Radioactive labeling of polypeptides

Abstract

To provide a method for labelling a polypeptide by radioactive metal ions by using click chemistry.SOLUTION: A method for labelling polypeptide by radioactive metal ions includes: providing a modified polypeptide including a polypeptide covalently bonded with a first click reaction partner; and providing a radioactive complex including the radioactive metal ions associated with a chelated portion, herein, the chelated portion includes chelator covalently bonded with a second click reaction partner; and bringing the modified polypeptide in contact with the radioactive complex under the condition enabling the polypeptide to be labelled by the radioactive metal ions by allowing the first click reaction partner to react with the second click reaction partner.SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] (Cross-reference of related applications) This application is a U.S. Patent Application filed on December 18, 2017, in accordance with Section 119(e) of the U.S. Patent Act. Priority is granted to Provisional Application No. 62 / 599,830, and the full disclosure thereof is referenced. This specification is more fully incorporated here.

[0003] (Field of invention) This invention relates to a method for radiolabeling polypeptides such as antibodies. For details, see the present invention. This describes a method for labeling polypeptides with radioactive metal ions using click chemistry. This invention relates to the present invention as well as to pharmaceutical compositions and uses of radiolabeled polypeptides. [Background technology]

[0004] Alpha particle-emitting radionuclides, due to their combination of high energy and short-range action, are considered to be effective in treating cancer. It is a very promising therapy, and it has the potential to be highly localized to tumor cells and to kill them powerfully. To serve (Kim, YSand MWBrechbiel, An overvie w of targeted alpha therapy.Tumour Biol, 2012.33(3):p.573-90). Antibodies, scaffold proteins, small molecules. Alpha-emissives that use ligands, aptamers, or other binding sites specific to cancer antigens Targeted delivery of radionuclides provides a method for selective delivery of radionuclides to tumors, thereby enhancing their efficacy. It provides a way to strengthen and mitigate off-target effects. In a typical implementation, the coupling part A radioactive complex is formed by attaching it to a chelator that is bonded to an alpha-emitting metal. Such examples involve using a monoclonal antibody (mAb) as a targeted ligand, and radioactive It generates what are known as immune complexes.

[0005] Actinium-225 ( 225 Ac) is an alpha-emitting radioactive material of particular interest for medical applications. It is an 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 is due to the formation of radioactive complexes. The length should be sufficient to facilitate, but should be matched to the circulating pharmacokinetics of delivery vehicles such as antibodies. It is short enough for that. 225 Ac's radioactive immune complexes are of particular interest. In addition , 225 Ac is a stable isotope. 209 Before reaching Bi, we eventually obtained four alpha particles. Its effectiveness increases as it decays during the release process. Another radioactive isotope for medical use is Both gamma-ray irradiation suitable for imaging and medium-energy beta-ray irradiation suitable for radiation therapy are used. Releases lutetium-177 ( 177It is Lu). 177 The Lu-labeled peptide demonstrates a reduction in normal tissue damage and 177 the Lu-label has been shown to be capable of using a single radioactive labeling agent for both therapy and imaging (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 for therapeutic purposes include, for example, beta emitters or al pha 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 Th, etc. Other radioisotopes used for imaging purposes include, for example, 62 Cu, 64 Cu, 67 Ga,68 Ga, 86 Y, 8 9 Zr, and 111 Examples include gamma-ray emitting radioactive isotopes such as 135c.

[0006] Previous clinical and preclinical programs were used for actinium chelation, 1, Using 4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) This has been used. However, the DOTA chelation of actinium may become difficult. It is known that (Deal, KA, et al., Improved in vivo stability of actinium-225 macrocyc lic complexes.J Med Chem,1999.42(15):p.2 988-92), in many cases, harsh conditions or high levels of DOTA per antibody are required. As a result, the two radioactive labeling methods known as "1-step" and "2-step" methods were developed. Different approaches are being used, each with its own drawbacks.

[0007] The "two-step" method, which involves two chemical steps including actinium, was the first to be developed. cDevitt,MR,et al.,Tumor therapy with t argeted atomic nanogenerators.Science,20 01.294(5546):p.1537-40). 225 Ac is used in 2M acetate buffer Use at 55°C to 60°C for 30 minutes to achieve a high radiochemical yield (approximately 9) at pH 4.5 to 5. 5% of the bifunctional chelator (BFC) DOTA-isothiocyanate (DOTA-SCN It was chelated by ) and then, 225Ac DOTA-SCN is reacted with a targeted antibody. Then, radioactive immune complexes were generated. The main drawback of the two-step method is that approximately 90% of the SCNs are targeted. Because it cannot withstand the recognition conditions, the input 225 Approximately 90% of Ac can be combined with antibodies. The problem is that it is compounded with DOTA in a non-reactive form that cannot be reacted with. As a result, the yield is low. (Typically only about 10%), not only is the cost higher, but the specific radioactivity also decreases, This may limit the effectiveness of the final complex.

[0008] The "one-step" method was recently 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 chemical reactions containing actinium. There is only one step in the process. DOTA-SCN was first conjugated to the antibody. Then, 225 A c is chelated to DOTA-mAb under mild conditions (37°C, pH 7.5), and maximally An 80% radiochemical yield was obtained. However, high levels of DOTA (approximately 10 per antibody) were observed. It was necessary to combine the above to achieve high yield. High chelator:antibody ratio (C In this case, species with a high DOTA:Ab ratio (DAR) may have reduced immunoreactivity. The possibility is high, and furthermore, the average DAR may be 10, 225 Ac is above average It may also be chelating a higher proportion of the population. Therefore, this method teeth, 225 There is a risk of linking Ac to at least the active fraction of the antibody-chelator complex. Furthermore, to avoid chelation of common metals such as iron, zinc, and copper, the metal-free formula is used. The antibody and DOTA-mAb complex must be handled under conditions that are not present, and the production process is very complex. This presents a major challenge.

[0009] Click chemistry is a chemical chemistry introduced by Sharpless in 2001. This approach involves rapidly and reliably generating substances by combining small amounts of units. This is a chemical method that has been adjusted to achieve this. 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. I want to be illuminated. Coupling reaction (some of which can be classified as "click chemistry") This includes, but is not limited to, esters from activated acids or acyl halogens, and thioesters. Formation of amides (e.g., peptide coupling); nucleophilic substitution reactions (e.g., halo Nucleophilic substitution of genides or ring opening of strained ring systems; azido-alkyl The hysgen cycloaddition reaction (for example, to form a 1,2,3-triazole linker) 1,3-dipolar cycloaddition reaction between azide and alkyne; thioline addition reaction; imine form Diels-Alder reaction between tetrazine and trans-cyclooctene (TCO) Examples include the addition reaction and the Michael addition reaction (e.g., the maleimide addition reaction).

[0010] Click chemistry reactions between alkynes and azides are typically 1,3-cyclo The addition of a copper catalyst is required to accelerate the addition reaction, specifically the copper-catalyzed azide-alkynecycloaddition. This is known as the (CuAAC) reaction. However, cyclooctin or cyclooc Click chemistry reactions between tin derivatives and azides typically require the addition of a copper catalyst. It is not required; instead, strain-promoting azide-alkyne cycloaddition reactions (SPAAC) are used. The process proceeds via (romoted 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, compared to random compounding, shows the effectiveness of ADCs and Because it has been proven that it can enhance both safety and effectiveness, antibody-drug conjugates (ADCs) (Agarwal) is a major area of ​​interest in the antibody-drug conjugate field. ,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): p.176-92). Similar safety and efficacy benefits are available with respect to radioactive immune complexes. It is thought that this can be achieved. [Overview of the Initiative] [Problems that the invention aims to solve]

[0012] As described above, the effect of producing stable radioimmune complexes with high specific activity and high yield The need for efficient methods still exists in the technology field. [Means for solving the problem]

[0013] This invention uses click chemistry to radiolabel polypeptides such as antibodies. This need is met by providing a method for using radioactive gold. It is necessary to reduce the use of the genera and in the process used to generate the initial radioactive complex While requiring only metallic conditions, the azide-modified antibody and the chelated moiety containing the alkyne group are used. A radioactive complex containing a radioactive metal ion associated with it is used in a click chemistry reaction. Stable radioimmunoassay with low chelator-to-antibody ratio (CAR) and high radiation chemi yield. The method of the present invention generates a composite. The method of the present invention enhances safety, effectiveness, and uniformity while generating radioactive immunoassay. Simplify previous methods for manufacturing epidemic complexes.

[0014] In a general embodiment, the present invention relates to a method for labeling polypeptides with radioactive metal ions. And the method is, a. Modified polypeptide containing a polypeptide covalently bonded to the first click reaction partner To provide a gift, b. To provide a radioactive complex containing a radioactive metal ion associated with the chelated portion, Here, the chelating portion covalently bonded to the second click reaction partner is a chelating agent. Including, c. The first click response partner reacts with the second click response partner. Therefore, under conditions that allow the polypeptide to be labeled with radioactive metal ions, modified polypeptide This includes contacting a lipeptide with a radioactive complex.

[0015] In another general embodiment, the present invention relates to radiolabeled polyp This invention relates to a pharmaceutical composition comprising a butylate and a pharmaceutically acceptable carrier.

[0016] In another general embodiment, the present invention relates to subjects requiring treatment for neoplastic diseases or disorders. A method for treating a tumorous disease or disorder, comprising administering the pharmaceutical composition of the present invention to a subject. Regarding methods including doing.

[0017] In another general embodiment, the present invention is a. Modified polypeptide containing a polypeptide covalently bonded to the first click reaction partner Do and, b. A radioactive complex containing a radioactive metal ion associated with the chelate portion, A radioactive complex containing a chelating agent whose chemical moiety is covalently bonded to a second click reaction partner. Regarding combinations or kits that include, Here, the combination or kit is for labeling polypeptides with radioactive metal ions. It will be used.

[0018] In other general embodiments, the present invention relates to radiolabeled polyp This relates to therapeutic or diagnostic agents containing ptide ("theranostic agents"). [Brief explanation of the drawing]

[0019] The above-mentioned "means for solving the problem" and the following "modes for carrying out the invention" are attached. This invention will be better understood when read in conjunction with the attached drawings. The present invention is shown in the drawings. It should be understood that this is not limited to the embodiment itself.

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

[0021] In the background of the invention, and throughout this specification, various publications, articles, and patents are cited. These references are included herein by reference. Each of these references is incorporated herein by reference in its entirety. The discussion of documents, operations, materials, devices, articles, etc. included in this specification is in the context of the present invention. This is to give a hint. Such considerations are based on the fact that any or all of these things are disclosed. Or, to allow any claimed invention to constitute part of the prior art. No.

[0022] Unless otherwise defined, all technical and scientific terms used herein are defined in this text. It has the same meaning as generally understood by those skilled in the art to which the invention belongs. In other words, any specific terms referenced herein shall have the meanings set forth herein. Yes. All patents, published patent applications and publications cited herein are by reference. It is incorporated as if the whole were in the same manner as described herein.

[0023] The singular forms "a," "an," and "the" as used in this specification and the appended claims. It should be noted that unless otherwise clearly stated in the context, the expression "" refers to multiple objects.

[0024] Throughout this specification and the following claims, unless otherwise required by context, the term "including" Variations such as "compose)" and "comprises" and "comprising" are, A specified integer or step, or a group of integers or steps, including any other integer. This means that it does not exclude steps, integers, or groups of steps. It will be understood. When used herein, the term "includes" is used with the term "contains" or It can be replaced with "include," or when used herein, The term can also be replaced with "possess".

[0025] When used herein, "consisting of" is used in the elements of the claims. Exclude any element, step, or component that is not present in this specification. "Qualitative" means that the material does not substantially affect the basic and novel features of the patent claims. No materials or steps are excluded. In order to change the scope of this disclosure, we may use terms such as "comprising," "containing," and "including." Either of the above terms, "including" or "having," can be replaced with the term "consisting of" or This can be replaced with "become essentially from".

[0026] As used herein, the connecting term "and / or" between multiple enumerated elements means It is understood to encompass both individual and combined options. For example, two If the elements are connected by "and / or", the first option is the second option without the second element. This refers to the applicability of element 1. The second option is that the second element is applicable without element 1. This refers to the fact that the first and second elements can be applied together. The third option refers to the fact that the first and second elements can be applied together. Any one of these options is equivalent to the term "and / or" as used herein. It is understood that the requirements are met. The simultaneous applicability of two or more of the options is also included in the meaning. It is understood that this is the case, and therefore the requirements of the term "and / or" are met.

[0027] To assist readers of this application, the description of the specification is divided into various paragraphs or sections. These are either directed towards or towards various embodiments of this application. These separations are paragraphs or Separating a section or embodiment from another paragraph or section or embodiment. It should not be considered that the description herein is applicable to a wide range of uses. On the contrary, a person skilled in the art would know that the description herein is not applicable to a wide range of uses. It encompasses all possible combinations of paragraphs, sentences, and other texts that have a path and can be conceived. You will understand that any consideration of any embodiment is merely illustrative. This indicates that the scope of this disclosure, including the claims, is limited to these embodiments. This is not intended to incite.

[0028] Click radiolabeling of polypeptides In contrast to known procedures, the method of the present invention is, for example, for objects that require it, for example, This invention provides an improved method for generating radioactive immune complexes suitable for medical applications in humans. In particular, the methods described herein are not limited to, 225 Ac, 111 In and 8 9 This process provides both high-yield chelation and low-DAR (Diameter-Resistant Arbitration) for metal ions containing Zr. This invention addresses the major limitations of current methods. Purpose (for example, 89 Zr or 111 (When labeled with In) or for therapeutic purposes (e.g., 225 Azide labels such as azide-mAb composites can be used in the production of (when labeled with Ac) It enables the production of a single batch of identified polypeptides, and radiolabeling allows for azide-labeled polypeptides. Adhering to the same site (or multiple sites) within a batch of material, site-specific modification or random a It can be obtained by any of the zid complexes. For example, in the case of random azid complexes, A sample of a batch of azide-labeled polypeptide containing a single distribution of zide modification sites is the present invention. Click chemistry can be used to radioactively label materials for different purposes.

