 79% 99% 82.0 279 82.0 3.0 Cetuximab (azide, 74% 75% 100% 51.3 290 51.3 3.6 randomly)

*Step 1 is the chelation of indium-111l DOTA-GA-DBCO * *Step 2 is the click reaction of the bifunctional chelate with the protein

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Table 5

Radiolabeling of Zr-89 anti-PSMA mAb with DFO-DBCO

mAb %RA conversions % RA purity (HPLC) Special, conc. (µCi/ml) Destiny, asset. (µCi/mg) Total RA (µCi) CAR anti-PSMA mAb (azide, randomly) 47% 100% 131 1171 262 2.4

Example 8. Click reaction of a modified mAb with DOTA-GA-DBCO

Randomly and site-specifically modified azido-mAbs (anti-PSMA mAbs, cetuximab, panitumumab, trastuzumab, pertuzumab) in an amount of 1-10 mg/ml were mixed with a 5x-20x excess of unchelated DOTA-GA-DBCO and incubated at room temperature or 37°C for 1-24 hours. The mAbs were desalted using Zeba Desalt spin columns (Thermo) and concentrated using an Amicon centrifugal concentrator (Millipore), re-diluted with buffer, and concentrated again to remove any remaining DBCO-DOTA. The completion of the click reaction of all free azides with DBCO-DOTA was confirmed by LC-MS.

Example 9. Click reaction of a modified mAb with DFO-DBCO

Azido-mAbs with randomly modified and site-specific mAbs at a concentration of 1-10 mg/ml were mixed with a 5-20-fold excess of unchelated DOTA-GA-DFO and incubated at room temperature or 37°C for 1-24 hours. The mAbs were desalted using Zeba Desalt spin columns (Thermo) and concentrated using an Amicon centrifugal concentrator (Millipore), re-diluted with buffer, and concentrated again to remove any remaining DBCO-DFO. The complete click reaction of all free azides with DBCO-DFO was confirmed by LC-MS.

Example 10: Cellular binding (FACS)

Cellular binding of azide-modified antibodies, DOTA-DBCO-azide-modified antibodies, and DFO-DBCO-azide-modified antibodies were compared with the parent mAbs for the conjugates described in Table 1. 1. Cell lines expressing the mAb target were treated with a range of antibody or conjugate concentrations and binding was measured by flow cytometry. Panitumumab and cetuximab conjugates were evaluated for binding to EGFR+ A431 cells. Herceptin and pertuzumab were assessed for binding to HER2+ SK-BR-3 cells. Anti-PSMA mAb was assessed for binding to PSMA+ C4-2b cells.

Cell lines: C4-2B cells, a human prostate adenocarcinoma cell line, were obtained from Janssen Oncology (Spring House, PA). A431 cells, a human epidermoid carcinoma cell line, and SK-BR-3 cells, a human breast cancer cell line, were obtained from Janssen BioTherapeutics (Spring House, PA) with cells originally obtained from ATCC (Manassas, Virginia). A suspension of EGFR negative human acute myeloid leukemia MOLM-13 cells was maintained in RPMI 1640+25 mM Hepes (Gibco) supplemented with 20% heat-inactivated fetal bovine serum (Gibco). Cells were grown in RPMI 1640+25 mM Hepes (Gibco, Waltham, MA) with 10% FBS (Gibco, Waltham, MA).

Flow Cytometry: Cells were separated from the flask using enzyme-free cell dissociation buffer (Gibco, Waltham, MA) and filtered through a 40-μm filter (Falcon). 5x104 cells were seeded per well in a 96-well U-bottom plate. Cells were incubated with conjugated antibody or parent antibody diluted in BSA staining buffer (BD Bioscience, San Jose, CA) for 1 hour at 4°C. Cells were washed twice with staining buffer. Cells were then incubated in the dark for 30 min at 4°C with Alexa Fluor 647-labeled anti-human IgG secondary antibody (Jackson ImmunoResearch Laboratories). Secondary antibodies were diluted 1:200 in 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. Cells were washed twice with staining buffer, then resuspended in a final volume of 25 μl/well in staining buffer and read on an iQue Screener flow cytometer (Intellicyt). ForeCyt software was used for data analysis. Live cells were determined by excluding events with strong nucleic acid staining. Mean fluorescence intensity (MFI) was determined on live cells and plotted in GraphPad Prism 7 (GraphPad Software) as the logarithm of antibody concentration versus MFI. A nonlinear regression curve fit was added to the data and EC ₅₀ values were calculated.

