Radiolabeling of polypeptides

Abstract

To provide improved methods of radiolabeling antibodies using click chemistry.SOLUTION: Provided is a method of labeling a polypeptide with a radiometal ion. The method comprises: a. providing a modified polypeptide comprising the polypeptide covalently linked to a first click reaction partner; b. providing a radiocomplex comprising the radiometal ion associated with a chelating moiety, the chelating moiety comprising a chelant covalently linked to a second click reaction partner; and c. contacting the modified polypeptide with the radiocomplex under a condition to allow the first click reaction partner to react with the second click reaction partner and to thereby label the polypeptide with the radiometal ion.SELECTED DRAWING: None

Description

[Technical Field]

[0001] (Reference to electronically submitted sequence listing) This application is filed on December 7, 2018, in accordance with the "Sequence Listing The sequence listing will be submitted electronically via EFS-Web as an ASCII sequence listing with the file name " The submitted sequence listing will be approximately 13.2 kB in size. The sequence listing provided is a part of the present specification and is incorporated herein by reference in its entirety.

[0002] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application is a joint venture of U.S. Patent Application No. 2017 / 0129999 filed on December 18, 2017, pursuant to 35 U.S.C. § 119(e). Priority is granted to Provisional Application No. 62 / 599,830, the entire disclosure of which is incorporated herein by reference. No. 60 / 699,493, filed on Oct. 1, 2003, and incorporated herein by reference.

[0003] FIELD OF THE INVENTION The present invention relates to a method for radiolabeling polypeptides such as antibodies. describes a method for labeling polypeptides with radioactive metal ions using click chemistry. The present invention also relates to pharmaceutical compositions and uses of radiolabeled polypeptides. [Background technology]

[0004] Alpha particle-emitting radionuclides are ideal for cancer treatment due to their combination of high energy and short-range action. These drugs are highly promising therapeutic options, offering the potential for potent killing, largely confined to tumor cells. Kim, YSand MWBrechbiel, An overvie w of targeted alpha therapy.Tumour Biol, 2012.33(3):p.573-90). Antibodies, scaffold proteins, small molecules Alpha-emitters using ligands, aptamers, or other binding moieties specific for cancer antigens Targeted delivery of radionuclides provides a method for selective delivery of radionuclides to tumors, improving their efficacy. In general practice, the binding moiety is attached to a chelator that binds to an alpha-emitting radioactive metal to form a radioactive complex. One such example is the use of a monoclonal antibody (mAb) as a targeting ligand to induce radioactivity They produce what are known as immune complexes.

[0005] Actinium-225( 225 Ac) are alpha-emitting radioactive materials of particular interest for medical applications. isotope (Miederer et al., Realizing the pot ential of the Actinium-225 radionuclide generator in targeted alpha particle the rapy applications.Adv Drug Deliv Rev,200 8.60(12):71-82). 225 The 10-day half-life of Ac allows for the formation of the radioconjugate. long enough to facilitate but match the circulating pharmacokinetics of the delivery vehicle, such as an antibody. It is short enough for 225 Radioimmunoconjugates of Ac are of particular interest. , 225 Ac is a stable isotope 209 Finally, four alpha particles are released before reaching Bi. Another radioisotope for medical use is It provides both gamma radiation suitable for imaging and medium-energy beta radiation suitable for radiation therapy. Emitting lutetium-177( 177Lu). 177 Lu-labeled peptides demonstrated significant reduction in tissue damage, 177 The Lu label is a single radiant for both treatment and imaging. It has been shown that it is possible to use sex markers (Kwekkeboom DJ ,et al.[177Lu-DOTAOTyr3]octreotate:compa rison with[111In-DTPAo]octreotide in pat ients.Eur J Nucl Med.2001;28:p.1319-1325 Other radioisotopes used in therapeutic applications include, for example, beta emitters or arsenic. emitters, such as thorium, radium, 32 P, 47 Sc, 67 Cu, 77 As, 89 Sr, 90 Y, 99 Tc, 105 Rh, 109 Pd, 111 Ag, 131 I, 153 Sm, 159 Gd, 165 Dy, 166 Ho, 169 Er, 186 Re, 188 Re, 1 94 Ir, 198 Au, 199 Au, 211 At, 212 Pb, 212 Bi, 213 Bi , 223 Ra, 255 Fm, and 227 Other examples of imaging applications include Th. Examples of radioisotopes include: 62 Cu, 64 Cu, 67 Ga,68 Ga, 86 Y, 8 9 Zr, and 111 gamma-ray emitting radioisotopes such as In.

[0006] Previous clinical and preclinical programs have focused on 1, 4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was used. However, DOTA chelation of actinium can be difficult. It is known that there is a vivo stability of actinium-225 macrocyc lic complexes.J Med Chem,1999.42(15):p.2 988-92), which often require harsh conditions or high levels of DOTA per antibody. As a result, two methods known as "one-step" and "two-step" radiolabeling methods are available. Different approaches have been used, each with its own drawbacks.

[0007] The "two-step" method, which involves two chemical steps involving actinium, was first developed (M cDevitt,MR,et al.,Tumor therapy with t argeted atomic nanogenerators.Science,20 01.294(5546):p.1537-40). 225 Ac, 2M acetate buffer High radiochemical yields (approximately 9%) were achieved at pH 4.5-5 using 55-60°C for 30 min. 5%) with bifunctional chelator (BFC) DOTA-isothiocyanate (DOTA-SCN ) and then chelated. 225AcDOTA-SCN reacted with targeting antibodies The main drawback of the two-step method is that approximately 90% of the SCNs are targeted. Because it cannot withstand the cognitive conditions, 225 Approximately 90% of Ac is conjugated to antibodies. The problem is that the DOTA is conjugated to an unreacted form that cannot be reacted with other compounds. This results in low yields. Not only is the cost lower (typically only about 10%), but the specific activity is also lower, resulting in This may limit the effectiveness of the final complex.

[0008] A "one-step" method has recently been developed for actinium (Maguire, WF ,et al.,Efficient 1-step radiolabeling o f monoclonal antibodies to high specificity activity with 225Ac for alpha-particle radioimmunotherapy of cancer.J Nucl Med, 2014.55(9):p.1492-8). This method involves the use of a chemical reaction containing actinium. There is only one reaction step: DOTA-SCN is first conjugated to the antibody. Then, 225 A c was chelated to DOTA-mAb under mild conditions (37°C, pH 7.5), resulting in a maximum A radiochemical yield of 80% was obtained. However, at high levels of DOTA (approximately 10 per antibody), It was necessary to conjugate a high chelator:antibody ratio (C or more) to achieve high yields. AR), in which case species with a high DOTA:Ab ratio (DAR) may have reduced immunoreactivity. Furthermore, the average DAR may be 10, 225 Ac is above average It is possible that chelation occurs to a higher proportion of the population. teeth, 225 There is a risk of linking Ac to at least an active fraction of the antibody-chelator conjugate. Additionally, metal-free formulations are used to avoid chelation of common metals such as iron, zinc, and copper. The antibody and DOTA-mAb conjugate must be handled under unfavorable conditions, which significantly impacts the production process. This poses significant challenges.

[0009] Click chemistry was introduced by Sharpless in 2001. approach, which produces substances quickly and reliably by bonding small units together For example, Kolb, Finn and Sha rpless Angewandte Chemie International E dition(2001)40:2004-2021;Evans,Australia See Journal of Chemistry (2007) 60:384-395 Coupling reactions (some of which can be classified as "click chemistry") Examples include, but are not limited to, esters from activated acids or acyl halides, thioesters, etc. amide formation (e.g., peptide coupling); nucleophilic substitution reactions (e.g., halo Nucleophilic substitution of hydrides or opening of strained ring systems; azido-alkoxy Huisgen cycloaddition with amines (e.g., to form 1,2,3-triazole linkers) 1,3-Dipolar Cycloaddition between Azides and Alkynes; Thiophosphorus Addition; Imine Form Diels-Alder reaction between tetrazine and trans-cyclooctene (TCO) and Michael addition reactions (e.g., maleimide addition reactions).

[0010] Click chemistry reactions between alkynes and azides typically involve the 1,3-cyclopentadiene Copper-catalyzed azide-alkyne cycloaddition requires the addition of a copper catalyst to promote the addition reaction. However, the CuAAC reaction is not a cyclooctyne or cyclooctyl reaction. Click chemistry reactions between tin derivatives and azides typically require the addition of a copper catalyst. Instead, strain-promoted azide-alkyne cycloaddition (SPAAC) is used. Promoted azide-alkyne cycloaddition (Debets, MF, e t al.,Bioconjugation with strained alken es and alkynes.Acc Chem Res,2011.44(9):p .805-15).

[0011] Site specificity is a key factor in improving the efficacy and safety of ADCs using a site-specific approach compared to random conjugation. Antibody-drug conjugates (ADCs) have been shown to enhance both safety and efficacy. This is a major area of ​​focus in the field of antibody-drug conjugates (Agarwal ,P.and CRBertozzi,Site-specific antibo dy-drug conjugates:the nexus of bioortho gonal chemistry, protein engineering, and drug development.Bioconjug Chem,2015.26( 2):pp.176-92). Similar safety and efficacy benefits have been observed for radioimmunoconjugates. It is believed that this can be achieved. Summary of the Invention [Problem to be solved by the invention]

[0012] As described above, the efficacy of generating stable radioimmunoconjugates with high specific activity and high yields was demonstrated. There remains a need in the art for efficient methods. [Means for solving the problem]

[0013] The present invention uses click chemistry to radiolabel polypeptides such as antibodies. This need is met by providing a method for using radioactive gold. It requires reduced use of metals and is therefore unnecessary in the process used to generate the initial radioactive complex. Azide-modified antibodies and chelating moieties containing alkyne groups, requiring only metal ionization Radioactive complexes containing radioactive metal ions associated with Stable radioimmunocomplexes with low chelator:antibody ratios (CARs) and high radiochemical yields The method of the present invention produces a radioimmunoglobulin complex with increased safety, efficacy, and uniformity. This simplifies previous methods for producing immunoconjugates.

[0014] In one general aspect, the invention relates to a method for labeling a polypeptide with a radioactive metal ion. And the method is a. A modified polypeptide comprising a polypeptide covalently linked to a first click reaction partner. and b. providing a radioactive complex comprising a radioactive metal ion associated with a chelating moiety; wherein the chelating moiety comprises a chelator covalently attached to a second click reaction partner. Including, c. The first click reaction partner reacts with the second click reaction partner. Thus, the modified polypeptide can be labeled with a radioactive metal ion under conditions that allow the polypeptide to be labeled with a radioactive metal ion. and contacting the polypeptide with a radioactive complex.

[0015] In another general aspect, the invention provides a method for producing a radiolabeled polypeptide prepared by the method of the invention. The present invention relates to a pharmaceutical composition comprising a peptide and a pharmaceutically acceptable carrier.

[0016] In another general aspect, the present invention provides a method for treating a neoplastic disease or disorder in a subject in need of treatment. A method for treating a neoplastic disease or disorder in a subject, comprising administering to the subject a pharmaceutical composition of the present invention. The present invention relates to a method comprising:

[0017] In another general aspect, the present invention provides a method for producing a method of manufacturing a semiconductor device comprising: a. A modified polypeptide comprising a polypeptide covalently linked to a first click reaction partner. Do and, b. A radioactive complex comprising a radioactive metal ion associated with a chelating moiety, a radioactive complex wherein the chelating moiety comprises a chelator covalently attached to a second click reaction partner; and a combination or kit comprising: The combination or kit comprises a compound for labeling a polypeptide with a radioactive metal ion. Used.

[0018] In another general aspect, the invention provides a method for producing a radiolabeled polypeptide prepared by the method of the invention. The present invention relates to therapeutic or diagnostic agents ("theranostic agents") comprising peptides. [Brief explanation of the drawings]

[0019] The above "Summary of the Invention" and the following "Description of the Invention" are attached. The present invention will be better understood when read in conjunction with the accompanying drawings, in which: It should be understood that the present invention is not limited to the precise embodiment.

[0020] The drawings are as follows: [Figure 1] 1 shows a schematic diagram of radiolabeling of antibodies according to the methods of the present invention. Random conjugation is shown in the diagram; a similar radiolabeling scheme is used when the azide is site-specifically conjugated to a monoclonal antibody (mAb). [Figure 2] 1 shows a synthetic scheme for an improved two-step preparation of 89Zr-DOTA-mAb via click chemistry according to one embodiment of the present application. [Figure 3] Figure 1 shows cell binding of In-111 radioimmunoconjugates, with bound radioactivity increasing with increasing cell number. In particular, Figure 1A shows binding of a PSMA-binding antibody ("PSMB127") In-111 radioimmunoconjugate and a human transferrin In-111 radioconjugate according to an embodiment of the present application to the human prostate cancer cell line C4-2B (PSMA+, transferrin receptor+), and Figure 1B shows binding of an EGFR-binding antibody, cetuximab, and panitumumab In-111 radioimmunoconjugate according to an embodiment of the present application to the human epidermoid carcinoma cell line A431 (EGFR+), as well as the lack of binding of these conjugates to the control (EGFR-) human AML cell line MOLM-13. [Figure 4] According to one embodiment of the present application, the kinetics of cellular internalization of In-111 in the human prostate cancer cell line C4-2B treated with anti-PSMA mAb In-111 radioimmunoconjugate are shown, where surface-bound In-111 was rapidly lost from the cell surface and redistributed intracellularly. [Figure 5A]Results are shown for a mouse tumor xenograft study in which mice were implanted with human prostate cancer LNCaP cells, and when tumors reached 100 mm, the mice were treated with a single dose of a click radiolabeled anti-PSMA mAb ("PSMB127") actinium radioconjugate according to an embodiment of the present application, a range of radioactivity, or an isotype control (a human IgG4 antibody that binds to a viral target not present in this system radioconjugate). Specifically, tumor volumes for each group are shown and plotted as the size at which less than half of the group survived. [Figure 5B] Results are shown for a mouse tumor xenograft study in which mice were implanted with human prostate cancer LNCaP cells, and when tumors reached 100 mm, the mice were treated with a single dose of a click radiolabeled anti-PSMA mAb ("PSMB127") actinium radioconjugate according to an embodiment of the present application, a range of radioactivity, or an isotype control (a human IgG4 antibody that binds to a viral target not present in this system radioconjugate). Specifically, the survival curve for the control mAb group is shown. [Figure 5C] Results are shown for a mouse tumor xenograft study in which mice were implanted with human prostate cancer LNCaP cells, and when tumors reached 100 mm, the mice were treated with a single dose of a click radiolabeled anti-PSMA mAb ("PSMB127") actinium radioconjugate according to an embodiment of the present application, a range of radioactivity, or an isotype control (a human IgG4 antibody that binds to a viral target not present in this system radioconjugate). Specifically, survival curves for the anti-PSMA mAb groups are shown. DETAILED DESCRIPTION OF THE INVENTION

[0021] In the background of the invention and throughout this specification, various publications, articles and patents are cited or Each of these references is incorporated herein by reference in its entirety. Any discussion of documents, operations, materials, devices, articles or the like which has been included in the specification is without limitation in the context of the present invention. Such discussion is intended to provide a general understanding of the invention and its applications. or admitted to constitute prior art to any claimed invention. It's not that.