[0029] The present invention method, called "click radiolabeling," relies on click chemistry. (1) A first click chemistry reaction partner, for example, an antibody containing an azide moiety. (2) to obtain modified polypeptides such as (2) radioactive metals associated with the chelated moiety On, for example, 225 Ac, 111 In, or 89 By obtaining a radioactive complex containing Zr And here, the chelate portion becomes the second click chemistry reaction partner, for example DOTA-dibenzocyclooctin (DOTA-DBCO) or deferoxamine-D It contains a chelating agent covalently bonded to an alkyne group such as BCO (DFO-DBCO), and (3) Strain-promoting azide-alkyne cycloaddition reaction between the azide moiety and the alkyne group (SP Click chemistry reaction partners for modified peptides such as AAC, and radioactive complexes This includes carrying out a reaction between the two.

[0030] The method of the present invention relates to the chelation of radioactive metals under low pH or high pH and / or high temperature conditions. This makes it possible to maximize efficiency, and this inactivates the alkyne reaction partner. This can be achieved without the risk of [unclearing]. Efficient cleavage between azide-mAb and radioactive complex Due to the fermentation and efficient SPAAC reaction, high radioactivity is achieved even with a low azide:mAb ratio. Radioactive immune complexes can be produced in high yield. The method of the present invention excludes trace metals. The only step that must be performed is the chelation of the chelated portion with radioactive metal ions. Therefore, the processes of antibody production, purification, and complexation must be carried out under metal-free conditions. There isn't one.

[0031] As used herein, the term "click chemistry" refers to Sharpless's approach. This refers to the introduced philosophy of chemistry, which involves combining small units containing reactive groups. This describes the chemistry tuned to rapidly and reliably generate covalent bonds (Kolb, above). (See et al.) Click chemistry does not refer to a specific reaction. However, this refers to a concept that includes, but is not limited to, reactions that mimic reactions found in nature. In some embodiments, the click chemistry reaction is modular and wide-ranging. Furthermore, it has a high chemical yield, produces harmless by-products, is stereospecific, and is a single reaction product. It exhibits a strong thermodynamic driving force for selecting the reaction with and / or under physiological conditions. It can be carried out. In some embodiments, the click chemistry reaction is performed with high atoms. It demonstrates economic efficiency, can be carried out under simple reaction conditions, and uses readily available starting materials and Use reagents, without using toxic solvents, or with harmless or easily removable solvents such as water. Using and / or simple raw materials by non-chromatographic methods such as crystallization or distillation Provides isolation of the product. In certain embodiments, the click chemistry reaction is performed on the azide (- Hüygen ring addition reaction between N3) and alkyne or alkyne moiety or 1,3-dipole addition This is a cyclization reaction, forming a 1,2,4-triazole linker.

[0032] In a general embodiment, the present invention relates to polypeptides, aptamers, or molecules with radioactive metal ions. Regarding a method for labeling child ligands, the method is: a. Modified polypeptide containing a polypeptide covalently bonded to the first click reaction partner To provide a gift, b. To provide a radioactive complex containing a radioactive metal ion associated with the chelated portion, Here, the chelating portion covalently bonded to the second click reaction partner is a chelating agent. Including, c. The first click response partner reacts with the second click response partner. Therefore, under conditions that allow the polypeptide to be labeled with radioactive metal ions, modified polypeptide This includes contacting a lipeptide with a radioactive complex.

[0033] As used herein, the term “polypeptide” means linked via peptide bonds. Polypropylene is composed of naturally occurring structural variants and their synthetic, non-naturally derived analogs. This refers to a polypeptide. The term "polypeptide" refers to a polypeptide of any size, structure, or function. Refers to. Typically, a polypeptide has a length of at least three amino acids. Polypeptide It may be of natural origin, recombinant, synthetic, or any combination thereof. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. In a preferred embodiment, the polypeptide is an antibody, preferably a monoclonal antibody. The body, or a fragment thereof, or an antigen-binding fragment thereof, etc. In a preferred embodiment, Therefore, the antibody or its fragment is specific to the cancer antigen. According to other embodiments, Lipeptides are genetically modified domains or scaffold proteins.

[0034] As used herein, the terms “antibody” or “immunoglobulin” are used in a broad sense. , immunoglobulin or antibody molecule containing polyclonal antibody, mouse, human, human compatible, hi Monoclonal antibodies, including those with antigen-binding fragments, and chimeric monoclonal antibodies. Contains clonal antibodies.

[0035] Generally, an antibody is a protein or peptide chain that exhibits binding specificity to a particular antigen. Hereinafter referred to as the "target". The structure of the antibody is publicly known. Immunoglobulins are Depending on the amino acid sequence of the heavy chain constant domain, there are five main classes: IgA, IgD, etc. IgE, IgG, and IgM can be assigned to these. IgA and IgG are iso Further subdivided into types IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. They are classified into five main classes or corresponding subclasses. The antibody of the present invention may be any of the following: IgG1, IgG2, IgG3, Alternatively, IgG4 is preferred. The antibody light chains of any vertebrate species are their constant-state Based on the main amino acid sequence, there are two distinctly different types, namely κ and λ. It can be assigned to one of the two. Therefore, the antibody of the present invention has a κ or λ light chain constant domain. It may contain . According to a particular embodiment, the antibody of the present invention is a mouse antibody or It contains the heavy chain and / or light chain constant regions of a human antibody. Each of the four IgG subclasses is: These effects pedal functions have different biological functions known to exist. Generally, this occurs through interaction with the Fc receptor (FcγR), or through the binding of C1q and complement. It is mediated by fixation. Binding to FcγR results in antibody-dependent cell-mediated cytolysis. However, binding to complement factors can lead to complement-mediated cell lysis. The antibody may have no effector function or only minimal effector function, but it may bind to FcRn. It retains that ability.

[0036] As used herein, the term “antigen-binding fragment” means, for example, a diabody. Fab, Fab', F(ab')2, Fv fragment, disulfide-stabilized Fv flag Ment(dsFv), (dsFv)2, bispecific dsFv(dsFv-dsFv'), Disulfide-stabilized diabody (ds diabody), single-chain antibody molecule (scFv), single One-domain antibody (sdab), scFv dimer (bivalent diabody), one or more CDRs Multiple specific antibodies formed from a portion of an antibody containing a nano-domain, camelized single-domain antibodies, nano-domain antibodies A di-domain antibody, a bivalent domain antibody, or an antibody that binds to an antigen but does not contain a complete antibody structure. This refers to any other antibody fragment, such as any other antibody fragment. The antigen-binding fragment is the parent antibody. Alternatively, it can bind to the same antigen to which the parent antibody fragment binds. When used in this specification, The term "single-chain antibody" refers to a single-chain antibody that is linked together by short peptides of approximately 15 to 20 amino acids. This refers to conventional single-chain antibodies in this field, including chain-variable regions and light-chain-variable regions. When used in this context, the term "single-domain antibody" refers to whether it includes the heavy chain variable region and the heavy chain constant region. This refers to conventional single-domain antibodies in this field, which either contain only the heavy chain variable region or only the heavy chain variable region.

[0037] As used herein, the terms “scaffold” or “scaffold protein” This refers to any protein that has a target-binding domain and can bind to a target. A cafold is a "framework" that is largely structural and makes specific contact with the target. It includes a "binding domain" that provides the binding. The binding domain of the scaffold is the scaffold It does not have to be defined by a single contiguous array of folds. In particular, a scaffold is It may also be part of a larger binding protein, and itself may be multiple scaffolds It may be part of a multimer-binding protein that includes [specific]. A particular binding protein may contain two or more [specific] proteins. In that it can bind to different epitopes, it is bispecific or multiplespecific. 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 a visit.

[0038] Any chemical or enzymatic modification of polypeptides known to those skilled in the art in consideration of this disclosure Using this method, the polypeptide of the present invention is covalently bonded to a first click reaction partner. It can be done. Primary ions present in the N-terminus and side chains of lysine residues of each polypeptide chain. The use of amine-reactive groups that react with amines in a random modification method for polypeptides is possible. Yes, it is possible. Examples of amine-reactive groups suitable for use in the present invention include, but are not limited to, N-H. Droxysuccinimide (NHS), substituted NHS, e.g., sulfo-NHS, isothiocyanate Examples include nates, as well as tetra and perfluorophenyl esters. Cysteine ​​residues The thiol-reactive group that reacts with thiols or sulfhydryls present in the side chain is polypept It can be used in the random modification of thiols. Suitable thiols for use in the present invention Examples of applicable groups include, but are not limited to, maleimide, haloacetyl, and phenyloxa. Examples include diazole sulfones. According to a preferred embodiment, the modified polypeptide is An electrophile covalently bonded to a click reaction partner (e.g., NHS-azide), It is obtained by reacting a lysine side chain, preferably an amino side chain.

[0039] The method of the present invention further enables the production of site-specific radiolabeled polypeptides. The click radiolabeling method utilizes an established method for site-specifically introducing azide groups into antibodies. This facilitates the site-specific generation of radioactive immune complexes (Li, X, e 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 using site-specific methods are available. Any method known in the field and known to those skilled in the art for site-specific labeling of antibodies is provided. The present invention may be used in consideration of this disclosure. Antibodies suitable for use in the present invention are site-specific. Examples of methods of modification include, but are not limited to, modified cysteine ​​residues (e.g., TH Incorporation of IOMAB® (trademark), non-natural amino acids or glycans (e.g., selenoside) Ingredients: p-AcPhe, formylglycine-producing enzyme (FGE, SMARTag®). (etc.), and enzymatic methods (e.g., glycotransferase, endoglycosidase, microbiome The use of substances or bacterial transglutaminases (MTG or BTG, saltase A, etc.) For example, according to a preferred embodiment, the modified polypeptide is the innermost GlcNAc Leaving it intact at Fc enables site-specific incorporation of azide sugars at that site, G lycINATOR (Genovis), etc., the core Gl of the Fc-glycosylation site of an antibody between cNac residues is trimmed by a bacterial endoglycosidase specific for β-1,4 linkages, an antibody or its antigen-binding fragment obtained by trimming. Then, the trimmed antibody or its antigen-binding fragment is reacted with an azide-labeled sugar such as UDP-N-azidoacetylgalactosamine (UDP-GalN az) or UDP-6-azido-6-deoxyGalNac in the presence of a glycosyltransferase such as GalT galactosyltransferase or GalNac transferase to obtain a modified antibody or its antigen-binding fragment. According to another preferred embodiment the modified polypeptide is an antibody or its antigen-binding fragment obtained by deglycosylating an antibody or its antigen-binding fragment with amidase. Then, the obtained deglycosylated antibody or its antigen-binding fragment is reacted with an azidoamine, preferably 3-azidopropylamine, 6-azidohexylamine, or any azide linker amine or any azidoalkylamine, azide-polyethylene glycol (PEG) -amine, etc., such as O-(2-aminoethyl)-O'-(2-azidoethyl) tetra ethylene glycol, O-(2-aminoethyl)-O'-(2-azidoethyl) pentaethylene glycol, O-(2-aminoethyl)-O'-(2-azidoethyl) triethylene glycol, etc., or reacted in the presence of microbial transglutaminase to obtain a modified antibody or its antigen-binding fragment. amine or any azidoalkylamine, azide-polyethylene glycol (PEG) -amine, etc., such as O-(2-aminoethyl)-O’-(2-azidoethyl) tetra ethylene glycol, O-(2-aminoethyl)-O’-(2-azidoethyl) pentaethylene glycol, O-(2-aminoethyl)-O’-(2-azidoethyl) triethylene glycol, etc., or reacted in the presence of microbial transglutaminase ethylene glycol, O-(2-aminoethyl)-O’-(2-azidoethyl) triethylene glycol, etc., or reacted in the presence of microbial transglutaminase to obtain a modified antibody or its antigen-binding fragment. to obtain a modified antibody or its antigen-binding fragment.

[0040] As used herein, the term "aptamer" means a substance that binds specifically to its target with high affinity. This refers to single-stranded oligonucleotides (single-stranded DNA or RNA molecules) that can perform this function. Ptamers can be used as molecules that target a variety of organic and inorganic substances.

[0041] As used herein, the term "small molecule ligand" refers to a low molecular weight organic compound. When used in this specification, small molecule ligands have a size of less than approximately 1000 daltons. It can refer to a compound that can be synthesized in a laboratory or is found in nature. It is possible.

[0042] When used herein, the terms “click reaction partner” or “click chemistry” are used. "Handle" refers to a reactant or reactive group that can be added to a click chemistry reaction. This refers to a click reaction partner, which is rarely found in naturally occurring biomolecules and is associated with a click reaction. Although chemically inert, for example, when reacted with an azide-reactive or alkyne-reactive group. In some cases, the reaction occurs under biologically relevant conditions, such as excessive heat or the absence of harsh reactants. The reaction can be carried out efficiently under cell culture conditions such as those listed below. Generally, clicker Mistry reactions involve fewer click reaction partners that can react with each other. Each requires two molecules. Such click reaction partners are reactive with each other. In this specification, these are referred to as click chemistry handle pairs or click chemistry pairs. In some embodiments, the click reaction partner may azid and strain. The alkyne is, for example, cyclooctyne, or any other alkyne. Other embodiments The click reaction partners are a reactive diene and a suitable tetrazine diene. For example, trans-cyclooctene, norbornene, or biscyclononene are crystalline It can be paired with a tetrazine diene derivative that is suitable as a reaction partner. Further embodiments So, tetrazole, when paired with an alkene that is not activated in the presence of ultraviolet light, In other embodiments, The click reaction partners are cysteine ​​and maleimide, for example, derived from peptides. The cysteine ​​(e.g., GGGC) is associated with a chelating agent (e.g., NOTA). It can be reacted with maleimide. Other suitable click chemistry handles are available. Known to vendors (for example, Spicer et al., Selective ch chemical protein modification.Nature Commu See nications.2014;5:p.4740. In other embodiments, Click response partners include phosphine and azide, among others. It is a click reaction component. In other embodiments, the click reaction partner is a dihydrogen such as tetrazine. En, and alkenes such as trans-cyclooctene (TCO) or norbornene. Diels-Alder reaction components. Exemplary click reaction partners are U.S. Patent Publication No. Application Publication No. 20130266512 and International Publication No. 2015073746 (Pamphlet) The relevant explanations for both click-response partners are listed in the retrieval section. As incorporated herein by reference. 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. According to another preferred embodiment, one of the first and second click reaction partners contains an alkene group and the other click reaction partner contains a diene. One of the reaction partners contains an alkene group and the other click reaction partner contains a diene.

