HK40032905A - Radiolabeling of polypeptides - Google Patents

Description

Radiolabelling of polypeptides

By electronsReference sequence list submitted by way

This application contains a Sequence listing submitted electronically via EFS-Web as an ASCII formatted Sequence listing with a file name of "Sequence _ L stating", a creation date of 2018, 12 months and 7 days, and a size of about 13.2 kB. submitted via EFS-Web is part of this specification and is incorporated herein by reference in its entirety.

Cross Reference to Related Applications

This application claims priority to U.S. provisional patent application 62/599,830 filed 2017, 12, 18, 35u.s.c. § 119(e), the disclosure of which is incorporated herein by reference in its entirety.

Technical Field

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

Background

Radionuclides emitting α particles have good promise for cancer therapy due to their combination of high energy and short range action, offering the potential for potent killing of primarily localized tumor cells (Kim, y.s. and m.w.brechbiel, Anoverview of targeted alpha therapy. tumor Biol, 2012.33 (3): pages 573-90.) targeted delivery of α emitters using antibodies, scaffold proteins, small molecule ligands, nucleic acid aptamers, or other binding moieties specific for cancer antigens, provides a method of selective delivery of radionuclides to tumors to enhance their efficacy and mitigate off-target effects.

Actinium-225 (²²⁵Ac) is a particularly interesting emitting α radioisotope for medical applications (Miederer et al, reading the potential of the Actinium-225 radioactive source generatorator intargeted alpha particle therapy applications.Adv Drug Deliv Rev，2008.60(12)：71-82)。²²⁵The 10-day half-life of Ac is long enough to facilitate radioconjugate production, but short enough to match the circulating pharmacokinetics of the delivery vehicle, such as an antibody. Therefore, the temperature of the molten metal is controlled,²²⁵ac radioactive immunoconjugates are of particular interest. In addition to this, the present invention is,²²⁵ac reaches stable isotope²⁰⁹Another radioisotope of interest for medical applications is lutetium 177 (lutetium 177) ((B))¹⁷⁷L u) which emit gamma radiation suitable for imaging and moderate energy β radiation suitable for radiation therapy¹⁷⁷L u-labeled peptides exhibit reduced normal tissue damage, and¹⁷⁷l u labeling allows treatment and imaging using a single radiolabel agent (Kwekkeboom DJ et al, [ 177L u-DOTAOTyr 3)]octreotate：comparison with[111In-DTPAo]Octreotide in tissues Eur JNucl Med.2001; 28: 1319-1325.) other radioisotopes for therapeutic use include, for example, β or α emitters such as, for example, thorium, radium, and,³²P、⁴⁷Sc、⁶⁷Cu、⁷⁷As、⁸⁹Sr、⁹⁰Y、⁹⁹Tc、¹⁰⁵Rh、¹⁰⁹Pd、¹¹¹Ag、¹³¹I、¹⁵³Sm、¹⁵⁹Gd、¹⁶⁵Dy、¹⁶⁶Ho、¹⁶⁹Er、¹⁸⁶Re、¹⁸⁸Re、¹⁹⁴Ir、¹⁹⁸Au、¹⁹⁹Au、²¹¹At、²¹²Pb、²¹²Bi、²¹³Bi、²²³Ra、²⁵⁵Fm and²²⁷th. Other radioisotopes for imaging applications include gamma-emitting radioisotopes, such as, for example⁶²Cu、⁶⁴Cu、⁶⁷Ga、⁶⁸Ga、⁸⁶Y、⁸⁹Zr and¹¹¹In。

previous clinical and preclinical procedures have primarily used 1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA) for actinide chelation. However, it is known that actinide DOTA chelation can be challenging (Deal, k.a. et al, Improved in vivo stability of actinoium-225 macrocycycycycycnic complexes j MedChem, 1999.42 (15): pages 2988-92), and often requires harsh conditions or high levels of DOTA per antibody. Thus, two different approaches, referred to as "one-step" and "two-step" radiolabelling approaches, have been utilized, each with its own drawbacks.

First a "two-step" process was developed, which involves 2 chemical steps involving actinium (McDeitt, M.R. et al, Tumorthopherapy with targeted atomic generators. science, 2001.294 (5546): pages 1537-40). Chelation with high radiochemical yield (about 95%) at 55 ℃ to 60 ℃ by a bifunctional chelating agent (BFC) DOTA-isothiocyanate (DOTA-SCN) at pH 4.5-5 using 2M acetate buffer²²⁵Ac 30 min. Then, make²²⁵Ac-DOTA-SCN reacts with the targeting antibody to produce a radioimmunoconjugate. The major drawback of the two-step process is that about 90% of the SCN does not survive the labeling conditions, so about 90% of the input²²⁵Ac is conjugated to a non-reactive form of DOTA that is not conjugated to an antibody. This not only results in low yields (typically only about 10%) and higher costs, but also results in a decrease in specific activity that can limit the efficacy of the final conjugate.

Recently, the "one-step" method has been developed for actinium (Maguire, W.F. et al, effective 1-steric analogs to high specific activity with 225Acfor alpha-specific radioimmunotherapy of cancer. J.J. Nucl Med, 2014.55 (9): pages 1492-8). The process has only 1 chemical reaction step involving actinium. The DOTA-SCN was first conjugated to the antibody. Then, the mixture is subjected to mild conditions (37 ℃ C., pH 7.5)²²⁵Ac chelates to DOTA-mAb, achieving radiochemical yields as high as 80%. However, it is necessary to conjugate high levels of DOTA (about 10 or more per antibody) to achieve high yields. High chelating agent: antibody Ratio (CAR), in this case high DOTA: Ab ratio (DAR), substances are more likely to have impaired immunoreactivity; further, while the average DAR may be 10, however²²⁵Ac may sequester the population at even higher ratios than the mean. Thus, the method exists²²⁵Ac-linked antibody chelationRisk of the least active part of the agent conjugate. Furthermore, it is necessary to treat the antibody and DOTA-mAb conjugate under metal-free conditions to avoid chelation of common metals such as iron, zinc and copper, which introduces significant challenges in the production process.

Click chemistry is a chemical method introduced by sharp in 2001 and describes chemistry that is modulated to generate substances quickly and reliably by linking small units together. See, e.g., Kolb, Finn and sharpless angelwaldte chemical Edition (2001) 40: 2004-2021; evans, Australian Journal of Chemistry (2007) 60: 384-395). Coupling reactions (some of which may be classified as "click chemistry") include, but are not limited to, the formation of esters, thioesters, amides (e.g., such as peptide couplings) from activated acids or halogenated acyl groups; nucleophilic displacement reactions (e.g., nucleophilic displacement such as halides or ring opening of strained ring systems); azide-alkyne Huisgen cycloaddition (e.g., 1, 3-dipolar cycloaddition between an azide and an alkyne to form a1, 2, 3-triazole linker); addition of mercaptoalkyne; imine formation; diels-alder reaction (Diels-alderection) between tetrazine and trans-cyclooctene (TCO); and Michael addition (e.g., maleimide addition).

Click chemistry reactions between alkynes and azides typically require the addition of a copper catalyst to promote the 1, 3-cycloaddition reaction, and are referred to as copper-catalyzed azido-alkyne cycloaddition (CuAAC) reactions. However, click chemistry reactions between cyclooctyne or cyclooctyne derivatives and azides typically do not require the addition of a copper catalyst, but rather proceed via strain-promoted azide-alkyne cycloaddition (SPAAC) (Debets, M.F. et al, Bioconjugation with structured aldehydes and lkynes. Acc Chem Res, 2011.44 (9): pages 805-15).

Site-specificity has become a key area of focus in the field of Antibody Drug Conjugates (ADCs) (Agarwal, P. and C. R. Bertozzi, Site-specific antibody-drugs: the new of biological chemistry, protein engineering, and drug reduction. bioconjugate chem, 2015.26 (2): pages 176-92), since it has been demonstrated that Site-specific methods can increase the efficacy and safety of ADCs compared to random conjugation. It is believed that similar safety and efficacy benefits may be achieved with radioactive immunoconjugates.

As indicated above, there remains a need in the art for efficient methods of producing stable radioimmunoconjugates with high specific activity and high yield.

Disclosure of Invention

The present invention fills this need by providing methods for radiolabeling polypeptides, such as antibodies, using click chemistry. In the methods of the invention, azide-modified antibodies and radioactive complexes comprising a radioactive metal ion associated with a chelating moiety containing an alkyne group are used in click chemistry reactions to produce antibodies with low chelator: antibody Ratio (CAR) and high radiochemical yield of stable radioimmunoconjugates, while requiring reduced use of radioactive metals and requiring only metal-free conditions in the step for generating the initial radioactive complex. The methods of the invention simplify previous methods for producing radioimmunoconjugates with increased safety, efficacy and uniformity.

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

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

b. providing a radioactive complex comprising the radiometal ion associated with a chelating moiety, wherein the chelating moiety comprises a chelator covalently linked to a second click reaction partner; and

c. contacting said modified polypeptide with said radioactive complex under conditions that allow said first click reaction partner to react with said second click reaction partner, thereby labeling said polypeptide with said radioactive metal ion.

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

In another general aspect, the present 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 of the present invention.

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

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

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

wherein the combination or kit will be used to label the polypeptide with the radioactive metal ion.

In other general aspects, the invention relates to a therapeutic or diagnostic agent ("diagnostic agent") comprising a radiolabeled polypeptide prepared by the method of the invention.

Drawings

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

In the drawings:

FIG. 1 shows a schematic representation of a radiolabeled antibody according to the method of the invention; random conjugation is shown in the figure, and a similar radiolabelling scheme was used when the azide was site-specifically conjugated to a monoclonal antibody (mAb);

FIG. 2 shows a pair of click chemistry, according to embodiments of the present application⁸⁹A synthetic protocol for an improved two-step preparation of Zr-DOTA-mAb;

figure 3 shows cell binding of In-111 radioimmunoconjugates: the bound radioactivity increased with increasing cell number; in particular:

FIG. 3A shows binding to human prostate cancer cell line C4-2B (PSMA +, transferrin receptor +) by a PSMA binding antibody ("PSMB 127") In-111 radioactive immunoconjugate and a human transferrin In-111 radioactive conjugate, according to embodiments of the present application; and is

FIG. 3B shows binding to human epidermoid carcinoma cell line A431(EGFR +) by EGFR-binding antibodies cetuximab (cetuximab) and panitumumab (panitumumab) In-111 radioimmunoconjugates and the lack of binding of these conjugates to the control (EGFR-) human AM L cell line MO L M-13, according to embodiments of the present application;

FIG. 4 shows the kinetics of cellular internalization of In-111 In human prostate cancer cell line C4-2B treated with anti-PSMA mAb In-111 radioimmunoconjugates, according to embodiments of the present application; surface-bound In-111 rapidly disappeared from the cell surface and redistributed within the cell; and is

FIG. 5 shows the results of a mouse tumor xenograft study, implanting human prostate cancer L NCaP cells into mice when tumors reached 100mm³When mice were treated with a series of active single doses of a click radiolabeled anti-PSMA mAb ("PSMB 127") actinium radioconjugate according to embodiments of the present application or an isotype control, human IgG4 antibody, that binds to a viral target not present in the systemic radioconjugate; in particular:

fig. 5A shows tumor volumes for each group; size until less than half of the groups remain;

fig. 5B shows survival curves for the control mAb group; and is

Fig. 5C shows the survival curves for the anti-PSMA mAb panel.

Detailed Description

Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, articles and the like which has been included in this specification is intended to provide a context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any invention disclosed or claimed.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Otherwise, certain terms referred to herein have the meanings set forth in the specification. All patents, published patent applications, and publications cited herein are hereby incorporated by reference as if fully set forth herein.

It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The term "comprising" as used herein may be replaced with the term "comprising" or "including", or sometimes with the term "having", as used herein.

As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claims. Whenever used herein in the context of one aspect or embodiment of the present invention, any of the foregoing terms "comprising," "including," and "having" may be substituted with the term "consisting of or" consisting essentially of.

As used herein, the connecting term "and/or" between a plurality of recited elements is understood to encompass both single and combined options. For example, where two elements are connected by "and/or," a first option means that the first element applies without the second element. The second option means that the second element is applied without the first element. A third option refers to the suitability of using the first and second elements together. Any of these options is understood to fall within the meaning and thus meet the requirements of the term "and/or" as used herein. Parallel applicability of more than one option is also understood to fall within the meaning and thus meet the requirements of the term "and/or".

To assist the reader of the present application, the specification has been divided into various paragraphs or sections, or directed to various embodiments of the present application. These divisions should not be considered as separating the essence of a paragraph or section or embodiment from the essence of another paragraph or section or embodiment. Rather, those skilled in the art will appreciate that the present description has broad application, and encompasses all combinations of individual sections, paragraphs, and sentences that are contemplated. The discussion of any embodiment is meant to be exemplary only, and is not intended to intimate that the scope of the disclosure, including the claims, is limited to these examples.