[0022] Unless otherwise defined, all technical and scientific terms used herein are defined by the It has the same meaning as commonly understood by a person skilled in the art to which the invention pertains. Unless otherwise specified, specific terms referred to herein shall have the meanings ascribed to them herein. All patents, published patent applications and publications cited herein are hereby incorporated by reference. No. 6,027,797, filed Dec. 1, 2004, which is incorporated herein in its entirety as if set forth herein.

[0023] As used in this specification and the appended claims, the singular forms "a," "an," and "the" It should be noted that "includes plural referents unless the context clearly indicates otherwise."

[0024] Throughout this specification and the claims that follow, unless the context otherwise requires, the term "comprises" "comprises" and variations such as "comprises" and "comprising" are used to refer to Including the specified integer or step or group of integers or steps, but any other integer or step does not mean to exclude any integer or step or group of steps. It will be understood that, as used herein, the term "comprises" is inclusive of the terms "contains" or "including" may be substituted for or as sometimes used herein. It can also be replaced with the term "having."

[0025] As used herein, "consisting of" refers to the As used herein, "excludes any element, step, or ingredient not included in the present invention." "Qualitative" means material that does not materially affect the basic and novel characteristics of the claim. As used herein in relation to an aspect or embodiment of the invention, the term "material" does not exclude any material or step. When used in conjunction with a reference to a specific subject, the terms "comprising," "containing," and "including" are used to vary the scope of the disclosure. Any of the above terms "including" and "having" may be replaced with the term "consisting of" or can be replaced with "consisting essentially of."

[0026] As used herein, the conjunctive term "and / or" between multiple listed elements means It is understood to encompass both individual and combined options. For example, When elements are connected by "and / or," the first option is the first option without the second option. The second option refers to the applicability of the second element without the first element. The third option indicates that the first and second elements are applicable together. Any one of these options is not intended to be construed as limiting the scope of the term "and / or" as used herein. The simultaneous applicability of two or more of the options is also implied. It is understood that the terms "and / or" are included in the preceding paragraphs and therefore satisfy the requirement of the term "and / or."

[0027] To assist the reader of this application, the description is divided into various paragraphs or sections. These separations are not intended to be limiting unless they are followed by a paragraph or a section. Separating a section or embodiment entity from another paragraph or section or embodiment entity On the contrary, those skilled in the art will appreciate that the description herein has broad application. It includes all possible combinations of paragraphs, paragraphs, and sentences. It will be understood that the discussion of any embodiment is meant to be exemplary only. It is to be understood that the scope of the present disclosure, including the claims, is limited to these examples. It is not intended to be suggestive.

[0028] Click radiolabeling of polypeptides In contrast to known procedures, the method of the present invention can be used to treat, for example, a subject in need thereof, e.g. Improved methods for the production of radioimmunoconjugates suitable for medical use in humans are provided. In particular, the methods described herein include, but are not limited to: 225 Ac, 111 In and 8 9 Provides a process for both high-yield chelation of metal ions, including Zr, and low DAR The present invention addresses a major limitation of current methods by providing radiolabeled diagnostic Purpose (e.g., 89 Zr or 111 In-labeled) or for therapeutic purposes (e.g. 225 Azide-labeled mAb conjugates can be used for the production of azide-labeled mAb (when labeled with Ac). The radiolabeling allows for the production of a single batch of azide-labeled polypeptide. The nucleotides are attached to the same site(s) within a batch of nucleotides, either site-specific or randomly. For example, in the case of random azide conjugation, A sample of a batch of azide-labeled polypeptides containing a single distribution of azide modification sites is prepared according to the method of the present invention. Click chemistry can be used to radiolabel for different purposes.

[0029] The method of the present invention, which relies on click chemistry and is referred to as "click radiolabeling," (1) a first click chemistry reaction partner, e.g., an antibody, comprising an azide moiety; and (2) obtaining a modified polypeptide, such as a radioactive metal ion, associated with a chelating moiety. On, for example, 225 Ac, 111 In, or 89 By obtaining radioactive complexes containing Zr, wherein the chelating moiety is linked to a second click chemistry reaction partner, e.g., , DOTA-dibenzocyclooctyne (DOTA-DBCO) or deferoxamine-D BCO (DFO-DBCO), and (3) Strain-promoted azide-alkyne cycloaddition reaction between the azide moiety and the alkyne group (SP A modified peptide is combined with a click chemistry reaction partner, such as a hydroxybenzoate (AAC), and a radioactive complex. and carrying out a reaction between

[0030] The method of the present invention involves chelation of radioactive metals under conditions of low or high pH and / or high temperature. This maximizes efficiency by deactivating the alkyne reaction partner. This can be achieved without the risk of inducing efficient cleavage between the azido-mAb and the radioactive complex. High radioactivity even at low azide:mAb ratios due to the azide-conjugated and efficient SPAAC reaction The method of the present invention allows the production of radioimmunoconjugates in high yields without trace metals. The only step required is chelation of the radioactive metal ion to the chelating moiety. Therefore, antibody production, purification, and conjugation steps must be carried out under metal-free conditions. There is no.

[0031] As used herein, the term "click chemistry" refers to the method described by Sharpless et al. refers to the chemical philosophy introduced by the Describe chemistry tailored to rapidly and reliably generate covalent bonds (Kolb, supra). (See, e.g., J. Med. et al.) Click chemistry does not refer to a specific reaction. However, it refers to a concept that includes, but is not limited to, reactions that mimic reactions found in nature. In some embodiments, click chemistry reactions are modular and comprehensive. It has high chemical yields, produces benign by-products, is stereospecific, and produces a single reaction product. exhibit a large thermodynamic driving force for reaction selection with and / or be carried out under physiological conditions In some embodiments, click chemistry reactions can be performed on high-atom It is economical, can be carried out under simple reaction conditions, and requires readily available starting materials and Use of reagents, no toxic solvents, or non-toxic or easily removed solvents such as water and / or simple preparation by non-chromatographic methods such as crystallization or distillation. In certain embodiments, the click chemistry reaction provides for the isolation of the product. N3) and alkyne or alkyne moiety in a Huisgen cycloaddition or 1,3-dipole attachment reaction This is a cyclization reaction to form a 1,2,4-triazole linker.

[0032] In a general aspect, the present invention provides a method for the preparation of a polypeptide, an aptamer, or a small molecule with a radioactive metal ion. A method for labeling a daughter ligand, the method comprising: a. A modified polypeptide comprising a polypeptide covalently linked to a first click reaction partner. and b. providing a radioactive complex comprising a radioactive metal ion associated with a chelating moiety; wherein the chelating moiety comprises a chelator covalently attached to a second click reaction partner. Including, c. The first click reaction partner reacts with the second click reaction partner. Thus, the modified polypeptide can be labeled with a radioactive metal ion under conditions that allow the polypeptide to be labeled with a radioactive metal ion. and contacting the polypeptide with a radioactive complex.

[0033] As used herein, the term "polypeptide" refers to a group of polypeptides linked together through peptide bonds. Polypeptides consisting of naturally occurring structural variants and their synthetic non-naturally occurring analogues. The term "polypeptide" refers to a polypeptide of any size, structure, or function. Typically, a polypeptide is at least three amino acids long. can 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, an antigen-binding fragment thereof, etc. In other embodiments, the antibody or fragment thereof is specific for a cancer antigen. A polypeptide is a genetically engineered domain or scaffold protein.

[0034] As used herein, the terms "antibody" or "immunoglobulin" are used broadly. Immunoglobulin or antibody molecules, including polyclonal antibodies, murine, human, human-adapted, human Monoclonal antibodies, including homogenized and chimeric monoclonal antibodies and antigen-binding fragments thereof Includes clonal antibodies.

[0035] In general, an antibody is a protein or peptide chain that exhibits binding specificity to a specific antigen. The structure of an antibody is known. Immunoglobulins are There are five major classes depending on the amino acid sequence of the heavy chain constant domain: IgA, IgD IgA and IgG can be assigned to the isotypes IgE, IgG, and IgM. It is further subdivided into types IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4. Thus, the antibodies of the present invention are classified into five major classes or corresponding subclasses: The antibodies of the present invention can be any of IgG1, IgG2, IgG3, Preferably, the antibody light chains of any vertebrate species are IgG4 or IgG5. Based on the main amino acid sequence, two distinct types are identified: kappa and lambda. Thus, the antibodies of the present invention can be assigned to either the κ or λ light chain constant domain. According to a particular embodiment, the antibody of the present invention is a murine antibody or Each of the four IgG subclasses contains a heavy and / or light chain constant region of a human antibody. They have different biological functions known as effector functions. These effector functions are generally mediated by interaction with Fc receptors (FcγR) or by binding of C1q and complement Binding to FcγRs leads to antibody-dependent cell-mediated cytolysis. Although the binding to complement factors can result in complement-mediated cell lysis, the compounds useful in the present invention The antibody may have no or minimal effector function but binds to FcRn retain that ability.

[0036] As used herein, the term "antigen-binding fragment" refers to, for example, a diabody, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv flag ment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), Disulfide-stabilized diabodies (ds diabodies), single-chain antibody molecules (scFv), single Single domain antibodies (sdab), scFv dimers (bivalent diabodies), one or more CDRs Multispecific antibodies formed from antibody portions containing domain antibodies, bivalent domain antibodies, or antibodies that bind to an antigen but do not comprise the complete antibody structure The term "antibody fragment" refers to an antibody fragment such as any other antibody fragment that does not bind to the parent antibody. or capable of binding to the same antigen as that bound by the parent antibody fragment. The term "single chain antibody" refers to a polymer chain consisting of multiple chains connected by short peptides of about 15 to about 20 amino acids. This refers to a conventional single-chain antibody in the art, which comprises a single chain variable region and a light chain variable region. As used herein, the term "single domain antibody" refers to a antibody that contains a heavy chain variable region and a heavy chain constant region. , or refers to conventional single domain antibodies in the art, which contain only the heavy chain variable region.

[0037] As used herein, the term "scaffold" or "scaffold protein" refers to any protein that has a target binding domain and is capable of binding to a target. The scaffold consists of a "framework" that is mostly structural and a target-specific binding domain. The binding domain of the scaffold is the binding domain of the scaffold. A scaffold does not have to be defined by a single contiguous sequence of scaffolds. In certain cases, a scaffold , may be part of a larger binding protein, which itself comprises multiple scaffolds A particular binding protein may be part of a multimeric binding protein comprising two or more Bispecific or multispecific in that they can bind to different epitopes. The scaffold may be derived from a single chain antibody, or the scaffold may be derived from an antibody. It doesn't have to be coming.

[0038] Any method for chemical or enzymatic modification of polypeptides known to those of skill in the art in light of this disclosure. The method is used to covalently attach a polypeptide of the invention to a first click reaction partner. The primary amino acids present at the N-terminus of each polypeptide chain and in the side chains of lysine residues can be Amine-reactive groups that react with amines can be used in a method for random modification of polypeptides. Examples of amine reactive groups suitable for use in the present invention include, but are not limited to, N-hydroxybenzoates. Hydroxysuccinimide (NHS), substituted NHS such as sulfo-NHS, isothiocyanate esters, and tetra- and perfluorophenyl esters. Cysteine ​​residues The polypeptide is provided with a thiol-reactive group that reacts with a thiol or sulfhydryl present in the side chain of the polypeptide. Suitable thiol reaction products for use in the present invention can be used in the random modification of thiols. Examples of reactive groups include, but are not limited to, maleimide, haloacetyl, and phenyloxa. According to a preferred embodiment, the modified polypeptide is an electrophile covalently attached to a click reaction partner (e.g., NHS-azide) of 1; It is obtained by reacting with the side chain of lysine, preferably the amino side chain.

[0039] The method of the present invention further allows for the production of site-specific radiolabeled polypeptides. The click radiolabeling method utilizes an established method for site-specifically introducing azide groups into antibodies. This facilitates the site-specific generation of radioimmunoconjugates (Li, X., et al., t al. Preparation of well-defined antibo dy-drug conjugates through glycan remode ling and strain-promoted azide-alkyne cy cloadditions.Angew Chem Int Ed Engl,2014 .53(28):p.7179-82;Xiao,H.,et al.,Genetic incorporation of multiple unnatural ami no acids into proteins in mammalian cell s.Angew Chem Int Ed Engl,2013.52(52):p.1 4080-3). Methods for attaching molecules to proteins or antibodies in a site-specific manner are well known in the art. Any method of site-specific labeling of antibodies known in the art and known to one of skill in the art can be used. In view of the present disclosure, antibodies suitable for use in the present invention may be used in the context of site-specific Examples of methods for differentially modifying include, but are not limited to, modified cysteine ​​residues (e.g., TH IOMAB™), incorporation of unnatural amino acids or glycans (e.g., selenocysteine) In, p-AcPhe, formylglycine generating enzyme (FGE, SMARTag™) etc.), and enzymatic methods (e.g., glycotransferases, endoglycosidases, microbial The use of bacterial transglutaminase (MTG or BTG), sortase A, etc. According to a preferred embodiment, the modified polypeptide has an innermost GlcNAc leaving the Fc intact, allowing site-specific incorporation of an azido sugar at that site, G Core Gl at the Fc-glycosylation site of antibodies, such as lycINATOR (Genovis) Bacterial endoglycosidase specific for β-1,4 linkages between cNac residues was used to An antibody or its antigen-binding fragment obtained by trimming the antibody or its antigen-binding fragment The trimmed antibody or antigen-binding fragment thereof is then , GalT galactosyltransferase or GalNac transferase In the presence of glycosyltransferase, UDP-N-azidoacetylgalactosamine (UDP-GalN) az) or UDP-6-azido 6-deoxyGalNAc. The modified antibody or antigen-binding fragment thereof can be obtained by the following steps. According to the method, the modified polypeptide is prepared by glycosylating an antibody or antigen-binding fragment thereof with an amidase. The antibody or antigen-binding fragment thereof is then obtained by cosylation of The resulting deglycosylated antibody or antigen-binding fragment thereof is reacted with an azidoamine, preferably is 3-azidopropylamine, 6-azidohexylamine, or any azido linker amine amine or any azidoalkylamine, azido-polyethylene glycol (PEG) -amines, for example, O-(2-aminoethyl)-O'-(2-azidoethyl)tetraamines Ethylene glycol, O-(2-aminoethyl)-O'-(2-azidoethyl)pentaenoic acid Ethylene glycol, O-(2-aminoethyl)-O'-(2-azidoethyl)triethylene or in the presence of microbial transglutaminase. The modified antibody or antigen-binding fragment thereof can be obtained by the method of the present invention.

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

[0041] As used herein, the term "small molecule ligand" refers to a low molecular weight organic compound. As used herein, a small molecule ligand is a compound having a size of less than about 1000 daltons. It can refer to compounds that can be synthesized in a laboratory or found in nature. This can be done.