[0043] As used herein, the terms "alkyne", "alkyne group" or "alkyne moiety" refer to a functional group containing a carbon-carbon triple bond. Examples of alkyne moieties include terminal alkynes and cyclic alkynes, preferably terminal alkynes and cyclic alkynes reactive with an azide group. A terminal alkyne has at least one hydrogen atom bonded to the triple bond carbon atom. A cyclic alkyne is a cycloalkyl ring containing one or more triple bonds. Examples of cyclic alkynes include, but are not limited to, bicyclononine (BCN), difluorocyclooctyne (DIFO), dibenzocyclooctyne (DIBO), keto-DIBO, biarylazacyclooctynone (BARAC), dibenzoazacyclooctyne (DIBAC), dimethoxyazacyclooctyne (DIMAC), dibenzocyclooctyne (DBCO), difluorobenzocyclooctyne (DIFBO), monobenzocyclooctyne (MOBO), and cyclooctyne and cyclooctyne derivatives such as tetramethoxy DIBO (TMDIBO). According to a preferred embodiment, one of the first and second click reaction partners comprises a cyclic alkyne, preferably DBCO. According to a preferred embodiment, the other click reaction partner comprises an azide, preferably NHS-azide. As used herein, the terms "alkyne", "alkyne group" or "alkyne moiety" refer to a functional group containing a carbon-carbon triple bond. Examples of alkyne moieties include terminal alkynes and cyclic alkynes, preferably terminal alkynes and cyclic alkynes reactive with an azide group. A terminal alkyne has at least one hydrogen atom bonded to the triple bond carbon atom. A cyclic alkyne is a cycloalkyl ring containing one or more triple bonds. Examples of cyclic alkynes include, but are not limited to, bicyclononine (BCN), difluorocyclooctyne (DIFO), dibenzocyclooctyne (DIBO), keto-DIBO, biarylazacyclooctynone (BARAC), dibenzoazacyclooctyne (DIBAC), dimethoxyazacyclooctyne (DIMAC), dibenzocyclooctyne (DBCO), difluorobenzocyclooctyne (DIFBO), monobenzocyclooctyne (MOBO), and cyclooctyne and cyclooctyne derivatives such as tetramethoxy DIBO (TMDIBO). FO), dibenzocyclooctyne (DIBO), keto-DIBO, biarylazacyclooctynone (BARAC), dibenzoazacyclooctyne (DIBAC), dimethoxyazacyclooctyne (DIMAC), dibenzocyclooctyne (DBCO), difluorobenzocyclooctyne (DIFBO), monobenzocyclooctyne (MOBO), and cyclooctyne and cyclooctyne derivatives such as tetramethoxy DIBO (TMDIBO). FO), dibenzocyclooctyne (DIBO), keto-DIBO, biarylazacyclooctynone (BARAC), dibenzoazacyclooctyne (DIBAC), dimethoxyazacyclooctyne (DIMAC), dibenzocyclooctyne (DBCO), difluorobenzocyclooctyne (DIFBO), monobenzocyclooctyne (MOBO), and cyclooctyne and cyclooctyne derivatives such as tetramethoxy DIBO (TMDIBO). (DIMAC), dibenzocyclooctyne (DBCO), difluorobenzocyclooctyne (DIFBO), monobenzocyclooctyne (MOBO), and cyclooctyne and cyclooctyne derivatives such as tetramethoxy DIBO (TMDIBO). (DIFBO), monobenzocyclooctyne (MOBO), and cyclooctyne and cyclooctyne derivatives such as tetramethoxy DIBO (TMDIBO). Examples of cyclooctyne and cyclooctyne derivatives such as tetramethoxy DIBO (TMDIBO). According to a preferred embodiment, one of the first and second click reaction partners comprises a cyclic alkyne, preferably DBCO. According to a preferred embodiment, one of the first and second click reaction partners comprises a cyclic alkyne, preferably DBCO. According to a preferred embodiment, the other click reaction partner comprises an azide, preferably NHS-azide.

[0044] As used herein, the term "diene" has two carbon-carbon double bonds, This refers to compounds in which the double bonds are located at the 1st and 3rd positions. The double bonds in dienes are cis or t It can be any of the lances. Examples of dienes include tetrazine or tetrazole groups. These are some examples, but they are not limited to these.

[0045] As used herein, the terms "alkene," "alkene group," or "alkene moiety" are defined as follows: This refers to unsaturated hydrocarbon molecules containing a carbon-carbon double bond. According to certain embodiments, Alkenes can contain 2 to 100 carbon atoms. An example of an alkene is norbol. Examples include, but are not limited to, ne and trans-cyclooctene (TCO). According to another preferred embodiment, one of the first and second click reaction partners is A It contains a ken group, preferably norbornene or TCO. According to a preferred embodiment, other k The rick reaction partner comprises a diene, preferably a tetrazine or tetrazole group.

[0046] As used herein, the term "covalent" means that at least one covalent bond is formed between two or more of the same materials. Then, the polypeptide attaches to the first click reaction partner, and the chelating agent is at least This means that it is also attached to a second click reaction partner via another covalent bond. The bond may be direct, i.e., without a linker, or indirect, i.e., without a linker. —You may also use that method.

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

[0048] As used herein, the term "radioactive metal ion" or "radioactive metal ion" refers to one or more isotopes of an element that emits particles and / or photons. Those skilled in the art considering the present disclosure can use any radioactive metal known to them in the present invention. Examples of radioactive metals suitable for use in the present invention include, but are not limited to, 32 P, 47 Sc, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 77 As, 86 Y, 89 Zr, 89 Sr, 90 Y, 99 Tc , 105 Rh, 109 Pd, 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 are included. When used in this specification, the term "diagnostic emitter" refers to radioactive metal ions useful in diagnostic or imaging applications. Examples of diagnostic emitters include, but are not limited to, Cu, 62 Cu, 64 Cu, 67 Ga, 68 Ga, 86 Y, 89 Zr, and 111 gamma emitters such as In are included. When used in this specification, the term "therapeutic emitter" refers to radioactive metal ions 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 are included. Preferred According to the embodiment, radioactive metal ions are 225 It is Ac. According to other embodiments, poly The peptides are labeled with non-metallic radiolabeling for use in pre-targeting or theranostic applications. It can be recognized. Examples of nonmetallic radioactive labels suitable for use in the present invention include, However, 125 I and 18 F is one example.

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

[0050] As used herein, the terms "chelating agent" or "chelator" are used in the following context: 225 Business etc. This refers to radioactive metals, or chemical compounds in which metals can be chelated via coordinate bonds. Taking the above into consideration, any chelating agent known to those skilled in the art can be used in the present invention. In one embodiment, the chelating agent includes a macrocyclic molecule. A macrocyclic molecule suitable for use in the present invention is Examples of chelating agents included, but not limited to, include deferoxamine (DFO) and ethylene. Examples include diaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA). In another embodiment, the chelating agent comprises an open-chain ligand. Suitable for use in the present invention. Examples of chelating agents containing open-chain ligands include, but are not limited to, 1,4,7,10-Te Traazacyclododecane-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 Azacyclopentanadecane-N,N',N'',N''',N''''-pentaacetate (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-teto Lapropionic acid (DOTPA), 1,4,8,11-tetraazacyclotetradecane-1 ,4,8,11-tetrapropionic acid (TETPA), and 1,4,7,10-tetraa One example is Zacyclododecane-1,4,7,10-tetramethylenephosphonate (DOTMP). It can be done. According to a preferred embodiment, the chelating agent is of formula (I):

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

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

[0053] [ka] It includes the structure.

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

[0055] [ka] Includes.

[0056] In one embodiment, the present invention relates to a method of the present invention that uses two or more radioactive metal ions This relates to a method for labeling polypeptides. For example, a polypeptide can be labeled with two radioactive metal ions. Methods for labeling are: a. Covalently bonded to the first click reaction partner and the second click reaction partner To provide modified polypeptides including polypeptides, b. Provides a first radioactive complex containing a first radioactive metal ion associated with the chelate portion. In this process, the chelate portion is covalently bonded to a third click reaction partner. Contains a chelating agent, c. Provides a second radioactive complex containing a second radioactive metal ion associated with the chelate portion. In this process, the chelate portion is covalently bonded to the fourth click reaction partner. Contains a rate-setting agent, d. The first click response partner reacts with the third click response partner, and the second When a click reaction partner reacts with a fourth click reaction partner, poly Under conditions that allow the peptide to be labeled with first and second radioactive metal ions, modification The method includes contacting a 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. The compound includes a group, and the other of the first and second click reaction partners includes 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 side of the toner contains diene.

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

[0059] The conditions for carrying out click chemistry reactions are known in the art, Any conditions for carrying out click chemistry reactions known to those skilled in the art in consideration of this disclosure This can be used in the present invention. Examples of conditions include, but are not limited to, pH 4 to 10. At temperatures ranging from 20°C to 70°C, modified polypeptides and released in a ratio of 1:1 to 1000:1. One method is to incubate the injection complex.

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

[0061] Pharmaceutical composition and therapeutic method The click radiolabeling method of the present invention may be improved into 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 , administer an azid-mAb to bind to target cells and eliminate it from circulation over time, or Remove with a removal agent. Subsequently, a radioactive complex is administered, and azide-mAbs bound to the target site are removed. Upon undergoing the SPAAC reaction, the remaining unbound radioactive complex is rapidly removed from 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). This Pre-targeting technology provides a method for enhancing the localization of radioactive metal ions at target sites. do.

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

[0063] As used herein, the term “carrier” includes any excipient, diluent, filler, salt, or b Flour, stabilizer, solubilizer, oil, lipid, lipid-containing vesicles, microspheres, liposome encapsulation This refers to other materials known in the art for use in excipients or pharmaceutical formulations. (e.g., carriers, excipients) Alternatively, it will be understood that the properties of the diluent are determined by the route of administration for the specific application. The term "pharmaceutically acceptable carrier" used in this context refers to the effect of the composition according to the present invention or This refers to a non-toxic material that does not interfere with the biological activity of the composition according to the present invention. According to a particular embodiment, In consideration of this disclosure, suitable for use in antibody-based or radiocomplex-based pharmaceutical compositions, Any pharmaceutically acceptable carrier can be used in the present invention.

[0064] According to specific embodiments, the compositions described herein are intended to be administered to a subject. Formulated to suit the route. For example, the compositions described herein are for intravenous, subcutaneous, It can be formulated for administration into muscles or tumors.

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

[0066] In another general embodiment, the present invention relates to subjects requiring treatment for neoplastic diseases or disorders. A method for treating a tumorous disease or disorder, comprising administering the pharmaceutical composition of the present invention to a subject. Regarding methods that include doing so.

[0067] According to a particular embodiment, the method of the present invention administers a therapeutically effective dose of the pharmaceutical composition of the present invention. This includes, wherein the composition targets cells associated with neoplastic disease or disorder. It contains a radiolabeled polypeptide for targeting, 225 From Ac and its daughter nuclides Alpha particles are delivered to target cells, causing cytotoxic effects on those cells, and To treat neoplastic diseases or disorders.

[0068] According to a particular embodiment, therapeutically effective amounts of modified polypeptides and radioactive complexes are used in different combinations. It is administered as a finished product.

[0069] As used herein, the term “therapeutic effective dose” means the desired biological or pharmacological dose for a subject. This refers to the amount of the active ingredient or component that elicits a response. The therapeutically effective dose is the amount of the active ingredient or component that elicits a response for the stated purpose. This can be determined by empirical and general methods. For example, if desired, in vitro Using this method, it can help determine the optimal dose range. The selection of a specific effective dose is... , the disease to be treated or prevented, the associated symptoms, the patient's weight, the patient's immune status, and the knowledge of a person skilled in the art Based on consideration of several factors, including other factors, by those skilled in the art (for example, clinical It can be determined by testing. Furthermore, the exact dosage used in the formulation depends on the route of administration and The treatment should be determined according to the severity of the disease, the doctor's judgment, and the individual patient's circumstances. Yes, the effective dose is estimated from dose-response curves derived from in vitro or animal model test systems. It is possible.

[0070] As used herein, the terms “treat” and “treating” Both "treatment" and "treatment" refer to radioactive metal ions in neoplastic diseases or disorders. At least one measurable condition related to a disease, disorder, or pathological condition in which the administration of n may be beneficial This is intended to refer to the improvement or restoration of physical parameters, and this applies to the subject. It is not always recognizable, but it may be recognizable in the case of the subject. The terms "treat," "treating," and "treatment" are Furthermore, it causes regression of a disease, disability, or pathological condition, prevents its progression, or at least reduces It can also refer to slowing down the progression. In certain embodiments, it means "to treat". "To treat" and "treatment" refer to neoplastic diseases or disorders, etc. One of the diseases, disorders, or conditions for which the administration of radioactive metal ions may be beneficial. Or it refers to the alleviation, prevention of progression or onset of two or more symptoms, or a reduction in their duration. In typical embodiments, "to treat," "to heal," and "treatment" refer to a disease, disorder, or illness. This refers to preventing the recurrence of the condition. In specific embodiments, this may be expressed as "treat," "treat," and "cure." "Treatment" refers to improving the survival rate of subjects with a disease, disability, or pathological condition. In specific embodiments... The terms "to treat," "to treat," and "treatment" refer to a disease, disorder, or pathological condition in the subject. It refers to the disappearance of something.