For all mAbs and conjugates tested, the original mAbs and modified mAbs demonstrated similar cellular binding (Table 6).

Table 6

EC50 (nM) binding of mAbs/conjugates to target cells

mAb mAb azide mAb-DOTA mAb-DFO Vectibix 1.3 1.3 0.9 Cetuximab 0.6 0.2 0.4 Herceptin 2.0 0.9 3.1 Pertuzumab 2.5 3.3 4.4 anti-PSMA mAb (azide, random) 5.5 4.7 5.4 4.6 anti-PSMA mAb (glycan) 0.7 0.5 0.7 MTG samples mAb Deglycosylated mAb azide mAb-DOTA mAb-DFO anti-PSMA mAb (MTG) 4.1 3.4 5.0 6.7 4.5

Example 11 Cellular Binding Assay with In-111

Cellular binding was measured by radiometric assay using In-111 radiolabeled proteins described in Table. 4. Anti-PSMA mAb and transferrin were tested on C4-2B cells (PSMA+ and transferrin+). Cetuximab and panitumumab were tested in A431 cells (EGFR+) and MOLM-13 cells (EGFR-).

Attached cells were separated using enzyme-free cell dissociation buffer (Gibco). Detached adherent cells and collected suspension cells were counted and washed with cold staining buffer (BD Biosciences). Various numbers of cells in 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 for 1 hour on ice. Cells were washed with cold PBS (Gibco) to remove unbound antibody and resuspended in 500 μl of cold PBS. Samples were transferred to scintillation tubes and cell-associated radioactivity was determined by gamma radiometry (Hidex Automatic Gamma Counter).

Counts per minute (cpm) from study samples were converted to μCi of In-111 using cpm values using linear regression obtained using known amounts of In-111-labeled protein. Bound μCi values were converted to bound moles using the following calculation: (Bound μCi/specific activity)/mAb or protein molecular weight. Each data point represents the average of two samples.

The In-111 click-labeled anti-PSMA mAb and In-111-transferrin bound to C4-2B cells, and the amount of cell-bound radioactivity increased with increasing cell number (Fig. 3A). Anti-EGFR antibodies labeled with In-111 using the click reaction, panitumumab and cetuximumab, bind to A431 cells, and the amount of cell-bound radioactivity increases with increasing number of cells; There was no specific binding of click-labeled anti-EGFR antibodies to negative control MOLM-13 cells (Fig. 3B).

Example 12 Cellular Indium Uptake Assay

In C4-2B cells, the internalization kinetics of In-111-labeled anti-PSMA mAb was determined.

Cells were seeded at 3 x 10 ⁶ cells per 60 mm diameter Petri dish (Corning) and placed in a humidified CO ₂ incubator at 37°C overnight. Inoculation media were removed and replaced with 2 ml of cold staining buffer (BD Biosciences). The Petri dishes were then placed on ice. Antibody labeled with 0.5 μCi In-111 was added to each Petri dish and incubated for 1 hour on ice. Cells were washed with cold PBS (Gibco) to remove unbound antibody from the cell surface. At various time points, cells were analyzed for surface membrane-associated radioactivity and intracellular radioactivity as described below.

Surface-bound radioactivity was removed using an acid scalding procedure: 1.5 ml of membrane scalding buffer (50 mM glycine, 150 mM NaCl, pH 2.7 with pepsin (Amresco) added to 25 μg/ml) was added to the cells and Petri dishes were incubated on ice for 15 minutes. The scalding buffer was transferred into scintillation tubes. The cells were washed with cold PBS, and the washing solutions were transferred to scintillation tubes. Radioactivity was determined by gamma radiometry (Hidex automatic gamma counter). The radioactivity associated with the surface membrane was determined as the sum of the washes with scalding buffer+PBS.