[0042] As used herein, the term "click reaction partner" or "click chemistry A "click handle" is a reactant or reactive group that can participate in a click chemistry reaction. Click reaction partners are rarely found in naturally occurring biomolecules and are often used to but reacted with, for example, an azide-reactive or alkyne-reactive group. In some cases, the reaction is carried out under biologically relevant conditions, e.g., in the absence of excessive heat or harsh reactants. The reaction can be carried out efficiently under cell culture conditions such as The Click reaction involves at least two reaction partners that can react with each other. Both require two molecules. These mutually reactive click reaction partners are , referred to herein as click chemistry handle pairs or click chemistry pairs. In some embodiments, the Click reaction partners are azides and strained groups. The alkyne may be a given alkyne, such as cyclooctyne, or any other alkyne. In the click reaction, the partners are a reactive diene and a suitable tetrazine dienophile. For example, trans-cyclooctene, norbornene, or biscyclononene are The tetrazine can be paired with a suitable dienophile as a reaction pair. In the study, tetrazole pairs with an unactivated alkene in the presence of UV light to form a In other embodiments, click reaction pairs can be created, which are referred to as "photo-click" reaction pairs. The click reaction partners are cysteine ​​and maleimide. The corresponding cysteine ​​(e.g., GGGC) is associated with a chelator (e.g., NOTA). Other suitable click chemistry handles can be used. known to the industry (e.g., Spicer et al., Selective ch chemical protein modification.Nature Commu (See nications.2014;5:p.4740). In other embodiments, In the click reaction, the reaction partners are Staudinger ligases such as phosphines and azides. In other embodiments, the click reaction partner is a diamine such as a tetrazine. ene, and alkenes such as trans-cyclooctene (TCO) or norbornene Diels-Alder reaction components. Exemplary click reaction partners are described in U.S. Pat. Application Publication No. 20130266512 and International Publication No. 2015073746 The relevant discussion of both click reaction partners is given in the references. According to a preferred embodiment, the first and second clicks One of the reaction partners contains an alkyne group, and the other click reaction partner contains an azide group. According to another preferred embodiment, one of the first and second click reaction partners comprises One contains an alkene group and the other click reaction partner contains a diene.

[0043] As used herein, the term "alkyne," "alkyne group," or "alkyne moiety" means The alkyne moiety includes terminal alkynes and ring alkynes. and cyclic alkynes, preferably terminal alkynes reactive with azide groups. Terminal alkynes have at least one hydrogen atom attached to a triple-bonded carbon atom. Cyclic alkynes are cycloalkyl rings that contain one or more triple bonds. Examples of cyclic alkynes are: Examples include, but are not limited to, bicyclononyne (BCN), difluorocyclooctyne (DI FO), dibenzocyclooctyne (DIBO), keto-DIBO, biaryl azacyclo Octynone (BARAC), dibenzoazacyclooctyne (DIBAC), dimethoxyaza Dibenzocyclooctyne (DIMAC), Dibenzocyclooctyne (DBCO), Difluorobenzyl Dibenzocyclooctyne (DIFBO), monobenzocyclooctyne (MOBO), and tetrabenzocyclooctyne Cyclooctyne and cyclooctyne derivatives such as trimethoxyDIBO (TMDIBO) According to a preferred embodiment, one of the first and second click reaction partners According to a preferred embodiment, the click reaction The other reaction partner comprises an azide, preferably NHS-azide.

[0044] As used herein, the term "diene" refers to a compound having two carbon-carbon double bonds, The double bond of a diene is either cis or tri. Examples of dienes include tetrazine or tetrazole groups. Examples include, but are not limited to:

[0045] As used herein, the term "alkene," "alkene group," or "alkene moiety" means refers to an unsaturated hydrocarbon molecule containing a carbon-carbon double bond. Alkenes can contain from 2 to 100 carbon atoms. Examples of alkenes include norbornane, Examples of cyclooctene include, but are not limited to, cyclooctene and trans-cyclooctene (TCO). According to another preferred embodiment, one of the first and second Click reaction partners is an alkyl group. According to a preferred embodiment, the compound contains an olefin group, preferably norbornene or TCO. The RIC reaction partner contains a diene, preferably a tetrazine or tetrazole group.

[0046] As used herein, the term "covalently bonded" means attached via at least one covalent bond. whereby the polypeptide is attached to a first click reaction partner and the chelator is attached to at least This means that the first covalent bond is also attached to a second click reaction partner via a covalent bond. The attachment may be direct, i.e., without a linker, or indirect, i.e., involving a linker. This may be done via a

[0047] As used herein, the term "linker" refers to a molecule that links a polypeptide or a chelator. refers to a chemical moiety that links a reactive partner to a reactive partner. Any linker can be used in the present invention. The linker can be, for example, a single covalent bond, a substituent, or a substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl moieties, polyethylene glycol a PEG linker, a peptide linker, a sugar-based linker, or a disulfide bond, or a cleavable phosphoryl group such as a protease cleavage site such as valine-citrulline-PAB. It can be a car.

[0048] As used herein, the term "radioactive metal ion" or "radioactive metal ion" refers to a metal ion that is refers to one or more isotopes of an element that emit particles and / or photons. Any radioactive metal known in the art can be used in the present invention. Examples of radioactive metals include, but are not limited to: 32 P, 47 Sc, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 77 As, 86 Y, 89 Zr, 89 Sr, 90 Y, 99 Tc , 105 Rh, 109 Pd, 111 Ag, 111 In, 117 Sn, 131 I, 153 S m, 159 Gd, 165 Dy, 166 Ho, 169 Er, 177 Lu, 186 Re, 18 8 Re, 194 Ir, 198 Au, 199 Au, 211 At, 212 Pb, 212 Bi, 213 Bi,223 Ra, 225 Ac, 227 Th, and 255 Fm is one example. As used herein, the term "diagnostic emitter" refers to a radioactive material useful in diagnostic or imaging applications. Refers to metal ions. Examples of diagnostic emitters include, but are not limited to: 62 Cu, 64 Cu, 67 Ga, 68 Ga, 86 Y, 89 Zr, and 111 Gamma emitters such as In As used herein, the term "therapeutic emitter" refers to a radiation emitter that emits radiation useful in therapeutic applications. Examples of therapeutic emitters include, but are not limited to, beta or alpha Emitters, such as thorium, radium, 32 P, 47 Sc, 67 Cu, 77 As, 89 Sr, 90 Y, 99 Tc, 105 Rh, 109 Pd, 111 Ag, 131 I, 153 Sm , 159 Gd, 165 Dy, 166 Ho, 169 Er, 177 Lu, 186 Re, 188 Re, 194 Ir, 198 Au, 199 Au, 211 At, 212 Pb, 212 Bi, 2 13 Bi, 223 Ra, 225 Ac, 255 Fm and 227 Th is preferred. According to an embodiment, the radioactive metal ion is 225 According to another embodiment, poly Peptides can be labeled with non-metallic radiolabels for use in pretargeting or theranostic applications. Examples of non-metallic radioactive labels suitable for use in the present invention include, but are not limited to, It won't happen, but 125 I and 18 F is one example.

[0049] The radiocomplexes described herein comprise a radioactive metal ion associated with a chelating moiety. According to an embodiment of the present invention, the chelating moiety is covalently attached to the Click reaction partner. These include chelating agents, sometimes referred to herein as "bifunctional chelators."

[0050] As used herein, the term "chelating agent" or "chelator" refers to a 225 Ac etc. This refers to a chemical compound in which a radioactive metal or metal can be chelated via a coordinate bond. In view of the above, any chelating agent known to those skilled in the art can be used in the present invention. In one embodiment, the chelating agent comprises a macrocycle. Macrocycles suitable for use in the present invention include Examples of chelating agents include, but are not limited to, deferoxamine (DFO), ethylene Diaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA) are examples. In another embodiment, the chelating agent comprises an open-chain ligand. Examples of chelating agents containing open-chain ligands include, but are not limited to, 1,4,7,10-tetramethyl- ... tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), 1,4, 7,10,13,16-Hexaazacyclohexadecane-N,N',N'',N''', N''',N'''''-Hexaacetic acid (HEHA), 1,4,7,10,13-penta Azacyclopentane-N,N',N'',N''',N''''-pentaacetic acid (P EPA), Macropa(Thiele et al., An Eighteen-M embered Macrocyclic Ligand for Actinium- 225 Targeted Alpha Therapy.Angew Chem In t Ed Engl.2017 Nov 13;56(46):p.14712-147 17), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tet Dipropionic acid (DOTPA), 1,4,8,11-tetraazacyclotetradecane-1 ,4,8,11-tetrapropionic acid (TETPA), and 1,4,7,10-tetrapropionic acid Examples include cyclododecane-1,4,7,10-tetramethylenephosphonic acid (DOTMP). According to a preferred embodiment, the chelating agent has formula (I):

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

[0052] According to a preferred embodiment, the chelating moiety has formula (II):

[0053] [ka] Includes the structure of

[0054] According to a preferred embodiment, the chelating moiety has formula (III):

[0055] [ka] Includes.

[0056] In one embodiment, the present invention provides a method for detecting radioactive metal ions with two or more radioactive metal ions using the method of the present invention. The present invention relates to a method for labeling a polypeptide, for example, labeling a polypeptide with two radioactive metal ions. The method of labeling is a. Covalently bonded to a first click reaction partner and a second click reaction partner providing a modified polypeptide comprising a polypeptide; b. providing a first radioactive complex comprising a first radioactive metal ion associated with a chelating moiety; wherein the chelating moiety is covalently attached to a third click reaction partner. Contains a chelating agent, c. providing a second radioactive complex comprising a second radioactive metal ion associated with a chelating moiety. wherein the chelating moiety is a chelating moiety covalently attached to a fourth click reaction partner. Contains a rate agent, d. The first click reaction partner reacts with a third click reaction partner to form a second The click reaction partner reacts with a fourth click reaction partner to form a poly Modification under conditions that allow the peptide to be labeled with a first and second radioactive metal ion. contacting the polypeptide with first and second radioactive complexes.

[0057] According to a preferred embodiment, one of the first and second click reaction partners is an alkyne. a group, the other of the first and second Click reaction partners comprises an azide, and the third and fourth one of the click reaction partners contains an alkene group, and the third and fourth click reaction partners The other of the toners contains a diene.

[0058] According to a preferred embodiment, the first or second radioactive metal ion is a diagnostic emitter; The other is a therapeutic emitter. According to a preferred implementation, the first and second radioactive metal ions Both are therapeutic emitters.

[0059] Conditions for carrying out click chemistry reactions are known in the art, Any conditions for carrying out click chemistry reactions known to one of skill in the art in light of the present disclosure. Examples of conditions that can be used in the present invention include, but are not limited to, pH 4 to 10 and and a temperature of 20°C to 70°C in a ratio of 1:1 to 1000:1. Incubating the radioactive complex.

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

[0061] Pharmaceutical compositions and methods of treatment The click radiolabeling method of the present invention may be modified to a pre-targeting approach (Kra eber-Bodere,F.,et al.,A pretargeting system tem for tumor PET imaging and radioimmunization otherapy. Front Pharmacol, 2015.6:p.54). First , administering the azido-mAb, allowing it to bind to target cells and clear from the circulation over time, or The radioactive complex is then administered to the target site, and the azide-mAbs bound to the target site are removed. Upon undergoing a SPAAC reaction with , the remaining unbound radioactive complex is rapidly cleared from the circulation. (Deal, KA, et al., Improved in stab vivo ility of actinium-225 macrocyclic comple xes.J Med Chem,1999.42(15):p.2988-92). Pre-targeting technology provides a method for enhancing radioactive metal ion localization at target sites in a subject. do.

[0062] Thus, in another general aspect, the present invention provides a radiation-sensitive composition prepared by the method of the present invention. The present invention relates to a pharmaceutical composition comprising a labeled polypeptide and a pharmaceutically acceptable carrier.

[0063] As used herein, the term "carrier" refers to any excipient, diluent, filler, salt, base, or the like. buffers, stabilizers, solubilizers, oils, lipids, lipid-containing vesicles, microspheres, liposomes Carriers, excipients, or other materials known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the diluent or diluent will depend on the route of administration for a particular application. The term "pharmaceutically acceptable carrier" as used herein means a pharmaceutical carrier that is effective or It refers to a non-toxic material that does not interfere with the biological activity of the composition according to the invention. In view of the present disclosure, suitable for use in antibody-based or radioconjugate-based pharmaceutical compositions: Any pharmaceutically acceptable carrier can be used in the present invention.

[0064] According to specific embodiments, the compositions described herein are administered to a subject in a manner contemplated by the present invention. For example, the compositions described herein can be administered intravenously, subcutaneously, or intravenously. It can be formulated to be suitable for intramuscular or intratumoral administration.

[0065] In certain embodiments, the modified polypeptide and the radioactive complex are of the same or different compositions. It can be administered at .

[0066] In another general aspect, the present invention provides a method for treating a neoplastic disease or disorder in a subject in need of treatment. A method for treating a neoplastic disease or disorder in a subject, comprising administering to the subject a pharmaceutical composition of the present invention. The present invention relates to a method comprising:

[0067] According to certain embodiments, the methods of the present invention comprise administering a therapeutically effective dose of a pharmaceutical composition of the present invention. wherein the composition targets cells associated with a neoplastic disease or disorder. and targeting the target polypeptide to 225 from Ac and its daughter nuclides Alpha particles are delivered to the target cells, causing a cytotoxic effect on the target cells, which to treat a neoplastic disease or disorder.

[0068] According to certain embodiments, the therapeutically effective amounts of the modified polypeptide and the radioconjugate are in different compositions. It is administered as a composition.

[0069] As used herein, the term "therapeutically effective amount" refers to a dose that provides a desired biological or pharmacological effect to a subject. A therapeutically effective amount refers to the amount of active ingredient or component that elicits a response. For example, the amount of the hydroxylase can be determined by an in vitro assay, if desired. The results can be used to help identify optimal dosage ranges. the disease to be treated or prevented, the accompanying symptoms, the patient's weight, the patient's immune status, and other factors known to those skilled in the art. The determination of the efficacy of the present invention may be made by a person skilled in the art (e.g., a clinical The exact dose to be used in the formulation can be determined by the route of administration and It depends on the severity of the disease and should be decided at the discretion of the doctor and according to the circumstances of each patient. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. It is possible.

[0070] As used herein, the terms "treat," "treating," and "treatment" both refer to the use of radioactive metal ions in the treatment of a neoplastic disease or disorder, such as at least one measurable variable associated with a disease, disorder, or condition that may benefit from administration of It is intended to refer to the improvement or restoration of a physical parameter in a subject. It is not necessarily recognizable, but it may be recognizable in the object. The terms "treat," "treating," and "treatment" are used interchangeably. It also causes regression of, prevents the progression of, or at least reduces the severity of, a disease, disorder, or condition. In certain embodiments, "treat" may refer to slowing the progression of a disease. "Treating" and "treatment" refer to treatment of a neoplastic disease or disorder, etc. a disease, disorder, or condition that may benefit from the administration of radioactive metal ions; It refers to the alleviation, prevention of the onset or progression of two or more symptoms, or the shortening of their duration. In certain embodiments, "treat," "treating," and "treatment" refer to the treatment of a disease, disorder, or condition. In specific embodiments, the terms "treat," "treat," and "cure" are used interchangeably. "Treatment" refers to improving the survival rate of a subject with a disease, disorder, or condition. "Treat," "treat," and "treatment" refer to the treatment of a disease, disorder, or condition in a subject. refers to the disappearance of

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

[0072] As used herein, the term "subject" refers to an animal, preferably a mammal. According to specific embodiments, the subject is a non-primate (e.g., camel, donkey, zebra, cow, , pigs, horses, goats, sheep, cats, dogs, rats, rabbits, guinea pigs, marmosets or mouse), or mammals, including primates (e.g., monkeys, chimpanzees, or humans). In a specific embodiment, the subject is a human.