[0071] Examples of neoplastic diseases or disorders include, but are not limited to, disseminated carcinoma, solid tumor carcinoma, hypertrophy, Coronary artery disease or vascular occlusive disease, diseases related to infected cells, microorganisms or viruses Or a disorder, or a disease or disorder related to inflammatory cells such as rheumatoid arthritis (RA). These are some examples.

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

[0073] Any dosing schedule for modified polypeptides and radioactive complexes may be used in consideration of this disclosure. This is possible. Generally, modified polypeptides and radioactive complexes are administered in different compositions. In this case, the radioactive complex can be administered at any time after the modified antibody has been administered.

[0074] According to a particular embodiment, a composition used in the treatment of a neoplastic disease or disorder is related to It can be used in combination with other active ingredients that are effective in treating neoplastic diseases or disorders. ru.

[0075] As used herein, the term “combined use” means the administration of two or more therapeutic agents to a subject and In this context, it refers to the use of multiple medications. The use of the term "combined use" implies that the treatment is The order in which the drugs are administered to the target is not limited. For example, the first therapeutic agent (for example, as described herein) The composition (which is used) is administered to the subject before the administration of the second therapeutic agent (for example, 5 minutes, 15 minutes, 30 minutes, 4 minutes). 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 Two weeks prior), simultaneously, or afterward (for example, 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 Administered after 1, 2, 3, 4, 5, 6, 8, or 12 weeks. It is possible.

[0076] In another general embodiment, the present invention relates to a radiolabeled antibody prepared by the method of the present invention and It includes a pharmaceutically acceptable carrier and preserves the immunological properties of the radiolabeled antibody. Regarding therapeutic agents.

[0077] As used herein, the term “theranostic” refers to either the diagnostic or therapeutic function. It refers to the ability to provide both diagnosis and treatment. In one embodiment, the theranostic agent provides both diagnosis and treatment. It provides the function of a theranostic agent, which is an active agent without diagnostic function. In yet another embodiment, the theranostic agent is useful for diagnosis but has no therapeutic function. It is not a drug.

[0078] According to a preferred embodiment, the radioactive metal ion is used in a diagnostic emitter, preferably 89 Zr In other preferred embodiments, the radioactive metal ion is a therapeutic emitter, preferably teeth 225 Ac is used. According to a preferred embodiment, the theranostic agent is used for diagnosis and treatment. It is used to provide both of these functions to those who need them.

[0079] Combinations and kits This specification provides combinations including the following: a. Modified polypeptide containing a polypeptide covalently bonded to the first click reaction partner Do and, b. A radioactive complex containing a radioactive metal ion associated with the chelate portion, A radioactive complex containing a chelating agent whose chemical moiety is covalently bonded to a second click reaction partner. Regarding combinations or kits that include, Here, the combination is used to label polypeptides with radioactive metal ions. .

[0080] According to a particular embodiment, the combination of the present invention involves a polypeptide with radioactive metal ions. This is a reaction mixture used for labeling. According to other embodiments, the combination is i Used to generate radiolabeled polypeptides in vitro or in vivo. It comes in a pack or kit. Upon request, it can be used for the manufacture, use, or sale of pharmaceutical or biological products. The combination must be accompanied by a notice or instruction in a format specified by the government agency regulating the sale. This warning applies to the manufacture, use, or sale of the product for human administration. This reflects the approval by the relevant agency. The combinations contained herein refer to the polypeptides described above. A method for labeling with radioactive metal ions, or a method for treating neoplastic diseases or disorders It can be used in methods for treating neoplastic diseases or disorders in the subject.

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

[0082] Embodiment 1 is a method for labeling polypeptides with radioactive metal ions, a. Modified polypeptide containing a polypeptide covalently bonded to the first click reaction partner To provide a gift, b. To provide a radioactive complex containing a radioactive metal ion associated with the chelated portion, Here, the chelating portion covalently bonded to the second click reaction partner is a chelating agent. Including, c. The first click response partner reacts with the second click response partner. Therefore, under conditions that allow the polypeptide to be labeled with radioactive metal ions, modified polypeptide This includes contacting a lipeptide with a radioactive complex.

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

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

[0085] Embodiment 2 is a configuration in which one of the first and second click reaction partners includes an alkyne group. This is the method according to Embodiment 1, wherein the other rick reaction partner comprises an azide.

[0086] Embodiment 3 is a first click reaction partner comprising an azide group and a second click reaction This is the method according to Embodiment 2, wherein the partner includes an alkyne group.

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

[0088] Embodiment 3b is a cyclic alkyne, preferably cyclooctyne or cyclo This is the method according to Embodiment 2 or 3, which includes an octin derivative.

[0089] Embodiment 3c is an embodiment in which the alkyne group contains bicyclononyne (BCN). This method is as described in application form 3b.

[0090] Embodiment 3d is an embodiment in which the alkyne group contains difluorinated cyclooctin (DIFO). This method is as described in application form 3b.

[0091] Embodiment 3e is an embodiment in which the alkyne group comprises dibenzocyclooctin (DIBO). The method is as described in 3b.

[0092] Embodiment 3f comprises an alkyne group containing biarylazacyclooctin (BARAC). This is the method described in Embodiment 3b.

[0093] Embodiment 3g contains an alkyne group containing dibenzoazacyclooctin (DIBAC), This method is as described in application form 3b.

[0094] Embodiment 3h contains an alkyne group comprising dimethoxyazacyclooctin (DIMAC), This is the method described in Embodiment 3b.

[0095] Embodiment 3i is an embodiment in which the alkyne group contains dibenzocyclooctin (DBCO). The method is as described in 3b.

[0096] Embodiment 3j contains an alkyne group comprising difluorobenzocyclooctin (DIFBO). This is the method described in Embodiment 3b.

[0097] Embodiment 3k is an embodiment in which the alkyne group contains monobenzocyclooctin (MOBO) This is the method described in state 3b.

[0098] Embodiment 3l is an embodiment in which the alkyne group contains tetramethoxyDIBO (TMDIBO). This is the method described in morphology 3b.

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

[0100] Embodiment 4 is a configuration in which one of the first and second click reaction partners includes an alkene group, This is the method according to Embodiment 1, wherein the other rick reaction partner comprises a diene.

[0101] Embodiment 4a is the same as Embodiment 4, wherein the diene contains a tetrazine or tetrazole group. It is a method.

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

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

[0104] Embodiment 5 is an embodiment in which the polypeptide is an antibody or its antigen-binding fragment. The method is one of the methods described in 1-4c.

[0105] Embodiment 6 is an embodiment in which the antibody is a monoclonal antibody or an antigen-binding fragment thereof. This method is described in the method shown in Application Form 5.

[0106] Embodiment 6a is a modified polypeptide in which one or more azide groups are randomly added to the polypeptide. The method according to any one of Embodiments 1 to 6, obtained by compounding the following: ru.

[0107] Embodiment 6b is a modified polypeptide that reacts site-specifically with the first click reaction partner. Embodiments 1-6 are modified antibodies or antigen-binding fragments thereof obtained by ingestion. The method described in any one of the following ways.

[0108] Embodiment 6c is a modified antibody or its antigen-binding fragment that is the Fc-glycosyl of the antibody. Bacteria specific to β-1,4 bonds between core GlcNac residues (sometimes multiple) at the site of transformation. Endoglycosidase is used to trim the antibody or its antigen-binding fragment, and then trimmin To obtain a converted antibody or its antigen-binding fragment, and a glycosyltransferase, preferably G Trimmed antibody or antigen in the presence of alT galactosyltransferase The binding fragment is reacted with an azide sugar, preferably a UDP-GalNaz azide sugar substrate. This is the method of Embodiment 6b, obtained by doing so.

[0109] Embodiment 6d is a modified antibody or its antigen-binding fragment, The lagment is deglycosylated with amidase, and a deglycosylated antibody or its antigen-binding agent is used. To obtain fragments, and to microorganisms deglycosylated antibodies or their antigen-binding fragments In the presence of transglutaminase, azidoamine, preferably 3-azidopropylamine, is produced. This is the method described in Embodiment 6b, obtained by reacting with n.

[0110] Embodiment 6e is a method in which the antibody is used to target human prostate-specific membrane antigen (PSMA) or its antigen-binding funnel. An antibody that binds to the ligant, preferably the antibody has the sequence of SEQ ID NO: 3, HC CDR1 Sequence, HC CDR2 sequence of sequence number 4, HC CDR3 sequence of sequence number 5, sequence number 6 The light chain (LC) CDR1 sequence of sequence number 7, the LC CDR2 sequence of sequence number 8 The method according to any one of embodiments 6 to 6d, which includes a CDR3 sequence.

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

[0112] Embodiment 7 describes a scenario in which radioactive metal ions are present. 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 of embodiments 1 to 6d One method is to use this method.

[0113] Embodiment 7a is a radioactive metal ion 225 Ac is any of embodiments 1 to 6d. One method is as follows.

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

[0115] Embodiment 7c is a radioactive metal ion 89Zr is any one of embodiments 1 to 6d The method described above is one of the following.

[0116] Embodiment 8 is a chelated portion that interacts with a second click reaction partner via a linker. The method is described in any one of embodiments 1 to 7c, wherein the bodies are covalently bonded.

[0117] Embodiment 9 has a sulfhydryl group covalently bonded to the first click reaction partner, and a side Electrophiles on a chain, preferably on or introduced into a polypeptide. The amino side chain of din is reacted to obtain a modified polypeptide, preferably an NHS-azide. The method according to any one of embodiments 1 to 8, further comprising the and

[0118] Embodiment 10 describes a modified polypeptide that is directly or via a linker converted to azide, tetradi One of Embodiments 1 to 9, comprising a polypeptide covalently bonded to a tetrazole group or a polypeptide. One method is as follows.

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

[0120] [ka] Structure (In the formula, R1, R2, R3, and R4 are each independently CHQCO2X, Q is independently hydrogen, C1-C4 alkyl, or (C1-C2 alkyl)phenyl. the law of nature, X is independently hydrogen, benzyl, or a C1-C4 alkyl group. Z is (CH2) n Y is, n is between 1 and 10. Y is an electrophilic or nucleophilic moiety covalently bonded to the second click reaction partner. Alternatively, Z is hydrogen; and R1, R2, R3, and R4 are each independently CHQCO2X. Q is independently hydrogen, C1-C4 alkyl, or (C1-C2 alkyl)phenyl. the law of nature, X is independently hydrogen, benzyl, C1-C4 alkyl, or a second click reaction part (These are electrophilic or nucleophilic parts covalently bonded to the toner.) The method is described in any one of Embodiments 1 to 10, including the method described in Embodiments 1 to 10.

[0121] Embodiment 12 has a chelate portion of formula (II):

[0122] [ka] The method is described in any one of Embodiments 1 to 11, including the structure of [the specified element].

[0123] Embodiment 12a preferably comprises a chelating agent having an open-chain ligand in the chelating portion. The chelate portion is given by equation (III):

[0124] [ka] The method is described in any one of Embodiments 1 to 11, including the structure of [the specified element].

[0125] Embodiment 12b has a chelated portion that 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''''-pentavinegar Acids (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- Acid (TETA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-te Trapropionic 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) A method comprising a chelating agent selected from the group, any one of embodiments 1 to 10. That is the case.

[0126] Embodiment 12c is a chelating agent comprising 1,4,7,10-tetraazacyclododecane-N The method according to Embodiment 12b, comprising ,N',N'',N'''-tetraacetic acid (DOTA) be.

[0127] Embodiment 12d is an embodiment in which the chelating agent contains deferoxamine (DFO), and Embodiment 12b This is the method used.

[0128] Embodiment 13 uses radioactive metal ions, preferably 225 Ac, 111 In or 89 Zr A method for labeling polypeptides, preferably antibodies or their antigen-binding fragments, using [a specific method]. And, a. Polypeptides covalently bonded to azide, tetrazine, or tetrazole groups, or antibodies Alternatively, a modified polypeptide containing the antigen-binding fragment, preferably a modified antibody or To provide the antigen-binding fragment, b. Radioactive metal ions, preferably associated with the chelated moiety. 225 Ac, 111 In or 89 To provide a radioactive complex containing Zr, wherein the chelate portion is an alkyne. or comprising a chelating agent covalently bonded to an alkene group, c. The reaction of an azide, tetrazine, or tetrazole group with an alkyne or alkene group. By doing so, polypeptides or antibodies or their antigen-binding fragments are subjected to radioactive metal ions. Preferably 225 Ac, 111 In, or 89 Under conditions that allow labeling with Zr , by contacting a modified polypeptide or antibody or its antigen-binding fragment with a radioactive complex. and, The chelating agent is given by formula (I):

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

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

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

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

[0133] Embodiment 13d contains radioactive metal ions, preferably 225 Ac, 111 In or 89 Z A method of labeling a polypeptide, preferably an antibody or its antigen-binding fragment, using r. It is a law, a. Polypeptides covalently bonded to azide, tetrazine, or tetrazole groups, or antibodies Alternatively, a modified polypeptide containing the antigen-binding fragment, preferably a modified antibody or To provide the antigen-binding fragment, b. Radioactive metal ions, preferably associated with the chelated moiety. 225 Ac, 111 In or 89 To provide a radioactive complex containing Zr, wherein the chelate portion is an alkyne. or comprising a chelating agent covalently bonded to an alkene group, c. The reaction of an azide, tetrazine, or tetrazole group with an alkyne or alkene group. By doing so, polypeptides or antibodies or their antigen-binding fragments are subjected to radioactive metal ions. Preferably 225 Ac, 111 In, or 89 Under conditions that allow labeling with Zr , by contacting a modified polypeptide or antibody or its antigen-binding fragment with a radioactive complex. and, Here, the chelating agent comprises an open-chain ligand, preferably deferoxamine (DFO). , that is the method.