Click radiolabeling of polypeptides

In contrast to known procedures, the methods of the present invention provide an improved method for producing a radioactive immunoconjugate suitable for, for example, medical applications in a subject (e.g., a human) in need thereof. In particular, the methods described herein provide for high yields of chelated metal ions (including, but not limited to²²⁵Ac、¹¹¹In and⁸⁹zr) and processes for low DAR address the major limitations of current approaches. The invention allows for the generation of a single batch of azide-labeled polypeptide, such as an azide-mAb conjugate, which can then be used for the generation of radiolabeled diagnostics (e.g., when used with⁸⁹Zr or¹¹¹In labeled) or treated (e.g., when administered with²²⁵Ac-labeling) wherein the radioactive label is attached at one or more of the same sites within the batch of azide-labeled polypeptides, which polypeptides may be obtained by site-specific modification or by random azide conjugation. For example, in the case of random azide conjugation, a batch of samples of azide-labeled polypeptides containing a single distribution of azide-modified sites can be radiolabeled using click chemistry of the invention toFor different purposes.

The methods of the invention that are click chemistry dependent and are referred to as "click radiolabelling" involve (1) obtaining a modified polypeptide, such as an antibody, comprising a first click chemistry reaction partner, e.g., an azide moiety; (2) obtaining a radioactive complex comprising a radioactive metal ion associated with a chelating moiety (e.g.,²²⁵Ac、¹¹¹in or⁸⁹Zr), wherein the chelating moiety comprises a chelating agent covalently linked to a second click chemistry reaction partner (e.g., an alkyne group), such as DOTA-dibenzocyclooctyne (DOTA-DBCO) or desferrioxamine-DBCO (DFO-DBCO); and (3) performing a reaction between a click chemistry reaction partner of the modified peptide and a radioactive complex, such as a strain-promoted alkyne-azide cycloaddition (SPAAC) between an azide moiety and an alkyne group.

The method of the invention allows chelation of radioactive metals under low or high pH and/or high temperature conditions to maximize efficiency, which can be achieved without the risk of inactivating alkyne reaction partners. Efficient chelation and efficient SPAAC reaction between azido-mAb and radioactive complexes allows the generation of radioactive immunoconjugates with high radioactive chemical yield, even though the azide: mAb ratio was low. In the method of the invention, the only step that must exclude trace metals is the chelation of the radioactive metal ion with the chelating moiety; the antibody production, purification and conjugation steps need not be performed under metal-free conditions.

As used herein, the term "click chemistry" refers to the chemical concept introduced by Sharpless, which describes chemistry that is tailored to rapidly and reliably generate covalent bonds by linking small units containing reactive groups together (see Kolb et al, supra). Click chemistry does not refer to a specific reaction, but rather refers to concepts including, but not limited to, reactions that mimic reactions that occur in nature. In some embodiments, click chemistry reactions are modular, broad in scope, give high chemical yields, generate innocuous byproducts, are stereospecific, exhibit a large thermodynamic driving force to facilitate reaction with a single reaction product, and/or can be performed under physiological conditions. In some embodiments, click chemistry reactions exhibit high atom economy, can be performed under simple reaction conditions, use readily available starting materials and reagents, use no toxic solvents or use benign or easily removable solvents, such as water, and/or provide simple product isolation by non-chromatographic methods such as crystallization or distillation. In certain embodiments, the click chemistry reaction is a Huisgen cycloaddition or a1, 3-dipolar cycloaddition between an azide (-N3) and an alkyne or alkyne moiety to form a1, 2, 4-triazole linker.

In one general aspect, the present invention relates to a method of labeling a polypeptide, nucleic acid aptamer, or small molecule ligand with a radioactive metal ion, the method comprising:

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

b. providing a radioactive complex comprising the radiometal ion associated with a chelating moiety, wherein the chelating moiety comprises a chelator covalently linked to a second click reaction partner; and

c. contacting said modified polypeptide with said radioactive complex under conditions that allow said first click reaction partner to react with said second click reaction partner, thereby labeling said polypeptide with said radioactive metal ion.

As used herein, the term "polypeptide" refers to a polymer composed of amino acid residues linked via peptide bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. The term refers to a polypeptide of any size, structure or function. Typically, a polypeptide is at least three amino acids long. The polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. According to a preferred embodiment, the polypeptide is an antibody, preferably a monoclonal antibody or a fragment thereof, such as an antigen binding fragment thereof. According to a preferred embodiment, the antibody or fragment thereof is specific for a cancer antigen. According to other embodiments, the polypeptide is an engineered domain or a scaffold protein.

As used herein, the term "antibody" or "immunoglobulin" is used broadly and includes immunoglobulins or antibody molecules, including polyclonal antibodies, monoclonal antibodies (including murine, human-adapted, humanized and chimeric monoclonal antibodies), and antigen-binding fragments thereof.

Generally, an antibody is a protein or peptide chain that exhibits binding specificity for a particular antigen (referred to herein as a "target"). Antibody structures are well known. Depending on the heavy chain constant domain amino acid sequence, immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG and IgM. IgA and IgG are further sub-classified into isotypes IgA1, IgA2, IgG1, IgG2, IgG3 and IgG 4. Thus, the antibodies of the invention can be any of the five main classes or corresponding subclasses. Preferably, the antibody of the invention is IgG1, IgG2, IgG3 or IgG 4. The light chain of an antibody of any vertebrate species can be assigned to one of two completely different types, κ and λ, based on the amino acid sequence of its constant domain. Thus, an antibody of the invention may contain a kappa or lambda light chain constant domain. According to a particular embodiment, the antibody of the invention comprises heavy and/or light chain constant regions from a mouse antibody or a human antibody. Each of the four IgG subclasses has a different biological function, which is referred to as an effector function. These effector functions are typically mediated by interaction with Fc receptors (Fc γ R) or by binding to C1q and fixing complement. Binding to Fc γ R results in antibody-dependent cell-mediated lysis, while binding to complement factors results in complement-mediated lysis. Antibodies useful in the invention may have no or minimal effector function, but retain their ability to bind FcRn.

As used herein, the term "antigen-binding fragment" refers to an antibody fragment, such as, for example, a diabody, Fab ', F (ab ')2, Fv fragment, disulfide 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 (diabody), multispecific antibody formed from a portion of an antibody comprising one or more CDRs, camelized single domain antibody, nanobody, domain antibody, bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise an intact antibody structure. The antigen binding fragment is capable of binding to the same antigen as the parent antibody or the antigen to which the parent antibody fragment binds. As used herein, the term "single chain antibody" refers to a single chain antibody as is conventional in the art, which 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. As used herein, the term "single domain antibody" refers to a conventional single domain antibody in the art that comprises a heavy chain variable region and a heavy chain constant region or 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 can bind to a target. The scaffold contains a "framework" which is largely structured, and a "binding domain" which contacts the target and provides specific binding. The binding domain of the scaffold need not be defined by one contiguous sequence of the scaffold. In some cases, the scaffold may be part of a larger binding protein, which itself may be part of a multimeric binding protein containing multiple scaffolds. Certain binding proteins may be bispecific or multispecific in that they may bind to two or more different epitopes. The scaffold may be derived from a single chain antibody, or the scaffold may not be derived from an antibody.

In light of the present disclosure, any method known to those skilled in the art for chemically or enzymatically modifying a polypeptide may be used to covalently attach a polypeptide of the present invention to a first click reaction partner. Amine reactive groups that react with primary amines present in the N-terminus of each polypeptide chain and in the side chains of lysine residues can be used in methods for randomly modifying polypeptides. Examples of amine reactive groups suitable for use in the present invention include, but are not limited to, N-hydroxysuccinimide (NHS), substituted NHS (such as sulfo-NHS), isothiocyanates, and tetrafluorophenyl and perfluorophenyl esters. Thiol-reactive groups that react with thiols or sulfhydryls present in the side chains of cysteine residues may be used in methods for randomly modifying polypeptides. Examples of thiol-reactive groups suitable for use in the present invention include, but are not limited to, maleimide, haloacetyl, and phenyl oxadiazole sulfone. According to a preferred embodiment, the modified polypeptide is obtained by reacting a side chain, preferably the amino side chain of lysine, with an electrophile covalently linked to a first click reaction partner, e.g. NHS-azide.

The click radiolabelling method of the invention facilitates the site-specific generation of radioimmunoconjugates by site-specifically mounting azide groups onto antibodies using established methods (L i, X. et al, Preparation of well-defined antibody-drugs conjugates through molecular remodelling and strain-protein a. angel Chem Int Ed, 2014.53 (28): pages 7179-82; Xiao, H. et al, Genetic engineering of multiple unknown amino acids in protein molecules, cell Chem em Ed Engl, 2013.52 (52): pages 14080-3. the site-specific radiolabelling method of the invention is suitable for the site-specific labelling of a protein or a molecule in the field according to the methods disclosed in the invention and is not limited to any of the methods known in the art (such as the methods disclosed in the art for site-specific labelling antibodies)^TM) The use of unnatural amino acids or glycans (e.g., selenocysteine, p-AcPhe, formylglycine generating enzymes (FGE, SMARTag)^TM) Etc.), and enzymatic methods (e.g., using glycosyltransferases, endoglycosidases, microbial or bacterial transglutaminase (MTG or BTG), transpeptidase a, etc.) according to preferred embodiments, the modified polypeptide is an antibody or antigen-binding fragment thereof obtained by trimming the antibody or antigen-binding fragment thereof with a bacterial endoglycosidase (such as glycinator (genovis)) specific for the β -1, 4 linkage between core GlcNac residues in the Fc glycosylation site of the antibody, which leaves the innermost GlcNac intact on the Fc, thereby allowing the azido saccharide to remain intact on the FcSite-specific incorporation is at this site. The trimmed antibody or antigen-binding fragment thereof can then be reacted with an azide-labeled sugar, such as UDP-N-azidoacetylgalactosamine (UDP-GalNaz) or UDP-6-azido 6-deoxygalnac, in the presence of a sugar transferase, such as GalT galactosyltransferase or GalNAc transferase, to obtain a modified antibody or antigen-binding fragment thereof. According to other preferred embodiments, the modified polypeptide is an antibody or antigen-binding fragment thereof obtained by deglycosylating 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-alkyl-amine, such as azido-polyethylene glycol (PEG) -amine, e.g., O- (2-aminoethyl) -O ' - (2-azidoethyl) tetraethylene glycol, O- (2-aminoethyl) -O ' - (2-azidoethyl) pentaethylene glycol, O- (2-aminoethyl) -O ' - (2-azidoethyl) triethylene glycol, or the like), or in the presence of microbial transglutaminase, to obtain a modified antibody or antigen-binding fragment thereof.

As used herein, the term "nucleic acid aptamer" refers to a single-stranded oligonucleotide (single-stranded DNA or RNA molecule) that can specifically bind its target with high affinity. Nucleic acid aptamers can be used as molecules targeting a variety of organic and inorganic materials.

As used herein, the term "small molecule ligand" refers to a low molecular weight organic compound. As used herein, a small molecule ligand may refer to a compound having a size of less than about 1000 daltons, and may be synthetic or naturally occurring in the laboratory.

As used herein, the term "click reaction partner" or "click chemistry handle" refers to a reactant or reactive group that can participate in a click chemistry reaction. The click reaction partner may be a moiety that is rarely present in and chemically inert to naturally occurring biomolecules, but, for example, when reacting with an azide-reactive or alkyne-reactive group, the reaction may occur effectively under biologically relevant conditions, for example, under cell culture conditions, such as in the absence of excess heat or harsh reactants. Generally, click chemistry reactions require at least two molecules that comprise click reaction partners that can react with each other. Such click reaction partners that react with each other are sometimes referred to herein as click chemistry handle pairs or click chemistry pairs. In some embodiments, the click reaction partner is 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, trans-cyclooctene, norbornene, or bicyclononene may be paired with a suitable tetrazine dienophile as a click reaction pair. In other embodiments, tetrazoles can serve as potential sources of nitrilimines that can pair with non-activated alkenes in the presence of ultraviolet light to produce click reaction pairs, known as "photo-click" reaction pairs. In other embodiments, the click reaction partners are cysteine and maleimide. For example, a cysteine from a peptide (e.g., GGGC) can be reacted with a maleimide associated with a chelator (e.g., NOTA). Other suitable click chemistry handles are known to those skilled in the art (see, e.g., Spicer et al, Selective chemical protein modification. Nature communications.2014; 5: page 4740). In other embodiments, the click reaction partner is a Staudinger ligation (Staudinger ligation) component, such as phosphines and azides. In other embodiments, the click reaction partner is a diels-alder reaction component, such as a diene, such as tetrazine, and an alkene, such as trans-cyclooctene (TCO) or norbornene. Exemplary click reaction partners are described in US20130266512 and WO2015073746, the relevant descriptions of click reaction partners in both of which are incorporated herein by reference. According to a preferred embodiment, one of the first click reaction partner and the second click reaction partner comprises an alkyne group and the other click reaction partner comprises an azide. According to other preferred embodiments, one of the first click reaction partner and the second click reaction partner comprises an alkene group and the other click reaction partner comprises a diene.