[0073] Any dosing schedule for the modified polypeptide and radioconjugate may be used in light of this disclosure. Generally, the modified polypeptide and the radioconjugate are administered in different compositions. If so, the radioconjugate can be administered any time after the modified antibody is administered.

[0074] According to a particular embodiment, the composition for use in the treatment of a neoplastic disease or disorder is It may be used in combination with other agents effective in treating the neoplastic disease or disorder. do.

[0075] As used herein, the term "combined" refers to the administration of two or more therapeutic agents to a subject. In the context of the present invention, the use of more than one therapeutic agent is referred to. The order in which the therapeutic agents are administered to a subject is not limited. For example, the first therapeutic agent (e.g., a therapeutic agent described herein) may be administered first. The composition to be administered may be administered at least 5 minutes, 15 minutes, 30 minutes, 4 hours, or more prior to administration of the second therapeutic agent to the subject. 5 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 7 2 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 1 2 weeks before), at the same time, 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 later) It is possible.

[0076] In another general aspect, the invention relates to radiolabeled antibodies prepared by the methods of the invention. and a pharmaceutically acceptable carrier, wherein the immunological properties of the radiolabeled antibody are preserved. , relating to theranostic agents.

[0077] As used herein, the term "theranostic" refers to both diagnostic and therapeutic functions. In one embodiment, theranostic agents are both diagnostic and therapeutic. In another embodiment, the theranostic agent is an active agent without a diagnostic function. In yet another embodiment, theranostic agents are useful for diagnosis but have no therapeutic function. It is a drug that does not have any side effects.

[0078] According to a preferred embodiment, the radioactive metal ion is a diagnostic emitter, preferably 89 Zr According to another preferred embodiment, the radioactive metal ions are therapeutic emitters, preferably teeth 225 According to a preferred embodiment, the theranostic agent is a diagnostic and therapeutic agent. It is used to provide both functions to those who need them.

[0079] Combinations and Kits Provided herein are combinations including: a. A modified polypeptide comprising a polypeptide covalently linked to a first click reaction partner. Do and, b. A radioactive complex comprising a radioactive metal ion associated with a chelating moiety, a radioactive complex wherein the chelating moiety comprises a chelator covalently attached to a second click reaction partner; and a combination or kit comprising: wherein the combination is used to label a polypeptide with a radioactive metal ion. .

[0080] According to a particular embodiment, the combination of the present invention comprises the step of activating a polypeptide with a radioactive metal ion. According to another embodiment, the combination is a reaction mixture used to label the n used to generate radiolabeled polypeptides in vitro or in vivo Optionally, a pack or kit for the manufacture, use or sale of a pharmaceutical or biological product. The combination shall be accompanied by warnings or instructions in a form specified by a government agency regulating sales. This notice may be used for the manufacture, use, or sale of any product for human administration. The combinations encompassed herein reflect the approval by the Agency of Science and Technology of the United States of America. with a radioactive metal ion, or for treating a neoplastic disease or disorder It can be used in a method for treating a neoplastic disease or disorder in a subject.

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

[0082] Embodiment 1 is a method for labeling a polypeptide with a radioactive metal ion, comprising: a. A modified polypeptide comprising a polypeptide covalently linked to a first click reaction partner. and b. providing a radioactive complex comprising a radioactive metal ion associated with a chelating moiety; wherein the chelating moiety comprises a chelator covalently attached to a second click reaction partner. Including, c. The first click reaction partner reacts with the second click reaction partner. Thus, the modified polypeptide can be labeled with a radioactive metal ion under conditions that allow the polypeptide to be labeled with a radioactive metal ion. and contacting the polypeptide with a radioactive complex.

[0083] Embodiment 1a is the method of embodiment 1, wherein the chelating agent comprises a macrocycle.

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

[0085] Embodiment 2 is a method in which one of the first and second click reaction partners comprises an alkyne group, and the click 2. The method of embodiment 1, wherein the other of the Lick reaction partners comprises an azide.

[0086] Embodiment 3 is a method for preparing a first Click reaction partner comprising an azide group and a second Click reaction partner comprising an azide group. 3. The method of embodiment 2, wherein the partner comprises an alkyne group.

[0087] Embodiment 3a is the method of embodiment 2 or 3, wherein the alkyne group comprises a terminal alkyne. be.

[0088] Embodiment 3b is an embodiment in which the alkyne group is a cyclic alkyne, preferably cyclooctyne or cyclooctyne. 4. The method of embodiment 2 or 3, comprising an octyne derivative.

[0089] Embodiment 3c is an embodiment in which the alkyne group comprises bicyclononyne (BCN). The method according to embodiment 3b.

[0090] Embodiment 3d is an embodiment in which the alkyne group comprises a difluorinated cyclooctyne (DIFO). The method according to embodiment 3b.

[0091] Embodiment 3e is an embodiment in which the alkyne group comprises dibenzocyclooctyne (DIBO). 3b.

[0092] Embodiment 3f is an embodiment in which the alkyne group comprises a biarylazacyclooctyne (BARAC). , the method according to embodiment 3b.

[0093] Embodiment 3g is an embodiment in which the alkyne group comprises dibenzoazacyclooctyne (DIBAC). The method according to embodiment 3b.

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

[0095] Embodiment 3i is an embodiment in which the alkyne group comprises dibenzocyclooctyne (DBCO). 3b.

[0096] Embodiment 3j is an embodiment in which the alkyne group comprises difluorobenzocyclooctyne (DIFBO). , the method according to embodiment 3b.

[0097] Embodiment 3k is an embodiment in which the alkyne group comprises monobenzocyclooctyne (MOBO). The method according to embodiment 3b.

[0098] Embodiment 31 is an embodiment in which the alkyne group comprises tetramethoxyDIBO (TMDIBO). The method according to embodiment 3b.

[0099] Embodiment 3m is any one of embodiments 2-3l, wherein the azide group comprises NHS-azide. This is the method described above.

[0100] Embodiment 4 is a method for preparing a click reaction in which one of the first and second click reaction partners comprises an alkene group. 2. The method of embodiment 1, wherein the other of the Rick reaction partners comprises a diene.

[0101] Embodiment 4a is the compound of embodiment 4, wherein the diene comprises a tetrazine or tetrazole group. It is a method.

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

[0103] Embodiment 4c is an embodiment in which the alkene group comprises trans-cyclooctene (TCO). 4 or 4a.

[0104] Embodiment 5 is an embodiment in which the polypeptide is an antibody or an antigen-binding fragment thereof. The method according to any one of 1 to 4c.

[0105] Embodiment 6 is an embodiment in which the antibody is a monoclonal antibody or an antigen-binding fragment thereof. The method according to embodiment 5.

[0106] Embodiment 6a is a modified polypeptide in which one or more azide groups are randomly added to the polypeptide. 7. The method according to any one of embodiments 1 to 6, wherein the compound is obtained by conjugating the compound to do.

[0107] Embodiment 6b is a method for preparing a modified polypeptide comprising the steps of: 6. A modified antibody or antigen-binding fragment thereof obtained by incorporating 10. The method according to claim 9, wherein

[0108] Embodiment 6c is a cross-sectional view of a modified antibody or antigen-binding fragment thereof, wherein the modified antibody or antigen-binding fragment thereof is an Fc-glycosyltransferase of the antibody. Bacteria specific for β-1,4 linkages between core GlcNac residue(s) at the glycosylation site Trimming the antibody or antigen-binding fragment thereof with endoglycosidase obtaining a glycosyltransferase, preferably a glycosyltransferase, or an antigen-binding fragment thereof; Trimmed antibody or its antigen in the presence of alT galactosyltransferase The linked fragment is reacted with an azido sugar, preferably a UDP-GalNaz azido sugar substrate. 6b. The method of embodiment 6b, wherein the method is obtained by:

[0109] Embodiment 6d is a modified antibody or antigen-binding fragment thereof, The fragment is deglycosylated with amidase to produce a deglycosylated antibody or its antigen-binding fragment. obtaining a deglycosylated antibody or antigen-binding fragment thereof, and infecting the microorganism In the presence of a transglutaminase, an azidoamine, preferably 3-azidopropylamine, is reacted with 6b. The method of embodiment 6b, wherein the compound is obtained by reacting with an amine.

[0110] Embodiment 6e is an embodiment in which the antibody binds to human prostate-specific membrane antigen (PSMA) or its antigen-binding fragment. Preferably, the antibody binds to the HC CDR1 fragment of the sequence of SEQ ID NO: 3. Sequence, HC CDR2 sequence of SEQ ID NO: 4, HC CDR3 sequence of SEQ ID NO: 5, SEQ ID NO: 6 a light chain (LC) CDR1 sequence of SEQ ID NO: 7, a LC CDR2 sequence of SEQ ID NO: 8, and a light chain (LC) CDR1 sequence of SEQ ID NO: 9. The method according to any one of embodiments 6 to 6d, comprising a CDR3 sequence.

[0111] Embodiment 6f is the antibody comprising the HC sequence of SEQ ID NO: 9 and the LC sequence of SEQ ID NO: 10. The method of embodiment 6e.

[0112] Embodiment 7 is a method for treating a radioactive metal ion comprising: 32 P,47 Sc, 67 Cu, 77 As, 89 S r, 90 Y, 99 Tc, 105 Rh, 109 Pd, 111 Ag, 131 I, 153 Sm, 159 Gd, 165 Dy, 166 Ho, 169 Er, 177 Lu, 186 Re, 188 R e. 194 Ir, 198 Au, 199 Au, 211 At, 212 Pb, 212 Bi, 21 3 Bi, 223 Ra, 225 Ac, 255 Fm, 227 Th, 62 Cu, 64 Cu, 67 Ga, 68 Ga, 86 Y, 89 Zr, or 111 In any one of embodiments 1 to 6d. One method is as follows.

[0113] Embodiment 7a is a radioactive metal ion 225 Any of embodiments 1-6d, wherein Ac is This is one method.

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

[0115] Embodiment 7c is a radioactive metal ion 89Any one of embodiments 1 to 6d, wherein Zr is The method is as follows.

[0116] Embodiment 8 is a method for preparing a chelating moiety, wherein the chelating moiety is connected to a second click reaction partner via a linker. The method of any one of embodiments 1 to 7c, wherein the covalent bond is

[0117] Embodiment 9 is directed to a hydroxyl group having a sulfhydryl group covalently attached to a first click reaction partner and a side An electrophile on the chain, preferably a linker on or introduced into the polypeptide and reacting the amino side chain of the azide to give a modified polypeptide, preferably an NHS-azide. 9. The method of any one of embodiments 1 to 8, further comprising:

[0118] Embodiment 10 is a modified polypeptide comprising, directly or via a linker, an azide, a tetrazide, 10. Any of embodiments 1-9, comprising a polypeptide covalently bound to a tetrazole or tetrazole group. This is one method.

[0119] Embodiment 11 is a method in which the chelating agent is a macrocycle, preferably a molecule of formula (I):

[0120] [ka] Structure (Wherein R1, R2, R3, and R4 are each independently CHQCO2X; Q is independently hydrogen, C1-C4 alkyl, or (C1-C2 alkyl)phenyl. the law of nature, X is independently hydrogen, benzyl, or C1-C4 alkyl; Z is (CH2) n Y, n is 1 to 10, Y is an electrophilic or nucleophilic moiety covalently attached to a second Click reaction partner; Alternatively, Z is hydrogen; and R1, R2, R3, and R4 are each independently CHQCO2X; Q is independently hydrogen, C1-C4 alkyl, or (C1-C2 alkyl)phenyl. the law of nature, X is independently hydrogen, benzyl, C1-C4 alkyl, or a second click reaction part. an electrophilic or nucleophilic moiety covalently bonded to the toner The method according to any one of embodiments 1 to 10, comprising:

[0121] Embodiment 12 is a method for treating a chelating moiety comprising administering to a patient a compound of formula (II):

[0122] [ka] 12. The method according to any one of embodiments 1 to 11, comprising the structure:

[0123] Embodiment 12a is a preferred embodiment in which the chelating moiety comprises a chelator having an open-chain ligand. Preferably, the chelating moiety has the formula (III):

[0124] [ka] 12. The method according to any one of embodiments 1 to 11, comprising the structure:

[0125] Embodiment 12b is an embodiment in which the chelating moiety is 1,4,7,10-tetraazacyclododecane -N,N',N'',N''''-tetraacetic acid (DOTA), deferoxamine (DFO), 1 ,4,7,10,13,16-Hexaazacyclohexadecane-N,N',N'',N' ',N'',N'''-Hexaacetic acid (HEHA), 1,4,7,10,13- Pentaazacyclopentanadecane-N,N',N'',N''',N''''-pentaaza Acid (PEPA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid ( DTPA), Macropa(Thiele et al., An Eighteen- Membered Macrocyclic Ligand for Actinium -225 Targeted Alpha Therapy.Angew Chem I nt Ed Engl.2017 Nov 13;56(46):p.14712-14 717), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid Acid (TETA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetramethyl Dodecanoic acid (DOTPA), 1,4,8,11-tetraazacyclotetradecane 1,4,8,11-tetrapropionic acid (TETPA) and 1,4,7,10-tetrapropionic acid From azacyclododecane-1,4,7,10-tetramethylenephosphonic acid (DOTMP) 11. The method of any one of embodiments 1 to 10, comprising a chelating agent selected from the group consisting of: is.

[0126] Embodiment 12c is an embodiment in which the chelating agent is 1,4,7,10-tetraazacyclododecane-N ,N',N'',N'''-tetraacetic acid (DOTA). be.

[0127] Embodiment 12d is a method for treating a chelating agent comprising: This is the method described above.

[0128] Embodiment 13 is directed to a radioactive metal ion, preferably 225 Ac, 111 In or 89 Zr Method for labeling a polypeptide, preferably an antibody or an antigen-binding fragment thereof, using And, a. A polypeptide or antibody covalently bound to an azide, tetrazine, or tetrazole group or a modified polypeptide, preferably a modified antibody, comprising an antigen-binding fragment thereof; providing an antigen-binding fragment thereof; b. a radioactive metal ion, preferably associated with a chelating moiety 225 Ac, 111 In or 89 providing a Zr-containing radiocomplex, wherein the chelating moiety is an alkyne; or a chelating agent covalently bonded to the alkene group; and c. Reaction of an azide, tetrazine, or tetrazole group with an alkyne or alkene group and reacting the polypeptide or antibody or antigen-binding fragment thereof with a radioactive metal ion, Preferably 225 Ac, 111 In, or 89 Under conditions that allow labeling with Zr contacting the modified polypeptide or antibody or antigen-binding fragment thereof with a radioactive complex; and, The chelating agent has the formula (I):

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

[0130] Embodiment 13a is an embodiment in which the chelating agent is 1,4,7,10-tetraazacyclododecane-N 14. The method of embodiment 13, comprising: do.