[0134] Embodiment 14 is an embodiment in which the chelating agent is covalently bonded to an alkyne or alkene group via a linker. This is a method according to any one of embodiments 13 to 13C.

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

[0136] Embodiment 16 describes a polypeptide, preferably an antibody or its antigen-binding fragment, Covalently bonded to the azid via a linker, as described in any one of embodiments 13 to 15. It is a method.

[0137] Embodiment 17 is a polypeptide which is an antibody or its antigen-binding fragment and is radioactive Metal ions, 225 Ac, 111 In or 89 The chelate part is Zr, and the chelate part is given by equation (II ):

[0138] [ka] This is the method according to Embodiment 13, which includes the structure of [the specified element].

[0139] Embodiment 17a is a polypeptide in which the antibody or antigen-binding fragment is emitted Metallic ions, 225 Ac, 111 In or 89 The chelate part is Zr, and the chelate part is given by equation (I II

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

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

[0142] Embodiment 17c is an antibody comprising the HC sequence of SEQ ID NO: 9 and the LC sequence of SEQ ID NO: 10. This is the method described in Embodiment 17b.

[0143] Embodiment 18 is a method for doubly labeling a polypeptide with two radioactive metal ions. hand, a. Covalently bonded to the first click reaction partner and the second click reaction partner To provide modified polypeptides including polypeptides, b. Provides a first radioactive complex containing a first radioactive metal ion associated with the chelate portion. In this process, the chelate portion is covalently bonded to a third click reaction partner. Contains a chelating agent, c. Provides a second radioactive complex containing a second radioactive metal ion associated with the chelate portion. In this process, the chelate portion is covalently bonded to the fourth click reaction partner. Contains a rate-setting agent, d. The first click response partner reacts with the third click response partner, and the second When a click reaction partner reacts with a fourth click reaction partner, poly Under conditions that allow the peptide to be labeled with first and second radioactive metal ions, modification The method includes contacting a polypeptide with first and second radioactive complexes.

[0144] Embodiment 19 is a configuration in which one of the first and second click reaction partners includes an alkyne group. The other of the first and second click response partners contains 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 The method according to Embodiment 18 is further comprising a diene.

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

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

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

[0148] Embodiment 21b is a therapeutic emitter, 32 P, 47 Sc, 67 Cu, 77 As, 89 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 The embodiment 20~ The method described in any one of 21a.

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

[0150] Embodiment 23 is a method for treating or diagnosing diseases or disorders, particularly neoplastic diseases or disorders. In a subject, a method for treating or diagnosing a disease or disorder, particularly a neoplastic disease or disorder. This method involves administering the composition of Embodiment 22 to a target.

[0151] Embodiment 24 is a pharmaceutical composition comprising two compositions that are administered sequentially, wherein the first composition is Embodiment 23 comprises a modified polypeptide, and the second component may be a radioactive complex (or multiple complexes). This is the method used.

[0152] Embodiment 25 was prepared by the method described in any one of Embodiments 1 to 21b. The present invention comprises a radiolabeled antibody and a pharmaceutically acceptable carrier, and the immunological properties of the radiolabeled antibody. It is a theranostic agent that is preserved.

[0153] Embodiment 26 is a diagnostic emitter in which radioactive metal ions are preferably 89 Zr is actual This is the theranostic agent described in application method 25.

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

[0155] Embodiment 27a is a radioactive metal ion 111 In the case of the ceramic described in embodiment 25, It is a nostic agent.

[0156] Embodiment 27b is a polypeptide that is an antiprostate agent of human prostate-specific membrane antigen (PSMA) or its antiprostate agent. The antibody binds to the proto-binding fragment, and preferably the antibody is an HC of the sequence of SEQ ID NO: 3. CDR1 sequence, HC CDR2 sequence of sequence number 4, HC CDR3 sequence of sequence number 5, The light chain (LC) CDR1 sequence of sequence number 6, the LC CDR2 sequence of sequence number 7, and the sequence number The cerano according to any one of embodiments 25 to 27a, which includes the LC CDR3 sequence of No. 8. It is a stick-type formulation.

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

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

[0159] [ka] It has the formula (IV) (DOTA-Ac-DBCO-protein), or, 22 5 Ac is 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 Embodiments 25-27c are substituted with other radioactive metal ions such as In. It is a theranostic agent as described in one of the following.

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

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

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

[0163] [ka] It has the formula (VI) (DFO-Zr DBCO-protein), or, 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, which are substituted with another radioactive metal ion such as In. It is a theranostic agent as described in one of the documents.

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

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

[0166] Embodiment 27h has a radiolabeled antibody with a chelator:antibody ratio (CAR) of less than 3. The theranostic agent described in any one of Embodiments 25 to 27 g.

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

[0168] Embodiment 28 is, a. Modified polypeptide containing a polypeptide covalently bonded to the first click reaction partner Do and, b. A radioactive complex containing a radioactive metal ion associated with the chelate portion, A radioactive complex containing a chelating agent whose chemical moiety is covalently bonded to a second click reaction partner. With regard to combinations or preferably kits including, Here, the combination is used to label polypeptides with radioactive metal ions. .

[0169] Embodiment 28a is a combination of Embodiment 28 or a chelating agent that includes a macrocyclic molecule. It is a set.

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

[0171] Embodiment 29 describes the reaction between the first and second click reaction partners in vitro. Embodiment 28 is used to label polypeptides with radioactive metal ions via This is the combination or kit described above.

[0172] Embodiment 30 describes the reaction between the first and second click response partners in vivo. Embodiment 28 is used to label polypeptides with radioactive metal ions via The combination or kit is as described.

[0173] Embodiment 31 includes a polypeptide covalently bonded to a first click reaction partner. This is a composition containing a decorative polypeptide.

[0174] Embodiment 32 includes a radioactive complex containing radioactive metal ions associated with the chelated portion. A composition in which the chelated portion is covalently bonded to a second click reaction partner. It is a composition containing an antiseptic agent.

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

[0176] Embodiment 32b is the composition according to 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 its antigen-binding fragment. A combination or kit described in any one of embodiments 28 to 30, or embodiment 31 or k is the composition described in 32.

[0178] Embodiment 33a is an antibody that targets human prostate-specific membrane antigen (PSMA) or its antigen-binding agent. The antibody can bind to the lagment, and preferably the antibody is an HC CDR of the sequence of SEQ ID NO: 3. Sequence 1, HC CDR2 sequence of sequence number 4, HC CDR3 sequence of sequence number 5, sequence number The light chain (LC) CDR1 sequence of sequence 6, the LC CDR2 sequence of sequence number 7, and the L of sequence number 8. The method according to embodiment 33, comprising a C CDR3 sequence.

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

[0180] Embodiment 33c is an antibody or its antigen-binding fragment that is covalently bound to an azide group. The combination or kit or composition described in Embodiment 33a or 33b.

[0181] Embodiment 33d is an antibody or its antigen-binding fragment randomly shared with an azide group. The combination, kit, or composition is that of Embodiment 33c.

[0182] Embodiment 33e is a model in which an antibody or its antigen-binding fragment is site-specifically coupled to an azide group. This is a combination, kit, or composition of Embodiment 33c that is bonded together.

[0183] Embodiment 33f is a method in which an antibody or its antigen-binding fragment is used to perform Fc-glycosylation of the antibody. Bacterial enzymes specific to β-1,4 bonds between core GlcNac residues (sometimes multiple) at the site The antibody or its antigen-binding fragment is trimmed with an endoglycosidase. To obtain an antibody or its antigen-binding fragment, and a glycosyltransferase, preferably Ga In the presence of lT galactosyltransferase, trimmed antibodies or their antigenic ligations The composite fragment is reacted with an azide sugar, preferably a UDP-GalNaz azide sugar substrate. The combination according to Embodiment 33e is covalently bonded to the azide group via a method including the following It is a combination, kit, or composition.

[0184] Embodiment 33g is a modified antibody or its antigen-binding fragment that binds to the antibody or its antigen. The fragment is deglycosylated with amidase, and then a deglycosylated antibody or its antigen is bound to it. To obtain a fragment, and to micronize the deglycosylated antibody or its antigen-binding fragment. In the presence of biological transglutaminase, azidoamine, preferably 3-azidopropylamine, is produced. The combination according to Embodiment 33e, obtained by a method including reacting with min. Alternatively, it may be a kit or a composition.

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

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

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

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

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

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

[0191] Embodiment 37a preferably comprises a chelating agent having an open-chain ligand in the chelating portion. The chelate portion is given by equation (III):

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

[0193] Embodiment 38 is an embodiment in which the polypeptide is covalently bonded to the azide via a linker. The combination, kit, or composition described in any one of Forms 34 to 37.

[0194] Embodiment 39 is a method for treating or diagnosing diseases or disorders, particularly neoplastic diseases or disorders. A law relating to the theranostic agent described in any one of embodiments 25 to 27d, or an actual This includes administering the combination described in any one of the administration methods 28 and 33-38. Hmm, that's the method.

[0195] Embodiment 40 describes a disease or disorder, particularly a neoplastic disease or disorder, and a disease or disorder, particularly a tumor. A method of treatment or diagnosis in a person requiring treatment or diagnosis of a ulcerative disease or disorder. This includes administering the composition of Embodiment 31 and the composition of Embodiment 32 to the target, preferably Alternatively, the polypeptide is an antibody, or this is the method. [Examples]

[0196] The following embodiments of the present invention are intended to further illustrate the essence of the present invention. The examples are not intended to limit the present invention, and the scope of the present invention is defined by the appended claims. I hope you understand this.

[0197] Example 1: Random conjugation of azide / handle to antibody Monoclonal antibody (mAb): Represented as "PSMB127," and referred to herein as "anti-PS." Human IgG4 that binds to human prostate-specific membrane antigen (PSMA), referred to as "MA mAb". The antibody consists of the heavy chain (HC) CDR1 sequence of sequence number 3 and the HC CDR2 sequence of sequence number 4. Column, HC CDR3 sequence of sequence number 5, light chain (LC) CDR1 sequence of sequence number 6, sequence number It has the LC CDR2 sequence of sequence number 7 and the LC CDR3 sequence of sequence number 8, and sequence number It has the HC sequence of sequence number 9 and the LC sequence of sequence number 10. It expresses an anti-PSMA mAb and targets Purification was performed using quasi-chromatography.

[0198] The human IgG4 S228P anti-PMSA mAb referred to herein as the "control mAb" The / F234A / L235A (IgG4-PAA) antibody isotype control is SEQ ID NO: 1 It has an HC sequence and an LC sequence of SEQ ID NO: 2. A commercially available antibody, trastuzumab (Herceptin) ), cetuximab (Erbitux), pertuzumab (Perjeta), and panitumumab (Vectibix) are Roche, Lilly, Roche, and Amgen respectively. I purchased it. Mouse anti-human Her2 mAb from BioXCell (catalog number BE02). Obtained from 77). Trastuzumab, pertuzumab, and anti-human Her2 mAb are human It binds to Her2. Cetuximab and panitumumab bind to human EGFR.

[0199] Compounding: 10 mM sodium acetate pH 5.2, phosphate-buffered saline pH 7, or other Add the antibody stock solution (1-10 mg / mL) in the compatibility buffer to a 20% (weight / weight) solution. Mixed with M sodium carbonate buffer pH9 to achieve a final pH of approximately 9. NHS-PEG Dissolve 4-azide (Thermo catalog number 26130) in DMSO to a final concentration of 1 Dilute to 00 mM, add 0.2% (weight / weight) of the stock solution, and add approximately 3 to 10 mAbs. A molar excess was generated. After incubating the reaction mixture at 22°C for 10 minutes, add 1M Tris Quenching was performed while adding (pH 7.5) to a final concentration of 50 mM Tris.

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

[0201] LC / MS analysis: Chelate: Antibody ratio (CAR) measured using Agilent PLRP-S color chromatograph. M (300-Angstrom, 2.1x150mm, catalog number PL1912-33) LC / MS analysis using an Agilent G6224 MS-TOF instrument equipped with 01) This was determined by (Table 1). The mass spectrum was obtained by m / for masses of 140-170 kDa. Using the maximum entropy algorithm over the z range of 2000 to 3200, decompression I applied the solution.

[0202] Analytical size exclusion chromatography (SEC): Analytical SEC is performed to isolate antibodies and antibodies We determined the oligomeric state of the complex and confirmed that the complex formation process did not result in aggregation. I did. Tosoh TSKgel G3000SWxl (Tosoh Bioscien Agilent 1200 series with 7.8mm x 30cm column (ce #08541) A Leeds HPLC was used. Mobile phase: 1×PBS, flow rate: 0.8 ml / ml, injection volume: 15 μl L, protein concentration 0.1-2 mg / mL.

[0203] [Table 1]

[0204] Example 2: Random conjugation of azide / handle to non-antibody polypeptide Non-antibody polypeptides: transferrin, human holo-transferrin, R&D S Purchased from ystems (catalog number 2914-HT), dissolved in water to make a concentration of 10 mg / mL. This was done. EGF, which is human epidermal growth factor, was used in Sino Biological I purchased it from (catalog number 10605-HNAE).

[0205] Compounding: 10 mM sodium acetate pH 5.2, phosphate-buffered saline pH 7, or other The stock solution of polypeptide (1-10 mg / mL) in the compatible buffer is diluted to 20% (by weight / weight). Mix with 1M sodium carbonate buffer pH9 (amount) to achieve a final pH of approximately 9. NHS- PEG4-azide (Thermo catalog number 26130) was dissolved in DMSO to obtain the final concentration. Dilute to 100 mM, add 0.2% (weight / weight) of the stock solution, and add approximately [amount missing] to the protein. A molar excess of 3-10 was generated. After incubating the reaction mixture at 22°C for 10 minutes, a 1M solution was produced. The final concentration was 50 mM while quenching with Tris pH 7.5. The compounding efficiency was... This is shown in Table 2.