As used herein, the term "alkyne," "alkyne group," or "alkyne moiety" refers to a functional group that includes a carbon-carbon triple bond. Alkyne moieties include terminal alkynes and cyclic alkynes, preferably those that react with azide groups. A terminal alkyne has at least one hydrogen atom bonded to a triple-bonded carbon atom. A cyclic alkyne is a cycloalkyl ring that contains one or more triple bonds. Examples of cyclic alkynes include, but are not limited to, cyclooctyne and cyclooctyne derivatives, such as Bicyclononylene (BCN), Difluorocyclooctyne (DIFO), Dibenzocyclooctyne (DIBO), keto-DIBO, diarylazacyclooctanone (BARAC), Dibenzoazacyclooctyne (DIBAC), Dimethoxyazacyclooctyne (DIMAC), Dibenzylcyclooctyne (DBCO), difluorobenzocyclooctyne (diffo), Monobenzocyclooctyne (MOBO), and tetramethoxydibo (tmdobo). According to a preferred embodiment, one of the first click reaction partner and the second click reaction partner comprises a cyclic alkyne, preferably DBCO. According to a preferred embodiment, the further click reaction partner comprises an azide, preferably NHS-azide.

As used herein, the term "diene" refers to a compound having two carbon-carbon double bonds, wherein these double bonds are conjugated in the 1, 3-position. The double bonds of the diene may be cis-or trans-form. Examples of dienes include, but are not limited to, tetrazine or tetrazole groups.

As used herein, the term "alkene," "olefinic group," or "olefinic moiety" refers to an unsaturated hydrocarbon molecule that contains a carbon-carbon double bond. According to particular embodiments, the olefin may comprise from 2 to 100 carbon atoms. Examples of alkenes include, but are not limited to, norbornene and trans-cyclooctene (TCO). According to other preferred embodiments, one of the first click reaction partner and the second click reaction partner comprises an olefinic group, preferably norbornene or TCO. According to a preferred embodiment, the further click reaction partner comprises a diene, preferably a tetrazine or tetrazole group.

As used herein, the term "covalently linked" means that the polypeptide is attached to the first click reaction partner via at least one covalent linkage, and the chelator is attached to the second click reaction partner via at least one covalent linkage. The linkage may be direct, i.e., without a linker, or indirect, i.e., via a linker.

As used herein, the term "linker" refers to a chemical moiety that links a polypeptide or chelator to a click reaction partner. Any suitable linker known to those skilled in the art may be used in the present invention in light of the present disclosure. The linker can be, for example, a single covalent bond, a substituted or unsubstituted alkyl group, 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 linkage or a protease cleavage site, such as valine-citrulline-PAB.

As used herein, the term "radioactive metal ion" or "radioactive metal ion" refers to one or more isotopes of an element that emit particles and/or photons. Any radioactive metal known to those skilled in the art may be used in the present invention in light of the present disclosure. Examples of radioactive metals suitable for use in the present invention include, but are not limited to³²P、⁴⁷Sc、⁶²Cu、⁶⁴Cu、⁶⁷Cu、⁶⁷Ga、⁶⁸Ga、⁷⁷As、⁸⁶Y、⁸⁹Zr、⁸⁹Sr、⁹⁰Y、⁹⁹Tc、¹⁰⁵Rh、¹⁰⁹Pd、¹¹¹Ag、¹¹¹In、¹¹⁷Sn、¹³¹I、¹⁵³Sm、¹⁵⁹Gd、¹⁶⁵Dy、¹⁶⁶Ho、¹⁶⁹Er、¹⁷⁷Lu、¹⁸⁶Re、¹⁸⁸Re、¹⁹⁴Ir、¹⁹⁸Au、¹⁹⁹Au、²¹¹At、²¹²Pb、²¹²Bi、²¹³Bi、²²³Ra、²²⁵Ac、²²⁷Th and²⁵⁵and Fm. As used herein, the term "diagnostic emitter" refers to a radioactive metal ion that can be used in diagnostic or imaging applications. Examples of diagnostic emitters include, but are not limited to, gamma emitters, such as⁶²Cu、⁶⁴Cu、⁶⁷Ga、⁶⁸Ga、⁸⁶Y、⁸⁹Zr and¹¹¹in. As used herein, the term "therapeutic emitter" refers to radioactive metal ions that can be used in therapeutic applications. Examples of therapeutic emitters include, but are not limited toLimited to β or α emitters, such as thorium, radium,³²P、⁴⁷Sc、⁶⁷Cu、⁷⁷As、⁸⁹Sr、⁹⁰Y、⁹⁹Tc、¹⁰⁵Rh、¹⁰⁹Pd、¹¹¹Ag、¹³¹I、¹⁵³Sm、¹⁵⁹Gd、¹⁶⁵Dy、¹⁶⁶Ho、¹⁶⁹Er、¹⁷⁷Lu、¹⁸⁶Re、¹⁸⁸Re、¹⁹⁴Ir、¹⁹⁸Au、¹⁹⁹Au、²¹¹At、²¹²Pb、²¹²Bi、²¹³Bi、²²³Ra、²²⁵Ac、²⁵⁵Fm and²²⁷th. According to a preferred embodiment, the radioactive metal ion is²²⁵Ac, is used. According to other embodiments, the polypeptide may be labeled with a non-metallic radioactive label for use in pre-targeting or diagnostic applications. Examples of non-metallic radioactive labels suitable for use in the present invention include, but are not limited to¹²⁵I and¹⁸F。

the radioactive complexes described herein comprise a radioactive metal ion associated with a chelating moiety. According to embodiments of the present invention, the chelating moiety comprises a chelating agent covalently linked to a click reaction partner, and is sometimes referred to herein as a "bifunctional chelating agent".

As used herein, the term "chelator (chelant)' or" chelator "refers to a radioactive metal, such as²²⁵Ac) or a compound to which a metal can chelate via coordination bonding. Any chelating agent known to those skilled in the art can be used in the present invention in light of the present disclosure. In one embodiment, the chelating agent comprises a macrocycle. Examples of macrocyclic-containing chelating agents suitable for use in the present invention include, but are not limited to, Desferoxamine (DFO), ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA). In another embodiment, the chelating agent comprises an open chain ligand. Examples of chelating agents containing open-chain ligands suitable for use in the present invention include, but are not limited to, 1, 4, 7, 10-tetraazacyclododecane-N, N ', N ", N '" -tetraacetic acid (DOTA), 1, 4, 7, 10, 13, 16-hexaazacyclohexadecane-N, N ', N ", N '", N "" ' -hexaacetic acid (HEHA), 1, 4, 7, 10, 13-pentaacetic acid (HEHA)azacyclopentadecane-N, N' -pentaacetic acid (PEPA), Macropa (Thiele et al, An Eighten-Membered Macrocyic L igand for Actinium-225 Targeted Alpha therapy, Angewchem Int Ed Engl.2017, 11 months and 13 days; 56(46,): 14712 th page 14717), 1, 4, 8, 11-tetraazacyclotetradecane-1, 4, 8, 11-tetraacetic acid (TETA), 1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetrapropionic acid (DOTPA), 1, 4, 8, 11-tetraazacyclotetradecane-1, 4, 8, 11-tetrapropionic acid (TETPA), and 1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraazacyclotetradecane-1, 10, 8, 11-tetrapropionic acid (TETPPA), and 1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetramethylenephosphonic acid (DOTMP), according to the preferred embodiments, the chelating agent comprises the structure of formula (I):

wherein R is₁、R₂、R₃And R₄Is independently CHQCO₂X, wherein

Q is independently hydrogen, C₁-C₄Alkyl or (C)₁-C₂Alkyl) phenyl, and

x is independently hydrogen, benzyl, C₁-C₄An alkyl group; and is

Z is (CH2)_nY is, wherein

n is 1 to 10, and

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

alternatively, Z is hydrogen; and is

R₁、R₂、R₃And R₄Is independently CHQCO₂X, wherein

Q is independently hydrogen, C₁-C₄Alkyl or (C)₁-C₂Alkyl) phenyl, and

x is independently hydrogen, benzyl, C₁-C₄An alkyl group or an electrophilic or nucleophilic moiety covalently linked to the second click reaction partner.

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

according to a preferred embodiment, the chelating moiety comprises a structure of formula (III):

in one embodiment, the invention relates to a method of labelling a polypeptide with two or more radioactive metal ions using the method of the invention. For example, methods for dual labeling of polypeptides with two radioactive metal ions include:

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 associated with a chelating moiety, wherein the chelating moiety comprises a chelator covalently linked to a third click reaction partner; and

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

d. contacting the modified polypeptide with the first and second radioactive complexes under conditions that allow the first click reaction partner to react with the third click reaction partner and the second click reaction partner to react with the fourth click reaction partner, thereby labeling the polypeptide with the first and second radioactive metal ions.

According to a preferred embodiment, one of the first click reaction partner and the second click reaction partner comprises an alkyne group and the other of the first click reaction partner and the second click reaction partner comprises an azide, and wherein one of the third click reaction partner and the fourth click reaction partner comprises an alkene group and the other of the third click reaction partner and the fourth click reaction partner comprises a diene.

According to a preferred embodiment, the first or second radioactive metal ion is a diagnostic emitter and the other is a therapeutic emitter. According to other preferred embodiments, both the first and second radioactive metal ions are therapeutic emitters.

Conditions for conducting click chemistry reactions are known in the art, and any condition known to one of skill in the art for conducting click chemistry reactions can be used in the present invention in light of the present disclosure. Examples of conditions include, but are not limited to, incubating the modified polypeptide and the radioactive complex at a ratio of 1: 1 to 1000: 1 at a pH of 4 to 10 and a temperature of 20 ℃ to 70 ℃.

For example, L C/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 by transient thin layer chromatography (e.g., iT L C-SG), and radiochemical purity can be determined by size exclusion HP L C.

Pharmaceutical compositions and methods of treatment

The click radiolabelling method of the invention may be modified to a pre-targeting method (Kraeber-Border, F. et al, A targeting system for tumor PET imaging and radioimmunotherapy. FrontPharmacol, 2015.6: page 54). First, the azido-mAb is administered, allowed to bind to the target cells and allow their clearance from the circulation over time, or removed with a scavenger. Subsequently, the radioactive complexes are administered and subjected to a SPAAC reaction with the azide-mAb bound at the target site, while the remaining unbound radioactive complexes are rapidly cleared from the circulation (Deal, K.A. et al, Improved in vivo stability of actinoium-225 macrocycliciccompounds. J. Med Chem, 1999.42 (15): pp. 2988-92). The pretargeting technique provides a method of enhancing the localisation of radioactive metal ions at a target site in a subject.

Thus, in another general aspect, the present invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a radiolabeled polypeptide prepared by the method of the invention.

As used herein, the term "carrier" refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid-containing vesicle, microsphere, liposome encapsulation, or other material known in the art for use in pharmaceutical formulations. It will be appreciated 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 interfere with the effect of, or the biological activity of, the composition according to the present invention. According to the present disclosure, any pharmaceutically acceptable carrier suitable for use in antibody-based or radioactive complex-based pharmaceutical compositions may be used in the present invention, according to particular embodiments.

According to particular embodiments, the compositions described herein are formulated in an intended route suitable for administration to a subject. For example, the compositions described herein can be formulated for intravenous, subcutaneous, intramuscular, or intratumoral administration.

According to particular embodiments, the modified polypeptide and the radioactive complex may be administered in the same or different compositions.

In another general aspect, the present 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 of the present invention.

According to a particular embodiment, the methods of the invention comprise 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, when targeted, results from²²⁵α particles of Ac and its progeny are delivered to target cells and exert a cytotoxic effect on them, thereby treatingTreating neoplastic diseases or disorders.

According to particular embodiments, therapeutically effective amounts of the modified polypeptide and the radioactive complex are administered in different compositions.

As used herein, the term "therapeutically effective amount" refers to the amount of an active ingredient or component that elicits a desired biological or pharmaceutical response in a subject. The therapeutically effective amount can be determined empirically and in a routine manner for the intended purpose. For example, in vitro assays may optionally be employed to help determine optimal dosage ranges. Selection of a particular effective dose can be determined by one of skill in the art (e.g., via clinical trials) based on consideration of several factors, including the disease to be treated or prevented, the symptoms involved, the weight of the patient, the immune status of the patient, and other factors known to those of skill. The precise dose to be employed in the formulation will also depend on the route of administration and the severity of the disease and should be decided according to the judgment of the physician and the circumstances of each patient. Effective doses can be derived from dose response curves derived from in vitro or animal model test systems.

As used herein, the terms "treating" and "treatment" are both intended to refer to the amelioration or reversal of at least one measurable physical parameter associated with a disease, disorder, or condition in which administration of radioactive metal ions would be beneficial, such as a neoplastic disease or disorder, which is not necessarily identifiable in a subject, but may be identifiable in a subject. The terms "treat" and "treating" may also refer to causing regression, preventing progression, or at least delaying progression of a disease, disorder, or condition. In particular embodiments, "treating" and "treatment" refer to alleviating, preventing the development or onset of, or reducing the duration of one or more symptoms associated with a disease, disorder, or condition in which administration of a radioactive metal ion would be beneficial, such as a neoplastic disease or disorder. In particular embodiments, "treating" and "treatment" refer to preventing the recurrence of a disease, disorder, or condition. In particular embodiments, "treating" and "treatment" refer to an increase in survival of a subject having a disease, disorder, or condition. In particular embodiments, "treating" and "treatment" refer to the elimination of a disease, disorder, or condition in a subject.