Intracellular radioactivity was analyzed by preparing cell lysates. After that

- 30 045797 th, as surface bound radioactivity was washed away with scalding buffer, and the cells were washed, 1.5 ml of 1 M NaOH (Teknova) was added to the cells, and the Petri dishes were incubated on ice for 5 minutes. Cell lysates with the surface stripped were transferred to scintillation tubes. Petri dishes were washed with cold PBS, and the washing solutions were transferred to scintillation tubes. Radioactivity was determined by gamma radiometry (Hidex automatic gamma counter). Intracellular radioactivity was determined as the sum of surface-scraped cell lysate wash solutions + PBS

For time=0 samples, cells were analyzed for surface membrane-associated 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 Petri dish after initial antibody binding, and the Petri dishes were placed in a humidified CO ₂ incubator at 37°C. At each time point, the Petri dishes were removed from the incubator and placed on ice. Cell culture media were transferred to scintillation tubes and cells were washed with cold PBS. PBS wash solutions were collected in scintillation tubes. Cells were analyzed for surface membrane-associated radioactivity and intracellular radioactivity as described below. At each time point, an unscalded sample was prepared by incubating cells with PBS instead of scalding buffer until the cells were lysed. These samples were used to evaluate the effectiveness of scalding and compare the results with scalded samples.

Cpm from study samples were converted to μCi of In-111 using the cpm value from a linear regression obtained using known amounts of In-111-labeled μAb. The percentage localization of In-111 mAb on the cell surface membrane (scalded samples) and inside cells (lysed samples) was determined using the following formula: % localized=100 * (sample μCi/average total μCi), where total μCi refers to the sum of all collected samples, including incubation media, PBS washes, glycine washes and lysed cells. Each data point represents the average of two samples.

Surface-bound In-111 quickly disappeared from the cell surface and was redistributed intracellularly. The scalding technique releases ~80% of the cell surface bound radioactivity at time 0; by the end of incubation, only 20% was released by scalding, and more than 60% of the radioactivity was in the cell lysate (Fig. 4).

Example 13 Efficacy in a murine tumor xenotransplantation model

Study to Determine the Optimal Dose of Anti-PSMA mkAt-DOTA-Ac-225: Male NSG mice (N=8 per group) were subcutaneously implanted with 10 ⁶ LNCaP cells and tumors grew to 100-150 mm ³ . Mice were intravenously administered a single dose of anti-PSMA mAb-azide-DOTA- ²²⁵ Ac or an isotype control, control mAb-azide-DOTA- ²²⁵ Ac, over a range of activities (10 nCi, 25 nCi, 70 nCi, 200 nCi). The administered dose per mouse was adjusted to 10 μg of protein with cold antibodies. Tumor size and body weight were measured twice a week. Animals were sacrificed when tumor size exceeded 1500 mm ³ or body weight loss exceeded 20%.

Anti-PSMA mkAt-DOTA-Ac-225 demonstrated tumor growth inhibition after a single dose, especially at higher radioactive doses, and was superior to isotype control at all doses (Fig. 5A). All doses of the control radioactive mAb conjugate had similar survival curves to the vehicle control (Fig. 5B; Table 7); The anti-PSMA mAb conjugate showed a clear dose-dependent effect in increasing survival with increasing radioactive dose (Fig. 5C; Table 7). the study was terminated after 209 days with 3 mice remaining, all from the anti-PSMA mAb 200 nCi group, and the study revealed no detectable tumors.