[0206] [Table 2]

[0207] Example 3: Site-specific incorporation of azido sugar into antibody glycan The antibody glycan contains the core GlcNac residue within the Fc glycosylation site (there may be multiple sites). GlycINATOR is a bacterial endoglycosidase specific to β-1,4 linkages between groups. (Genovis) trims the image and later uses it for site-specific incorporation of azidosaccharides. This was achieved by leaving the innermost GlcNac on Fc. More specifically, the column GlycINATOR immobilized on agarose beads filled with (Genovis) It was equilibrated in Tris-buffered saline pH 7.4 (TBS). 5-10 mg / mL Add 1 mL of the mAb to the resin and incubate on a rocker at room temperature for 1 hour. Elution was achieved by rotating the column at 100Xg for 1 minute. The column was then eluted with 0.5 mL of TBS. Elution was performed three times. The eluate containing the trimmed mAbs was pooled and supplied with buffer. The additive (Genovis) is used as a UDP-GALNaz azide sugar substrate and GalT galactosin It was added together with the transferase enzyme. The reaction mixture was stirred overnight at 30°C. A precise azide mAb is extracted using an mAb selection column (GE) in an AKTA Avant instrument. The product was manufactured. Azide modification was confirmed by LC-MS, and it was determined that the CAR was exactly 2.

[0208] Example 4: By microbial transglutaminase (MTG) of 3-azidopropylamine Site-specific implantation As described, the azide group is essentially site-specifically positioned at position Gln295 relative to the antibody. (Dennler et al. 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 mAbs, with the innermost GlcNac residue and high mannose, hybrid The cleavage between the rhizome and the asparagine residue of the complex oligosaccharide is preserved (for example, Both Fc Asn297 and non-preserved (e.g., Fab N-glycan) Complete deglycosylation of all N-glycans, including N-glycans derived from the lycosylated site. Rapid PNGase F (New Deglycosylated using England Biolabs. Sodium acetate buffer ( 10 mL of 1 mg / mL antibody in pH 5.2 was mixed with 5 μL of PNGase F in a 37-degree Celsius solution. The solution was incubated overnight at °C, and deglycosylation was confirmed by LC-MS. PNGase F was removed by 4 cycles of enrichment, and then diluted with an Amicon device (50kDa cutoff). It was explained. A compound of 3-azidopropylamine (3-APA; click chemistry tool). For the chemical treatment, we added 2% (by weight) of 0.5M HEPES pH 7.5. Therefore, the deglycosylated mAb (0.5-1 mg / mL) was adjusted to a pH of 7-7.5. 100 Equivalent amounts of 3-APA are used with activin TI transglutaminase (Ajino), which is MTG. The reaction mixture was added along with 5-10% by weight / volume. The reaction mixture was incubated at 37°C for 1-4 hours. After vaping, use standard chromatography on an mAbSelect Sure column. Azide-modified mAbs were purified. The complex was manually characterized by LC-MS, and the CAR was 2 It was determined that this was the case.

[0209] Example 5: Chelation of radioactive metals into a bifunctional chelator (BFC) 225 Synthesis of Ac-DOTA-Ga-DBCO: 225 Ac(NO3)3 to Oak R Purchased from Idge National Laboratory. 1,4,7,10- Tetraazacyclododecane, 1-(glutaric acid)-4,7,10-triacetate-(3-amine Custom blend of no-propanoic acid dibenzocyclo-octin (DOTA-Ga-DBCO) Successfully achieved. The synthesis was described in Bernhard et al. Chem. Eur. J. 2012, 18. Based on 7834-7841. DBCO-amine(3-amino-1-[(5-aza- 3,4:7,8-Dibenzocycloocta-1-in)-5-yl]-1-propanone(S igma was reacted with DOTA-GA anhydrous, and the product was purified by reverse-phase HPLC.

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

[0211] 111 Synthesis of In-DOTA-GA-DBCO: in 0.05M HCl 111 Inc I purchased l3 from GE Healthcare. Tetraacetic acid in a plastic vial. Methylammonium (1M 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) In the solution of (L), add 0.05N HCl 111 InCl3 (5 μL, Capintec) Measurements using the CRC-55TW dose calibrator showed 104.3 μCi and 0.0022 nm. (ol) was added. The pH of the mixture was observed to be approximately 5.5 using pH paper. Place the vial on a shaking block at 60°C and 290 rpm for 30 minutes, then allow it to return to room temperature. It was cooled.

[0212] 89 Synthesis of Zr-DFO-DBCO: 89 Zr oxalate from 3D Imaging I bought it. DFO-DBCO is Macrocyclics (Plano, Texas, Catalonia). Purchased from log number B-773), dissolved in DMSO to 0.5 mg / mL, and diluted with water for 25 minutes. Diluted to μg / mL.

[0213] Transfer 2 mCi of Zr-89 to a metal-free microcentrifuge tube and add 1 M oxalic acid. The total volume was then adjusted to 80 μL. 12 μL of 2M potassium carbonate was added in 2 μL increments and mixed. The mixture was stirred at the tip of the pipette until bubbling stopped. Next, 120 μL of 1M HE was added. PES was added, followed by 300 μL of water. The pH of the solution was tested, and p was adjusted as needed. An additional 2M potassium carbonate was added to raise H to 6-6.5. 136 μL Add DFO-DBCO stock (3.4 μg) and incubate the reaction mixture at room temperature for 1 hour. I did that. I spread 0.5 μL of the reaction mixture onto a TLC Green strip (Biodex). The sample was then eluted with 20% NaCl for analysis. After elution, most of the Zr-89 was found to be present. To ensure that it stays within the baseline, PerkinElmer Cyclon The strip was scanned with the e Plus phosphor imager, and the DFO-DBCO was used to capture the image. The rating system was shown.

[0214] 89 Synthesis of Zr-DOTA-GA-DBCO: In water, Sep-pak Light Q MA Strong Anion Exchange Cartridge (Acrylic Acid / Acrylamide Copo) 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) [The substance] was added, followed by 0.9% physiological saline (10 mL), and then water (10 mL). In 0.0M oxalic acid (2 μL, 290 μCi)89 A solution of Zr(ox)2 is prepared by Precond It was added to the ionized cartridge. Then, the cartridge was put in deionized water (20 ml). After washing to remove excess oxalic acid, use 1.0 M HCl (aqueous solution) (100 μL each). 89 ZrCl4 was eluted from the column, with a recovery rate of 248 μCi (86%) and a total volume of 400 μCi. A μl was obtained, and the majority of the radioactivity was in fraction 3. The combined organic extract was then evaporated to dryness. It was dry and hardened.

[0215] 10 μL DOTA-GA-DBCO (1.0 mg / mL metal-free water, 10 μL (g, 13.6 nmol) 89 It was added to ZrCl4 (268 μCi, 50 μL). 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., Zirconi 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):p.2309-2314). Then, incubate the solution at 90°C for 60 minutes. It was incubated. 89 The yield of the Zr-DOTA-GA-DBCO complex is 1% NH4OH SPC25 column eluted in solution (Sigma Aldrich part number SPC251) It was determined to be 98% by 20-50G. (Not chelated) 89 Zr is on the column It remains there,89 The Zr-DOTA-GA-DBCO complex is eluted.

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

[0217] Anti-PSMA mAb- in PBS or other compatible buffer (10-20 mg / mL) Dibenzo-[1,2,3]-triazoloazosin-Ga-DOTA-225Ac (site-specific) Heterogeneous, CAR=2): Random or site-specific azide-modified antibody (site-specific, CAR=2) Alternatively, randomly generated (average CAR of 1-4) as described. 225 Ac-DO It was added to a solution of TA-GA-DBCO. The final pH of the mixture was approximately 6.5 according to pH paper. The reaction solution was gently stirred, and 15 mL of NaOAc buffer was added, 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 purification at GE Healthcare. Transfer the eluate to the reservoir of the pre-conditioned PD-10 column using a pipette. The reaction was collected in a plastic tube. The reaction vial was washed with NaOAc buffer (0.2 ml). The washing solution was pipetteed into the reservoir of the Pd-10 column (three times with L), and the eluate was collected. NaOAc buffer is continuously applied to the reservoir of the PD-10 column, and the eluate is poured into a plastic container. Collect the eluate in a plastic tube, 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 recovered fraction is determined using a citrate-H2O-MeOH solution as the mobile phase. Evaluated using iTLC-SG (Agilent). Pure fraction (may contain multiple fractions). The final product was obtained by combining the two components in 10 mM NaOAc buffer. The product solution was then chemically mixed. The chemical and radiochemical purity was analyzed by HPLC. Using a standard curve, the generated The antibody concentration in the solution was measured by UV absorption. Then, Capintec CRC-5 The radioactivity of the product solution was quantified using a 5TW dose calibrator.

[0219] AC-225 chelation quality control: Diethylene triphosphate is used as a quality control measure for the purified product. A chelation challenge using amine pentaacetic acid (DTPA) was performed. 10 mM Na5 DTPA aqueous solution, 225 Add to a sample solution containing Ac-labeled mAb and [DPT A / [mAb] was set to 500-1,000. A 50 mM Na5DTPA aqueous solution was used. 2 25 Add to an aliquot of the purified product in a solution containing Ac-labeled mAb, and [DPTA The mAb / mA ratio was set to 50,000-100,000. The two mixtures were heated at room temperature and 29 The mixture was placed on a shaking block at 0 rpm for 30 minutes. The mixture was then spotted onto the iTLC-SG. The cell was expanded using citrate-H2O-MeOH as the mobile phase. Under these conditions, free 225 Ac moved to the solvent front and bound 225 Ac-mAb remains at baseline.

[0220] Anti-PSMA mAb-dibenzo-[1,2,3]-triazoloazosin-GA-DOT A- 111 Synthesis of In: Random or site-directed azide-modified antibodies in 10 mM NaOAc PSMA mAb (site-specific, CAR=2 or random, mean CAR of 1-4) Generated exactly as shown. 111 It was added to a solution of Ac-DOTA-GA-DBCO. The solution was gently stirred and left at room temperature for 2 hours before passing it through the PD-10 column. A PD-10 column is used with 15 mL of NaOAc buffer, 10 mM, pH 6, depending on the column. The solution was passed through 6.5°C for preconditioning, and then the washing solution was discarded. Next, the reaction was mixed. The substance is pipetted into the reservoir of a pre-conditioned PD-10 column and dissolved. The effluent was collected in a plastic tube. The reaction vial was washed with NaOAc buffer (0. Transfer the washing solution (2 mL, 3 times) to the reservoir of the PD-10 column using a pipette, and collect the eluate. The NaOAc buffer was continuously applied to the reservoir of the PD-10 column, and the eluate was obtained. The eluate is collected in plastic tubes, and approximately 1 mL of eluate is added to each tube until a total of 10 mL of eluate has been collected. The liquid was collected.

[0221] The purity of each recovered fraction was determined using a 10 mM EDTA aqueous solution (pH=5~6) as the mobile phase. The samples were evaluated using iTLC-SG. The pure fractions (which may include multiple fractions) were combined. The final product was obtained in 10 mM NaOAc buffer. The product solution was chemically and radioactively analyzed. Linear chemical purity was analyzed by HPLC. Using a standard curve, the antibody in the product solution was analyzed. Body concentration was measured by UV absorption. Then, Capintec CRC-55TW dose meter The radioactivity of the product solution was quantified using a calibrator.

[0222] In-111 chelation quality control: DTPA is used for quality control of the purified product. Using a chelation challenge: a 10 mM Na5DTPA aqueous solution was used. 111 In marker When added to a sample solution containing a recognition mAb, the ratio of [DTPA] / [mAb] = 1000~ The value was set to 10,000. The mixture was placed on a shaking block at room temperature and 290 rpm for 30 minutes. The mixture was spotted onto an iTLC-SG, and a 10 mM EDTA aqueous solution (pH=5~6) was added. ) was deployed as the mobile phase. 111 In or loosely bonded 111 Transfer to the solvent front. Moved and firmly connected 111 In-mAb remains at baseline.

[0223] Anti-PSMA mAb-dibenzo-[1,2,3]-triazoloazosin-DFO-ZR -89 synthesis 10 mM HEPES, 50 mM NaCl, pH 7.5, or other compatible buffer. Randomly modified anti-PSMA mAbs (average CAR) within the same site, similar procedure (Can be administered by heterozygous azide-mAb) 800 μg, prepared as described. Achieved 89 Add to Zr-DFO-DBCO solution and incubate at 37°C for 1.5 hours. Afterward, the sample was passed through a PD-10 column. The PD-10 column was then passed through 15 mL of isotonic saline. The mixture was passed through the column and the washing solution was discarded. Next, the reaction mixture was pre-conditioned. The eluate was pipetteed into the reservoir of the PD-10 column, and the eluate was collected in a plastic tube. Physiological saline was continuously applied to the reservoir of the PD-10 column, and the eluate was extracted into a plastic tube. Collect the eluate in one tube, and 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 the material remaining on the column was measured using a dose calibrator. The peaks of the product, usually fractions 4-7, were pooled to obtain the final product. The product solution was chemically analyzed. The radiochemical purity was analyzed by HPLC. Using a standard curve, the product solution was analyzed. The antibody concentration in the solution was measured by UV absorption. A dose calibrator was used to measure the concentration of the product solution. The radioactivity was quantified.