Examples of neoplastic diseases or disorders include, but are not limited to, disseminated cancer, solid tumor cancer, hypertrophy, coronary artery disease or vascular occlusive disease, diseases or disorders associated with infected cells, microorganisms or viruses, or diseases or disorders associated with inflammatory cells, such as Rheumatoid Arthritis (RA).

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

Any dosing schedule for the modified polypeptide and radioactive complex may be used in accordance with the present disclosure. 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.

According to particular embodiments, the compositions for treating neoplastic diseases or disorders can be used in combination with other agents effective in treating the relevant neoplastic diseases or disorders.

As used herein, the term "combination" in the context of administering two or more therapies to a subject refers to the use of more than one therapy. The use of the term "in combination" does not limit the order in which the therapies are administered to a subject. For example, a first therapy (e.g., a composition described herein) can be administered prior to (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 before), concurrently with, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the second therapy is administered to the subject.

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

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

According to a preferred embodiment, the radioactive metal ion is a diagnostic emitter, preferably⁸⁹Zr. According to other preferred embodiments, the radioactive metal ion is a therapeutic emitter, preferably a therapeutic emitter²²⁵Ac, is used. According to a preferred embodiment, the diagnostic agent is used to provide both diagnostic and therapeutic functions to a subject in need thereof.

Combination and kit

Provided herein is a combination comprising:

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

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

wherein the combination is to be used for labelling a polypeptide with a radioactive metal ion.

According to a particular embodiment, the combination of the invention is a reaction mixture for labeling a polypeptide with a radioactive metal ion. According to other embodiments, the combination is a package or kit for producing a radiolabeled polypeptide in vitro or in vivo. The combination may optionally be accompanied by a notice or instructions in the form of government regulations governing the manufacture, use or sale of the pharmaceutical or biological product reflecting approval by the regulatory agency of manufacture, use or sale for human administration. The combinations encompassed herein are useful 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.

Detailed description of the preferred embodiments

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

Embodiment 1 is a method of labeling a polypeptide with a radioactive metal ion, the method comprising:

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

b. providing a radioactive complex comprising the radiometal ion associated with a chelating moiety, wherein the chelating moiety comprises a chelator covalently linked to a second click reaction partner; and

c. contacting said modified polypeptide with said radioactive complex under conditions that allow said first click reaction partner to react with said second click reaction partner, thereby labeling said polypeptide with said radioactive metal ion.

Embodiment 1a is the method of embodiment 1, wherein the chelator comprises a macrocycle.

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

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

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

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

Embodiment 3b is the method of embodiment 2 or 3, wherein the alkyne group comprises a cyclic alkyne, preferably a cyclooctyne or a cyclooctyne derivative.

Embodiment 3c is the method of embodiment 3b, wherein the alkyne group comprises Bicyclononene (BCN).

Embodiment 3d is the method of embodiment 3b, wherein the alkyne group comprises a difluorinated cyclooctyne (DIFO).

Embodiment 3e is the method of embodiment 3b, wherein the alkyne group comprises Dibenzocyclooctyne (DIBO).

Embodiment 3f is the method of embodiment 3b, wherein the alkyne group comprises a diaryl azacyclooctanone (BARAC).

Embodiment 3g is the method of embodiment 3b, wherein the alkyne group comprises a Dibenzoazacyclooctyne (DIBAC).

Embodiment 3h is the method of embodiment 3b, wherein the alkyne group comprises dimethoxy azacyclooctyne (DIMAC).

Embodiment 3i is the method of embodiment 3b, wherein the alkyne group comprises Dibenzylcyclooctyne (DBCO).

Embodiment 3j is the method of embodiment 3b, wherein the alkyne group comprises difluorobenzocyclooctyne (diffbo).

Embodiment 3k is the method of embodiment 3b, wherein the alkyne group comprises a Monobenzocyclooctyne (MOBO).

Embodiment 31 is the method of embodiment 3b, wherein the alkyne group comprises tetramethoxy dibo (tmdobo).

Embodiment 3m is the method of any one of embodiments 2 to 31, wherein the azide group comprises NHS-azide.

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

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

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

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

Embodiment 5 is the method of any one of embodiments 1 to 4c, wherein the polypeptide is an antibody or 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 the method of any one of embodiments 1 to 6, wherein the modified polypeptide is obtained by random conjugation of one or more azide groups to the polypeptide.

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

Embodiment 6c is the method of embodiment 6b, wherein the modified antibody or antigen-binding fragment thereof is obtained by cleaving the antibody or antigen-binding fragment thereof with a bacterial endoglycosidase specific for the β -1, 4 linkage between one or more core GlcNac residues in one or more Fc glycosylation sites of the antibody to obtain a cleaved antibody or antigen-binding fragment thereof, and reacting the cleaved antibody or antigen-binding fragment thereof with an azido sugar, preferably a UDP-GalNaz azido sugar substrate, in the presence of a glycosyltransferase, preferably a GalT galactosyltransferase.

Embodiment 6d is the method of embodiment 6b, wherein the modified antibody or antigen-binding fragment thereof is obtained by deglycosylating the antibody or antigen-binding fragment thereof with an amidase to obtain a deglycosylated antibody or antigen-binding fragment thereof, and reacting the deglycosylated antibody or antigen-binding fragment thereof with an azidoamine, preferably 3-azidopropylamine, in the presence of microbial transglutaminase.

Embodiment 6e is the method of any one of embodiments 6 to 6d, wherein the antibody is an antibody or antigen-binding fragment thereof that binds human Prostate Specific Membrane Antigen (PSMA), preferably the antibody comprises the HC CDR1 sequence of SEQ ID NO: 3, the HC CDR2 sequence of SEQ ID NO: 4, the HC CDR3 sequence of SEQ ID NO: 5, the light chain (L C) CDR1 sequence of SEQ ID NO: 6, the L C CDR2 sequence of SEQ ID NO: 7, and the L C CDR3 sequence of SEQ ID NO: 8.

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

Embodiment 7 is the method of any one of embodiments 1 to 6d, wherein the radioactive metal ion is³²P、⁴⁷Sc、⁶⁷Cu、⁷⁷As、⁸⁹Sr、⁹⁰Y、⁹⁹Tc、¹⁰⁵Rh、¹⁰⁹Pd、¹¹¹Ag、¹³¹I、¹⁵³Sm、¹⁵⁹Gd、¹⁶⁵Dy、¹⁶⁶Ho、¹⁶⁹Er、¹⁷⁷Lu、¹⁸⁶Re、¹⁸⁸Re、¹⁹⁴Ir、¹⁹⁸Au、¹⁹⁹Au、²¹¹At、²¹²Pb、²¹²Bi、²¹³Bi、²²³Ra、²²⁵Ac、²⁵⁵Fm、²²⁷Th、⁶²Cu、⁶⁴Cu、⁶⁷Ga、⁶⁸Ga、⁸⁶Y、⁸⁹Zr or¹¹¹In。

Embodiment 7a is the method of any one of embodiments 1 to 6d, wherein the radioactive metal ion is²²⁵Ac。

Embodiment 7b is the method of any one of embodiments 1 to 6d, wherein the radioactive metal ion is¹¹¹In。

Embodiment 7c is the method of any one of embodiments 1 to 6d, wherein the radioactive metal ion is⁸⁹Zr。

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

Embodiment 9 is the method of any one of embodiments 1 to 8, further comprising reacting an electrophile located on or introduced to a side chain of the polypeptide, preferably an amino side chain of lysine, with a thiol group covalently linked to the first click reaction partner to obtain the modified polypeptide, preferably a NHS-azide.

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

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

wherein R is₁、R₂、R₃And R₄Is independently CHQCO₂X, wherein

Q is independently hydrogen, C₁-C₄Alkyl or (C)₁-C₂Alkyl) phenyl, and

x is independently hydrogen, benzyl, C₁-C₄An alkyl group; and is

Z is (CH2)_nY is, wherein

n is 1 to 10, and

y is an electrophilic or nucleophilic moiety covalently linked to the second click reaction partner;

alternatively, Z is hydrogen; and is

R₁、R₂、R₃And R₄Is independently CHQCO₂X, wherein

Q is independently hydrogen, C₁-C₄Alkyl or (C)₁-C₂Alkyl) phenyl, and

x is independently hydrogen, benzyl, C₁-C₄An alkyl group or an electrophilic or nucleophilic moiety covalently linked to the second click reaction partner.

Embodiment 12 is the method of any one of embodiments 1 to 11, wherein the chelating moiety comprises a structure of formula (II):

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

embodiment 12b is the method of any one 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 "" ' -hexaacetic acid (HEHA), 1, 4, 7, 10, 13-pentaazacyclopentadecane-N, N ', N ", N '", N "" -pentaacetic acid (PEPA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), Macropa (Thiele et al, Anighte-Membered Macrocydic L igoic acid for nigrum-225 targetated Alraquinone cheiram. Andegem. Ed.7. for example, Tetraazatetradecanephosphonic acid (TPA), Tetraazatetradecanepropionic acid (TPA-1, 4, 11-tetraazacyclododecane-N ', 4, 7, 10-tetraazacyclododecane-1, 4-11, 11-tetraazacyclododecane-1, 4-TETA ', N ' "-pentaacetic acid (PEPA), TPA,", N ' "-pentaacetic acid (TPA, N '" -pentaacetic acid (TPE ' ", TPE, E '" -E ' ", TPE '", E, III ' ", E.

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

Embodiment 12d is the method of embodiment 12b, wherein the chelator comprises Desferoxamine (DFO).

Embodiment 13 is a method of using radioactive metal ions, preferably²²⁵Ac、¹¹¹In or⁸⁹A method of Zr labeling a polypeptide, preferably an antibody or antigen binding fragment thereof, said method comprising:

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

b. providing a radioactive complex comprising said radioactive metal ion associated with a chelating moiety, preferably²²⁵Ac、¹¹¹In or⁸⁹Zr, wherein the chelating moiety comprises a chelating agent covalently attached to an alkyne or alkene group; and

c. contacting the modified polypeptide or antibody or antigen-binding fragment thereof with the radioactive complex under conditions that allow the azide, tetrazine or tetrazole group to react with the alkyne or the olefin group, thereby contacting the modified polypeptide or antibody or antigen-binding fragment thereof with the radioactive metal ion, preferably with the radioactive metal ion²²⁵Ac、¹¹¹In or⁸⁹Zr labeling the polypeptide or antibody or antigen binding fragment thereof, wherein the chelating agent comprises the structure of formula (I):

wherein R is₁、R₂、R₃And R₄Is independently CHQCO₂X, wherein

Q is independently hydrogen, C₁-C₄Alkyl or (C)₁-C₂Alkyl) phenyl, and

x is independently hydrogen, benzyl, C₁-C₄An alkyl group; and is

Z is (CH2)_nY is, wherein

n is 1 to 10, and

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

alternatively, Z is hydrogen; and is

R₁、R₂、R₃And R₄Is independently CHQCO₂X, wherein

Q is independently hydrogen, C₁-C₄Alkyl or (C)₁-C₂Alkyl) phenyl, and

x is independently hydrogen, benzyl, C₁-C₄An alkyl group or an electrophilic or nucleophilic moiety covalently linked to the alkyne group.

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

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

embodiment 13d is a method of using radioactive metal ions, preferably²²⁵Ac、¹¹¹In or⁸⁹A method of Zr labeling a polypeptide, preferably an antibody or antigen binding fragment thereof, said method comprising:

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

b. providing a radioactive complex comprising the radiometal associated with a chelating moietyIon, preferably²²⁵Ac、¹¹¹In or⁸⁹Zr, wherein the chelating moiety comprises a chelating agent covalently attached to an alkyne or alkene group; and

c. contacting the modified polypeptide or antibody or antigen-binding fragment thereof with the radioactive complex under conditions that allow the azide, tetrazine, or tetrazole groups to react with the alkyne or the olefin groups, thereby labeling the polypeptide or antibody or antigen-binding fragment thereof with the radioactive metal ion, preferably 225Ac, 111In, or 89Zr,

wherein the chelating agent comprises an open-chain ligand, preferably Deferoxamine (DFO).

Embodiment 14 is the method of any one of embodiments 13-13 c, wherein the chelator is covalently attached to the alkyne or alkene group via a linker.

Embodiment 15 is the method of any one of embodiments 13 to 14, further comprising reacting an electrophile located on the polypeptide, preferably the antibody or antigen-binding fragment thereof, or introduced onto a side chain of the polypeptide, preferably an amino side chain of lysine, with a thiol group covalently attached to an azide, preferably NHS-azide, to obtain the modified polypeptide or antibody or antigen-binding fragment thereof.