[0131] Embodiment 13b is directed to a compound having a chelating moiety of formula (II):

[0132] [ka] 14. The method of embodiment 13, comprising the structure:

[0133] Embodiment 13d is directed to a radioactive metal ion, preferably 225 Ac, 111 In or 89 Z r to label a polypeptide, preferably an antibody or an antigen-binding fragment thereof. It is a law, a. A polypeptide or antibody covalently bound to an azide, tetrazine, or tetrazole group or a modified polypeptide, preferably a modified antibody, comprising an antigen-binding fragment thereof; providing an antigen-binding fragment thereof; b. a radioactive metal ion, preferably associated with a chelating moiety 225 Ac, 111 In or 89 providing a Zr-containing radiocomplex, wherein the chelating moiety is an alkyne; or a chelating agent covalently bonded to the alkene group; and c. Reaction of an azide, tetrazine, or tetrazole group with an alkyne or alkene group and reacting the polypeptide or antibody or antigen-binding fragment thereof with a radioactive metal ion, Preferably 225 Ac, 111 In, or 89 Under conditions that allow labeling with Zr contacting the modified polypeptide or antibody or antigen-binding fragment thereof with a radioactive complex; and, wherein the chelating agent comprises an open-chain ligand, preferably deferoxamine (DFO). , method.

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

[0135] Embodiment 15 is directed to a sulfhydryl group covalently attached to an azide, preferably an NHS-azide. and an electrophile on a side chain, preferably on a polypeptide or polypeptide, preferably The amino side chain of lysine introduced into the antibody or antigen-binding fragment thereof is reacted, obtaining the modified polypeptide, or the antibody or antigen-binding fragment thereof, The method according to any one of embodiments 13 to 14.

[0136] Embodiment 16 is a method for treating a leukemia, in which the polypeptide, preferably an antibody or antigen-binding fragment thereof, is 16. The method of any one of embodiments 13 to 15, wherein the azide is covalently attached to the azide via a linker. It is a method.

[0137] Embodiment 17 is a method for treating a cancer, comprising administering to a subject therapies ... the antibodies or antigen-binding fragments thereof, and The metal ions are 225 Ac, 111 In or 89 Zr, and the chelating moiety is of formula (II ):

[0138] [ka] 14. The method of embodiment 13, comprising the structure:

[0139] Embodiment 17a is a method in which the polypeptide is an antibody or antigen-binding fragment thereof and The reactive metal ion is 225 Ac, 111 In or 89 Zr, and the chelating moiety is of the formula (I II):

[0140] [ka] The method of embodiment 13c, comprising the structure:

[0141] Embodiment 17b is a method for treating a prostate cancer, in which the polypeptide is human prostate-specific membrane antigen (PSMA) or an antibody thereof. an antibody that binds to the original binding fragment, preferably the antibody is an antibody having the HC of the sequence SEQ ID NO: 3 CDR1 sequence, HC CDR2 sequence of SEQ ID NO: 4, HC CDR3 sequence of SEQ ID NO: 5, The light chain (LC) CDR1 sequence of SEQ ID NO: 6, the LC CDR2 sequence of SEQ ID NO: 7, and The method of any one of embodiments 13 to 17a, comprising the LC CDR3 sequence of No. 8.

[0142] Embodiment 17c is a method for producing an antibody comprising the HC sequence of SEQ ID NO: 9 and the LC sequence of SEQ ID NO: 10. , the method according to embodiment 17b.

[0143] Embodiment 18 is a method for dual-labeling a polypeptide with two radioactive metal ions. hand, a. Covalently bonded to a first click reaction partner and a second click reaction partner providing a modified polypeptide comprising a polypeptide; b. providing a first radioactive complex comprising a first radioactive metal ion associated with a chelating moiety; wherein the chelating moiety is covalently attached to a third click reaction partner. Contains a chelating agent, c. providing a second radioactive complex comprising a second radioactive metal ion associated with a chelating moiety. wherein the chelating moiety is a chelating moiety covalently attached to a fourth click reaction partner. Contains a rate agent, d. The first click reaction partner reacts with a third click reaction partner to form a second The click reaction partner reacts with a fourth click reaction partner to form a poly Modification under conditions that allow the peptide to be labeled with a first and second radioactive metal ion. contacting the polypeptide with first and second radioactive complexes.

[0144] Embodiment 19 is directed to a method for preparing a hydroxyl group comprising the steps of: The other of the first and second click reaction partners contains an azide, and the third and fourth click One of the reaction partners contains an alkene group, and the other of the third and fourth click reaction partners 19. The method of embodiment 18, wherein the catalyst comprises a diene.

[0145] Embodiment 20 is a method in which the first or second radioactive metal ion is a diagnostic emitter and the other is a therapeutic 20. The method of embodiment 18 or 19, wherein the emitter is an emitter.

[0146] Embodiment 21 is an embodiment in which both the first and second radioactive metal ions are therapeutic emitters. 20. The method according to aspect 18 or 19.

[0147] Embodiment 21a is a further embodiment in which the diagnostic emitter comprises: 62 Cu, 64 Cu, 67 Ga, 68 Ga, 86 Y, 89 Zr, or 111 22. The method of embodiment 20 or 21, wherein In is In.

[0148] Embodiment 21b is an embodiment in which the treatment emitter comprises: 32 P, 47 Sc, 67 Cu, 77 As, 89 S r, 90 Y, 99 Tc, 105 Rh, 109 Pd, 111 Ag, 131 I, 153 Sm, 159 Gd, 165 Dy, 166 Ho, 169 Er, 177 Lu, 186 Re, 188 R e. 194 Ir, 198 Au, 199 Au, 211 At, 212 Pb, 212 Bi, 21 3 Bi, 223 Ra, 225 Ac, 255 Fm, or 227 Embodiments 20 to 24 are 21a.

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

[0150] Embodiment 23 is directed to a method for treating or diagnosing a disease or disorder, particularly a neoplastic disease or disorder, in need of treatment or diagnosis. The present invention relates to a method for treating or diagnosing a disease or disorder, particularly a neoplastic disease or disorder, in a subject. 23. A method comprising administering the composition of embodiment 22 to a subject.

[0151] Embodiment 24 is a pharmaceutical composition comprising two compositions administered sequentially, the first composition being 23. The first comprises a modified polypeptide and the second comprises a radioactive complex(es). This is the method described above.

[0152] Embodiment 25 is prepared by the method of any one of embodiments 1 to 21b. a radiolabeled antibody and a pharmaceutically acceptable carrier, and It is a conserved theranostic agent.

[0153] Embodiment 26 is directed to a method in which the radioactive metal ion is a diagnostic emitter, preferably 89 Zr, Theranostic agent according to embodiment 25.

[0154] Embodiment 27 is directed to a method in which a radioactive metal ion is a therapeutic emitter, preferably 225 Ac, The theranostic agent according to embodiment 25.

[0155] Embodiment 27a is a radioactive metal ion 111 26. The method of claim 25, wherein the It is a gnostic agent.

[0156] Embodiment 27b is a method for treating a psoriasis in which the polypeptide is human prostate-specific membrane antigen (PSMA) or an antibody thereof. an antibody that binds to the original binding fragment, preferably the antibody is an antibody having the HC of the sequence SEQ ID NO: 3 CDR1 sequence, HC CDR2 sequence of SEQ ID NO: 4, HC CDR3 sequence of SEQ ID NO: 5, The light chain (LC) CDR1 sequence of SEQ ID NO: 6, the LC CDR2 sequence of SEQ ID NO: 7, and 27a. The serranolide of any one of embodiments 25 to 27a, comprising the LC CDR3 sequence of No. 8. It is a stick formulation.

[0157] Embodiment 27c is a method in which the theranostic agent comprises the HC sequence of SEQ ID NO: 9 and the HC sequence of SEQ ID NO: 10 27b. The method of embodiment 27b, comprising an LC sequence.

[0158] Embodiment 27d is a radiolabeled antibody having Formula (IV):

[0159] [ka] or having the formula (IV) (DOTA-Ac-DBCO-protein) 22 5 Ac, 32 P, 47 Sc, 67 Cu, 77 As, 89 Sr, 90 Y, 99 Tc, 10 5 Rh, 109 Pd, 111 Ag, 131 I, 153 Sm, 159 Gd, 165 Dy, 1 66 Ho, 169 Er, 177 Lu, 186 Re, 188 Re, 194 Ir, 198 Au , 199 Au, 211 At, 212 Pb, 212 Bi, 213 Bi,223 Ra, 255 Fm, 227 Th, 62 Cu, 64 Cu, 67 Ga, 68 Ga, 86 Y, 89 Zr, or 111 Any of embodiments 25 to 27c, wherein the radioactive metal ion is substituted with another radioactive metal ion, such as In. The theranostic agent is one described in any one of the above.

[0160] Embodiment 27e is an embodiment in which the radiolabeled antibody has formula (V):

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

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

[0163] [ka] or having the formula (VI) (DFO-Zr DBCO-protein) 89 Z r is, 32 P, 47 Sc, 67 Cu, 77 As, 89 Sr, 90 Y, 99 Tc, 105 R h, 109 Pd, 111 Ag, 131 I, 153 Sm, 159 Gd, 165 Dy, 166 Ho, 169 Er,177 Lu, 186 Re, 188 Re, 194 Ir, 198 Au, 1 99 Au, 211 At, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 225 Ac , 255 Fm, 227 Th, 62 Cu, 64 Cu, 67 Ga, 68 Ga, 86 Y, or 1 11 Any of embodiments 25-27c, wherein the compound is substituted with another radioactive metal ion, such as In. or a theranostic agent described in one of the above.

[0164] Embodiment 27g is a radiolabeled antibody having Formula (VII):

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

[0166] Embodiment 27h is a method for treating a rhodopsin-related ... The theranostic agent according to any one of embodiments 25 to 27g.

[0167] Embodiment 27i is an embodiment in which the radiolabeled antibody has a chelator:antibody ratio (CAR) of 2. The theranostic agent according to any one of Forms 25 to 27h.

[0168] Embodiment 28 is a. A modified polypeptide comprising a polypeptide covalently linked to a first click reaction partner. Do and, b. A radioactive complex comprising a radioactive metal ion associated with a chelating moiety, a radioactive complex wherein the chelating moiety comprises a chelator covalently attached to a second click reaction partner; and a combination or preferably a kit comprising: wherein the combination is used to label a polypeptide with a radioactive metal ion. .

[0169] Embodiment 28a is the combination or chelating agent of embodiment 28, wherein the chelating agent comprises a macrocycle. It is a .

[0170] Embodiment 28b is a combination of embodiment 28, wherein the chelating agent comprises an open-chain ligand. That is it.

[0171] Embodiment 29 is directed to a method for preparing a reaction between a first and a second click reaction partner in vitro. 28. The method of claim 27, wherein the polypeptide is used to label the polypeptide with a radioactive metal ion via The combination or kit according to claim 1.

[0172] Embodiment 30 is directed to a method for determining the reaction between the first and second click reaction partners in vivo. 29. The method of claim 28, wherein the method is used to label a polypeptide with a radioactive metal ion via The combination or kit described herein.

[0173] Embodiment 31 is a modified antibody comprising a polypeptide covalently linked to a first click reaction partner. A composition comprising a decorative polypeptide.

[0174] Embodiment 32 includes a radioactive complex comprising a radioactive metal ion associated with a chelating moiety. The composition, wherein the chelating moiety is a chelator covalently attached to a second click reaction partner. The composition includes a stimulating agent.

[0175] Embodiment 32a is the composition of embodiment 32, wherein the chelating agent comprises a macrocycle. do.

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

[0177] Embodiment 33 is an embodiment in which the polypeptide is an antibody or an antigen-binding fragment thereof. The combination or kit according to any one of embodiments 28 to 30, or embodiment 31 or The composition is preferably the composition described in 32.

[0178] Embodiment 33a is a method for treating a leukemia in which the antibody binds to human prostate-specific membrane antigen (PSMA) or an antigen-binding fragment thereof. Preferably the antibody is capable of binding to the HC CDR fragment of the sequence of SEQ ID NO: 3. 1 sequence, HC CDR2 sequence of SEQ ID NO: 4, HC CDR3 sequence of SEQ ID NO: 5, SEQ ID NO: 6, the LC CDR1 sequence of SEQ ID NO: 7, and the L CDR2 sequence of SEQ ID NO: 8. 34. The method of embodiment 33, comprising a C CDR3 sequence.

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

[0180] Embodiment 33c is an embodiment in which the antibody or antigen-binding fragment thereof is covalently linked to an azide group. The combination or kit or composition according to embodiment 33a or 33b,

[0181] Embodiment 33d is an embodiment in which the antibody or antigen-binding fragment thereof is covalently linked randomly to the azide group. The combination or kit or composition of embodiment 33c is linked.

[0182] Embodiment 33e is an embodiment in which the antibody or antigen-binding fragment thereof is site-specifically conjugated to an azide group. The combination or kit or composition of embodiment 33c is covalently linked.

[0183] Embodiment 33f is a method for treating an Fc-glycosylated antibody or antigen-binding fragment thereof. Bacterial enzyme specific for β-1,4 linkages between core GlcNAc residue(s) at the site Trimming the antibody or antigen-binding fragment thereof with isoglycosidase and obtaining an antibody or antigen-binding fragment thereof, and In the presence of 1T galactosyltransferase, the trimmed antibody or its antigen-binding The resulting fragment is reacted with an azido sugar, preferably a UDP-GalNaz azido sugar substrate. The combination of embodiment 33e, wherein the azido group is covalently attached via a method comprising: It is a combination, kit or composition.

[0184] Embodiment 33g is a method for treating a leukemia, encephalopathy, or encephalopathy syndrome, in which the modified antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof. The fragment is deglycosylated with an amidase to produce a deglycosylated antibody or its antigen-binding fragment. and obtaining a deglycosylated antibody or antigen-binding fragment thereof. Azidoamine, preferably 3-azidopropyl amine, is reacted with azidoamine in the presence of biological transglutaminase. a combination according to embodiment 33e, obtained by a process comprising reacting Or a kit or composition.

[0185] Embodiment 34 is an embodiment in which the radioactive metal ion is 225 Ac, 111 In or 89 Including Zr A combination, kit or composition according to any one of embodiments 33 to 33g.