[0224] Anti-PMSA mAb-DOTA- 89 Zr synthesis 20mMHEPES 50mM NaClpH7.5 (10.1mg / mL, 200μ Azide-modified antiPS modified by random azide complexation in L (approximately 13.5 nmol) MA mAb complexes (CAR=1~4) were generated as described above. 89 Zr-DOTA- It was added to the GA-DBCO solution. The final pH of the mixture was adjusted to 7.0. 3 Incubate at 7°C for 2 hours, then add 0.9% physiological saline, HEPES buffer, or It was purified using either PBS and a PD-10 column (GE Healthcare), and 6 4% 89 Zr-DOTA-mAb was obtained.

[0225] Quality control of Zr-89 chelation: For Zr-DFO-mAb, the purified product... These were analyzed by HPLC alone. These complexes were challenged with DTPA or EDTA. Sometimes Zr-89 was not retained. Zr-DOTA-mAb with EDTA at 33mM. The sample was then added and incubated overnight at room temperature. The sample was then passed through a PD-10 column. Analysis of the complex after the challenge revealed that it retained 80% of its radioactivity. Ta.

[0226] Example 7: Analytical characterization of click-labeled radioactive composites Determination of radiochemical transformation Radiochemical conversion (%RA conversion; see Tables 3-5) is performed using iTLC-SG (silica gelatin). Binderless glass microfiber chromatography paper impregnated with SG (Sulfur) is used. The %RA conversion value was determined by simple thin-layer chromatography (iTLC). The product of the radiation signals of the product peaks (with different retention times for the radioactive starting material and by-products) The minute value is calculated by multiplying all radiation signal peaks present between the baseline and the solvent front. It is calculated by dividing by the value obtained when dividing. Then, the proportion of this product This is expressed as a conversion rate.

[0227] For products incorporating Ac-225, the amount is approximately 0.1 to 1 μCi. 225 Includes Ac Place the sample solution onto the baseline of the iTLC-SG strip, approximately 2 cm from the bottom edge. The iTLC-SG strip was placed in a citric acid-water-methanol mobile phase (20 mL 0 Use 0.4M trisodium citrate / 3mL and 2N HCl / 2.3mL (MeOH). The samples were spread out, dried at room temperature, and stored for at least 6 hours before analysis (225Ac and all daughter nuclides). (It reaches its secular equilibrium). Using the 99mTc setting, the Bioscan AR2000 emission iTLC-SG was scanned using a linear TLC imaging scanner.

[0228] For In-111 chelate, approximately 2 cm from the bottom edge of the iTLC-SG strip... At the baseline of the pu, approximately 0.5 to 2.5 μCi 111 A sample solution containing In is spotted. I used 10 mM EDTA pH 5-6 as the mobile phase and iTLC-SG strip. After unfolding, it was dried at room temperature. Using the In-111 setting, Bioscan A Using the R2000 radioactive TLC imaging scanner, we scanned the dry iTLC-SG. Ta.

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

[0230] Determining the radiochemical purity of radiolabeled proteins. Radiochemical purity (%RA purity) of Ac-225 and In-111 chelates (see Tables 3-4). The (to be illuminated) was determined by SE-HPLC (size exclusion HPLC). Ac-22 Regarding item 5, Tosoh TSKgel column (G3000SW x 17.8mm x 30 Using a column (cm, 5μm), the column was prepared with DPBS buffer (×1, calcium and magnesium) Elution was performed using a hydrate (without um). Flow rate: 0.7 mL / min, operation for 20 minutes, room temperature. HPLC Next, the eluate is collected in pre-numbered vials, and each vial is subjected to an elution process for 0.5 minutes or 1 minute. The liquid fraction was recovered. The vial containing the eluate was left at room temperature for more than 6 hours. 225 That This made it possible to reach a permanent equilibrium with the daughter nuclide. Then, the radioactivity in each vial was adjusted by Ca Counted using a Pintec CRC-55TW well counter. Based on the radioactivity in the vial. Then, the radioactive chromatogram was reconstructed.

[0231] Regarding In-111, it is a Tosoh TSKgel column (G3000SW×17). A column (8mm x 30cm, 5μm) was used. The column was treated with DPBS buffer (x1, calcium Elution was performed using a solution (without um and magnesium). Flow rate: 0.7 mL / min, operation for 20 minutes. Room temperature. The above HPLC system and Perkin Elmer radioactive flow detector Rad Radioactivity detection is performed using the iomatic 625TR, with In-111 settings. Ultima Flo™ for use with a 0.5 mL flow cell and a flow rate of 1.4 mL / min. They offer M cocktails.

[0232] For Zr-89 (see Table 5), use the Tosoh TSKgel column (G A 3000SW x 17.8mm x 30cm (5μm) column was used. Elution was performed with physiological saline. Flow rate: 1 mL / min, operation for 20 minutes, room temperature. The above HPLC system... Beckman flow connected to the stem and Bioscan Flow Count instrument - A flow-through detector was used to detect radioactivity.

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

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

[0235] [Table 5]

[0236] Example 8: Click reaction between modified mAb and DOTA-GA-DBCO Random and site-specific azide mAbs (anti-PSMA mAbs) of 1-10 mg / mL Cetuximab, panitumumab, tratuzumab, and pertuzumab are administered in excess of 5 to 20 times the normal dose. Mix with unchelated DOTA-GA-DBCO and leave at room temperature or 37°C for 1 to 24 hours. Intermittent incubation was performed. The mAb was desalted using a Zeba desalting and dehydration column (Thermo), and A The solution is concentrated using a microcentrifuge (Millipore), then re-diluted with buffer, and concentrated again. It was reduced and all remaining DBCO-DOTA was removed. All free azide DBCO-DOTA The complete click response with TA was confirmed by LC-MS.

[0237] Example 9: Click reaction between modified mAb and DFO-DBCO Random and site-specific anti-PSMA mAb azide-mAbs at 1-10 mg / mL, Mix with 5 to 20 times the excess unchelated DOTA-GA-DFO, at room temperature or The mAbs were incubated at 37°C for 1 to 24 hours. The mAbs were then desalted and dehydrated using a Zeba desalting column (The Desalting was performed using RMO, and the solution was concentrated using an Amicon centrifuge (Millipore), followed by buffering. The solution was re-diluted and then concentrated again to remove all remaining DBCO-DFO. All free argillaceous ash was removed. The complete click reaction between D and DBCO-DFO was confirmed by LC-MS.

[0238] Example 10: Cell Binding (FACS) Azide-modified antibodies, DOTA-DBCO-azide-modified antibodies, and DFO-DBCO-azide-modified antibodies. The cell binding of modified antibodies was compared to that of the parent mAbs of the complexes listed in Table 1. The cell line was treated with an antibody or complex within a specified concentration range, and the binding was determined by flow cytometry. The measurement was performed by applying panitumumab and cetuximab complexes to EGFR+A431 cells. We evaluated the following: Herceptin and pertuzumab were administered to HER2+SK-BR-3 cells. The binding was evaluated. The binding of anti-PSMA mAbs to PSMA+C4-2b cells was evaluated. I evaluated it.

[0239] Cell line: C4-2B cells, human prostate cancer cell line, Janssen Oncology ( Obtained from Springhouse, Pennsylvania. A431 is a human epidermal carcinoma cell line. Cells, and the human breast cancer cell line SK-BR-3 cells, were originally used in ATCC (Virginia Janssen BioTherapeutics uses cells derived from Manassas, Argentina. Obtained from Springhouse, Pennsylvania. EGFR receptor-negative MOLM-1 3. Human acute myeloid leukemia suspension cells were supplemented with 20% heat-inactivated fetal bovine serum (Gibco). The cells were maintained in RPMI1640 + 25mM Hepes (Gibco). RPMI1640+25m including %FBS (Gibo, Waltham, Massachusetts) It was propagated using M HEPES (Gibo, Waltham, Massachusetts).

[0240] Flow cytometry: Enzyme-free cell dissociation buffer (Gibco, Massachusetts) Detach the cells from the flask using a 40 μm filter (Fa) (Waltham, Setts). Filtered with lcon. 5 x 10 per well on a 96-well U-bottom plate. 4 individual cells The cells were seeded. The cells were stained with BSA staining buffer (BD Bioscience, California). Incubate the combined antibody or parent antibody, diluted in San Jose, Niagara, at 4°C for 1 hour. The cells were washed twice with staining buffer. Then, the cells were left in the dark at 4°C for 30 minutes. AlexaFluor647 tagged anti-human IgG secondary antibody (Jackson Immuno) Incubated with noResearch Laboratories. Secondary antibody The body contains 3% donkey serum (Rockland Immunochemicals). Diluted to 1:200 with staining buffer. During the last 10 minutes of incubation, S YTOX (trademark) Green Nucleic Acid Stain (ThermoF The cells were treated with isher at a final concentration of 30 nM. The cells were washed twice with staining buffer. Afterward, resuspend in a final volume of 25 μL / well in staining buffer and use iQue Scree The data was read using a NER flow cytometer (Intellicyt). To exclude events with high nucleic acid staining, ForeCyt software was used. Viable cells were determined from this. Mean fluorescence intensity (MFI) was used. Determined in cells, the logarithm of antibody concentration vs. MFI is obtained using GraphPad Prism 7(G The graph was created using raphPad Software. A nonlinear regression curve was fitted to the data. In addition, EC 50 The value was calculated.

[0241] For all mAbs and complexes tested, the parent mAb and modified mAb showed similar cell aggregation. The results were shown (Table 6).

[0242] [Table 6]

[0243] Example 11: In-111 cell binding assay Cell binding was measured radiometrically using the In-111 radiolabeled protein listed in Table 4. Measurements were taken using method (I). Anti-PSMA mAbs and transferrin were used on C4-2B cells (PSMA). Tested with + and transferrin+) Cetuximab and panitumumab were tested with A431. The study was conducted on EGFR-positive (EGFR+) and MOLM-13 (EGFR-) cells.

[0244] Adherent cells were detached using enzyme-free cell dissociation buffer (Gibco). The cells and recovered suspension cells were counted and stained with cold staining buffer (BD Biosciences Washed with ). Various numbers of cells in 200 μL of staining buffer were added to a microcentrifuge tube. They were placed on ice. 0.5 μCi of In-111 labeled protein was added to each tube and left on ice for 1 The cells were incubated for 2 hours. The cells were washed with cold PBS (Gibco) to remove unbound antibodies. The sample was then resuspended in 500 μL of cold PBS. The sample was transferred to a counting vial and gamma counted. Cell-associated emission (Hidex Automatic Gamma Counter) The radiation power was measured.

[0245] The counts per minute (CPM) from the test sample are calculated using a known amount of In- Using linear regression constructed with 111-labeled protein, the CPM value was used in In-1 Converted to 11 μCi. The μCi boundary value was calculated using the following method to determine the molar bond (Moles Converted to Bound: (μCi binding / specific activity) / MW mAb or protein. Each data point represents the mean ± SD of the triple reaction.

[0246] Click-labeled In-111 anti-PSMA mAb and In-111 transferrin are It bound to C4-2B cells, and cell-associated radioactivity increased as the number of cells increased (Figure 3A). Panitumumab and cetuximab, which are Rick-labeled In-111 anti-EGFR antibodies, are A431. It bound to cells, and as the number of cells increased, cell-associated radioactivity increased. (Negative control: MOLM-13) In cells, specific binding of the click-labeled anti-EGFR antibody was not detected (Figure 3B).

[0247] Example 12: Indium Cell Uptake Assay Dynamics of In-111 click-labeled anti-PSMA mAb internalization in C4-2B cells We made that decision.

[0248] Place the cells in a 60mm dish (Corning), 3 x 10 6 Seed in cells, 37 The samples were placed overnight in a humidified CO2 incubator at °C. The seeding medium was removed, and 2 mL of cold staining was placed in a container. Replaced with fur (BD Bioscience). Then, place the dish on ice. 0.5 μCi of In-111 labeled antibody was added to each dish, and the ink was left on ice for 1 hour. The cells were vaccinated. The cells were washed with cold PBS (Gibco) to remove unbound antibodies from the cell surface. They left. As described below, at various points in time, the cells were subjected to surface membrane-bound radioactivity and intracellular emission. We performed an assay to assess the radioactivity.

[0249] Surface-bound radioactivity was stripped using an acid washing and stripping procedure: 1.5 mL Stripping buffer (50ml glycine, 150ml NaCl pH 2.7, Add pepsin (Amresco) to cells at a concentration of up to 25 μg / mL, and The mixture was incubated on ice for 15 minutes. Stripping buffer was added to the counting vial. The cells were transferred to a gamma meter. The cells were washed with cold PBS, and the washing solution was transferred to a counting vial. Radioactivity was measured using a gamma meter. The assay was performed using the Hidex Automatic Gamma Counter. The surface film-bound radioactivity was determined as the sum of the stripping buffer and PBS washing solution. Ta.

[0250] Intracellular radioactivity was assayed by preparing cell lysates: surface-bound radioactivity was measured. After tripping and washing the cells, add 1.5 mL of 1 M NaOH (Teknova) to the cells. In addition, the dish was incubated on ice for 5 minutes. Surface strip cell lysates were then prepared. Transferred to a counting vial. Washed the dish with cold PBS and transferred the washing solution to the counting vial. Radioactivity The gamma counter (Hidex Automatic Gamma Counter) The assay was performed. Intracellular radioactivity was measured as the sum of surface strip cell lysates + PBS wash. It's decided.

[0251] For the sample at time=0, the cells were examined for surface membrane-bound radioactivity immediately after initial antibody binding on ice. And intracellular radioactivity was assayed. Sun tests were performed for 10 minutes, 30 minutes, 1 hour, and 2 hours. Regarding the pull, after initial antibody binding, 3 mL of cell culture medium is added to each dish, and the dish The dish was placed in a 37°C humidified CO2 incubator. At each stage, the dish was incubated... Removed from the container and placed on ice. Transferred the cell culture medium to a counting vial and the cells were placed in cold PBS. Washed. The PBS wash solution was collected in a counting vial. The cells were prepared as described, surface cleaned. Membrane-bound radioactivity and intracellular radioactivity were assayed. At each time point, before cell lysis, strips were used. By incubating cells with PBS instead of stripping buffer, stripping Unpinned samples were prepared. These samples were used for stripping. Efficiency was evaluated, and the results were compared with those of stripped samples.