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

Embodiment 17 is the method of embodiment 13, wherein the polypeptide is an antibody or antigen-binding fragment thereof and the radioactive metal ion is²²⁵Ac、¹¹¹In or⁸⁹Zr, and the chelating moiety comprises a structure of formula (II):

embodiment 17a is the method of embodiment 13c, wherein the polypeptide is an antibody or antigen-binding fragment thereof, theThe radioactive metal ion is²²⁵Ac、¹¹¹In or⁸⁹Zr, and the chelating moiety comprises a structure of formula (III):

embodiment 17b is the method of any one of embodiments 13 to 17a, wherein the polypeptide is an antibody that binds human Prostate Specific Membrane Antigen (PSMA) or an antigen-binding fragment thereof, preferably the antibody comprises the HC CDR1 sequence of SEQ ID NO: 3, the HC CDR2 sequence of SEQ ID NO: 4, the HC CDR3 sequence of SEQ ID NO: 5, the light chain (L C) CDR1 sequence of SEQ ID NO: 6, the L C CDR2 sequence of SEQ ID NO: 7, and the L C CDR3 sequence of SEQ ID NO: 8.

Embodiment 17C is the method of embodiment 17b, wherein the antibody comprises the HC sequence of SEQ ID NO 9 and the L C sequence of SEQ ID NO 10.

Embodiment 18 is a method of dual labeling a polypeptide with two radioactive metal ions, the method 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 associated with a chelating moiety, wherein the chelating moiety comprises a chelator covalently linked to a third click reaction partner; and

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

d. contacting said modified polypeptide with said first and second radioactive complexes under conditions that allow said first click reaction partner to react with said third click reaction partner and said second click reaction partner to react with said fourth click reaction partner, thereby labeling said polypeptide with said first and second radioactive metal ions.

Embodiment 19 is the method of embodiment 18, wherein one of the first click reaction partner and the second click reaction partner comprises an alkyne group and the other of the first click reaction partner and the second click reaction partner comprises an azide, and wherein one of the third click reaction partner and the fourth click reaction partner comprises an olefin group and the other of the third click reaction partner and the fourth click reaction partner comprises 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 of embodiment 18 or 19, wherein both the first and second radioactive metal ions are therapeutic emitters.

Embodiment 21a is the method of embodiment 20 or 21, wherein the diagnostic emitter is⁶²Cu、⁶⁴Cu、⁶⁷Ga、⁶⁸Ga、⁸⁶Y、⁸⁹Zr or¹¹¹In。

Embodiment 21b is the method of any one of embodiments 20 to 21a, wherein the therapeutic emitter is³²P、⁴⁷Sc、⁶⁷Cu、⁷⁷As、⁸⁹Sr、⁹⁰Y、⁹⁹Tc、¹⁰⁵Rh、¹⁰⁹Pd、¹¹¹Ag、¹³¹I、¹⁵³Sm、¹⁵⁹Gd、¹⁶⁵Dy、¹⁶⁶Ho、¹⁶⁹Er、¹⁷⁷Lu、¹⁸⁶Re、¹⁸⁸Re、¹⁹⁴Ir、¹⁹⁸Au、¹⁹⁹Au、²¹¹At、²¹²Pb、²¹²Bi、²¹³Bi、²²³Ra、²²⁵Ac、²⁵⁵Fm or²²⁷Th。

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

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 compositions to be administered sequentially, a first composition comprising the modified polypeptide and a second composition comprising one or more radioactive complexes.

Embodiment 25 is a diagnostic agent comprising a pharmaceutically acceptable carrier and a radiolabeled antibody prepared by the method according to any one of embodiments 1 to 21b, wherein the immunological properties of the radiolabeled antibody are retained.

Embodiment 26 is the therapeutic agent of embodiment 25, wherein the radioactive metal ion is a diagnostic emitter, preferably a diagnostic emitter⁸⁹Zr。

Embodiment 27 is the therapeutic agent of embodiment 25, wherein the radioactive metal ion is a therapeutic emitter, preferably a therapeutic emitter²²⁵Ac。

Embodiment 27a is the therapeutic agent according to embodiment 25, wherein the radioactive metal ion is 111 In.

Embodiment 27b is the diagnostic and therapeutic agent of any one of embodiments 25 to 27a, wherein the polypeptide is an antibody that binds human Prostate Specific Membrane Antigen (PSMA) or an antigen binding fragment thereof, preferably the antibody comprises the HC CDR1 sequence of SEQ ID No. 3, the HC CDR2 sequence of SEQ ID No. 4, the HC CDR3 sequence of SEQ ID No. 5, the light chain (L C) CDR1 sequence of SEQ ID No. 6, the L C CDR2 sequence of SEQ ID No. 7, and the L C CDR3 sequence of SEQ ID No. 8.

Embodiment 27C is the therapeutic agent of embodiment 27b, wherein the antibody comprises the HC sequence of SEQ ID No. 9 and the L C sequence of SEQ ID No. 10.

Embodiment 27d is the diagnostic agent of any one of embodiments 25 to 27c, wherein the radiolabeled antibody has the formula of formula (IV):

formula (IV) (DOTA-Ac-DBCO-protein), alternatively, the²²⁵Ac is substituted by another radioactive metal ion such as³²P、⁴⁷Sc、⁶⁷Cu、⁷⁷As、⁸⁹Sr、⁹⁰Y、⁹⁹Tc、¹⁰⁵Rh、¹⁰⁹Pd、¹¹¹Ag、¹³¹I、¹⁵³Sm、¹⁵⁹Gd、¹⁶⁵Dy、¹⁶⁶Ho、¹⁶⁹Er、¹⁷⁷Lu、¹⁸⁶Re、¹⁸⁸Re、¹⁹⁴Ir、¹⁹⁸Au、¹⁹⁹Au、²¹¹At、²¹²Pb、²¹²Bi、²¹³Bi、²²³Ra、²⁵⁵Fm、²²⁷Th、⁶²Cu、⁶⁴Cu、⁶⁷Ga、⁶⁸Ga、⁸⁶Y、⁸⁹Zr or¹¹¹In。

Embodiment 27e is the diagnostic agent of any one of embodiments 25 to 27c, wherein the radiolabeled antibody has the formula of formula (V):

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

Embodiment 27f is the diagnostic agent of any one of embodiments 25 to 27c, wherein the radiolabeled antibody has the formula of formula (VI):

formula (VI) (DFO-Zr-DBCO-protein), alternatively, the⁸⁹Zr is replaced by another radioactive metal ion such as³²P、⁴⁷Sc、⁶⁷Cu、⁷⁷As、⁸⁹Sr、⁹⁰Y、⁹⁹Tc、¹⁰⁵Rh、¹⁰⁹Pd、¹¹¹Ag、¹³¹I、¹⁵³Sm、¹⁵⁹Gd、¹⁶⁵Dy、¹⁶⁶Ho、¹⁶⁹Er、¹⁷⁷Lu、¹⁸⁶Re、¹⁸⁸Re、¹⁹⁴Ir、¹⁹⁸Au、¹⁹⁹Au、²¹¹At、²¹²Pb、²¹²Bi、²¹³Bi、²²³Ra、²²⁵Ac、²⁵⁵Fm、²²⁷Th、⁶²Cu、⁶⁴Cu、⁶⁷Ga、⁶⁸Ga、⁸⁶Y is or¹¹¹In。

Embodiment 27g is the diagnostic agent of any one of embodiments 25 to 27c, wherein the radiolabeled antibody has the formula of formula (VII):

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

Embodiment 27h is the therapeutic agent of any one of embodiments 25-27 g, wherein the radiolabeled antibody has a chelator of less than 3: antibody Ratio (CAR).

Embodiment 27i is the diagnostic agent of any one of embodiments 25 to 27h, wherein the radiolabeled antibody has a chelator of 2: antibody Ratio (CAR).

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

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

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

wherein said combination is to be used for labelling said polypeptide with said radioactive metal ion.

Embodiment 28a is the combination or kit of embodiment 28, wherein the chelator comprises a macrocycle.

Embodiment 28b is the combination or kit of embodiment 28, wherein the chelating agent comprises an open-chain ligand.

Embodiment 29 is a combination or kit according to embodiment 28 to be used for labelling said polypeptide with said radioactive metal ion via an in vitro reaction between said first click reaction partner and said second click reaction partner.

Embodiment 30 is a combination or kit according to embodiment 28 to be used for labelling said polypeptide with said radioactive metal ion via an in vivo reaction between said first click reaction partner and said second click reaction partner.

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 associated with a chelating moiety, wherein the chelating moiety comprises a chelator covalently linked to a second click reaction partner.

Embodiment 32a is the composition of embodiment 32, wherein the chelator comprises a macrocycle.

Embodiment 32b is the composition of embodiment 32, wherein the chelating agent comprises an open-chain ligand.

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

Embodiment 33a is the combination or kit or composition of embodiment 33, wherein the antibody is capable of binding human Prostate Specific Membrane Antigen (PSMA) or an antigen binding fragment thereof, preferably the antibody comprises the HC CDR1 sequence of SEQ ID NO: 3, the HC CDR2 sequence of SEQ ID NO: 4, the HC CDR3 sequence of SEQ ID NO: 5, the light chain (L C) CDR1 sequence of SEQ ID NO: 6, the L C CDR2 sequence of SEQ ID NO: 7, and the L C CDR3 sequence of SEQ ID NO: 8.

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

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

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

Embodiment 33f is the combination or kit or composition of embodiment 33e, wherein the antibody or antigen-binding fragment thereof is covalently attached to the azido group via a method comprising cleaving the antibody or antigen-binding fragment thereof with a bacterial endoglycosidase specific for the β -1, 4 linkage between one or more core GlcNac residues in one or more Fc glycosylation sites of the antibody to obtain a cleaved antibody or antigen-binding fragment thereof, and reacting the cleaved antibody or antigen-binding fragment thereof with an azido sugar, preferably a UDP-GalNaz azido sugar substrate, in the presence of a glycosyltransferase, preferably a GalT galactosyltransferase.

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

Embodiment 34 is the combination, kit or composition of any one of embodiments 33 to 33g, wherein the radioactive metal ion comprises²²⁵Ac、¹¹¹In or⁸⁹Zr。

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

wherein R is₁、R₂、R₃And R₄Is independently CHQCO₂X, wherein

Q is independently hydrogen, C₁-C₄Alkyl or (C)₁-C₂Alkyl) phenyl, and

x is independently hydrogen, benzyl, C₁-C₄An alkyl group; and is

Z is (CH2)_nY is, wherein

n is 1 to 10, and

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

alternatively, Z is hydrogen; and is

R₁、R₂、R₃And R₄Is independently CHQCO₂X, wherein

Q is independently hydrogen, C₁-C₄Alkyl or (C)₁-C₂Alkyl) phenyl, and

x is independently hydrogen, benzyl, C₁-C₄An alkyl group or an electrophilic or nucleophilic moiety covalently linked to the alkyne group.

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

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

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

embodiment 38 is the combination, kit, or composition of any one of embodiments 34 to 37, wherein the polypeptide is covalently attached to the 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 diagnostic agent according to any one of embodiments 25 to 27d or a combination according to any one of embodiments 28 and 33 to 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 according to embodiment 31 and a composition according to embodiment 32, preferably the polypeptide is an antibody.

Examples

The following examples of the present invention are intended to further illustrate the nature of the invention. 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 conjugation of azide/handle to antibody

Monoclonal antibodies (mAb)A human IgG4 antibody that binds human Prostate Specific Membrane Antigen (PSMA), designated herein as an "anti-PSMA mAb", having the Heavy Chain (HC) CDR1 sequence of SEQ ID NO: 3, the HC CDR2 sequence of SEQ ID NO: 4, the HC CDR3 sequence of SEQ ID NO: 5, the light chain (L C) CDR1 sequence of SEQ ID NO: 6, the L C CDR2 sequence of SEQ ID NO: 7, and the L C CDR3 sequence of SEQ ID NO: 8, and having the HC sequence of SEQ ID NO: 9 and the L C sequence of SEQ ID NO: 10.

Human IgG 4S 228P/F234A/L235A (IgG4-PAA) antibody isotype control anti-PMSA mAb (referred to herein as "control mAb") has the HC sequence of SEQ ID NO: 1 and L C sequence of SEQ ID NO: 2 commercial antibodies trastuzumab (trastuzumab) (Herceptin), cetuximab (cetuximab) (Erbitux), pertuzumab (pertuzumab) (Perjeta) and panitumumab (panitumumab) (vicktibix (Vectibix)) were purchased from Roche, L illy, Roche and amgen, mouse anti-human Her2 mAb was obtained from BioXCell (catalog No. 027be 7), trastuzumab, pertuzumab and anti-human Her2 bind to human EGFR and panitumumab 2.

ConjugationMixing a stock solution of antibody (1mg/M L-10 mg/M L) in 10mM sodium acetate pH 5.2, phosphate buffered saline pH 7, or other compatible buffer with 20% (v/v) 1M sodium carbonate buffer pH 9 to a final pH. of about 9 NHS-PEG 4-azide (Thermo catalog No. 26130) was dissolved in DMSO to a final concentration of 100mM, and 0.2% (v/v) of the stock solution was added to create a molar excess of about 3-10 relative to the mAb the reaction was incubated at 22 ℃ for 10 minutes before quenching to a final concentration of 50mM Tris by addition of 1M Tris pH 7.5.