Table 7

Median survival

Dose anti-PSMA mAb Isotype control 10 nCi 61 days 46 days 25 nCi 82 days 48 days 70 nCi 92.5 days 50 days 200 nCi 126.5 days 39.5 days Carrier medium 46

It is intended that the embodiments of the invention are illustrative only and those skilled in the art will recognize or be able to determine, using no more than routine experimentation, numerous equivalents to the specific procedures of the invention. All such equivalents are intended to be included within the scope of this invention and are covered

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Claims (31)

the attached claims. All references (including patent applications, patents and publications) contained herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or application the patent has been specifically and individually indicated to be incorporated by reference in its entirety in all respects. CLAIM 1. A method for labeling a polypeptide with a radioactive metal ion, including: a. providing a modified polypeptide comprising a polypeptide covalently linked to a first click reaction partner; b. providing a radioactive complex containing a radioactive metal ion associated with a chelating moiety, wherein the chelating moiety comprises a chelating agent covalently linked to a second click reaction partner; And c. contacting the modified polypeptide with a radioactive complex under conditions that allow a first click reaction partner to react with a second click reaction partner to thereby label the polypeptide with a radioactive metal ion, in which one of the first and second click reaction partners contains an alkyne group and the other click partner contains an azide, or wherein one of the first and second click partners contains an alkene group and the other click partner contains a diene. 2. The method of claim 1, wherein the polypeptide is an antibody or an antigen-binding fragment thereof. 3. The method of claim 2, wherein the antibody is a monoclonal anti-PSMA antibody. 4. The method according to any one of claims 1 to 3, wherein the radioactive metal ion is ²²⁵ Ac, ¹ⁿ In or ⁸⁹ Zr. 5. The method according to any one of claims 1 to 3, further comprising reacting an electrophile on the side chain with a sulfhydryl group covalently linked to the first click reaction partner to obtain a 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 inclusion of a first click reaction partner. 7. The method of claim 6, wherein the modified antibody or antigen-binding fragment thereof is produced by a method comprising adjusting the antibody or antigen-binding fragment thereof with a bacterial endoglycosidase specific for the β-1,4-linkage between the core GlcNac residue at the antibody Fc glycosylation site for producing a tailored antibody or an antigen-binding fragment thereof, and reacting the tailored antibody or an antigen-binding fragment thereof with an azide-labeled sugar in the presence of a glycotransferase such as Galactosyltransferase GalT or GalNAc transferase, thereby obtaining a modified antibody or antigen-binding fragment thereof. 8. The method of claim 7, wherein the azide-labeled sugar is UDP-N-azidoacetylgalactosamine (UDP-GalNaz) or UDP-6-azido-6-deoxy GalNAc. 9. The method of claim 7, wherein the glycosyltransferase is a GalT galactosyltransferase or a GalNAc transferase. 10. The method of claim 6, wherein the modified antibody or antigen-binding fragment thereof is produced by a method comprising deglycosylation of the antibody or antigen-binding fragment thereof with an amidase to produce a deglycosylated antibody or antigen-binding fragment thereof, and reacting the deglycosylated antibody or antigen-binding fragment thereof with an azidoamine in the presence of transglutaminase. microorganisms to thereby obtain a modified polypeptide. 11. The method according to claim 10, in which 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. 12. The method of claim 1, wherein the modified polypeptide comprises a polypeptide covalently linked, directly or via a linker, to an azide, tetrazine or tetrazole group. 13. The method according to claim 1, in which the chelating agent contains a macrocycle having the structure of formula (I): - 32 045797 formula (I) wherein each of R1, R2, R3 and _R4 independently represents CHQCO2X, where Q is independently hydrogen, C1- _C4 alkyl or (C1-C2 alkyl)phenyl, and X independently represents hydrogen, benzyl, C1- _C4 alkyl; And Z is (CH2) _n Y, where n is 1-10, and Y is an electrophilic or nucleophilic moiety covalently linked to a second click reaction partner; alternatively, Z is hydrogen; and R1, R2, R3 and _R4 are each independently CHQCO2X, where Q is independently hydrogen, _C1 - _C4 alkyl or (C1-C2 alkyl)phenyl, and X independently represents hydrogen, benzyl, C1- _C4 alkyl, or an electrophilic or nucleophilic moiety covalently bonded to a second click reaction partner; alternatively, the chelating agent contains an open chain ligand. 