[0186] Embodiment 35 is an embodiment wherein the chelating agent is a macrocycle, preferably of formula (I):

[0187] [ka] Structure (Wherein R1, R2, R3, and R4 are each independently CHQCO2X; Q is independently hydrogen, C1-C4 alkyl, or (C1-C2 alkyl)phenyl. the law of nature, X is independently hydrogen, benzyl, or C1-C4 alkyl; Z is (CH2) n Y, n is 1 to 10, Y is an electrophilic or nucleophilic moiety covalently attached to the alkyne group; Alternatively, Z is hydrogen; and R1, R2, R3, and R4 are each independently CHQCO2X; Q is independently hydrogen, C1-C4 alkyl, or (C1-C2 alkyl)phenyl. the law of nature, X is independently hydrogen, benzyl, C1-C4 alkyl, or covalently bonded to an alkyne group. (electrophilic or nucleophilic moiety) 35. The combination, kit or composition of embodiment 34, comprising:

[0188] Embodiment 36 is an embodiment in which the electrophilic or nucleophilic moiety is covalently attached to the alkyne group via a linker. 36. The combination, kit or composition according to embodiment 35,

[0189] Embodiment 37 is directed to a compound having a chelating moiety of formula (II):

[0190] [ka] 37. The combination, kit or composition according to embodiment 35 or 36, comprising the structure:

[0191] Embodiment 37a is a preferred embodiment in which the chelating moiety comprises a chelator having an open-chain ligand. The chelating moiety has the formula (III):

[0192] [ka] 35. The combination, kit or composition of embodiment 34, comprising the structure:

[0193] Embodiment 38 is an embodiment in which the polypeptide is covalently attached to the azide via a linker. A combination, kit or composition according to any one of Aspects 34 to 37.

[0194] Embodiment 39 is directed to a method for treating or diagnosing a disease or disorder, particularly a neoplastic disease or disorder. The method of any one of embodiments 25 to 27d, or Administering a combination according to any one of embodiments 28 and 33 to 38 to a subject. Hmm, that's the method.

[0195] Embodiment 40 is directed to treating a disease or disorder, particularly a neoplastic disease or disorder, with a and a method for treating or diagnosing a neoplastic disease or disorder in a subject in need thereof. and administering to a subject the composition of embodiment 31 and the composition of embodiment 32, preferably or a method wherein the polypeptide is an antibody. [Example]

[0196] The following examples of the present invention are intended to further illustrate the principles of the present invention. The examples do not limit the invention, the scope of which is defined by the appended claims. I would like you to understand this.

[0197] Example 1: Random conjugation of azide / handle to antibody Monoclonal antibody (mAb): Denoted "PSMB127" and herein referred to as "anti-PS Human IgG4 that binds to human prostate-specific membrane antigen (PSMA), referred to as "MA mAb" The antibody has a heavy chain (HC) CDR1 sequence of SEQ ID NO: 3, a HC CDR2 sequence of SEQ ID NO: 4, HC CDR3 sequence of SEQ ID NO: 5, light chain (LC) CDR1 sequence of SEQ ID NO: 6, SEQ ID NO: 7, and the LC CDR3 sequence of SEQ ID NO: 8, The HC sequence of SEQ ID NO: 9 and the LC sequence of SEQ ID NO: 10. It was purified using semi-chromatographic methods.

[0198] Human IgG4 S228P, an anti-PMSA mAb, referred to herein as the "control mAb" / F234A / L235A (IgG4-PAA) antibody isotype control is SEQ ID NO: 1 The commercially available antibody trastuzumab (Herceptin ), cetuximab (Erbitux), pertuzumab (Perjeta), and panitumumab (Vectibix) from Roche, Lilly, Roche, and Amgen, respectively. Mouse anti-human Her2 mAb was purchased from BioXCell (catalog no. BE02 77). Trastuzumab, pertuzumab, and anti-human Her2 mAb were obtained from human Binds to Her2. Cetuximab and panitumumab bind to human EGFR.

[0199] Complexation: 10 mM sodium acetate pH 5.2, phosphate buffered saline pH 7, or other Stock solutions of antibodies (1–10 mg / mL) in compatible buffer were diluted with 20% (wt / wt) of 1 The final pH was adjusted to approximately 9 by mixing with M sodium carbonate buffer pH 9. 4-Azide (Thermo Cat. No. 26130) was dissolved in DMSO to a final concentration of 1 00 mM and add 0.2% (wt / wt) of the stock solution to obtain approximately 3-10 A molar excess was generated. The reaction was incubated at 22°C for 10 minutes, then diluted with 1M Tris The solution was quenched by adding Tris (pH 7.5) to a final concentration of 50 mM.

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

[0201] LC / MS analysis: Chelator:antibody ratio (CAR) was determined using an Agilent PLRP-S column. 300-Angstrom, 2.1x150mm, Catalog Number PL1912-33 LC / MS analysis using an Agilent G6224 MS-TOF instrument equipped with The mass spectra were determined by m / s for masses of 140-170 kDa (Table 1). Decompression using a maximum entropy algorithm over the z range 2000-3200 Solution.

[0202] Analytical size exclusion chromatography (SEC): Analytical SEC was performed to separate the antibodies and The oligomeric state of the complex was determined, confirming that the complexation process did not result in aggregation Tosoh TSKgel G3000SWxl (Tosoh Bioscience Agilent 1200 system equipped with a 7.8 mm x 30 cm column (ce #08541) A Leeds HPLC was used. The mobile phase was 1x PBS, the flow rate was 0.8 ml / ml, and the injection volume was 15 μL. L, protein concentration 0.1–2 mg / mL.

[0203] [Table 1]

[0204] Example 2: Random conjugation of azide / handle to non-antibody polypeptides Non-antibody polypeptides: Transferrin, human holo-transferrin, R&D S Systems (catalog number 2914-HT) and dissolved in water to 10 mg / mL Human epidermal growth factor (EGF) was developed by Sino Biological (Cat. No. 10605-HNAE).

[0205] Complexation: 10 mM sodium acetate pH 5.2, phosphate buffered saline pH 7, or other Stock solutions of polypeptides (1–10 mg / mL) in compatible buffers are diluted to 20% (wt / wt) (volume) of 1M sodium carbonate buffer pH 9 to a final pH of approximately 9. PEG4-azide (Thermo Cat. No. 26130) was dissolved in DMSO to a final concentration The concentration was adjusted to 100 mM, and 0.2% (wt / wt) of the stock solution was added to obtain a concentration of approximately A molar excess of 3 to 10 was generated. The reaction was incubated at 22°C for 10 min, then diluted to 1 M The final concentration was 50 mM, followed by quenching with Tris pH 7.5. Shown in Table 2.

[0206] [Table 2]

[0207] Example 3: Site-specific incorporation of azido sugars into antibody glycans Antibody glycans are bound to core GlcNac residues within the Fc glycosylation site(s). GlycINATOR, a bacterial endoglycosidase specific for β-1,4 intergroup linkages (Genovis) and later used for site-specific incorporation of azido sugars. The innermost GlcNac was left intact on the Fc. GlycINATOR immobilized on agarose beads packed in (Genovis) Equilibrated in Tris-buffered saline pH 7.4 (TBS). 5-10 mg / mL Add 1 mL of mAb to the resin and incubate on a rocker for 1 hour at room temperature. The column was eluted by spinning at 100×g for 1 minute. The eluates containing the trimmed mAb were pooled and diluted in the supplied buffer. Additive (Genovis) was added to the UDP-GALNaz azido-sugar substrate and GalT galactosidase. The reaction mixture was stirred overnight at 30°C. A typical azidomAb was purified using a mAb-selective column (GE) on an AKTA Avant instrument. The azide modification was confirmed by LC-MS, and the CAR was determined to be exactly 2.

[0208] Example 4: Microbial transglutaminase (MTG) of 3-azidopropylamine Site-specific integration The azide group is site-specifically placed relative to the antibody at position Gln295, as described. Transglutaminase-based chemo-enzymatic conjugation approach elds homogeneous antibody-drug conjugate s.Bioconj Chem 2014 Mar 19;25(3):p.569-7 8) Anti-PSMA mAb was applied to the innermost GlcNac residue and the high mannose, hybridized The cleavage occurs between the nucleotide and the asparagine residue of complex oligosaccharides, which are conserved (e.g., Both conserved (e.g., Fc Asn297) and non-conserved (e.g., Fab N-glycans) Complete deglycosylation of all N-glycans, including those derived from glycosylation sites Rapid PNGase F (New The solution was deglycosylated using sodium acetate buffer ( 10 mL of 1 mg / mL antibody in PBS (pH 5.2) was mixed with 5 μL of PNGase F for 37 min. The mixture was incubated at 4°C overnight, and deglycosylation was confirmed by LC-MS. F was removed by four cycles of concentration and diluted with an Amicon device (50 KDa cutoff). The conjugation of 3-azidopropylamine (3-APA; click chemistry tool) For the precipitation, 2% (w / w) of 0.5M HEPES pH 7.5 was added. Deglycosylated mAb (0.5-1 mg / mL) was adjusted to pH 7-7.5. An equivalent amount of 3-APA was added to MTG, activin TI transglutaminase (Ajino The reaction was incubated at 37°C for 1 to 4 hours. After incubation, the mAbSelect Sure column was purified using standard chromatography techniques. The azide-modified mAb was purified using LC-MS. The conjugate was manually characterized by LC-MS, and CAR was found to be 2 It was determined that this was the case.

[0209] Example 5: Chelation of radiometals to bifunctional chelators (BFCs) 225 Synthesis of Ac-DOTA-Ga-DBCO: 225 Ac(NO3)3 in Oak R It was purchased from the Idge National Laboratory. Tetraazacyclododecane, 1-(glutaric acid)-4,7,10-triacetic acid-(3-amino) Dibenzocyclooctyne (DOTA-Ga-DBCO) was custom synthesized. The synthesis was carried out by Bernhard et al. Chem. Eur. J. 2012, 18 Based on DBCO-amine (3-amino-1-[(5-aza- 3,4:7,8-Dibenzocyclooct-1-yn)-5-yl]-1-propanone (S DOTA-GA (Igma) was reacted with DOTA-GA anhydride, and the product was purified by reverse-phase HPLC.

[0210] Actinium-225 quantification was performed using a Capintec CRC-55TW dose calibrator. This is achieved using data, which allows 225 Dissolve Ac(NO3)3 in 0.1N HCl The solution was dissolved to prepare a 10 mCi / mL solution. Ammonium (1 M solution, 7.5 μL, 7.5 μmol), DOTA-GA-DBCO( 1 mg / mL aqueous solution, 2.5 μL, 3.4 nmol) and NaOH (0.1 N, 2.5 μ L, 0.25 μmol) solution, 225 Ac(NO3)3 (0.1N HCl 5μL The pH of the mixture was 10 mCi / mL, 50 μCi, 0.0038 nmol. The pH was observed to be about 6.5 by pH paper. The vial was heated at 80°C and 290 rpm. The vial was allowed to cool to room temperature by placing it on a shaking block for 30 minutes.

[0211] 111 Synthesis of In-DOTA-GA-DBCO: in 0.05 M HCl 111 InC l3 was purchased from GE Healthcare. Tetraacetate in a plastic vial Methylammonium (1 M solution, 7.5 μL, 7.5 μmol), DOTA-GA-DB CO (1 mg / mL aqueous solution, 2.5 μL, 3.4 nmol) and HCl (0.1 N, 5 μL) L) in 0.05N HCl 111 InCl3 (5 μL, Capintec 104.3 μCi, 0.0022 nm measured with a CRC-55TW dose calibrator The pH of the mixture was observed to be about 5.5 by pH paper. Place the vial on a shaking block at 60 °C and 290 rpm for 30 min and allow the vial to warm to room temperature. Cooled.

[0212] 89 Synthesis of Zr-DFO-DBCO: 89 Zr oxalate from 3D Imaging DFO-DBCO was purchased from Macrocyclics, Plano, Texas, USA. (Log No. B-773), dissolved in DMSO to 0.5 mg / mL, and diluted with water to 25 Diluted to μg / mL.

[0213] Transfer 2 mCi of Zr-89 to a metal-free microcentrifuge tube and add 1 M oxalic acid. Add 12 μL of 2M potassium carbonate in 2 μL increments and mix. The mixture was stirred with a pipette tip until bubbling stopped. Then, 120 μL of 1 M HE PES was added, followed by 300 μL of water. The pH of the solution was tested and adjusted as needed. Additional 2 M potassium carbonate was added to raise the H to 6–6.5. 136 μL DFO-DBCO stock (3.4 μg) was added and the reaction was incubated at room temperature for 1 hour. 0.5 μL of the reaction was spotted onto a TLC Green strip (Biodex). After elution, most of the Zr-89 was To ensure that the PerkinElmer Cyclon Scan the strips with the e Plus phosphor imager to confirm the DFO-DBCO capsid. The rating was shown.

[0214] 89 Synthesis of Zr-DOTA-GA-DBCO: Sep-pak Light Q in water MA strong anion exchange cartridge (acrylic acid / acrylamide copolymer on diol silica) Rimmer, surface sensitivity: C(O)NH(CH2)3N(CH3)3 + Cl - , pore diameter 300Å , particle size 37-55 μm, ion exchange capacity 230 μeq / gram), MeCN (6 mL) was added, followed by 0.9% saline (10 mL) and then water (10 mL). in 0.0 M oxalic acid (2 μL, 290 μCi)89 The Zr(ox)2 solution was pre-concentrated. The cartridge was then filled with deionized water (20 ml). After washing with 1.0 M HCl (aqueous) (100 μL each) to remove excess oxalic acid, 89 ZrCl4 was eluted from the column with a recovery of 248 μCi (86%) in a total volume of 400 mL. μl was taken, with the majority of the radioactivity in fraction 3. The combined organic extracts were then evaporated to dryness. It dried and solidified.

[0215] 10 μL of DOTA-GA-DBCO (1.0 mg / mL in metal-free water, 10 μL g, 13.6 nmol) 89 ZrCl4 (268 uCi, 50 μL) was added. TA-GA-DBCO / 89 The Zr solution was diluted with 150 μL of 1.0 M HEPES. The pH of the mixture was adjusted to pH 7.5 (Pandya et al., Zirconia um tetraazamacrocycle complexes display extraordinary stability and provide a ne w strategy for zirconium-89-based radiop pharmaceutical development.Chem Sci.2017 Mar 1;8(3):pp.2309-2314). The solution was then incubated at 90°C for 60 minutes. Incubated. 89 The yield of Zr-DOTA-GA-DBCO complex was 1% NH4OH SPC25 column (Sigma Aldrich part number SPC251) eluted with 98% determined by 20-50G). Not chelated 89 Zr is on the column It remains89 The Zr-DOTA-GA-DBCO complex is eluted.

[0216] Example 6: Synthesis of click-labeled radioconjugates of anti-PSMA mAbs See Figures 1 and 2 for a schematic diagram of radiolabeled antibodies according to the methods of the present invention. .

[0217] Anti-PSMA mAb- in PBS or other compatible buffer (10-20 mg / mL) Dibenzo-[1,2,3]-triazoloazocine-Ga-DOTA-225Ac (site specific Random or site-specific azide-modified antibody (site-specific, CAR=2) or random, mean CAR of 1–4) were generated as described 225 Ac-DO The final pH of the mixture was approximately 6.5 by pH paper. The reaction solution was gently stirred and 15 mL of NaOAc buffer, 10 mM, pH 6. PD-10 column preconditioned with ~6.5 or another compatible buffer The reaction mixture was left at room temperature for 3 hours before being purified by HPLC (GE Healthcare). Pipette the eluate into the reservoir of a preconditioned PD-10 column. The reaction vial was washed with NaOAc buffer (0.2 ml). The washing solution was pipetted into the reservoir of the Pd-10 column, and the eluate was collected. The NaOAc buffer was applied sequentially to the reservoir of the PD-10 column, and the eluate was then added to the plate. Collect the eluate in stick tubes, adding approximately 1 mL of eluate to each tube until a total of 10 mL of eluate has been collected. It was recovered.