Purification of: the azide-mAb conjugates are purified and exchanged to compatible buffers (PBS; 20mM HEPES 150mM, NaCl pH 7.5; or 10mM sodium acetate pH 5.2) using methods such as Zeba desalting column with 7K molecular weight cut-off (Thermo), dialysis; standard protein a affinity chromatography; or another compatible method. After purification, the conjugate was concentrated to 1 using an Amicon concentrator (Millipore) with a molecular weight cut-off of 50K0mg/mL-20mg/mL。

L C/MS analysisDetermination of the chelator to antibody ratio (CAR) by L C/MS analysis Using an Agilent G6224 MS-TOF instrument equipped with an Agilent P L RP-S column (300 angstroms, 2.1mmx150 mm; catalog number P L1912-.

Analytical Size Exclusion Chromatography (SEC)Agilent 1200 series HP L C equipped with a Tosoh TSKgel G3000SWxl (Tosoh bioscience #08541)7.8mm × 30cm column, mobile phase 1 XPBS, flow rate 0.8ml/ml, injection volume 15 μ L, protein concentration 0.1mg/m L-2 mg/m L was used.

Table 1: conjugation efficiency

Antibodies	Isoforms	CAR
			anti-PSMA mAbs	Human IgG4	2.4
Control mAbs	Human IgG4	2.0
			Trastuzumab	Humanized IgG1	2.5
Cetuximab	Chimeric human/mouse IgG1	3.6
			Pertuzumab	Humanized IgG1	2.3
Panitumumab	Human IgG2	3.0
			Anti-human Her2	Mouse IgG2a	3.1

Example 2: random conjugation of azide/handle to non-antibody polypeptide

Non-antibody polypeptide transferrin (human holotransferrin) was purchased from R & D Systems (catalog No. 2914-HT) and dissolved in water to 10mg/m L EGF (human Epidermal Growth Factor (EGF)) was purchased from Sino Biological (catalog No. 10605-HNAE).

Conjugation A stock solution of the polypeptide (1mg/M L-10 mg/M L) in 10mM sodium acetate pH 5.2, phosphate buffered saline pH 7, or other compatible buffer is mixed with 20% (v/v) 1M sodium carbonate buffer pH 9 to a final pH. of about 9 NHS-PEG 4-azide (Thermo catalog No. 26130) is dissolved in DMSO to a final concentration of 100mM, and 0.2% (v/v) of the stock solution is added to produce a molar excess of about 3-10 relative to the protein the reaction is incubated at 22 ℃ for 10 minutes followed by quenching with 1M Tris pH 7.5 to a final concentration of 50 mM. the conjugation efficiency is shown in Table 2.

Table 2: conjugation efficiency

Protein	Chelating agent: protein ratio
		Transferrin	3.6
EGF	1.1

Example 3: site-specific incorporation of azido sugars into antibody glycans

The antibody glycans were trimmed with a bacterial endoglycosidase GlycINATOR (Genovis) specific for the β -1, 4 linkage between core GlcNac residues in one or more Fc glycosylation sites, leaving the innermost GlcNAc intact on the Fc, which could then be used for site-specific incorporation of azido sugars.more specifically, immobilized GlycINATOR on agarose beads packed into a column (Genovis) was equilibrated in Tris Buffered Saline (TBS) pH 7.4. 5mg/m L-10 mg/m L mAb at 1m L was added to the resin and incubated on a rocker arm for 1 hour at room temperature, the mAb was eluted by rotating 100x g for 1 minute. the column was re-eluted 3 times with 0.5m L. the eluates containing the trimmed mAb were pooled and the buffer additive provided (Genovis) was added along with UDP-GalNaz azido sugar substrate and GalT galactosyltransferase (GalT) and the reaction was incubated on a Select galactosyltransferase at 30 ℃ with a Select modification column (ATC-ATC. 25. Final Azide modification mAb was determined by a final assay in which was performed overnight.

Example 4: site-specific incorporation of 3-azidopropylamines using Microbial Transglutaminase (MTG)

The anti-PSMA mAb was deglycosylated with an amidase Rapid PNA (New England Biolabs) which cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid and complex oligosaccharides and allowed for complete deglycosylation and release of all N-glycans including N-glycans from both conserved (e.g., Asn Fc 297) glycosylation sites and non-conserved (e.g., Fab N-glycan) glycosylation sites and non-conserved (e.g., N-glycan) glycosylation sites and removal of 10M 54 in a pH 5 modified antibody at position Gln295 site specifically mounted on the antibody, essentially as described (Dennler et al, Transglutaminase-based chemotherapy-enzyme conjugation, bioconjugated oligosaccharides, 3 months 19; 25 (3): pages 569-78.) and purification of the anti-PSMA mAb by addition of a standard PNA mAb 7-7, 7. mu.7. C7. alpha. 5. alpha. PNA-5. alpha. PNA conjugate, 34. alpha. 5. alpha. and 7. alpha. 5. gamma. 5. gamma. was added by a loop-7. centrifugation, and incubated with a standard PNA-7. gamma. 35. gamma. 7. gamma. medium, and purified by a loop method at pH 7, 3. 20. 7. 20. gamma. 7. overnight, 7. 20. gamma. medium, 7. gamma. for removal, 7. gamma. for overnight, 7. gamma. medium, and concentration, as described by.

Example 5: chelation of radioactive metals with bifunctional chelators (BFC)

²²⁵Synthesis of Ac-DOTA-GA-DBCO：²²⁵Ac(NO₃)₃Custom Synthesis of 1, 4, 7, 10-tetraazacyclododecane, 1- (glutarate) -4, 7, 10-triacetic acid- (3-amino-propionic acid) dibenzocyclooctyne (DOTA-GA-DBCO), available from Oak Ridge National L laboratory, based on Bernhard et al, chem. Eur. J.2012, 18, 7834 Across 7841 DBCO-amine (3-amino-1- [ (5-aza-3, 4: 7, 8-dibenzocycloocta-1-yne) -5-yl)]-1-propanone, Sigma) was reacted with DOTA-GA anhydride and the product was purified by reverse phase HP L C.

Quantification of actinium-225 was accomplished using a Capintec CRC-55TW dose calibrator, and thus will²²⁵Ac(NO₃)3 dissolved in 0.1N HCl to make a 10mCi/M L solution to a solution of tetramethylammonium acetate (1M solution, 7.5. mu. L, 7.5. mu. mol), DOTA-GA-DBCO (1mg/M L in water, 2.5. mu. L, 3.4nmol) and NaOH (0.1N, 2.5. mu. L, 0.25. mu. mol) in a plastic vial was added²²⁵Ac(NO₃)₃(10mCi/m L in 0.1N HCl, 5. mu. L, 50. mu. Ci, 0.0038 nmol.) the pH of the mixture was observed to be about 6.5 by pH paper the vial was placed on a shaking block at 80 ℃ and 290rpm for 30min and the vial was allowed to cool to room temperature.

¹¹¹Synthesis of In-DOTA-GA-DBCO: in 0.05M HCl¹¹¹InCl3 from GE healthcare to a solution of tetramethylammonium acetate (1M solution, 7.5. mu. L, 7.5. mu. mol), DOTA-GA-DBCO (1mg/M L in water, 2.5. mu. L, 3.4nmol) and HCl (0.1N, 5. mu. L) in a plastic vial was added 0.05N HCl¹¹¹InCl₃(5 μ L, measured in a Capintec CRC-55TW dose calibrator as 104.3 μ Ci, 0.0022 nmol.) the pH of the mixture was observed to be about 5.5 by pH paper the vial was placed on a shaking block at 60 ℃ and 290rpm for 30min and the vial was allowed to cool to room temperature.

⁸⁹Synthesis of Zr-DFO-DBCO：⁸⁹Zr oxalate was purchased from 3D imaging. DFO-DBCO from Macrocyclics (Plano, TX Cat. B-773), dissolved in DMSO to 0.5mg/m L, and diluted to 25. mu.g/m L in water.

2mCi of Zr-89 was transferred to a metal free microcentrifuge tube and 1M oxalic acid was added to reach a total volume of 80 μ L. 12 μ L of 2M potassium carbonate was added in 2 μ L increments and the mixture was stirred with the pipette tip until bubbling stopped.120 μ L of 1M HEPES was then added followed by 300 μ L of water. the pH of the test solution and if necessary additional 2M potassium carbonate was added to raise the pH to 6-6.5. 136 μ L of DFO-DBCO stock (3.4 μ g) was added and the reaction was incubated at room temperature for 1 hour. chelate was analyzed by spotting 0.5 μ L of the reaction on a T L C Green strip (Biodex) and eluting with 20% NaCl.

⁸⁹Synthesis of Zr-DOTA-GA-DBCOTo a Waters Sep-pak L light QMA Strong anion exchange column (acrylic acid/acrylamide copolymer on glycol silica, surface functional group: C (O)) NH (CH)₂)₃N(CH₃)₃ ⁺Cl^-，Pore size, particle size 37 μm-55 μm, 230 μ eq/g ion exchange capacity) MeCN (6m L) was added followed by 0.9% brine (10m L) and then water (10m L)⁸⁹Zr(ox)₂(2. mu. L, 290. mu. Ci) in 1.0M oxalic acid was added to the preconditioning column, the column was then washed with deionized water (20ml) to remove excess oxalic acid, after which the column was washed with 1.0M HCl (aq) (100. mu. L each time)⁸⁹ZrCl₄Elute from the column to give a total volume of 400 μ L, recover 248 μ Ci (86%), mostly active in fraction 3.

Add 10 μ L DOTA-GA-DBCO (1.0mg/m L in metal free water, 10 μ g, 13.6nmo1) to⁸⁹ZrCl₄(268uCi, 50 μ L.) DOTA-GA-DBCO is/are combined with a suitable solvent for use in a sample⁸⁹The Zr solution was diluted in 1.0M HEPES 150. mu. L the pH of the mixture was adjusted to pH 7.5(Pandya et al, Zirconium tetrahazacyclic compounds displaypurity stability and precursor a new growth for Zirconium-89-basedridopharmaceutical degradation. chem Sci.2017, 3/month 1; 8 (2309) 2314.) the solution was then incubated at 90 ℃ for 60min by incubation with 1% NH₄OH solution eluted SPC25 column (Sigma Aldrich part number SPC25120-50G), assay⁸⁹The yield of the Zr-DOTA-GA-DBCO complex was 98%. Not chelated⁸⁹Zr is left on the column, and⁸⁹the Zr-DOTA-GA-DBCO complex was eluted.

Example 6: synthesis of click-labeled radioconjugates of anti-PSMA mAbs

See figures 1 and 2 for schematic illustrations of a radiolabelled antibody according to the method of the invention.

anti-PSMA mAb-dibenzo- [1, 2, 3]-triazoloazacin-GA-DOTA-225 Ac (site-specific, CAR ═ 2) Synthesis of (2)Random or site-specific azide-modified antibodies (site-specific, CAR 2 or random, average CAR between 1 and 4) in PBS or other compatible buffers (10mg/m L-20 mg/m L) were added to the solution as describedProduced by²²⁵Ac-DOTA-GA-DBCO solution the final pH of the mixture was about 6.5 by pH paper, the reaction solution was gently stirred and allowed to stand at room temperature for 3h, then purified using a PD-10 column (GE Healthcare) preconditioned with 15m L NaOAc buffer (10mM, pH6-6.5) or another compatible buffer, the reaction mixture was removed into the reservoir of the preconditioned PD-10 column and the eluate was collected in a plastic tube, the reaction vial was washed with NaOAc buffer (0.2m L× 3), the wash solution was removed into the reservoir of the PD-10 column and the eluate was collected, NaOAc buffer was continuously applied to the reservoir of the PD-10 column and the eluate was collected in a plastic tube, with about 1m L eluate per tube being collected until a total of 10m L eluate was collected.

Using citrate-H₂The purity of each fraction collected was assessed by iT L C-sg (agilent) as the mobile phase combining one or more pure fractions to give the final product in 10mM NaOAc buffer the chemical and radiochemical purity of the product solution was analyzed by HP L C the antibody concentration in the product solution was determined by UV absorption using a standard curve followed by quantification of the activity of the product solution using a capentec CRC-55TW dose calibrator.

Quality control of Ac-225 sequestration: diethylenetriaminepentaacetic acid (DTPA) was used as a quality control for the purified product, with chelation attack. Adding 10mM Na₅Adding DTPA aqueous solution to the mixture containing²²⁵Sample solution of Ac-labeled mAb, until [ DTPA ]]/[mAb]500-. 50mM Na₅Adding DTPA aqueous solution to the mixture containing²²⁵In aliquots of purified product in a solution of Ac-labeled mAb, until [ DTPA [ ]]/[mAb]50,000-100,000 at room temperature and 290rpm, the two mixtures were placed on a shaking block for 30min, spotted on iT L C-SG, and treated with citrate-H₂O-MeOH solution developed color as the mobile phase. Under these conditions, free²²⁵Ac migrates to the solvent front and binds²²⁵The Ac-mAb remained at baseline.