14. The method according to claim 1, in which the chelating moiety contains the structure of formula (II): formula (II) or structure of formula (III): 15. The method of claim 1, wherein said modified polypeptide is a modified antibody or an antigen-binding fragment thereof, and said polypeptide covalently linked to the first click reaction partner is an antibody or an antigen-binding fragment thereof covalently linked to an azide, tetrazine or a tetrazole group; said radioactive complex containing a radioactive metal ion bound to a chelating moiety is a radioactive complex containing ²²⁵ Ac, ^m In or ⁸⁹ Zr bound to a chelating moiety, and said chelating agent covalently bound to a second click reaction partner is is a chelating agent covalently bonded to an alkyne or alkene group; and said contacting of the modified antibody or antigen-binding fragment thereof with a radioactive complex under conditions allowing an azide, tetrazine or tetrazole group to react with an alkyne or alkene group to thereby label the antibody or antigen-binding fragment thereof with ²²⁵ Ac, ^m In or ⁸⁹ Zr, wherein the chelating agent contains the structure of formula (I): - 33 045797 formula (I) wherein each of R1, R2, _R3 and R4 independently represents CHQCO2X, where Q is independently hydrogen, C1-C4 alkyl or ( _C1 - _C2 alkyl)phenyl, and X independently represents hydrogen, benzyl, C1-C4 alkyl; And Z represents (CH ₂ ) _n Y, where n is 1-10, and Y is an electrophilic or nucleophilic moiety covalently linked to an alkyne group; alternatively, Z is hydrogen; and each of R1, R2, _R3 and R4 independently represents CHQCO2X, where Q is independently hydrogen, C1-C4 alkyl or ( _C1 - _C2 alkyl)phenyl, and X independently represents hydrogen, benzyl, C1-C4 alkyl, or an electrophilic or nucleophilic moiety covalently bonded to an alkyne group, or alternatively, the chelating agent contains an open chain ligand. 16. The method of claim 15, further comprising reacting a side chain electrophile with a sulfhydryl group covalently linked to an azide, tetrazine or tetrazole group to produce a modified antibody or antigen binding fragment thereof. 17. The method of claim 15, wherein the modified antibody or antigen-binding fragment thereof is produced by site-specific incorporation of a first click reaction partner. 18. The method of claim 17, wherein the modified antibody or antigen-binding fragment thereof is produced by a method comprising adjusting the antibody or antigen-binding fragment thereof with a bacterial endoglycosidase specific for the β-1,4-linkage between the core GlcNac residue at the antibody Fc glycosylation site for producing a customized antibody or an antigen-binding fragment thereof, and reacting the customized antibody or an antigen-binding fragment thereof with an azide-labeled sugar in the presence of a glycotransferase, thereby obtaining a modified antibody or an antigen-binding fragment thereof. 19. The method of claim 18, wherein the azide-labeled sugar is UDP-N-azidoacetyl-lactosamine (UDP-GalNaz) or UDP-6-azido-6-deoxy GalNAc. 20. The method according to claim 18, characterized in that the glycosyltransferase is selected from galactosyltransferase GalT or GalNAc transferase. 21. The method of claim 15, wherein the modified antibody or antigen-binding fragment thereof is produced by a method comprising deglycosylation of the antibody or antigen-binding fragment thereof with an amidase to produce a deglycosylated antibody or antigen-binding fragment thereof, and reacting the deglycosylated antibody or antigen-binding fragment thereof with an azidoamine, in the presence of transglutaminase of microorganisms, thereby obtaining a modified polypeptide. 22. The method according to claim 21, in which 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. 23. The method according to claim 15, in which the chelating moiety contains the structure of formula (II): formula (II) or structure of formula (III): - 34 045797 formula (III). 