[0218] The purity of each collected fraction was confirmed by using a citrate-HO-MeOH solution as the mobile phase. The pure fraction(s) were evaluated by iTLC-SG (Agilent). The resulting mixture was combined to give the final product in 10 mM NaOAc buffer. The product was analyzed for chemical and radiochemical purity by HPLC. The antibody concentration in the sample solution was measured by UV absorption. The radioactivity of the product solution was quantified using a 5TW dose calibrator.

[0219] Quality control of AC-225 chelation: Diethylenetriamine was used as a quality control for the purified product. A chelation challenge using diamine pentaacetic acid (DTPA) was used. DTPA aqueous solution, 225 Add the Ac-labeled mAb to the sample solution and A / mAb = 500-1,000. A 50 mM Na5DTPA solution was 2 25 To an aliquot of the purified product in solution containing Ac-labeled mAb, [DPTA The two mixtures were incubated at room temperature and 29 The mixture was placed on a shaking block at 0 rpm for 30 minutes. The mixture was spotted onto iTLC-SG. Citrate-H2O-MeOH was used as the mobile phase. 225 Ac moves to the solvent front and binds 225 Ac-mAb remains at baseline.

[0220] Anti-PSMA mAb-dibenzo-[1,2,3]-triazoloazocine-GA-DOT A- 111 Synthesis of In: Random or site-specific azide-modified anti- PSMA mAb (site-specific, CAR=2 or random, mean CAR of 1-4) was recorded. Generated as described 111 Ac-DOTA-GA-DBCO solution was added. The reaction solution was gently stirred and allowed to stand at room temperature for 2 hours before being passed through a PD-10 column. The PD-10 column was loaded with 15 mL of NaOAc buffer, 10 mM, pH 6. The reaction mixture was then preconditioned by passing it through 6.5°C, and the washings were discarded. The mixture was pipetted into the reservoir of a preconditioned PD-10 column and dissolved. The exudate was collected in a plastic tube. The reaction vial was washed with NaOAc buffer (0. (2 mL x 3), pipette the wash solution into the reservoir of the PD-10 column and collect the eluate. The NaOAc buffer was applied sequentially to the reservoir of the PD-10 column, and the eluate was Collect the eluate into plastic tubes and add approximately 1 mL of eluate to each tube until a total of 10 mL of eluate has been collected. The liquid was collected.

[0221] The purity of each collected fraction was confirmed by elution with 10 mM EDTA aqueous solution (pH 5-6) as the mobile phase. The pure fraction(s) were combined and evaluated by iTLC-SG using The final product was obtained in 10 mM NaOAc buffer. The product solution was subjected to chemical and radiochemical analysis. The product solution was analyzed for its radiochemical purity by HPLC. A standard curve was used to determine the level of the antibody in the product solution. The concentration of thrombin was measured by UV absorption. The radioactivity of the product solution was quantified using a calibrator.

[0222] Quality control of In-111 chelation: Use of DTPA as a quality control for purified products. A chelation challenge was used: 10 mM Na5DTPA in water, 111 In mark The DTPA / mAb ratio was increased to 1000. The mixture was placed on a shaking block at room temperature and 290 rpm for 30 minutes. The mixture was spotted on iTLC-SG and washed with 10 mM EDTA aqueous solution (pH = 5-6). ) was used as the mobile phase. 111 In or loosely bound 111 moves to the solvent front Moving and tightly coupled 111 In-mAb remains at baseline.

[0223] Anti-PSMA mAb-dibenzo-[1,2,3]-triazoloazocine-DFO-ZR Synthesis of -89 10mM HEPES 50mM NaCl pH 7.5, or other compatible buffer The average CAR of random azide-modified anti-PSMA mAb (1-4) in the same medium was determined by the same procedure for site specificity. 800 μg of mAb (which can be performed with a specific azido-mAb) was prepared as described. accomplished 89 Add Zr-DFO-DBCO solution and incubate at 37 °C for 1.5 hours. The PD-10 column was then washed with 15 mL of isotonic saline. The reaction mixture was then added to the column and the wash was discarded. The solution was pipetted into the reservoir of a PD-10 column, and the eluate was collected in a plastic tube. Saline was continuously applied to the reservoir of the PD-10 column, and the eluate was collected in a plastic tube. Collect approximately 0.5 mL of eluate in each tube until a total of 10 mL of eluate has been collected. The radioactivity of each fraction and of the material remaining on the column was measured using a dose calibrator. The product peak, usually fractions 4-7, was pooled to obtain the final product. The product solution was analyzed for radiochemical purity by HPLC. The antibody concentration in the product solution was measured by UV absorption. The radioactivity was quantified.

[0224] Anti-PMSA mAb-DOTA- 89 Synthesis of Zr 20mMHEPES 50mM NaClpH7.5 (10.1mg / mL, 200μ Azide-modified anti-PS modified by random azide conjugation in 1000 kJ / L (approximately 13.5 nmol) MA mAb conjugates (CAR = 1–4) were generated as described above. 89 Zr-DOTA- The reaction solution was added to the GA-DBCO solution. The final pH of the mixture was adjusted to 7.0. Incubate at 7°C for 2 hours, followed by 0.9% saline, HEPES buffer, or The 6-well plate was purified on a PD-10 column (GE Healthcare) in either PBS or PBS. 4% product 89 Zr-DOTA-mAb was obtained.

[0225] Quality control of Zr-89 chelation: For Zr-DFO-mAb, the purified product These conjugates were analyzed by HPLC only. When Zr-DOTA-mAb was added to 33 mM EDTA, Zr-89 was not retained. The challenge was performed with the addition of 1000 mg of ATP and incubated overnight at room temperature. Analysis of the complex after challenge with HCl revealed that it retained 80% of its radioactivity. Ta.

[0226] Example 7: Analytical characterization of click-labeled radioconjugates Determination of radiation chemical transformation Radiochemical conversion (% RA conversion; see Tables 3-5) was measured by iTLC-SG (silica gel electrophoresis). A binderless glass microfiber chromatography paper impregnated with SG was used. The %RA conversion values ​​were determined by simplified thin layer chromatography (iTLC) using The product of the radioactive signals of the product peaks (of different retention times of the radioactive starting material and by-products) The minute value is calculated by integrating all the radioactive signal peaks present between the baseline and the solvent front. The ratio of this product is then calculated by dividing the value obtained by the product ratio. is expressed as a conversion rate.

[0227] For products incorporating Ac-225, approximately 0.1-1 μCi 225 Contains Ac The sample solution was spotted on the baseline of the iTLC-SG strip, approximately 2 cm from the bottom edge. The iTLC-SG strip was then washed with citric acid-water-methanol mobile phase (20 mL 0 0.4M trisodium citrate / 3mL 2N HCl / 2.3mL MeOH) The samples were then developed, dried at room temperature, and stored for a minimum of 6 hours before analysis (225Ac and all daughter nuclides). (The secular equilibrium is reached.) Using the 99mTc setting, the Bioscan AR2000 radiation The iTLC-SG was scanned using a line TLC imaging scanner.

[0228] For the In-111 chelate, the iTLC-SG strip was approximately 2 cm from the lower end. Approximately 0.5 to 2.5 μCi of 111 The sample solution containing In was spotted. iTLC-SG strip analysis was performed using 10 mM EDTA pH 5-6 as the mobile phase. After spreading, the plates were allowed to dry at room temperature. Scan the dried iTLC-SG using an R2000 radiological TLC imaging scanner. Ta.

[0229] For Zr-89 chelates, %RA conversion was determined by counting with a dose calibrator. As shown, the radioactivity for the product peak was calculated as the total activity including the radioactivity in the PD-10 column. It was determined by dividing by the radioactivity.

[0230] Determine the radiochemical purity of radiolabeled proteins The radiochemical purity of Ac-225 and In-111 chelates (%RA purity, see Tables 3 and 4) The cleavage of Ac-22 (see reference) was determined by SE-HPLC (size exclusion HPLC). For 5, a Tosoh TSKgel column (G3000SW x 17.8 mm x 30 cm, 5 μm) and fill the column with DPBS buffer (1× calcium and magnesium Elution was performed with 0.7 mL / min (containing no sodium). Flow rate: 0.7 mL / min, 20 min run, room temperature. HPLC Afterwards, the eluate was collected in pre-numbered vials, and each vial was subjected to 0.5 or 1 minute of elution. The liquid fraction was collected. The vial containing the eluate was left at room temperature for more than 6 hours. 225 Ac is The radioactivity in each vial was then allowed to reach permanent equilibrium with the daughter nuclide. The wells were counted using a Pintec CRC-55TW well counter. The radiochromatogram was reconstructed.

[0231] For In-111, a Tosoh TSKgel column (G3000SW x 17. The column was filled with DPBS buffer (1×, calcium iodide). Elution was performed with 0.7 mL / min (containing no methyl or magnesium). Flow rate: 0.7 mL / min, 20 min run, Room temperature. The HPLC system described above and a Perkin Elmer radioactive flow detector Rad Radioactivity detection using the iomatic 625TR, using the In-111 setting Ultima Flo™ with a 0.5 mL flow cell and a flow rate of 1.4 mL / min It features M cocktails.

[0232] For Zr-89 (see Table 5), a Tosoh TSKgel column (G The column was loaded with a citrate buffer. Elution was performed with buffered saline. Flow rate: 1 mL / min, 20 min run, room temperature. Beckman Flow Count instrument connected to the stem and Bioscan Flow Count instrument -Radioactivity was detected using a flow-through detector.

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

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

[0235] [Table 5]

[0236] Example 8: Click reaction of modified mAb with DOTA-GA-DBCO Random and site-specific azido-mAb (anti-PSMA mAb, cetuximab, panitumumab, trastuzumab, pertuzumab) in a 5- to 20-fold excess Mix with unchelated DOTA-GA-DBCO and incubate at room temperature or 37°C for 1-24 hours. The mAb was desalted using a Zeba desalting column (Thermo) and incubated for 1 h. Concentrate in a micron centrifuge (Millipore), re-dilute with buffer, and re-concentrate. All free DBCO-DOTA was removed by condensation. The complete click reaction with TA was confirmed by LC-MS.

[0237] Example 9: Click reaction of modified mAb with DFO-DBCO Random and site-specific anti-PSMA mAb azide-mAb at 1-10 mg / mL Mix with a 5- to 20-fold excess of unchelated DOTA-GA-DFO and incubate at room temperature or The mAb was incubated at 37°C for 1 to 24 hours. The solution was desalted using a centrifuge (Millipore) and concentrated in an Amicon centrifuge (Millipore). The solution was rediluted with ethanol and reconcentrated to remove any remaining DBCO-DFO. The complete click reaction of the decamethionine with DBCO-DFO was confirmed by LC-MS.

[0238] Example 10: Cell Binding (FACS) Azide-modified antibody, DOTA-DBCO-azide-modified antibody, and DFO-DBCO-azide Cell binding of the modified antibodies was compared to the parent mAb of the conjugates listed in Table 1. Cell lines containing the antibody or conjugate were treated with a range of concentrations and binding was monitored by flow cytometry. The binding of panitumumab and cetuximab conjugates to EGFR+ A431 cells was measured. Herceptin and pertuzumab were administered to HER2+ SK-BR-3 cells. Anti-PSMA mAb was evaluated for binding to PSMA+C4-2b cells. and evaluated it.

[0239] Cell line: C4-2B cells, a human prostate cancer cell line, were purchased from Janssen Oncology ( The human epidermoid carcinoma cell line, A431, was obtained from the University of Pennsylvania (Spring House, PA). Cells and the human breast cancer cell line, SK-BR-3 cells, were originally obtained from ATCC (Virginia Janssen BioTherapeutics, which uses cells derived from (Spring House, PA). EGFR receptor-negative MOLM-1 3 Human acute myeloid leukemia cells were cultured in suspension in 20% heat-inactivated fetal bovine serum (Gibco). The cells were maintained in RPMI1640 + 25 mM Hepes (Gibco). RPMI 1640 + 25 ml containing 100% FBS (Gibo, Waltham, MA) M HEPES (Gibo, Waltham, MA) was used for growth.

[0240] Flow cytometry: Enzyme-free cell dissociation buffer (Gibco, Massachusetts) Cells were detached from the flask using a 40 μm filter (Fa 5x10 per well in a 96-well U-bottom plate. 4 individual cells The cells were then plated in BSA staining buffer (BD Bioscience, California). Incubate with conjugated or parent antibodies diluted in PBS (San Jose, CA) for 1 hour at 4°C. The cells were washed twice with staining buffer. Then, the cells were incubated at 4°C in the dark for 30 minutes. AlexaFluor647-tagged anti-human IgG secondary antibody (Jackson Immunosorbent Assays) The secondary antibodies were incubated with IgG1 (BioResearch Laboratories). The body was incubated with 3% donkey serum (Rockland Immunochemicals) Diluted 1:200 in staining buffer. For the final 10 min incubation, YTOX (trademark) Green Nucleic Acid Stain (ThermoF The cells were washed twice with staining buffer. Afterwards, resuspend in staining buffer in a final volume of 25 μL / well and apply to the iQue Screen The data were read on a NER flow cytometer (Intellicyt). ForeCyt software was used to exclude events with high nucleic acid staining. The mean fluorescence intensity (MFI) was used to determine the number of live cells. The antibody concentration was determined in cells and expressed as the logarithm of MFI vs. antibody concentration using GraphPad Prism 7 (G The data were graphed using GraphPad Software. A nonlinear regression curve fit was performed on the data. In addition, EC 50 values ​​were calculated.

[0241] For all mAbs and conjugates tested, the parent and modified mAbs exhibited similar cell binding activity. The results were consistent (Table 6).

[0242] [Table 6]

[0243] Example 11: In-111 cell binding assay Cell binding was assayed radiometrically using In-111 radiolabeled proteins listed in Table 4. Anti-PSMA mAb and transferrin were administered to C4-2B cells (PSMA Cetuximab and panitumumab were tested in A431 cells. The antibody was tested on EGFR+ and MOLM-13 cells (EGFR-).

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

[0245] The counts per minute (CPM) from the test sample was calculated using a known amount of In- CPM values ​​were used with a linear regression generated using 111-labeled proteins to determine In-1 The μCi limits were converted to 11 μCi. The μCi limits were converted to molar binding (Moles The MW was converted to (μCi bound / specific activity): (μCi bound / specific activity) / MW mAb or protein. Each data point is the mean ± SD of triplicate reactions.

[0246] Click-labeled In-111 anti-PSMA mAb and In-111 transferrin It bound to C4-2B cells, and cell-associated radioactivity increased with increasing cell number (Fig. 3A). The In-111-labeled anti-EGFR antibodies panitumumab and cetuximab were identified as A431 The radioactivity bound to the cells and increased with increasing cell number. No specific binding of the click-labeled anti-EGFR antibody was detected in the cells (Fig. 3B).