¹¹¹anti-PSMA mAb-dibenzo- [1, 2, 3]-Synthesis of triazoloazacin-GA-DOTA-In:random in 10mM NaOAc orSite-specific azide-modified anti-PSMA mAb (site-specific, CAR 2 or random, average CAR between 1 and 4) was added to generated as described¹¹¹The reaction solution was gently stirred and allowed to stand at room temperature for 2h before it was passed through the PD-10 column, the PD-10 column was preconditioned by passing 15m L of NaOAc buffer (10mM, pH6-6.5) through the column, and the wash was removed, then the reaction mixture was transferred to the reservoir of the preconditioned PD-10 column and the eluate was collected In a plastic tube, the reaction vial was washed with NaOAc buffer (0.2m L× 3), the wash was transferred to the reservoir of the PD-10 column and the eluate was collected, NaOAc buffer was continuously applied to the reservoir of the PD-10 column and the eluate was collected In a plastic tube, with about 1m L of eluate per tube until a total of 10m L of eluate was collected.

The purity of each fraction collected was assessed by iT L C-SG using 10mM aqueous EDTA (pH 5-6) as the mobile phase one or more pure fractions were combined to give the final product in 10mM NaOAc buffer.

Quality control of In-111 chelation: DTPA was used as quality control for the purified product, with chelation attack: adding 10mM Na₅Adding DTPA aqueous solution to the mixture containing¹¹¹In sample solution of In-labeled mAb until [ DTPA]/[mAb]1000 ℃ 10,000. the mixture was placed on a shaking block for 30min at room temperature and 290rpm, spotted on iT L C-SG and developed with 10mM aqueous EDTA (pH 5-6) as the mobile phase¹¹¹In or loosely bound¹¹¹In migrates to the solvent front and is tightly bound¹¹¹The In-mAb remained at baseline.

anti-PSMA mAb-dibenzo- [1, 2, 3]Synthesis of (E) -triazoloazacin-DFO-Zr-89

800 μ g random azide-modified anti-PSMA mAb (average CAR between 1 and 4; sites available for the same procedure)Specific azido-mAb) was added to the generated as described in 10mM HEPES 50mM NaCl pH 7.5 or other compatible buffer⁸⁹Zr-DFO-DBCO solution and incubation at 37 ℃ for 1.5 hours before passing it through the PD-10 column, preconditioning the PD-10 column by passing 15m L isotonic saline through the column and removing the wash solution, then removing the reaction mixture into the reservoir of the preconditioned PD-10 column and collecting the eluate in a plastic tube, continuously applying saline to the reservoir of the PD-10 column and collecting the eluate in a plastic tube, wherein 0.5m L of eluate is collected per tube until a total of 10m L of eluate is collected, determining the activity of each fraction and the material remaining on the column with a dose calibrator, combining the product peaks (typically fractions 4-7) to give the final product, analyzing the chemical and radioactive chemical purity of the product solution by HP L C.

⁸⁹Synthesis of anti-PMSA mAb-DOTA-Zr

Azide-modified anti-PSMA mAb conjugate (CAR ═ 1-4) modified by random azide conjugation in 20mM HEPES 50mM NaCl pH 7.5(10.1mg/m L, 200 μ L, about 13.5nmol) was added to the generated as described above⁸⁹Zr-DOTA-GA-DBCO solution. The final pH of the mixture was adjusted to 7.0. The reaction solution was incubated at 37 ℃ for 2h, followed by purification on a PD-10 column (GE Healthcare) with 0.9% saline, HEPES buffer or PBS to give 64% of the product⁸⁹Zr-DOTA-mAb。

Quality control of Zr-89 chelation for Zr-DFO-mAb, purified product was analyzed by HP L C only, these conjugates did not retain Zr-89 when challenged with DTPA or EDTA, Zr-DOTA-mAb was challenged by addition of EDTA to 33mM and incubated overnight at room temperature, conjugates were analyzed by running on a PD-10 column after challenge and found to retain 80% of radioactivity.

Example 7: analytical characterization of click-labeled radioconjugates

Determination of radiochemical conversion

The radiochemical conversion (RA conversion%; see tables 3 to 5) was determined by iT L C-SG (transient thin layer chromatography using binderless glass microfiber chromatography paper impregnated with Silica Gel (SG) (iT L C).; RA conversion% was calculated by dividing the integrated value of the radio signal of the product peak (different retention times of radioactive starting material and by-products) by the value obtained when integrating all the radio signal peaks present between the baseline and the solvent front.

For products incorporating Ac-225, it will contain about 0.1. mu. Ci to 1. mu. Ci²²⁵Sample solutions of Ac were spotted on the base line of the iT L C-SG strips approximately 2cm from the bottom edge the iT L C-SG strips were developed using citrate-water-methanol mobile phase (20M L0.4M trisodium citrate/3M L2N HCl/2.3M L MeOH), allowed to dry at room temperature and stored for a minimum of 6h before analysis (reaching a long-term equilibrium of 225Ac and all nuclear daughter) the iT L C-SG was scanned using a Bioscan AR2000 radioactive T L C imaging scanner set with 99 mTc.

For In-111 chelates, one will contain about 0.5. mu. Ci-2.5. mu. Ci¹¹¹Sample solutions of In were spotted on a baseline of iT L C-SG strips approximately 2cm from the bottom edge, iT L C-SG strips were developed using 10mM sodium EDTA pH 5-6 as the mobile phase and then allowed to dry at room temperature, the dried iT L C-SG was scanned using a Bioscan AR2000 radioactive T L C imaging scanner set with In-111.

For the Zr-89 chelate, RA conversion was determined by dividing the activity on the product peak by the total activity (including activity in PD-10 column), which was determined by counting with a dose calibrator.

Determination of radiochemical purity of radiolabeled proteins

The radiochemical purity (RA purity; "see tables 3-4) of the Ac-225 and In-111 chelates was determined by SE-HP L C (size exclusion HP L C.) for Ac-225, a Tosoh TSKgel column (G3000SWx7.8mm × 30cm, 5um) was used, the column was eluted with DPBS buffer (1X, calcium and magnesium free), the flow rate was 0.7m L/min; run for 20 min; room temperature.) after HP L C, the eluate was collected In pre-numbered vials, with 0.5min or 1min of elution being collected for each vialAnd (4) liquid fraction. The vial containing the eluent was allowed to stand at room temperature for > 6h to allow²²⁵Ac reaches long-term equilibrium with its daughter nuclides. The activity in each vial was then counted in a Capintec CRC-55TW well counter. The radiochromatogram was reconstructed based on the activity in the vial.

For In-111, a Tosoh TSKgel column (G3000SWxl7.8mm × 30cm, 5um), elution of the column with DPBS buffer (1X, calcium and magnesium free), flow rate: 0.7M L/min, 20min run, room temperature, radioactive detection was accomplished using the above-described HP L C system and Perkinelmer wireless amperometric detector radiometric 625TR, and equipped with a 0.5M L flow cell, using an In-111 setting and an Ultima FloTM M mixture, flow rate of 1.4M L/min.

For Zr-89 (see Table 5), a Tosoh TSKgel column (G3000SWxl7.8mm × 30cm, 5um) was used, the column was eluted with citric acid buffered saline, Flow rate: 1m L/min, 20min run, room temperature, radioactivity detection was accomplished using the HP L C system described above and a Beckman Flow-through detector coupled to a Bioscan Flow Count instrument.

Table 3: actinium-225 radiolabelled proteins

Step 1 is the chelation of actinium-225 to DOTA-GA-DBCO

Step 2 is a click reaction of bifunctional chelate complexes with proteins

Table 4: indium-111 radiolabeled proteins

Step 1 is the chelation of indium-111 to DOTA-GA-DBCO

Step 2 is a click reaction of bifunctional chelate complexes with proteins

Table 5: zr-8 with DFO-DBCO9 radiolabeled anti-PSMA mAb

Example 8: click reaction of modified mAbs with DOTA-GA-DBCO

1mg/m L-10 mg/m L of random and site-specific azido-mAb (anti-PSMA mAb, cetuximab, panitumumab, trastuzumab, pertuzumab) was mixed with 5x to 20x excess of unchelated DOTA-GA-DBCO and incubated at room temperature or 37 ℃ for 1-24 hours.

Example 9: click reaction of modified mAbs with DFO-DBCO

1mg/m L-10 mg/m L of random and site specific anti-PSMA mAb azido-mAb was mixed with 5x to 20x excess of unchelated DOTA-GA-DFO and incubated at room temperature or 37 ℃ for 1-24 hours the mAb was desalted with a Zeba desalting spin column (Thermo) and concentrated with an Amicon centrifugal concentrator (Millipore), re-diluted with buffer, and re-concentrated to remove any remaining DBCO-DFO complete click reaction of all free azides with DBCO-DFO was confirmed by L C-MS.

Example 10: cell binding (FACS)

The cellular binding of azide-modified antibodies, DOTA-DBCO-azide-modified antibodies and DFO-DBCO-azide-modified antibodies was compared to the parent mabs of the conjugates described in table 1. Cell lines expressing mAb targets are treated with a range of concentrations of antibody or conjugate, and binding is measured by flow cytometry. The binding of panitumumab and cetuximab conjugates to EGFR + a431 cells was evaluated. Binding of herceptin and pertuzumab to HER2+ SK-BR-3 cells was evaluated. anti-PSMA mAbs were evaluated for binding to PSMA + C4-2b cells.

Cell lines: human prostate cancer cell line C4-2B cell lineCells were obtained from Janssen Oncology (Spring House, PA.) human epidermoid carcinoma cell line A431 cells and human breast carcinoma cell line SK-BR-3 cells were obtained from Janssen Biotherapeutics (Spring House, PA), the cells of which were native to ATCC (Manassas, VA.) EGFR receptor negative MO L M-13 human acute myeloid leukemia suspension cells were maintained in RPMI1640+25mM Hepes (Gibco) supplemented with 20% heat inactivated fetal bovine serum (Gibco.) cells were grown in RPMI1640+25mM Hepes (Gibco, Waltham, MA) with 10% FBS (Gibco, Waltham, MA).

Flow cytometry: cells were isolated from flasks using enzyme-free cell dissociation buffer (Gibco, Waltham, Ma) and filtered through a 40um filter (Falcon). Inoculate 5x10 per well in a 96-well u-shaped bottom plate⁴Cells were incubated with conjugated or parent antibodies diluted in BSA staining buffer (BD Bioscience, San Jose, Calif.) for 1 hour at 4 deg.C, cells were washed twice with staining buffer, then cells were incubated with AlexaFluor 647-labeled anti-human IgG secondary antibody (Jackson ImmunoResearch L antibodies) at 4 deg.C in the dark for 30min, secondary antibody was diluted 1: 200 in staining buffer containing 3% donkey serum (Rockland Immunochemicals), SYX TOX was diluted 1: 200 for the last 10 minutes of incubation^TMGreen Nucleic Acid Stain (ThermoFisher) was added to cells at a final concentration of 30nM, cells were washed twice with staining buffer, then resuspended in staining buffer at a final volume of 25 μ L/well and read on an iQue Screener flow cytometer (Intellicy), data were analyzed using ForeCyt Software, live cells were determined by excluding events with high Nucleic Acid staining, Mean Fluorescence Intensity (MFI) of live cells was determined and plotted as log-versus-MFI of antibody concentration in GraphPad Prism 7(GraphPad Software), nonlinear regression curve fitting was added to the data and EC was calculated₅₀The value is obtained.

The parent mAb and the modified mAb showed similar cell binding for all mabs and conjugates tested (table 6).

Table 6: EC50(nM) for mAb/conjugate binding to target cells

Example 11: in-111 cell binding assay

anti-PSMA mAb and transferrin were tested on C4-2B cells (PSMA + and transferrin +) and cetuximab and panitumumab were tested on a431 cells (EGFR +) and MO L M-13 cells (EGFR-) using In-111 radiolabeled proteins described In table 4, using radiometric assays.

Adherent cells were isolated with enzyme-free cell dissociation buffer (Gibco) adherent cells and collected suspension cells were counted and washed with cold staining buffer (BD Biosciences) various numbers of cells In 200 μ L staining buffer were added to microcentrifuge tubes and placed on ice 0.5 μ Ci of In-111 labeled protein was added to each tube and incubated on ice for 1 hour.

The Counts Per Minute (CPM) of the study samples were converted to μ Ci of In-111 using linearly regressed CPM values established with proteins labeled with known amounts of In-111. The μ Ci binding values were converted to moles bound using the following calculation: (μ Ci binding/specific activity)/MW mAb or protein. Each data point is the average of duplicate samples.

Click-labeled In-111 anti-PSMA mAb and In-111 transferrin bind to C4-2B cells, where the amount of cell-associated radioactivity increases with increasing cell number (fig. 3A) click-labeled In-111 anti-EGFR antibodies panitumumab and cetuximab bind to a431 cells, where the amount of cell-associated radioactivity increases with increasing cell number, and no specific binding of the click-labeled anti-EGFR antibody was detected with the negative control MO L M-13 cells (fig. 3B).

Example 12: indium cell uptake assay

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

The cells were treated with 3 × 10⁶Individual cells/60 mm petri dish (corning) were seeded and placed in humidified CO at 37 deg.C₂The inoculation medium was removed overnight In the incubator and replaced with 2m L cold staining buffer (BD Biosciences). the dishes were then placed on ice.0.5 μ Ci of In-111 labeled antibody was added to each dish and incubated on ice for 1 hour.

Surface bound radioactivity was stripped using an acid wash stripping procedure 1.5m L strip buffer (50mM glycine, 150mM NaCl pH 2.7, and pepsin (Amresco) added to 25 μ g/m L) was added to the cells and the dishes were incubated on ice for 15 minutes.