[0252] CPM from test samples was prepared using a known amount of In-111 labeled mAb. The CPM values ​​from the shape regression were used to convert to μCi In-111. On the cell surface membrane (stri Localization of In-111 mAb in lysed samples and intracellular samples. The occupancy rate (%) is calculated using the following formula: Localization rate % = 100 * (Sample μCi / Average total μCi) The method used to determine the total amount of μCi is the incubation medium, PBS washing solution, and glycine. This refers to the total of all recovered samples, including rinsed and lysed cells. Each data point The result is the mean ± SD of the triple reaction.

[0253] Surface-bound In-111 rapidly disappeared from the cell surface and was redistributed within the cell. The tripping technique releases approximately 80% of the cell surface-related radioactivity at time 0, and the ink By the end of the ovation, only 20% had been released by stripping, and the radioactivity Over 60% were found in the cell lysates (Figure 4).

[0254] Example 13: Efficacy in a mouse tumor xenograft model Dose determination study of anti-PSMA mAb-DOTA-AC-225: Male NSG mice (1 10 (n=8 per group) 6 LNCap cells were subcutaneously transplanted to reduce the tumor size to 100-150 mm. 3 to They were propagated. Mice were given radiation levels (10nCi, 25nCi, 70nCi, 200nCi). i) Anti-PSMA mAb-azid-DOTA- 225 Ac or isotype control, control mAb-Azid-DOTA- 225A single dose of Ac was administered intravenously. Per mouse The injection volume was adjusted to a total of 10 μg of protein using cold antibodies. Tumor measurement and body weight were monitored for one week. Recorded twice in between. Tumor size was 1,500 mm. 3 Did you euthanize the animals when they exceeded a certain threshold? They were euthanized when their weight loss exceeded 20%.

[0255] Anti-PSMA mAb-DOTA-Ac-225 is particularly effective for tumors after a single dose of high-dose radiation therapy. It exhibits growth inhibition and shows superior tumor growth inhibition compared to isotype controls at all doses. Figure 5A). Both control mAb radioactive complexes at all doses exhibited survival curves similar to those of the vehicle control. (Figure 5B; Table 7); anti-PSMA mAb complexes showed increased survival rates as radiation dose increased. The levels increased, showing a clear dose-response (Figure 5C; Table 7). When the study was terminated after 209 days, All three mice remained from the anti-PSMA mAb200nCi group and had detectable tumors. It did not show that.

[0256] [Table 7]

[0257] The embodiments of the present invention are intended to be merely illustrative, and those skilled in the art will not be able to specify the invention. To recognize or confirm numerous equivalents to the procedure using only routine experiments. This will be possible. All such equivalents are considered to be within the scope of the present invention, and the following This is included in the claims of the patent.

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

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

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

[12] The method according to

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

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

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

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

change

change

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

change

[17] The method according to

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

[18] The method according to

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

[19] The method according to

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

[20] The method according to

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

[21] The method according to

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

[22] The method according to

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

[23] The method according to

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

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

change

change

[16] above, including the structure of .

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

[26] The method according to

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

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

[16] above, and a pharmaceutically acceptable carrier.

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

[27] above to the subject.

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

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

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

change

change

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

[29] , selected from In.

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

Claims

1. A method for labeling an antibody or its antigen-binding fragment with a radioactive metal ion, a. To provide an antibody or antigen-binding fragment thereof covalently bound to a first click reaction partner, Here, the first click reaction partner contains an azide, and the antibody or its antigen-binding fragment covalently bound to the first click reaction partner is obtained by site-specific integration of the first click reaction partner through reaction with an azide-labeled sugar or azidoamine. b. To provide a radioactive complex comprising a radioactive metal ion and a chelated moiety coordinated to the radioactive metal ion, wherein the chelated moiety comprises a chelating agent covalently bonded to a second click reaction partner, and the second click reaction partner comprises an alkyne group to which strain has been induced. c. Contacting the antibody or antigen-binding fragment covalently bound to the first click reaction partner with the radioactive complex, Here, the contact is carried out without a copper catalyst, and the antibody or its antigen-binding fragment is labeled with the radioactive metal ion by reacting the first click reaction partner with the second click reaction partner. Includes, The chelating agent is of formula (I): 【Chemistry 1】 (In the formula, Z is hydrogen; R １ , R ２ , R ３ and R ４ Each of them operates independently as CHQCO ２ X is, Q is independently H, or a linker (CH ２ ), ｐ -C(O)-NH-(CH ２ ), ｑ -C(O) (where p = q = 2), and X is independently hydrogen, benzyl, or C １ ~C ４ Includes a macrocycle having an alkyl structure, Alternatively, the chelating agent comprises deferoxamine-YY, where YY is a linker covalently bonded to the second click reaction partner and the chelating agent, where YY is C(O)-(CH ２ ) ｍ -C(O)(m=2) method.

2. The method according to claim 1, wherein the antibody is an antibody that binds to human prostate-specific membrane antigen (PSMA) or an antigen-binding fragment thereof, and comprises the heavy chain (HC) complementarity-determining region (CDR) 1 sequence of SEQ ID NO: 3, the HC CDR 2 sequence of SEQ ID NO: 4, the HC CDR 3 sequence of SEQ ID NO: 5, the light chain (LC) CDR 1 sequence of SEQ ID NO: 6, the LC CDR 2 sequence of SEQ ID NO: 7, and the LC CDR 3 sequence of SEQ ID NO:

8.

3. The aforementioned radioactive metal ions ２２５ Ac, １１１ In, or ８９ The method according to claim 1, wherein the material is Zr.

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

5. The method according to claim 4, wherein the azide-labeled sugar is UDP-N-azidoacetylgalactosamine (UDP-GalNaZ) or UDP-6-azido6-deoxyGalNAc.

6. The method according to claim 4, wherein the glycosyltransferase is GalT galactosyltransferase or GalNActransferase.

7. The method according to claim 1, wherein the antibody or antigen-binding fragment covalently bound to a first click reaction partner is obtained by a method comprising: deglycosylating the antibody or antigen-binding fragment with amidase to obtain a deglycosylated antibody or antigen-binding fragment; and reacting the deglycosylated antibody or antigen-binding fragment with azidoamine in the presence of microbial transglutaminase to obtain the antibody or antigen-binding fragment covalently bound to the first click reaction partner.

8. The method according to claim 7, wherein the azidoamine is selected from 3-azidopropylamine, 6-azidohexylamine, O-(2-aminoethyl)-O'-(2-azidoethyl)tetraethylene glycol, O-(2-aminoethyl)-O'-(2-azidoethyl)pentaethylene glycol, and O-(2-aminoethyl)-O'-(2-azidoethyl)triethylene glycol.

9. The chelate portion is defined by formula (II): 【Chemistry 2】 Structure, or formula (III): 【Transformation 3】 The method according to claim 1, comprising the structure of

10. The method according to claim 3, wherein the antibody or antigen-binding fragment covalently bound to a first click reaction partner is obtained by a method comprising: trimming the antibody or antigen-binding fragment with a bacterial endoglycosidase specific to the β-1,4 linkage between core GlcNac residues in the Fc-glycosylation site of the antibody to obtain a trimmed antibody or antigen-binding fragment; and reacting the trimmed antibody or antigen-binding fragment with an azide-labeled sugar in the presence of a glycosyltransferase to obtain the antibody or antigen-binding fragment covalently bound to a first click reaction partner.

11. The method according to claim 10, wherein the azide-labeled sugar is UDP-N-azidoacetylgalactosamine (UDP-GalNaZ) or UDP-6-azido6-deoxyGalNAc.

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

13. The method according to claim 3, wherein the antibody or antigen-binding fragment covalently bound to a first click reaction partner is obtained by a method comprising: deglycosylating the antibody or antigen-binding fragment with amidase to obtain a deglycosylated antibody or antigen-binding fragment; and reacting the deglycosylated antibody or antigen-binding fragment with azidoamine in the presence of microbial transglutaminase to obtain the antibody or antigen-binding fragment covalently bound to a first click reaction partner.

14. The method according to claim 13, wherein the azidoamine is selected from 3-azidopropylamine, 6-azidohexylamine, O-(2-aminoethyl)-O'-(2-azidoethyl)tetraethylene glycol, O-(2-aminoethyl)-O'-(2-azidoethyl)pentaethylene glycol, and O-(2-aminoethyl)-O'-(2-azidoethyl)triethylene glycol.

15. The chelate portion is defined by formula (II): 【Chemistry 4】 Structure or formula (III): 【Transformation 5】 The method according to claim 3, including the structure.

16. A method for preparing a pharmaceutical composition comprising a radiolabeled polypeptide and a pharmaceutically acceptable carrier, comprising: preparing a radiolabeled polypeptide by the method of Claim 1; and obtaining the pharmaceutical composition using the radiolabeled polypeptide and a pharmaceutically acceptable carrier.

17. A method for preparing a theranostic agent comprising a radiolabeled antibody and a pharmaceutically acceptable carrier, wherein the immunological properties of the radiolabeled antibody are preserved, the method comprising: preparing a radiolabeled polypeptide by the method of Claim 1; and obtaining the theranostic agent using the radiolabeled polypeptide and a pharmaceutically acceptable carrier.

18. Formula (VIII): 【Transformation 6】 Or formula (IX): 【Transformation 7】 A method for preparing a theranostic agent having the structure of, the method comprising preparing the theranostic agent by the method of claim 1.

19. The aforementioned radioactive metal ions ３２ P, ４７ Sc, ６７ Cd, ７７ As, ８９ Sr, ９０ Y, ９９ Tc, １０５ Rh, １０９ Pd, １１１ Ag, １３１ I, １５３ Sm, １５９ Gd, １６５ Dy, １６６ Ho, １６９ Er, １７７ Lu, １８６ Re, １８８ Re, １９４ Ir, １９８ Au, １９９ Au, ２１１ At, ２１２ Pb, ２１２ Bi, ２１３ Bi, ２２３ Ra, ２２５ Ac, ２５５ Fm, ２２７ Th, ６２ Cd, ６４ Cd, ６７ Ga, ６８ Ga, ８６ Y, ８９ Zr, or １１１ The method according to claim 17, selected from In.

20. It is a combination, a. A modified polypeptide containing a polypeptide covalently bonded to a first click reaction partner, b. A radioactive complex comprising a radioactive metal ion associated with a chelated portion, wherein the chelated portion comprises a chelating agent covalently bonded to a second click reaction partner, The chelating agent is of formula (I): 【Transformation 8】 (In the formula, Z is hydrogen; R1, R2, R3, and R4 are each independently CHQCO2X, Q is independently H, or a linker (CH₂)p-C(O)-NH-(CH₂)q-C(O) (p=q=2) covalently bonded to the second click reaction partner and the chelating agent, X independently comprises a macrocycle having a structure of hydrogen, benzyl, or C1-C4 alkyl. Alternatively, the chelating agent comprises deferoxamine-YY, where YY is a linker covalently bonded to the second click reaction partner and the chelating agent, where YY is C(O)-(CH2)m-C(O)(m=2), The combination is used to label the polypeptide with the radioactive metal ion.

21. The method according to claim 3, wherein the antibody is an antibody or antigen-binding fragment thereof that binds to human prostate-specific membrane antigen (PSMA), and comprises the heavy chain (HC) complementarity-determining region (CDR) 1 sequence of SEQ ID NO: 3, the HC CDR 2 sequence of SEQ ID NO: 4, the HC CDR 3 sequence of SEQ ID NO: 5, the light chain (LC) CDR 1 sequence of SEQ ID NO: 6, the LC CDR 2 sequence of SEQ ID NO: 7, and the LC CDR 3 sequence of SEQ ID NO:

8.

22. When the antibody or antigen-binding fragment covalently bound to a first click reaction partner is obtained by reacting it with an azide-labeled sugar, the antibody or antigen-binding fragment covalently bound to the first click reaction partner is obtained by a method comprising: trimming the antibody or antigen-binding fragment with a bacterial endoglycosidase specific to the β-1,4 linkage between core GlcNac residues in the Fc-glycosylation site of the antibody to obtain a trimmed antibody or antigen-binding fragment; and reacting the trimmed antibody or antigen-binding fragment with an azide-labeled sugar in the presence of a glycosyltransferase, wherein the azide-labeled sugar is UDP-N-azidoacetylgalactosamine (UDP-GalNaz) or UDP-6-azido6-deoxyGalNAc, and the glycosyltransferase is GalT galactosyltransferase or GalNAc transferase. When the antibody or antigen-binding fragment covalently bound to a first click reaction partner is obtained by reacting with an azidoamine, the antibody or antigen-binding fragment covalently bound to the first click reaction partner is obtained by a method comprising: deglycosylating the antibody or antigen-binding fragment with amidase to obtain a deglycosylated antibody or antigen-binding fragment; and reacting the deglycosylated antibody or antigen-binding fragment with an azidoamine in the presence of microbial transglutaminase, wherein the azidoamine is selected from 3-azidopropylamine, 6-azidohexylamine, O-(2-aminoethyl)-O'-(2-azidoethyl)tetraethylene glycol, O-(2-aminoethyl)-O'-(2-azidoethyl)pentaethylene glycol, and O-(2-aminoethyl)-O'-(2-azidoethyl)triethylene glycol. The chelate portion is defined by formula (II): 【Chemistry 9】 Structure, or formula (III): 【Chemistry 10】 The method according to claim 1, comprising the structure of

23. The method according to claim 22, wherein the radioactive metal ion is a diagnostic emitter.

24. The method according to claim 22, wherein the radioactive metal ion is a therapeutic emitter.

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