[0247] Example 12: Indium cellular uptake assay Internalization kinetics of In-111 click-labeled anti-PSMA mAb in C4-2B cells It was decided that:

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

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

[0250] Intracellular radioactivity was assayed by preparing cell lysates: surface-bound radioactivity was analyzed by scanning After tripping and washing the cells, add 1.5 mL of 1 M NaOH (Teknova) to the cells. The surface stripped cell lysate was added to the dish and incubated on ice for 5 minutes. The plates were washed with cold PBS and the washes were transferred to counting vials. By gamma counting (Hidex Automatic Gamma Counter) Intracellular radioactivity was assayed as the sum of surface strip cell lysate + PBS wash. It has been decided.

[0251] For time = 0 samples, cells were assayed for surface membrane-bound radioactivity immediately after initial antibody binding on ice. and intracellular radioactivity. For the pull, 3 mL of cell culture medium was added to each dish after the initial antibody binding. The dishes were placed in a humidified CO2 incubator at 37°C. The cell culture medium was transferred to a counting vial and the cells were washed with cold PBS. The PBS wash was collected in a counting vial. The cells were then surface-washed as described. Membrane-bound and intracellular radioactivity was assayed. At each time point, streptavidin was added prior to cell lysis. By incubating the cells in PBS instead of stripping buffer, Unstripped samples were prepared. These samples were used to The efficiency was evaluated and the results were compared with the stripped sample.

[0252] CPMs from test samples were analyzed using a line generated using a known amount of In-111 labeled mAb. The CPM values ​​from the shape regression were converted to μCi In-111. Localization of In-111 mAb in the lysed and capped samples The localization rate (%) was calculated using the following formula: localization rate % = 100 * (sample μCi / average total μCi) where total μCi is the total of the incubation medium, PBS wash, glycine Each data point represents the sum of all collected samples, including rinse and lysed cells. , mean ± SD of triplicate reactions.

[0253] Surface-bound In-111 was rapidly lost from the cell surface and redistributed intracellularly. The tripping technique releases approximately 80% of the cell surface-associated radioactivity at time 0, resulting in the release of ink. By the end of incubation, only 20% was released by stripping, More than 60% was in the cell lysate (Fig. 4).

[0254] Example 13: Efficacy in a mouse tumor xenograft model Dose determination study of anti-PSMA mAb-DOTA-AC-225: Male NSG mice (1 10 (n=8 per group) 6 LNCap cells were subcutaneously implanted, and tumors were grown to 100–150 mm 3 to Mice were given a range of radioactivity (10 nCi, 25 nCi, 70 nCi, 200 nCi). i) Anti-PSMA mAb-azide-DOTA- 225 Ac or isotype control, control mAb-azide-DOTA- 225A single dose of Ac was injected intravenously per mouse. The injection volume was adjusted to a total of 10 μg of protein with cold antibody. Tumor measurements and body weights were taken at 1 week. The tumor size was 1,500 mm 3 Did you euthanize animals when the or when body weight loss exceeded 20%.

[0255] Anti-PSMA mAb-DOTA-Ac-225 significantly improved tumor survival, especially after a single dose of high radiation. showed growth inhibition and superior tumor growth inhibition to isotype control at all doses ( (Figure 5A). Both doses of the control mAb radioconjugate produced survival curves similar to those of the vehicle control. (Figure 5B; Table 7); the anti-PSMA mAb conjugates increased survival with increasing radiation dose. The dose-response curves increased significantly, demonstrating a clear dose response (Fig. 5C; Table 7). Three mice, all from the anti-PSMA mAb 200 nCi group, remained with detectable tumors. did not show any.

[0256] [Table 7]

[0257] The embodiments of the present invention are intended to be merely exemplary, and those skilled in the art will appreciate that certain aspects of the present invention may be readily apparent to those skilled in the art. Numerous equivalents to the procedures herein will be recognized or ascertainable using no more than routine experimentation. Any such equivalents are considered to be within the scope of this invention and are included hereinafter. This is included in the scope of the claims.

[0258] All references cited herein (including patent applications, patents, or publications) are hereby incorporated by reference in their entirety. By reference to the patent or patent application, each individual document or patent or patent application is deemed to be of a different class for all purposes. to the same extent as if each such application were specifically and individually indicated to be incorporated by reference in its entirety. 1006 / 000222, filed on Oct. 1, 2006, which is incorporated herein by reference for all purposes.

Claims

1. 1. A method for labeling a polypeptide with a radioactive metal ion, comprising: a. a modified polypeptide comprising said polypeptide covalently linked to a first click reaction partner; Providing petites, b. Providing a radioactive complex comprising said radioactive metal ion associated with a chelating moiety. wherein the chelating moiety is a chelating group covalently attached to a second click reaction partner. Contains a thickening agent, c. The first click reaction partner reacts with the second click reaction partner. This allows the polypeptide to be labeled with the radioactive metal ion. and contacting said modified polypeptide with said radioactive complex under conditions comprising: 。

2. one of the first and second Click reaction partners comprises an alkyne group, the other of the reaction partners comprises an azide, or the first and second Click reaction partners wherein one of the Click reaction partners comprises an alkene group and the other of the Click reaction partners comprises a diene.

1. The method according to claim 1.

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

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

5. The radioactive metal ion is ２２５ Ac, １１１ In, or ８９ Zr 5. The method according to any one of claims 4 to 4.

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

1. The method according to claim 1.

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

8. The modified antibody or antigen-binding fragment thereof is Bacterial endoglycosidase specific for β-1,4 linkages between core GlcNAc residues, either by antibody or trimming the antigen-binding fragment to obtain the trimmed antibody or its antigen-binding fragment obtaining a fragment and galactosyltransferase GalT or GalNa The trimmed antibody or its anti-antibody is subjected to the treatment in the presence of a glycosyltransferase such as glycosyltransferase. The modified antibody or its antigen is prepared by reacting the original binding fragment with an azide-labeled sugar.

8. The method of claim 7, wherein the binding fragment is obtained by a method comprising:

9. The azide-labeled sugar is UDP-N-azidoacetylgalactosamine (UDP-GalN). az) or UDP-6-azido 6-deoxyGalNAc. 。

10. The glycosyltransferase is a GalT galactosyltransferase or a GalNAc trans The method of claim 8, wherein the enzyme is spherase.

11. The modified antibody or antigen-binding fragment thereof comprises an antibody or antigen-binding fragment thereof amidase to obtain a deglycosylated antibody or antigen-binding fragment thereof. and subjecting said deglycosylated antibody or antigen-binding fragment thereof to a microbial transfection. The modified polypeptide is reacted with azidoamine in the presence of glutaminase. The method of claim 7, wherein the compound is obtained by a method comprising obtaining a tide.

12. The azidoamine is 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 The method of claim 11 .

13. The modified polypeptide is linked, either directly or via a linker, to an azide, tetrazine, or tet 2. The method of claim 1, comprising the polypeptide covalently attached to a benzoyl group.

14. The chelating agent has the formula (I): 【Chemical 1】 (In the formula, R １ , R ２ , R ３ and R ４ are each independently CHQCO ２ X, Q is independently hydrogen, C １ ~C ４ Alkyl or (C １ ~C ２ alkyl)phenyl the law of nature, X is independently hydrogen, benzyl, C １ ~C ４ is alkyl, Z is (CH2) ｎ Y, n is 1 to 10; Y is an electrophilic or nucleophilic moiety covalently attached to the second Click reaction partner. can be, Alternatively, Z is hydrogen; and R １ , R ２ , R ３ and R ４ are each independently CHQCO ２ X, Q is independently hydrogen, C １ ~C ４ Alkyl or (C １ ~C ２ alkyl)phenyl can be, X is independently hydrogen, benzyl, C １ ~C ４ alkyl, or the second click reaction an electrophilic or nucleophilic moiety covalently bound to a reaction partner, Alternatively, the chelating agent comprises an open-chain ligand.

10. The method of claim 1, comprising a macrocycle having the structure:

15. The chelating moiety has the formula (II): 【Chemistry 2】 or a structure of formula (III): 【Chemistry 3】 The method of claim 1 , comprising the structure:

16. ２２５ A Ｃ , １１１ In, or ８９ Labeling antibodies or antigen-binding fragments thereof with Zr A method of a. An antibody or its antigen-binding fragment covalently bound to an azide, tetrazine, or tetrazole group providing a modified antibody or antigen-binding fragment thereof comprising a fragment thereof; b. Associated with a chelating moiety ２２５ A Ｃ , １１１ In or ８９ Zr-containing radioactive complexes wherein the chelating moiety is covalently bonded to an alkyne or alkene group. a chelating agent, and c. The azide, tetrazine, or tetrazole group reacts with the alkyne or alkene group. The antibody or antigen-binding fragment thereof is reacted with ２２５ A Ｃ , １１１ In , or ８９ The modified antibody or its antigen-binding fragment is then subjected to labeling with Zr under conditions that allow the antibody to be labeled with Zr. contacting the fragment with a radioactive complex; The chelating agent has the formula (I): 【Chemistry 4】 (In the formula, R １ , R ２ , R ３ and R ４ are each independently CHQCO ２ X, Q is independently hydrogen, C １ ~C ４ Alkyl or (C １ ~C ２ alkyl)phenyl the law of nature, X is independently hydrogen, benzyl, C １ ~C ４ is alkyl, Z is (CH2) ｎ Y, n is 1 to 10; Y is an electrophilic or nucleophilic moiety covalently bonded to the alkyne group; Alternatively, Z is hydrogen; and R １ , R ２ , R ３ and R ４ are each independently CHQCO ２ X, Q is independently hydrogen, C １ ~C ４ Alkyl or (C １ ~C ２ alkyl)phenyl and X is independently hydrogen, benzyl, C １ ~C ４ alkyl or covalent to the alkyne group a covalently bound electrophilic or nucleophilic moiety, Alternatively, the chelating agent comprises an open-chain ligand. The method includes the structure of

17. A sulfur atom covalently attaching an electrophile on the side chain to the azide, tetrazine, or tetrazole group. and reacting the modified antibody or antigen-binding fragment thereof with a hydroxyl group to obtain the modified antibody or antigen-binding fragment thereof.

17. The method of claim 16, comprising:

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

19. The modified antibody or antigen-binding fragment thereof is Bacterial endoglycosidase specific for β-1,4 linkages between core GlcNAc residues, either by antibody or trimming the antigen-binding fragment to obtain the trimmed antibody or its antigen-binding fragment and obtaining a trimmed antibody or a fragment thereof in the presence of a glycosyltransferase. The modified antibody or its antigen-binding fragment is reacted with an azide-labeled sugar to form a modified antibody or its antigen-binding fragment.

20. The method of claim 18, wherein the binding fragment is obtained by a method comprising:

20. The azide-labeled sugar is UDP-N-azidoacetylgalactosamine (UDP-GalN). az) or UDP-6-azido 6-deoxyGalNAc. Law.

21. The glycosyltransferase is a GalT galactosyltransferase or a GalNAc trans 20. The method of claim 19, wherein the enzyme is selected from the group consisting of spherases.

22. The modified antibody or antigen-binding fragment thereof comprises an antibody or antigen-binding fragment thereof amidase to obtain a deglycosylated antibody or antigen-binding fragment thereof. and subjecting said deglycosylated antibody or antigen-binding fragment thereof to a microbial transfection. The modified polypeptide is reacted with azidoamine in the presence of glutaminase. The method of claim 16, wherein the compound is obtained by a method comprising obtaining a tide.

23. The azidoamine is 3-azidopropylamine, 6-azidohexylamine, O-( 2-aminoethyl)-O'-(2-azidoethyl)tetraethylene glycol, O-(2 -aminoethyl)-O'-(2-azidoethyl)pentaethylene glycol, and O-( 2-aminoethyl)-O'-(2-azidoethyl)triethylene glycol 23. The method of claim 22.

24. The chelating moiety has the formula (II): 【Chemistry 5】 or formula (III): 【Chemistry 6】 17. The method of claim 16, comprising the structure:

25. 1. A method for dual labeling a polypeptide with two radioactive metal ions, comprising: a. Covalently bonded to a first click reaction partner and a second click reaction partner providing a modified polypeptide comprising said polypeptide; b. a first radioactive complex comprising said first radioactive metal ion associated with a chelating moiety; wherein the chelating moiety is covalently linked to a third click reaction partner. containing a bound chelating agent, c. a second radioactive complex comprising said second radioactive metal ion associated with a chelating moiety. wherein the chelating moiety is covalently linked to a fourth click reaction partner. containing a chelating agent incorporated therein, d. The first click reaction partner reacts with the third click reaction partner. the second click reaction partner reacts with the fourth click reaction partner. and labeling the polypeptide with the first and second radioactive metal ions. contacting the modified polypeptide with the radioactive complex under conditions that allow Including, a method.

26. one of the first and second Click reaction partners comprises an alkyne group, the other of the second click reaction partners contains an azide, and the third and fourth click reactions one of the partners comprises an alkene group, and the other of the third and fourth click reaction partners one of the two radioactive metal ions comprises a diene, the first or second radioactive metal ion is a diagnostic emitter, and the other one of the first and second radioactive metal ions is a therapeutic emitter, or both of the first and second radioactive metal ions are therapeutic emitters.

26. The method of claim 25,

27. A radiolabeled polypeptide prepared by the method of claim 1 or 16 and a pharmaceutical and an acceptable carrier.

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

29. A radiolabeled antibody prepared by the method of claim 1 or 16 and a pharmaceutically acceptable carrier. and a carrier capable of preserving the immunological properties of the radiolabeled antibody. Drug.

30. 10. Theranostic agent prepared by the method of claim 1, Formula (VIII): 【Chemistry 7】 Or formula (IX): 【Chemistry 8】 Theranostic agent having the structure:

31. The radioactive metal ion is ３２ P. ４７ Sc, ６７ Cu, ７７ As, ８９ Sr, ９０ Y 、 ９９ Tc、 １０５ Rh、 １０９ Pd、 １１１ Ag、 １３１ I、 １５３ Sm、 １５９ Gd 、 １６５ Dy １６６ H １６９ Er、 １７７ Lu １８６ ee １８８ ee １９４ -r、 １９８ Oh １９９ Oh ２１１ At ２１２ Pb ２１２ Bi ２１３ Bi ２ ２３ Ra, ２２５ Ac, ２５５ Fm, ２２７ Th、 ６２ Cu, ６４ Cu, ６７ Ga, ６８ Ga, ８６ Y. ８９ Zr, or １１１ 30. Theranos of claim 29, wherein the Tick ​​agent.

32. It is a combination, a. A modified polypeptide comprising a polypeptide covalently linked to a first click reaction partner. Do and, b. A radiocomplex comprising a radioactive metal ion associated with a chelating moiety, said chelating moiety the chelating moiety comprises a chelator covalently attached to a second click reaction partner; a complex, The combination is used to label the polypeptide with the radioactive metal ion. A combination that can be done.

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