Intracellular radioactivity was determined by preparing cell lysates by adding 1.5M L1M NaOH (Teknova) to the cells after stripping surface bound radioactivity and washing the cells, and incubating the dishes on ice for 5 minutes.

For the time-0 sample, the surface membrane bound radioactivity and intracellular radioactivity of the cells were measured immediately after initial antibody binding on ice for the 10 min, 30min, 1 hr, and 2 hr samples, 3m L cell culture medium was added to each dish after initial antibody binding and the dishes were placed in 37 ℃ humidified CO₂An incubator. At each time point, the petri dish was removed from the incubator and placed on ice. Cell culture medium was transferred to counting vials and cells were washed with cold PBS. Collect PBS wash into the meterIn several vials. The surface membrane bound radioactivity and intracellular radioactivity of the cells were determined as described. At each time point, an unstripped sample was generated by incubating the cells with PBS prior to cell lysis, rather than with a stripping buffer. These samples were used to evaluate the stripping efficiency and the results were compared to the stripped samples.

CPM of the study samples was converted to μ Ci In-111 using CPM values from linear regression established with known amounts of In-111 labeled mAb. The percentage of In-111mAb localization In cell surface membrane (stripped sample) and intracellular (lysed sample) was determined using the following formula: localization,% 100 (sample μ Ci/mean total μ Ci), where total μ Ci refers to the addition of all collected samples, including incubation medium, PBS wash, glycine rinse, and lysed cells. Each data point is the average of duplicate samples.

Surface-bound In-111 rapidly disappeared from the cell surface and redistributed within the cell. The stripping technique released about 80% of the cell surface-associated radioactivity at time 0; by the end of the incubation, only 20% was released by stripping and more than 60% of the radioactivity was in the cell lysate (fig. 4).

Example 13: efficacy in mouse tumor xenograft models

Dose range study of anti-PSMA mAb-DOTA-Ac-225: male NSG mice (N ═ 8 per group) were implanted 10 subcutaneously⁶L NCaP cells, and growing the tumor to 100mm³-150mm³. Mice were injected intravenously with a single dose of anti-PSMA mAb-azide-DOTA-²²⁵Ac or isotype control, control mAb-azide-DOTA-²²⁵Ac, is used. The injection dose per mouse is up to a total of 10 μ g of protein with cold antibodies. Tumor size and body weight were measured twice a week. When the tumor size exceeds 1,500mm³At times, or when weight loss exceeded 20%, the animals were euthanized.

The anti-PSMA mAb-DOTA-Ac-225 showed tumor growth inhibition after a single administration, particularly at higher radioactive doses, and outperformed the isotype control at all doses (fig. 5A). All doses of control mAb radioconjugates had similar survival curves to vehicle controls (FIG. 5B; Table 7); the anti-PSMA mAb conjugates showed significant dose response, with survival increasing with increasing radioactive dose (fig. 5C; table 7). The study was terminated after 209 days, where 3 mice remained, all from the anti-PSMA mAb 200nCi group, and no detectable tumors were shown.

Table 7: median survival rate

Embodiments of the invention are intended to be exemplary only, and those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific procedures of the invention. All such equivalents are considered to be within the scope of the invention and are encompassed by the following claims.

All references, including patent applications, patents, and publications, cited herein are hereby incorporated by reference in their entirety and for all purposes to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety and for all purposes.

Claims (32)

1. A method of labeling a polypeptide with a radioactive metal ion, the method comprising:

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

b. providing a radioactive complex comprising the radiometal ion associated with a chelating moiety, wherein the chelating moiety comprises a chelator covalently linked to a second click reaction partner; and

c. contacting said modified polypeptide with said radioactive complex under conditions that allow said first click reaction partner to react with said second click reaction partner, thereby labeling said polypeptide with said radioactive metal ion.

2. The method of claim 1, wherein one of the first click reaction partner and the second click reaction partner comprises an alkyne group and the other click reaction partner comprises an azide, or wherein one of the first click reaction partner and the second click reaction partner comprises an alkene group and the other click reaction partner comprises a diene.

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

4. The method of claim 3, wherein the antibody is an anti-PSMA monoclonal antibody.

5. The method of any one of claims 1 to 4, wherein the radioactive metal ion is²²⁵Ac、¹¹¹In or⁸⁹Zr。

6. The method of any one of claims 1 to 4, further comprising reacting an electrophile on a side chain with a thiol group covalently linked to the first click reaction partner to obtain the modified polypeptide.

7. The method according to any one of claims 1 to 5, wherein the modified polypeptide is a modified antibody or antigen-binding fragment thereof obtained by site-specific incorporation of the first click reaction partner.

8. The method of claim 7, wherein the modified antibody or antigen-binding fragment thereof is obtained by a method comprising cleaving an antibody or antigen-binding fragment thereof with a bacterial endoglycosidase specific for an β -1, 4 linkage between core GlcNac residues in an Fc glycosylation site of the antibody to obtain a cleaved antibody or antigen-binding fragment thereof, and reacting the cleaved antibody or antigen-binding fragment thereof with an azide-labeled sugar in the presence of a glycosyltransferase, such as GalT galactosyltransferase or GalNAc transferase, to obtain the modified antibody or antigen-binding fragment thereof.

9. The method of claim 8, wherein the azide-labeled sugar is UDP-N-azidoacetylgalactosamine (UDP-GalNaz) or UDP-6-azido 6-deoxygalnac.

10. The process of claim 8, wherein the glycosyltransferase is a GalT galactosyltransferase or a GalNAc transferase.

11. The method of claim 7, wherein the modified antibody or antigen-binding fragment thereof is obtained by a method comprising: deglycosylating an antibody or antigen-binding fragment thereof with an amidase to obtain a deglycosylated antibody or antigen-binding fragment thereof, and reacting the deglycosylated antibody or antigen-binding fragment thereof with azidoamine in the presence of microbial transglutaminase, thereby obtaining the modified polypeptide.

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

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

14. The method of claim 1, wherein the chelator comprises a macrocycle having the structure of formula (I):

wherein R is₁、R₂、R₃And R₄Is independently CHQCO₂X, wherein

Q is independently hydrogen, C₁-C₄Alkyl or (C)₁-C₂Alkyl) phenyl, and

x is independently hydrogen, benzyl, C₁-C₄An alkyl group; and is

Z is (CH2)_nY is, wherein

n is 1 to 10, and

y is an electrophilic or nucleophilic moiety covalently linked to the second click reaction partner;

alternatively, Z is hydrogen; and is

R₁、R₂、R₃And R₄Is independently CHQCO₂X, wherein

Q is independently hydrogen, C₁-C₄Alkyl or (C)₁-C₂Alkyl) phenyl, and

x is independently hydrogen, benzyl, C₁-C₄An alkyl group or an electrophilic or nucleophilic moiety covalently linked to the second click reaction partner;

alternatively, the chelating agent comprises an open chain ligand.

15. The method of claim 1, wherein the chelating moiety comprises a structure of formula (II):

or a structure of formula (III):

16. one kind is used²²⁵Ac、¹¹¹In or⁸⁹A method of Zr-labeled antibody or antigen-binding fragment thereof, the method comprising:

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

b. providing a radioactive complex, said radioactive complexThe substance comprising a chelating moiety associated therewith²²⁵Ac、¹¹¹In or⁸⁹Zr, wherein the chelating moiety comprises a chelating agent covalently attached to an alkyne or alkene group; and

c. contacting the modified antibody or antigen-binding fragment thereof with the radioactive complex under conditions that allow the azide, tetrazine, or tetrazole group to react with the alkyne or the olefin group²²⁵Ac、¹¹¹In or⁸⁹Zr labels the antibody or antigen-binding fragment thereof,

wherein the chelating agent comprises the structure of formula (I):

wherein R is₁、R₂、R₃And R₄Is independently CHQCO₂X, wherein

Q is independently hydrogen, C₁-C₄Alkyl or (C)₁-C₂Alkyl) phenyl, and

x is independently hydrogen, benzyl, C₁-C₄An alkyl group; and is

Z is (CH2)_nY is, wherein

n is 1 to 10, and

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

alternatively, Z is hydrogen; and is

R₁、R₂、R₃And R₄Is independently CHQCO₂X, wherein

Q is independently hydrogen, C₁-C₄Alkyl or (C)₁-C₂Alkyl) phenyl, and

x is independently hydrogen, benzyl, C₁-C₄An alkyl group or an electrophilic or nucleophilic moiety covalently linked to the alkyne group,

or, alternatively, the chelator comprises an open-chain ligand.

17. The method of claim 16, further comprising reacting an electrophile on a side chain with a thiol group covalently linked to the azide, tetrazine, or tetrazole group to obtain the modified antibody, or antigen-binding fragment thereof.

18. The method of claim 16, wherein the modified antibody or antigen-binding fragment thereof is obtained by site-specific incorporation into the first click reaction partner.

19. The method of claim 18, wherein the modified antibody or antigen-binding fragment thereof is obtained by a method comprising cleaving an antibody or antigen-binding fragment thereof with a bacterial endoglycosidase specific for an β -1, 4 linkage between core GlcNac residues in an Fc glycosylation site of the antibody to obtain a cleaved antibody or antigen-binding fragment thereof, and reacting the cleaved antibody or antigen-binding fragment thereof with an azide-labeled sugar in the presence of a glycosyltransferase, thereby obtaining the modified antibody or antigen-binding fragment thereof.

20. The method of claim 19, wherein the azide-labeled sugar is UDP-N-azidoacetylgalactosamine (UDP-GalNaz) or UDP-6-azido 6-deoxygalnac.

21. The process of claim 19, wherein the glycosyltransferase is selected from a GalT galactosyltransferase or a GalNAc transferase.

22. The method of claim 16, wherein the modified antibody or antigen-binding fragment thereof is obtained by a method comprising: deglycosylating an antibody or antigen-binding fragment thereof with an amidase to obtain a deglycosylated antibody or antigen-binding fragment thereof, and reacting the deglycosylated antibody or antigen-binding fragment thereof with azidoamine in the presence of microbial transglutaminase, thereby obtaining the modified polypeptide.

23. The process of claim 22, wherein the azidoamine is selected from the group consisting of 3-azidopropylamine, 6-azidohexylamine, O- (2-aminoethyl) -O ' - (2-azidoethyl) tetraethylene glycol, O- (2-aminoethyl) -O ' - (2-azidoethyl) pentaethylene glycol, and O- (2-aminoethyl) -O ' - (2-azidoethyl) triethylene glycol.

24. The method of 16, wherein the chelating moiety comprises a structure of formula (II):

or a structure of formula (III):

25. a method of dual labeling a polypeptide with two radioactive metal ions, the method 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 associated with a chelating moiety, wherein the chelating moiety comprises a chelator covalently linked to a third click reaction partner; and

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

d. contacting said modified polypeptide with said radioactive complex under conditions that allow said first click reaction partner to react with said third click reaction partner and said second click reaction partner to react with said fourth click reaction partner, thereby labeling said polypeptide with said first and second radioactive metal ions.

26. The method of claim 25, wherein one of the first click reaction partner and the second click reaction partner comprises an alkyne group and the other of the first click reaction partner and the second click reaction partner comprises an azide, and wherein one of the third click reaction partner and the fourth click reaction partner comprises an olefin group and the other of the third click reaction partner and the fourth click reaction partner comprises a diene, and wherein the first radioactive metal ion or the second radioactive metal ion is a diagnostic emitter and the other is a therapeutic emitter, or wherein both the first radioactive metal ion and the second radioactive metal ion are therapeutic emitters.

27. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a radiolabeled polypeptide prepared by the method according to claim 1 or 16.

28. A method of treating a neoplastic disease or disorder in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 27.

29. A diagnostic agent comprising a pharmaceutically acceptable carrier and a radiolabeled antibody prepared by the method according to claim 1 or 16, wherein the immunological properties of the radiolabeled antibody are retained.

30. A diagnostic agent prepared by the method of claim 1, having

A structure of formula (VIII):

or a structure of formula (IX):

31. the diagnostic and therapeutic agent according to claim 29, wherein the radioactive metal ion is selected from the group consisting of³²P、⁴⁷Sc、⁶⁷Cu、⁷⁷As、⁸⁹Sr、⁹⁰Y、⁹⁹Tc、¹⁰⁵Rh、¹⁰⁹Pd、¹¹¹Ag、¹³¹I、¹⁵³Sm、¹⁵⁹Gd、¹⁶⁵Dy、¹⁶⁶Ho、¹⁶⁹Er、¹⁷⁷Lu、¹⁸⁶Re、¹⁸⁸Re、¹⁹⁴Ir、¹⁹⁸Au、¹⁹⁹Au、²¹¹At、²¹²Pb、²¹²Bi、²¹³Bi、²²³Ra、²²⁵Ac、²⁵⁵Fm、²²⁷Th、⁶²Cu、⁶⁴Cu、⁶⁷Ga、⁶⁸Ga、⁸⁶Y、⁸⁹Zr or¹¹¹In。

32. A combination, comprising:

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

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

wherein said combination is to be used for labelling said polypeptide with said radioactive metal ion.