24. The method of claim 1, wherein said polypeptide covalently linked to the first click reaction partner is also linked to the second click reaction partner; said radioactive complex is a first radioactive complex, and said radioactive complex is a first radioactive metal ion bound to a chelating moiety, wherein the chelating moiety comprises a chelating agent covalently bound to a third click reaction partner; additionally includes: providing a second radioactive complex comprising a second radioactive metal ion linked to a chelating moiety, wherein the chelating moiety comprises a chelating agent covalently linked to a fourth click reaction partner; wherein said contacting comprises contacting the modified polypeptide with radioactive complexes under conditions allowing the first click partner to react with a third click partner, and the second click partner to react with a fourth click partner, so as to label the polypeptide with first and second radioactive metal ions, and in which one of the first and second click reaction partners contains an alkyne group, and the other of the first and second click reaction partners contains an azide, and in which one of the third and the fourth click partner contains an alkene group, and the other of the third and fourth click partners contains a diene. 25. The method of claim 24, wherein the first or second radioactive metal ion is a diagnostic emitter and the other is a therapeutic emitter, or wherein both the first and second radioactive metal ions are therapeutic emitters. 26. A pharmaceutical composition containing a radiolabeled polypeptide obtained by the method according to claim 1 or 15, and a pharmaceutically acceptable carrier, in which the chelating agent specified in claim 1 contains a macrocycle having the structure of formula (I): formula (I) wherein R1, _R2 , R3 and _R4 are each independently CHQCO2X, wherein Q is independently hydrogen, C1- _C4 alkyl or (C1-C2 alkyl)phenyl, and X is independently hydrogen, benzyl, C1- _C4 alkyl; and Z is (CH ₂ ) _n Y, where n is 1-10, and Y is an electrophilic or nucleophilic moiety covalently linked to a second click reaction partner; alternatively, Z is hydrogen; and each of R1, _R2 , R3 and _R4 is independently CHQCO2X, where Q is independently hydrogen, C1- _C4 alkyl or ( _C1 - _C2 alkyl)phenyl, and X independently represents hydrogen, benzyl, C1- _C4 alkyl, or an electrophilic or nucleophilic moiety covalently bonded to a second click reaction partner; or alternatively, the chelating agent contains an open chain ligand containing deferoxamine (DFO). 27. A method of treating a neoplastic disease or disorder in a subject in need thereof, comprising administering to the subject a pharmaceutical composition according to claim 26. 28. A theranostic agent containing a radiolabeled antibody obtained by the method according to claim 1 or 15, and a pharmaceutically acceptable carrier in which the immunological properties of the radiolabeled antibody are preserved, in which the chelating agent specified in claim 1 contains macro- - 35 045797 Z cycle having the structure of formula (I): formula (I) wherein each of R1, R2, R3 and R4 independently represents CHQCO2X, where Q is independently hydrogen, C1-C4 alkyl or ( _C1 - _C2 alkyl)phenyl, and X independently represents hydrogen, benzyl, C1-C4 alkyl; And Z is (CH ₂ )nY, where n is 1-10, and Y is an electrophilic or nucleophilic moiety covalently linked to a second click reaction partner; alternatively, Z is hydrogen; and each of R1, R2, R3 and R4 independently represents CHQCO2X, where Q is independently hydrogen, C1-C4 alkyl or ( _C1 - _C2 alkyl)phenyl, and X independently represents hydrogen, benzyl, C1-C4 alkyl, or an electrophilic or nucleophilic moiety covalently linked to a second click reaction partner; or alternatively, the chelating agent contains an open chain ligand containing deferoxamine (DFO). 29. Polypeptide theranostic agent obtained by the method according to claim 1, having the structure of formula (VIII): or formula (IX): About n-A. Η 'W Ν .0 Ν Ν' χ protein . Q Z'P N.H. 30. Theranostic agent according to claim 29, in which the radioactive metal ion is selected from ³² P, ⁴⁷ Sc, ⁶⁷ Cu, ⁷⁷ As, ⁸⁹ Sr, ⁹⁰ Y, ⁹⁹ Tc, ¹⁰⁵ Rh, ¹⁰⁹ Pd, ^m 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 ^m In. 31. Combination containing: a. a modified polypeptide comprising a polypeptide covalently linked to a first click reaction partner; And b. a radioactive complex containing a radioactive metal ion associated with a chelating moiety, wherein the chelating moiety comprises a chelating agent covalently linked to a second click reaction partner; wherein the combination is intended for use in labeling a polypeptide with a radioactive ion